CN101273066B - Water-soluble chitosan nano particle for delivering carcinostatic agent and method for producing the same - Google Patents

Water-soluble chitosan nano particle for delivering carcinostatic agent and method for producing the same Download PDF

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CN101273066B
CN101273066B CN2005800515276A CN200580051527A CN101273066B CN 101273066 B CN101273066 B CN 101273066B CN 2005800515276 A CN2005800515276 A CN 2005800515276A CN 200580051527 A CN200580051527 A CN 200580051527A CN 101273066 B CN101273066 B CN 101273066B
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water
soluble chitosan
nano particle
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carcinostatic agent
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CN101273066A (en
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罗在云
郑特来
张美卿
丁映日
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Industry Academy Cooperation Foundation of SCNU
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0024—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Glucans; (beta-1,3)-D-Glucans, e.g. paramylon, coriolan, sclerotan, pachyman, callose, scleroglucan, schizophyllan, laminaran, lentinan or curdlan; (beta-1,6)-D-Glucans, e.g. pustulan; (beta-1,4)-D-Glucans; (beta-1,3)(beta-1,4)-D-Glucans, e.g. lichenan; Derivatives thereof
    • C08B37/0027—2-Acetamido-2-deoxy-beta-glucans; Derivatives thereof
    • C08B37/003—Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/722—Chitin, chitosan
    • 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
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The present invention relates to a water soluble chitosan nanoparticle (WSC-NP) for delivering an anticancer agent and a preparing method thereof, more precisely, a water soluble chitosan nanoparticle for delivering an anticancer agent which has function of targeting on a wanted area by introducing a functional group in the location of highly reactive amine group and becomes an excellent gene carrier with the use of water soluble chitosan since the water soluble chitosan itself can combined with DNA having a negative electric charge(-) owing to the very strong positive electric charge(+) of its amine group, and a preparing method thereof.; Therefore, a water-soluble chitosan nanoparticle for delivering an anticancer agent of the present invention can effectively envelope paclitaxel by introducing hydrophilic and hydrophobic groups in the position of highly reactive amine group of the water-soluble chitosan. A water soluble chitosan nanopaclitaxel prepared as the above has an excellentre-dispersion force, after freeze-drying, in distilled water and has an outstanding anticancer effect with its accumulation in tumor cells greater than that of the other anticancer agent carriers.

Description

Be used for water-soluble chitosan nano particle of delivering carcinostatic agent and preparation method thereof
Background of invention
Invention field
The present invention relates to water-soluble chitosan nano particle (WSC-NP) for delivering carcinostatic agent and preparation method thereof, more accurately, relate to water-soluble chitosan nano particle for delivering carcinostatic agent and preparation method thereof, described water-soluble chitosan nano particle has the function in the required zone of target by introducing functional group at height reactive amine base location, and because the very strong positive charge (+) of its amido, described water-soluble chitosan itself can be combined with the DNA with negative charge (-), therefore described water-soluble chitosan nano particle utilizes water-soluble chitosan (WSC) and becomes splendid genophore.
Description of the Prior Art
Chitosan is to form by the β of the pyranose monomer of glycosamine-Isosorbide-5-Nitrae key, and it has the glycosamine residue above 5,000.Its molecular weight surpasses 1,000,000.As the biological polymer that belongs to the polysaccharide with polycation, chitosan from fishery products such as Crustaceans (Crustacea) as extracting crab or shrimp and the squid.It has and Mierocrystalline cellulose, a kind of polysaccharide, and similar molecular structure, this shows that it has fabulous biocompatibility, and is not subjected to immunoreactive repulsion.Therefore, chitosan has been widely used in medicine industry, and is safety food by the U.S. FDA testing identity recently.Therefore, estimate that chitosan will become the related substances that is applicable to biological industry and biomedical industries in 21 century.
Especially, known chitosan with molecular weight of from 20,000 to 100,000 scopes has very strong physiologically active, and therefore described chitosan can be for the production of heath food, food and drink, makeup, health and medical supplies.
Although the above-mentioned promising characteristic of chitosan, but since with the strong hydrogen bond of adjacent molecule, it is indissoluble in water, and must use organic acid, comprise lactic acid, acetic acid, propionic acid, formic acid, xitix and tartrate, and mineral acid, comprise hydrochloric acid, nitric acid and sulfuric acid, dissolve chitosan, so it not yet is successfully used to biological industry.In order to overcome this problem, the inventor has developed water-soluble chitosan, and has applied for patent (Korean Patent Application No. 2001-0059282 and 2001-0070052).
In above-mentioned patent, reported and had 1,000～100, the preparation method of the pure water soluble unhindered amina chitosan of the molecular weight of 000Da scope, described method comprises step: 1) the organic or inorganic acid salt solution of chitosan oligosaccharide is processed with trialkylamine, 2) organic solvent is added in the mentioned solution, to separate the organic or inorganic acid that is connected with the chitosan oligosaccharide that forms the triol amine salt, and then, recovery does not have the chitosan oligosaccharide of organic or inorganic salt, 3) will there be the solution mineral acid treatment of chitosan oligosaccharide of acid, and carry out purifying by gac/ion exchange resin column, acquisition has the pure water soluble unhindered amina chitosan of 1,000～100,000Da molecular weight.
Although compare with other conventional carcinostatic agent, has fabulous antitumour activity by U.S. FDA in the taxol of proof in 1992, but it has the severe side effect problem, this side effect is because taxol is hydrophobic so that its disperses in water, produces to be used as injection therefore it must be dispersed in the mixing solutions of polyoxyethylenated castor oil (polyethoxye carburetion) and ethanol (50: 50).
Since insoluble in water, taxol, and a kind of crude substance with antitumour activity has limitation as in the application of injection.Therefore, it was suggested many so that the injection of taxol becomes possible system.For example, with biodegradable and be combined with taxol with the human serum albumin (HSA) of the strong combination of taxol, then according to ordinary method sonication, high-pressure homogenization and microfluid, produce applicable emulsion as injection.
At U.S. Patent number 5,439,686,5,498,421,5,560,933 and WO 94/18954 in reported by VivoRx drugmaker and utilized the combination of the taxol sonication technology of the mean particle size that is lower than 10 μ m is provided and produces and human serum albumin particle and the applicable injection for preparing.Yet the preparation method who reports in above-mentioned patent can not be used on the large technical scale.And the particle that is obtained by aforesaid method can not be administered to the patient too greatly.
At U.S. Patent number 5,916,596,6,096, also carried out other report among 331, WO 98/14174 and the WO 99/00113, described report relates to by the powder dissolution of freeze-drying is obtained to have the taxol of the mean size that is lower than 0.2 μ m in aseptic 0.9%NaCl solution and aseptic human serum albumin nanometer-emulsion.According to above-mentioned report, the particle in the nanometer-emulsion that obtains by high-pressure homogenization is homogeneous in size, and pass by not form nanoparticle precipitate in time, and this represents the stability of nanometer-emulsion.Yet, can't be used as plant-scale injection by mix the taxol suspensoid for preparing with human serum albumin.
Therefore, the inventor has studied and has utilized chitosan as the method for the paclitaxel carrier that successfully is applicable to medicine industry, reason is when using, and chitosan has fabulous biocompatibility and do not repelled by immune response, and has finished the present invention by setting up described method.
Summary of the invention
An object of the present invention is to provide water-soluble chitosan nano particle and preparation method thereof, described water-soluble chitosan nano particle can utilize the chitosan of the high water soluble with free amino as paclitaxel carrier.
To achieve these goals, the invention provides water-soluble chitosan nano particle of effective delivering carcinostatic agent and preparation method thereof, wherein with the basal component of water-soluble chitosan as the carcinostatic agent carrier, owing to having the amido of the chitosan of very strong positive charge (+), by being combined with the DNA with negative charge (-), water-soluble chitosan itself becomes outstanding genophore, and described carrier is by introducing the function that functional group provides the needed zone of target in highly reactive free amino.
The accompanying drawing summary
To understand best the application of the preferred embodiment of the invention with reference to the accompanying drawings, wherein:
Fig. 1 is the figure that shows the MTT test-results of WSC,
Fig. 2～3rd, a picture group of the FT-IR spectrum of the WSC that demonstration and MEPG p-NP put together,
Fig. 4～6th shows WSC's that wherein hydrophilic radical MEPG p-NP and hydrophobic grouping cholesterol chloro-formiate put together 1One picture group of H-NMR spectrum,
Fig. 7 and Fig. 8 are the photos that shows the surface of the WSC-NP of the present invention that takes with TEM and AFM,
Fig. 9～10th is presented at before the envelope paclitaxel (Figure 10) thermoanalytical result's a picture group behind (Fig. 9) and envelope paclitaxel,
Figure 11～12nd shows the mass analysis result's of the taxol be encapsulated among the WSC-NPT and standard taxol a picture group,
Figure 13 is the figure that shows the mouse survival rate of processing according to different pharmaceutical,
Figure 14 is the figure that is presented to tumor growth behind the mouse drug administration,
Figure 15 is the figure that is presented to changes in weight behind the mouse drug administration of transplantation tumor.
DESCRIPTION OF THE PREFERRED
Hereinafter, the present invention will be described in more detail.
The invention provides a kind of water-soluble chitosan nano particle for delivering carcinostatic agent (WSC-NP), it prepares by introduce hydrophobic grouping such as cholesterol and the p-nitrophenyl carbonate of methoxyl group PEG (MPEG p-NP) in the water-soluble chitosan chain.
The cholesterol chloro-formiate of introducing seals taxol to be delivered; a kind of hydrophobic drug; and MPEG increases the solvability of cholesterol; this causes so that taxol circulates in blood for a long time, and protects described nano particle to avoid the attack of reticuloendothelial system (RES) and scavenger cell.
The present invention also is provided for sending the preparation method of the water-soluble nanoparticles of the carcinostatic agent that comprises hydrophobic core, and described method comprises the steps:
1) the p-nitrophenyl carbonate of methoxyl group PEG (MPEG p-NP) is added in the water-soluble chitosan, with the formation amido linkage, and eliminate by product, p-nitrophenyl group; With
2) cholesterol chloro-formiate is added in the above-mentioned reaction soln, between the free amino of described water-soluble chitosan and cholesterol chloro-formiate, to form another amido linkage.
By eliminate p-nitrophenyl group, step 1 with frozen water dialysis) by product, and by eliminating residual by product fully with the dehydrated alcohol purifying.
In the present invention, in order to produce the water-soluble chitosan nanopaclitaxel with cancer therapy effect, the p-nitrophenyl carbonate of hydrophilic group methoxy PEG (MPEG p-NP) and hydrophobic grouping cholesterol chloro-formiate are combined with the free amino of water-soluble chitosan, and obtain water-soluble chitosan nano particle.Then, taxol is sealed effectively the water-soluble chitosan nanopaclitaxel that acquisition can easily be dispersed in water again (WSC-NPT) with described nano particle.
More accurately, described water-soluble chitosan nanopaclitaxel can be such as following production.In the first step, the p-nitrophenyl carbonate of methoxyl group PEG (MPEG p-NP) is added in the water-soluble chitosan, with the formation amido linkage, and by eliminating by product " p-nitrophenyl group " in 48 hours with the frozen water dialysis.Residual by product is by eliminating fully with dehydrated alcohol purification reaction solution.In next step, cholesterol chloro-formiate is added in the above-mentioned reaction soln, to form another amido linkage between free amino and cholesterol chloro-formiate, this acquisition has the water-soluble chitosan nano particle (square case 1) of hydrophobic core.At last, for above-mentioned reaction soln provides carcinostatic agent (taxol), then, described kit is enclosed in the cholesterol, the hydrophobic grouping of above-mentioned water-soluble chitosan nano particle, this causes the generation of water-soluble chitosan nanopaclitaxel.
＜scheme 1 〉
The water-soluble chitosan that is used as the carcinostatic agent carrier among the present invention is fit to delivering carcinostatic agent, because it contains the hyperergy free amino.
Different from other preparation method who uses the organic solvent that the mankind are harmful to, the preparation of water-soluble chitosan nano particle of the present invention is carried out in PBS (phosphate-buffered saline) solution (pH 7.0～8.0) under mild conditions, and this shows that this process is safety and simple.
Usually known, polymeric medicine is more than gathering in normal cell in tumour cell, and reason is its EPR (perviousness of enhancing and stick effect).Compare with other conventional carcinostatic agent carrier, use in the present invention the water-soluble chitosan nano particle of water-soluble chitosan preparation also more than gathering in normal cell in tumour cell, this illustrates its splendid antitumous effect.Usually, cancer cell is sourer a little than flanking cell.Water-soluble chitosan with 6.5pKa is stable better in weak acid, and this shows that it can target on cancer cells rather than normal cell.Therefore, described water-soluble chitosan nano particle accumulates in the cancer cell.
The water-soluble chitosan nanopaclitaxel that wherein comprises taxol is characterised in that in the splendid redispersion power in distilled water after the lyophilize, and to have other advantage of natural polymerization material such as water-soluble chitosan.
Now exemplary, nonrestrictive embodiment of the present invention will be described more fully hereinafter.Yet the present invention can embody with many different forms, and should not be interpreted as being subject to exemplary in this paper.
Embodiment 1: preparation water-soluble chitosan nano particle 1
Having the water-soluble chitosan that 18kDa molecular weight and 87% takes off the acetyl degree is provided by KITTOLIFE company limited.The p-nitrophenyl carbonate of methoxyl group PEG (MPEG p-NP), hydrophilic segment, available from Sigma company (Sigma Co.), and cholesterol chloro-formiate, hydrophobic grouping is available from aldrich company (Aldrich Co.).Dialysis tubing (MWCO 12,000) is purchased from frequency spectrum company (Spectrum Co.).All other chemical all are SILVER REAGENT and in statu quo use.
The preparation of water-soluble chitosan nano particle is carried out in following step.In step 1, the 0.1g water-soluble chitosan is dissolved among the 15ml PBS (pH 7.0～8.0), then stirred one hour.The p-nitrophenyl carbonate of 0.5g methoxyl group PEG (MPEG p-NP) is dissolved among the PBS (pH 8.0), when stirring solution, it is slowly splashed in the above-mentioned chitosan reaction solution.After two hours, described solution is used molecular retention 12, the dialysis tubing of 000g/mol was to cold distilled water dialysis 48 hours, and this causes being formed on limpid product (clear product) and the by product in the beds of precipitation in the supernatant.With those product separations, and with the lyophilize of limpid supernatant layer.
The white product that will obtain by lyophilize in above-mentioned steps 1 is dissolved among the 15ml PBS (pH 7.0～8.0), then stirs one hour.Cholesterol chloro-formiate is dissolved in 1～2ml dry DMF with 1.0 parts of per 10 parts of chitosan glycosamine monomers.Stirring described reaction soln in 2 hours, cholesterol chloro-formiate solution is slowly splashed in the above-mentioned reaction soln.When finishing reaction, used distill water dialysis 24 hours.Then with 10,000rpm centrifugal 10 minutes, then use 0.45 μ m syringe filter to filter and lyophilize.Obtain end product, white water-soluble chitosan nano particle.
Embodiment 2: preparation water-soluble chitosan nano particle 2
Except using 1.5 parts of cholesterol chloro-formiates of per 10 parts of chitosan glycosamine monomers, use with the same procedure described in above-described embodiment 1 to prepare water-soluble chitosan nano particle.
Embodiment 3: preparation water-soluble chitosan nano particle 3
Except using 1.8 parts of cholesterol chloro-formiates of per 10 parts of chitosan glycosamine monomers, use with the same procedure described in above-described embodiment 1 to prepare water-soluble chitosan nano particle.
Embodiment 4: preparation water-soluble chitosan nanopaclitaxel 1
The 25mg water-soluble chitosan nano particle of preparation in above-described embodiment 1 is dispersed among the 10mlPBS (pH 7.4), then stirred 30 minutes at 40 ℃.The 4mg taxol is dissolved in the 2ml ethanol, is slowly dropped in the mentioned solution, make in this course water type acoustic wave oscillator carry out sonication.By using excellent type acoustic wave oscillator again to mentioned solution sonication 2 seconds, it is repeated 10 times.In PBS (pH 7.4), dialyse.At second day, in distilled water, dialysed again 12 hours, then centrifugal.Then, product is filtered by 0.45 μ m syringe filter, and lyophilize, the water-soluble chitosan nanopaclitaxel that wherein comprises taxol produced.
Embodiment 5: preparation water-soluble chitosan nanopaclitaxel 2
Except the WSC-NP that uses preparation in above-described embodiment 2, use with the identical method described in above-described embodiment 4 to prepare water-soluble chitosan nanopaclitaxel.
Embodiment 6: preparation water-soluble chitosan nanopaclitaxel 3
Except the WSC-NP that uses preparation in above-described embodiment 3, use with the identical method described in above-described embodiment 4 to prepare water-soluble chitosan nanopaclitaxel.
Experimental example 1:MTT detects
Carry out MTT and detect to study the toxicity of the water-soluble chitosan that uses among the present invention.With the 293T cell with 5 * 10 4Individual cells/well places 96 holes dull and stereotyped, then cultivates 24 hours.
Concentration with 0.1～100 μ g/ml adds the water-soluble chitosan with Different Weight molecular-weight average (Mw) (being respectively 10,000,20,000 and 50,000) in every hole, then cultivated 4 hours at 37 ℃.Then 50 μ l MTT to wherein adding with the concentration preparation of 3mg/ml further cultivated 4 hours at 37 ℃.Remove supernatant.In each hole of 96 hole flat boards, add 100 μ l DMSO, it was placed 10 minutes.Then, use micro-plate reader (VERSA MAX) result of study.Calculate cell survival by following formula 1.
＜formula 1 〉
Cell viability (%)=(OD 570(sample)/OD 570(contrast)) * 100
OD 570(sample) means the OD value measured and OD in the hole of processing with water-soluble chitosan 570(contrast) means the OD value measured in the hole with the processing of PBS damping fluid only.Test-results is illustrated among Fig. 1.As shown in fig. 1, have 10,000-50, the water-soluble chitosan of the weight average molecular weight of 000 scope does not have toxicity.
Experimental example 2:FT-IR and 1The measurement of H-NMR spectrum
Synthetic for the water-soluble chitosan nano particle of modifying with hydrophobic grouping and hydrophilic radical of identifying in above-described embodiment 1 preparation, use FT-IR (Shimadzu, FT-IR 8700) and 1H-NMR spectrometer (Bruker, DRX-500MHz).For 1H-NMR measures, and water-soluble chitosan nano particle is dissolved in CDCl with the concentration of 10mg/ml 3In, and spectrum carries out at 298K.In order to study the constitutional features of the water-soluble chitosan nano particle of preparation in above-described embodiment 1, at CDCl 3And D 2Detect among the O 1H-NMR spectrum.The result is illustrated in Fig. 2～3 and Fig. 4～6.
Fig. 2 shows the FT-IR spectrum of water-soluble chitosan, and wherein the peak of the amido of glycosamine monomer and amide group is clear and differently be presented at 1539cm respectively -1And 1635cm -1As shown in FIG. 3, MPEG is incorporated into causes in the water-soluble chitosan near 2880cm -1The place observes the specific peak of aliphatic series-CH of MPEG, near 1539cm -1The amido peak intensity at place, a specific peak of water-soluble chitosan, reduced by the amido linkage between the carbonyl of the free amino of water-soluble chitosan and MPEG, and compare with the peak intensity of amido, by amido linkage new between water-soluble chitosan and MPEG, the strength increase of the specific peak of acid amides.Therefore, this result shows that MPEG is successfully conjugated in the water-soluble chitosan.
The water-soluble chitosan that Fig. 4～6 expression usefulness hydrophilic radical MPEG and hydrophobic group cholesterol are modified 1H-NMR spectrum.Especially, Fig. 4 demonstration shows that it is that WSC-NP is being dissolved in CDCI at the spectrum at the peak of the responseless cholesterol at about 1.5～0.5ppm place 3In after do not remove residual cholesterol and record.Especially, the 1.8ppm regional observation to strong peak show that by with cholesterol a kind of hydrophobic grouping is connected on the water-soluble chitosan chain and has formed hydrophobic core.That is, the key of the parent mass peak of hydrophobic type cholesterol by the amount of polymers of water-soluble chitosan and MPEG hinders, so that these cholesterol are formed on the hydrophobic core at 1.8ppm place, produces there the strong characteristic peak of cholesterol.Fig. 5 shows WSC-NP's 1H-NMR spectrum, it is record after eliminating unreacted remaining cholesterol with the described particle of ether purifying.As described in as shown in the figure, disappear at the peak of the cholesterol at 1.5～0.5ppm place, and clearly manifest the specific peak at 1.8ppm place, this shows and has formed described hydrophobic core.Fig. 6 is presented at water-soluble chitosan nano particle is dissolved in D 2Obtain after among the O 1H-NMR spectrum, it also confirms to have formed hydrophobic core in WSC-NP.As described in as shown in the figure, the specific peak completely dissolve of cholesterol, and only observe the characteristic peak of water-soluble chitosan and MPEG, this proves that described hydrophobic core forms.All the above results show that all MPEG and cholesterol successfully are connected on the chain of water-soluble chitosan.
Experimental example 3: the morphologic observation of water-soluble chitosan nano particle
The form of WSC-NP of the present invention TEM (transmission electron microscope; JEOL JEM-2000FX-II) and AFM (atomic force microscope; PARK ' s science the CP (PARK ' s ScienceAutoprobe CP) that automatically pops one's head in) checks.A chitosan nano particle that is suspended in 0.01% phospho-wolframic acid (PTA) is placed on the carbon film that is coated on the copper grid for TEM.For the AFM imaging analysis, the chitosan nano particle of the 0.1mg/ml in the distilled water is placed the silicon water surface, and at room temperature be used in 12 and 24kHz between the cantilever frequency of vibrating observe.Fig. 7 shows the surface structure with the water-soluble chitosan nano particle of TEM research.Fig. 7 shows the TEM photo of water-soluble chitosan nano particle.As shown in these photos, the shape of water-soluble chitosan nano particle is spherical, and size is in diameter 30-150nm scope.When the constant rate of MPEG and glycosamine unit, the ratio increase of cholesterol is more, because the hydrophobicity increase causes the size of chitosan nano particle less.Fig. 8 shows that the AFM of water-soluble chitosan nano particle observes.In these photos, confirm that described water-soluble chitosan nano particle has spherical smooth surface, and because described particle is by the cholesterol multiviscosisty, so the size of described particle is along with cholesterol, a kind of hydrophobic grouping, replacement increase and reduce, this is the result who comes to the same thing who obtains with tem analysis.
Experimental example 4: photon correlation spectroscopy (PCS) is measured
Photon correlation spectroscopy (PCS) uses the Zetasizer 3000 (Malvern instrument, Britain) of the He-Ne laser beam with 633nm wavelength to measure, to determine the surface charge of WSC-NP of the present invention.The result is presented in the table 1.
＜table 1 〉
WSC-NPs Proportion of composing (WSC: MPEG: cholesterol) Granular size (nm) CAC (mol) ζ-electromotive force (mV)
Embodiment 1 10∶2∶1.0 100～150 1.0×10 -5 22.4
Embodiment 2 10∶2∶1.5 50～100 0.6×10 -5 18.2
Embodiment 3 10∶2∶1.8 30～70 0.4×10 -5 15.3
With water-soluble chitosan MEPG, a kind of hydrophilic radical, and cholesterol, a kind of hydrophobic grouping is modified, with envelope paclitaxel, a kind of hydrophobicity carcinostatic agent.As shown in Table 1, granular size reduces along with the increase of cholesterol replacement, and critical aggregate concentration (CAC) also reduces.The result shows, works as cholesterol, when a kind of level of hydrophobic grouping increases, even forms nano particle under lower concentration.As the result that Zeta electric potential detects, confirm to have reduced the positive charge of water-soluble chitosan by using MPEG and cholesterol substituted amido.
Experimental example 5: poor formula scanning calorimetry (DSC) is measured
Use TA instrument (E.I.Du Pont Company (Dupont), TA 2000) under nitrogen gas stream, to measure the Tm of taxol and water-soluble chitosan nanopaclitaxel.Sweep velocity is 10 ℃/minute, and scope is defined as 20～350 ℃.The result is presented in Fig. 9～10.Fig. 9～10 are presented at before the envelope paclitaxel (Fig. 9) and (Figure 10) thermoanalytical result afterwards.Usually, respectively at 50 ℃ and 215 ℃ of Tm that detect water-soluble chitosan nano particle and taxol.Detect the Tm (referring to Fig. 9) that does not wherein contain the WSC-NP of taxol at about 50 ℃.Simultaneously, therein in the situation of the water-soluble chitosan nanopaclitaxel of envelope paclitaxel, at about 50 ℃ of Tm that detect WSC-NP, simultaneously at the about 240 ℃ Tm (referring to Figure 10) that observe taxol.The Tm that is included in the taxol in the water-soluble chitosan nanopaclitaxel is higher than the Tm of original paclitaxel (215 ℃), and this may be because taxol is encapsulated in the hydrophobic core of water-soluble chitosan nano particle fully.
Experimental example 6: the HPLC of the taxol of quantitatively sealing measures
Use phenomenex sphereclone 5micro ODS (2) 250 * 4.6mm posts and 75% methyl alcohol as mutually mobile, quantitatively be encapsulated in the taxol in the water-soluble chitosan nanopaclitaxel of preparation in above-described embodiment 4 by HPLC.Simultaneously, at 1.5ml/ minute, and temperature was 50 ℃ with flow set.And use Ultraviolet Detector (226nm) to be used for measuring.Taxol is dissolved in the ethanol, makes 20 μ l and is used for injection.The result is presented in the table 2.
＜table 2 〉
WSC-NPT Proportion of composing (WSC: MPEG: cholesterol) Load content (% by weight) Load efficiency (% by weight)
Embodiment 4 10∶2∶1.0 4.4 13
Embodiment 5 10∶2∶1.5 10 13
Embodiment 6 10∶2∶1.8 18 26
Table 2 is presented at load content and the load efficiency of the taxol of sealing in the water-soluble chitosan nanopaclitaxel for preparing among above-described embodiment 4～embodiment 6.The load content of taxol and load efficiency be along with cholesterol, a kind of hydrophobic grouping, the increase of the replacement in carrying the water-soluble chitosan chain of MPEG and increasing.And the result is by passing through quantitatively reconfirming of HPLC shown in Figure 11～12.
As shown in Figure 11～12, Figure 12 taxol characteristic peak detected at 3.4 minutes, and Figure 11 shows the quantitative taxol that is encapsulated in the water-soluble chitosan nanopaclitaxel.The result shows, the load content of taxol increases along with the increase of cholesterol level, and this is to be caused by the hydrophobic interaction between water-soluble chitosan nano particle and the cholesterol.
Experimental example 7: the antitumor efficacy in the CT-26 of tumor inducing mouse model
With female BALB/c mouse (weight in average: 20g) subcutaneous injection CT-26 mouse source (mourine) tumour cell (5 * 10 under the side of body 4Individual cell/mouse).When tumor growth is big or small to 3mm * 3mm, mouse is divided into two groups: experimental group and control group.Every group has 8 mouse with tumour, and with they marks on their ear, is used for observing.Transplanted rear 15 days, and began to use (intravenous injection) medicine and vehicle.Every kind of trial drug is used with the low dosage of 2mg/kg and the high dosage of 10mg/kg, uses 4 times totally 12 days in per 3 days.Control group is only used vehicle (polyoxyethylenated castor oil: dewatered ethanol, 1: 1 volume ratio).Check the mortality ratio of mouse every day, and use slide calliper rule formula measuring apparatus with the growth of 2 or 3 days time interval measurement tumour.Calculate gross tumor volume by following formula 2.
＜formula 2 〉
Gross tumor volume: mm 3=(long * wide 2)/2
In two weeks after tumour transplatation, medicine or vehicle are expelled in the tail vein of mouse.Figure 13 is the figure that shows the survival rate of the mouse of distinguishing administered with paclitaxel and water-soluble chitosan nanopaclitaxel.Control group mice is got killed sooner than any other group.The mouse of administered with high dose (10mg/kg) is dead more faster within 40 days than other three groups, but too different to each other with 3 groups survival rate of 2mg/kg and 10mg/kg taxol and the processing of 2mg/kg water-soluble chitosan nanopaclitaxel respectively.The experimental group of processing with high dosage (10mg/kg) water-soluble chitosan nanopaclitaxel shows the highest survival rate at whole experimental session in group.
Figure 14 shows that taxol and water-soluble chitosan nanopaclitaxel are to the figure of the antitumour activity of CT-26 tumor model.The 15th day measurement gross tumor volume behind tumor cell transplantation.When two kinds of samples, taxol and water-soluble chitosan nanopaclitaxel, when using with the low dosage of 2mg/kg, not too large difference of gross tumor volume between them.Yet, when described medicine is used with the high dosage of 10mg/kg, compare with other group of only processing with vehicle, 2mg/kg taxol and 2mg/kg water-soluble chitosan nanopaclitaxel respectively, significantly reducing with the gross tumor volume in the mouse of taxol treatment.When taxol was used with 10mg/kg, the growth of tumour was inhibited admirably, but after 30 days, tumour begins again growth.As shown in Figure 14, the antitumour activity of water-soluble chitosan nanopaclitaxel is the highest with the dosage of 10mg/kg.When administration of water soluble chitosan nano taxol, tumor growth stably was inhibited nearly 38 days, but began growth after 40 days.
Figure 15 is the figure that is presented to changes in weight behind the mouse drug administration of transplantation tumor.As described as shown in FIG, the changes in weight of (contrast, 2mg/kg taxol treatment group, 2mg/kg water-soluble chitosan nanopaclitaxel treatment group) is significantly not different between group.Yet the weight of the mouse of processing with the water-soluble chitosan nanopaclitaxel of high dosage greatly reduces.The result shows, reduces the size of tumour with high dosage administration of water soluble chitosan nano taxol, and this causes the inhibition to the weight increase.
The water-soluble chitosan nanopaclitaxel of low dosage can not suppress tumor growth, and does not also affect more survival rate, but the medicine of high dosage improves survival rate, and reduces gross tumor volume, is better than original paclitaxel.In a word, the water-soluble chitosan nanopaclitaxel that is higher than the high dosage of 10mg/kg has fabulous antitumour activity.
Such as hereinbefore elaboration, by utilizing water-soluble chitosan that hydrophilic radical and hydrophobic grouping are incorporated into hyperergy free amino zone, water-soluble chitosan nano particle for delivering carcinostatic agent of the present invention is used for producing successfully with carcinostatic agent, taxol is encapsulated in water-soluble chitosan nanopaclitaxel wherein.Owing to being based on natural polymer " water-soluble chitosan ", it produces, so described water-soluble chitosan nanopaclitaxel has advantages of many polymkeric substance.At length, after lyophilize, it easily redispersion in distilled water, and accumulate in the tumour cell, be better than other carcinostatic agent carrier.In addition, water-soluble chitosan stability in weak acid is better, so that particle target on cancer cells of the present invention rather than normal cell, causes nano particle to accumulate in better in the cancer cell, and this shows that described particle has fabulous antitumour activity.
It should be appreciated by those skilled in the art, in aforementioned description disclosed idea and specific embodiment can be easily with making improvements or the basis of other embodiment of design implementation identical purpose of the present invention.Those skilled in the art be also to be understood that such equivalent embodiments does not deviate from the spirit and scope of the present invention that propose in accompanying claim.

Claims (4)

1. the water-soluble chitosan nano particle (WSC-NP) that is used for delivering carcinostatic agent, water-soluble chitosan wherein: the methoxyl group PEG: the ratio of cholesterol is 10: 2: 1～1.8, described water-soluble chitosan nano particle for delivering carcinostatic agent is by introducing cholesterol chloro-formiate at water-soluble chitosan (WSC) chain and the p-nitrophenyl carbonate of methoxyl group PEG (MPEG p-NP) prepares, and wherein said carcinostatic agent is taxol.
2. the preparation method of the water-soluble chitosan nano particle for delivering carcinostatic agent claimed in claim 1, described method comprises the steps:
1) MPEG p-NP is joined in the water-soluble chitosan to form amido linkage, and eliminate by product, p-nitrophenyl group; With
2) cholesterol chloro-formiate is joined in the above-mentioned reaction soln, between the free amino of described water-soluble chitosan and cholesterol chloro-formiate, to form another amido linkage.
3. the preparation method who is used for as described in claim 2 the water-soluble chitosan nano particle of delivering carcinostatic agent, wherein with described p-nitrophenyl group, step 1) by product, eliminate by dialysing with frozen water, and residual by product is passed through with dehydrated alcohol purifying and fully elimination.
4. be used for as described in claim 2 the preparation method of the water-soluble chitosan nano particle of delivering carcinostatic agent, wherein said preparation process is carried out in PBS solution.
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