A targeted nano-microsphere drug delivery system, preparation method and application

Abstract

The present invention discloses a targeted nano-microsphere drug delivery system, a preparation method and an application. The preparation of the drug delivery system includes forming nano-micelles by mixing a drug, a chitosan-deoxycholic acid-mannose graft and / or a biotin-vitamin E polyethylene glycol succinate graft, so as to achieve targeted aggregation of the drug at the site of colon inflammation; then encapsulating the nano-micelles with calcium alginate to prepare a pH-responsive composite microsphere, so as to achieve site-specific drug release in the colon and be used for oral administration to treat ulcerative colitis. The drug is preferably asiatic acid. The nano-microsphere drug delivery system has a controllable particle size distribution, can reduce the destruction of the drug in the stomach, and has a certain colon-targeting effect, and is a novel drug delivery system for ulcerative colitis.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a targeted nano-microsphere drug delivery system, a preparation method and application of the system in preparing a drug for treating ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a common inflammatory disease of the colon. Due to its repeated course of disease, it is difficult to cure and has a tendency to become cancerous, which seriously endangers public health. At present, the first-line treatment drugs for UC mainly include anti-inflammatory drugs, immunosuppressants and biological products, but the overall efficacy is not satisfactory, and the toxic side effects are large (nephrotoxicity, hepatotoxicity, neurotoxicity, etc.). In addition, due to the diarrhea caused by ulcerative colitis, the drug delivery system with particles larger than 200μm (including clinically used drugs) will have a shortened retention time in the gastrointestinal tract, resulting in reduced efficacy. Therefore, it is necessary to develop a new drug delivery system that can deliver drugs to the inflammatory area of ​​the colon in a targeted manner to improve efficacy and reduce side effects.

[0003] Traditional Chinese medicine is a treasure of Chinese culture with a long history. Chinese herbal medicines and their extracts with multiple pharmacological activities are widely used in inflammatory-related diseases and are the first choice for supplementary and alternative treatment drugs for colon inflammation. Asiatic acid (AA) is a pentacyclic triterpene acid isolated from the medicinal plant Centella asiatica L. Urban. It has multiple pharmacological effects such as anti-inflammatory, antioxidant, and anti-cancer, and has potential preventive and therapeutic effects on colon inflammation. However, due to the low water solubility, rapid elimination in the body, short half-life, and extremely low oral bioavailability of asiatic acid, its clinical application is greatly limited. In order to solve the above problems, patent CN 105477001B makes it into solid lipid nanoparticles of asiatic acid tromethamine salt, which improves the solubility and oral absorption of asiatic acid, but it involves structural modification and may have safety hazards. Nanocarriers for delivering traditional Chinese medicines such as asiatic acid have been reported, such as patents CN 103054804A and CN 102784096A, which respectively disclose asiatic acid solid dispersions and microemulsions. The laboratory also prepared asiatic acid micelles and nanolipid carriers (CN 107638388B and CN 105919976B) in the early stage, and the oral bioavailability was significantly improved. However, the above nanoformulations have the disadvantages of insufficient targeting and inability to release drugs in a targeted manner, and thus cannot provide local drug concentrations that are cytotoxic, resulting in low drug enrichment in the inflammatory site, and even increasing the systemic blood drug concentration while causing more serious systemic side effects, and the in vivo efficacy also needs to be confirmed.

[0004] Surface modification of nanocarriers with specific targeting ligands is an effective method to increase local drug retention and enrichment. During the pathological process of ulcerative colitis, a large number of macrophages are activated and highly expressed in the colonic inflammatory response. Mannose receptor is a recognition receptor mainly present on the surface of macrophage membranes. Therefore, mannose-modified polymeric micelles can be used as macrophage-targeting molecules and selectively accumulate at the inflammatory site. In addition, the intestine is the main site for drug absorption and utilization. Therefore, drug delivery systems targeting intestinal epithelial cells have become another important strategy to improve the bioavailability of oral drugs.

[0005] In addition, due to the special environment and complexity of the gastrointestinal tract, many drugs and nanocarriers are degraded by gastric acid and proteases before reaching the colon. Therefore, oral nano-systems must overcome the barrier of instability in the stomach. Sodium alginate is a natural polysaccharide with good adhesiveness, biodegradability and biocompatibility. Due to the presence of a large number of carboxyl groups in its structure, the molecular segments of sodium alginate contract in an acidic environment and expand in neutral and alkaline environments, showing pH sensitivity. In addition, the G units of sodium alginate react with metal ions such as Ca 2+ to form a dense network structure, which can protect the encapsulated drugs from the destruction of gastric acid and fully release them in the intestinal environment. Summary of the Invention

[0006] The object of the present invention is to provide a targeted nano-microsphere drug delivery system, a preparation method and its application in the preparation of drugs for treating ulcerative colitis in view of the deficiencies of the prior art.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A targeted nano-microsphere drug delivery system, wherein the targeted nano-microsphere drug delivery system at least comprises nano-micelles, and the nano-micelles are composed of a mixture of a drug, a chitosan-deoxycholic acid-mannose graft and / or a biotin-vitamin E polyethylene glycol succinate graft.

[0009] The mechanism of the present invention to play a targeted role in colitis is as follows:

[0010] The present invention utilizes the structural characteristics of the self-assembly of chitosan-deoxycholic acid-mannose graft and biotin-vitamin E polyethylene glycol succinate graft into nano-micelles in water, which can be used as a carrier material for the nano-system and simultaneously have the functions of targeting macrophages and intestinal epithelial cells at the inflammatory site, thereby increasing the local drug concentration in the colonic lesion tissue.

[0011] Further, the drug is asiatic acid.

[0012] Further, the particle size of the nano-micelles is 20-150 nm, the encapsulation efficiency is greater than 90%, and the drug loading is greater than 12%.

[0013] Furthermore, the mass ratio of the drug, chitosan-deoxycholic acid-mannose graft copolymer, and biotin-vitamin E polyethylene glycol succinate graft copolymer is 1.5:0 to 10:0 to 10. Among them, the mass of at least one of the chitosan-deoxycholic acid-mannose graft copolymer and the biotin-vitamin E polyethylene glycol succinate graft copolymer is not 0.

[0014] Furthermore, the chitosan-deoxycholic acid-mannose graft copolymer is prepared by the following method:

[0015] Activate the carboxyl group on deoxycholic acid, and then drop the activated deoxycholic acid solution into the chitosan aqueous solution for reaction grafting to obtain chitosan-deoxycholic acid graft copolymer;

[0016] Add 4-isothiocyanatophenyl-α-D-mannopyranoside solution to the chitosan-deoxycholic acid aqueous solution for reaction grafting to obtain chitosan-deoxycholic acid-mannose graft copolymer.

[0017] Furthermore, the method for activating the carboxyl group on deoxycholic acid is specifically as follows: Dissolve deoxycholic acid and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in an ethanol-acetone (volume ratio 3:7) mixed solvent. Among them, the concentration of deoxycholic acid is 18 mg / ml, and the molar ratio of deoxycholic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:3. Magnetically stir for 1 h in a water bath at 60-65 °C to activate the carboxyl group.

[0018] Furthermore, the concentration of the activated deoxycholic acid solution is 18 mg / ml, the concentration of the chitosan aqueous solution is 15 mg / ml, the concentration of the 4-isothiocyanatophenyl-α-D-mannopyranoside solution is 20 mg / ml, and the concentration of the chitosan-deoxycholic acid aqueous solution is 8.3 mg / ml.

[0019] Furthermore, the molar ratio of deoxycholic acid to chitosan monomer is 1:4, and the molar ratio of 4-isothiocyanatophenyl-α-D-mannopyranose to chitosan-deoxycholic acid monomer is 1:3.

[0020] Furthermore, the biotin-vitamin E polyethylene glycol succinate graft copolymer is prepared by the following method:

[0021] Activate the terminal carboxyl group of biotin, and then add vitamin E polyethylene glycol succinate for mixed reaction grafting to obtain biotin-vitamin E polyethylene glycol succinate graft copolymer.

[0022] Furthermore, the method for activating the terminal carboxyl group of biotin is specifically as follows:

[0023] Dissolve biotin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine in dimethyl sulfoxide and stir to activate the terminal carboxyl group of biotin; the molar ratio of biotin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 4-dimethylaminopyridine is 1:1:0.8, and the concentration of biotin is 25 mg / ml.

[0024] Furthermore, the targeted nano-microsphere drug delivery system further comprises sodium alginate microspheres for coating the nano micelles.

[0025] By further encapsulating the nano micelles with alginate to form a nano-microsphere complex, taking advantage of the pH-sensitive characteristics of alginate (in an acidic environment, the molecular chain segments contract, which can protect the stability of the encapsulated drug in gastric acid; in a neutral and slightly alkaline environment, ionization occurs, and the encapsulated drug is slowly released), the formed composite microspheres have pH responsiveness. This nano-microsphere system can reduce the release of the drug in the stomach and small intestine, while achieving sustained and slow drug release in the colonic environment, having the effect of targeted drug release at the colonic site, further increasing the effective drug accumulation at the colonic site, achieving effective treatment of ulcerative colitis, and reducing systemic side effects at the same time.

[0026] Furthermore, the particle size of the nano-microsphere complex is 40 μm, and the drug loading amount is greater than 8%.

[0027] A preparation method of the above system, and the method is as follows:

[0028] Prepare an aqueous solution of chitosan-deoxycholic acid-mannose graft and / or biotin-vitamin E polyethylene glycol succinate graft as the carrier aqueous solution and the drug solution, add the drug solution to the carrier aqueous solution, and mix evenly to obtain nano micelles.

[0029] Furthermore, the concentrations of both the carrier aqueous solution and the drug solution are 5 mg / ml, and they are mixed in proportion according to requirements.

[0030] Furthermore, it further comprises:

[0031] Take a Span 80 solution with a concentration of 20 mg / ml. Under mechanical stirring, use a syringe to dropwise add 20 ml of a sodium alginate solution with a concentration of 20 mg / ml onto the liquid surface, and stir and emulsify in a water bath at 40 - 45 °C; then use a syringe to add 30 ml of the nano micelle dispersion solution below the liquid surface and stir to mix evenly; then, dropwise add 30 ml of a calcium chloride solution with a concentration of 100 mg / ml for crosslinking; centrifuge the mixed solution to take the lower layer precipitate, wash and dry it to obtain targeted nano-microsphere particles with sodium alginate microspheres coating the nano micelles.

[0032] An application of the above system in the preparation of drugs for treating ulcerative colitis.

[0033] The beneficial effects of the present invention are as follows: The present invention is simple to operate, has controllable quality, good reproducibility, and does not require chemical structure modification. The particle size of the asiatic acid nanosystem prepared by the present invention is 20 - 150 nm, the encapsulation efficiency is greater than 90%, and the drug loading amount is greater than 12%; the particle size of the obtained asiatic acid nano-microsphere system is 40 μm, and the drug loading amount is greater than 8%. In addition, the nano-microsphere system has good targeting effect on the colon inflammatory site and good safety, solving the problems that traditional nano-drugs cannot target the colon and the local drug concentration is low, and can be used for targeted treatment of colon inflammatory diseases, having good market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is the synthesis route (A) and 1H NMR spectrum (B) of chitosan-deoxycholic acid-mannose graft; in the figure, a represents chitosan, b represents deoxycholic acid, c represents chitosan-deoxycholic acid graft, d represents 4-isothiocyanatophenyl-α-D-mannopyranoside, and e represents chitosan-deoxycholic acid-mannose graft;

[0035] Figure 2 is the synthesis route (A) and 1H NMR spectrum (B) of biotin-vitamin E polyethylene glycol succinate graft; in the figure, a represents vitamin E polyethylene glycol succinate, and b represents biotin-vitamin E polyethylene glycol succinate graft;

[0036] Figure 3 is the particle size distribution diagram (A); potential diagram (B); transmission electron microscope image (C) of the nanomicelles in Example 5;

[0037] Figure 4 is the result diagram of the uptake characteristics of the nanomicelles in Examples 3 - 7 in Raw 264.7 macrophages and Caco-2 cells: (A) is the quantitative uptake diagram of the micelles in Raw 264.7 macrophages; (B) is the quantitative uptake diagram of the micelles in Raw 264.7 macrophages pre-saturated with mannose; (C) is the quantitative uptake diagram of the micelles in Caco-2 cells; (D) is the quantitative uptake diagram of the micelles in Caco-2 cells pre-saturated with biotin; (E) is the fluorescence diagram of the uptake of the micelles in Raw 264.7 macrophages and Caco-2 cells;

[0038] Figure 5 is the in vitro release curve diagram (A) and scanning electron microscope image (B) of the nano-microspheres soaked in different pH media for 2 h; in the figure, a represents the unsoaked microspheres, b represents the microspheres soaked in pH 1.2 solution for 2 h, c represents the microspheres soaked in pH 6.8 buffer for 2 h, and d represents the microspheres soaked in pH 7.4 buffer for 2 h;

[0039] Figure 6In vivo organ distribution diagrams of nano - micelles and nano - microspheres; (A) Fluorescence images of the gastrointestinal tract of mice at different time points (6 h, 12 h, 24 h); (B) Fluorescence images of the heart, liver, spleen, lung, and kidney of mice at different time points (6 h, 12 h, 24 h); (C) Semi - quantitative fluorescence statistical chart of the colon; (D) Semi - quantitative fluorescence statistical chart of the liver;

[0040] Figure 7 Pharmacodynamic evaluation results of asiatic acid nano - microspheres in a colitis model mice; among them, (A) Schematic diagram of colitis induction and treatment process; (B) Body weight change curve; (C) Disease activity index; (D) Comparative photos of the colon length of mice; (E) Quantitative analysis of the colon length; (F and G) Representative H&E staining diagrams of colon tissues in each group (10×, F; 20×, G); (H) Representative PAS staining diagram of colon tissues; (I) Immunohistochemical ZO - 1 analysis diagram; (J) Immunohistochemical Claudin - 1 analysis diagram; (K) Immunofluorescence diagram of neutrophil infiltration in colon sections (green represents neutrophils; blue represents cell nuclei); (L) Immunofluorescence diagram of macrophage infiltration in the colon mucosa (red represents macrophages; blue represents cell nuclei);

[0041] Figure 8 H&E staining diagrams of main organs;

[0042] Figure 9 Results of the effect of asiatic acid nano - microspheres on inflammatory factors in colitis model mice; among them, (A) Changes in inflammatory factors in colon tissues; (B) Changes in inflammatory factors in serum; (C) Changes in mRNA expression in colon tissues. Detailed implementation mode

[0043] The novel targeted nano - microsphere drug delivery system for treating ulcerative colitis of the present invention, the targeted nano - microsphere drug delivery system at least includes nano - micelles, and the nano - micelle system contains the following components: asiatic acid, chitosan - deoxycholic acid - mannose graft copolymer and / or biotin - vitamin E polyethylene glycol succinate graft copolymer, etc.; the targeted nano - microsphere drug delivery system of the present invention also includes alginate microspheres for coating the nano - micelles, and the microsphere system contains the following components: asiatic acid nano - micelle dispersion, span 80, sodium alginate, calcium chloride.

[0044] The preparation method of the above - mentioned novel targeted nano - microsphere drug delivery system for treating ulcerative colitis includes the following processes:

[0045] (1) Prepare an aqueous solution of chitosan - deoxycholic acid - mannose graft copolymer and / or biotin - vitamin E polyethylene glycol succinate graft copolymer as the carrier aqueous solution:

[0046] (1.1) Synthesis of chitosan-deoxycholic acid-mannose graft copolymer:

[0047] Activation of the carboxyl group on deoxycholic acid: Dissolve deoxycholic acid and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in ethanol-acetone (volume ratio 3:7) with concentrations of 18 mg / ml and 26.4 mg / ml respectively. The molar ratio of deoxycholic acid to 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:3. Stir magnetically in a water bath at 60 ± 5 °C for 1 h to activate the carboxyl group.

[0048] Then slowly add it dropwise to the chitosan aqueous solution and continue the reaction for 12 h; Dialyze with deionized water to remove free deoxycholic acid and organic solvents, and freeze-dry the dialysis solution to obtain the chitosan-deoxycholic acid graft copolymer. Generally, the concentration of the chitosan aqueous solution is 15 mg / ml, and the grafting rate of deoxycholic acid can be adjusted by adjusting the molar ratio of deoxycholic acid to chitosan monomer.

[0049] Take another 4-isothiocyanatophenyl-α-D-mannopyranoside dissolved in dimethyl sulfoxide and slowly add it to the chitosan-deoxycholic acid aqueous solution. The molar ratio of 4-isothiocyanatophenyl-α-D-mannopyranose to chitosan-deoxycholic acid monomer is 1:3. Stir at room temperature for 24 h, dialyze with deionized water, and freeze-dry to obtain the chitosan-deoxycholic acid-mannose graft copolymer; Similarly, the grafting rate of mannose can be adjusted by adjusting the molar ratio of 4-isothiocyanatophenyl-α-D-mannopyranose to chitosan-deoxycholic acid monomer. Among them, the concentration of the 4-isothiocyanatophenyl-α-D-mannopyranoside solution is generally 20 mg / ml, and the concentration of the chitosan-deoxycholic acid aqueous solution is 8.3 mg / ml.

[0050] (1.2) Synthesis of biotin-vitamin E polyethylene glycol succinate graft copolymer:

[0051] Dissolve biotin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine in dimethyl sulfoxide with concentrations of 25 mg / ml, 20 mg / ml and 10 mg / ml respectively. The molar ratio of biotin, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to 4-dimethylaminopyridine is 1:1:0.8. Stir magnetically to activate the terminal carboxyl group of biotin. Then add vitamin E polyethylene glycol succinate (TPGS) and continue magnetic stirring for 48 h; After dialysis with deionized water for 48 h, freeze-dry to obtain the biotin-vitamin E polyethylene glycol succinate graft copolymer; Similarly, the grafting rate of biotin can be adjusted by adjusting the molar ratio of vitamin E polyethylene glycol succinate to biotin.

[0052] (2) Preparation of targeted drug-loaded micelles: Take the chitosan-deoxycholic acid-mannose graft solution (5 mg / ml) and the biotin-vitamin E polyethylene glycol succinate graft solution (5 mg / ml), and mix them evenly; under magnetic stirring at 600 r / min, add the asiatic acid methanol solution (5 mg / ml), stir for 15 min, evaporate the organic solvent under reduced pressure, and centrifuge at 2000 r / min for 5 min to obtain the asiatic acid nanomicelle dispersion; among them, the chitosan-deoxycholic acid-mannose graft and the biotin-vitamin E polyethylene glycol succinate graft can be mixed evenly in any mass ratio; the particle size of the obtained asiatic acid drug-loaded micelles is 20-150 nm, the surface potential is 18-40 mV, the encapsulation efficiency is greater than 90%, and the drug loading is greater than 12%.

[0053] (3) Preparation of targeted drug-loaded nano-microspheres: Take the Span 80 solution with a concentration of 20 mg / ml, and dropwise add the sodium alginate solution with a concentration of 20 mg / ml (viscosity 200±20 mPa·s) on the liquid surface with a syringe under mechanical stirring, and stir and emulsify at 40±5 °C in a water bath for 2 hours; then slowly add the drug-loaded micelle dispersion under the liquid surface with a syringe and stir mechanically for 15 minutes; then, dropwise add the calcium chloride solution with a concentration of 100 mg / ml and crosslink for 30 minutes; centrifuge the mixed solution at 9000 rpm for 10 minutes, take the lower layer precipitate, wash it repeatedly with petroleum ether 4 times, and then dry it in vacuo to obtain the targeted nano-microsphere particles; the particle size of the asiatic acid drug-loaded nano-microspheres is 40 μm, and the drug loading is greater than 8%.

[0054] The present invention will be described in detail below with reference to the drawings and embodiments, and the purpose and effect of the present invention will become more obvious.

[0055] In the specific implementation manner of the technical solution of the present invention, the main reagents and materials used are introduced as follows: Asiatic Acid (purchased commercially, purity > 98%), Chitosan (purchased commercially, molecular weight 126 KDa, deacetylation degree 88.6%, purity > 90%), Deoxycholic acid (purchased commercially, purity > 98%), 4-Isothiocyanatophenylα-D-Mannopyranoside (Man, purchased commercially, purity > 99%), Biotin (purchased commercially, purity > 99%), Vitamin E Polyethylene Glycol Succinate (TPGS, purchased commercially, vitamin E content > 28%), Sodium alginate (viscosity 200±20 mPa·s, purchased commercially, purity > 90%).

[0056] Example 1: Synthesis of Chitosan-Deoxycholic Acid-Mannose Graft Copolymer

[0057] As Figure 1 shown, 4.5 g of deoxycholic acid and 6.6 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in 250 ml of ethanol-acetone (volume ratio 3:7), and the carboxyl group was activated by magnetic stirring in a water bath at 60±5 °C for 1 h to obtain an organic phase. 7.5 g of chitosan (molecular weight 126 KDa) was dissolved in 500 ml of deionized water to obtain a chitosan aqueous solution. The organic phase was slowly added dropwise to the chitosan aqueous solution, and the reaction was continued for 12 h. Free deoxycholic acid and organic solvents were removed by dialysis with deionized water, and the dialysis solution was freeze-dried to obtain a chitosan-deoxycholic acid graft copolymer.

[0058] 0.15 g of 4-isothiocyanatophenyl-α-D-mannopyranoside was dissolved in 7.5 ml of dimethyl sulfoxide to obtain a 4-isothiocyanatophenyl-α-D-mannopyranoside solution, and 0.25 g of the chitosan-deoxycholic acid graft copolymer was dissolved in 30 ml of deionized water to obtain a chitosan-deoxycholic acid aqueous solution. The 4-isothiocyanatophenyl-α-D-mannopyranoside solution was slowly added to the chitosan-deoxycholic acid aqueous solution, and the mixture was stirred at room temperature for 24 h. The mixture was dialyzed with deionized water and freeze-dried to obtain a chitosan-deoxycholic acid-mannose graft copolymer. The structure was confirmed by 1H NMR spectroscopy ( Figure 1 B). Characteristic peaks of chitosan were observed in a (chitosan), c (chitosan-deoxycholic acid graft copolymer) and e (chitosan-deoxycholic acid-mannose graft copolymer): δ1.9 (-COCH3), δ2.9-4.9 (hydrogens on the benzene ring). The multiplets at δ0.6, 0.8 and 0.9 were the three methyl peaks of deoxycholic acid, which were visible in both b (deoxycholic acid) and c (chitosan-deoxycholic acid graft copolymer). In addition, the carboxyl peak of deoxycholic acid at δ12 in b was not observed in a (chitosan) and c (chitosan-deoxycholic acid graft copolymer), indicating that deoxycholic acid had been successfully grafted onto chitosan. Characteristic peaks of 4-isothiocyanatophenyl-α-D-mannopyranoside included hydrogens on the benzene ring (δ7.0-7.2) and acetal on the sugar ring (δ5.5), which were also visible at the same chemical shifts in e (chitosan-deoxycholic acid-mannose graft copolymer), indicating that 4-isothiocyanatophenyl-α-D-mannopyranoside had been successfully grafted. According to the 1H NMR spectrum, the grafting rate of deoxycholic acid was 6.8% and the grafting rate of mannose was 21%.

[0059] Example 2: Synthesis of Biotin-Vitamin E Polyethylene Glycol Succinate Graft Copolymer

[0060] Weigh 1.5 g of biotin, 1.2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.6 g of 4-dimethylaminopyridine and dissolve them in 60 ml of dimethyl sulfoxide. Magnetically stir to activate the carboxyl group at the end of biotin. Then add 3.0 g of vitamin E polyethylene glycol succinate (TPGS) and continue magnetic stirring for 48 h. After dialyzing the mixture with deionized water for 48 h, freeze-dry to obtain the biotin-vitamin E polyethylene glycol succinate graft. The structure was confirmed by nuclear magnetic resonance hydrogen spectrum ( Figure 2 B). Compared with a (vitamin E polyethylene glycol succinate), the characteristic proton peaks of biotin such as δ 2.34 (2H, brs), 3.10 (1H, brs), 4.32 (1H, m), 6.35 - 6.40 (2H, =N-H, brs) appeared in b (biotin-vitamin E polyethylene glycol succinate graft), confirming that biotin has been successfully attached to the TPGS backbone. The grafting rate of biotin was calculated to be 26% based on the nuclear magnetic resonance hydrogen spectrum.

[0061] Example 3: Preparation of targeted drug-loaded micelles

[0062] Weigh 50 mg of chitosan-deoxycholic acid-mannose graft, dissolve it in 10 ml of deionized water to obtain the carrier aqueous solution. Weigh another 7.5 mg of asiatic acid and dissolve it in 1.5 ml of methanol to obtain the asiatic acid solution. Under magnetic stirring at 600 r / min, add the asiatic acid solution to the carrier aqueous solution, stir for 15 min, evaporate the organic solvent under reduced pressure, and centrifuge at 2000 r / min for 5 min to obtain the asiatic acid nano-micelle dispersion.

[0063] The particle size of this nano-dispersion was 130.1 ± 6.5 nm, and the potential was 37.9 ± 1.5 mV (Malvern Zetasizer Nano-ZS90, the same below); the encapsulation efficiency was 97.5 ± 4.8% (encapsulation efficiency = amount of drug in nanoparticles (mg) / amount of drug dosed (mg) × 100%, the same below); the drug loading was 12.7 ± 0.6% (drug loading = amount of drug in nanoparticles (mg) / total amount of carrier and drug dosed (mg) × 100%, the same below).

[0064] Example 4: Preparation of targeted drug-loaded micelles

[0065] Weigh 37.5 mg of chitosan-deoxycholic acid-mannose graft copolymer, dissolve it in 7.5 ml of deionized water to obtain an aqueous solution of chitosan-deoxycholic acid-mannose graft copolymer; weigh 12.5 mg of biotin-vitamin E polyethylene glycol succinate graft copolymer, dissolve it in 2.5 ml of deionized water to obtain an aqueous solution of biotin-vitamin E polyethylene glycol succinate graft copolymer, and mix the aqueous solution of chitosan-deoxycholic acid-mannose graft copolymer and the aqueous solution of biotin-vitamin E polyethylene glycol succinate graft copolymer to obtain a carrier aqueous solution. Separately, weigh 7.5 mg of asiatic acid, dissolve it in 1.5 ml of methanol to obtain an asiatic acid solution. Under magnetic stirring at 600 r / min, add the asiatic acid solution to the carrier aqueous solution, stir for 15 min, evaporate the organic solvent under reduced pressure, and centrifuge at 2000 r / min for 5 min to obtain an asiatic acid nano-micelle dispersion.

[0066] It was determined that the particle size of this nano-dispersion was 79.2 ± 16.7 nm, the zeta potential was 23.4 ± 1.4 mV; the encapsulation efficiency was 93.1 ± 1.4%; the drug loading was 12.2 ± 0.2%.

[0067] Example 5: Preparation of targeted drug-loaded micelles

[0068] Weigh 25 mg of chitosan-deoxycholic acid-mannose graft copolymer, dissolve it in 5 ml of deionized water to obtain an aqueous solution of chitosan-deoxycholic acid-mannose graft copolymer; weigh 25 mg of biotin-vitamin E polyethylene glycol succinate graft copolymer, dissolve it in 5 ml of deionized water to obtain an aqueous solution of biotin-vitamin E polyethylene glycol succinate graft copolymer, and mix the aqueous solution of chitosan-deoxycholic acid-mannose graft copolymer and the aqueous solution of biotin-vitamin E polyethylene glycol succinate graft copolymer to obtain a carrier aqueous solution. Separately, weigh 7.5 mg of asiatic acid, dissolve it in 1.5 ml of methanol to obtain an asiatic acid solution. Under magnetic stirring at 600 r / min, add the asiatic acid solution to the carrier aqueous solution, stir for 15 min, evaporate the organic solvent under reduced pressure, and centrifuge at 2000 r / min for 5 min to obtain an asiatic acid nano-micelle dispersion.

[0069] It was determined that the particle size of this nano-dispersion was 37.8 ± 7.1 nm, the zeta potential was 21.9 ± 1.6 mV; the encapsulation efficiency was 99.3 ± 7.5%; the drug loading was 13.0 ± 1.0%.

[0070] Example 6: Preparation of targeted drug-loaded micelles

[0071] Weigh 12.5 mg of chitosan-deoxycholic acid-mannose graft copolymer, dissolve it in 2.5 ml of deionized water to obtain an aqueous solution of chitosan-deoxycholic acid-mannose graft copolymer; weigh 37.5 mg of biotin-vitamin E polyethylene glycol succinate graft copolymer, dissolve it in 7.5 ml of deionized water to obtain an aqueous solution of biotin-vitamin E polyethylene glycol succinate graft copolymer, and mix the aqueous solution of chitosan-deoxycholic acid-mannose graft copolymer and the aqueous solution of biotin-vitamin E polyethylene glycol succinate graft copolymer to obtain a carrier aqueous solution. Additionally, weigh 7.5 mg of asiatic acid, dissolve it in 1.5 ml of methanol to obtain an asiatic acid solution. Under magnetic stirring at 600 r / min, add the asiatic acid solution to the carrier aqueous solution, stir for 15 min, evaporate the organic solvent under reduced pressure, and centrifuge at 2000 r / min for 5 min to obtain an asiatic acid nano-micelle dispersion.

[0072] It was determined that the particle size of this nano-dispersion was 26.8 ± 1.5 nm, the zeta potential was 20.4 ± 2.4 mV; the encapsulation efficiency was 95.6 ± 3.5%; the drug loading was 12.5 ± 0.5%.

[0073] Example 7: Preparation of targeted drug-loaded micelles

[0074] Weigh 50 mg of biotin-vitamin E polyethylene glycol succinate graft copolymer, dissolve it in 10 ml of deionized water to obtain an aqueous solution of biotin-vitamin E polyethylene glycol succinate graft copolymer, as the carrier aqueous solution. Additionally, weigh 7.5 mg of asiatic acid, dissolve it in 1.5 ml of methanol to obtain an asiatic acid solution. Under magnetic stirring at 600 r / min, add the asiatic acid solution to the carrier aqueous solution, stir for 15 min, evaporate the organic solvent under reduced pressure, and centrifuge at 2000 r / min for 5 min to obtain an asiatic acid nano-micelle dispersion.

[0075] It was determined that the particle size of this nano-dispersion was 22.8 ± 3.5 nm, the zeta potential was 18.3 ± 0.4 mV; the encapsulation efficiency was 97.6 ± 1.7%; the drug loading was 12.7 ± 0.2%.

[0076] Example 8: Preparation of targeted drug-loaded nano-microspheres

[0077] Take 40 ml of Span 80 solution with a concentration of 20 mg / ml. Under mechanical stirring, add 20 ml of sodium alginate solution with a concentration of 20 mg / ml dropwise onto the liquid surface using a syringe. Stir and emulsify at 40 ± 5 °C in a water bath for 2 hours. Then, slowly add 30 ml of drug-loaded micelle dispersion under the liquid surface using a syringe, and stir mechanically for 15 minutes; then, add 30 ml of calcium chloride solution with a concentration of 100 mg / ml dropwise and crosslink for 30 minutes; centrifuge the mixed solution at 9000 rpm for 10 minutes, take the lower-layer precipitate, wash it repeatedly with petroleum ether 4 times, and then dry it under vacuum to obtain targeted nano-microsphere particles. Scanning electron microscopy shows that the microspheres are nearly circular with a particle size of 40 μm. The drug loading of asiatic acid is determined by high-performance liquid chromatography to be 8.3 ± 1.0% (drug loading = amount of drug in microspheres (mg) / weight of microspheres (mg) × 100%).

[0078] Example 9: Investigation of cell uptake characteristics

[0079] Using coumarin 6 as a fluorescent probe, replacing asiatic acid in Examples 3 - 7 respectively, the uptake of nano-micelles in Caco-2 and Raw 264.7 cells was studied. Caco-2 and Raw 264.7 cells were respectively inoculated into 12-well plates (2 × 10 5 cells per well) and incubated for 12 h, then fluorescently labeled nano-micelles in Examples 3 - 7 were added (final concentration of C6 was 10 μg / ml), and incubated at 37 °C for 5 min, 15 min, 30 min, 60 min, and 120 min respectively. Immediately wash the cells with PBS at 4 °C, add cell lysate, freeze-thaw at -80 °C 3 times repeatedly, and then measure the fluorescence intensity and protein concentration in the centrifuged supernatant, and calculate the uptake percentage. Meanwhile, in order to study the targeting function of the micelles, Caco-2 and Raw 264.7 cells were inoculated into 12-well plates with the same density and in the above steps. After incubation for 12 h, Caco-2 cells were pre-incubated with mannose solution (5 mg / ml) for 2 h, and Raw 264.7 was pre-incubated with biotin solution (5 mg / ml) for 2 h. Then add fluorescently labeled micelles in Examples 3 - 7 and incubate together for 2 h. The remaining operations are the same as above. Figure 4 A shows that after incubation of Examples 3 - 7 in Raw 264.7 macrophages for 2 h, the fluorescence of Example 3 is the strongest, and that of Example 5 is the second. After pre-saturating Raw 264.7 cells with mannose, the fluorescence intensity decreases rapidly (p < 0.01, Figure 4 B). While in Caco-2 cells, Example 7 shows the highest fluorescence intensity( Figure 4 C), and there is no significant difference in the fluorescence intensity between Example 5 and Example 7. After pre-saturating with biotin, the intracellular fluorescence of Example 5 and Example 7 decreases significantly after 1 h (p < 0.001, Figure 4D). These results demonstrate that mannose- or biotin-modified nanoparticles can improve the uptake efficiency in macrophages and colon cells.

[0080] In addition, to visually compare the uptake characteristics, Caco-2 and Raw 264.7 cells were seeded at the same density onto 12-well plates containing glass coverslips. After incubation for 12 h, the cells were pre-saturated with biotin or mannose solution. Then, fluorescently labeled micelles of Examples 3 - 7 (final concentration of C6 was 10 μg / ml) were added and co-incubated for 2 h. The cells were fixed with 4% paraformaldehyde for 15 min and stained with Hoechst 33258 (nuclear dye, 10 μg / ml) and DiI (cell membrane dye, 10 μg / ml) for 10 min respectively. The glass coverslips were inverted onto glass slides and fluorescence was observed. Figure 4 E analyzed the positions of the cell nucleus (blue fluorescence), cell membrane (red fluorescence), and C6 (green fluorescence). The fluorescence of the nanomicelles of Examples 3, 5, and 7 was stronger inside Caco-2 cells. After adding mannose, the intracellular fluorescence value in Example 3 showed no obvious change, while the uptake amount in Example 7 decreased significantly. The uptake ability in Raw 264.7 macrophages showed that after pre-saturation with mannose, the intracellular fluorescence intensity in Example 3 decreased significantly. These results indicate that biotin-modified nanomicelles have an obvious promoting effect on the uptake by intestinal cells, and mannose-modified nanomicelles have a certain targeted uptake ability for macrophages.

[0081] Example 10: In vitro release investigation

[0082] The drug-loaded nanomicelles obtained in "Example 5", the drug-loaded nano-microspheres obtained in "Example 8", and asiatic acid solution (about 1 mg of drug) were placed in a dialysis bag (molecular weight cut-off of 7000), sealed, and then immersed in 20 ml of hydrochloric acid solution (pH 1.2, 1 vol% SDS, simulating the gastric acid environment) at 37 °C with constant shaking at 100 rpm. After 2 h, the release medium was changed to PBS at pH 6.8 (containing 1 vol% SDS, simulating the duodenal environment) with constant shaking at 37 °C. After 6 h, the release medium was changed to PBS at pH 7.4 (containing 1 vol% SDS, simulating the colon environment). Samples of the entire release medium were taken at 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 2 d, 3 d, 4 d, and 7 d, and fresh release medium of the same volume was immediately replenished. The results showed ( Figure 5A), The cumulative release amount of asiatic acid raw material reached nearly 20% in 2 hours and reached the end point (101.2%) within 24 hours; the release of the drug from the nanomicelles was relatively slow, with 10.1% and 20.4% released at 2 hours and 6 hours respectively, and it was basically completely released (92.6%) within 4 days; the release of the drug from the nano-microspheres was slower than the previous two groups. The cumulative release amount at 2 hours in the pH 1.2 solution was only 5.4%, and 78% of the drug was cumulatively released in 7 days, indicating that the nano-microspheres have the characteristics of pH sensitivity and slow drug release.

[0083] The morphological changes of the microspheres during the drug release process were further studied. The nano-microspheres obtained in "Example 8" were soaked in hydrochloric acid solution with pH 1.2, PBS with pH 6.8, and PBS with pH 7.4 for 2 hours respectively, then the residual particles were taken out, sputter-coated with gold, and observed under a scanning electron microscope. The results showed ( Figure 5 B), The nano-microspheres obtained in "Example 8" remained intact spherical after being soaked in the solution with pH 1.2 for 2 hours; in the buffer with pH 6.8, the outer layer of the microspheres was damaged; after being soaked in the buffer with pH 7.4 for 2 hours, the morphology of the microspheres became irregular, indicating that the nano-microspheres have the characteristics of pH response, which helps to protect the encapsulated drug in the acidic environment.

[0084] Example 11: Investigation of in vivo targeting distribution characteristics

[0085] Using the near-infrared dye DiR as a fluorescence probe, asiatic acid in Example 5 and Example 8 was replaced respectively to study the in vivo targeting distribution characteristics of the nanomicelles and nano-microspheres in colitis mice. The mice were randomly divided into 3 groups and were intragastrically administered with free DiR, DiR-loaded nanomicelles, and DiR-loaded nano-microspheres respectively, and the dose was 3 mg / kg based on DiR. The mice were euthanized at 6 hours, 12 hours, and 24 hours after administration respectively. The heart, liver, spleen, lung, kidney, and the whole gastrointestinal tract were collected and photographed with an in vivo imaging system. The fluorescence intensities were compared at the wavelengths of λEx720nm and λEm 790nm.

[0086] As Figure 6 A and Figure 6As shown in C, after intragastric administration of free DiR to mice, strong fluorescence appeared in the stomach. Subsequently, the fluorescence gradually moved towards the intestine, accompanied by a decrease in fluorescence intensity. After 24 h, only weak fluorescence was visible. After 6 h of administration of DiR / nanomicelles, strong fluorescence was shown in the stomach of mice, which might be due to DiR adsorbed on the surface of the nanomicelles or the release of the drug from the nanomicelles in the stomach. At 12 h, the fluorescence intensity in the colon gradually increased, indicating that the micelles could accumulate in the colon directionally. After 24 h, a small amount of fluorescence was still visible in the cecum and colon, indicating that DiR was slowly released from the micelles and the retention time in vivo was longer than that in the free DiR group. Interestingly, at 6 h, 12 h, and 24 h, the fluorescence intensity of the DiR / nano-microsphere group in the cecum and colon was higher than that of the nanomicelle group, which proved that the calcium alginate microspheres protected the encapsulated micelles and prevented premature release of the drug in the stomach. In addition, due to the bioadhesive effect of alginate, the retention time of the drug in the intestine was further prolonged. When the outer shell of the calcium alginate microspheres decomposed in the intestine, the micelles were released and the drug was slowly released. Therefore, the nano-microspheres had the characteristics of precisely delivering the drug to the inflamed site of the colon and releasing the drug at a fixed point.

[0087] Meanwhile, the fluorescence signals of the heart, liver, spleen, lung, and kidney of mice at each time point were compared ( Figure 6 B and Figure 6 D). After oral administration of free DiR, strong fluorescence was observed in the liver and weak fluorescence in the kidney, and no fluorescence was distributed in other organs. Due to the targeting effect of chitosan-deoxycholic acid-mannose graft and biotin-vitamin E polyethylene glycol succinate graft, the fluorescence intensity of DiR / nanomicelles decreased in the liver and increased in the intestine. On the other hand, due to the characteristics of calcium alginate, the accumulation of the DiR / nano-microsphere group in the colon increased further, resulting in a decrease in fluorescence in the liver. These results also confirmed that the prepared calcium alginate microspheres had the ability to specifically accumulate in the colon tissue after oral administration.

[0088] Example 12: In vivo pharmacodynamic investigation of mice with colitis

[0089] The DSS-induced mouse model is a typical animal model of colitis, with pathological characteristics similar to those of humans, such as weight loss, colon shortening, destruction of the colonic epithelial layer, and infiltration of inflammatory cells (Hoffmann, M.; Schwertassek, U.; Seydel, A.; Weber, K.; Falk, W.; Hauschildt, S.; Lehmann, J. A refined and translationally relevant model of chronic DSS colitis in BALB / c mice. Laboratory Animals 2018, 52(3), 240-252.). A chronic colitis model of Balb / c mice (male) was induced using 3 cycles. As Figure 7 shown in A, each cycle consisted of 7 consecutive days of drinking an aqueous solution of DSS at a concentration of 30 mg / ml, followed by 7 consecutive days of drinking water. Subsequently, the mice with colitis models were randomly divided into 3 groups, and a normal group was set up simultaneously: 1) normal group (healthy mice), 2) model group (DSS-induced colitis mice), 3) asiatic acid raw material group (DSS-induced colitis mice were orally administered asiatic acid at a dose of 30 mg / kg per day), 4) asiatic acid nano-microsphere group (DSS-induced colitis mice were orally administered the nano-micelles of Example 8 at a dose of 30 mg / kg per day). During the entire experiment, the body weight, fecal consistency, and fecal bleeding of the animals were monitored twice a week. The disease activity index (DAI) was used to comprehensively evaluate the severity of colitis in mice, including a comprehensive evaluation of weight loss (0-4), fecal consistency status (1-3), and fecal bleeding (1-3). The weight loss scores were as follows: 0, <1%; 1, 1%-5%; 2, 5%-10%; 3, 10%-15%; 4 >15%. The fecal scores were as follows: 1, good granule shape; 2, soft stools, semi-formed, not sticking to the anus; 3, loose stools sticking to the anus. The bleeding scores were: 1 point, no bleeding; 2, occult bleeding; 3, severe bleeding. At the end of the experiment, the mice were sacrificed, and the entire colon (from the cecum to the anus) and major organs were excised. Corresponding colon sections were taken for histological analysis, and the cytokine levels were measured. Blood samples were collected, and the cytokine levels were detected using the corresponding ELISA kits.

[0090] As Figure 7 shown in B, the body weight of normal mice continued to increase during the experiment, while the body weight of the model group mice fluctuated significantly and recovered slightly after the drug withdrawal. This may be due to intestinal damage caused by oral administration of DSS, resulting in weight loss in the model group. The body weight increase of the asiatic acid nano-microsphere group mice was greater than that of the asiatic acid raw material group, indicating a certain therapeutic effect on colitis. Compared with normal mice, the DAI of DSS-treated mice was significantly increased, and the colon was shortened ( Figure 7C-E). In contrast, the DAI of the asiatic acid nano-microsphere group was lower, and the colon length was close to that of the normal group (p > 0.05). As Figure 7 Shown in F-G, there were no obvious inflammations or destructions in the H&E stained sections of the colon of healthy mice. However, the model group showed obvious inflammatory features, such as intestinal epithelial destruction, goblet cell loss, and a large number of inflammatory cell infiltrations in the lamina propria. The morphology of the asiatic acid nano-microsphere group was basically normal, with only mild inflammation and few inflammatory cell infiltrations, effectively improving the abnormal lesions of the colon tissue.

[0091] The intestinal epithelial barrier is crucial in the progression of ulcerative colitis. Colonic goblet cells produce and secrete mucus to maintain the mucosal barrier of the colon. The reduction of tight junction proteins (ZO-1 and Claudin-1) will increase the intestinal permeability, leading to colonic barrier dysfunction. Figure 7 Shown in H, compared with normal cells, the goblet cells in the model group were severely absent, and the number of intact cells decreased. The asiatic acid nano-microsphere group had significant improvements in the reduction of goblet cells and the restoration of mucosal integrity. As Figure 7 Shown in I and 7J, the immunohistochemical staining of ZO-1 and Claudin-1 showed that the immune response in the model group was the weakest, indicating that the intestinal mucosal barrier was damaged. The expressions of ZO-1 and Claudin-1 in the normal group and the asiatic acid nano-microsphere group were significantly increased. It was proved that the nano-microspheres had a protective effect on the intestinal barrier of colitis mice.

[0092] MPO is a peroxidase hydrogen peroxide reductase and an important marker for detecting the severity of mucosal neutrophil infiltration. As Figure 7 Shown in K, 7L, a large number of neutrophils and macrophages infiltrated the colonic mucosa of DSS mice. The inflammatory cell infiltration in the asiatic acid nano-microsphere group was significantly reduced compared with the model group and the asiatic acid raw material group, indicating that the targeted microspheres could improve the treatment effect of colitis.

[0093] In addition, no obvious pathological changes were found in the H&E staining of the main organs of the mice in the asiatic acid nano-microsphere group ( Figure 8 ), which confirmed the safety of oral administration of the nano-microspheres and provided a prerequisite for potential clinical transformation.

[0094] From the perspective of the quantitative detection level of inflammatory factors, the secretion of IL-6, TNF-α, and IL-1β in the colonic tissue of colitis mice was significantly higher than that of the control group, and IL-10 was significantly lower than that of the control group (p < 0.0001, Figure 9 A). These levels in the asiatic acid nano-microsphere group showed significant changes compared with those of the untreated colitis mice (p < 0.001). The levels of IL-6 and TNF-α in the serum of the mice in the asiatic acid nano-microsphere group were significantly lower than those in the model group (p < 0.001, Figure 9 B), which was consistent with the results of the gene expression levelFigure 9 C). All of the above results indicate that the prepared calcium alginate microspheres have good anti-colitis effects.

[0095] The present invention provides a preparation method of a novel targeted nano-microsphere drug delivery system for treating ulcerative colitis. By means of two targeted carrier materials, asiatic acid is encapsulated to form a nano-system, and further encapsulated with calcium alginate to form a nano-microsphere composite system for oral targeted treatment of ulcerative colitis. The preparation process adopted by the present invention is simple, has good reproducibility, the materials are easily available, is suitable for industrial production, does not involve drug structure modification, the particle size distribution of the drug delivery system is controllable, and has good practicability and economy.

Claims

1. A targeted nano-microsphere drug delivery system, characterized in that, the targeted nano-microsphere drug delivery system at least comprises nano-micelles, and the nano-micelles are composed of a mixture of a drug, a chitosan-deoxycholic acid-mannose graft and / or a biotin-vitamin E polyethylene glycol succinate graft; the targeted nano-microsphere drug delivery system further comprises alginate microspheres for coating the nano-micelles; the drug is asiatic acid; the chitosan-deoxycholic acid-mannose graft is obtained by the following method: Activating the carboxyl group on deoxycholic acid, and then dropping the activated deoxycholic acid solution into an aqueous chitosan solution for reaction grafting to obtain a chitosan-deoxycholic acid graft; Adding a 4-isothiocyanatophenyl-α-D-mannopyranoside solution to an aqueous chitosan-deoxycholic acid solution for reaction grafting to obtain a chitosan-deoxycholic acid-mannose graft; The biotin-vitamin E polyethylene glycol succinate graft is obtained by the following method: Activating the terminal carboxyl group of biotin, and then adding vitamin E polyethylene glycol succinate for mixing reaction grafting to obtain a biotin-vitamin E polyethylene glycol succinate graft.

2. The system according to claim 1, characterized in that, the mass ratio of the drug, the chitosan-deoxycholic acid-mannose graft, and the biotin-vitamin E polyethylene glycol succinate graft is 1.5:0 to 10:0 to 10; wherein, the mass of at least one of the chitosan-deoxycholic acid-mannose graft and the biotin-vitamin E polyethylene glycol succinate graft is not 0.

3. The system according to claim 1, characterized in that, the molar ratio of deoxycholic acid to chitosan monomer is 1:4, and the molar ratio of 4-isothiocyanatophenyl-α-D-mannopyranose to chitosan-deoxycholic acid monomer is 1:

3.

4. A preparation method of the system according to any one of claims 1-3, characterized in that, the method is: Preparing an aqueous solution of a chitosan-deoxycholic acid-mannose graft and / or a biotin-vitamin E polyethylene glycol succinate graft as a carrier aqueous solution and a drug solution, adding the drug solution to the carrier aqueous solution, and mixing evenly to obtain nano-micelles.

5. The preparation method according to claim 4, characterized in that, further comprising: Taking a Span 80 solution with a concentration of 20 mg / ml, adding 20 ml of an alginate solution with a concentration of 20 mg / ml dropwise on the liquid surface with mechanical stirring, stirring and emulsifying in a water bath at 40-45 °C; then adding 30 ml of a nano-micelle dispersion liquid under the liquid surface with a syringe, and stirring and mixing evenly; then, dropping 30 ml of a calcium chloride solution with a concentration of 100 mg / ml for crosslinking; centrifuging the mixed solution to take the lower layer precipitate for washing and drying to obtain targeted nano-microsphere particles coated with nano-micelles by alginate microspheres.

6. Use of the system according to any one of claims 1-3 in the preparation of a drug for treating ulcerative colitis.

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