Preparation and application of PD-L1 nano inhibitor for enhancing cervical cancer immunotherapy effect

Abstract

The invention discloses preparation and application of a PD-L1 nano-inhibitor capable of enhancing the immunotherapy effect of cervical cancer, and belongs to the field of nano-drugs. According to the invention, PDA-coated double-high-Z metal nanoparticles, GdPt-coated PDA for short, are synthesized by using a solvothermal method and a solution oxidation method. Compared with the prior art, the GdPt-coated PDA has an excellent immunotherapy effect and good biological safety. The GdPt (at) PDA not only can realize immunotherapy by down-regulating the expression level of PD-L1, but also can improve the tumor immunosuppression microenvironment and promote immune cell infiltration in tumors through combined radiotherapy, and cooperates with alphaCTLA-4 to realize dual-immune checkpoint blocking therapy, thereby successfully inhibiting the growth of primary tumors and distant tumors.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technologies, and particularly to the preparation and application of a PD-L1 nano-inhibitor that can enhance the effect of cervical cancer immunotherapy. Background Art

[0002] Cervical cancer is one of the most common malignant tumors in the female reproductive system. In 2022, there were more than 660,000 new cases of cervical cancer globally. Early-stage cervical cancer usually has a good prognosis. Unfortunately, locally advanced cervical cancer is often accompanied by medium- to high-risk recurrence factors such as lymph node metastasis and parametrial invasion, and the 5-year overall survival rate is less than 70%. In recent years, immune checkpoint therapy targeting Programmed cell death-ligand 1 (PD-L1) and Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) has received great attention from researchers due to its remarkable effect in tumor treatment.

[0003] Tumor cells bind to the Programmed cell death protein 1 (PD-1) receptor on the surface of T cells through the highly expressed PD-L1 protein on their surface, transmitting an "immune brake" signal, inhibiting the activity of T cells and inducing their apoptosis. PD-L1 inhibitors can specifically bind to PD-L1, preventing its interaction with PD-1, thereby relieving the functional inhibition of T cells. This process enables T cells to re-recognize and attack tumor cells, forming specific immune killing. In cervical cancer, high-risk Human papillomavirus (HPV) infection (such as HPV16 / 18) can promote PD-L1 expression by integrating viral genes (E6 / E7), helping tumors evade immune surveillance. PD-L1 inhibitors can reverse HPV-induced immune suppression, re-expose virus-related antigens, and activate specific T cell responses. Currently, the PD-L1 inhibitors used clinically to treat cervical cancer are mainly atezolizumab, etc. However, most of these immune checkpoint inhibitors have limitations such as low response rates, severe immune-related adverse reactions, and heavy economic burdens on patients, restricting their clinical application effects.

[0004] As an emerging treatment modality, nano-drugs have the following advantages compared to traditional immune antibodies. First, the small size of nanoparticles can penetrate abnormally proliferating tumor blood vessels, overcoming the limitation that traditional antibodies are difficult to penetrate solid tumors. Second, nano-drugs can usually act synergistically with traditional treatment methods such as radiotherapy and chemotherapy. By inducing immunogenic cell death (ICD) and releasing tumor antigens, they achieve the effect of "immunogenic death + immune checkpoint inhibition". In addition, nano-drugs usually have a long drug action time, which can continuously activate T cells and extend immune memory. Finally, through reasonable modification in the design of nano-drugs, the drug side effects can be reduced. Through pH-responsive design, the drug is released only in the tumor microenvironment, which can further reduce systemic toxicity. Therefore, the development of effective PD-L1 nano-inhibitors has broad prospects for the immunotherapy of cervical cancer.

[0005] Due to the low single-agent efficacy of PD-L1 inhibitors (the objective response rate is about 14%-20%), combination therapy is usually required to improve the efficacy. Clinically, PD-L1 inhibitors are often combined with radiotherapy. Radiotherapy can directly kill tumor cells and release tumor-associated antigens, enhancing the immune system's recognition of tumors and thus enhancing the effect of PD-L1 inhibitors. A series of clinical studies have shown that the combination of radiotherapy and immunotherapy is expected to become the standard treatment for locally advanced cervical cancer. However, this combination treatment approach faces some challenges. On the one hand, due to radiotherapy exacerbating the hypoxic microenvironment of tumors, the damage of X-rays to deoxyribonucleic acid (DNA) is limited. On the other hand, the immune resistance induced by the increased PD-L1 expression level after radiotherapy and the immunosuppressive microenvironment in tumors hinder the effect of combination therapy. Therefore, there is still an urgent need for effective multifunctional adjuvants to improve the efficacy of immunotherapy for cervical cancer.

[0006] The hypoxic microenvironment of tumors can make the free radicals generated by X-rays become unstable, reducing the degree of DNA damage. In addition, the vascular damage caused by radiotherapy will further exacerbate the hypoxia of tumors and activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α can stimulate the expression of DNA-dependent protein kinase, repair the DNA double-strand breaks caused by ionizing radiation, and make tumor cells resistant to radiotherapy. High-Z metal nanoparticles such as platinum (Pt) and gadolinium (Gd) are commonly used radiosensitizers, which can enhance the DNA damage caused by X-rays by increasing the emission of secondary electrons. Pt nanoparticles have catalase-like activity and can catalyze the excessive hydrogen peroxide in the tumor microenvironment to generate oxygen, thus alleviating hypoxia and enhancing the DNA damage effect of radiotherapy. Low levels of immune cell infiltration and the immunosuppressive microenvironment of tumors are another important reason for the failure of radioimmunotherapy. Low levels of immune cell infiltration mainly refer to insufficient numbers of cytotoxic T lymphocytes (CTLs). Dendritic cells (DCs) are considered the basis for establishing the "tumor immune cycle" and are effective CTL stimulants. Certain cytokines in the tumor microenvironment can damage the antigen-presenting ability of DCs by inhibiting DC cell maturation or differentiating DCs into monocytes lacking tumor-presenting function. Highly efficient radiosensitizers can induce ICD, and then release damage-associated molecular patterns (DAMPs) such as calreticulin (CRT), high mobility group box 1 (HMGB1), and adenosine triphosphate (ATP). DAMPs can promote DC activation by releasing different signals and ultimately enhance the effect of immunotherapy by increasing the infiltration level of CTLs. However, radiotherapy will exacerbate the hypoxic microenvironment of tumors, and HIF-1α selectively upregulates PD-L1 by binding to the hypoxia response element in the proximal promoter of PD-L1, thereby reducing the killing effect of CTLs. Therefore, increasing the supply of oxygen in tumors to downregulate the expression level of PD-L1 is an effective strategy to enhance the effect of radio-immunotherapy.

[0007] CTLA-4 is another inhibitory checkpoint molecule that is highly expressed on activated T cells and regulatory T cells. The expression of CTLA-4 is induced after T cell activation and inhibits the uncontrolled expansion of activated T cells. Although both anti-CTLA-4 and anti-programmed cell death protein 1 (PD-1) / PD-L1 therapies can enhance the body's immune response, their mechanisms of action are different. Specifically, CTLA-4 antibodies mainly enhance the clonal proliferation and migration of T cells by relieving the signals that inhibit T cell activation. PD-1 acts downstream of the T cell receptor signal by recruiting and activating protein tyrosine phosphatases 1 and 2 to regulate the effector phase of T cell responses. PD-1 / PD-L1 antibodies are mainly used to maintain the killing activity of CTLs against tumors. Therefore, the mechanisms of action of the two immune checkpoint inhibitors are complementary and act at different stages of the "tumor immune cycle". Immunotherapy targeting both the PD-L1 and CTLA-4 targets is expected to achieve an "immunotherapy effect of 1 + 1 > 2". Summary of the Invention

[0008] In view of the deficiencies of the prior art, in this invention, we constructed a polydopamine (PDA)-coated bimetallic nanoparticle (GdPt@PDA) with PD-L1 inhibition and immune therapy enhancement functions. This nanoparticle has excellent catalase-like activity and can react with the excessive hydrogen peroxide in tumors to release oxygen, thereby downregulating the expression level of HIF-1α. Since HIF-1α can activate its transcription by directly binding to the promoter region of the PD-L1 gene, GdPt@PDA can downregulate the expression level of PD-L1 by inhibiting the expression of HIF-1α and is an effective PD-L1 inhibitor. In addition, GdPt@PDA in combination with radiotherapy can trigger a strong ICD, and the released DAMPs can enhance immune cell infiltration through multiple signals, thereby enhancing the anti-tumor immune response. In a mouse cervical cancer model, GdPt@PDA combined with radiotherapy can improve the tumor immunosuppressive microenvironment and inhibit PD-L1 expression, and achieve dual immune checkpoint blockade therapy in combination with CTLA-4 antibodies.

[0009] To achieve the above-mentioned invention objectives, the present invention provides the following technical solutions.

[0010] The present invention discloses a PD-L1 nano-inhibitor for enhancing cervical cancer immunotherapy, characterized in that the inhibitor is a polydopamine-coated bimetallic nanoparticle with high atomic numbers Z, wherein the bimetallic nanoparticle with high atomic numbers Z is Pt and Gd.

[0011] The present invention also discloses a preparation method of the above-mentioned PD-L1 nano-inhibitor, which is characterized by comprising the following steps: Step 1, GdPt solution: Add 25 mg of gadolinium acetylacetonate and 25 mg of platinum acetylacetonate into 20 mL of diethylene glycol and stir for 40 min. Then add 0.4 g of polyethyleneimine into the above solution and stir for 20 min. Then add 0.5 mL of triethanolamine and stir for 30 min. After stirring, transfer the above solution into a reaction kettle and react at 200 °C for 24 h. Then dilute the obtained reaction solution by 10 times, centrifuge, discard the supernatant, and wash the precipitate three times with pure water and anhydrous ethanol respectively to obtain the GdPt solution. Take 1 mL of the solution, centrifuge and dry it, weigh it, and calculate the mass concentration; Step 2, PD-L1 nano-inhibitor GdPt@PDA: Add 4 mg of GdPt prepared in Step 1 into 20 mL of 10 mM Tris-HCl solution (pH = 8.5) and stir for 30 min. Then slowly drop the mixture prepared by adding 5 mg of dopamine hydrochloride into 20 mL of 10 mM Tris-HCl solution into the above stirred solution. Stir at 25 °C, 600 rpm, and in the dark for 6 h. Then centrifuge the obtained solution, discard the supernatant, and wash the precipitate twice with pure water and anhydrous ethanol respectively to obtain the GdPt@PDA solution, and store it in pure water.

[0012] Further, in Step (1), the stirring condition is stirring at 80 °C and 600 rpm; the centrifugation condition is centrifugation at 11,000 rpm for 8 min.

[0013] Further, in Step (2), the stirring condition is stirring at 25 °C and 600 rpm; the centrifugation condition is centrifugation at 11,000 rpm for 8 min.

[0014] The present invention also discloses the application of the above-mentioned PD-L1 nano-inhibitor or GdPt@PDA prepared by the preparation method of the PD-L1 nano-inhibitor described in any one of the above in the preparation of drugs for treating cervical cancer.

[0015] Further, the drug includes being used in combination with radiotherapy, which reverses the up-regulation of PD-L1 expression induced by radiotherapy and realizes the drug use.

[0016] Further, the drug also includes being combined with an αCTLA-4 antibody to form a dual immune checkpoint blockade therapy.

[0017] Preferably, the dual immune checkpoint blockade therapy can simultaneously inhibit the growth of in-situ tumors and distant metastatic tumors and induce an immune memory effect.

[0018] The present invention also discloses the application of GdPt@PDA prepared by the preparation method of the above-mentioned PD-L1 nano-inhibitor or the PD-L1 nano-inhibitor described in any one of the above in the preparation of a drug for improving the tumor immune microenvironment.

[0019] Furthermore, in the application described in any one of the above, the drug further comprises a pharmaceutically or immunologically acceptable carrier or excipient.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0021] The present invention has successfully prepared a nano-drug GdPt@PDA that can inhibit the expression level of PD-L1 in cervical cancer. GdPt@PDA has high stability and good biocompatibility. GdPt@PDA can down-regulate the expression level of HIF-1α by alleviating the hypoxia of tumor cells, thereby inhibiting the expression level of PD-L1 on cervical cancer cells and effectively realizing immunotherapy. At the same time, GdPt@PDA can also induce ICD and release DAMPs by combining with radiotherapy to improve the tumor immune microenvironment, and cooperate with the immune checkpoint inhibitor αCTLA-4 to achieve dual immune checkpoint blockade, improve the immunotherapy effect, and successfully inhibit the metastasis of tumors.

[0022] The present invention provides a new idea for the immunotherapy of cervical cancer and brings new hope for achieving the long-term control and cure of cervical cancer. Through the strategy of dual immune checkpoint blockade, GdPt@PDA shows great potential in the treatment of cervical cancer and is expected to become an effective anti-tumor treatment plan. In the future, this patent provides important support for carrying out clinical experiments and further clinical applications based on this invention. By further optimizing and improving the preparation method and use conditions of GdPt@PDA, we are expected to push it into clinical application, thereby providing more individualized and effective treatment options for cervical cancer patients. Brief Description of the Drawings

[0023] Figure 1 This is the PD-L1 nano-inhibitor - GdPt@PDA prepared by the present invention. Among them, A is the transmission electron microscope photograph of GdPt@PDA in Example 1, and the scale bar is 500 nm; B is the transmission electron microscope photograph of GdPt@PDA in Example 1, and the scale bar is 20 nm.

[0024] Figure 2 In Example 2, after treatment with GdPt@PDA at different concentrations (0, 10, 20, 40 μg / mL), flow cytometry was used to evaluate the uptake level of HeLa cells for GdPt@PDA at 0, 1, and 4 h.

[0025] Figure 3In Example 2, after treatment with 80 μg / mL of GdPt@PDA, an inductively coupled plasma optical emission spectrometer was used to evaluate the uptake level of GdPt@PDA by HeLa cells at 0, 1, 2, 4, and 8 h.

[0026] Figure 4 In Example 3, the viability of HeLa cells measured by the MTT method after treatment with different concentrations (0, 40, 80, 160 μg / mL) of GdPt@PDA and different doses of X-ray (0, 3, 6 Gy).

[0027] Figure 5 To verify the effect of different concentrations of GdPt@PDA on the expression levels of HIF-1α and PD-L1 in HeLa cells in vitro. Among them, A is the expression level of HIF-1α in HeLa cells after treatment with different concentrations (0, 40, 80, 160 μg / mL) of GdPt@PDA in Example 4; B is the expression level of PD-L1 in HeLa cells after treatment with different concentrations (0, 40, 80, 160 μg / mL) of GdPt@PDA in Example 4.

[0028] Figure 6 To verify the mechanism of GdPt@PDA combined with radiotherapy in improving the tumor immune microenvironment in vitro. Among them, A is the mean fluorescence intensity of CRT in HeLa cells of different treatment groups (control group, GdPt@PDA group, radiotherapy group, and GdPt@PDA + radiotherapy combination treatment group) in Example 5; B is the mean fluorescence intensity of HMGB1 in HeLa cells of different treatment groups (control group, GdPt@PDA group, radiotherapy group, and GdPt@PDA + radiotherapy combination treatment group) in Example 5.

[0029] Figure 7 In Example 5, the protein expression levels of HIF-1α and PD-L1 in HeLa cells of different treatment groups (control group, GdPt@PDA group, radiotherapy group, and GdPt@PDA + radiotherapy combination treatment group).

[0030] Figure 8 To verify the inhibitory effect of GdPt@PDA combined with radiotherapy on subcutaneous transplanted tumors in mice in vivo. Among them, A is the tumor volume change curve of C57BL / 6 mice in different treatment groups (control group, GdPt@PDA group, radiotherapy group, and GdPt@PDA + radiotherapy combination treatment group) in Example 6; B is the post-treatment tumor photos of C57BL / 6 mice in different treatment groups (control group, GdPt@PDA group, radiotherapy group, and GdPt@PDA + radiotherapy combination treatment group) in Example 6.

[0031] Figure 9For the expression levels of PD-L1, CRT, and HMGB1 in tumors of different treatment groups (control group, GdPt@PDA group, radiotherapy group, and GdPt@PDA + radiotherapy combination treatment group) in Example 7, the scale bar is 50 μm.

[0032] Figure 10 To verify the inhibitory effect of GdPt@PDA combined with radiotherapy on orthotopic tumors in vivo. Among them, A is the curve of the change in orthotopic tumor volume of C57BL / 6 mice in different treatment groups (control group, radiotherapy group, radiotherapy + GdPt@PDA combination treatment group, radiotherapy + αCTLA-4 combination treatment group, radiotherapy + GdPt@PDA + αCTLA-4 combination treatment group) in Example 8; B is the photo of the orthotopic tumor after treatment of C57BL / 6 mice in different treatment groups (control group, radiotherapy group, radiotherapy + GdPt@PDA combination treatment group, radiotherapy + αCTLA-4 combination treatment group, radiotherapy + GdPt@PDA + αCTLA-4 combination treatment group) in Example 8.

[0033] Figure 11 To verify the inhibitory effect of GdPt@PDA combined with radiotherapy on distant tumors in vivo. Among them, A is the curve of the change in distant tumor volume of C57BL / 6 mice in different treatment groups (control group, radiotherapy group, radiotherapy + GdPt@PDA combination treatment group, radiotherapy + αCTLA-4 combination treatment group, radiotherapy + GdPt@PDA + αCTLA-4 combination treatment group) in Example 8; B is the photo of the distant tumor after treatment of C57BL / 6 mice in different treatment groups (control group, radiotherapy group, radiotherapy + GdPt@PDA combination treatment group, radiotherapy + αCTLA-4 combination treatment group, radiotherapy + GdPt@PDA + αCTLA-4 combination treatment group) in Example 8.

[0034] Figure 12 For Example 9, flow cytometry was used to detect the proportions of DC cells, CD8+ T cells, and CD4+ T cells in distant tumors of C57BL / 6 mice in different treatment groups (control group, radiotherapy group, radiotherapy + GdPt@PDA combination treatment group, radiotherapy + αCTLA-4 combination treatment group, radiotherapy + GdPt@PDA + αCTLA-4 combination treatment group).

[0035] Figure 13 For Example 10, H&E staining was used to evaluate the safety of GdPt@PDA combined with radiotherapy on various important organs of C57BL / 6 mice, and the scale bar is 100 μm. Detailed implementation manners

[0036] The following is a further detailed description of the present invention with specific embodiments. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following embodiments. All technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0037] Unless otherwise specified, the reagents and materials used in the present invention are all commercially available.

[0038] Example 1.

[0039] In this example, GdPt@PDA was constructed by the following method, which is

[0040] (1) Add 25 mg of gadolinium acetylacetonate and 25 mg of platinum acetylacetonate to 20 mL of diethylene glycol, and stir at 80 °C and 600 rpm for 40 min. Then add 0.4 g of polyethyleneimine to the above solution, and stir at 80 °C and 600 rpm for 20 min. Then add 0.5 mL of triethanolamine, and stir at 80 °C and 600 rpm for 30 min. After stirring, transfer the above solution into a reaction kettle and react at 200 °C for 24 h. After the reaction, dilute the original solution 10 times in pure water, centrifuge at 11000 rpm for 8 min, remove the supernatant, and resuspend with pure water. Wash three times with pure water and anhydrous ethanol respectively to obtain a GdPt solution, which is stored in pure water. Take 1 mL of the solution, centrifuge, dry and weigh it to calculate the mass concentration.

[0041] (2) Add 4 mg of GdPt to 20 mL of 10 mM Tris-Hcl solution (pH = 8.5), and stir at 25 °C and 600 rpm for 30 min. Then add 5 mg of dopamine hydrochloride to 20 mL of 10 mM Tris-Hcl solution, and slowly drop it into the above solution, and stir at 25 °C, 600 rpm and in the dark for 6 h. After the reaction, centrifuge the original solution at 11000 rpm for 8 min, remove the supernatant, and resuspend with pure water. Wash twice with pure water and anhydrous ethanol respectively to obtain a GdPt@PDA solution, which is stored in pure water.

[0042] (3) Use a transmission electron microscope (HT7800, Hitachi, Japan) to perform morphological characterization on the obtained GdPt@PDA, as Figure 1 shown, GdPt is a nanoparticle with a particle size of about 30 - 40 nm. After being wrapped with PDA, GdPt@PDA is distributed in clusters. The electron microscope results indicate that we have successfully prepared GdPt@PDA.

[0043] Example 2.

[0044] The purpose of this example is to verify the uptake effect of GdPt@PDA by HeLa cells in vitro, and the method is as follows.

[0045] (1) Seed HeLa cells in a 6-well plate (3×10 5 cells / well) and culture for 24 h. Subsequently, treat the cells with GdPt@PDA-Cy5.5 at different concentrations (0, 10, 20, and 40 μg / mL) for 1 h or 4 h. After treatment, wash the cells with PBS, digest with trypsin, and finally detect by flow cytometry (Attune NxT, Thermo Fisher Scientific, USA). As Figure 2 shown, with the increase of concentration and time, the uptake of GdPt@PDA by HeLa cells increases.

[0046] (2) Seed HeLa cells in a 6-well plate (3×10 5 cells / well) and culture for 24 h. Subsequently, treat with 80 μg / mL of GdPt@PDA for 0, 1, 2, 4, and 8 h respectively. After treatment, wash the cells with PBS, digest with trypsin, treat with aqua regia nitrification, and finally detect by inductively coupled plasma emission spectrometer (G8018A, Agilent, USA). As Figure 3 shown, with the increase of time, the uptake of GdPt@PDA by HeLa cells increases and reaches the peak at 4 h.

[0047] Example 3.

[0048] The purpose of this example is to verify the killing effect of GdPt@PDA on cervical cancer cells in vitro, and the method is as follows.

[0049] Detect the killing effect of GdPt@PDA combined with X-ray on cervical cancer cell lines by MTT method. Seed HeLa cells in a 96-well plate (100 μL per well, containing 10,000 cells) and culture for 24 h. Subsequently, co-incubate the cells with GdPt@PDA at different concentrations (0, 20, 40, 80, and 160 μg / mL) for 6 h. After replacing the fresh medium, irradiate with different doses (0, 3, and 6 Gy) of X-ray respectively. After continuing to culture for 48 h, add MTT solution (20 μL, 5 mg / mL) and co-incubate for 4 h. Subsequently, discard the MTT solution, add 150 μL of DMSO and shake for about 15 min. Use a microplate reader (CMax Plus, Molecular Devices, USA) to measure the absorbance. As Figure 4As shown, when the concentration of GdPt@PDA was 80 and 160 μg / mL, the cell viability was approximately 78.76% and 70.61%, indicating that GdPt@PDA had a certain inhibitory ability on the proliferation of HeLa cells. In addition, when the concentration of GdPt@PDA reached 160 μg / mL and the X-ray dose was 6 Gy, the viability of HeLa cells decreased to 42.95% (when irradiated with 6 Gy of X-ray alone, the cell viability was approximately 91.35%), indicating that GdPt@PDA also had a significant radiosensitizing effect.

[0050] The radiotherapy in this example was completed by an X-ray biological irradiator (RS2000 Pro, Rod Source technologies Asia Limited, USA).

[0051] Example 4.

[0052] The purpose of this example was to verify the effects of different concentrations of GdPt@PDA on the expression levels of HIF-1α and PD-L1 in HeLa cells in vitro. The method was as follows: HeLa cells were seeded in culture dishes (3×10 5 cells / dish), and after 24 h of culture, different concentrations of GdPt@PDA (0, 40, 80, 160 μg / mL) were added respectively. After further culturing for 24 h, the cells were lysed with RIPA lysis buffer to extract total protein, and the protein concentration was measured using a BCA protein concentration detection kit. The proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a PVDF membrane. After blocking with 5% skim milk at room temperature for 2 h, they were incubated overnight at 4 °C with GAPDH (internal reference), HIF-1α and PD-L1 primary antibodies in sequence; washed 3 times with TBST (10 min each time), and then incubated with the corresponding HRP-labeled secondary antibody at room temperature for 2 h. Finally, the protein bands were detected by chemiluminescence. As Figure 5 shown in FIGS. 5A and 5B, when the concentration of GdPt@PDA was 40, 80, 160 μg / mL, the expression levels of HIF-1α in HeLa cells decreased to 0.64, 0.53 and 0.39 times that of the control group, respectively, and the expression levels of PD-L1 decreased to 0.88, 0.66 and 0.46 times that of the control group, respectively. Therefore, with the increase in the concentration of GdPt@PDA, the expression levels of both HIF-1α and PD-L1 in HeLa cells gradually decreased, demonstrating that GdPt@PDA effectively inhibited PD-L1 by regulating HIF-1α.

[0053] Example 5.

[0054] The purpose of this example was to verify the mechanism of GdPt@PDA combined with radiotherapy in improving the tumor immune microenvironment in vitro. The method was as follows.

[0055] (1) Seed HeLa cells in confocal dishes (3×10 5 cells / dish) and culture for 24 h, then perform the following treatments respectively: treat with 0 or 80 μg / mL GdPt@PDA for 6 h; receive 0 or 6 Gy X-ray irradiation. After 24 h of treatment, perform the following steps in sequence: ① fix with 4% paraformaldehyde for 30 min; ② permeabilize with 0.2% TritonX-100 for 30 min; ③ block with 5% BSA for 1 h; ④ incubate with CRT or HMGB1 primary antibody overnight at 4 °C; ⑤ incubate with corresponding fluorescent secondary antibody for 2 h at room temperature in the dark (wash 3 times with PBS between each step). Finally, observe using a confocal laser scanning microscope (DMi8, Leica, Germany). As Figure 6 shown in A and 6B, the fluorescence intensity of CRT in the GdPt@PDA + radiotherapy combined treatment group was the highest (about 10.27 times that of the control group), while the fluorescence intensity of HMGB1 was the lowest (about 0.42 times that of the control group), indicating that GdPt@PDA + radiotherapy successfully induced ICD, promoted the exposure of CRT and the release of HMGB1.

[0056] (2) Seed HeLa cells in culture dishes (3×10 5 cells / dish), and perform different treatments after 24 h of culture (the treatment method is the same as above). Continue to culture for 24 h, lyse the cells with RIPA lysis buffer to extract total protein, and measure the protein concentration using a BCA protein concentration detection kit. Separate the proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer them to a PVDF membrane. After blocking with 5% skim milk at room temperature for 2 h, incubate with GAPDH (internal reference), HIF-1α and PD-L1 primary antibodies overnight at 4 °C in sequence; wash 3 times with TBST (10 min each time), and then incubate with HRP-labeled secondary antibody of the corresponding species for 2 h at room temperature. Finally, detect the protein bands using chemiluminescence method. As Figure 7 shown, the expression levels of PD-L1 in the GdPt@PDA group, radiotherapy group and GdPt@PDA + radiotherapy group were about 0.73, 1.17 and 0.86 times that of the control group respectively, and the expression levels of HIF-1α were about 0.67, 1.37 and 0.83 times that of the control group respectively. It shows that GdPt@PDA effectively reversed the up-regulation of PD-L1 expression induced by X-ray by regulating the HIF-1α-PD-L1 axis.

[0057] Example 6.

[0058] The purpose of this example is to verify the inhibitory effect of GdPt@PDA combined with radiotherapy on subcutaneous transplanted tumors in mice in vivo, and the method is as follows.

[0059] Seed TC-1 cells (2×10 6Cells / mouse) were subcutaneously injected into the flanks of female C57BL / 6 mice to establish a xenograft tumor model. When the average tumor volume reached 80 - 100 mm 3 At this time, the tumor-bearing mice were randomly divided into four groups (n = 5): control group, GdPt@PDA group, radiotherapy group, and GdPt@PDA + radiotherapy combination treatment group. Treatment was started according to the predetermined protocol: On the 0th and 4th days after treatment, GdPt@PDA (100 μL, 10 mg / kg) was injected via the tail vein. Four hours after injection, local tumor irradiation was performed with 6 Gy of ionizing radiation. The tumor volume was measured every two days (calculation formula: tumor volume = 0.5 × long diameter × short diameter 2 ). The experiment was terminated on the 10th day of treatment. After sacrificing the mice, the tumor tissues were surgically removed and photographed for record. As Figure 8 shown in A and 8B, the average tumor volumes of the control group, GdPt@PDA group, radiotherapy group, and GdPt@PDA + radiotherapy combination treatment group were 425.69, 338.18, 270.83, and 147.83 mm 3 respectively. The tumor volume of the GdPt@PDA + radiotherapy combination treatment group was the smallest, indicating that GdPt@PDA has good tumor inhibitory effects and excellent radiotherapy sensitization effects.

[0060] Example 7.

[0061] The purpose of this example is to verify the mechanism of GdPt@PDA in inhibiting PD-L1 and improving the tumor immune microenvironment in vivo, and the method is as follows.

[0062] Tumor tissues of the tumor-bearing mice in Example 6 were collected and made into sections for immunohistochemical staining. The steps included baking the sections, dewaxing, exposing antigenic determinants, incubating with primary antibodies, blocking, DAB oxidation and color development, hematoxylin counterstaining, dehydration, and mounting. The primary antibodies used included PD-L1, CRT, and HMGB1. As Figure 9 shown, the results showed that the expression level of PD-L1 in the radiotherapy group was higher than that in the control group, while the combined use of GdPt@PDA effectively reversed this trend. In addition, compared with other groups, the exposure of CRT in the GdPt@PDA + radiotherapy group increased, and the expression of HMGB1 in the cell nucleus decreased. These results indicate that the degradation of PD-L1 mediated by GdPt@PDA and the enhanced release of DAMPs (CRT exposure and HMGB1 secretion) jointly improved the tumor immune microenvironment.

[0063] Example 8.

[0064] The purpose of this example is to verify the inhibitory effect of GdPt@PDA combined with radiotherapy on distant tumors in vivo, and the method is as follows.

[0065] TC-1 cells (2 × 10 6TC-1 cells (2×10 6 The remote tumor model was established by subcutaneously injecting 100 cells / mouse into the contralateral abdomen of mice. 3 At the time of treatment, the tumor-bearing mice were randomly divided into five groups (n=4): control group, radiotherapy group, GdPt@PDA+radiotherapy group, αCTLA-4+radiotherapy group, and GdPt@PDA+αCTLA-4+radiotherapy group. Treatment was started according to the predetermined plan: GdPt@PDA (100μL, 10mg / kg) was injected through the tail vein on the 0th, 4th, and 10th day after treatment, and 6Gy ionizing radiation was irradiated to the tumor locally 4h after injection; αCTLA-4 (200μg / mouse) was injected intraperitoneally on the 1st, 5th, and 11th day after treatment. The tumor volume was measured every three days (calculation formula: tumor volume = 0.5×long diameter×short diameter 2 The experiment was terminated on the 18th day of treatment, and the mice were killed and the tumor tissues were surgically removed and photographed. Figure 10 As shown in A and 10B, the average tumor volumes of the control group, radiotherapy group, GdPt@PDA+radiotherapy group, αCTLA-4+radiotherapy group, and GdPt@PDA+αCTLA-4+radiotherapy group were 886.78, 524.29, 361.14, 342.23, and 176.75 mm, respectively. 3 The in situ tumor volume in the GdPt@PDA+αCTLA-4+radiotherapy group was the smallest, indicating that GdPt@PDA combined with radiotherapy effectively enhanced the immunotherapy effect of αCTLA-4. Figure 11 As shown in A and 11B, the average tumor volumes of the control group, radiotherapy group, GdPt@PDA+radiotherapy group, αCTLA-4+radiotherapy group, and GdPt@PDA+αCTLA-4+radiotherapy group were 623.14, 473.67, 447.21, 389.60, and 210.16 mm, respectively. 3 The distant tumor volume in the GdPt@PDA+αCTLA-4+radiotherapy group was the smallest, indicating that GdPt@PDA combined with radiotherapy effectively induced the distant effect and synergized with αCTLA-4 to successfully inhibit the growth of metastatic tumors.

[0066] The αCTLA-4 used in this example, namely InVivoMAb anti-mouse CTLA-4 (CD152), was purchased from BioXCell (Lebanon, USA).

[0067] Example 9.

[0068] The purpose of this example is to verify in vivo whether GdPt@PDA combined with radiotherapy promotes immune cell infiltration through the remote effect, and the method is.

[0069] For immune cell analysis, distant tumor tissues of the tumor-bearing mice in Example 8 were collected. After digestion with collagenase A and hyaluronidase at 37 °C for 40 min, red blood cells were removed using red blood cell lysis buffer, and the single-cell suspension was resuspended with HBSS to a final volume of 1 mL. For intracellular cytokine staining, tumor-derived cells were permeabilized with 0.1% TritonX-100 for 15 min, and the cell density was adjusted to 5×10 6 / mL. 100 μL of the cell suspension was incubated with fluorescently labeled antibodies (CD45, CD3, CD4, CD8, CD11c, CD86) in the dark. Data were collected using a flow cytometer (Aurora, Cytek Biosciences, USA), and the proportions of immune cell subsets were analyzed. As Figure 12 shown, the proportions of DC cells, CD8 + T cells, and CD4 + T cells in the GdPt@PDA + αCTLA-4 + radiotherapy group were the highest (35.5%, 25.2%, and 34.9%, respectively), indicating that this treatment regimen successfully promoted the maturation of DC cells, thereby increasing the infiltration of immune cells such as CD8 and CD4 in the tumor and enhancing the immunotherapeutic effect of αCTLA-4.

[0070] Example 10.

[0071] The purpose of this example is to verify the biosafety of GdPt@PDA, and the method is as follows.

[0072] To evaluate systemic toxicity, after sacrificing the mice in Example 6, the heart, liver, spleen, lung, and kidney tissues were completely excised and quickly fixed in 4% paraformaldehyde solution for 24 h. After dehydration with gradient ethanol and paraffin embedding, 4-μm-thick sections were prepared and stained with hematoxylin-eosin. As Figure 13 shown, no obvious damage was observed in the important organs of the mice, indicating that GdPt@PDA has good biosafety.

[0073] The above are only the preferred embodiments of the present invention and are not used to limit the patent scope of the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A PD-L1 nano-inhibitor for enhancing the immunotherapy of cervical cancer, characterized in that, The inhibitor is polydopamine-coated high-Z metal nanoparticles, where the high-Z metal is Pt and Gd. The GdPt@PDA has catalase-like activity and can catalyze the decomposition of hydrogen peroxide in the tumor microenvironment into oxygen, thereby alleviating hypoxia and downregulating the expression levels of HIF-1α and PD-L1.

2. The preparation method of the PD-L1 nano-inhibitor according to claim 1, characterized in that, It includes the following steps: Step 1, GdPt solution: Add 25 mg of gadolinium acetylacetonate and 25 mg of platinum acetylacetonate to 20 mL of diethylene glycol and stir for 40 min. Then add 0.4 g of polyethyleneimine to the above solution and stir for 20 min. Next, add 0.5 mL of triethanolamine and stir for 30 min. After stirring, transfer the above solution to a reaction kettle and react at 200 °C for 24 h. Then dilute the obtained reaction solution by 10 times, centrifuge, discard the supernatant, and wash the precipitate three times with pure water and anhydrous ethanol respectively to obtain the GdPt solution. Take 1 mL of the GdPt solution, centrifuge, dry, and weigh it to calculate the mass concentration. Step 2, PD-L1 nano-inhibitor GdPt@PDA: Add 4 mg of GdPt prepared in Step 1 to 20 mL of 10 mM Tris-Hcl solution (pH = 8.5) and stir for 30 min. Then slowly drop the mixture prepared by adding 5 mg of dopamine hydrochloride to 20 mL of 10 mM Tris-Hcl solution into the above stirred solution. Stir at 25 °C, 600 rpm, and in the dark for 6 h. Then centrifuge the obtained solution, discard the supernatant, and wash the precipitate twice with pure water and anhydrous ethanol respectively to obtain the GdPt@PDA solution, which is stored in pure water.

3. The preparation method according to claim 2, characterized in that, The stirring condition in Step (1) is to stir at 80 °C and 600 rpm; the centrifugation condition is to centrifuge at 11000 rpm for 8 min.

4. The preparation method according to claim 2, characterized in that, The stirring condition in Step (2) is to stir at 25 °C and 600 rpm; the centrifugation condition is to centrifuge at 11000 rpm for 8 min.

5. Use of GdPt@PDA prepared by the PD-L1 nano-inhibitor according to Claim 1 or the preparation method of the PD-L1 nano-inhibitor according to any one of Claims 2 to 4 in the preparation of a cervical cancer treatment drug.

6. Use according to claim 5 in a medicament, characterized in that, The drug includes being used in combination with radiotherapy, which reverses the radiotherapy-induced upregulation of PD-L1 expression to achieve the drug use.

7. The application according to claim 5, wherein The drug also includes being combined with an αCTLA-4 antibody to form a dual immune checkpoint blockade therapy.

8. The application according to claim 7, wherein The dual immune checkpoint blockade therapy can simultaneously inhibit the growth of in-situ tumors and distant metastatic tumors and induce an immune memory effect.

9. Use of GdPt@PDA prepared by the PD-L1 nano-inhibitor according to Claim 1 or the preparation method of the PD-L1 nano-inhibitor according to any one of Claims 2 to 4 in the preparation of a drug for improving the tumor immune microenvironment.

10. The application according to any one of claims 5 to 9, characterized in that, The drug also includes a pharmaceutically or immunologically acceptable carrier or excipient.

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