Use of a borosilicate bioglass hydrogel for the preparation of a material for inhibiting the recurrence of melanoma after surgery

Abstract

This invention discloses the application of borosilicate bioglass hydrogel in the preparation of materials for inhibiting postoperative recurrence of melanoma. A borosilicate bioglass suspension and Fe3O4 nanoparticle suspension are added to an amino-modified hyaluronic acid solution and mixed thoroughly. Then, an aldehyde-modified hyaluronic acid solution is added, and a cross-linking reaction is carried out at room temperature to obtain the borosilicate bioglass hydrogel. The borosilicate bioglass hydrogel is used to prepare materials for inhibiting postoperative recurrence of melanoma. The material can inhibit the growth of melanoma in a mouse model of postoperative recurrence, increase the proportion of mature dendritic cells, promote dendritic cell maturation, and increase cytotoxic CD8. + T cells and CD4 + The proportion of T cells in tumors and spleen can be increased to enhance the killing effect of T cells, promote the polarization of macrophages in tumor sites to M1 type, reduce the proportion of M2 type macrophages, and downregulate the content of regulatory T cells and myeloid-derived suppressor cells in tumor sites.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and relates to the application of a borosilicate bioglass hydrogel in the preparation of materials to inhibit postoperative recurrence of melanoma. Background Technology

[0002] Melanoma is a highly aggressive malignant tumor, and current clinical strategies primarily rely on surgical resection for its treatment. Although diagnostic techniques, treatment levels, and postoperative care have continuously improved, the recurrence rate in stage II patients remains above 30%. Immunotherapy offers a promising new approach to suppressing postoperative tumor recurrence. However, low infiltration of immune cells at the tumor site, insufficient exposure to tumor antigens leading to a low immune response rate, and the immunosuppressive characteristics of the tumor microenvironment all contribute to this. This immunosuppressive microenvironment promotes the production of immunosuppressive cytokines and the formation of immunosuppressive networks by immunosuppressive cells such as M2 tumor-associated macrophages, regulatory T cells (Tregs), and tumor-derived myeloid-derived suppressor cells (MDSCs), thus facilitating tumor immune escape. Furthermore, the accumulation of lactic acid produced by tumor cell glycolysis at the tumor site further exacerbates immunosuppression, impacting postoperative treatment outcomes. Neutralizing tumor acidity can reshape the immunosuppressive microenvironment and enhance anti-tumor immunity. Materials such as bicarbonate have been shown to effectively neutralize lactic acid produced by tumor metabolism. However, bicarbonate is metabolized and diffuses rapidly in the body, making it difficult to regulate the tumor immunosuppressive microenvironment for extended periods. Repeated injections may also cause metabolic alkalosis. Therefore, there is an urgent need to find an immunotherapy platform that can sustainably neutralize the tumor acidic microenvironment, reverse immunosuppression, and activate the anti-tumor immune response to inhibit postoperative tumor recurrence.

[0003] Bioactive glass is a bioactive, compositionally tunable alkaline material. Its main chemical composition is SiO2·CaO·P2O5, exhibiting good biocompatibility and wide application in tissue repair and bone defect treatment. Furthermore, the functionalization of bioactive glass for bacterial infection or anti-tumor purposes is currently a research focus. Bioactive glasses such as silicate glasses exhibit alkalinity through silicate hydrolysis, neutralizing tumor acidity; however, this degradation rate is relatively slow. Borosilicate bioglasses, due to their faster degradation rate, may more quickly modulate the tumor acidic microenvironment, more efficiently reshape the tumor immune microenvironment, enhance the efficacy of tumor immunotherapy, and thus inhibit tumor growth. However, the tumor-killing properties of borosilicate bioglasses are insufficient. To improve their tumor-killing effect, current research focuses on doping borosilicate bioglasses with ions or combining them with nanoparticles to meet the killing requirements. Iron oxide is an ideal chemokine with good biocompatibility. In the acidic tumor microenvironment, it can catalyze the Fenton reaction, thereby inducing immunogenic cell death and activating anti-tumor immune responses. Combining the alkalinity of borosilicate bioglass with the chemokinetic therapy mediated by the Fenton reaction via iron oxide offers an ideal method for regulating the immune microenvironment while enhancing the immune response. To achieve long-term regulation of the tumor immune microenvironment and inhibit tumor recurrence, a sustained-release carrier is needed. Hyaluronic acid, a glycosaminoglycan distributed in the extracellular matrix of connective tissue cells, possesses good biocompatibility and low immunogenicity, making it a common hydrogel substrate. Under the stimulation of an acidic tumor microenvironment with high hyaluronidase expression, hyaluronic acid hydrogels responsively degrade, releasing borosilicate bioglass with long-term acid-resistant properties and the chemokinetic agent iron oxide. This regulates the immunosuppressive microenvironment and activates the immune response. The combination of these two agents holds promise for improving the efficacy of immunotherapy and its application in inhibiting postoperative tumor recurrence.

[0004] Based on the above, it is necessary to construct a borosilicate bioglass hydrogel that inhibits postoperative recurrence of melanoma. This hydrogel can reverse tumor immunosuppression by neutralizing the acidic tumor microenvironment and activate anti-tumor immune responses by combining immunogenic cell death induced by chemokinetics, thereby improving the efficacy of immunotherapy and inhibiting postoperative recurrence of melanoma. Summary of the Invention

[0005] In view of this, the present invention provides an application of borosilicate bioglass hydrogel in the preparation of materials for inhibiting postoperative recurrence of melanoma.

[0006] The present invention specifically provides the following technical solution:

[0007] An application of borosilicate bioglass hydrogel in the preparation of materials for inhibiting postoperative recurrence of melanoma is disclosed. The preparation method of the borosilicate bioglass hydrogel is as follows: Borosilicate bioglass suspension and Fe3O4 nanoparticle suspension are added to an amino-modified hyaluronic acid solution and mixed evenly. Then, an aldehyde-modified hyaluronic acid solution is added, and a cross-linking reaction is carried out at room temperature to obtain the borosilicate bioglass hydrogel. The borosilicate bioglass hydrogel is used to prepare materials for inhibiting postoperative recurrence of melanoma. The material can inhibit the growth of melanoma in a mouse model of postoperative recurrence, increase the proportion of mature dendritic cells, promote dendritic cell maturation, and increase cytotoxic CD8. + T cells and CD4 + The proportion of T cells in tumors and spleen can be increased to enhance the killing effect of T cells, promote the polarization of macrophages in tumor sites to M1 type, reduce the proportion of M2 type macrophages, and downregulate the content of regulatory T cells and myeloid-derived suppressor cells in tumor sites.

[0008] Furthermore, the volume ratio of the aminated hyaluronic acid solution, the aldehyde-modified hyaluronic acid solution, the borosilicate bioglass suspension, and the Fe3O4 nanoparticle suspension is 12:2:1:1; the concentration of the borosilicate bioglass suspension is 3%-5% w / v, and the concentration of the Fe3O4 nanoparticle suspension is 250 μg / mL.

[0009] Furthermore, the concentration of the amino-modified hyaluronic acid solution is 10–30 mg / mL; the concentration of the aldehyde-modified hyaluronic acid solution is 100–300 mg / mL.

[0010] Furthermore, the grafting rate of the amino-modified hyaluronic acid is 25%–40%, and the degree of aldehyde modification of the aldehyde-modified hyaluronic acid is 40%–50%.

[0011] Furthermore, the Fe3O4 nanoparticles have a particle size of approximately 70-90 nm.

[0012] Furthermore, the borosilicate bioglass has a particle size of 11-45 μm.

[0013] Furthermore, the cross-linking reaction takes 0.5 to 5 minutes.

[0014] Furthermore, the preparation method of the amino-modified hyaluronic acid is as follows: 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine chloride and oxaloacetylhydrazine are added to a 2-morpholine ethanesulfonic acid solution of hyaluronic acid, and the mixture is reacted in an oil bath at 25℃~56℃ for 24h~48h, then dialyzed, and then freeze-dried to obtain amino-modified hyaluronic acid with a grafting rate of 25%~40%.

[0015] Furthermore, the preparation method of the aldehyde-modified hyaluronic acid is as follows: sodium periodate is added to an aqueous solution of hyaluronic acid under light-protected conditions and reacted for 6 to 24 hours. The reaction is terminated with ethylene glycol, dialyzed, and then freeze-dried to obtain aldehyde-modified hyaluronic acid with a degree of aldehyde modification of 40 to 50%.

[0016] Furthermore, the preparation method of the Fe3O4 nanoparticles is as follows: Ferric chloride hexahydrate and sodium citrate dihydrate are completely dissolved in ethylene glycol, then sodium acetate trihydrate and deionized water are added and the mixture is stirred until homogeneous. The mixture is then subjected to a hydrothermal reaction at 180℃~220℃ for 7h~9h. The nanoparticles are then washed alternately with deionized water and ethanol to obtain Fe3O4 nanoparticles.

[0017] The beneficial effects of this invention are as follows:

[0018] This invention incorporates borosilicate bioglass and Fe3O4 into a hyaluronic acid hydrogel. The borosilicate bioglass efficiently degrades to release silicate ions, neutralizing lactic acid produced by tumor metabolism and reversing the tumor's immunosuppressive microenvironment. Fe3O4 responds to the acidic tumor microenvironment, catalyzing the Fenton reaction, inducing immunogenic cell death, and activating an anti-tumor immune response. The borosilicate bioglass hydrogel constructed in this invention, which inhibits postoperative recurrence of melanoma, combines chemokinetic-induced immunogenic cell death with neutralization of tumor acidity to reverse immunosuppression, enhancing the efficacy of immunotherapy and providing a novel material combination for inhibiting postoperative recurrence of melanoma. This invention is the first to propose the application of borosilicate bioglass in inhibiting postoperative recurrence of melanoma, providing new ideas and directions for the development of novel materials for inhibiting postoperative recurrence of melanoma. Attached Figure Description

[0019] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided:

[0020] Figure 1 The 1H NMR spectra of HA, HA-NH2, and HA-CHO are... 1 H NMR).

[0021] Figure 2 These are scanning electron microscope (SEM) images of borosilicate bioactive glass and transmission electron microscope (TEM) images of Fe3O4.

[0022] Figure 3 This is a scanning electron microscope image of the borosilicate bioglass hydrogel that inhibits postoperative recurrence of melanoma according to the present invention.

[0023] Figure 4 This is a test graph showing the ability of the borosilicate bioglass hydrogel of the present invention to regulate pH under the condition of pH=6.5, which inhibits postoperative recurrence of melanoma.

[0024] Figure 5 This is an evaluation diagram of the killing effect of the borosilicate bioglass hydrogel, which inhibits postoperative recurrence of melanoma according to the present invention, on mouse skin melanoma cells (B16F10).

[0025] Figure 6 This is an image showing the effect of the borosilicate bioglass hydrogel of the present invention on inhibiting postoperative recurrence of melanoma.

[0026] Figure 7 This is a cell flow cytometry diagram showing the in vivo immunotherapy mechanism of borosilicate bioglass hydrogel, which inhibits postoperative recurrence of melanoma, in the treatment of melanoma recurrence after surgery. Detailed Implementation

[0027] The preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0028] Example 1

[0029] 1. Prepare amino-modified hyaluronic acid (HA-NH2) based on hyaluronic acid with a molecular weight of 20kDa-40kDa, as follows:

[0030] 0.625 g of hyaluronic acid (HA) with a molecular weight of 20 kDa-40 kDa was completely dissolved in a 50 mmol / L solution of 2-morpholine ethanesulfonic acid. 0.4275 g of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine chloride was added to the mixture, and the mixture was stirred for 0.5 h. Then, 1.35 g of oxalic acid dihydrazide was added, and the reaction was carried out in an oil bath at 56 °C for 48 h. The reaction solution was then transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 1000 Da and dialyzed in deionized water for 3 days. After dialysis, the liquid in the dialysis bag was freeze-dried to obtain the product, amino-modified hyaluronic acid (HA-NH2), which was then dried and stored at 4 °C.

[0031] Figure 1 Curve A is the 1H NMR spectrum of HA, where ζ = 1.9 ppm is the methyl proton peak in the acetyl group on the side chain of HA, and the broad absorption peak of ζ = 3.2-3.8 ppm is the characteristic peak produced by the H on the sugar ring in the HA molecule. Figure 1Curve B is the 1H NMR spectrum of HA-NH2, where ζ = 1.9 ppm represents the methyl proton peak on the acetamino group of the N-acetyl-d-glucosamine residue on the HA side chain; ζ = 1.4 and 1.6 ppm and ζ = 2.1 and 2.3 ppm represent the methylene proton peaks in the introduced ADH. The degree of substitution of HA-NH2 was obtained by the ratio of the integrated area of ​​the methyl proton peak in the acetyl group of the HA side chain at ζ = 1.9 ppm to the methylene proton peaks in the ADH at ζ = 1.4 and 1.6 ppm and ζ = 2.1 and 2.3 ppm. The results show that the amino grafting rate in HA-NH2 is approximately 32.62%.

[0032] 2. The following steps were taken to prepare aldehyde-modified hyaluronic acid (HA-CHO) based on hyaluronic acid with a molecular weight of 100 kDa:

[0033] 2.5 g of HA with a molecular weight of 100 kDa was dissolved in deionized water. 25 mL of a 26.65 g / L sodium periodate solution was added under light-protected conditions, and the mixture was stirred for 24 h. The reaction was then terminated by adding 0.6875 mL of ethylene glycol. The reaction solution was then transferred to a dialysis bag (MWCO 1000) and dialyzed against deionized water for 3 days. After dialysis, the liquid in the dialysis bag was freeze-dried to obtain aldehyde-modified hyaluronic acid. Figure 1 Curve C is the 1H NMR spectrum of HA-CHO, where ζ = 4.9 ppm represents the proton peak of the introduced hemiacetal. The degree of substitution of HA-CHO was determined by hydroxylamine hydrochloride-pH titration, and the results showed that the degree of aldehyde in HA-CHO was approximately 46%.

[0034] 3. The borosilicate bioglass (BG) was characterized by scanning electron microscopy, such as... Figure 2 As shown in Figure A, the particle size can be obtained as 11-45 μm.

[0035] 4. Preparation of Fe3O4 nanoparticles, the steps are as follows:

[0036] 0.325 g of ferric chloride hexahydrate and 0.2 g of sodium citrate dihydrate were dissolved in 20 mL of ethylene glycol and stirred for 0.5 h. Then, 1.2 g of sodium acetate trihydrate and 0.3 mL of deionized water were added to the mixture and stirred until homogeneous. The solution was transferred to a hydrothermal reactor and reacted at 200 °C for 8 h. The resulting nanoparticles were washed three times alternately with deionized water and ethanol to obtain Fe3O4 nanoparticles. The transmission electron microscopy (TEM) image of the obtained Fe3O4 nanoparticles is shown below. Figure 2 As shown in Figure B, the particle size of the Fe3O4 nanoparticles is approximately 70-90 nm.

[0037] 5. Preparation of borosilicate bioglass hydrogel to inhibit postoperative recurrence of melanoma, the steps are as follows:

[0038] 1) Dissolve HA-NH2 in deionized water to obtain a 20 mg / mL HA-NH2 solution, and dissolve HA-CHO in deionized water to obtain a 200 mg / mL HA-CHO solution. The volume ratio of HA-NH2 solution to HA-CHO solution is 6:1.

[0039] 2) Prepare a 3% w / v borosilicate bioglass suspension and a 250 μg / mL Fe3O4 suspension, and disperse them thoroughly by ultrasonication.

[0040] 3) Add borosilicate bioglass suspension and Fe3O4 suspension to HA-NH2 solution and mix thoroughly. Then add HA-CHO solution for cross-linking and react at room temperature for about 2 minutes to form a borosilicate bioglass hydrogel that inhibits postoperative recurrence of melanoma. The volume ratio of each component (HA-NH2 solution: HA-CHO solution: borosilicate bioglass suspension: Fe3O4 suspension) is 12:2:1:1.

[0041] Figure 3 Scanning electron microscope (SEM) images of borosilicate bioglass hydrogel used to inhibit postoperative recurrence of melanoma, magnified 100x (left) and 500x (right). Figure 3 As can be seen, the hydrogel has a porous structure, with borosilicate bioglass and Fe3O4 dispersed in the hyaluronic acid hydrogel matrix.

[0042] Example 2: Investigation into the optimal concentration of borosilicate bioglass suspension

[0043] A total of 5 experimental groups were set up: HA group, HA / Fe group, HA / 1BG / Fe group, HA / 3BG / Fe group, and HA / 5BG / Fe group, as detailed below:

[0044] 1) HA / 3BG / Fe group: HA / 3BG / Fe was prepared according to step 5 of Example 1. HA / 3BG / Fe was soaked in 3 mL of PBS with pH = 6.5 and the pH of the extract was measured at different time points.

[0045] 2) HA group: The operation is basically the same as the HA / 3BG / Fe group, except that borosilicate bioglass (BG) and Fe3O4 are not doped in the hydrogel.

[0046] 3) HA / Fe group: The operation is basically the same as that of HA / 3BG / Fe group, except that the concentration of borosilicate bioglass (BG) in the hydrogel is 0% w / v.

[0047] 4) HA / 1BG / Fe group: The operation is basically the same as that of HA / 3BG / Fe group, except that the concentration of borosilicate bioglass (BG) in the hydrogel is 1% w / v.

[0048] 5) HA / 5BG / Fe group: The operation is basically the same as that of HA / 3BG / Fe group, except that the concentration of borosilicate bioglass (BG) in the hydrogel is 5% w / v.

[0049] A pH of 6.5 can simulate the pH of an acidic tumor microenvironment, which may also represent the pH of the residual tumor environment after incomplete tumor resection. The ability of HA / BG / Fe with different borosilicate bioglass contents to regulate pH at pH 6.5 is as follows: Figure 4 As shown.

[0050] from Figure 4 As can be seen, the pH of the HA extract remained unchanged at the measurement endpoint, indicating that the hydrogel carrier itself does not have the ability to regulate pH. The pH of the HA / 1BG / Fe extract was approximately 8.12, and the pH of the HA / 5BG / Fe extract was approximately 8.94, demonstrating that the pH of the hydrogel extract increased with the increase of borosilicate bioglass content. The pH of the HA / 3BG / Fe extract was approximately 8.87, and the pH of the HA / 5BG / Fe extract did not increase significantly compared to HA / 3BG / Fe, indicating that further increasing the borosilicate bioglass content in the hydrogel did not show a more significant ability to regulate pH when 3% w / v BG was already added. The borosilicate bioglass hydrogel that inhibits postoperative recurrence of melanoma can release ions to hydrolyze and neutralize protons, constructing an alkaline microenvironment. This borosilicate bioglass doping level-dependent alkalinity has the potential to regulate the acidic tumor microenvironment.

[0051] Example 3: Investigation into the optimal concentration of Fe3O4 suspension

[0052] The killing effect of HA / BG / Fe doped with different concentrations (50, 75, 100, 250 μg / mL) of Fe3O4 on mouse skin melanoma (B16F10) cells was evaluated. Details are as follows:

[0053] 1) Preparation of HA and HA / BG / Fe doped with different concentrations (50, 75, 100, 250 μg / mL) of Fe3O4.

[0054] 2) After seeding B16F10 cells in 96-well plates and allowing them to adhere for 24 hours, the original culture medium was replaced, and different hydrogels were added. Normal culture medium served as the positive control, and sterile water as the negative control. Cells were cultured for another 24 hours. After removing the culture medium and hydrogels, serum-free medium containing 5% thiazolyl blue (MTT) was added under dark conditions. After incubation for 4 hours, the MTT solution was discarded, and DMSO was added. The absorbance was read using a microplate reader to calculate cell viability.

[0055] The evaluation of the killing effect of the borosilicate bioglass hydrogel that inhibits postoperative recurrence of melanoma on B16F10 cells in this invention is as follows: Figure 5 As shown.

[0056] from Figure 5 As can be seen, the borosilicate bioglass hydrogel that inhibits postoperative recurrence of melanoma exhibits a Fe3O4 concentration-dependent killing effect. With the increase of Fe3O4 doping, the survival rate of B16F10 cells gradually decreases. When 250 μg / mL Fe3O4 is doped into the hydrogel, the survival rate of B16F10 cells is less than 20%, proving that HA / BG / Fe has a good effect on killing melanoma cells.

[0057] Example 4: Evaluation of the effect of hydrogel on postoperative recurrence of melanoma in mouse skin.

[0058] To evaluate the effect of the borosilicate bioglass hydrogel of the present invention, which inhibits postoperative recurrence of melanoma, on the recurrence of mouse cutaneous melanoma, we compared different materials used in a mouse cutaneous melanoma cell-luciferase-labeled (B16F10-Luc) postoperative recurrence treatment model. The flowchart of this model is shown below. Figure 6 As shown in A): When mice were subcutaneously inoculated with B16F10-Luc cells, the tumors grew to approximately 300 mm on day 10. 3 The tumor was surgically removed, and a borosilicate bioglass hydrogel designed to inhibit postoperative melanoma recurrence was implanted. Tumor growth was continuously monitored. All mice were sacrificed on day 22.

[0059] The specific procedure was as follows: 10 mg of [unspecified substance] was injected into the right hind leg of a female C57BL / 6N mouse. 7 B16F10-Luc cells were used to observe tumor development. After 10 days, when the tumor volume was approximately 300 mm², [the tumor was observed to be progressing]. 3 At that time, 99% of the tumor was surgically removed, and different treatments were administered, including the PBS group, HA group, HA / BG group, HA / Fe group, and HA / BG / Fe group, as detailed below:

[0060] 1) PBS group: only PBS solution is added.

[0061] 2) HA / BG / Fe group: prepared according to step 5 of Example 1, thus obtaining HA / BG / Fe.

[0062] 3) HA group: The preparation method is basically the same as that of HA / BG / Fe group, except that BG and Fe3O4 are not doped in the hydrogel.

[0063] 4) HA / BG group: The preparation method is basically the same as that of HA / BG / Fe group, except that Fe3O4 is not doped in the hydrogel.

[0064] 5) HA / Fe group: The preparation method is basically the same as that of HA / BG / Fe group, the only difference is that BG is not added to the hydrogel.

[0065] Tumor volume and body weight were monitored in mice every two days. On days 9, 10, 12, 18, and 22, mice were injected intraperitoneally with D-fluorescein for small animal fluorescence imaging, and fluorescence intensity was recorded. On day 17, one mouse from each group was sacrificed, and flow cytometry analysis was performed on the tumors and organs. The remaining mice were monitored for tumor growth and body weight. All mice were sacrificed on day 22, and the experiment was terminated.

[0066] The formula for calculating mouse tumor volume is as follows:

[0067] Mouse tumor volume = 0.5 × length × width 2 .

[0068] Figure 6 B) Tumor growth curves for different treatment groups. By day 10, the tumor volume in each group reached approximately 300 mm. 3 After 99% of the tumor was removed, the tumor volume was considered to be 0 mm. 3 And different treatments were administered.

[0069] from Figure 6 As shown in B), on day 22, the HA / BG group (approximately 1800 mm) 3 ) and HA / Fe group (approximately 1250mm) 3 The tumor size of the PBS group (approximately 2500 mm) was significantly smaller than that of the PBS group. 3 ) and HA group (approximately 2000 mm) 3 The smaller size indicates that BG and Fe3O4 have a certain inhibitory effect on postoperative recurrence of melanoma; compared with other groups, the HA / BG / Fe group had a consistently smaller tumor volume after tumor resection and treatment, with a tumor volume of approximately 250 mm on day 22. 3 This indicates that the borosilicate bioglass hydrogel of the present invention, which inhibits postoperative recurrence of melanoma, has a more significant potential to inhibit tumor recurrence after surgery for melanoma in mouse skin.

[0070] Figure 6C) Fluorescence imaging images of mice in different treatment groups. Higher fluorescence intensity indicates a larger tumor volume in the mouse. On day 9, fluorescence was present in all groups, confirming the successful establishment of the mouse skin melanoma model. After tumor resection (day 10), no biofluorescence signal was observed in any group of mice. On day 12, tumors in all groups showed varying degrees of recurrence, but due to the small size of the tumors, fluorescence signals could not be observed within the set fluorescence intensity range. On day 18, mice in the PBS, HA, HA / BG, and HA / Fe groups showed different sizes of fluorescence imaging areas, with the fluorescence areas in the HA / BG and HA / Fe groups being significantly smaller than those in the PBS and HA groups. This indicates that BG and Fe3O4 have a certain consistent effect on postoperative recurrence of melanoma, which is consistent with... Figure 6 B) Correspondingly, at 22 days, the HA / BG / Fe group had the smallest fluorescence area, demonstrating that the borosilicate bioglass hydrogel of this invention, which inhibits postoperative recurrence of melanoma, effectively suppresses tumor recurrence and further growth.

[0071] Figure 6 D) Statistical graphs of fluorescence imaging in mice of different treatment groups. Figure 6 As can be seen from D), the borosilicate bioglass hydrogel for inhibiting postoperative recurrence of melanoma in this invention exhibits a fluorescence intensity (approximately 1600 p / sec / cm). 2 The tumor density ( / sr) in the HA / BG / Fe group was significantly lower than that in the other groups, indicating that the tumor size in the HA / BG / Fe group at 22 days was smaller than that in the PBS group (approximately 15700 p / sec / cm). 2 / sr), HA group (approximately 10300p / sec / cm) 2 / sr), HA / BG group (approximately 8350p / sec / cm) 2 / sr) and HA / Fe group (approximately 6300 p / sec / cm) 2 Therefore, the hydrogel of the present invention has a significant inhibitory effect on melanoma growth.

[0072] Combination Figure 6 B) Figure 6 C) and Figure 6 D) It can be seen that the borosilicate bioglass hydrogel of the present invention, which inhibits postoperative recurrence of melanoma, can inhibit postoperative recurrence of melanoma after treatment, and its anti-tumor effect is better than that of the control group and the treatment strategy of adding only BG or Fe3O4.

[0073] To further illustrate the impact of borosilicate bioglass hydrogels on the tumor immune microenvironment in inhibiting postoperative recurrence of melanoma, the content of mature dendritic cells (DCs) in the lymph node region was analyzed by flow cytometry. Figure 7 A) T cell content in tumor and spleen sites ( Figure 7 B and C), M1 / M2 macrophage content in the tumor site ( Figure 7 D) and regulatory T cells (Tregs) Figure 7 E) and tumor-derived myeloid suppressor cells (MDSCs) Figure 7 F content.

[0074] from Figure 7 As can be seen from A, mature DCs (CD11c) in the HA / Fe group + CD80 + CD86 + The proportion of mature DCs in the HA / BG / Fe group (27.7%) was higher than that in the PBS group (10.9%), HA group (13.9%), and HA / BG group (15.7%), indicating that Fe3O4 doped in the hydrogel can mediate the Fenton reaction, generate highly toxic ·OH, kill tumor cells, promote the release of tumor-associated antigens through a chemokinetic pathway, thereby inducing ICD, promoting DC maturation, and thus improving the efficiency of the immune response. The proportion of mature DCs in the HA / BG / Fe group (37.2%) was even higher than that in the HA / Fe group. This may be because the borosilicate bioglass reverses tumor immunosuppression, further enhancing the effect of Fe3O4 in inducing ICD through chemokinetics, thereby further promoting DC maturation.

[0075] from Figure 7 Figures B and C show that doping the hydrogel with borosilicate bioglass and Fe3O4 can increase CD8 levels in tumor and spleen sites. + T (34.5% and 26.4% respectively) and CD4 + The increased T cell content (18.2% and 40.2% respectively) improved the infiltration of T cells at the tumor site, effectively activating anti-tumor immunity and systemic immune response.

[0076] To further illustrate the ability of borosilicate bioglass hydrogels to modulate the tumor immune microenvironment and inhibit postoperative recurrence of melanoma, the content of M1 and M2 macrophages in the tumor site was measured, and the results were obtained. Figure 7 D. From Figure 7 As shown in Figure D, compared to the PBS group (M1 macrophage percentage: 5.91%; M2 macrophage percentage: 34.2%), treatment with the borosilicate bioglass hydrogel of this invention, which inhibits postoperative recurrence of melanoma, to a certain extent increased the number of M1 macrophages (CD11b). + F4 / 80CD86 + The proportion of M2 macrophages (CD11b) was downregulated (25.1%). + F4 / 80CD206 + The proportion (14.9%) indicates that HA / BG / Fe has the ability to polarize macrophages into the M1 anti-tumor type.

[0077] To further illustrate the effect of borosilicate bioglass hydrogel in reversing tumor immunosuppression and inhibiting postoperative recurrence of melanoma, the content of Tregs in the tumor site was detected, and the test results are as follows: Figure 7 As shown in E, the content of MDSCs in the tumor site was detected, and the test results are as follows. Figure 7 The content is shown in F.

[0078] The content of Tregs was 35.0% in the PBS group, 33.8% in the HA group, 25.1% in the HA / BG group, 23.1% in the HA / Fe group, and 11.3% in the HA / BG / Fe group.

[0079] The content of MDSCs was 37.8% in the PBS group, 36.6% in the HA group, 29.5% in the HA / BG group, 22.8% in the HA / Fe group, and 14.5% in the HA / BG / Fe group.

[0080] from Figure 7 As can be seen from E and 7F, doping the hydrogel with borosilicate bioglass (HA-BG group) or Fe3O4 (HA-Fe group) can downregulate Tregs (CD3+). + CD4 + FoxP3 + ) and MDSCs (CD45) + CD11b + Gr-1 + The high H+ content may be due to the fact that borosilicate bioglass can release silicate ions in an acidic environment. These silicate ions hydrolyze and neutralize excess H+ in the tumor microenvironment. + It can reverse tumor immunosuppression; while Fe3O4 can induce immunogenic cell death through a chemokinetic pathway, which can transform immunosuppressed "cold" tumors into immunologically active "hot" tumors, thereby downregulating the content of immunosuppressive cells.

[0081] After treatment in the HA / BG / Fe group, compared with the other groups, the content of Tregs and MDSCs in the tumor can be further downregulated, indicating that the borosilicate bioglass hydrogel that inhibits postoperative recurrence of melanoma in this invention can effectively regulate the tumor immunosuppressive microenvironment and reverse tumor immunosuppression.

[0082] The above experimental results show that the borosilicate bioglass hydrogel of the present invention, which inhibits postoperative recurrence of melanoma, can induce immunogenic cell death and activate tumor immune response through chemical kinetic pathways while regulating the tumor immune microenvironment. This immune-enhancing effect can increase the proportion of cytotoxic T cells in tumors and spleen, promote dendritic cell maturation, induce M1 polarization of macrophages in tumors, and inhibit postoperative recurrence of tumors.

[0083] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. Use of a borosilicate bioglass hydrogel for the preparation of a material for inhibiting the post-surgical recurrence of melanoma, characterized in that, The preparation method of the borosilicate bioglass hydrogel is that: the amino hyaluronic acid solution is added with the borosilicate bioglass suspension and the Fe3O4 nanoparticle suspension, after being uniformly mixed, the aldehyde hyaluronic acid solution is added, and the cross-linking reaction is carried out at room temperature to obtain the borosilicate bioglass hydrogel.

2. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The volume ratio of the amino hyaluronic acid solution, the aldehyde hyaluronic acid solution, the borosilicate bioglass suspension and the Fe3O4 nanoparticle suspension is 12:2:1:1; the concentration of the borosilicate bioglass suspension is 3%-5% w / v, and the concentration of the Fe3O4 nanoparticle suspension is 250 μg / mL.

3. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The concentration of the amino hyaluronic acid solution is 10-30 mg / mL; and the concentration of the aldehyde hyaluronic acid solution is 100-300 mg / mL.

4. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The grafting rate of the amino hyaluronic acid is 25%-40%, and the aldehyde degree of the aldehyde hyaluronic acid is 40%-50%.

5. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The particle size of the Fe3O4 nanoparticle is 70-90 nm.

6. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The particle size of the borosilicate bioglass is 11-45 μm.

7. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The time of the cross-linking reaction is 0.5-5 min.

8. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The preparation method of the amino hyaluronic acid is that: the 2-morpholinoethanesulfonic acid solution of hyaluronic acid is added with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride and oxalyl dihydrazide, and after being reacted in an oil bath at 25 °C-56 °C for 24 h-48 h, dialysis is carried out, and then freeze-drying is carried out to obtain the amino hyaluronic acid with a grafting rate of 25%-40%.

9. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The preparation method of the aldehyde hyaluronic acid is that: under light-proof conditions, sodium periodate is added into the hyaluronic acid aqueous solution to react for 6 h-24 h, the reaction is terminated with ethylene glycol, dialysis is carried out, and then freeze-drying is carried out to obtain the aldehyde hyaluronic acid with an aldehyde degree of 40-50%.

10. Use of a borosilicate bioglass hydrogel according to claim 1 for the preparation of a material for inhibiting the recurrence of melanoma after surgery, characterized in that, The preparation method of the Fe3O4 nanoparticle is that: after ferric chloride hexahydrate and sodium citrate dihydrate are completely dissolved in ethylene glycol, sodium acetate trihydrate and deionized water are added and continue to be stirred until the system is uniform, and then the hydrothermal reaction is carried out at 180 °C-220 °C for 7 h-9 h, the nanoparticles are washed with deionized water and ethanol alternately to obtain the Fe3O4 nanoparticle.

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