Hydrogel as well as preparation method and application thereof

Abstract

The invention discloses hydrogel as well as a preparation method and application thereof, belongs to the technical field of medical materials, and solves the problems of high recurrence rate and low curative effect of a treatment strategy of a diabetic wound in long-term treatment in the prior art. The method comprises the following steps: mixing a glutaminase inhibitor and a nano drug loading system, centrifuging, and collecting the precipitate to obtain a glutaminase inhibitor carrier, the drug loading system having oxidation resistance and drug loading capacity; mixing the modified hyaluronic acid with a gelatin solution to obtain gel; and adding the glutaminase inhibitor and the carrier into the gel, co-stirring, and adjusting the pH value to 7-8 to obtain the hydrogel. The hydrogel provided by the invention realizes specific removal of senescent cells from the source and remodeling of the ecological niche of the wound surface at the same time, so as to promote reepithelization and collagen deposition of diabetic tissues.

Description

Technical Field

[0001] The present invention relates to the technical field of medical materials, and more specifically to a hydrogel and a preparation method and application thereof. Background Art

[0002] Type 2 diabetes is a global chronic disease that can cause systemic tissue and organ lesions and serious complications in patients. Diabetic ulcers (diabetic wounds) are one of the most common types of ulcers. Diabetic wounds are characterized by a complex degenerative microenvironment, such as hyperglycemia, high reactive oxygen species (ROS), and excessive inflammation. Current treatments for diabetic wounds, such as lifestyle care, oral medication, injection medication, surgical debridement, and local decompression, are unable to achieve sustained, effective, and broad-spectrum effects. Since the vicious cycle between cell aging and inflammation is the key reason for wounds to heal, many research hotspots focus on improving the wound microenvironment through bioactive materials to promote wound healing, and improving the wound microenvironment through the introduction of functional materials such as antioxidants, anti-inflammatory agents, and continuous hemostasis and immunomodulation to accelerate the healing of skin wounds.

[0003] However, with the release of bioactive molecules or the consumption of functional groups, the existing strategies for improving the microenvironment of diabetic wounds to promote their healing mostly show problems such as high recurrence, high cost, and low efficacy in long-term treatment. Summary of the invention

[0004] The present invention provides a hydrogel and a preparation method and application thereof, which are used to solve the problems of high recurrence rate and low efficacy in long-term treatment of diabetic wounds in the prior art.

[0005] In a first aspect, the present invention provides a method for preparing a hydrogel, comprising the following steps: mixing a glutaminase inhibitor and a nano drug delivery system, centrifuging, collecting the precipitate, and obtaining a glutaminase inhibitor@carrier, wherein the drug delivery system has both antioxidant properties and drug loading capacity; mixing modified hyaluronic acid and a gelatin solution to obtain a gel; adding the glutaminase inhibitor@carrier to the gel, stirring, and adjusting the pH to 7-8 to obtain the hydrogel.

[0006] As a possible implementation method, the glutaminase inhibitor is any one of BPTES, Telaglenastat, IPN-60090 or a combination of several thereof; and / or, the modified hyaluronic acid is thiol-modified hyaluronic acid, and the thiol-modified hyaluronic acid method is any one of cystamine conjugation-thiolation modification method, lipoic acid hydrazide modification method, dithiodihydrazide modification method, and alkyl ester modification method; and / or, the nano drug delivery system is any one of polyphenol-based nanoparticles such as polydopamine particles, EGCG nanoparticles, and tannic acid nanoparticles.

[0007] As a possible implementation, the glutaminase inhibitor is BPTES; the modified hyaluronic acid is thiol-modified hyaluronic acid; and the nano drug delivery system is polydopamine.

[0008] As a possible implementation method, dopamine hydrochloride (DA) is added to a Tris 8.5 solution (50 mM, pH 8.5) with a final concentration of 2 mg / mL, stirred at room temperature in the dark, and centrifuged to collect the precipitate to obtain polydopamine (PDA), and PDA is used as the nanodrug delivery system; BPTES is used as the glutaminase inhibitor; the BPTES and the PDA are mixed, and the precipitate is collected by centrifugation to obtain BPTES@PDA; thiol-modified hyaluronic acid (HASH) is prepared by amide reaction to prepare a HASH solution; the HASH solution and a gelatin (Gel) solution are mixed, mixed with the BPTES@PDA, and the pH is adjusted to form a stable hydrogel BPTES@HASG-PDA, i.e., the hydrogel.

[0009] As a possible implementation method, the HASH preparation step includes: adding 3 times the amount of EDC and NHS of HA to a hyaluronic acid (HA, 100000MW, 14-17mg / mL) solution, stirring, and keeping the solution pH at about 6.5 to obtain a first mixed solution; adding 3 times the amount of HA to the first mixed solution, cystamine dihydrochloride, keeping the solution pH at 5-6, and continuing to stir for 36 hours at room temperature to obtain a second mixed solution; adding dithiothreitol, which has a 5-fold amount of HA in an anaerobic environment, to the second mixed solution, keeping the solution pH at 8.5, and continuing to stir for 7-12 hours at room temperature to obtain a third mixed solution; purifying the third mixed solution by dialyzing (MWCO 3500) with deionized water at pH 3.5, the dialysis time is more than 3d, and freeze-drying under vacuum for 96h to obtain the purified thiol-modified hyaluronic acid.

[0010] As a possible implementation, the molar ratio of the nano drug delivery system and the glutaminase inhibitor is 10:0.1-0.2; and / or, the solvent of the glutaminase inhibitor@carrier is a mixed solution of water and dimethyl sulfoxide, wherein the volume ratio of water to dimethyl sulfoxide is 1-2:1; and / or, in terms of g / mL, the ratio of the glutaminase inhibitor@carrier to the gel is 0.5-2:100.

[0011] In a second aspect, the present invention provides a hydrogel prepared by the preparation method described in any possible implementation manner of the first aspect.

[0012] As a possible implementation manner, the hydrogel is an in situ adhesion co-crosslinked hydrogel.

[0013] In a third aspect, the present invention provides a use of a hydrogel prepared by the preparation method described in any possible implementation of the first aspect or a hydrogel described in any possible implementation of the second aspect in the preparation of a drug that promotes wound healing.

[0014] As a possible implementation method, the hydrogel is used to specifically induce apoptosis of senescent cells.

[0015] As a possible implementation, the wound is caused by an aging-related degenerative disease.

[0016] As a possible implementation, the aging-related degenerative disease is diabetes.

[0017] The present invention provides a hydrogel, which is a thiol-modified hyaluronic acid-gelatin-dopamine nanoparticle integrated in-situ co-crosslinked hydrogel (HASG-PDA). Among them, the thiol and quinone groups can enhance the adhesion of the hydrogel to the skin, and the PDA (polydopamine) particles improve the mechanical properties of the hydrogel, while providing additional antioxidant activity, removing reactive oxygen, and improving the microenvironment. During cell aging, insoluble macromolecules in lysosomes cause lysosomal membrane damage, and hydrogen ions leak into cells, causing a decrease in intracellular pH. PDA particles are π-π stacked and loaded with BPTES (glutaminase inhibitor), which specifically inhibits glutamine enzymatic hydrolysis so that excess hydrogen ions cannot be neutralized, thereby causing intracellular acidosis and promoting apoptosis of senescent cells. The apoptosis of senescent cells also promotes the rejuvenation of the cell pool and the re-epithelialization of wound tissue, providing a more favorable new method for promoting diabetic wound healing. Compared with the prior art, the present invention recognizes that the vicious cycle between cell senescence and inflammation is the key reason why wounds are difficult to heal. It specifically removes senescent cells from the source while reshaping the wound niche, thereby promoting re-epithelialization of diabetic tissue and collagen deposition.

[0018] The hydrogel prepared by the present invention has strong adhesion and can specifically induce apoptosis of local senescent fibroblasts by inhibiting glutamine hydrolysis, and synergistically accelerate wound cell rejuvenation, tissue re-epithelialization and collagen deposition through antioxidant effects. The present invention is expected to promote wound healing in diabetic patients and has great clinical potential in treating diabetic wounds.

[0019] The present invention provides the use of hydrogel in the preparation of a drug for promoting wound healing. Since a moist and breathable environment is more conducive to wound repair, and hydrogel dressings have great potential in clinical wound treatment due to their bioadhesive properties, excellent water absorption properties, three-dimensional porous structure and adjustable degradation rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 The present invention provides a schematic diagram of the hydrogel preparation process according to an embodiment of the present invention, wherein dopamine represents dopamine, Polymerization represents polymerization, PDA represents polydopamine nanoparticles, BPTES represents glutaminase inhibitor, π-πStacking represents π-π stacking, HA-SH represents thiol-modified hyaluronic acid, Gel represents gelatin, -NH2 represents amino group, and Tissue represents tissue.

[0022] Figure 2 Schematic diagram of SEM characterization of PDA and BPTES@PDA provided in the embodiments of the present invention and the result diagram of sulfur element EDS analysis.

[0023] Figure 3 Schematic diagram of SEM characterization of hydrogel HASG, hydrogel HASG-PDA and hydrogel BPTES@HASG-PDA provided in embodiments of the present invention.

[0024] Figure 4 High-resolution XPS analysis results of the N spectra of the HASG hydrogel (left) and HASG-PDA hydrogel (right) provided in the embodiments of the present invention.

[0025] Figure 5 The swelling analysis results of HASG and HASG-PDA hydrogels provided in the embodiments of the present invention and the release spectrum results of BPTES@HASG-PDA hydrogels in simulated body fluids are presented, wherein the result data are expressed as mean ± standard deviation (*p<0.05, **p<0.01, ***p<0.001).

[0026] Figure 6 The results of adhesion analysis of HASG and HASG-PDA hydrogels provided in the embodiments of the present invention (n=4), wherein the result data are expressed as mean±standard deviation (*p<0.05, **p<0.01, ***p<0.001).

[0027] Figure 7 The results of apoptosis experiments provided in the embodiments of the present invention, wherein the first row is non-aging L929, and the second row is aging L929.

[0028] Figure 8The cell viability experiment results (n=6) provided in the embodiments of the present invention, wherein the left figure is the cell viability result of 1 day, and the right figure is the cell viability result of 3 days.

[0029] Fig. 9 The image results of the healing of the wound on the back of rats after surgery in the clinical experiment provided in the embodiment of the present invention.

[0030] Fig.10 In the clinical experiment provided in the embodiment of the present invention, the image results of the wound healing on the back of rats after surgery are fitted.

[0031] Fig.11 Representative images of HE staining and Masson staining of postoperative rat back wound tissue sections in the clinical experiment provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] In order to solve the problems of high recurrence rate and low efficacy in long-term treatment of diabetic wounds in the prior art, an embodiment of the present invention provides a hydrogel preparation experiment. Figure 1 The schematic diagram of the preparation process of the hydrogel provided by the embodiment of the present invention is shown as an example. Figure 1 As shown, the embodiment of the present invention provides the preparation of the hydrogel BPTES@HASG-PDA, and is verified by SEM analysis, XPS analysis, swelling analysis and other analysis methods, that the embodiment of the present invention successfully prepares the target product, and the obtained target product can achieve long-term sustained release of BPTES.

[0034] Furthermore, the embodiment of the present invention provides an application effect verification experiment of the hydrogel BPTES@HASG-PDA. It can be seen that BPTES@HASG-PDA can achieve effective removal of senescent cells under the premise of maintaining the normal function of non-senescent cells. BPTES@HASG-PDA showed the strongest wound healing ability on the back of diabetic rats, accelerating tissue re-epithelialization and collagen deposition. It shows that the hydrogel provided by the present invention can successfully promote diabetic wound healing, and this integrated hydrogel has great clinical potential in the treatment of diabetic wounds.

[0035] Further, the embodiment of the present invention provides a clinical experiment of hydrogel BPTES@HASG-PDA. In order to improve the wound microenvironment, the study found that biomaterials with ROS scavenging function are a new trend to accelerate diabetic wound healing and regeneration. For example, by introducing antioxidant materials to scavenge reactive oxygen species, skin cells are protected from high ROS-mediated death and proliferation inhibition, while mediating the polarization of macrophages to an anti-inflammatory phenotype to promote wound healing. The design of anti-inflammatory materials is used to reduce the expression of persistent inflammatory factors, avoid extracellular matrix deposition and interfere with the wound tissue remodeling process, and is conducive to wound healing. There are also studies that propose to balance the bioactivity of macrophages, endothelial cells and fibroblasts by regulating the pH microenvironment of the diabetic wound site, thereby accelerating cell proliferation and remodeling at the diabetic wound site. Designing scaffolds and particle platforms can regulate immune cells by delivering immunomodulators, thereby improving inflammatory diseases and accelerating diabetic wound healing. However, with the release of bioactive molecules or the consumption of functional groups, these strategies mostly show problems such as high recurrence, high cost, and low efficacy in long-term treatment.

[0036] Studies have found that diabetes is closely related to cell aging. The development of diabetes will accelerate the aging of various tissues of patients and promote the accumulation of senescent cells in the ecological niche. The accumulation of senescent cells in turn aggravates the formation of a degenerative inflammatory microenvironment, thereby mediating the production of secondary aging. This vicious cycle is the key to the difficulty in wound healing. The embodiment of the present invention specifically inhibits the inability of glutamine hydrolysis to neutralize excess hydrogen ions, thereby causing acidosis in senescent cells and promoting their apoptosis. The apoptosis of senescent cells also promotes the rejuvenation of the cell pool and the re-epithelialization of wound tissue. The hydrogel developed by the present invention adheres to the wound site in situ, promotes the apoptosis of senescent cells through sustained-release of drugs, and provides a more favorable new method for promoting diabetic wound healing.

[0037] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0038] Example 1

[0039] This example provides a hydrogel preparation experiment.

[0040] BPTES and nanoparticles of PDA were mixed, mixed in a rotary mixer in the dark for 24 h, centrifuged at 11000 rpm for 10 min, and the precipitate was collected to obtain nanoparticles of BPTES@PDA;

[0041] The thiol-modified hyaluronic acid (HASH) was dissolved in PBS solvent to prepare a HASH solution with a final concentration of 5% (w / v), and the pH was adjusted to 7.5 to obtain a hydrogel HASH; the hydrogel HASH and a gelatin (Gel) solution with a final concentration of 10% were mixed in a volume ratio of 2:1, and the pH was adjusted to 7.5 to obtain a hydrogel HASG;

[0042] The PDA and HASG colloidal solutions were mixed at a ratio of 1:100 (w / v), and the pH was adjusted to 7.5, and the hydrogel BPTES@HASG-PDA was quickly obtained within 5 seconds;

[0043] BPTES@PDA and HASG colloidal solution were mixed in a ratio of 1:100 (w / v), the pH was adjusted to 7.5, and the hydrogel BPTES@HASG-PDA was quickly obtained within 5 seconds.

[0044] Comparative Example: Tissue adhesion properties of HA-PEG (hydrazide-modified hyaluronic acid and four-arm polyethylene glycol with OPA end groups) hydrogels, which were sandwiched between two pieces of pig skin and their adhesion strength was measured by shear test. When the polymer concentration increased from 4% to 7% (w / v), the adhesion strength of the hydrogel increased significantly from 18.0±1.8kPa to 27.6±3.9kPa. However, when the concentration was further increased to 10% (w / v), the adhesion increased only slightly to 30.3±4.6kPa. (DOI:10.1126 / sciadv.adh4327)

[0045] In the present study, the maximum adhesion strength of HAGS-PDA hydrogel (46.55±7.21 kPa) was significantly higher than that of dopamine-coupled HA hydrogel (~10 kPa), photo-cross-linked HA hydrogel (~13 kPa), or commercially available fibrin glue (2-40 kPa).

[0046] Example 2

[0047] This example provides a hydrogel performance characterization experiment.

[0048] The PDA and BPTES@PDA, hydrogel HASG, hydrogel HASG-PDA and hydrogel BPTES@HASG-PDA in Example 1 were analyzed by SEM, and the results were as follows: Figure 2-3 The results shown. Figure 2 It can be seen that both PDA and BPTES@PDA nanoparticles are spherical and tightly aggregated, and drug loading does not affect the particle structure. EDS images show that sulfur is evenly distributed on BPTES@PDA nanoparticles, indicating the successful loading of drug BPTES. Figure 3 It can be seen that a three-dimensional porous structure can be observed in each hydrogel sample, and aggregated PDA nanoparticles appear on the surface of HASG-PDA and BPTES@HASG-PDA. With the addition of nanoparticles, the surface of the hydrogel becomes rough, and the covalent bonding of quinone groups with other groups increases the cross-linking density of the hydrogel and reduces the pore size.

[0049] XPS analysis was performed on the HASG hydrogel and HASG-PDA hydrogel in Example 1, and the following results were obtained: Figure 4 The results shown in the figure; the hydrogel swelling analysis and the drug release analysis of BPTES@HASG-PDA hydrogel were performed, and the results were as follows Figure 5 The results shown; the hydrogel adhesion analysis was performed and the results were as follows Figure 6 Results shown.

[0050] Depend on Figure 4 It can be seen that there are three peaks, including NH2, *NH 3+ The fitting of HASG-PDA with *CO-NH indicated that there was no chemical bond between the original Gel and HA-SH. However, the five peaks including *NH2, *NH 3+ , *C=N, *CO-NH and *C-NH-C, indicating that covalent bonds are formed between HASH, PDA and Gel matrix through Michael reaction or Schiff base reaction. Figure 5 It can be seen that the three groups of HASG-PDA hydrogels have a smaller swelling rate than HASG, so HASG-PDA hydrogel can effectively absorb exudate for a long time. The swelling rate of HASG-PDA hydrogel decreases with the increase of PDA content, which may be because the internal structure of the hydrogel becomes tighter with the increase of PDA content. The samples were immersed in a mixed solution of PBS and DMSO (1:9, v / v, pH 7.4) to simulate the conditions of the body fluid microenvironment. BPTES was gradually released before 7 days, and the release rate decreased with the number of days. It began to release slowly after 7 days, indicating that the hydrogel can achieve long-term sustained release of BPTES. Figure 6 It can be seen that the adhesion of HASG+PDA is significantly higher than that of HASG, and increases with the increase of PDA content.

[0051] Example 3

[0052] This embodiment provides an application experiment of a hydrogel.

[0053] 2×10 4 aging L929 and 2×10 4 Non-senescent L929 cells were inoculated in well plates, and the sample extracts were added and cultured in an incubator. The sample extracts were replaced every 2 days, and the cells were taken out at the designated time for apoptosis (Annexin V-FITC method) and cell viability (CCK8 method) experiments.

[0054] In the present invention, the hydrogel HASG, hydrogel HASG-PDA and hydrogel BPTES@HASG-PDA in Example 1 were used as test sample extracts, and apoptosis analysis and cell viability detection at different time points were performed on aged L929 and non-aged L929. Figure 7 Annexin V-FITC analysis results shown and Figure 8 The cell viability results are shown. Figure 7 and Figure 8 It can be seen that BPTES@HASG-PDA can significantly induce apoptosis of senescent L929 and has no obvious cytotoxicity to non-senescent L929, which confirms that BPTES@HASG-PDA can effectively eliminate senescent cells while maintaining the normal functions of non-senescent cells.

[0055] Example 4

[0056] This embodiment provides a clinical experiment of a hydrogel.

[0057] Each rat was intraperitoneally injected with streptozotocin (STZ, diabetic animal model) at a dose of 45 mg / kg to establish a diabetic rat model. The SD rats were 8 weeks old when they were first injected with STZ to induce diabetes. Hyperglycemia was confirmed when the blood sugar level was stabilized above 300 mg / dL. A full-thickness skin defect model was established after the blood sugar level was stabilized for one week, and the blood sugar of the SD rats was recorded until the wound was completely healed. The diabetic rats were randomly divided into 4 groups (n=5). After the rats were anesthetized and shaved, two complete skin defect wounds (10 mm in diameter) were formed on the back of each rat. The hydrogel HASG, hydrogel HASG-PDA and hydrogel BPTES@HASG-PDA in Example 1 were respectively used as wound dressings and adhered in situ to the skin defects of the rats. At the same time, a blank control group was set up and the rats were cultured under the same conditions. Record at the corresponding time points. The following is obtained: Fig. 9 and Fig.10 The wound healing diagram shown in the figure and Fig.11 Staining diagram shown.

[0058] Depend on Fig. 9 , Fig.10 and Fig.11 It can be seen that compared with other groups, BPTES@HASG-PDA showed the strongest wound healing ability on the back of diabetic rats, accelerating tissue re-epithelialization and collagen deposition. This shows that the hydrogel provided by the present invention can successfully promote diabetic wound healing, and this integrated hydrogel has great clinical potential in the treatment of diabetic wounds.

[0059] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0060] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing a hydrogel, characterized in that: The following steps are involved: The glutaminase inhibitor and the nano drug delivery system are mixed and then centrifuged to collect the precipitate to obtain the glutaminase inhibitor@carrier, wherein the drug delivery system has both antioxidant properties and drug loading capacity; The modified hyaluronic acid and gelatin solution are mixed to obtain a gel; the glutaminase inhibitor@carrier is added to the gel and stirred, and the pH is adjusted to 7-8 to obtain the hydrogel.

2. The preparation method according to claim 1, characterized in that: The glutaminase inhibitor is any one or a combination of BPTES, Telaglenastat, and IPN-60090; And / or, the modified hyaluronic acid is thiol-modified hyaluronic acid, and the thiol-modified hyaluronic acid method is any one of cystamine conjugation-thiolation modification method, lipoic acid hydrazide modification method, dithiodihydrazide modification method, and alkyl ester modification method; And / or, the nano drug delivery system is any one of polyphenol-based nanoparticles such as polydopamine nanoparticles, EGCG nanoparticles, and tannic acid nanoparticles.

3. The preparation method according to claim 1, characterized in that: The glutaminase inhibitor is BPTES; the modified hyaluronic acid is thiol-modified hyaluronic acid; and the nano drug delivery system is polydopamine.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The molar ratio of the nano drug delivery system to the glutaminase inhibitor is 10:0.1-0.2; And / or, the solvent of the glutaminase inhibitor@carrier is a mixed solution of water and dimethyl sulfoxide, wherein the volume ratio of water to dimethyl sulfoxide is 1 to 2:1; And / or, in terms of g / mL, the ratio of the glutaminase inhibitor@carrier to the gel is 0.5 to 2:

100.

5. A hydrogel prepared by the preparation method according to any one of claims 1 to 4.

6. The hydrogel according to claim 5, characterized in that The hydrogel is an in-situ adhesion co-crosslinked hydrogel.

7. Use of a hydrogel prepared by the preparation method according to any one of claims 1 to 4 or a hydrogel according to any one of claims 5 to 6 in the preparation of a drug for promoting wound healing.

8. The use according to claim 7, characterized in that: The hydrogel is used to specifically induce apoptosis of senescent cells.

9. The use according to claim 7, characterized in that: The wound is caused by aging-related degenerative diseases.

10. The use according to claim 9, characterized in that: The aging-related degenerative disease is diabetes.

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