Acid-resistant nutrient-carrying nanomicelles and their preparation method

Abstract

This invention provides an acid-resistant nutrient-carrying nanomicelles and its preparation method, comprising: (1) dissolving ursolic acid, 1-hydroxybenzotriazole, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride in a solvent and stirring to obtain an activated ester; (2) reacting an amine-containing cationic polymer with the activated ester obtained in step (1) to obtain an amphiphilic cationic polymer; (3) mixing the amphiphilic cationic polymer with a lipid-soluble active substance in a good solvent, removing the good solvent by rotary reflux in the dark to obtain nutrient-carrying nanomicelles; and (4) mixing the polyanionic polymer solution with the nutrient-carrying nanomicelles in water to obtain acid-resistant nutrient-carrying nanomicelles. The nanomicelles prepared by this invention can withstand gastric digestion and gel and aggregate in the enteritis area of ​​mice by prolonging the intestinal retention time.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and new medicine, and in particular to an acid-resistant nutrient-carrying nanomicelle and its preparation method. Background Technology

[0002] Various fat-soluble food bioactive ingredients exist in nature, such as curcumin from plants and astaxanthin from animals. These natural bioactive ingredients typically possess good anti-inflammatory and antioxidant activities, and have the potential to improve gastrointestinal inflammation. However, their poor water solubility and instability (due to the complex environment of the gastrointestinal tract) greatly limit the application of food bioactive ingredients in the biomedical field.

[0003] Currently, oral administration is the common route of administration for these fat-soluble food-derived active ingredients. Researchers have made significant progress in improving the intestinal cell bioavailability of these ingredients through various strategies, such as esterification, microencapsulation, and nanocarriers. Esterification involves modifying the ketone rings at both ends of astaxanthin with polyunsaturated fatty acids to form precursor esters, and then restoring the astaxanthin structure through in vivo enzymatic metabolism, thereby improving its bioavailability. Zhou et al. investigated the effects of mono / diesters, graft chain length, and unsaturation degree by simulating different esterification structures, significantly improving the stability of astaxanthin and proposing a new approach from the perspective of molecular structure modification (FoodHydrocolloid. 2020, 110). Microencapsulation utilizes the hydrophobic structure within microcapsules to load fat-soluble active ingredients, achieving protection and sustained release through microencapsulation, thus improving the intestinal cell bioavailability of these ingredients. Zhang et al. uniformly dispersed lutein in a modified starch and sucrose matrix, encapsulated it with corn starch, and prepared lutein microcapsules using spray drying technology. The prepared lutein microcapsules can directly dissolve lutein in water to form a homogeneous liquid, which improves the solubility and storage stability of lutein and increases its bioavailability, with a relative bioavailability of 139.1% (Food Science and Technology Research, 2015, 21(4):503-507). Nanocarrier delivery further expands the delivery methods of lipid-soluble active ingredients. Hu et al. prepared acylated ovalbumin (AOVA) nanogels through acylation modification and thermally induced self-assembly. Compared with unchemically acylated natural ovalbumin (NOVA) nanogels, curcumin encapsulated in AOVA nanogels showed higher encapsulation efficiency (93.64%) and slower sustained release under simulated gastrointestinal conditions (Food Chemistry, 2021, 355, 129635).

[0004] In summary, reported delivery strategies for fat-soluble food-derived active ingredients mainly focus on improving their water dispersibility and stability. Among these, the astaxanthin esterification strategy emphasizes molecular modification but lacks research on in vitro and in vivo gastrointestinal digestion and metabolism. In vivo pharmacokinetic studies of lutein loaded with microcapsules showed no significant difference in lutein content in the blood before and after loading, and the microencapsulation process often results in micron-sized capsules, which is not conducive to absorption by intestinal epithelial cells. While nanoemulsions and nanomicelles significantly improve the size of the delivery system, they still cannot overcome the problem of low bioavailability after oral administration.

[0005] One of the main characteristics of the gastrointestinal physiological barrier is its variable pH environment: the stomach is strongly acidic (pH = 1–2), while the small intestine is weakly acidic to neutral (pH = 6.5–7.5). The strongly acidic environment of the stomach directly affects the stability of nanodelivery systems, causing premature release and decomposition of nutrients (destruction of molecular structure). Currently, publicly available oral delivery carriers have not yet addressed both the water solubility of lipophilic nutrients and the structural instability of nanodelivery systems under strongly acidic conditions. Summary of the Invention

[0006] The purpose of this invention is to fill the gaps in fat-soluble nutrient transport technology and provide an acid-resistant nutrient nanomicelle that is resistant to the gastrointestinal barrier and has inflammatory-responsive adhesion, as well as its preparation method. The prepared nanomicelles can withstand gastric digestion and gel and aggregate in the enteritis area of ​​mice by prolonging the intestinal retention time.

[0007] This invention is achieved through the following technical solution:

[0008] The method for preparing acid-resistant nutrient-carrying nanomicelles according to the present invention includes the following steps:

[0009] (1) Ursolic acid (UC), 1-hydroxybenzotriazole (HOBT) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl) were fully dissolved in dimethylformimide (DMF), and stirred to allow them to react fully to obtain a solution of activated ester;

[0010] (2) The amino-containing cationic polymer is fully dissolved in a DMF solution containing 0.1% to 5% (v / v) acetic acid, and mixed with the solution of the activated ester obtained in step (1) above. The mixture is stirred to carry out a full amide reaction. After washing with ether and rotary evaporation under negative pressure, an amphiphilic cationic polymer is obtained.

[0011] (3) Dissolve the amphiphilic cationic polymer molecules fully in a good solvent, add the lipophilic active substance and mix well, evaporate and reflux in the dark to remove the good solvent, and redissolve in ultrapure water or other buffer solutions to obtain a nutrient-loaded nanomicelle solution.

[0012] (4) Stir the aqueous solution of polyanionic polymer with the nutrient-carrying nanomicelle solution obtained in step (3) above in an ice bath to obtain acid-resistant nutrient-carrying nanomicelles.

[0013] Further, in step (1), the molar ratio of ursolic acid to 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1-6):(1-6).

[0014] Further, in step (2), the amino-containing cationic polymer is one or more of chitosan, ε-polylysine, α-polylysine and polyacetylimide, wherein the molecular weight range of chitosan is 3 to 200 kDa, the molecular weight range of ε-polylysine and α-polylysine is 3 to 20 kDa, and the molecular weight range of polyacetylimide is 1 to 10 kDa.

[0015] Further, in step (2), the molar ratio of the amino-containing cationic polymer to ursolic acid in the solution of step (1) is 1:(0.05-0.8) based on the molar amount of amino groups.

[0016] Furthermore, in step (2), the reaction temperature is 4 to 70°C, the reaction time is greater than 4 hours, and the stirring speed is 200 to 800 rpm.

[0017] Furthermore, in step (3), the good solvent is any one of methanol, dichloromethane, acetonitrile, dimethylformimide and dimethyl sulfoxide or a mixture of them in any proportion.

[0018] Furthermore, in step (3), the fat-soluble active substance is astaxanthin (AST), curcumin (CUR), lutein (LUT), lutein ester (LUTe), or linoleic acid (LA).

[0019] Furthermore, in step (3), the mass ratio of the amphiphilic cationic polymer to the lipid-soluble active substance is 1:(0.01-0.2).

[0020] Furthermore, in step (3), the light-protected reflux time is 30 min to 3 h.

[0021] Further, in step (4), the solvent for the polyanionic polymer includes water and physiological saline, and the mass ratio of the polyanionic polymer to the nutrient-carrying nanomicelles is (0.01-0.2):1.

[0022] Furthermore, in step (4), the polyanionic polymer is sodium hyaluronate (HA), sodium alginate (SA), or sodium carboxymethyl cellulose (CMC).

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This invention, for the first time, explores and develops a nutrient-carrying nanomicelle resistant to gastric juice digestion, using ursolic acid, an amino-containing cationic polymer, and a polyanionic polymer as raw materials. The amino-containing cationic polymer, after modification with ursolic acid, forms amphiphilic nanomicelles with a positively charged surface, while the negatively charged polyanionic polymer binds to it via an electrostatic self-assembly reaction. When this nanomicelle is transported to the stomach, the highly acidic environment of the gastric juice (pH≈1-2) does not affect the nanostructure. Furthermore, when the nanomicelle is transported to the intestine, the intestinal environment has a pH≈5.5-6.0, within which the isoelectric point of the amino-containing cationic polymer falls, leading to gradual structural dissociation and adhesion to the enteritis area to form a gel. Compared to gastric juice-intolerant nanodelivery systems, this nanomicelle can protect the nutrients it carries during transport to the stomach after oral administration until the change in pH in the intestinal environment causes the nanomicelle structure to dissociate, releasing the nutrients and allowing their bioactivity to be exerted. This invention, using astaxanthin—a natural nutrient that effectively improves the inflammatory environment—as an example, intervened in a mouse model of ulcerative colitis. Results showed that it significantly improved the inflammatory condition in the mouse ulcerative colitis model, highlighting its higher delivery efficiency compared to gastric juice-intolerant nanodelivery systems. It protects the nutrient from gastric juice degradation, prolongs its intestinal retention time, and gels and aggregates in the inflammatory area, greatly improving nutrient bioavailability and exhibiting good biocompatibility. This invention has enormous market potential in the medical field and broad prospects for application and development in other new directions. Attached Figure Description

[0025] Figure 1 This is a synthetic route diagram of the modified chitosan nanomicelles in this invention.

[0026] Figure 2 These are high-resolution transmission electron microscopy (TEM) images of modified chitosan nanomicelles.

[0027] Figure 3 This is the result of dynamic light scattering (DLS) of modified chitosan nanomicelles.

[0028] Figure 4 This is the result of particle size change of modified chitosan nanomicelles under gastric juice (pH=1) conditions.

[0029] Figure 5 This is the result of pH stability of modified chitosan nanomicelles.

[0030] Figure 6 It affects the intestinal injury coefficient in a mouse model of ulcerative colitis (UC) after treatment.

[0031] Figure 7 This refers to the change in colon length in the UC mouse model after treatment.

[0032] Figure 8 This refers to the change in spleen coefficient in a UC mouse model after treatment.

[0033] Figure 9 This is an evaluation of the inflammatory-responsive adhesion properties of modified chitosan nanomicelles.

[0034] Figure 10 This study investigates the effect of modified chitosan nanomicelles on the activity of intestinal epithelial cells. Detailed Implementation

[0035] To further understand the present invention, the present invention will be described below with reference to embodiments. These descriptions are only for further explaining the features and advantages of the present invention and are not intended to limit the claims of the present invention.

[0036] In this invention, the gastric juice-resistant, acid-resistant nutrient-carrying nanomicelles are obtained by self-assembly of an amphiphilic cationic polymer and a lipid-soluble active substance, followed by the formation of non-covalent bonds with a polyanionic polymer.

[0037] Taking chitosan, astaxanthin, and sodium hyaluronate as examples, the reaction process of this invention is explained as follows: Figure 1 As shown. Modified chitosan is prepared by forming amide bonds between chitosan and ursolic acid under the action of 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; the modified chitosan is fully dissolved in acetone, mixed with astaxanthin in a certain proportion, refluxed in the dark for a period of time, the organic solvent acetone is removed, and it is redissolved in water to obtain astaxanthin-loaded chitosan nanoparticles (CSU@AST); under ice bath conditions, the astaxanthin-loaded chitosan nanoparticles are fully mixed with sodium hyaluronate in a certain proportion to obtain the final astaxanthin-loaded nanomicelles (HA-CSU@AST).

[0038] When the nanomicelles are taken orally, astaxanthin is protected in the highly acidic environment of the gastric juice until it reaches the intestines. Due to the change in pH, the nanomicelle structure is stimulated to dissociate and is released, thus exerting its biological activity.

[0039] The present invention will be further described in detail below with reference to the embodiments.

[0040] Example 1

[0041] (1) Synthesis of modified chitosan nanoparticles (CSU)

[0042] Usolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (0.54 g, 2 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.767 g, 2 mmol) were dissolved in DMF and stirred at 4°C for 12 h at 400 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. Chitosan with a molecular weight of 10 kDa was added (based on the molar amount of amino groups on the chitosan chain, the molar ratio of chitosan to ursolic acid was 1:0.6), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain CSU.

[0043]

[0044] (2) Synthesis of astaxanthin-modified chitosan nanomicelles (CSU@AST)

[0045] Dissolve 1g of CSU completely in methanol. Add astaxanthin at a CSU to astaxanthin mass ratio of 1:0.1. Reflux in the dark for 0.5 hours, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain CSU@AST. The color of its aqueous solution differs from that of the CSU aqueous solution; the former is a clear orange-red, while the latter is lemon yellow.

[0046] (3) Preparation of acid-resistant astaxanthin-modified chitosan nanomicelles (HA-CSU@AST)

[0047] Under ice bath conditions, 1.1 mg of AST@CSU and 100 μg of sodium hyaluronate were dissolved in pure water and stirred for 30 min to obtain HA-CSU@AST micelles.

[0048] Example 2

[0049] (1) Synthesis of modified chitosan nanoparticles (CSU)

[0050] Ursolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (2.16 g, 8 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.068 g, 8 mmol) were dissolved in DMF and stirred at 37°C for 10 h at a stirring speed of 800 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 200 kDa of chitosan was added (based on the molar amount of amino groups on the chitosan chain, the molar ratio of chitosan to ursolic acid was 1:0.8), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain CSU.

[0051] (2) Astaxanthin-loaded modified chitosan nanomicelles (CSU@AST)

[0052] Dissolve 1g of CSU completely in methanol, add astaxanthin at a CSU to astaxanthin mass ratio of 1:0.2, reflux for 3 hours in the dark, remove the organic solvent by rotary evaporation, and redissolve in water to obtain CSU@AST. The color of its aqueous solution differs from that of the CSU aqueous solution; the former is a clear orange-red, while the latter is lemon yellow.

[0053] (3) Preparation of acid-resistant astaxanthin-modified chitosan nanomicelles (HA-CSU@AST)

[0054] Under ice bath conditions, 1 mg of CSU@AST and 200 μg of sodium hyaluronate were dissolved in physiological saline and stirred for 30 min to obtain HA-CSU@AST micelles.

[0055] Example 3

[0056] (1) Synthesis of modified chitosan nanoparticles (CSU)

[0057] Usolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (3.24 g, 12 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.136 g, 12 mmol) were dissolved in DMF and stirred at 70°C for 5 h at a stirring speed of 200 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 3 kDa chitosan was added (based on the molar amount of amino groups on the chitosan chain, the molar ratio of chitosan to ursolic acid was 1:0.05), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain CSU.

[0058] (2) Astaxanthin-loaded modified chitosan nanomicelles (CSU@AST)

[0059] Dissolve 1g of CSU completely in methanol. Add astaxanthin at a CSU to astaxanthin mass ratio of 1:0.01. Reflux in the dark for 1 hour, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain CSU@AST. The color of its aqueous solution differs from that of the CSU aqueous solution; the former is a clear orange-red, while the latter is lemon yellow.

[0060] (3) Preparation of acid-resistant astaxanthin-modified chitosan nanomicelles (HA-CSU@AST)

[0061] Under ice bath conditions, 1 mg of CSU@AST and 10 μg of sodium hyaluronate were dissolved in pure water and stirred for 30 min to obtain HA-CSU@AST micelles.

[0062] Example 4

[0063] (1) Synthesis of modified ε-polylysine nanoparticles (ε-PLU)

[0064] Usolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (0.54 g, 2 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.767 g, 2 mmol) were dissolved in DMF and stirred at 4°C for 12 h at 400 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 10 kDa of ε-polylysine was added (based on the molar amount of the amino group on ε-polylysine, the molar ratio of ε-polylysine to ursolic acid was 1:0.6), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain ε-PLU.

[0065] (2) Curcumin-modified ε-polylysine nanomicelles (ε-PLU@CUR)

[0066] Dissolve 1g of ε-PLU completely in dichloromethane. Add curcumin at a mass ratio of ε-PLU to curcumin of 1:0.1. Reflux in the dark for 0.5 hours, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain ε-PLU@CUR. The color of its aqueous solution differs from that of the ε-PLU aqueous solution; the former is a colorless and transparent solution, while the latter is a bright orange-yellow solution.

[0067] (3) Preparation of acid-resistant curcumin-modified ε-polylysine nanomicelles (SA-ε-PLU@CUR)

[0068] Under ice bath conditions, 1.1 mg ε-PLU@CUR and 100 μg sodium alginate were dissolved in physiological saline and stirred for 30 min to obtain SA-ε-PLU@CUR micelles.

[0069] Example 5

[0070] (1) Synthesis of modified ε-polylysine nanoparticles (ε-PLU)

[0071] Usolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (2.16 g, 8 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.068 g, 8 mmol) were dissolved in DMF and stirred at 37°C for 10 h at a stirring speed of 800 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 20 kDa of ε-polylysine was added (based on the molar amount of the amino group on ε-polylysine, the molar ratio of ε-polylysine to ursolic acid was 1:0.8), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain ε-PLU.

[0072] (2) Curcumin-modified ε-polylysine nanomicelles (ε-PLU@CUR)

[0073] Dissolve 1g of ε-PLU completely in dichloromethane. Add curcumin at a mass ratio of ε-PLU to curcumin of 1:0.2. Reflux in the dark for 3 hours, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain ε-PLU@CUR. The color of its aqueous solution differs from that of the ε-PLU aqueous solution; the former is a colorless and transparent solution, while the latter is a bright orange-yellow solution.

[0074] (3) Preparation of acid-resistant curcumin-modified ε-polylysine nanomicelles (SA-ε-PLU@CUR)

[0075] Under ice bath conditions, 1 mg ε-PLU@CUR and 200 μg sodium alginate were dissolved in pure water and stirred for 30 min to obtain SA-ε-PLU@CUR micelles.

[0076] Example 6

[0077] (1) Synthesis of modified ε-polylysine nanoparticles (ε-PLU)

[0078] Usolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (3.24 g, 12 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.136 g, 12 mmol) were dissolved in DMF and stirred at 70 °C for 5 h at a stirring speed of 200 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 3 kDa of ε-polylysine was added (based on the molar amount of the amino group on ε-polylysine, the molar ratio of ε-polylysine to ursolic acid was 1:0.05), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain ε-PLU.

[0079] (2) Curcumin-modified ε-polylysine nanomicelles (ε-PLU@CUR)

[0080] Dissolve 1g of ε-PLU completely in dichloromethane. Add curcumin at a mass ratio of ε-PLU to curcumin of 1:0.01. Reflux in the dark for 1 hour, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain ε-PLU@CUR. The color of its aqueous solution differs from that of the ε-PLU aqueous solution; the former is a colorless and transparent solution, while the latter is a bright orange-yellow solution.

[0081] (3) Preparation of acid-resistant curcumin-modified ε-polylysine nanomicelles (SA-ε-PLU@CUR)

[0082] Under ice bath conditions, 1 mg of ε-PLU@CUR and 10 μg of sodium alginate were dissolved in pure water and stirred for 30 min to obtain SA-ε-PLU@CUR micelles.

[0083] Example 7

[0084] (1) Synthesis of modified α-polylysine nanoparticles (α-PLU)

[0085] Usolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (0.54 g, 2 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.767 g, 2 mmol) were dissolved in DMF and stirred at 4°C for 12 h at 400 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 10 kDa of α-polylysine was added (based on the molar amount of the amino group on α-polylysine, the molar ratio of α-polylysine to ursolic acid was 1:0.6), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain α-PLU.

[0086] (2) Lutein-loaded modified α-polylysine nanomicelles (α-PLU@LUT)

[0087] Dissolve 1g of α-PLU completely in dimethylformimide. Add curcumin at a mass ratio of α-PLU to curcumin of 1:0.1. Reflux in the dark for 0.5h, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain α-PLU@LUT. The color of its aqueous solution differs from that of the α-PLU aqueous solution; the former is a colorless and transparent solution, while the latter is a clear, light yellow solution.

[0088] (3) Preparation of acid-resistant lutein-modified α-polylysine nanomicelles (CMC-α-PLU@LUT)

[0089] Under ice bath conditions, 1.1 mg α-PLU@CUR and 100 μg sodium carboxymethyl cellulose were dissolved in physiological saline and stirred for 30 min to obtain CMC-α-PLU@LUT micelles.

[0090] Example 8

[0091] (1) Synthesis of modified α-polylysine nanoparticles (α-PLU)

[0092] Usolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (2.16 g, 8 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.068 g, 8 mmol) were dissolved in DMF and stirred at 37°C for 10 h at a stirring speed of 800 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 20 kDa of α-polylysine was added (based on the molar amount of the amino group on α-polylysine, the molar ratio of α-polylysine to ursolic acid was 1:0.8), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain α-PLU.

[0093] (2) Lutein-loaded α-polylysine nanomicelles (α-PLU@LUTe)

[0094] Dissolve 1g of α-PLU completely in dimethylformimide. Add lutein ester at a mass ratio of 1:0.2 (α-PLU to lutein ester). Reflux in the dark for 3 hours, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain α-PLU@LUTe. The color of its aqueous solution differs from that of the α-PLU aqueous solution; the former is a colorless and transparent solution, while the latter is a clear, light yellow solution.

[0095] (3) Preparation of acid-resistant lutein ester modified α-polylysine nanomicelles (CMC-α-PLU@LUTe)

[0096] Under ice bath conditions, 1 mg of α-PLU@LUTe and 200 μg of sodium carboxymethyl cellulose were dissolved in pure water and stirred for 30 min to obtain CMC-α-PLU@LUTe micelles.

[0097] Example 9

[0098] (1) Synthesis of modified α-polylysine nanoparticles (α-PLU)

[0099] Usolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (3.24 g, 12 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.136 g, 12 mmol) were dissolved in DMF and stirred at 70 °C for 5 h at a stirring speed of 200 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 3 kDa of α-polylysine was added (based on the molar amount of the amino group on α-polylysine, the molar ratio of α-polylysine to ursolic acid was 1:0.05), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain α-PLU.

[0100] (2) Lutein-loaded α-polylysine nanomicelles (α-PLU@LUTe)

[0101] Dissolve 1g of α-PLU completely in dimethylformimide. Add lutein ester at a mass ratio of 1:0.01 (α-PLU to lutein ester). Reflux in the dark for 1 hour, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain α-PLU@LUTe. The color of its aqueous solution differs from that of the α-PLU aqueous solution; the former is a colorless and transparent solution, while the latter is a clear, light yellow solution.

[0102] (3) Preparation of acid-resistant lutein-modified α-polylysine nanomicelles (CMC-α-PLU@LUTe)

[0103] Under ice bath conditions, 1 mg of α-PLU@CUR and 10 μg of sodium carboxymethyl cellulose were dissolved in pure water and stirred for 30 min to obtain CMC-α-PLU@LUTe micelles.

[0104] Example 10

[0105] (1) Synthesis of modified polyacetylimide nanoparticles (PIU)

[0106] Ursolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (0.54 g, 2 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.767 g, 2 mmol) were dissolved in DMF and stirred at 4°C for 12 h at 400 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 5 kDa polyacetylimide was added (based on the molar amount of amino groups on the polyacetylimide, the molar ratio of polyacetylimide to ursolic acid was 1:0.6), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain PIU.

[0107] (2) Linoleic acid-modified polyacetylimide nanoparticles (PIU@LA)

[0108] Dissolve 1 g of PIU completely in dimethyl sulfoxide. Add linoleic acid at a mass ratio of PIU to linoleic acid of 1:0.1. Reflux in the dark for 0.5 h, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain PIU@LA. The color of its aqueous solution is not significantly different from that of the PIU aqueous solution.

[0109] (3) Preparation of acid-resistant linoleic acid-modified polyacetylimine nanomicelles (CMC-PIU@LA)

[0110] Under ice bath conditions, 1.1 mg of PIU@LA and 100 μg of sodium carboxymethyl cellulose were dissolved in physiological saline and stirred for 30 min to obtain CMC-PIU@LA micelles.

[0111] Example 11

[0112] (1) Synthesis of modified polyacetylimide nanoparticles (PIU)

[0113] Ursolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (2.16 g, 8 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (3.068 g, 8 mmol) were dissolved in DMF and stirred at 37°C for 10 h at a stirring speed of 800 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 10 kDa of polyacetylimide was added (based on the molar amount of amino groups on the polyacetylimide, the molar ratio of polyacetylimide to ursolic acid was 1:0.8), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain PIU.

[0114] (2) Linoleic acid-modified polyacetylimide nanoparticles (PIU@LA)

[0115] Dissolve 1 g of PIU completely in dimethyl sulfoxide. Add linoleic acid at a PIU to linoleic acid mass ratio of 1:0.2. Reflux in the dark for 3 hours, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain PIU@LA. The color of its aqueous solution does not change significantly compared to the aqueous solution of PIU.

[0116] (3) Preparation of acid-resistant linoleic acid-modified polyacetylimine nanomicelles (CMC-PIU@LA)

[0117] Under ice bath conditions, 1 mg PIU@LA and 200 μg sodium carboxymethyl cellulose were dissolved in pure water and stirred for 30 min to obtain CMC-PIU@LA micelles.

[0118] Example 12

[0119] (1) Synthesis of modified polyacetylimide nanoparticles (PIU)

[0120] Ursolic acid (1 g, 2 mmol), 1-hydroxybenzotriazole (3.24 g, 12 mmol), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (6.136 g, 12 mmol) were dissolved in DMF and stirred at 70 °C for 5 h at a stirring speed of 200 rpm. Separately, a certain amount of N,N-dimethylformamide was taken, and the pH of the solution was adjusted to approximately 3–5 with acetic acid. 1 kDa of polyacetylimide was added (based on the molar amount of amino groups on the polyacetylimide, the molar ratio of polyacetylimide to ursolic acid was 1:0.05), and the mixture was stirred at room temperature until no visible solid particles remained. After thoroughly mixing the two solutions, an appropriate amount of diethyl ether was added for washing, and the organic solvent was removed by rotary evaporation to obtain PIU.

[0121] (2) Linoleic acid-modified polyacetylimide nanoparticles (PIU@LA)

[0122] Dissolve 1g of PIU completely in dimethyl sulfoxide. Add linoleic acid at a mass ratio of PIU to linoleic acid of 1:0.01. Reflux in the dark for 1 hour, then remove the organic solvent by rotary evaporation. Redissolve in water to obtain PIU@LA. The color of its aqueous solution is not significantly different from that of the PIU aqueous solution.

[0123] (3) Preparation of acid-resistant linoleic acid-modified polyacetylimine nanomicelles (CMC-PIU@LA)

[0124] Under ice bath conditions, 1 mg of PIU@LA and 10 μg of sodium carboxymethyl cellulose were dissolved in pure water and stirred for 30 min to obtain CMC-PIU@LA micelles.

[0125] Example 13 (taking Example 1 as an example)

[0126] Properties and characterization of HA-CSU@AST micelles

[0127] (1) Morphology and size of HA-CSU@AST micelles

[0128] Figure 2 These are high-resolution transmission electron microscope images of HA-CSU@AST micelles. The images show that HA-CSU@AST is approximately spherical with a uniform diameter of (98.74±15.36 nm). Figure 3 The dynamic light scattering results of HA-CSU@AST micelles are consistent with high-resolution transmission electron microscopy images.

[0129] (2) Evaluation of the stability of HA-CSU@AST micelles

[0130] The HA-CSU@AST micelle aqueous solution remains stable under strong acid conditions (pH=1), and its size is measured at predetermined time intervals, allowing for continuous monitoring of hydrodynamic dimensions using DLS. Figure 4 The results showed that the micelle nanostructures could maintain a stable size distribution for 6 hours at pH=1, which is twice the average gastric transit time in healthy individuals.

[0131] Example 14 (taking Example 1 as an example)

[0132] (1) pH sensitivity evaluation of HA-CSU@AST micelles

[0133] Based on the pH characteristics of the human gastrointestinal tract, HA-CSU@AST (AST 100mg*mL) -1The particles were treated with PBS solutions of different pH values ​​(1, 3.5, 5.5, 6.5, and 7.5) for 1 hour each. The particle size was measured before and after treatment. Figure 5 The results showed that at pH=1 and pH=3.5, i.e., when simulating the gastric environment, the particle size of the nanomicelles did not change significantly compared with that without acid treatment; at pH=5.5, i.e., when simulating the intestinal environment, the nanomicelles aggregated due to the gradual dissociation of the polyanion layer, resulting in a significant increase in particle size; at pH=6.5 and pH=7.5, i.e., in a neutral environment, the particle size of the nanomicelles did not change.

[0134] (2) In vivo therapeutic effects of HA-CSU@AST micelles

[0135] Using a DSS-induced ulcerative colitis model as an example, 6-8 week old C57BL / 6 mice were selected. Mice were given sodium dextran sulfate (2.5% w / v) in a 2.5% aqueous solution for free access to water for 7 days, inducing typical symptoms of ulcerative colitis such as diarrhea with bloody stools and weight loss, thus obtaining the UC model. Furthermore, the treatment period was set at 15 days, during which HA-CSU@AST was administered 6 times via gavage. Mice were divided into 6 groups (n=7 per group): Control group, DSS group, Free Ast group, HA-CSU group, CSU@AST group, and HA-CSU@AST group.

[0136] Throughout the treatment period, mice were observed regularly and scored. Figure 6 The changes in disease activity index scores for each group of mice are shown in the figure. As can be seen from the figure, both the HA-CSU@AST group and the CSU@AST group significantly reduced the disease activity index of the UC model compared with the Free AST group; in addition, the disease activity index score of the HA-CSU@AST group was significantly lower than that of the CSU@AST group, further indicating that HA plays a role in enabling the long-term retention of drug-loaded nanomicelles in the enteritis area.

[0137] After the treatment cycle ended, the mice were euthanized and dissected, and their colons were harvested and their lengths measured for comparison. Colon length is one of the important indicators for evaluating the degree of inflammation in the UC model. Figure 7 The image shows a comparison of colon length in each group of mice. Next, the spleen was harvested, its weight was measured, and its spleen index was calculated. Clinically, the spleen index, the ratio of spleen weight to body weight, is one of the indicators reflecting the degree of inflammation in the body. Figure 8 Comparison of spleen indices among different groups of mice. Figure 7-8 Comparative analysis showed that the HA-CSU@AST group significantly increased the colon length of the UC model compared with the Free AST group; and the HA-CSU@AST group significantly improved the inflammation status of mice.

[0138] (3) Retention effect of HA-CSU@AST micelles at the site of inflammation

[0139] In mice with a DSS-induced ulcerative colitis (UC) model, colonic tissue was collected 12 hours after oral administration of HA-CSU@Cy5.5 for frozen sectioning. Hochest blue fluorescent labeling of cell nuclei (a), inflammatory probe green fluorescent labeling of inflammatory areas (b), and HA-CSU@AST nanomicelles loaded with red fluorescent dye Cy5.5 (c) were used for imaging. Figure 9 In Figure d, the signal of cy5.5 largely overlaps with the inflammatory marker region, indicating that HA-CSU@AST has highly efficient inflammatory-responsive adhesion properties.

[0140] Example 15 (taking Example 1 as an example)

[0141] Biocompatibility evaluation of HA-CSU@AST micelles

[0142] Mouse intestinal epithelial cells were treated with different concentrations of HA-CSU@AST for 24 hours, and the cell viability was detected using a CCK-8 assay kit. The results are as follows: Figure 10 It can be seen that within the range of 0-25 μg / mL, HA-CSU@AST has no significant effect on the survival rate of rat intestinal epithelial cells, and has good biocompatibility.

[0143] This invention, for the first time, explores and develops an acid-resistant nutrient-carrying nanomicelle that is resistant to gastric juice digestion, using ursolic acid, cationic polymers (e.g., chitosan), and polyanionic polymers (e.g., sodium hyaluronate) as raw materials. Compared to gastric juice-intolerant nanodelivery systems, this nanomicelle protects the nutrients it carries during transport to the stomach after oral administration, until it reaches the intestinal environment where pH changes cause the nanomicelle structure to dissociate, releasing the nutrients and allowing their bioactivity to be realized.

[0144] Compared to a healthy state, the gastrointestinal environment under inflammatory conditions is far more demanding. This nanomicelle, loaded with astaxanthin—a natural nutrient that effectively improves the inflammatory environment—was used to intervene in a mouse model of ulcerative colitis. It significantly improved the inflammatory state in the mouse model, highlighting its higher delivery efficiency compared to gastric juice-intolerant nanodelivery systems. It protects nutrients from gastric juice degradation, prolongs their intestinal retention time, and gels and aggregates in the inflammatory area, exhibiting excellent biocompatibility. This invention has enormous market potential in the medical field and broad prospects for application and development in other new directions.

Claims

1. A method for preparing acid-resistant nutrient-carrying nanomicelles, characterized in that, Includes the following steps: (1) Usolic acid, 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were dissolved in a solvent and stirred to react, thus obtaining an activated ester; (2) The amino-containing cationic polymer is reacted with the activated ester obtained in step (1) to obtain an amphiphilic cationic polymer; wherein the amino-containing cationic polymer is chitosan; the molecular weight range of the chitosan is 3~200 Kda; the molar ratio of the amino-containing cationic polymer to ursolic acid is 1:(0.05~0.8) based on the molar amount of amino groups. (3) The amphiphilic cationic polymer and the lipophilic active substance are mixed in a good solvent, and the good solvent is removed by rotary evaporation in the dark to obtain nutrient-loaded nano micelles; wherein the lipophilic active substance is astaxanthin; the mass ratio of the amphiphilic cationic polymer to the lipophilic active substance is 1: (0.01~0.2). (4) Acid-resistant nutrient-carrying nanomicelles are prepared by mixing a polyanionic polymer solution with nutrient-carrying nanomicelles in water; wherein the polyanionic polymer is sodium hyaluronate and the mass ratio of the polyanionic polymer to the nutrient-carrying nanomicelles is (0.01~0.2):

1.

2. The method for preparing acid-resistant nutrient-carrying nanomicelles according to claim 1, characterized in that, In step (1), the molar ratio of ursolic acid to 1-hydroxybenzotriazole and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1: (1~6): (1~6).

3. Acid-resistant nutrient-carrying nanomicelles obtained by the preparation method according to any one of claims 1 to 2.

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