CN100417417C - Surface-modified hydrophobically modified chitosan oligosaccharide polymer drug-loaded micelles and preparation method thereof - Google Patents

Abstract

The present invention provides a medicine carrying micelle modified by a hydrophobic modified chitosan oligosaccharide polymer on the surface. The micelle is prepared by grafting chitosan with the average molecular weight of 1.5kD to 51kD with fatty acid of C10 to C22. In the present invention, bifunctional organic small molecules are used for modifying the surface of amido or hydroxyl on chitosan oligosaccharide molecules on the surface of a polymer on the basis of the micelle of a hydrophobic modified chitosan oligosaccharide polymer; the instability of the diluted polymer micelle is improved by bridging chemical bonds among molecules on the surface of the polymer micelle; meanwhile, the original loose structure of the surface of the polymer micelle is changed into a dense net-shaped structure by bridging chemical bonds among molecules on the surface of the polymer micelle so as to reduce the unique sudden medicine release of the polymer micelle and achieve the purpose of the slow medicine release control. The present invention has the advantage of reasonable design, and the provided medicine carrying micelle is a nanometer carrier with good organelle target and can be used in the fields of life science and pharmacy.

Description

Surface-modified hydrophobically modified chitosan oligosaccharide polymer drug-loaded micelles and preparation method thereof

technical field

The invention belongs to the surface modification of polymer micelles, and relates to the surface modification of hydrophobically modified chitosan oligosaccharide polymer micelles by bifunctional organic small molecules, and the control of the surface modification degree of polymer micelles to achieve polymer Sustained and controlled release properties of drug-loaded micelles.

Background technique

Polymeric micelles (PMs) are a new class of nanocarriers that are being developed in recent years, with the advantages of solubilization, targeting, low toxicity and long circulation. It is formed spontaneously from amphiphilic polymers in an aqueous environment and has a unique core-membrane (shell) structure. Its internal core can provide storage for insoluble drugs, peptide and protein drugs and genes, and the hydrophilic outer membrane can be modified in physical and chemical properties to achieve targeted distribution in vivo, avoid phagocytosis by monocytes and macrophages, and improve biofilm transport etc. The polymer micelle itself can gather into the tumor tissue through the "enhanced permeability and retention effect".

Polymeric micelles are nanomicelles with dynamic equilibrium characteristics that spontaneously form when the concentration of amphiphilic polymers in solution is higher than their critical aggregation concentration. In vivo studies have found that there is a problem of stability after the polymer drug delivery micelles are diluted with body fluids; and increasing the concentration of the polymer will lead to high toxicity of the carrier itself. At the same time, the polymer micelle is formed by the hydrophobic interaction force between the hydrophobic segments of the amphiphilic polymer, and the solubilization of the lipophilic drug by the polymer micelle is also due to the hydrophobicity between the drug and the amphiphilic polymer. As a result of the hydrophobic interaction force between the sexual chain segments, the hydrophobic interaction force is a kind of physical force (van der Waals force), so the drug release of polymer drug-loaded micelles generally has the problems of burst release and rapid drug release. Although polymer micelles can have targeting and long-term circulation functions, their burst release and rapid drug release characteristics greatly reduce the targeting of drugs in polymer micelles to tissues, cells and organelles. Therefore, the clinical application of polymer drug-loaded micelles is still blank.

Chitosan is a cationic polymer composed of glucosamine, which has good biocompatibility, low toxicity and biodegradability. Chitosan is widely used in pharmaceutical excipients, hemostatic materials, tissue engineering scaffolds, etc. Large molecular weight chitosan is insoluble in water under physiological pH conditions (7.2-7.4), while low molecular weight chitosan (chitosan oligosaccharide, CSO) degraded by enzymes has good water solubility. After chain fatty acid graft modification, it can spontaneously form polymer micelles of 5-1000nm in aqueous solution, and has the function of nuclear transport. The polymer micelle has been applied to the carrier of lipophilic and biomacromolecular drugs such as protein and DNA.

Contents of the invention

The first object of the present invention is to provide surface-modified hydrophobically modified chitosan oligosaccharide polymer drug-loaded micelles. The micelles are composed of chitosan with an average molecular weight of 1.5kD-51kD and fatty acids of C ₁₀ -C ₂₂ grafted to obtain , the amino substitution degree of chitosan in the graft is 1%-50%, and the critical micelle concentration is 0.01-0.1mg/ml. On the basis of hydrophobically modified chitosan oligosaccharide polymer micelles, the present invention uses bifunctional organic small molecules to carry out surface chemical modification on the amino groups or hydroxyl groups on the polymer surface chitosan oligosaccharide molecules. bridging the chemical bonds between the polymer micelles to improve the instability of the diluted polymer micelles; at the same time, by bridging the chemical bonds between the surface molecules of the polymer micelles, the original loose structure of the polymer micelles surface is changed to form a denser The network structure is used to reduce the sudden release of drugs unique to polymer micelles, and to achieve the purpose of slow and controlled release of drugs. Drugs used as surface-modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles are lipophilic drugs, such as paclitaxel and the like.

The second object of the present invention is to provide a preparation method for surface-modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles, which is achieved by the following scheme:

(1) Preparation of chitosan oligosaccharide: get commercially available molecular weight chitosan (90-95% degree of deacetylation) of high molecular weight of 450kDa, stir and dissolve at 55-60 DEG C and pH5.0, press cellulase and Chitosan ratio 0.5:100 (w/w) was added to cellulase for degradation, and impurities were removed by filtration. According to the requirements of use, from the ultrafiltration membranes of 1kDa, 10kDa, 50kDa, 100kDa, and 200kDa respectively, select a suitable ultrafiltration molecular weight membrane ultrafiltration fractionation, and the ultrafiltrate is freeze-dried to obtain a molecular weight less than 200kDa (preferably chitosan oligosaccharide is 1 ~ 50kDa), the degree of deacetylation is greater than 80% low molecular weight chitosan oligosaccharide, the molecular weight is determined by gel permeation chromatography.

(2) Preparation of hydrophobically modified chitosan oligosaccharides: Take the above aqueous solution of chitosan oligosaccharides, control the temperature at 50° C. to 90° C. °C, react for 5-48 hours. The final reaction solution is purified by dialysis and freeze-dried to obtain hydrophobically modified chitosan oligosaccharide; the fatty acid is selected from any one of capric acid, myristic acid, palmitic acid, oleic acid, stearic acid, and behenic acid.

(3) Preparation of lipophilic drug-loaded hydrophobic modified chitosan oligosaccharide micelles: take hydrophobic modified chitosan oligosaccharide, add appropriate amount of distilled water, and ultrasonically disperse in a water bath to obtain a micellar solution, take the micellar solution, add lipophilic drug or its Solution, probe ultrasonic, get hydrophobic modified chitosan oligosaccharide drug-loaded micelles.

(4) Surface modification of drug-loaded micelles: take the drug-loaded micelles solution, add quantitative (controlling the molar ratio of hydrophobically modified chitosan oligosaccharides and bifunctional small organic molecules to 1:1-100) bifunctional small organic molecules , the solution is magnetically stirred at room temperature for a certain period of time, and the surface-modified hydrophobic modified chitosan oligosaccharide drug-loaded micelles are obtained.

The bifunctional organic small molecule includes a bifunctional organic small molecule surface modifier that can react with the amino group on the chitosan oligosaccharide: glutaraldehyde, genipin, dibasic fatty acid, ethylene glycol glycidyl ether, etc.; And a bifunctional organic small molecule surface modifier that can react with the hydroxyl group on the chitosan oligosaccharide: isophorone-diisocyanate.

The structural formula of described glutaraldehyde is:

The structural formula of the genipin is:

The structural formula of described ethylene glycol glycidyl ether is:

The dibasic fatty acid: as the structural formula of lauric acid is:

The structural formula of described isophorone-diisocyanate is:

The material of the surface-modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles used in the present invention is the micelles materials disclosed in patent application numbers 200510050798.1 and 200610050516.2. The composition of the micelles is: Chitosan with an average molecular weight of 1.5kD-51kD is grafted with C ₁₀ -C ₂₂ fatty acids, the amino substitution degree of chitosan in the graft is 1%-50%, and the critical micelle concentration is 0.01 ~0.1mg/ml. Achieved through the following schemes:

(1) Preparation of chitosan oligosaccharide: get commercially available molecular weight chitosan (90-95% degree of deacetylation) of high molecular weight of 450kDa, stir and dissolve at 55-60 DEG C and pH5.0, press cellulase and Chitosan ratio 0.5:100 (w/w) was added to cellulase for degradation, and impurities were removed by filtration. According to the requirements of use, from ultrafiltration membranes with molecular weights of 1kDa, 10kDa, 50kDa, 100kDa, and 200kDa, select a suitable ultrafiltration membrane for ultrafiltration classification, and the ultrafiltrate is freeze-dried to obtain a molecular weight less than 200kDa (preferably chitosan oligosaccharide is 1～200kDa). 50kDa), the degree of deacetylation is greater than 80% low molecular weight chitosan oligosaccharide, the molecular weight is determined by gel permeation chromatography.

(2) Preparation of hydrophobically modified chitosan oligosaccharides: Take the above aqueous solution of chitosan oligosaccharides, control the temperature at 50° C. to 90° C. °C, react for 5 to 48 hours. The final reaction solution is purified by dialysis and freeze-dried to obtain hydrophobically modified chitosan oligosaccharide; the fatty acid is selected from any one of capric acid, myristic acid, palmitic acid, oleic acid, stearic acid, and behenic acid.

(3) Preparation of hydrophobically modified chitosan oligosaccharide micelles loaded with lipophilic drugs: take hydrophobic modified chitosan oligosaccharides, add appropriate amount of distilled water, and ultrasonically disperse in a water bath to obtain a micellar solution.

The benefits of the present invention are:

(1) The provided functional organic small molecule modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles is a nanocarrier with excellent organelle targeting and can be applied in the fields of life sciences and pharmaceuticals. The carrier can be used for targeted therapy of drugs and improve the curative effect of drugs.

(2) In the present invention, the micelle surface modification technology adopted improves the instability of the polymer micelle after dilution by bridging the chemical bonds between the surface molecules of the polymer micelle; The intermolecular chemical bond bridging changes the original loose structure on the surface of the polymer micelle to form a denser network structure, which can reduce the unique drug burst release of the polymer micelle and achieve the purpose of sustained and controlled release.

(3) The preparation method of the bifunctional organic small molecule modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles is simple. The main raw material selected is chitosan oligosaccharide, which is derived from crustaceans in nature and has low toxicity and biodegradable characteristics.

Description of drawings

Figure 1 is the in vitro release curves of paclitaxel-loaded hydrophobically modified chitosan oligosaccharide micelles with different degrees of surface modification.

Figure 2 is the in vitro release curves of paclitaxel-loaded hydrophobically modified chitosan oligosaccharide micelles with different surface modification degrees.

Figure 3 is the in vitro release curves of paclitaxel-loaded hydrophobically modified chitosan oligosaccharide micelles with different surface modification degrees.

Figure 4 is the in vitro release curves of paclitaxel-loaded hydrophobically modified chitosan oligosaccharide micelles with different degrees of surface modification.

Detailed ways

The present invention is described further in conjunction with embodiment and accompanying drawing.

Embodiment one

1. Preparation of chitosan oligosaccharide

Add 6g of chitosan (average molecular weight 450kDa) to 200mL of 1.25% (v/v) hydrochloric acid aqueous solution, stir and dissolve at 55-60°C, adjust the pH to 5.0 with dilute ammonia water or dilute hydrochloric acid, press cellulase and shell Cellulase was added to the glycan ratio of 0.5:100 (w/w), and the reaction time was controlled for 8 hours. The reaction product was centrifuged at 4000r×min ^-1 for 10 minutes, and the supernatant was pretreated with a 0.45 μm microporous membrane to Molecular weight ultrafiltration membrane ultrafiltration fractionation, ultrafiltrate freeze-drying to obtain chitosan oligosaccharides with a certain molecular weight. And the average molecular weight was determined to be 50.6 kDa by gel permeation chromatography.

2. Preparation of hydrophobically modified chitosan oligosaccharides

Get above-mentioned chitosan oligosaccharide 5.0g, accurately weighed. After adding 40ml of double distilled water and stirring to dissolve, add 30mg of carbodiimide and stir to dissolve. Add 0.35g of stearic acid into 10ml of methanol solution, ultrasonically dissolve it, then add it into the above chitosan oligosaccharide solution, under the condition of 400r·min ⁻¹ magnetic stirring, control the temperature at 60°C, and the reaction time is more than 24h. The final reaction solution was placed in a dialysis bag and dialyzed against double distilled water for 24 hours to remove the reaction by-products. Dialysate was freeze-dried to prepare hydrophobically modified chitosan oligosaccharides.

The degree of amino substitution of chitosan oligosaccharides after hydrophobic modification was determined by trinitrobenzenesulfonic acid method. Get different amounts of chitosan oligosaccharides (0.5 ~ 9mg), accurately weighed, respectively dissolved in 2ml of double distilled water, add 2ml of 4% (w/v) sodium bicarbonate solution and 0.1% (w/v) tris Nitrobenzenesulfonic acid 2ml, incubate at 37°C for 2h. Add 2ml of 2N hydrochloric acid, shake well, measure the absorbance at a wavelength of 344nm, and prepare a standard curve. Get above-mentioned chitosan oligosaccharide 4mg to be dissolved in 2ml redistilled water, operate in the same way, measure the absorption value at 344nm wavelength place, calculate its substitution degree according to standard curve to be 5%.

3. Preparation of hydrophobically modified chitosan oligosaccharide drug-loaded micelles

Take 10 mg of hydrophobically modified chitosan oligosaccharide and weigh it accurately. Add an appropriate amount of double-distilled water and ultrasonically disperse in a water bath for 10 minutes, and set the volume to 100ml to obtain a hydrophobic modified chitosan oligosaccharide micellar solution.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of the hydrophobically modified chitosan oligosaccharide micelles is 74.6nm; the surface potential is 53.2±0.1mV.

Take 5 ml of the micellar solution, and add a quantitative volume of paclitaxel standard methanol solution (concentration: 1 mg/ml). After the methanol was removed by suction filtration under reduced pressure, the probe was sonicated 50 times in an ice bath (400W, 2s on, 3s off), to obtain the drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of the hydrophobically modified chitosan oligosaccharide drug-loaded micelles is 175.1nm; the surface potential is 58.7±0.1mV. The encapsulation rate of the drug is 94.82%.

4. Surface modification of drug-loaded micelles: Take 5ml of drug-loaded micelles solution, add quantitative glutaraldehyde, make the molar ratio of polymer to glutaraldehyde 1:10 and 1:20, stir magnetically at room temperature for 4 Hours, surface modification of the drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of hydrophobic modified chitosan oligosaccharide drug-loaded micelles with the molar ratio of polymer and glutaraldehyde being 1:10 is 131.6nm; the surface potential is 55.6±0.1mV; the encapsulation efficiency of the drug is 97.49 %. The average particle diameter of hydrophobic modified chitosan oligosaccharide drug-loaded micelles with the molar ratio of polymer and glutaraldehyde being 1:20 is 112.6nm; the surface potential is 44.6±0.1mV; the encapsulation efficiency of the drug is 97.27 %.

5. In vitro release of drug-loaded micelles: Take 2ml of drug-loaded micelles or surface-modified drug-loaded micelles, seal them in a dialysis bag (molecular weight 7000), and place them in 30ml of 2M sodium salicylate PBS (7.4) release medium Release in vitro in the medium, take samples at regular intervals, pass the sample through 0.22 μm microporous membrane, and measure the drug concentration of the filtrate by HPLC (chromatographic column: C18 column, mobile phase: acetonitrile: water=50:50, detection wavelength: 227nm, injection volume: 20 μ l , flow rate: 1ml/min.). Add or replace the release medium after sampling to keep the volume of the release medium constant. The results are shown in Figure 1. The results showed that with the increase of the surface modification degree of the polymer micelles, the release rate of the drug was significantly slowed down.

Embodiment two

1. Preparation of chitosan oligosaccharide

Add 6g of chitosan (average molecular weight 450kDa) to 200mL of 1.25% (v/v) hydrochloric acid aqueous solution, stir and dissolve at 55-60°C, adjust the pH to 5.0 with dilute ammonia water or dilute hydrochloric acid, press cellulase and shell Cellulase was added to the glycan ratio of 0.5:100 (w/w), and the reaction time was controlled for 8 hours. The reaction product was centrifuged at 4000r×min ^-1 for 10 minutes, and the supernatant was pretreated with a 0.45 μm microporous membrane to Molecular weight ultrafiltration membrane ultrafiltration fractionation, ultrafiltrate freeze-drying to obtain chitosan oligosaccharides with a certain molecular weight. And the average molecular weight was determined to be 50.6 kDa by gel permeation chromatography.

2. Preparation of hydrophobically modified chitosan oligosaccharides

Get above-mentioned chitosan oligosaccharide 5.0g, accurately weighed. After adding 40ml of double distilled water and stirring to dissolve, add 120mg of carbodiimide and stir to dissolve. Add 1.4g of stearic acid to 10ml of methanol solution, ultrasonically dissolve it, then add it to the above chitosan oligosaccharide solution, under the condition of 400r·min ⁻¹ magnetic stirring, control the temperature at 60°C, and the reaction time is more than 24h. The final reaction solution was placed in a dialysis bag and dialyzed against double distilled water for 24 hours to remove the reaction by-products. Dialysate was freeze-dried to prepare hydrophobically modified chitosan oligosaccharides.

The degree of amino substitution of chitosan oligosaccharides after hydrophobic modification was determined by trinitrobenzenesulfonic acid method. Get different amounts of chitosan oligosaccharides (0.5 ~ 9mg), accurately weighed, respectively dissolved in 2ml of double distilled water, add 2ml of 4% (w/v) sodium bicarbonate solution and 0.1% (w/v) tris Nitrobenzenesulfonic acid 2ml, incubate at 37°C for 2h. Add 2ml of 2N hydrochloric acid, shake well, measure the absorbance at a wavelength of 344nm, and prepare a standard curve. Get above-mentioned chitosan oligosaccharide 4mg to be dissolved in 2ml redistilled water, operate in the same way, measure the absorption value at 344nm wavelength place, calculate its substitution degree according to standard curve to be 12%.

3. Preparation of hydrophobically modified chitosan oligosaccharide drug-loaded micelles

Take 10 mg of hydrophobically modified chitosan oligosaccharide and weigh it accurately. Add an appropriate amount of double-distilled water and ultrasonically disperse in a water bath for 10 minutes, and set the volume to 100ml to obtain a hydrophobic modified chitosan oligosaccharide micellar solution.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of the hydrophobically modified chitosan oligosaccharide micelles is 67.7nm; the surface potential is 52.1±0.1mV.

Take 5 ml of the micellar solution, and add a quantitative volume of paclitaxel standard methanol solution (concentration: 1 mg/ml). After the methanol was removed by suction filtration under reduced pressure, the probe was sonicated 50 times in an ice bath (400W, 2s on, 3s off), to obtain the drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of the hydrophobically modified chitosan oligosaccharide drug-loaded micelles is 117.8nm; the surface potential is 56.8±0.1mV. The encapsulation rate of the drug is 97.11%.

4. Surface modification of drug-loaded micelles: Take 5ml of drug-loaded micelles solution, add a certain amount of glutaraldehyde to it, make the molar ratio of polymer to glutaraldehyde 1:10 and 1:20, stir magnetically at room temperature After 4 hours, the surface modification of the drug-loaded micelles was carried out.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle size of hydrophobic modified chitosan oligosaccharide drug-loaded micelles with a molar ratio of polymer to glutaraldehyde of 1:10 is 126.5nm; the surface potential is 56.8±0.1mV; the encapsulation efficiency of the drug is 96.8 %. The average particle size of the surface-modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles with a molar ratio of polymer and glutaraldehyde of 1:20 is 77.7nm; the surface potential is 46.7±0.1mV; the encapsulation efficiency of the drug is 97. %.

5. In vitro release of drug-loaded micelles: Take 2ml of drug-loaded micelles or surface-modified drug-loaded micelles, seal them in a dialysis bag (molecular weight 7000), and place them in 30ml of 2M sodium salicylate PBS (7.4) release medium Released in vitro, samples were taken at regular intervals, the samples were passed through a 0.22 μm microporous membrane, and the drug concentration of the filtrate was determined by HPLC. Add or replace the release medium after sampling to keep the volume of the release medium constant. See Figure 2 for the results. The results also showed that with the increase of the surface modification degree of the polymer micelles, the release rate of the drug was significantly slowed down.

Embodiment Three

1. Preparation of chitosan oligosaccharide

Add 6g of chitosan (average molecular weight 450kDa) to 200mL of 1.25% (v/v) hydrochloric acid aqueous solution, stir and dissolve at 55-60°C, adjust the pH to 5.0 with dilute ammonia water or dilute hydrochloric acid, and mix with cellulase Chitosan ratio 0.5:100 (w/w) was added to cellulase, and after controlling the reaction time for 8 hours, the reaction product was centrifuged at 4000r×min ^for 10 minutes, and the supernatant was pretreated with a 0.45 μm microporous membrane to Ultrafiltration membranes with different molecular weights are fractionated by ultrafiltration, and the ultrafiltrate is freeze-dried to obtain chitosan oligosaccharides with a certain molecular weight. And the average molecular weight was determined to be 50.6 kDa by gel permeation chromatography.

2. Preparation of hydrophobically modified chitosan oligosaccharides

Get above-mentioned chitosan oligosaccharide 5.0g, accurately weighed. After adding 40ml of double-distilled water and stirring to dissolve, add 300mg of carbodiimide, and stir to dissolve. Add 3.5g of stearic acid to 10ml of methanol solution, ultrasonically dissolve it, then add it to the above chitosan oligosaccharide solution, under the condition of 400r·min ⁻¹ magnetic stirring, control the temperature at 60°C, and the reaction time is more than 24h. The final reaction solution was placed in a dialysis bag and dialyzed against double distilled water for 24 hours to remove the reaction by-products. Dialysate was freeze-dried to prepare hydrophobically modified chitosan oligosaccharides.

The degree of amino substitution of chitosan oligosaccharides after hydrophobic modification was determined by trinitrobenzenesulfonic acid method. Get different amounts of chitosan oligosaccharides (0.5 ~ 9mg), accurately weighed, respectively dissolved in 2ml of double distilled water, add 2ml of 4% (w/v) sodium bicarbonate solution and 0.1% (w/v) tris Nitrobenzenesulfonic acid 2ml, incubate at 37°C for 2h. Add 2ml of 2N hydrochloric acid, shake well, measure the absorbance at a wavelength of 344nm, and prepare a standard curve. Get above-mentioned chitosan oligosaccharide 4mg to be dissolved in 2ml redistilled water, operate in the same way, measure the absorption value at 344nm wavelength place, calculate its substitution degree according to standard curve to be 40%.

3. Preparation of hydrophobically modified chitosan oligosaccharide drug-loaded micelles

Take 10 mg of hydrophobically modified chitosan oligosaccharide and weigh it accurately. Add an appropriate amount of double-distilled water and ultrasonically disperse in a water bath for 10 minutes, and set the volume to 100ml to obtain a hydrophobic modified chitosan oligosaccharide micellar solution.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of the hydrophobically modified chitosan oligosaccharide micelles is 31.4nm; the surface potential is 39.0±0.1mV.

Take 5 ml of the micellar solution, and add a quantitative volume of paclitaxel standard methanol solution (concentration: 1 mg/ml). After the methanol was removed by suction filtration under reduced pressure, the probe was sonicated 50 times in an ice bath (400W, 2s on, 3s off), to obtain the drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of the hydrophobically modified chitosan oligosaccharide drug-loaded micelles is 57.3nm; the surface potential is 53.2±0.1mV. The encapsulation rate of the drug was 97.04%. 4. Surface modification of drug-loaded micelles: Take 5ml of drug-loaded micelles solution, add a certain amount of glutaraldehyde to it, make the molar ratio of polymer to glutaraldehyde 1:1 and 1:100, and stir magnetically at room temperature After 4 hours, the surface modification of the drug-loaded micelles was carried out. The effects of different curing ratios on the particle size, potential and drug encapsulation efficiency of the formed cured drug-loaded micelles are shown in Table 1.

Table 1. Effect of curing ratio on particle size, potential and drug encapsulation efficiency of cured drug-loaded micelles

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle size of the surface-modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles with a molar ratio of polymer to glutaraldehyde of 1:1 is 35.7nm; the surface potential is 38.0±0.1mV. The encapsulation rate of the drug is 97.83%. The average particle diameter of hydrophobic modified chitosan oligosaccharide drug-loaded micelles with the molar ratio of polymer and glutaraldehyde being 1:10 is 67.2nm; the surface potential is 49.8±0.1mV; the encapsulation efficiency of the drug is 98.77 %. The average particle diameter of hydrophobic modified chitosan oligosaccharide drug-loaded micelles with the molar ratio of polymer and glutaraldehyde being 1:20 is 35.8nm; the surface potential is 44.0±0.1mV; the encapsulation efficiency of the drug is 99.14 %. The average particle diameter of hydrophobic modified chitosan oligosaccharide drug-loaded micelles with the molar ratio of polymer and glutaraldehyde being 1:100 is 27.5nm; the surface potential is 18.0±0.1mV; the encapsulation efficiency of the drug is 99.36 %.

5. In vitro release of drug-loaded micelles: Take 2ml of drug-loaded micelles or surface-modified drug-loaded micelles, seal them in a dialysis bag (molecular weight 7000), and place them in 30ml of 2M sodium salicylate PBS (7.4) release medium Released in vitro, samples were taken at regular intervals, the samples were passed through a 0.22 μm microporous membrane, and the drug concentration of the filtrate was determined by HPLC. Add or replace the release medium after sampling to keep the volume of the release medium constant. See Figure 3 for the results. The results also showed that with the increase of the surface modification degree of the polymer micelles, the release rate of the drug was significantly slowed down.

Embodiment Four

1. Preparation of chitosan oligosaccharide

Add 6g of chitosan (average molecular weight 450kDa) to 200mL of 1.25% (v/v) hydrochloric acid aqueous solution, stir and dissolve at 55-60°C, adjust the pH to 5.0 with dilute ammonia water or dilute hydrochloric acid, press cellulase and shell Cellulase was added to the glycan ratio of 0.5:100 (w/w), and the reaction time was controlled for 8 hours. The reaction product was centrifuged at 4000r×min ^-1 for 10 minutes, and the supernatant was pretreated with a 0.45 μm microporous membrane to Molecular weight ultrafiltration membrane ultrafiltration fractionation, ultrafiltrate freeze-drying to obtain chitosan oligosaccharides with a certain molecular weight. And the average molecular weight was determined to be 50.6 kDa by gel permeation chromatography.

2. Preparation of hydrophobically modified chitosan oligosaccharides

Get above-mentioned chitosan oligosaccharide 5.0g, accurately weighed. After adding 40ml of double-distilled water and stirring to dissolve, add 300mg of carbodiimide, and stir to dissolve. Add 3.5g of stearic acid to 10ml of methanol solution, ultrasonically dissolve it, then add it to the above chitosan oligosaccharide solution, under the condition of 400r·min ⁻¹ magnetic stirring, control the temperature at 60°C, and the reaction time is more than 24h. The final reaction solution was placed in a dialysis bag and dialyzed against double distilled water for 24 hours to remove the reaction by-products. Dialysate was freeze-dried to prepare hydrophobically modified chitosan oligosaccharides.

The degree of amino substitution of chitosan oligosaccharides after hydrophobic modification was determined by trinitrobenzenesulfonic acid method. Get different amounts of chitosan oligosaccharides (0.5 ~ 9mg), accurately weighed, respectively dissolved in 2ml of double distilled water, add 2ml of 4% (w/v) sodium bicarbonate solution and 0.1% (w/v) tris Nitrobenzenesulfonic acid 2ml, incubate at 37°C for 2h. Add 2ml of 2N hydrochloric acid, shake well, measure the absorbance at a wavelength of 344nm, and prepare a standard curve. Get above-mentioned chitosan oligosaccharide 4mg to be dissolved in 2ml redistilled water, operate in the same way, measure the absorption value at 344nm wavelength place, calculate its substitution degree according to standard curve to be 40%.

3. Preparation of hydrophobically modified chitosan oligosaccharide drug-loaded micelles

Take 10 mg of hydrophobically modified chitosan oligosaccharide and weigh it accurately. Add an appropriate amount of double-distilled water and ultrasonically disperse in a water bath for 10 minutes, and set the volume to 100ml to obtain a hydrophobic modified chitosan oligosaccharide micellar solution.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of the hydrophobically modified chitosan oligosaccharide micelles is 31.4nm; the surface potential is 39.0±0.1mV.

Take 5 ml of the micellar solution, and add a quantitative volume of paclitaxel standard methanol solution (concentration: 1 mg/ml). After the methanol was removed by suction filtration under reduced pressure, the probe was sonicated 50 times in an ice bath (400W, 2s on, 3s off), to obtain the drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle diameter of the hydrophobically modified chitosan oligosaccharide drug-loaded micelles is 57.3nm; the surface potential is 53.2±0.1mV. The encapsulation rate of the drug was 97.04%.

4. Surface modification of drug-loaded micelles: Take 5ml of drug-loaded micelles solution, add quantitative isophorone-diisocyanate to it, so that the molar ratio of polymer to isophorone-diisocyanate is 1:10 and 1 : 20, magnetically stirred at room temperature for 4 hours to carry out surface modification on the drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The average particle size of the surface-modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles with a molar ratio of polymer and isophorone-diisocyanate of 1:10 is 53.4nm; the surface potential is 34.7±0.1mV; The sealing rate is 96.75%. The average particle size of the surface-modified hydrophobically modified chitosan oligosaccharide drug-loaded micelles with a molar ratio of polymer and isophorone-diisocyanate of 1:20 is 43.1nm; the surface potential is 35.2±0.1mV; The sealing rate is 98.34%.

5. In vitro release of drug-loaded micelles: Take 2ml of drug-loaded micelles or surface-modified drug-loaded micelles, seal them in a dialysis bag (molecular weight 7000), and place them in 30ml of 2M sodium salicylate PBS (7.4) release medium Released in vitro, samples were taken at regular intervals, the samples were passed through a 0.22 μm microporous membrane, and the drug concentration of the filtrate was determined by HPLC. Add or replace the release medium after sampling to keep the volume of the release medium constant. See Figure 4 for the results. The results also showed that with the increase of the surface modification degree of the polymer micelles, the release rate of the drug was significantly slowed down.

Embodiment five

1. Preparation of oligochitosaccharides, hydrophobically modified oligochitosaccharides, and drug-loaded micelles of hydrophobically modified oligochitosaccharides is the same as in Examples 1 to 4.

2. Surface modification of drug-loaded micelles: Take 5ml of drug-loaded micelles solution, add 1ml of genipin ethanol solution to make the molar ratio of polymer to genipin 1:20, stir magnetically at room temperature for 4 hours , to modify the surface of drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The surface modified hydrophobically modified chitosan oligosaccharide drug-loaded micelle has an average particle diameter of 41.8nm; a surface potential of 43.2±0.1mV; and a drug encapsulation efficiency of 98.15%.

Embodiment six

1. Preparation of oligochitosaccharides, hydrophobically modified oligochitosaccharides, and drug-loaded micelles of hydrophobically modified oligochitosaccharides is the same as in Examples 1 to 4.

2. Surface modification of drug-loaded micelles: Take 5ml of drug-loaded micelles solution, add 1ml of ethanol solution of lauric acid to make the molar ratio of polymer to lauric acid 1:20, stir magnetically at room temperature for 4 hours , to modify the surface of drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The surface modified hydrophobically modified chitosan oligosaccharide drug-loaded micelle has an average particle size of 37.6nm; a surface potential of 45.2±0.1mV; and a drug encapsulation efficiency of 98.97%.

Embodiment seven

1. Preparation of oligochitosaccharides, hydrophobically modified oligochitosaccharides, and drug-loaded micelles of hydrophobically modified oligochitosaccharides is the same as in Examples 1 to 4.

2. Surface modification of drug-loaded micelles: take 5ml of drug-loaded micelles solution, add 1ml of ethylene glycol glycidyl ether ethanol solution, make the molar ratio of polymer to ethylene glycol glycidyl ether be 1:20, in Magnetic stirring was carried out at room temperature for 4 hours to modify the surface of the drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The surface modified hydrophobically modified chitosan oligosaccharide drug-loaded micelle has an average particle diameter of 45.3nm; a surface potential of 34.5±0.1mV; and a drug encapsulation efficiency of 99.23%.

Embodiment eight

1. Preparation of oligochitosaccharides, hydrophobically modified oligochitosaccharides, and drug-loaded micelles of hydrophobically modified oligochitosaccharides is the same as in Examples 1 to 4.

2. Surface modification of drug-loaded micelles: Take 5ml of drug-loaded micelles solution, add 1ml of isophorone-diisocyanate ethanol solution to it, so that the molar ratio of polymer to isophorone-diisocyanate is 1:20 , and magnetically stirred at room temperature for 4 hours to modify the surface of the drug-loaded micelles.

Zetasizer 3000HS analyzer was used to measure particle size and surface potential. The surface modified hydrophobically modified chitosan oligosaccharide drug-loaded micelle has an average particle size of 56.4nm; a surface potential of 31.4±0.1mV; and a drug encapsulation efficiency of 98.67%.

Without further elaboration, it is believed that one skilled in the art can, using the preceding disclosure, utilize the present invention to its fullest extent. Accordingly, the foregoing preferred specific embodiments are to be understood as illustrative only, and are not intended to limit the scope of the invention in any way.

Claims (5)

1. surface-modified hydrophobically modified oligochitosan medicine carrying micelle, the consisting of of micelle: the oligochitosan of mean molecule quantity 1.5kD～51kD and C ₁₀～C ₂₂The grafting of fatty acid, the amino group substitution degree of oligochitosan is 1%～50% in the grafting, critical micelle concentration is 0.01～0.1mg/ml, it is characterized in that: amino on the polymer surfaces oligochitosan molecule or hydroxyl are carried out chemical modification with the bi-functional organic molecule, wherein said bi-functional organic molecule is selected glutaraldehyde for use, genipin, the binary fatty acid, diglycidyl ether of ethylene glycol, in isophorone-vulcabond any, described chemical modification is to get the medicine carrying micelle solution, add bi-functional organic molecule solution, the mol ratio of control hydrophobic modified chitin and bi-functional organic molecule is that 1: 1～100,15～50 ℃ lower magnetic forces stirred 2～24 hours.

2. surface-modified hydrophobically modified oligochitosan medicine carrying micelle according to claim 1 is characterized in that: the medicine as surface-modified hydrophobically modified oligochitosan medicine carrying micelle is a lipophilic drugs.

3. surface-modified hydrophobically modified oligochitosan medicine carrying micelle according to claim 2 is characterized in that: as the choice of drug paclitaxel of surface-modified hydrophobically modified oligochitosan medicine carrying micelle.

4. the preparation method of the arbitrary described surface-modified hydrophobically modified oligochitosan medicine carrying micelle of claim 1-3, (1) getting molecular weight is 450kDa, the high-molecular weight chitosan of 90～95% deacetylations, 90～95% deacetylations, stirring and dissolving under 55～60 ℃ and pH5.0 condition, by cellulase and chitosan weight ratio is 0.5: 100 adding cellulose degraded, remove by filter impurity, with ultrafilter membrane ultrafiltration classification, the filtrate lyophilization, get the low-molecular-weight oligochitosan of molecular weight less than 200kDa, get above-mentioned oligochitosan aqueous solution, according to oligochitosan, fatty acid, crosslinked coupling agent carbodiimide mol ratio 1: 1～50: 1～50 is controlled 50 ℃～90 ℃, reacts 5～48 hours, end reaction liquid dialysis purification, lyophilization obtains hydrophobic modified chitin; It is characterized in that further comprising the steps of:

(2) get hydrophobic modified chitin, the adding distilled water is an amount of, the water-bath ultra-sonic dispersion, get micelle solution, get micelle solution, add lipophilic drugs or its solution, it is ultrasonic to pop one's head in, get hydrophobic modified chitin medicine carrying micelle, get the medicine carrying micelle solution, add bi-functional organic molecule solution, the mol ratio of control hydrophobic modified chitin and bi-functional organic molecule is 1: 1～100,15～50 ℃ of lower magnetic forces stirred 2～24 hours, got surface-modified hydrophobically modified oligochitosan medicine carrying micelle.

5. the preparation method of surface-modified hydrophobically modified oligochitosan medicine carrying micelle according to claim 4 is characterized in that: it is that 1～50kDa prepares hydrophobic modified chitin that the described oligochitosan aqueous solution of step (1) is selected molecular weight for use.

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