Heparin-like hydrogel microspheres for postoperative wound repair of melanoma, their preparation method and application

Abstract

This invention discloses a heparin-like hydrogel microsphere for melanoma postoperative wound repair, its preparation method, and its application, belonging to the field of medical dressing technology. The preparation of the heparin-like hydrogel microsphere for melanoma postoperative wound repair includes the following steps: dissolving KOSMA and GelMA in water to prepare a GelMA solution; adding a photoinitiator to form a precursor hydrogel solution; using the precursor hydrogel solution as the dispersed phase and castor oil as the continuous phase to prepare hydrogel microspheres; collecting the effluent; exposing it to ultraviolet light for curing; washing; and drying to obtain the final product. When used for melanoma postoperative wound care, these hydrogel microspheres can safely promote wound healing, specifically eliminate residual tumor cells, reduce the postoperative local recurrence rate, and can also serve as drug carriers to achieve synergistic therapeutic effects; providing new research ideas and technical strategies for integrated care of melanoma postoperative wounds and tumor prevention and control.

Description

Technical Field

[0001] This invention relates to a heparin-like hydrogel microsphere for postoperative wound repair of melanoma, its preparation method and application, belonging to the field of medical dressing technology. Background Technology

[0002] Melanoma is one of the most aggressive malignant tumors in dermatology, and its core treatment strategy has always relied on standardized surgical procedures. Acral melanomas account for more than 60% of cases, and they commonly occur in special anatomical locations such as the soles of the feet, palms, and nail beds. This unique distribution pattern directly determines the higher degree of specialization and complexity in the management of postoperative wounds in melanoma surgery.

[0003] The primary goal of surgical intervention is to achieve complete radical resection of the primary tumor lesion while maximizing the preservation of limb function. In clinical practice, the extent of resection for melanoma is determined on an individualized basis based on the depth of tumor invasion. However, for areas with unique anatomical structures and high functional requirements, such as the face and extremities, extensive surgical resection can easily lead to severe local tissue defects and limited limb function. The appropriateness of the resection extent is directly related to the patient's long-term prognosis. Balancing radical tumor resection with local functional preservation is currently a core clinical challenge in the surgical treatment of melanoma.

[0004] In terms of local adjuvant therapy, clinical trials have been conducted on the local application of the Toll-like receptor 7 / 8 (TLR7 / 8) agonist imiquimod, which exerts an anti-tumor effect by activating the local innate and adaptive immune system. This approach is mainly suitable for in situ melanomas such as those on the face that cannot tolerate extensive surgical resection, as well as for postoperative adjuvant therapy of oral mucosal melanomas.

[0005] The standardized care of postoperative wounds after melanoma surgery heavily relies on wound dressings. Traditional dressings only serve as a physical barrier, providing basic functions such as wound protection and absorbing exudate. However, current specialized postoperative dressings for melanoma are gradually breaking through the limitations of single functions and transforming into a synergistic treatment system that combines anti-tumor effects with wound healing promotion. Numerous cutting-edge studies have been conducted in this area. For example, some researchers have used coaxial electrospinning technology to construct a bifunctional nanofiber wound dressing for postoperative melanoma treatment. This involves incorporating nanoparticles loaded with 5-fluorouracil (5-FU) into a biodegradable polylactic-co-glycolic acid (PLGA) coated fiber core, creating a core-shell structured MBG-U CSF nanofiber. This achieves local sustained release of the chemotherapy drug 5-FU while simultaneously promoting wound healing. Another study constructed a nanocomposite hydrogel wound patch, which encapsulates methotrexate-loaded doped mesoporous bioactive glass in a sulfobetaine-polyacrylamide-carboxymethyl chitosan hydrogel system through thermal polymerization. The aim is to simultaneously address the dual clinical challenges of high tumor recurrence rate and poor wound healing after melanoma surgery.

[0006] Although some research and exploration has been conducted on novel functional dressings for postoperative wound care in melanoma surgery, existing postoperative dressings for melanoma are mostly biologically inert, and their anti-tumor function mainly relies on the loading of exogenous drugs. However, traditional drug delivery systems often face limitations such as uncontrolled release kinetics (e.g., burst release effect), poor drug stability, and cytotoxicity caused by excessively high local concentrations; furthermore, postoperative wound care for melanoma presents core clinical challenges such as high tumor recurrence and difficulty in tissue healing. Summary of the Invention

[0007] To address the key issues of poor wound healing quality, high recurrence rate of local tumors after melanoma surgery, and the lack of regulatory effects on the biological behavior of melanoma by traditional functional dressings that rely solely on loading chemotherapy drugs, this invention provides heparin-like hydrogel microspheres for melanoma postoperative wound repair, along with their preparation method and application. Heparin, a naturally derived high-molecular-weight polysaccharide, is a classic anticoagulant with mature clinical applications. However, direct application of heparin to the wound may pose the following risks. First, anticoagulation can interfere with the construction of the early fibrin temporary matrix, which is the physical basis for guiding fibroblast migration and granulation tissue filling; its absence will directly lead to the stagnation of re-epithelialization. Second, the local anticoagulant environment is highly susceptible to inducing persistent bleeding and hematoma formation, which not only provides a breeding ground for pathogens and increases the risk of infection but also creates physical pressure, hindering the ingrowth of new microvessels. Furthermore, the risk of systemic bleeding caused by the absorption of large doses of anticoagulants into the systemic circulation through the wound is also a significant safety hazard in clinical application.

[0008] Based on the aforementioned limitations, this invention employs a heparin-based polysaccharide structure that lacks anticoagulant activity but retains highly efficient anti-inflammatory and anti-tumor activities. Using this structure as the core substrate, hydrogel microspheres specifically designed for melanoma postoperative wound care are prepared. When used for melanoma postoperative wound care, these hydrogel microspheres can safely promote wound healing, specifically eliminate residual tumor cells, reduce the postoperative local recurrence rate, and can also serve as drug carriers to achieve synergistic therapeutic effects. This provides new research ideas and technical strategies for integrated care of melanoma postoperative wounds and tumor prevention and control.

[0009] To achieve the above objectives, the following technical solution is provided: The purpose of this invention is to provide a method for preparing heparin-like hydrogel microspheres for postoperative wound repair in melanoma surgery, the method comprising the following steps: (1) Dissolve KOSMA and GelMA in water to prepare a GelMA solution; then add a photoinitiator to the solution and stir until homogeneous to form a precursor hydrogel solution; (2) Using the precursor hydrogel solution obtained in step (1) as the dispersed phase and castor oil as the continuous phase, hydrogel microspheres were prepared using a single-channel injection pump and a T-type microfluidic chip. The effluent was collected and then quickly exposed to ultraviolet light for curing. After washing and drying, heparin-like hydrogel microspheres were obtained.

[0010] In one embodiment, the KOSMA in step (1) is obtained by sequentially subjecting KOS to an amidation reaction and a methacrylation modification.

[0011] In one embodiment, the KOSMA is prepared by reacting O-sulfated K5 polysaccharide KOS with cystamine dihydrochloride to obtain KOS-Cys; then reacting it with MA to obtain KOSMA.

[0012] In one embodiment, the preparation of the KOSMA specifically includes the following steps: (1) Dissolve K5 polysaccharide in anhydrous DMF, then add sulfur trioxide pyridine complex, stir the reaction at room temperature under nitrogen atmosphere, stop the reaction, collect the reaction solution, transfer it to a dialysis bag for dialyzing, freeze dry, and obtain O-sulfated K5 polysaccharide (KOS). (2) Dissolve KOS in water, then add EDC·HCl and NHS to adjust the pH to 4.5~5.4, activate the carboxyl group in an ice bath environment, add cystamine dihydrochloride, maintain the pH of the solution at 6.8~7.0, react overnight at room temperature, and measure that the pH remains basically unchanged at 6.8~7.0. Stop the reaction, collect the product, dialyze, and freeze dry to obtain KOS-Cys (KC). (3) Under light-protected conditions, the amide reaction product KC was placed in a round-bottom flask, PBS was added, and it was dissolved in an oil bath at 50 °C. After the reactant KC was completely dissolved, MA was slowly added dropwise at 50 °C with vigorous stirring. After the reaction, the product was collected into a dialysis bag, dialyzed, and lyophilized to obtain KOSMA.

[0013] In one embodiment, the amount of GelMA in the GelMA solution in step (1) is 5%, w / v, g / mL.

[0014] In one embodiment, the amount of KOSMA in the GelMA solution in step (1) is 1~3%, w / v, g / mL; preferably 1.5~2.5%; more preferably 2.5%.

[0015] In one embodiment, the photoinitiator in step (1) is LAP.

[0016] In one embodiment, the amount of photoinitiator in step (1) is 0.1~0.25%, w / v, g / mL.

[0017] In one embodiment, the stirring temperature in step (1) is 35~40°C.

[0018] In one embodiment, the flow rate of the dispersed phase in step (2) is 1~2 mL / h.

[0019] In one embodiment, the flow rate of the mobile phase in step (2) is 10~15 ml / h.

[0020] In one embodiment, the dispersed phase inlet of step (2) is a glass capillary with an inner diameter of 0.58 mm, and the mobile phase inlet is a dispensing needle with an inner diameter of 0.8 mm.

[0021] In one embodiment, the outflow process of the effluent in step (2) must always be maintained on an ice-water bath, and the temperature sensitivity of gelatin is used to pre-gel it at low temperature to avoid the uncrosslinked precursor hydrogel monodisperse droplets from fusing together.

[0022] In one embodiment, the UV curing conditions in step (2) are: 405 nm, 25 mW / cm². 2 , 60s.

[0023] In one embodiment, the washing in step (2) is performed using anhydrous ethanol and deionized water.

[0024] In one embodiment, the drying in step (2) specifically involves pre-freezing at -60~-80℃ for 6~8 hours, followed by freeze-drying at -20~-40℃ for 2~3 days.

[0025] The present invention also provides heparin-like hydrogel microspheres prepared by the method described above.

[0026] This invention also provides the application of the heparin-like hydrogel microspheres described above in biomedical materials.

[0027] In one embodiment, the biomedical material includes a drug carrier or a wound dressing.

[0028] The present invention also provides an application of the heparin-like hydrogel microspheres described above in the preparation of wound dressings for postoperative care of melanoma.

[0029] In one embodiment, the heparin-like hydrogel microspheres can effectively inhibit the growth of melanoma cells.

[0030] Beneficial effects: The heparin-like hydrogel microspheres prepared in this invention for postoperative wound repair in melanoma surgery have the following advantages: (1) The hydrogel microspheres have good biocompatibility with key cells for wound repair (fibroblasts and keratinocytes) and can safely support the skin tissue repair process; at the same time, they can specifically and significantly inhibit the proliferation and growth of melanoma cells. When used on postoperative wounds of melanoma, they can not only effectively accelerate wound healing and improve repair efficiency, but also have a specific clearing and inhibitory effect on melanoma cells that may remain in the wound. Animal experiments further verified that the hydrogel microspheres can significantly reduce the local recurrence rate of melanoma after surgery, while effectively improving the wound healing speed and repair quality.

[0031] (2) The heparin-based hydrogel microspheres constructed in this invention are not simply drug carriers. They possess endogenous biological activity that directly inhibits melanoma growth. At the same time, the microsphere structure retains excellent drug loading and controlled release capabilities, and has great potential to be used in combination with other anti-tumor drugs to achieve synergistic effects, providing a new idea and strategy for postoperative wound care of melanoma. Attached Figure Description

[0032] Figure 1 The images show the morphology, structure, and diameter distribution of heparin-like hydrogel microspheres; (a) optical microscope images of the microspheres in the oil and aqueous phases; (b) diameter distribution data of the microspheres in the aqueous phase. Figure 2 Scanning electron microscope images of heparin-like hydrogel microspheres at different sizes; Figure 3 The following are graphs showing the cytotoxicity test data of heparin-like hydrogel microspheres on different cell types: (a) MTT results of NIH-3T3 cells at 1 day and 2 days; (b) MTT results of HaCat cells at 1 day and 2 days; (c) MTT results of B16 cells at 1 day and 2 days. Figure 4 The following figures illustrate the postoperative recovery of mice with heparin-like hydrogel microspheres after melanoma surgery: (a) dissected mouse melanoma; (b) photographs of mouse wound healing at different time points; (c) statistical graph of mouse wound area at different time points; (d) statistical graph of mouse wound healing rate at different time points; (e) statistical graph of mouse weight change at different time points; and (f) statistical graph of recurrent melanoma volume at different time points. Figure 5 Images showing the results of fluorescence staining and quantification of mouse skin and lungs; (a) fluorescence micrographs of skin and lungs; (b) quantitative results of skin fluorescence; (c) quantitative results of lung fluorescence. Figure 6 A synthetic route diagram for KOSMA; Figure 7 Graphs showing toxicity assessment data for different cell types; (a) MTT results for NIH-3T3 cells; (b) MTT results for HaCat cells; (c) MTT results for L929 cells; (d) MTT results for B16 cells. Detailed Implementation

[0033] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0034] Example 1 The synthesis methods for KOSMA include the following: Synthetic routes are as follows... Figure 6 As shown; Preparation of O-sulfated K5 polysaccharide: 500 mg of K5 polysaccharide was weighed and dissolved in 8 mL of anhydrous DMF. Then, 8 mL of DMF solution containing 1.2 g of sulfur trioxide pyridine complex was added. The reaction was stirred at room temperature under nitrogen for 24 h. The reaction was then stopped, the reaction solution was collected, transferred to a 3.5 kDa dialysis bag and dialyzed for 3 days. The solution was then lyophilized to obtain O-sulfated K5 polysaccharide (KOS). Amide reaction: Weigh 1 g KOS (3.4 W, approximately 5 mmol) and dissolve it in a round-bottom flask containing 30 mL of permeate water. Stir magnetically until fully dissolved. Add 958.5 mg (5 mmol) EDC·HCl and 575 mg (5 mmol) NHS to the solution. Adjust the pH to 4.5–5.4 with 2 M hydrochloric acid solution. Activate the carboxyl group in an ice bath for 1 h. Add 1689 mg (7.5 mmol) cystamine dihydrochloride. Maintain the pH of the solution at 6.8–7.0 with 2 M sodium hydroxide solution. React overnight at room temperature. If the pH remains relatively constant at 6.8–7.0, stop the reaction. Collect the product into a 3.5 kDa dialysis bag and dialyze for 3 days. Freeze-dry to obtain KOS-Cys (KC). Preparation of heparin-like polysaccharide methacrylated: Weigh 200 mg of the amide reaction product KC into a 50 mL round-bottom flask, add 10 mL of PBS, and dissolve in an oil bath at 50 °C for 10 min. After the KC is completely dissolved, slowly add 1 mL of MA (0.2 mL / min) dropwise at 50 °C with vigorous stirring for 24 h. Stop the reaction, collect the product into a 3.5 kDa dialysis bag, dialyze for 3 days, and lyophilize to obtain KOSMA. The reaction and dialysis processes must be conducted in the dark.

[0035] Result characterization Cytotoxicity assessment of K5, KOS, KOS-Cys, and KOSMA The cytotoxicity of K5 polysaccharide and its derivatives was validated using mouse melanoma cells (B16), human immortalized keratinocytes (HaCat), mouse fibroblasts (L929), and mouse embryonic fibroblasts (NIH-3T3). Specifically, certain amounts of K5, KOS, KOS-Cys, and KOSMA polysaccharides were respectively prepared into 1640 complete culture medium solutions of 10 μg / ml, 50 μg / ml, 100 μg / ml, and 150 μg / ml, and then filtered through a 0.22 μm filter membrane for sterilization. Well-grown B16 cells were then cultured at 5 × 10⁻⁶ cells / year. 4The seeding density was 100 μl / ml in 96-well plates, i.e., 5 × 10⁶ μl per well. 3 Cells were cultured at 37°C and 5% CO2 for 24 hours until they were fully adhered. The culture medium was carefully aspirated to minimize the differences between groups caused by the operation. Different concentrations of 1640 polysaccharide culture medium (100 μl per well) were added. After culturing at 37°C and 5% CO2 for 24 hours, the culture medium was carefully aspirated. Under the dark, 100 μl of 0.5 mg / ml thiazolyl blue solution was added to each well and cultured for another 4 hours. The 96-well plate was removed and the solution was carefully aspirated. 100 μl of DMSO was added to each well and cultured at 37°C and 100 rpm for 15 minutes. After the formazan was completely dissolved, the absorbance was measured at 490 nm using an ELISA reader. Cell viability was then calculated according to formula (4). The MTT assay procedures for HaCat, L929, and NIH-3T3 cells were the same as those for B16 cells. The main difference was that the complete culture medium for HaCat and L929 cells consisted of DEAE basal medium and fetal bovine serum, while the complete culture medium for NIH-3T3 cells consisted of DEAE basal medium and newborn calf serum. All other procedures were identical. The relative viability of cells in each experimental group was calculated using a formula, and the results are expressed as mean ± standard deviation.

[0036]

[0037] In the formula, As is the absorbance value of the experimental group, Ab is the absorbance value of the solvent group (DMSO), and Ac is the absorbance value of the control group.

[0038] Results Analysis The MTT assay, also known as the MTT colorimetric assay, is a method for detecting cell viability and growth. Its principle is that succinate dehydrogenase in the mitochondria of living cells reduces exogenous MTT to water-insoluble blue-purple formazan crystals, which are then deposited in the cells. Dead cells lack this function. Dimethyl sulfoxide (DMSO) dissolves the formazan in the cells, and the absorbance is measured at 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader, indirectly reflecting the number of living cells. Within a certain cell number range, the amount of MTT crystals formed is directly proportional to the cell number. By culturing NIH-3T3, HaCat, L929, and B16 cells in a polysaccharide-based culture medium for 24 hours, the toxicity of methacrylated heparin-like polysaccharides to normal cells and the persistence of their cytotoxic effect on B16 cells were determined. Figure 7 As shown in (a), (b), and (c), with the increase of polysaccharide concentration, the viability of different cells in each group was above 80%, and the cell viability of the methacrylated heparin-like polysaccharide group was also above 80%, indicating that the methacrylated heparin-like polysaccharide does not produce toxicity to normal cells. Figure 7As shown in (b), B16 cells began to exhibit toxicity at a KOS concentration of 50 μg / ml, with a cell survival rate of 65.36 ± 5.17%. The survival rate gradually decreased with increasing concentration. After further modification with KOS polysaccharide, the cell viability of the KOS-Cys and KOSMA groups was not significantly different from that of the KOS group. Therefore, it can be concluded that the methacrylated heparin-like polysaccharide still has a killing effect on B16.

[0039] Example 2 A method for preparing heparin-like hydrogel microspheres includes the following: Using the KOSMA prepared in Example 1, four microspheres with different concentrations were prepared: 5% GelMA (5G), 5% GelMA + 1.5% KOSMA (5G + 1.5K), 5% GelMA + 2.0% KOSMA (5G + 2K), and 5% GelMA + 2.5% KOSMA (5G + 2.5K). The specific process is as follows: (1) Methacrylic anhydride-modified gelatin GelMA (5%, w / v, g / mL) was dissolved in deionized water with KOSMA (1.5%, w / v, g / mL), KOSMA (2.0%, w / v, g / mL), and KOSMA (2.5%, w / v, g / mL), respectively, and then a 5% (w / v, g / mL) GelMA solution was prepared. Then, 0.25% (w / v, g / mL) photoinitiator (LAP) was added, and the mixture was stirred at 37 °C until the solid completely formed 5G-LAP, 5G1.5K-LAP, 5G2K-LAP, and 5G2.5K-LAP solutions, which formed the precursor hydrogel solution of 5G, 5G1.5K, 5G2K, and 5G2.5K microspheres. (2) Using the precursor hydrogel solution as the dispersed phase (flow rate 2 mL / h) and castor oil as the continuous phase (flow rate 10 mL / h), hydrogel microspheres were prepared using a single-channel syringe pump and a T-type microfluidic chip. The inlet of the mobile phase of the chip was a dispensing needle with an inner diameter of 0.8 mm, and the inlet of the dispersed phase was a glass capillary with an inner diameter of 0.58 mm. The process of collecting the effluent must be kept on an ice-water bath. The temperature sensitivity of gelatin was used to pre-gel it at low temperature to avoid the uncrosslinked precursor hydrogel monodisperse droplets from fusing together. After collection, the microspheres were quickly exposed to ultraviolet light (405 nm, 25 mW / cm2) for 60 s to solidify the precursor hydrogel microspheres into microspheres. The microspheres were soaked in anhydrous ethanol and repeatedly washed until the castor oil was completely removed. Then, they were soaked in deionized water for 2 h to remove the residual LAP and continued to be washed with water until the ethanol was completely removed. The washed microspheres were pre-frozen at -80 ℃ for 6 h and then freeze-dried for 2-3 days to obtain the final product. Structural characterization 1. The prepared microspheres were placed in a glass dish and their morphology was photographed under a 10x microscope. The microsphere size was then statistically analyzed using ImageJ software to obtain preliminary microsphere morphology results. Further microsphere morphology images were taken using a scanning electron microscope (SEM). Specifically, the microspheres were attached to conductive adhesive to fix them to the sample stage. Any loose microspheres were then blown away with a syringe bulb. Gold was then sputtered onto the microspheres using a 10mA current, and images of the microspheres were acquired from different fields of view.

[0040] The results are as follows Figure 1 and Figure 2 As shown, when freshly prepared microspheres in castor oil were directly photographed under an optical microscope, it was observed that the transparency of the GK microspheres gradually decreased with increasing KOSMA concentration, and the distribution of microspheres in each group was uniform. After thoroughly washing the microspheres with ethanol and deionized water, placing them in deionized water and photographing them directly under a microscope revealed that the transparency of the cleaned microspheres increased while maintaining their spherical shape. ImageJ software was used to analyze the diameter of the microspheres. The diameters of the three groups of microspheres were uniformly distributed and conformed to a normal distribution, with diameters of 233.34±15.65, 241.59±8.49, 264.71±15.44, and 269.62±15.65, respectively. It can be seen that the microsphere diameter gradually increases with increasing KOSMA concentration, possibly because the hydrophilic groups such as sulfonic acid groups in KOSMA enhance the water absorption capacity of the microspheres, leading to a larger diameter.

[0041] 2. Cytotoxicity assessment of GK microspheres The cytotoxicity of KG microspheres was verified using B16, HaCat, and NIH-3T3 cells.

[0042] The specific method involves taking a certain amount of 5G, 5G1.5K, 5G2K, and 5G2.5K hydrogel microspheres, freeze-drying them, sterilizing them, and then incubating them in 1640 complete culture medium at an extraction concentration of 100 mg / ml at 37°C for 24 hours. Well-grown B16 cells are then incubated at 5 × 10⁻⁶ cells / ml. 4 The seeding density was 100 μl / ml in 96-well plates, i.e., 5 × 10⁶ μl per well. 3 Cells were cultured at 37°C and 5% CO2 for 24 hours until they were fully adhered. The culture medium was carefully aspirated to minimize inter-group differences caused by handling. 100 μl of GK microsphere extract (for each group) was added, and the cells were cultured at 37°C and 5% CO2 for 24 hours. The culture medium was carefully aspirated, and 100 μl of 0.5 mg / ml thiazolyl blue solution was added to each well under dark conditions. The cells were cultured for another 4 hours. The 96-well plate was removed, the solution was carefully aspirated, and 100 μl of DMSO was added to each well. The cells were cultured at 37°C and 100 rpm for 15 minutes with shaking. After the formazan was completely dissolved, the absorbance was measured at 570 nm using a microplate reader. Cell viability was then calculated according to the formula.

[0043]

[0044] In the formula, As is the absorbance value of the experimental group, Ab is the absorbance value of the solvent group (DMSO), and Ac is the absorbance value of the control group.

[0045] The procedure for the MTT assay in HaCat, L929, and NIH-3T3 cells is the same as that in the MTT assay in B16 cells. The difference is that the complete culture medium for HaCat and L929 cells is DEAE basal medium and fetal bovine serum, while the complete culture medium for NIH-3T3 cells is composed of DEAE basal medium and newborn calf serum. All other procedures are exactly the same.

[0046] NIH-3T3, HaCat, and B16 cells were cultured for 1 and 2 days using different GK microsphere extracts, and cytotoxicity was assessed based on the cell survival rate of each group.

[0047] The results are as follows Figure 3 The cell viability of groups (a) and (b) shown is above 80%, indicating that GK microspheres do not have cytotoxicity against normal fibroblasts and keratinocytes, but still have a killing effect on B16 cells. Figure 3 (c) The survival rate of B16 cells was below 80% on both the first and second days. Furthermore, with the increase of GK microsphere concentration, the survival rate of cells in the 5G2.5K group was below 50%, indicating that GK microspheres still have a good killing effect and have almost no effect on normal cells. This suggests that GK microspheres can specifically target melanoma and have good biocompatibility with normal cells.

[0048] Example 3 The application of GK microspheres in the anti-tumor effect of postoperative wounds in mouse melanoma is as follows: A melanoma tumor model was constructed using 6-week-old female C57 mice. The model construction method was as follows: B16 cells were cultured in batches, then digested and counted to obtain a final density of 2×10⁻⁶ cells. 7 Single-cell suspensions were prepared at 100 μl / ml, centrifuged to remove the culture medium, and the cells were resuspended in PBS while maintaining the cell density. The cell suspension was then subcutaneously injected into the back of C57 mice (100 μl / mouse) using a sterile syringe. A round bulge was observed on the back of the mouse immediately after injection. The mice were weighed and their weight recorded every two days after inoculation. Tumors began to appear around day 7 after inoculation, and tumor volume was measured every two days until the tumor reached 200-300 mm. 3 The entire process takes about 14 days. The patients are randomly divided into 3 groups (n=6) to undergo melanoma surgery. A small incision is made along the edge of the tumor with scissors, and then the entire tumor is cut off along the outline of the tumor and the tumor epithelium is removed for photographing.

[0049] After the surgical wounds of mice with melanoma were repaired, they were divided into three groups: a PBS control group, a 5G microsphere group, and a 5G 2.5K microsphere group. 10mg of lyophilized microspheres were weighed from each group and sterilized. The microspheres were then swollen with PBS and applied to the surgical wounds of the melanoma patients. PBS was added to the control group. The wounds were then bandaged with gauze and tape to prevent the microspheres from falling off and the mice from biting each other. The dressings were changed on the 3rd day. Photos were taken on the 1st, 3rd, 5th, 7th, and 14th days to record the changes in the tumor wounds before and after treatment. A time-varying graph of tumor volume was also plotted between different groups over time. The weight of the mice was measured every 2 days to plot the weight changes over time. The tumor volume was calculated using formula (1), and the results were expressed as mean ± standard deviation. Six parallel groups were set up for each experiment. The wound healing rate was calculated using formula (2), and the results were expressed as mean ± standard deviation. Six parallel groups were set up for each experiment.

[0050] (1) In the formula, W represents the tumor width and L represents the tumor length.

[0051] (2) In the formula, S0 represents the wound area on day 0, and St represents the wound area on day t.

[0052] Results Analysis Tumors in mice grew to 200-300 mm 3 In between, surgery was performed to remove the tumor, and then a complete photograph was taken. Figure 4 (a) After applying different groups of microspheres, the wounds were bandaged. Photos were taken at 0, 1, 3, 5, 7, 11, and 14 days to assess wound healing and melanoma recurrence. Figure 4 As shown in (b), the wound healing rate of the three groups generally reached over 90% within 10 days. However, around day 5, tumor recurrence began to occur in the wounds of the control group and the 5G group, and the tumors rapidly increased in size. By day 9, the wounds also began to enlarge (Figures (c) and (d)), and the patients experienced weight loss (Figure e). By day 11, the tumors had grown to 200 mm. 3 (Figure (f)), and both groups of mice began to die, but the mice in the 5G2.5K microsphere group showed almost no tumor recurrence, their weight returned to normal, and their wounds also healed normally. This was in stark contrast to the control group and the 5G group, which also shows that the 5G2.5K microspheres have a significant effect and play a good role in preventing postoperative recurrence of melanoma.

[0053] The S100b protein is closely related to melanoma and is one of the most important and commonly used serum tumor markers in melanoma diagnosis and treatment. Melanoma originates from melanocytes in the skin. During embryonic development, melanocytes and nerve cells (homogeneous, both originating from the neural crest) are naturally capable of expressing members of the S100 protein family. When melanocytes undergo malignant transformation into melanoma, this expression function is abnormally amplified and activated. Therefore, staining for S100b protein is used to assess the degree of malignancy in different groups of melanomas. Considering the possibility of lung metastasis, fluorescent staining was also performed on the lungs of mice.

[0054] The results are as follows Figure 5 As shown in Figure (a), the red fluorescence of the control group and the 5G group is significantly stronger, while the red fluorescence of the 5G2.5K group is very weak. The results of quantitative fluorescence analysis (Figures (b) and (c)) are consistent with those in Figure (a), indicating that the 5G2.5K microspheres effectively inhibit the growth of melanoma cells, which is consistent with the previous H&E staining and TUNEL staining results.

[0055] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing heparin-like hydrogel microspheres for postoperative wound repair in melanoma surgery, characterized in that, The method includes the following steps: (1) Dissolve KOSMA and GelMA in water to prepare a GelMA solution; then add a photoinitiator to the solution and stir until homogeneous to form a precursor hydrogel solution; The KOSMA is obtained by reacting O-sulfated K5 polysaccharide KOS with cystamine dihydrochloride to obtain KOS-Cys, followed by a reaction with MA modification. (2) Using the precursor hydrogel solution obtained in step (1) as the dispersed phase and castor oil as the continuous phase, hydrogel microspheres were prepared using a single-channel injection pump and a T-type microfluidic chip. The effluent was collected, exposed to ultraviolet light for curing, washed, and dried to obtain heparin-like hydrogel microspheres.

2. The method according to claim 1, characterized in that, The preparation of the KOSMA specifically includes the following steps: (1) Dissolve K5 polysaccharide in anhydrous DMF, then add sulfur trioxide pyridine complex, stir the reaction at room temperature under nitrogen atmosphere, stop the reaction, collect the reaction solution, transfer it to a dialysis bag for dialyzing, freeze dry, and obtain O-sulfated K5 polysaccharide KOS. (2) Dissolve KOS in water, then add EDC·HCl and NHS to adjust the pH to 4.5~5.4, activate the carboxyl group in an ice bath environment, add cystamine dihydrochloride, maintain the pH of the solution at 6.8~7.0, react overnight at room temperature, and measure that the pH remains basically unchanged at 6.8~7.

0. Stop the reaction, collect the product, dialyze, and freeze dry to obtain KOS-Cys; (3) Under light-protected conditions, the amide reaction product KOS-Cys was placed in a round-bottom flask, and PBS was added to completely dissolve it. Then, MA was slowly added dropwise to react. After the reaction, the product was collected into a dialysis bag, dialyzed, and lyophilized to obtain KOSMA.

3. The method according to claim 1, characterized in that, The amount of photoinitiator in step (1) is 0.1~0.25%, w / v, g / mL.

4. The method according to claim 1, characterized in that, The amount of GelMA in the GelMA solution in step (1) is 5%, w / v, g / mL.

5. The method according to claim 1, characterized in that, The amount of KOSMA in the GelMA solution in step (1) is 1~3%, w / v, g / mL.

6. The method according to claim 1, characterized in that, The UV curing conditions in step (2) are: 405 nm, 25 mW / cm². 2 , 60s.

7. The heparin-like hydrogel microspheres prepared by the method according to any one of claims 1 to 6.

8. The application of the heparin-like hydrogel microspheres according to claim 7 in biomedical materials.

9. The application according to claim 8, characterized in that, The biomedical materials mentioned include drug carriers or wound dressings.

10. The use of the heparin-like hydrogel microspheres of claim 7 in the preparation of wound dressings for postoperative care of melanoma.

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