CN105326810A - Folic acid modified chitosan micro-capsule with reducing responsiveness and preparation method thereof - Google Patents

Abstract

The invention relates to a reduction-responsive chitosan microcapsule modified by folic acid and a preparation method thereof. The capsule wall is modified by folic acid and has a reduction-responsive disulfide bond structure. The modified chitosan cross-linked membrane is the capsule wall, and the oil phase loaded with hydrophobic drugs is the core material. Firstly, chitosan was modified with folic acid and thiol-containing compounds or biomolecules, and then the oil phase loaded with hydrophobic drugs and chitosan aqueous solution modified with folic acid and sulfhydryl-containing compounds or biomolecules were subjected to ultrasonic radiation to obtain Chitosan drug-loaded microcapsules with folic acid and disulfide bond structure in the capsule wall. The preparation method is fast and simple, efficient and environmentally friendly, with a wide range of sources of materials, the product is pure and non-toxic, and has a large drug loading capacity. It can be widely used in the coating of hydrophobic drugs, and the targeted delivery of drugs can be realized by using the targeting of folic acid and the use of disulfide bonds. The reduction responsiveness can realize the controlled release of drugs, which has a good application prospect.

Description

A kind of folic acid-modified chitosan microcapsule with reduction responsiveness and preparation method thereof

technical field

The invention relates to a biomedical material, in particular to a folic acid-modified microcapsule with reduction responsiveness and a preparation method.

Background technique

Microcapsules are micro-containers or packages with a specific geometric structure composed of capsule-forming substances. Due to their special structure, variability in performance and wide range of applications, they are widely used in the fields of agriculture, food and biomedicine. Significant application. There are various materials for preparing microcapsules, mainly natural polymer materials (proteins, lipids, etc.) or synthetic polymer materials (PMMA, polylactic acid, etc.), or inorganic compounds (SiO ₂ , CaCO ₃ , etc.). Among them, natural polymer materials have important application value in the preparation of microcapsules due to their good biocompatibility, non-toxicity and easy degradation. There are many methods for preparing microcapsules. According to the mechanism of encapsulation, they can be roughly divided into three categories: physical methods (spray drying method, air suspension method, vacuum evaporation deposition method, etc.), physical and chemical methods (water phase separation method, capsule core exchange method, etc.) method, extrusion method, etc.) and chemical methods (in-situ polymerization method, interfacial polymerization method, molecular encapsulation method, etc.). In recent years, some new microencapsulation techniques have emerged, such as surface grafting, template polymerization, and layer-by-layer self-assembly. These technologies are all microencapsulation methods based on chemical reactions, which have the characteristics of controllable structure, adjustable performance, and easy endowment of various characteristic functions. However, the operation process of these methods is cumbersome, time-consuming, and impurities are easily introduced, which limits their practical applicability.

The method of using ultrasonic radiation to prepare protein microcapsules was first invented by American scientists Suslick et al. in the early 1990s. They used high-intensity ultrasonic radiation to encapsulate gas or water-insoluble liquids with proteins to prepare protein microcapsules. . Because this method is simple to operate, efficient, fast and environmentally friendly, and the obtained microcapsules have good biocompatibility, it has potential application value in the fields of targeted drug technology and the like. Patent CN101953817B uses sonochemical method to synthesize a microcapsule carrying oil-soluble substances, and the capsule wall material is protein or polymer. The preparation of folic acid-modified microcapsules has been reported in recent years, and has potential applications in the fields of drug targeting technology, but there is no report on the preparation of reduction-responsive folic acid-modified chitosan microcapsules by ultrasonic method.

Contents of the invention

The object of the present invention is to aim at the deficiencies of the above-mentioned prior art, to provide a kind of highly efficient and concise preparation of folic acid-modified chitosan microcapsules with reduction responsiveness;

The purpose of the present invention is to provide a preparation method of folic acid-modified chitosan microcapsules with reduction responsiveness.

Principle of the present invention: Utilize the catalysis of high-intensity ultrasonic waves at the oil/water interface to promote the cross-linking of the modified sulfhydryl groups on chitosan, forming a cross-link with the coexistence of a reduction-responsive disulfide bond structure and a folic acid modified structure membrane, and embedding the oil phase loaded with hydrophobic drugs to form a drug-loaded microcapsule with the cross-linked membrane of chitosan modified by sulfhydryl groups as the capsule wall and the oil phase loaded with hydrophobic drugs as the core material. Folate-modified chitosan microcapsules with reduction responsiveness, the functionalized chitosan cross-linked membrane as the capsule wall, and the oil phase loaded with hydrophobic drugs as the capsule core.

The purpose of the present invention is achieved by the following technical solutions

A folic acid-modified chitosan microcapsule with reduction responsiveness is formed by ultrasonic irradiation of the oil phase loaded with hydrophobic drugs and chitosan aqueous solution modified with folic acid and thiol-containing compounds or biomolecules. The chitosan cross-linked film modified by disulfide bond cross-linked folic acid is the capsule wall, and the oil phase loaded with hydrophobic medicine is the drug-loading microcapsule as the core material, and the size of the microcapsule is 0.2-50 μm.

The mercapto-containing compound or biomolecule is mercapto-alkanoic acid [HS-(CH ₂ ) _n -COOH, number of alkyl groups n≥1], 2-amino-5-mercaptobenzoic acid, 5-mercapto-1H-tetra azole-1-acetic acid, 2-mercaptonicotinic acid, mercaptosuccinic acid, 5-mercaptotetrazolo-1-acetic acid, cysteine and its derivatives or glutathione and its derivatives.

The hydrophobic drug is one or more of paclitaxel, lomustine, ginsenoside, camptothecin or silymarin.

The oil phase is various animal oils, vegetable oils, microbial oils, mineral oils, silicone oils, or other non-toxic liquid organic substances incompatible with water that are available in biomedicine.

A preparation method of folic acid-modified chitosan with reduction-responsive microcapsules, comprising the following steps:

a, dissolving chitosan in acetic acid and sodium acetate buffer solution of pH=5.0 to prepare a certain concentration of chitosan aqueous solution;

b. Compounds or biomolecules containing mercapto groups and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are used in a molar ratio of 1:1:1 Dissolve, prepare an aqueous solution with a concentration of 0.1-1.0 mg/ml, and activate for 20 minutes;

c. Under nitrogen protection, mix the above two solutions with a volume ratio of 5:1-1:5, stir and react for 12 hours, and repeatedly centrifuge and wash to remove unreacted thiol-containing compounds or biomolecules to obtain thiol-containing modified chitosan aqueous solution;

d. Dissolve folic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in a molar ratio of 1:2:2, and the preparation concentration is 0.1 -1.0mg/ml aqueous solution, protected from light for 1h;

e. Mixing an aqueous chitosan solution with a pH=5.0 and a folic acid activation solution at a volume ratio of 5:1-1:5 in the dark, stirring and reacting for 12 hours, to obtain an aqueous chitosan solution containing folic acid modification;

f, according to the ratio of 1:1-20:1, mix the chitosan aqueous solution containing thiol modification and the chitosan aqueous solution containing folic acid modification;

g, measure the above-mentioned mixed chitosan aqueous solution by a certain volume ratio and place the oil phase containing the hydrophobic drug together in an ice-water bath and mix uniformly;

h. Then place the ultrasonic probe at the oil-water two-phase interface, and act for a period of time under a certain ultrasonic power;

i. After adjusting the pH of the reaction solution to about 9-10, carry out centrifugation and washing until the product is neutral, and obtain a chitosan cross-linked membrane with a disulfide bond structure modified by folic acid as the capsule wall, and use oil containing hydrophobic drugs Drug-loaded microcapsules with phase as core material.

The ultrasonic intensity is 100-900W, and the ultrasonic time is 0.5-30min.

The concentration of the chitosan aqueous solution is 0.1-3.0mg/ml.

The volume ratio of the mixed chitosan aqueous solution to the oil phase containing hydrophobic medicine is 1:1-20:1.

Beneficial effects: the method of the present invention can directly load folic acid on the wall of the microcapsule, combine folic acid targeting, disulfide bond reduction responsiveness and chitosan biocompatibility, and realize the targeting of hydrophobic drugs Transmission and controllable release; the product is pure and non-toxic, with a large drug loading capacity, which can be widely used in the coating of hydrophobic drugs, using the targeting of folic acid to realize the targeted delivery of drugs and the reduction responsiveness of disulfide bonds to realize drug delivery. The controllable release of the preparation method is simple, efficient and quick, and it is not easy to introduce impurities.

detailed description:

Below in conjunction with embodiment the present invention is described in further detail:

A folic acid-modified chitosan microcapsule with reduction responsiveness is formed by ultrasonic irradiation of the oil phase loaded with hydrophobic drugs and chitosan aqueous solution modified with folic acid and thiol-containing compounds or biomolecules. The chitosan cross-linked film modified by disulfide bond cross-linked folic acid is the capsule wall, and the oil phase loaded with hydrophobic medicine is the drug-loading microcapsule as the core material, and the size of the microcapsule is 0.2-50 μm.

The mercapto-containing compound or biomolecule is mercapto-alkanoic acid [HS-(CH ₂ ) _n -COOH, number of alkyl groups n≥1], 2-amino-5-mercaptobenzoic acid, 5-mercapto-1H-tetra azole-1-acetic acid, 2-mercaptonicotinic acid, mercaptosuccinic acid, 5-mercaptotetrazolo-1-acetic acid, cysteine and its derivatives or glutathione and its derivatives.

The hydrophobic drug is one or more of paclitaxel, lomustine, ginsenoside, camptothecin or silymarin.

The oil phase is various animal oils, vegetable oils, microbial oils, mineral oils, silicone oils, or other non-toxic liquid organic substances incompatible with water that are available in biomedicine.

A preparation method of folic acid-modified chitosan with reduction-responsive microcapsules, comprising the following steps:

a, dissolving chitosan in acetic acid and sodium acetate buffer solution of pH=5.0 to prepare a certain concentration of chitosan aqueous solution;

b. Compounds or biomolecules containing mercapto groups and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are used in a molar ratio of 1:1:1 Dissolve, prepare an aqueous solution with a concentration of 0.1-1.0 mg/ml, and activate for 20 minutes;

c. Under nitrogen protection, mix the above two solutions with a volume ratio of 5:1-1:5, stir and react for 12 hours, and repeatedly centrifuge and wash to remove unreacted thiol-containing compounds or biomolecules to obtain thiol-containing modified chitosan aqueous solution;

d. Dissolve folic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in a molar ratio of 1:2:2, and the preparation concentration is 0.1 -1.0mg/ml aqueous solution, protected from light for 1h;

e. Mixing an aqueous chitosan solution with a pH=5.0 and a folic acid activation solution at a volume ratio of 5:1-1:5 in the dark, stirring and reacting for 12 hours, to obtain an aqueous chitosan solution containing folic acid modification;

f, according to the ratio of 1:1-20:1, mix the chitosan aqueous solution containing thiol modification and the chitosan aqueous solution containing folic acid modification;

g, measure the above-mentioned mixed chitosan aqueous solution by a certain volume ratio and place the oil phase containing the hydrophobic drug together in an ice-water bath and mix uniformly;

h. Then place the ultrasonic probe at the oil-water two-phase interface, and act for a period of time under a certain ultrasonic power;

i. After adjusting the pH of the reaction solution to about 9-10, carry out centrifugation and washing until the product is neutral, and obtain a chitosan cross-linked membrane with a disulfide bond structure modified by folic acid as the capsule wall, and use oil containing hydrophobic drugs Drug-loaded microcapsules with phase as core material.

The ultrasonic intensity is 100-900W, and the ultrasonic time is 0.5-30min.

The concentration of the chitosan aqueous solution is 0.1-3.0mg/ml.

The volume ratio of the mixed chitosan aqueous solution to the oil phase containing hydrophobic medicine is 1:1-20:1.

Example 1

In acetic acid and sodium acetate buffer solution of pH=5.0, chitosan was dissolved to prepare 0.1 mg/ml chitosan aqueous solution; cysteine hydrochloride and 1-(3-dimethylaminopropyl)-3 -Ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:1:1, and an aqueous solution with a concentration of 0.1 mg/ml was prepared, and activated for 20 minutes; under nitrogen protection, the volume ratio was 1 : 1 Mix the above two solutions and stir for 12 hours, centrifuge and wash repeatedly to remove unreacted cysteine hydrochloride to obtain an aqueous solution of chitosan containing thiol modification; mix folic acid and 1-(3-dimethylaminopropyl )-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:2:2, and an aqueous solution with a concentration of 0.1/ml was prepared, and activated for 1 hour in the dark; Mix the chitosan aqueous solution and the folic acid activation solution with a pH=5.0 at a ratio of 5:1, stir and react for 12 hours to obtain an aqueous solution of chitosan modified with folic acid; mix the aqueous solution of chitosan modified with a sulfhydryl group with Folic acid-modified chitosan aqueous solution; measure the above-mentioned mixed chitosan aqueous solution and soybean oil containing hydrophobic drug paclitaxel at a volume ratio of 5:1 and place them in an ice-water bath and mix them evenly; then place the ultrasonic probe at the oil-water two-phase interface , under the ultrasonic power of 100W for 30min; adjust the pH of the reaction solution to about 9-10, then carry out centrifugal washing until the product is neutral, and obtain a chitosan cross-linked membrane with a disulfide bond structure modified by folic acid as the capsule wall , drug-loaded microcapsules with the oil phase containing hydrophobic drugs as the core material.

Example 2

Dissolve chitosan in acetic acid and sodium acetate buffer solution with pH=5.0 to prepare 0.5 mg/ml chitosan aqueous solution; mercaptoundecanoic acid [HS-(CH ₂ ) _n -COOH, alkyl number n=10 ] and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are dissolved in a molar ratio of 1:1:1, and the preparation concentration is 0.1mg/ml Under the protection of nitrogen, the above two solutions were mixed and stirred for 12 hours with a volume ratio of 1:5, and the unreacted mercaptoundecanoic acid was removed by repeated centrifugal washing to obtain an aqueous solution of chitosan modified with a thiol group; Dissolve folic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide at a molar ratio of 1:2:2, and the prepared concentration is 1.0mg/ ml of aqueous solution, protected from light for 1h; under light-shielding, mix chitosan aqueous solution and folic acid activation solution with pH=5.0 in a volume ratio of 2:1, stir and react for 12h, and obtain the chitosan aqueous solution containing folic acid modification; according to 2: Mix the chitosan aqueous solution containing sulfhydryl group modification and the chitosan aqueous solution containing folic acid modification in a ratio of 1; measure the above-mentioned mixed chitosan aqueous solution and the hydroxyl silicone oil containing hydrophobic drug lomustine at a volume ratio of 1:2 Mix evenly in an ice-water bath; then place the ultrasonic probe at the oil-water two-phase interface, and act for 8 minutes under the ultrasonic power of 300W; adjust the pH of the reaction solution to about 9-10, and then carry out centrifugal washing until the product is neutral, and the product containing The folic acid-modified chitosan cross-linked membrane with disulfide bond structure is the capsule wall, and the oil phase containing hydrophobic drugs is the drug-loading microcapsule as the core material.

Example 3

Chitosan was dissolved in acetic acid and sodium acetate buffer solution of pH=5.0 to prepare 0.3 mg/ml chitosan aqueous solution; mercaptopropionic acid [HS-(CH ₂ ) _n -COOH, alkyl number n=2] and 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:1:1 to prepare an aqueous solution with a concentration of 1.0mg/ml , activated for 20min; under the protection of nitrogen, the above two solutions were mixed and stirred for 12 hours with a volume ratio of 5:1, and the unreacted mercaptopropionic acid was removed by repeated centrifugation and washing to obtain an aqueous solution of chitosan modified with a thiol group; folic acid and 1 -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:2:2 to prepare an aqueous solution with a concentration of 0.5mg/ml, Protect from light and activate for 1 hour; mix chitosan aqueous solution with pH=5.0 and folic acid activation solution at a volume ratio of 2:1 in the dark, stir and react for 12 hours to obtain a chitosan aqueous solution modified with folic acid; mix according to the ratio of 5:1 An aqueous solution of chitosan containing thiol modification and an aqueous solution of chitosan containing folic acid modification; measure the above-mentioned mixed aqueous solution of chitosan and peanut oil containing hydrophobic drug ginsenoside at a volume ratio of 1:5 and place them in an ice-water bath and mix uniformly; Then place the ultrasonic probe at the oil-water two-phase interface, and act for 6 minutes under the ultrasonic power of 500W; adjust the pH of the reaction solution to about 9-10, and then carry out centrifugal washing until the product is neutral, and obtain folic acid-modified product with disulfide bond The chitosan cross-linked film of the structure is the capsule wall, and the oil phase containing the hydrophobic drug is the drug-loading microcapsule as the core material.

Example 4

Chitosan was dissolved in acetic acid and sodium acetate buffer solution with pH=5.0 to prepare a 1.0 mg/ml chitosan aqueous solution; mercaptohexadecanoic acid [HS-(CH ₂ ) _n -COOH, alkyl number n=15 ] and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are dissolved in a molar ratio of 1:1:1, and the preparation concentration is 0.2mg/ml Under the protection of nitrogen, the above two solutions were mixed and stirred for 12 hours with a volume ratio of 1:5, and the unreacted mercaptohexadecanoic acid was removed by repeated centrifugal washing to obtain an aqueous solution of chitosan containing thiol modification; Dissolve folic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide at a molar ratio of 1:2:2, and the prepared concentration is 0.5mg/ ml of aqueous solution, protected from light for 1h; under light-shielding, mix chitosan aqueous solution and folic acid activation solution of pH=5.0 with a volume ratio of 2:1 in dark place, stir and react for 12h, and obtain the chitosan aqueous solution containing folic acid modification; according to 10: Mix the chitosan aqueous solution containing thiol modification and the chitosan aqueous solution containing folic acid modification in a ratio of 1; measure the above-mentioned mixed chitosan aqueous solution and olive oil containing hydrophobic drug camptothecin at a volume ratio of 10:1 Mix evenly in an ice-water bath; then place the ultrasonic probe at the oil-water two-phase interface, and act for 15 minutes under the ultrasonic power of 300W; adjust the pH of the reaction solution to about 9-10, and then carry out centrifugal washing until the product is neutral to obtain folic acid-containing The modified chitosan cross-linked film with disulfide bond structure is the capsule wall, and the oil phase containing hydrophobic drugs is the drug-loading microcapsule as the core material.

Example 5

Chitosan was dissolved in acetic acid and sodium acetate buffer solution of pH=5.0 to prepare 3.0mg/ml chitosan aqueous solution; thioglycolic acid [HS-(CH ₂ ) _n -COOH, alkyl number n=1] and -(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:1:1 to prepare an aqueous solution with a concentration of 1.0 mg/ml, Activation for 20min; under the protection of nitrogen, the above two solutions were mixed and stirred for 12 hours with a volume ratio of 1:3, and the unreacted thioglycolic acid was removed by repeated centrifugation and washing to obtain an aqueous solution of chitosan modified with a thiol group; folic acid and 1-( 3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:2:2, and an aqueous solution with a concentration of 1.0mg/ml was prepared, protected from light Activate for 1 hour; mix chitosan aqueous solution with pH = 5.0 and folic acid activation solution at a volume ratio of 1:3 in the dark, stir and react for 12 hours to obtain an aqueous solution of chitosan modified with folic acid; mix thiol-containing Modified chitosan aqueous solution and modified chitosan aqueous solution containing folic acid; Measure the above-mentioned mixed chitosan aqueous solution and the seaweed oil containing hydrophobic drug silymarin in an ice-water bath and mix uniformly in a volume ratio of 20:1; Place the ultrasonic probe at the oil-water two-phase interface and act for 5 minutes under the ultrasonic power of 600W; adjust the pH of the reaction solution to about 9-10, then carry out centrifugal washing until the product is neutral, and obtain folic acid-modified product with disulfide bond structure The chitosan cross-linked film is the capsule wall, and the drug-loading microcapsule takes the oil phase containing hydrophobic drugs as the core material.

Example 6

In acetic acid and sodium acetate buffer solution of pH=5.0, chitosan is dissolved to prepare 0.3 mg/ml chitosan aqueous solution; 2-amino-5-mercaptobenzoic acid and 1-(3-dimethylaminopropyl) -3-Ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:1:1, and an aqueous solution with a concentration of 0.5 mg/ml was prepared and activated for 20 minutes; Mix the above two solutions at 5:3 and stir for 12 hours, then centrifuge and wash repeatedly to remove unreacted 2-amino-5-mercaptobenzoic acid to obtain a thiol-modified chitosan aqueous solution; mix folic acid and 1-(3-di Dissolve methylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at a molar ratio of 1:2:2, prepare an aqueous solution with a concentration of 0.6 mg/ml, and activate it in the dark for 1 hour; Mix chitosan aqueous solution with pH=5.0 and folic acid activation solution at a volume ratio of 2:1 in the dark, stir and react for 12 hours to obtain an aqueous solution of chitosan modified with folic acid; Polysaccharide aqueous solution and chitosan aqueous solution modified with folic acid; measure the above-mentioned mixed chitosan aqueous solution and hydroxyl silicone oil containing hydrophobic drug paclitaxel in an ice-water bath and mix uniformly in a volume ratio of 20:1; then place the ultrasonic probe in At the oil-water two-phase interface, under the ultrasonic power of 900W, act for 0.5min; adjust the pH of the reaction solution to about 9-10, and then carry out centrifugal washing until the product is neutral, and obtain folic acid-modified chitosan with a disulfide bond structure The sugar cross-linked film is the capsule wall, and the oil phase containing hydrophobic drugs is the drug-loaded microcapsule as the core material.

Example 7

In acetic acid and sodium acetate buffer solution of pH=5.0, chitosan is dissolved to prepare 0.3 mg/ml chitosan aqueous solution; 5-mercapto-1H-tetrazolium-1-acetic acid and 1-(3-dimethyl Aminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:1:1, and an aqueous solution with a concentration of 0.1-1.0 mg/ml was prepared and activated for 20 minutes; Under the protection of nitrogen, the above two solutions were mixed with a volume ratio of 3:1, stirred and reacted for 12 hours, and the unreacted 5-mercapto-1H-tetrazolium-1-acetic acid was removed by repeated centrifugal washing to obtain an aqueous solution of chitosan modified with a mercapto group. ; Dissolve folic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in a molar ratio of 1:2:2, and the prepared concentration is 0.3mg /ml of the aqueous solution, protected from light for 1h; under the dark, the chitosan aqueous solution and the folic acid activation solution were mixed with a volume ratio of 1:1, stirred and reacted for 12 hours, and the chitosan aqueous solution containing folic acid modification was obtained; according to 1 Mix the chitosan aqueous solution containing thiol modification and the chitosan aqueous solution containing folic acid modification in a ratio of 1; measure the above-mentioned mixed chitosan aqueous solution and soybean oil containing hydrophobic drug paclitaxel in an ice-water bath in a volume ratio of 5:1 Then, place the ultrasonic probe at the oil-water two-phase interface, and act for 6 minutes under the ultrasonic power of 500W; adjust the pH of the reaction solution to about 9-10, and then carry out centrifugal washing until the product is neutral, and obtain folic acid-modified The chitosan cross-linked membrane with disulfide bond structure is the capsule wall, and the oil phase containing hydrophobic drugs is the drug-loading microcapsule as the core material.

Example 8

In acetic acid and sodium acetate buffer solution of pH=5.0, chitosan was dissolved to prepare 0.5 mg/ml chitosan aqueous solution; 2-mercaptonicotinic acid and 1-(3-dimethylaminopropyl)-3-ethane Carbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:1:1, and an aqueous solution with a concentration of 1.0 mg/ml was prepared, and activated for 20 minutes; under nitrogen protection, the volume ratio was 2:1 The above two solutions were mixed and stirred for 12 hours, and the unreacted 2-mercaptonicotinic acid was removed by repeated centrifugation and washing to obtain an aqueous solution of chitosan modified with a thiol group; folic acid and 1-(3-dimethylaminopropyl)-3- Dissolve ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at a molar ratio of 1:2:2, prepare an aqueous solution with a concentration of 0.3 mg/ml, and activate for 1 hour in the dark; : 1 mixed chitosan aqueous solution and folic acid activation solution with pH=5.0, stirred and reacted for 12h, obtained chitosan aqueous solution containing folic acid modification; mixed chitosan aqueous solution containing sulfhydryl group modification and folic acid modified Chitosan aqueous solution; measure the above-mentioned mixed chitosan aqueous solution and soybean oil containing hydrophobic drug paclitaxel in an ice-water bath according to the volume ratio of 5:1 and mix evenly; then place the ultrasonic probe at the oil-water two-phase interface, Under the ultrasonic power of 6min; adjust the pH of the reaction solution to about 9-10, then carry out centrifugation and washing until the product is neutral, and obtain a chitosan cross-linked membrane with a disulfide bond structure modified by folic acid as the capsule wall, to contain The oil phase of the hydrophobic drug is the drug-loaded microcapsule of the core material.

Example 9

Dissolve chitosan in acetic acid and sodium acetate buffer solution with pH=5.0 to prepare 0.1 mg/ml chitosan aqueous solution; mix mercaptosuccinic acid and 1-(3-dimethylaminopropyl)-3-ethyl carbon Dissolve diimine hydrochloride and N-hydroxysuccinimide at a molar ratio of 1:1:1, prepare an aqueous solution with a concentration of 0.1 mg/ml, activate for 20 minutes; mix the above at a volume ratio of 1:1 under nitrogen protection The two solutions were stirred and reacted for 12 hours, and the unreacted mercaptosuccinic acid was removed by repeated centrifugation and washing to obtain an aqueous solution of chitosan modified with a mercapto group; Dissolve imine hydrochloride and N-hydroxysuccinimide at a molar ratio of 1:2:2, prepare an aqueous solution with a concentration of 0.3 mg/ml, and activate for 1 hour in the dark; mix the pH at a volume ratio of 1:1 in the dark =5.0 chitosan aqueous solution and folic acid activation solution, stirring reaction 12h, obtain the chitosan aqueous solution containing folic acid modification; mix the chitosan aqueous solution containing sulfhydryl group modification and the chitosan aqueous solution containing folic acid modification according to the ratio of 1:1 Measure the above-mentioned mixed chitosan aqueous solution and the soybean oil containing hydrophobic drug paclitaxel in an ice-water bath and mix uniformly in a volume ratio of 5:1; then the ultrasonic probe is placed at the oil-water two-phase interface, under the ultrasonic power of 500W Act for 6 minutes; adjust the pH of the reaction solution to about 9-10, then carry out centrifugation and washing until the product is neutral, and obtain a chitosan cross-linked membrane with a disulfide bond structure modified by folic acid as the capsule wall. Drug-loaded microcapsules whose oil phase is the core material.

Example 10

In acetic acid and sodium acetate buffer solution of pH=5.0, chitosan is dissolved to prepare 0.3 mg/ml chitosan aqueous solution; 5-mercapto tetrazolo-1-acetic acid and 1-(3-dimethylaminopropyl )-3-Ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:1:1, and an aqueous solution with a concentration of 0.1 mg/ml was prepared and activated for 20 minutes; Mix the above two solutions with a ratio of 1:5, stir and react for 12 hours, and repeatedly centrifuge and wash to remove unreacted 5-mercaptotetrazol-1-acetic acid to obtain an aqueous solution of chitosan modified with a thiol group; mix folic acid and 1-(3 -Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:2:2, and an aqueous solution with a concentration of 0.3mg/ml was prepared and activated in the dark 1h; mix chitosan aqueous solution with pH = 5.0 and folic acid activation solution at a volume ratio of 1:1 in the dark, and stir for 12 hours to obtain a chitosan aqueous solution containing folic acid modification; mix thiol-containing modified chitosan aqueous solution and chitosan aqueous solution containing folic acid modification; measure the above-mentioned mixed chitosan aqueous solution and soybean oil containing hydrophobic drug paclitaxel in an ice-water bath and mix uniformly in a volume ratio of 5:1; Place it at the oil-water two-phase interface, and act under the ultrasonic power of 500W for 6 minutes; adjust the pH of the reaction solution to about 9-10, and then perform centrifugal washing until the product is neutral, and obtain folic acid-modified chitosan with a disulfide bond structure The sugar cross-linked film is the capsule wall, and the oil phase containing hydrophobic drugs is the drug-loaded microcapsule as the core material.

Example 11

In acetic acid and sodium acetate buffer solution of pH=5.0, chitosan was dissolved to prepare 0.5 mg/ml chitosan aqueous solution; glutathione and 1-(3-dimethylaminopropyl)-3-ethyl Dissolve carbodiimide hydrochloride and N-hydroxysuccinimide at a molar ratio of 1:1:1, prepare an aqueous solution with a concentration of 0.1 mg/ml, activate for 20 minutes; mix at a volume ratio of 1:5 under nitrogen protection The above two solutions were stirred and reacted for 12 hours, and the unreacted glutathione was removed by repeated centrifugation and washing to obtain an aqueous solution of chitosan modified with sulfhydryl groups; folic acid and 1-(3-dimethylaminopropyl)-3-ethyl Dissolve carbodiimide hydrochloride and N-hydroxysuccinimide at a molar ratio of 1:2:2, prepare an aqueous solution with a concentration of 0.3 mg/ml, and activate it in the dark for 1 hour; in the dark, use a volume ratio of 1:1 Mix the chitosan aqueous solution with pH=5.0 and the folic acid activation solution, stir and react for 12 hours to obtain the chitosan aqueous solution containing folic acid modification; mix the chitosan aqueous solution containing thiol modification and the chitosan modified folic acid modification according to the ratio of 1:1 Sugar aqueous solution; measure the above-mentioned mixed chitosan aqueous solution and soybean oil containing hydrophobic drug camptothecin in an ice-water bath and mix uniformly in a volume ratio of 5:1; then place the ultrasonic probe at the oil-water two-phase interface, Under the ultrasonic power of 6min; adjust the pH of the reaction solution to about 9-10, then carry out centrifugation and washing until the product is neutral, and obtain a chitosan cross-linked membrane with a disulfide bond structure modified by folic acid as the capsule wall, to contain The oil phase of the hydrophobic drug is the drug-loaded microcapsule of the core material.

Example 12

In acetic acid and sodium acetate buffer solution of pH=5.0, chitosan is dissolved to prepare 0.2mg/ml chitosan aqueous solution; Acetylcysteine and 1-(3-dimethylaminopropyl)-3-ethane Carbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in a molar ratio of 1:1:1, and an aqueous solution with a concentration of 1.0 mg/ml was prepared and activated for 20 minutes; under nitrogen protection, the volume ratio was 5:1 The above two solutions were mixed and stirred for 12 hours, and the unreacted acetylcysteine was removed by repeated centrifugation and washing to obtain an aqueous solution of chitosan modified with sulfhydryl groups; folic acid and 1-(3-dimethylaminopropyl)-3- Dissolve ethylcarbodiimide hydrochloride and N-hydroxysuccinimide at a molar ratio of 1:2:2, prepare an aqueous solution with a concentration of 0.3 mg/ml, and activate for 1 hour in the dark; : 1 mixed chitosan aqueous solution and folic acid activation solution with pH=5.0, stirred and reacted for 12h, obtained chitosan aqueous solution containing folic acid modification; mixed chitosan aqueous solution containing sulfhydryl group modification and folic acid modified Chitosan aqueous solution; Measure the above-mentioned mixed chitosan aqueous solution and the hydroxyl silicone oil containing hydrophobic drug paclitaxel in an ice-water bath and mix uniformly in a volume ratio of 5:1; then place the ultrasonic probe at the oil-water two-phase interface. Under the ultrasonic power of 500W, act for 6 minutes; adjust the pH of the reaction solution to about 9-10, then carry out centrifugal washing until the product is neutral, and obtain a chitosan cross-linked membrane with a disulfide bond structure modified by folic acid as the capsule wall, and use Drug-loaded microcapsules with the oil phase containing hydrophobic drugs as the core material.

Claims (8)

1. the chitosan microcapsules with reduction response of a modified with folic acid, it is characterized in that, by carrying out ultrasonic radiation to the oil phase being mounted with hydrophobic drug and the chitosan aqueous solution of modifying through folic acid and the compound containing sulfydryl or biomolecule, formed with the chitose crosslinked membrane of the modified with folic acid of disulfide bond crosslinking as cyst wall, to be loaded with the drug-loading microcapsule of oil phase for core of hydrophobic drug, the size of microcapsule is at 0.2-50 μm.

2. according to the chitosan microcapsules with reduction response of a kind of modified with folic acid according to claim 1, it is characterized in that, the described compound containing sulfydryl or biomolecule are sulfydryl alkanoic acid [HS-(CH ₂) _n-COOH, alkyl number n>=1], 2-amino-5-mercaptobenzoic acid, 5-sulfydryl-1H-tetrazole-1-acetic acid, 2-mercaptonicotinic acid, mercapto succinic acid, 5-mercapto-tetrazole also-1-acetic acid, cysteine and derivant thereof or glutathion and derivant thereof.

3. according to the chitosan microcapsules with reduction response of a kind of modified with folic acid according to claim 1, it is characterized in that, described hydrophobic drug is one or more in paclitaxel, lomustine, ginsenoside, camptothecine or silymarin medicine.

4. according to a kind of modified with folic acid described in right 1 have reduction response chitosan microcapsules, it is characterized in that, described oil phase be biological medicine can various animal oil, vegetable oil, microbial grease, mineral oil, silicone oil, or other and the inconsistent nontoxic liquid state organics of water.

5. a preparation method with the chitosan of the microcapsule of reduction response for modified with folic acid, is characterized in that, comprise the following steps:

A, in the acetic acid and sodium acetate buffer of pH=5.0, chitosan is dissolved and prepare certain density chitosan aqueous solution;

B, the compound containing sulfydryl or biomolecule and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and N-hydroxy-succinamide are dissolved with mol ratio 1:1:1, compound concentration is the aqueous solution of 0.1-1.0mg/ml, activation 20min;

C, be that 5:1-1:5 mixes above-mentioned two kinds of solution stirring and reacts 12 hours with volume ratio under nitrogen protection, the unreacted compound containing sulfydryl of centrifuge washing removing or biomolecule, obtain the chitosan aqueous solution containing sulfydryl modification repeatedly;

D, folic acid and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, N-hydroxy-succinamide are dissolved with mol ratio 1:2:2, compound concentration is the aqueous solution of 0.1-1.0mg/ml, lucifuge activation 1h;

Be chitosan aqueous solution and the folic acid activating solution of 5:1-1:5 mixing pH=5.0 with volume ratio under e, lucifuge, stirring reaction 12h, obtains the chitosan aqueous solution containing modified with folic acid;

F, according to the ratio mixing of 1:1-20:1 containing the chitosan aqueous solution of sulfydryl modification with containing the chitosan aqueous solution of modified with folic acid;

G, according to a certain volume measure above-mentioned mixed shell water solution and containing hydrophobic drug oil phase together be placed in ice-water bath mix homogeneously;

H, again ultrasonic probe is placed in oil-water two-phase interfaces place, under certain ultrasonic power, acts on a period of time;

I, to regulate after about pH to 9-10 carry out centrifuge washing to product for neutral to reactant liquor, the chitose crosslinked membrane with disulfide bond pattern obtained containing modified with folic acid is cyst wall, the drug-loading microcapsule being core with the oil phase containing hydrophobic drug.

6., according to the preparation method with the chitosan microcapsules of reduction response of a kind of modified with folic acid according to claim 5, it is characterized in that, described ultrasound intensity is 100-900W, and ultrasonic time is 0.5-30min.

7., according to the preparation method with the chitosan microcapsules of reduction response of a kind of modified with folic acid described in right 5, it is characterized in that, the concentration of described chitosan aqueous solution is 0.1-3.0mg/ml.

8., according to the preparation method with the chitosan microcapsules of reduction response of a kind of modified with folic acid described in right 5, it is characterized in that, described chitosan aqueous solution is 1:1-20:1 with the volume ratio of the oil phase containing hydrophobic drug.