Preparation method and application of GelMA Cryogel microspheres loaded with resveratrol nanocrystals

Abstract

The present invention is applicable to the fields of materials and pharmaceutical technology, and provides a preparation method and application of GelMA Cryogel microspheres loaded with resveratrol nanocrystals. In this method, RES nanocrystals are loaded in GelMA Cryogels to construct a RES sustained-release system. RES nanocrystals are present on the surface of microspheres, the surface of microsphere pores, and inside the microsphere skeleton, achieving efficient loading and triple release. GelMA Cryogels can not only promote cell adhesion, but also control the absorption rate and in vivo effect of RES. As the microspheres degrade, the RGD sequence decreases, the competitive effect weakens, and at the same time, the RES inside the microspheres is further released to play a sustained biological role. Therefore, the GelMA Cryogels sustained-release system loaded with RES nanocrystals can play a role in regulating inflammation and angiogenesis microenvironment in a sustained and stable manner at various stages of diabetic chronic skin wound healing.

Description

Technical Field

[0001] The invention belongs to the technical field of materials and pharmaceuticals, and in particular relates to a preparation method and application of GelMACryogel microspheres carrying resveratrol nanocrystals. Background Art

[0002] With the improvement of people's living standards, the prevalence of diabetes has increased year by year, becoming one of the most important chronic metabolic diseases in the world. The threat of diabetes to human health is related to many complications, including but not limited to cardiovascular and cerebrovascular diseases, chronic renal failure, peripheral neuropathy, and chronic refractory skin wounds in diabetic states. According to statistics, the cost of treating chronic diabetic skin wounds accounts for about 25%-50% of the total cost of diabetes treatment, which brings a heavy burden to the patients themselves and social medical care. PDGF-BB supplementation therapy is the only approved clinical treatment strategy for chronic wounds. Although it can effectively promote the healing of chronic diabetic wounds, it is expensive and has been pointed out to have the risk of increasing the incidence of malignant tumors, so its clinical application is limited. Therefore, it is very necessary to invent a safe, effective and economical treatment method for chronic diabetic wounds. The wound healing process can be summarized into four processes: hemostasis, inflammation, proliferation, and maturation. The macrophage polarization disorder caused by the hyperglycemic environment prevents the pro-inflammatory M1 macrophages from smoothly transforming into the anti-inflammatory and reparative M2 macrophages. This disorder not only leads to the formation of an "inflammatory waterfall" and overexpression of pro-inflammatory factors, but also greatly reduces the angiogenic regulatory factors (such as VEGF) produced by M2 macrophages. At the same time, many studies have pointed out that diabetes leads to disordered secretion of cytokines related to angiogenesis, and the balance between pro-angiogenesis in the early stage of trauma and anti-angiogenesis in the vascular maturation stage is broken, which ultimately leads to disordered wound healing. Therefore, regulating the inflammatory microenvironment and restoring orderly vascular regeneration are key links in the treatment of chronic diabetic skin wounds.

[0003] In recent years, natural compounds that regulate host inflammatory responses have received widespread attention. Resveratrol (trans-3,4,5-trihydroxystilbene, resveratrol, RES) is a plant-derived polyphenol compound that is mainly found in black grapes, red wine, berries and peanut skins. It can exert anti-inflammatory, anti-oxidative stress, anti-tumor, cardioprotective and aging-delaying effects by regulating NF-kB, MAPK and other signaling pathways in the body. However, rapid absorption, low bioavailability and low water solubility are key factors that limit the in vivo application of resveratrol. Studies have shown that the oral bioavailability of resveratrol is less than 5%, and its solubility in water is about 0.03 g·L -1 Therefore, optimizing the application of RES and increasing its bioavailability have become research hotspots, and synthesizing resveratrol into nanocrystals is the only choice to improve its bioavailability.

[0004] At present, most of the improved application methods of resveratrol can generally improve its bioavailability to a certain extent, but there are still problems such as low content of active ingredients, low loading rate, unstable effect and potential biological toxicity. How to achieve precise delivery of resveratrol, improve its bioavailability while ensuring its good biocompatibility and stable efficacy is of great significance to promoting the clinical application of resveratrol. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide a preparation method and application of GelMA Cryogel microspheres loaded with resveratrol nanocrystals, aiming to solve the problems raised in the above background technology.

[0006] The embodiment of the present invention is implemented as follows: a method for preparing GelMA Cryogel microspheres loaded with resveratrol nanocrystals comprises the following steps:

[0007] Step 1, heat 50ml PBS water bath to 50℃, add 5g pig skin gelatin, and stir continuously at 400rpm in 50℃ water bath for about 1-2 hours until completely dissolved;

[0008] Step 2, slowly and dropwise add 6 ml of methacrylic anhydride to the gelatin solution at a rate of less than 0.5 ml / min, and continue stirring at 400 rpm for 3 h. After the substitution reaction is completed, a GelMA hydrogel precursor solution is obtained;

[0009] Step 3, adding a small amount of NaOH solution to the GelMA hydrogel precursor solution after the reaction several times, and measuring the pH of the solution, adjusting the pH to 7, and the reaction gradually stopped;

[0010] Step 4: After the reaction is stopped, the GelMA hydrogel precursor solution is transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 D, and dialyzed in distilled water at 40° C. for 7 days, with deionized water replaced every 3 hours to remove excess methacrylic anhydride, harmful substances and other impurities;

[0011] Step 5: After the dialysis is completed, the reaction product is placed in a refrigerator at -80°C for overnight freezing, placed in a freeze dryer, and freeze-dried for 48 hours to obtain a white porous foam-like GelMA hydrogel precursor;

[0012] Step 6, weigh 20 mg of resveratrol powder, add 1 ml of anhydrous ethanol, and repeatedly blow and beat to dissolve to obtain a RES solution;

[0013] Step 7, add 2.5 ml of anhydrous ethanol, 100 ul of ammonia water, 100 ul of formaldehyde and the RES solution completely dissolved in the previous step to 12 ml of deionized water, place on a magnetic stirring device and stir at 500 rpm at room temperature for 90 min to obtain a RES nanocrystal solution;

[0014] Step 8: Centrifuge at 15000 rpm for 15 min, discard the supernatant, add 5 ml of deionized water, sonicate for 5 min, resuspend the RES nanocrystals, repeat this step three times, remove residual ethanol, ammonia and formaldehyde, and freeze-dry the solid after centrifugation for 12 h to obtain RES nanocrystal powder;

[0015] Step 9, weigh 100 mg of GelMA hydrogel precursor and 2 mg of RES nanocrystals and dissolve them in 1 ml of PBS solution containing 1 mg Tween 60 and 100 ul LAP, protect from light, place in a 45°C water bath, shake repeatedly to fully dissolve, add the solution to 10 ml of liquid paraffin containing 10 mg Span80, stir at room temperature, 300 rpm, protect from light for 30 min, transfer to an ice water bath and continue stirring at 300 rpm, protect from light for 30 min, to form a short-term physical crosslink, put the solution into a -20°C refrigerator, freeze for 30 min, then immerse in liquid nitrogen for 10 min to terminate ice crystal growth, and irradiate at 405 nm for 45 min to obtain GelMA Cryogels microspheres loaded with RES nanocrystals for photocrosslinking;

[0016] Step 10: Wash the microspheres three times with PBS and freeze-dry for 24 h to obtain GelMACryogels microsphere powder loaded with RES nanocrystals.

[0017] Another purpose of an embodiment of the present invention is to provide an application of GelMA Cryogel microspheres loaded with resveratrol nanocrystals. Based on the GelMA Cryogel microspheres loaded with resveratrol nanocrystals prepared by the above method, the GelMA Cryogel microspheres loaded with resveratrol nanocrystals are used to prepare a drug for promoting the healing of chronic refractory wounds.

[0018] According to a further technical solution, the chronic refractory wound is specifically a skin wound under diabetic conditions.

[0019] The present invention provides a method for preparing and applying a GelMA Cryogel microsphere loaded with resveratrol nanocrystals. The method loads RES nanocrystals in GelMA Cryogels to construct a RES sustained-release system. RES nanocrystals are present on the surface of microspheres, on the surface of microsphere pores, and inside the microsphere skeleton, achieving efficient loading and triple release. Since GelMACryogels contains abundant RGD sequences, it can not only promote cell adhesion, but also compete with RES nanocrystals for binding to cell surface integrin αvβ3, thereby controlling the absorption rate and in vivo effect of RES. As the microspheres degrade, the RGD sequence decreases, the competitive effect weakens, and the RES inside the microspheres is further released to exert a sustained biological effect. Therefore, the GelMA Cryogels sustained-release system loaded with RES nanocrystals can play a role in regulating inflammation and angiogenesis microenvironment in a sustained and stable manner at various stages of diabetic chronic skin wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Microscopic images of RES nanocrystals and GelMA Cryogels microspheres loaded with RES nanocrystals (A is a scanning electron microscope image of RES nanocrystals; B is a scanning electron microscope image of the overall view of GelMA Cryogels microspheres loaded with RES nanocrystals; C is a scanning electron microscope image of the surface of GelMA Cryogels microspheres loaded with RES nanocrystals);

[0021] Figure 2 is the particle size analysis result of RES nanocrystals;

[0022] Figure 3 Zeta potential diagrams of RES nanocrystals, GelMA Cryogels microspheres, and GelMACryogels microspheres loaded with RES nanocrystals;

[0023] Figure 4 Fourier transform infrared spectra of RES nanocrystals, GelMA Cryogels microspheres and GelMACryogels microspheres loaded with RES nanocrystals;

[0024] Figure 5 Results of cell viability test with RC at concentrations of 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml and 10 mg / ml (a: 1 day; b: 3 days);

[0025] Figure 6 Live-dead staining results of RC at application concentrations of 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml, and 10 mg / ml (a: 1 day; b: 3 days; scale bar = 200 um);

[0026] Figure 7 The results of skeleton staining with RC at concentrations of 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml and 10 mg / ml (a: 1 day; b: 3 days; scale bar = 200 um);

[0027] Figure 8 ROS scavenging experiments and quantitative analysis for CMS, RES, and RC;

[0028] Fig. 9 The experimental results of ABTS free radical scavenging of CMS, RES and RC;

[0029] Fig.10 The immunofluorescence staining results and quantitative analysis of the inflammatory-related cytokines TNF-α, IL-1β, and IL-6 in CMS, RES, and RC;

[0030] Fig.11 The immunofluorescence staining results and quantitative analysis of the inflammatory-related cytokines IL-10, Arg-1, and TGF-β in CMS, RES, and RC;

[0031] Fig.12 CD86 in CMS, RES and RC measured by flow cytometry + and CD206 + Cell percentage;

[0032] Fig.13 The results of cell scratch assay using HUVECs;

[0033] Fig.14 The results of Transwell chamber experiments and tube formation experiments using HUVECs;

[0034] Fig.15 The immunofluorescence staining results of angiogenesis-related cytokines in CMS, RES and RC under high glucose culture conditions;

[0035] Fig.16 To investigate the wound healing status and quantitative analysis of the non-infected skin defect model in diabetic mice;

[0036] Fig.17 Results of local HE and Masson staining of skin wounds in each group;

[0037] Fig.18 Immunofluorescence staining and quantitative analysis of pro-inflammatory factors (IL-1β and TNF-α);

[0038] Fig.19Immunofluorescence staining and quantitative analysis of anti-inflammatory factors (IL-10 and TGF-β);

[0039] Fig. 20 Immunofluorescence staining and quantitative analysis of angiogenesis-related factor (VEGF);

[0040] Fig.21 Specialized H-type blood vessels (CD31 + / EMCN + )Co-immunofluorescence staining results and quantitative analysis;

[0041] Fig. 22 The present invention provides a flowchart of a method for preparing GelMA Cryogel microspheres loaded with resveratrol nanocrystals according to an embodiment of the present invention. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0043] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0044] like Fig. 22 As shown, a method for preparing a GelMACryogel microsphere loaded with resveratrol nanocrystals provided by one embodiment of the present invention comprises the following steps:

[0045] Step 1. Heat 50 ml of phosphate buffered saline (PBS) in a water bath to 50°C, add 5 g of pig skin gelatin (Type A), and stir continuously at 400 rpm in a 50°C water bath for about 1-2 hours until completely dissolved;

[0046] Step 2, slowly and dropwise adding 6 ml of methacrylic anhydride (MA) to the gelatin solution at a rate of less than 0.5 ml / min, and continuing stirring at 400 rpm for 3 h. After the substitution reaction is completed, a GelMA hydrogel precursor solution is obtained;

[0047] Step 3, adding a small amount of NaOH solution to the GelMA hydrogel precursor solution after the reaction several times, and measuring the pH of the solution, adjusting the pH to 7, and the reaction gradually stopped;

[0048] Step 4: After the reaction is stopped, the GelMA hydrogel precursor solution is transferred to a dialysis bag with a molecular weight cutoff of 8000-14000 D, and dialyzed in distilled water at 40° C. for 7 days, with deionized water replaced every 3 hours to remove excess methacrylic anhydride, harmful substances and other impurities;

[0049] Step 5: After the dialysis is completed, the reaction product is placed in a refrigerator at -80°C for overnight freezing, placed in a freeze dryer, and freeze-dried for 48 hours to obtain a white porous foam-like GelMA hydrogel precursor;

[0050] Step 6, weigh 20 mg of resveratrol powder, add 1 ml of anhydrous ethanol, and repeatedly blow to dissolve to obtain a resveratrol (RES) solution;

[0051] Step 7, add 2.5 ml of anhydrous ethanol, 100 ul of ammonia water, 100 ul of formaldehyde and the RES solution completely dissolved in the previous step to 12 ml of deionized water, place on a magnetic stirring device and stir at 500 rpm at room temperature for 90 min to obtain a RES nanocrystal solution;

[0052] Step 8: Centrifuge at 15000 rpm for 15 min, discard the supernatant, add 5 ml of deionized water, sonicate for 5 min, resuspend the RES nanocrystals, repeat this step three times, remove residual ethanol, ammonia and formaldehyde, and freeze-dry the solid after centrifugation for 12 h to obtain RES nanocrystal powder;

[0053] Step 9, weigh 100 mg of GelMA hydrogel precursor and 2 mg of RES nanocrystals and dissolve them in 1 ml of PBS solution containing 1 mg of Tween 60 and 100 ul of LAP (2.5 wt%), protect from light, place in a 45°C water bath, shake repeatedly to fully dissolve, add the solution to 10 ml of liquid paraffin containing 10 mg of Span80, stir at room temperature, 300 rpm, protect from light for 30 min, transfer to an ice water bath and continue stirring at 300 rpm, protect from light for 30 min, to form a short-term physical crosslink, put the solution into a -20°C refrigerator, freeze for 30 min, then immerse in liquid nitrogen for 10 min to terminate ice crystal growth, and irradiate at 405 nm for 45 min to obtain GelMA Cryogels microspheres loaded with RES nanocrystals for photocrosslinking;

[0054] Step 10: Wash the microspheres three times with PBS and freeze-dry for 24 h to obtain GelMACryogels microsphere powder loaded with RES nanocrystals.

[0055] like Figure 1 As shown, RES nanocrystals are regular spheres with a diameter of about 120 nm. GelMACryogels loaded with RES nanocrystals are spherical, and RES nanocrystals can be observed on their surface.

[0056] The particle size analysis results of RES nanocrystals are shown in Figure 2 As shown, the results show that the particle size of RES nanocrystals is about 125.5 nm.

[0057] Zeta potential of RES nanocrystals, GelMA Cryogels microspheres and GelMA Cryogels microspheres loaded with RES nanocrystals Figure 3 As shown in (RES represents RES nanocrystals; CMS represents GelMA Cryogels microspheres; RC represents GelMA Cryogels microspheres loaded with RES nanocrystals), Figure 3 It can be seen that RES nanocrystals are positively charged and CMS microspheres are negatively charged. With the loading of RES nanocrystals, the negative charge carried by RC decreases.

[0058] Fourier transform infrared spectra of RES nanocrystals, GelMA Cryogels microspheres and GelMA Cryogels microspheres loaded with RES nanocrystals are shown in Figure 2. Figure 4 As shown in the figure (RES represents RES nanocrystals; CMS represents GelMA Cryogels microspheres; RC represents GelMA Cryogels microspheres loaded with RES nanocrystals), all characteristic peaks of RES and CMS can be clearly observed in the RC group and no new absorption peaks appear, proving that RES crystals are successfully loaded onto GelMA Cryogels microspheres, and the structure of RES does not change before and after loading, and the pharmacological activity is not affected.

[0059] Another embodiment of the present invention provides an application of GelMA Cryogel microspheres loaded with resveratrol nanocrystals. Based on the GelMA Cryogel microspheres loaded with resveratrol nanocrystals prepared by the above method, the GelMA Cryogel microspheres loaded with resveratrol nanocrystals are used to prepare a drug for promoting the healing of chronic refractory wounds, specifically a drug for promoting the healing of skin wounds under diabetic conditions. In vitro experiments are performed to verify the effectiveness of the GelMA Cryogel microspheres loaded with resveratrol nanocrystals in related applications.

[0060] The results of cell viability test with RC (GelMA Cryogels microspheres loaded with RES nanocrystals) at concentrations of 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml and 10 mg / ml are shown in the figure. Figure 5 As shown (a: 1 day; b: 3 days). The cell activity decreased with the increase of RC application concentration. When the application concentration was 2.5 mg / ml or 5 mg / ml, the cell activity was higher than 75%. When the application concentration was increased to 7.5 mg / ml or 10 mg / ml, the cell activity decreased significantly.

[0061] The live-dead staining results of RC (GelMA Cryogels microspheres loaded with RES nanocrystals) at concentrations of 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml and 10 mg / ml are shown in the figure. Figure 6 As shown, a: 1 day; b: 3 days (scale bar = 200um). Figure 6 As shown, when the applied concentration was 2.5 mg / ml or 5 mg / ml, there were almost no dead cells, and when the applied concentration was increased to 7.5 mg / ml or 10 mg / ml, the number of dead cells increased significantly.

[0062] The results of skeleton staining with RC (GelMA Cryogels microspheres loaded with RES nanocrystals) at concentrations of 2.5 mg / ml, 5 mg / ml, 7.5 mg / ml and 10 mg / ml are shown in the figure. Figure 7 As shown, a: 1 day, b: 3 days (scale bar = 200um). Figure 7 As shown in the figure, when the applied concentration is 2.5mg / ml or 5mg / ml, the cytoskeleton is stretched and the cells are in good condition. When the applied concentration is increased to 7.5mg / ml or 10mg / ml, the cytoskeleton is obviously wrinkled. In summary, 50mg / ml is selected as the optimal applied concentration of RC.

[0063] ROS scavenging experiments and quantitative analysis of CMS, RES and RC Figure 8 As shown (scale bar = 100um). The ROS scavenging experiment was used to evaluate the in vitro antioxidant properties of the material. The results showed that RC could significantly scavenge ROS generated by LPS (i.e., bacterial lipopolysaccharide) stimulation, and the scavenging effect was better than that of the RES group.

[0064] The results of ABTS free radical scavenging experiments of CMS, RES and RC are as follows Fig. 9 The results showed that RC and RES had ABTS free radical scavenging ability comparable to that of the standard positive control group of 0.4mmol / L VitC, and there was no statistical difference between the RC group and the RES group, proving that loading RES nanocrystals in GelMA Cryogels did not affect its antioxidant stress effect.

[0065] Immunofluorescence staining results and quantitative analysis of inflammatory cytokines TNF-α, IL-1β and IL-6 in CMS, RES and RC (scale bar = 100um) Fig.10 As shown, the immunofluorescence staining results and quantitative analysis of inflammatory cytokines IL-10, Arg-1, and TGF-β in CMS, RES, and RC (scale bar = 100 μm) are shown. Fig.11The results showed that RC and RES treatments significantly reversed the increased expression of pro-inflammatory factors (TNF-α, IL-1β, IL-6) caused by LPS stimulation, and promoted the expression of anti-inflammatory factors (IL-10, Arg-1, TGF-β), effectively improved the inflammatory state of macrophages, and exerted excellent in vitro anti-inflammatory effects. In addition, the anti-inflammatory effect of the RC group was better than that of the RES group.

[0066] Flow cytometry was used to measure the expression of CD86 in CMS, RES and RC + 、CD206 + Cell proportion (CD86 + CD206 is a surface marker of M1 macrophages. + is a surface marker of M2 macrophages). Fig.12 As shown in the figure, the application of RC and RES reduced the proportion of M1 macrophages and increased the proportion of M2 macrophages, which could significantly improve the inflammatory state of macrophages, and the effect of RC was more obvious than that of RES.

[0067] HUVECs were used to perform a cell scratch test to characterize the migration ability of endothelial cells and to show the in vitro angiogenesis potential of different treatment groups. Cell migration was photographed under a microscope at 0, 6, 12, 24, and 36 hours (scale bar = 100um) and quantitatively analyzed. The results are shown in Fig.13 HG: high glucose treatment group; MA: mannitol-treated hyperosmotic control group; AGEs: advanced glycation end products treatment group; and CMS, RES, and RC treatment groups.

[0068] The results showed that compared with the control group, the migration rate of endothelial cells in the high glucose treatment group was significantly slowed down, while the migration rate in the MA group was no different from that in the control group, indicating that the difference was caused by high glucose rather than the high osmotic pressure secondary to high glucose; similarly, the AGEs group also experienced a similar slowdown in endothelial cell migration rate as the high glucose group, indicating that the advanced glycation end products may dominate the high glucose environment.

[0069] Regarding the effect on the migration rate of endothelial cells, the migration rate of endothelial cells treated with high glucose was improved after the addition of RES and RC, especially in the RC group. The migration rate of endothelial cells was equivalent to that of the blank group, and was even faster than that of the blank group in the sixth hour. This shows that the application of RES and RC can reverse the damage of the high glucose environment to the migration rate of endothelial cells, and the effect of RC is more significant.

[0070] HUVECs were used to perform Transwell chamber experiments (scale bar = 100um) and tube formation experiments (scale bar = 200um) to characterize the migration and tubule formation abilities of endothelial cells, and quantitative analysis was performed to count the number of migrated cells, the number of nodes, and the total length of the tubes. The results are shown in Fig.14 HG: high glucose treatment group; MA: mannitol-treated hyperosmotic control group; AGEs: advanced glycation end products treatment group; and CMS, RES, and RC treatment groups.

[0071] The results showed that the effects of each treatment method on cell migration ability were the same as those in the scratch test. High glucose treatment and AGEs treatment significantly weakened the tube formation function of endothelial cells, while the tube formation of the hypertonic treatment group was not affected, which indicates that the accumulation of advanced glycation products caused by high glucose treatment not only affects the migration function of endothelial cells, but also impairs their tube formation and differentiation ability, while RES and RC treatment can significantly reverse this damage and improve the in vitro angiogenesis potential of endothelial cells, with the most significant effect in the RC group.

[0072] The results of immunofluorescence staining of angiogenesis-related cytokines in CMS, RES and RC under high glucose culture conditions (scale bar = 100um) are shown in Fig.15 The results showed that under the action of RES and RC, compared with the blank group, the expression of angiogenesis-related factors (VEGF, CD31, HIF-1α) of endothelial cells in a high glucose environment increased significantly, and the effect of RC was the most significant, which indicates that RC and RES can promote the differentiation of endothelial cells and achieve pro-angiogenesis activity in vitro.

[0073] A non-infected skin defect model was established in diabetic mice. Local wound photos were taken on days 0, 2, 4, 7, 10, and 14 to observe the wound healing process. Fitting graphs of wound healing areas at different times were established and quantitatively analyzed. The results are shown in Table 1. Fig.16 shown.

[0074] The results showed that compared with the blank control group (normal mice without diabetes), the wound healing speed of the db group (untreated diabetes group) was significantly slowed down. When the wounds of diabetic mice were treated with RES and RC, the wound healing speed was accelerated, significantly higher than that of the db group, and the effect of the RC group was the most significant. This shows that the application of RC in vivo can effectively improve the healing state of diabetic refractory wounds. This may be attributed to its excellent anti-inflammatory and angiogenic activity.

[0075] The results of local HE and Masson staining of skin wounds in each group are as follows Fig.17 As shown. Compared with the blank control group, the number of local inflammatory cells in the wound of the db group was higher, and the number of new blood vessels and collagen fiber deposition was lower; after RES and RC treatment, the local inflammatory cell infiltration of the wound was significantly reduced, the number of new blood vessels increased, and mature and abundant collagen fiber deposition was observed in the wound area, and the effect of RC was the most significant, while CMS alone had no such effect. In summary, RC can effectively improve the healing state of refractory wounds under diabetic conditions and promote the healing and repair process of wounds.

[0076] Immunofluorescence staining and quantitative analysis of pro-inflammatory cytokines (IL-1β and TNF-α) Fig.18 As shown, immunofluorescence staining and quantitative analysis of anti-inflammatory factors (IL-10 and TGF-β) are shown in Fig.19 As shown, immunofluorescence staining and quantitative analysis of angiogenesis-related factor (VEGF) are shown in Fig. 20 As shown, specialized H-type blood vessels (CD31 + / EMCN + ) and quantitative analysis of co-immunofluorescence staining results. Fig.21 shown.

[0077] The results showed that compared with the blank group, the expression of pro-inflammatory factors (IL-1β and TNF-α) in the db group was significantly increased, and the expression of anti-inflammatory factors (IL-10 and TGF-β) and angiogenesis-related factors (VEGF) was significantly reduced. The expression of these factors was mainly concentrated in the wound center. The application of RES and RC can effectively reduce the expression of IL-1β and TNF-α, increase the expression of IL-10, TGF-β and VEGF, and RC has the most significant effect, which indicates that RC has excellent anti-inflammatory and angiogenic functions. Many studies have shown that H-type blood vessels are a type of blood vessels that express CD31 at the same time. + With EMCN + The specialized blood vessels not only determine the speed and potential of bone regeneration during bone defect repair, but are also closely related to the speed of soft tissue healing. Therefore, we characterized the proportion of H-type blood vessels in the wound site by co-immunofluorescence staining. Compared with the blank group, the percentage of H-type blood vessels in the db group was significantly reduced, while the application of RC increased the H-type blood vessel ratio, indicating that RC promotes the healing of diabetic refractory wounds by promoting angiogenesis and increasing the proportion of H-type blood vessels.

[0078] 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 and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing GelMA Cryogel microspheres loaded with resveratrol nanocrystals, characterized in that: The following steps are involved: Step 1, preparing a GelMA hydrogel precursor; Step 2, preparing RES nanocrystal powder; Step 3, weigh 100 mg of GelMA hydrogel precursor and 2 mg of RES nanocrystals and dissolve them in 1 ml of PBS solution containing 1 mg Tween 60 and 100 ul LAP, protect from light, place in a 45°C water bath, shake repeatedly to fully dissolve, add the solution to 10 ml of liquid paraffin containing 10 mg Span80, stir at room temperature, 300 rpm, protect from light for 30 min, transfer to an ice water bath and continue stirring at 300 rpm, protect from light for 30 min, to form a short physical crosslink, put the solution into a -20°C refrigerator, freeze for 30 min, then immerse in liquid nitrogen for 10 min to terminate ice crystal growth, and irradiate at 405 nm for 45 min to obtain GelMA Cryogels microspheres loaded with RES nanocrystals for photocrosslinking; Step 4, washing the microspheres three times with PBS, freeze-drying for 24 h, and obtaining GelMA Cryogels microsphere powder loaded with RES nanocrystals; The step 1 comprises the following specific steps: Step 1.1, heat 50 ml PBS to 50°C in a water bath, add 5 g pig skin gelatin, and stir continuously at 400 rpm in a 50°C water bath for 1-2 hours until completely dissolved to obtain a gelatin solution; Step 1.2, slowly and dropwise adding 6 ml of methacrylic anhydride to the gelatin solution at a rate of less than 0.5 ml / min, and continuing stirring at 400 rpm for 3 h, after the substitution reaction is completed, a GelMA hydrogel precursor solution is obtained; Step 1.3, add a small amount of NaOH solution to the GelMA hydrogel precursor solution after the reaction several times, and measure the pH of the solution. When the pH is adjusted to 7, the reaction gradually stops; Step 1.4, transfer the GelMA hydrogel precursor solution after the reaction is stopped to a dialysis bag with a molecular weight cutoff of 8000-14000 D, and dialyze in distilled water at 40°C for 7 days, replacing the deionized water every 3 hours; Step 1.5: After the dialysis is completed, the reaction product is placed in a refrigerator at -80°C overnight, placed in a freeze dryer, and freeze-dried for 48 hours to obtain a white porous foam-like GelMA hydrogel precursor; The step 2 comprises the following specific steps: Step 2.1, weigh 20 mg of resveratrol powder, add 1 ml of anhydrous ethanol, and repeatedly blow and beat to dissolve to obtain RES solution; Step 2.2, add 2.5 ml of anhydrous ethanol, 100 ul of ammonia water, 100 ul of formaldehyde and the RES solution completely dissolved in the previous step to 12 ml of deionized water, place on a magnetic stirring device and stir at 500 rpm at room temperature for 90 min to obtain a RES nanocrystal solution; Step 2.3, centrifuge at 15000rpm for 15min, discard the supernatant, add 5ml of deionized water, sonicate for 5min, resuspend the RES nanocrystals, repeat this step three times to remove residual ethanol, ammonia and formaldehyde, and freeze-dry the solid after centrifugation for 12h to obtain RES nanocrystal powder.

2. An application of GelMA Cryogel microspheres loaded with resveratrol nanocrystals, wherein the GelMA Cryogel microspheres loaded with resveratrol nanocrystals are prepared by the method for preparing GelMA Cryogel microspheres loaded with resveratrol nanocrystals according to claim 1, characterized in that: The GelMA Cryogel microspheres loaded with resveratrol nanocrystals are used to prepare a drug for promoting the healing of chronic refractory wounds.

3. The use of GelMA Cryogel microspheres loaded with resveratrol nanocrystals according to claim 2, characterized in that: The chronic refractory wound is a skin wound under diabetic conditions.

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