Gel long-acting slow-release agent and preparation method thereof

Abstract

The invention relates to the technical field of biological materials, and particularly discloses a gel long-acting slow-release agent and a preparation method thereof. The slow-release agent is obtained by firstly grafting neuropeptide on chitosan, then grafting self-assembled small peptide with the neuropeptide and finally mixing with nano zinc oxide, the amino acid sequence of the neuropeptide SP is RPKPQQFFGLM, and the amino acid sequence of the self-assembled peptide Nap-beta is NapFFVLEGG. The hydrogel is loose in structure and compact in arrangement, proliferation and adhesion of cells are promoted through a net-shaped micropore interconnected structure, rapid loss of gel moisture is avoided, hydrogel cross-linked micelles can be increased through embedded nanometer ZnO, the mechanical performance of the gel is improved, and the function of long-acting slow release of neuropeptide is achieved. The accumulative release amount of the neuropeptide SP is not less than 75% within 72 hours in a phosphate buffer solution with the pH value of 7.2-7.4, and the sustained release agent can be used as a continuous drug release system to ensure the local drug concentration.

Description

Technical Field

[0001] The invention belongs to the technical field of biomaterials, and particularly relates to a long-acting sustained-release gel and a preparation method thereof. Background Art

[0002] With the increasing incidence of chronic diseases such as diabetes and the accelerating global aging population, the incidence of chronic skin wounds is increasing annually. Currently, treatments for chronic wounds rely primarily on ultrasonic hydrotherapy debridement, negative pressure aspiration, platelet-rich plasma therapy, and topical cytokine, stem cell, and drug administration. However, these treatments are limited by their high cost, limited efficacy, and the potential for new infections. Notably, among these treatments, topical administration offers unique advantages, such as high drug concentration, minimal systemic toxicity, and minimal side effects, making it widely used. Several biomaterials, such as foams, sponges, nanofibers, and hydrogels, have been developed as wound dressings. However, most dressings serve solely as biological delivery vehicles and physical covering matrices, resulting in poor efficacy in treating chronic wounds. This is due to the holistic nature of the chronic wound healing process and the multifactorial nature of autoimmune disorders, which lead to persistent, long-lasting inflammation and further complicate treatment. Excessive inflammation is the primary culprit for wound healing difficulties. Without effective treatments, refractory wounds will ultimately lead to sepsis or amputation, posing a serious threat to public health.

[0003] Inflammation is a double-edged sword for wound healing. In chronic wounds, immune dysfunction leads to severe tissue necrosis and oxidative stress, stimulating macrophage aggregation and sustained pro-inflammatory activity. This disrupts the inflammatory balance, creating a self-sustaining vicious cycle that ultimately leads to prolonged wound healing. Therefore, designing a local multifunctional drug delivery system to alleviate wound oxidative stress and restore immune regulation is an effective strategy to improve the deficiencies of current dressings and promote chronic wound healing.

[0004] In this context, the search for a substance that can restore immune regulation and participate in the overall wound healing process is crucial to overcome the current difficulties faced by wound repair. In previous studies, we screened and identified the neuropeptide Substance P (SP), which is involved in multiple wound healing processes. Previous studies have shown that SP exerts immunomodulatory effects through the G protein-coupled neurokinin receptor 1 (NK-1R), promoting early inflammation, inducing neutrophil migration to the site of inflammation, promoting mononuclear macrophage polarization, exerting anti-inflammatory effects, and promoting the migration and proliferation of epidermal and endothelial cells during the proliferative phase, thereby inducing tissue granulation and angiogenesis. In short, SP, through the action of cytokines, influences and regulates skin immunity and participates in multiple wound repair processes. However, when soluble SP is directly injected into damaged tissue, it is easily degraded by enzymes, making it impossible to maintain local concentration. To address this issue, it is necessary to find a superior deformable material that can act as a covering matrix while continuously releasing SP.

[0005] In view of this, it is meaningful to develop a sustained drug release hydrogel system to ensure local drug concentration. Summary of the Invention

[0006] The purpose of the present invention is to provide a gel long-acting sustained-release agent and a preparation method thereof to overcome the defects of skin wound dressings in the prior art in view of the above-mentioned deficiencies of the prior art.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The first aspect of the present invention is to provide a method for preparing a long-acting sustained-release gel, comprising the following steps:

[0009] Step S1, activating the neuropeptide SP with EDC and NHS, adding the activated SP to an acetate buffer solution containing chitosan, stirring, dialyzing, and freeze-drying to obtain chitosan CS-SP grafted with neuropeptide; the amino acid sequence of the neuropeptide SP is RPKPQQFFGLM;

[0010] Step S2: In the presence of catalysts EDC and NHS, the CS-SP prepared in step S1 was dissolved in an aqueous solution, DMSO was added to dissolve the self-assembling peptide Nap-β, the mixture was stirred for reaction, dialyzed, and freeze-dried to obtain a self-assembled chitosan drug-loaded hydrogel grafted with neuropeptide, which was designated as CS-SP-Nap-β.

[0011] The amino acid sequence of the self-assembling peptide Nap-β is NapFFVLEGG;

[0012] Step S3, dissolving the CS-SP-Nap-β obtained in step S2 in water, dispersing nano-ZnO in water, and then mixing the obtained CS-SP-Nap-β solution and nano-ZnO solution to obtain a mixed solution, which is allowed to stand to obtain a self-assembled chitosan drug-loaded hydrogel grafted with neuropeptide, recorded as CS-SP-Nap-β@ZnO, which is the gel long-acting sustained-release agent.

[0013] Furthermore, in step S1, the molar ratio of neuropeptide SP, EDC and NHS is (0.4-0.7):15.6:17.4.

[0014] Furthermore, the mass ratio of neuropeptide SP to chitosan is (0.8-1.5):500.

[0015] Furthermore, the volume concentration of the acetic acid aqueous solution is 1.5% to 2%; the pH value of the acetate buffer solution containing the chitosan is 4 to 5; and the mixture is stirred at 2° C. to 4° C. overnight.

[0016] Furthermore, the activation process is to add neuropeptide SP, EDC and NHS into an aqueous solution and stir at 2°C to 4°C for 0.5 to 2 hours.

[0017] Furthermore, in step S2, the mass ratio of neuropeptide SP, chitosan and small peptide Nap-β is (0.8-1.5):500:(4.5-5.5).

[0018] Furthermore, in step S3, the concentration of CS-SP-Nap-β in the mixed solution is 0.025-0.0375 g / mL, and the concentration of ZnO is 1-5 μg / mL.

[0019] Furthermore, the dialysis process in steps S1 and S2 is: dialyzing in an aqueous solution for 70 to 80 hours using a dialysis bag with a molecular weight cut-off of 1000 Da.

[0020] Furthermore, in step S2, the stirring reaction temperature is 2° C. to 4° C., and the stirring reaction time is 5 to 8 hours.

[0021] The second aspect of the present invention is a long-acting sustained-release gel prepared by the above-mentioned preparation method.

[0022] Furthermore, using a phosphate buffer solution with a pH of 7.2 to 7.4 as a dissolution medium, the cumulative release of neuropeptide SP within 72 hours is not less than 75%.

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

[0024] (1) The present invention provides a long-acting sustained-release gel, which is obtained by first grafting a neuropeptide onto chitosan, then grafting a self-assembling peptide onto the neuropeptide, and finally mixing it with nano-zinc oxide. The amino acid sequence of the neuropeptide SP is RPKPQQFFGLM, and the amino acid sequence of the self-assembling peptide Nap-β is NapFFVLEGG. The hydrogel has a loose structure and a densely packed network of interconnected micropores, which promotes cell proliferation and adhesion and prevents rapid water loss in the gel.

[0025] (2) The nano-ZnO contained in the long-acting sustained-release gel provided by the present invention can increase the cross-linked micelles in the hydrogel and improve the mechanical properties of the gel. The chitosan composite hydrogel containing nano-ZnO can achieve long-term sustained release of neuropeptides. Furthermore, nano-ZnO, as a metal cofactor for oxidases, can regulate oxidative stress in the wound microenvironment.

[0026] (3) The long-acting sustained-release gel provided by the present invention has a cumulative release of no less than 75% of neuropeptide SP within 72 hours in a phosphate buffer solution with a pH of 7.2 to 7.4, and can serve as a sustained drug release system to ensure local drug concentration.

[0027] (4) The preparation method provided by the present invention is simple to operate and has mild experimental conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is the infrared spectrum of the CS-SP-Nap-β co-block in Example 1;

[0029] Figure 2 This is the UV spectrum of the CS-SP-Nap-β@ZnO drug-loaded hydrogel in Example 1;

[0030] Figure 3 Scanning electron microscopy images of CS-SP-Nap-β and CS-SP-Nap-β@ZnO in Example 1;

[0031] Figure 4 This is a transmission electron microscopy image of the CS-SP-Nap-β@ZnO head in Example 1;

[0032] Figure 5 Amplitude scanning diagram of CS-SP-Nap-β@ZnO drug-loaded hydrogel at different concentration ratios;

[0033] Figure 6 This is the self-recovery scanning diagram of CS-SP-Nap-β@ZnO drug-loaded hydrogel at different concentration ratios;

[0034] Figure 7 This is the frequency scanning diagram of CS-SP-Nap-β@ZnO drug-loaded hydrogel at different concentration ratios;

[0035] Figure 8 is the cumulative release rate of SP of CS-SP-Nap-β, CS-SP-Nap-β@ZnO (0.5%), and CS-SP-Nap-β@ZnO (1%);

[0036] Figure 9 The swelling rate results of CS-SP-Nap-β@ZnO at different concentrations;

[0037] Figure 10 This is the result diagram of the moisturizing rate of CS-SP-Nap-β@ZnO at different concentrations;

[0038] Figure 11 This is the degradation rate result diagram of CS-SP-Nap-β@ZnO at different concentrations;

[0039] Figure 12 The graph shows the effects of neuropeptide SP, simple Nap-β, carrier CS-Nap-β and nano-ZnO on cell viability;

[0040] Figure 13The graph shows the blood compatibility results of neuropeptide SP, simple Nap-β, carrier CS-Nap-β, nano-ZnO and hydrogel CS-SP-Nap-β, CS-SP-Nap-β@ZnO. DETAILED DESCRIPTION

[0041] To make the objects, technical solutions, and advantages of the present invention more apparent, the following describes the specific embodiments of the present invention in further detail with reference to the specific examples and accompanying drawings. Where specific test methods, instruments, or conditions are not specified in the examples, the methods or conditions described in the literature in the art or the product specifications were used. Where the manufacturer of the reagents or instruments is not specified, all are commercially available conventional products.

[0042] The biomaterials used in this invention are as follows: neuropeptide SP (sequence: RPKPQQFFGLM) and self-assembling peptide Nap-β (sequence: NapFFVLEGG) were synthesized by Dangang Biotechnology Co., Ltd.; nano-zinc oxide (particle size: 50±10 nm) was purchased from Solebo; chitosan (deacetylation >90%, Mw ≈200 kDa) was purchased from Maclean; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl), N-hydroxysuccinamide (NHS), glacial acetic acid, and dimethyl sulfoxide (DMSO) were purchased from Aladdin Biotechnology Co., Ltd.; calcein and propidium iodide (PI), lipopolysaccharide (LPS), interferon-γ (IFN-γ), interleukin-4 (IL-4), and Hostchest were all purchased from Beyotime Biotechnology Co., Ltd. The CCK-8 kit and ELISA kit used in the experiments were purchased from Xinbosheng Biotechnology Co., Ltd. The EDU kit was purchased from SIGMA-ALDRICH.

[0043] The English abbreviations in this invention are explained as follows:

[0044] SP: neuropeptide SP;

[0045] Nap-β: self-assembling peptide Nap-β;

[0046] CS: chitosan;

[0047] EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0048] NHS: N-hydroxysuccinamide.

[0049] The present invention provides a long-acting sustained-release gel preparation, which is prepared by using a self-assembled neuropeptide-grafted chitosan drug-loaded hydrogel, and the specific steps include:

[0050] (1) Neuropeptide SP was activated by EDC and NHS, and the activated SP was added to an acetate buffer solution containing chitosan, stirred, dialyzed, and freeze-dried to obtain chitosan CS-SP grafted with neuropeptide, wherein the mass ratio of neuropeptide SP to chitosan was 0.8-1.5:500; wherein the activation process was as follows: neuropeptide SP, EDC and NHS were added to an aqueous solution, stirred at 2°C to 4°C for 2 h; the concentration of the acetate buffer solution was 1.5% to 2% (v / v), the pH value of the acetate buffer solution containing chitosan was 4-5, and stirred at 2°C to 4°C overnight; and dialyzed in an aqueous solution using a dialysis bag with MWCO=1000 for 70-80 h.

[0051] (2) In the presence of catalysts EDC and NHS, the CS-SP prepared in step (1) is dissolved in an aqueous solution, DMSO is added to dissolve the self-assembling peptide Nap-β, the reaction is stirred, and the mixture is dialyzed to obtain a self-assembling chitosan drug-loaded hydrogel CS-SP-Nap-β grafted with neuropeptide, which is freeze-dried. In step S2, the mass ratio of neuropeptide SP, chitosan, and small peptide Nap-β is 0.8-1.5:500:4.5-5.5; the stirring reaction temperature is 2°C-4°C, the stirring reaction time is 5-8h, and the mixture is dialyzed in an aqueous solution using a dialysis bag with MWCO=1000 for 70-80 hours.

[0052] (3) The freeze-dried CS-SP-Nap-β in step (2) is dissolved in water, nano-ZnO is sonicated in the aqueous solution, the obtained CS-SP-Nap-β solution and the nano-ZnO solution are stirred together, and allowed to stand to obtain a self-assembled chitosan drug-loaded hydrogel CS-SP-Nap-β@ZnO grafted with neuropeptide, wherein the concentration of CS-SP-Nap-β is 0.025-0.0375 g / mL and the concentration of ZnO is 1-5 μg / mL.

[0053] The CS-SP-Nap-β@ZnO prepared by the above method have similar chemical structures and drug release properties.

[0054] Release determination:

[0055] 1 mg / mL SP was diluted to 5-500 μg / mL solutions, and the absorbance at the maximum absorption wavelength was measured. Next, to detect the release of SP from the gel group, CS-SP-Nap-β and CS-SP-Nap-β@ZnO hydrogels of varying concentrations (20 mg, n=3) were placed in a 3500 kDa dialysis bag, added with PBS (3 mL), and immersed in PBS (30 mL) at 37°C and shaken at 250 rpm. At predetermined time points, 1 mL of release buffer was aspirated for analysis, and 1 mL of fresh PBS was added to maintain the same volume.

[0056] 8 mg of the freeze-dried CS-SP-Nap-β@ZnO hydrogel of the present invention was immersed in a constant temperature aqueous solution at 37°C. At different time points, the immersed hydrogel was taken out to remove surface moisture, and the mass was weighed and recorded to study the swelling and degradation properties of the hydrogel.

[0057] The CS-SP-Nap-β@ZnO hydrogel of the present invention was applied to skin cells and macrophages to detect cell viability. Specifically, in the cell survival experiment, the amount of CS-SP-Nap-β@ZnO hydrogel loaded was 20 μl per well, and PBS (pH = 7.2-7.4) containing SP was added, and the SP concentration ranged from 2 μg / mL to 20 μg / mL.

[0058] Example 1

[0059] This embodiment provides a method for preparing a long-acting sustained-release gel (0.5% ZnO-CS-SP-Nap-β (1%)), the specific steps of which are as follows:

[0060] Step S1: Dissolve 0.5 g of chitosan (CS) in glacial acetic acid (0.2 mM, 50 ml) and stir overnight to prepare a CS solution. Dissolve 1 mg of neuropeptide SP in 50 ml of deionized water and add a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 3 mg) and N-hydroxysuccinamide (NHS, 2 mg) for 2 hours to activate its carboxyl (-COOH) groups to form a stable active ester. The prepared CS solution is then added to the reaction system, and the mixture is stirred at 4°C overnight. The resulting solution is designated CS-SP.

[0061] Step S2: 5 mg of the self-assembling peptide (Nap-β) was dissolved in an appropriate amount of dimethyl sulfoxide (DMSO). The activation was carried out in the same manner as described above for the carboxyl group activation step. After activation, the peptide was added to the CS-SP solution that had reacted overnight at 4°C and stirred for 8 h. The peptide was then transferred to a dialysis bag (MWCO = 1000 Da, Tono Biotech, SP132638). After dialysis, the product was freeze-dried and designated as CS-SP-Nap-β.

[0062] In step S3, 0.25 g of CS-SP-Nap-β was added to 5 ml of deionized water, slowly stirred overnight, and allowed to stand at 37°C. The gelation of the hydrogel was assessed using a tube inversion test. In this test, if no visual fluidity was observed within 1 minute of inversion, hydrogel formation was confirmed. A nanozinc oxide solution was prepared by dispersing 40 μg of nanozinc oxide in 3 ml of deionized water. The CS-SP-Nap-β hydrogel loaded with nanozinc oxide (ZnO) was prepared by adding the hydrogel obtained above to the nanozinc oxide solution and continuing to stir for 30 minutes. The ZnO concentration was 5 μg / ml. The product was freeze-dried for subsequent use.

[0063] Example 2

[0064] This embodiment provides a method for preparing a long-acting sustained-release gel (0.75% ZnO-CS-SP-Nap-β (1%)), the specific steps of which are as follows:

[0065] Step S1: Dissolve 0.5 g of chitosan (CS) in glacial acetic acid (0.2 mM, 50 ml) and stir overnight to prepare a CS solution. Dissolve 1 mg of neuropeptide SP in 50 ml of deionized water and add a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 3 mg) and N-hydroxysuccinamide (NHS, 2 mg) for 2 hours to activate its carboxyl (-COOH) groups to form a stable active ester. The prepared CS solution is then added to the reaction system, and the mixture is stirred at 4°C overnight. The resulting solution is designated CS-SP.

[0066] Step S2: 5 mg of the self-assembling peptide (Nap-β) was dissolved in an appropriate amount of dimethyl sulfoxide (DMSO). The activation was carried out in the same manner as described above for the carboxyl group activation step. After activation, the peptide was added to the CS-SP solution that had reacted overnight at 4°C and stirred for 8 h. The peptide was then transferred to a dialysis bag (MWCO = 1000 Da, Tono Biotech, SP132638). After dialysis, the product was freeze-dried and designated as CS-SP-Nap-β.

[0067] In step S3, 0.25 g of CS-SP-Nap-β was added to 8 ml of deionized water, slowly stirred overnight, and allowed to stand at 37°C. The gelation of the hydrogel was assessed using a tube inversion test. In the tube inversion test, if no visual fluidity was observed within 1 minute of inversion, hydrogel formation was confirmed. A nanozinc oxide solution was prepared by dispersing 60 μg of nanozinc oxide in 3 ml of deionized water. The CS-SP-Nap-β hydrogel loaded with nanozinc oxide (ZnO) was prepared by adding the hydrogel obtained above to the nanozinc oxide solution and continuing to stir for 30 minutes. The ZnO concentration was 7.5 μg / ml. The product was freeze-dried for subsequent use.

[0068] Example 3

[0069] This embodiment provides a method for preparing a long-acting sustained-release gel (1% ZnO-CS-SP-Nap-β (1%)), the specific steps of which are as follows:

[0070] Step S1: Dissolve 0.5 g of chitosan (CS) in glacial acetic acid (0.2 mM, 50 ml) and stir overnight to prepare a CS solution. Dissolve 1 mg of neuropeptide SP in 50 ml of deionized water and add a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC, 3 mg) and N-hydroxysuccinamide (NHS, 2 mg) for 2 hours to activate its carboxyl (-COOH) groups to form a stable active ester. The prepared CS solution is then added to the reaction system, and the mixture is stirred at 4°C overnight. The resulting solution is designated CS-SP.

[0071] Step S2: 5 mg of the self-assembling peptide (Nap-β) was dissolved in an appropriate amount of dimethyl sulfoxide (DMSO). The activation was carried out in the same manner as described above for the carboxyl group activation step. After activation, the peptide was added to the CS-SP solution that had reacted overnight at 4°C and stirred for 8 h. The peptide was then transferred to a dialysis bag (MWCO = 1000 Da, Tono Biotech, SP132638). After dialysis, the product was freeze-dried and designated as CS-SP-Nap-β.

[0072] In step S3, 0.25 g of CS-SP-Nap-β was added to 8 ml of deionized water, slowly stirred overnight, and allowed to stand at 37°C. The gelation of the hydrogel was assessed using a tube inversion test. In the tube inversion test, if no visual fluidity was observed within 1 minute of inversion, hydrogel formation was confirmed. 80 μg of nanozinc oxide was dispersed in 3 ml of deionized water to obtain a nanozinc oxide solution. The CS-SP-Nap-β hydrogel loaded with nanozinc oxide (ZnO) was prepared by adding the hydrogel obtained above to the nanozinc oxide solution and continuing to stir for 30 minutes. The ZnO concentration was 10 μg / ml. The product was freeze-dried for subsequent use.

[0073] refer to Figure 1 , is the infrared spectrum of CS-SP-Nap-β coblock;

[0074] refer to Figure 2 , is the UV spectrum of CS-SP-Nap-β@ZnO drug-loaded hydrogel;

[0075] refer to Figure 3 , which are scanning electron micrographs of CS-SP-Nap-β and CS-SP-Nap-β@ZnO. Scanning electron microscopy shows that CS-SP-Nap-β forms a cross-linked network structure after self-assembly. After loading 0.5% ZnO, the micelle cross-linking increases, showing a tightly arranged porous structure. Compared with the dense sheet morphology of 1% ZnO-CS-SP-Nap-β, this network of interconnected micropores is more conducive to cell proliferation and adhesion, and avoids rapid loss of gel water.

[0076] refer to Figure 4, which is a transmission electron microscopy image of the CS-SP-Nap-β@ZnO head. The adhesion of nanoparticles can be seen more intuitively under TEM. Nano-zinc oxide particles adhere to the surface of the gel and are evenly distributed with a particle size of 30 to 40 nm.

[0077] In order to better illustrate the sustained-release performance of the CS-SP-Nap-β hydrogel loaded with nano zinc oxide (ZnO) provided by the present invention, the inventors also conducted the following research:

[0078] (1) Rheological research

[0079] In rheological experiments, the self-assembly process of CS-SP-Nap-β in solution was observed using a time-sweep mode. Subsequently, hydrogels prepared with varying concentrations of Nap-β and ZnO (as shown in Table 1) were subjected to three rheometer-based sweeps: amplitude, frequency, and self-healing performance. The amplitude sweep of the hydrogels was conducted under strain conditions ranging from 0.1% to 100%, with no normal force applied and a strain interval of 14 seconds. Self-healing was tested using an oscillation-rotation-oscillation mode, with alternating strain values set according to the amplitude sweep results. For the CS-SP-Nap-β gel, the alternating strain values were 20%, 180%, and 20%, and for the CS-SP-Nap-β@ZnO, the alternating strain values were 0.1%, 100%, and 0.1%. Two cycles were performed with an alternating interval of 10 data points and a fixed angular frequency of 1 rad / s. The frequency sweep was conducted with a fixed strain of 0.1% and an angular frequency of 0.1 to 100 rad / s.

[0080] Table 1.

[0081]

[0082] like Figure 5 As shown in the amplitude scanning mode, it can be seen that the linear viscoelastic range of 0.5% Nap-β-CS-SP gel is small and is destroyed at a strain of 1%, indicating that the cross-linked network of the self-assembled small peptide at this concentration is unstable, and 1% ZnO-CS-SP-Nap-β is too brittle due to excessive cross-linking and is also destroyed at a strain of 1%. 1% Nap-β-CS-SP, 0.5% ZnO-CS-SP-Nap-β (1%) gel has a relatively stable linear viscoelastic range at 0.1% to 100% strain. However, compared with 1% Nap-β-CS-SP, the linear viscoelastic range of the gel becomes narrower after loading nano-zinc oxide. This is mainly because nano-zinc oxide increases the interpenetrating network while also increasing the brittleness of the gel ( Figure 6As shown), this result was also proved by the self-recovery performance test. Two groups of gels with good linear viscoelastic range, 1% Nap-β-CS-SP and 0.5% ZnO-CS-SP-Nap-β (1%), were selected for self-recovery cycle test. Both groups of gels were able to recover to the gel state in a short time after being damaged by stress. It is worth noting that the strain of 1% Nap-β-CS-SP was 180%, which was greater than 0.5% Nps ZnO-CS-SP-Nap-β, and it was still able to recover by itself, proving that 1% Nap-β-CS-SP has stronger toughness. Next, the two groups of gels were scanned in a fixed angular frequency range. As the frequency increased, the storage modulus (G′) was always greater than the loss modulus (G″), showing good stability. At the same time, the storage modulus (G′) and loss modulus (G″) of CS-SP-Nap-β@ZnO gel were almost greater than those of CS-SP-Nap-β, indicating that Zn 2+ It is indeed possible to increase the cross-linking of the gel and reduce the gel concentration of the material. The greater the degree of cross-linking of the gel, the greater the strength of the material ( Figure 7 shown).

[0083] (2) Release rate study

[0084] The CS-SP-Nap-β@ZnO hydrogel of the present invention was placed in a phosphate buffer solution (pH = 7.2-7.4) and placed in a dialysis bag. The release of neuropeptides was measured, and samples were taken at different time points. The buffer was replenished to generate drug release curves. A standard concentration curve was used to quantitatively determine the release of substance P (SP) from different hydrogel groups. Specifically, SP was dissolved in phosphate buffer (pH = 7.2-7.4) at a concentration of 5-500 μg / mL to prepare an SP solution, and the absorbance at the wavelength of maximum absorption was measured. CS-SP-Nap-β and CS-SP-Nap-β@ZnO hydrogels of varying concentrations (20 mg, n = 3) were placed in a 3500 kDa dialysis bag and immersed in 30 mL of fresh phosphate buffer (pH = 7.2-7.4). The mixture was stirred at 37°C and 250 rpm. At predetermined time intervals, 1 mL of release buffer was collected for analysis, and an equal amount of fresh phosphate buffer saline (pH = 7.2-7.4) was added to maintain a constant volume.

[0085] exist Figure 8The results show that the release of SP was detected in all three groups of materials. The cumulative release curve shows that the cumulative release amounts of SP-containing hydrogels with different material ratios of CS-SP-Nap-β, CS-SP-Nap-β@ZnO (0.5%) and CS-SP-Nap-β@ZnO (1%) were 72%, 79% and 62%, respectively. The hydrogel in the CS-SP-Nap-β@ZnO (0.5%) group released slightly faster within 1-8 hours, with a cumulative release amount of 30%. After incubation in PBS for 3 days, the unreleased amounts of SP in CS-SP-Nap-β and CS-SP-Nap-β@ZnO were 28%, 21% and 38%, respectively. This may be because a small amount of drug is locked in the hydrogel network and cannot be released, and will be released along with the degradation of the hydrogel. In summary, the results show that the cumulative release of SP in the CS-SP-Nap-β@ZnO (0.5%) group is higher than that in the other two groups, indicating that the addition of a certain concentration of nano-ZnO increases the SP release curve. The drug release experiment shows that the hydrogel can continuously release SP within 72 hours, which can play a long-term role in the wound.

[0086] (3) Study on swelling rate and moisture retention

[0087] The swelling ratio (SR) and moisture retention (WR) of gelled hydrogel materials of different concentrations at 37°C were tested by weighing. First, the freeze-dried gel was weighed and recorded as the initial weight (W0). Then, a certain initial mass of the material was immersed in a 37°C constant temperature aqueous solution. At different time points, the soaked hydrogel was removed to remove the surface moisture and the weight was recorded (Wt). Three sets of parallel experiments were repeated and the average value was taken. Finally, the swelling ratio was calculated according to the following formula:

[0088] SR%=[(Wt-W0) / W0]×100.

[0089] The moisture retention test experiment was similar to the above. After the hydrogel was prepared, the initial wet mass (M0) was recorded. The hydrogel was placed in a container and exposed to air to dry. The mass (Mt) was weighed at different time points. Three sets of parallel experiments were repeated. The WR was calculated as follows:

[0090] WR%=Mt / M0×100.

[0091] In the degradation experiments, a constant weight (W0) of freeze-dried hydrogel was weighed and immersed in 8 ml of phosphate-buffered saline (PBS) and incubated on a 37°C shaker. At different times, the remaining material was freeze-dried and weighed (Wt). Three replicates were performed. The residual weight (WR) was calculated using the same method as in the moisture retention experiments. The results were analyzed and plotted using Origin 2021.

[0092] like Figure 9The swelling test results shown in the figure show that the abundant hydrophilic groups and internal space will cause the gel to absorb water and swell rapidly. The initial swelling ratios of the two groups of gels with the largest difference in cross-linking, 0.5% Nap-β-CS-SP and 1% ZnO-CS-SP-Nap-β, are 487.95% and 178.78%, respectively. As time increases, the difference in their swelling ratios remains large. The 0.5% Nap-β-CS-SP gel collapsed and dispersed after 3.5 hours, while the 1% ZnO-CS-SP-Nap-β always maintained the lowest swelling ratio, indicating that self-assembly strength and gel curing are important factors affecting the water absorption and swelling ability of the gel. The 0.5% Nap-β-CS-SP gel has a low degree of cross-linking and a large internal void. It absorbs more water per unit volume and has the highest swelling ratio. However, its mechanical strength is low. The addition of nano-zinc oxide improves the cross-linking of the simple self-assembled network gel, but its swelling ability is low. The swelling ratios of the two groups of gels in the middle, 1% Nap-β-CS-SP and 0.5% ZnO-CS-SP-Nap-β, were similar within 5 h, and the swelling ratios tended to be stable after 3 h. 2+ Due to the presence of the induced second micelles, the swelling ratio of 0.5% ZnO-CS-SP-Nap-β was lower than that of 1% Nap-β-CS-SP. The final swelling ratios of the other four groups of gels increased to 695%, 512%, 590%, and 521% respectively, indicating that the cross-linking density of the gel will also affect the swelling rate to a certain extent.

[0093] Figure 10 In the room temperature weight loss experiment, the relative residual mass ratios of the four groups of gels, 1% Nap-β-CS-SP, 0.5% Nap-β-CS-SP, 0.5% ZnO-CS-SP-Nap-β and 1% ZnO-CS-SP-Nap-β, after 72 hours were 8.7%, 5.2%, 5.8% and 2.1%, respectively. The water content of each group of gels was the same. However, due to their different swelling equilibrium ratios, at the same mass, gels with low cross-linking degree had more voids to store water. When the gel was denser, the lower porosity reduced its water retention capacity. This result was also shown in our experiment. It is worth noting that the 1% Nap-β-CS-SP gel showed the lowest water loss rate, indicating that low cross-linking degree can increase water retention to a certain extent.

[0094] In addition, from Figure 11In vitro degradation results showed that the hydrogel material initially absorbed water and swelled, increasing in mass. The mass ratio then began to decrease. After 24 hours, the mass ratio decreased at a faster rate as the gel strength decreased. During the first three days, the composite hydrogel lost weight rapidly, indicating severe disruption of the three-dimensional network structure. This resulted in the release of some of the hydrogel's water, causing the hydrogel to break off and disintegrate into fragments at the edges. The rate of weight loss then slowed. After 20 days of degradation, the mass ratios of the 1% Nap-β-CS-SP, 0.5% Nap-β-CS-SP, 0.5% ZnO-CS-SP-Nap-β, and 1% ZnO-CS-SP-Nap-β gels decreased by 315, 457, 375, and 312%, respectively, compared to those at 24 hours. The hydrogel completely dissociated, and the 0.5% Nap-β-CS-SP with the lowest crosslinking level degraded fastest, which would cause the peptide embedded in the hydrogel to diffuse and dissipate quickly, preventing localized efficacy. The 1% ZnO-CS-SP-Nap-β hydrogel degraded the slowest, hindering the release of the encapsulated substance. In the aforementioned characterization experiments, a comprehensive analysis of mechanical, swelling, and degradation tests revealed that gels synthesized with 1% Nap-β and 0.5% ZnO exhibited excellent swelling and moisturizing properties, as well as suitable degradation properties. CS-Nap-β, CS-SP-Nap-β, and CS-SP-Nap-β@ZnO gels were synthesized at these concentrations in the following experiments.

[0095] (4) Cytotoxicity studies

[0096] Human fibroblasts (HSF) were cultured in a 96-well plate at 3 × 10 4 pieces / cm 2 Cells were inoculated at a density of 100 μg / mL and co-cultured with the two cell lines in 96-well plates for 48 h. The cells were treated with a CCK-8 kit, and the absorbance was measured at 540 nm using a microplate reader. The changes in cell viability were analyzed using GraphPad Prism 8 software.

[0097] Figure 12 As can be seen in the figure, different groups of materials were treated with cells at concentrations of 2μg / ml, 5μg / ml, 10μg / ml, and 20μg / ml to assess cytotoxicity. For HSF, 20μg / ml of nano-zinc oxide had a certain inhibitory effect on cell growth, while the other groups did not inhibit cell viability. In the SP and CS-SP groups, compared with the control group, cell viability gradually increased with increasing peptide concentration, reaching the highest cell viability at an SP concentration of 10μg / ml.

[0098] (6) Biocompatibility studies.

[0099] The blood compatibility of the hydrogel was evaluated by hemolysis experiment. In short, blood was collected from the rat heart, collected into sodium heparin blood collection tubes, and centrifuged at 4°C and 3000rpm for 15min. The supernatant was discarded, and the red blood cells were resuspended in an appropriate amount of phosphate buffered saline (PBS), and the centrifugation was continued for 5min. The washing was repeated until the supernatant had no obvious red color. The red blood cells were diluted with PBS to a 2% red blood cell suspension for later use. At the same time, hydrogel materials of different concentrations were prepared, 1ml of sample was mixed with 1mL of red blood cells, and incubated at 37°C for 3h. The experiment also included a negative control (PBS) and a positive control (0.5% Triton X-100) to calculate the hemolysis rate. All samples were centrifuged at 3500rpm for 5min, and the upper solution was taken and the absorbance at 540nm was detected by an enzyme reader.

[0100] like Figure 13 As shown, 10 μg / ml SP, 10 μg / ml Nap-β, 5 μg / ml ZnO, CS-Nap-β, CS-SP-Nap-β and CS-SP-Nap-β@ZnO gels were incubated with red blood cells for 3 hours without showing hemolysis phenomenon, and the hemolysis rates were all less than 5%, indicating no hemolytic reaction.

[0101] Comparative Example 1

[0102] According to a previous report (Wang X, et al. Journal of biomedical materials research Part A, 2023, 111(3): 404-14.), Wang X's research group used a chemical cross-linking method to prepare a carboxymethyl chitosan-gelatin hydrogel to co-deliver SP (substance P) and DMOG (dimethyloxyacryloylglycine). The report did not specify the specific release rate of SP. However, the hydrogel system CS-SP-Nap-β@ZnO obtained by our invention achieved a cumulative release of SP of more than 75% within 72 hours. The delivery of SP uses a simple chemical cross-linking method instead of a simple loading method, which is simple to operate and has mild experimental conditions. This chitosan self-assembled hydrogel can more accurately and long-term release drugs in the wound microenvironment.

[0103] In the absence of conflict, the above embodiments and features in the embodiments may be combined with each other.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing a long-acting sustained-release gel, characterized in that: The following steps are involved: S1. Activating neuropeptide SP with EDC and NHS, adding the activated neuropeptide SP to an acetic acid aqueous solution containing chitosan, stirring, dialyzing, and freeze-drying to obtain chitosan CS-SP grafted with neuropeptide; the amino acid sequence of the neuropeptide SP is RPKPQQFFGLM; S2. In the presence of catalysts EDC and NHS, the CS-SP prepared in step S1 was dissolved in an aqueous solution, DMSO was added to dissolve the self-assembling peptide Nap-β, the mixture was stirred for reaction, dialyzed, and freeze-dried to obtain a self-assembled chitosan drug-loaded hydrogel grafted with neuropeptide, which was designated as CS-SP-Nap-β. The amino acid sequence of the self-assembling peptide Nap-β is NapFFVLEGG; S3. Dissolve the CS-SP-Nap-β obtained in step S2 in water, disperse the nano-ZnO in water, and then mix and stir the obtained CS-SP-Nap-β solution and the nano-ZnO solution to obtain a mixed solution, and let it stand to obtain a self-assembled chitosan drug-loaded hydrogel grafted with neuropeptide, recorded as CS-SP-Nap-β@ZnO, which is the gel long-acting sustained-release agent.

2. The preparation method according to claim 1, wherein In step S1, the molar ratio of neuropeptide SP, EDC and NHS is (0.4-0.7):15.6:17.4, and the mass ratio of neuropeptide SP to chitosan is (0.8-1.5):

500.

3. The preparation method according to claim 2, wherein The volume concentration of the acetic acid aqueous solution is 1.5% to 2%; the pH value of the acetate buffer solution containing chitosan is 4 to 5; and the mixture is stirred at 2 to 4° C. overnight.

4. The preparation method according to claim 2, wherein The activation process is as follows: neuropeptide SP, EDC and NHS are added to an aqueous solution and stirred at 2°C to 4°C for 0.5 to 2 hours.

5. The preparation method according to claim 1, wherein In step S2, the mass ratio of neuropeptide SP, chitosan and small peptide Nap-β is (0.8-1.5):500:(4.5-5.5).

6. The preparation method according to claim 1, wherein In step S3, the concentration of CS-SP-Nap-β in the mixed solution is 0.025-0.0375 g / mL, and the concentration of ZnO is 1-5 μg / mL.

7. The preparation method according to claim 1, wherein The dialysis process in steps S1 and S2 is: dialyzing in an aqueous solution using a dialysis bag with a molecular weight cut-off of 1000 Da for 70 to 80 hours.

8. The preparation method according to claim 1, wherein In step S2, the stirring reaction temperature is 2° C. to 4° C., and the stirring reaction time is 5 to 8 hours.

9. A long-acting sustained-release gel prepared by the method according to any one of claims 1 to 7.

10. The long-acting sustained-release gel according to claim 9, characterized in that: Using phosphate buffer solution with a pH of 7.2 to 7.4 as the dissolution medium, the cumulative release of neuropeptide SP within 72 hours is not less than 75%.

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