Lipoic acid grafted hyaluronic acid derivative, preparation method and application

Abstract

The invention relates to the technical field of hyaluronic acid derivatives and nano drug delivery systems, in particular to a lipoic acid grafted hyaluronic acid derivative, a preparation method and application, and provides a lipoic acid grafted hyaluronic acid derivative with a structure that lipoic acid is grafted with hyaluronic acid through a thioketal bond. A lipoic acid grafted hyaluronic acid derivative loaded IAA drug delivery system is constructed, can be gathered at a colitis disease part through intravenous injection, and is actively absorbed by cells by virtue of the affinity effect of hyaluronic acid in a lipoic acid grafted hyaluronic acid derivative structure on CD44 receptors on the surfaces of inflammatory cells; according to the present invention, with the application of the IAA in the cell, the responsive structure degradation occurs in the intracellular high oxidative stress environment, and the IAA is delivered into the cell in the positioning manner, such that the IAA inhibits the inflammation generation by activating the AhR and the downstream oxidative stress and inflammatory reaction related pathway so as to promote the mucous membrane repair, and the problem that the colitis treatment drug cannot accurately act on the focus in the prior art is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of hyaluronic acid derivatives and nano drug delivery systems, specifically to a lipoic acid-grafted hyaluronic acid derivative, a preparation method and applications thereof, and in particular to an active oxygen-responsive lipoic acid-grafted hyaluronic acid derivative, a preparation method thereof and applications thereof as a drug delivery carrier in the treatment of ulcerative colitis. Background Art

[0002] Ulcerative colitis (UC) is a chronic, nonspecific inflammatory disease affecting the rectal and colonic mucosa. UC begins with persistent stimulation of the intestinal mucosa by antigens in the intestinal contents, leading to a defective mucosal barrier. This in turn allows intestinal flora to invade and further activate the immune system, inducing the expression of large amounts of inflammatory mediators and reactive oxygen species (ROS) through both cellular and humoral immunity. These mediators are toxic to intestinal epithelial cells, inducing cell death, and promote lymphocyte infiltration, T cell proliferation, and differentiation in the blood, exacerbating the inflammatory response. Therefore, suppressing excessive inflammatory responses and promoting restoration of intestinal mucosal structure and function are key to achieving UC treatment goals.

[0003] Hyaluronic acid (HA) is a natural, water-soluble, anionic glycosaminoglycan composed of N-acetyl-D-glucosamine and D-glucuronic acid linked by alternating β-(1-4) glycosidic bonds. It is a major component of the extracellular matrix and exhibits excellent biocompatibility. Functional groups within the HA structure, such as carboxyl groups, are highly chemically reactive and can be combined with a variety of compounds through conjugation, chemical bonding, or cross-linking to construct bioactive HA derivatives. Furthermore, studies have shown that HA not only possesses inherent anti-inflammatory and immunomodulatory properties but also has a strong affinity for the transmembrane glycoprotein CD44, which is highly expressed on the surface of inflammatory cells (inflammatory epithelial cells and activated macrophages). Therefore, the key to achieving effective therapeutic effects in ulcerative colitis is to construct amphiphilic polymer derivatives based on HA and use them as drug delivery vehicles that bind to the CD44 ligand-receptor affinity, leading to high-throughput uptake by inflammatory cells and precise drug release. Summary of the Invention

[0004] In view of the problem that colitis treatment drugs in the prior art cannot accurately act on the lesions, the present invention provides a lipoic acid-grafted hyaluronic acid derivative, a preparation method and application.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: The present invention provides a thioctic acid grafted hyaluronic acid derivative. The thioctic acid grafted hyaluronic acid derivative is a structure in which thioctic acid is grafted onto a hyaluronic acid via a thioketal bond.

[0006] A method for preparing the above-mentioned lipoic acid-grafted hyaluronic acid derivative is characterized by comprising: S1: Prepare TK-LA paste and TK-LA solution using lipoic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysuccinimide solution and propane-2, 2-diylbis(sulfonamide)diethylamine solution; S2: Prepare a lipoic acid-grafted hyaluronic acid derivative using TK-LA solution, hyaluronic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution.

[0007] Optionally, in S1, the molar ratio of lipoic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and propane-2, 2-diylbis(sulfonamide)diethylamine is (1:2:2:1) to (1:2:2:2).

[0008] Optionally, in S1, the solvents of the lipoic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysuccinimide solution and TK-LA solution are all dichloromethane, the concentration of the lipoic acid solution is 0.05-0.1 mol / L, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 0.01-0.5 mol / L, the concentration of the N-hydroxysuccinimide solution is 0.1-0.2 mol / L, and the concentration of the TK-LA solution is 10-20 mg / mL; the solvent of the propane-2, 2-diylbis(sulfonamide)diethylamine solution is methanol, and the concentration of the propane-2, 2-diylbis(sulfonamide)diethylamine solution is 0.1-0.2 mol / L.

[0009] Optionally, in S2, the solvent of the hyaluronic acid solution is dimethylformamide, and the concentration is 0.5-1 mg / mL; the solvent of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is dichloromethane, and the solution concentration is 0.1-0.5 mol / L; the solvent of the N-hydroxysuccinimide solution is dichloromethane, and the solution concentration is 0.1-0.5 mol / L.

[0010] Optionally, in S2, the molar ratio of hyaluronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and propane-2, 2-diylbis(sulfonamide)diethylamine (TK-NH2) is (1:2:2:1) to (1:2:2:2).

[0011] Use of the lipoic acid-grafted hyaluronic acid derivative described above in the preparation of a drug for treating ulcerative colitis.

[0012] The use of the lipoic acid-grafted hyaluronic acid derivative as a drug delivery carrier.

[0013] Optionally, the lipoic acid-grafted hyaluronic acid derivative is used as a drug delivery carrier, and the loaded drug is indole-3-acetic acid.

[0014] Optionally, the drug loading of the lipoic acid-grafted hyaluronic acid derivative is 25% to 40%, and the encapsulation efficiency is 65% to 80%, calculated by mass percentage.

[0015] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a lipoic acid-grafted hyaluronic acid derivative, wherein the lipoic acid-grafted hyaluronic acid derivative is a hyaluronic acid structure grafted with lipoic acid via a thioketal bond. Hyaluronic acid can recognize and bind to specific receptors on the surface of intestinal epithelial cells, such as the CD44 receptor, thereby achieving targeted drug delivery. Since lipoic acid is a natural disulfide compound containing a disulfide five-membered ring in its structure, it has high electron density and electrophilicity, as well as strong antioxidant and biocompatibility. Therefore, the lipoic acid-grafted hyaluronic acid derivative has an amphiphilic structure and can form nanoparticles through self-assembly in aqueous solution, which can be used as a drug delivery carrier. At the same time, since the thioketal (TK) bond can be oxidatively cleaved under the stimulation of ROS, thereby releasing the coupled active molecule, targeted drug release can be achieved. Therefore, the nanoparticles constructed by the lipoic acid-grafted hyaluronic acid derivative can simultaneously rely on the ROS-responsive cleavage of the thioketal in its structure and the lipoic acid-responsive glutathione (GSH) The lipoic acid grafted hyaluronic acid derivatives can enhance their hydrophilicity and undergo structural depolymerization, thereby releasing the loaded drugs in a targeted manner. This property enables the nanoparticles constructed by lipoic acid grafted hyaluronic acid derivatives to achieve responsive drug release in the strong oxidative stress environment within inflammatory tissue cells. In addition, hyaluronic acid itself has the function of promoting intestinal barrier repair. In the treatment of ulcerative colitis, not only can targeted drug delivery and targeted drug release be achieved, but the lipoic acid grafted hyaluronic acid derivatives with this structure can also enhance the integrity of the intestinal barrier and reduce intestinal permeability, thereby preventing harmful substances and bacteria from entering the blood circulation and reducing inflammatory responses, providing a reliable direction for the effective treatment of ulcerative colitis.

[0016] The present invention provides a method for preparing the above-mentioned lipoic acid-grafted hyaluronic acid derivative. The method comprises preparing a TK-LA paste and a TK-LA solution by using a lipoic acid solution, a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, an N-hydroxysuccinimide solution, and a propane-2, 2-diylbis(sulfonamide)diethylamine solution. The lipoic acid-grafted hyaluronic acid derivative is then prepared by using the TK-LA solution, the hyaluronic acid solution, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and the N-hydroxysuccinimide solution. The method is simple and the reaction conditions are mild. The structure of the prepared lipoic acid-grafted hyaluronic acid derivative includes lipoic acid, a thioketal bond, and a hyaluronic acid structure. Lipoic acid itself is a powerful antioxidant that can scavenge free radicals and reduce the damage of oxidative stress to cells. Hyaluronic acid has good biocompatibility and moisturizing properties, which helps protect cells and Tissues can synergistically enhance antioxidant and anti-inflammatory effects by combining with hyaluronic acid. The thioketal bond can break under the influence of high levels of ROS in the inflammatory site, thereby achieving tropic release of drugs. This method uses a chemical grafting method to combine zinc sulfate and hyaluronic acid through a thioketal bond. The prepared lipoic acid-grafted hyaluronic acid derivative can not only achieve targeted delivery and release of drugs, but also hyaluronic acid, as a natural polymer material, has good biocompatibility and biodegradability, which helps to improve the bioavailability of drugs and reduce side effects. It can not only be used for targeted drug delivery of ulcerative colitis, but also provides technical support for research in other fields that require antioxidant, anti-inflammatory or controlled drug release.

[0017] For example, the use of the above-mentioned lipoic acid-grafted hyaluronic acid derivative in the preparation of a drug for the treatment of ulcerative colitis. Since the lipoic acid-grafted hyaluronic acid derivative itself has good anti-inflammatory, oxidative, intestinal barrier repair and targeted drug delivery effects, the ulcerative colitis drug prepared using the lipoic acid-grafted hyaluronic acid derivative can accurately act on the intestinal lesions, has few side effects, high safety, and is more reliable in clinical application.

[0018] As mentioned above, lipoic acid-grafted hyaluronic acid derivatives are used as drug delivery carriers. In response to the degree of inflammation at the site of colonic inflammation and the pathological characteristics of the intestinal mucosa during the onset of ulcerative colitis, the present invention constructs a drug delivery system using lipoic acid-grafted hyaluronic acid derivatives as carriers and loaded with indole-3-acetic acid (IAA). The drug can be injected intravenously and accumulate at the site of colonic inflammation through the enhanced permeability and retention (EPR) effect. The drug is then actively taken up by the cells due to the affinity of hyaluronic acid in the structure of the lipoic acid-grafted hyaluronic acid derivatives for the CD44 receptor on the surface of inflammatory cells. The drug undergoes responsive structural degradation in the high oxidative stress environment within the cells, and IAA is delivered to the cells in a targeted manner. IAA activates the aryl hydrocarbon receptor (AhR) and its downstream pathways related to oxidative stress and inflammatory response, thereby inhibiting inflammation and promoting mucosal repair. In addition, the ROS cleavage of the thioketal bond in the structure of lipoic acid-grafted hyaluronic acid derivatives and the strong antioxidant effect of lipoic acid can effectively eliminate excess ROS in cells. The anti-inflammatory effect of IAA and the antioxidant and targeted delivery effects of lipoic acid-grafted hyaluronic acid derivatives synergize with each other, which can provide new ideas and methods for the treatment of ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The present invention is a schematic flow chart of a method for preparing a lipoic acid-grafted hyaluronic acid derivative.

[0020] Figure 2 This is a diagram of the synthesis process of a lipoic acid-grafted hyaluronic acid derivative of the present invention.

[0021] Figure 3 is the ratio of LA, TK-NH2 and TK-LA in Example 1 1 H NMR spectrum (Figure A) and the range of 0-4 ppm 1 H-NMR spectrum (Figure B).

[0022] Figure 4 The ratio of HA and HTL in the range of 0-4 ppm in Example 1 is 1 H NMR spectroscopy.

[0023] Figure 5 The figure shows the scavenging efficiency of HA, HTL, and HTO on active oxygen species in Example 1 (n = 3, mean ± SD).

[0024] Figure 6 Figure 2 shows the particle size diagram (Figure A), potential diagram (Figure B) and transmission electron microscopy image (Figure C) of IAA@HTL nanoparticles.

[0025] Figure 7 Cumulative release curves of IAA@HTL nanoparticles in different release media (n = 3, mean ± SD).

[0026] Figure 8 Figure 2 shows the uptake of different rhodamine 123 (R123) preparations by RAW264.7 cells (scale bar: 100 μm).

[0027] Figure 9 Figure 2 shows the uptake of different Rhodamine 123 (R123) preparations by HT-29 cells (scale bar: 100 μm).

[0028] Figure 10 Effects of IAA@HTL nanoparticles containing different concentrations of IAA on the survival rate of HT-29 cells (n = 3, mean ± SD).

[0029] Figure 11 The results of intracellular reactive oxygen species determination in RAW264.7 cells and HT-29 cells after different treatments (scale bar: 50 μm).

[0030] Figure 12 Effects of different IAA preparations on macrophage (RAW264.7) polarization.

[0031] Figure 13 The results of different inflammatory factors in macrophages (RAW264.7) are shown. A shows the test results of tumor necrosis factor-α (TNF-α), B shows the test results of interleukin-1β (IL-1β), C shows the test results of interleukin-10 (IL-10), and D shows the test results of transforming growth factor-β1 (TGF-β1). n = 3, mean ± SD. Compared with the LPS group, ns indicates no significant difference. ** p <0.01, *** p <0.001.

[0032] Figure 14 Figure 3. Body weight (Figure A), disease activity index (DAI, Figure B), and colon length (Figure C) of mice, n = 6, mean ± SD. NS indicates no significant difference compared with the DSS group. * p <0.05, *** p <0.001.

[0033] Figure 15Figure 3 shows the results of the determination of myeloperoxidase (MPO, Figure A), superoxide dismutase (SOD, Figure B), and malondialdehyde (MDA, Figure C) in the colon tissues of mice in different treatment groups, n = 3, mean ± SD. Compared with the DSS group, ns indicates no significant difference. * p <0.05, *** p <0.001.

[0034] Figure 16 The results of hematoxylin and eosin staining of colon tissues of mice in different treatment groups (scale bar: 100 μm).

[0035] Figure 17 Figure 1 shows the results of a hemolysis assay using IAA@HTL nanoparticles. Figure A shows the effect of different IAA@HTL nanoparticle concentrations on rat erythrocyte membrane integrity; Figure B shows the hemolysis rate of different IAA@HTL nanoparticle concentrations. The above concentrations are calculated based on IAA concentration. n = 3, mean ± SD.

[0036] Figure 18 The results of hematoxylin and eosin staining of the main organs and tissues of mice in different treatment groups (scale bar: 50 μm). DETAILED DESCRIPTION

[0037] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0038] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0039] All features, such as values, amounts, contents, and concentrations, described herein as numerical ranges or percentage ranges are provided for simplicity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0040] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0041] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0042] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0043] The following examples were prepared using conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or according to the conditions recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art. In the present specification and the following examples, unless otherwise specified, "%" indicates percentage by weight, "part" indicates parts by weight, and "ratio" indicates weight ratio.

[0044] The present invention will be further described in detail below with reference to specific embodiments, which are intended to explain the present invention rather than to limit it.

[0045] Reactive oxygen species (ROS) is a general term for a range of molecular oxygen derivatives. Physiological levels of ROS are widely involved in regulating cellular signaling. During ulcerative colitis (UC), mucosal epithelial cells and immune cells, under the influence of inflammatory factors, upregulate the expression of multiple oxidases and induce mitochondrial dysfunction, leading to the massive production of ROS. These ROS antagonize the endogenous antioxidant system, leading to oxidative stress. High levels of ROS not only promote the activation of multiple inflammatory pathways but also exacerbate cell damage and necrosis, thereby increasing intestinal mucosal permeability and the invasion of pathogens. Therefore, timely clearance of excess ROS in inflamed colonic tissue is essential for suppressing the inflammatory response and repairing the intestinal mucosal barrier.

[0046] Lipoic acid (LA) is a naturally occurring disulfide compound containing a five-membered disulfide ring. It possesses high electron density and electrophilicity, enabling it to scavenge free radicals. LA is converted to dihydrolipoic acid (DHLA) in the body. Both LA and DHLA possess potent antioxidant properties, acting synergistically to scavenge a variety of reactive oxygen species and nitrogen species, including hydroxyl radicals, singlet oxygen, hypochlorous acid, and peroxynitrite. Hence, it is known as a universal antioxidant. LA exhibits excellent biocompatibility. In response to high glutathione concentrations within inflammatory cells, LA cleaves disulfide bonds within its structure and converts them to sulfhydryl groups, thereby transforming itself from a hydrophobic state to a hydrophilic state. This property enables nano-drug delivery systems constructed with LA-based redox-responsive polymers to not only release drugs in response to GSH but also exploit LA's reactive oxygen species scavenging function. Furthermore, high oxidative stress in the intestine can serve as a stimulus for responsive drug release in ROS-responsive drug delivery systems. A variety of ROS-responsive functional groups, such as thioethers, thioketals, and boronates, can be used to construct amphiphilic drug delivery systems. Thioketals, due to their stability under both acidic and alkaline conditions, are widely used as carbonyl protecting groups in organic synthesis. Under ROS stimulation, TK can be oxidatively cleaved, releasing the coupled active molecule. This responsive cleavage property has led to its widespread application in oxidation-responsive drug delivery vectors.

[0047] Tryptophan (Trp), the only essential amino acid with an indole structure, serves as a crucial biosynthetic precursor in microbial and host metabolism. Its metabolic pathways and products play a key role in regulating intestinal immune tolerance, maintaining commensal microbial homeostasis, and suppressing inflammatory responses. Indole derivatives produced by intestinal microbiota metabolism of Trp can act as ligands to activate the ubiquitously distributed AhR, thereby regulating downstream biological processes including cell proliferation, differentiation, and immune regulation, ultimately maintaining intestinal mucosal homeostasis. Among them, indole-3-acetic acid, a bacterial metabolite of Trp, can specifically activate AhR. When IAA is transported to intestinal epithelial and immune cells, it not only inhibits apoptosis and necrosis through AhR activation but also significantly reduces inflammation and promotes intestinal mucosal barrier repair by inducing the activation of antioxidant stress pathways downstream of AhR and inhibiting inflammatory pathways. Based on this, the development of an efficient drug delivery system that includes hyaluronic acid, zinc sulfate, and ROS-responsive functional groups can target the delivery of IAA to the epithelial cells and macrophages of the inflamed intestinal mucosa, thereby achieving precise regulation of related signaling pathways. This is the key to inhibiting the progression of inflammation and accelerating mucosal repair.

[0048] The present invention discloses a lipoic acid-grafted hyaluronic acid derivative. The lipoic acid-grafted hyaluronic acid derivative is a hyaluronic acid structure grafted with lipoic acid via a thioketal bond. Nanoparticles constructed with the lipoic acid-grafted hyaluronic acid derivative can simultaneously undergo structural depolymerization by virtue of the ROS-responsive cleavage of the thioketal in its structure and the lipoic acid's response to glutathione (GSH) to enhance its hydrophilicity, thereby releasing the loaded drug in a targeted manner. This characteristic enables the nanoparticles constructed with the lipoic acid-grafted hyaluronic acid derivative to achieve responsive drug release in the strong oxidative stress environment within inflammatory tissue cells, while enhancing the integrity of the intestinal barrier and reducing intestinal permeability, thereby preventing harmful substances and bacteria from entering the blood circulation and alleviating the inflammatory response.

[0049] See also Figure 1 and Figure 2 The present invention also provides a method for preparing the above-mentioned lipoic acid-grafted hyaluronic acid derivative, comprising: S1: Prepare TK-LA paste and TK-LA solution using lipoic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, and propane-2,2-diylbis(sulfonamide)diethylamine solution, specifically as follows: Dissolve lipoic acid (LA) in dichloromethane (DCM) to prepare a lipoic acid solution with a concentration of 0.05-0.1 mol / L (denoted as LA solution); 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) was dissolved in DCM to prepare a 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution (referred to as EDC·HCl solution) with a concentration of 0.01 to 0.5 mol / L. Dissolve N-hydroxysuccinimide (NHS) in DCM to prepare an N-hydroxysuccinimide solution with a concentration of 0.1-0.2 mol / L (denoted as NHS solution); Dissolve propane-2,2-diylbis(sulfonamide)diethylamine (TK-NH2) in methanol to prepare a 0.1-0.2 mol / L propane-2,2-diylbis(sulfonamide)diethylamine solution (referred to as TK-NH2 solution); At room temperature and under a nitrogen atmosphere, a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution was added to a thioctic acid solution until the solution was reduced to half, and the mixture was stirred for 25 to 30 minutes to activate the carboxyl group of the thioctic acid. Then, an N-hydroxysuccinimide solution was added, and stirring was continued for 25 to 30 minutes. Then, a propane-2,2-diylbis(sulfonamide)diethylamine solution was added to allow the amino group in the structure to undergo an amidation reaction with the carboxyl group of the thioctic acid. The reaction time was 24 to 72 hours. The molar ratio of thioctic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and propane-2,2-diylbis(sulfonamide)diethylamine was (1:2:2:1) to (1:2:2:2). After the amidation reaction is completed, cold ether is added to the above solution, and the solution is allowed to stand at 2°C to 8°C for 8 to 24 hours to precipitate impurities, and the filtrate is filtered and dried to obtain a yellow paste, namely TK-LA paste; The TK-LA paste was dissolved in DCM to prepare a TK-LA solution with a concentration of 10-20 g / mL.

[0050] S2: Using TK-LA solution, hyaluronic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution, prepare a lipoic acid-grafted hyaluronic acid derivative, specifically: Dissolve hyaluronic acid (HA) in dimethylformamide (DMF) to prepare a hyaluronic acid solution with a concentration of 0.5-1 mg / mL (denoted as HA solution); Dissolve EDC·HCl in DCM to prepare a 0.1-0.5 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution; Dissolve NHS in DCM to prepare a 0.1-0.5 mol / L N-hydroxysuccinimide solution; A 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and a hyaluronic acid solution are mixed and stirred for 15 to 30 minutes to activate the carboxyl groups of the hyaluronic acid, and then an N-hydroxysuccinimide solution is added. After stirring for 15 to 30 minutes, a TK-LA solution is added to allow the amino groups in its structure to undergo an amidation reaction with the carboxyl groups of the hyaluronic acid. The reaction time is 12 to 24 hours. The molar ratio of the hyaluronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and propane-2, 2-diylbis(sulfonamide)diethylamine (TK-NH2) is (1:2:2:1) to (1:2:2:2). After the reaction is completed, cold ether is added to the reaction solution, and the reaction solution is allowed to stand for 8 to 24 hours at 2°C to 8°C. The solution is filtered, and the filtrate is added to a dialysis bag, dialyzed in deionized water, and freeze-dried to obtain a white flocculent solid, which is the lipoic acid-grafted hyaluronic acid derivative. Preferably, the dialysis bag has a molecular weight cutoff of 3500Da; the dialysis time is 12 to 24 hours, and the deionized water is replaced every 4 to 8 hours during the dialysis process.

[0051] The critical micelle concentration of lipoic acid grafted hyaluronic acid derivatives was determined to be 20~25 mg / L by pyrene fluorescence method. Example 1 Lipoic acid (LA, 0.1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.2 mmol), and N-hydroxysuccinimide (NHS, 0.2 mmol) were dissolved in DCM to obtain 0.07 mol / L LA solution, 0.25 mol / L EDC·HCl solution, and 0.15 mol / L NHS solution, respectively. At room temperature and under nitrogen protection, the EDC·HCl solution and the NHS solution were added to the LA solution in sequence with continuous stirring. Subsequently, propane-2, 2-diylbis(sulfonamide)diethylamine (TK-NH2, 0.15 mmol) was dissolved in methanol to prepare a TK-NH2 solution with a concentration of 0.15 mol / L. The solution was added to the mixture of the above-mentioned EDC·HCl solution, NHS solution, and LA solution, and the reaction was continued with stirring for 12 h. After the reaction was completed, chilled ether was added, and the mixture was allowed to stand at 4°C to precipitate impurities. After separation by filtration, the filtrate was placed in a fume hood to dry to obtain a yellow paste of TK-LA.

[0052] TK-LA paste (0.04 mmol) was dissolved in DCM to obtain a 15 g / mL TK-LA solution. Hyaluronic acid (HA, 0.02 mmol) was dissolved in DMF to prepare a 0.7 mg / mL HA solution. EDC·HCl (0.04 mmol) was dissolved in DCM to prepare a 0.3 mol / L EDC·HCl solution. NHS (0.04 mmol) was dissolved in DCM to prepare a 0.3 mol / L NHS solution. Subsequently, EDC·HCl solution 1 and NHS solution were added to the HA solution in sequence. After stirring for 20 minutes, the TK-LA solution was added and the reaction was stirred for 12 hours. After completion of the reaction, the reaction solution was transferred to a dialysis bag, dialyzed against deionized water, and freeze-dried to obtain a white flocculent solid product, a lipoic acid-grafted hyaluronic acid derivative (HTL). The critical micelle concentration of HTL was determined to be 25.22 mg / L using pyrene fluorescence. See also Figure 3, HTL and its intermediate products in the preparation process were characterized by hydrogen nuclear magnetic resonance spectroscopy ( 1 H-NMR) characterization and comparison, Figure 3 In A, a, b, and c are LA, TK-NH2, and LA-TK, respectively. 1 H-NMR spectrum. LA-TK is synthesized via an acylation reaction between the carboxyl group (-COOH) of LA and the amino group (-NH2) of TK-NH2. In the spectrum of LA, a characteristic peak for the active hydrogen of -COOH appears at 12 ppm; in the spectrum of TK-NH2, a singlet peak (s) for the active hydrogen of NH2 appears at 12 ppm. After the reaction, the -COOH peak at 12 ppm in the spectrum of LA-TK disappears, and the -NH2 peak at 8 ppm transforms from a singlet (s) to a triplet (t), indicating the formation of an amide bond between -COOH and -NH2. Simultaneously, the symmetric structure of -NH2 transforms into the asymmetric structure of LA-TK, causing the -NH- of the amide bond to undergo spin splitting due to the influence of the adjacent methyl group, resulting in a triplet peak (t), preliminarily confirming the successful synthesis of LA-TK. Figure 3 B shows the LA, TK-NH2, LA-TK in the range of 0-4ppm 1 In the TK-NH2 spectrum, the 2.84-2.98 ppm band is attributed to the four methylene groups between S and N (H-j1 and H-k1, Hj2, and H-k2), showing two dd peaks. After the reaction, no characteristic peaks appear in this region in the LA-TK spectrum. However, a multiplet (m) appears at 2.59 ppm, integrated for 4H and attributed to H-k1 and H-k2. H-j1 and H-j2 shift to 3.0-3.5 ppm and overlap with other peaks. The positions of the methyl proton peaks (H i and H i2) in LA-TK are consistent with those in TK-NH2. In addition, the characteristic peaks of the -LA portion of LA-TK correspond to the LA spectrum: 1.32-1.62 ppm are attributed to the three connected methylene groups (Hg, Hf, and Hh); the two proton peaks of the methylene Hd in the dithiolane are located at 2.37 ppm and 1.84 ppm respectively; the methylene He connected to the carbonyl group shifts to 2.17 ppm due to the decrease in electron cloud density; 3.09-3.18 ppm are attributed to the methine Hb and methylene H-c2, and 3.56 ppm is attributed to H-c1. The above results further confirm the successful synthesis of LA-TK. The spectrum of LA-TK is interpreted as follows: 1H-NMR (400MHz, C2D6OS, δ): 3.56 (1H, m, H-c1), 3.12 (6H, m, H-c2, Hb, H-j1, H-j2), 2.59(4H, m, H-k1, H-k2), 2.37 (1H, dt, J=6.3, 12.3 Hz, H-d1), 2.17 (2H, t, J=7.3Hz, He), 1.84 (1H, J=6.3, 12.3 Hz H-d2), 1.32-1.62 (12H, m, Hf, Hg, Hh,H-i1, H-i2).

[0053] See also Figure 4 , respectively, of HA and HA-TK-LA (HTL) 1 The H-NMR spectrum of HA shows proton peaks of the methine and methylene groups in the six-membered ring structure in the 3.0-3.6 ppm range. The HTL spectrum retains the characteristic peaks of HA within the same chemical shift range. The signal peaks of the HTL side chain LA-TK are assigned as follows: 3.57 ppm corresponds to the methylene proton H-c1, which is attached to the sulfur atom of the dithiolane ring in the LA structure; the overlapping peaks in the 3.04-3.18 ppm range include H-c2 (another proton of the dithiolane methylene group), Hb (the methine group), and the methylene groups H-j1 and H-j2 in TK-NH2. The peak at 2.59 ppm is assigned to the methylene groups H-k1 and H-k2 attached to the -NH- group in TK-NH2. Two proton peaks of the methylene group Hd of the dithiolane ring in the LA structure appear at 2.39 ppm and 1.83 ppm, respectively. The triplet (t) at 2.06 ppm corresponds to the methylene group He attached to the carbonyl group. The multiple peaks at 1.28-1.64 ppm include the methylene groups Hf, Hg, and Hh in LA and the methyl groups H-i1 and H-i2 in TK-NH2. 1 All proton signals in the H-NMR spectrum accurately correspond to the structures of HA and LA-TK, confirming the successful synthesis of the HTL polymer. The spectrum of HA-TK-LA (HTL) is: 1H-NMR (400 MHz, C2D6OS, δ): 3.57 (1H, m, H-c1), 3.04-3.18 (6H, m,H-c2, Hb, H-j1, H-j2), 2.59 (4H, m, H-k1, H-k2), 2.39 (1H, dt, J=6.3, 12.3Hz, H-d1), 2.06 (2H, t, J=7.3 Hz, He), 1.83 (1H, J=6.3, 12.3 Hz H-d2), 1.28-1.64 (12H, m, Hf, Hg, Hh, H-i1, H-i2).

[0054] To test the ROS-scavenging ability of the HTL prepared in this example, HTL was dissolved in deionized water and prepared into HTL solutions with concentrations ranging from 0.1 to 2.0 mg / mL, calculated based on the relative HA content. Additionally, HA solutions and a control compound, HA-TK-OA (denoted as HTO, where OA is oleic acid), were prepared in deionized water at the same concentration range, calculated based on the relative HA content. The synthesis of HTO follows the following steps: oleic acid (OA) reacts with propane-2,2-diylbis(sulfonamide)diethylamine (TK-NH2) in the presence of EDC·HCl / NHS to form TK-OA, which is then condensed with hyaluronic acid (HA) in the presence of EDC·HCl / NHS to yield HTO. To an EP tube, 0.5 mL of a 0.4 mmol / L H2O2 solution and 0.5 mL of a 0.4 mmol / L FeCl3 solution were added, followed by 200 μL of the HTL solution, and the mixture was allowed to stand for 10 minutes. Then, 25 μL of 0.5 mg / mL 3,3',5,5'-tetramethylbenzidine (TMB) solution was added, the mixture was centrifuged, the supernatant was collected, and the absorbance was measured at a wavelength of 450 nm using a UV spectrophotometer. The clearance rate was calculated using the following formula: :

[0055] Among them, the sample group was added with H2O2, FeCl3, different HA derivatives (HA, HTL and HTO) and TMB, and its absorbance was recorded as ; The control group was added with H2O2, FeCl3 and different HA derivatives (HA, HTL and HTO), and its absorbance was recorded as ; The blank group was the one with only TMB added, and its absorbance was recorded as .

[0056] The experimental results can be found in Figure 5HA and HTO both showed some ROS scavenging activity, confirming that HA itself has ROS scavenging function. However, LA-modified HTL showed even better ROS scavenging ability, with a scavenging rate exceeding 90%. This result fully confirms that the introduction of LA can significantly enhance the ROS scavenging efficiency of HTL.

[0057] Example 2 Lipoic acid (LA, 0.1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.2 mmol), and N-hydroxysuccinimide (NHS, 0.2 mmol) were dissolved in DCM to obtain 0.05 mol / L LA solution, 0.01 mol / L EDC·HCl solution, and 0.1 mol / L NHS solution, respectively; at room temperature and under nitrogen protection, the EDC·HCl solution and the NHS solution were added to the LA solution in sequence with continuous stirring. Subsequently, propane-2, 2-diylbis(sulfonamide)diethylamine (TK-NH2, 0.1 mmol) was dissolved in methanol to prepare a TK-NH2 solution with a concentration of 0.1 mol / L. The solution was added to the mixture of the above-mentioned EDC·HCl solution, NHS solution, and LA solution, and the reaction was continued with stirring for 18 h. After the reaction was completed, chilled ether was added and the mixture was allowed to stand at 2°C to precipitate impurities. After separation by filtration, the filtrate was placed in a fume hood to dry to obtain a yellow paste of TK-LA.

[0058] TK-LA paste (0.02 mmol) was dissolved in DCM to obtain a 10 g / mL TK-LA solution. Hyaluronic acid (HA, 0.02 mmol) was dissolved in DMF to prepare a 0.5 mg / mL HA solution. EDC·HCl (0.04 mmol) was dissolved in DCM to prepare a 0.1 mol / L EDC·HCl solution. NHS (0.04 mmol) was dissolved in DCM to prepare a 0.1 mol / L NHS solution. Subsequently, EDC·HCl solution 1 and NHS solution were added sequentially to the HA solution. After stirring for 30 minutes, the TK-LA solution was added and the reaction was stirred for 15 hours. After completion of the reaction, the reaction solution was transferred to a dialysis bag, dialyzed against deionized water, and freeze-dried to obtain a white flocculent solid product, a lipoic acid-grafted hyaluronic acid derivative (denoted as HTL).

[0059] Example 3 Lipoic acid (LA, 0.1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.2 mmol), and N-hydroxysuccinimide (NHS, 0.2 mmol) were dissolved in DCM to obtain 0.1 mol / L LA solution, 0.5 mol / L EDC·HCl solution, and 0.2 mol / L NHS solution, respectively. At room temperature and under nitrogen protection, the EDC·HCl solution and the NHS solution were added to the LA solution in sequence with continuous stirring. Subsequently, propane-2, 2-diylbis(sulfonamide)diethylamine (TK-NH2, 0.2 mmol) was dissolved in methanol to prepare a TK-NH2 solution with a concentration of 0.2 mol / L. The solution was added to the mixture of the above-mentioned EDC·HCl solution, NHS solution, and LA solution, and the reaction was continued with stirring for 24 h. After the reaction was completed, chilled ether was added and the mixture was allowed to stand at 6°C to precipitate impurities. After separation by filtration, the filtrate was placed in a fume hood to dry to obtain a yellow paste of TK-LA.

[0060] TK-LA paste (0.04 mmol) was dissolved in DCM to obtain a 20 g / mL TK-LA solution. Hyaluronic acid (HA, 0.02 mmol) was dissolved in DMF to prepare a 1 mg / mL HA solution. EDC·HCl (0.04 mmol) was dissolved in DCM to prepare a 0.5 mol / L EDC·HCl solution. NHS (0.04 mmol) was dissolved in DCM to prepare a 0.5 mol / L NHS solution. Subsequently, EDC·HCl solution 1 and NHS solution were added to the HA solution in sequence. After stirring for 25 minutes, the TK-LA solution was added and the reaction was stirred for 18 hours. After completion of the reaction, the reaction solution was transferred to a dialysis bag, dialyzed against deionized water, and freeze-dried to obtain a white flocculent solid product, a lipoic acid-grafted hyaluronic acid derivative (denoted as HTL).

[0061] Example 4 Lipoic acid (LA, 0.1 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.2 mmol), and N-hydroxysuccinimide (NHS, 0.2 mmol) were dissolved in DCM to obtain 0.08 mol / L LA solution, 0.1 mol / L EDC·HCl solution, and 0.16 mol / L NHS solution, respectively. At room temperature and under nitrogen protection, the EDC·HCl solution and the NHS solution were added to the LA solution in sequence with continuous stirring. Subsequently, propane-2, 2-diylbis(sulfonamide)diethylamine (TK-NH2, 0.13 mmol) was dissolved in methanol to prepare a TK-NH2 solution with a concentration of 0.17 mol / L. The solution was added to the mixture of the above-mentioned EDC·HCl solution, NHS solution, and LA solution, and the reaction was continued with stirring for 24 h. After the reaction was completed, chilled ether was added and the mixture was allowed to stand at 8°C to precipitate impurities. After separation by filtration, the filtrate was placed in a fume hood to dry to obtain a yellow paste of TK-LA.

[0062] TK-LA paste (0.03 mmol) was dissolved in DCM to obtain a 17 g / mL TK-LA solution. Hyaluronic acid (HA, 0.02 mmol) was dissolved in DMF to prepare a 0.7 mg / mL HA solution. EDC·HCl (0.04 mmol) was dissolved in DCM to prepare a 0.4 mol / L EDC·HCl solution. NHS (0.04 mmol) was dissolved in DCM to prepare a 0.4 mol / L NHS solution. Subsequently, EDC·HCl solution 1 and NHS solution were added to the HA solution in sequence. After stirring for 15 minutes, the TK-LA solution was added and the reaction was stirred for 24 hours. After completion of the reaction, the reaction solution was transferred to a dialysis bag, dialyzed against deionized water, and freeze-dried to obtain a white flocculent solid product, a lipoic acid-grafted hyaluronic acid derivative (denoted as HTL).

[0063] For example, the use of the above-mentioned lipoic acid-grafted hyaluronic acid derivative in the preparation of a drug for the treatment of ulcerative colitis. Since the lipoic acid-grafted hyaluronic acid derivative itself has good anti-inflammatory, oxidative, intestinal barrier repair and targeted drug delivery effects, the ulcerative colitis drug prepared using the lipoic acid-grafted hyaluronic acid derivative can accurately act on the intestinal lesions, has few side effects, high safety, and is more reliable in clinical application.

[0064] As mentioned above, the lipoic acid grafted hyaluronic acid derivative is used as a drug delivery carrier. Preferably, the lipoic acid grafted hyaluronic acid derivative is used as a drug delivery carrier, and the loaded drug is indole-3-acetic acid. The loaded drug is indole-3-acetic acid, the drug loading capacity of the lipoic acid grafted hyaluronic acid derivative is 25% to 40%, and the encapsulation efficiency is 65% to 80%.

[0065] The specific application method is: The prepared lipoic acid-grafted hyaluronic acid derivative (HTL) was dissolved in tetrahydrofuran (THF) to prepare an HTL solution with a concentration of 200-500 μg / mL. The solution was added dropwise into deionized water under stirring, and the volume ratio of the HTL solution to deionized water was (1:5) to (1:7.5). The mixture was stirred for 0.5-2 hours, and the THF in the solution was removed by rotary evaporation at 45°C-70°C, and then dried to obtain HTL nanoparticles.

[0066] IAA and HTL nanoparticles were mixed and dissolved in THF to make the concentration of IAA in the solution be 200-500 μg / mL and the concentration of HTL nanoparticles be 200-500 g / mL. The solution was added dropwise into deionized water under stirring conditions. THF in the solution was removed by rotary evaporation at 45°C-70°C, and the solution was filtered and dried to obtain drug-loaded nanoparticles.

[0067] Example 5 The HTL prepared in Example 1 was dissolved in THF to prepare an HTL solution with a concentration of 400 μg / mL. The solution was added dropwise into deionized water under stirring, with a volume ratio of the HTL solution to deionized water of 1:6. The mixture was stirred for 1.5 h. The THF in the solution was removed by rotary evaporation at 50° C., and the HTL nanoparticles were obtained by drying.

[0068] IAA and HTL nanoparticles were mixed and dissolved in THF to a concentration of 200 μg / mL IAA and 200 g / mL HTL nanoparticles in the solution. The solution was added dropwise into deionized water under stirring. THF was removed from the solution by rotary evaporation at 50°C. The solution was filtered through a 0.22 μm microporous filter membrane and dried to obtain drug-loaded nanoparticles, designated as IAA@HTL nanoparticles.

[0069] The IAA@HTL nanoparticle solution before filtration was placed in a laser particle size analyzer and the particle size was measured using dynamic light scattering. The measurement cell temperature was set at 25°C. In addition, a portion of the sample solution was dripped onto a copper grid. After the sample dried, the surface morphology of the particles was observed using a transmission electron microscope (TEM). Figure 6 As shown, the particle size of IAA@HTL nanoparticles is about 129 nm ( Figure 6 A), the surface potential is -22 mV ( Figure 6B) TEM observation results show that the drug-loaded nanoparticles are spherical in structure, with a particle size distribution range of 100-150 nm, which is basically consistent with the results of laser particle size analysis. TEM images further show that the nanoparticles have a clear hydration layer on the surface and a dense solid core ( Figure 6 C). In addition, the results of HPLC test showed that the drug loading capacity and encapsulation efficiency of the nanoparticles for IAA were 36% and 73%, respectively.

[0070] The IAA@HTL nanoparticles prepared in this example were subjected to an in vitro responsive release curve test. 5 mL of the IAA@HTL nanoparticle aqueous solution was placed in a 3500Da dialysis bag and placed in 45 mL of PBS buffer (pH 7.4), phosphate-buffered saline (PBS) buffer containing 10 mM H2O2 (pH 7.4), or PBS buffer containing 10 mM glutathione (pH 7.4). Incubate in a water bath shaker at 37°C and 100 rpm for 48 hours. At a fixed time point, 200 μL of sample was taken from the same position of the dialysis device, and an equal volume of dialysis medium was added after sampling. The sample was analyzed by high-performance liquid chromatography, and the cumulative release rate was calculated and the release curve was drawn. The experimental results are shown in Figure 2. Figure 7 As shown, IAA@HTL releases slowly in PBS buffer, with a cumulative release rate of approximately 20% within 48 hours. However, in PBS buffer containing 10 mM GSH, the cumulative release rate significantly increases to over 50%, and in PBS buffer containing 10 mM H2O2, the cumulative release rate further increases to over 80%. This phenomenon demonstrates that HTL can undergo specific structural changes in response to GSH and H2O2: GSH induces a hydrophilic-hydrophobic shift in the LA group; H2O2 triggers the cleavage of the TK bond, ultimately accelerating the release of IAA. Notably, the H2O2 response significantly promotes IAA release compared to the GSH response.

[0071] The IAA@HTL nanoparticles prepared in this example were subjected to a cell uptake experiment. The same method was used to prepare the IAA@HTL nanoparticles in Example 5, but the nanoparticles were loaded with the fluorescent molecule Rhodamine 123 (R123). HT-29 cells and RAW264.7 macrophages were cultured at a rate of 2×10 5The cells were seeded at a density of 100 / well in a 6-well plate and incubated with a culture medium containing 100 ng / mL lipopolysaccharide (LPS) for 24 hours to induce M1 macrophage polarization. To evaluate the competitive inhibitory uptake effect of HA, a culture medium solution containing 1 mg / mL HA was added to one of the groups at the same time. After 24 hours, the culture medium was discarded and replaced with a culture medium containing free R123 or loaded R123IAA@HTL nanoparticles, and cultured for another 4 hours. Subsequently, the culture medium was discarded, the cells were washed three times with 4°C PBS, fixed with 4% paraformaldehyde solution for 15 minutes, and then the cell nuclei were stained with 1 μg / mL Hoechst 33342 staining solution. After washing with PBS three times, the cells were observed under an inverted fluorescence microscope. The results are shown in Figure 2. Figure 8 and Figure 9 As shown, there was no difference in the uptake of free R123 and R123@HTL by untreated cells. However, after incubation with LPS, HT-29 and RAW264.7 cells uptake R123@HTL significantly more strongly than free R123, indicating that under inflammatory conditions, upregulation of cell surface CD44 receptor expression is conducive to HA-mediated active targeting. However, when HA is pretreated, the prior binding of HA to the CD44 receptor hinders the binding of R123@HTL to CD44, resulting in a reduced uptake effect.

[0072] For the cytotoxicity study of IAA@HTL nanoparticles, HT-29 macrophages in the logarithmic growth phase were obtained, digested, and then blown into a single cell suspension. The cell density was adjusted to 5×10 4 In a 96-well plate, the first column was designated as a blank control (no cells were seeded), and 100 μL of cell suspension was added to each of the remaining wells. After incubation in an incubator for 24 hours, the medium was replaced with LPS-containing medium and cultured for another 24 hours. The medium was then aspirated and discarded. 200 μL of blank medium was added to the blank and negative control groups, respectively. The remaining groups were then added with 200 μL of medium containing different concentrations of IAA@HTL nanoparticles (measured in IAA concentration). Each group was plated in triplicate. After another 24 hours of incubation, the medium was discarded, and 180 μL of serum-free medium and 20 μL of 5 mg / mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution were added to each well. The plates were incubated at 37°C for 4 hours. Discard the liquid in the wells and add 150 μL of dimethyl sulfoxide to each well. Shake on a shaker for 10 minutes to fully dissolve the crystals. Use a microplate reader to measure the absorbance (OD) of each well at 490 nm and calculate the cell survival rate according to the following formula :

[0073] Among them, the sample group contained different concentrations of IAA@HTL nanoparticles, which was recorded as OD sample The blank control group was filled with complete medium of the same volume as the experimental group and no cells were inoculated, and the OD blank The negative control group was not given any treatment and was recorded as OD negative The experimental results are as follows. Figure 10 As shown, the addition of LPS successfully induced a cellular inflammatory response, resulting in a significant decrease in HT-29 cell survival. However, after the addition of IAA@HTL, cell survival increased in a dose-dependent manner. At an IAA concentration of 16 μg / mL, cell survival increased to over 80%, demonstrating that IAA@HTL nanoparticles can effectively protect epithelial cells and significantly reduce cell damage and death caused by the inflammatory response.

[0074] HT-29 cells and RAW264.7 macrophages were cultured at a rate of 1×10 5 The cells were seeded at a density of 100 μmol / L in a 6-well plate. After culturing in an incubator for 24 h, the culture medium was discarded and replaced with a culture medium containing 200 μmol / L H2O2 for a further 24 h. The culture medium was then discarded and culture medium containing free IAA solution, blank HTL nanoparticles or IAA@HTL nanoparticles was added, and the incubation continued for 6 h. The cells were washed three times with pre-cooled PBS, and a culture medium containing 10 μmol / L DCFH-DA was added to each well and incubated at 37°C in the dark for 30 min. Finally, the cells were washed three times with PBS, and the changes in intracellular fluorescence intensity were observed and recorded using an inverted fluorescence microscope. The experimental results are shown in Figure 2. Figure 11 As shown in the results, after H2O2 treatment, the levels of reactive oxygen species in both cells increased significantly. IAA alone had no obvious effect on ROS scavenging, while LA-based HTL could effectively reduce the level of intracellular ROS. On this basis, IAA@HTL showed more significant reactive oxygen species scavenging ability and effectively inhibited the intracellular oxidative stress response.

[0075] RAW264.7 macrophages were cultured at a rate of 1 × 10 5The cells were seeded at a density of 1000 cells / well in a 6-well plate. After culturing for 24 h, the culture medium was discarded and replaced with culture medium containing free IAA, blank HTL nanoparticles or IAA@HTL nanoparticles (measured in IAA concentration), and the incubation continued for 4 h. The cells were collected and incubated with PE-labeled rat anti-mouse CD86 antibody and FITC-labeled rat anti-mouse CD11b antibody at room temperature for 15 min. The fluorescence signal was detected by flow cytometry, and the data was analyzed using Flowjo 10.6 software to determine the expression level of M1 macrophage marker CD86. Using the same method, PE-labeled rat anti-mouse CD206 antibody and FITC-labeled rat anti-mouse CD11b antibody were incubated with the cells for 15 min to determine the expression level of M2 macrophage marker CD206. The experimental results are shown in Figure 2. Figure 12 As shown, after LPS induction, RAW264.7 cells exhibited significant M1 phenotype characteristics. IAA, HTL nanoparticles, and IAA@HTL nanoparticles all inhibited M1 polarization to varying degrees, with IAA@HTL exhibiting a significantly stronger inhibitory effect than either IAA or HTL alone. Following IL-4 induction, cells polarized toward the M2 phenotype. IAA and HTL nanoparticles did not exhibit significant M2 polarization induction compared to the blank control group, while IAA@HTL nanoparticles exhibited an M2 polarization-inducing effect comparable to IL-4. These results demonstrate that IAA@HTL nanoparticles can effectively inhibit M1 polarization of macrophages and promote their polarization toward the anti-inflammatory M2 phenotype.

[0076] RAW264.7 macrophages in the logarithmic growth phase were digested and pipetted into single suspended cells. The cells were counted using a hemocytometer and the number of cells was 1×10 4 Cells were seeded at a density of 100 cells / well in a 12-well plate and cultured for 24 hours before the culture medium was discarded. 2 mL of culture medium was added to the blank group, and culture medium containing LPS was added to the other groups to a final concentration of 200 μg / mL to induce an acute inflammatory response in the cells. The total protein concentration was determined using a BCA kit, and then culture medium containing free IAA solution or different IAA nanoparticles was added. After incubation for 12 hours, the expression of TNF-α, IL-1β, IL-10, and TGF-β1 was determined using an ELISA kit. The experimental results are shown in Figure 2. Figure 13 As shown in the results, compared with the blank control group, LPS treatment significantly promoted the onset of inflammatory responses (upregulating proinflammatory factors TNF-α and IL-1β and inhibiting anti-inflammatory factors IL-10 and TGF-β1). IAA alone failed to significantly improve the expression of inflammatory factors. HTL nanoparticles alone inhibited proinflammatory factors to a certain extent and promoted the expression of anti-inflammatory factors. However, IAA@HTL nanoparticles significantly inhibited the expression of proinflammatory factors and promoted the expression of anti-inflammatory factors. These results indicate that IAA@HTL can effectively regulate the inflammatory response of macrophages, thereby alleviating inflammatory damage.

[0077] The efficacy of IAA@HTL nanoparticles against DSS colitis in mice was evaluated by oral administration of distilled water (5%, w / v A colitis model was established using a 24-hour oral gavage. Mice were divided into a blank control group (healthy mice), a model group, a free IAA group, a HTL nanoparticle group, and an IAA@HTL nanoparticle group. The blank control and model groups were given equal amounts of distilled water, while the remaining groups were given the corresponding therapeutic drugs according to the experimental protocol. Mouse body weight, mental status, fecal characteristics, and occult blood were recorded daily, and the Disease Activity Index (DAI) score was used to assess disease activity. After 7 days of continuous oral administration, mice were sacrificed by cervical dislocation on the 8th day. The colon was dissected, its length measured, and then cut open along the mesentery. The contents were flushed with ice-cold saline. Tissue from the inflamed colon was weighed, minced, and homogenized. Myeloperoxidase (MPO) activity, superoxide dismutase (SOD), and malondialdehyde (MDA) levels were measured. Part of the inflamed colon tissue was fixed with 4% formaldehyde solution, embedded in paraffin, and stained with hematoxylin and eosin (H&E) to observe villus length, crypt depth, and inflammatory infiltration, and to assess the degree of mucosal damage. Experimental data showed that compared with the DSS model group, IAA@HTL nanoparticles significantly promoted the recovery of mouse weight and effectively reduced the DAI score. The colon length of the mice was restored to a level close to that of the normal control group ( Figure 14 ). MPO activity, as a specific indicator of the degree of neutrophil infiltration, can objectively reflect the inflammatory state. The IAA@HTL nanoparticles treatment group showed the most significant inhibitory effect on MPO activity. In terms of oxidative stress indicators, SOD, as a key antioxidant enzyme in cells, its activity level can characterize the body's ability to scavenge free radicals; MDA, as the end product of lipid peroxidation, its content can reflect the degree of cell membrane lipid peroxidation. The experimental results showed that compared with IAA or HTL nanoparticles, IAA@HTL nanoparticles significantly upregulated the expression level of SOD and effectively inhibited the expression of MDA, thereby significantly improving the oxidative stress state of colon tissue ( Figure 15 ). Histopathological observation results showed that IAA@HTL had a more significant effect than a single component in reducing colon mucosal damage and inflammatory cell infiltration ( Figure 16 The above research results confirmed that IAA@HTL effectively promoted the recovery of colon tissue structure and function by synergistically inhibiting inflammatory response and alleviating oxidative stress damage.

[0078] For safety evaluation, a hemolysis experiment of the nanoparticles was first conducted. Fresh whole blood of rats was collected, and red blood cells were collected after centrifugation. The blood was washed three times with normal saline, and then a 2% red blood cell suspension was prepared with isotonic PBS (pH 7.4). Different concentrations of IAA@HTL nanoparticle solutions (measured in IAA concentration) were prepared using PBS. 500 μL of the solution was mixed with 500 μL of red blood cell suspension, incubated in a 37°C water bath for 4 hours, and the supernatant was collected after centrifugation. The absorbance was measured at 540 nm using a UV spectrophotometer. The experiment was repeated three times, with isotonic PBS buffer as the negative control and deionized water as the positive control. The hemolysis rate (HR) of the nanoparticles was calculated according to the following formula:

[0079] Where A sample , A negative and A positive are the absorbance values ​​of the sample group, negative control group and positive control group at a wavelength of 540nm. Figure 17 As shown in the figure, as the concentration of IAA@HTL solution increased from 5 μg / mL to 45 μg / mL, the supernatant remained colorless and transparent, no hemolysis or red blood cell aggregation was observed, and the hemolysis rate was always maintained below 5%. This result indicates that PEG-CNPs have good blood compatibility.

[0080] Histopathological observation: Fifteen male ICR mice were randomly divided into six groups (n=3), one of which was a normal group and the other five were treated groups. The normal group was injected with normal saline via the tail vein, while the treated groups were injected with IAA, HTL and IAA@HTL nanoparticles via the tail vein, respectively. Seven days after administration, the mice were killed, and the main organs (heart, liver, spleen, lung, and kidney) were separated and fixed in 10% paraformaldehyde fixative. After paraffin embedding, sections were prepared and H&E stained. Finally, a medical digital slice scanner was used to observe and record the pathological changes of the main organs of the mice. The experimental results are shown in the figure. Figure 18 As shown in the results, IAA@HTL nanoparticles had no obvious damage to the heart, liver, spleen, lung and kidney of mice, indicating that they have good biocompatibility.

[0081] In summary, the present invention provides a lipoic acid-grafted hyaluronic acid derivative, a preparation method and an application thereof, targeting the degree of inflammation at the site of colon inflammation and the pathological characteristics of the intestinal mucosa during the onset of ulcerative colitis. A lipoic acid-grafted hyaluronic acid derivative having a structure in which lipoic acid is grafted onto hyaluronic acid via a thioketal bond is prepared, and a drug delivery system of the lipoic acid-grafted hyaluronic acid derivative loaded with IAA is constructed. The derivative can be accumulated at the site of colon inflammation via intravenous injection, and then actively taken up by the cells due to the affinity of hyaluronic acid in the structure of the lipoic acid-grafted hyaluronic acid derivative for the CD44 receptor on the surface of inflammatory cells. Responsive structural degradation occurs in the high oxidative stress environment within the cell, and IAA is delivered to the cell in a targeted manner. IAA inhibits inflammation by activating AhR and its downstream pathways related to oxidative stress and inflammatory response, thereby promoting mucosal repair. The anti-inflammatory effect of IAA synergizes with the antioxidant and targeted delivery effects of the lipoic acid-grafted hyaluronic acid derivative, providing new ideas and methods for the treatment of ulcerative colitis.

[0082] The above description is merely a preferred embodiment of the present invention and is not intended to impose any limitation on the technical solution of the present invention. Those skilled in the art should understand that, without departing from the spirit and principles of the present invention, the technical solution can also be subjected to several simple modifications and replacements, and these modifications and replacements are also within the scope of protection covered by the claims.

Claims

1. A lipoic acid-grafted hyaluronic acid derivative, characterized in that: The lipoic acid grafted hyaluronic acid derivative is a lipoic acid grafted hyaluronic acid structure via a thioketal bond.

2. A method for preparing the lipoic acid-grafted hyaluronic acid derivative according to claim 1, characterized in that: include: S1: Prepare TK-LA paste and TK-LA solution using lipoic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysuccinimide solution and propane-2, 2-diylbis(sulfonamide)diethylamine solution; S2: Prepare a lipoic acid-grafted hyaluronic acid derivative using TK-LA solution, hyaluronic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution and N-hydroxysuccinimide solution.

3. The method for preparing the lipoic acid-grafted hyaluronic acid derivative according to claim 2, wherein In S1, the molar ratio of lipoic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and propane-2, 2-diylbis(sulfonamide)diethylamine is (1:2:2:1) to (1:2:2:2).

4. The method for preparing the lipoic acid-grafted hyaluronic acid derivative according to claim 2, wherein In S1, the solvents of the lipoic acid solution, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, N-hydroxysuccinimide solution, and TK-LA solution are all dichloromethane, the concentration of the lipoic acid solution is 0.05-0.1 mol / L, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is 0.01-0.5 mol / L, the concentration of the N-hydroxysuccinimide solution is 0.1-0.2 mol / L, and the concentration of the TK-LA solution is 10-20 mg / mL; the solvent of the propane-2,2-diylbis(sulfonamide)diethylamine solution is methanol, and the concentration of the propane-2,2-diylbis(sulfonamide)diethylamine solution is 0.1-0.2 mol / L.

5. The method for preparing the lipoic acid-grafted hyaluronic acid derivative according to claim 2, wherein: In S2, the solvent of the hyaluronic acid solution is dimethylformamide, and the concentration is 0.5-1 mg / mL; the solvent of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution is dichloromethane, and the solution concentration is 0.1-0.5 mol / L; the solvent of the N-hydroxysuccinimide solution is dichloromethane, and the solution concentration is 0.1-0.5 mol / L.

6. The method for preparing the lipoic acid-grafted hyaluronic acid derivative according to claim 2, wherein In S2, the molar ratio of hyaluronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide, and propane-2, 2-diylbis(sulfonamide)diethylamine (TK-NH2) is (1:2:2:1) to (1:2:2:2).

7. Use of the lipoic acid-grafted hyaluronic acid derivative according to claim 1 in the preparation of a drug for treating ulcerative colitis.

8. Use of the lipoic acid-grafted hyaluronic acid derivative according to claim 1 as a drug delivery carrier.

9. The use according to claim 8, characterized in that The lipoic acid-grafted hyaluronic acid derivative is used as a drug delivery carrier, and the loaded drug is indole-3-acetic acid.

10. The use according to claim 8, characterized in that Calculated by mass percentage, the drug loading amount of the lipoic acid grafted hyaluronic acid derivative is 25% to 40%, and the encapsulation efficiency is 65% to 80%.

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