Fatty acid supramolecular hydrogel constructed through hydrophilic and hydrophobic effects as well as preparation method and application of fatty acid supramolecular hydrogel

Abstract

The invention relates to a supramolecular hydrogel formed by self-assembly of hydrophilic macromolecules modified by fatty acids, the hydrophilic macromolecules are selected from natural polysaccharides, other hydrophilic polymers and blends of the polysaccharides or the hydrophilic polymers, and the fatty acids comprise saturated and unsaturated fatty acids. The supramolecular hydrogel disclosed by the invention has a multi-stage structure of a microscopic nano lipid micelle-macroscopic porous gel network, and has high affinity with cartilage while having good lubricating property. The micelle can be used for delivery and slow release of loaded hydrophobic drugs, released nano-scale and micron-scale micelles are easy to be ingested by cells, and the delivery efficiency of the drugs is improved. The supramolecular hydrogel is simple in preparation method, does not need to use a cross-linking agent, and has injectability, self-healing property and shape adaptability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical polymer materials, and particularly relates to a fatty acid supramolecular hydrogel constructed by hydrophilic-hydrophobic interaction, a preparation method thereof, and an application thereof. Background Art

[0002] Traditional hydrogel networks are mostly crosslinked by covalent bonds to form a network structure, that is, the intermolecular forces between molecular chains are formed by covalent bonds, and two-dimensional molecular chains are crosslinked into a three-dimensional network. This kind of hydrogel has good mechanical properties and a relatively stable chemical structure. However, since the hydrogel is formed by covalent bonds between molecular chains, it is difficult for the molecular chains to slip under stress, and the self-healing performance of the hydrogel is poor, which limits its application in the field of biomedical engineering.

[0003] Constructing a dynamic hydrogel crosslinked by non-covalent bonds helps to simulate the microenvironment of human tissues and is of great significance for the development of biomedical materials. Supramolecular gels refer to three-dimensional gels constructed by supramolecular forces, including physical interactions such as hydrogen bonds, host-guest interactions, ion coordination interactions, hydrophilic-hydrophobic interactions, and van der Waals forces. Without changing the macroscopic structure, the molecular chains of physically crosslinked hydrogels can be recombined and slipped, so they have good self-healing and dynamics, are closer to the microstructure of the extracellular matrix, and thus simulate its physiological functions. Supramolecular hydrogels are crosslinked by non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, host-guest interactions, etc.) to form a flexible network, which has good dynamics and reversibility, and can realize the slip and recombination of molecular chains without affecting the overall structure of the hydrogel. The dynamic crosslinked structure of supramolecular hydrogels can not only effectively encapsulate drugs, but also control the release of drugs, avoid the "burst release" phenomenon of traditional hydrogels, and provide a more stable and long-acting drug delivery.

[0004] In biological tissues and organs, there are substances with natural lubricating effects. For example, cartilage can maintain a very low friction coefficient for a long time under high contact stress. The simulation and substitution of these natural lubricating functions by hydrogel materials have become one of the focuses of researchers. At present, clinically, attempts are made to inject hyaluronic acid into the joint cavity to promote joint cartilage lubrication and relieve the pain of arthritis patients. However, due to the presence of hyaluronidase in the joint cavity, the half-life of hyaluronic acid lubricant in the joint cavity is only 1-2 days, and patients need to receive injections frequently. A common idea for researching new lubricating materials is to construct a hydrogel to inhibit the degradation of hyaluronic acid and achieve long-term lubrication of the material. At present, hydrogel microspheres and supramolecular hydrogels crosslinked by non-covalent bonds have become hot research directions. However, the current lubricating hydrogel system still needs to be broken through in terms of load-bearing capacity, lubrication life, and lubrication sustainability under limited lubricants.

[0005] Existing supramolecular hydrogels have shown significant potential in the fields of drug delivery and tissue engineering. However, there are still many limitations in the encapsulation and sustained release of hydrophobic drugs and joint lubrication. First of all, the construction of existing supramolecular hydrogels mostly relies on the addition of cross-linking agents to induce gelation. This method not only increases the complexity of material preparation but also may lead to the problem of cross-linking agent residues. In clinical applications, cross-linking agents often lack good biocompatibility and may cause inflammatory reactions after use, which further limits their long-term use in biomedical engineering. In addition, the use of cross-linking agents may also affect the drug release dynamics. Especially for hydrophobic drugs, cross-linking agent residues may affect the stability and sustained release effect of drugs.

[0006] For hydrophobic drugs, the hydrophilic structure of the hydrogel itself makes it difficult to effectively encapsulate hydrophobic drugs, resulting in low drug loading efficiency. Although the preparation of hydrogel microspheres on the micron or nanometer scale can improve the loading capacity of hydrophobic drugs and shows potential in joint lubrication and drug delivery, the preparation process of microspheres is highly complex and not conducive to large-scale application and promotion. Summary of the Invention

[0007] Aiming at the problems in the prior art, one of the purposes of the present invention is to provide a new supramolecular hydrogel constructed by hydrophilic-hydrophobic interaction, which is self-assembled from hydrophilic macromolecules modified by fatty acids. The hydrophilic macromolecules include, but are not limited to, natural polysaccharides such as hyaluronic acid, alginic acid, chitosan, pectin, gelatin, polyethylene glycol and other hydrophilic polymers, as well as blends of these natural polysaccharides or other hydrophilic polymers. The fatty acid-modified hydrophilic macromolecules are cross-linked through hydrophilic-hydrophobic interaction to form a hydrogel. The fatty acid is a saturated or unsaturated fatty acid, including but not limited to oleic acid, such as linolenic acid, stearic acid, palmitic acid, lauric acid, etc.

[0008] In some embodiments, the solid content (mass percentage) of the hydrogel is greater than or equal to 1%, and in some preferred embodiments, the solid content of the hydrogel is 1%-10%.

[0009] In some embodiments, the synthesis steps of the fatty acid-modified hydrophilic macromolecules include:

[0010] a. Synthesize macromolecules grafted with tetrabutylammonium hydroxide;

[0011] b. Dissolve the hydrophilic macromolecules grafted with tetrabutylammonium hydroxide in anhydrous DMSO. After complete dissolution, add 4-dimethylaminopyridine (0.75-1.5eq) and di-tert-butyl dicarbonate (0.5-1.5eq), and at the same time add fatty acid (2-3eq), and heat and stir for reaction; dialyze the reaction solution with DMSO and deionized water, and freeze-dry to obtain the fatty acid-modified macromolecules.

[0012] In some embodiments, the degree of substitution of the fatty acid-modified hydrophilic macromolecule is 50%-100%.

[0013] In some embodiments of the present invention, the method for preparing a fatty acid-modified hydrophilic macromolecule hydrogel includes: adding the fatty acid-modified hydrophilic macromolecule to an aqueous phase and standing to obtain a hydrogel. In certain embodiments, the aqueous phase is water or an aqueous solution, including but not limited to physiological saline, buffer solution, cell or tissue culture medium, body fluid (such as synovial fluid, tears, intestinal fluid, etc.).

[0014] In certain embodiments, the mass fraction of the fatty acid-modified macromolecule added to the aqueous phase is 1%-10%.

[0015] The fatty acid-modified macromolecule hydrogel forms a multi-level structure of microscopic nano-lipid micelles - macroscopic porous gel network. During the degradation of the hydrogel, this multi-level assembly structure releases nano-scale and micron-scale fatty acid-modified macromolecule micelles. At the same time, the hydrophobic microdomains in the hydrogel can efficiently encapsulate hydrophobic drugs. While the drug loading efficiency is improved, the nano- to micron-scale hydrogel micelles released by the hydrogel are more easily taken up by cells, enhancing the effect of drug delivery.

[0016] Another object of the present application is to provide the application of the fatty acid-modified macromolecule hydrogel as a lubricant or in drug delivery. The nano-micron multi-level structure of the hydrogel of the present invention has a good lubricating effect, significantly reducing the friction coefficient between the interfacial joints.

[0017] Another object of the present application is to provide the application of a supramolecular hydrogel in the preparation of a drug, wherein the supramolecular hydrogel is formed by self-assembly of a linolenic acid-modified hydrophilic macromolecule, and the drug is used for treating joint inflammation and promoting cartilage regeneration. In certain embodiments, the hydrophilic macromolecule is hyaluronic acid.

[0018] The technical solution of the present invention has at least the following technical effects:

[0019] (1) The present invention innovatively utilizes the hydrophobicity of fatty acids and modifies them on the main chain of a macromolecule with good biocompatibility, so that the modified molecular chain forms a hydrophobic region through the assembly of fatty acid side chains, thereby generating intermolecular interactions to form a supramolecular hydrogel, which has the advantages of not requiring a cross-linking agent, injectability, self-healing property and shape adaptability.

[0020] (2) The supramolecular hydrogel constructed by hydrophilic-hydrophobic self-assembly has a microscopic multi-level structure and exhibits good lubricating effects, and can be used for the treatment of diseases caused by lubrication disorders such as arthritis. This structure not only simplifies the preparation process, but also does not require a cross-linking agent, improving the biological safety.

[0021] (3) When the hydrogel of the present invention degrades, it releases low-solid components at the micro-nano scale, which is helpful for the uptake of cells in the application environment and will further exert the biological effects of grafted fatty acids. The specific effects are determined by the properties of the grafted fatty acids.

[0022] (4) The hydrophobic microdomains in the supramolecular hydrogel can encapsulate hydrophobic drugs to achieve long-term slow release, solving the problems of low efficiency and unstable release of traditional hydrogels for loading hydrophobic drugs, and having both lubricating and drug delivery functions. The hyaluronic acid hydrogel modified with fatty acids has the ability to encapsulate and slowly release hydrophobic drugs and can be used for the slow release of hydrophobic drugs at specific sites such as the knee joint. Description of the Drawings

[0023] Figure 1 a) 1H NMR spectrum of hyaluronic acid molecule 1 b) 1H NMR spectrum of hyaluronic acid molecule modified with linolenic acid 1 1H NMR spectrum.

[0024] Figure 2 1H NMR spectrum of hyaluronic acid molecule modified with oleic acid 1 1H NMR spectrum.

[0025] Figure 3 [[ID=,23]]Schematic diagram of gel formation of fatty acid-modified hyaluronic acid hydrogel (HA-OA, HA-LA).

[0026] Figure 4 Rheological test of HA-OA hydrogel: a) Variation of the viscosity of the hydrogel with the angular frequency, b) Variation of the modulus of the hydrogel with the scanning time, c) Variation of the modulus of the hydrogel with the scanning frequency, d) indicating that the hydrogel has self-healing properties.

[0027] Figure 5 Fluorescence image of 3T3 fibroblasts.

[0028] Figure 6 Degradation rate curve of linolenic acid-modified hyaluronic acid hydrogel with a mass fraction of 3%-5%.

[0029] Figure 7 Slow release curves of oleic acid-modified hyaluronic acid hydrogel encapsulating salicylic acid and lauric acid respectively.

[0030] Figure 8 Electron micrograph of the microstructure of linolenic acid-modified hyaluronic acid hydrogel.

[0031] Figure 9 Static and dynamic friction coefficients of linolenic acid-modified hyaluronic acid hydrogel and its control.

[0032] Figure 10 Static and dynamic friction coefficients of linolenic acid-modified hyaluronic acid hydrogel with different solid contents.

[0033] Figure 11 Fluorescence image of the affinity between linolenic acid-modified hyaluronic acid hydrogel and the cartilage joint surface.

[0034] Figure 12 Results of dynamic light scattering (DSL) test on the particle size of the supernatant after HA-LA was soaked for 7 days.

[0035] Figure 13 Confocal laser scanning microscope images of chondrocytes co-cultured with fluorescently labeled HA-LA and HA.

[0036] Figure 14 RNA expression after OA chondrocytes uptake nano-sized and micron-sized HA-LA micelles. Detailed implementation mode

[0037] Unless otherwise specified, the reagents used in the embodiments of this application are all commercially available products. The cell sources used in the experiments are all from the Cell Bank of the Chinese Academy of Sciences.

[0038] Fatty acids are usually hydrophobic. In the present invention, fatty acids are used to modify hydrophilic macromolecules, thereby constructing a new type of amphiphilic polymer material. When this amphiphilic polymer is dissolved in water, due to the hydrophobicity of the side chain groups, the main chain is driven to assemble into a nano-lipid micelle structure. As the solid content increases, the fatty acids on different molecular chains will entangle and assemble together, thereby forming a network structure and forming a hydrogel with a multi-level structure. The micro-micelle structure - macro-gel structure formed by self-assembly through the hydrophobic interaction of the fatty acid side chains can effectively reduce the friction coefficient between articular cartilages, and it is a new type of lubricating gel material with good lubricating properties and a simple preparation method.

[0039] Taking hyaluronic acid as an example of the macromolecular material, the preparation method of the supramolecular hydrogel of the present invention includes the following steps:

[0040] 1) Synthesis of HA-TBA: Modify hyaluronic acid with tetrabutylammonium hydroxide (TBA-OH) to facilitate subsequent reactions in organic solvents.

[0041] a. Dissolve sodium hyaluronate (HA-Na) powder in deionized water with stirring at room temperature, add H + exchange resin, stir at room temperature, filter off the resin, and test the pH of the solution until the pH is in the range of 2-3;

[0042] b. Stir and add tetrabutylammonium hydroxide (TBA-OH) dropwise to the above acidified solution until the pH is 7;

[0043] c. Lyophilize the above neutralized solution to obtain a white foam, which is HA-TBA.

[0044] 2) Synthesis of Fatty Acid-Modified Hyaluronic Acid

[0045] a. Dissolve HA-TBA (1 eq) in anhydrous DMSO. After complete dissolution, add the catalyst 4-dimethylaminopyridine (0.75 - 1.5 eq) and di-tert-butyl dicarbonate (0.5 - 1.5 eq).

[0046] b. Simultaneously add fatty acid (2 - 3 eq) and stir the reaction at 45 °C.

[0047] c. Terminate the reaction when it reaches a certain extent.

[0048] d. Dialyze the reaction solution with DMSO and deionized water, and then completely freeze-dry it to obtain a white spongy material, which is fatty acid-modified hyaluronic acid.

[0049] Among them, in step 1)a, the mass of the added resin is two to three times the mass of sodium hyaluronate, and the stirring duration at room temperature is 6 - 10 hours. In step 2)a, the dissolution temperature of the HA-TBA in anhydrous DMSO is 40 - 70 °C. In step 2)a, the catalysis duration of the catalyst at room temperature is 10 min - 30 min. In step 2)b, the reaction duration at room temperature is 12 - 24 h.

[0050] In some embodiments, the fatty acid-modified hyaluronic acid molecules crosslinked by hydrophilic and hydrophobic interactions have the following structural formula (taking oleic acid-modified hyaluronic acid hydrogel as an example):

[0051]

[0052] The relevant chemical reactions of the present invention proceed according to the following chemical reaction formula.

[0053]

[0054] Example 1 Preparation of Fatty Acid-Modified Hyaluronic Acid

[0055] 1) Weigh 1 g of sodium hyaluronate powder, dissolve it in 50 ml of deionized water under stirring at room temperature, add 3 g of H ion exchange resin, filter after stirring at room temperature for 6 h, test the pH to be 2, drop in tetrabutylammonium hydroxide until neutral, and freeze-dry to obtain HA-TBA.

[0056] Weigh 200 mg of HA-TBA into a 50-ml round-bottom flask, add 30 ml of anhydrous DMSO, and stir to dissolve at 40 °C. At the same time, weigh 300 mg of 4-dimethylaminopyridine and 200 μL of BOC anhydride, and add them to the reaction system. Add 273 μl of linolenic acid to the flask containing HA-TBA, and stir and catalyze at 40 °C for 20 min, then react at room temperature for 24 h. After that, load the reaction solution into a dialysis bag with a molecular weight cut-off of 3500 Kda, and dialyze with DMSO as the dialysis solution for 3 days, changing the dialysis solution every day. Then dialyze with pure water as the dialysis solution for three days, changing the dialysis solution three times a day. After lyophilization, HA-LA is obtained. Dissolve 30 mg of HA-LA in 1000 μL of deionized water, stir, and let it stand for a period of time to form a gel.

[0057] It can be seen from 1 the 1H NMR spectrum (see Figure 1 b)) that compared with the hyaluronic acid molecule ( Figure 1 a)), a new absorption peak appears at a chemical shift of 2.8 in the reaction product, which is the absorption peak of the hydrogen atoms on the olefin structure of linolenic acid, indicating that linolenic acid has been successfully modified onto the hyaluronic acid molecular chain.

[0058] 2) Weigh 0.5 g of sodium hyaluronate powder, dissolve it in 50 ml of deionized water under stirring at room temperature, add 2 g of H + exchange resin, stir at room temperature for 6 h, then filter. The measured pH is 2. Dropwise add tetrabutylammonium hydroxide until neutral, and lyophilize to obtain HA-TBA.

[0059] Weigh 500 mg of HA-TBA into a 250-ml round-bottom flask, add 50 ml of anhydrous DMSO, and stir to dissolve at 45 °C. At the same time, weigh 65 mg of 4-dimethylaminopyridine and 193 μL of BOC anhydride, and add them to the reaction system. Add an appropriate amount of oleic acid to the flask containing HA-TBA, and stir and catalyze at 45 °C for 20 min, then react at room temperature for 24 h. After that, load the reaction solution into a dialysis bag with a molecular weight cut-off of 3500 Kda, and dialyze with DMSO as the dialysis solution for 3 days, changing the dialysis solution every day. Then dialyze with 10% brine as the dialysis solution for 1 day. Finally, dialyze with pure water as the dialysis solution for 3 days, changing the dialysis solution three times a day. After lyophilization, HA-OA is obtained. Dissolve 10 mg of HA-OA in 1 mL of deionized water, stir, and let it stand at 4 °C for a period of time to form a gel.

[0060] It can be seen from the 1H NMR spectrum (see Figure 2 ) that compared with the hyaluronic acid molecule ( Figure 1 a)), a new absorption peak appears at a chemical shift of 5.4 in the reaction product, which is the absorption peak of the hydrogen atoms on the olefin structure of oleic acid, indicating that oleic acid has been successfully modified onto the hyaluronic acid molecular chain.

[0061] Example 2 Gelation Properties of Fatty Acid-Modified Hyaluronic Acid Hydrogel

[0062] Dissolve the fatty acid-modified hyaluronic acid prepared according to the method of Example 1 in deionized water and let it stand in a refrigerator at 4°C overnight. Due to the hydrophobic interaction between fatty acid molecules, self-assembly forms hydrophobic microdomains, and thus the fatty acid-modified hyaluronic acid hydrogel ( Figure 3 ) can be obtained. Hydrogels can be obtained using various fatty acid-modified hyaluronic acids, such as oleic acid, linolenic acid, stearic acid, etc.

[0063] The gelation of fatty acid-modified hyaluronic acids with different solid contents in water was experimentally studied using oleic acid-modified hyaluronic acid (HA-OA), and the results are shown in Table 1.

[0064] Table 1

[0065] Solid content State 0.1％ Solution 0.5％ Solution 1％ Hydrogel >1％ Hydrogel

[0066] The results of rheological tests on the HA-OA hydrogel using a rotational rheometer are as Figure 4 shown. Since this hydrogel forms a gel through hydrophilic-hydrophobic interactions, and rheological tests show that as the shear rate increases, the viscosity of the hydrogel continuously decreases (see Figure 4 a), which represents shear-thinning characteristics, so the hydrogel has good injectability, Figure 3 and this property is also confirmed. Figure 4 b, 4c show the changes in the modulus of the hydrogel with the scanning time and scanning frequency, Figure 4 d indicates that the hydrogel has self-healing properties.

[0067] Example 3 Biocompatibility and Stability of Fatty Acid-Modified Hyaluronic Acid Hydrogel

[0068] Fatty acids such as linolenic acid are widely present in the human body and have good biocompatibility. After grafting onto hyaluronic acid, they will not be toxic to cells. Immerse 1 mL of the HA-LA hydrogel in 20 mL of DMEM medium and let it stand at 4°C to obtain the extract. As shown by the fluorescence microscope observation results Figure 5 after adding 3% of the HA-LA hydrogel extract, the number of 3T3 fibroblasts has no obvious difference from that of the normal culture group. This shows that the hydrogel of the present invention has good biocompatibility.

[0069] Analyze the degradation rate of linolenic acid-modified hyaluronic acid hydrogel with a solid content of 3%-5%. The degradation test of HA-LA hydrogel uses HA-LA hydrogels with solid content gradients of 3%, 4%, and 5% (wt / v), and the time variable is set to 1 day, 3 days, 5 days, and 7 days. After preparing 50 μL of each HA-LA hydrogel with a specific solid content in a mold, transfer it to a 24-well plate (n = 3) and immerse it in 2 mL of PBS. At the specified time points, weigh the content after complete solid freeze-drying. The degradation rate of the hydrogel in PBS within 7 days is approximately 30% (see Figure 6 ), and it has good stability. Through experiments, it is found that this hydrogel can be retained in the body for more than 2 weeks.

[0070] Example 4 Sustained Release of Drugs from Fatty Acid-Modified Hyaluronic Acid Hydrogel

[0071] In this example, fatty acid hyaluronic acid hydrogel was used to encapsulate salicylic acid and lauric acid respectively, and the drug sustained release ability was investigated. The experimental method is as follows:

[0072] 1) Salicylic acid:

[0073] Drug loading - Take 10 mg of the freeze-dried product of HA-OA (oleic acid-modified hyaluronic acid hydrogel) and salicylic acid powder, add them to a 15 ml centrifuge tube, and add 1 ml of DI water. Centrifuge at 1000 rpm for 1 min and place it at 4 °C overnight to form a gel.

[0074] Sustained release experiment - Add 2 ml of PBS solution to the 15 ml centrifuge tube loaded with salicylic acid. At 0 h, 2 h, 4 h, 6 h, 10 h, 29 h, 32 h, and 51 h, aspirate 300 μl of the solution and supplement 300 μl of PBS solution. Dilute the aspirated sustained release solution to 4 ml with PBS solution and measure the absorbance at 230 nm.

[0075] 2) Lauric acid:

[0076] Drug loading - Dissolve lauric acid powder in 50 μl of absolute ethanol. Take 10 mg of the freeze-dried product of HA-OA and lauric acid ethanol solution, add them to a 15 ml centrifuge tube, and add 1 ml of DI water. Centrifuge at 1000 rpm for 1 min and place it at 4 °C overnight to form a gel.

[0077] Sustained release experiment - Add 2 ml of PBS solution to the 15 ml centrifuge tube loaded with salicylic acid. At 0 h, 2 h, 4 h, 6 h, 10 h, 24 h, 39 h, 48 h, and 75 h, aspirate 300 μl of the solution and supplement 300 μl of PBS solution. Dilute the aspirated sustained release solution to 4 ml with PBS solution and measure the absorbance at 220 nm.

[0078] The sustained release curves obtained from the sustained release experiments are as Figure 7As shown. The hydrophobic microdomains in the hydrogel can efficiently encapsulate hydrophobic drugs, achieving long-acting and sustained release, and solving the problems of low efficiency in loading hydrophobic drugs and unstable release in traditional hydrogels.

[0079] Example 5 Microstructure of Fatty Acid-Modified Hyaluronic Acid

[0080] By observing the microstructure of hydrogels with solid contents of 1%, 3%, 4%, and 5% through electron microscopy, the fatty acid-modified hyaluronic acid hydrogel can form a multi-level assembly structure of microscopic nano-lipid micelle structure - macroscopic porous gel structure. Figure 8 The microstructure of 3% HA-LA hydrogel is shown as follows.

[0081] Example 6 Lubricating Performance of Fatty Acid-Modified Hyaluronic Acid Hydrogel

[0082] In this example, the lubricating performance of the hydrogel was investigated. The test method for the lubricating effect of the fatty acid-modified hyaluronic acid hydrogel is as follows.

[0083] Before the test started, a plastic petri dish was fixed to the bottom plate of the rheometer using cyanoacrylate. Then, a porcine cartilage sample was adhered to the upper plate of the rheometer and another one was adhered to the petri dish, ensuring that the two pieces of cartilage were concentrically aligned to ensure the accuracy of the measurement. Next, a lubricating fluid was added to the petri dish to fully wet the cartilage contact interface. Subsequently, the lubricating system to be tested was evenly applied to the surface of the cartilage to make it evenly distributed in the friction area. The upper plate was slowly lowered until the two pieces of cartilage came into contact, and an additional downward pressure was applied to ensure that the cartilage reached the strain conditions required for the test. In the next 60 min, a fixed strain was maintained for stress relaxation treatment. Subsequently, the upper plate was rotated counterclockwise by 720°, and then rotated clockwise by 720°. Different pre-slip times (1200 s, 120 s, 12 s, 1.2 s) were set before the rotation. During the entire rotation process, the shear rate was maintained at approximately 0.121 s -1 , and at the same time, the friction coefficient was calculated by measuring the torque and normal force. The calculation formula is:

[0084]

[0085] Using the above method, in this example, 3% HA solution and a physically blended solution of 3% HA and LA (HA in 3% HA+LA is 3% by mass percentage, and LA is the same amount as the grafted LA in 3% solid content HA-LA) were tested simultaneously as a comparison. The multi-level gel structure assembled by fatty acid-modified hyaluronic acid can effectively reduce the dynamic and static friction coefficients (as shown in Figure 9 ). The test results for HA-LA hydrogels with different solid contents are as shown in Figure 10 , which are 3%, 4%, and 5% of HA-LA respectively. As the solid content increases, the dynamic and static friction coefficients decrease.

[0086] Through the lubrication effect test, it can be seen that the multi-level structure of the hydrogel of the present invention has good lubrication effect, which can improve the affinity and binding force with the articular cartilage interface, provide an interfacial hydration layer, and significantly reduce the friction coefficient between the articular interfaces.

[0087] This example also investigated the affinity of the fatty acid-modified hyaluronic acid hydrogel with the joint surface. HA and HA-LA were labeled with red fluorescence and co-incubated on the cartilage surface. 0.1 mg of rhodamine B isothiocyanate (RITC) powder was added to 100 mL of HA solution and HA-LA solution with a mass-volume concentration of 0.1% (wt / v) respectively, and continuously stirred in the dark at room temperature for 24 h to prevent photobleaching. Subsequently, the solutions were dialyzed and freeze-dried to obtain RITC-labeled HA (RITC-HA) and RITC-labeled HA-LA (RITC-HA-LA). Porcine cartilage was purchased from the market and cut into the same thickness and size using a mold. Then, the cartilage samples were co-incubated with 3% RITC-HA and RITC-HA-LA hydrogels at room temperature overnight. After the incubation, the cartilage samples were cut along the vertical axis by cryosectioning technique, and the fluorescence intensity of RITC-labeled on the cartilage surface was observed using an inverted fluorescence microscope. Fluorescence images were collected under 555 nm excitation light. Quantitative analysis was performed using ImageJ, and the average fluorescence intensity was measured to evaluate the binding ability of HA-LA on the cartilage surface ( Figure 11 as shown). From the experimental results, it can be seen that HA-LA can penetrate deeper into the joint surface and form strong hydrophobic binding.

[0088] Example 7 Experiment on the uptake of fatty acid-modified hyaluronic acid by cells

[0089] The macroscopically assembled multi-level gel of the present invention continuously forms and releases micron- and nano-scale assembled micelles during the degradation process, which can be taken up by chondrocytes and exert the biological effects of grafted fatty acids.

[0090] a. After 50 μL of 3% HA-LA was prepared in a mold, it was transferred to a 24-well plate (n = 3) and immersed in 2 mL of PBS. The 3% HA-LA hydrogel was soaked in PBS for 7 days, and then the supernatant was taken. The particle size of the supernatant was tested by dynamic light scattering (DSL). It was found that nano- to micron-scale assembled structures appeared (such as Figure 12 as shown).

[0091] b. Rat chondrocytes were seeded at a density of 1×10 4Cells were seeded in 24-well plates at a density of, and the culture medium was DMEM containing 10% FBS and 1% p / s. After culturing for 24 h, 0.1% (wt / v) RITC-HA and RITC-HA-LA were added respectively. After culturing the cells for another 1 day, the cells were fixed and the nuclei were stained, and then the uptake of RITC-HA and RITC-HA-LA by chondrocytes was observed by confocal laser scanning microscopy. The results are shown as Figure 13 follows. After co-culture with fluorescently labeled HA-LA and HA, chondrocytes significantly took up HA-LA but did not take up free coiled HA macromolecules, indicating that micro- and nano-scale assembly driven by lipid hydrophobic interaction is the key to cell uptake. This experiment confirmed that the nano-lipid structure formed after the dissociation and recombination of fatty acid-modified hyaluronic acid hydrogel contributed to cell uptake.

[0092] The fatty acid-modified hyaluronic acid complex showed an assembled structure of nano-lipid micelles at low solid content. With the increase of solid content, it underwent secondary assembly and formed a macroscopic three-dimensional porous gel through network entanglement. The low-solid-content fragments generated during the degradation and wear of the gel were released in the form of nano-lipid micelles and could be taken up by cells. If the fatty acid was defined as an unsaturated fatty acid with anti-inflammatory function, it could exhibit intracellular anti-inflammatory efficacy.

[0093] c. PCR verification was carried out after OA chondrocytes took up the micro- and nano-assembled structures of HA-LA

[0094] An osteoarthritis (OA) model of chondrocytes was established by adding IL-1β (1 ng / mL) to chondrocytes. The effective concentration of LA for inhibiting OA progression was determined to be 10 μM. The exact mass of HA-LA dissolved in the culture medium was determined to be 0.02% (wt / v) according to the substitution degree calculated by 1 HNMR. The HA+LA group was prepared by adding equal amounts of HA and LA (dissolved in 100 μL of ethanol) to the culture medium respectively. In other experimental groups, 100 μL of ethanol was added to control the variables.

[0095] In the experiment, chondrocytes were seeded in 6-well plates at a density of 1.8×10 5 cells / well and cultured in normal medium (Control), HA+LA group or HA-LA group for 4 days. Subsequently, the cells were stimulated with IL-1β (1 ng / mL) for 48 h, and then total RNA was extracted for quantitative detection.

[0096] Total RNA was extracted from cultured chondrocytes using Trizol reagent according to the instructions. After extraction, the purity and concentration of RNA were evaluated by measuring the absorbance at 260 nm and 280 nm. Subsequently, 2 μg of RNA was reverse-transcribed using HiScript III RT SuperMix. cDNA was amplified using Taq Pro Universal SYBR qPCR Master Mix. The mRNA expression levels were detected with specific primers and normalized to Gapdh using the 2-ΔΔCT method. The results of several RNA expression experiments tested are as Figure 14 shown. Since linolenic acid has an anti-inflammatory effect and hyaluronic acid promotes chondrogenesis, the hydrogel in this example has a synergistic effect of the two on cartilage, and OA chondrocytes show a tendency to promote cartilage regeneration and inhibit matrix degradation.

Claims

1. A fatty acid supramolecular hydrogel constructed by hydrophilic-hydrophobic interaction, which is formed by self-assembly of hydrophilic macromolecules modified with fatty acids. The hydrophilic macromolecules are selected from natural polysaccharides, other hydrophilic polymers, and blends of the natural polysaccharides and hydrophilic polymers. The fatty acids include saturated fatty acids and unsaturated fatty acids.

2. The supramolecular hydrogel according to claim 1, wherein The hydrophilic macromolecules include hyaluronic acid, alginic acid, chitosan, pectin, gelatin, polyethylene glycol, and the fatty acids include oleic acid, linolenic acid, stearic acid, palmitic acid, lauric acid.

3. The supramolecular hydrogel according to claim 1 or 2, characterized in that, The solid content of the supramolecular hydrogel is greater than or equal to 1%.

4. The supramolecular hydrogel according to claim 3, wherein The solid content of the supramolecular hydrogel is 1% - 10%.

5. The supramolecular hydrogel according to claim 1, wherein The supramolecular hydrogel has a multi-level structure of microscopic nano-lipid micelles - macroscopic porous gel network.

6. A preparation method of a fatty acid supramolecular hydrogel constructed by hydrophilic-hydrophobic interaction, comprising the steps of: a) Synthesizing a hydrophilic macromolecule grafted with tetrabutylammonium hydroxide; b) Dissolving the hydrophilic macromolecule grafted with tetrabutylammonium hydroxide in anhydrous DMSO, adding 4-dimethylaminopyridine and di-tert-butyl dicarbonate, and simultaneously adding fatty acids, heating and stirring for reaction; dialyzing the reaction solution and freeze-drying to obtain a fatty acid-modified hydrophilic macromolecule; c) Adding the fatty acid-modified hydrophilic macromolecule to an aqueous phase and standing to obtain a hydrogel.

7. The preparation method according to claim 6, characterized in that The aqueous phase is water or an aqueous solution, including but not limited to normal saline, buffer solution, cell or tissue culture medium, body fluid.

8. Use of the supramolecular hydrogel according to any one of claims 1-7 as a lubricant.

9. Use of the supramolecular hydrogel according to any one of claims 1-7 in drug delivery.

10. Application of supramolecular hydrogel in the preparation of drugs, characterized in that, The supramolecular hydrogel is formed by self-assembly of a hydrophilic macromolecule modified with linolenic acid, and the drug is used for treating joint inflammation and promoting cartilage regeneration.

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