Preparation method of hydrogel conjugate for treating arthritis

Abstract

The invention discloses a preparation method of a hydrogel conjugate for treating arthritis, which comprises the following steps: step 1, weighing HA-H powder, stirring and dissolving the HA-H powder with deionized water at room temperature, and slowly adding Ca (OAc) 2 into an HA solution; 2, after the reaction is completed, dialyzing in deionized water by using a dialysis bag until all inorganic impurities are removed, and freeze-drying the purified aqueous solution to obtain an HA-Ca complex; 3, an HA-Ca aqueous solution, an Alg-Na aqueous solution and an Ica solution are prepared respectively; 4, the HA-Ca aqueous solution and the Alg-Na aqueous solution are mixed, and the Ica solution is added; and 5, carrying out vortex oscillation, so as to obtain the injectable HA-Ca-Alg (at) Ica hydrogel loaded with Ica. The preparation method has the advantages that 1, the biocompatibility and the biodegradability are good; 2, the HA-Ca complex is combined with the Alg hydrogel, so that a stable compound structure is formed; 3, stable gel can be formed during in-vivo injection; 4, as a carrier, the compound has relatively high drug loading performance; and 5, directional release of the medicine can be realized.

Description

Technical Field

[0001] The invention belongs to the technical field of drug preparation, and in particular relates to a method for preparing a hydrogel conjugate for arthritis treatment. Background Art

[0002] Knee osteoarthritis (OA) is a common chronic disease in the middle-aged and elderly, characterized by the progressive loss of articular cartilage and excessive bone proliferation, which causes patients to experience symptoms such as chronic joint pain, stiffness and limited mobility. According to statistics, about 300 million people suffer from knee OA worldwide, which seriously affects the physical and mental health and quality of life of patients. With the aging of my country's population, the number of patients with knee OA is increasing, and its incidence and disability rate are also on the rise, which has attracted great attention and attention.

[0003] In the field of traditional Chinese medicine, icariin, as an important drug, is widely used in the treatment of strengthening muscles and bones and nourishing the liver and kidneys. It has a significant effect on regulating chondrocytes, which can increase bone formation, promote osteogenic differentiation, inhibit bone resorption, reduce bone loss, etc., and is of great significance for delaying the progression of osteoarthritis. Sodium hyaluronate (sodium hyaluronate) is a high-molecular linear mucopolysaccharide widely present in human connective tissue and body fluids. It is the main component of synovial fluid and cartilage matrix. It forms a complex with protein and is distributed in synovial fluid. It adheres to the surface of articular cartilage and synovial membrane, reducing the friction between tissues in the joint cavity. As a natural polymer, hyaluronic acid can reduce immune response to a certain extent when implanted in the body. At the same time, because it can simulate the mechanical properties of the extracellular matrix and can be degraded by enzymes, it is sufficient to withstand the pressure of surrounding tissues in the body without deformation or collapse. It is currently widely used as a degradable biomaterial in clinical practice. Studies have shown that intra-articular injection of hyaluronic acid can reduce chondrocyte apoptosis and increase chondrocyte proliferation. It can be used as the main component of drugs for the treatment of knee osteoarthritis. As a three-dimensional network-like porous material, hydrogel can be loaded with therapeutic elements such as drugs, cells, cytokines, etc. Its high water content can provide an ideal environment for cell survival. When hydrogel materials containing therapeutic elements are applied to pathological locations such as tissues and organs, it not only plays the role of a bionic scaffold, but also releases the contained therapeutic elements to help tissue recovery. As a natural polysaccharide, sodium alginate can easily form hydrogels with divalent cations to improve the mechanical properties and adhesion of hyaluronic acid hydrogels, so it can be used as a tissue engineering scaffold to transport cells and bioactive molecules. As a three-dimensional network-like porous material, hydrogel can be loaded with therapeutic elements such as drugs, cells, cytokines, etc. Its high water content can provide an ideal environment for cell survival. When hydrogel materials containing therapeutic elements are applied to pathological locations such as tissues and organs, it not only plays the role of a bionic scaffold, but also releases the contained therapeutic elements to help tissue recovery.

[0004] The existing synthesis technology is to combine through tert-butyloxycarbonyl, and the specific method is as follows: obtain tert-butyloxycarbonylglycine tert-butyl ester through glycine amino protection, add icariin to a mixed solution of tetrahydrofuran and pyridine, stir continuously until completely dissolved, add a certain amount of tert-butyloxycarbonylglycine tert-butyl ester, slowly add dicyclohexylcarbodiimide and dimethylaminopyridine tetrahydrofuran solution, stir the reaction at room temperature, add deionized water, filter, wash, and remove insoluble substances as much as possible; concentrate the solution through a rotary evaporator, and then precipitate the concentrated solution in water, dissolve the precipitate in a mixed solution of THF and pyridine; add water, freeze overnight, remove insoluble substances, concentrate, and add water to precipitate. Repeat this three times to obtain a dehydration condensation combination of icariin and tert-butyloxycarbonylglycine tert-butyl ester. Add to a certain amount of trifluoroacetic acid and DCM mixed solution, stir at room temperature for 1-2h; remove DCM by rotary evaporator, dissolve the obtained solid with ethyl acetate, wash with 5% sodium bicarbonate and adjust the pH to 8-9, remove the solvent, and obtain the third step reaction product aminoicariin. Add hyaluronic acid to distilled water, slowly add aminoicariin dropwise thereto, and add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide thereto. Adjust the pH of the solution to 4.5, stir the reaction at room temperature for 20min, then adjust the pH to 5.5, and stir at room temperature for 24h. Use column chromatography to separate and obtain the final product, i.e., the combination of icariin and hyaluronic acid.

[0005] This technology has the following shortcomings: 1. The operation is cumbersome and time-consuming; 2. Product purity issues: Using tert-butyloxycarbonyl to protect glycine may introduce impurities or produce side reactions, resulting in reduced purity of the synthesized product. This may affect the reliability of subsequent experiments and the interpretability of the results. 3. Reaction selectivity: After icariin is added to a mixed solution of tetrahydrofuran and pyridine, competitive reactions with other functional groups may occur, resulting in the formation of undesirable by-products and reducing the selectivity and purity of the target product. 4. Optimization of experimental conditions: The choice of reaction conditions may need to be further optimized, including the choice of solvent, temperature, reaction time, etc., to improve the efficiency of the reaction and the purity of the product.

[0006] The present invention provides a method for preparing a hydrogel conjugate for treating arthritis, provides a new material for treating arthritis, brings more effective treatment options to patients with knee osteoarthritis, and improves their quality of life and activity function. Summary of the invention

[0007] The above technical objectives of the present invention are achieved through the following technical solutions: a method for preparing a hydrogel conjugate for arthritis treatment, comprising the following steps:

[0008] A method for preparing a hydrogel conjugate for treating arthritis comprises the following steps:

[0009] Step 1: Weigh HA-H powder, stir and dissolve it in deionized water at room temperature to obtain a HA solution, then slowly add Ca(OAc)2 into the HA solution, and stir the mixture overnight;

[0010] Step 2: After the reaction is completed, dialyze in deionized water using a dialysis bag to remove all inorganic impurities, and freeze-dry the purified aqueous solution to obtain the HA-Ca complex for further use;

[0011] Step 3: Use ultrapure water to prepare HA-Ca aqueous solution with a mass concentration of 3%±0.5% and Alg-Na aqueous solution with a mass concentration of 3%±0.5%, and use DMSO to prepare Ica solution;

[0012] Step 4: Mix the HA-Ca aqueous solution and the Alg-Na aqueous solution, and add the Ica solution;

[0013] Step 5: Obtain injectable HA-Ca-Alg@Ica hydrogel loaded with Ica by vortex shaking.

[0014] Furthermore, in the step 1, HA-H is in powder form, and the amount of Ca(OAc)2 is 1 mol per 500 ml of deionized water, and the amount of HA-H powder is 0.005 mol.

[0015] Furthermore, the stirring in step 1 is carried out at 40°C.

[0016] Furthermore, the concentration of the Ica solution is 2 mg / mL, 4 mg / mL or 6 mg / mL.

[0017] Furthermore, the vortex shaking time in step 5 is 20±5 minutes.

[0018] Furthermore, the mixing volume ratio of the HA-Ca aqueous solution, the Alg-Na aqueous solution and the Ica solution is 50:50:1.

[0019] Compared with traditional treatment methods, this technology has the following advantages:

[0020] 1. The hydrogel complex of icariin and hyaluronic acid can exert the effects of both at the same time, thereby achieving comprehensive treatment with multiple therapeutic effects.

[0021] 2. The hydrogel conjugate provided by the present invention can be used for intra-articular injection. In this way, the drug can be directly delivered to the damaged joint tissue, thereby increasing the local concentration of the treatment and enhancing the treatment effect.

[0022] 3. This method has good safety and tolerability, can effectively reduce patients' discomfort and treatment risks, and improve treatment compliance and long-term efficacy.

[0023] Advantages in drug preparation:

[0024] 1. Drug compatibility: HA-Ca complex and Alginate (Alg) hydrogel are both natural products with good biocompatibility and biodegradability. They are not likely to cause immune response and tissue rejection and are suitable for in vivo injection.

[0025] 2. Structural stability: By combining the HA-Ca complex with the Alg hydrogel, a stable complex structure is formed, which is beneficial to maintaining the structural stability of the hydrogel and the loading stability of the drug.

[0026] 3. Injectability: HA-Ca-Alg hydrogel has a certain viscosity and fluidity, can form a stable gel when injected in vivo, has good injectability, and is not prone to tissue damage and uneven injection.

[0027] 4. Drug loading capacity: HA-Ca-Alg hydrogel as a carrier has a high drug loading capacity and can effectively load the target drug into the gel structure, thereby improving the stability and sustained release effect of the drug.

[0028] 5. Directed release: HA-Ca-Alg hydrogel can achieve directed release of drugs. By adjusting the composition and structure of the hydrogel, the drug can be slowly released in the body, prolonging the drug's action time in the body and improving the therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of preparing injectable hydrogels for HA-Ca and Alg-Na;

[0030] Figure 2 Preparation of injectable hydrogel HA-Ca-Alg for HA-Ca and Alg-Na, and images after inversion and freeze drying;

[0031] Figure 3 Images of HA-Ca-Alg@Ica hydrogel after inversion and freeze-drying.

[0032] Figure 4 Scanning electron microscope (SEM) images of HA-Ca and HA-Ca-Alg at different magnifications;

[0033] Figure 5 Energy dispersive X-ray spectra (EDS) and elemental distribution maps of HA-Ca and HA-Ca-Alg;

[0034] Figure 6 is the degradation rate of HA-Ca-Alg hydrogel in phosphate buffered saline (PBS) at 37°C;

[0035] Figure 7 Figure 2 is the degradation rate of the release curve of HA-Ca-Alg@Ica hydrogel containing different concentrations of icariin in PBS. DETAILED DESCRIPTION

[0036] The following is combined with Figure 1-3 The present invention is further described in detail with the following embodiments:

[0037] 1. Material preparation: Sodium alginate (Alg-Na), Jiangsu Peptide Biotechnology Co., Ltd.; hyaluronic acid, Xi'an Wanfang Biotechnology Co., Ltd.; calcium acetate (Ca(OAc)2), Jinan Anmu Biotechnology Co., Ltd.; icariin, Chengdu Huaxia Chemical Reagent Co., Ltd.

[0038] Example 1

[0039] HA-Ca-Alg hydrogel was prepared according to the following steps. Specifically, 5 g of HA-H powder was weighed and dissolved with 500 mL of deionized water at room temperature by stirring, and then 176.2 g (Ca(OAc)2) was weighed and slowly added to the HA solution. The mixture was stirred overnight at 40 degrees Celsius. After the reaction was completed, the solution was dialyzed in deionized water for 5 days using a dialysis bag to remove all inorganic impurities. The purified aqueous solution was freeze-dried to obtain a HA-Ca complex (yield 4.9 g). Thereafter, separate HA-Ca and Alg-Na aqueous solutions were prepared, each with a concentration of 3 wt%, referred to as solution A and solution B, respectively. 1 mL of solution A and 1 mL of solution B were taken in a ratio of 1:1, mixed and cross-linked after a brief vortex until a HA-Ca-Alg hydrogel was formed. In order to prepare hydrogels containing different concentrations of icariin, DMSO was used to prepare Ica solutions (solution C) with concentrations of 0.5, 1, and 2 mg / mL. During the above cross-linking process, 20 μL of icariin solution was immediately added to obtain injectable HA-Ca-Alg@Ica hydrogels containing Ica with concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively, which were named HA-Ca-Alg@Ica50, HA-Ca-Alg@Ica100, and HA-Ca-Alg@Ica200.

[0040] Field emission scanning electron microscopy (FE-SEM, Sigma 300, Zeiss, Germany) was used to characterize the microstructural morphology of HA-Ca and HA-Ca-Alg.

[0041] Energy dispersive spectroscopy (EDS, Oxford X-MaxN50, Oxford Instruments) was used to characterize the chemical composition of HA-Ca and HA-Ca-Alg.

[0042] In addition, to evaluate the release behavior of the loaded Ica hydrogel sample (HA-Ca-Alg@Ica), 1 mL of PBS was added to 1 mL of HA-Ca-Alg@Ica and incubated at 37 °C. The PBS solution was collected at the predetermined time point and supplemented with fresh PBS to continue incubation. The absorbance of Ica in the sample was measured at 300 nm using a multifunctional microplate reader (spectraMax i3x, Molecular Devices). The concentration of icariin was calculated based on the standard calibration curve of Ica.

[0043] Results: Preparation and characterization of hydrogels

[0044] Hydrogels, characterized by their cross-linked polymer structures, have attracted extensive attention as potential scaffolds for targeted delivery of therapeutic agents. Various mechanisms and structures allow drugs to be loaded into hydrogels. Figure 1 Schematic illustration of the injectable hydrogel (HA-Ca-Alg). The Ha-Ca complex is the precursor of the hydrogel and is synthesized by a direct acid-base reaction between hyaluronic acid (HA-H) and calcium acetate. Calcium acetate is used to form the HA-Ca-Alg hydrogel as a source of the main calcium ions, which are essential for the cross-linking process. These ions interact with the carboxylic acid groups of hyaluronic acid and sodium alginate to promote gelation. The internal gelation achieved by calcium acetate eliminates the need for external calcium salts such as or, simplifies the preparation process, and ensures a uniform distribution of calcium ions within the hydrogel. In addition, calcium ions enhance the mechanical properties of the hydrogel by forming ionic bonds with the carboxylic acid groups, thereby improving the hardness and stability of the hydrogel network. Subsequently, the injectable hydrogel (HA-Ca-Alg) was successfully prepared using the internal diffusion technique, involving the mixing of HA-Ca solution (solution A, 3%) with Alg-Na solution (solution B, 3%). In this case, the HA-Ca molecules that transport calcium ions act as two external donors and polysaccharide building blocks, resulting in the formation of a complex polymer network next to the Alg-Na molecules. When solutions A and B were mixed rapidly and homogeneously, the polymer chains of water-soluble HA-Ca(in) and Alg-Na collided, leading to robust cross-linking via the divalency of the carboxylate groups in the two cyclic polysaccharide chains.

[0045] Table 1 Primer sequences

[0046]

[0047] like Figure 2Preparation of injectable hydrogels HA-Ca-Alg for HA-Ca and Alg-Na, and images after inversion and freeze drying; Figure 3 Images of HA-Ca-Alg@Ica hydrogel after inversion and freeze-drying.

[0048] Scanning electron microscopy was used to observe the microstructure of the hydrogel in the freeze-dried samples. Figure 4 As shown, HA-Ca, as a precursor of hydrogel, presents an amorphous scaffold structure. When HA-Ca and Alg-Na polymerize to form HA-Ca-Alg, a honeycomb porous network structure appears with high porosity and a specific surface area of ​​approximately 100 μm. It is worth noting that this porous network structure also provides a suitable microenvironment for the delivery of the drug ingredient epi mediosine and ensures the transport of nutrients and oxygen, making cell penetration, migration and proliferation possible. In addition, the elemental composition and distribution level of the samples were analyzed using an energy spectrometer. Figure 5 As shown, HA-Ca collected elements C (46.66%), O (46.38%) and Ca (6.96%), while HA-Ca-Alg not only collected elements C (46.64%), O (47.46%) and Ca (2.98%), but also collected Na (2.91%). The element distribution map confirmed that the characteristic elements were uniformly distributed in the hydrogel HA-Ca-Alg.

[0049] The hydrolytic degradation behavior of hydrogels is critical to their use in biomedical applications. In the field of tissue engineering, hydrogels act as scaffolds to promote cell growth. It is critical that their degradation rate is consistent with the rate of new tissue formation. This ensures that the hydrogel is safely degraded when no longer needed, thereby minimizing potential adverse effects on nearby tissues. Additionally, within the field of drug delivery, the degradation properties of hydrogels enable their use as drug carriers for controlled drug release. The hydrolytic degradation behavior of HA-Ca-Alg hydrogels was quantified using a weighing method, and the change in the mass of the hydrogel samples during the hydrolysis process reflects their degradation kinetics. Figure 6 In the results, the degradation rate of HA-Ca-Alg hydrogel was slow in the first 4 days. In contrast, the hydrolytic degradation rate became faster in the next 7 days. In the last 5 days, the degradation rate gradually decreased. By the 16th day, the hydrogel sample reached a completely degraded state, at which time the measured value of the sample mass was close to zero, indicating that all degradable components had been decomposed. Figure 7 The release kinetics of icariin at different concentrations in HA-Ca-Alg hydrogels were demonstrated. The experimental data showed that

[0050] The release profiles of icariin from HA-Ca-Alg@Ica50, HA-Ca-Alg@Ica100, and HA-Ca-Alg@Ica200 groups all showed similar trends. A slightly faster release rate of icariin was observed on the first day, followed by a gradual slowdown in the following days, reflecting a controlled and sustained drug release pattern. This release pattern can be attributed to the hydrolytic degradation behavior of the hydrogel matrix. This is because the escape of the hydrophobic icariin molecules from the hydrophilic hydrogel phase to the hydrophilic PBS phase can only be achieved by water intrusion into the hydrogel drug carrier followed by hydrolytic degradation. The initial burst release of icariin can provide an immediate therapeutic effect, which is essential for reducing inflammation and pain in the early stages of treatment. The subsequent sustained release phase ensures a continuous supply of icariin, which is essential for maintaining therapeutic levels over a longer period of time. This is particularly important for chronic diseases such as osteoarthritis, which require long-term management. The controlled release of icariin reduces the risk of potential side effects associated with high local concentrations of the drug, thereby enhancing the safety of the treatment. In addition, the continued presence of icariin supports ongoing chondrocyte proliferation and matrix synthesis, which are essential for cartilage regeneration and repair. In summary, the release pattern of icariin from HA-Ca-Alg hydrogels provides a significant advance in the treatment of knee osteoarthritis by providing a rapid and sustained therapeutic effect. This release mechanism enhances the overall efficacy of the hydrogel formulation in promoting cartilage regeneration and reducing inflammation.

[0051] Summary: After synthesis, the HA-Ca complex was cross-linked with Alg-Na by internal diffusion technology to form HA-Ca-Alg hydrogel. Scanning electron microscopy showed that HA-Ca was an amorphous scaffold structure, and HA-Ca-Alg was a honeycomb porous network structure. Elemental energy spectrum analysis showed that the elements were evenly distributed.

[0052] The hydrolysis degradation experiment showed that HA-Ca-Alg hydrogel was completely degraded within 16 days. The drug release experiment showed that hydrogels loaded with different concentrations of drug (icariin) all showed an initial rapid release and subsequent slow and sustained release pattern. This release pattern is beneficial for the treatment of knee osteoarthritis.

[0053] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A method for preparing a hydrogel conjugate for treating arthritis, characterized in that: The following steps are involved: Step 1: Weigh HA-H powder, stir and dissolve it in deionized water at room temperature to obtain a HA solution, then slowly add Ca(OAc)2 into the HA solution, and stir the mixture overnight; Step 2: After the reaction is completed, dialyze in deionized water using a dialysis bag to remove all inorganic impurities, and freeze-dry the purified aqueous solution to obtain the HA-Ca complex for further use; Step 3: Use ultrapure water to prepare HA-Ca aqueous solution with a mass concentration of 3%±0.5% and Alg-Na aqueous solution with a mass concentration of 3%±0.5%, and use DMSO to prepare Ica solution; Step 4: Mix the HA-Ca aqueous solution and the Alg-Na aqueous solution, and add the Ica solution; Step 5: Obtain injectable HA-Ca-Alg@Ica hydrogel loaded with Ica by vortex shaking.

2. The method for preparing the hydrogel conjugate for arthritis treatment according to claim 1, characterized in that: In the step 1, HA-H is in powder form, and the amount of Ca(OAc)2 is 1 mol per 500 ml of deionized water, and the amount of HA-H powder is 0.005 mol.

3. The method for preparing the hydrogel conjugate for arthritis treatment according to claim 1, characterized in that: The stirring in step 1 is carried out at 40°C.

4. The method for preparing the hydrogel conjugate for arthritis treatment according to claim 1, characterized in that: The concentration of the Ica solution is 2 mg / mL, 4 mg / mL or 6 mg / mL.

5. The method for preparing the hydrogel conjugate for arthritis treatment according to claim 1, characterized in that: The vortex shaking time in step 5 is 20±5 minutes.

6. The method for preparing the hydrogel conjugate for arthritis treatment according to claim 1, characterized in that: The mixing volume ratio of the HA-Ca aqueous solution, the Alg-Na aqueous solution and the Ica solution is 50:50:1.

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