CN1055417C - Nucleus-shell structure protein high-molecule microball and its manufacturing method - Google Patents
Nucleus-shell structure protein high-molecule microball and its manufacturing method Download PDFInfo
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- CN1055417C CN1055417C CN95111487A CN95111487A CN1055417C CN 1055417 C CN1055417 C CN 1055417C CN 95111487 A CN95111487 A CN 95111487A CN 95111487 A CN95111487 A CN 95111487A CN 1055417 C CN1055417 C CN 1055417C
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- microsphere
- polylactic acid
- protein
- albumin
- nucleocapsid structure
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Abstract
The present invention relates to a biodegradable macromolecule microsphere with a core-shell structure, which is composed of a core with albumin as a substrate, and a shell with synthesized biodegradable macromolecules as substrates. The preparation of the microsphere comprises the following two steps carried out in sequence: 1, an albumin microsphere with a particle diameter smaller than 3 mum is prepared in a water/oil system; 2, a macromolecular microsphere with the albumin microsphere as a core and synthesized macromolecules as a shell is prepared in the water/oil system, wherein the particle diameter of the macromolecular microsphere is smaller than 5 mum. In step 1, active protein, such as insulin, is added. The active protein accounts for 40 wt% of the microsphere. The present invention can provide a preparation of insulin with long acting time of medicine effect and convenience for taking the medicine.
Description
The present invention relates to a kind of protein drug carrier, can be used for preparing the target controlling and releasing protein drug, belong to biological medical polymer material and target controlling and releasing administration field.
At present, the carrier as the microsphere sustained-release drug system mainly contains protein microsphere and synthetic high polymer microsphere two big classes.This two classes microsphere respectively has its pluses and minuses.The substrate of protein microsphere is generally albumin and collagen protein.Because proteic hydrophilic, thereby more easily in conjunction with some hydrophilic medicaments.The method of its medicine carrying can have multiple.The medicine amount of carrying of bibliographical information albumin microsphere can reach 40% (US Patent, 4,671,954,1987).Yet protein microsphere but has the too fast shortcoming of unstable in vivo degradation speed.The substrate of synthetic high polymer microsphere is generally the macromolecule of polylactic acid series.Can degrade in vivo because of this family macromolecule, toxic and side effects is little, and is stable in vivo, is difficult for by the enzyme effect, and gastrointestinal systems is insensitive, but oral administration.But this kind material mostly is lipophile greatly, so it is low that some hydrophilic medicaments are carried difficulty (most of cancer therapy drugs are hydrophilic) drug loading, particularly to the more apparent difficulty of carrying of some hydrophilic proteins.Polylactic acid series microsphere to the albuminous amount of carrying less than 5% (Vaccine, 1991,9 (10), 768-771), proteic the carrying of external membrane is about 0.1% (Vaccine, 1994,12 (5), 138), be the 2IU/100mg microsphere to the amount of carrying of insulin, this must influence dosage and curative effect.
The objective of the invention is to improve polymer microsphere carries and controlled-release effect active medicine is proteic.The advantages of protein microsphere and synthetic high polymer microsphere is got up, prepare a kind of novel nucleocapsid structure and carry protein microsphere.The nuclear matrix of this microsphere is an albumen, and conchiolin is the polylactic acid macromolecule, and up to 80%, its effective active albumen amount of carrying reaches 40% of microsphere gross weight to its proteic weight content in nucleocapsid structure.The synthetic high polymer conchiolin of this microsphere becomes the protective layer of pyrenoids substrate; albumen substrate and activated protein stability have in vivo wherein been strengthened; no matter per os or injection all can reach the purpose of target administration; be a kind of ideal target controlling and releasing preparation, be useful for the target controlling and releasing administration of the protein drug of insulin type.
Technology contents of the present invention: 1. adopting macromolecule organic solution is disperse medium, and five carbochain hydrocarbon of band dialdehyde base are the albumin microsphere that the emulsion chemistry cross-linking method preparation of cross-linking agent contains the activated protein medicine; 2. adopt poly--D, L-lactic acid is conchiolin, be dissolved in organic solvent, 1. the protein microsphere that obtains is dispersed in poly--D, in the L-lactic acid organic solution, will contains the poly--D of protein microsphere, the L-lactic acid solution dropwise joins in the water disperse medium, continuous stirring 12-14 hour, slow solvent evaporated, just getting nuclear matrix with distilled water eccysis disperse medium is that albumin, conchiolin are the nucleocapsid structure microsphere of polylactic acid.
During for the nucleocapsid structure polymer microsphere of nuclear, should be anhydrous state at the preparation protein microsphere,, increase encapsulation ratio to improve the affinity between protein microsphere and the polylactic acid as the protein microsphere of its nuclear.Before the parcel protein microsphere, protein microsphere should be immersed in and be no less than 12 hours in the polylactic acid solution.
Containing the poly--D of protein microsphere, the L-lactic acid solution joins before the water disperse medium, needs to disperse this mixed system with the ultrasonic dispersing instrument.
The nuclear matrix of microsphere of the present invention is selected for use has better water miscible human serum albumin, bovine serum albumin, ovalbumin etc., accounts for the 50-90% of Tot Prot, and the activated protein that it wrapped up accounts for the 10-50% of Tot Prot.
In the preparation process of protein microsphere of the present invention, protein concentration is 10-30% in the protein solution.
Protein microsphere preparation process among the present invention: organic decentralized photo is toluene/chloroform (T/C) solution of polymethyl methacrylate (PMMA) or poly(propylene oxide)-oxirane (PEO-PPO) copolymer.The molecular weight of PMMA is 10
4-10
5Scope, PEO-PPO is P
55E
33P
56Type.The concentration of macromolecule in organic solution is 20-25%.
The preparation process of protein microsphere of the present invention comprises the jitter time of protein solution in organic solution and is no less than 15 minutes, and the response time after dialdehyde five carbochain hydrocarbon add is no less than 4 hours, adds the aldehyde radical of excess ethyl alcohol amine with capping subsequently.Response time after ethanolamine adds is no less than 1 hour.
The dialdehyde five carbochain hydrocarbon that use among the present invention are glutaraldehyde, use its aqueous solution commodity of 25%.Glutaraldehyde water solution and proteic proportioning are 1-2ml/300mg albumen.
Albumen substrate nuclear of the present invention, its particle diameter is no more than 3 μ m.
Conchiolin as microsphere of the present invention is poly--D, L-lactic acid (PLA) series, and such material comprises poly--D, L-lactic acid-ethanol (PLGA), poly--D, L-lactic acid-Polyethylene Glycol (PLEG), poly--D, L-lactic acid-6-caprolactone copolymers such as (PLACL) and poly--D, L-lactic acid homopolymer.The molecular weight of polymer is 10
4-10
5Scope.
As the preparation technology of microsphere of the present invention, the weight ratio of albumin and polylactic acid is 20-80: 20-80.
Preparing required water disperse medium as microsphere of the present invention is polyvinyl alcohol (PVA) aqueous solution, and its concentration is between 4-10%.
Adopt magnetic stirrer as function input equipment of the present invention, the mixing speed scope is 600-1000 rev/min.
The nucleocapsid structure microspherulite diameter that the present invention makes is controlled at the 0-10 mu m range.
Adopt the present invention can make polymer microsphere with greater activity protein content.This microsphere is typical nucleocapsid structure, bio-compatible, biodegradable, realizes target administration to organism reticuloendothelial system tissue by oral or injection.Zoopery shows that the insulin active in the HAM (6IU/100mg) of the prepared insulin-containing of employing chemical crosslink technique is not affected.After 20 minutes, White Rabbit blood glucose is reduced to 2mg/ml (normal value is 5mg/ml) to White Rabbit administration (80mg microsphere/kg body weight), and after 5 hours, blood glucose is reduced to about 1mg/ml.
Good effect of the present invention is: because the present invention has adopted the nucleocapsid structure mode to construct microsphere, guaranteed the activated protein content in the carrier, overcome single poly-lactic acid material and wrapped up proteic low encapsulation ratio, strengthened the practicality of protein microsphere.Because the particle diameter of protein microsphere distributes within the specific limits, the thickness of the shell of its nucleocapsid structure also is in certain distribution, and the erosion of microsphere is broken in the different time and taken place, thereby produces the dispose procedure at intermittence gradually, reaches the purpose of slow release.So microsphere of the present invention is applicable to that preparation insulin controlled release carrier is because treating diabetes.The administration cycle of multiple administering mode and prolongation can alleviate patient's misery.
Be embodiments of the invention below:
Embodiment 1: the preparation of albumen substrate nuclear.Accurately measure insulin solutions (40IU/ml) 2ml.Accurately take by weighing human serum albumin 300mg and join in the insulin solutions, treat its dissolving.Toluene/chloroformic solution the 15ml of measuring concentration and be 20% PMMA stirs with the rotating speed of magnetic stirrer with 250 rev/mins in three-neck flask.Under the stirring condition, dropwise add consoluet albumin-insulin solution.Measure 25% glutaraldehyde water solution 1ml, add 1ml toluene again, glutaraldehyde/toluene mixture liquid was vibrated on vortex mixer 5 minutes, leave standstill afterwards, after treating layering, with suction pipe draw the upper strata saturated the toluene solution of glutaraldehyde join in the three-neck flask, at this moment, the jitter time of protein solution in organic solution is no less than 15 minutes.Add after the glutaraldehyde, with ground bottleneck jam-pack, mixing speed increases to 400 rev/mins; Cross-linking reaction begins, and continues 5 hours, adds the 1ml ethanolamine then, seals unreacted free aldehyde, after 1 hour, and stopped reaction.Product is used toluene, acetone, distilled water wash successively.Behind the wash clean, use the acetone dehydrate, the 292mg that weighs, productive rate 95%, optical microscope observe particle diameter down less than 3 μ m.
Embodiment 2: the parcel protein microsphere becomes the nucleocapsid structure microsphere.Accurately weighing PLEG500mg is dissolved in the 10ml dichloromethane, treats that it dissolves fully, and exsiccant protein microsphere 292mg is joined in the organic solution, in ultrasonic oscillator, disperses vibration, takes out to keep spending the night.Measure 6% PVA aqueous solution 15ml, in the wide mouthed bottle of the 100ml that packs into, stir with magnetic stirrer.Under stirring condition, the PLEG solution that is dispersed with protein microsphere is dropwise joined in the water disperse medium, rotating speed increases to 600 rev/mins, keeps this speed 30 minutes, and speed is reduced to 250 rev/mins then, stirs 12 hours, treats that solvent evaporation finishes, and uses distilled water wash.Washing process carries out in centrifuge, 10000 rev/mins of centrifugal speeds, and centrifugation time 5 minutes repeats 8 times.Collecting precipitation, lyophilizing is weighed, 650mg, productive rate 82.3%, microscopically are observed particle diameter less than 5 μ m.
Claims (5)
1. biodegradable, a biocompatible albumen polymer microsphere is characterized in that its structure is a nuclear-shell mould, and particle diameter is below 5 μ m, and nuclear matrix is an albumin, and conchiolin is polylactic acid-based macromolecule, and nuclear matrix carries effective active albumen.
2. according to the microsphere of claim 1, it is characterized in that adopting human serum albumin, bovine serum albumin Pseudobulbus Bletillae (Rhizoma Bletillae) ovalbumin etc. as the albumin of nuclear matrix, the weight content in nucleocapsid structure is 20-80%.
3. according to the microsphere of claim 1, it is characterized in that adopting polylactic acid (PLA) as the polylactic acid-based macromolecule of conchiolin, gather (lactic acid-ethanol) (PLGA), polylactic acid-polyglycol (PLEG) and poly-(lactic acid-6-caprolactone) be polylactic acid-based homopolymerization or copolymer (PLACL), and the content in nucleocapsid structure is 20-80%.
4. according to the microsphere of claim 1,2 or 3, it is characterized in that the bioactive albumen of tool, the weight content that comprises insulin or vaccine antigen is the 0-40% of whole nucleocapsid structure total amount.
5. method for preparing microsphere according to claim 1 may further comprise the steps successively:
1. adopting macromolecular solution is that disperse medium and glutaraldehyde are the protein microsphere that the chemical cross-linking agent preparation contains effective activated protein or medicine.
2. serve as that to adopt the emulsion solvent evaporation technique to prepare shell in the water macromolecular solution be polylactic acid-based high molecular nucleocapsid structure polymer microsphere to nuclear with the protein microsphere that makes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95111487A CN1055417C (en) | 1995-09-05 | 1995-09-05 | Nucleus-shell structure protein high-molecule microball and its manufacturing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN95111487A CN1055417C (en) | 1995-09-05 | 1995-09-05 | Nucleus-shell structure protein high-molecule microball and its manufacturing method |
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| Publication Number | Publication Date |
|---|---|
| CN1144714A CN1144714A (en) | 1997-03-12 |
| CN1055417C true CN1055417C (en) | 2000-08-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN95111487A Expired - Fee Related CN1055417C (en) | 1995-09-05 | 1995-09-05 | Nucleus-shell structure protein high-molecule microball and its manufacturing method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4671954A (en) * | 1983-12-13 | 1987-06-09 | University Of Florida | Microspheres for incorporation of therapeutic substances and methods of preparation thereof |
| EP0377477A1 (en) * | 1989-01-04 | 1990-07-11 | Brocades Pharma B.V. | Process for microencapsulation |
| EP0556917A1 (en) * | 1992-02-18 | 1993-08-25 | Akzo Nobel N.V. | A process for the preparation of biologically active material-containing polymeric microcapsules |
-
1995
- 1995-09-05 CN CN95111487A patent/CN1055417C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4671954A (en) * | 1983-12-13 | 1987-06-09 | University Of Florida | Microspheres for incorporation of therapeutic substances and methods of preparation thereof |
| EP0377477A1 (en) * | 1989-01-04 | 1990-07-11 | Brocades Pharma B.V. | Process for microencapsulation |
| EP0556917A1 (en) * | 1992-02-18 | 1993-08-25 | Akzo Nobel N.V. | A process for the preparation of biologically active material-containing polymeric microcapsules |
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| Publication number | Publication date |
|---|---|
| CN1144714A (en) | 1997-03-12 |
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