Drug-loaded hydrogel for tumor immunotherapy as well as preparation method and application of drug-loaded hydrogel

Abstract

The invention belongs to the field of anti-tumor materials, and provides drug-loaded hydrogel for tumor immunotherapy as well as a preparation method and application of the drug-loaded hydrogel. The drug-loaded injectable hydrogel is composed of matrix hydrogel, polypolyphenol nanoparticles and an immune checkpoint inhibitor, the polypolyphenol nanoparticles and the immune checkpoint inhibitor are distributed in the matrix hydrogel, the polypolyphenol nanoparticles are nanoparticles formed by self-assembly of polyethylene glycol, one end of the polyethylene glycol is connected with epigallocatechin gallate, and the other end of the polyethylene glycol is connected with the immune checkpoint inhibitor. The polypolyphenol nanoparticles inhibit expression of tumor cell hypoxia-inducible factors by inhibiting expression of tumor cell hypoxia-inducible factors and further inhibit expression of tumor cell PD-L1 antibodies, so that the sensitivity degree of tumor cells to immune checkpoint inhibitors is improved. The tumor inhibition effect is enhanced through the synergistic effect of the polypolyphenol nanoparticles and the immune checkpoint inhibitor, and meanwhile, the toxicity of the polypolyphenol nanoparticles to normal cells is relatively low; the problems that in the prior art, chemotherapy drugs and immunotherapy drugs are combined for use, the toxic and side effects of the chemotherapy drugs are large, and drug resistance is generated can be solved.

Description

Technical Field

[0001] The invention belongs to the field of anti-tumor materials and relates to a drug-loaded hydrogel for tumor immunotherapy and a preparation method and application thereof. Background Art

[0002] Cancer is a major public health problem worldwide. According to statistics, there will be nearly 20 million new cancer cases and 9.7 million deaths from cancer in 2022. Currently, the main method for treating cancer clinically is systemic chemotherapy, but chemotherapy drugs have strong toxic side effects, which can damage normal tissues and organs and seriously impair the patient's quality of life. With the development of technology, tumor immunotherapy has made certain breakthroughs and has become an effective treatment method.

[0003] Although some tumors have strong immunogenicity and can be treated with immunotherapy through cytokines, vaccines, adoptive T cells, and immune checkpoint inhibitors, some tumors are still in a state of low immunogenicity. At the same time, the aerobic glycolysis metabolism of tumors will accumulate a large amount of lactic acid inside the tumor tissue, thereby producing an immunosuppressive microenvironment; and in order to escape the attack of the immune system, the surface of tumor cells will overexpress some proteins, such as PD-L1, to inhibit the activity of T cells. Studies have shown that some chemotherapy drugs (such as doxorubicin) can cause immunogenic death of tumor cells, promote the release of tumor-associated antigens and activate anti-tumor immunity. Although chemotherapy drugs can be combined with immunotherapy drugs, the toxic side effects of chemotherapy itself and the drug resistance caused by chemotherapy are still a major problem. Therefore, there is still a need to explore low-toxic immune-activating drugs to improve the immunosuppressive microenvironment of tumor tissues, thereby coordinating immunotherapy methods and improving the effectiveness of tumor treatment. Summary of the invention

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

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0006] The invention discloses a drug-loaded injectable hydrogel for tumor immunotherapy, which is composed of a matrix hydrogel and polyphenol nanoparticles and immune checkpoint inhibitors distributed in the matrix hydrogel; the polyphenol nanoparticles are nanoparticles formed by self-assembly of polyethylene glycol with one end connected to epigallocatechin gallate; the matrix hydrogel is formed by 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 antibody in tumor cells, thereby enhancing the sensitivity of tumor cells to immune checkpoint inhibitors and achieving synergistic inhibition of tumors.

[0007] In the technical solution of the above-mentioned drug-loaded injectable hydrogel, 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 blinatumomab.

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

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

[0010]

[0011] In the technical scheme of the above-mentioned drug-loaded injectable hydrogel, the polyethylene glycol with one end connected to epigallocatechin gallate can be prepared by referring to the prior art. Generally, the polyethylene glycol with one end connected to epigallocatechin gallate is obtained by the Michael addition reaction of the thiol group of the polyethylene glycol with one end group being a thiol group and the phenolic hydroxyl group of epigallocatechin gallate, and the molar ratio of the polyethylene glycol with one end group being a thiol group to epigallocatechin gallate is controlled to be 1:(1-2), and the molecular weight of the polyethylene glycol with one end group being a thiol group as the modification base is preferably 1000-10000 g / mol.

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

[0013] In the technical scheme of the above-mentioned drug-loaded injectable hydrogel, based on the difference in hydrophilicity between the polyethylene glycol segment and the epigallocatechin gallate segment in the polyethylene glycol with one end connected to epigallocatechin gallate, the polyethylene glycol with one end connected to epigallocatechin gallate can self-assemble to form polyphenol nanoparticles. Generally, polyphenol nanoparticles can be prepared by a thin film hydration method, that is, the polyethylene glycol with one end connected to epigallocatechin gallate is dissolved with an organic solvent, the solvent is removed to obtain a polyethylene glycol film with one end connected to epigallocatechin gallate, and then an aqueous medium is added and fully reacted, and then filtered with an aqueous filter membrane.

[0014] In the technical scheme of the above-mentioned drug-loaded injectable hydrogel, the mass ratio of the immune checkpoint inhibitor to the polyphenol nanoparticles in the drug-loaded injectable hydrogel is determined according to the specific immune checkpoint inhibitor selected in actual application. After the immune checkpoint inhibitor is selected, the mass ratio of the immune checkpoint inhibitor to the polyphenol nanoparticles can be determined by experiment. Usually, in the drug-loaded injectable hydrogel, the mass ratio of the immune checkpoint inhibitor to the polyphenol nanoparticles is (0.1-10):1, preferably (0.2-5):1, and more preferably (0.5-2):1. In the drug-loaded injectable hydrogel, the concentration of polyphenol nanoparticles is not less than 2 mg / mL, and the concentration of immune checkpoint inhibitors 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 technical solution of the above-mentioned drug-loaded injectable hydrogel, the modified natural polysaccharide is obtained by grafting a modified substance onto a natural polysaccharide. Generally, the molecular weight of the natural polysaccharide as the basis for modification is 0.1 to 900 MDa, the grafting rate of the modified substance onto the natural polysaccharide is 10% to 80%, and the grafting rate of the modified substance onto the natural polysaccharide is preferably 20% to 60%. Furthermore, the natural polysaccharide can be hyaluronic acid, chitosan, dextran, alginic acid, etc., and the modified substance can be dopamine, thiol, gallic acid.

[0016] The present invention also provides a method for preparing the above-mentioned drug-loaded injectable hydrogel for tumor immunotherapy, which comprises 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, and the dispersion is converted into a gel state.

[0018] In the technical solution of the above preparation method, the modified natural polysaccharide in the dispersion can be oxidatively self-crosslinked by adding an oxidant and / or adjusting the pH value of the dispersion.

[0019] The present invention verifies through cell experiments that polyphenol nanoparticles can inhibit the proliferation, migration and invasion of B16F10 cells and have low toxicity to L929 cells. Polyphenol nanoparticles can increase the activity of mitochondrial respiratory chain complex enzyme I in B16F10 cells, thereby enhancing mitochondrial activity, increasing ATP generation, and promoting the generation of ROS.

[0020] The present invention has been confirmed by animal experiments: the drug-loaded injectable hydrogel loaded with polyphenol nanoparticles and the immune checkpoint inhibitor BMS-1 ​​is administered to the tumor wound site of postoperative B16F10 tumor-bearing mice, which can effectively inhibit the recurrence of melanoma. The transcriptome results of the recurrent tumor tissue show that the drug-loaded injectable hydrogel loaded with polyphenol nanoparticles up-regulates the oxidative phosphorylation-related pathways and down-regulates the MAPK, PI3K-Akt, and HIF-1 signaling pathways; by performing flow cytometry, immunofluorescence staining, PCR, WB, etc. on the tumor tissue, the results confirm that the drug-loaded injectable hydrogel loaded with polyphenol nanoparticles and the immune checkpoint inhibitor BMS-1 ​​up-regulates the expression of mitochondrial respiratory chain complex enzyme I-related genes and proteins, and inhibits the expression of HIF-1α. In addition, in the in situ tumor model and the distal metastasis model, the drug-loaded injectable hydrogel loaded with polyphenol nanoparticles and the immune checkpoint inhibitor BMS-1 ​​can effectively inhibit tumor growth and metastasis, and activate anti-tumor immunity.

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

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

[0023] 1. The present invention provides a drug-loaded injectable hydrogel for tumor immunotherapy, which is composed of a matrix hydrogel and polyphenol nanoparticles and immune checkpoint inhibitors distributed in the matrix hydrogel; the polyphenol nanoparticles are nanoparticles formed by self-assembly of polyethylene glycol connected to epigallocatechin gallate at one end; the matrix hydrogel is formed by oxidative self-crosslinking of an aqueous solution of modified natural polysaccharides. The present invention has been experimentally confirmed that the polyphenol nanoparticles in the drug-loaded injectable hydrogel can activate the mitochondrial function of tumor cells, stimulate the oxidative phosphorylation process of 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, thereby increasing the sensitivity of tumor cells to immune checkpoint inhibitors. The present invention enhances the inhibitory effect on tumors through the synergistic effect of polyphenol nanoparticles and immune checkpoint inhibitors, and the polyphenol nanoparticles have low toxicity to normal cells, which can overcome the problems of large toxic side effects and drug resistance of chemotherapy drugs in the existing technology of combining chemotherapy drugs with immunotherapy drugs.

[0024] 2. The drug-loaded injectable hydrogel for tumor immunotherapy provided by the present invention loads polyphenol nanoparticles and immune checkpoint inhibitors into a modified natural polysaccharide hydrogel. The hydrogel has good biocompatibility, variable morphology, and a simple administration method. When used, it is locally injected into the tumor site and can be applied to various forms of tumor treatment, such as in situ tumor treatment, postoperative tumor treatment, and distant metastasis treatment, etc., and has the potential for application in multiple scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 Figure a is the TEM image of PE-NPs and a partial enlarged image of the TEM image. Figure 2 Figure b is the particle size distribution diagram of PE-NPs. Figure 2 Figure c is the surface potential of PE-NPs.

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

[0028] Figure 4 Figures a and b are pictures of free EGCG (Free EGCG) and PE-NPs inhibiting the lateral migration of B16F10 cells and the scratch distance test results, respectively.

[0029] Figure 5 Figure a is a schematic diagram of the test process of Example 5. Figure 5 Figure b is the test result of the longitudinal migration rate of free EGCG and PE-NPs inhibiting the longitudinal migration of B16F10 cells. Figure 5 Figures c1 to c3 are pictures of the control group, EGCG experimental group and PE-NPs experimental group inhibiting the longitudinal migration of B16F10 cells.

[0030] Figure 6 Figure a is a schematic diagram of the test process of Example 6. Figure 6 Figure b is the test result of cell invasion rate of free EGCG and PE-NPs inhibiting B16F10 cell invasion. Figure 6 Figures c1 to c3 are pictures of the control group, EGCG experimental group and PE-NPs experimental group inhibiting B16F10 cell invasion.

[0031] Figure 7 Figures a and b are the intracellular mitochondrial staining and semi-quantitative results after PE-NPs acted on B16F10 cells.

[0032] Figure 8 The effect of PE-NPs on the mitochondrial respiratory chain complex of tumor cells, among which, Figure a is coenzyme I (NAD + Figure 1 is the test result of the content of 1,4-dihydronaphthyl ester (NADH), Figure 2 is the test result of the activity of succinate dehydrogenase (SDH), Figure 3 is the test result of the activity of coenzyme Q-cytochrome c reductase, and Figure 4 is the test result of the activity of adenosine triphosphatase.

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

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

[0035] Fig.11 The a graph is the KEGG significantly upregulated entries. Fig.11 Figure b is a heat map of differentially expressed genes in KEGG upregulated entries. Fig.11 Figure c is a heat map of significantly differentially expressed genes in the oxidative phosphorylation pathway. Fig.11 Figure d is a bubble chart of significant differences in GO pathways. Fig.11 The e graph is the chord graph of differentially expressed genes in the GO pathway. Fig.11 The f graph is the KEGG significantly down-regulated entries, Fig.11Figure g is a heat map of significantly differentially expressed genes in the MAPK, PI3K-AKT and HIF signaling pathways.

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

[0037] Fig.13 Figure a is the curve of tumor volume changing with time. Fig.13 Figure b is the tumor weight. Fig.13 Figure c is a photo of the tumor. Fig.13 Figure d is the immunofluorescence staining of CD4 and CD8 proteins in the recurrent tumor tissue.

[0038] Fig.14 Figure a is a flow cytometric graph of the CD8+T cell infiltration ratio detected by flow cytometry. Fig.14 Figure b is a statistical chart of the CD8+T cell infiltration ratio detected by flow cytometry. Fig.14 Figure c is a flow cytometric graph of Treg cell infiltration ratio detected by flow cytometry. Fig.14 Figure d is a statistical graph of the Treg cell infiltration ratio detected by flow cytometry.

[0039] Fig.15 Figure a is a flow cytometric graph of DC cell maturation detected by flow cytometry. Fig.15 Figure b is a statistical diagram of DC cell maturation detected by flow cytometry. Fig.15 Figure c is a flow cytometric graph of the proportion of memory T cells detected by flow cytometry. Fig.15 Figure d is a statistical graph of the proportion of central memory T cells detected by flow cytometry. Fig.15 Figure e is a statistical graph of the proportion of effector memory T cells detected by flow cytometry.

[0040] Fig.16 Figure a shows the tumor volume of the contralateral tumor. Fig.16 Figure b shows the tumor mass of the contralateral tumor. Fig.16 Figure c shows the tumor inhibition rate of the contralateral tumor. Fig.16 Figure d is a picture of the contralateral tumor. Fig.16 Figure e is the CD8 protein immunofluorescence staining of the contralateral tumor tissue. Fig.16 Figure f is a flow cytometric graph of the CD8+T cell infiltration ratio in the contralateral tumor detected by flow cytometry. Fig.16 Figure g is a statistical graph of the proportion of CD8+T cell infiltration in the contralateral tumor detected by flow cytometry. Fig.16 Figure h is the immunofluorescence staining of CD4 and Foxp3 proteins in the contralateral tumor tissue. Fig.16 Figure i is a flow cytometric graph of the Treg cell infiltration ratio in the contralateral tumor detected by flow cytometry. Fig.16 Figure j is a statistical graph of the Treg cell infiltration ratio in the contralateral tumor detected by flow cytometry. DETAILED DESCRIPTION

[0041] The following examples further illustrate the drug-loaded hydrogel for tumor immunotherapy provided by the present invention and its preparation method and application. In the following embodiments, the anti-tumor effect of the drug-loaded hydrogel for tumor immunotherapy is described by taking dopamine-modified hyaluronic acid hydrogel as the matrix hydrogel as an example, but the present invention is not limited to the use of polyphenol nanoparticles and immune checkpoint inhibitors loaded in dopamine-modified hyaluronic acid hydrogels. It is necessary to point out that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. The technical personnel in the relevant field make some non-essential improvements and adjustments to the present invention according to the above-mentioned invention content for specific implementation, which still belongs to the scope of protection of the present invention.

[0042] Example 1

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

[0044]

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

[0046] The EGCG solution and the PEG-SH solution were mixed in equal volumes, stirred and reacted at room temperature for 7 hours, and then glacial acetic acid was added to adjust the pH value to 4.0 to terminate the reaction. 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, and finally freeze-dried to obtain PEG-EGCG.

[0047] Weigh 5 mg of EGCG standard and freeze-dried PEG-EGCG, respectively, dissolve them in 0.5 mL of heavy water, and transfer them to NMR tubes with a pipette to measure the NMR hydrogen spectrum of the samples ( 1 H-NMR), the results are as follows Figure 1 As shown, according to the comparative analysis of the characteristic peaks in the spectrum, it can be seen that PEG-EGCG was successfully prepared in this example.

[0048] Example 2

[0049] In this example, PEG-EGCG prepared in Example 1 was used to prepare polyphenol nanoparticles (PE-NPs) by thin film hydration method, and the steps were as follows:

[0050] Dissolve PEG-EGCG in chloroform to obtain a PEG-EGCG solution with a concentration of 4 mg / mL, transfer it to a rotary evaporator, use a rotary evaporator to evaporate the solvent chloroform under reduced pressure, and form a homogeneous film at the bottom of the rotary evaporator. Add deionized water to the rotary evaporator, the amount of water added is the same as the amount of chloroform used to dissolve PEG-EGCG, incubate at 45°C under nitrogen for 24 hours, and finally filter with a 0.45μm water filter to obtain a PE-NPs aqueous solution. The PE-NPs aqueous solution can be freeze-dried to obtain PE-NPs, 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 the particle size pool. The particle size and polydispersity index (PDI) of PE-NPs were measured 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. The Zeta potential was detected using DLS. Each sample was measured 3 times in parallel, and the measurement temperature was 25°C. The morphology of PE-NPs was observed using a transmission electron microscope. The test results are shown in the figure. Figure 2 As shown, Figure 2 Figure a is the TEM image of PE-NPs and a partial enlarged image of the TEM image. Figure 2 Figure b is the particle size distribution diagram of PE-NPs. Figure 2 Figure c is the surface potential of PE-NPs. Figure 2 It can be seen that the DLS particle size test shows that the particle size of PE-NPs is 135±1.98nm, and the PDI is less than 0.3, indicating that the particle size distribution of PE-NPs is relatively uniform, and its surface ζ potential is about -16mV. The TEM image shows that PE-NPs are spherical with a hydration shell. The TEM image shows that the particle size of PE-NPs is about 120nm, which is close to the DLS measurement results.

[0052] Example 3

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

[0054] B16F10 and L929 cells in the logarithmic growth phase were collected and counted at 5×10 3 100 μL of culture medium containing different concentrations (10, 20, 30, 40, 50 and 60 μg / mL) of free EGCG or PE-NPs were added respectively. Incubate in the incubator for 48 hours, remove the plate and wash the cells with PBS buffer, and then measure the survival rate of B16F10 and L929 cells with CCK-8 kit. The specific operation is as follows: after absorbing the original culture medium, add CCK-8 dilution (10 times the original solution) and place it in the incubator to incubate in the dark for 2 to 4 hours, and use an enzyme reader to detect the absorbance of the reaction solution at a wavelength of 450 nm. The wells with 100 μL of blank culture medium but no cells are used as blank wells, the wells with cells added with blank culture medium are used as control wells, and the wells with cells added with culture medium containing EGCG or PE-NPs are used as experimental wells. The cell survival rate is calculated according to the following formula:

[0055] Cell survival rate = [(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 test results of the cytotoxicity of different concentrations of free EGCG and PE-NPs polyphenol nanoparticles on L929 cells and B16F10 cells are shown in Figure 3 As shown, Figure 3 The Control in the table represents the control well. Figure 3 It can be seen that the cytotoxicity of PE-NPs is concentration-dependent. For in vitro cell experiments, PE-NPs have good biosafety when the concentration is ≤60 μg / mL, while free EGCG shows stronger cytotoxicity.

[0058] Example 4

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

[0060] The scratch test was used to evaluate the lateral migration ability of B16F10 cells. B16F10 cells in the logarithmic growth phase were collected and cultured at 2×10 5 / well were inoculated in a 24-well plate and incubated overnight. When the cells in the well plate grew to 90% density, a 200μL pipette tip was used to draw a uniform straight line vertically in the middle of the well plate, and then the cells were washed with room temperature PBS buffer, and the well plate was gently shaken to wash away the fallen cells. A free EGCG experimental group, a PE-NPs experimental group and a control group (Control) were set up, wherein the free EGCG experimental group was added with 0.5mL of culture medium (0.1% serum) with a free EGCG concentration of 10μg / mL, the PE-NPs experimental group was added with 0.5mL of culture medium (0.1% serum) with a PE-NPs concentration of 10μg / mL, and the control group was added with 0.5mL of blank culture medium (0.1% serum) without EGCG and PE-NPs. At different time points (0, 5, 9 and 12h), photos were taken under bright field with an inverted microscope, and the straight line width at different time points was quantified using Image J software, and the scratch distance was calculated according to the following formula:

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

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

[0063] Free EGCG and PE-NPs inhibit the lateral migration of B16F10 cells and the scratch distance test results are shown in Figure 2. Figure 4 As shown. Figure 4 It can be seen that with the increase of incubation time, only a small amount of cell migration occurred in the PE-NPs experimental group and the free EGCG experimental group, while the cell migration distance in the control group (Control) increased significantly and was basically in a state of rapid healing at 12h. At 12h, the scratch distance of the control group was only 52.6%±12.7% of the distance at 0h, the scratch distance of the free EGCG experimental group was 91.7%±1.5% of the distance at 0h, and the scratch distance of the PE-NPs experimental group was 81.4±5.8% of the distance at 0h. It can be seen that both the PE-NPs experimental group and the free EGCG experimental group can significantly inhibit the lateral migration of cells.

[0064] Example 5

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

[0066] Transwell migration assay was used to evaluate the longitudinal migration ability of B16F10 cells. The schematic diagram of the test process is shown in Figure 5 As shown in Figure a. B16F10 cells in the logarithmic growth phase were collected and cultured at 3×10 5 Each well was inoculated in a 6-well plate. A free EGCG experimental group, a PE-NPs experimental group and a control group (Control) were set up. After the cells in the free EGCG experimental group adhered to the wall, the original culture medium was replaced with a culture medium (0.1% serum) with a free EGCG concentration of 10 μg / mL. After the cells in the PE-NPs experimental group adhered to the wall, the original culture medium was replaced with a culture medium (0.1% serum) with a PE-NPs concentration of 10 μg / mL. After the cells in the control group adhered to the wall, the original culture medium was replaced with a blank culture medium (0.1% serum) without EGCG and PE-NPs. After each group was incubated for 24 hours, the cells in each group were collected and resuspended in fresh culture medium (0.1% serum) and 3×10 5 At a cell density of 100 μL / mL, 100 μL of cell suspension was added to the Transwell chamber, and 500 μL of complete culture medium (10% serum) was added outside the chamber for induction. After incubation for another 24 hours, the cells under the Transwell chamber membrane were washed twice with PBS, and the cells that did not migrate in the chamber membrane were wiped with a cotton swab, fixed with 4% paraformaldehyde for 20 minutes, and stained with 0.1% crystal violet solution for 20 minutes. After washing twice with PBS buffer, the cells that migrated to the lower side of the Transwell chamber were photographed under an inverted microscope in the bright field. After the photo was taken, it was decolorized with 33% acetic acid solution for 1 hour, and the 33% acetic acid solution was used as the blank group (Blank). The absorbance value of each group of decolorization solution at a wavelength of 570nm was detected by an enzyme marker, and the longitudinal migration rate of the cells (Migration rate) was calculated according to the following formula:

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

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

[0069] Figure 5 Figure b is the test result of the longitudinal migration rate of free EGCG and PE-NPs inhibiting the longitudinal migration of B16F10 cells. Figure 5 Figures c1 to c3 are pictures of the control group, EGCG experimental group, and PE-NPs experimental group inhibiting the longitudinal migration of B16F10 cells (i.e., the photographic results of cells that migrated to the lower side of the Transwell chamber). Figure 5 It can be seen that only a few cells in the free EGCG experimental group and the PE-NPs experimental group migrated to the lower side of the Transwell chamber, while in the control group, it can be seen that the number of cells migrating to the lower side of the chamber increased significantly, and the cells were connected to each other and spread into sheets. From the data of cell longitudinal migration rate, the migration rate of the free EGCG experimental group was 43.2%±3.1%, and the migration rate of the PE-NPs experimental group was 33.8%±5.9%. Both PE-NPs and free EGCG have the ability to significantly inhibit the longitudinal migration of cells, and the ability of PE-NPs to inhibit the longitudinal migration of cells is better than that of free EGCG.

[0070] Example 6

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

[0072] Transwell invasion assay was used to evaluate the invasive ability of B16F10 cells. The schematic diagram of the test process is shown in Figure 6As shown in Figure a. First, spread a layer of Matrigel inside the Transwell chamber: thaw Matrigel in a 4°C refrigerator the night before, and pre-cool the well plate, pipette tip, and serum-free medium used in the experiment in a 4°C refrigerator for 2 hours. Dilute Matrigel 5 times with serum-free medium, pipette 60 μL and evenly drip it inside the Transwell chamber, then carefully transfer the well plate to the incubator and incubate for 1 to 2 hours until the Matrigel changes from a fluid state to a gel state. The free EGCG experimental group, PE-NPs experimental group and control group (Control) were set up. After the cells in the free EGCG experimental group adhered to the wall, the original culture medium was replaced with a culture medium (0.1% serum) with a free EGCG concentration of 10 μg / mL. After the cells in the PE-NPs experimental group adhered to the wall, the original culture medium was replaced with a culture medium (0.1% serum) with a PE-NPs concentration of 10 μg / mL. After the cells in the control group adhered to the wall, the original culture medium was replaced with a blank culture medium (0.1% serum) without EGCG and PE-NPs. After each group was incubated for 24 hours, the cells in each group were collected and resuspended in fresh culture medium (0.1% serum) and 3×10 5 At a cell density of 100 μL / mL, 100 μL of cell suspension was added to the Transwell chamber, and 500 μL of complete culture medium (10% serum) was added outside the chamber for induction. After incubation for another 24 hours, the cells under the Transwell chamber membrane were washed twice with PBS, and the cells that did not migrate in the chamber membrane were wiped off with a cotton swab, fixed with 4% paraformaldehyde for 20 minutes, and stained with 0.1% crystal violet solution for 20 minutes. After washing twice with PBS buffer, the cells that invaded the lower side of the Transwell chamber were photographed under an inverted microscope in the bright field. After the photo was taken, it was decolorized with 33% acetic acid solution for 1 hour, and the 33% acetic acid solution was used as the blank group (Blank). The absorbance value of the decolorization solution of each group at a wavelength of 570nm was detected by an enzyme marker, and the cell invasion rate (Invasion rate) was calculated according to the following formula:

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

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

[0075] Figure 6Figure b is the test result of cell invasion rate of free EGCG and PE-NPs inhibiting B16F10 cell invasion. Figure 6 Figures c1 to c3 are pictures of the control group, EGCG experimental group, and PE-NPs experimental group inhibiting B16F10 cell invasion (i.e., the photographic results of cells invading the lower side of the Transwell chamber). Figure 6 It can be seen that only a few cells in the PE-NPs experimental group and the free EGCG experimental group invaded the lower side of the Transwell chamber, while in the control group, it can be seen that the number of cells migrating to the lower side of the Transwell chamber increased significantly. From the cell invasion rate data, the invasion rate of the free EGCG experimental group was 42.3% ± 4.8%, and the invasion rate of the PE-NPs experimental group was 32.7% ± 3.6%, indicating that PE-NPs has a better effect in inhibiting cell invasion.

[0076] Example 7

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

[0078] B16F10 cells in the logarithmic growth phase were collected and counted at 2.5×10 4 The cells were inoculated at a density of 100 μg / mL / well in a confocal culture dish and placed in an incubator for overnight incubation. The original culture medium was discarded. The sterilized culture medium containing PE-NPs (PE-NPs concentration was 60 μg / mL) was added as the experimental group. The blank culture medium without PE-NPs was added as the control group (Control). After 48 hours of continuous culture, the original culture medium was discarded and the cells were washed twice with PBS buffer. 200 nmol / L 37°C preheated mitochondrial red fluorescent probe ( RedCMXRos) staining for 30 minutes, and 15 minutes later, 1 mg / mL Hochest dye was added and incubated for 15 minutes. After the staining, the staining solution was replaced with fresh PBS buffer and placed under a confocal laser scanning microscope (CLSM) for observation and photography. Mitochondrial fluorescent probe (Ex / Em=579 / 599nm) was used to label mitochondria, and red fluorescence was observed under CLSM; Hochest (Ex / Em=346 / 460nm) was used to label cell nuclei, and blue fluorescence was observed under CLSM.

[0079] Image J software was used to calculate the fluorescence intensity of the experimental group and the control group samples, and the mitochondrial activity in each group of samples was compared. Three parallel samples were tested for each group. The test results are shown in Figure 2. Figure 7 As shown, Figure 7 Figure a is a CLSM image, and Figure 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 example, the effect of the PE-NPs prepared in Example 2 on the activity of the mitochondrial respiratory complex of the mitochondria of B16F10 cells was investigated.

[0082] According to the instructions of the kit (Solebo), the coenzyme I NAD (H) content detection kit, succinate dehydrogenase (SDH) activity detection kit, mitochondrial respiratory chain complex III activity detection kit and mitochondrial respiratory chain complex V activity detection kit were used to detect and analyze the activity of mitochondrial respiratory complexes in B16F10 cells. The experimental process is described by taking the coenzyme I NAD (H) content detection kit as an example, as follows:

[0083] B16F10 cells in the logarithmic growth phase were collected and counted at 1.8×10 6 The cells were inoculated in a culture dish at a density of 1000 μg / dish. Sterilized culture medium containing PE-NPs (PE-NPs concentration of 60 μg / mL) was added as the experimental group, and blank culture medium without PE-NPs was added as the control group. The cells were incubated for 48 hours, washed twice with PBS buffer, digested and collected into 5 million cells in a 2mL EP tube, 500 μL of acid / alkaline extract was added, and the cells were ultrasonically disrupted (200W, ultrasonic 2s, stop 1s), then placed in boiling water for 5 minutes, cooled on ice, centrifuged (4°C, 10000g) for 10 minutes, 200 μL of supernatant was taken, and an equal volume of alkaline / acidic extract was added to neutralize it, quickly mixed with a pipette, centrifuged again (4°C, 10000g) for 10 minutes, and the supernatant was placed on ice for later use. According to the test instructions, add samples and test reagents one by one in order, pipette and mix evenly, use a microplate reader to detect the absorbance of the reaction solution at a wavelength of 570nm, and 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 is coenzyme I (NAD + Figure 1 is the test result of the content of NADH, Figure 2 is the test result of the activity of succinate dehydrogenase (SDH), Figure 3 is the test result of the activity of coenzyme Q-cytochrome c reductase, and Figure 4 is the test result of the activity of adenosine triphosphatase. Figure 8It can be seen that PE-NPs significantly increased the activity of respiratory chain complex I (NAD + / NADH), but had no significant effect on the activities of other enzymes, indicating that after PE-NPs entered B16F10 cells, they acted on mitochondrial respiratory chain complex I and accelerated the process of electron transfer from NADH to oxygen.

[0085] Example 9

[0086] In this example, 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 and counted at 1.5×10 5 The cells were inoculated at a density of 100 / well in a 12-well plate and incubated overnight until the cells adhered. A free EGCG experimental group, a PE-NPs experimental group and a control group (Control) were set up. After the cells adhered to the wall, the original culture medium in the free EGCG experimental group was replaced with a culture medium with a free EGCG concentration of 60 μg / mL. After the cells adhered to the wall in the PE-NPs experimental group, the original culture medium was replaced with a culture medium with a PE-NPs concentration of 60 μg / mL. After the cells adhered to the wall in the control group, the original culture medium was replaced with a blank culture medium without EGCG and PE-NPs. Continue to incubate for 24 hours, collect the cells into a flow tube, wash twice with PBS buffer, and incubate with anti-mouse CD274-PE antibody (0.5 μg / tube) in the dark for 30 minutes. After washing twice with PBS buffer, a certain volume of PBS buffer was added to resuspend the cells for on-machine detection. The flow cytometry experiments involved in the present invention were all detected using a high-sensitivity flow cytometer (FACSAria III, BD|SENSORS, Germany), and the flow data were analyzed using Flowjo software.

[0088] Fig. 9 The results of the test on the inhibition of PD-L1 expression on the surface of B16F10 cells by free EGCG and PE-NPs are shown in Figure 1. Figure a is a flow cytometry chart, and Figure b is a statistical chart of the average fluorescence intensity. Fig. 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 by Schiff base reaction, and the synthesis route and steps are as follows:

[0091]

[0092] (1) Hyaluronic acid (HA, Mw = 1000 kDa) was dissolved in deionized water to obtain an 8 mg / mL HA solution. Sodium periodate solution was slowly added dropwise to the HA solution. The amount of sodium periodate solution added should satisfy the mass ratio of sodium periodate to HA of 1.07:1. The mixture was reacted at room temperature in the dark for 2 h. Ethylene glycol was then added and stirred for 2 h to remove unreacted sodium periodate. The resulting reaction solution was dialyzed in ultrapure water for 3 days and freeze-dried to obtain oxidized hyaluronic acid (OHA).

[0093] (2) OHA was dissolved in a PBS buffer solution with a pH value of 5 to form a 10 mg / mL OHA solution, and dopamine hydrochloride (Dopa) in an amount equimolar to OHA was added to the OHA solution. The reaction was allowed to react at room temperature for 10 h. The resulting reaction solution was dialyzed in pure water for 2 days and freeze-dried to obtain HA-Dopa.

[0094] Weigh 5 mg of HA, Dopa standard, OHA and HA-Dopa, add 0.5 mL of heavy water, shake with a vortexer to dissolve, and transfer to NMR tubes for testing. 1 H-NMR was used to measure the nuclear magnetic hydrogen spectrum. The results were as follows Fig.10 As shown by Fig.10 It can be seen that the peak of HA-Dopa at 7ppm is the characteristic peak of catechol aromatic protons, and the peaks at 3.1 and 2.8ppm are the characteristic peaks of catechol methylene protons, proving that dopamine groups have been successfully grafted onto HA. 2.8 / (2×A 1.9 ) was used to calculate the grafting rate of dopamine in HA-Dopa, where A 2.8 and A 1.9 The results are the integration of the peak areas at the chemical shifts of 2.8 ppm (catechol methylene) and 1.9 ppm (methyl group of HA), respectively. The results show that the grafting rate of dopamine in the HA-Dopa prepared in this example is about 24%.

[0095] Embodiment 11

[0096] In this example, a drug-loaded hydrogel (PE-NPs@PD / HD) containing a PD-1 / PD-L1 inhibitor and PE-NPs 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 fully dispersed. Then, an equal volume of a NaOH solution containing sodium periodate (pH = 8.5, wherein the concentration of sodium periodate was 2.825 mg / mL) was added to the HA-Dopa solution and fully mixed to obtain a gel precursor solution. 50 μL of the gel precursor solution was injected into a homemade silicone mold (with a diameter of 8 mm and a height of 2 mm), and allowed to stand until HA-Dopa cross-linked and the gel precursor solution was converted into a gel state, and then demolded to obtain PE-NPs@PD / HD.

[0098] The volume of each piece of PE-NPs@PD / HD is 50 μL, the concentration of cross-linked HA-Dopa in PE-NPs@PD / HD is 30 mg / mL, and each piece of PE-NPs@PD / HD 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, and then an equal volume of a NaOH solution containing sodium periodate (pH=8.5, wherein the concentration of sodium periodate was 2.825 mg / mL) was added to the HA-Dopa solution, and the mixture was thoroughly mixed to obtain a gel precursor solution. 50 μL of the gel precursor solution was injected into a homemade silica gel mold (with a diameter of 8 mm and a height of 2 mm), and the mixture was allowed to stand until HA-Dopa cross-linked and the gel precursor solution was converted into a gel state, and then the mold was demolded to obtain HD.

[0102] The volume of each HD was 50 μL, and the concentration of cross-linked HA-Dopa in the HD was 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 fully dispersed. Then, an equal volume of a NaOH solution containing sodium periodate (pH=8.5, wherein the concentration of sodium periodate was 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 homemade silica gel mold (with a diameter of 8 mm and a height of 2 mm), and allowed to stand until HA-Dopa cross-linked and the gel precursor solution was converted into a gel state, and then demolded to obtain PE-NPs / HD.

[0106] The volume of each piece of PE-NPs / HD is 50 μL, the concentration of cross-linked HA-Dopa in PE-NPs / HD is 30 mg / mL, and each piece of PE-NPs / HD contains 100 μg of PE-NPs, that is, 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, fully dispersed and dissolved, and then an equal volume of a NaOH solution containing sodium periodate (pH=8.5, wherein the concentration of sodium periodate was 2.825 mg / mL) was added to the HA-Dopa solution, and the mixture was fully mixed to obtain a gel precursor solution. 50 μL of the gel precursor solution was injected into a homemade silicone mold (with a diameter of 8 mm and a height of 2 mm), and the mixture was allowed to stand until HA-Dopa cross-linked and the gel precursor solution was converted into a gel state, and then the mold was demolded to obtain PD / HD.

[0110] The volume of each PD / HD is 50 μL, the concentration of cross-linked HA-Dopa in PD / HD is 30 mg / mL, and each PD / HD contains 50 μg of BMS-1, that is, the concentration of BMS-1 ​​in PD / HD is 1 mg / mL.

[0111] Example 12

[0112] In this example, the intrinsic mechanism of the drug-loaded hydrogel containing PE-NPs (PE-NPs / HD) in inhibiting postoperative tumor recurrence was investigated.

[0113] B16F10 cells in the logarithmic growth phase were collected and inoculated subcutaneously on the right ventral back of each mouse (inoculation dose, 1×10 6The health status of the mice and the size of the melanoma were observed every other day. The tumor volume reached 80 mm 7 days after inoculation. 3 About, at this time, subsequent surgical resection experiments can be carried out. Gently cut the connection along the edge of the tumor, carefully remove the tumor, and leave a residual tumor with a width of about 2 mm. Sterilize the PE-NPs / HD prepared in Comparative Example 2, cover the sterilized PE-NPs / HD on the residual tumor and suture the wound, and use this situation as the experimental group. The case where the wound was sutured directly was used as the control group. Finally, a layer of Tegaderm Film (3M) breathable dressing was applied to the surgical site to prevent the sutures from falling off after surgery. After the operation, when the mice wake up and are in a normal active state, put them back in the cage and continue to be raised in an SPF environment.

[0114] The recurrent tumors on the 14th day after surgery were analyzed by transcriptome sequencing. Fig.11 As shown, Fig.11 The a graph is the KEGG significantly upregulated entries. Fig.11 Figure b is a heat map of differentially expressed genes in KEGG upregulated entries. Fig.11 Figure c is a heat map of significantly differentially expressed genes in the oxidative phosphorylation pathway. Fig.11 Figure d is a bubble chart of significant differences in GO pathways. Fig.11 The e graph is the chord graph of differentially expressed genes in the GO pathway. Fig.11 The f graph is the KEGG significantly down-regulated entries, Fig.11 The g graph is a heat map of significantly differentially expressed genes in the MAPK, PI3K-AKT, and HIF signaling pathways. Among the upregulated entries in KEGG, the oxidative phosphorylation and heat production pathways were significantly upregulated, such as Fig.11 As shown in Figures a and b, the heat map of significantly differentially expressed genes in the oxidative phosphorylation pathway was analyzed. Compared with the control group, PE-NPs / HD significantly upregulated the expression of related genes such as Ndufa4, Cox5a, Atp5g1, Ndufa2 and Uqcr11. Fig.11 As shown in Figure c, PE-NPs / HD increased the expression of complex subunits on mitochondria, which is consistent with the results of Example 8. In addition, through GO analysis, PE-NPs / HD significantly upregulated the 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, ATP metabolism and other related biological processes, such as Fig.11 As shown in Figures d and e, these are closely related to mitochondrial activity. Among the KEGG downregulated pathways, PE-NPs / HD inhibited tyrosine kinase inhibitor resistance, MAPK, HIF-1, ErbB, PI3K-AKT, EGFR, and mTOR signaling pathways, such as Fig.11As shown in Figures f and g, these pathways are directly or indirectly related to the expression of PD-L1, which also explains why PE-NPs can downregulate the expression of PD-L1 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 example, the ability and mechanism of drug-loaded hydrogels (PE-NPs@PD / HD) containing PD-1 / PD-L1 inhibitors and PE-NPs to inhibit postoperative tumor recurrence were verified.

[0117] B16F10 cells in the logarithmic growth phase were collected and inoculated subcutaneously on the right ventral back of each mouse (inoculation dose, 1×10 6 The health status of the mice and the size of the melanoma were observed every other day. The tumor volume reached 80 mm 7 days after inoculation. 3 At this time, a subsequent surgical excision experiment can be performed. Gently cut the connection along the edge of the tumor and carefully remove the tumor, leaving a residual tumor with a width of about 2mm.

[0118] The HD, PE-NPs / HD, PD / HD and PE-NPs@PD / HD prepared in Comparative Examples 1 to 3 and Example 11 were sterilized respectively, and the sterilized HD, PE-NPs / HD, PD / HD and PE-NPs@PD / HD were covered on the residual tumor and the wound was sutured. This situation was used as different experimental groups (HD group, PE-NPs / HD group, PD / HD group and PE-NPs@PD / HD group). The situation where the wound was directly sutured was used as the control group (sham control group, referred to as Sham or Control). Finally, a layer of Tegaderm Film (3M) breathable dressing was applied to the surgical site to prevent the sutures from falling off after surgery. After the operation, when the mice woke up and were in a normal active state, they were put back into the cage and continued to be raised in an SPF environment to observe the recurrence of tumors in the mice.

[0119] On the 14th day after surgery, the recurrent tumors were collected and analyzed by PCR, Western Blot, and immunofluorescence. Fig.12 As shown, Fig.12 Figure a is a photo of the tumors of mice in each experimental group and control group. Fig.12 Figure b shows the tumor volume of mice in each experimental group and control group. Fig.12 Figure c is the immunofluorescence staining image of CD31 and Ki67 in recurrent tumor tissue. Fig.12 Figure d shows the gene expression level of Ndufa4 in recurrent tumor tissues detected by PCR. Fig.12Figure e is an immunofluorescence staining image of Ndufa4 in recurrent tumor tissues. Fig.12 Figure f is the result of Western blot detection of Ndufa4 protein expression in recurrent tumor tissues. Fig.12 Figure g shows the result of detecting the gene expression level of HIF in recurrent tumor tissues by PCR. Fig.12 Figure h is an immunofluorescence staining image of HIF-1α in recurrent tumor tissue. Fig.12 Figure 1 shows the results of Western blot detection of HIF protein expression in recurrent tumor tissues. Fig.12 It can be seen that the recurrent tumor volume and weight in the sham control group were the largest, and there was no significant difference 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 could inhibit the growth of recurrent tumors to varying degrees, among which the tumor volume in the PE-NPs@PD / HD group was the smallest. Fig.12 Figures a to b show that PE-NPs and PD-1 / PD-L1 inhibitors have a synergistic effect in inhibiting tumor growth. In terms of inhibiting tumor growth, the combination of PE-NPs and PD-1 / PD-L1 inhibitors has a significantly better inhibitory effect than the use of either alone, and inhibits the formation and proliferation of tumor tissue angiogenesis. Fig.12 As shown in Figures d to i, PCR, Western Blot and immunofluorescence analysis of recurrent tumor tissues showed that the PE-NPs / HD group and PE-NPs@PD / HD group significantly reduced the expression of Ndufa-4 and HIF-1α genes and proteins in tumor tissues.

[0120] Embodiment 14

[0121] In this example, 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 inoculated subcutaneously on the right ventral back of each mouse (inoculation dose, 1×10 6cells / mouse). The health status and melanoma size of the mice were observed every other day. A visible tumor was formed on the 7th day after inoculation, and the mice were randomly divided and numbered. A PE-NPs / HD group, a PE-NPs@PD / HD group and a control group were set up. The PE-NPs / HD group and the PE-NPs@PD / HD group were injected with the gel precursor solution of PE-NPs / HD and PE-NPs@PD / HD prepared in Comparative Example 2 and Example 11, respectively. The gel precursor solution will be gelled at the injection site after injection. The control group was injected with PBS buffer, that is, 50 μL of the gel precursor solution or PBS buffer solution for preparing PE-NPs / HD or PE-NPs@PD / HD was injected next to the tumor each time. A total of 3 injection treatments were performed, and the time of the first administration was recorded as Day 0. The second administration (Day 2) was performed 2 days after the first administration, and the third administration (Day 4) was performed 4 days after the first administration. The tumor volume of tumor-bearing mice was recorded. Eight days after the last administration, the mice were euthanized, the tumors were removed, a portion was weighed, fixed with paraformaldehyde, embedded in paraffin, and sliced. The tumor tissue was immunohistofluorescence stained to mark T cells. The results are shown in Fig.13 shown.

[0123] Fig.13 Figure a is the curve of tumor volume changing with time. Fig.13 Figure b is the tumor weight. Fig.13 Figure c is a photo of the tumor. Fig.13 Figure d is the immunofluorescence staining of CD4 and CD8 proteins in the recurrent tumor tissue. Fig.13 It can be seen that the PE-NPs@PD / HD group had the best tumor inhibition effect, and the combination of PE-NPs and PD-1 / PD-L1 inhibitors produced more CD8T cell infiltration.

[0124] Another part of the tumor tissue was subjected to immune cell flow cytometry, and the lymphocytes in the tumor tissue were extracted using the tumor infiltrating tissue lymphocyte isolation kit (Solebo): prepare a 6-well plate and add 5mL of whole blood and tissue diluent. After the mouse was euthanized, the tumor was taken out, crushed with the piston head of a sterile syringe and passed through a 100μm mesh into the whole blood and tissue diluent. Then the single cell suspension was slowly dripped into a 15mL centrifuge tube (5mL of separation solution was added in advance). Due to the certain density difference between the two, the boundary layer can be clearly seen. Centrifuge at 900g for 30min. After the centrifugation, obvious stratification can be seen: the cells in the centrifuge tube are divided into four layers from top to bottom, namely the diluent layer, the milky white lymphocyte layer, the transparent separation liquid layer and the red blood cell layer. Then carefully pipette the middle milky white lymphocyte layer (the second layer) into another clean and sterile 15mL centrifuge tube, add 10mL of cell washing solution, and centrifuge at 250g for 10min. Then discard the supernatant, add 5mL PBS solution to wash the cells twice, centrifuge at 250g for 10min each time, and collect the lymphocytes at the bottom. Stain with anti-mouse CD3-PE, anti-mouse CD8-percp / cy5.5, anti-mouse CD4-FITC, anti-mouse CD25-bv510 and anti-mouse Foxp3-AF647 antibodies for analysis: transfer the above cells to a flow cytometry tube, add 100μL anti-mouse Zombie-APC-CY7 antibody dilution (diluted 200 times), incubate in the dark for 20min, and perform live / dead cell staining. After washing once with PBS buffer, stain the cell membrane with antibodies anti-mouse CD3-PE, anti-mouse CD8-percp / cy5.5, anti-mouse CD4-FITC and anti-mouse CD25-bv510 (all 0.5μg / tube) and incubate in the dark for 30min. Wash twice with PBS buffer, add 1mL of fixative to fix the cells for 45 minutes, then add 1mL of nuclear membrane breaking solution directly to it, centrifuge at 350g for 6 minutes, and discard the supernatant. Add 2mL of nuclear membrane breaking solution to resuspend the cells, blow with a gun to mix, and then centrifuge (350g, 6min). Discard the supernatant, stain the cell nucleus with anti-mouse Foxp3-AF647 (0.5μg / tube), and incubate in the dark for 40 minutes. Finally, wash twice with PBS buffer, transfer the cells to the flow tube and add a certain volume of PBS buffer to resuspend them for on-machine detection, and use Flowjo software to analyze the flow data. The experimental results are as follows Fig.14 shown.

[0125] Fig.14Figure a is a flow cytometric graph of the CD8+T cell infiltration ratio detected by flow cytometry. Fig.14 Figure b is a statistical chart of the CD8+T cell infiltration ratio detected by flow cytometry. Fig.14 Figure c is a flow cytometric graph of Treg cell infiltration ratio detected by flow cytometry. Fig.14 Figure d is a statistical graph of the Treg cell infiltration ratio detected by flow cytometry. Fig.14 The results showed that the tumor tissues of the PE-NPs@PD / HD group had higher CD8 + The proportion of T cells and a lower proportion of Treg cells.

[0126] The spleen lymphocyte isolation kit (Solebo) was used to extract lymphocytes from mouse spleen tissue. The specific operation steps were consistent with the extraction of tumor tissue infiltrating lymphocytes. Subsequently, anti-mouse CD11c-PE, anti-mouseCD86-APC and anti-mouse CD80-FITC antibodies were used to investigate the maturation of DC cells in the spleen; anti-mouse CD3-PE, anti-mouse CD8-FITC, anti-mouse CD62L-bv421 and anti-mouse CD44-AF647 antibodies were used to investigate the immune memory effect of T cells in the spleen. The specific staining steps are as above. The experimental results are as follows. Fig.15 shown.

[0127] Fig.15 Figure a is a flow cytometric graph of DC cell maturation detected by flow cytometry. Fig.15 Figure b is a statistical diagram of DC cell maturation detected by flow cytometry. Fig.15 Figure c is a flow cytometric graph of the proportion of memory T cells detected by flow cytometry. Fig.15 Figure d is a statistical graph of the proportion of central memory T cells detected by flow cytometry. Fig.15 Figure e is a statistical graph of the proportion of effector memory T cells detected by flow cytometry. Fig.15 It can be seen that the 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] Embodiment 15

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

[0130] B16F10 cells in the logarithmic growth phase were collected and concentrated to a concentration of 1×10 7The cells were resuspended in sterile PBS buffer and then subcutaneously inoculated with B16F10 cells (50 μL / mouse) on the right side of the back of each mouse. On the 4th day after inoculation, B16F10 cells were inoculated at the same dose on the left side of the back of the mouse to simulate melanoma metastasis. On the 5th day after inoculation, a macroscopic tumor (about 50 mm) was formed on the right side of the back of the mouse. 3 ), the mice were randomly divided into groups and numbered. A PE-NPs / HD group, a PE-NPs@PD / HD group and a control group were set up. The PE-NPs / HD group and the PE-NPs@PD / HD group were injected with the gel precursors of PE-NPs / HD and PE-NPs@PD / HD prepared in Comparative Example 2 and Example 11, respectively. The gel precursors would form a gel at the injection site after injection. The control group was injected with PBS buffer, that is, 50 μL of PE-NPs / HD or PE-NPs@PD / HD or PBS buffer was injected next to the tumor of the right abdominal back each time. A total of 3 injection treatments were performed. The time of the first administration was recorded as Day 0. The second administration (Day 2) was performed 2 days after the first administration, and the third administration (Day 4) was performed 4 days after the first administration. The health status of the mice and the growth trend of the melanoma on the left were observed every other day, and the infiltration of immune cells in the left tumor tissue was detected. The specific steps and operations were the same as those in Example 14. The test results of this example are shown in the following figure. Fig.16 shown.

[0131] Fig.16 Figure a shows the tumor volume of the contralateral tumor. Fig.16 Figure b shows the tumor mass of the contralateral tumor. Fig.16 Figure c shows the tumor inhibition rate of the contralateral tumor. Fig.16 Figure d is a picture of the contralateral tumor. Fig.16 Figure e is the CD8 protein immunofluorescence staining of the contralateral tumor tissue. Fig.16 Figure f is a flow cytometric graph of the CD8+T cell infiltration ratio in the contralateral tumor detected by flow cytometry. Fig.16 Figure g is a statistical graph of the proportion of CD8+T cell infiltration in the contralateral tumor detected by flow cytometry. Fig.16 Figure h is the immunofluorescence staining of CD4 and Foxp3 proteins in the contralateral tumor tissue. Fig.16 Figure i is a flow cytometric graph of the Treg cell infiltration ratio in the contralateral tumor detected by flow cytometry. Fig.16 Figure j is a statistical graph of the proportion of Treg cell infiltration in the contralateral tumor detected by flow cytometry. Fig.16 It can be seen that both the PE-NPs / HD group and the PE-NPs@PD / HD group can inhibit the formation and development of the contralateral tumor and promote the proliferation of CD8 +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 the distant metastasis of tumors.

Claims

1. A drug-loaded injectable hydrogel for tumor immunotherapy, characterized in that: The drug-loaded injectable hydrogel consists of a matrix hydrogel and polyphenol nanoparticles and immune checkpoint inhibitors distributed in the matrix hydrogel; the polyphenol nanoparticles are nanoparticles formed by self-assembly of polyethylene glycol with one end connected to epigallocatechin gallate; the matrix hydrogel is formed by 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, thereby enhancing the sensitivity of tumor cells to immune checkpoint inhibitors and achieving synergistic inhibition of tumors.

2. The drug-loaded injectable hydrogel for tumor immunotherapy according to claim 1, characterized in that: The immune checkpoint inhibitor is a PD-1 / PD-L1 inhibitor.

3. The drug-loaded injectable hydrogel for tumor immunotherapy according to claim 2, characterized in that: The PD-1 / PD-L1 inhibitor includes any one of BMS-1, BMS-202, BMS-1166, nivolumab, pembrolizumab, atezolizumab, ipilimumab and blinatumomab.

4. The drug-loaded injectable hydrogel for tumor immunotherapy according to any one of claims 1 to 3, characterized in that: The tumor cells are melanoma cells.

5. The drug-loaded injectable hydrogel for tumor immunotherapy according to any one of claims 1 to 3, characterized in that: The particle size of the polyphenol nanoparticles is 100-300 nm.

6. The drug-loaded injectable hydrogel for tumor immunotherapy according to any one of claims 1 to 3, characterized in that: In the drug-loaded injectable hydrogel, the mass ratio of the immune checkpoint inhibitor to the polyphenol nanoparticles is (0.1-10):

1.

7. The drug-loaded injectable hydrogel for tumor immunotherapy according to claim 6, characterized in that: In the drug-loaded injectable hydrogel, the concentration of polyphenol nanoparticles is not less than 2 mg / mL, and the concentration of immune checkpoint inhibitors is not less than 0.5 mg / mL.

8. The method for preparing the drug-loaded injectable hydrogel for tumor immunotherapy according to any one of claims 1 to 7, characterized in that: The following steps are involved: 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, and the dispersion is converted into a gel state.

9. The method for preparing the drug-loaded injectable hydrogel for tumor immunotherapy according to claim 8, characterized in that: The modified natural polysaccharide in the dispersion is oxidatively self-crosslinked by adding an oxidant and / or adjusting the pH value of the dispersion.

10. Use of the drug-loaded injectable hydrogel for tumor immunotherapy according to any one of claims 1 to 7 in the preparation of drugs for treating solid tumors.

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