CA2745339C - Coating method - Google Patents
Coating method Download PDFInfo
- Publication number
- CA2745339C CA2745339C CA2745339A CA2745339A CA2745339C CA 2745339 C CA2745339 C CA 2745339C CA 2745339 A CA2745339 A CA 2745339A CA 2745339 A CA2745339 A CA 2745339A CA 2745339 C CA2745339 C CA 2745339C
- Authority
- CA
- Canada
- Prior art keywords
- projections
- coating
- patch
- coating solution
- delivery
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000576 coating method Methods 0.000 title claims abstract description 363
- 239000011248 coating agent Substances 0.000 claims abstract description 348
- 238000000034 method Methods 0.000 claims abstract description 113
- 239000000463 material Substances 0.000 claims abstract description 83
- 238000001035 drying Methods 0.000 claims abstract description 28
- 239000000427 antigen Substances 0.000 claims description 167
- 108091007433 antigens Proteins 0.000 claims description 167
- 102000036639 antigens Human genes 0.000 claims description 167
- 239000007789 gas Substances 0.000 claims description 118
- 239000002105 nanoparticle Substances 0.000 claims description 32
- 239000004094 surface-active agent Substances 0.000 claims description 32
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 102000004169 proteins and genes Human genes 0.000 claims description 28
- 239000002671 adjuvant Substances 0.000 claims description 26
- 230000008685 targeting Effects 0.000 claims description 24
- 239000003795 chemical substances by application Substances 0.000 claims description 22
- 239000003814 drug Substances 0.000 claims description 22
- 210000002615 epidermis Anatomy 0.000 claims description 22
- 241000700605 Viruses Species 0.000 claims description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 18
- 241000894006 Bacteria Species 0.000 claims description 13
- 238000009826 distribution Methods 0.000 claims description 12
- 239000003623 enhancer Substances 0.000 claims description 12
- 229940124597 therapeutic agent Drugs 0.000 claims description 12
- 239000013566 allergen Substances 0.000 claims description 10
- 150000001875 compounds Chemical class 0.000 claims description 10
- 230000002209 hydrophobic effect Effects 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
- 102000039446 nucleic acids Human genes 0.000 claims description 9
- 108020004707 nucleic acids Proteins 0.000 claims description 9
- 150000007523 nucleic acids Chemical class 0.000 claims description 9
- 239000007787 solid Substances 0.000 claims description 9
- 210000000434 stratum corneum Anatomy 0.000 claims description 9
- 229920002134 Carboxymethyl cellulose Polymers 0.000 claims description 7
- 210000000981 epithelium Anatomy 0.000 claims description 7
- 210000004072 lung Anatomy 0.000 claims description 7
- 229910052751 metal Inorganic materials 0.000 claims description 7
- 239000002184 metal Substances 0.000 claims description 7
- 239000002245 particle Substances 0.000 claims description 7
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 claims description 6
- 229920001817 Agar Polymers 0.000 claims description 5
- 229920000936 Agarose Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 210000000270 basal cell Anatomy 0.000 claims description 5
- 150000002739 metals Chemical class 0.000 claims description 5
- 210000004877 mucosa Anatomy 0.000 claims description 5
- 210000004927 skin cell Anatomy 0.000 claims description 5
- 108010010803 Gelatin Proteins 0.000 claims description 4
- 108091005461 Nucleic proteins Proteins 0.000 claims description 4
- 239000008273 gelatin Substances 0.000 claims description 4
- 229920000159 gelatin Polymers 0.000 claims description 4
- 235000019322 gelatine Nutrition 0.000 claims description 4
- 235000011852 gelatine desserts Nutrition 0.000 claims description 4
- 244000045947 parasite Species 0.000 claims description 4
- 238000001069 Raman spectroscopy Methods 0.000 claims description 3
- 229910052786 argon Inorganic materials 0.000 claims description 3
- 239000011261 inert gas Substances 0.000 claims description 3
- 239000002096 quantum dot Substances 0.000 claims description 3
- 238000004416 surface enhanced Raman spectroscopy Methods 0.000 claims description 3
- 229920006184 cellulose methylcellulose Polymers 0.000 claims 1
- 239000000243 solution Substances 0.000 description 174
- 210000003491 skin Anatomy 0.000 description 89
- 210000004027 cell Anatomy 0.000 description 47
- 229960005486 vaccine Drugs 0.000 description 43
- 108010058846 Ovalbumin Proteins 0.000 description 36
- 108020004414 DNA Proteins 0.000 description 33
- 210000000612 antigen-presenting cell Anatomy 0.000 description 30
- 235000018102 proteins Nutrition 0.000 description 26
- 230000001580 bacterial effect Effects 0.000 description 23
- 239000010410 layer Substances 0.000 description 21
- 229940092253 ovalbumin Drugs 0.000 description 19
- 230000003612 virological effect Effects 0.000 description 19
- 239000000203 mixture Substances 0.000 description 16
- 241000699670 Mus sp. Species 0.000 description 15
- 206010028980 Neoplasm Diseases 0.000 description 15
- 210000001519 tissue Anatomy 0.000 description 15
- 239000007788 liquid Substances 0.000 description 14
- 102000005962 receptors Human genes 0.000 description 14
- 108020003175 receptors Proteins 0.000 description 14
- 240000001307 Myosotis scorpioides Species 0.000 description 13
- 230000028993 immune response Effects 0.000 description 13
- 241000699666 Mus <mouse, genus> Species 0.000 description 12
- 229920002873 Polyethylenimine Polymers 0.000 description 12
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 12
- 239000010931 gold Substances 0.000 description 12
- 229910052737 gold Inorganic materials 0.000 description 12
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 11
- 230000000975 bioactive effect Effects 0.000 description 11
- -1 can also be used Substances 0.000 description 11
- 102000040430 polynucleotide Human genes 0.000 description 11
- 108091033319 polynucleotide Proteins 0.000 description 11
- 239000002157 polynucleotide Substances 0.000 description 11
- 239000010703 silicon Substances 0.000 description 11
- 229910052710 silicon Inorganic materials 0.000 description 11
- 101710154606 Hemagglutinin Proteins 0.000 description 10
- 101710093908 Outer capsid protein VP4 Proteins 0.000 description 10
- 101710135467 Outer capsid protein sigma-1 Proteins 0.000 description 10
- 101710176177 Protein A56 Proteins 0.000 description 10
- 230000002538 fungal effect Effects 0.000 description 10
- 239000000185 hemagglutinin Substances 0.000 description 10
- 229910001868 water Inorganic materials 0.000 description 10
- 108020004705 Codon Proteins 0.000 description 9
- 239000003570 air Substances 0.000 description 9
- 238000010586 diagram Methods 0.000 description 9
- 238000009792 diffusion process Methods 0.000 description 9
- 229940079593 drug Drugs 0.000 description 9
- 239000012530 fluid Substances 0.000 description 9
- 230000001965 increasing effect Effects 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 230000004044 response Effects 0.000 description 9
- 239000003981 vehicle Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 229940021995 DNA vaccine Drugs 0.000 description 8
- 210000004207 dermis Anatomy 0.000 description 8
- 206010022000 influenza Diseases 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 229940023143 protein vaccine Drugs 0.000 description 8
- 238000001878 scanning electron micrograph Methods 0.000 description 8
- 101150029707 ERBB2 gene Proteins 0.000 description 7
- 101710100968 Receptor tyrosine-protein kinase erbB-2 Proteins 0.000 description 7
- 102100030086 Receptor tyrosine-protein kinase erbB-2 Human genes 0.000 description 7
- 238000000708 deep reactive-ion etching Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000007850 fluorescent dye Substances 0.000 description 7
- 238000002649 immunization Methods 0.000 description 7
- 229960003971 influenza vaccine Drugs 0.000 description 7
- 102000004196 processed proteins & peptides Human genes 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 239000000758 substrate Substances 0.000 description 7
- 238000002255 vaccination Methods 0.000 description 7
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical group CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 6
- 241001502567 Chikungunya virus Species 0.000 description 6
- 229920002307 Dextran Polymers 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 108010056995 Perforin Proteins 0.000 description 6
- 102000004503 Perforin Human genes 0.000 description 6
- 201000011510 cancer Diseases 0.000 description 6
- 210000004443 dendritic cell Anatomy 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000013604 expression vector Substances 0.000 description 6
- 230000003053 immunization Effects 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 230000035515 penetration Effects 0.000 description 6
- 229920000642 polymer Polymers 0.000 description 6
- 229930182490 saponin Natural products 0.000 description 6
- 235000017709 saponins Nutrition 0.000 description 6
- 230000008961 swelling Effects 0.000 description 6
- 239000003053 toxin Substances 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 241000193738 Bacillus anthracis Species 0.000 description 5
- 108010041986 DNA Vaccines Proteins 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 5
- CTKXFMQHOOWWEB-UHFFFAOYSA-N Ethylene oxide/propylene oxide copolymer Chemical compound CCCOC(C)COCCO CTKXFMQHOOWWEB-UHFFFAOYSA-N 0.000 description 5
- 241000725303 Human immunodeficiency virus Species 0.000 description 5
- 240000007472 Leucaena leucocephala Species 0.000 description 5
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 5
- 206010037742 Rabies Diseases 0.000 description 5
- 238000003491 array Methods 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 239000001768 carboxy methyl cellulose Substances 0.000 description 5
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 5
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 5
- 238000004624 confocal microscopy Methods 0.000 description 5
- 238000003618 dip coating Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 238000007918 intramuscular administration Methods 0.000 description 5
- 239000007927 intramuscular injection Substances 0.000 description 5
- 201000001441 melanoma Diseases 0.000 description 5
- 239000000546 pharmaceutical excipient Substances 0.000 description 5
- 229920001993 poloxamer 188 Polymers 0.000 description 5
- 229940044519 poloxamer 188 Drugs 0.000 description 5
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 5
- 150000007949 saponins Chemical class 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 231100000765 toxin Toxicity 0.000 description 5
- 108700012359 toxins Proteins 0.000 description 5
- 238000012546 transfer Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 206010012186 Delayed delivery Diseases 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241000233866 Fungi Species 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 206010013023 diphtheria Diseases 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 4
- 229960005542 ethidium bromide Drugs 0.000 description 4
- 238000007654 immersion Methods 0.000 description 4
- 230000003308 immunostimulating effect Effects 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 210000001821 langerhans cell Anatomy 0.000 description 4
- 210000003463 organelle Anatomy 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 210000000605 viral structure Anatomy 0.000 description 4
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- PIGTXFOGKFOFTO-PPEDVFHSSA-N CC1(C)CC[C@@]2([C@H](O)C[C@]3(C)C(=CC[C@@H]4[C@@]5(C)CCC(O[C@@H]6O[C@@H]([C@@H](O)[C@H](O)[C@H]6O)C(O)=O)[C@@](C)(C=O)[C@@H]5CC[C@@]34C)[C@@H]2C1)C(O)=O Chemical compound CC1(C)CC[C@@]2([C@H](O)C[C@]3(C)C(=CC[C@@H]4[C@@]5(C)CCC(O[C@@H]6O[C@@H]([C@@H](O)[C@H](O)[C@H]6O)C(O)=O)[C@@](C)(C=O)[C@@H]5CC[C@@]34C)[C@@H]2C1)C(O)=O PIGTXFOGKFOFTO-PPEDVFHSSA-N 0.000 description 3
- 102100025570 Cancer/testis antigen 1 Human genes 0.000 description 3
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 3
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 3
- 201000009182 Chikungunya Diseases 0.000 description 3
- 206010009944 Colon cancer Diseases 0.000 description 3
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 3
- 206010014596 Encephalitis Japanese B Diseases 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 241000606768 Haemophilus influenzae Species 0.000 description 3
- 208000017604 Hodgkin disease Diseases 0.000 description 3
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 3
- 101000856237 Homo sapiens Cancer/testis antigen 1 Proteins 0.000 description 3
- 201000005807 Japanese encephalitis Diseases 0.000 description 3
- 241000710842 Japanese encephalitis virus Species 0.000 description 3
- 108091034117 Oligonucleotide Proteins 0.000 description 3
- 102100040283 Peptidyl-prolyl cis-trans isomerase B Human genes 0.000 description 3
- 201000005702 Pertussis Diseases 0.000 description 3
- 206010039491 Sarcoma Diseases 0.000 description 3
- 208000005718 Stomach Neoplasms Diseases 0.000 description 3
- 206010043376 Tetanus Diseases 0.000 description 3
- 206010058874 Viraemia Diseases 0.000 description 3
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 235000010419 agar Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000003719 b-lymphocyte Anatomy 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000008045 co-localization Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 108010048032 cyclophilin B Proteins 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000000151 deposition Methods 0.000 description 3
- 210000005069 ears Anatomy 0.000 description 3
- 206010017758 gastric cancer Diseases 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000010255 intramuscular injection Methods 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 208000014018 liver neoplasm Diseases 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 108091070501 miRNA Proteins 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 201000005962 mycosis fungoides Diseases 0.000 description 3
- 208000002154 non-small cell lung carcinoma Diseases 0.000 description 3
- 239000011368 organic material Substances 0.000 description 3
- 230000037361 pathway Effects 0.000 description 3
- 108010044156 peptidyl-prolyl cis-trans isomerase b Proteins 0.000 description 3
- 210000000680 phagosome Anatomy 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 238000004626 scanning electron microscopy Methods 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000007480 spreading Effects 0.000 description 3
- 238000003892 spreading Methods 0.000 description 3
- 201000011549 stomach cancer Diseases 0.000 description 3
- 239000012646 vaccine adjuvant Substances 0.000 description 3
- 229940124931 vaccine adjuvant Drugs 0.000 description 3
- 238000009736 wetting Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- 102100038222 60 kDa heat shock protein, mitochondrial Human genes 0.000 description 2
- 208000024893 Acute lymphoblastic leukemia Diseases 0.000 description 2
- 208000014697 Acute lymphocytic leukaemia Diseases 0.000 description 2
- 241000256844 Apis mellifera Species 0.000 description 2
- 206010004593 Bile duct cancer Diseases 0.000 description 2
- 206010005003 Bladder cancer Diseases 0.000 description 2
- 208000003174 Brain Neoplasms Diseases 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 2
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 206010008342 Cervix carcinoma Diseases 0.000 description 2
- 108010058432 Chaperonin 60 Proteins 0.000 description 2
- 101710098119 Chaperonin GroEL 2 Proteins 0.000 description 2
- 208000006332 Choriocarcinoma Diseases 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 101000609762 Gallus gallus Ovalbumin Proteins 0.000 description 2
- 208000032612 Glial tumor Diseases 0.000 description 2
- 206010018338 Glioma Diseases 0.000 description 2
- 241000590002 Helicobacter pylori Species 0.000 description 2
- 241000700721 Hepatitis B virus Species 0.000 description 2
- 208000005176 Hepatitis C Diseases 0.000 description 2
- 208000009889 Herpes Simplex Diseases 0.000 description 2
- 208000007514 Herpes zoster Diseases 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 241000228402 Histoplasma Species 0.000 description 2
- 208000021519 Hodgkin lymphoma Diseases 0.000 description 2
- 241000282414 Homo sapiens Species 0.000 description 2
- 241000701806 Human papillomavirus Species 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 208000008839 Kidney Neoplasms Diseases 0.000 description 2
- 108700027766 Listeria monocytogenes hlyA Proteins 0.000 description 2
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 2
- 108091054437 MHC class I family Proteins 0.000 description 2
- 102000043129 MHC class I family Human genes 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 101710085938 Matrix protein Proteins 0.000 description 2
- 201000005505 Measles Diseases 0.000 description 2
- 241000712079 Measles morbillivirus Species 0.000 description 2
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 2
- 101710127721 Membrane protein Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000186359 Mycobacterium Species 0.000 description 2
- 241000187479 Mycobacterium tuberculosis Species 0.000 description 2
- 208000001894 Nasopharyngeal Neoplasms Diseases 0.000 description 2
- 206010061306 Nasopharyngeal cancer Diseases 0.000 description 2
- 206010030155 Oesophageal carcinoma Diseases 0.000 description 2
- 206010033128 Ovarian cancer Diseases 0.000 description 2
- 206010061535 Ovarian neoplasm Diseases 0.000 description 2
- 208000002193 Pain Diseases 0.000 description 2
- 241001631646 Papillomaviridae Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 229920000954 Polyglycolide Polymers 0.000 description 2
- 208000006664 Precursor Cell Lymphoblastic Leukemia-Lymphoma Diseases 0.000 description 2
- 206010060862 Prostate cancer Diseases 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 102100038358 Prostate-specific antigen Human genes 0.000 description 2
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 206010038389 Renal cancer Diseases 0.000 description 2
- 201000000582 Retinoblastoma Diseases 0.000 description 2
- 240000005384 Rhizopus oryzae Species 0.000 description 2
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 2
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 2
- 206010041067 Small cell lung cancer Diseases 0.000 description 2
- 108020004459 Small interfering RNA Proteins 0.000 description 2
- 208000021712 Soft tissue sarcoma Diseases 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 208000024313 Testicular Neoplasms Diseases 0.000 description 2
- 206010057644 Testis cancer Diseases 0.000 description 2
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 description 2
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 2
- 208000008383 Wilms tumor Diseases 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 230000005875 antibody response Effects 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 230000001640 apoptogenic effect Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000560 biocompatible material Substances 0.000 description 2
- 239000003114 blood coagulation factor Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 208000025997 central nervous system neoplasm Diseases 0.000 description 2
- 201000010881 cervical cancer Diseases 0.000 description 2
- 239000008199 coating composition Substances 0.000 description 2
- 239000011247 coating layer Substances 0.000 description 2
- 238000013270 controlled release Methods 0.000 description 2
- 210000000736 corneocyte Anatomy 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 210000000172 cytosol Anatomy 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000007598 dipping method Methods 0.000 description 2
- 239000000428 dust Substances 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 239000012645 endogenous antigen Substances 0.000 description 2
- 210000001163 endosome Anatomy 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 239000000147 enterotoxin Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000001508 eye Anatomy 0.000 description 2
- 229940037467 helicobacter pylori Drugs 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 208000005252 hepatitis A Diseases 0.000 description 2
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000004957 immunoregulator effect Effects 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000011147 inorganic material Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 201000010982 kidney cancer Diseases 0.000 description 2
- 208000032839 leukemia Diseases 0.000 description 2
- 201000005202 lung cancer Diseases 0.000 description 2
- 208000020816 lung neoplasm Diseases 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 210000004940 nucleus Anatomy 0.000 description 2
- 201000008106 ocular cancer Diseases 0.000 description 2
- 230000000242 pagocytic effect Effects 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 244000052769 pathogen Species 0.000 description 2
- 239000008177 pharmaceutical agent Substances 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 239000004633 polyglycolic acid Substances 0.000 description 2
- 229920006254 polymer film Polymers 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 230000001177 retroviral effect Effects 0.000 description 2
- 201000005404 rubella Diseases 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 229910052709 silver Inorganic materials 0.000 description 2
- 239000004332 silver Substances 0.000 description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 201000003120 testicular cancer Diseases 0.000 description 2
- 229910052719 titanium Inorganic materials 0.000 description 2
- 239000010936 titanium Substances 0.000 description 2
- 230000003614 tolerogenic effect Effects 0.000 description 2
- 206010044412 transitional cell carcinoma Diseases 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 239000003656 tris buffered saline Substances 0.000 description 2
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 description 2
- 241000701161 unidentified adenovirus Species 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 201000005112 urinary bladder cancer Diseases 0.000 description 2
- 210000003934 vacuole Anatomy 0.000 description 2
- PJRSUKFWFKUDTH-JWDJOUOUSA-N (2s)-6-amino-2-[[2-[[(2s)-2-[[(2s,3s)-2-[[(2s)-2-[[2-[[(2s)-2-[[(2s)-6-amino-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[(2-aminoacetyl)amino]-4-methylsulfanylbutanoyl]amino]propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]propanoyl]amino]acetyl]amino]propanoyl Chemical compound CSCC[C@H](NC(=O)CN)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(N)=O PJRSUKFWFKUDTH-JWDJOUOUSA-N 0.000 description 1
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- LKKMLIBUAXYLOY-UHFFFAOYSA-N 3-Amino-1-methyl-5H-pyrido[4,3-b]indole Chemical compound N1C2=CC=CC=C2C2=C1C=C(N)N=C2C LKKMLIBUAXYLOY-UHFFFAOYSA-N 0.000 description 1
- 208000030507 AIDS Diseases 0.000 description 1
- 241000238876 Acari Species 0.000 description 1
- 241001019659 Acremonium <Plectosphaerellaceae> Species 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 208000031261 Acute myeloid leukaemia Diseases 0.000 description 1
- 208000006468 Adrenal Cortex Neoplasms Diseases 0.000 description 1
- 239000012099 Alexa Fluor family Substances 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- 102400000310 Alpha-dystroglycan Human genes 0.000 description 1
- 208000037540 Alveolar soft tissue sarcoma Diseases 0.000 description 1
- 244000036975 Ambrosia artemisiifolia Species 0.000 description 1
- 235000003129 Ambrosia artemisiifolia var elatior Nutrition 0.000 description 1
- 208000004881 Amebiasis Diseases 0.000 description 1
- 206010001980 Amoebiasis Diseases 0.000 description 1
- 206010061424 Anal cancer Diseases 0.000 description 1
- 241001465677 Ancylostomatoidea Species 0.000 description 1
- 201000003076 Angiosarcoma Diseases 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 208000007860 Anus Neoplasms Diseases 0.000 description 1
- 241001351995 Aphomia sociella Species 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 101100504181 Arabidopsis thaliana GCS1 gene Proteins 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 241000239290 Araneae Species 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 206010003571 Astrocytoma Diseases 0.000 description 1
- 206010003594 Ataxia telangiectasia Diseases 0.000 description 1
- 101800001288 Atrial natriuretic factor Proteins 0.000 description 1
- 102400001282 Atrial natriuretic peptide Human genes 0.000 description 1
- 101800001890 Atrial natriuretic peptide Proteins 0.000 description 1
- 102100022717 Atypical chemokine receptor 1 Human genes 0.000 description 1
- 102100035526 B melanoma antigen 1 Human genes 0.000 description 1
- 208000010839 B-cell chronic lymphocytic leukemia Diseases 0.000 description 1
- 208000003950 B-cell lymphoma Diseases 0.000 description 1
- 208000032791 BCR-ABL1 positive chronic myelogenous leukemia Diseases 0.000 description 1
- 208000004429 Bacillary Dysentery Diseases 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 201000001178 Bacterial Pneumonia Diseases 0.000 description 1
- 206010004146 Basal cell carcinoma Diseases 0.000 description 1
- 241000235579 Basidiobolus Species 0.000 description 1
- 206010004272 Benign hydatidiform mole Diseases 0.000 description 1
- 241001465178 Bipolaris Species 0.000 description 1
- 241000335423 Blastomyces Species 0.000 description 1
- 241001674044 Blattodea Species 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000013585 Bombesin Human genes 0.000 description 1
- 108010051479 Bombesin Proteins 0.000 description 1
- 241000255789 Bombyx mori Species 0.000 description 1
- 206010005949 Bone cancer Diseases 0.000 description 1
- 208000018084 Bone neoplasm Diseases 0.000 description 1
- 241000588832 Bordetella pertussis Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 208000003508 Botulism Diseases 0.000 description 1
- 206010006143 Brain stem glioma Diseases 0.000 description 1
- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 description 1
- 108010061299 CXCR4 Receptors Proteins 0.000 description 1
- 102000012000 CXCR4 Receptors Human genes 0.000 description 1
- 102000000905 Cadherin Human genes 0.000 description 1
- 108050007957 Cadherin Proteins 0.000 description 1
- 102000055006 Calcitonin Human genes 0.000 description 1
- 108060001064 Calcitonin Proteins 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 206010007279 Carcinoid tumour of the gastrointestinal tract Diseases 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 241000255930 Chironomidae Species 0.000 description 1
- 241000498849 Chlamydiales Species 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 208000005243 Chondrosarcoma Diseases 0.000 description 1
- 208000010833 Chronic myeloid leukaemia Diseases 0.000 description 1
- 241000983417 Chrysomya bezziana Species 0.000 description 1
- 241001668502 Cladophialophora carrionii Species 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 241000193468 Clostridium perfringens Species 0.000 description 1
- 241000193449 Clostridium tetani Species 0.000 description 1
- 241000254173 Coleoptera Species 0.000 description 1
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 1
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 1
- 102100025680 Complement decay-accelerating factor Human genes 0.000 description 1
- 206010053138 Congenital aplastic anaemia Diseases 0.000 description 1
- 206010052465 Congenital poikiloderma Diseases 0.000 description 1
- 241001480517 Conidiobolus Species 0.000 description 1
- 101710139375 Corneodesmosin Proteins 0.000 description 1
- 241000557626 Corvus corax Species 0.000 description 1
- 241000186216 Corynebacterium Species 0.000 description 1
- 241001315231 Cricotopus trifasciatus Species 0.000 description 1
- 206010011409 Cross infection Diseases 0.000 description 1
- 241001527609 Cryptococcus Species 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 241000223208 Curvularia Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 206010011968 Decreased immune responsiveness Diseases 0.000 description 1
- 208000001490 Dengue Diseases 0.000 description 1
- 206010012310 Dengue fever Diseases 0.000 description 1
- 241000702421 Dependoparvovirus Species 0.000 description 1
- 208000008334 Dermatofibrosarcoma Diseases 0.000 description 1
- 206010057070 Dermatofibrosarcoma protuberans Diseases 0.000 description 1
- 241000238710 Dermatophagoides Species 0.000 description 1
- 108010061629 Dermatophagoides pteronyssinus antigen p 1 Proteins 0.000 description 1
- 108010061608 Dermatophagoides pteronyssinus antigen p 2 Proteins 0.000 description 1
- 208000008743 Desmoplastic Small Round Cell Tumor Diseases 0.000 description 1
- 206010064581 Desmoplastic small round cell tumour Diseases 0.000 description 1
- 101100216227 Dictyostelium discoideum anapc3 gene Proteins 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 208000006402 Ductal Carcinoma Diseases 0.000 description 1
- 108010071885 Dystroglycans Proteins 0.000 description 1
- 108010031111 EBV-encoded nuclear antigen 1 Proteins 0.000 description 1
- 241001115402 Ebolavirus Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 208000001976 Endocrine Gland Neoplasms Diseases 0.000 description 1
- 206010014733 Endometrial cancer Diseases 0.000 description 1
- 206010014759 Endometrial neoplasm Diseases 0.000 description 1
- 101001095863 Enterobacteria phage T4 RNA ligase 1 Proteins 0.000 description 1
- 101710126487 Envelope glycoprotein B Proteins 0.000 description 1
- 206010014967 Ependymoma Diseases 0.000 description 1
- 101800003838 Epidermal growth factor Proteins 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 241001480035 Epidermophyton Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108090000394 Erythropoietin Proteins 0.000 description 1
- 102000003951 Erythropoietin Human genes 0.000 description 1
- 101000867232 Escherichia coli Heat-stable enterotoxin II Proteins 0.000 description 1
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- 208000000461 Esophageal Neoplasms Diseases 0.000 description 1
- 208000006168 Ewing Sarcoma Diseases 0.000 description 1
- 241000223664 Exophiala jeanselmei Species 0.000 description 1
- 241000306559 Exserohilum Species 0.000 description 1
- 101150048576 FIM3 gene Proteins 0.000 description 1
- 241000045671 Falciformispora senegalensis Species 0.000 description 1
- 201000001342 Fallopian tube cancer Diseases 0.000 description 1
- 208000013452 Fallopian tube neoplasm Diseases 0.000 description 1
- 201000004939 Fanconi anemia Diseases 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 201000008808 Fibrosarcoma Diseases 0.000 description 1
- 101710154643 Filamentous hemagglutinin Proteins 0.000 description 1
- 201000006353 Filariasis Diseases 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 1
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 1
- 241000122862 Fonsecaea Species 0.000 description 1
- 241000122864 Fonsecaea pedrosoi Species 0.000 description 1
- 241000223221 Fusarium oxysporum Species 0.000 description 1
- 241000427940 Fusarium solani Species 0.000 description 1
- 208000022072 Gallbladder Neoplasms Diseases 0.000 description 1
- 201000003741 Gastrointestinal carcinoma Diseases 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- 244000168141 Geotrichum candidum Species 0.000 description 1
- 235000017388 Geotrichum candidum Nutrition 0.000 description 1
- 241000699694 Gerbillinae Species 0.000 description 1
- 102400000321 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 description 1
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 description 1
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 description 1
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 108010068370 Glutens Proteins 0.000 description 1
- 108010051696 Growth Hormone Proteins 0.000 description 1
- 206010066476 Haematological malignancy Diseases 0.000 description 1
- 208000001258 Hemangiosarcoma Diseases 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 208000033640 Hereditary breast cancer Diseases 0.000 description 1
- 241000487062 Histoplasma capsulatum var. capsulatum Species 0.000 description 1
- 241001354006 Histoplasma capsulatum var. duboisii Species 0.000 description 1
- 101000678879 Homo sapiens Atypical chemokine receptor 1 Proteins 0.000 description 1
- 101000874316 Homo sapiens B melanoma antigen 1 Proteins 0.000 description 1
- 101000856022 Homo sapiens Complement decay-accelerating factor Proteins 0.000 description 1
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 description 1
- 101001076292 Homo sapiens Insulin-like growth factor II Proteins 0.000 description 1
- 101000578784 Homo sapiens Melanoma antigen recognized by T-cells 1 Proteins 0.000 description 1
- 101001005720 Homo sapiens Melanoma-associated antigen 4 Proteins 0.000 description 1
- 101000961414 Homo sapiens Membrane cofactor protein Proteins 0.000 description 1
- 101000581981 Homo sapiens Neural cell adhesion molecule 1 Proteins 0.000 description 1
- 101000835745 Homo sapiens Teratocarcinoma-derived growth factor 1 Proteins 0.000 description 1
- 101000823316 Homo sapiens Tyrosine-protein kinase ABL1 Proteins 0.000 description 1
- 241000308514 Hortaea werneckii Species 0.000 description 1
- 241000714260 Human T-lymphotropic virus 1 Species 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 208000006937 Hydatidiform mole Diseases 0.000 description 1
- 208000037147 Hypercalcaemia Diseases 0.000 description 1
- 108010007403 Immediate-Early Proteins Proteins 0.000 description 1
- 108010043496 Immunoglobulin Idiotypes Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100025947 Insulin-like growth factor II Human genes 0.000 description 1
- 102100034349 Integrase Human genes 0.000 description 1
- 102100022337 Integrin alpha-V Human genes 0.000 description 1
- 102100022297 Integrin alpha-X Human genes 0.000 description 1
- 108010047852 Integrin alphaVbeta3 Proteins 0.000 description 1
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 1
- 102000015271 Intercellular Adhesion Molecule-1 Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 206010061252 Intraocular melanoma Diseases 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- 102100031413 L-dopachrome tautomerase Human genes 0.000 description 1
- 101710093778 L-dopachrome tautomerase Proteins 0.000 description 1
- 108010001831 LDL receptors Proteins 0.000 description 1
- 241000526687 Lacazia loboi Species 0.000 description 1
- 108010000851 Laminin Receptors Proteins 0.000 description 1
- 102000002297 Laminin Receptors Human genes 0.000 description 1
- 206010069698 Langerhans' cell histiocytosis Diseases 0.000 description 1
- 206010023825 Laryngeal cancer Diseases 0.000 description 1
- 241000190144 Lasiodiplodia theobromae Species 0.000 description 1
- 208000007764 Legionnaires' Disease Diseases 0.000 description 1
- 208000018142 Leiomyosarcoma Diseases 0.000 description 1
- 241000222732 Leishmania major Species 0.000 description 1
- 208000004554 Leishmaniasis Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 201000011062 Li-Fraumeni syndrome Diseases 0.000 description 1
- 241000144128 Lichtheimia corymbifera Species 0.000 description 1
- 206010061523 Lip and/or oral cavity cancer Diseases 0.000 description 1
- 206010062038 Lip neoplasm Diseases 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 1
- 241000736227 Lucilia sericata Species 0.000 description 1
- 102000009151 Luteinizing Hormone Human genes 0.000 description 1
- 108010073521 Luteinizing Hormone Proteins 0.000 description 1
- 208000016604 Lyme disease Diseases 0.000 description 1
- 208000031422 Lymphocytic Chronic B-Cell Leukemia Diseases 0.000 description 1
- 206010025282 Lymphoedema Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 108010010995 MART-1 Antigen Proteins 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 241001444203 Madurella mycetomatis Species 0.000 description 1
- 241000555688 Malassezia furfur Species 0.000 description 1
- 208000004059 Male Breast Neoplasms Diseases 0.000 description 1
- 208000032271 Malignant tumor of penis Diseases 0.000 description 1
- 241000375796 Medicopsis romeroi Species 0.000 description 1
- 208000000172 Medulloblastoma Diseases 0.000 description 1
- 108010071463 Melanoma-Specific Antigens Proteins 0.000 description 1
- 102000007557 Melanoma-Specific Antigens Human genes 0.000 description 1
- 102100025077 Melanoma-associated antigen 4 Human genes 0.000 description 1
- 208000002030 Merkel cell carcinoma Diseases 0.000 description 1
- 108010057081 Merozoite Surface Protein 1 Proteins 0.000 description 1
- 206010027406 Mesothelioma Diseases 0.000 description 1
- 241001480037 Microsporum Species 0.000 description 1
- 208000003445 Mouth Neoplasms Diseases 0.000 description 1
- 208000005647 Mumps Diseases 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 101100481584 Mus musculus Tlr1 gene Proteins 0.000 description 1
- 241000257159 Musca domestica Species 0.000 description 1
- 241000204031 Mycoplasma Species 0.000 description 1
- 201000003793 Myelodysplastic syndrome Diseases 0.000 description 1
- 208000033761 Myelogenous Chronic BCR-ABL Positive Leukemia Diseases 0.000 description 1
- 208000033776 Myeloid Acute Leukemia Diseases 0.000 description 1
- 208000014767 Myeloproliferative disease Diseases 0.000 description 1
- 208000012266 Needlestick injury Diseases 0.000 description 1
- 208000034176 Neoplasms, Germ Cell and Embryonal Diseases 0.000 description 1
- 241001547451 Neoscytalidium dimidiatum Species 0.000 description 1
- 241000322250 Neotestudina rosatii Species 0.000 description 1
- 108090000028 Neprilysin Proteins 0.000 description 1
- 102000003729 Neprilysin Human genes 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000015336 Nerve Growth Factor Human genes 0.000 description 1
- 102000007339 Nerve Growth Factor Receptors Human genes 0.000 description 1
- 108010032605 Nerve Growth Factor Receptors Proteins 0.000 description 1
- 102100027347 Neural cell adhesion molecule 1 Human genes 0.000 description 1
- 206010029260 Neuroblastoma Diseases 0.000 description 1
- 208000009905 Neurofibromatoses Diseases 0.000 description 1
- 102000019315 Nicotinic acetylcholine receptors Human genes 0.000 description 1
- 108050006807 Nicotinic acetylcholine receptors Proteins 0.000 description 1
- 208000004485 Nijmegen breakage syndrome Diseases 0.000 description 1
- 208000015914 Non-Hodgkin lymphomas Diseases 0.000 description 1
- 208000010505 Nose Neoplasms Diseases 0.000 description 1
- 206010029803 Nosocomial infection Diseases 0.000 description 1
- 102000011931 Nucleoproteins Human genes 0.000 description 1
- 108010061100 Nucleoproteins Proteins 0.000 description 1
- 241000243985 Onchocerca volvulus Species 0.000 description 1
- 241001421411 Onychocola Species 0.000 description 1
- 206010057444 Oropharyngeal neoplasm Diseases 0.000 description 1
- 241000150452 Orthohantavirus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 108060006580 PRAME Proteins 0.000 description 1
- 102000036673 PRAME Human genes 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 241000526686 Paracoccidioides brasiliensis Species 0.000 description 1
- 208000000821 Parathyroid Neoplasms Diseases 0.000 description 1
- 108090000445 Parathyroid hormone Proteins 0.000 description 1
- 102000003982 Parathyroid hormone Human genes 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000002471 Penile Neoplasms Diseases 0.000 description 1
- 206010034299 Penile cancer Diseases 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 108010081690 Pertussis Toxin Proteins 0.000 description 1
- 241001531356 Phialophora verrucosa Species 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 241001326499 Piedraia hortae Species 0.000 description 1
- 208000007913 Pituitary Neoplasms Diseases 0.000 description 1
- 206010035148 Plague Diseases 0.000 description 1
- 108010001014 Plasminogen Activators Proteins 0.000 description 1
- 102000001938 Plasminogen Activators Human genes 0.000 description 1
- 241000223960 Plasmodium falciparum Species 0.000 description 1
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 1
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 1
- 101710183389 Pneumolysin Proteins 0.000 description 1
- 206010035718 Pneumonia legionella Diseases 0.000 description 1
- 208000000474 Poliomyelitis Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 101710194807 Protective antigen Proteins 0.000 description 1
- 101710192141 Protein Nef Proteins 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- 241000235525 Rhizomucor pusillus Species 0.000 description 1
- 241000606651 Rickettsiales Species 0.000 description 1
- 208000000791 Rothmund-Thomson syndrome Diseases 0.000 description 1
- 241000710799 Rubella virus Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 208000004337 Salivary Gland Neoplasms Diseases 0.000 description 1
- 206010061934 Salivary gland cancer Diseases 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 206010039438 Salmonella Infections Diseases 0.000 description 1
- 241000825258 Scopulariopsis brevicaulis Species 0.000 description 1
- 206010040070 Septic Shock Diseases 0.000 description 1
- 208000009359 Sezary Syndrome Diseases 0.000 description 1
- 208000021388 Sezary disease Diseases 0.000 description 1
- 241000607764 Shigella dysenteriae Species 0.000 description 1
- 241000607762 Shigella flexneri Species 0.000 description 1
- 206010040550 Shigella infections Diseases 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 241000254179 Sitophilus granarius Species 0.000 description 1
- 208000000453 Skin Neoplasms Diseases 0.000 description 1
- 102100038803 Somatotropin Human genes 0.000 description 1
- 101001039853 Sonchus yellow net virus Matrix protein Proteins 0.000 description 1
- 241001149963 Sporothrix schenckii Species 0.000 description 1
- 241000713675 Spumavirus Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 241000193996 Streptococcus pyogenes Species 0.000 description 1
- 108010011834 Streptolysins Proteins 0.000 description 1
- 101710137302 Surface antigen S Proteins 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 108091008874 T cell receptors Proteins 0.000 description 1
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 1
- 208000031673 T-Cell Cutaneous Lymphoma Diseases 0.000 description 1
- 208000000389 T-cell leukemia Diseases 0.000 description 1
- 208000028530 T-cell lymphoblastic leukemia/lymphoma Diseases 0.000 description 1
- 241000255588 Tephritidae Species 0.000 description 1
- 102100026404 Teratocarcinoma-derived growth factor 1 Human genes 0.000 description 1
- 108010055044 Tetanus Toxin Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 208000000728 Thymus Neoplasms Diseases 0.000 description 1
- 208000024770 Thyroid neoplasm Diseases 0.000 description 1
- 208000002474 Tinea Diseases 0.000 description 1
- 241000130764 Tinea Species 0.000 description 1
- 208000007712 Tinea Versicolor Diseases 0.000 description 1
- 206010056131 Tinea versicolour Diseases 0.000 description 1
- 102000002689 Toll-like receptor Human genes 0.000 description 1
- 108020000411 Toll-like receptor Proteins 0.000 description 1
- 206010044248 Toxic shock syndrome Diseases 0.000 description 1
- 231100000650 Toxic shock syndrome Toxicity 0.000 description 1
- 241000223996 Toxoplasma Species 0.000 description 1
- 201000005485 Toxoplasmosis Diseases 0.000 description 1
- 108010009583 Transforming Growth Factors Proteins 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 241000045663 Trematosphaeria grisea Species 0.000 description 1
- 206010044608 Trichiniasis Diseases 0.000 description 1
- 241000223238 Trichophyton Species 0.000 description 1
- 241000223230 Trichosporon Species 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 102000005937 Tropomyosin Human genes 0.000 description 1
- 108010030743 Tropomyosin Proteins 0.000 description 1
- 241000223109 Trypanosoma cruzi Species 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 108010061610 Tva receptor Proteins 0.000 description 1
- 102000046255 Type III Sodium-Phosphate Cotransporter Proteins Human genes 0.000 description 1
- 108091006286 Type III sodium-phosphate co-transporters Proteins 0.000 description 1
- 208000037386 Typhoid Diseases 0.000 description 1
- 102100022596 Tyrosine-protein kinase ABL1 Human genes 0.000 description 1
- 206010046431 Urethral cancer Diseases 0.000 description 1
- 206010046458 Urethral neoplasms Diseases 0.000 description 1
- 208000008385 Urogenital Neoplasms Diseases 0.000 description 1
- 102000012349 Uroplakins Human genes 0.000 description 1
- 108010061861 Uroplakins Proteins 0.000 description 1
- 208000002495 Uterine Neoplasms Diseases 0.000 description 1
- 201000005969 Uveal melanoma Diseases 0.000 description 1
- 108010000134 Vascular Cell Adhesion Molecule-1 Proteins 0.000 description 1
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 241000711975 Vesicular stomatitis virus Species 0.000 description 1
- 241000256856 Vespidae Species 0.000 description 1
- 208000014070 Vestibular schwannoma Diseases 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 241000219977 Vigna Species 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108010059722 Viral Fusion Proteins Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000713325 Visna/maedi virus Species 0.000 description 1
- 208000004354 Vulvar Neoplasms Diseases 0.000 description 1
- 208000033559 Waldenström macroglobulinemia Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000001015 X-ray lithography Methods 0.000 description 1
- 101001001642 Xenopus laevis Serine/threonine-protein kinase pim-3 Proteins 0.000 description 1
- 208000003152 Yellow Fever Diseases 0.000 description 1
- 241000607479 Yersinia pestis Species 0.000 description 1
- UZQJVUCHXGYFLQ-AYDHOLPZSA-N [(2s,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-4-[(2r,3r,4s,5r,6r)-4-[(2s,3r,4s,5r,6r)-3,5-dihydroxy-6-(hydroxymethyl)-4-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxy-3,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-6-(hy Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O)O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2[C@@]1(C=O)C)C)(C)CC(O)[C@]1(CCC(CC14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O[C@H]4[C@@H]([C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)[C@H](O)[C@@H](CO)O4)O)[C@H](O)[C@@H](CO)O3)O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O UZQJVUCHXGYFLQ-AYDHOLPZSA-N 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 208000004064 acoustic neuroma Diseases 0.000 description 1
- 208000017733 acquired polycythemia vera Diseases 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 208000002517 adenoid cystic carcinoma Diseases 0.000 description 1
- 102000030621 adenylate cyclase Human genes 0.000 description 1
- 108060000200 adenylate cyclase Proteins 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 208000008524 alveolar soft part sarcoma Diseases 0.000 description 1
- 239000012080 ambient air Substances 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 235000003484 annual ragweed Nutrition 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000001455 anti-clotting effect Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 201000011165 anus cancer Diseases 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000013011 aqueous formulation Substances 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 229940065181 bacillus anthracis Drugs 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 229920002988 biodegradable polymer Polymers 0.000 description 1
- 239000004621 biodegradable polymer Substances 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- DNDCVAGJPBKION-DOPDSADYSA-N bombesin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1NC2=CC=CC=C2C=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C(C)C)C1=CN=CN1 DNDCVAGJPBKION-DOPDSADYSA-N 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 235000006263 bur ragweed Nutrition 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 208000002458 carcinoid tumor Diseases 0.000 description 1
- NSQLIUXCMFBZME-MPVJKSABSA-N carperitide Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)=O)[C@@H](C)CC)C1=CC=CC=C1 NSQLIUXCMFBZME-MPVJKSABSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 208000019065 cervical carcinoma Diseases 0.000 description 1
- 210000003679 cervix uteri Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 201000002797 childhood leukemia Diseases 0.000 description 1
- 208000011654 childhood malignant neoplasm Diseases 0.000 description 1
- 210000001612 chondrocyte Anatomy 0.000 description 1
- 208000032852 chronic lymphocytic leukemia Diseases 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 229940047120 colony stimulating factors Drugs 0.000 description 1
- 235000003488 common ragweed Nutrition 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 108010047295 complement receptors Proteins 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 201000007241 cutaneous T cell lymphoma Diseases 0.000 description 1
- 208000017763 cutaneous neuroendocrine carcinoma Diseases 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 238000002716 delivery method Methods 0.000 description 1
- 239000000412 dendrimer Substances 0.000 description 1
- 210000001787 dendrite Anatomy 0.000 description 1
- 229920000736 dendritic polymer Polymers 0.000 description 1
- 208000025729 dengue disease Diseases 0.000 description 1
- 238000005137 deposition process Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- PSLWZOIUBRXAQW-UHFFFAOYSA-M dimethyl(dioctadecyl)azanium;bromide Chemical compound [Br-].CCCCCCCCCCCCCCCCCC[N+](C)(C)CCCCCCCCCCCCCCCCCC PSLWZOIUBRXAQW-UHFFFAOYSA-M 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000004070 electrodeposition Methods 0.000 description 1
- 230000012202 endocytosis Effects 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 210000001842 enterocyte Anatomy 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 108700004025 env Genes Proteins 0.000 description 1
- 210000003979 eosinophil Anatomy 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 229940105423 erythropoietin Drugs 0.000 description 1
- 201000004101 esophageal cancer Diseases 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- 238000005530 etching Methods 0.000 description 1
- 229960003699 evans blue Drugs 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 201000008819 extrahepatic bile duct carcinoma Diseases 0.000 description 1
- 208000024519 eye neoplasm Diseases 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 229940028334 follicle stimulating hormone Drugs 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 210000000285 follicular dendritic cell Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108700004026 gag Genes Proteins 0.000 description 1
- 201000010175 gallbladder cancer Diseases 0.000 description 1
- 210000000973 gametocyte Anatomy 0.000 description 1
- 150000002270 gangliosides Chemical class 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 208000003884 gestational trophoblastic disease Diseases 0.000 description 1
- 201000007116 gestational trophoblastic neoplasm Diseases 0.000 description 1
- 201000006592 giardiasis Diseases 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 235000021312 gluten Nutrition 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 229940047650 haemophilus influenzae Drugs 0.000 description 1
- 201000009277 hairy cell leukemia Diseases 0.000 description 1
- 210000003128 head Anatomy 0.000 description 1
- 201000010536 head and neck cancer Diseases 0.000 description 1
- 208000014829 head and neck neoplasm Diseases 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000002607 hemopoietic effect Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 208000025581 hereditary breast carcinoma Diseases 0.000 description 1
- 201000008298 histiocytosis Diseases 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- 230000000887 hydrating effect Effects 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 230000005661 hydrophobic surface Effects 0.000 description 1
- 230000000148 hypercalcaemia Effects 0.000 description 1
- 208000030915 hypercalcemia disease Diseases 0.000 description 1
- 201000006866 hypopharynx cancer Diseases 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940068935 insulin-like growth factor 2 Drugs 0.000 description 1
- 230000002608 insulinlike Effects 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 210000005133 interdigitating dendritic cell Anatomy 0.000 description 1
- 229940047122 interleukins Drugs 0.000 description 1
- 201000002313 intestinal cancer Diseases 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 208000022013 kidney Wilms tumor Diseases 0.000 description 1
- 210000004684 kidney tubule cell Anatomy 0.000 description 1
- 210000001865 kupffer cell Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 206010023841 laryngeal neoplasm Diseases 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 201000006721 lip cancer Diseases 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 206010024627 liposarcoma Diseases 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 241000238565 lobster Species 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 229940040129 luteinizing hormone Drugs 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 208000002502 lymphedema Diseases 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000001755 magnetron sputter deposition Methods 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 201000003175 male breast cancer Diseases 0.000 description 1
- 208000010907 male breast carcinoma Diseases 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 208000026045 malignant tumor of parathyroid gland Diseases 0.000 description 1
- 208000016847 malignant urinary system neoplasm Diseases 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000012768 mass vaccination Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 210000002752 melanocyte Anatomy 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 229920000609 methyl cellulose Polymers 0.000 description 1
- 239000001923 methylcellulose Substances 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 230000002025 microglial effect Effects 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000000921 morphogenic effect Effects 0.000 description 1
- 238000000465 moulding Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 206010051747 multiple endocrine neoplasia Diseases 0.000 description 1
- 208000010805 mumps infectious disease Diseases 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000003387 muscular Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 206010028537 myelofibrosis Diseases 0.000 description 1
- 210000003098 myoblast Anatomy 0.000 description 1
- DDOVBCWVTOHGCU-QMXMISKISA-N n-[(e,2s,3r)-3-hydroxy-1-[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxynonadec-4-en-2-yl]octadecanamide Chemical compound CCCCCCCCCCCCCCCCCC(=O)N[C@H]([C@H](O)\C=C\CCCCCCCCCCCCCC)CO[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O DDOVBCWVTOHGCU-QMXMISKISA-N 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 208000037830 nasal cancer Diseases 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 201000008026 nephroblastoma Diseases 0.000 description 1
- 229940053128 nerve growth factor Drugs 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 208000007538 neurilemmoma Diseases 0.000 description 1
- 201000004931 neurofibromatosis Diseases 0.000 description 1
- 108091008685 nuclear receptors type I Proteins 0.000 description 1
- 108091008686 nuclear receptors type II Proteins 0.000 description 1
- 102000027507 nuclear receptors type II Human genes 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- 229920002113 octoxynol Polymers 0.000 description 1
- 201000002575 ocular melanoma Diseases 0.000 description 1
- VQWNELVFHZRFIB-UHFFFAOYSA-N odn 1826 Chemical compound O=C1NC(=O)C(C)=CN1C(O1)CC(O)C1COP(O)(=O)OC1CC(N2C(NC(=O)C(C)=C2)=O)OC1COP(O)(=O)OC1CC(N2C3=C(C(NC(N)=N3)=O)N=C2)OC1COP(O)(=O)OC1CC(N2C(N=C(N)C=C2)=O)OC1COP(O)(=O)OC1CC(N2C3=NC=NC(N)=C3N=C2)OC1COP(O)(=O)OC1CC(N2C3=C(C(NC(N)=N3)=O)N=C2)OC1COP(O)(=O)OC1CC(N2C(NC(=O)C(C)=C2)=O)OC1COP(O)(=O)OC1CC(N2C(N=C(N)C=C2)=O)OC1COP(O)(=O)OC1CC(N2C(N=C(N)C=C2)=O)OC1COP(O)(=O)OC1CC(N2C(NC(=O)C(C)=C2)=O)OC1COP(O)(=O)OC(C(O1)COP(O)(=O)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=O)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=O)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=O)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=O)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=O)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=O)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=O)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=O)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(O)=O)CC1N1C=C(C)C(=O)NC1=O VQWNELVFHZRFIB-UHFFFAOYSA-N 0.000 description 1
- 208000002042 onchocerciasis Diseases 0.000 description 1
- 201000005443 oral cavity cancer Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 201000006958 oropharynx cancer Diseases 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 201000002530 pancreatic endocrine carcinoma Diseases 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- 239000000199 parathyroid hormone Substances 0.000 description 1
- 201000001219 parotid gland cancer Diseases 0.000 description 1
- 239000013618 particulate matter Substances 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 108010021753 peptide-Gly-Leu-amide Proteins 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 108010021711 pertactin Proteins 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 208000005814 piedra Diseases 0.000 description 1
- 230000003114 pinocytic effect Effects 0.000 description 1
- 201000002511 pituitary cancer Diseases 0.000 description 1
- 201000000508 pityriasis versicolor Diseases 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 108700004029 pol Genes Proteins 0.000 description 1
- 229920001481 poly(stearyl methacrylate) Polymers 0.000 description 1
- 208000037244 polycythemia vera Diseases 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229940068977 polysorbate 20 Drugs 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 208000025638 primary cutaneous T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 208000003476 primary myelofibrosis Diseases 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 235000009736 ragweed Nutrition 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012429 release testing Methods 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 210000000844 retinal pigment epithelial cell Anatomy 0.000 description 1
- 208000013860 rhabdoid tumor of the kidney Diseases 0.000 description 1
- 201000009410 rhabdomyosarcoma Diseases 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 210000003079 salivary gland Anatomy 0.000 description 1
- 206010039447 salmonellosis Diseases 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 201000004409 schistosomiasis Diseases 0.000 description 1
- 206010039667 schwannoma Diseases 0.000 description 1
- 210000003786 sclera Anatomy 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 235000015170 shellfish Nutrition 0.000 description 1
- 229940007046 shigella dysenteriae Drugs 0.000 description 1
- 201000005113 shigellosis Diseases 0.000 description 1
- 108010061514 sialic acid receptor Proteins 0.000 description 1
- 201000000849 skin cancer Diseases 0.000 description 1
- 201000008261 skin carcinoma Diseases 0.000 description 1
- 201000002314 small intestine cancer Diseases 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 206010062261 spinal cord neoplasm Diseases 0.000 description 1
- 210000003046 sporozoite Anatomy 0.000 description 1
- 206010041823 squamous cell carcinoma Diseases 0.000 description 1
- 208000017572 squamous cell neoplasm Diseases 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 201000009862 superficial mycosis Diseases 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 206010042863 synovial sarcoma Diseases 0.000 description 1
- 229940118376 tetanus toxin Drugs 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 230000002992 thymic effect Effects 0.000 description 1
- 201000009377 thymus cancer Diseases 0.000 description 1
- 201000002510 thyroid cancer Diseases 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 208000003982 trichinellosis Diseases 0.000 description 1
- 201000007588 trichinosis Diseases 0.000 description 1
- 229940031418 trivalent vaccine Drugs 0.000 description 1
- 208000029387 trophoblastic neoplasm Diseases 0.000 description 1
- 201000002311 trypanosomiasis Diseases 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
- 201000008297 typhoid fever Diseases 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 210000000689 upper leg Anatomy 0.000 description 1
- 201000004435 urinary system cancer Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
- 208000037965 uterine sarcoma Diseases 0.000 description 1
- 206010046885 vaginal cancer Diseases 0.000 description 1
- 208000013139 vaginal neoplasm Diseases 0.000 description 1
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 1
- 229940118696 vibrio cholerae Drugs 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 201000005102 vulva cancer Diseases 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D—PROCESSES FOR APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
- B05D3/00—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials
- B05D3/04—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials by exposure to gases
- B05D3/0466—Pretreatment of surfaces to which liquids or other fluent materials are to be applied; After-treatment of applied coatings, e.g. intermediate treating of an applied coating preparatory to subsequent applications of liquids or other fluent materials by exposure to gases the gas being a non-reacting gas
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0023—Drug applicators using microneedles
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
- A61M37/00—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin
- A61M37/0015—Other apparatus for introducing media into the body; Percutany, i.e. introducing medicines into the body by diffusion through the skin by using microneedles
- A61M2037/0046—Solid microneedles
Abstract
A method of coating a material onto projections provided on a patch. The method includes applying a coating solution containing the material to at least the projections and drying the coating solution to at least the projections using a gas flow.
Description
2 PCT/AU2008/001903 COATING METHOD
Background of the Invention The present invention relates to a method of coating and in particular to coating projections provided on a patch.
Description of the Prior Art The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
It is known to provide patches including a number of projections thereon to allow bioactive material to be administered to a subject. Such arrays of projections or needles on a patch are an increasingly effective way of delivering therapeutic agents or biomarkers since there is minimal or no pain, little- or no injury from the needle and highly reduced possibility of cross infection. The solid projections or needles on a patch can be coated with drugs or macromolecules. These can be subsequently delivered to a desired target by the penetration of the projections or needles into the skin.
For example, W02005/072630 describes devices for delivering bioactive materials and other stimuli to living cells, methods of manufacture of the device and various uses of the device, including a number of medical applications. The device comprises a plurality of projections which can penetrate a body surface so as to deliver the bioactive material or stimulus to the required site. The projections are typically solid and the delivery end section of the projection is so dimensioned as to be capable of insertion into targeted cells or specific sites to deliver the bioactive material or stimulus without appreciable damage to the targeted cells or specific sites therein.
Various methods of coating patches are also known. For example, microprojection arrays are known to be coated by being dipped into a coating solution reservoir through dip-holes at the same spacing as the microneedles in the array (Harvinder S. Gill and Mark R
Prausnitz, Journal of Controlled Release, 117 (2007) 227-237 and Harvinder S. Gill and Mark R
Prausnitz, Pharmaceutical Research, 24 (2007) 1369-1380). The coating solution contains carboxymethylcellulose (CMC) sodium salt, poloxamer 188 and a suitable drug.
The size of the projection is around 700 m in length, 160 m in width and 50 m in thickness. The distance between projections is over a few mm.
Microneedle arrays can also be coated with a drug by partial immersion in aqueous formulations containing drug and polysorbate 20 (Michel Cormier, Bonny Johnson, Mahmoud Ameri, Kofi Nyam, Luz Libiran, Dee Dee Zhang, Pete Daddona, Journal of Controlled Release 97 (2004) 503-511). Each microneedle is arrowhead-shaped with a length of 200 m, a maximal width of 170 m, and a thickness of 35 m. The density of projections is 321 projections/cm2.
Microprojection arrays are also known to be coated by immersion in an aqueous solution of ovabulmin or OVA (James A. Matriano, Michel Cormier, Juanita Johnson, Wendy A.
Young, Margaret Buttery, Kofi Nyam, and Peter E. Daddona, Pharmaceutical Research, 19 (2002) 63-70). The arrays were air-dried for 1 h at ambient conditions. The length of each microprojection is 330 m. The density of projections is 190 projections/cm2.
W002/074173 and US-6,855,372 describe an apparatus and method for selectively applying an agent-containing liquid coating to skin piercing microprojections (10). The coating solution is applied to the skin piercing microprojections (10) using a coating technique which selectively coats only the skin piercing microprojections (10) and not the substrate (12) from which the microprojections (10) extend, and then dried. The coating method includes providing an agent-containing coating liquid and conveying the liquid to a liquid holding surface having a coating transfer region. The depth of the coating liquid at the coating transfer region is precisely controlled. The microprojections are then immersed to a predetermined level in the coating liquid. The liquid that coats the microprojections (10) is then dried to form a solid agent-containing coating on the microprojections (10).
US2005/197308 relates to a pharmaceutical agent delivery device having a skin piercing protrusion that is typically about 100 to 400 m in length. The protrusion can be coated with a solid biodegradable reservoir medium containing the pharmaceutical agent.
Background of the Invention The present invention relates to a method of coating and in particular to coating projections provided on a patch.
Description of the Prior Art The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that the prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
It is known to provide patches including a number of projections thereon to allow bioactive material to be administered to a subject. Such arrays of projections or needles on a patch are an increasingly effective way of delivering therapeutic agents or biomarkers since there is minimal or no pain, little- or no injury from the needle and highly reduced possibility of cross infection. The solid projections or needles on a patch can be coated with drugs or macromolecules. These can be subsequently delivered to a desired target by the penetration of the projections or needles into the skin.
For example, W02005/072630 describes devices for delivering bioactive materials and other stimuli to living cells, methods of manufacture of the device and various uses of the device, including a number of medical applications. The device comprises a plurality of projections which can penetrate a body surface so as to deliver the bioactive material or stimulus to the required site. The projections are typically solid and the delivery end section of the projection is so dimensioned as to be capable of insertion into targeted cells or specific sites to deliver the bioactive material or stimulus without appreciable damage to the targeted cells or specific sites therein.
Various methods of coating patches are also known. For example, microprojection arrays are known to be coated by being dipped into a coating solution reservoir through dip-holes at the same spacing as the microneedles in the array (Harvinder S. Gill and Mark R
Prausnitz, Journal of Controlled Release, 117 (2007) 227-237 and Harvinder S. Gill and Mark R
Prausnitz, Pharmaceutical Research, 24 (2007) 1369-1380). The coating solution contains carboxymethylcellulose (CMC) sodium salt, poloxamer 188 and a suitable drug.
The size of the projection is around 700 m in length, 160 m in width and 50 m in thickness. The distance between projections is over a few mm.
Microneedle arrays can also be coated with a drug by partial immersion in aqueous formulations containing drug and polysorbate 20 (Michel Cormier, Bonny Johnson, Mahmoud Ameri, Kofi Nyam, Luz Libiran, Dee Dee Zhang, Pete Daddona, Journal of Controlled Release 97 (2004) 503-511). Each microneedle is arrowhead-shaped with a length of 200 m, a maximal width of 170 m, and a thickness of 35 m. The density of projections is 321 projections/cm2.
Microprojection arrays are also known to be coated by immersion in an aqueous solution of ovabulmin or OVA (James A. Matriano, Michel Cormier, Juanita Johnson, Wendy A.
Young, Margaret Buttery, Kofi Nyam, and Peter E. Daddona, Pharmaceutical Research, 19 (2002) 63-70). The arrays were air-dried for 1 h at ambient conditions. The length of each microprojection is 330 m. The density of projections is 190 projections/cm2.
W002/074173 and US-6,855,372 describe an apparatus and method for selectively applying an agent-containing liquid coating to skin piercing microprojections (10). The coating solution is applied to the skin piercing microprojections (10) using a coating technique which selectively coats only the skin piercing microprojections (10) and not the substrate (12) from which the microprojections (10) extend, and then dried. The coating method includes providing an agent-containing coating liquid and conveying the liquid to a liquid holding surface having a coating transfer region. The depth of the coating liquid at the coating transfer region is precisely controlled. The microprojections are then immersed to a predetermined level in the coating liquid. The liquid that coats the microprojections (10) is then dried to form a solid agent-containing coating on the microprojections (10).
US2005/197308 relates to a pharmaceutical agent delivery device having a skin piercing protrusion that is typically about 100 to 400 m in length. The protrusion can be coated with a solid biodegradable reservoir medium containing the pharmaceutical agent.
-3-However, the coating quality of these techniques can be poor as a large area around the edges and the tips of the projections remain poorly coated.
Furthermore, previous systems have focussed on coating large and very sparsely packed projections. Such techniques often prove to be unsuccessful when coating small and densely packed projections, which are often hydrophobic, reducing the effectiveness of traditional coating techniques. Hydrophobic properties occur when such type of microstructures are patterned on a hydrophobic substrate. Consequently, coating using straightforward immersion often results in projections being uncoated.
Attempts to overcome poor coating by attaching thiols to microprojection patches, with DNA
and positively charged polymers then being deposited in alternate layers, have been tried.
The DNA amount deposited on patches increased exponentially with the increase of number of DNA layers on patches. However, in-vitro release experiments showed that the release in phosphate-buffered saline (PBS) solution was extremely slow. For example, 12 layers of DNA on both projections and base of each patch can only release 2.25 g DNA
after overnight dipping in 1.5 M NaCl solution (physiological salt concentration is only 0.15 M or 0.9%). Whilst no release of DNA can be detected after overnight dipping of coated patches in 0.15 M NaCl solution.
For successful vaccine delivery systems, effective dry coating of the vaccine on the patch projections in a controlled manner, followed by the rapid, subsequent release of an effective amount of the vaccine in the skin after application of the patch, is required.
Further, whilst it is desirable to employ patches that have smaller projections or needles, effectively coating these using existing techniques is difficult.
Summary of the Present Invention The present invention seeks to substantially overcome, or at least ameliorate, one or more disadvantages of existing arrangements.
In a first broad form the present invention seeks to provide a method of coating a material onto projections provided on a patch, wherein the method includes:
a) applying a coating solution containing the material to at least the projections; and, b) drying the coating solution using a gas flow.
Furthermore, previous systems have focussed on coating large and very sparsely packed projections. Such techniques often prove to be unsuccessful when coating small and densely packed projections, which are often hydrophobic, reducing the effectiveness of traditional coating techniques. Hydrophobic properties occur when such type of microstructures are patterned on a hydrophobic substrate. Consequently, coating using straightforward immersion often results in projections being uncoated.
Attempts to overcome poor coating by attaching thiols to microprojection patches, with DNA
and positively charged polymers then being deposited in alternate layers, have been tried.
The DNA amount deposited on patches increased exponentially with the increase of number of DNA layers on patches. However, in-vitro release experiments showed that the release in phosphate-buffered saline (PBS) solution was extremely slow. For example, 12 layers of DNA on both projections and base of each patch can only release 2.25 g DNA
after overnight dipping in 1.5 M NaCl solution (physiological salt concentration is only 0.15 M or 0.9%). Whilst no release of DNA can be detected after overnight dipping of coated patches in 0.15 M NaCl solution.
For successful vaccine delivery systems, effective dry coating of the vaccine on the patch projections in a controlled manner, followed by the rapid, subsequent release of an effective amount of the vaccine in the skin after application of the patch, is required.
Further, whilst it is desirable to employ patches that have smaller projections or needles, effectively coating these using existing techniques is difficult.
Summary of the Present Invention The present invention seeks to substantially overcome, or at least ameliorate, one or more disadvantages of existing arrangements.
In a first broad form the present invention seeks to provide a method of coating a material onto projections provided on a patch, wherein the method includes:
a) applying a coating solution containing the material to at least the projections; and, b) drying the coating solution using a gas flow.
-4-Typically the method includes at least one of.
a) distributing the coating solution over the projections at least in part using the gas flow; and, b) moving coating solution on patches to wet all projections using a gas flow, thereby coating at least part of the projections.
Typically the method includes selecting coating properties to thereby control the distribution of coating over the projections.
Typically coating properties are selected so that at least one of.
a) at least tips of the projections are coated; and, b) at least target sections of the projections are coated.
Typically the projections are provided on a surface of the patch, and wherein the method includes selecting coating properties to thereby vary at least one of:
a) an amount of coating on a surface of the patch; and, b) an amount of coating on the projections.
Typically the coating properties include at least one of a) a gas flow rate;
b) patch properties;
c) coating solution properties; and, d) a drying time.
Typically the patch properties include at least one of.
a) projection size;
b) projection shape;
c) projection spacing; and, d) projection materials.
Typically the coating solution properties include at least one of:
a) a surface tension; and, b) a viscosity.
Typically the material includes at least one of:
a) distributing the coating solution over the projections at least in part using the gas flow; and, b) moving coating solution on patches to wet all projections using a gas flow, thereby coating at least part of the projections.
Typically the method includes selecting coating properties to thereby control the distribution of coating over the projections.
Typically coating properties are selected so that at least one of.
a) at least tips of the projections are coated; and, b) at least target sections of the projections are coated.
Typically the projections are provided on a surface of the patch, and wherein the method includes selecting coating properties to thereby vary at least one of:
a) an amount of coating on a surface of the patch; and, b) an amount of coating on the projections.
Typically the coating properties include at least one of a) a gas flow rate;
b) patch properties;
c) coating solution properties; and, d) a drying time.
Typically the patch properties include at least one of.
a) projection size;
b) projection shape;
c) projection spacing; and, d) projection materials.
Typically the coating solution properties include at least one of:
a) a surface tension; and, b) a viscosity.
Typically the material includes at least one of:
-5-a) nanoparticles;
b) a nucleic acid or protein;' c) an antigen, allergen, or adjuvant;
d) parasites, bacteria, viruses, or virus-like particles;
e) quantum dots, SERS tags, Raman tags or other nanobiosensors;
f) metals or metallic compounds; and, g) molecules, elements or compounds.
Typically the coating solution includes a therapeutic agent.
Typically the therapeutic agent is at least one of a) DNA having a concentration of between 0.01 mg/ml and 5 mg/ml; and, b) protein having a concentration of between 0.01 mg/ml and 50 mg/ml Typically the coating solution includes at least one of:
a) a viscosity enhancer;
b) a surfactant; and, c) an adjuvant.
Typically the adjuvant acts as a surfactant.
Typically at least one of:
a) the viscosity agent is 0% to 90% of the coating solution; and, b) the surfactant is 0% to 90% of the coating solution.
Typically the viscosity agent is at least one of MC, CMC, gelatin, agar, and agarose.
Typically the coating solution has a viscosity of between 10"3 Pa=S and 1 Pa-S.
Typically the coating solution has a viscosity of 0.01-0.06 Pa=S
Typically the coating solution has a surface tension of between 0.023 N/m and 0.073 N/m.
Typically the coating solution has a surface tension of 0.03-0.04 N/m.
Typically the gas flow has a gas flow rate of between 6m/s and 10 m/s.
b) a nucleic acid or protein;' c) an antigen, allergen, or adjuvant;
d) parasites, bacteria, viruses, or virus-like particles;
e) quantum dots, SERS tags, Raman tags or other nanobiosensors;
f) metals or metallic compounds; and, g) molecules, elements or compounds.
Typically the coating solution includes a therapeutic agent.
Typically the therapeutic agent is at least one of a) DNA having a concentration of between 0.01 mg/ml and 5 mg/ml; and, b) protein having a concentration of between 0.01 mg/ml and 50 mg/ml Typically the coating solution includes at least one of:
a) a viscosity enhancer;
b) a surfactant; and, c) an adjuvant.
Typically the adjuvant acts as a surfactant.
Typically at least one of:
a) the viscosity agent is 0% to 90% of the coating solution; and, b) the surfactant is 0% to 90% of the coating solution.
Typically the viscosity agent is at least one of MC, CMC, gelatin, agar, and agarose.
Typically the coating solution has a viscosity of between 10"3 Pa=S and 1 Pa-S.
Typically the coating solution has a viscosity of 0.01-0.06 Pa=S
Typically the coating solution has a surface tension of between 0.023 N/m and 0.073 N/m.
Typically the coating solution has a surface tension of 0.03-0.04 N/m.
Typically the gas flow has a gas flow rate of between 6m/s and 10 m/s.
-6-Typically the method includes selecting a gas flow rate in accordance with gas properties.
Typically the gas properties include a gas density.
Typically the gas flow includes at least one of.
a) nitrogen;
b) argon;
c) air flow; and, d) an inert gas.
Typically the gas flow is induced at least in part by extracting gas from a container containing the patch.
Typically the method includes coating the projections a number of times.
Typically the method includes:
a) coating the surface a first time using a first set of coating parameters;
and, b) coating the surface at least a second time using a second set of coating parameters different to the first set of coating parameters.
Typically the method includes applying between 5 and 15 d of coating solution to the patch.
Typically the patch has a surface area of approximately 0.16 cm2.
Typically the projections have a density of between 1,000-30,000 projections/cm2.
Typically the projections have a density of 20,000 projections/cm2 Typically the projections have a length of between 10 to 400 m.
Typically the projections have a length of 90 m Typically the projections have a radius of curvature of greater than 1 m.
Typically the projections have a radius of curvature greater than 5 m.
Typically the projections include a support section and a targeting section.
Typically the gas properties include a gas density.
Typically the gas flow includes at least one of.
a) nitrogen;
b) argon;
c) air flow; and, d) an inert gas.
Typically the gas flow is induced at least in part by extracting gas from a container containing the patch.
Typically the method includes coating the projections a number of times.
Typically the method includes:
a) coating the surface a first time using a first set of coating parameters;
and, b) coating the surface at least a second time using a second set of coating parameters different to the first set of coating parameters.
Typically the method includes applying between 5 and 15 d of coating solution to the patch.
Typically the patch has a surface area of approximately 0.16 cm2.
Typically the projections have a density of between 1,000-30,000 projections/cm2.
Typically the projections have a density of 20,000 projections/cm2 Typically the projections have a length of between 10 to 400 m.
Typically the projections have a length of 90 m Typically the projections have a radius of curvature of greater than 1 m.
Typically the projections have a radius of curvature greater than 5 m.
Typically the projections include a support section and a targeting section.
-7-Typically the targeting section has a diameter of less than at least one of:
a) 50 gm; and, b) 100 gm;
c) 150 gm; and, d) 400 gm.
Typically a length for the targeting section is at least:
a) less than 50 gm; and, b) less than 100 gm; and, c) less than 300 gm.
Typically a length for the support section is at least one of:
a) for epidermal delivery < 200 gm;
b) for dermal cell delivery < 1000 gm;
c) for delivery to basal cells in the epithelium of the mucosa 600-800 gm;
and, d) for lung delivery of the order of 100 gm.
Typically a length for the support section is at least one of:
a) for epidermal delivery greater than the thickness of the Stratum Corneum;
b) for dermal cell delivery greater than the thickness of epidermis;
c) for delivery to basal cells in the epithelium of the mucosa greater than a thickness of upper epithelium; and, d) for lung delivery of the order of 100 gm in this case.
Typically the projections are solid.
Typically the projections are non-porous and non-hollow.
Typically the patch is at least one of:
a) hydrophobic; and, b) hydrophilic.
In a second broad form the present invention seeks to provide a method of coating a material onto projections provided on a patch, wherein the method includes:
a) applying a coating solution containing the material to at least the projections; and, -g-b) distributing the coating solution, over the projections at least in part using a gas flow.
Typically the method further includes drying the coating solution using the gas flow.
In a third broad form the present invention seeks to provide a coating solution for coating a material onto projections on a patch, the coating solution including Quillaja saponins acting as a surfactant and a vaccine adjuvant.
Typically the Quillaja saponins include at least one of QA, QS-21, QS-7 and other purified saponin adjuvants.
Typically the coating solution includes an adjuvant that is an Immunostimulating complex.
Typically the Immunostimulating complex includes ISCOMATRIX.
Typically the coating solution includes at least one of:
a) a viscosity enhancer;
b) a surfactant; and, c) an adjuvant.
In a fourth broad form the present invention seeks to provide a coating solution for coating a material onto projections on a patch, the coating solution including nanoparticles.
Typically the nanoparticles are multilayered nanoparticles.
Typically the nanoparticles includes layers including at least one of:
a) cell targeting molecules; and, b) cell-entry facilitating molecules.
Typically the nanoparticles include layers including intracellular targeting molecules.
In a fifth broad form the present invention seeks to provide a patch for use in medical procedures, the patch including a number of projections thereon, the projections having a coating applied thereto using the method of the first broad form of the invention.
In a sixth broad form the present invention seeks to provide a method performing a medical procedure, the method including applying a patch to 'a subject, the patch being a patch according to the fifth broad form of the invention.
Typically the method includes hydrating a surface of the subject and applying the patch to the hydrated surface.
It will be appreciated that the broad forms of the invention may be used individually or in combination.
Brief Description of the Drawings An example of the present invention will now be described with reference to the accompanying drawings, in which: -Figures IA and lB are schematic side and plan views, respectively, of an example of device for delivery of material to targets within a body;
Figure I C is a schematic diagram of an example of the device of Figure 1 A in use;
Figures 1D to IF are schematic diagrams of examples of projections used in the device of Figure IA;
Figures 2A and 2B are schematic plan views of examples of a fluid spreading out and of a droplet forming on a hydrophobic patch, respectively;
Figures 2C and 2D are schematic side views of the examples of Figures 2A and 2B in a Wenzel state;
Figures 2E and 2F are schematic side views of the examples of Figures 2A and 2B in a Cassie state;
Figure 3 is a graph of an example of a relationship between a coating ratio and a gas flow rate;
Figure 4 is an example of a secondary electron image of a patch having a gold coating;
Figures 5A and 5B are schematic diagrams of a first example of apparatus for providing gas flow;
Figures 5C and 5D are schematic diagrams of a second example of apparatus for providing gas flow;
Figure 6A is a schematic diagram view of a third example of apparatus for providing gas flow;
Figure 6B is a schematic diagram view of a fourth example of apparatus for providing gas flow;
Figures 7A and 7B are schematic diagrams illustrating the transfer of coating material to a subject, in use;
Figures 8A and 8B are schematic diagrams of an example of a well provided at the base of a projection;
Figures 9A and 9C show examples of secondary electron images of patches with 60 m and 90 m long projections, respectively;
Figures 9B and 9D show examples of corresponding backscattered electron images for the patches of Figures 9A and 9C, respectively;
Figures 9E and 9F show examples of scanning electron microscopy (SEM) images of 60 m long projections dip coated and dried in air;
Figures 10A and 10B show examples of SEM images of 35 m long projections before and after coating, respectively, using a gas flow;
Figures 10C and 10D show examples of SEM images of 60 m long projections before and after coating, respectively, using a gas flow;
Figures 1OE and 10F show examples of secondary and backscattered electron images, respectively, of 90 m long projections after coating using a gas flow;
Figures 11A, 11B and 11C show examples of individual 35 m long projections before coating, after coating using a gas flow and an overlay of the images, respectively;
Figures 11D, 11E and 11F show examples of individual 60 m long projections before coating, after coating using a gas flow and an overlay of the images, respectively;
Figures 11G, 11H and 111 show fluorescence images of individual 90 m long projections from a DiD coating, the reflection and an overlay of the images, respectively;
Figure 12A shows an example of an SEM image of a patch coated using a gas flow;
Figures 12B and 12C show example of secondary and backscattered electron high-magnification images of projections coated using a gas flow;
Figures 13A to 13D show examples of secondary electron images for patches coated with OVA DNA vaccine on 90 m projections with concentrations of MC of 0%, 0.5%, 1%
and 2.5%, respectively;
Figures 14A and 14B show examples of secondary electron and backscattered electron images, respectively, for patches coated with OVA protein vaccine on 90 m projections, with concentrations of QA of 0.2%;
Figures 14C and 14D show examples of secondary electron and backscattered electron images, respectively, for patches coated with OVA protein vaccine on 90 m projections, with concentrations of QA of 1%; , Figures 15A and 15B show examples of secondary electron and backscattered electron images, respectively, for an example of the tip of the patch coated with of OVA protein on 90 m projections;
Figures 15C and 15D show examples of secondary electron and backscattered electron images, respectively, for an example of the patch coated by applying 10 gl of OVA protein coating solution dried in air;
Figure 16 shows an example of patches and measured local delivery characteristics in mouse epidermis;
Figure 17A is a graph of an example of release intensity values from a 70kDa payload in living skin;
Figure J 7B is a graph of an example of release diffusion coefficients kinetics from a 70 kDa payload in living skin;
Figure 17C is a schematic diagram illustrating an interrogation space for the measurements of Figures 17A and 17B;
Figure 18 is an example of comparative results of serum samples for five mice vaccinated with chicken egg albumin protein using a syringe and needle, or a protein coated patch;
Figure 19A is a graph showing an example of ELISA antibody reactivity for different intramuscular needle and syringe vaccine doses, and for 0.04 g vaccine delivered using a patch having projections coated using a gas flow;
Figure 19B shows graphs of example of Hemagglutinin Inhibition assays (HI) performed for different intramuscular needle and syringe vaccine doses, and for 0.04 ug vaccine delivered using a patch having projections coated using a gas flow for Wisconsin A, Malaysia B, and New Caledonia A;
Figure 20 shows graphs of examples of total IgG, IgGl and IgG2a responses induced by coated nanopatches;
Figure 21A shows examples of (a) the morphology of a patch, (b)-(d) the projections on the patch, (e)-(f) the patch after being antigen coated, (g)-(h) the coated patch after being applied on mouse ear for antigen delivery, (i)-(m) the penetration of the coated patch on mouse ear skin, and (n) the delivery of coating in the mouse ear skin;
Figure 21B shows examples of (a)-(c) the delivery of coating in mouse skin and the following diffusion after the coating being delivered in mouse ear skin, and (d)-(g) the migration of cells after the mouse ear being treated by antigen coated nanopatches;
Figure 21C shows an example of a nanopatch generated immune response and protection from Chikungunya viral challenge; and, Figures 22A and 22B show an example of the size distribution of PEI/DNA
nanoparticles (N:P ratio of 5:1);
Figures 22C and 22D show an example of the coating of polyethylenimine (PEI)/DNA
nanoparticles on patch projections before and after use respectively;
Figure 22E shows an example agarose gel analysis for original and reconstituted PEI:DNA
nanoparticles for a variety of formulations including different N:P ratios (0:1, 5:1, and 9:1);
and, Figures 22F and 22G are example transfection images obtained using the patch of Figure 22C.
Detailed Description of the Preferred Embodiments An example of a device for delivering material to targets within a body will now be described with reference to Figures 1A to 1F.
In this example, the device is in the form of patch 100 having a number of projections 110 provided on a surface 121 of a substrate 120. The projections 110 and substrate 120 may be formed from any suitable material, but in one example, are formed from a silicon type material, allowing the device to be fabricated using processes such as vapour deposition, silicon etching, Deep Reactive Ion Etching (DRIE), or the like. The projections are therefore typically solid, non-porous and non-hollow, although this is not essential.
In the example shown, the patch has a width W and a breadth B with the projections 110 being separated by spacing S.
In use, the patch 100 is positioned against a surface of a subject, allowing the projections to enter the surface and provide material to one or more targets therein. An example of this is shown in Figure I C.
In this example, the patch 100 is urged against a subject's skin shown generally at 150, so that the projections 110 pierce the Stratum Corneum 160, and enter the Viable Epidermis 170 to reach targets of interest, shown generally at 180. However, this is not essential and the patch can be used to deliver material to any part or region in the subject.
It will be appreciated that the projections can have a variety of shapes, and examples of suitable projection shapes are shown in more detail in Figures 1D, lE and IF.
In one example, the projection includes a targeting section 111, intended to deliver the material or stimulus to targets within the body, and a support section 112 for supporting the targeting section 111. However, this is not essential, and a single element may be used.
In the example of Figure 1D, the projection is formed from a conically shaped member, which tapers gradually along its entire length. In this example, the targeting section 111 is therefore defined to be the part of the projection having a diameter of less than d2.
In Figures lE and IF, the structure of the projection may vary along its length to provide a defined targeting section l II with a designed structure. In the example of Figure IE, the targeting section 111 is- in the form of a substantially cylindrical shape, such that the diameter dl is approximately equal to the diameter d2, with a tapered support section, such that the diameter d2 is smaller than the diameter d3. In contrast, in the example of Figure IF, the targeting section 111 is. in the form of taper such that the diameter di is smaller than the diameter d2, with a cylindrical support section, such that the diameter d2 is substantially equal to the diameter d3.
In general, the support section 112 has a length a, whilst the targeting section 111 has a length 1. The diameter of the tip is indicated by d1, whilst the diameter of the support section base is given by d3.
In use, the device can be used to deliver material to specific targets within the body or more generally to the blood supply, or, tissue within the body and the configuration of the device will tend to depend on its intended use.
Thus, for example, if the patch is configured so as to ensure material is delivered to specific targets such as cells, then it may be necessary to select a more specific arrangement of projections than if delivery is provided more, generally to the blood. To achieve this, the device can be provided with a particular configuration of patch parameters to ensure specific targeting. The patch parameters can include the number of projections N, the spacing S
between projections, and the projection size and shape. This is described in more detail in co-pending application US SN-11 /496053.
In one specific example, a patch having a surface area of approximately 0.16 cm2 has projections provided at a density of between 1,000-30,000 projections/cm2, and typically at a density of approximately 20,000 projections/cm2. However, alternative dimensions can be used. ' For example, a patch for an animal such as a mouse may have a surface area of 0.32 to 0.48 cm2, whereas as a patch for a human may have a surface area of approximately 1 cm2.
A variety of surface areas can be achieved by mounting a suitable number and arrangement of patches on a common substrate.
The projections typically have a length of between 10 to 200 gm and typically 90 gm with a radius of curvature of greater than 1 gm and more typically greater than 5 gm.
However, it will be appreciated that other dimensions may be used.
If distinct targeting section and support sections are provided, the targeting section typically has a diameter of less than 1 gm and more typically less than 0.5 gm. The length of the targeting section is typically less than 100 gm, less than 10 gm and typically less than 5 gm.
The length of the support section typically varies depending on the location of the target within the subject. Example lengths include less than 200 gm for epidermal delivery, less than 1000 gm for dermal cell delivery, 600-800 gm for delivery to basal cells in the epithelium of the mucosa and approximately 100 gm for lung delivery.
In order to allow delivery of material to the subject, it is necessary to provide a coating on at least the projections. In one example, coating is achieved by applying a solution containing the material to at least the projections. This may be achieved in any one of a number of manners. Thus, for example, the solution can be applied by dripping the solution onto the patch. Alternatively however other techniques may be used, such as immersion of the patch in solution.
In one example, the gas flow can be used to help ensure even distribution of material over the entire patch. This is particularly useful when the combination of patch and coating solution properties prevent the coating solution from wetting the projections. When coating solution is applied to a surface it can either spread out, or remain as a droplet, and can also fill the space between the projections (known as a "Wenzel" state), or rest on the top of the projections (known as a "Cassie" state). Examples of this will now be described will respect to Figures 2A to 2F.
In the example of Figure 2A the coating solution has properties, such as surface tension and viscosity that allow the coating solution 200 to spread out over the patch 100. In the example of Figure 2B the properties are such that prevents the solution 200 spreading out over the patch and projections. In this example, when solution is applied to the patch, the solution forms a droplet 210.
Examples of these scenarios in the Wenzel and Cassie states are shown in Figures 2C to 2F.
As shown in Figure 2C, the coating solution has spread out in the Wenzel state, so that the coating solution 200 flows over the surface 121 of the patch 100 between the projections 110.
As a result, it is possible to completely immerse the projections 110 by simply adding more solution until the solution level 201 rises above the level of the projections 110.
In the example of Figure 2D, the coating has remained confined in the Wenzel state. Despite being in the Wenzel state, not all of the projections 110 are completely wetted.
In the example of Figure 2E, even though the coating solution has spread out, but by virtue of being in the Cassie state, not all of the projections 110 are completely wetted as the droplet rests on top of the projections 110. Similarly, in the example of Figure 2F, as the coating solution is in the Cassie state, again not all of the projections 110 are completely wetted as the droplet rests on top of the projections 110.
Accordingly, in some instances, the projections 110 can remain un-immersed, meaning they will not be coated when the solution dries. However, using the gas flow, this can urge the coating solution around the surface of the patch, thereby ensuring that the projections are completely wetted.
Thus, in some of the example patch configurations described above, the patch is hydrophobic so that the contact angle of coating solution on patches is greater than 90 degrees, meaning the coating solution can not spread on patches.
In this case, gas flow allows a small volume of coating solution to be distributed over the patch to thereby thoroughly wet all projections. This avoids the need to immerse the entire patch surface in coating solution as well as allowing a small volume of coating solution to be distributed over the patch to thoroughly wet all projections, thereby reducing the amount of coating solution required to coat a,patch.
In one example, when coat 0.16 cm2 patches with 60 m needles, over 20 l coating solution is needed to cover all projections. However, the using of gas flow can control the movement of 6 l coating solution to wet all projections and achieve uniform coating.
Even in the event that coating solution initially wets the projections, previous drying techniques often leave the projections uncoated. The reason for this is that the coating solution covers many projections due to capillary action, and slowly disperses from the projections during drying under ambient .conditions. During the slow drying process, the coating solution drips off from the projections to the base of patches, meaning the projections will not be coated once the coating solution dries. This is undesirable as it reduces the ability of the patch to deliver material to a subject. In particular, maximising coating on the projections increases the rate of transfer of material to the subject, as well as maximising the amount of material on the patch that is delivered.
Accordingly, in one example, the coating solution is dried using a gas flow, to thereby remove the coating solution between projections, reduce the drying time and consequently reduce the chance of coating solution dispersing from the projections, and thereby ensure that the projections remain coated as the coating solution dries.
The gas flow could also be provided in a variety of manners. For example, this could be achieved by using a gas jet directed towards the patch. Whilst any gas may be used, in one example the gas is nitrogen as this is substantially inert and will not therefore react with the solution, whilst also being readily available. It will be appreciated that other inert gases, such as argon, can also be used, as well as air flow or other types of gas flow. In one example, the gas selected will depend on the reactivity of the coating material. As an alternative to the use of a gas jet however, flow could be induced by extracting gas from a container containing the patch.
When performing the coating process it is typical to select coating properties, such as gas flow rate, ' solution properties such as the solution viscosity and surface tension, and optionally a drying time, to thereby control the distribution of coating over the projections 110.
For example, the degree to which the projections are wetted will also depend on the coating solution properties. Thus, for example, if a higher viscosity solution is used, this will tend to adhere more strongly to the projections, and hence allow a greater thickness of coating to be achieved. However, a higher viscosity coating solution may require an increased gas flow to allow adequate distribution over the patch.
In the case of surface tension, if the surface tension is too great, the coating solution will not be effective at wetting the projections, reducing the effectiveness of coating. A lower surface tension will increase the ability of the coating solution to wet the projections, allowing better coating, although too low a surface tension and the coating solution can rest primarily on the surface of patches reducing coating of the projection tips.
In addition to this, the solution properties will also have an impact on the drying process. For example, if a thicker viscosity coating solution is used this reduces the likelihood of coating run-off during the drying process, but may increase the drying time.
Additional control is also achieved using the gas flow rate. Thus, a higher gas flow rate can increase the degree to which coating solution is distributed on the patch, and/or can reduce the drying time.
Appropriate selection of the coating properties can be used to ensure at least the projections are coated, as well'as to allow the thickness of coating on the projections to be controlled.
This can also be used to vary properties such as the relative amounts of coating on the patch surface 121 and on the projections 110, which can be characterised by a coating ratio based on a ratio of an amount of coating on the projections 110 against an amount of coating on the patch surface 121.
It will also be appreciated that the degree to which the patch is hydrophobic will depend on the patch configuration and in particular, on patch parameters such as the projection size and shape and the projection spacing S. Accordingly, when performing a coating process, it is typical to first determine patch properties and then use this information to allow appropriate coating properties to be selected.
In general the coating solution includes at least a material such as a therapeutic agent and examples of suitable materials include:
= nanoparticles;
= a nucleic acid or protein;
= an antigen, allergen, or adjuvant;
= parasites, bacteria, viruses,,or virus-like particles;
= quantum dots, SERS tags, Raman tags or other nanobiosensors;
= metals or metallic compounds; and, = molecules, elements or compounds.
Examples of preferred formulations include, a solution containing DNA having a concentration of between 0.01 mg/ml and 5 mg/ml or protein having a concentration of between 0.01 and 50 mg/ml.
The agent or other material is typically- either dissolved in a suitable solvent or held in suspension in a suitable carrier fluid, as will be appreciated by those skilled in the art. In one example, the solvent is acetone, although alternatively water or other suitable solvents can be used. The resulting surface tension in pure acetone solution and pure aqueous solution is between 0.023 N/m (acetone) and 0.073 N/m (water).
The solution properties are also typically controlled through the addition of one or more other agents such as a viscosity enhancer, a surfactant, and an adjuvant. It will be appreciated that other additives such as detergents may also be used. These ingredients can be provided in a range of different- concentrations. For example, the viscosity enhancer or surfactant can form between 0% and 90% of the coating solution.
A range of different viscosity enhancers can be used and examples include MC, CMC, gelatin, agar, and agarose and any other viscosity agents. The solution typically has a viscosity of between 10"3 Pa-S and 1 Pa-S. In one example, using a coating solution containing 1-2% MC, which results in suitable uniform coatings, resulting in a viscosity within the range 0.011 (1%) - 0.055 (2%) Pa-S.
Similarly, a range of different surfactants can be used to modify the surface tension of the coating solution, such as any surfactant or any suitable agent that changes surface tension, and that is biocompatible at a low concentration.
Surfactants are wetting agents that lower the surface tension of a liquid, allowing easier spreading, and lower the interfacial tension between two liquids. The term 'surfactant' is a blend of "surface acting agent". Surfactants are usually organic compounds that are amphiphilic, meaning they contain both hydrophobic groups ("tails") and hydrophilic groups ("heads"). Therefore, they are soluble in both organic solvents and water.
Surfactants may be used as the surface tension of the coating solution becomes dominant on a micron-scale, so the surfactant reduces the surface tension of the solution, which helps solution, wet the surface of patch projections, thereby improving coating quality.
Furthermore, a viscosity enhancer can increase the viscosity of coating solution and therefore increase the thickness of coating.
Example coating solutions will be described in more detail below.
Once the coating solution has been formed, the patch can be coated either by dripping the coating solution onto the patch or by immersing the patch in the coating solution. Typically, the amount of coating solution used to coat a patch is between 5 l to 15 l, for patches similar to those outlined above.
Once the coating solution is deposited on the patch, the patch- may be allowed to rest, for example in a sealed environment, to assist with wetting of projections, although this is not essential and may depend on the nature of the deposition process. Following this, or otherwise, a gas jet is used to evenly disperse the coating solution over the patch surface, and/or to dry the coating solution.
In general, the gas jet should be of sufficient diameter to completely encompass the patch.
Accordingly, in one example, the diameter should be about 1.5 times and even 2 times as big as the largest patch dimension.
Typically a flow rate of between 6-10 m/s is used to distribute and/or dry the coating solution, however, this will depend on the solution properties. However a gas jet of a higher flow rate can also be used to remove excess coating solution, and the gas flow rate used may depend on gas properties, such the density of the gas.
An example of the coating ratio against gas flow rate is shown in Figure 3, for a coating solution having a viscosity of between 0 - 0.05 Pa-S and a surface tension of 0.023 - 0.073 N/m. For this coating solution, a suitable range of gas flow rate is about 6-8 m/s. A faster flow rate, around 10 m/s gas flow, can be used to remove excess coating solution, whilst a reduced gas flow rate has a reduced effect on the coating. Whilst different flow rates may be required for coating solutions having different coating properties, in general, a flow rate of 6-
a) 50 gm; and, b) 100 gm;
c) 150 gm; and, d) 400 gm.
Typically a length for the targeting section is at least:
a) less than 50 gm; and, b) less than 100 gm; and, c) less than 300 gm.
Typically a length for the support section is at least one of:
a) for epidermal delivery < 200 gm;
b) for dermal cell delivery < 1000 gm;
c) for delivery to basal cells in the epithelium of the mucosa 600-800 gm;
and, d) for lung delivery of the order of 100 gm.
Typically a length for the support section is at least one of:
a) for epidermal delivery greater than the thickness of the Stratum Corneum;
b) for dermal cell delivery greater than the thickness of epidermis;
c) for delivery to basal cells in the epithelium of the mucosa greater than a thickness of upper epithelium; and, d) for lung delivery of the order of 100 gm in this case.
Typically the projections are solid.
Typically the projections are non-porous and non-hollow.
Typically the patch is at least one of:
a) hydrophobic; and, b) hydrophilic.
In a second broad form the present invention seeks to provide a method of coating a material onto projections provided on a patch, wherein the method includes:
a) applying a coating solution containing the material to at least the projections; and, -g-b) distributing the coating solution, over the projections at least in part using a gas flow.
Typically the method further includes drying the coating solution using the gas flow.
In a third broad form the present invention seeks to provide a coating solution for coating a material onto projections on a patch, the coating solution including Quillaja saponins acting as a surfactant and a vaccine adjuvant.
Typically the Quillaja saponins include at least one of QA, QS-21, QS-7 and other purified saponin adjuvants.
Typically the coating solution includes an adjuvant that is an Immunostimulating complex.
Typically the Immunostimulating complex includes ISCOMATRIX.
Typically the coating solution includes at least one of:
a) a viscosity enhancer;
b) a surfactant; and, c) an adjuvant.
In a fourth broad form the present invention seeks to provide a coating solution for coating a material onto projections on a patch, the coating solution including nanoparticles.
Typically the nanoparticles are multilayered nanoparticles.
Typically the nanoparticles includes layers including at least one of:
a) cell targeting molecules; and, b) cell-entry facilitating molecules.
Typically the nanoparticles include layers including intracellular targeting molecules.
In a fifth broad form the present invention seeks to provide a patch for use in medical procedures, the patch including a number of projections thereon, the projections having a coating applied thereto using the method of the first broad form of the invention.
In a sixth broad form the present invention seeks to provide a method performing a medical procedure, the method including applying a patch to 'a subject, the patch being a patch according to the fifth broad form of the invention.
Typically the method includes hydrating a surface of the subject and applying the patch to the hydrated surface.
It will be appreciated that the broad forms of the invention may be used individually or in combination.
Brief Description of the Drawings An example of the present invention will now be described with reference to the accompanying drawings, in which: -Figures IA and lB are schematic side and plan views, respectively, of an example of device for delivery of material to targets within a body;
Figure I C is a schematic diagram of an example of the device of Figure 1 A in use;
Figures 1D to IF are schematic diagrams of examples of projections used in the device of Figure IA;
Figures 2A and 2B are schematic plan views of examples of a fluid spreading out and of a droplet forming on a hydrophobic patch, respectively;
Figures 2C and 2D are schematic side views of the examples of Figures 2A and 2B in a Wenzel state;
Figures 2E and 2F are schematic side views of the examples of Figures 2A and 2B in a Cassie state;
Figure 3 is a graph of an example of a relationship between a coating ratio and a gas flow rate;
Figure 4 is an example of a secondary electron image of a patch having a gold coating;
Figures 5A and 5B are schematic diagrams of a first example of apparatus for providing gas flow;
Figures 5C and 5D are schematic diagrams of a second example of apparatus for providing gas flow;
Figure 6A is a schematic diagram view of a third example of apparatus for providing gas flow;
Figure 6B is a schematic diagram view of a fourth example of apparatus for providing gas flow;
Figures 7A and 7B are schematic diagrams illustrating the transfer of coating material to a subject, in use;
Figures 8A and 8B are schematic diagrams of an example of a well provided at the base of a projection;
Figures 9A and 9C show examples of secondary electron images of patches with 60 m and 90 m long projections, respectively;
Figures 9B and 9D show examples of corresponding backscattered electron images for the patches of Figures 9A and 9C, respectively;
Figures 9E and 9F show examples of scanning electron microscopy (SEM) images of 60 m long projections dip coated and dried in air;
Figures 10A and 10B show examples of SEM images of 35 m long projections before and after coating, respectively, using a gas flow;
Figures 10C and 10D show examples of SEM images of 60 m long projections before and after coating, respectively, using a gas flow;
Figures 1OE and 10F show examples of secondary and backscattered electron images, respectively, of 90 m long projections after coating using a gas flow;
Figures 11A, 11B and 11C show examples of individual 35 m long projections before coating, after coating using a gas flow and an overlay of the images, respectively;
Figures 11D, 11E and 11F show examples of individual 60 m long projections before coating, after coating using a gas flow and an overlay of the images, respectively;
Figures 11G, 11H and 111 show fluorescence images of individual 90 m long projections from a DiD coating, the reflection and an overlay of the images, respectively;
Figure 12A shows an example of an SEM image of a patch coated using a gas flow;
Figures 12B and 12C show example of secondary and backscattered electron high-magnification images of projections coated using a gas flow;
Figures 13A to 13D show examples of secondary electron images for patches coated with OVA DNA vaccine on 90 m projections with concentrations of MC of 0%, 0.5%, 1%
and 2.5%, respectively;
Figures 14A and 14B show examples of secondary electron and backscattered electron images, respectively, for patches coated with OVA protein vaccine on 90 m projections, with concentrations of QA of 0.2%;
Figures 14C and 14D show examples of secondary electron and backscattered electron images, respectively, for patches coated with OVA protein vaccine on 90 m projections, with concentrations of QA of 1%; , Figures 15A and 15B show examples of secondary electron and backscattered electron images, respectively, for an example of the tip of the patch coated with of OVA protein on 90 m projections;
Figures 15C and 15D show examples of secondary electron and backscattered electron images, respectively, for an example of the patch coated by applying 10 gl of OVA protein coating solution dried in air;
Figure 16 shows an example of patches and measured local delivery characteristics in mouse epidermis;
Figure 17A is a graph of an example of release intensity values from a 70kDa payload in living skin;
Figure J 7B is a graph of an example of release diffusion coefficients kinetics from a 70 kDa payload in living skin;
Figure 17C is a schematic diagram illustrating an interrogation space for the measurements of Figures 17A and 17B;
Figure 18 is an example of comparative results of serum samples for five mice vaccinated with chicken egg albumin protein using a syringe and needle, or a protein coated patch;
Figure 19A is a graph showing an example of ELISA antibody reactivity for different intramuscular needle and syringe vaccine doses, and for 0.04 g vaccine delivered using a patch having projections coated using a gas flow;
Figure 19B shows graphs of example of Hemagglutinin Inhibition assays (HI) performed for different intramuscular needle and syringe vaccine doses, and for 0.04 ug vaccine delivered using a patch having projections coated using a gas flow for Wisconsin A, Malaysia B, and New Caledonia A;
Figure 20 shows graphs of examples of total IgG, IgGl and IgG2a responses induced by coated nanopatches;
Figure 21A shows examples of (a) the morphology of a patch, (b)-(d) the projections on the patch, (e)-(f) the patch after being antigen coated, (g)-(h) the coated patch after being applied on mouse ear for antigen delivery, (i)-(m) the penetration of the coated patch on mouse ear skin, and (n) the delivery of coating in the mouse ear skin;
Figure 21B shows examples of (a)-(c) the delivery of coating in mouse skin and the following diffusion after the coating being delivered in mouse ear skin, and (d)-(g) the migration of cells after the mouse ear being treated by antigen coated nanopatches;
Figure 21C shows an example of a nanopatch generated immune response and protection from Chikungunya viral challenge; and, Figures 22A and 22B show an example of the size distribution of PEI/DNA
nanoparticles (N:P ratio of 5:1);
Figures 22C and 22D show an example of the coating of polyethylenimine (PEI)/DNA
nanoparticles on patch projections before and after use respectively;
Figure 22E shows an example agarose gel analysis for original and reconstituted PEI:DNA
nanoparticles for a variety of formulations including different N:P ratios (0:1, 5:1, and 9:1);
and, Figures 22F and 22G are example transfection images obtained using the patch of Figure 22C.
Detailed Description of the Preferred Embodiments An example of a device for delivering material to targets within a body will now be described with reference to Figures 1A to 1F.
In this example, the device is in the form of patch 100 having a number of projections 110 provided on a surface 121 of a substrate 120. The projections 110 and substrate 120 may be formed from any suitable material, but in one example, are formed from a silicon type material, allowing the device to be fabricated using processes such as vapour deposition, silicon etching, Deep Reactive Ion Etching (DRIE), or the like. The projections are therefore typically solid, non-porous and non-hollow, although this is not essential.
In the example shown, the patch has a width W and a breadth B with the projections 110 being separated by spacing S.
In use, the patch 100 is positioned against a surface of a subject, allowing the projections to enter the surface and provide material to one or more targets therein. An example of this is shown in Figure I C.
In this example, the patch 100 is urged against a subject's skin shown generally at 150, so that the projections 110 pierce the Stratum Corneum 160, and enter the Viable Epidermis 170 to reach targets of interest, shown generally at 180. However, this is not essential and the patch can be used to deliver material to any part or region in the subject.
It will be appreciated that the projections can have a variety of shapes, and examples of suitable projection shapes are shown in more detail in Figures 1D, lE and IF.
In one example, the projection includes a targeting section 111, intended to deliver the material or stimulus to targets within the body, and a support section 112 for supporting the targeting section 111. However, this is not essential, and a single element may be used.
In the example of Figure 1D, the projection is formed from a conically shaped member, which tapers gradually along its entire length. In this example, the targeting section 111 is therefore defined to be the part of the projection having a diameter of less than d2.
In Figures lE and IF, the structure of the projection may vary along its length to provide a defined targeting section l II with a designed structure. In the example of Figure IE, the targeting section 111 is- in the form of a substantially cylindrical shape, such that the diameter dl is approximately equal to the diameter d2, with a tapered support section, such that the diameter d2 is smaller than the diameter d3. In contrast, in the example of Figure IF, the targeting section 111 is. in the form of taper such that the diameter di is smaller than the diameter d2, with a cylindrical support section, such that the diameter d2 is substantially equal to the diameter d3.
In general, the support section 112 has a length a, whilst the targeting section 111 has a length 1. The diameter of the tip is indicated by d1, whilst the diameter of the support section base is given by d3.
In use, the device can be used to deliver material to specific targets within the body or more generally to the blood supply, or, tissue within the body and the configuration of the device will tend to depend on its intended use.
Thus, for example, if the patch is configured so as to ensure material is delivered to specific targets such as cells, then it may be necessary to select a more specific arrangement of projections than if delivery is provided more, generally to the blood. To achieve this, the device can be provided with a particular configuration of patch parameters to ensure specific targeting. The patch parameters can include the number of projections N, the spacing S
between projections, and the projection size and shape. This is described in more detail in co-pending application US SN-11 /496053.
In one specific example, a patch having a surface area of approximately 0.16 cm2 has projections provided at a density of between 1,000-30,000 projections/cm2, and typically at a density of approximately 20,000 projections/cm2. However, alternative dimensions can be used. ' For example, a patch for an animal such as a mouse may have a surface area of 0.32 to 0.48 cm2, whereas as a patch for a human may have a surface area of approximately 1 cm2.
A variety of surface areas can be achieved by mounting a suitable number and arrangement of patches on a common substrate.
The projections typically have a length of between 10 to 200 gm and typically 90 gm with a radius of curvature of greater than 1 gm and more typically greater than 5 gm.
However, it will be appreciated that other dimensions may be used.
If distinct targeting section and support sections are provided, the targeting section typically has a diameter of less than 1 gm and more typically less than 0.5 gm. The length of the targeting section is typically less than 100 gm, less than 10 gm and typically less than 5 gm.
The length of the support section typically varies depending on the location of the target within the subject. Example lengths include less than 200 gm for epidermal delivery, less than 1000 gm for dermal cell delivery, 600-800 gm for delivery to basal cells in the epithelium of the mucosa and approximately 100 gm for lung delivery.
In order to allow delivery of material to the subject, it is necessary to provide a coating on at least the projections. In one example, coating is achieved by applying a solution containing the material to at least the projections. This may be achieved in any one of a number of manners. Thus, for example, the solution can be applied by dripping the solution onto the patch. Alternatively however other techniques may be used, such as immersion of the patch in solution.
In one example, the gas flow can be used to help ensure even distribution of material over the entire patch. This is particularly useful when the combination of patch and coating solution properties prevent the coating solution from wetting the projections. When coating solution is applied to a surface it can either spread out, or remain as a droplet, and can also fill the space between the projections (known as a "Wenzel" state), or rest on the top of the projections (known as a "Cassie" state). Examples of this will now be described will respect to Figures 2A to 2F.
In the example of Figure 2A the coating solution has properties, such as surface tension and viscosity that allow the coating solution 200 to spread out over the patch 100. In the example of Figure 2B the properties are such that prevents the solution 200 spreading out over the patch and projections. In this example, when solution is applied to the patch, the solution forms a droplet 210.
Examples of these scenarios in the Wenzel and Cassie states are shown in Figures 2C to 2F.
As shown in Figure 2C, the coating solution has spread out in the Wenzel state, so that the coating solution 200 flows over the surface 121 of the patch 100 between the projections 110.
As a result, it is possible to completely immerse the projections 110 by simply adding more solution until the solution level 201 rises above the level of the projections 110.
In the example of Figure 2D, the coating has remained confined in the Wenzel state. Despite being in the Wenzel state, not all of the projections 110 are completely wetted.
In the example of Figure 2E, even though the coating solution has spread out, but by virtue of being in the Cassie state, not all of the projections 110 are completely wetted as the droplet rests on top of the projections 110. Similarly, in the example of Figure 2F, as the coating solution is in the Cassie state, again not all of the projections 110 are completely wetted as the droplet rests on top of the projections 110.
Accordingly, in some instances, the projections 110 can remain un-immersed, meaning they will not be coated when the solution dries. However, using the gas flow, this can urge the coating solution around the surface of the patch, thereby ensuring that the projections are completely wetted.
Thus, in some of the example patch configurations described above, the patch is hydrophobic so that the contact angle of coating solution on patches is greater than 90 degrees, meaning the coating solution can not spread on patches.
In this case, gas flow allows a small volume of coating solution to be distributed over the patch to thereby thoroughly wet all projections. This avoids the need to immerse the entire patch surface in coating solution as well as allowing a small volume of coating solution to be distributed over the patch to thoroughly wet all projections, thereby reducing the amount of coating solution required to coat a,patch.
In one example, when coat 0.16 cm2 patches with 60 m needles, over 20 l coating solution is needed to cover all projections. However, the using of gas flow can control the movement of 6 l coating solution to wet all projections and achieve uniform coating.
Even in the event that coating solution initially wets the projections, previous drying techniques often leave the projections uncoated. The reason for this is that the coating solution covers many projections due to capillary action, and slowly disperses from the projections during drying under ambient .conditions. During the slow drying process, the coating solution drips off from the projections to the base of patches, meaning the projections will not be coated once the coating solution dries. This is undesirable as it reduces the ability of the patch to deliver material to a subject. In particular, maximising coating on the projections increases the rate of transfer of material to the subject, as well as maximising the amount of material on the patch that is delivered.
Accordingly, in one example, the coating solution is dried using a gas flow, to thereby remove the coating solution between projections, reduce the drying time and consequently reduce the chance of coating solution dispersing from the projections, and thereby ensure that the projections remain coated as the coating solution dries.
The gas flow could also be provided in a variety of manners. For example, this could be achieved by using a gas jet directed towards the patch. Whilst any gas may be used, in one example the gas is nitrogen as this is substantially inert and will not therefore react with the solution, whilst also being readily available. It will be appreciated that other inert gases, such as argon, can also be used, as well as air flow or other types of gas flow. In one example, the gas selected will depend on the reactivity of the coating material. As an alternative to the use of a gas jet however, flow could be induced by extracting gas from a container containing the patch.
When performing the coating process it is typical to select coating properties, such as gas flow rate, ' solution properties such as the solution viscosity and surface tension, and optionally a drying time, to thereby control the distribution of coating over the projections 110.
For example, the degree to which the projections are wetted will also depend on the coating solution properties. Thus, for example, if a higher viscosity solution is used, this will tend to adhere more strongly to the projections, and hence allow a greater thickness of coating to be achieved. However, a higher viscosity coating solution may require an increased gas flow to allow adequate distribution over the patch.
In the case of surface tension, if the surface tension is too great, the coating solution will not be effective at wetting the projections, reducing the effectiveness of coating. A lower surface tension will increase the ability of the coating solution to wet the projections, allowing better coating, although too low a surface tension and the coating solution can rest primarily on the surface of patches reducing coating of the projection tips.
In addition to this, the solution properties will also have an impact on the drying process. For example, if a thicker viscosity coating solution is used this reduces the likelihood of coating run-off during the drying process, but may increase the drying time.
Additional control is also achieved using the gas flow rate. Thus, a higher gas flow rate can increase the degree to which coating solution is distributed on the patch, and/or can reduce the drying time.
Appropriate selection of the coating properties can be used to ensure at least the projections are coated, as well'as to allow the thickness of coating on the projections to be controlled.
This can also be used to vary properties such as the relative amounts of coating on the patch surface 121 and on the projections 110, which can be characterised by a coating ratio based on a ratio of an amount of coating on the projections 110 against an amount of coating on the patch surface 121.
It will also be appreciated that the degree to which the patch is hydrophobic will depend on the patch configuration and in particular, on patch parameters such as the projection size and shape and the projection spacing S. Accordingly, when performing a coating process, it is typical to first determine patch properties and then use this information to allow appropriate coating properties to be selected.
In general the coating solution includes at least a material such as a therapeutic agent and examples of suitable materials include:
= nanoparticles;
= a nucleic acid or protein;
= an antigen, allergen, or adjuvant;
= parasites, bacteria, viruses,,or virus-like particles;
= quantum dots, SERS tags, Raman tags or other nanobiosensors;
= metals or metallic compounds; and, = molecules, elements or compounds.
Examples of preferred formulations include, a solution containing DNA having a concentration of between 0.01 mg/ml and 5 mg/ml or protein having a concentration of between 0.01 and 50 mg/ml.
The agent or other material is typically- either dissolved in a suitable solvent or held in suspension in a suitable carrier fluid, as will be appreciated by those skilled in the art. In one example, the solvent is acetone, although alternatively water or other suitable solvents can be used. The resulting surface tension in pure acetone solution and pure aqueous solution is between 0.023 N/m (acetone) and 0.073 N/m (water).
The solution properties are also typically controlled through the addition of one or more other agents such as a viscosity enhancer, a surfactant, and an adjuvant. It will be appreciated that other additives such as detergents may also be used. These ingredients can be provided in a range of different- concentrations. For example, the viscosity enhancer or surfactant can form between 0% and 90% of the coating solution.
A range of different viscosity enhancers can be used and examples include MC, CMC, gelatin, agar, and agarose and any other viscosity agents. The solution typically has a viscosity of between 10"3 Pa-S and 1 Pa-S. In one example, using a coating solution containing 1-2% MC, which results in suitable uniform coatings, resulting in a viscosity within the range 0.011 (1%) - 0.055 (2%) Pa-S.
Similarly, a range of different surfactants can be used to modify the surface tension of the coating solution, such as any surfactant or any suitable agent that changes surface tension, and that is biocompatible at a low concentration.
Surfactants are wetting agents that lower the surface tension of a liquid, allowing easier spreading, and lower the interfacial tension between two liquids. The term 'surfactant' is a blend of "surface acting agent". Surfactants are usually organic compounds that are amphiphilic, meaning they contain both hydrophobic groups ("tails") and hydrophilic groups ("heads"). Therefore, they are soluble in both organic solvents and water.
Surfactants may be used as the surface tension of the coating solution becomes dominant on a micron-scale, so the surfactant reduces the surface tension of the solution, which helps solution, wet the surface of patch projections, thereby improving coating quality.
Furthermore, a viscosity enhancer can increase the viscosity of coating solution and therefore increase the thickness of coating.
Example coating solutions will be described in more detail below.
Once the coating solution has been formed, the patch can be coated either by dripping the coating solution onto the patch or by immersing the patch in the coating solution. Typically, the amount of coating solution used to coat a patch is between 5 l to 15 l, for patches similar to those outlined above.
Once the coating solution is deposited on the patch, the patch- may be allowed to rest, for example in a sealed environment, to assist with wetting of projections, although this is not essential and may depend on the nature of the deposition process. Following this, or otherwise, a gas jet is used to evenly disperse the coating solution over the patch surface, and/or to dry the coating solution.
In general, the gas jet should be of sufficient diameter to completely encompass the patch.
Accordingly, in one example, the diameter should be about 1.5 times and even 2 times as big as the largest patch dimension.
Typically a flow rate of between 6-10 m/s is used to distribute and/or dry the coating solution, however, this will depend on the solution properties. However a gas jet of a higher flow rate can also be used to remove excess coating solution, and the gas flow rate used may depend on gas properties, such the density of the gas.
An example of the coating ratio against gas flow rate is shown in Figure 3, for a coating solution having a viscosity of between 0 - 0.05 Pa-S and a surface tension of 0.023 - 0.073 N/m. For this coating solution, a suitable range of gas flow rate is about 6-8 m/s. A faster flow rate, around 10 m/s gas flow, can be used to remove excess coating solution, whilst a reduced gas flow rate has a reduced effect on the coating. Whilst different flow rates may be required for coating solutions having different coating properties, in general, a flow rate of 6-
8 m/s is acceptable for most coating solutions. If a coating solution is applied on 60 m projection patches and dried in ambient air, the coating will tend to remain exclusively on the patch surface 121. However, if 10 pl of coating solution containing 2% MC, 2%
OVA
protein and 0.2% QA is applied on 90 m projection patches and dry with a nitrogen jet, 120 g OVA protein will be coated onto projections and 40 pg OVA protein will be coated onto base, using a gas flow in the range 6-8 m/s.
Accordingly, the above described examples provide method for coating therapeutic agents including vaccines on to projections on a patch, to thereby allow for their rapid release when the patch is applied to a subject. The method provides substantially uniform and controllable coating of therapeutic agents like DNA or protein vaccine onto the patches, even in circumstances when the patches are hydrophobic. The method can be applied to any form of patch but is especially suited for patches having projections that are shorter than 200 m and separated by 10-1000 m.
Further variations and options will now be described.
For example, the patch and/or projections can be coated with a thin layer of a suitable metal, prior to application of the coating solution. The reason for this is that metals tend to have, relative to the native silicon or other patch material, a high surface energy, which in turn helps assist with the coating process. In one example, the metal layer is gold, although other suitable metals may be used. An example of a gold coating on a silicon projection is shown in Figure 4. Gold coating forms a nanostructure on silicon projection. The thickness is about 400-1500 nm and the size of gold particles is about 200-400 nm. This structure together with the projection arrangement provides a very hydrophobic surface.
As described above, the coating solution is typically selected to have a suitable viscosity and surface tension. This may be achieved using viscosity enhancers and surfactants to control the coating solution properties. However, use of surfactants is not essential and in one example, a vaccine coating can be achieved using MC without requiring surfactants.
However, if CMC is used for coating, the addition of surfactants is preferred.
As mentioned above, the surfactant can be any suitable agent such as poloxomer 188, triton-X 100, NP40, QA or any surfactant that is biocompatible at a low concentration. The concentration of the surfactant is from about wt. 0% to about 90% of the coating solution, depending on the required solution properties.
A vaccine adjuvant may also be added to the coating solution for enhancing immune response to vaccines. In one example, the adjuvants used include Quillaja saponins, such as QA, QS-21, QS-7 or other purified saponin adjuvants. Use of QA and other similar saponin adjuvants can be particularly beneficial as QA not only acts as a surfactant for coating purposes but also as the vaccine adjuvant. Furthermore, due to QA
effectiveness in reducing the surface tension of the coating solution, this can in turn help in reducing the amount of excipients used for coating.
Other amphipathic immunostimulatory compounds such as dimethyldioctadecylammonium bromide or chemically modified immunostimulatory molecules to give surfactant properties can also be employed.
The viscosity agent can be selected from MC, CMC, gelatin, agar, agarose or any other viscosity agent, which can be any substance that modifies the viscosity of the coating solution. The concentration of the viscosity agent is typically from about wt.
0% to about 90% of the coating solution.
Whilst a range of therapeutic agents can be used, in one. example the agents are vaccines.
The vaccine can be composed of DNA or protein and can also contain an adjuvant. The concentration of DNA in the coating solution can be from 0.01 mg/ml to 5 mg/ml. The concentration of protein in the coating solution can be from 0.01 to 50 mg/ml.
The material can include nanoparticles to provide a nanodelivery system. For example the coating can include DNA containing nanoparticles.
In one example, the nanoparticles are multilayered nanoparticles. Outermost layers of the nanoparticles can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest.
Molecular biosensors can be used to confirm the presence of expected molecules as a surrogate molecule for signs of infection, for activation in radiation damage, or other criteria, prior to delivery of counter-measure molecules such as vaccines, drugs, or gene therapy. The biosensors can also be used as a feedback control mechanism to control the proper amount of vaccine/drug/gene delivery for each cell.
Further, the nanodelivery system can be used to restrict any cells from encountering the drug unless that cell is specifically targeted. Successful targeting can be verified by 3D
multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.
This example can be used to provide a nanomedical system and method that can be used for diagnostics, therapeutics, vaccines, or a combination thereof by use of a multilayered nanoparticle system. The multilayered nanoparticle system can built on a nanoparticle core of bio-polymer, polystyrene, silica, gold, iron, or other material.
The concentration, viscosity and surface tension will all influence the thickness, morphology and payload of coating. In the most preferred embodiments, the thickness of the coated vaccines can be from 10 nm to 10 m.
The amount of resulting dry coating on the projections can be controlled by the concentrations of excipients in coating solution, as well as the surface area of the projections, although as mentioned above, selection of an appropriate surfactant, such as QA can avoid the need for unnecessary excipients.
The coating solution can be applied in several ways. In one example, the projections are completely submersed in the coating solution, although alternatively a defined volume of coating solution can be applied to the patch, the amount of which can. vary depending on the patch area.
Once the coating solution is applied, the projections and/or the patch are dried. The gas flow can be used to move the coating solution over the patch surface 121, to thereby ensure all the projections 110 are coated. For example, the gas jet can be used to move the coating solution from one edge of the patch to another opposing edge of the patch, by suitable direction of the gas jet. Additionally, and/or alternatively, the gas flow can be used to dry the coating solution on the projections quickly so the coating solution remains on the projections until they are dry. By using the gas drying technique, this ensures that coating is evenly distributed on the projections.
It will be appreciated that in some instances it may be desirable to coat the projections but not the base of the patch itself, for example to control the rate of delivery of the material, and to help reduce excessive usage of coating solution. This can be achieved using a coating solution of proper viscosity and surface tension and a defined drying process.
Specific examples of this will be described in more detail below.
In order to allow the coating solution to be distributed over the patch, it is typical to direct the gas flow over the patch in an appropriate manner. An example of apparatus for achieving this will now be described with reference to Figures 5A and 5B.
In this example, the gas flow is generated by a gas jet expelled from a nozzle 500. In one example, the nozzle is coupled via a tube 501 to a gas source 502, such as a compressed gas cylinder, a compressor, or the like. This allows the gas source 502 to supply gas to the nozzle 500, via the tube 501, thereby causing a gas jet to be emitted from the nozzle 500 in a direction substantially parallel to a nozzle axis 510. In one example, the gas source 502 includes a control 503, such as a flow rate valve, that allows the flow rate of the gas from the nozzle to be controlled.
As shown in Figure 5A, initially the coating solution is applied to the surface 120 on one side of the patch 100 near the edge 100B. The nozzle 500 is then aligned with the fluid on the patch, and aimed so as to direct the coating solution towards the other edge 100A of the patch 100. The nozzle 500 is generally aimed so that the nozzle axis 510 is at an angle a relative to a plane 511 containing the patch substrate 120.
Adjustment of the angle a can be used to control the rate at which the fluid is moved across the patch, allowing coating solutions of different viscosities to be moved across the patch prior to drying. It will be appreciated that additional distribution control can also be achieved by adjusting the gas flow rate, although this in turn has an impact on drying rate.
Accordingly, it is generally desirable to balance the distribution rate and drying rate for the coating solution by appropriate selection of an appropriate gas flow rate and angle a, which will in turn depend on the viscosity and surface tension of the coating solution. Typically however the angle a is in the region of 0 to 45 , and more typically 10 to 30 , and more typically about 20 .
In addition to this, the position of the nozzle 500 can also be adjusted to help distribute coating evenly over the patch 100. This can include moving the nozzle in a direction parallel to the edge 100B, to thereby ensure that coating is distributed across the entire patch width, as well as to move the nozzle in a direction perpendicular to the edge 100B, to thereby move solution along the length of the patch, as shown in Figure 5B.
In use, the nozzle 500 may also be held in position by a support arrangement 504, which can be any form of suitable support, such as an arm including a clamp, or the like. The support may be capable of manipulation, to allow the position of the nozzle 500 relative to the patch 100 to be adjusted. Thus, in one example, the support 504 could be in the form of a computer controlled arm, such as a robot arm, thereby allowing computer control of the coating process.
It will be appreciated that in addition to the above, multiple gas jets may be used to induce movement and/or drying of the coating solution. Furthermore, the multiple gas jets could be provided at different angles a, as well as at different orientations relative to the patch, to thereby enhance the distribution or drying effect.
Apparatus of this form can also be adapted to allow "Multiple patches to be coated during a single process. An example of such apparatus will now be described with reference to Figures 5C and 5D.
In this example, the apparatus is formed from a base 550 for supporting a number of patches 551, typically provided in an array. The apparatus includes two supports 552, for supporting two arms 560, 570, which are mounted to allow movement of the arms in the direction of arrow 580. The first arm 560 includes a coating solution delivery system including a nozzle 561 for depositing coating solution on the patches 551. The second arm including a gas delivery system including a gas nozzle 571. In use, the nozzle 561 and the gas nozzle 571 are movably mounted to allow lateral movement of the nozzles 561, 571 in the direction of the arrows 5 81.
Movement of the arms 560, 570 and the nozzles 561, 571, gas flow rate and coating solution delivery are typically achieved using a computer controlled drive system, shown generally at 590. This allows coating solution and gas flow to be delivered to the patches 551. This can be achieved collectively, or 'by delivery to each of the patches in turn. In either case, this allows coating solution to be applied, optionally distributed over the patches and dried.
In the example shown, a single. respective nozzle 561, 571 is used to deliver coating solution and gas flow. However, multiple nozzles may be provided. Additionally, or alternatively the coating solution and gas delivery systems can be incorporated into a single arm. A further alternative is to provide nozzle systems that extend across an entire length of the arm allowing coating solution and gas to be applied to multiple patches simultaneously.
Further examples of apparatus for providing a gas flow will now be described with reference to Figure 6A and 6B.
In the example of Figure 6A, the apparatus includes a housing 600 having a cavity 602 for containing a patch. In one example, the container is generally sealed to allow a pressure differential to be established between the inside and outside of the housing 600. This can be achieved by coupling the housing to a gas source 610 via a connecting tube 611, allowing the pressure within the housing 600 to be increased to a suitable level. Once this has been reached, a release valve 601 can be activated, allowing gas to escape from the housing 600 through the valve 601. This in turn generates a gas flow, as shown by the arrow 603. The gas flow can be directed utilising appropriate baffles provided on inner surfaces of the housing 600 as required.
As an alternative to pressurising the container however, a further option is to replace the gas source 610 with a vacuum pump, allowing air or another gas within the cavity 605 to be extracted, to thereby generate a gas flow.
In either case, it will be appreciated that appropriate positioning of the patch 100 within the housing 600, together with a suitable pressure differential, and hence suitable gas flow, can be used to ensure the patch is appropriately coated.
in the example of Figure 6B, an alternative design of container 650 is shown.
In this example, the container includes an opening 651 to allow a cavity 652 to be coupled to a vacuum pump 660, via a connecting tube 661. The patch 100 is supported in the cavity 652 above a lower surface of the cavity 653, using a suitable support 654. The patch 100 is also positioned below the opening 651. Consequently, when air or another gas is evacuated from the housing 650, a gas flow is generated as shown by the arrows 670. As the gas flows around the patch 100 turbulence causes air flow over the entire patch surface, thereby helping to distribute and/or dry the coating solution. It will be appreciated that as an alternative, the cavity 652 can be pressurised in a manner similar to that described above with respect to Figure 6A.
In one example, only the projections are coated. Consequently, when the patch is placed on the skin, substantially all of the coated therapeutic agent can be rapidly delivered into the skin from the projections. As a result, this can be used where rapid delivery of an agent is required.
However, there are cases where it is required for agent to also be coated to the base. As one example, where some delay is required for delivery of a therapeutic agent, the agent can also be coated onto the patch substrate or base 120. The agent coated on the projections can achieve fast delivery in skin for a first dose, while those coated on the patch base 120 can slowly permeate into the subject's skin through holes made by the projections thereby providing for further dose(s).
As another example, such arrangements may be used when it is desirable to deliver higher amounts of payload into the skin over and above the amount coated on the projections. In this case, the additional payload on the base of the patch can be hydrated (e.g. by fluid within the skin moving through holes generated by the projections with a capillary action) and released, a "depot effect" for higher delivery dose.
An example of this will now be described with reference to Figures 7A and 7B.
In the example of Figure 7A, the patch 100 includes coating 710 provided on the projection 110, and coating 720 on the surface 121. Initially, when the patch 100 is applied to a subject, the projections 110 extend through the skin 700. The skin typically deforms in a region immediately surrounding the projection, with the skin bowing down away from the patch surface 121.
Upon insertion into the skin 700, coating 710 on the tip of the projections 110 below the skin surface 700, will immediately begin to hydrate and dissolve, thereby being dispersed into the subject, as shown by the arrows 730.
In addition to this, fluid from the subject will gradually flow into the coating 710 at the base of the projection 110, and coating 720 on the surface 121, as shown by the arrows 735, thereby hydrate the fluid. This will in turn cause fluid to diffuse into the subject, as shown by the arrows 740.
A further effect that can contribute to the delivery of material from the patch surface 121 is a squeezing effect, caused by the resilience of the skin 720, which urges the skin upward as shown by the arrow 750, which in turn urges hydrated material in the direction of the arrow 755, thereby increasing delayed delivery to the subject.
It will therefore be appreciated that controlling the coating ratio can therefore be used to manipulate the amount and rate which material is delivered to the subject. By maximising the coating on the projections, this maximises rapid delivery of material.
However, by increasing the amount of coating on the surface 121, this increases the delayed delivery of material.
Further delayed delivery of material can be achieved by further increasing the amount of material on the surface. This can be achieved using a projection configuration as shown in Figures 8A and 8B.
In this example, the surface 121 includes a raised annular portion 821 surrounding the base of a projection 110, thereby providing a well for containing addition coating solution.
Accordingly, in this instance, the coating 820 on the surface 821 can be of an increased thickness in the region immediately surrounding the base of the projection 110. This enhances the delayed delivery of material to the subject.
In one example, the projections can be coated a single time. In a further example, the projections can be coated a number of times. This can be used to allow a required thickness of coating to be achieved. In addition to this however, this allows different coating regimes to be used, which in turn allows greater control over the coating process.
Thus, for example, if coating is carried out using a first set of appropriate coating properties, then the coating can be confined primarily to the tips of the projections. A
second coating procedure can then be performed in order to allow the entire projection to be coated. This can be used to ensure that the tip includes a suitable amount of material to maximise the efficacy of the delivery process.
The above described processes therefore allow projections to be dry coated with material. In one example, this is achieved by using a gas flow to move or distribute coating solution over the patch to thereby ensure that all projections are wetted prior to drying.
In another example, this is achieved by using as gas flow to dry coating solution more rapidly than can be achieved under ambient conditions, thereby ensuring that coating solution remains on the projections during the drying process. It will be appreciated that the moving and drying steps can be performed simultaneously.
By dry coating the projections of the patch, this ensures that material on the projections is rapidly delivered directly to the subject. This maximises the proportion of coating material effectively delivered to the subject, which in turn reduces the amount of material required in order to produce a biological effect within the subject.
In addition to the above, appropriate selection of coating properties, such as gas flow rate, drying time, and solution properties can be used to further control the coating process. In particular, this can be used to control the thickness of the coating applied to the projections.
The projections can be coated with DNA or protein vaccines. However, in addition to this, many other reagents can be coated using this process including both inorganic and organic materials. Example coatings used include inorganic materials such as EtBr, or organic materials such Evans blue, Dextran, DiD, or the like.
Consequently, the resulting patch can provide small and densely packed projections that can be uniformly and controllably coated. This allows vaccines or other agents to be subsequently delivered to highly immunologically sensitive cells within the epidermis, or to the blood or muscular tissue as required.
In use, the coated and dried projection patches are applied to the skin of a mammal by placing the patch on the skin. The coated and dried projection patches can be tested on skin or skin analogs and the conditions for optimal coating release determined.
These conditions include patch application time, force, velocity, strain-rate of insertion, temperature, humidity, location, and skin pretreatment. This process can be done in vitro, ex vivo or in vivo.
It will be appreciated that the final release of the therapeutic agent can also be influenced by several of the coating properties such as the inclusion,of excipients and viscosity enhancers, as well as the coating thickness, and testing again allows optimum coating properties such as those outlined above, to be determined.
The in vitro method utilizes a thin polymer film to approximate the stratum corneum (SC), or outer layer of skin. The film can be polycarbonate, polyethylene, or any other film that has physical characteristics that approximate those of the SC. Beneath the polymer is an absorbent material that can be filter paper, polymer mesh, or any other soft and inert material that does not bind the vaccine or coating material. This material is then moistened with water, tris buffered saline (TBS), phosphate buffered saline (PBS), or any other liquid that can dissolve the coating material. The device is then applied to the polycarbonate and the projections pierce the top layer of polymer film. The liquid in the absorbent layer can then dissolve the dry coating. Once the device is removed the absorbent layer is flushed with the liquid. The elutate is then quantified and the device release calculated. The coated and dried projections patches can be applied to this testing environment under many varied conditions to optimize release.
The ex vivo release assay can be used to assess release from the coated and dried projection patches and employ skin. A patch of skin is dissected from a donor (i.e.
mouse, pig, rat, human) and kept at -20 C for less than 7 days prior to use. The skin is warmed to 37 C and the patches coated as outlined above are applied under a variety of conditions. The patches can be coated with fluorescent dyes such as FITC, Evans Blue, Propidium Iodide, Ethidium Bromide, Alexa Fluor dyes. The patches can also be coated with DNA or proteins that are labelled with fluorescent dyes. Alternately, the patches can be coated with fluorescent dye labelled polymers like dextran, agarose, agar or any other biocompatible polymer that approximates the size, shape, and chemical nature of DNA and protein vaccines.
The release of these fluorescently labelled agents in skin can be monitored by methods including multi-photon/confocal microscopy, fluorescence microscopy, spectrofluorometer, and flow cytometry. Multi-Photon/Confocal microscopy can give real time, 3D
patch release information that is necessary for optimizing the device coating and application.
In in vivo release testing, a coated projection patch is applied to the skin.
After the application, analysis was carried out as discussed for the ex vivo testing protocol.
Alternately, a portion of the skin treated with the projection patch is excised. The outer layer of the skin is peeled and trimmed as required. The skin is snap frozen in liquid nitrogen and then pulverized to a fine powder.
For DNA vaccine delivery, the DNA is extracted with a Qiagen extraction kit and a standard curve employed to determine the amount of DNA with semi-quantitative Polymerase chain reaction (PCR).
A number of specific examples will now be described. For the purpose of these examples the general coating procedure used was as follows:
= Patches are cleaned in glycerol:H20 (1:1) for 10 minutes and then flushed with plenty of water;
= Cleaned patches are dried with nitrogen blow;
= Coating solution is made of MC, poloxamer 188 or QA, and different concentration of vaccine (0.01 mg/ml - 50 mg/ml), the concentrations of chemicals being adjusted to suit different requirements;
= 5-15 microliters of coating solution is dropped onto each patch; and, = Patches are dried under nitrogen flow as described above.
During application the skin of the subject is typically hydrated to ease application of the patch, and increase hydration of the coating, thereby enhancing delivery.
Example 1 The projection patches are cleaned in a mixture of glycerol and water in a 1:1 ratio for 10 minutes and then flushed with plenty of water. The patches are then dried with nitrogen blow. Example of cleaned and uncoated projections are shown in Figures 9A to 9D, which show secondary electron and backscattered electron images for patches with 60 m and 90 m long projections, respectively.
A coating solution containing a viscosity enhancer (MC), a surfactant (QA or poloxamer 188) and different concentrations of vaccine (OVA protein or DNA) is prepared. The compositions are set out in Table 1. All percentages are weight percentages of the total compositions unless otherwise indicated.
Table 1 MC wt. 0 - 2.5%
QA or poloxamer 188 wt. 0 - 1%
OVADNA wt.0-0.5%
OVA protein wt. 0 - 5%
microliters of the coating solutions are dropped onto each patch prepared as described above. A gas jet is used to control the movement of coating solution on patches so the liquid can wet the projections without being stuck on patches and covering many projections. In the meantime, the coating solution can be adsorbed and dried on the projections.
In this example, to provide a comparison, the patches of Figures 9A and 9C
were treated using a classical dip coating approach. Four patches, having totally over 14,000 projections, were coated with a solution containing 10 mg/ml of CMC (viscosity enhancer), 10 mg/ml of poloxamer 188 (surfactant) and 2 mg/ml of OVA DNA (active agent). Patches were dipped into the solution for 10 seconds and dried in air for 1 hour. The morphology of coated patches was then observed by SEM, with the results being shown in Figures 9E and 9F.
Figure 9E shows that no coating is present on the projections. Instead, the coating solution has been exclusively dried on the base the patch. From the magnified image of a single projection, shown in Figure 9F, the sputter coated gold particles can still be clearly observed, which also confirms that no coating has been obtained on the projection. This highlights that a dip-coating technique is not effective when applied to very small and densely packed projections. This experiment was repeated when MC, QA and OVA protein were used in coating solution at different concentrations and the results were similar. In other words, no coating or very little coating can be obtained on projections by using the dip-coating technique.
Gas jet drying was used to coat vaccines on patches and SEM was employed to characterise the morphology of the coating. Figures 10A and 10C show baseline secondary electron images of uncoated patches, with 30, 60 and 90 m long projections, respectively.
The patches were then coated using a,coating solution composed of 20 mg/ml of MC, 2 mg/ml of Quil-A and 2 mg/ml of OVA DNA vaccine, which was dried using the gas flow technique outlined above. The respective SEM images of the coated patches are shown in Figures 10B, 10D, WE and 10F, which highlight how the effective thickness of the projection increases, due to the coating of a consistent layer. The coating layer is up to 5 m thick.
The image in Figure 1OF is a backscattered electron image, which also confirms that the projections are uniformly coated. In this regard after coating, projections presenting dark BSE signals are seen due to the presence of organic materials with low atomic numbers, i.e.
carbon, oxygen, and hydrogen, on the surface of projections. In comparison, the base of the patch still has bright BSE signal after coating, which suggests that the coating on the base is very thin (- 1 m).
In Figures 11 A to 11I, the coating on selected, individual projections are shown in more detail. Figures 11A, 11B and 11C show an individual 35 pm long projection before coating, after coating, and an overlay of the two images, respectively. With a longer, 60 m projection, these images are respectively shown in the same series in Figures I1D, 11E and 11F. From these figures, the coating layer on the projections can be clearly observed.
With consistent coating of projections established, the next stage is to demonstrate that biologically active (or relevant) material was uniformly coated on the projections, to show that the projections are not only other excipients, such as, viscosity enhancers, surfactants, or the like. In this regard, Figures 11G, 11H and 11I show the fluorescence from a DiD coating on 90 m projections, a reflection from the projections and an overlay- image, respectively.
These figures demonstrate that surrogates for active materials in the form of fluorescent dyes can be uniformly coated on projections, as shown the fluorescence from DiD.
Following this, the work was extended to demonstrate that the coating process is robust and broadly applicable to many active entities, including ethidium bromide (EtBr), OVA protein vaccine, OVA DNA vaccine, fluorescent dyes (dextran and DiD) and. flu virus on projections.
The selection of coated compounds spans from low molecular weight molecules (a few hundred Daltons) to high molecular weight molecules (a few million Daltons).
In all cases, coatings were reproducibly applied onto projections on the patches.
Figure 12A shows an example of an SEM image of a patch uniformly coated with protein using a gas flow. The secondary and backscattered electron images of Figures 12B and 12C
highlight the even coating.
Accordingly, the gas jet coating can achieve uniform coating on projections and can rapidly coat large numbers of projections.
Example 2 In this example, the projections are coated in accordance with Example 1, however with the concentration of MC in coating solution being adjusted from 0 to 2.5% while the concentration of QA and OVA protein is kept to be 0.2% and 1%, respectively.
Figures 13A to 13D, show secondary electron images for patches coated with OVA
DNA
vaccine on 90 m projections with concentrations of MC of 0%, 0.5%, 1% and 2.5%
respectively. The coating is pretty uniform for all samples, but the coating thickness is different for coating solutions containing different concentration of MC. This is shown in Table 2, which shows the coating thickness on the middle cylindrical part of projections for coating solutions containing different concentration of MC.
Table 2 MC concentration Coating thickness 0 <0.25gm 0.5% 1.42 0.18 m 1% 2.10 0.18 m 2.5% 4.00 0.50 m Example 3 Projections are coated in accordance as described in Example 1 but with a concentration of QA in coating solution of 0.2% or 1% while the concentration of MC and OVA
protein is kept to be 2% and 1%, respectively.
Figures 14A to 14D show secondary electron and backscattered electron images for patches coated with OVA protein vaccine on 90 m projections, with concentrations of QA of 0.2%
and 1%, respectively. The coating is again pretty uniform for all samples, but the coating thickness on the base is different for the different QA concentrations.
When the concentration of QA is 0.2%, the coating on base is very thin (<1 m), so very bright backscattered electron signal can be detected from the base, as shown in Figure 12B.
When` the concentration of QA increases to 1%, the coating on base starts to be thicker (> 2 m). Therefore, the backscattered electron signal from gold under the OVA
protein coating is difficult to be detected and the base looks dark in backscattered electron image of Figure 14D.
Example 4 In this example, the projections are coated with OVA protein in accordance with Example 1, but with the coating solution containing water and ethanol (2:1), 1% OVA and 1% MC.
Figures 15A and 15B show a secondary electron and backscattered electron images for an example of the patch coated with of OVA protein on 90 .xm projections. It can be seen the coating is mainly on the top part of projections. From the backscattered electron image, it can be seen that the top part of projections look dark while the bottom part of projections and base are bright. This further confirms that the coating is mainly on the top part of projections.
After addition of ethanol, the surface tension of the coating solution is very low, so it can well wet projections. Therefore, less amount of coating solution (<6 l) is enough for coating a patch, which will reduce the cost of vaccine coating required to coat a patch.
To further confirm that the tip coating effect is caused by using a gas jet flow, Figures 15C
and 15D show examples of secondary electron and backscattered electron images for an example of the patch coated by applying 10 41 of OVA protein coating solution and drying in air. It can be seen that the coating is mainly on base and little coating is on some part of projections.
Example 5 Hardness and Young's modulus are two important mechanical properties for vaccine coating.
In order to deliver vaccine into the skin, coating should be robust enough to pierce into skin without wiping off. Preferably, values of hardness and Young's modulus of vaccine coating should be larger than those of skin. Hardness and reduced modulus have been measured for silicon patch, gold coated silicon patch, OVA protein coating and mouse ear skin. Results are shown in Table 3.
Young's modulus can be calculated from reduced modulus. Young's modulus describes tensile elasticity, or the tendency of an object to deform along an axis when opposing forces are applied along that axis; it is defined as the ratio of tensile stress to tensile strain. Hardness is the characteristic of a solid material expressing its resistance to permanent deformation.
From the results, it can be found that the values of hardness and reduced modulus of OVA
protein vaccine coating are much higher than those of mouse ear skin or porcine skin. It is a clear evidence of robust coating, which should be able to pierce into skin without wiping off.
Table 3 Hardness (GPa) Reduced modulus (GPa) Silicon patch 12 0.47 173 5 OVA coated patch 0.14-0.19 3.2-3.8 Porcine skin stratum Dry SC: 0.1-0.3 corneum (SC)* Wet SC: 0.01-0.05 *. Yuan Y. and Verma R., Colloids and Surfaces B 48 (2006) 6-12 It should be noted that in the case of the porcine skin stratum that the application of the patch was performed under differing conditions. In this regard, the patch was initially applied with the skin in a dry state, yielding a higher reduced modulus for the coating than when the skin is wet. The reason for this is the fluid on the skin hydrates the coating, reducing adherence of the coating to the projection. In some circumstances, this can be beneficial as it assists rapid delivery of the all the coating material to the subject.
Example 6 In this example, a coated patch is tested using a conventional commercially-available influenza vaccine (trivalent vaccine (Fluvax 2007) CSL, Ltd, Melbourne Australia;
consisting of viruses New Caledonia A, Wisconsin A and Malaysia B)) to assess the local delivery of vaccine within the skin (viable epidermis and dermis), as shown in Figure 16.
Figure 16 shows that applying patches, coated with the influenza vaccine, to the skin (for 15 minutes) achieves targeted delivery to the skin viable epidermis and underlying dermis.
Within the viable epidermis the co-localization of vaccine to targeted immunologically-sensitive cells is very high (at 40%). Furthermore, the overall payload delivered within the skin is accurately quantified at 19.9 5.7 ng (per patch).
In particular, Figure 16 shows an example of patches and measured local delivery characteristics in the mouse epidermis. Patches (a) were fabricated to the projection length of 90 gm (with Deep Reactive Ion Etching; at the Rutherford Appleton Laboratories, by Derek Jenkins) and then dry-coated with vaccine and photographed with SEM (b) and (c).
Once coated, the patch was applied to the skin and Cryo-SEM was used to visualize the skin during patch application (d). By labelling Fluvax with a fluorescent dye (Cy3), shown at 1600 in (e) and (h), confocal microscopy was used to examine. co-localization (arrow heads in Panel (h)) of vaccine with MHCII, shown at 1610 in (f) and (h) containing cells (e) to (h).
The patch was applied at 1.89 m/s and held in place with 500g for 15 minutes, penetrating to 27.7 gm (which is deeper than the epidermis thickness of 17 gm. The images (e) to (h) are a projected z-stack of the surface of the mouse skin (a hair can be seen at 1620 in (f) and (h) as a large diagonal bar) to the depth of 46 gm (which is well into the mouse ear dermis).
The dense nuclei in (g) and (h), stained with Hoechst 33342, in the epidermis were used to determine the epidermal and dermal boundary. Successful vaccine targeting to key epidermal cells (MHC Class II stained, including Langerhans cells) can be seen in two of the five vaccine deposition sites within Panel (h), highlighted with white arrow heads.
Several parameters were quantified through confocal image analysis (i) to (m) and are shown as per mm2 unless otherwise noted. Nine areas in three patched ears were imaged and analyzed for all but the last two graphs (1) and (m). The final two graphs (1) and (m) show quantification of delivered vaccine payload in skin by patches. Patched mouse ears were then homogenized and used in a quantitative dot-blot, using the generated standard curve, on five mice ears. The mass of vaccine delivered per projection was determined by measuring the integrated density of nine single projections and calculating the percentage of fluorescence per projection. This was then used in conjunction with the total delivery mass to calculate the mass of Fluvax delivered per projection.
Example 7 In this example, ex vivo release kinetics of 70kDa dextran coated 60 m nanoprojections were determined. These data were captured over 40 minutes in living skin using fluorescent microscopy.
Projections were coated with 20 gg of rhodamine labelled dextran (70 kDa) as a surrogate for ovalbumin in 2% melthylcellulose. Confocal imaging commenced immediately after patch application with the patch in place.
4D release kinetics from 70kDa payload in living skin are shown in Figures 17A
and 17B.
Figure 17A shows raw intensity values over 42 minutes, whereas Figure 17B
shows the calculated diffusion coefficient over the first 15 minutes.
The data were gathered from 20 m above the tip 1710 of projection 1700, as shown by the arrowhead 1720 in Figure 17C. The projection 1700 is pointing down and the region 1730 represents payload release. The colored cubes 1740 (2 m3 and 2 m away from the projection) show the 3D space that is being analyzed.
Example 8 In this example, groups of five C57BL/6 female mice aged 6 to 8 weeks were vaccinated with chicken egg albumin (Ovalbumin) protein either intramuscularly using the conventional syringe and needle, or onto the interior part of the ear skin using protein coated patch. The coating solution contains 10 mg/ml of MC, 10 mg/ml of OVA and 2 mg/ml of QA.
The area of each patch is 0.16 cm2. One patch per each ear was used in the vaccinations (i.e. a total of 2 patches per mouse). The patch was inserted into the skin at a speed of 1.96 m/s. The patch was kept for a further 5 minutes for the coated vaccine to be released. After 21 days, mice were bled and sera collected.
The serum samples were assayed by Enzyme-Linked ImmunoSorbent Assay (ELISA) using plates coated with Ovalbumin. Intramuscular immunised mice were injected with 6 g of OVA protein per mouse. MNP patch immunized mice were anesthetised and a single patch was applied to each ear, resulting in a total of 4.4 1.4 g of OVA protein delivered per mouse. The antibody levels of mice, including unimmunised, intramuscular immunised and coated patch immunised mice, are shown in Figure 18.
The data shown in Figure 18 demonstrates that much greater immune responses can be achieved by using coated patches at a similar dose with conventional needle and syringe.
Example 9 Following example 6, with these patch local skin delivery attributes established, the resultant systemic immune responses generated in mice were measured, with the results being shown in Figure 19A. The patch mice data were compared against needle and syringe intramuscular injection controls. Using needle and syringe (gauge.29 needle) intramuscular injection, a range of doses were delivered to the mouse caudal thigh muscle (0 (control), 0.04, 0.08, 0.8, and 6.0 g corresponding to the total HA as stated by the manufacturer (CSL
Ltd, Melbourne, Australia). Mice were bled 63 days after one immunisation.
Firstly, as shown in Figure 19A, the ELISA antibody reactivity (performed using sera with doubling serial dilutions starting from 1:100 up to 1:12800) was compared for the intramuscular needle and syringe doses compared with 0.04 g delivered with two patches.
The results show patch delivery (0.04 g) achieves similar antibody levels as generated by =6.0 gg delivered by IM injection.
Notably, it will be appreciated that although this establishes a dose reduction of a factor of 150, it is not specific to vaccination against influenza.
Thus, to measure relative levels of influenza protection, a heamagglutinin inhibition (HI) assay was used on the mice sera samples, with results being shown in Figure 19B. In particular, Hemagglutinin Inhibition assays (HI), were performed using the sera at different dilutions against each of the virus types (Wisconsin A, Malaysia B, and New Caledonia A).
Clearly, for all three stains of influenza, patch delivery (0.04 g) achieved HI levels equivalent to those generated by 6.0 g delivered by IM injection (p=0.357, 0.488 and 0.128 respectively for Wisconsin A, Malaysia B and New Caledonia A). This data shows the patch achieves a surrogate for vaccination protection against the influenza vaccine, with just a 1/150 of the dose delivered with the conventional needle and syringe.
Accordingly, it will be appreciated that dose reductions up to 150x could be achieved when influenza vaccine ((Fluvax 2007 ) was delivered directly to the dermis/epidermis of mice using the patch described herein. In this particular example, the patch includes densely packed projections (average 90 m in length) dry coated with the vaccine. This type of device is ideal for administering influenza vaccine in the case of a pandemic, not only because of the dose reduction achieved but the possibility of mass vaccinations by self administration of the vaccine. Notably, it will be appreciated that the device described can be extended to other types of vaccinations.
Thus, this example illustrates that a patch coated as described above shows may overcome the issues with using syringes and needles to vaccinate. In particular, a conventional influenza vaccine was delivered (Fluvax 2007 ) to C57BL/6 mice and the results showed that the patch delivery achieves equivalent immune responses as those induced by injection but with a dose reduced by a factor of 150. Accordingly, the patch as described in this example, can overcome key shortcomings of existing vaccine delivery technologies.
Example 10 In this example, two groups of 4 C57BL/6 female mice were immunised once with Fluvax coated patch or with Fluvax + CpG (ODN 1826) adjuvant. Mice were bled 2 weeks after one vaccination, and antigen specific total IgG, IgG1 and IgG2a levels were measured using ELISA. The results shown in Figure 20 demonstrate that a total reversal of IgGl and IgG2a responses when the adjuvant is included.
Accordingly, this example shows that the Th2 bias (Low antigen specific IgG2a/IgGl levels) shown by the use of the coated patch could be changed to Thl type of response, which may increase the CTL activity. This may be important in the case of cross protection to a different strain of the virus.
Example 11 Following the above example, a further example is used to investigate the ability to vaccinate subjects in more detail.
The present example combines patch and gene gun technology into a small scale device by allowing a gene gun to be used in patch application.
In this example, the patches are created through DRIE and contained 3364 individual projections that are 30 m wide at the base and between 45 and 130 m in length as shown in Figures 21A(c) and (d). The overall patch dimensions are 5x5 mm, as shown in Figures 21A(a) and (b).
The projection spacing are selected to match the distribution and depth of antigen presenting cells of the epidermis. Notably, these patches are not widely spaced and are typically short (<0.5 mm). The patches may also be made by deep reactive ion etching, so that they can be composed of silica and coated with a thin (-100 nm) gold layer.
The patch projections are -coated with the above described nitrogen jet drying method that results in a consistent and robust layer of antigen and/or adjuvant as shown in Figures 21A(e) and (f).
It will be appreciated that the gas jet coating method can provide numerous advantages. In one particular example, the method creates a dry-coating formulation that is typically robust enough to use with different antigens and adjuvants. Notably, dip-coating techniques are difficult to use in this instance as the presently described patch has densely packed patch projections, and dip coating followed by air drying often leads to a thick layer of dried material at the base and not the patch projections.
After removal, the coating on the patch projections was removed (g) and (h).
During patch application the skin is penetrated (i) to (m) (in (i) to (k) the bars indicate 1.00, 0.10, and 0.01 mm, respectively) by the projections and the strata compressed at the puncture site. The penetration of the skin by the coated patch projections resulted in the delivery of antigens to the epidermis and the upper-dermis ((n), bar is 100 and 10 m in the panel and inset, respectively).
The coated patch can then be applied with an anchored spring device that drives the patch into the skin at 1.8 m/s, where it can remain for up to 10 minutes. As shown in the SEM
images of Figures 21A(g) and (h) that the majority of coating is removed from the patch. The arrowheads are identifying corneocytes that have remained with the device. The high magnification image in Figure 21A(h) illustrates that the majority of the coating has been removed during the application process.
Furthermore, Figures 21A(i) to 21A(k) show increased magnification of the ventral side of a mouse ear that was snap frozen during patch application. These cryo-SEM images show the penetration of the individual patch projections into the surface of the, skin.
The depth of penetration and shape of the skin during patch application can be seen in the cryo-fractured skin photographed at an angle in Figure 21 A(1) and 21 A(m).
In particular, Figure 21A(m) is a single penetration site with an upturned corneocytes at the top; from this image one can appreciate that the patch can penetrate easily through the epidermis and into the dermis.
Once the patch penetrates the skin, the dried vaccine formulation can release from the patch projections and remain in the skin. This was monitored by having Fluvax 2007 fluorescently labelled, so that sections of the skin revealed the release pattern of a dry-coated vaccine delivered by the patch. In this example the fluorescent labelling is shown at 2120 in Figure 21A(n). In this image, the top row of nuclei 2110 highlight the epidermis with the vaccine shown at 2120 being seen through the epidermis and into the dermis.
The inset in Figure 21A(n) shows an overlay of each deposit site with dotted lines highlighting the strata boundaries(S, stratum corneum; E, epidermis; and D, dermis). In this image this highlights the ability of the patch to deliver antigen to both the epidermis and the upper dermis. One observation from the Figure 21A(n) is that the deposit of antigen does not appear to retain the cone shape of the patch projection, nor a cylindrical pattern; but rather resembles amorphous diffusion.
The diffusion of fluorescently labelled antigens was observed and analyzed using live confocal microscopy. Thus, a patch coated with fluorescently labeled dextran was applied to freshly excised skin and immediately imaged in 3D every minute for' over 15 minutes, with the resulting diffusion of the released material being rendered in 3D and shown in Figure 21B(a) to (c). These show that within 10 minutes the majority of diffusion had occurred. The data also indicated that the diffusion radius was approximately 1 to 2 cell diameters. This range is useful due to the even distribution of antigen presenting cells in the epidermis.
Accordingly, this highlights the ability to deliver antigen directly to antigen presenting cells in the epidermis. Having observed the delivery, release, and diffusion of antigen in the areas where antigen presenting cells were located,,it was also noted that three days after the patch delivered antigen to the skin, the antigen presenting cells (MHCII positive) were gone form the patch area but remained outside the patch projection free margin, as shown in Figure 21B(d). In particular, this shows a series of stitched images from the patch area to the margin and into the untreated region of the skin.
This observation led to further tests with patch delivered ovalbumin (OVA).
Quantification of MHCII positive cells over time revealed a rapid decline in the number of epidermal antigen presenting cells within three days, as shown in Figure 21B(e) and (f).
The number of antigen deposit sites was also tallied and showed greater that 86% (or >2800 projections per patch) of the patch projections delivered antigen into the skin.
Notably, one day after patch application the number of MHCII and antigen co-localization dropped more than the number of MHCII cells, as shown in Figures 21B(f) and (g). This implies that those MHCII positive cells that were in contact with the released antigen migrated quicker than those further away. Together these observations may indicate a mechanism through which the patch could deliver antigen directly to cells with MHCII; and those cells could carry the antigen to the lymph nodes for presentation. The number of MHCII positive cells that were exposed to antigen and their migration away from the application area leads to systemic immune response studies to confirm vaccination.
Influenza antigen from the commercially available vaccine, Fluvax was used for testing the patch delivery device of this example. The coating formulation contained 4 micrograms Hemagglutinin (HA) and 100 micrograms MC per patch. The coated patches are shown in Figures 21A(e) and 21A(f). A release assay based on fluorescently labeled Fluvax showed that this configuration of patch delivered approximately 20 ng HA per device.
Notably, this small amount of antigen is enough to generate strong IgG
production after 14 days (Figure 21C(a), solid line, solid triangles). For comparison, the antibody response of patch is much greater than that from an implant containing 40 ng HA and 100 microgram MC
(Figure 21C(a), dashed line, solid squares). Unimmunized sera is shown as a solid line in Figure 21C(a) to 21C(c).
Immune responses from patch delivered Fluvax was also compared to intramuscular injection of 0.04 micrograms HA (Fluvax ). However, the 40 ng HA injected dose was weaker than the patch (p=0.008) (Figure 21C(b)). Thus, it will be appreciated that these data values indicate that patch vaccination can result in a strong immune response with a well known and strong antigen.
According to a further example, the patch technology described herein was tested with an untested antigen from a globally important emerging disease without a commercial antigen.
Chikungunya virus antigen was made by irradiating cultured virus from the 2005-Reunion Island out break. The irradiated virus was then coated onto the patch at 5 micrograms killed virus, 100 micrograms MC, and 6 micrograms QA (or 20 micrograms CpG). Only a single patch was applied per animal.
After 14 days a strong immune response could be seen from the group with QA as shown in Figure 21C(c). The patch-QA response was significantly greater than the subcutaneously injected positive control that contained 5 micrograms killed virus and 6 micrograms QA to p=0.0017. The CpG adjuvant group showed a weaker response than the subcutaneous positive control, but the response was obviously higher than the subcutaneous injected 5 micrograms killed virus with no adjuvant, the negative control. Both the QA
and the CpG
patch groups elicited immune responses but this positive result did not indicate protection status.
After confirming the antibody response to the patch delivered Chikungunya antigen, it is determined whether or not the patch induced the protection of virus-neutralizing antibodies.
The success of immunization depends on the ability of the individual to resist a challenge.
Two months after immunization, live Chikungunya virus challenges were carried out. The virus was injected into the feet and this results in foot swelling and viraemia in naive individuals. Mouse models of Chikungunya infection show that the viral replication induces the expression of MCP-1. MCP-1 is a known proinflammatory gene that helps to recruit macrophages and thus the result is inflammation and swelling at the site of alphaviral injection that can be documented by measuring foot swelling. The results indicated that while the patch group with CpG did decrease swelling, only the patch group with QA
was statistically significant from the sham immunized group and indistinguishable from the untreated controls.
Notably, foot swelling is a good but rough measure of the inflammatory response. However, the viraemia data shows clear protection from Chikungunya virus challenge in the patch group with QA group. This group showed no appreciable foot swelling nor was there any virus recovered from the sera after challenge. The peak viral titers were found at day 2 which is historically consistent. The sham immunized group had a mean TCID50 of 3.3 loglo which was much higher than the patch group with CpG as an adjuvant, to TCIDS0 of 1.4. loglo. The TCID50 from the patch group with QA had no detectible viraemia and was significantly different. from the sham group, to p=0.001. Thus, it will be appreciated that patch immunization can completely protect from Chikungunya virus challenge.
Accordingly, the above example highlights that dry-coating antigens with or without adjuvant onto patch projections that have been specifically designed to target immune cells of the skin have the capacity to protect against viral infection. The patch is simple to use and quite small compared to a needle and syringe. Thus, it will be appreciated that there is no risk of needle stick injury with this device.
Thus, it will be appreciated that the patch described herein, in one example, can provide technology which has the capacity to effectively deliver antigen directly to antigen presenting cells, thereby eliciting a strong, protective immune response that holds up against challenge.
The coating methodology also developed has worked well with a variety of formulations including Influenza vaccine and killed Chikungunya virus; with and without adjuvants. The antigens were targeted to the immune cells of the skin and MHCII positive cells have been observed migrating in response to patch immunization. This immunization also led to strong and long lasting immunity to Chikungunya virus challenge. Thus, the patch described herein can provide an effective, next generation device for effective immunization.
Accordingly, this highlights that the coated patch provides a vaccine delivery method that is economical and efficient to prevent emerging, endemic, and enzootic diseases before they cause health and economic tragedies.
Example 12 In this example, epidermal targeted transfection with- a projection patch dry-coated with DNA
containing nanoparticles is performed.
The size distribution of PEI:DNA nanoparticles is shown in Figures 22A and 22B.
Nanoparticles were produced at a N:P of 5:1 with PEI (25k linear) and pEGFP
DNA in ultra-pure water.
A coating solution containing methylcellulose and PEI/DNA nanoparticles was used to coat projection patches following Example 1. The morphology of the coated patches and the coated patches after being applied on mouse ear for 15 minutes for nanoparticle delivery to the mouse ear skin is shown in Figures 22C and 22D, respectively.
After dry coating, the coated patches were dipped in water to get reconstituted PEI/DNA
nanoparticles. The aim was to confirm that the nanoparticles did not aggregate after coating process. Agarose gel analysis was performed on original and reconstituted PEI:DNA
nanoparticles for a variety of formulations including different N:P ratios (0:1, 5:1, and 9:1).
The results in Figure 22E show that dried and reconstituted PEI:DNA
nanoparticles still retain their supramolecular structure and do not release free DNA despite a change in size.
This is evidenced by positive staining in the well of both reconstituted samples.
Finally, patches coated with PEI/DNA nanoparticles were used to deliver nanoparticles into mouse ear skin for transfection study. The resulting transfection image is shown in Figures 22F and 22G. Figure 22F shows that cells with dendrites can be transfected by PEI/DNA
nanoparticles delivered by coated Nanopatches. Figure 22G shows that the transfection is in the epidermal layer of mouse ear skin.
A number of further variations and options for use with the above described devices will now be described.
Herein, the terms "projection", "micro-nanoprojection", "nanoneedle", "nanoprojection", "needle", "rod" etc are used interchangeably to describe the projections.
A further feature is that the projections may be used for delivery not only through the skin but through other body surfaces, including mucosal surfaces, to cellular sites below the outer layer or layers of such surfaces. The term "internal site", as used herein, is to be understood as indicating a site below the outer layer(s) of skin and other tissues for which the devices of the present invention are to be used.
The device is suitable for intracellular delivery. The device is suitable for delivery to specific organelles within cells. Examples of organelles to which the device can be applied include a cell nucleus, or endoplasmic reticulum, for example.
In one example the device is provided having a needle support section, that is to say the projections comprise a suitable support section, of sufficient length to reach the desired site and a (needle) delivery end section having a length no greater than 20 microns and a maximum width no greater than 5 microns, preferably no greater than 2 microns.
In one example, the maximum width of the delivery end section is no greater than 1000 nm, even more preferably the maximum width of the delivery end section is no greater than 500 nm.
In a further example, the device is for mucosal delivery. This device may have a needle support section, that is to say the projections comprise a suitable support section, of sufficient length to reach the desired site, such as of length at least 100 microns and a (needle) delivery end section having a length no greater than 20 microns and a maximum width no greater than microns, preferably no greater than 2 microns.
In one example, the device of the invention is for delivery to lung, eye, cornea, sclera or other internal organ or tissue. In a further example, the device is for in-vitro delivery to tissue, cell cultures, cell lines, organs, artificial tissues and tissue engineered products. This device typically has a needle support section, that is to say the projections comprise a suitable support section, of length at least 5 microns and a needle delivery end section having a length no greater than 20 microns and a maximum width no greater than 5 microns, preferably no greater than 2 microns.
In one example, the device comprises projections in which the (needle) delivery end section and support length, that is to say the "needle support section", is coated with a bioactive material across the whole or part of its length. The (needle) delivery end section and support length may be coated'on selective areas thereof. This may depend upon the bioactive material being used or the target selected for example.
In a further example, a bioactive material is releasably incorporated into the material of which the needle, or projection, is composed. All, or part of the projection may be constructed of a biocompatible, biodegradable polymer (such as Poly Lactic Acid (PLA), PolyGlycolic Acid (PGA) or PGLA or Poly Glucleic Acid), which is formulated with the bioactive material of choice. The projections may then be inserted into the appropriate target site and, as they dissolve, the bioactive material will enter the organelle(s)/cells.
Examples of bioactive materials, which are not intended to be limiting with respect to the invention include polynucleotides and nucleic acid or protein molecules, antigens, allergens, adjuvants, molecules, elements or compounds. In addition, the device may be coated with materials such as biosensors, nanosensors or MEMS.
Illustrative material that can be delivered may include any or more of. small chemical or biochemical compounds including drugs, metabolites, amino acids, sugars, lipids, saponins, and hormones; macromolecules such as complex carbohydrates, phospholipids, peptides, polypeptides, peptidomimetics, and nucleic acids; or other organic (carbon containing) or inorganic molecules; and particulate matter including whole cells, bacteria, viruses, virus-like particles, cell membranes, dendrimers and liposomes.
The material can be selected from nucleic acids, illustrative examples of which include DNA, RNA, sense oligonucleotides, antisense oligonucleotides, ribozymes, small interfering oligonucleotides (siRNAs), micro RNAs (miRNAs), repeat associated RNAs (rasiRNA), effector RNAs (eRNAs), and any other oligonucleotides known in the art, which inhibit transcription and/or translation of a mutated or - other detrimental protein.
In illustrative examples of this type, the nucleic acid is in the form of an expression vector from which a polyilucleotide of interest is expressible. The polynucleotide of interest may encode a polypeptide or an effector nucleic acid molecule such as sense or antisense oligonucleotides, siRNAs, miRNAs and eRNAs.
The material can be selected from peptides or polypeptides, illustrative examples of which include insulin, proinsulin, follicle stimulating hormone, insulin like growthfactor-l, insulin like growth factor-2, platelet derived growth factor, epidermal growth factor, fibroblast growth factors, nerve growth factor, colony stimulating factors, transforming growth factors, tumor necrosis factor, calcitonin, parathyroid hormone, growth hormone, bone morphogenic protein, erythropoietin, hemopoietic growth factors, luteinizing hormone, glucagon, glucagon likepeptide-1, anti-angiogenic proteins, clotting factors, anti-clotting factors, atrial natriuretic factor, plasminogen activators, bombesin, thrombin, enkephalinase, vascular endothelial growth factor, interleukins, viral antigens, non-viral antigens, transport proteins, and antibodies.
The material can be selected from receptor ligands. Illustrative examples of receptors include Fc receptor, heparin sulfate receptor, vitronectin receptor, Vcam-1 receptor, hemaglutinin receptor, Pvr receptor, Icam-1 receptor, decay-accelerating protein (CD55) receptor, Car (coxsackievirus-aderiovirus) receptor, integrin receptor, sialic acid receptor, HAVCr-1 receptor, low-density lipoprotein receptor, BGP (biliary glycoprotien) receptor, aminopeptidease N receptor, MHC class-1 receptor, laminin receptor, nicotinic acetylcholine receptor, CD56 receptor, nerve growth factor receptor, CD46 receptor, asialoglycoprotein receptor Gp-2, alpha-dystroglycan receptor, galactosylceramide receptor, Cxcr4 receptor, Glvrl receptor, Ram-1 receptor, Cat receptor, Tva receptor, BLVRcp1 receptor, MHC class-2 receptor, toll-like receptors (such as TLR-1 to -6) and complement receptors.
The material can be selected from antigens including endogenous antigens produced by a host that is the subject of the stimulus or material delivery or exogenous antigens that are foreign to that host. The antigens may be in the form of soluble peptides or polypeptides or polynucleotides from which an expression product (e.g., protein or RNA) is producible.
Suitable endogenous antigens include, but are not restricted to, cancer or tumor antigens.
Non-limiting examples of cancer or tumor antigens include antigens from a cancer or tumor selected from ABL1 proto-oneogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS
tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma, oesophageal cancer, Ewing's Sarcoma, Extra-Hepatic Bile Duct Cancer, Eye Cancer, Eye: Melanoma, Retinoblastoma, Fallopian Tube cancer, Fanconi anemia, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell tumors, gestational-trophoblastic-disease, glioma, gynecological cancers, haematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhans' cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant-rhabdoid tumor of kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer (NSCLC), ocular cancers, esophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral-neuroectodermal tumours, pituitary cancer, polycythemia vera, prostate cancer, rare cancers and associated disorders,. renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, 'Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom's macroglobulinemia, Wilms' tumor. In certain examples, the cancer or tumor relates to melanoma. Illustrative examples of melanoma-related antigens include melanocyte differentiation antigen (e.g., gplOO, MART, Melan-A/MART-1, TRP-l, Tyros, TRP2, MCIR, MUC1F, MUC1R or a combination thereof) and melanoma-specific antigens (e.g., BAGE, GAGE-I, gplOOIn4, MAGE-1 (e.g., GenBank Accession No.
and AA494311), MAGE-3, MAGE4, PRAME, TRP21N2, NYNSO1a, NYNSO1b, LAGEI,, p97 melanoma antigen (e.g., GenBank Accession No. M12154) p5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides, cdc27, p2lras, gp100Pme1117 or a combination thereof.
Other tumour-specific antigens include, but are not limited to: etv6, amll, cyclophilin b (acute lymphoblastic leukemia); Ig-idiotype (B cell lymphoma); E-cadherin, a-catenin, 13-catenin, y-catenin, p l20ctn (glioma); p2lras (bladder cancer); p2lras (biliary cancer); MUC
family, HER2/neu, c-erbB-2 (breast cancer); p53, p2lras (cervical carcinoma);
p2lras, HER2/neu, c-erbB-2, MUC family, Cripto- 1 protein, Pim-1 protein (colon carcinoma);
Colorectal associated antigen (CRC)-CO17-lA/GA733, APC (colorectal cancer);
carcinoembryonic antigen (CEA) (colorectal cancer; choriocarcinoma);
cyclophilin b (epithelial cell cancer); HER2/neu, c-erbB-2, ga733 glycoprotein (gastric cancer); a-fetoprotein (hepatocellular cancer); Imp-1, EBNA-1 (Hodgkin's lymphoma); CEA, MAGE-3, NY-ESO-1 (lung cancer); cyclophilin b (lymphoid cell-derived leukemia); MUC
family, p2lras (myeloma); HER2/neu, c-erbB-2 (non-small cell lung -carcinoma); Imp-1, EBNA-l (nasopharyngeal cancer); MUC family, HER2/neu, c-erbB-2, MAGE-A4, NY-ESO-1 (ovarian cancer); Prostate Specific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB-2, ga733 glycoprotein (prostate cancer);
HER2/neu, c-erbB-2 (renal cancer); viral products such as human papillomavirus proteins (squamous cell cancers of the cervix and esophagus); NY-ESO-1 (testicular cancer); and HTLV-1 epitopes (T cell leukemia).
Foreign antigens are suitably selected from transplantation antigens, allergens as well as antigens from pathogenic organisms. Transplantation antigens can be derived from donor cells or tissues from e.g., heart, lung, liver, pancreas, kidney, neural graft components, or from the donor antigen-presenting cells bearing MHC loaded with self antigen in the absence of exogenous antigen.
Non-limiting examples of allergens include Fel d 1 (i.e., the feline skin and salivary gland allergen of the domestic cat Felis domesticus, the amino acid sequence of which is disclosed International Publication WO 91/06571), Der p I, Der p II, Der if or Der fiI
(i.e., the major protein allergens from the house dust mite dermatophagoides, the amino acid sequence of which is disclosed in International Publication WO 94/24281). Other allergens may be derived, for example from the following: grass, tree and weed (including ragweed) pollens;
fungi and moulds; foods such as fish, shellfish, crab, lobster, peanuts, nuts, wheat gluten, eggs and milk; stinging insects such as bee, wasp, and hornet and the chirnomidae (non-biting midges); other insects such as the housefly, fruitfly, sheep blow fly, screw worm fly, grain weevil, silkworm, honeybee, non-biting midge larvae, bee moth larvae, mealworm, cockroach and larvae of Tenibrio molitor beetle; spiders and mites, including the house dust mite; allergens found in the dander, urine, saliva, blood or other bodily fluid of mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat, guinea pig, mouse and gerbil; airborne particulates in general; latex; and protein surfactant additives.
The material can be pathogenic organisms such as, but are not limited to, viruses, bacteria, fungi parasites, algae and protozoa and amoebae. Illustrative viruses include viruses responsible for diseases including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C
(e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Epstein-Barr virus and other herpesviruses such as papillomavirus, Ebola virus, influenza virus, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No.
M24444), hantavirus, Sendai virus, respiratory syncytial virus, othromyxoviruses, vesicular stomatitis virus, visna virus, cytomegalovirus and human immunodeficiency virus (HIV) (e.g., GenBank Accession No. U18552). Any suitable antigen derived from such viruses are useful in the practice of the present invention. For example, illustrative retroviral antigens derived from HIV include, but are not limited to, antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV
components.
Illustrative examples of hepatitis viral antigens include, but are not limited to, antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA.
Illustrative examples of influenza viral antigens include; but are not limited to, antigens such as hemagglutinin and neurarninidase and other influenza viral components.
Illustrative examples of measles viral antigens include, but are not limited to, antigens such as the measles virus fusion protein and other measles virus components. Illustrative examples of rubella viral antigens include, but are not limited to, antigens such as proteins El and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components. Illustrative examples of cytomegaloviral antigens include, but are not limited to, antigens such as envelope glycoprotein B and other cytomegaloviral antigen components. Non-limiting examples of respiratory syncytial viral antigens include antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components. Illustrative examples of herpes simplex viral antigens include, but are not limited to, antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components. Nori-limiting examples of varicella zoster viral antigens include antigens such as 9PI, gpll, and other varicella zoster viral antigen components.
Non-limiting examples of Japanese encephalitis viral antigens include antigens such as proteins E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80%E, and other Japanese encephalitis viral antigen components. Representative examples of rabies viral antigens include, but are not limited to, antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. Illustrative examples of papillomavirus antigens include, but are not limited to, the L1 and L2 capsid proteins as well as the E6/E7 antigens associated with cervical cancers, See Fundamental Virology, Second Edition, eds. Fields, B.N.
and Knipe, D.M., 1991, Raven Press, New York, for additional examples of viral antigens.
Illustrative examples of fungi include Acremonium spp., Aspergillus spp., Basidiobolus spp., Bipolaris spp., Blastomyces derinatidis, Candida spp., Cladophialophora carrionii, Coccoidiodes immitis, Conidiobolus spp., Cryptococcus spp., Curvularia spp., Epidermophyton spp., Exophiala jeanselmei, Exserohilum spp., Fonsecaea cornpacta, Fonsecaea pedrosoi, Fusarium oxysporum, Fusarium solani, Geotrichum candidum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Hortaea werneckii, Lacazia loboi, Lasiodiplodia theobromae, Leptosphaeria senegalensis, Madurella grisea, Madurella mycetomatis, Malassezia furfur, Microsporum spp., Neotestudina rosatii, Onychocola , canadensis, Paracoccidioides brasiliensis, Phialophora verrucosa, Piedraia hortae, Piedra iahortae, Pityriasis versicolor, Pseudallesheria boydii, Pyrenochaeta romeroi, Rhizopus arrhizus, Scopulariopsis brevicaulis, Scytalidium dimidiatum, Sporothrix schenckii, Trichophyton spp., Trichosporon spp., Zygomcete fungi, Absidia corymbifera, Rhizomucor pusillus and Rhizopus arrhizus. Thus, representative fungal antigens that can be used in the compositions and methods of the present invention include, but are not limited to, candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as'spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.
Illustrative examples of bacteria include bacteria that are responsible for diseases including, but not restricted to, diphtheria (e.g., Corynebacterium diphtheria), pertussis (e.g., Bordetella pertussis, GenBank Accession No. M35274), tetanus (e.g., Clostridium tetani, GenBank Accession No. M64353), tuberculosis (e.g., Mycobacterium tuberculosis), bacterial pneumonias (e.g., Haemophilus influenzae.), cholera (e.g., Vibrio cholerae), anthrax (e.g., Bacillus anthracis), typhoid, plague, shigellosis (e.g., Shigella dysenteriae), botulism (e.g., Clostridium botulinuni), salmonellosis (e.g., GenBank Accession No.
L03833), peptic ulcers (e.g., Helicobacter pylori), Legionnaire's Disease, Lyme disease (e.g., GenBank Accession No. U59487), Other pathogenic bacteria include Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. Thus, bacterial antigens which can be used in the compositions and methods of the invention include, but are not limited to: pertussis bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, F M2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diphtheria bacterial antigens such as diphtheria toxin or toxoid and other diphtheria bacterial antigen components;
tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components, streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components;
Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30kDa major secreted protein, antigen 85A and other mycobacterial antigen components;
Helicobacter pylori bacterial antigen components, pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pnermiococcal bacterial antigen components; Haemophilus influenza bacterial antigens such as capsular polysaccharides and other Haemophilus influenza bacterial antigen components;
anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as rompA and other rickettsiae bacterial antigen component. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.
Illustrative examples of protozoa include protozoa that are responsible for diseases including, but not limited to, malaria (e.g., GenBank Accession No. X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. LOS 198), toxoplasmosis, trypanosomiasis, leishmaniasis, giardiasis (GenBank Accession No. M33641), amoebiasis, filariasis (e.g., GenBank Accession No.
J03266), borreliosis, and trichinosis. Thus, protozoal antigens which can be used in the compositions and methods of the invention include, but are not limited to:
plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, blood-stage antigen pf 155/RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and trypanosoma cruzi antigens such as the 75-77kDa antigen, the 56kDa antigen and other trypanosomal antigen components.
The material can be toxin components acting as antigens. Illustrative examples of toxins include, but are not restricted to, staphylococcal enterotoxins, toxic shock syndrome toxin;
retroviral antigens (e.g., antigens derived from HIV), streptococcal antigens, staphylococcal enterotoxin-A (SEA), staphylococcal enterotoxin-B (SEB), staphylococcal enterotoxin1_3 (SE1_3), staphylococcal enterotoxin-D (SED), staphylococcal enterotoxin-E
(SEE) as well as toxins derived from mycoplasma, mycobacterium, and herpes viruses.
In specific examples, the antigen is delivered to antigen-presenting cells.
Such antigen-presenting cells include professional or facultative antigen-presenting cells.
Professional antigen-presenting cells function physiologically to present antigen in a form that is recognised by specific T cell receptors so as to stimulate or anergise a T
lymphocyte or B
lymphocyte mediated immune response. Professional antigen-presenting cells not only process and present antigens in the context of the major histocompatability complex (MHC), but also possess the additional immunoregulatory molecules required to complete T cell activation or induce a tolerogenic response. Professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Kupffer cells, microglia, T
cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells. Non-professional or facultative antigen-presenting cells typically lack one or more of the immunoregulatory molecules required to complete T
lymphocyte activation or anergy. Examples of non-professional or facultative antigen-presenting cells include, but are not limited to, activated T lymphocytes, eosinophils, keratinocytes, astrocytes, follicular cells, microglial cells, thymic cortical cells, endothelial cells, Schwann cells, retinal pigment epithelial cells, myoblasts, vascular smooth muscle cells, chondrocytes, enterocytes, thymocytes, kidney tubule cells and fibroblasts. In some examples, the antigen-presenting cell is selected from monocytes, macrophages, B lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells. In certain advantageous examples, the antigen-presenting cell expresses CD11c and includes a dendritic cell or Langerhans cell. In some examples the antigen-presenting cell stimulates an immune response. In other examples, the antigen-presenting cell induces a tolerogenic response.
The delivery of exogenous antigen to an antigen-presenting cell can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous antigen to the endogenous processing pathway of antigen-presenting cells, especially dendritic cells. These methods include insertion of antigen into pH-sensitive liposomes (Zhou and Huang, 1994, Immunomethods, 4:229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (Moore et al., 1988, Cell, 54:777-785), coupling of antigens to potent adjuvants (Aichele et al., 1990, J Exp.
Med., 171: 1815-1820; Gao et al., 1991, J Immunol., 147: 3268-3273; Schulz et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 991-993; Kuzu et al., 1993, Euro. J. Immunol., 23: 1397-1400; and Jondal et al., 1996, Immunity 5: 295-302) and apoptotic cell delivery of antigen (Albert et al. 1998, Nature 392:86-89; Albert et al. 1998, Nature Med. 4:1321-1324; and in International Publications WO 99/42564 and WO 01/85207). Recombinant bacteria (eg. E.
coli) or transfected host mammalian cells may be pulsed onto dendritic cells (as particulate antigen, or apoptotic bodies respectively) for antigen delivery. Recombinant chimeric virus-like particles (VLPs) have also been used as vehicles for delivery of exogenous heterologous antigen to the MHC class I processing pathway of a dendritic cell line (Bachmann et al,, .1996, Eur. J. Immunol., 26(11): 2595-2600).
Alternatively, or in addition, an antigen may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the antigen into the cytosol of an antigen-presenting cell of the invention for delivery to the MHC class I pathway. Exemplary cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997, Vaccine 15(3): 248-256 and U.S. Patent No.
6,352,697), phospholipases (see, e.g., Camilli et al., 1991, J. Exp. Med. 173: 751-754), pore-forming toxins (e.g., an a-toxin), natural cytolysins of gram-positive bacteria, such as listeriolysin 0 (LLO, e.g., Mengaud et al., 1988, Infect. Immun. 56: 766-772 and Portnoy et al., 1992, Infect.
Immun. 60: 2710-2717), streptolysin 0 (SLO, e.g., Palmer et al., 1998, Biochemistry 37(8):
2378-2383) and perfringolysin 0 (PFO, e.g., Rossjohn et al., Cell 89(5): 685-692). Where the antigen-presenting cell is phagosomal, acid activated cytolysins may be advantageously used.
For example, listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH
conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al., Infect. Immun.
1992, 60: 2710-2717).
The' cytolysin may be provided together with a pre-selected antigen in the form of a single composition or may be provided as a separate composition, for 'contacting the antigen-presenting cells. In one example, the cytolysin is fused or otherwise linked to the antigen, wherein the fusion or linkage permits' the delivery of the antigen to the cytosol of the target cell. In another example, the cytolysin and antigen are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast. Suitably, when the delivery vehicle is a microbial delivery vehicle, the delivery vehicle is non-virulent. In a preferred example of this type, the delivery vehicle is a non-virulent bacterium, as for example described by Portnoy et al. in U.S. Patent No.
6,287,556, comprising a first polynucleotide encoding a non-secreted functional cytolysin operably linked to a regulatory polynucleotide which expresses the cytolysin in the bacterium, and a second polynucleotide encoding one or more pre-selected antigens. Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc. A
wide variety of nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterised strains, particularly laboratory strains of E. coli, such as MC4100, MC1061, DH5a, etc. Other bacteria that can be engineered for the invention include well-characterised, nonvirulent, non-pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc. In a particular example, the bacteria are attenuated to be non-replicative, non-integrative into the host cell genome, and/or non-motile inter- or intra-cellularly.
The coated patches described above can be used to deliver one or more antigens to virtually any antigen-presenting cell capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells. In examples when the delivery vehicle is a microbe, the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen-presenting cell vacuole (including phagosomes and endosomes).
In other examples, the antigen is produced inside the antigen-presenting cell by introduction of a suitable expression vector as for example described above. The antigen-encoding portion of the expression vector may comprise a naturally-occurring sequence or a variant thereof, which has been engineered using recombinant techniques. In one example of a variant, the codon composition of an antigen-encoding polynucleotide is modified to permit enhanced expression of the antigen in a target cell or tissue of choice using methods as set forth in detail in International Publications WO 99/02694 and WO 00/42215. Briefly, these methods are based on the observation that translational efficiencies of different codons vary between different cells or tissues and that these differences can be exploited, together with codon composition of a gene, to regulate expression of a protein in a particular cell or tissue type.
Thus, for the construction of codon-optimised polynucleotides, at least one existing codon of a parent polynucleotide is replaced with a synonymous codon that has a higher translational, efficiency in a target cell or tissue than the existing codon it replaces.
Although it is preferable to replace all the existing codons of a parent nucleic acid molecule with synonymous codons which have that higher translational efficiency, this is not necessary because increased expression can be accomplished even with partial replacement. Suitably, the replacement step affects 5, 10, 15, 20, 25, 30%, more preferably 35, 40, 50, 60, 70% or more of the existing codons of a parent polynucleotide.
The expression vector for introduction into the antigen-presenting cell will be compatible therewith such that the antigen-encoding polynucleotide is expressible by the cell. For example, expression vectors of this type can be derived from viral DNA
sequences including, but not limited to, adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses. Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000, Curr. Opin. Biotechnol, 11(2):205-208), Vigna and Naldini (2000, J. Gene Med. 2(5):308-316), Kay, et al. (2001, Nat. Med. 7(l):33-40), Athanasopoulos, et al. (2000, Int. J. Mol. Med. 6(4):363-375) and Walther and Stein (2000, Drugs 60(2):249-271).
In one aspect, the device is provided in the form of a patch containing a plurality of needles (projections) for application to a body surface. A multiplicity of projections can allow multiple cells and organelles to be targeted and provided with a material at the same time.
The patch may be of any suitable shape, such as square or round for example.
The overall number of projections per patch depends upon the particular application in which the device is to be used. Preferably, the patch has at least 10 needles per mm, and more preferably at least 100 needles per mm2. Considerations and specific examples of such a patch are provided in more detail below.
Examples of specific manufacturing steps used to fabricate the device are described in greater detail below. In one preferred aspect, the device of the invention is constructed from biocompatible materials such as Titanium, Gold, Silver or Silicon, for example. This may be the entire device, or alternatively it may only be the projections or the delivery end section of the projections which are made from the biocompatible materials.
One manufacturing method for the device utilises the Deep Reactive Ion Etching (DRIE) of the patterns direct from silicon wafers, see the construction section below.
Another manufacturing method for the device utilises manufacturing from a male template constructed with X-ray lithography, electrodeposition and moulding (LIGA). The templates are then multiply inserted into a soft polymer to produce a plurality of masks. The masks are then vacuum deposited/sputtered with the material of choice for the nanoprojections, such as titanium, gold, silver, or tungsten. Magnetron sputtering may also be applied, see the construction section below.
An alternative means for producing masks is, with 2 photon Stereolithography, a technique which is known in the art and is described in more detail below.
In one example, the device is constructed of silicon.
The device may be for a single use or may be used and then recoated with the same or. a different bioactive material or other stimulus, for example.
In one example, the device comprises projections which are of differing lengths and/or diameters (or thicknesses depending on the shape of the projections) to allow targeting of different targets within the same use of the device.
Persons skilled in the art will appreciate that numerous variations and modifications will become apparent. All such variations and modifications which become apparent to persons skilled in the art, should be considered to fall within the spirit and scope that the invention broadly appearing before described.
OVA
protein and 0.2% QA is applied on 90 m projection patches and dry with a nitrogen jet, 120 g OVA protein will be coated onto projections and 40 pg OVA protein will be coated onto base, using a gas flow in the range 6-8 m/s.
Accordingly, the above described examples provide method for coating therapeutic agents including vaccines on to projections on a patch, to thereby allow for their rapid release when the patch is applied to a subject. The method provides substantially uniform and controllable coating of therapeutic agents like DNA or protein vaccine onto the patches, even in circumstances when the patches are hydrophobic. The method can be applied to any form of patch but is especially suited for patches having projections that are shorter than 200 m and separated by 10-1000 m.
Further variations and options will now be described.
For example, the patch and/or projections can be coated with a thin layer of a suitable metal, prior to application of the coating solution. The reason for this is that metals tend to have, relative to the native silicon or other patch material, a high surface energy, which in turn helps assist with the coating process. In one example, the metal layer is gold, although other suitable metals may be used. An example of a gold coating on a silicon projection is shown in Figure 4. Gold coating forms a nanostructure on silicon projection. The thickness is about 400-1500 nm and the size of gold particles is about 200-400 nm. This structure together with the projection arrangement provides a very hydrophobic surface.
As described above, the coating solution is typically selected to have a suitable viscosity and surface tension. This may be achieved using viscosity enhancers and surfactants to control the coating solution properties. However, use of surfactants is not essential and in one example, a vaccine coating can be achieved using MC without requiring surfactants.
However, if CMC is used for coating, the addition of surfactants is preferred.
As mentioned above, the surfactant can be any suitable agent such as poloxomer 188, triton-X 100, NP40, QA or any surfactant that is biocompatible at a low concentration. The concentration of the surfactant is from about wt. 0% to about 90% of the coating solution, depending on the required solution properties.
A vaccine adjuvant may also be added to the coating solution for enhancing immune response to vaccines. In one example, the adjuvants used include Quillaja saponins, such as QA, QS-21, QS-7 or other purified saponin adjuvants. Use of QA and other similar saponin adjuvants can be particularly beneficial as QA not only acts as a surfactant for coating purposes but also as the vaccine adjuvant. Furthermore, due to QA
effectiveness in reducing the surface tension of the coating solution, this can in turn help in reducing the amount of excipients used for coating.
Other amphipathic immunostimulatory compounds such as dimethyldioctadecylammonium bromide or chemically modified immunostimulatory molecules to give surfactant properties can also be employed.
The viscosity agent can be selected from MC, CMC, gelatin, agar, agarose or any other viscosity agent, which can be any substance that modifies the viscosity of the coating solution. The concentration of the viscosity agent is typically from about wt.
0% to about 90% of the coating solution.
Whilst a range of therapeutic agents can be used, in one. example the agents are vaccines.
The vaccine can be composed of DNA or protein and can also contain an adjuvant. The concentration of DNA in the coating solution can be from 0.01 mg/ml to 5 mg/ml. The concentration of protein in the coating solution can be from 0.01 to 50 mg/ml.
The material can include nanoparticles to provide a nanodelivery system. For example the coating can include DNA containing nanoparticles.
In one example, the nanoparticles are multilayered nanoparticles. Outermost layers of the nanoparticles can include cell targeting and cell-entry facilitating molecules. The next layer can include intracellular targeting molecules for precise delivery of the nanoparticle complex inside the cell of interest.
Molecular biosensors can be used to confirm the presence of expected molecules as a surrogate molecule for signs of infection, for activation in radiation damage, or other criteria, prior to delivery of counter-measure molecules such as vaccines, drugs, or gene therapy. The biosensors can also be used as a feedback control mechanism to control the proper amount of vaccine/drug/gene delivery for each cell.
Further, the nanodelivery system can be used to restrict any cells from encountering the drug unless that cell is specifically targeted. Successful targeting can be verified by 3D
multispectral confocal microscopy. These single cell molecular morphology measurements can be extended from individual cells, to other cells in a tissue in tissue monolayers or tissue sections.
This example can be used to provide a nanomedical system and method that can be used for diagnostics, therapeutics, vaccines, or a combination thereof by use of a multilayered nanoparticle system. The multilayered nanoparticle system can built on a nanoparticle core of bio-polymer, polystyrene, silica, gold, iron, or other material.
The concentration, viscosity and surface tension will all influence the thickness, morphology and payload of coating. In the most preferred embodiments, the thickness of the coated vaccines can be from 10 nm to 10 m.
The amount of resulting dry coating on the projections can be controlled by the concentrations of excipients in coating solution, as well as the surface area of the projections, although as mentioned above, selection of an appropriate surfactant, such as QA can avoid the need for unnecessary excipients.
The coating solution can be applied in several ways. In one example, the projections are completely submersed in the coating solution, although alternatively a defined volume of coating solution can be applied to the patch, the amount of which can. vary depending on the patch area.
Once the coating solution is applied, the projections and/or the patch are dried. The gas flow can be used to move the coating solution over the patch surface 121, to thereby ensure all the projections 110 are coated. For example, the gas jet can be used to move the coating solution from one edge of the patch to another opposing edge of the patch, by suitable direction of the gas jet. Additionally, and/or alternatively, the gas flow can be used to dry the coating solution on the projections quickly so the coating solution remains on the projections until they are dry. By using the gas drying technique, this ensures that coating is evenly distributed on the projections.
It will be appreciated that in some instances it may be desirable to coat the projections but not the base of the patch itself, for example to control the rate of delivery of the material, and to help reduce excessive usage of coating solution. This can be achieved using a coating solution of proper viscosity and surface tension and a defined drying process.
Specific examples of this will be described in more detail below.
In order to allow the coating solution to be distributed over the patch, it is typical to direct the gas flow over the patch in an appropriate manner. An example of apparatus for achieving this will now be described with reference to Figures 5A and 5B.
In this example, the gas flow is generated by a gas jet expelled from a nozzle 500. In one example, the nozzle is coupled via a tube 501 to a gas source 502, such as a compressed gas cylinder, a compressor, or the like. This allows the gas source 502 to supply gas to the nozzle 500, via the tube 501, thereby causing a gas jet to be emitted from the nozzle 500 in a direction substantially parallel to a nozzle axis 510. In one example, the gas source 502 includes a control 503, such as a flow rate valve, that allows the flow rate of the gas from the nozzle to be controlled.
As shown in Figure 5A, initially the coating solution is applied to the surface 120 on one side of the patch 100 near the edge 100B. The nozzle 500 is then aligned with the fluid on the patch, and aimed so as to direct the coating solution towards the other edge 100A of the patch 100. The nozzle 500 is generally aimed so that the nozzle axis 510 is at an angle a relative to a plane 511 containing the patch substrate 120.
Adjustment of the angle a can be used to control the rate at which the fluid is moved across the patch, allowing coating solutions of different viscosities to be moved across the patch prior to drying. It will be appreciated that additional distribution control can also be achieved by adjusting the gas flow rate, although this in turn has an impact on drying rate.
Accordingly, it is generally desirable to balance the distribution rate and drying rate for the coating solution by appropriate selection of an appropriate gas flow rate and angle a, which will in turn depend on the viscosity and surface tension of the coating solution. Typically however the angle a is in the region of 0 to 45 , and more typically 10 to 30 , and more typically about 20 .
In addition to this, the position of the nozzle 500 can also be adjusted to help distribute coating evenly over the patch 100. This can include moving the nozzle in a direction parallel to the edge 100B, to thereby ensure that coating is distributed across the entire patch width, as well as to move the nozzle in a direction perpendicular to the edge 100B, to thereby move solution along the length of the patch, as shown in Figure 5B.
In use, the nozzle 500 may also be held in position by a support arrangement 504, which can be any form of suitable support, such as an arm including a clamp, or the like. The support may be capable of manipulation, to allow the position of the nozzle 500 relative to the patch 100 to be adjusted. Thus, in one example, the support 504 could be in the form of a computer controlled arm, such as a robot arm, thereby allowing computer control of the coating process.
It will be appreciated that in addition to the above, multiple gas jets may be used to induce movement and/or drying of the coating solution. Furthermore, the multiple gas jets could be provided at different angles a, as well as at different orientations relative to the patch, to thereby enhance the distribution or drying effect.
Apparatus of this form can also be adapted to allow "Multiple patches to be coated during a single process. An example of such apparatus will now be described with reference to Figures 5C and 5D.
In this example, the apparatus is formed from a base 550 for supporting a number of patches 551, typically provided in an array. The apparatus includes two supports 552, for supporting two arms 560, 570, which are mounted to allow movement of the arms in the direction of arrow 580. The first arm 560 includes a coating solution delivery system including a nozzle 561 for depositing coating solution on the patches 551. The second arm including a gas delivery system including a gas nozzle 571. In use, the nozzle 561 and the gas nozzle 571 are movably mounted to allow lateral movement of the nozzles 561, 571 in the direction of the arrows 5 81.
Movement of the arms 560, 570 and the nozzles 561, 571, gas flow rate and coating solution delivery are typically achieved using a computer controlled drive system, shown generally at 590. This allows coating solution and gas flow to be delivered to the patches 551. This can be achieved collectively, or 'by delivery to each of the patches in turn. In either case, this allows coating solution to be applied, optionally distributed over the patches and dried.
In the example shown, a single. respective nozzle 561, 571 is used to deliver coating solution and gas flow. However, multiple nozzles may be provided. Additionally, or alternatively the coating solution and gas delivery systems can be incorporated into a single arm. A further alternative is to provide nozzle systems that extend across an entire length of the arm allowing coating solution and gas to be applied to multiple patches simultaneously.
Further examples of apparatus for providing a gas flow will now be described with reference to Figure 6A and 6B.
In the example of Figure 6A, the apparatus includes a housing 600 having a cavity 602 for containing a patch. In one example, the container is generally sealed to allow a pressure differential to be established between the inside and outside of the housing 600. This can be achieved by coupling the housing to a gas source 610 via a connecting tube 611, allowing the pressure within the housing 600 to be increased to a suitable level. Once this has been reached, a release valve 601 can be activated, allowing gas to escape from the housing 600 through the valve 601. This in turn generates a gas flow, as shown by the arrow 603. The gas flow can be directed utilising appropriate baffles provided on inner surfaces of the housing 600 as required.
As an alternative to pressurising the container however, a further option is to replace the gas source 610 with a vacuum pump, allowing air or another gas within the cavity 605 to be extracted, to thereby generate a gas flow.
In either case, it will be appreciated that appropriate positioning of the patch 100 within the housing 600, together with a suitable pressure differential, and hence suitable gas flow, can be used to ensure the patch is appropriately coated.
in the example of Figure 6B, an alternative design of container 650 is shown.
In this example, the container includes an opening 651 to allow a cavity 652 to be coupled to a vacuum pump 660, via a connecting tube 661. The patch 100 is supported in the cavity 652 above a lower surface of the cavity 653, using a suitable support 654. The patch 100 is also positioned below the opening 651. Consequently, when air or another gas is evacuated from the housing 650, a gas flow is generated as shown by the arrows 670. As the gas flows around the patch 100 turbulence causes air flow over the entire patch surface, thereby helping to distribute and/or dry the coating solution. It will be appreciated that as an alternative, the cavity 652 can be pressurised in a manner similar to that described above with respect to Figure 6A.
In one example, only the projections are coated. Consequently, when the patch is placed on the skin, substantially all of the coated therapeutic agent can be rapidly delivered into the skin from the projections. As a result, this can be used where rapid delivery of an agent is required.
However, there are cases where it is required for agent to also be coated to the base. As one example, where some delay is required for delivery of a therapeutic agent, the agent can also be coated onto the patch substrate or base 120. The agent coated on the projections can achieve fast delivery in skin for a first dose, while those coated on the patch base 120 can slowly permeate into the subject's skin through holes made by the projections thereby providing for further dose(s).
As another example, such arrangements may be used when it is desirable to deliver higher amounts of payload into the skin over and above the amount coated on the projections. In this case, the additional payload on the base of the patch can be hydrated (e.g. by fluid within the skin moving through holes generated by the projections with a capillary action) and released, a "depot effect" for higher delivery dose.
An example of this will now be described with reference to Figures 7A and 7B.
In the example of Figure 7A, the patch 100 includes coating 710 provided on the projection 110, and coating 720 on the surface 121. Initially, when the patch 100 is applied to a subject, the projections 110 extend through the skin 700. The skin typically deforms in a region immediately surrounding the projection, with the skin bowing down away from the patch surface 121.
Upon insertion into the skin 700, coating 710 on the tip of the projections 110 below the skin surface 700, will immediately begin to hydrate and dissolve, thereby being dispersed into the subject, as shown by the arrows 730.
In addition to this, fluid from the subject will gradually flow into the coating 710 at the base of the projection 110, and coating 720 on the surface 121, as shown by the arrows 735, thereby hydrate the fluid. This will in turn cause fluid to diffuse into the subject, as shown by the arrows 740.
A further effect that can contribute to the delivery of material from the patch surface 121 is a squeezing effect, caused by the resilience of the skin 720, which urges the skin upward as shown by the arrow 750, which in turn urges hydrated material in the direction of the arrow 755, thereby increasing delayed delivery to the subject.
It will therefore be appreciated that controlling the coating ratio can therefore be used to manipulate the amount and rate which material is delivered to the subject. By maximising the coating on the projections, this maximises rapid delivery of material.
However, by increasing the amount of coating on the surface 121, this increases the delayed delivery of material.
Further delayed delivery of material can be achieved by further increasing the amount of material on the surface. This can be achieved using a projection configuration as shown in Figures 8A and 8B.
In this example, the surface 121 includes a raised annular portion 821 surrounding the base of a projection 110, thereby providing a well for containing addition coating solution.
Accordingly, in this instance, the coating 820 on the surface 821 can be of an increased thickness in the region immediately surrounding the base of the projection 110. This enhances the delayed delivery of material to the subject.
In one example, the projections can be coated a single time. In a further example, the projections can be coated a number of times. This can be used to allow a required thickness of coating to be achieved. In addition to this however, this allows different coating regimes to be used, which in turn allows greater control over the coating process.
Thus, for example, if coating is carried out using a first set of appropriate coating properties, then the coating can be confined primarily to the tips of the projections. A
second coating procedure can then be performed in order to allow the entire projection to be coated. This can be used to ensure that the tip includes a suitable amount of material to maximise the efficacy of the delivery process.
The above described processes therefore allow projections to be dry coated with material. In one example, this is achieved by using a gas flow to move or distribute coating solution over the patch to thereby ensure that all projections are wetted prior to drying.
In another example, this is achieved by using as gas flow to dry coating solution more rapidly than can be achieved under ambient conditions, thereby ensuring that coating solution remains on the projections during the drying process. It will be appreciated that the moving and drying steps can be performed simultaneously.
By dry coating the projections of the patch, this ensures that material on the projections is rapidly delivered directly to the subject. This maximises the proportion of coating material effectively delivered to the subject, which in turn reduces the amount of material required in order to produce a biological effect within the subject.
In addition to the above, appropriate selection of coating properties, such as gas flow rate, drying time, and solution properties can be used to further control the coating process. In particular, this can be used to control the thickness of the coating applied to the projections.
The projections can be coated with DNA or protein vaccines. However, in addition to this, many other reagents can be coated using this process including both inorganic and organic materials. Example coatings used include inorganic materials such as EtBr, or organic materials such Evans blue, Dextran, DiD, or the like.
Consequently, the resulting patch can provide small and densely packed projections that can be uniformly and controllably coated. This allows vaccines or other agents to be subsequently delivered to highly immunologically sensitive cells within the epidermis, or to the blood or muscular tissue as required.
In use, the coated and dried projection patches are applied to the skin of a mammal by placing the patch on the skin. The coated and dried projection patches can be tested on skin or skin analogs and the conditions for optimal coating release determined.
These conditions include patch application time, force, velocity, strain-rate of insertion, temperature, humidity, location, and skin pretreatment. This process can be done in vitro, ex vivo or in vivo.
It will be appreciated that the final release of the therapeutic agent can also be influenced by several of the coating properties such as the inclusion,of excipients and viscosity enhancers, as well as the coating thickness, and testing again allows optimum coating properties such as those outlined above, to be determined.
The in vitro method utilizes a thin polymer film to approximate the stratum corneum (SC), or outer layer of skin. The film can be polycarbonate, polyethylene, or any other film that has physical characteristics that approximate those of the SC. Beneath the polymer is an absorbent material that can be filter paper, polymer mesh, or any other soft and inert material that does not bind the vaccine or coating material. This material is then moistened with water, tris buffered saline (TBS), phosphate buffered saline (PBS), or any other liquid that can dissolve the coating material. The device is then applied to the polycarbonate and the projections pierce the top layer of polymer film. The liquid in the absorbent layer can then dissolve the dry coating. Once the device is removed the absorbent layer is flushed with the liquid. The elutate is then quantified and the device release calculated. The coated and dried projections patches can be applied to this testing environment under many varied conditions to optimize release.
The ex vivo release assay can be used to assess release from the coated and dried projection patches and employ skin. A patch of skin is dissected from a donor (i.e.
mouse, pig, rat, human) and kept at -20 C for less than 7 days prior to use. The skin is warmed to 37 C and the patches coated as outlined above are applied under a variety of conditions. The patches can be coated with fluorescent dyes such as FITC, Evans Blue, Propidium Iodide, Ethidium Bromide, Alexa Fluor dyes. The patches can also be coated with DNA or proteins that are labelled with fluorescent dyes. Alternately, the patches can be coated with fluorescent dye labelled polymers like dextran, agarose, agar or any other biocompatible polymer that approximates the size, shape, and chemical nature of DNA and protein vaccines.
The release of these fluorescently labelled agents in skin can be monitored by methods including multi-photon/confocal microscopy, fluorescence microscopy, spectrofluorometer, and flow cytometry. Multi-Photon/Confocal microscopy can give real time, 3D
patch release information that is necessary for optimizing the device coating and application.
In in vivo release testing, a coated projection patch is applied to the skin.
After the application, analysis was carried out as discussed for the ex vivo testing protocol.
Alternately, a portion of the skin treated with the projection patch is excised. The outer layer of the skin is peeled and trimmed as required. The skin is snap frozen in liquid nitrogen and then pulverized to a fine powder.
For DNA vaccine delivery, the DNA is extracted with a Qiagen extraction kit and a standard curve employed to determine the amount of DNA with semi-quantitative Polymerase chain reaction (PCR).
A number of specific examples will now be described. For the purpose of these examples the general coating procedure used was as follows:
= Patches are cleaned in glycerol:H20 (1:1) for 10 minutes and then flushed with plenty of water;
= Cleaned patches are dried with nitrogen blow;
= Coating solution is made of MC, poloxamer 188 or QA, and different concentration of vaccine (0.01 mg/ml - 50 mg/ml), the concentrations of chemicals being adjusted to suit different requirements;
= 5-15 microliters of coating solution is dropped onto each patch; and, = Patches are dried under nitrogen flow as described above.
During application the skin of the subject is typically hydrated to ease application of the patch, and increase hydration of the coating, thereby enhancing delivery.
Example 1 The projection patches are cleaned in a mixture of glycerol and water in a 1:1 ratio for 10 minutes and then flushed with plenty of water. The patches are then dried with nitrogen blow. Example of cleaned and uncoated projections are shown in Figures 9A to 9D, which show secondary electron and backscattered electron images for patches with 60 m and 90 m long projections, respectively.
A coating solution containing a viscosity enhancer (MC), a surfactant (QA or poloxamer 188) and different concentrations of vaccine (OVA protein or DNA) is prepared. The compositions are set out in Table 1. All percentages are weight percentages of the total compositions unless otherwise indicated.
Table 1 MC wt. 0 - 2.5%
QA or poloxamer 188 wt. 0 - 1%
OVADNA wt.0-0.5%
OVA protein wt. 0 - 5%
microliters of the coating solutions are dropped onto each patch prepared as described above. A gas jet is used to control the movement of coating solution on patches so the liquid can wet the projections without being stuck on patches and covering many projections. In the meantime, the coating solution can be adsorbed and dried on the projections.
In this example, to provide a comparison, the patches of Figures 9A and 9C
were treated using a classical dip coating approach. Four patches, having totally over 14,000 projections, were coated with a solution containing 10 mg/ml of CMC (viscosity enhancer), 10 mg/ml of poloxamer 188 (surfactant) and 2 mg/ml of OVA DNA (active agent). Patches were dipped into the solution for 10 seconds and dried in air for 1 hour. The morphology of coated patches was then observed by SEM, with the results being shown in Figures 9E and 9F.
Figure 9E shows that no coating is present on the projections. Instead, the coating solution has been exclusively dried on the base the patch. From the magnified image of a single projection, shown in Figure 9F, the sputter coated gold particles can still be clearly observed, which also confirms that no coating has been obtained on the projection. This highlights that a dip-coating technique is not effective when applied to very small and densely packed projections. This experiment was repeated when MC, QA and OVA protein were used in coating solution at different concentrations and the results were similar. In other words, no coating or very little coating can be obtained on projections by using the dip-coating technique.
Gas jet drying was used to coat vaccines on patches and SEM was employed to characterise the morphology of the coating. Figures 10A and 10C show baseline secondary electron images of uncoated patches, with 30, 60 and 90 m long projections, respectively.
The patches were then coated using a,coating solution composed of 20 mg/ml of MC, 2 mg/ml of Quil-A and 2 mg/ml of OVA DNA vaccine, which was dried using the gas flow technique outlined above. The respective SEM images of the coated patches are shown in Figures 10B, 10D, WE and 10F, which highlight how the effective thickness of the projection increases, due to the coating of a consistent layer. The coating layer is up to 5 m thick.
The image in Figure 1OF is a backscattered electron image, which also confirms that the projections are uniformly coated. In this regard after coating, projections presenting dark BSE signals are seen due to the presence of organic materials with low atomic numbers, i.e.
carbon, oxygen, and hydrogen, on the surface of projections. In comparison, the base of the patch still has bright BSE signal after coating, which suggests that the coating on the base is very thin (- 1 m).
In Figures 11 A to 11I, the coating on selected, individual projections are shown in more detail. Figures 11A, 11B and 11C show an individual 35 pm long projection before coating, after coating, and an overlay of the two images, respectively. With a longer, 60 m projection, these images are respectively shown in the same series in Figures I1D, 11E and 11F. From these figures, the coating layer on the projections can be clearly observed.
With consistent coating of projections established, the next stage is to demonstrate that biologically active (or relevant) material was uniformly coated on the projections, to show that the projections are not only other excipients, such as, viscosity enhancers, surfactants, or the like. In this regard, Figures 11G, 11H and 11I show the fluorescence from a DiD coating on 90 m projections, a reflection from the projections and an overlay- image, respectively.
These figures demonstrate that surrogates for active materials in the form of fluorescent dyes can be uniformly coated on projections, as shown the fluorescence from DiD.
Following this, the work was extended to demonstrate that the coating process is robust and broadly applicable to many active entities, including ethidium bromide (EtBr), OVA protein vaccine, OVA DNA vaccine, fluorescent dyes (dextran and DiD) and. flu virus on projections.
The selection of coated compounds spans from low molecular weight molecules (a few hundred Daltons) to high molecular weight molecules (a few million Daltons).
In all cases, coatings were reproducibly applied onto projections on the patches.
Figure 12A shows an example of an SEM image of a patch uniformly coated with protein using a gas flow. The secondary and backscattered electron images of Figures 12B and 12C
highlight the even coating.
Accordingly, the gas jet coating can achieve uniform coating on projections and can rapidly coat large numbers of projections.
Example 2 In this example, the projections are coated in accordance with Example 1, however with the concentration of MC in coating solution being adjusted from 0 to 2.5% while the concentration of QA and OVA protein is kept to be 0.2% and 1%, respectively.
Figures 13A to 13D, show secondary electron images for patches coated with OVA
DNA
vaccine on 90 m projections with concentrations of MC of 0%, 0.5%, 1% and 2.5%
respectively. The coating is pretty uniform for all samples, but the coating thickness is different for coating solutions containing different concentration of MC. This is shown in Table 2, which shows the coating thickness on the middle cylindrical part of projections for coating solutions containing different concentration of MC.
Table 2 MC concentration Coating thickness 0 <0.25gm 0.5% 1.42 0.18 m 1% 2.10 0.18 m 2.5% 4.00 0.50 m Example 3 Projections are coated in accordance as described in Example 1 but with a concentration of QA in coating solution of 0.2% or 1% while the concentration of MC and OVA
protein is kept to be 2% and 1%, respectively.
Figures 14A to 14D show secondary electron and backscattered electron images for patches coated with OVA protein vaccine on 90 m projections, with concentrations of QA of 0.2%
and 1%, respectively. The coating is again pretty uniform for all samples, but the coating thickness on the base is different for the different QA concentrations.
When the concentration of QA is 0.2%, the coating on base is very thin (<1 m), so very bright backscattered electron signal can be detected from the base, as shown in Figure 12B.
When` the concentration of QA increases to 1%, the coating on base starts to be thicker (> 2 m). Therefore, the backscattered electron signal from gold under the OVA
protein coating is difficult to be detected and the base looks dark in backscattered electron image of Figure 14D.
Example 4 In this example, the projections are coated with OVA protein in accordance with Example 1, but with the coating solution containing water and ethanol (2:1), 1% OVA and 1% MC.
Figures 15A and 15B show a secondary electron and backscattered electron images for an example of the patch coated with of OVA protein on 90 .xm projections. It can be seen the coating is mainly on the top part of projections. From the backscattered electron image, it can be seen that the top part of projections look dark while the bottom part of projections and base are bright. This further confirms that the coating is mainly on the top part of projections.
After addition of ethanol, the surface tension of the coating solution is very low, so it can well wet projections. Therefore, less amount of coating solution (<6 l) is enough for coating a patch, which will reduce the cost of vaccine coating required to coat a patch.
To further confirm that the tip coating effect is caused by using a gas jet flow, Figures 15C
and 15D show examples of secondary electron and backscattered electron images for an example of the patch coated by applying 10 41 of OVA protein coating solution and drying in air. It can be seen that the coating is mainly on base and little coating is on some part of projections.
Example 5 Hardness and Young's modulus are two important mechanical properties for vaccine coating.
In order to deliver vaccine into the skin, coating should be robust enough to pierce into skin without wiping off. Preferably, values of hardness and Young's modulus of vaccine coating should be larger than those of skin. Hardness and reduced modulus have been measured for silicon patch, gold coated silicon patch, OVA protein coating and mouse ear skin. Results are shown in Table 3.
Young's modulus can be calculated from reduced modulus. Young's modulus describes tensile elasticity, or the tendency of an object to deform along an axis when opposing forces are applied along that axis; it is defined as the ratio of tensile stress to tensile strain. Hardness is the characteristic of a solid material expressing its resistance to permanent deformation.
From the results, it can be found that the values of hardness and reduced modulus of OVA
protein vaccine coating are much higher than those of mouse ear skin or porcine skin. It is a clear evidence of robust coating, which should be able to pierce into skin without wiping off.
Table 3 Hardness (GPa) Reduced modulus (GPa) Silicon patch 12 0.47 173 5 OVA coated patch 0.14-0.19 3.2-3.8 Porcine skin stratum Dry SC: 0.1-0.3 corneum (SC)* Wet SC: 0.01-0.05 *. Yuan Y. and Verma R., Colloids and Surfaces B 48 (2006) 6-12 It should be noted that in the case of the porcine skin stratum that the application of the patch was performed under differing conditions. In this regard, the patch was initially applied with the skin in a dry state, yielding a higher reduced modulus for the coating than when the skin is wet. The reason for this is the fluid on the skin hydrates the coating, reducing adherence of the coating to the projection. In some circumstances, this can be beneficial as it assists rapid delivery of the all the coating material to the subject.
Example 6 In this example, a coated patch is tested using a conventional commercially-available influenza vaccine (trivalent vaccine (Fluvax 2007) CSL, Ltd, Melbourne Australia;
consisting of viruses New Caledonia A, Wisconsin A and Malaysia B)) to assess the local delivery of vaccine within the skin (viable epidermis and dermis), as shown in Figure 16.
Figure 16 shows that applying patches, coated with the influenza vaccine, to the skin (for 15 minutes) achieves targeted delivery to the skin viable epidermis and underlying dermis.
Within the viable epidermis the co-localization of vaccine to targeted immunologically-sensitive cells is very high (at 40%). Furthermore, the overall payload delivered within the skin is accurately quantified at 19.9 5.7 ng (per patch).
In particular, Figure 16 shows an example of patches and measured local delivery characteristics in the mouse epidermis. Patches (a) were fabricated to the projection length of 90 gm (with Deep Reactive Ion Etching; at the Rutherford Appleton Laboratories, by Derek Jenkins) and then dry-coated with vaccine and photographed with SEM (b) and (c).
Once coated, the patch was applied to the skin and Cryo-SEM was used to visualize the skin during patch application (d). By labelling Fluvax with a fluorescent dye (Cy3), shown at 1600 in (e) and (h), confocal microscopy was used to examine. co-localization (arrow heads in Panel (h)) of vaccine with MHCII, shown at 1610 in (f) and (h) containing cells (e) to (h).
The patch was applied at 1.89 m/s and held in place with 500g for 15 minutes, penetrating to 27.7 gm (which is deeper than the epidermis thickness of 17 gm. The images (e) to (h) are a projected z-stack of the surface of the mouse skin (a hair can be seen at 1620 in (f) and (h) as a large diagonal bar) to the depth of 46 gm (which is well into the mouse ear dermis).
The dense nuclei in (g) and (h), stained with Hoechst 33342, in the epidermis were used to determine the epidermal and dermal boundary. Successful vaccine targeting to key epidermal cells (MHC Class II stained, including Langerhans cells) can be seen in two of the five vaccine deposition sites within Panel (h), highlighted with white arrow heads.
Several parameters were quantified through confocal image analysis (i) to (m) and are shown as per mm2 unless otherwise noted. Nine areas in three patched ears were imaged and analyzed for all but the last two graphs (1) and (m). The final two graphs (1) and (m) show quantification of delivered vaccine payload in skin by patches. Patched mouse ears were then homogenized and used in a quantitative dot-blot, using the generated standard curve, on five mice ears. The mass of vaccine delivered per projection was determined by measuring the integrated density of nine single projections and calculating the percentage of fluorescence per projection. This was then used in conjunction with the total delivery mass to calculate the mass of Fluvax delivered per projection.
Example 7 In this example, ex vivo release kinetics of 70kDa dextran coated 60 m nanoprojections were determined. These data were captured over 40 minutes in living skin using fluorescent microscopy.
Projections were coated with 20 gg of rhodamine labelled dextran (70 kDa) as a surrogate for ovalbumin in 2% melthylcellulose. Confocal imaging commenced immediately after patch application with the patch in place.
4D release kinetics from 70kDa payload in living skin are shown in Figures 17A
and 17B.
Figure 17A shows raw intensity values over 42 minutes, whereas Figure 17B
shows the calculated diffusion coefficient over the first 15 minutes.
The data were gathered from 20 m above the tip 1710 of projection 1700, as shown by the arrowhead 1720 in Figure 17C. The projection 1700 is pointing down and the region 1730 represents payload release. The colored cubes 1740 (2 m3 and 2 m away from the projection) show the 3D space that is being analyzed.
Example 8 In this example, groups of five C57BL/6 female mice aged 6 to 8 weeks were vaccinated with chicken egg albumin (Ovalbumin) protein either intramuscularly using the conventional syringe and needle, or onto the interior part of the ear skin using protein coated patch. The coating solution contains 10 mg/ml of MC, 10 mg/ml of OVA and 2 mg/ml of QA.
The area of each patch is 0.16 cm2. One patch per each ear was used in the vaccinations (i.e. a total of 2 patches per mouse). The patch was inserted into the skin at a speed of 1.96 m/s. The patch was kept for a further 5 minutes for the coated vaccine to be released. After 21 days, mice were bled and sera collected.
The serum samples were assayed by Enzyme-Linked ImmunoSorbent Assay (ELISA) using plates coated with Ovalbumin. Intramuscular immunised mice were injected with 6 g of OVA protein per mouse. MNP patch immunized mice were anesthetised and a single patch was applied to each ear, resulting in a total of 4.4 1.4 g of OVA protein delivered per mouse. The antibody levels of mice, including unimmunised, intramuscular immunised and coated patch immunised mice, are shown in Figure 18.
The data shown in Figure 18 demonstrates that much greater immune responses can be achieved by using coated patches at a similar dose with conventional needle and syringe.
Example 9 Following example 6, with these patch local skin delivery attributes established, the resultant systemic immune responses generated in mice were measured, with the results being shown in Figure 19A. The patch mice data were compared against needle and syringe intramuscular injection controls. Using needle and syringe (gauge.29 needle) intramuscular injection, a range of doses were delivered to the mouse caudal thigh muscle (0 (control), 0.04, 0.08, 0.8, and 6.0 g corresponding to the total HA as stated by the manufacturer (CSL
Ltd, Melbourne, Australia). Mice were bled 63 days after one immunisation.
Firstly, as shown in Figure 19A, the ELISA antibody reactivity (performed using sera with doubling serial dilutions starting from 1:100 up to 1:12800) was compared for the intramuscular needle and syringe doses compared with 0.04 g delivered with two patches.
The results show patch delivery (0.04 g) achieves similar antibody levels as generated by =6.0 gg delivered by IM injection.
Notably, it will be appreciated that although this establishes a dose reduction of a factor of 150, it is not specific to vaccination against influenza.
Thus, to measure relative levels of influenza protection, a heamagglutinin inhibition (HI) assay was used on the mice sera samples, with results being shown in Figure 19B. In particular, Hemagglutinin Inhibition assays (HI), were performed using the sera at different dilutions against each of the virus types (Wisconsin A, Malaysia B, and New Caledonia A).
Clearly, for all three stains of influenza, patch delivery (0.04 g) achieved HI levels equivalent to those generated by 6.0 g delivered by IM injection (p=0.357, 0.488 and 0.128 respectively for Wisconsin A, Malaysia B and New Caledonia A). This data shows the patch achieves a surrogate for vaccination protection against the influenza vaccine, with just a 1/150 of the dose delivered with the conventional needle and syringe.
Accordingly, it will be appreciated that dose reductions up to 150x could be achieved when influenza vaccine ((Fluvax 2007 ) was delivered directly to the dermis/epidermis of mice using the patch described herein. In this particular example, the patch includes densely packed projections (average 90 m in length) dry coated with the vaccine. This type of device is ideal for administering influenza vaccine in the case of a pandemic, not only because of the dose reduction achieved but the possibility of mass vaccinations by self administration of the vaccine. Notably, it will be appreciated that the device described can be extended to other types of vaccinations.
Thus, this example illustrates that a patch coated as described above shows may overcome the issues with using syringes and needles to vaccinate. In particular, a conventional influenza vaccine was delivered (Fluvax 2007 ) to C57BL/6 mice and the results showed that the patch delivery achieves equivalent immune responses as those induced by injection but with a dose reduced by a factor of 150. Accordingly, the patch as described in this example, can overcome key shortcomings of existing vaccine delivery technologies.
Example 10 In this example, two groups of 4 C57BL/6 female mice were immunised once with Fluvax coated patch or with Fluvax + CpG (ODN 1826) adjuvant. Mice were bled 2 weeks after one vaccination, and antigen specific total IgG, IgG1 and IgG2a levels were measured using ELISA. The results shown in Figure 20 demonstrate that a total reversal of IgGl and IgG2a responses when the adjuvant is included.
Accordingly, this example shows that the Th2 bias (Low antigen specific IgG2a/IgGl levels) shown by the use of the coated patch could be changed to Thl type of response, which may increase the CTL activity. This may be important in the case of cross protection to a different strain of the virus.
Example 11 Following the above example, a further example is used to investigate the ability to vaccinate subjects in more detail.
The present example combines patch and gene gun technology into a small scale device by allowing a gene gun to be used in patch application.
In this example, the patches are created through DRIE and contained 3364 individual projections that are 30 m wide at the base and between 45 and 130 m in length as shown in Figures 21A(c) and (d). The overall patch dimensions are 5x5 mm, as shown in Figures 21A(a) and (b).
The projection spacing are selected to match the distribution and depth of antigen presenting cells of the epidermis. Notably, these patches are not widely spaced and are typically short (<0.5 mm). The patches may also be made by deep reactive ion etching, so that they can be composed of silica and coated with a thin (-100 nm) gold layer.
The patch projections are -coated with the above described nitrogen jet drying method that results in a consistent and robust layer of antigen and/or adjuvant as shown in Figures 21A(e) and (f).
It will be appreciated that the gas jet coating method can provide numerous advantages. In one particular example, the method creates a dry-coating formulation that is typically robust enough to use with different antigens and adjuvants. Notably, dip-coating techniques are difficult to use in this instance as the presently described patch has densely packed patch projections, and dip coating followed by air drying often leads to a thick layer of dried material at the base and not the patch projections.
After removal, the coating on the patch projections was removed (g) and (h).
During patch application the skin is penetrated (i) to (m) (in (i) to (k) the bars indicate 1.00, 0.10, and 0.01 mm, respectively) by the projections and the strata compressed at the puncture site. The penetration of the skin by the coated patch projections resulted in the delivery of antigens to the epidermis and the upper-dermis ((n), bar is 100 and 10 m in the panel and inset, respectively).
The coated patch can then be applied with an anchored spring device that drives the patch into the skin at 1.8 m/s, where it can remain for up to 10 minutes. As shown in the SEM
images of Figures 21A(g) and (h) that the majority of coating is removed from the patch. The arrowheads are identifying corneocytes that have remained with the device. The high magnification image in Figure 21A(h) illustrates that the majority of the coating has been removed during the application process.
Furthermore, Figures 21A(i) to 21A(k) show increased magnification of the ventral side of a mouse ear that was snap frozen during patch application. These cryo-SEM images show the penetration of the individual patch projections into the surface of the, skin.
The depth of penetration and shape of the skin during patch application can be seen in the cryo-fractured skin photographed at an angle in Figure 21 A(1) and 21 A(m).
In particular, Figure 21A(m) is a single penetration site with an upturned corneocytes at the top; from this image one can appreciate that the patch can penetrate easily through the epidermis and into the dermis.
Once the patch penetrates the skin, the dried vaccine formulation can release from the patch projections and remain in the skin. This was monitored by having Fluvax 2007 fluorescently labelled, so that sections of the skin revealed the release pattern of a dry-coated vaccine delivered by the patch. In this example the fluorescent labelling is shown at 2120 in Figure 21A(n). In this image, the top row of nuclei 2110 highlight the epidermis with the vaccine shown at 2120 being seen through the epidermis and into the dermis.
The inset in Figure 21A(n) shows an overlay of each deposit site with dotted lines highlighting the strata boundaries(S, stratum corneum; E, epidermis; and D, dermis). In this image this highlights the ability of the patch to deliver antigen to both the epidermis and the upper dermis. One observation from the Figure 21A(n) is that the deposit of antigen does not appear to retain the cone shape of the patch projection, nor a cylindrical pattern; but rather resembles amorphous diffusion.
The diffusion of fluorescently labelled antigens was observed and analyzed using live confocal microscopy. Thus, a patch coated with fluorescently labeled dextran was applied to freshly excised skin and immediately imaged in 3D every minute for' over 15 minutes, with the resulting diffusion of the released material being rendered in 3D and shown in Figure 21B(a) to (c). These show that within 10 minutes the majority of diffusion had occurred. The data also indicated that the diffusion radius was approximately 1 to 2 cell diameters. This range is useful due to the even distribution of antigen presenting cells in the epidermis.
Accordingly, this highlights the ability to deliver antigen directly to antigen presenting cells in the epidermis. Having observed the delivery, release, and diffusion of antigen in the areas where antigen presenting cells were located,,it was also noted that three days after the patch delivered antigen to the skin, the antigen presenting cells (MHCII positive) were gone form the patch area but remained outside the patch projection free margin, as shown in Figure 21B(d). In particular, this shows a series of stitched images from the patch area to the margin and into the untreated region of the skin.
This observation led to further tests with patch delivered ovalbumin (OVA).
Quantification of MHCII positive cells over time revealed a rapid decline in the number of epidermal antigen presenting cells within three days, as shown in Figure 21B(e) and (f).
The number of antigen deposit sites was also tallied and showed greater that 86% (or >2800 projections per patch) of the patch projections delivered antigen into the skin.
Notably, one day after patch application the number of MHCII and antigen co-localization dropped more than the number of MHCII cells, as shown in Figures 21B(f) and (g). This implies that those MHCII positive cells that were in contact with the released antigen migrated quicker than those further away. Together these observations may indicate a mechanism through which the patch could deliver antigen directly to cells with MHCII; and those cells could carry the antigen to the lymph nodes for presentation. The number of MHCII positive cells that were exposed to antigen and their migration away from the application area leads to systemic immune response studies to confirm vaccination.
Influenza antigen from the commercially available vaccine, Fluvax was used for testing the patch delivery device of this example. The coating formulation contained 4 micrograms Hemagglutinin (HA) and 100 micrograms MC per patch. The coated patches are shown in Figures 21A(e) and 21A(f). A release assay based on fluorescently labeled Fluvax showed that this configuration of patch delivered approximately 20 ng HA per device.
Notably, this small amount of antigen is enough to generate strong IgG
production after 14 days (Figure 21C(a), solid line, solid triangles). For comparison, the antibody response of patch is much greater than that from an implant containing 40 ng HA and 100 microgram MC
(Figure 21C(a), dashed line, solid squares). Unimmunized sera is shown as a solid line in Figure 21C(a) to 21C(c).
Immune responses from patch delivered Fluvax was also compared to intramuscular injection of 0.04 micrograms HA (Fluvax ). However, the 40 ng HA injected dose was weaker than the patch (p=0.008) (Figure 21C(b)). Thus, it will be appreciated that these data values indicate that patch vaccination can result in a strong immune response with a well known and strong antigen.
According to a further example, the patch technology described herein was tested with an untested antigen from a globally important emerging disease without a commercial antigen.
Chikungunya virus antigen was made by irradiating cultured virus from the 2005-Reunion Island out break. The irradiated virus was then coated onto the patch at 5 micrograms killed virus, 100 micrograms MC, and 6 micrograms QA (or 20 micrograms CpG). Only a single patch was applied per animal.
After 14 days a strong immune response could be seen from the group with QA as shown in Figure 21C(c). The patch-QA response was significantly greater than the subcutaneously injected positive control that contained 5 micrograms killed virus and 6 micrograms QA to p=0.0017. The CpG adjuvant group showed a weaker response than the subcutaneous positive control, but the response was obviously higher than the subcutaneous injected 5 micrograms killed virus with no adjuvant, the negative control. Both the QA
and the CpG
patch groups elicited immune responses but this positive result did not indicate protection status.
After confirming the antibody response to the patch delivered Chikungunya antigen, it is determined whether or not the patch induced the protection of virus-neutralizing antibodies.
The success of immunization depends on the ability of the individual to resist a challenge.
Two months after immunization, live Chikungunya virus challenges were carried out. The virus was injected into the feet and this results in foot swelling and viraemia in naive individuals. Mouse models of Chikungunya infection show that the viral replication induces the expression of MCP-1. MCP-1 is a known proinflammatory gene that helps to recruit macrophages and thus the result is inflammation and swelling at the site of alphaviral injection that can be documented by measuring foot swelling. The results indicated that while the patch group with CpG did decrease swelling, only the patch group with QA
was statistically significant from the sham immunized group and indistinguishable from the untreated controls.
Notably, foot swelling is a good but rough measure of the inflammatory response. However, the viraemia data shows clear protection from Chikungunya virus challenge in the patch group with QA group. This group showed no appreciable foot swelling nor was there any virus recovered from the sera after challenge. The peak viral titers were found at day 2 which is historically consistent. The sham immunized group had a mean TCID50 of 3.3 loglo which was much higher than the patch group with CpG as an adjuvant, to TCIDS0 of 1.4. loglo. The TCID50 from the patch group with QA had no detectible viraemia and was significantly different. from the sham group, to p=0.001. Thus, it will be appreciated that patch immunization can completely protect from Chikungunya virus challenge.
Accordingly, the above example highlights that dry-coating antigens with or without adjuvant onto patch projections that have been specifically designed to target immune cells of the skin have the capacity to protect against viral infection. The patch is simple to use and quite small compared to a needle and syringe. Thus, it will be appreciated that there is no risk of needle stick injury with this device.
Thus, it will be appreciated that the patch described herein, in one example, can provide technology which has the capacity to effectively deliver antigen directly to antigen presenting cells, thereby eliciting a strong, protective immune response that holds up against challenge.
The coating methodology also developed has worked well with a variety of formulations including Influenza vaccine and killed Chikungunya virus; with and without adjuvants. The antigens were targeted to the immune cells of the skin and MHCII positive cells have been observed migrating in response to patch immunization. This immunization also led to strong and long lasting immunity to Chikungunya virus challenge. Thus, the patch described herein can provide an effective, next generation device for effective immunization.
Accordingly, this highlights that the coated patch provides a vaccine delivery method that is economical and efficient to prevent emerging, endemic, and enzootic diseases before they cause health and economic tragedies.
Example 12 In this example, epidermal targeted transfection with- a projection patch dry-coated with DNA
containing nanoparticles is performed.
The size distribution of PEI:DNA nanoparticles is shown in Figures 22A and 22B.
Nanoparticles were produced at a N:P of 5:1 with PEI (25k linear) and pEGFP
DNA in ultra-pure water.
A coating solution containing methylcellulose and PEI/DNA nanoparticles was used to coat projection patches following Example 1. The morphology of the coated patches and the coated patches after being applied on mouse ear for 15 minutes for nanoparticle delivery to the mouse ear skin is shown in Figures 22C and 22D, respectively.
After dry coating, the coated patches were dipped in water to get reconstituted PEI/DNA
nanoparticles. The aim was to confirm that the nanoparticles did not aggregate after coating process. Agarose gel analysis was performed on original and reconstituted PEI:DNA
nanoparticles for a variety of formulations including different N:P ratios (0:1, 5:1, and 9:1).
The results in Figure 22E show that dried and reconstituted PEI:DNA
nanoparticles still retain their supramolecular structure and do not release free DNA despite a change in size.
This is evidenced by positive staining in the well of both reconstituted samples.
Finally, patches coated with PEI/DNA nanoparticles were used to deliver nanoparticles into mouse ear skin for transfection study. The resulting transfection image is shown in Figures 22F and 22G. Figure 22F shows that cells with dendrites can be transfected by PEI/DNA
nanoparticles delivered by coated Nanopatches. Figure 22G shows that the transfection is in the epidermal layer of mouse ear skin.
A number of further variations and options for use with the above described devices will now be described.
Herein, the terms "projection", "micro-nanoprojection", "nanoneedle", "nanoprojection", "needle", "rod" etc are used interchangeably to describe the projections.
A further feature is that the projections may be used for delivery not only through the skin but through other body surfaces, including mucosal surfaces, to cellular sites below the outer layer or layers of such surfaces. The term "internal site", as used herein, is to be understood as indicating a site below the outer layer(s) of skin and other tissues for which the devices of the present invention are to be used.
The device is suitable for intracellular delivery. The device is suitable for delivery to specific organelles within cells. Examples of organelles to which the device can be applied include a cell nucleus, or endoplasmic reticulum, for example.
In one example the device is provided having a needle support section, that is to say the projections comprise a suitable support section, of sufficient length to reach the desired site and a (needle) delivery end section having a length no greater than 20 microns and a maximum width no greater than 5 microns, preferably no greater than 2 microns.
In one example, the maximum width of the delivery end section is no greater than 1000 nm, even more preferably the maximum width of the delivery end section is no greater than 500 nm.
In a further example, the device is for mucosal delivery. This device may have a needle support section, that is to say the projections comprise a suitable support section, of sufficient length to reach the desired site, such as of length at least 100 microns and a (needle) delivery end section having a length no greater than 20 microns and a maximum width no greater than microns, preferably no greater than 2 microns.
In one example, the device of the invention is for delivery to lung, eye, cornea, sclera or other internal organ or tissue. In a further example, the device is for in-vitro delivery to tissue, cell cultures, cell lines, organs, artificial tissues and tissue engineered products. This device typically has a needle support section, that is to say the projections comprise a suitable support section, of length at least 5 microns and a needle delivery end section having a length no greater than 20 microns and a maximum width no greater than 5 microns, preferably no greater than 2 microns.
In one example, the device comprises projections in which the (needle) delivery end section and support length, that is to say the "needle support section", is coated with a bioactive material across the whole or part of its length. The (needle) delivery end section and support length may be coated'on selective areas thereof. This may depend upon the bioactive material being used or the target selected for example.
In a further example, a bioactive material is releasably incorporated into the material of which the needle, or projection, is composed. All, or part of the projection may be constructed of a biocompatible, biodegradable polymer (such as Poly Lactic Acid (PLA), PolyGlycolic Acid (PGA) or PGLA or Poly Glucleic Acid), which is formulated with the bioactive material of choice. The projections may then be inserted into the appropriate target site and, as they dissolve, the bioactive material will enter the organelle(s)/cells.
Examples of bioactive materials, which are not intended to be limiting with respect to the invention include polynucleotides and nucleic acid or protein molecules, antigens, allergens, adjuvants, molecules, elements or compounds. In addition, the device may be coated with materials such as biosensors, nanosensors or MEMS.
Illustrative material that can be delivered may include any or more of. small chemical or biochemical compounds including drugs, metabolites, amino acids, sugars, lipids, saponins, and hormones; macromolecules such as complex carbohydrates, phospholipids, peptides, polypeptides, peptidomimetics, and nucleic acids; or other organic (carbon containing) or inorganic molecules; and particulate matter including whole cells, bacteria, viruses, virus-like particles, cell membranes, dendrimers and liposomes.
The material can be selected from nucleic acids, illustrative examples of which include DNA, RNA, sense oligonucleotides, antisense oligonucleotides, ribozymes, small interfering oligonucleotides (siRNAs), micro RNAs (miRNAs), repeat associated RNAs (rasiRNA), effector RNAs (eRNAs), and any other oligonucleotides known in the art, which inhibit transcription and/or translation of a mutated or - other detrimental protein.
In illustrative examples of this type, the nucleic acid is in the form of an expression vector from which a polyilucleotide of interest is expressible. The polynucleotide of interest may encode a polypeptide or an effector nucleic acid molecule such as sense or antisense oligonucleotides, siRNAs, miRNAs and eRNAs.
The material can be selected from peptides or polypeptides, illustrative examples of which include insulin, proinsulin, follicle stimulating hormone, insulin like growthfactor-l, insulin like growth factor-2, platelet derived growth factor, epidermal growth factor, fibroblast growth factors, nerve growth factor, colony stimulating factors, transforming growth factors, tumor necrosis factor, calcitonin, parathyroid hormone, growth hormone, bone morphogenic protein, erythropoietin, hemopoietic growth factors, luteinizing hormone, glucagon, glucagon likepeptide-1, anti-angiogenic proteins, clotting factors, anti-clotting factors, atrial natriuretic factor, plasminogen activators, bombesin, thrombin, enkephalinase, vascular endothelial growth factor, interleukins, viral antigens, non-viral antigens, transport proteins, and antibodies.
The material can be selected from receptor ligands. Illustrative examples of receptors include Fc receptor, heparin sulfate receptor, vitronectin receptor, Vcam-1 receptor, hemaglutinin receptor, Pvr receptor, Icam-1 receptor, decay-accelerating protein (CD55) receptor, Car (coxsackievirus-aderiovirus) receptor, integrin receptor, sialic acid receptor, HAVCr-1 receptor, low-density lipoprotein receptor, BGP (biliary glycoprotien) receptor, aminopeptidease N receptor, MHC class-1 receptor, laminin receptor, nicotinic acetylcholine receptor, CD56 receptor, nerve growth factor receptor, CD46 receptor, asialoglycoprotein receptor Gp-2, alpha-dystroglycan receptor, galactosylceramide receptor, Cxcr4 receptor, Glvrl receptor, Ram-1 receptor, Cat receptor, Tva receptor, BLVRcp1 receptor, MHC class-2 receptor, toll-like receptors (such as TLR-1 to -6) and complement receptors.
The material can be selected from antigens including endogenous antigens produced by a host that is the subject of the stimulus or material delivery or exogenous antigens that are foreign to that host. The antigens may be in the form of soluble peptides or polypeptides or polynucleotides from which an expression product (e.g., protein or RNA) is producible.
Suitable endogenous antigens include, but are not restricted to, cancer or tumor antigens.
Non-limiting examples of cancer or tumor antigens include antigens from a cancer or tumor selected from ABL1 proto-oneogene, AIDS related cancers, acoustic neuroma, acute lymphocytic leukemia, acute myeloid leukemia, adenocystic carcinoma, adrenocortical cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part sarcoma, anal cancer, angiosarcoma, aplastic anemia, astrocytoma, ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer, bone cancers, bowel cancer, brain stem glioma, brain and CNS
tumors, breast cancer, CNS tumors, carcinoid tumors, cervical cancer, childhood brain tumors, childhood cancer, childhood leukemia, childhood soft tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, colorectal cancers, cutaneous T-cell lymphoma, dermatofibrosarcoma protuberans, desmoplastic small round cell tumor, ductal carcinoma, endocrine cancers, endometrial cancer, ependymoma, oesophageal cancer, Ewing's Sarcoma, Extra-Hepatic Bile Duct Cancer, Eye Cancer, Eye: Melanoma, Retinoblastoma, Fallopian Tube cancer, Fanconi anemia, fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers, germ cell tumors, gestational-trophoblastic-disease, glioma, gynecological cancers, haematological malignancies, hairy cell leukemia, head and neck cancer, hepatocellular cancer, hereditary breast cancer, histiocytosis, Hodgkin's disease, human papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma, kidney cancer, Langerhans' cell histiocytosis, laryngeal cancer, leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer, liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer, malignant-rhabdoid tumor of kidney, medulloblastoma, melanoma, Merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer, multiple endocrine neoplasia, mycosis fungoides, myelodysplastic syndromes, myeloma, myeloproliferative disorders, nasal cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma, neurofibromatosis, Nijmegen breakage syndrome, non-melanoma skin cancer, non-small-cell-lung-cancer (NSCLC), ocular cancers, esophageal cancer, oral cavity cancer, oropharynx cancer, osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal cancer, parathyroid cancer, parotid gland cancer, penile cancer, peripheral-neuroectodermal tumours, pituitary cancer, polycythemia vera, prostate cancer, rare cancers and associated disorders,. renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, 'Rothmund-Thomson syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary syndrome, skin cancer, small cell lung cancer (SCLC), small intestine cancer, soft tissue sarcoma, spinal cord tumors, squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma, testicular cancer, thymus cancer, thyroid cancer, transitional-cell-cancer-(bladder), transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic cancer, urethral cancer, urinary system cancer, uroplakins, uterine sarcoma, uterus cancer, vaginal cancer, vulva cancer, Waldenstrom's macroglobulinemia, Wilms' tumor. In certain examples, the cancer or tumor relates to melanoma. Illustrative examples of melanoma-related antigens include melanocyte differentiation antigen (e.g., gplOO, MART, Melan-A/MART-1, TRP-l, Tyros, TRP2, MCIR, MUC1F, MUC1R or a combination thereof) and melanoma-specific antigens (e.g., BAGE, GAGE-I, gplOOIn4, MAGE-1 (e.g., GenBank Accession No.
and AA494311), MAGE-3, MAGE4, PRAME, TRP21N2, NYNSO1a, NYNSO1b, LAGEI,, p97 melanoma antigen (e.g., GenBank Accession No. M12154) p5 protein, gp75, oncofetal antigen, GM2 and GD2 gangliosides, cdc27, p2lras, gp100Pme1117 or a combination thereof.
Other tumour-specific antigens include, but are not limited to: etv6, amll, cyclophilin b (acute lymphoblastic leukemia); Ig-idiotype (B cell lymphoma); E-cadherin, a-catenin, 13-catenin, y-catenin, p l20ctn (glioma); p2lras (bladder cancer); p2lras (biliary cancer); MUC
family, HER2/neu, c-erbB-2 (breast cancer); p53, p2lras (cervical carcinoma);
p2lras, HER2/neu, c-erbB-2, MUC family, Cripto- 1 protein, Pim-1 protein (colon carcinoma);
Colorectal associated antigen (CRC)-CO17-lA/GA733, APC (colorectal cancer);
carcinoembryonic antigen (CEA) (colorectal cancer; choriocarcinoma);
cyclophilin b (epithelial cell cancer); HER2/neu, c-erbB-2, ga733 glycoprotein (gastric cancer); a-fetoprotein (hepatocellular cancer); Imp-1, EBNA-1 (Hodgkin's lymphoma); CEA, MAGE-3, NY-ESO-1 (lung cancer); cyclophilin b (lymphoid cell-derived leukemia); MUC
family, p2lras (myeloma); HER2/neu, c-erbB-2 (non-small cell lung -carcinoma); Imp-1, EBNA-l (nasopharyngeal cancer); MUC family, HER2/neu, c-erbB-2, MAGE-A4, NY-ESO-1 (ovarian cancer); Prostate Specific Antigen (PSA) and its antigenic epitopes PSA-1, PSA-2, and PSA-3, PSMA, HER2/neu, c-erbB-2, ga733 glycoprotein (prostate cancer);
HER2/neu, c-erbB-2 (renal cancer); viral products such as human papillomavirus proteins (squamous cell cancers of the cervix and esophagus); NY-ESO-1 (testicular cancer); and HTLV-1 epitopes (T cell leukemia).
Foreign antigens are suitably selected from transplantation antigens, allergens as well as antigens from pathogenic organisms. Transplantation antigens can be derived from donor cells or tissues from e.g., heart, lung, liver, pancreas, kidney, neural graft components, or from the donor antigen-presenting cells bearing MHC loaded with self antigen in the absence of exogenous antigen.
Non-limiting examples of allergens include Fel d 1 (i.e., the feline skin and salivary gland allergen of the domestic cat Felis domesticus, the amino acid sequence of which is disclosed International Publication WO 91/06571), Der p I, Der p II, Der if or Der fiI
(i.e., the major protein allergens from the house dust mite dermatophagoides, the amino acid sequence of which is disclosed in International Publication WO 94/24281). Other allergens may be derived, for example from the following: grass, tree and weed (including ragweed) pollens;
fungi and moulds; foods such as fish, shellfish, crab, lobster, peanuts, nuts, wheat gluten, eggs and milk; stinging insects such as bee, wasp, and hornet and the chirnomidae (non-biting midges); other insects such as the housefly, fruitfly, sheep blow fly, screw worm fly, grain weevil, silkworm, honeybee, non-biting midge larvae, bee moth larvae, mealworm, cockroach and larvae of Tenibrio molitor beetle; spiders and mites, including the house dust mite; allergens found in the dander, urine, saliva, blood or other bodily fluid of mammals such as cat, dog, cow, pig, sheep, horse, rabbit, rat, guinea pig, mouse and gerbil; airborne particulates in general; latex; and protein surfactant additives.
The material can be pathogenic organisms such as, but are not limited to, viruses, bacteria, fungi parasites, algae and protozoa and amoebae. Illustrative viruses include viruses responsible for diseases including, but not limited to, measles, mumps, rubella, poliomyelitis, hepatitis A, B (e.g., GenBank Accession No. E02707), and C
(e.g., GenBank Accession No. E06890), as well as other hepatitis viruses, influenza, adenovirus (e.g., types 4 and 7), rabies (e.g., GenBank Accession No. M34678), yellow fever, Epstein-Barr virus and other herpesviruses such as papillomavirus, Ebola virus, influenza virus, Japanese encephalitis (e.g., GenBank Accession No. E07883), dengue (e.g., GenBank Accession No.
M24444), hantavirus, Sendai virus, respiratory syncytial virus, othromyxoviruses, vesicular stomatitis virus, visna virus, cytomegalovirus and human immunodeficiency virus (HIV) (e.g., GenBank Accession No. U18552). Any suitable antigen derived from such viruses are useful in the practice of the present invention. For example, illustrative retroviral antigens derived from HIV include, but are not limited to, antigens such as gene products of the gag, pol, and env genes, the Nef protein, reverse transcriptase, and other HIV
components.
Illustrative examples of hepatitis viral antigens include, but are not limited to, antigens such as the S, M, and L proteins of hepatitis B virus, the pre-S antigen of hepatitis B virus, and other hepatitis, e.g., hepatitis A, B, and C, viral components such as hepatitis C viral RNA.
Illustrative examples of influenza viral antigens include; but are not limited to, antigens such as hemagglutinin and neurarninidase and other influenza viral components.
Illustrative examples of measles viral antigens include, but are not limited to, antigens such as the measles virus fusion protein and other measles virus components. Illustrative examples of rubella viral antigens include, but are not limited to, antigens such as proteins El and E2 and other rubella virus components; rotaviral antigens such as VP7sc and other rotaviral components. Illustrative examples of cytomegaloviral antigens include, but are not limited to, antigens such as envelope glycoprotein B and other cytomegaloviral antigen components. Non-limiting examples of respiratory syncytial viral antigens include antigens such as the RSV fusion protein, the M2 protein and other respiratory syncytial viral antigen components. Illustrative examples of herpes simplex viral antigens include, but are not limited to, antigens such as immediate early proteins, glycoprotein D, and other herpes simplex viral antigen components. Nori-limiting examples of varicella zoster viral antigens include antigens such as 9PI, gpll, and other varicella zoster viral antigen components.
Non-limiting examples of Japanese encephalitis viral antigens include antigens such as proteins E, M-E, M-E-NS 1, NS 1, NS 1-NS2A, 80%E, and other Japanese encephalitis viral antigen components. Representative examples of rabies viral antigens include, but are not limited to, antigens such as rabies glycoprotein, rabies nucleoprotein and other rabies viral antigen components. Illustrative examples of papillomavirus antigens include, but are not limited to, the L1 and L2 capsid proteins as well as the E6/E7 antigens associated with cervical cancers, See Fundamental Virology, Second Edition, eds. Fields, B.N.
and Knipe, D.M., 1991, Raven Press, New York, for additional examples of viral antigens.
Illustrative examples of fungi include Acremonium spp., Aspergillus spp., Basidiobolus spp., Bipolaris spp., Blastomyces derinatidis, Candida spp., Cladophialophora carrionii, Coccoidiodes immitis, Conidiobolus spp., Cryptococcus spp., Curvularia spp., Epidermophyton spp., Exophiala jeanselmei, Exserohilum spp., Fonsecaea cornpacta, Fonsecaea pedrosoi, Fusarium oxysporum, Fusarium solani, Geotrichum candidum, Histoplasma capsulatum var. capsulatum, Histoplasma capsulatum var. duboisii, Hortaea werneckii, Lacazia loboi, Lasiodiplodia theobromae, Leptosphaeria senegalensis, Madurella grisea, Madurella mycetomatis, Malassezia furfur, Microsporum spp., Neotestudina rosatii, Onychocola , canadensis, Paracoccidioides brasiliensis, Phialophora verrucosa, Piedraia hortae, Piedra iahortae, Pityriasis versicolor, Pseudallesheria boydii, Pyrenochaeta romeroi, Rhizopus arrhizus, Scopulariopsis brevicaulis, Scytalidium dimidiatum, Sporothrix schenckii, Trichophyton spp., Trichosporon spp., Zygomcete fungi, Absidia corymbifera, Rhizomucor pusillus and Rhizopus arrhizus. Thus, representative fungal antigens that can be used in the compositions and methods of the present invention include, but are not limited to, candida fungal antigen components; histoplasma fungal antigens such as heat shock protein 60 (HSP60) and other histoplasma fungal antigen components; cryptococcal fungal antigens such as capsular polysaccharides and other cryptococcal fungal antigen components; coccidiodes fungal antigens such as'spherule antigens and other coccidiodes fungal antigen components; and tinea fungal antigens such as trichophytin and other coccidiodes fungal antigen components.
Illustrative examples of bacteria include bacteria that are responsible for diseases including, but not restricted to, diphtheria (e.g., Corynebacterium diphtheria), pertussis (e.g., Bordetella pertussis, GenBank Accession No. M35274), tetanus (e.g., Clostridium tetani, GenBank Accession No. M64353), tuberculosis (e.g., Mycobacterium tuberculosis), bacterial pneumonias (e.g., Haemophilus influenzae.), cholera (e.g., Vibrio cholerae), anthrax (e.g., Bacillus anthracis), typhoid, plague, shigellosis (e.g., Shigella dysenteriae), botulism (e.g., Clostridium botulinuni), salmonellosis (e.g., GenBank Accession No.
L03833), peptic ulcers (e.g., Helicobacter pylori), Legionnaire's Disease, Lyme disease (e.g., GenBank Accession No. U59487), Other pathogenic bacteria include Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. Thus, bacterial antigens which can be used in the compositions and methods of the invention include, but are not limited to: pertussis bacterial antigens such as pertussis toxin, filamentous hemagglutinin, pertactin, F M2, FIM3, adenylate cyclase and other pertussis bacterial antigen components; diphtheria bacterial antigens such as diphtheria toxin or toxoid and other diphtheria bacterial antigen components;
tetanus bacterial antigens such as tetanus toxin or toxoid and other tetanus bacterial antigen components, streptococcal bacterial antigens such as M proteins and other streptococcal bacterial antigen components; gram-negative bacilli bacterial antigens such as lipopolysaccharides and other gram-negative bacterial antigen components;
Mycobacterium tuberculosis bacterial antigens such as mycolic acid, heat shock protein 65 (HSP65), the 30kDa major secreted protein, antigen 85A and other mycobacterial antigen components;
Helicobacter pylori bacterial antigen components, pneumococcal bacterial antigens such as pneumolysin, pneumococcal capsular polysaccharides and other pnermiococcal bacterial antigen components; Haemophilus influenza bacterial antigens such as capsular polysaccharides and other Haemophilus influenza bacterial antigen components;
anthrax bacterial antigens such as anthrax protective antigen and other anthrax bacterial antigen components; rickettsiae bacterial antigens such as rompA and other rickettsiae bacterial antigen component. Also included with the bacterial antigens described herein are any other bacterial, mycobacterial, mycoplasmal, rickettsial, or chlamydial antigens.
Illustrative examples of protozoa include protozoa that are responsible for diseases including, but not limited to, malaria (e.g., GenBank Accession No. X53832), hookworm, onchocerciasis (e.g., GenBank Accession No. M27807), schistosomiasis (e.g., GenBank Accession No. LOS 198), toxoplasmosis, trypanosomiasis, leishmaniasis, giardiasis (GenBank Accession No. M33641), amoebiasis, filariasis (e.g., GenBank Accession No.
J03266), borreliosis, and trichinosis. Thus, protozoal antigens which can be used in the compositions and methods of the invention include, but are not limited to:
plasmodium falciparum antigens such as merozoite surface antigens, sporozoite surface antigens, circumsporozoite antigens, gametocyte/gamete surface antigens, blood-stage antigen pf 155/RESA and other plasmodial antigen components; toxoplasma antigens such as SAG-1, p30 and other toxoplasmal antigen components; schistosomae antigens such as glutathione-S-transferase, paramyosin, and other schistosomal antigen components; leishmania major and other leishmaniae antigens such as gp63, lipophosphoglycan and its associated protein and other leishmanial antigen components; and trypanosoma cruzi antigens such as the 75-77kDa antigen, the 56kDa antigen and other trypanosomal antigen components.
The material can be toxin components acting as antigens. Illustrative examples of toxins include, but are not restricted to, staphylococcal enterotoxins, toxic shock syndrome toxin;
retroviral antigens (e.g., antigens derived from HIV), streptococcal antigens, staphylococcal enterotoxin-A (SEA), staphylococcal enterotoxin-B (SEB), staphylococcal enterotoxin1_3 (SE1_3), staphylococcal enterotoxin-D (SED), staphylococcal enterotoxin-E
(SEE) as well as toxins derived from mycoplasma, mycobacterium, and herpes viruses.
In specific examples, the antigen is delivered to antigen-presenting cells.
Such antigen-presenting cells include professional or facultative antigen-presenting cells.
Professional antigen-presenting cells function physiologically to present antigen in a form that is recognised by specific T cell receptors so as to stimulate or anergise a T
lymphocyte or B
lymphocyte mediated immune response. Professional antigen-presenting cells not only process and present antigens in the context of the major histocompatability complex (MHC), but also possess the additional immunoregulatory molecules required to complete T cell activation or induce a tolerogenic response. Professional antigen-presenting cells include, but are not limited to, macrophages, monocytes, B lymphocytes, cells of myeloid lineage, including monocytic-granulocytic-DC precursors, marginal zone Kupffer cells, microglia, T
cells, Langerhans cells and dendritic cells including interdigitating dendritic cells and follicular dendritic cells. Non-professional or facultative antigen-presenting cells typically lack one or more of the immunoregulatory molecules required to complete T
lymphocyte activation or anergy. Examples of non-professional or facultative antigen-presenting cells include, but are not limited to, activated T lymphocytes, eosinophils, keratinocytes, astrocytes, follicular cells, microglial cells, thymic cortical cells, endothelial cells, Schwann cells, retinal pigment epithelial cells, myoblasts, vascular smooth muscle cells, chondrocytes, enterocytes, thymocytes, kidney tubule cells and fibroblasts. In some examples, the antigen-presenting cell is selected from monocytes, macrophages, B lymphocytes, cells of myeloid lineage, dendritic cells or Langerhans cells. In certain advantageous examples, the antigen-presenting cell expresses CD11c and includes a dendritic cell or Langerhans cell. In some examples the antigen-presenting cell stimulates an immune response. In other examples, the antigen-presenting cell induces a tolerogenic response.
The delivery of exogenous antigen to an antigen-presenting cell can be enhanced by methods known to practitioners in the art. For example, several different strategies have been developed for delivery of exogenous antigen to the endogenous processing pathway of antigen-presenting cells, especially dendritic cells. These methods include insertion of antigen into pH-sensitive liposomes (Zhou and Huang, 1994, Immunomethods, 4:229-235), osmotic lysis of pinosomes after pinocytic uptake of soluble antigen (Moore et al., 1988, Cell, 54:777-785), coupling of antigens to potent adjuvants (Aichele et al., 1990, J Exp.
Med., 171: 1815-1820; Gao et al., 1991, J Immunol., 147: 3268-3273; Schulz et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 991-993; Kuzu et al., 1993, Euro. J. Immunol., 23: 1397-1400; and Jondal et al., 1996, Immunity 5: 295-302) and apoptotic cell delivery of antigen (Albert et al. 1998, Nature 392:86-89; Albert et al. 1998, Nature Med. 4:1321-1324; and in International Publications WO 99/42564 and WO 01/85207). Recombinant bacteria (eg. E.
coli) or transfected host mammalian cells may be pulsed onto dendritic cells (as particulate antigen, or apoptotic bodies respectively) for antigen delivery. Recombinant chimeric virus-like particles (VLPs) have also been used as vehicles for delivery of exogenous heterologous antigen to the MHC class I processing pathway of a dendritic cell line (Bachmann et al,, .1996, Eur. J. Immunol., 26(11): 2595-2600).
Alternatively, or in addition, an antigen may be linked to, or otherwise associated with, a cytolysin to enhance the transfer of the antigen into the cytosol of an antigen-presenting cell of the invention for delivery to the MHC class I pathway. Exemplary cytolysins include saponin compounds such as saponin-containing Immune Stimulating Complexes (ISCOMs) (see e.g., Cox and Coulter, 1997, Vaccine 15(3): 248-256 and U.S. Patent No.
6,352,697), phospholipases (see, e.g., Camilli et al., 1991, J. Exp. Med. 173: 751-754), pore-forming toxins (e.g., an a-toxin), natural cytolysins of gram-positive bacteria, such as listeriolysin 0 (LLO, e.g., Mengaud et al., 1988, Infect. Immun. 56: 766-772 and Portnoy et al., 1992, Infect.
Immun. 60: 2710-2717), streptolysin 0 (SLO, e.g., Palmer et al., 1998, Biochemistry 37(8):
2378-2383) and perfringolysin 0 (PFO, e.g., Rossjohn et al., Cell 89(5): 685-692). Where the antigen-presenting cell is phagosomal, acid activated cytolysins may be advantageously used.
For example, listeriolysin exhibits greater pore-forming ability at mildly acidic pH (the pH
conditions within the phagosome), thereby facilitating delivery of vacuole (including phagosome and endosome) contents to the cytoplasm (see, e.g., Portnoy et al., Infect. Immun.
1992, 60: 2710-2717).
The' cytolysin may be provided together with a pre-selected antigen in the form of a single composition or may be provided as a separate composition, for 'contacting the antigen-presenting cells. In one example, the cytolysin is fused or otherwise linked to the antigen, wherein the fusion or linkage permits' the delivery of the antigen to the cytosol of the target cell. In another example, the cytolysin and antigen are provided in the form of a delivery vehicle such as, but not limited to, a liposome or a microbial delivery vehicle selected from virus, bacterium, or yeast. Suitably, when the delivery vehicle is a microbial delivery vehicle, the delivery vehicle is non-virulent. In a preferred example of this type, the delivery vehicle is a non-virulent bacterium, as for example described by Portnoy et al. in U.S. Patent No.
6,287,556, comprising a first polynucleotide encoding a non-secreted functional cytolysin operably linked to a regulatory polynucleotide which expresses the cytolysin in the bacterium, and a second polynucleotide encoding one or more pre-selected antigens. Non-secreted cytolysins may be provided by various mechanisms, e.g., absence of a functional signal sequence, a secretion incompetent microbe, such as microbes having genetic lesions (e.g., a functional signal sequence mutation), or poisoned microbes, etc. A
wide variety of nonvirulent, non-pathogenic bacteria may be used; preferred microbes are relatively well characterised strains, particularly laboratory strains of E. coli, such as MC4100, MC1061, DH5a, etc. Other bacteria that can be engineered for the invention include well-characterised, nonvirulent, non-pathogenic strains of Listeria monocytogenes, Shigella flexneri, mycobacterium, Salmonella, Bacillus subtilis, etc. In a particular example, the bacteria are attenuated to be non-replicative, non-integrative into the host cell genome, and/or non-motile inter- or intra-cellularly.
The coated patches described above can be used to deliver one or more antigens to virtually any antigen-presenting cell capable of endocytosis of the subject vehicle, including phagocytic and non-phagocytic antigen-presenting cells. In examples when the delivery vehicle is a microbe, the subject methods generally require microbial uptake by the target cell and subsequent lysis within the antigen-presenting cell vacuole (including phagosomes and endosomes).
In other examples, the antigen is produced inside the antigen-presenting cell by introduction of a suitable expression vector as for example described above. The antigen-encoding portion of the expression vector may comprise a naturally-occurring sequence or a variant thereof, which has been engineered using recombinant techniques. In one example of a variant, the codon composition of an antigen-encoding polynucleotide is modified to permit enhanced expression of the antigen in a target cell or tissue of choice using methods as set forth in detail in International Publications WO 99/02694 and WO 00/42215. Briefly, these methods are based on the observation that translational efficiencies of different codons vary between different cells or tissues and that these differences can be exploited, together with codon composition of a gene, to regulate expression of a protein in a particular cell or tissue type.
Thus, for the construction of codon-optimised polynucleotides, at least one existing codon of a parent polynucleotide is replaced with a synonymous codon that has a higher translational, efficiency in a target cell or tissue than the existing codon it replaces.
Although it is preferable to replace all the existing codons of a parent nucleic acid molecule with synonymous codons which have that higher translational efficiency, this is not necessary because increased expression can be accomplished even with partial replacement. Suitably, the replacement step affects 5, 10, 15, 20, 25, 30%, more preferably 35, 40, 50, 60, 70% or more of the existing codons of a parent polynucleotide.
The expression vector for introduction into the antigen-presenting cell will be compatible therewith such that the antigen-encoding polynucleotide is expressible by the cell. For example, expression vectors of this type can be derived from viral DNA
sequences including, but not limited to, adenovirus, adeno-associated viruses, herpes-simplex viruses and retroviruses such as B, C, and D retroviruses as well as spumaviruses and modified lentiviruses. Suitable expression vectors for transfection of animal cells are described, for example, by Wu and Ataai (2000, Curr. Opin. Biotechnol, 11(2):205-208), Vigna and Naldini (2000, J. Gene Med. 2(5):308-316), Kay, et al. (2001, Nat. Med. 7(l):33-40), Athanasopoulos, et al. (2000, Int. J. Mol. Med. 6(4):363-375) and Walther and Stein (2000, Drugs 60(2):249-271).
In one aspect, the device is provided in the form of a patch containing a plurality of needles (projections) for application to a body surface. A multiplicity of projections can allow multiple cells and organelles to be targeted and provided with a material at the same time.
The patch may be of any suitable shape, such as square or round for example.
The overall number of projections per patch depends upon the particular application in which the device is to be used. Preferably, the patch has at least 10 needles per mm, and more preferably at least 100 needles per mm2. Considerations and specific examples of such a patch are provided in more detail below.
Examples of specific manufacturing steps used to fabricate the device are described in greater detail below. In one preferred aspect, the device of the invention is constructed from biocompatible materials such as Titanium, Gold, Silver or Silicon, for example. This may be the entire device, or alternatively it may only be the projections or the delivery end section of the projections which are made from the biocompatible materials.
One manufacturing method for the device utilises the Deep Reactive Ion Etching (DRIE) of the patterns direct from silicon wafers, see the construction section below.
Another manufacturing method for the device utilises manufacturing from a male template constructed with X-ray lithography, electrodeposition and moulding (LIGA). The templates are then multiply inserted into a soft polymer to produce a plurality of masks. The masks are then vacuum deposited/sputtered with the material of choice for the nanoprojections, such as titanium, gold, silver, or tungsten. Magnetron sputtering may also be applied, see the construction section below.
An alternative means for producing masks is, with 2 photon Stereolithography, a technique which is known in the art and is described in more detail below.
In one example, the device is constructed of silicon.
The device may be for a single use or may be used and then recoated with the same or. a different bioactive material or other stimulus, for example.
In one example, the device comprises projections which are of differing lengths and/or diameters (or thicknesses depending on the shape of the projections) to allow targeting of different targets within the same use of the device.
Persons skilled in the art will appreciate that numerous variations and modifications will become apparent. All such variations and modifications which become apparent to persons skilled in the art, should be considered to fall within the spirit and scope that the invention broadly appearing before described.
Claims (42)
1) A method of coating a material onto projections provided on a patch, wherein the method includes:
a) applying a coating solution containing the material to at least the projections; and, b) using a gas flow to:
i) distribute the applied coating solution, at least in part, over the projections; and, ii) dry the coating solution.
a) applying a coating solution containing the material to at least the projections; and, b) using a gas flow to:
i) distribute the applied coating solution, at least in part, over the projections; and, ii) dry the coating solution.
2) A method according to claim 1, wherein the method includes moving coating solution on the patch to wet all the projections using the gas flow, thereby coating at least part of the projections.
3) A method according to claim 1 or claim 2, wherein the method includes selecting coating properties to thereby control the distribution of coating over the projections.
4) A method according to claim 3, wherein coating properties are selected so that at least one of:
a) at least tips of the projections are coated; and, b) at least target sections of the projections are coated.
a) at least tips of the projections are coated; and, b) at least target sections of the projections are coated.
5) A method according to claim 3 or claim 4, wherein the projections are provided on a surface of the patch, and wherein the method includes selecting coating properties to thereby vary at least one of:
a) an amount of coating on a surface of the patch; and, b) an amount of coating on the projections.
a) an amount of coating on a surface of the patch; and, b) an amount of coating on the projections.
6) A method according to any one of the claims 3 to 5, wherein the coating properties include at least one of:
a) a gas flow rate;
b) patch properties;
c) coating solution properties; and, d) a drying time.
a) a gas flow rate;
b) patch properties;
c) coating solution properties; and, d) a drying time.
7) A method according to claim 6, wherein the patch properties include at least one of:
a) projection size;
b) projection shape;
c) projection spacing; and, d) projection materials.
a) projection size;
b) projection shape;
c) projection spacing; and, d) projection materials.
8) A method according to claim 6 or claim 7, wherein the coating solution properties include at least one of:
a) a surface tension; and, b) a viscosity.
a) a surface tension; and, b) a viscosity.
9) A method according to any one of the claims 1 to 8, wherein the material includes at least one of:
a) nanoparticles;
b) a nucleic acid or protein;
c) an antigen, allergen, or adjuvant;
d) parasites, bacteria, viruses, or virus-like particles;
e) quantum dots, SERS tags, Raman tags or other nanobiosensors;
0 metals or metallic compounds; and, g) molecules, elements or compounds.
a) nanoparticles;
b) a nucleic acid or protein;
c) an antigen, allergen, or adjuvant;
d) parasites, bacteria, viruses, or virus-like particles;
e) quantum dots, SERS tags, Raman tags or other nanobiosensors;
0 metals or metallic compounds; and, g) molecules, elements or compounds.
10) A method according to any one of the claims 1 to 9, wherein the coating solution includes a therapeutic agent.
11)A method according to claim 10, wherein the therapeutic agent is at least one of.
a) DNA having a concentration of between 0.01 mg/m1 and 5 mg/m1; and, b) protein having a concentration of between 0.01 ing/m1 and 50 mg/ml
a) DNA having a concentration of between 0.01 mg/m1 and 5 mg/m1; and, b) protein having a concentration of between 0.01 ing/m1 and 50 mg/ml
12) A method according to any one of the claims 1 to 11, wherein the coating solution includes at least one of:
a) a viscosity enhancer;
b) a surfactant; and, c) an adjuvant.
a) a viscosity enhancer;
b) a surfactant; and, c) an adjuvant.
13) A method according to claim 12, wherein the adjuvant acts as a surfactant.
14) A method according to claim 12 or claim 13, wherein at least one of:
a) the viscosity agent is 0% to 90% of the coating solution; and, b) the surfactant is 0% to 90% of the coating solution.
a) the viscosity agent is 0% to 90% of the coating solution; and, b) the surfactant is 0% to 90% of the coating solution.
15)A method according to any one of the claims 12 to 14, wherein the viscosity agent is at least one of MC, CMC, gelatin, agar, and agarose.
16)A method according to any one of the claims 1 to 15, wherein the coating solution has a viscosity of between 10 -3 Pa.cndot.S and 1 Pa.cndot.S.
17) A method according to claim 16, wherein the coating solution has a viscosity of 0.01-0.06 Pa.cndot.S
18) A method according to any one of the claims 1 to 17, wherein the coating solution has a surface tension of between 0.023 N/m and 0.073 N/m.
19)A method according to claim 18, wherein the coating solution has a surface tension of 0.03-0.04 N/m.
20)A method according to any one of the claims 1 to 19, wherein the gas flow has a gas flow rate of between 6m/s and 10 m/s.
21)A method according to any one of the claims 1 to 20, wherein the method includes selecting a gas flow rate in accordance with gas properties.
22)A method according to claim 21, wherein the gas properties include a gas density.
23)A method according to any one of the claims 1 to 22, wherein the gas flow includes at least one of:
a) nitrogen;
b) argon;
c) air flow; and, d) an inert gas.
a) nitrogen;
b) argon;
c) air flow; and, d) an inert gas.
24)A method according to any one of the claims 1 to 23, wherein the gas flow is induced at least in part by extracting gas from a container containing the patch.
25)A method according to any one of the claims 1 to 24, wherein the method includes coating the projections a number of times.
26)A method according to claim 25, wherein the method includes:
a) coating the surface a first time using a first set of coating parameters;
and, b) coating the surface at least a second time using a second set of coating parameters different to the first set of coating parameters.
a) coating the surface a first time using a first set of coating parameters;
and, b) coating the surface at least a second time using a second set of coating parameters different to the first set of coating parameters.
27) A method according to any one of the claims 1 to 26, wherein the method includes applying between 5 and 15 µI of coating solution to the patch.
28) A method according to any one of the claims 1 to 27, wherein the patch has a surface area of approximately 0.16 cm2.
29)A method according to any one of the claims 1 to 28, wherein the projections have a density of between 1,000-30,000 projections/cm2.
30) A method according to claim 29, wherein the projections have a density of 20,000 projections/cm2
31)A method according to any one of the claims 1 to 30, wherein the projections have a length of between 10 to 400 µm.
32)A method according to claim 31, wherein the projections have a length of 90 µm
33)A method according to any one of the claims 1 to 32, wherein the projections have a radius of curvature of greater than 1 µm.
34)A method according to claim 33, wherein the projections have a radius of curvature greater than 5 nm.
35) A method according to any one of the claims 1 to 34, wherein the projections include a support section and a targeting section.
36) A method according to claim 35, wherein the targeting section has a diameter of less than at least one of:
a) 50 µm; and, b) 100 µm;
c) 150 µm; and, d) 400 µm.
a) 50 µm; and, b) 100 µm;
c) 150 µm; and, d) 400 µm.
37) A method according to claim 35 or claim 36, wherein a length for the targeting section is at least:
a) less than 50 µm; and, b) less than 100 µm; and, c) less than 300 µm.
a) less than 50 µm; and, b) less than 100 µm; and, c) less than 300 µm.
38) A method according to any one of the claims 35 to 37, wherein a length for the support section is at least one of:
a) for epidermal delivery < 200 µm;
b) for dermal cell delivery < 1000 µm;
c) for delivery to basal cells in the epithelium of the mucosa 600-800 µm; and, d) for lung delivery of the order of 100 µm.
a) for epidermal delivery < 200 µm;
b) for dermal cell delivery < 1000 µm;
c) for delivery to basal cells in the epithelium of the mucosa 600-800 µm; and, d) for lung delivery of the order of 100 µm.
39) A method according to any one of the claims 35 to 38, wherein a length for the support section is at least one of:
a) for epidermal delivery greater than the thickness of the Stratum Corneum;
b) for dermal cell delivery greater than the thickness of epidermis;
c) for delivery to basal cells in the epithelium of the mucosa greater than a thickness of upper epithelium; and, d) for lung delivery of the order of 100 µm in this case.
a) for epidermal delivery greater than the thickness of the Stratum Corneum;
b) for dermal cell delivery greater than the thickness of epidermis;
c) for delivery to basal cells in the epithelium of the mucosa greater than a thickness of upper epithelium; and, d) for lung delivery of the order of 100 µm in this case.
40)A method according to any one of the claims 1 to 39, wherein the projections are solid.
41)A method according to any one of the claims 1 to 40, wherein the projections are non-porous and non-hollow.
42)A method according to any one of the claims 1 to 41, wherein the patch is at least one of:
a) hydrophobic; and, b) hydrophilic.
a) hydrophobic; and, b) hydrophilic.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2007907092 | 2007-12-24 | ||
AU2007907092A AU2007907092A0 (en) | 2007-12-24 | Coating method | |
PCT/AU2008/001903 WO2009079712A1 (en) | 2007-12-24 | 2008-12-23 | Coating method |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2745339A1 CA2745339A1 (en) | 2009-07-02 |
CA2745339C true CA2745339C (en) | 2016-06-28 |
Family
ID=40800583
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2745339A Active CA2745339C (en) | 2007-12-24 | 2008-12-23 | Coating method |
Country Status (5)
Country | Link |
---|---|
US (2) | US9220678B2 (en) |
EP (1) | EP2231257A4 (en) |
AU (1) | AU2008341030B2 (en) |
CA (1) | CA2745339C (en) |
WO (1) | WO2009079712A1 (en) |
Families Citing this family (39)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB0402131D0 (en) | 2004-01-30 | 2004-03-03 | Isis Innovation | Delivery method |
ES2817249T3 (en) | 2007-04-16 | 2021-04-06 | Corium Inc | Microneedle matrices obtained by dissolution and casting containing an active principle |
US9220678B2 (en) | 2007-12-24 | 2015-12-29 | The University Of Queensland | Coating method |
EP2247527A4 (en) | 2008-02-07 | 2014-10-29 | Univ Queensland | Patch production |
CA2760680A1 (en) | 2008-05-23 | 2009-11-26 | The University Of Queensland | Analyte detection by microneedle patch with analyte selective reagents |
EP2405827B1 (en) | 2009-03-10 | 2020-05-13 | The Johns Hopkins University | Biological tissue connection and repair devices |
US20110006458A1 (en) * | 2009-04-24 | 2011-01-13 | Corium International, Inc. | Methods for manufacturing microprojection arrays |
CA2796965C (en) | 2010-04-28 | 2019-04-16 | Kimberly-Clark Worldwide, Inc. | Method for increasing permeability of an epithelial barrier |
WO2011135532A2 (en) | 2010-04-28 | 2011-11-03 | Kimberly-Clark Worldwide, Inc. | Composite microneedle array including nanostructures thereon |
CA2797205C (en) | 2010-04-28 | 2019-04-16 | Kimberly-Clark Worldwide, Inc. | Medical devices for delivery of sirna |
RU2585159C2 (en) | 2010-04-28 | 2016-05-27 | Кимберли-Кларк Ворлдвайд, Инк. | Device for delivering drug used in rheumatoid arthritis |
CA2798145C (en) | 2010-05-04 | 2022-10-18 | Corium International, Inc. | Method and device for transdermal delivery of parathyroid hormone using a microprojection array |
US9943673B2 (en) | 2010-07-14 | 2018-04-17 | Vaxxas Pty Limited | Patch applying apparatus |
US8696637B2 (en) | 2011-02-28 | 2014-04-15 | Kimberly-Clark Worldwide | Transdermal patch containing microneedles |
US8636696B2 (en) | 2011-06-10 | 2014-01-28 | Kimberly-Clark Worldwide, Inc. | Transdermal device containing microneedles |
JP2013052202A (en) * | 2011-09-01 | 2013-03-21 | Kosumedei Seiyaku Kk | Dna vaccine microneedle |
EP2765927B1 (en) | 2011-10-12 | 2021-02-24 | Vaxxas Pty Limited | Delivery device |
CA2850931A1 (en) | 2011-10-27 | 2013-05-02 | Kimberly-Clark Worldwide, Inc. | Implantable devices for delivery of bioactive agents |
US20170246439A9 (en) | 2011-10-27 | 2017-08-31 | Kimberly-Clark Worldwide, Inc. | Increased Bioavailability of Transdermally Delivered Agents |
AU2012328037B2 (en) | 2011-10-27 | 2017-11-02 | Sorrento Therapeutics, Inc. | Transdermal delivery of high viscosity bioactive agents |
US10010676B2 (en) | 2012-03-13 | 2018-07-03 | Becton Dickinson France | Method of manufacture for a miniaturized drug delivery device |
RU2015111197A (en) * | 2012-08-30 | 2016-10-20 | Тейдзин Лимитед | MATRIX OF MICRO-NEEDLES COATED WITH A MEDICINE COMPOSITION |
WO2014100750A1 (en) | 2012-12-21 | 2014-06-26 | Corium International, Inc. | Microarray for delivery of therapeutic agent and methods of use |
EP2968887B1 (en) | 2013-03-12 | 2022-05-04 | Corium, Inc. | Microprojection applicators |
WO2014151654A1 (en) | 2013-03-15 | 2014-09-25 | Corium International, Inc. | Microarray for delivery of therapeutic agent and methods of use |
EP4194028A1 (en) | 2013-03-15 | 2023-06-14 | Corium Pharma Solutions, Inc. | Multiple impact microprojection applicators |
AU2014237279B2 (en) | 2013-03-15 | 2018-11-22 | Corium Pharma Solutions, Inc. | Microarray with polymer-free microstructures, methods of making, and methods of use |
EP3188714A1 (en) | 2014-09-04 | 2017-07-12 | Corium International, Inc. | Microstructure array, methods of making, and methods of use |
CA3204959A1 (en) | 2015-02-02 | 2016-08-11 | Vaxxas Pty Limited | Microprojection array applicator and method |
JP6317690B2 (en) | 2015-03-03 | 2018-04-25 | 富士フイルム株式会社 | Transdermal absorption sheet and method for producing the same |
JP6482323B2 (en) * | 2015-03-03 | 2019-03-13 | 富士フイルム株式会社 | Transdermal absorption sheet |
FR3033941B1 (en) * | 2015-03-18 | 2017-04-21 | Commissariat Energie Atomique | FUEL CELL CHARACTERIZATION APPARATUS TEST CELL AND METHOD FOR MANUFACTURING SUCH A TEST CELL |
WO2017004067A1 (en) | 2015-06-29 | 2017-01-05 | Corium International, Inc. | Microarray for delivery of therapeutic agent, methods of use, and methods of making |
WO2017045031A1 (en) | 2015-09-18 | 2017-03-23 | Vaxxas Pty Limited | Microprojection arrays with microprojections having large surface area profiles |
US20180264244A1 (en) | 2015-09-28 | 2018-09-20 | Vaxxas Pty Limited | Microprojection arrays with enhanced skin penetrating properties and methods thereof |
WO2018176102A1 (en) | 2017-03-31 | 2018-10-04 | Vaxxas Pty Limited | Device and method for coating surfaces |
CA3065371A1 (en) | 2017-06-13 | 2018-12-20 | Vaxxas Pty Limited | Quality control of substrate coatings |
EP4218893A1 (en) | 2017-08-04 | 2023-08-02 | Vaxxas Pty Limited | Compact high mechanical energy storage and low trigger force actuator for the delivery of microprojection array patches (map) |
CN114903843B (en) * | 2022-06-02 | 2023-01-13 | 优微(珠海)生物科技有限公司 | Microneedle preparation, microneedle patch and preparation method thereof |
Family Cites Families (159)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2213830A (en) | 1938-12-10 | 1940-09-03 | Anastasi John Joseph | Suturing and ligating instrument |
US2881500A (en) | 1958-07-03 | 1959-04-14 | Charles W Furness | Corneal clamp |
DE3484951D1 (en) | 1983-10-14 | 1991-09-26 | Sumitomo Pharma | EXTENDED PREPARATIONS WITH DELAYED DELIVERY. |
US4702799A (en) * | 1985-09-03 | 1987-10-27 | Nestec S.A. | Dryer and drying method |
ES2174822T3 (en) | 1989-11-03 | 2002-11-16 | Immulogic Pharma Corp | A REACTIVE FELINE PROTEIN WITH HUMAN T-CELLS (TRFP) ISOLATED FROM DOMESTIC POWDER AND USES FOR THE SAME. |
US5201992A (en) | 1990-07-12 | 1993-04-13 | Bell Communications Research, Inc. | Method for making tapered microminiature silicon structures |
US5527288A (en) | 1990-12-13 | 1996-06-18 | Elan Medical Technologies Limited | Intradermal drug delivery device and method for intradermal delivery of drugs |
WO1994024281A1 (en) | 1993-04-14 | 1994-10-27 | Immulogic Pharmaceutical Corporation | T cell epitopes of the major allergens from dermatophagoides (house dust mite) |
AT397458B (en) | 1992-09-25 | 1994-04-25 | Avl Verbrennungskraft Messtech | SENSOR ARRANGEMENT |
US5449064A (en) | 1994-03-03 | 1995-09-12 | University Of Kansas | On-line interface and valve for capillary electophoresis system |
US5457041A (en) | 1994-03-25 | 1995-10-10 | Science Applications International Corporation | Needle array and method of introducing biological substances into living cells using the needle array |
CA2149943C (en) | 1994-05-23 | 1999-07-13 | Kwang Kyun Jang | Skin perforating device for transdermal medication |
AUPM873294A0 (en) | 1994-10-12 | 1994-11-03 | Csl Limited | Saponin preparations and use thereof in iscoms |
US5499474A (en) | 1994-11-28 | 1996-03-19 | Knooihuizen; Louis D. | Method and apparatus for liquid application |
WO1996037256A1 (en) | 1995-05-22 | 1996-11-28 | Silicon Microdevices, Inc. | Micromechanical patch for enhancing the delivery of compounds through the skin |
US6969635B2 (en) | 2000-12-07 | 2005-11-29 | Reflectivity, Inc. | Methods for depositing, releasing and packaging micro-electromechanical devices on wafer substrates |
AU3399197A (en) | 1996-06-18 | 1998-01-07 | Alza Corporation | Device for enhancing transdermal agent delivery or sampling |
CA2217134A1 (en) | 1996-10-09 | 1998-04-09 | Sumitomo Pharmaceuticals Co., Ltd. | Sustained release formulation |
JP4153999B2 (en) | 1996-12-20 | 2008-09-24 | アルザ・コーポレーション | Compositions and methods for enhancing transdermal agent flow |
WO1998028038A1 (en) | 1996-12-24 | 1998-07-02 | Alza Corporation | Method and device for controlling mammalian reproductive cycle |
US5859937A (en) | 1997-04-04 | 1999-01-12 | Neomecs Incorporated | Minimally invasive sensor |
KR100219848B1 (en) | 1997-04-22 | 1999-09-01 | 이종수 | Method and apparatus for the structural analysis of a structure |
US5928207A (en) | 1997-06-30 | 1999-07-27 | The Regents Of The University Of California | Microneedle with isotropically etched tip, and method of fabricating such a device |
AUPP807899A0 (en) | 1999-01-08 | 1999-02-04 | University Of Queensland, The | Codon utilization |
CA2296067C (en) | 1997-07-09 | 2008-10-07 | The University Of Queensland | Nucleic acid sequence and method for selectively expressing a protein in a target cell or tissue |
NL1008315C2 (en) | 1998-02-16 | 1999-08-25 | Stichting Fund Ond Material | Microdialysis probe integrated with Si chip. |
JP2002504322A (en) | 1998-02-20 | 2002-02-12 | ザ ロックフェラー ユニバーシティー | Apoptotic cell-mediated antigen presentation to dendritic cells |
JP3372862B2 (en) | 1998-03-25 | 2003-02-04 | 株式会社日立製作所 | Biological fluid mass spectrometer |
US6503231B1 (en) | 1998-06-10 | 2003-01-07 | Georgia Tech Research Corporation | Microneedle device for transport of molecules across tissue |
JP2002517300A (en) | 1998-06-10 | 2002-06-18 | ジョージア テック リサーチ コーポレイション | Microneedle devices and methods of manufacture and uses thereof |
GB9815819D0 (en) | 1998-07-22 | 1998-09-16 | Secr Defence | Transferring materials into cells and a microneedle array |
US6004815A (en) | 1998-08-13 | 1999-12-21 | The Regents Of The University Of California | Bacteria expressing nonsecreted cytolysin as intracellular microbial delivery vehicles to eukaryotic cells |
US6414501B2 (en) | 1998-10-01 | 2002-07-02 | Amst Co., Ltd. | Micro cantilever style contact pin structure for wafer probing |
US6610382B1 (en) | 1998-10-05 | 2003-08-26 | 3M Innovative Properties Company | Friction control article for wet and dry applications |
US20020004041A1 (en) | 1999-02-19 | 2002-01-10 | Albert Matthew L. | Methods for abrogating a cellular immune response |
EP1187653B1 (en) | 1999-06-04 | 2010-03-31 | Georgia Tech Research Corporation | Devices for enhanced microneedle penetration of biological barriers |
US6743211B1 (en) | 1999-11-23 | 2004-06-01 | Georgia Tech Research Corporation | Devices and methods for enhanced microneedle penetration of biological barriers |
US6312612B1 (en) | 1999-06-09 | 2001-11-06 | The Procter & Gamble Company | Apparatus and method for manufacturing an intracutaneous microneedle array |
US6256533B1 (en) | 1999-06-09 | 2001-07-03 | The Procter & Gamble Company | Apparatus and method for using an intracutaneous microneedle array |
US6299621B1 (en) | 1999-06-18 | 2001-10-09 | Novare Surgical Systems, Inc. | Surgical clamp pads with elastomer impregnated mesh |
EP1065823A1 (en) | 1999-06-29 | 2001-01-03 | Siemens Aktiengesellschaft | Method for monitoring the bit transmission quality in a packet oriented transmission |
DE19935165A1 (en) | 1999-07-28 | 2001-02-01 | Roche Diagnostics Gmbh | Method and arrangement for determining the concentration of glucose in a body fluid |
US6835184B1 (en) | 1999-09-24 | 2004-12-28 | Becton, Dickinson And Company | Method and device for abrading skin |
AU2573801A (en) | 1999-11-02 | 2001-05-14 | University Of Hawaii | Method for fabricating arrays of micro-needles |
EP1239916B1 (en) | 1999-12-10 | 2005-11-23 | ALZA Corporation | Device and method for enhancing microprotrusion skin piercing |
US6558361B1 (en) | 2000-03-09 | 2003-05-06 | Nanopass Ltd. | Systems and methods for the transport of fluids through a biological barrier and production techniques for such systems |
US6565532B1 (en) | 2000-07-12 | 2003-05-20 | The Procter & Gamble Company | Microneedle apparatus used for marking skin and for dispensing semi-permanent subcutaneous makeup |
US6537242B1 (en) | 2000-06-06 | 2003-03-25 | Becton, Dickinson And Company | Method and apparatus for enhancing penetration of a member for the intradermal sampling or administration of a substance |
US6540675B2 (en) | 2000-06-27 | 2003-04-01 | Rosedale Medical, Inc. | Analyte monitor |
US6589202B1 (en) | 2000-06-29 | 2003-07-08 | Becton Dickinson And Company | Method and apparatus for transdermally sampling or administering a substance to a patient |
GB0017999D0 (en) | 2000-07-21 | 2000-09-13 | Smithkline Beecham Biolog | Novel device |
US6749575B2 (en) | 2001-08-20 | 2004-06-15 | Alza Corporation | Method for transdermal nucleic acid sampling |
IL155583A0 (en) * | 2000-10-26 | 2003-11-23 | Alza Corp | Transdermal drug delivery devices having coated microprotrusions |
WO2002064193A2 (en) | 2000-12-14 | 2002-08-22 | Georgia Tech Research Corporation | Microneedle devices and production thereof |
US6663820B2 (en) | 2001-03-14 | 2003-12-16 | The Procter & Gamble Company | Method of manufacturing microneedle structures using soft lithography and photolithography |
WO2002074173A1 (en) | 2001-03-16 | 2002-09-26 | Alza Corporation | Method and apparatus for coating skin piercing microprojections |
IL158480A0 (en) | 2001-04-20 | 2004-05-12 | Alza Corp | Microprojection array having a beneficial agent containing coating |
US20020193729A1 (en) | 2001-04-20 | 2002-12-19 | Cormier Michel J.N. | Microprojection array immunization patch and method |
US6591124B2 (en) | 2001-05-11 | 2003-07-08 | The Procter & Gamble Company | Portable interstitial fluid monitoring system |
DE60213976T2 (en) | 2001-06-08 | 2007-04-26 | Becton Dickinson And Co. | DEVICE FOR MANIPULATING NEEDLES OR POLISHING ARRAY |
US6767341B2 (en) | 2001-06-13 | 2004-07-27 | Abbott Laboratories | Microneedles for minimally invasive drug delivery |
US6557849B2 (en) | 2001-06-21 | 2003-05-06 | Da La Rue International | Sheet handling apparatus |
AT412060B (en) | 2001-07-06 | 2004-09-27 | Schaupp Lukas Dipl Ing Dr Tech | METHOD FOR MEASURING CONCENTRATIONS IN LIVING ORGANISMS BY MEANS OF MICRODIALYSIS AND AND DEVICE FOR IMPLEMENTING THIS METHOD |
US6881203B2 (en) | 2001-09-05 | 2005-04-19 | 3M Innovative Properties Company | Microneedle arrays and methods of manufacturing the same |
US20040087992A1 (en) | 2002-08-09 | 2004-05-06 | Vladimir Gartstein | Microstructures for delivering a composition cutaneously to skin using rotatable structures |
CA2500452A1 (en) | 2001-09-28 | 2003-04-03 | Biovalve Technologies, Inc. | Switchable microneedle arrays and systems and methods relating to same |
AU2002367965A1 (en) | 2001-11-06 | 2003-12-31 | Dermal Systems International Inc. | High throughput methods and devices for assaying analytes in a fluid sample |
US20030199810A1 (en) | 2001-11-30 | 2003-10-23 | Trautman Joseph Creagan | Methods and apparatuses for forming microprojection arrays |
HUP0402605A2 (en) | 2001-12-20 | 2005-06-28 | Alza Corporation | Skin-piercing microprojections having piercing depth control |
US6908453B2 (en) | 2002-01-15 | 2005-06-21 | 3M Innovative Properties Company | Microneedle devices and methods of manufacture |
DE10209763A1 (en) | 2002-03-05 | 2003-10-02 | Bosch Gmbh Robert | Device and method for anisotropic plasma etching of a substrate, in particular a silicon body |
AU2003222691A1 (en) | 2002-04-30 | 2003-11-17 | Morteza Shirkhanzadeh | Arrays of microneedles comprising porous calcium phosphate coating and bioactive agents |
US6945952B2 (en) | 2002-06-25 | 2005-09-20 | Theraject, Inc. | Solid solution perforator for drug delivery and other applications |
EP1534376B1 (en) | 2002-06-25 | 2016-08-31 | Theraject, Inc. | Rapidly dissolving micro-perforator for drug delivery and other applications |
DK1523355T3 (en) | 2002-07-22 | 2019-04-23 | Becton Dickinson Co | PLASTER-LIKE INFUSION DEVICE |
US7081118B2 (en) | 2002-08-22 | 2006-07-25 | Helmut Weber | Medical tool |
US8062573B2 (en) | 2002-09-16 | 2011-11-22 | Theraject, Inc. | Solid micro-perforators and methods of use |
US7045069B2 (en) | 2002-11-14 | 2006-05-16 | Gennady Ozeryansky | Microfabrication method based on metal matrix composite technology |
WO2004080275A2 (en) | 2003-03-06 | 2004-09-23 | Applied Medical Resources Corporation | Spring clip and method for assembling same |
DE10328730B4 (en) | 2003-06-25 | 2006-08-17 | Micronas Gmbh | Method for producing a microarray and device for providing a carrier for a microarray with coating materials |
JP2007508914A (en) | 2003-10-24 | 2007-04-12 | アルザ・コーポレーシヨン | Apparatus and method for facilitating transdermal drug delivery |
CN1897882A (en) | 2003-10-28 | 2007-01-17 | 阿尔扎公司 | Method and apparatus for reducing the incidence of tobacco use |
CA2543641A1 (en) | 2003-10-31 | 2005-05-19 | Alza Corporation | Self-actuating applicator for microprojection array |
EP1740256A4 (en) | 2003-11-10 | 2011-06-29 | Agency Science Tech & Res | Microneedles and microneedle fabrication |
WO2005049108A2 (en) | 2003-11-13 | 2005-06-02 | Alza Corporation | System and method for transdermal delivery |
US7753888B2 (en) | 2003-11-21 | 2010-07-13 | The Regents Of The University Of California | Method and/or apparatus for puncturing a surface for extraction, in situ analysis, and/or substance delivery using microneedles |
IL160033A0 (en) | 2004-01-25 | 2004-06-20 | Transpharma Medical Ltd | Transdermal delivery system for polynucleotides |
WO2005072360A2 (en) | 2004-01-27 | 2005-08-11 | Buyerleverage | System and method for granting deposit-contingent e-mailing rights |
GB0402131D0 (en) | 2004-01-30 | 2004-03-03 | Isis Innovation | Delivery method |
US20080312669A1 (en) | 2004-03-31 | 2008-12-18 | Vries Luc De | Surgical instrument and method |
US7591806B2 (en) | 2004-05-18 | 2009-09-22 | Bai Xu | High-aspect-ratio microdevices and methods for transdermal delivery and sampling of active substances |
EP1773444B1 (en) | 2004-06-10 | 2017-09-20 | 3M Innovative Properties Company | Patch application device and kit |
TWI246929B (en) | 2004-07-16 | 2006-01-11 | Ind Tech Res Inst | Microneedle array device and its fabrication method |
US7524430B2 (en) | 2004-09-10 | 2009-04-28 | Lexmark International, Inc. | Fluid ejection device structures and methods therefor |
JP2008520367A (en) | 2004-11-18 | 2008-06-19 | スリーエム イノベイティブ プロパティズ カンパニー | Non-skin-type microneedle array applicator |
US7846488B2 (en) * | 2004-11-18 | 2010-12-07 | 3M Innovative Properties Company | Masking method for coating a microneedle array |
WO2006101459A1 (en) | 2005-03-23 | 2006-09-28 | Agency For Science, Technology And Research | Microneedles |
WO2006108185A1 (en) | 2005-04-07 | 2006-10-12 | 3M Innovative Properties Company | System and method for tool feedback sensing |
US20060253079A1 (en) | 2005-04-25 | 2006-11-09 | Mcdonough Justin | Stratum corneum piercing device |
US20070270738A1 (en) | 2005-04-25 | 2007-11-22 | Wu Jeffrey M | Method of treating ACNE with stratum corneum piercing patch |
US20060264783A1 (en) | 2005-05-09 | 2006-11-23 | Holmes Elizabeth A | Systems and methods for monitoring pharmacological parameters |
US8043250B2 (en) | 2005-05-18 | 2011-10-25 | Nanomed Devices, Inc. | High-aspect-ratio microdevices and methods for transdermal delivery and sampling of active substances |
WO2006138719A2 (en) | 2005-06-17 | 2006-12-28 | Georgia Tech Research Corporation | Coated microstructures and method of manufacture thereof |
WO2007002123A2 (en) * | 2005-06-21 | 2007-01-04 | Alza Corporation | Method and device for coating a continuous strip of microprojection members |
AU2006261898B2 (en) | 2005-06-27 | 2011-11-03 | 3M Innovative Properties Company | Microneedle array applicator device |
US20070027474A1 (en) | 2005-07-15 | 2007-02-01 | Jeffrey Lasner | Pressure limiting forceps |
JP2009502261A (en) | 2005-07-25 | 2009-01-29 | ナノテクノロジー ビクトリア ピーティーワイ リミテッド | Microarray device |
CA2629393C (en) | 2005-09-06 | 2014-06-10 | Theraject, Inc. | Solid solution perforator containing drug particle and/or drug-adsorbed particles |
WO2007040938A1 (en) | 2005-09-30 | 2007-04-12 | Tti Ellebeau, Inc. | Functionalized microneedles transdermal drug delivery systems, devices, and methods |
CA2628546A1 (en) * | 2005-11-09 | 2007-05-18 | Pharmexa A/S | Therapeutic vaccines targeting hmgb1 |
WO2007061964A1 (en) | 2005-11-18 | 2007-05-31 | 3M Innovative Properties Company | Methods for coating microneedles |
US8524303B2 (en) | 2005-11-23 | 2013-09-03 | The Coca-Cola Company | High-potency sweetener composition with phytosterol and compositions sweetened therewith |
WO2007070004A2 (en) | 2005-12-14 | 2007-06-21 | Silex Microsystems Ab | Methods for making micro needles and applications thereof |
US7658728B2 (en) | 2006-01-10 | 2010-02-09 | Yuzhakov Vadim V | Microneedle array, patch, and applicator for transdermal drug delivery |
GB0600795D0 (en) | 2006-01-16 | 2006-02-22 | Functional Microstructures Ltd | Method of making microneedles |
JP5028872B2 (en) | 2006-03-02 | 2012-09-19 | 凸版印刷株式会社 | Manufacturing method of needle-shaped body |
US20070224252A1 (en) | 2006-03-27 | 2007-09-27 | Trautman Joseph C | Microprojections with capillary control features and method |
WO2007124411A1 (en) | 2006-04-20 | 2007-11-01 | 3M Innovative Properties Company | Device for applying a microneedle array |
WO2007127815A2 (en) | 2006-04-25 | 2007-11-08 | Alza Corporation | Microprojection array application with multilayered microprojection member for high drug loading |
JP2009535122A (en) | 2006-04-25 | 2009-10-01 | アルザ コーポレイション | Application of microprojection array with shaped microprojections for high drug loading |
WO2007127976A2 (en) | 2006-05-01 | 2007-11-08 | Georgia Tech Research Corporation | Particle based molding |
US7425465B2 (en) | 2006-05-15 | 2008-09-16 | Fujifilm Diamatix, Inc. | Method of fabricating a multi-post structures on a substrate |
WO2008011625A2 (en) | 2006-07-21 | 2008-01-24 | Georgia Tech Researh Corporation | Microneedle devices and methods of drug delivery or fluid withdrawal |
KR100793615B1 (en) | 2006-07-21 | 2008-01-10 | 연세대학교 산학협력단 | A biodegradable solid type microneedle and methods for preparing it |
JPWO2008020632A1 (en) | 2006-08-18 | 2010-01-07 | 凸版印刷株式会社 | Microneedle and microneedle patch |
WO2008053481A1 (en) | 2006-11-01 | 2008-05-08 | Svip 6 Llc | Microneedle arrays |
KR20080051342A (en) | 2006-12-05 | 2008-06-11 | 연세대학교 산학협력단 | A microneedle device and methods for applicating it |
US8684968B2 (en) | 2006-12-29 | 2014-04-01 | Aktivpak, Inc. | Hypodermic drug delivery reservoir and apparatus |
DE102007002832A1 (en) | 2007-01-19 | 2008-07-24 | Robert Bosch Gmbh | Method for manufacturing device with arrangement of micro-needles, involves preparing silicon semiconductor substrate, whose surface is applied and structured with masking layer |
WO2008091602A2 (en) | 2007-01-22 | 2008-07-31 | Corium International, Inc. | Applicators for microneedle arrays |
US20090017210A1 (en) | 2007-07-09 | 2009-01-15 | Andrianov Alexander K | Methods and systems for coating a microneedle with a dosage of a biologically active compound |
CN101808588B (en) | 2007-09-28 | 2012-12-19 | 贝尔法斯特女王大学 | Delivery device and method |
JP5419702B2 (en) | 2007-10-18 | 2014-02-19 | 久光製薬株式会社 | Microneedle device |
JP5538897B2 (en) | 2007-11-21 | 2014-07-02 | 株式会社バイオセレンタック | Preparation for body surface application, and preparation holding sheet for body surface application |
GB0725018D0 (en) | 2007-12-21 | 2008-01-30 | Univ Cardiff | Monitoring system for microneedle delivery |
US9220678B2 (en) | 2007-12-24 | 2015-12-29 | The University Of Queensland | Coating method |
CN101214395A (en) | 2008-01-02 | 2008-07-09 | 西南交通大学 | Inorganic material surface biological method |
EP2247527A4 (en) | 2008-02-07 | 2014-10-29 | Univ Queensland | Patch production |
CA2760680A1 (en) | 2008-05-23 | 2009-11-26 | The University Of Queensland | Analyte detection by microneedle patch with analyte selective reagents |
CN101297989B (en) | 2008-06-19 | 2010-06-23 | 上海交通大学 | Batch preparation of hollow micro-needle based on molding |
JPWO2010001671A1 (en) | 2008-06-30 | 2011-12-15 | 久光製薬株式会社 | Microneedle device and method for increasing the efficacy of influenza vaccine by microneedle device |
EP2344234A4 (en) | 2008-10-16 | 2015-07-29 | Univ Queensland | A method and associated apparatus for coating projections on a patch assembly |
US20110021996A1 (en) | 2008-12-18 | 2011-01-27 | Miti Systems Inc. | Structure of micro-needle with side channel and manufacturing method thereof |
EP2379160B1 (en) | 2008-12-22 | 2014-09-10 | The University Of Queensland | Patch production |
US20120078293A1 (en) | 2009-03-27 | 2012-03-29 | Technion Research & Development Foundation Ltd. | Applicators for patches and adhesives |
US20110006458A1 (en) | 2009-04-24 | 2011-01-13 | Corium International, Inc. | Methods for manufacturing microprojection arrays |
JP5918700B2 (en) | 2010-01-29 | 2016-05-18 | アイコン メディカル コーポレーション | Biodegradable protrusions on inflatable devices |
WO2011105496A1 (en) | 2010-02-24 | 2011-09-01 | 久光製薬株式会社 | Micro-needle device |
WO2011116388A1 (en) | 2010-03-19 | 2011-09-22 | Nanostar Health Corporation | Body fluid sampling/fluid delivery device |
CA2796965C (en) | 2010-04-28 | 2019-04-16 | Kimberly-Clark Worldwide, Inc. | Method for increasing permeability of an epithelial barrier |
CN105662530B (en) | 2010-05-04 | 2018-07-20 | 考里安国际公司 | Micropin applicator |
US9943673B2 (en) | 2010-07-14 | 2018-04-17 | Vaxxas Pty Limited | Patch applying apparatus |
EP2654582B1 (en) | 2010-12-22 | 2017-05-31 | Valeritas, Inc. | Microneedle patch applicator |
JP6265740B2 (en) | 2011-10-06 | 2018-01-24 | 久光製薬株式会社 | applicator |
IN2014CN02637A (en) | 2011-10-12 | 2015-08-07 | 3M Innovative Properties Co | |
EP2765927B1 (en) | 2011-10-12 | 2021-02-24 | Vaxxas Pty Limited | Delivery device |
EP2906284B8 (en) | 2012-10-10 | 2021-01-20 | Kindeva Drug Delivery L.P. | Force-controlled applicator for applying a microneedle device to skin |
EP4194028A1 (en) | 2013-03-15 | 2023-06-14 | Corium Pharma Solutions, Inc. | Multiple impact microprojection applicators |
CA3204959A1 (en) | 2015-02-02 | 2016-08-11 | Vaxxas Pty Limited | Microprojection array applicator and method |
-
2008
- 2008-12-23 US US12/810,298 patent/US9220678B2/en active Active
- 2008-12-23 CA CA2745339A patent/CA2745339C/en active Active
- 2008-12-23 EP EP20080865041 patent/EP2231257A4/en not_active Withdrawn
- 2008-12-23 AU AU2008341030A patent/AU2008341030B2/en active Active
- 2008-12-23 WO PCT/AU2008/001903 patent/WO2009079712A1/en active Application Filing
-
2015
- 2015-11-12 US US14/939,726 patent/US10022322B2/en active Active
Also Published As
Publication number | Publication date |
---|---|
EP2231257A1 (en) | 2010-09-29 |
US9220678B2 (en) | 2015-12-29 |
WO2009079712A1 (en) | 2009-07-02 |
US10022322B2 (en) | 2018-07-17 |
AU2008341030A1 (en) | 2009-07-02 |
CA2745339A1 (en) | 2009-07-02 |
US20110059150A1 (en) | 2011-03-10 |
EP2231257A4 (en) | 2013-11-06 |
US20160058697A1 (en) | 2016-03-03 |
AU2008341030B2 (en) | 2014-04-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10022322B2 (en) | Coating method | |
US8734697B2 (en) | Patch production | |
US20110288484A1 (en) | Method and associated apparatus for coating projections on a patch assembly | |
US20200405331A1 (en) | Delivery device | |
US9943673B2 (en) | Patch applying apparatus | |
US20210170152A1 (en) | Delivery device |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20131119 |