Drug-loaded hydrogel for tumor immunotherapy and preparation method and application thereof

Abstract

The application belongs to the field of antitumor materials, and provides a drug-loaded hydrogel for tumor immunotherapy and a preparation method and application thereof. The drug-loaded injectable hydrogel is composed of a base hydrogel and polyphenol nanoparticles and an immune checkpoint inhibitor distributed in the base hydrogel, the polyphenol nanoparticles are nanoparticles self-assembled by polyethylene glycol connected to epigallocatechin gallate at one end, the polyphenol nanoparticles inhibit the expression of tumor cell hypoxia inducible factor, and then inhibit the expression of tumor cell PD-L1 antibody, so as to improve the sensitivity of tumor cells to the immune checkpoint inhibitor. The application strengthens the inhibition effect on tumors through the synergistic effect of the polyphenol nanoparticles and the immune checkpoint inhibitor, and the polyphenol nanoparticles have low toxicity to normal cells, so that the problems of great toxicity and side effects of chemotherapeutic drugs and drug resistance in the combined use of chemotherapeutic drugs and immunotherapeutic drugs in the prior art can be overcome.

Description

Technical Field

[0001] This invention belongs to the field of antitumor materials, and relates to drug-loaded hydrogels for tumor immunotherapy, their preparation methods and applications. Background Technology

[0002] Cancer is a major public health problem worldwide. Statistics show that in 2022, there were nearly 20 million new cancer cases globally, and 9.7 million people died from cancer. Currently, the main clinical treatment for cancer is systemic chemotherapy. However, chemotherapy drugs have strong toxic side effects, damaging normal tissues and organs and severely impairing patients' quality of life. With technological advancements, tumor immunotherapy has made breakthroughs and has become an effective treatment method.

[0003] While some tumors possess strong immunogenicity and can be treated with immunotherapy using cytokines, vaccines, adoptive T cells, and immune checkpoint inhibitors, others remain in a state of low immunogenicity. Simultaneously, the aerobic glycolysis of tumors leads to the accumulation of large amounts of lactic acid within the tumor tissue, creating an immunosuppressive microenvironment. Furthermore, to evade the immune system, tumor cells overexpress proteins such as PD-L1, inhibiting T cell activity. Studies have shown that some chemotherapy drugs (such as doxorubicin) induce immunogenic cell death in tumor cells, promoting the release of tumor-associated antigens and activating anti-tumor immunity. Although chemotherapy drugs can be combined with immunotherapy drugs, the toxic side effects of chemotherapy itself and the development of chemotherapy-induced resistance remain significant challenges. Therefore, there is still a need to explore low-toxicity immune-activating drugs to improve the immunosuppressive microenvironment of tumor tissues, thereby synergizing with immunotherapy and enhancing the efficacy of tumor treatment. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a drug-loaded hydrogel for tumor immunotherapy, its preparation method and application, which enhances the sensitivity of tumor cells to immune checkpoint inhibitors through polyphenol nanoparticles, improves the immunotherapy effect of tumors, and achieves synergistic inhibition of tumors.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by the present invention is as follows:

[0006] A drug-loaded injectable hydrogel for tumor immunotherapy comprises a matrix hydrogel and polyphenol nanoparticles and immune checkpoint inhibitors distributed within the matrix hydrogel. The polyphenol nanoparticles are formed by the self-assembly of polyethylene glycol with one end connected to epigallocatechin gallate. The matrix hydrogel is formed by the oxidative self-crosslinking of an aqueous solution of modified natural polysaccharides. The polyphenol nanoparticles inhibit the expression of hypoxia-inducible factor in tumor cells, thereby inhibiting the expression of PD-L1 antibodies in tumor cells, thus enhancing the sensitivity of tumor cells to immune checkpoint inhibitors and achieving synergistic tumor suppression.

[0007] In the above-mentioned drug-loaded injectable hydrogel technology, the immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor. Further, the PD-1 / PD-L1 inhibitor includes, but is not limited to, any one of BMS-1, BMS-202, BMS-1166, nivolumab, pembrolizumab, atezolizumab, ipilimumab, and bonatetumab.

[0008] In the above-mentioned drug-loaded injectable hydrogel technical solution, the tumor cells are melanoma cells.

[0009] In the above-mentioned drug-loaded injectable hydrogel technical solution, the polyethylene glycol with one end connected to epigallocatechin gallate has the following structural formula:

[0010]

[0011] In the above-mentioned drug-loaded injectable hydrogel technical solution, the polyethylene glycol with epigallocatechin gallate at one end can be prepared with reference to existing technology. Typically, the polyethylene glycol with epigallocatechin gallate at one end is obtained by a Michael addition reaction between the thiol group of polyethylene glycol with a thiol end group and the phenolic hydroxyl group of epigallocatechin gallate. The molar ratio of the polyethylene glycol with a thiol end group to the epigallocatechin gallate is controlled to be 1:(1-2), and the molecular weight of the polyethylene glycol with a thiol end group as the modification basis is preferably 1000-10000 g / mol.

[0012] In the above-mentioned drug-loaded injectable hydrogel technical solution, the particle size of the polyphenol nanoparticles is 100-300 nm.

[0013] In the aforementioned drug-loaded injectable hydrogel technology, based on the hydrophilicity difference between the polyethylene glycol fragment and the epigallocatechin gallate fragment in polyethylene glycol with one end connected to epigallocatechin gallate, the polyethylene glycol with one end connected to epigallocatechin gallate can self-assemble into polyphenol nanoparticles. Typically, polyphenol nanoparticles can be prepared by a thin-film hydration method, which involves dissolving the polyethylene glycol with one end connected to epigallocatechin gallate in an organic solvent, removing the solvent to obtain a polyethylene glycol membrane with one end connected to epigallocatechin gallate, then adding an aqueous medium and allowing it to react fully, followed by filtration through an aqueous filter membrane.

[0014] In the above-mentioned drug-loaded injectable hydrogel technical solution, the mass ratio of immune checkpoint inhibitor to polyphenol nanoparticles in the drug-loaded injectable hydrogel is determined according to the specific immune checkpoint inhibitor selected in actual application. After selecting the immune checkpoint inhibitor, the mass ratio of immune checkpoint inhibitor to polyphenol nanoparticles can be determined experimentally. Typically, the mass ratio of immune checkpoint inhibitor to polyphenol nanoparticles in the drug-loaded injectable hydrogel is (0.1–10):1, preferably (0.2–5):1, and more preferably (0.5–2):1. The concentration of polyphenol nanoparticles in the drug-loaded injectable hydrogel is not less than 2 mg / mL, and the concentration of immune checkpoint inhibitor is not less than 0.5 mg / mL. In specific applications, the concentrations of polyphenol nanoparticles and immune checkpoint inhibitors in the drug-loaded injectable hydrogel can be adjusted according to actual application requirements.

[0015] In the above-mentioned drug-loaded injectable hydrogel technical solution, the modified natural polysaccharide is obtained by grafting a modifying substance onto a natural polysaccharide. Typically, the molecular weight of the natural polysaccharide used as the modification base is 0.1–900 MDa, and the grafting rate of the modifying substance onto the natural polysaccharide is 10%–80%, preferably 20%–60%. Further, the natural polysaccharide can be hyaluronic acid, chitosan, dextran, alginate, etc., and the modifying substance can be dopamine, thiol, or gallic acid.

[0016] The present invention also provides a method for preparing the above-mentioned drug-loaded injectable hydrogel for tumor immunotherapy, the method comprising the following steps:

[0017] The modified natural polysaccharide is dissolved in a pharmaceutically acceptable aqueous solvent, and then polyphenol nanoparticles and immune checkpoint inhibitors are added and fully dispersed to obtain a dispersion. Conditions are applied to cause the modified natural polysaccharide in the dispersion to undergo oxidative self-crosslinking, thereby transforming the dispersion into a gel state, which is the final product.

[0018] In the above preparation method, the modified natural polysaccharides in the dispersion can undergo oxidative self-crosslinking by adding an oxidant and / or adjusting the pH value of the dispersion.

[0019] This invention demonstrates through cell experiments that polyphenol nanoparticles can inhibit the proliferation, migration, and invasion of B16F10 cells, and exhibit low cytotoxicity against L929 cells. Polyphenol nanoparticles can increase the activity of mitochondrial respiratory chain complex I in B16F10 cells, thereby enhancing mitochondrial activity, increasing ATP production, and promoting ROS generation.

[0020] This invention demonstrates through animal experiments that administering a drug-loaded injectable hydrogel containing polyphenol nanoparticles and the immune checkpoint inhibitor BMS-1 ​​to the tumor wound site in postoperative B16F10 tumor-bearing mice effectively inhibits melanoma recurrence. Transcriptomic results from recurrent tumor tissues show that the drug-loaded injectable hydrogel containing polyphenol nanoparticles upregulated oxidative phosphorylation-related pathways and downregulated MAPK, PI3K-Akt, and HIF-1 signaling pathways. Flow cytometry, immunofluorescence staining, PCR, and Western blotting of tumor tissues confirmed that the drug-loaded injectable hydrogel containing polyphenol nanoparticles and the immune checkpoint inhibitor BMS-1 ​​upregulated the expression of mitochondrial respiratory chain complex I-related genes and proteins and inhibited HIF-1α expression. Furthermore, in both orthotopic tumor models and distant metastasis models, the drug-loaded injectable hydrogel containing polyphenol nanoparticles and the immune checkpoint inhibitor BMS-1 ​​effectively inhibited tumor growth and metastasis and activated anti-tumor immunity.

[0021] Based on the above experimental results, the present invention also provides the application of the above-mentioned drug-loaded injectable hydrogel for tumor immunotherapy in the preparation of drugs for treating solid tumors, including but not limited to melanoma.

[0022] Compared with the prior art, the technical solution provided by the present invention has the following beneficial technical effects:

[0023] 1. This invention provides a drug-loaded injectable hydrogel for tumor immunotherapy, comprising a matrix hydrogel and polyphenol nanoparticles and immune checkpoint inhibitors distributed within the matrix hydrogel. The polyphenol nanoparticles are polyethylene glycol nanoparticles self-assembled from epigallocatechin gallate at one end. The matrix hydrogel is formed by the oxidative self-crosslinking of an aqueous solution of modified natural polysaccharides. Experiments have demonstrated that the polyphenol nanoparticles in this drug-loaded injectable hydrogel can activate mitochondrial function in tumor cells, stimulate oxidative phosphorylation in tumor cells, generate more ROS, and inhibit the expression of HIF-1α in tumor cells, thereby reducing the expression of PD-L1 antibodies in tumor cells and enhancing the sensitivity of tumor cells to immune checkpoint inhibitors. This invention enhances the tumor-suppressive effect through the synergistic effect of polyphenol nanoparticles and immune checkpoint inhibitors. Simultaneously, the polyphenol nanoparticles exhibit low toxicity to normal cells, overcoming the problems of significant toxic side effects and drug resistance associated with combining chemotherapy drugs and immunotherapy drugs in existing technologies.

[0024] 2. The drug-loaded injectable hydrogel for tumor immunotherapy provided by this invention loads polyphenol nanoparticles and immune checkpoint inhibitors into a modified natural polysaccharide hydrogel. The hydrogel has good biocompatibility, variable morphology, and simple administration method. It can be locally injected into the tumor site during use and can be applied to various forms of tumor treatment, such as in situ tumor treatment, postoperative tumor treatment, and treatment of distant metastases, etc., and has the potential for multi-scenario application. Attached Figure Description

[0025] Figure 1 It is EGCG and PEG-EGCG 1 H-NMR spectrum.

[0026] Figure 2 Figure a shows a TEM image of PE-NPs and a magnified view of a portion of the TEM image. Figure 2 Figure b is the particle size distribution of PE-NPs. Figure 2 Figure c shows the surface potential of PE-NPs.

[0027] Figure 3 These are the results of cytotoxicity tests on B16F10 and L929 cells using free EGCG and PE-NPs.

[0028] Figure 4 Figures a and b show the results of the scratch distance test, respectively, of the inhibition of lateral migration of B16F10 cells by free EGCG and PE-NPs.

[0029] Figure 5 Figure a is a schematic diagram of the test process in Example 5. Figure 5 Figure b shows the results of a cell longitudinal migration rate assay demonstrating the inhibition of B16F10 cell longitudinal migration by free EGCG and PE-NPs. Figure 5 Images c1-c3 show the inhibition of longitudinal migration of B16F10 cells in the control group, EGCG experimental group, and PE-NPs experimental group.

[0030] Figure 6 Yes, Figure a is a schematic diagram of the test process in Example 6. Figure 6 Figure b shows the cell invasion rate assay results of free EGCG and PE-NPs inhibiting B16F10 cell invasion. Figure 6 Images c1-c3 show the inhibition of B16F10 cell invasion in the control group, EGCG experimental group, and PE-NPs experimental group.

[0031] Figure 7 Figures a and b show the intracellular mitochondrial staining and semi-quantitative results after PE-NPs were applied to B16F10 cells.

[0032] Figure 8 This diagram illustrates the effects of PE-NPs on mitochondrial respiratory chain complexes in tumor cells. Figure a shows the effect of coenzyme I (NAD). + The results of the NDH content test are shown in Figure b, the results of the succinate dehydrogenase (SDH) activity test are shown in Figure c, the results of the coenzyme Q-cytochrome c reductase activity test are shown in Figure d, and the results of the adenosine triphosphate (ATP) activity test are shown in Figure d.

[0033] Figure 9 The results show the test results of free EGCG and PE-NPs inhibiting the expression of PD-L1 on the surface of B16F10 cells. Figure a is the flow cytometry detection graph, and Figure b is the statistical graph of the average fluorescence intensity.

[0034] Figure 10 It includes HA, Dopa standard, OHA, and HA-Dopa. 1 H-NMR spectrum.

[0035] Figure 11 Figure a shows the entries that are significantly upregulated by KEGG. Figure 11 Figure b is a heatmap of differentially expressed genes in the KEGG upregulated category. Figure 11 Figure c is a heatmap of differentially expressed genes in the oxidative phosphorylation pathway. Figure 11 The d-plot is a bubble chart showing significant differences in the GO pathway. Figure 11 The diagram shown is a string diagram of differentially expressed genes in the GO pathway. Figure 11 The f-plot shows the items that were significantly downregulated by KEGG. Figure 11The g-plot is a heatmap of significantly differentially expressed genes in the MAPK, PI3K-AKT, and HIF signaling pathways.

[0036] Figure 12 Figure a shows photographs of tumors in mice from each experimental and control group. Figure 12 Figure b shows the tumor volume of mice in each experimental group and the control group. Figure 12 Image c shows the immunofluorescence staining image of CD31 and Ki67 in recurrent tumor tissue. Figure 12 Figure d shows the gene expression level of Ndufa4 in recurrent tumor tissue detected by PCR. Figure 12 Image e is an immunofluorescence staining image of Ndufa4 in recurrent tumor tissue. Figure 12 Figure f shows the results of Western blot analysis of Ndufa4 protein expression levels in recurrent tumor tissues. Figure 12 The g-plot shows the results of PCR detection of HIF gene expression levels in recurrent tumor tissue. Figure 12 Image h is an immunofluorescence staining image of HIF-1α in recurrent tumor tissue. Figure 12 Figure i shows the results of Western blot analysis of HIF protein expression levels in recurrent tumor tissue.

[0037] Figure 13 Figure a shows the curve of tumor volume change over time. Figure 13 Image b shows the tumor weight. Figure 13 Image C is a photograph of the tumor. Figure 13 Image d is an immunofluorescence staining image of CD4 and CD8 proteins in recurrent tumor tissue.

[0038] Figure 14 Figure a is a flow cytometry plot showing the proportion of CD8+ T cell infiltration as detected by flow cytometry. Figure 14 Figure b is a statistical graph showing the proportion of CD8+ T cell infiltration as detected by flow cytometry. Figure 14 Figure c is a flow cytometry plot showing the proportion of Treg cell infiltration as detected by flow cytometry. Figure 14 The d-plot is a statistical graph showing the proportion of Treg cell infiltration detected by flow cytometry.

[0039] Figure 15 Figure a is a flow cytometry plot of DC cell maturation as detected by flow cytometry. Figure 15 Figure b is a statistical graph of DC cell maturation detected by flow cytometry. Figure 15 Figure c is a flow cytometry plot showing the proportion of memory T cells as detected by flow cytometry. Figure 15 The d-plot is a statistical graph showing the proportion of central memory T cells as detected by flow cytometry. Figure 15 The figure shown in Figure e is a statistical graph of the proportion of effector memory T cells detected by flow cytometry.

[0040] Figure 16 Figure a shows the volume of the contralateral tumor. Figure 16 Figure b shows the quality of the contralateral tumor. Figure 16 Figure c shows the tumor inhibition rate of the contralateral tumor. Figure 16 Image d is an image of the tumor on the opposite side. Figure 16 Figure e shows the immunofluorescence staining of CD8 protein in the contralateral tumor tissue. Figure 16 Figure f is a flow cytometry plot showing the proportion of CD8+ T cell infiltration in the contralateral tumor as detected by flow cytometry. Figure 16 The g-plot is a statistical graph showing the proportion of CD8+ T cell infiltration in the contralateral tumor as detected by flow cytometry. Figure 16 The h-figure is an immunofluorescence staining image of CD4 and Foxp3 proteins in the contralateral tumor tissue. Figure 16 Figure i is a flow cytometry plot showing the proportion of Treg cell infiltration in the contralateral tumor as detected by flow cytometry. Figure 16 Figure j is a statistical graph showing the proportion of Treg cell infiltration in the contralateral tumor as detected by flow cytometry. Detailed Implementation

[0041] The following examples further illustrate the drug-loaded hydrogel for tumor immunotherapy provided by the present invention, its preparation method, and its application. Specifically, the examples use dopamine-modified hyaluronic acid hydrogel as the matrix hydrogel to illustrate the antitumor effect of the drug-loaded hydrogel for tumor immunotherapy. However, the present invention is not limited to the use of polyphenol nanoparticles or immune checkpoint inhibitors loaded onto dopamine-modified hyaluronic acid hydrogels. It should be noted that the following examples are only for further illustration of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the invention are still within the scope of protection of the present invention.

[0042] Example 1

[0043] In this embodiment, polyphenols are prepared via Michael addition reaction, specifically polyethylene glycol (PEG-EGCG) with one end linked to epigallocatechin gallate. The synthetic route and specific steps are as follows:

[0044]

[0045] Epigallocatechin gallate (EGCG) was dissolved in a mixed solvent of phosphate buffer (PBS) and anhydrous dimethyl sulfoxide (DMSO) (PBS to DMSO volume ratio 1:1) to obtain an EGCG solution with a concentration of 0.94 mg / mL; polyethylene glycol (PEG-SH, average relative molecular weight of 5000 g / mol) with one end modified with a thiol group was dissolved in PBS buffer to obtain a PEG-SH solution with a concentration of 5 mg / mL.

[0046] Equal volumes of EGCG solution and PEG-SH solution were mixed and stirred at room temperature for 7 hours. Then, glacial acetic acid was added to adjust the pH to 4.0, and the reaction was terminated. The resulting reaction solution was transferred to a dialysis bag (MWCO = 2000) and dialyzed in deionized water for 3 days to remove excess unreacted EGCG. Finally, it was freeze-dried to obtain PEG-EGCG.

[0047] Weigh 5 mg each of EGCG standard and freeze-dried PEG-EGCG, dissolve them separately in 0.5 mL of heavy water, and transfer them to NMR tubes using a pipette to determine the 1H NMR spectrum of the samples. 1 H-NMR), the results are as follows Figure 1 As shown, based on the comparative analysis of the characteristic peaks in the spectrum, it can be seen that PEG-EGCG was successfully prepared in this embodiment.

[0048] Example 2

[0049] In this embodiment, polyphenol nanoparticles (PE-NPs) were prepared using PEG-EGCG prepared in Example 1 via a thin-film hydration method, as follows:

[0050] PEG-EGCG was dissolved in chloroform to obtain a PEG-EGCG solution with a concentration of 4 mg / mL. The solution was transferred to a rotary evaporator and evaporated under reduced pressure to remove the chloroform solvent, resulting in a homogeneous thin film at the bottom of the flask. Deionized water was added to the flask in the same amount as the chloroform used to dissolve the PEG-EGCG. The mixture was incubated at 45°C for 24 hours under nitrogen atmosphere. Finally, the solution was filtered through a 0.45 μm aqueous filter to obtain an aqueous solution of PE-NPs. PE-NPs can be obtained by freeze-drying this aqueous solution, or the PE-NPs aqueous solution can be used directly.

[0051] A certain amount of PE-NPs aqueous solution was diluted 10 times with deionized water and added to a particle size analyzer. The particle size and polydispersity index (PDI) of the PE-NPs were determined using a Malvern dynamic light scattering particle size analyzer (DLS). A certain amount of PE-NPs aqueous solution was diluted 10 times with deionized water and placed in a potential cell. Its zeta potential was detected using DLS. Each sample was measured in triplicate, and the measurement temperature was 25℃. The morphology of the PE-NPs was observed using a transmission electron microscope. The test results are as follows. Figure 2 As shown, Figure 2 Figure a shows a TEM image of PE-NPs and a magnified view of a portion of the TEM image. Figure 2 Figure b is the particle size distribution of PE-NPs. Figure 2 Figure c shows the surface potential of PE-NPs. Figure 2 The DLS particle size analysis showed that the particle size of PE-NPs was 135±1.98nm, and the PDI was less than 0.3, indicating that the particle size distribution of PE-NPs was relatively uniform. The surface zeta potential was about -16mV. The TEM image showed that PE-NPs were spherical and had a hydration shell. The TEM image showed that the particle size of PE-NPs was about 120nm, which was close to the DLS measurement results.

[0052] Example 3

[0053] In this embodiment, the cytotoxicity of PE-NPs prepared in Example 2 on B16F10 cells and L929 cells was tested.

[0054] Collect B16F10 and L929 cells in logarithmic growth phase at a concentration of 5 × 10⁻⁶ cells / year. 3 Cells were seeded per well in 96-well plates. After cell attachment, the original culture medium was discarded, and 100 μL of culture medium containing different concentrations (10, 20, 30, 40, 50, and 60 μg / mL) of free EGCG or PE-NPs was added. The plates were incubated for 48 h. The plates were then removed, and the cells were washed with PBS buffer. The viability of B16F10 and L929 cells was then determined using a CCK-8 assay kit. Specifically, after removing the original culture medium, CCK-8 dilution buffer (10-fold dilution) was added, and the plates were incubated in the dark for 2–4 h. The absorbance of the reaction solution at 450 nm was measured using a microplate reader. Wells containing 100 μL of blank culture medium but without cells were designated as blank wells; wells containing cells and blank culture medium were designated as control wells; and wells containing cells and culture medium containing EGCG or PE-NPs were designated as experimental wells. Cell viability was calculated using the following formula:

[0055] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%

[0056] In the above formula, Ab is the OD value of the blank well, Ac is the OD value of the control well, and As is the OD value of the experimental well.

[0057] The results of the cytotoxicity tests of different concentrations of free EGCG and PE-NPs polyphenol nanoparticles on L929 cells and B16F10 cells are as follows: Figure 3 As shown, Figure 3 In this context, "Control" represents the reference hole, which is... Figure 3 It is known that the cytotoxicity of PE-NPs is concentration-dependent. For in vitro cell experiments, PE-NPs have good biosafety when the concentration is ≤60μg / mL, while free EGCG shows stronger cytotoxicity.

[0058] Example 4

[0059] In this embodiment, the effect of PE-NPs prepared in Example 2 on the lateral migration of B16F10 cells was investigated.

[0060] The scratch assay was used to evaluate the lateral migration ability of B16F10 cells. B16F10 cells in logarithmic growth phase were collected and subjected to a scratch assay at a concentration of 2 × 10⁶ cells / cells. 5 Cells were seeded per well in 24-well plates and incubated overnight. When the cells reached 90% confluence, a uniform vertical line was drawn down the center of each well using a 200 μL pipette tip. The cells were then washed with room temperature PBS buffer, and the plates were gently shaken to remove any loose cells. Three experimental groups were set up: a free EGCG group, a PE-NPs group, and a control group. The free EGCG group received 0.5 mL of 10 μg / mL free EGCG culture medium (0.1% serum), the PE-NPs group received 0.5 mL of 10 μg / mL PE-NPs culture medium (0.1% serum), and the control group received 0.5 mL of blank culture medium (0.1% serum) without EGCG and PE-NPs. Images were taken at different time points (0, 5, 9, and 12 h) using an inverted microscope under bright field. The line width at each time point was quantified using ImageJ software, and the scratch distance was calculated using the following formula:

[0061] Scratch distance (%) = W t / W0×100%

[0062] In the above formula, W t W represents the width of the scratch at time t, and W0 represents the width of the scratch at time 0 (the original scratch width).

[0063] Images and scratch distance test results of B16F10 cell lateral migration inhibition by free EGCG and PE-NPs are shown below. Figure 4 As shown. By Figure 4 It was observed that with increasing incubation time, only a small amount of cell migration occurred in the PE-NPs and free EGCG experimental groups, while the cell migration distance in the control group significantly increased and was essentially in a rapid healing state at 12 h. At 12 h, the scratch distance in the control group was only 52.6% ± 12.7% of the distance at 0 h, the scratch distance in the free EGCG experimental group was 91.7% ± 1.5% of the distance at 0 h, and the scratch distance in the PE-NPs experimental group was 81.4% ± 5.8% of the distance at 0 h. Therefore, both the PE-NPs and free EGCG experimental groups significantly inhibited lateral cell migration.

[0064] Example 5

[0065] In this embodiment, the effect of PE-NPs prepared in Example 2 on the longitudinal migration of B16F10 cells was investigated.

[0066] The Transwell migration assay was used to evaluate the longitudinal migration ability of B16F10 cells. A schematic diagram of the assay process is shown below. Figure 5 As shown in Figure a. B16F10 cells in logarithmic growth phase were collected at a concentration of 3 × 10⁻⁶ cells / cells. 5 Cells were seeded per well in 6-well plates. Three experimental groups were set up: a free EGCG group, a PE-NPs group, and a control group. In the free EGCG group, after cell adhesion, the original culture medium was replaced with medium containing 10 μg / mL free EGCG (0.1% serum). In the PE-NPs group, after cell adhesion, the original culture medium was replaced with medium containing 10 μg / mL PE-NPs (0.1% serum). In the control group, after cell adhesion, the original culture medium was replaced with blank medium (0.1% serum) without EGCG and PE-NPs. After incubation for 24 hours, cells were collected and resuspended in fresh culture medium (0.1% serum) at a concentration of 3 × 10⁶ cells / well. 5 Cells were added at a density of 100 μL / mL to Transwell chambers, followed by 500 μL of complete culture medium (10% serum) for induction. After 24 h of incubation, cells below the Transwell chamber membrane were washed twice with PBS, and unmigrated cells were removed from the chamber membrane with cotton swabs. Cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet solution for 20 min. After washing twice with PBS buffer, cells that had migrated to the lower side of the Transwell chamber were photographed under a bright field microscope. After photographing, cells were destained with 33% acetic acid solution for 1 h. The 33% acetic acid solution was used as a blank control. The absorbance of each destained solution at 570 nm was measured using a microplate reader, and the cell migration rate was calculated using the following formula:

[0067] Cell longitudinal migration rate (%) = (A Experiment –A Blank ) / (A Control –A Blank )×100%

[0068] Among them, A Experiment A represents the absorbance value of the experimental group. Control A represents the absorbance value of the control group. Blank This represents the absorbance value of the blank group.

[0069] Figure 5 Figure b shows the results of a cell longitudinal migration rate assay that showed the inhibition of B16F10 cell longitudinal migration by free EGCG and PE-NPs. Figure 5 Images c1-c3 show the results of images (i.e., images of cells that have migrated to the lower side of the Transwell chamber) showing the inhibition of B16F10 cell longitudinal migration in the control group, EGCG experimental group, and PE-NPs experimental group. Figure 5 It was observed that only a small number of cells migrated to the lower side of the Transwell chamber in both the free EGCG and PE-NPs experimental groups, while the control group showed a significantly increased number of cells migrating to the lower side of the chamber, with cells interconnected and spreading out in sheets. Regarding longitudinal cell migration rates, the migration rate in the free EGCG experimental group was 43.2% ± 3.1%, while the migration rate in the PE-NPs experimental group was 33.8% ± 5.9%. Both PE-NPs and free EGCG significantly inhibited longitudinal cell migration, with PE-NPs showing a superior inhibitory effect compared to free EGCG.

[0070] Example 6

[0071] In this embodiment, the effect of PE-NPs prepared in Example 2 on the invasion of B16F10 cells was investigated.

[0072] The Transwell invasion assay was used to assess the invasive ability of B16F10 cells. A schematic diagram of the assay procedure is shown below. Figure 6As shown in Figure a. First, a layer of Matrigel was laid inside the Transwell chamber: the Matrigel was thawed overnight in a 4°C freezer, and the well plates, pipette tips, and serum-free culture medium used in the experiment were pre-cooled at 4°C for 2 hours. The Matrigel was diluted 5 times with serum-free culture medium, and 60 μL was evenly added to the inside of the Transwell chamber. Then, the well plates were carefully transferred to an incubator and incubated for 1–2 hours, until the Matrigel changed from a fluid state to a gel state. Three experimental groups were set up: a free EGCG group, a PE-NPs group, and a control group. In the free EGCG group, after cell adhesion, the original culture medium was replaced with medium containing 10 μg / mL free EGCG (0.1% serum). In the PE-NPs group, after cell adhesion, the original culture medium was replaced with medium containing 10 μg / mL PE-NPs (0.1% serum). In the control group, after cell adhesion, the original culture medium was replaced with blank medium (0.1% serum) without EGCG and PE-NPs. After incubation for 24 hours, cells from each group were collected and resuspended in fresh culture medium (0.1% serum) at a concentration of 3 × 10⁶ cells / mL. 5 Cells were added at a density of 100 μL / mL to Transwell chambers, followed by 500 μL of complete culture medium (10% serum) for induction. After 24 h of incubation, cells below the Transwell chamber membrane were washed twice with PBS, and unmigrated cells were removed from the chamber membrane with cotton swabs. Cells were fixed with 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet solution for 20 min. After washing twice with PBS buffer, cells invading the lower side of the Transwell chamber were photographed under a bright field microscope. After photographing, cells were destained with 33% acetic acid solution for 1 h. The 33% acetic acid solution was used as a blank control. The absorbance of each destained solution at 570 nm was measured using a microplate reader, and the cell invasion rate was calculated using the following formula:

[0073] Cell invasion rate (%) = (A Experiment –A Blank ) / (A Control –A Blank )×100%

[0074] Among them, A Experiment A represents the absorbance value of the experimental group. Control A represents the absorbance value of the control group. Blank This represents the absorbance value of the blank group.

[0075] Figure 6Figure b shows the cell invasion rate assay results of free EGCG and PE-NPs inhibiting B16F10 cell invasion. Figure 6 Images c1-c3 show the results of photographs taken of cells that have invaded the lower part of the Transwell chamber, representing the control group, EGCG experimental group, and PE-NPs experimental group, respectively, inhibiting B16F10 cell invasion. Figure 6 It was observed that only a small number of cells invaded the lower side of the Transwell chamber in both the PE-NPs and free EGCG experimental groups, while a significantly larger number of cells migrated to the lower side of the Transwell chamber in the control group. Regarding cell invasion rate data, the invasion rate of the free EGCG experimental group was 42.3% ± 4.8%, while that of the PE-NPs experimental group was 32.7% ± 3.6%, indicating that PE-NPs had a better inhibitory effect on cell invasion.

[0076] Example 7

[0077] In this embodiment, the effect of PE-NPs prepared in Example 2 on mitochondrial activity in B16F10 cells was investigated.

[0078] B16F10 cells in the logarithmic growth phase were collected at a concentration of 2.5 × 10⁻⁶. 4 Seeds were inoculated at a density of 100 PE-NPs / mL / well in confocal culture dishes and incubated overnight. The original culture medium was then discarded. The experimental group consisted of culture medium containing sterilized PE-NPs (PE-NPs concentration of 60 μg / mL). The control group consisted of blank culture medium without PE-NPs. After 48 hours of further incubation, the original culture medium was discarded, and the samples were washed twice with PBS buffer. 200 nmol / L preheated mitochondrial red fluorescent probe (PE-NPs) at 37°C was then added. Red CMXRos staining was performed for 30 min, followed by incubation with 1 mg / mL Hochest dye for 15 min. After staining, the staining solution was replaced with fresh PBS buffer, and the sample was observed and photographed under a confocal laser scanning microscope (CLSM). Mitochondrial fluorescent probes (Ex / Em = 579 / 599 nm) were used to label mitochondria, exhibiting red fluorescence under CLSM; Hochest (Ex / Em = 346 / 460 nm) was used to label the cell nucleus, exhibiting blue fluorescence under CLSM.

[0079] The fluorescence intensity of the experimental and control samples was statistically analyzed using ImageJ software to compare mitochondrial activity in each group. Three replicates were tested for each group. The test results are as follows: Figure 7 As shown, Figure 7 Image a is a CLSM image, and image b is the average fluorescence intensity of red fluorescence. Figure 7It can be seen that after PE-NPs treatment, there are more mitochondria distributed in the cells, and the average fluorescence intensity of red fluorescence is also significantly increased, that is, the cells treated with PE-NPs have stronger mitochondrial fluorescence intensity. The experimental results of this example show that PE-NPs can enhance the activity of mitochondria in B16F10 cells.

[0080] Example 8

[0081] In this embodiment, the effect of PE-NPs prepared in Example 2 on the activity of mitochondrial respiratory complex in B16F10 cells was investigated.

[0082] Following the instructions of the kits (Solepro), the activities of mitochondrial respiratory complexes in B16F10 cells were detected and analyzed using the Coenzyme I NAD(H) content assay kit, the Succinate Dehydrogenase (SDH) activity assay kit, the Mitochondrial Respiratory Chain Complex III activity assay kit, and the Mitochondrial Respiratory Chain Complex V activity assay kit. The experimental procedure is illustrated below using the Coenzyme I NAD(H) content assay kit as an example:

[0083] B16F10 cells in the logarithmic growth phase were collected at a concentration of 1.8 × 10⁻⁶. 6 Cells were seeded at a density of cells / dish in culture dishes. A sterilized culture medium containing PE-NPs (PE-NPs concentration 60 μg / mL) was added as the experimental group, while a blank culture medium without PE-NPs was added as the control group. Cells were incubated for 48 h, washed twice with PBS buffer, digested, and 5 million cells were collected into 2 mL EP tubes. 500 μL of acid / alkaline extraction buffer was added, and the cells were sonicated (200 W, 2 s sonication, 1 s pause). The cells were then boiled in boiling water for 5 min, cooled on ice, and centrifuged (4℃, 10000g) for 10 min. 200 μL of the supernatant was collected, and an equal volume of alkaline / acidic extraction buffer was added to neutralize it. The mixture was quickly mixed with a pipette and centrifuged again (4℃, 10000g) for 10 min. The supernatant was then stored on ice for later use. According to the test instructions, add the sample and test reagents one by one in sequence, mix them evenly with a pipette, and then use an ELISA reader to detect the absorbance value of the reaction solution at a wavelength of 570nm. Calculate the corresponding NAD+ / NADH content based on the concentration of the standard.

[0084] The test results of this embodiment are as follows: Figure 8 As shown, figure a represents coenzyme I (NAD). + The results of the NDH (non-reductase) content test are shown in Figure b, the activity test results of succinate dehydrogenase (SDH) are shown in Figure c, the activity test results of coenzyme Q-cytochrome c reductase are shown in Figure d, and the activity test results of adenosine triphosphate (ATP) are shown in Figure d. Figure 8It can be seen that PE-NPs significantly increased the activity of respiratory chain complex I (NAD). + The PE-NPs act on mitochondrial respiratory chain complex I after entering B16F10 cells, accelerating the transfer of electrons from NADH to oxygen, while having no significant effect on the activity of other enzymes.

[0085] Example 9

[0086] In this embodiment, the effect of PE-NPs prepared in Example 2 on the expression of PD-L1 on the surface of B16F10 cells was investigated.

[0087] B16F10 cells in the logarithmic growth phase were collected at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 12-well plates and incubated overnight until adherence. Three experimental groups were set up: a free EGCG group, a PE-NPs group, and a control group. In the free EGCG group, after cell adherence, the original culture medium was replaced with medium containing 60 μg / mL free EGCG. In the PE-NPs group, after cell adherence, the original culture medium was replaced with medium containing 60 μg / mL PE-NPs. In the control group, after cell adherence, the original culture medium was replaced with blank medium without EGCG and PE-NPs. After incubation for 24 hours, cells were collected into flow cytometry tubes, washed twice with PBS buffer, and then incubated for 30 minutes in the dark with anti-mouse CD274-PE antibody (0.5 μg / tube). After washing twice with PBS buffer, the cells were resuspended in PBS buffer for flow cytometry analysis. All flow cytometry experiments involved in this invention were performed using a high-sensitivity flow cytometer (FACSAriaⅢ, BD|SENSORS, Germany), and the flow cytometry data were analyzed using Flowjo software.

[0088] Figure 9 The results show the assay results of free EGCG and PE-NPs inhibiting PD-L1 expression on the surface of B16F10 cells. Figure a shows the flow cytometry results, and figure b shows the statistical graph of the average fluorescence intensity. Figure 9 It can be seen that, compared with the control group and the free EGCG experimental group, the PE-NPs experimental group significantly reduced the expression of PD-L1 antibody on the surface of B16F10 cells, that is, PE-NPs can inhibit the expression of PD-L1 antibody on the surface of B16F10 cells.

[0089] Example 10

[0090] In this embodiment, dopamine-modified hyaluronic acid (HA-Dopa) was synthesized via the Schiff base reaction. The synthetic route and steps are as follows:

[0091]

[0092] (1) Dissolve hyaluronic acid (HA, Mw = 1000 kDa) in deionized water to obtain an 8 mg / mL HA solution. Slowly add sodium periodate solution to the HA solution. The amount of sodium periodate solution added should meet the mass ratio of sodium periodate to HA of 1.07:1. React at room temperature in the dark for 2 h. Then add ethylene glycol and continue stirring for 2 h to remove unreacted sodium periodate. Dialyze the resulting reaction solution in ultrapure water for 3 days and freeze dry to obtain oxidized hyaluronic acid (OHA).

[0093] (2) Dissolve OHA in PBS buffer with pH 5 to form a 10 mg / mL OHA solution. Add an equimolar amount of dopamine hydrochloride (Dopa) to the OHA solution and react at room temperature for 10 h. Dialyze the resulting reaction solution in pure water for 2 days and freeze dry to obtain HA-Dopa.

[0094] Weigh 5 mg each of HA, Dopa standards, OHA, and HA-Dopa, add 0.5 mL of heavy water to each, dissolve by vortexing, and then transfer to NMR tubes for analysis. 1 The results of the 1H NMR spectroscopy were as follows: Figure 10 As shown, by Figure 10 It can be seen that the peak at 7 ppm of HA-Dopa is the characteristic peak of the aromatic protons of catechol, and the peaks at 3.1 and 2.8 ppm are the characteristic peaks of the methylene protons of catechol, proving that the dopamine group was successfully grafted onto HA. According to the degree of substitution (DS) = 3 × A 2.8 / (2×A 1.9 To calculate the grafting rate of dopamine in HA-Dopa, A 2.8 and A 1.9 The integrals of the peak areas at chemical shifts of 2.8 ppm (catechol methylene) and 1.9 ppm (HA methyl) indicate that the grafting rate of dopamine in the HA-Dopa prepared in this example is approximately 24%.

[0095] Example 11

[0096] In this embodiment, a drug-loaded hydrogel containing PD-1 / PD-L1 inhibitors and PE-NPs (PE-NPs@PD / HD) was prepared.

[0097] The HA-Dopa prepared in Example 10 was dissolved in pure water to obtain a 60 mg / mL HA-Dopa solution. The PD-1 / PD-L1 inhibitor BMS-1 ​​and the PE-NPs prepared in Example 2 were added to the HA-Dopa and dispersed thoroughly. Then, an equal volume of NaOH solution containing sodium periodate (pH = 8.5, with a sodium periodate concentration of 2.825 mg / mL) was added to the HA-Dopa solution and mixed thoroughly to obtain a gel precursor solution. 50 μL of the gel precursor solution was injected into a self-made silicone mold (diameter 8 mm, height 2 mm) and allowed to stand until the HA-Dopa crosslinked and the gel precursor solution was converted into a gel state. The mold was then removed to obtain PE-NPs@PD / HD.

[0098] Each PE-NPs@PD / HD has a volume of 50 μL, a cross-linked HA-Dopa concentration of 30 mg / mL, and contains 50 μg of BMS-1 ​​and 100 μg of PE-NPs. That is, the concentration of BMS-1 ​​in PE-NPs@PD / HD is 1 mg / mL and the concentration of PE-NPs is 2 mg / mL.

[0099] Comparative Example 1

[0100] In this comparative example, a blank hydrogel (HD) was prepared.

[0101] The HA-Dopa prepared in Example 10 was dissolved in pure water to obtain a 60 mg / mL HA-Dopa solution. Then, an equal volume of NaOH solution containing sodium periodate (pH = 8.5, with a sodium periodate concentration of 2.825 mg / mL) was added and mixed thoroughly to obtain a gel precursor solution. 50 μL of the gel precursor solution was injected into a self-made silicone mold (8 mm in diameter and 2 mm in height). The mold was allowed to stand until the HA-Dopa crosslinked and transformed the gel precursor solution into a gel state. The mold was then removed to obtain HD.

[0102] Each HD block has a volume of 50 μL, and the concentration of cross-linked HA-Dopa in the HD is 30 mg / mL.

[0103] Comparative Example 2

[0104] In this comparative example, a drug-loaded hydrogel containing PE-NPs (PE-NPs / HD) was prepared.

[0105] The HA-Dopa prepared in Example 10 was dissolved in pure water to obtain a 60 mg / mL HA-Dopa solution. The PE-NPs prepared in Example 2 were added to the HA-Dopa and dispersed thoroughly. Then, an equal volume of NaOH solution containing sodium periodate (pH = 8.5, with a sodium periodate concentration of 2.825 mg / mL) was added to the HA-Dopa solution and mixed thoroughly to obtain a gel precursor solution. 50 μL of the gel precursor solution was injected into a self-made silicone mold (diameter 8 mm, height 2 mm) and allowed to stand until the HA-Dopa crosslinked and transformed the gel precursor solution into a gel state. The mold was then demolded to obtain PE-NPs / HD.

[0106] Each PE-NPs / HD has a volume of 50 μL, the concentration of cross-linked HA-Dopa in PE-NPs / HD is 30 mg / mL, and each PE-NPs / HD contains 100 μg of PE-NPs, which means the concentration of PE-NPs in PE-NPs / HD is 2 mg / mL.

[0107] Comparative Example 3

[0108] In this comparative example, a drug-loaded hydrogel (PD / HD) containing a PD-1 / PD-L1 inhibitor was prepared.

[0109] The HA-Dopa prepared in Example 10 was dissolved in pure water to obtain a 60 mg / mL HA-Dopa solution. The PD-1 / PD-L1 inhibitor BMS-1 ​​was added to the HA-Dopa and fully dispersed and dissolved. Then, an equal volume of NaOH solution containing sodium periodate (pH = 8.5, with a sodium periodate concentration of 2.825 mg / mL) was added to the HA-Dopa solution and mixed thoroughly to obtain a gel precursor solution. 50 μL of the gel precursor solution was injected into a self-made silicone mold (diameter 8 mm, height 2 mm) and allowed to stand until the HA-Dopa crosslinked and transformed the gel precursor solution into a gel state. The mold was then demolded to obtain PD / HD.

[0110] Each PD / HD has a volume of 50 μL, the concentration of cross-linked HA-Dopa in the PD / HD is 30 mg / mL, and each PD / HD contains 150 μg of BMS-1, which means the concentration of BMS-1 ​​in the PD / HD is 1 mg / mL.

[0111] Example 12

[0112] In this embodiment, the intrinsic mechanism by which drug-loaded hydrogels containing PE-NPs (PE-NPs / HD) inhibit postoperative tumor recurrence was investigated.

[0113] B16F10 cells in the logarithmic growth phase were collected and subcutaneously inoculated into the right ventral dorsal region of each mouse (inoculation dose: 1 × 10⁻⁶ cells). 6(1 cell / mouse). The health status and melanoma size of the mice were observed every other day. The tumor volume reached 80 mm after 7 days of inoculation. 3 At this point, subsequent surgical resection experiments can be performed. The connecting point is gently cut along the tumor edge, and the tumor is carefully removed, leaving a residual tumor approximately 2 mm wide. The PE-NPs / HD prepared in Comparative Example 2 is sterilized, and the sterilized PE-NPs / HD is applied to the residual tumor, and the wound is sutured. This is the experimental group. The case where the wound is directly sutured is the control group. Finally, a layer of Tegaderm Film (3M) breathable dressing is applied to the surgical site to prevent suture dislodgement after surgery. After surgery, once the mice have recovered and are in a normal active state, they are returned to their cages and continued to be housed in an SPF-grade environment.

[0114] Reference transcriptome sequencing analysis was performed on the recurrent tumor on postoperative day 14, and the results are as follows: Figure 11 As shown, Figure 11 Figure a shows the entries that are significantly upregulated by KEGG. Figure 11 Figure b is a heatmap of differentially expressed genes in the KEGG upregulated category. Figure 11 Figure c is a heatmap of differentially expressed genes in the oxidative phosphorylation pathway. Figure 11 The d-plot is a bubble chart showing significant differences in the GO pathway. Figure 11 The diagram shown is a string diagram of differentially expressed genes in the GO pathway. Figure 11 The f-plot shows the items that were significantly downregulated by KEGG. Figure 11 The g-plot is a heatmap of significantly differentially expressed genes in the MAPK, PI3K-AKT, and HIF signaling pathways. Among the KEGG-upregulated entries, oxidative phosphorylation and thermogenesis pathways are significantly upregulated, such as... Figure 11 Figures a and b show a heatmap analysis of differentially expressed genes in the oxidative phosphorylation pathway. Compared to the control group, PE-NPs / HD significantly upregulated the expression of related genes such as Ndufa4, Cox5a, Atp5g1, Ndufa2, and Uqcr11. Figure 11 Figure c shows that PE-NPs / HD increases the expression of mitochondrial complex subunits, consistent with the results of Example 8. Furthermore, GO analysis revealed that PE-NPs / HD significantly upregulated related biological processes such as electron transport chain, NADH dehydrogenase complex assembly (mitochondrial complex I), mitochondrial respiratory chain complex assembly, mitochondrial protein complex and electron transfer activity, response to reactive oxygen species, and ATP metabolism. Figure 11 As shown in Figures d and e, these are all closely related to mitochondrial activity. Among the KEGG-downregulated pathways, PE-NPs / HD inhibit tyrosine kinase inhibitor resistance, and signaling pathways such as MAPK, HIF-1, ErbB, PI3K-AKT, EGFR, and mTOR are involved. Figure 11As shown in Figures f and g, these pathways are directly or indirectly related to PD-L1 expression, which explains why PE-NPs can downregulate PD-L1 expression on the surface of tumor cells in Example 9, especially the HIF-1 signaling pathway, which is also related to mitochondrial respiration.

[0115] Example 13

[0116] In this embodiment, the ability and mechanism of drug-loaded hydrogel containing PD-1 / PD-L1 inhibitors and PE-NPs (PE-NPs@PD / HD) to inhibit postoperative tumor recurrence were verified.

[0117] B16F10 cells in the logarithmic growth phase were collected and subcutaneously inoculated into the right ventral dorsal region of each mouse (inoculation dose: 1 × 10⁻⁶ cells). 6 (1 cell / mouse). The health status and melanoma size of the mice were observed every other day. The tumor volume reached 80 mm after 7 days of inoculation. 3 At this point, a subsequent surgical resection experiment can be performed. Gently cut along the edge of the tumor at the junction, carefully remove the tumor, leaving a residual tumor approximately 2mm wide.

[0118] HD, PE-NPs / HD, PD / HD, and PE-NPs@PD / HD prepared in Comparative Examples 1-3 and Example 11 were sterilized. The sterilized HD, PE-NPs / HD, PD / HD, and PE-NPs@PD / HD were then applied to the residual tumor sites, and the wounds were sutured. This arrangement served as different experimental groups (HD group, PE-NPs / HD group, PD / HD group, and PE-NPs@PD / HD group). The case where the wound was directly sutured served as the control group (sham control group, abbreviated as Sham or Control). Finally, a layer of Tegaderm Film (3M) breathable dressing was applied to the surgical site to prevent suture dislodgement after surgery. After surgery, once the mice were awake and in a normal active state, they were returned to their cages and continued to be housed in an SPF-grade environment to observe tumor recurrence.

[0119] Recurrent tumors were collected on the 14th day after surgery and analyzed by PCR, Western blotting, and immunofluorescence. The results are as follows: Figure 12 As shown, Figure 12 Figure a shows photographs of tumors in mice from each experimental and control group. Figure 12 Figure b shows the tumor volume of mice in each experimental group and the control group. Figure 12 Image c shows the immunofluorescence staining image of CD31 and Ki67 in recurrent tumor tissue. Figure 12 Figure d shows the gene expression level of Ndufa4 in recurrent tumor tissue detected by PCR. Figure 12Image e is an immunofluorescence staining image of Ndufa4 in recurrent tumor tissue. Figure 12 Figure f shows the results of Western blot analysis of Ndufa4 protein expression levels in recurrent tumor tissues. Figure 12 The g-plot shows the results of PCR detection of HIF gene expression levels in recurrent tumor tissue. Figure 12 Image h is an immunofluorescence staining image of HIF-1α in recurrent tumor tissue. Figure 12 Figure i shows the results of Western blot analysis of HIF protein expression levels in recurrent tumor tissue. Figure 12 It was found that the sham surgery control group had the largest recurrent tumor volume and weight. There was no significant difference in tumor size between the HD group and the Sham group, indicating that the HA-Dopa gel carrier material had no effect on tumor growth. The PE-NPs / HD group, PD / HD group, and PE-NPs@PD / HD group all inhibited the growth of recurrent tumors to varying degrees. Among them, the PE-NPs@PD / HD group had the smallest tumor volume. Figure 12 Figures a-b show that PE-NPs and PD-1 / PD-L1 inhibitors have a synergistic effect in inhibiting tumor growth. In inhibiting tumor growth, the combination of PE-NPs and PD-1 / PD-L1 inhibitors produced a significantly better inhibitory effect compared to using either one alone, and also inhibited tumor angiogenesis and proliferation. Furthermore, as shown in... Figure 12 As shown in Figures d-i, PCR, Western Blot, and immunofluorescence analyses of recurrent tumor tissues showed that the PE-NPs / HD group and the PE-NPs@PD / HD group significantly reduced the expression of Ndufa-4 and HIF-1α genes and proteins in tumor tissues.

[0120] Example 14

[0121] In this embodiment, the immune activation effect of drug-loaded hydrogels containing PD-1 / PD-L1 inhibitors and PE-NPs (PE-NPs@PD / HD) in tumor-bearing mice was investigated.

[0122] B16F10 cells in the logarithmic growth phase were collected and subcutaneously inoculated into the right ventral dorsal region of each mouse (inoculation dose: 1 × 10⁻⁶ cells). 6(Cells / mouse). The health status and melanoma size of mice were observed every other day. Grossly visible tumors formed on day 7 after inoculation. Mice were randomly grouped and numbered. PE-NPs / HD group, PE-NPs@PD / HD group, and control group were set up. The PE-NPs / HD group and PE-NPs@PD / HD group were injected with the gel precursor solution used in Comparative Example 2 and Example 11 to prepare PE-NPs / HD and PE-NPs@PD / HD, respectively. The gel precursor solution would gel at the injection site after injection. The control group was injected with PBS buffer, i.e., 50 μL of the gel precursor solution or PBS buffer used to prepare PE-NPs / HD or PE-NPs@PD / HD was injected next to the tumor each time. A total of 3 injection treatments were performed. The time of the first administration was recorded as Day 0, the second administration was performed 2 days after the first administration (Day 2), and the third administration was performed 4 days after the first administration (Day 4). Tumor volume was recorded in tumor-bearing mice. Eight days after the last administration, the mice were euthanized, and the tumors were removed. A portion was weighed, fixed with paraformaldehyde, embedded in paraffin, and sectioned. Immunohistofluorescence staining was performed on the tumor tissue to label T cells. The results are as follows: Figure 13 As shown.

[0123] Figure 13 Figure a shows the curve of tumor volume change over time. Figure 13 Image b shows the tumor weight. Figure 13 Image C is a photograph of the tumor. Figure 13 Image d is an immunofluorescence staining image of CD4 and CD8 proteins in recurrent tumor tissue. Figure 13 It can be seen that the PE-NPs@PD / HD group has the best tumor suppression effect, and the combination of PE-NPs with PD-1 / PD-L1 inhibitors produced more CD8 T cell infiltration.

[0124] Another portion of the tumor tissue was subjected to immunocytometry analysis. Lymphocytes were extracted from the tumor tissue using a tumor-infiltrating tissue lymphocyte separation kit (Solepro): A 6-well plate was prepared, and 5 mL of whole blood and tissue diluent was added. The tumors of mice were euthanized, crushed using the plunger of a sterile syringe, and passed through a 100 μm sieve into the whole blood and tissue diluent. The single-cell suspension was then slowly added dropwise to a 15 mL centrifuge tube (with 5 mL of separation buffer added beforehand). Due to the density difference between the two, the boundary layer was clearly visible. The tube was centrifuged at 900 g for 30 min. After centrifugation, a clear stratification was observed: from top to bottom, the cells in the centrifuge tube were divided into four layers: a diluent layer, a milky white lymphocyte layer, a clear separation buffer layer, and a red blood cell layer. The middle milky white lymphocyte layer (the second layer) was then carefully aspirated into another clean, sterile 15 mL centrifuge tube, and 10 mL of cell washing buffer was added. The tube was then centrifuged at 250 g for 10 min. The supernatant was then discarded, and the cells were washed twice with 5 mL of PBS solution, centrifuged at 250 g for 10 min each time, and the basal lymphocytes were collected. Analysis was performed using anti-mouse CD3-PE, anti-mouse CD8-percp / cy5.5, anti-mouse CD4-FITC, anti-mouse CD25-bv510, and anti-mouse Foxp3-AF647 antibodies. The cells were transferred to flow cytometry tubes, and 100 μL of anti-mouse Zombie-APC-CY7 antibody dilution buffer (200-fold dilution) was added. The cells were incubated in the dark for 20 min for live / dead cell staining. After washing once with PBS buffer, the cell membranes were stained with anti-mouse CD3-PE, anti-mouse CD8-percp / cy5.5, anti-mouse CD4-FITC, and anti-mouse CD25-bv510 antibodies (0.5 μg / tube each), and incubated in the dark for 30 min. Cells were washed twice with PBS buffer, fixed with 1 mL of fixative for 45 min, and then 1 mL of nuclear membrane lysis buffer was added directly. The cells were centrifuged at 350 g for 6 min, and the supernatant was discarded. Cells were resuspended in 2 mL of nuclear membrane lysis buffer, mixed by pipetting, and centrifuged again (350 g, 6 min). The supernatant was discarded, and the cell nuclei were stained with anti-mouse Foxp3-AF647 antibody (0.5 μg / tube) and incubated in the dark for 40 min. Finally, cells were washed twice with PBS buffer, transferred to flow cytometry tubes, resuspended with a certain volume of PBS buffer, and analyzed using Flowjo software. Experimental results are as follows: Figure 14 As shown.

[0125] Figure 14Figure a is a flow cytometry plot showing the proportion of CD8+ T cell infiltration as detected by flow cytometry. Figure 14 Figure b is a statistical graph showing the proportion of CD8+ T cell infiltration as detected by flow cytometry. Figure 14 Figure c is a flow cytometry plot showing the proportion of Treg cell infiltration as detected by flow cytometry. Figure 14 The d-plot is a statistical graph showing the proportion of Treg cell infiltration detected by flow cytometry. Figure 14 The results showed that the tumor tissue of the PE-NPs@PD / HD group had a higher CD8 content. + The proportion of T cells and an even lower proportion of Treg cells.

[0126] Lymphocytes were extracted from mouse spleen tissue using a spleen lymphocyte isolation kit (Solepro), following the same procedures as for extracting infiltrative lymphocytes from tumor tissue. Subsequently, staining with anti-mouse CD11c-PE, anti-mouse CD86-APC, and anti-mouse CD80-FITC antibodies was used to investigate the maturation of dendritic cells (DCs) in the spleen; staining with anti-mouse CD3-PE, anti-mouse CD8-FITC, anti-mouse CD62L-bv421, and anti-mouse CD44-AF647 antibodies was used to investigate the immune memory effect of T cells in the spleen. The specific staining procedures are as described above. Experimental results are as follows. Figure 15 As shown.

[0127] Figure 15 Figure a is a flow cytometry plot of DC cell maturation as detected by flow cytometry. Figure 15 Figure b is a statistical graph of DC cell maturation detected by flow cytometry. Figure 15 Figure c is a flow cytometry plot showing the proportion of memory T cells as detected by flow cytometry. Figure 15 The d-plot is a statistical graph showing the proportion of central memory T cells as detected by flow cytometry. Figure 15 The figure e is a statistical graph showing the proportion of effector memory T cells as detected by flow cytometry. Figure 15 It is known that PE-NPs@PD / HD group can promote the maturation of DC cells and activate memory T cells, forming a long-lasting tumor immune effect.

[0128] Example 15

[0129] In this embodiment, the effect of drug-loaded hydrogels containing PD-1 / PD-L1 inhibitors and PE-NPs (PE-NPs@PD / HD) on inhibiting distant tumors was investigated.

[0130] Collect B16F10 cells in the logarithmic growth phase and concentrate the cells to a concentration of 1×10⁻⁶. 7B16F10 cells were injected subcutaneously at a concentration of 10 cells / mL and resuspended in sterile PBS buffer. Then, 50 μL of B16F10 cells were injected subcutaneously into the right ventral dorsal region of each mouse. On day 4 post-inoculation, the same dose of B16F10 cells was injected into the left ventral dorsal region of the mice to simulate melanoma metastasis. Grossly visible tumors (approximately 50 mm) formed on day 5 post-inoculation in the right ventral dorsal region of the mice. 3 Mice were randomly grouped and numbered. A PE-NPs / HD group, a PE-NPs@PD / HD group, and a control group were established. The PE-NPs / HD group and the PE-NPs@PD / HD group were injected with gel precursor solutions prepared in Comparative Example 2 and Example 11, respectively. The gel precursor solutions gelled at the injection site after injection. The control group was injected with PBS buffer. Specifically, 50 μL of PE-NPs / HD, PE-NPs@PD / HD, or PBS buffer was injected adjacent to the tumor on the right side of the abdomen and back each time. A total of three injection treatments were performed. The first administration was designated Day 0, the second administration was given two days after the first administration (Day 2), and the third administration was given four days after the first administration (Day 4). The health status of the mice and the growth trend of the left-sided melanoma were observed every other day, and the infiltration of immune cells in the left-sided tumor tissue was detected. The specific steps and operations were the same as in Example 14. The test results of this example are as follows: Figure 16 As shown.

[0131] Figure 16 Figure a shows the volume of the contralateral tumor. Figure 16 Figure b shows the quality of the contralateral tumor. Figure 16 Figure c shows the tumor inhibition rate of the contralateral tumor. Figure 16 Image d is an image of the tumor on the opposite side. Figure 16 Figure e shows the immunofluorescence staining of CD8 protein in the contralateral tumor tissue. Figure 16 Figure f is a flow cytometry plot showing the proportion of CD8+ T cell infiltration in the contralateral tumor as detected by flow cytometry. Figure 16 The g-plot is a statistical graph showing the proportion of CD8+ T cell infiltration in the contralateral tumor as detected by flow cytometry. Figure 16 The h-figure is an immunofluorescence staining image of CD4 and Foxp3 proteins in the contralateral tumor tissue. Figure 16 Figure i is a flow cytometry plot showing the proportion of Treg cell infiltration in the contralateral tumor as detected by flow cytometry. Figure 16 Figure j is a statistical graph showing the proportion of Treg cell infiltration in the contralateral tumor as detected by flow cytometry. Figure 16 It can be seen that both the PE-NPs / HD group and the PE-NPs@PD / HD group can inhibit the generation and development of contralateral tumors and promote CD8 in tumor tissue. +The infiltration of T cells and the reduction of the proportion of Treg cells indicate that PE-NPs@PD / HD can activate long-term anti-tumor immune memory and inhibit distant metastasis of tumors.

Claims

1. A drug-loaded injectable hydrogel for tumor immunotherapy, characterized in that, This drug-loaded injectable hydrogel consists of a matrix hydrogel, polyphenol nanoparticles distributed within the matrix hydrogel, and an immune checkpoint inhibitor. The polyphenol nanoparticles are formed by the self-assembly of polyethylene glycol with one end linked to epigallocatechin gallate. The matrix hydrogel is formed by the oxidative self-crosslinking of an aqueous solution of modified natural polysaccharides. The polyphenol nanoparticles inhibit the expression of hypoxia-inducible factor in tumor cells, thereby inhibiting the expression of PD-L1 protein in tumor cells, thus enhancing the sensitivity of tumor cells to immune checkpoint inhibitors and achieving synergistic tumor suppression. The tumor cells are melanoma cells. The immune checkpoint inhibitor is the PD-1 / PD-L1 inhibitor BMS-1. The structural formula of the polyethylene glycol with one end linked to epigallocatechin gallate is: ， In this drug-loaded injectable hydrogel, the mass ratio of immune checkpoint inhibitor to polyphenol nanoparticles is (0.1~10):1, the concentration of polyphenol nanoparticles is not less than 2 mg / mL, and the concentration of immune checkpoint inhibitor is not less than 0.5 mg / mL. This drug-loaded injectable hydrogel was prepared by the following method: The modified natural polysaccharide is dissolved in a pharmaceutically acceptable aqueous solvent, and then polyphenol nanoparticles and immune checkpoint inhibitors are added and fully dispersed to obtain a dispersion. Conditions are applied to cause the modified natural polysaccharide in the dispersion to undergo oxidative self-crosslinking, thereby transforming the dispersion into a gel state, which is the final product.

2. The drug-loaded injectable hydrogel for tumor immunotherapy according to claim 1, characterized in that, The polyphenol nanoparticles have a particle size of 100~300 nm.

3. The drug-loaded injectable hydrogel for tumor immunotherapy according to claim 1, characterized in that, The modified natural polysaccharides in the dispersion are oxidized and self-crosslinked by adding oxidants and / or adjusting the pH of the dispersion.

4. The use of the drug-loaded injectable hydrogel for tumor immunotherapy as described in any one of claims 1 to 3 in the preparation of a medicament for treating melanoma.

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