CN116270490A - Targeted nano-microsphere drug delivery system, preparation method and application - Google Patents
Targeted nano-microsphere drug delivery system, preparation method and application Download PDFInfo
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- CN116270490A CN116270490A CN202310306278.0A CN202310306278A CN116270490A CN 116270490 A CN116270490 A CN 116270490A CN 202310306278 A CN202310306278 A CN 202310306278A CN 116270490 A CN116270490 A CN 116270490A
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Abstract
The invention discloses a targeting nano-microsphere drug delivery system, a preparation method and application thereof, wherein the drug delivery system preparation comprises a nano micelle formed by mixing a drug, chitosan-deoxycholic acid-mannose grafts and/or biotin-vitamin E polyethylene glycol succinate grafts, so as to realize targeted aggregation of the drug at a colon inflammation part; and then the nano micelle is wrapped by calcium alginate to prepare the composite microsphere with pH responsiveness, so that the colon site-specific drug release is realized, and the composite microsphere is used for oral administration to treat ulcerative colitis. The drug is preferably asiatic acid. The nanometer-microsphere drug delivery system has controllable particle size distribution, can reduce the damage of drugs in the stomach, has a certain colon targeting effect, and is a novel ulcerative colitis drug delivery system.
Description
Technical Field
The invention relates to the technical field of pharmaceutical preparations, in particular to a targeting nano-microsphere drug delivery system, a preparation method and application thereof in preparing a medicament for treating ulcerative colitis.
Background
Ulcerative colitis (Ulcerative Colitis, UC) is a common inflammatory disease of the colon that is difficult to cure due to repeated attacks of disease course and has a tendency to become cancerous, severely jeopardizing public health. Currently, first-line therapeutic drugs for UC are mainly anti-inflammatory drugs, immunosuppressants and biological products, but the overall therapeutic effect is not very satisfactory, and the toxic and side effects are large (nephrotoxicity, hepatotoxicity, neurotoxicity, etc.). In addition, due to diarrhea caused by ulcerative colitis, the residence time of drug delivery systems (including clinically used drugs) with particles larger than 200 μm in the gastrointestinal tract can be shortened, resulting in reduced efficacy. Therefore, there is a need for new drug delivery systems that can deliver drugs targeted to areas of colonic inflammation, improve efficacy and reduce side effects.
The traditional Chinese medicine is a magnificent of Chinese culture and has a long history. The Chinese herbal medicine and the extract thereof with various pharmacological activities have wide application in inflammation-related diseases, and are the first choice of medicines for supplementing and replacing colon inflammation. Asiatic Acid (AA) is a pentacyclic triterpenic acid separated from centella asiatica (Centella asiatica L. Urban) which is a medicinal plant, has various pharmacological effects of anti-inflammatory, antioxidant, anticancer and the like, and has potential preventive and therapeutic effects on enteritis. However, asiatic acid has a low water solubility, rapid elimination in vivo, short half-life, and extremely low oral bioavailability, and its clinical application is greatly limited. In order to solve the above problems, the patent CN 105477001B prepares the solid lipid nanoparticle of tromethamine salt of centella asiatica, which improves the solubility and oral absorption of centella asiatica, but involves structural modification, and may have potential safety hazard. Nano-carriers for delivering traditional Chinese medicines such as asiatic acid are reported, and as patent CN 103054804A and patent CN 102784096A respectively disclose drug delivery systems such as asiatic acid solid dispersion and microemulsion; centella asiatica micelle and nano lipid carrier (CN 107638388B, CN 105919976B) are also prepared in the early stage of the laboratory, and the oral bioavailability is obviously improved. However, the above nano-preparation has the defects of insufficient targeting and incapability of positioning and releasing the drug, so that local drug concentration with cytotoxicity cannot be achieved, the enrichment amount of the drug at an inflammation part is low, and serious systemic side effects can be caused when the systemic blood drug concentration is even improved, and the in-vivo efficacy is still to be confirmed.
Modification of specific targeting ligands on nanocarrier surfaces is an effective method to increase localized drug retention and enrichment. In the pathological course of ulcerative colitis, a large number of macrophages are activated and are highly expressed in the inflammatory response of the colon. Mannose receptors are recognition receptors that are predominantly present on the surface of macrophage membranes. Thus, mannose-modified polymeric micelles can be used as macrophage targeting molecules, selectively accumulating at sites of inflammation. In addition, the intestinal tract is the main site of drug absorption and utilization, and thus, intestinal epithelial cell targeted drug delivery systems have become another important strategy for improving the bioavailability of oral drugs.
In addition, due to the particular environment and complexity of the gastrointestinal tract, many drugs and nanocarriers are degraded by gastric acid and proteases before reaching the colon. Thus, oral nanosystems must overcome the barrier to intragastric instability. Sodium alginate is a natural polysaccharide with good adhesiveness, biodegradability and biocompatibility. Because the structure contains a large amount of carboxyl groups, the molecular chain segment of the sodium alginate contracts in an acidic environment and expands in a neutral and alkaline environment, and has pH sensitivity. In addition, G units of sodium alginate are linked to Ca 2+ The plasma reaction forms a dense network structure, which can protect the wrapped medicine from being damaged by gastric acid and fully release in the intestinal environment.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and provides a targeting nano-microsphere drug delivery system, a preparation method and application thereof in preparing medicines for treating ulcerative colitis.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the targeted nano-microsphere drug delivery system at least comprises nano-micelles, wherein the nano-micelles are formed by mixing a drug, chitosan-deoxycholic acid-mannose grafts and/or biotin-vitamin E polyethylene glycol succinate grafts.
The invention plays a role in colonitis targeting by the following mechanism:
the invention utilizes the structural characteristics that chitosan-deoxycholic acid-mannose grafts and biotin-vitamin E polyethylene glycol succinate grafts are self-assembled into nano-micelles in water, can be used as a carrier material of a nano system, and has the function of targeting macrophages and intestinal epithelial cells at inflammation positions, thereby improving the local drug concentration of colon lesion tissues.
Further, the medicine is asiatic acid.
Further, the particle size of the nano micelle is 20-150 nm, the encapsulation efficiency is more than 90%, and the drug loading rate is more than 12%.
Further, the mass ratio of the medicine to the chitosan-deoxycholic acid-mannose graft to the biotin-vitamin E polyethylene glycol succinate graft is 1.5:0-10:0-10. Wherein the mass of at least one of chitosan-deoxycholic acid-mannose graft and biotin-vitamin E polyethylene glycol succinate graft is not 0.
Further, the chitosan-deoxycholic acid-mannose graft is prepared by the following method:
activating carboxyl on deoxycholic acid, and then dripping the activated deoxycholic acid solution into chitosan aqueous solution for reaction grafting to obtain chitosan-deoxycholic acid graft;
adding the 4-isothiocyanate-alpha-D-mannopyranoside solution into chitosan-deoxycholic acid aqueous solution for reaction grafting to obtain the chitosan-deoxycholic acid-mannose graft.
Further, the method for activating the carboxyl group on deoxycholic acid specifically comprises the following steps: dissolving deoxycholic acid and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride in an ethanol-acetone (volume ratio is 3:7) mixed solvent, wherein the concentration of deoxycholic acid is 18mg/ml, the molar ratio of deoxycholic acid to 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:3, and magnetically stirring in a water bath at 60-65 ℃ for 1h to activate carboxyl.
Further, the concentration of the deoxycholic acid solution after activation was 18mg/ml, the concentration of the chitosan aqueous solution was 15mg/ml, the concentration of the 4-isothiocyanatophenyl-alpha-D-mannopyranoside solution was 20mg/ml, and the concentration of the chitosan-deoxycholic acid aqueous solution was 8.3mg/ml.
Further, the mol ratio of deoxycholic acid to chitosan monomer is 1:4, and the mol ratio of 4-isothiocyanate-alpha-D-mannopyranose to chitosan-deoxycholic acid monomer is 1:3.
Further, the biotin-vitamin E polyethylene glycol succinate graft is prepared and obtained by the following method:
activating the terminal carboxyl of biotin, adding vitamin E polyethylene glycol succinate, mixing, reacting and grafting to obtain the biotin-vitamin E polyethylene glycol succinate graft.
Further, the method for activating the terminal carboxyl group of the biotin specifically comprises the following steps:
dissolving biotin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine in dimethyl sulfoxide, and stirring to activate terminal carboxyl of the biotin; wherein the mol ratio of biotin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine is 1:1:0.8, and the concentration of biotin is 25mg/ml.
Further, the targeting nano-microsphere drug delivery system also comprises sodium alginate microspheres for coating the nano-micelles.
The nano-microsphere compound is formed by further encapsulating nano-micelle with alginate, the pH sensitive characteristic of the alginate (molecular chain segment is contracted in an acidic environment to protect the stability of the encapsulated drug in gastric acid, ionization phenomenon occurs in neutral and slightly alkaline environments to slowly release the encapsulated drug) is utilized, so that the formed compound microsphere has pH responsiveness, the release of the drug in the stomach and small intestine can be reduced, the sustained and slow release of the drug in the colon environment is realized, the effect of targeted drug release in the colon is realized, the effective drug accumulation in the colon is further increased, the ulcerative colitis is effectively treated, and the systemic side effect is reduced.
Further, the particle size of the nano-microsphere composite is 40 mu m, and the drug loading is more than 8%.
The preparation method of the system comprises the following steps:
preparing an aqueous solution of chitosan-deoxycholic acid-mannose graft and/or biotin-vitamin E polyethylene glycol succinate graft as a carrier aqueous solution and a drug solution, adding the drug solution into the carrier aqueous solution, and uniformly mixing to obtain the nano micelle.
Further, the concentration of the carrier aqueous solution and the drug solution is 5mg/ml, and the carrier aqueous solution and the drug solution are mixed according to the requirement in proportion.
Further, the method further comprises the following steps:
taking span 80 solution with the concentration of 20mg/ml, dropwise adding 20ml of sodium alginate solution with the concentration of 20mg/ml on the liquid surface by using a syringe under mechanical stirring, and stirring and emulsifying under water bath with the temperature of 40-45 ℃; adding 30ml of nano micelle dispersion liquid under the liquid surface by using a syringe, and stirring and uniformly mixing; then, 30ml of calcium chloride solution with the concentration of 100mg/ml is added dropwise for crosslinking; and centrifuging the mixed solution, taking out the lower layer precipitate, cleaning and drying to obtain the targeted nano microsphere particles coated with the sodium alginate microspheres and nano micelles.
An application of the system in preparing a medicament for treating ulcerative colitis.
The beneficial effects of the invention are as follows: the invention has simple operation, controllable quality and good repeatability, and does not need chemical structure modification. The particle size of the asiatic acid nano system prepared by the invention is 20-150 nm, the encapsulation efficiency is more than 90%, and the drug loading rate is more than 12%; the obtained asiatic acid nanometer-microsphere system has a particle size of 40 μm and a drug loading rate of more than 8%. In addition, the nano-microsphere system has good targeting effect on colon inflammation parts, good safety, solves the problems that the traditional nano medicine cannot target the colon, the local medicine concentration is low and the like, can be used for targeted treatment of colon inflammatory diseases, and has good market prospect.
Drawings
FIG. 1 shows a synthetic route (A) and a nuclear magnetic resonance hydrogen spectrum (B) of chitosan-deoxycholic acid-mannose grafts; in the figure, a represents chitosan, b represents deoxycholic acid, c represents chitosan-deoxycholic acid graft, D represents 4-isothiocyanate-alpha-D-mannopyranoside, and e represents chitosan-deoxycholic acid-mannose graft;
FIG. 2 shows the synthetic route (A) and nuclear magnetic resonance hydrogen spectrum (B) for biotin-vitamin E polyethylene glycol succinate grafts; in the figure, a represents vitamin E polyethylene glycol succinate and b represents biotin-vitamin E polyethylene glycol succinate grafts;
FIG. 3 is a particle size distribution diagram (A) of the nano-micelle of example 5; a potential map (B); a transmission electron microscope image (C);
FIG. 4 is a graph showing the results of uptake characteristics of nanomicelles of examples 3 to 7 in Raw 264.7 macrophages and Caco-2 cells: (A) Quantitative uptake patterns of micelles on Raw 264.7 macrophages; (B) Quantitative uptake profile of micelles in mannose pre-saturated Raw 264.7 macrophages; (C) is a quantitative uptake map of micelles in Caco-2 cells; (D) Quantitative uptake patterns of micelles in biotin-presaturated Caco-2 cells; (E) Fluorescence images of micelle uptake in Raw 264.7 macrophages and Caco-2 cells;
FIG. 5 is a scanning electron microscope (B) of in vitro release profile (A) and nano-microspheres immersed for 2h in different pH media; in the figure, a represents non-soaked microspheres, b represents microspheres soaked in a pH 1.2 solution for 2 hours, c represents microspheres soaked in a pH 6.8 buffer for 2 hours, and d represents microspheres soaked in a pH 7.4 buffer for 2 hours;
FIG. 6 is an in vivo organ profile of nanomicelle and nano-microsphere; (A) Fluorescence images of the gastrointestinal tract of mice at different time points (6 h, 12h, 24 h); (B) Fluorescence images of mice heart, liver, spleen, lung, kidney at different time points (6 h, 12h, 24 h); (C) is a colon semi-quantitative fluorescence statistical map; (D) a semi-quantitative fluorescence statistical map of the liver;
FIG. 7 is a graph showing the results of pharmacodynamic evaluation of centella asiatica nano-microspheres in a colitis model mouse; wherein, (A) is a schematic diagram of the induction and treatment process of colitis; (B) is a weight change curve; (C) is a disease activity index; (D) is a comparison of colon length of mice; (E) is a quantitative analysis of colon length; (F and G) staining representative graphs (10X, F; 20X, G) for each set of colon tissue H & E; (H) staining representative figures for colon tissue PAS; (I) is an immunohistochemical ZO-1 analysis chart; (J) is an immunohistochemical Claudin-1 profile; (K) Immunofluorescence of neutrophil infiltration for colon sections (green for neutrophils; blue for nuclei); (L) is a colonic intramucosal macrophage infiltration immunofluorescence graph (red for macrophages; blue for nuclei);
FIG. 8 is an H & E staining chart of major viscera;
FIG. 9 is a graph showing the effect of asiatic acid nano-microspheres on inflammatory factors in mice with colon inflammation model, wherein (A) is the change of inflammatory factors in colon tissue; (B) is a change in inflammatory factors in serum; (C) is the variation in mRNA expression in colon tissue.
Detailed Description
The invention relates to a novel targeting nano-microsphere drug delivery system for treating ulcerative colitis, which at least comprises nano micelle, wherein a nano micelle system comprises the following components: centella asiatica, chitosan-deoxycholic acid-mannose grafts, biotin-vitamin E polyethylene glycol succinate grafts, and the like; the targeted nano-microsphere drug delivery system also comprises sodium alginate microspheres for coating nano micelles, wherein the microsphere system comprises the following components: the snow accumulating oxalic acid nano micelle dispersion liquid, span 80, sodium alginate and calcium chloride.
The preparation method of the novel targeted nano-microsphere drug delivery system for treating ulcerative colitis comprises the following steps:
(1) Preparing an aqueous solution of chitosan-deoxycholic acid-mannose graft and/or biotin-vitamin E polyethylene glycol succinate graft as a carrier aqueous solution:
(1.1) Synthesis of chitosan-deoxycholic acid-mannose graft:
activation of carboxyl groups on deoxycholic acid: deoxycholic acid and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride are dissolved in ethanol-acetone (volume ratio 3:7) to obtain the concentration of 18mg/ml and 26.4mg/ml respectively, the mol ratio of deoxycholic acid to 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1:3, and the mixture is magnetically stirred in a water bath at 60+/-5 ℃ for 1h to activate carboxyl.
Slowly dripping the mixture into the chitosan water solution, and continuing to react for 12 hours; and (3) dialyzing with deionized water to remove free deoxycholic acid and an organic solvent, and freeze-drying the dialyzate to obtain the chitosan-deoxycholic acid graft. In general, the concentration of the chitosan aqueous solution is 15mg/ml, and the grafting rate of deoxycholic acid can be adjusted by adjusting the molar ratio of deoxycholic acid to chitosan monomer.
Dissolving 4-isothiocyanate-alpha-D-mannopyranoside in dimethyl sulfoxide, slowly adding into chitosan-deoxycholate aqueous solution, stirring at room temperature for 24h with the molar ratio of 4-isothiocyanate-alpha-D-mannopyranose to chitosan-deoxycholate monomer being 1:3, dialyzing with deionized water, and freeze-drying to obtain chitosan-deoxycholate-mannose graft; similarly, the grafting ratio of mannose can be adjusted by adjusting the molar ratio of 4-isothiocyanatophenyl-alpha-D-mannopyranose to chitosan-deoxycholic acid monomer. Wherein, the concentration of the 4-isothiocyanate-alpha-D-mannopyranoside solution is generally 20mg/ml, and the concentration of the chitosan-deoxycholate aqueous solution is 8.3mg/ml.
(1.2) Synthesis of Biotin-vitamin E polyethylene glycol succinate graft:
biotin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 4-dimethylaminopyridine are dissolved in dimethyl sulfoxide at concentrations of 25mg/ml, 20mg/ml and 10mg/ml respectively, the molar ratio of biotin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride to 4-dimethylaminopyridine is 1:1:0.8, and the biotin terminal carboxyl group is activated by magnetic stirring. Adding vitamin E polyethylene glycol succinate (TPGS), and continuing to magnetically stir for 48 hours; dialyzing with deionized water for 48h, and freeze-drying to obtain biotin-vitamin E polyethylene glycol succinate graft; likewise, the grafting ratio of biotin can be adjusted by adjusting the molar ratio of vitamin E polyethylene glycol succinate to biotin.
(2) Preparing a targeted drug-loaded micelle: mixing chitosan-deoxycholic acid-mannose graft solution (5 mg/ml) with biotin-vitamin E polyethylene glycol succinate graft solution (5 mg/ml); adding asiatic acid methanol solution (5 mg/ml) under magnetic stirring at 600r/min, stirring for 15min, evaporating the organic solvent under reduced pressure, and centrifuging at 2000r/min for 5min to obtain asiatic acid nano micelle dispersion; wherein, the chitosan-deoxycholic acid-mannose graft and the biotin-vitamin E polyethylene glycol succinate graft can be uniformly mixed according to any mass ratio; the particle diameter of the obtained asiatic acid drug-loaded micelle is 20-150 nm, the surface potential is 18-40 mV, the encapsulation efficiency is more than 90%, and the drug loading rate is more than 12%.
(3) Preparation of targeted drug-loaded nano-microspheres: taking span 80 solution with the concentration of 20mg/ml, dropwise adding sodium alginate solution with the concentration of 20mg/ml (with the viscosity of 200+/-20 mpa x s) on the liquid surface by using a syringe under mechanical stirring, and stirring and emulsifying for 2 hours in a water bath with the temperature of 40+/-5 ℃; slowly adding the drug-loaded micelle dispersion liquid under the liquid surface by using a syringe, and mechanically stirring for 15 minutes; then, dropwise adding a calcium chloride solution with the concentration of 100mg/ml, and crosslinking for 30 minutes; centrifuging the mixed solution at 9000rpm for 10 minutes, taking the lower layer precipitate, adding petroleum ether, repeatedly cleaning for 4 times, and vacuum drying to obtain targeted nano-microsphere particles; the particle diameter of asiatic acid drug-loaded nano-microsphere is 40 mu m, and the drug-loaded quantity is more than 8%.
The objects and effects of the present invention will become more apparent from the following detailed description of the present invention with reference to the accompanying drawings and examples.
In the specific implementation mode of the technical scheme of the invention, the main reagents and materials adopted are as follows: asiatic Acid (commercially available with purity > 98%), chitosan (Chitosan, commercially available with molecular weight 126kDa, degree of deacetylation 88.6%, purity > 90%), deoxycholic Acid (Deoxycholic Acid, commercially available with purity > 98%), 4-isothiocyanatophenolide (4-isothiophenyl alpha-D-Mannopyranoside, man, commercially available with purity > 99%), biotin (Biotin, commercially available with purity > 99%), vitamin E polyethylene glycol succinate (Vitamin E Polyethylene Glycol Succinate, TPGS, commercially available with vitamin E content > 28%), sodium Alginate (Alginate sodium, viscosity 200+ -20 mpa, commercially available with purity > 90%).
Example 1: synthesis of chitosan-deoxycholic acid-mannose graft
As shown in FIG. 1, 4.5g of deoxycholic acid and 6.6g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride are weighed and dissolved in 250ml of ethanol-acetone (volume ratio 3:7), and the mixture is magnetically stirred in a water bath at 60+ -5 ℃ for 1h to activate carboxyl groups, so as to obtain an organic phase. Weighing 7.5g of chitosan (with the molecular weight of 126 kDa), and adding 500ml of deionized water for dissolution to obtain a chitosan aqueous solution. The organic phase was slowly added dropwise to the aqueous chitosan solution and the reaction continued for 12h. And (3) dialyzing with deionized water to remove free deoxycholic acid and an organic solvent, and freeze-drying the dialyzate to obtain the chitosan-deoxycholic acid graft.
Weighing 0.15g of 4-phenyl isothiocyanate-alpha-D-mannopyranoside, dissolving in 7.5ml of dimethyl sulfoxide to obtain a 4-phenyl isothiocyanate-alpha-D-mannopyranoside solution, dissolving 0.25g of chitosan-deoxycholic acid graft in 30ml of deionized water to obtain a chitosan-deoxycholic acid aqueous solution, slowly adding the 4-phenyl isothiocyanate-alpha-D-mannopyranoside solution into the chitosan-deoxycholic acid aqueous solution, stirring at room temperature for 24 hours, dialyzing the mixture with deionized water, and freeze-drying to obtain the chitosan-deoxycholic acid-mannose graft. The characteristic peaks of the chitosan were confirmed by nuclear magnetic resonance hydrogen spectrum (FIG. 1B), and δ1.9 (-COCH 3), δ2.9-4.9 (hydrogen on benzene ring) were seen in a (chitosan), c (chitosan-deoxycholic acid graft) and e (chitosan-deoxycholic acid-mannose graft). The multiple peaks at δ0.6, 0.8 and 0.9 are the three methyl peaks of deoxycholic acid, visible in both b (deoxycholic acid) and c (chitosan-deoxycholic acid grafts). Furthermore, δ12 in b is the carboxyl peak of deoxycholic acid, but this peak is not seen in both a (chitosan) and c (chitosan-deoxycholic acid grafts), indicating that deoxycholic acid has been successfully grafted onto chitosan. Characteristic peaks of 4-isothiocyanatophenyl-alpha-D-mannopyranoside include hydrogen on the benzene ring (delta 7.0-7.2) and acetal on the sugar ring (delta 5.5), which is also seen at the same chemical shift of e (chitosan-deoxycholic acid-mannose graft), indicating that 4-isothiocyanatophenyl-alpha-D-mannopyranoside has been successfully grafted. And according to the nuclear magnetic resonance hydrogen spectrogram, the grafting rate of deoxycholic acid is 6.8%, and the grafting rate of mannose is 21%.
Example 2: synthesis of biotin-vitamin E polyethylene glycol succinate graft
1.5g of biotin, 1.2g of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and 0.6g of 4-dimethylaminopyridine were weighed and dissolved in 60ml of dimethyl sulfoxide, and the carboxyl group at the tail end of biotin was activated by magnetic stirring. Then, 3.0g of vitamin E polyethylene glycol succinate (TPGS) was added thereto, and the mixture was magnetically stirred for 48 hours. And dialyzing the mixture with deionized water for 48 hours, and freeze-drying to obtain the biotin-vitamin E polyethylene glycol succinate graft. The structure was confirmed by nuclear magnetic resonance hydrogen spectroscopy (fig. 2B). Compared with a (vitamin E polyethylene glycol succinate), b (biotin-vitamin E polyethylene glycol succinate graft) shows characteristic proton peaks belonging to biotin such as delta 2.34 (2H, brs), 3.10 (1H, brs), 4.32 (1H, m), 6.35-6.40 (2H, =N-H, brs) and the like, and the successful grafting of biotin to TPGS skeleton is confirmed. And the grafting rate of the biotin is calculated to be 26% according to a nuclear magnetic resonance hydrogen spectrogram.
Example 3: preparation of targeted drug-loaded micelle
50mg of chitosan-deoxycholic acid-mannose graft is weighed, and 10ml of deionized water is added for dissolution to be used as a carrier aqueous solution. And weighing 7.5mg of asiatic acid, and adding 1.5ml of methanol for dissolving to obtain asiatic acid solution. Adding asiatic acid solution into carrier water solution under magnetic stirring at 600r/min, stirring for 15min, evaporating under reduced pressure to remove organic solvent, and centrifuging at 2000r/min for 5min to obtain asiatic acid nano micelle dispersion.
The particle size of the Nano dispersion liquid is 130.1+/-6.5 nm, the potential is 37.9+/-1.5 mV (Markov Zetasizer Nano-ZS90, the following is the same); the encapsulation efficiency was 97.5±4.8% (encapsulation efficiency=drug amount (mg) in nanoparticle)/dose amount (mg) ×100%, the same applies below); the drug loading was 12.7±0.6% (drug loading=drug amount in nanoparticle (mg)/total drug input amount of carrier and drug (mg) ×100%, the same applies below).
Example 4: preparation of targeted drug-loaded micelle
Weighing 37.5mg of chitosan-deoxycholic acid-mannose graft, and adding 7.5ml of deionized water for dissolution to obtain chitosan-deoxycholic acid-mannose graft aqueous solution; 12.5mg of biotin-vitamin E polyethylene glycol succinate graft is weighed, 2.5ml of deionized water is added for dissolution to obtain a biotin-vitamin E polyethylene glycol succinate graft aqueous solution, and the chitosan-deoxycholic acid-mannose graft aqueous solution and the biotin-vitamin E polyethylene glycol succinate graft aqueous solution are uniformly mixed to be used as a carrier aqueous solution. And weighing 7.5mg of asiatic acid, and adding 1.5ml of methanol for dissolving to obtain asiatic acid solution. Adding asiatic acid solution into carrier water solution under magnetic stirring at 600r/min, stirring for 15min, evaporating under reduced pressure to remove organic solvent, and centrifuging at 2000r/min for 5min to obtain asiatic acid nano micelle dispersion.
The particle size of the nano dispersion liquid is 79.2+/-16.7 nm, and the potential is 23.4+/-1.4 mV; the encapsulation efficiency is 93.1+/-1.4%; the drug loading rate is 12.2+/-0.2 percent.
Example 5: preparation of targeted drug-loaded micelle
Weighing 25mg of chitosan-deoxycholic acid-mannose graft, and adding 5ml of deionized water for dissolution to obtain chitosan-deoxycholic acid-mannose graft aqueous solution; weighing 25mg of biotin-vitamin E polyethylene glycol succinate graft, adding 5ml of deionized water for dissolution to obtain biotin-vitamin E polyethylene glycol succinate graft aqueous solution, and uniformly mixing chitosan-deoxycholic acid-mannose graft aqueous solution and biotin-vitamin E polyethylene glycol succinate graft aqueous solution to obtain carrier aqueous solution. And weighing 7.5mg of asiatic acid, and adding 1.5ml of methanol for dissolving to obtain asiatic acid solution. Adding asiatic acid solution into carrier water solution under magnetic stirring at 600r/min, stirring for 15min, evaporating under reduced pressure to remove organic solvent, and centrifuging at 2000r/min for 5min to obtain asiatic acid nano micelle dispersion.
The particle size of the nano dispersion liquid is determined to be 37.8+/-7.1 nm, and the potential is determined to be 21.9+/-1.6 mV; the encapsulation efficiency is 99.3+/-7.5%; the drug loading rate is 13.0+/-1.0 percent.
Example 6: preparation of targeted drug-loaded micelle
Weighing 12.5mg of chitosan-deoxycholic acid-mannose graft, and adding 2.5ml of deionized water for dissolution to obtain an aqueous solution of the chitosan-deoxycholic acid-mannose graft; weighing 37.5mg of biotin-vitamin E polyethylene glycol succinate graft, adding 7.5ml of deionized water for dissolution to obtain a biotin-vitamin E polyethylene glycol succinate graft aqueous solution, and uniformly mixing the chitosan-deoxycholic acid-mannose graft aqueous solution and the biotin-vitamin E polyethylene glycol succinate graft aqueous solution to serve as a carrier aqueous solution. And weighing 7.5mg of asiatic acid, and adding 1.5ml of methanol for dissolving to obtain asiatic acid solution. Adding asiatic acid solution into carrier water solution under magnetic stirring at 600r/min, stirring for 15min, evaporating under reduced pressure to remove organic solvent, and centrifuging at 2000r/min for 5min to obtain asiatic acid nano micelle dispersion.
The particle size of the nano dispersion liquid is 26.8+/-1.5 nm, and the potential is 20.4+/-2.4 mV; the encapsulation efficiency is 95.6+/-3.5%; the drug loading rate is 12.5+/-0.5 percent.
Example 7: preparation of targeted drug-loaded micelle
50mg of biotin-vitamin E polyethylene glycol succinate graft is weighed, 10ml of deionized water is added for dissolution to obtain biotin-vitamin E polyethylene glycol succinate graft aqueous solution as carrier aqueous solution. And weighing 7.5mg of asiatic acid, and adding 1.5ml of methanol for dissolving to obtain asiatic acid solution. Adding asiatic acid solution into carrier water solution under magnetic stirring at 600r/min, stirring for 15min, evaporating under reduced pressure to remove organic solvent, and centrifuging at 2000r/min for 5min to obtain asiatic acid nano micelle dispersion.
The particle size of the nano dispersion liquid is 22.8+/-3.5 nm, and the potential is 18.3+/-0.4 mV; the encapsulation efficiency is 97.6+/-1.7%; the drug loading rate is 12.7+/-0.2 percent.
Example 8: preparation of targeted drug-loaded nano microsphere
Taking 40ml of span 80 solution with the concentration of 20mg/ml, dropwise adding 20ml of sodium alginate solution with the concentration of 20mg/ml on the liquid surface by using a syringe under mechanical stirring, and stirring and emulsifying for 2 hours in a water bath with the temperature of 40+/-5 ℃. Slowly adding 30ml of the drug-loaded micelle dispersion liquid under the liquid surface by using a syringe, and mechanically stirring for 15 minutes; then, 30ml of calcium chloride solution with the concentration of 100mg/ml is added dropwise for crosslinking for 30 minutes; and (3) centrifuging the mixed solution at 9000rpm for 10 minutes, taking the precipitate at the lower layer, adding petroleum ether, repeatedly cleaning for 4 times, and drying in vacuum to obtain the targeted nano microsphere particles. The microspheres are observed to be nearly circular by adopting a scanning electron microscope, and the particle size is 40 mu m. The high performance liquid chromatography determines the asiatic acid drug loading of 8.3 + -1.0% (drug loading = amount of drug in microsphere (mg)/weight of microsphere (mg). Times.100%).
Example 9: investigation of cell uptake characteristics
The uptake of nanomicelles in Caco-2 and Raw 264.7 cells was studied using coumarin 6 as a fluorescent probe instead of asiatic acid in examples 3 to 7, respectively. Caco-2 and Raw 264.7 cells were seeded into 12-well plates (2X 10 per well) 5 Cells) were incubated for 12h, then the fluorescent-labeled nanomicelles of examples 3 to 7 (C6 final concentration 10. Mu.g/ml) were added, and incubated at 37℃for 5min, 15min, 30min, 60min, 120min, respectively. Immediately using 4 ℃ PBS washing cells, adding cell lysate, 80 ℃ repeatedly freezing and thawing for 3 times, measuring the fluorescence intensity and protein concentration in the supernatant, and calculating the uptake percentage. Meanwhile, to investigate the targeting function of micelles, caco-2 and Raw 264.7 cells were seeded onto 12-well plates at the same density and the above procedure. After 12h incubation, caco-2 cells were pre-incubated with mannose solution (5 mg/ml) for 2h and Raw 264.7 with biotin solution (5 mg/ml) for 2h. The micelles of examples 3 to 7, which were fluorescently labeled, were then added and incubated for 2 hours. The rest of the operations are the same as described above. FIG. 4A shows that examples 3-7 fluoresce the most strongly in example 3 and example 5 times after 2h incubation of Raw 264.7 macrophages. After pre-saturation of Raw 264.7 cells with mannose, the fluorescence intensity rapidly decreased (p < 0.01, FIG. 4B). Whereas in Caco-2 cells, example 7 showed the highest fluorescence intensity (FIG. 4C), and there was no significant difference in fluorescence intensity between example 5 and example 7. After pre-saturation with biotin, the intracellular fluorescence of example 5 and example 7 decreased significantly after 1h (p < 0.001, fig. 4D). These results demonstrate that nanoparticle mannose or biotin modification can increase uptake efficiency in macrophages and colon cells.
In addition, for visual comparison of uptake characteristics, caco-2 and Raw 264.7 cells were seeded at the same density on 12-well plates containing glass discs. After 12h incubation, the cells were pre-saturated with biotin or mannose solution. The fluorescently labeled example 3-7 micelles (C6 final concentration 10. Mu.g/ml) were then added and incubated for 2h. Cells were fixed with 4% paraformaldehyde for 15min and stained with Hoechst 33258 (nuclear dye, 10. Mu.g/ml) and DiI (cell membrane dye, 10. Mu.g/ml) for 10min, respectively. The glass disc was inverted onto a glass slide and fluorescence was observed. FIG. 4E analyzes the positions of the cell nucleus (blue fluorescence), cell membrane (red fluorescence) and C6 (green fluorescence). Examples 3, 5, 7 nanomicelles fluoresce more strongly in Caco-2 cells. After addition of mannose, there was no significant change in intracellular fluorescence values in example 3, while the uptake in example 7 was significantly reduced. The uptake capacity in Raw 264.7 macrophages showed a significant decrease in intracellular fluorescence intensity after pre-saturation with mannose in example 3. These results show that the nano micelle has obvious promotion effect on the uptake of intestinal cells after being modified by biotin, and has certain targeted uptake capacity on macrophages after being modified by mannose.
Example 10: in vitro Release investigation
The drug-loaded nano-micelle obtained in "example 5", the drug-loaded nano-microsphere obtained in "example 8" and asiatic acid solution (about 1mg of drug) were placed in a dialysis bag (molecular weight cut-off 7000), and after sealing, 20ml of hydrochloric acid solution (pH 1.2,1vol% SDS, simulated gastric acid environment) was put in, and the mixture was shaken at a constant temperature of 37℃and 100 rpm. After 2h the release medium was changed to PBS (containing 1vol% SDS, simulating the duodenal environment) at pH 6.8 and shaken at 37 ℃. After 6h the release medium was changed to PBS at pH 7.4 (1 vol% SDS, simulating the colonic environment). Taking out all release mediums respectively at 0.5h, 1h, 2h, 3h, 4h, 6h, 8h, 10h, 12h, 24h, 2d, 3d, 4d and 7d, and immediately supplementing the same volume of fresh release mediums. The result shows (figure 5A) that the accumulated release amount of asiatic acid raw material is close to 20% in 2 hours and reaches the end point (101.2%) in 24 hours; the release of the drug from the nano-micelle is relatively slow, 10.1 percent and 20.4 percent of the drug are released respectively in 2 hours and 6 hours, and the drug is basically released completely (92.6 percent) in 4 days; the release of the drug from the nano-microsphere is slower than the first two groups, the accumulated release amount of the drug in the pH 1.2 solution for 2 hours is only 5.4 percent, and the accumulated release amount of the drug for 7 days is 78 percent, which indicates that the nano-microsphere has the characteristics of pH sensitivity and slow drug release.
The morphological change of the microsphere in the drug release process is further studied. The nano-microspheres obtained in "example 8" were soaked in hydrochloric acid solution at pH 1.2, PBS at pH 6.8 and PBS at pH 7.4 for 2 hours, respectively, and then the residual particles were taken out, sprayed with gold, and observed under a scanning electron microscope. The results show (fig. 5B), the nano-microspheres obtained in "example 8" remained completely spherical after 2 hours of immersion in a solution having a pH of 1.2; in a buffer solution with pH of 6.8, the outer layer of the microsphere is destroyed; after 2h of immersion in pH 7.4 buffer, the morphology of the microspheres became irregular, indicating that the nano-microspheres have pH response properties that help protect the encapsulated drug in an acidic environment.
Example 11: in vivo targeted distribution characteristic investigation
The in vivo targeting distribution characteristics of the nanomicelles and the nano-microspheres in colitis mice were studied using near infrared dye DiR as fluorescent probe instead of asiatic acid in example 5 and example 8, respectively. Mice were randomly divided into 3 groups and given by gavage to free DiR, diR-loaded nanomicelles and DiR-loaded nano-microspheres, respectively, at a dose of 3mg/kg as DiR. Mice were euthanized at 6h, 12h, 24h, respectively, after dosing. The heart, liver, spleen, lung, kidney and whole gastrointestinal tract were collected and photographed using an in vivo imaging system. Fluorescence intensities were compared at λEx720nm and λEm 790nm wavelengths.
As shown in fig. 6A and 6C, after the mice were given free DiR by lavage, stronger fluorescence appeared in the stomach, and subsequently, the fluorescence gradually moved to the intestinal tract with a decrease in fluorescence intensity. After 24h, only weak fluorescence was visible. After 6h of DiR/nanomicelle administration, strong fluorescence was shown in the stomach of mice, which may be that DiR or nanomicelle adsorbed on the surface of nanomicelle released the drug in the stomach. At 12h, the fluorescence intensity of the colon was gradually increased, indicating that micelles could be directionally aggregated in the colon. After 24h, small amounts of fluorescence were still visible in the cecum and colon, indicating slow release of DiR from the micelles and prolonged residence time in vivo compared to free DiR groups. Interestingly, at 6h, 12h and 24h, the DiR/nano-microsphere set had higher fluorescence intensity in the cecum and colon than the nano-micelle set, demonstrating that the calcium alginate microspheres protected the encapsulated micelles, preventing premature release of the drug in the stomach. Furthermore, the residence time of the drug in the intestine is further prolonged due to bioadhesion of the alginate. When the shell of the calcium alginate microsphere is decomposed in the intestinal tract, the micelle is released, and the medicine is slowly released. Therefore, the nano-microsphere has the characteristics of accurately delivering the medicine to the inflammation part of colon and releasing the medicine at fixed points.
Meanwhile, the fluorescence signals of the heart, liver, spleen, lung, kidney of the mice at each time point were compared (fig. 6B and 6D). After the free DiR is orally taken, liver fluorescence is strong, kidney fluorescence is weak, and other organs have no fluorescence distribution. Due to the targeting effect of chitosan-deoxycholic acid-mannose grafts and biotin-vitamin E polyethylene glycol succinate grafts, the fluorescence intensity of DiR/nanomicelle in liver is reduced, while that in intestinal tract is increased. On the other hand, due to the nature of calcium alginate, the accumulation of DiR/nano-microsphere sets at the colon site further increases, resulting in a decrease of fluorescence in the liver. These results also confirm that the prepared calcium alginate microspheres have the ability to specifically accumulate in colon tissue after oral administration.
Example 12: in vivo pharmacodynamics investigation of colitis mice
DSS-induced mouse models are typical animal models of colitis, with pathological features similar to humans, such as weight loss, colon shortening, colonic epithelial destruction and inflammatory cell infiltration (Hoffmann, m.; schwertassek, u.; seydel, a.; weber, k.; falk, w.; hauschildt, s.; lehmann, j. Arefined and translationally relevant model of chronic DSS colitis in BALB/c microcomputer laboratory Animals 2018,52 (3), 240-252). A model of chronic colitis in Balb/c mice (males) was induced using 3 cycles, each cycle containing 7 consecutive days of DSS aqueous solution at a drinking concentration of 30mg/ml followed by 7 consecutive days of drinking water, as shown in FIG. 7A. Subsequently, the colitis model mice were randomly divided into 3 groups, while normal groups were set: 1) normal group (healthy mice), 2) model group (DSS-induced colitis mice), 3) asiatic acid raw material group (DSS-induced colitis mice orally administered with asiatic acid daily at a dose of 30 mg/kg), 4) asiatic acid nano-microsphere group (DSS-induced colitis mice orally administered daily with nano-micelle of example 8, at a dose of 30 mg/kg). Animals were monitored twice weekly for body weight, fecal consistency, and fecal bleeding throughout the course of the experiment. The severity of colitis in mice was comprehensively evaluated using Disease Activity Index (DAI), including comprehensive evaluation of weight loss (0-4), stool consistency status (1-3), and stool hemorrhage (1-3). The weight loss scores were as follows: 0, <1%; 1. 1% -5%; 2. 5% -10%; 3. 10% -15%;4 to 15 percent. Fecal scores were as follows: 1. the particle shape is good; 2. the feces are pasty and semi-shaped, and are not adhered to anus; 3. the loose stool sticks to the anus. Bleeding score: 1 min, no bleeding; 2. recessive hemorrhage; 3. severe bleeding. Mice were sacrificed at the end of the experiment and the entire colon (from cecum to anus) and major viscera were excised. Corresponding colon sections were taken for histological analysis and cytokine levels were determined. Blood samples were collected and cytokine levels were detected using the corresponding ELISA kit.
As shown in fig. 7B, normal mice body weight increased continuously during the course of the experiment, while model group mice had significant body weight fluctuation and slightly recovered body weight after withdrawal. This may be due to intestinal damage caused by oral DSS, resulting in weight loss in the model group. The weight gain of asiatic acid nano-microsphere group mice is larger than that of asiatic acid raw material group, which shows that the asiatic acid nano-microsphere group mice have a certain colonitis treatment effect. DAI was significantly elevated and colon shortened in DSS treated mice compared to normal mice (FIGS. 7C-E). In contrast, asiatic acid nano-microsphere group mice had lower DAI and colon length close to normal group (p > 0.05). As shown in fig. 7F-G, there was no significant inflammation or destruction in the colon H & E stained sections of healthy mice. However, the model group showed significant inflammatory features such as intestinal epithelial destruction, goblet cell loss, and massive inflammatory cell infiltration of the lamina propria. The asiatic acid nano-microsphere group has basically normal morphology, only has low inflammation and less inflammatory cell infiltration, and effectively improves abnormal lesions of colon tissues.
The intestinal epithelial barrier is critical in the progression of ulcerative colitis. The goblet cells of the colon produce and secrete mucus to maintain the mucosal barrier of the colon. The reduction of Claudin-1 and zon-1 increases intestinal permeability, leading to colonic barrier dysfunction. Figure 7H shows that the model group goblet cells were severely depleted and the number of intact cells decreased compared to normal cells. The asiatic acid nano-microsphere group has remarkable improvement in the aspects of goblet cell reduction and mucosa integrity recovery. As shown in FIGS. 7I and 7J, ZO-1 and Claudin-1 immunohistochemical staining showed the least immune response in the model group, indicating that the intestinal mucosal barrier was disrupted. The expression of ZO-1 and Claudin-1 in the normal group and asiatic acid nano-microsphere group is obviously increased. The nano-microsphere has the protective effect on the intestinal barrier of mice with enteritis.
MPO is a hydrogen peroxide oxidoreductase and is an important marker for detecting the severity of mucosal neutrophil infiltration. As shown in fig. 7K and 7L, the infiltration of a large number of neutrophils and macrophages in the colon mucosa of DSS mice, and the infiltration of inflammatory cells in the asiatic acid nano-microsphere group is obviously reduced compared with that in the model group and the asiatic acid raw material group, which indicates that the targeting microsphere can improve the treatment effect of colitis.
In addition, the H & E staining of the main organs of the asiatic acid nano-microsphere group mice has no obvious pathological change (figure 8), which proves the safety of the oral administration of the nano-microsphere and provides a precondition for potential clinical transformation.
From the quantitative detection level of inflammatory factors, the secretion of IL-6, TNF-alpha and IL-1β in colonic tissues of colonic mice of colitis is significantly higher than that of the control group, and IL-10 is significantly lower than that of the control group (p < 0.0001, FIG. 9A). These levels of asiatic acid nano-microsphere groups were all significantly altered (p < 0.001) compared to the untreated group of colitis mice. IL-6 and TNF-alpha levels in serum of asiatic acid nano-microsphere group mice were significantly reduced compared with the model group (p < 0.001, FIG. 9B), consistent with the results of gene expression levels (FIG. 9C). The results show that the prepared calcium alginate microspheres have good anti-colonitis effect.
The invention provides a preparation method of a novel targeting nano-microsphere drug delivery system for treating ulcerative colitis, which comprises the steps of preparing an asiatic acid nano system by means of two targeting carrier material packages, and further preparing a nano-microsphere composite system by using calcium alginate packages, and is used for oral targeting treatment of ulcerative colitis. The preparation process adopted by the invention is simple, the reproducibility is good, the materials are easy to obtain, the invention is suitable for industrial production, the invention does not involve the transformation of a medicine structure, the particle size distribution of a medicine delivery system is controllable, and the invention has good practicability and economy.
Claims (10)
1. The targeted nano-microsphere drug delivery system is characterized by at least comprising nano-micelles, wherein the nano-micelles are formed by mixing a drug, chitosan-deoxycholic acid-mannose grafts and/or biotin-vitamin E polyethylene glycol succinate grafts.
2. The system of claim 1, wherein the drug is asiatic acid.
3. The system of claim 1, wherein the mass ratio of the drug, chitosan-deoxycholic acid-mannose graft, biotin-vitamin E polyethylene glycol succinate graft is 1.5:0-10:0-10.
4. The system according to claim 1, wherein the chitosan-deoxycholic acid-mannose graft is prepared by:
activating carboxyl on deoxycholic acid, and then dripping the activated deoxycholic acid solution into chitosan aqueous solution for reaction grafting to obtain chitosan-deoxycholic acid graft;
adding the 4-isothiocyanate-alpha-D-mannopyranoside solution into chitosan-deoxycholic acid aqueous solution for reaction grafting to obtain the chitosan-deoxycholic acid-mannose graft.
5. The system of claim 4, wherein the molar ratio of deoxycholic acid to chitosan monomer is 1:4, 4-phenyl-alpha-D-mannopyranose to chitosan-deoxycholic acid monomer is 1:3.
6. The system of claim 1, wherein the biotin-vitamin E polyethylene glycol succinate graft is prepared by the following method:
activating the terminal carboxyl of biotin, adding vitamin E polyethylene glycol succinate, mixing, reacting and grafting to obtain the biotin-vitamin E polyethylene glycol succinate graft.
7. The system of claim 1, wherein the targeted nano-microsphere drug delivery system further comprises sodium alginate microspheres for coating the nano-micelles.
8. A method of preparing the system of any one of claims 1-6, comprising:
preparing an aqueous solution of chitosan-deoxycholic acid-mannose graft and/or biotin-vitamin E polyethylene glycol succinate graft as a carrier aqueous solution and a drug solution, adding the drug solution into the carrier aqueous solution, and uniformly mixing to obtain the nano micelle.
9. The method of manufacturing according to claim 8, further comprising:
taking span 80 solution with the concentration of 20mg/ml, dropwise adding 20ml of sodium alginate solution with the concentration of 20mg/ml on the liquid surface by using a syringe under mechanical stirring, and stirring and emulsifying under water bath with the temperature of 40-45 ℃; adding 30ml of nano micelle dispersion liquid under the liquid surface by using a syringe, and stirring and uniformly mixing; then, 30ml of calcium chloride solution with the concentration of 100mg/ml is added dropwise for crosslinking; and centrifuging the mixed solution, taking out the lower layer precipitate, cleaning and drying to obtain the targeted nano microsphere particles coated with the sodium alginate microspheres and nano micelles.
10. Use of the system of any one of claims 1-7 in the manufacture of a medicament for the treatment of ulcerative colitis.
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