Preparation method of tumor microenvironment response type nano drug delivery system

Abstract

The invention relates to a preparation method of a tumor microenvironment response type nano drug delivery system, which comprises the following steps: forming MnO2 nanoclusters through biomineralization induction under an alkaline condition, modifying HSA nanoparticles, then loading Ce6 through a carbodiimide method, and finally combining with a PD-L1 antibody by adopting a copper-free click chemistry method to obtain nanoparticles HMC and anti-PD-L1. The nanoparticle can be retained in a tumor tissue in a passive targeting manner through an EPR effect, and is decomposed into Mn < 2 + >, O2 and a small molecule drug through a high H2O2 and low pH microenvironment of the tumor tissue, so that the tumor permeability is greatly enhanced, the tumor immunogenicity is enhanced by combining sonodynamic therapy, the release of a tumor specific antigen and the aging of antigen presenting cells are promoted, and the tumor treatment effect is enhanced. The infiltration of tumor T cells is increased, and cold tumors are changed into hot tumors; the nanoparticles can generate O2 in situ at the same time, and the immunosuppressive ability and drug resistance caused by tumor hypoxia are overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine preparation, and in particular to a method for preparing a tumor microenvironment responsive nano drug delivery system. Background Art

[0002] Immunotherapy is a new approach to tumor treatment that trains or stimulates the host's innate immune system to identify and attack tumor cells. However, most cancer patients do not benefit from these treatments. The main reasons are: (1) insufficient production of tumor antigens, which makes it difficult to stimulate the body's immune system to produce a large number of cytotoxic T cells to kill tumor cells; (2) low efficiency of tumor antigen expression, which reduces the number of mature dendritic cells that form tumor antigen presenting cells; (3) immunosuppression caused by hypoxia, low pH, and high H2O2 in the tumor microenvironment. These problems will weaken the efficacy of immunotherapy and hinder its clinical application. Therefore, how to improve the production and presentation efficiency of tumor immune antigens has become the main problem facing tumor immunotherapy.

[0003] In response to the above problems, recent studies have found that immunotherapy combined with chemotherapy, radiotherapy, photodynamic therapy, sonodynamic therapy and other tumor treatment methods can not only directly kill tumor cells, but also induce immunogenic cell necrosis, that is, necrotic tumor cells release tumor immune antigens and trigger immune responses, improve T cell responses of different tumors, and promote the transformation of "cold tumors" into "hot tumors" with immunogenicity, improve the patient's immunotherapy response rate, and induce immune responses to treat primary and metastatic tumors. Sonodynamic therapy (SDT) is a promising method for treating malignant tumors. It can penetrate the surface of the skin and focus on deep target tissues. It can activate sonosensitizers to produce ROS to kill tumor cells, induce ICD effects, increase the infiltration of immune effector cells, and effectively improve the patient's treatment response rate. However, the tumor immunosuppressive microenvironment will destroy the cytotoxic T cells produced by the ICD effect, inhibiting the effect of tumor immunotherapy. PD-L1 inhibitors can overcome the tumor immunosuppressive microenvironment and are a new type of anticancer immunotherapy that is currently attracting much attention and widely studied around the world. It can block the binding of PD-L1 highly expressed on the surface of tumor cells with PD-1 of T cells, enhance T cells' recognition of tumor cells, activate their attack and killing functions, and mobilize the body's own immune function to achieve anti-tumor effects.

[0004] However, the complex structure of tumor tissues and their high heterogeneity not only provide a driving force for tumor invasion and metastasis, but also become a biological barrier for the delivery of many anticancer drugs in cancer treatment. Although the nano drug delivery system technology can use the EPR effect to pass through tumor blood vessels and deliver to the tumor tissue gap, the macromolecular nano drug delivery system is difficult to penetrate the complex and dense extracellular matrix of the tumor, and the highly heterogeneous microenvironment in the tumor causes the fluid pressure in the tumor tissue gap to increase, and the extracellular matrix is ​​tortuous and dense, so that the drug is only enriched in the local peripheral tumor area, and it is difficult to penetrate to the deep tumor. Therefore, the nano drug delivery system loaded with PD-L1 antibodies cannot fully inhibit the PD-L1 expressed by tumor cells, and tumor cells will still turn on the defense system to "deceive" T cells, thereby successfully avoiding the recognition and attack of T cells. How to promote the nano drug delivery system loaded with PD-L1 antibodies to effectively penetrate the highly heterogeneous interstitial tissue of the tumor, reach the deep part of the tumor tissue, and fully and efficiently bind to the PD-L1 expressed on tumor cells is a key problem that needs to be solved urgently. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for preparing a tumor microenvironment responsive nano-drug delivery system, which improves drug delivery efficiency, enhances tumor immunogenicity, overcomes the immunosuppressive microenvironment, and thereby improves the tumor immunotherapy effect.

[0006] To achieve the above object, the technical solution adopted by the present invention is to provide a method for preparing a tumor microenvironment responsive nano drug delivery system, wherein the nano drug delivery system is a conjugate of PD-L1 antibody, MnO2 and Ce6 coupled to HSA nanoparticles, and the preparation method of the nano drug delivery system comprises the following steps:

[0007] (1) First, HSA nanoparticles were dissolved in deionized water to prepare a HSA solution with a concentration of 10 mg / mL, KMnO4 solution was added to the HSA solution, and the reaction was continued in a water bath at 25°C for 4 hours, and the obtained HSA-manganese particles were dialyzed and ultrafiltered for later use;

[0008] (2) reacting HSA-manganese particles and DBCO-NHS at room temperature overnight; then removing free DBCO-NHS by dialysis ultrafiltration to obtain DBCO-modified HSA-manganese particles, i.e., HSA-manganese particles-DBCO;

[0009] (3) Weigh 15 mg of Ce6 powder, 9.6 mg of EDC, and 10.9 mg of NHS, take 1 mL of DMSO to completely dissolve the three, and stir for 4 hours in a dark place to obtain a Ce6 activated sample; take HSA-manganese particle-DBCO and 0.2 mL of Ce6 activated sample and stir overnight, then dialyze and ultrafilter to remove free Ce6 to obtain HSA-manganese ion-Ce6 surface modified DBCO, i.e. DBCO-HMC;

[0010] (4) activating the thiol group of the PD-L1 antibody using Traut's reagent, and then stirring with N3-PEG-Mal to obtain N3-PEG-PD-L1 antibody;

[0011] (5) The N3-PEG-PD-L1 antibody was subjected to a copper-free click reaction with DBCO-HMC to couple the PD-L1 antibody to the HSA nanoparticles, thereby obtaining the nanoparticles HMC@anti-PD-L1.

[0012] A further improvement to the above technical solution is that in step (1), 150 mg of HSA nanoparticles are dissolved in 15 mL of deionized water to prepare an HSA solution with a concentration of 10 mg / mL, and 3 mL of a 20 mM KMnO4 solution is added to the HSA solution.

[0013] A further improvement to the above technical solution is to detect the obtained nanoparticles HMC@anti-PD-L1 to determine whether the HSA nanoparticles are successfully loaded with MnO2, Ce6 and PD-L1 antibodies; the detection content includes the morphological structure, particle size, potential, optical properties, drug encapsulation rate of the nanoparticles HMC@anti-PD-L1 and biosafety detection in cells and in vivo.

[0014] A further improvement to the above technical solution is that the particle size of the nanoparticles HMC@anti-PD-L1 was measured to be 409.08 to 508.32 nm using the DLS method.

[0015] A further improvement to the above technical solution is that the nanoparticles HMC@anti-PD-L1 were observed to be spherical in shape using TEM.

[0016] A further improvement to the above technical solution is that the results of X-ray photoelectron spectroscopy analysis showed that the peaks of Mn(2p1 / 2) and Mn(2p3 / 2) were detected at binding energies of around 652.9 eV and 641.8 eV, indicating that the Mn element in the synthesized MnO2 was in valence state IV.

[0017] A further improvement to the above technical solution is that the encapsulation efficiency and drug loading of MnO2 in the nanoparticles HMC@anti-PD-L1 were measured by ICP-OES, which were 63% and 1.21% respectively; the encapsulation efficiency and drug loading of PD-L1 antibody in the nanoparticles HMC@anti-PD-L1 were measured by ELISA experiments, which were 78.98% and 1.46% respectively; and the encapsulation efficiency and drug loading of Ce6 in the nanoparticles HMC@anti-PD-L1 were found to be 48% and 3.1% respectively by UV-visible absorption spectrum analysis.

[0018] A further improvement to the above technical solution is to perform HE staining on major organs to study the potential toxicity of the nanoparticle HMC@anti-PD-L1. The HE staining results did not reveal any histopathological abnormalities, lesions, or degeneration.

[0019] The above technical solution is further improved by changing the physical and chemical properties of nanoparticles HMC@antiPD-L1, such as structure, particle size, potential, etc., in vitro in a simulated tumor microacid environment and H2O2 environment, and the Mn 2+ , qualitative and quantitative analysis of O2 release is used to determine whether the nanoparticles HMC@anti-PD-L1 have the ability of responsive release in the micro-acidic environment of the tumor.

[0020] The beneficial effects of the present invention are:

[0021] The tumor microenvironment responsive nano drug delivery system of the present invention uses HSA nanoparticles as carriers, forms MnO2 nanoclusters by biomineralization in alkaline conditions, modifies HSA nanoparticles, and then loads Ce6 by carbodiimide method, and finally combines PD-L1 antibody by copper-free click chemistry method to obtain nanoparticles HMC@anti-PD-L1. This nanoparticle can be retained in tumor tissue by "passive targeting" through the EPR effect, and decomposed into Mn in the high H2O2 and low pH microenvironment of tumor tissue. 2+ , O2 and small molecule drugs, on the one hand, the nanoparticles are gradually degraded into small-sized (<10nm) personalized therapeutic albumin drug complexes, which greatly enhance the intratumor permeability, improve the sonodynamic therapy to synergistically enhance the immunogenicity of the tumor, promote the release of tumor-specific antigens and the maturation of antigen-presenting cells, increase the infiltration of tumor T cells, and turn the "cold tumor" with low immunogenicity into the "hot tumor" with high immunogenicity; on the other hand, the nanoparticles can also generate O2 in situ at the same time, overcome the immunosuppression and drug resistance caused by tumor hypoxia, and further enhance the efficacy of sonodynamic therapy. In addition, Ce6, O2 and Mn 2+ The release of can be achieved through magnetic resonance imaging to achieve visualized tumor precision treatment under molecular imaging monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the particle size distribution diagram of nanoparticles HMC@anti-PD-L1.

[0023] Figure 2 It is the potential diagram of nanoparticles HMC@anti-PD-L1.

[0024] Figure 3 This is a transmission electron microscopy image of the nanoparticle HMC@anti-PD-L1.

[0025] Figure 4This is the X-ray photoelectron spectrum of nanoparticles HMC@anti-PD-L1.

[0026] Figure 5 are the UV-vis-NIR absorption spectra of nanoparticles HC, nanoparticles HM, and nanoparticles HMC@anti-PD-L1.

[0027] Figure 6 This is the hydrodynamic diameter curve of nanoparticles HMC@anti-PD-L1 in PBS solution for 7 days.

[0028] Figure 7 These are the TEM images of nanoparticles HMC@anti-PD-L1 at different times under weak acid / H2O2 conditions.

[0029] Figure 8 This is a curve of particle size change of nanoparticles HMC@anti-PD-L1 at different times under weak acid / H2O2 conditions.

[0030] Fig. 9 This is a graph showing the changes in the retention amount of MnO2 in the nanoparticles HMC@anti-PD-L1 in different solutions.

[0031] Fig.10 This is a graph showing the changes in drug release of nanoparticles HMC@anti-PD-L1 at different times under normal physiological conditions and simulated tumor microenvironment.

[0032] Fig.11 This is a graph showing the changes in oxygen release of nanoparticles HMC@anti-PD-L1 in different solutions.

[0033] Fig.12 This is a macroscopic diagram of oxygen release of different reagents in a simulated tumor microenvironment.

[0034] Fig.13 This is a graph showing the changes in ROS production of nanoparticles HMC@anti-PD-L1 in different solutions.

[0035] Fig.14 This is a graph showing changes in cell activity at different concentrations of nanoparticles HMC@anti-PD-L1.

[0036] Fig.15 This is a diagram showing the deep delivery effect of nanoparticles HMC@anti-PD-L1 in 4T1 3D cell spheroids under weak acid / H2O2 conditions.

[0037] Fig.16A This is a diagram showing the deep delivery effect of nanoparticles HMC@anti-PD-L1 into tumor tissues via intratumoral injection.

[0038] Fig. 16BThis is a diagram showing the deep delivery effect of nanoparticles HMC@anti-PD-L1 into tumor tissue after intravenous injection.

[0039] Fig.17A This is a schematic diagram of the experimental design for evaluating the distal effects of nanoparticles HMC@anti-PD-L1 combined with sonodynamic therapy.

[0040] Fig. 17B It is the growth curve of orthotopic tumors in mice of different treatment groups.

[0041] Fig. 17C It is the growth curve of distal tumors in mice of different treatment groups.

[0042] Fig.17D It is the survival curve of mice in different treatment groups.

[0043] Fig.17E It is the body weight change curve of mice in different treatment groups.

[0044] Fig.18 These are the CRT immunofluorescence sections in the tumors of different treatment groups.

[0045] Fig.19 These are the flow cytometric graphs and statistical analysis graphs of immune T cells in tumors of different treatment groups.

[0046] Fig. 20 This is the ELISA analysis of intratumoral immune cytokines in different treatment groups.

[0047] Fig.21A This is a schematic diagram of immunotherapy in a tumor model after tumor treatment.

[0048] Fig. 21B This is a graph showing the growth curve of tumor size in different treatment groups after tumor re-immunotherapy.

[0049] Fig. 21C This is a graph showing changes in body weight during tumor re-immunotherapy in different treatment groups.

[0050] Fig.21D This is a graph of the survival rates of tumors in different treatment groups after tumor re-immunotherapy.

[0051] Fig. 22 Figure 2 is the flow cytometric plot and statistical analysis of tumor immune memory T cells in different treatment groups. DETAILED DESCRIPTION

[0052] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] The present invention relates to a method for preparing a tumor microenvironment responsive nano drug delivery system, wherein the nano drug delivery system is a conjugate of PD-L1 antibody, MnO2 and Ce6 coupled to HSA nanoparticles.

[0054] The preparation method of the nano drug delivery system comprises the following steps:

[0055] (1) First, 150 mg of HSA (human serum albumin) nanoparticles were dissolved in 15 mL of deionized water to prepare a 10 mg / mL HSA solution, 3 mL of a 20 mM KMnO4 solution was added to the HSA solution, and the reaction was continued in a 25°C water bath for 4 hours. The obtained HSA-manganese particles were dialyzed and ultrafiltered for later use;

[0056] (2) reacting HSA-manganese particles and DBCO-NHS (diphenylcyclooctyne-active ester) at room temperature overnight; then removing free DBCO-NHS by dialysis ultrafiltration to obtain DBCO-modified HSA-manganese particles, i.e., HSA-manganese particles-DBCO;

[0057] (3) Weigh 15 mg of Ce6 powder, 9.6 mg of EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride), and 10.9 mg of NHS (N-hydroxysuccinimide), and dissolve them completely in 1 mL of DMSO (dimethyl sulfoxide), and stir for 4 hours in a dark place to obtain a Ce6 activated sample; stir HSA-manganese particle-DBCO and 0.2 mL of Ce6 activated sample overnight, and then remove free Ce6 by dialysis ultrafiltration to obtain HSA-manganese particle-Ce6 surface modified DBCO, namely DBCO-HMC;

[0058] (4) activating the thiol group of the PD-L1 antibody using Traut's (2-iminothiolane hydrochloride) reagent, and then stirring with N3 (azido)-PEG-Mal to obtain N3-PEG-PD-L1 antibody;

[0059] (5) N3-PEG-PD-L1 antibody and DBCO-HMC were subjected to a copper-free click reaction to couple the PD-L1 antibody to the HSA nanoparticles, thereby obtaining the nanoparticles HMC@anti-PD-L1;

[0060] (6) The obtained nanoparticles HMC@anti-PD-L1 were tested to determine whether the HSA nanoparticles were successfully loaded with MnO2, Ce6 and PD-L1 antibodies; the changes in the physical and chemical properties of the nanoparticles HMC@antiPD-L1, such as structure, particle size, potential, etc., under in vitro simulated tumor microacid environment and H2O2 environment, as well as the changes in MnO2, Ce6 and PD-L1 antibodies were detected. 2+ , qualitative and quantitative analysis of O2 release is used to determine whether the nanoparticles HMC@anti-PD-L1 have the ability of responsive release in the micro-acidic environment of the tumor.

[0061] In step (6), the detection content of the nanoparticle HMC@anti-PD-L1 includes the morphological structure, particle size, potential, optical properties, drug encapsulation efficiency and biosafety detection in cells and in vivo of the nanoparticle HMC@anti-PD-L1.

[0062] Combination Figure 1 As shown, the particle size of nanoparticles HMC@anti-PD-L1 was measured by DLS method to be 409.08~508.32nm.

[0063] Combination Figure 2 As shown, the potential of the nanoparticle HMC@anti-PD-L1 was measured by Zeta to be -33.6 to -25.8 mV.

[0064] Combination Figure 3 As shown, TEM observed that the nanoparticles HMC@anti-PD-L1 were spherical in shape, with good dispersion and a particle size mainly distributed around 400 nm. Several gray-black particles were visible on the nanoparticles HMC@anti-PD-L1, indicating that the PD-L1 antibody was successfully encapsulated in the nanoparticles HMC@anti-PD-L1.

[0065] Combination Figure 4 As shown, the results of X-ray photoelectron spectroscopy analysis showed that the peaks of Mn(2p1 / 2) and Mn(2p3 / 2) were detected at binding energies of around 652.9 eV and 641.8 eV, indicating that the Mn element in the synthesized MnO2 was in valence state IV, indicating that MnO2 was loaded on the nanoparticles HMC@anti-PD-L1.

[0066] Figure 5The UV-vis-NIR absorption spectra of nanoparticles HC (conjugate of Ce6 coupled to HSA nanoparticles), nanoparticles HM (conjugate of MnO2 coupled to HSA nanoparticles) and nanoparticles HMC@anti-PD-L1. Compared with nanoparticles HC, nanoparticles HMC@anti-PD-L1 show a new absorption band near 300-400nm, which may be attributed to the surface plasmon band of MnO2; while compared with nanoparticles HM, nanoparticles HMC@anti-PD-L1 still have the characteristic peaks of Ce6 at 403nm and 660nm, further confirming that Ce6 has been successfully coupled to the surface of HSA nanoparticles, and at the same time indicating that nanoparticles HMC@anti-PD-L1 retains the optical properties of Ce6 and has the potential to be used as enhanced fluorescence imaging. Figure 6 As shown, DLS measured the hydrodynamic diameter of the nanoparticles dispersed in PBS solution containing 10% serum at 37°C for 7 days. The results showed that the nanoparticles HMC@anti-PD-L1 had good colloidal stability, which is expected to prolong the circulation time in vivo and accumulate at the tumor site through passive targeting, thereby achieving diagnosis and treatment of the tumor site.

[0067] The drug encapsulation efficiency mainly tests the encapsulation rate and drug loading amount of MnO2, Ce6 and PD-L1 antibody in the nanoparticles HMC@anti-PD-L1.

[0068] The encapsulation efficiency and drug loading of MnO2 in the nanoparticles HMC@anti-PD-L1 were measured by ICP-OES to be 63% and 1.21%, respectively;

[0069] The ELISA experiment measured the encapsulation efficiency and drug loading of PD-L1 antibody in the nanoparticles HMC@anti-PD-L1 to be 78.98% and 1.46%, respectively.

[0070] The free Ce6 sample has a characteristic absorption peak at 660nm in the UV-visible absorption spectrum. The absorbance value A at 660nm is plotted against the concentration C to obtain a standard curve. The curve is linearly fitted to obtain the standard curve equation: y = 0.1248x-0.0051, R2 = 0.999. The results show that it has good linearity in the concentration range of 0.125μg / ml to 4μg / ml without obvious interference. It was calculated that the encapsulation efficiency and drug loading of Ce6 in the nanoparticle HMC@anti-PD-L1 were 48% and 3.1%, respectively.

[0071] In order to further understand the state changes of nanoparticles HMC@anti-PD-L1 in the simulated tumor microenvironment, we performed TEM and DLS characterization after weak acid / H2O2 treatment. Figure 7As shown in Figure 2, after being treated with pH 6.5 / H2O2, the morphology of HMC@anti-PD-L1 nanoparticles changed and the particle size became smaller. The DLS hydration particle size distribution characterization obtained consistent results. Figure 8 It can be seen that the particle size of the dual-responsive nanoparticles HMC@anti-PD-L1 gradually decreased after being treated with the simulated in vitro tumor microenvironment solution. + Reactivity and degradation behavior of MnO2 in different media of nanoparticles HMC@anti-PD-L1. Fig. 9 As shown, the degradation of MnO2 can be determined by the decrease of its UV-vis characteristic absorption band. In the case of pH 6.5 and H2O2, the absorbance of MnO2 in the nanoparticles HMC@anti-PD-L1 decreases rapidly, which can further promote the decomposition of MnO2. In contrast, MnO2 in the nanoparticles HMC@anti-PD-L1 appears to be quite stable at pH 7.4. Based on the above characteristics, MnO2 in HMC@anti-PD-L1 can also react and degrade under H2O2 and acidic conditions. According to the reaction formula: H2O2+2H + +MnO2=2H2O+O2+Mn 2+ , which can release O2 and Mn 2+ , which is conducive to the "change from large to small" of nanoparticles HMC@anti-PD-L1, dispersing PD-L1 antibodies and Ce6 drugs, making it easier for drugs to penetrate deeply into the highly heterogeneous extracellular matrix of TNBC, and at the same time achieving in situ production of O2 in the tumor microenvironment. It can be seen that the dual-responsive nanoparticles HMC@anti-PD-L1 have undergone significant changes in state under weak acidity / H2O2, and have tumor microenvironment responsiveness.

[0072] Next, we further explored the performance of the nanoparticles HMC@anti-PD-L1 in releasing drugs in a weakly acidic / H2O2 state.

[0073] In order to evaluate the release of weak acid / H2O2 nanoparticles HMC@anti-PD-L1, Ce6, Mn 2+ And the release of PD-L1 antibody in vitro. Fig.10 It can be seen that Ce6 and Mn 2+ The release of PD-L1 antibody under normal physiological conditions (pH 7.4) was low, and the drug release after 24 hours of incubation was less than 20%, with the lowest degree of drug release, and the drug was almost still in the nanoparticles HMC@anti-PD-L1. When the buffer conditions simulated the tumor microenvironment (weak acid / H2O2), with the extension of treatment time, Ce6, Mn 2+The amount of drug released by PD-L1 antibody increased significantly, and weak acid, H2O2, and MnO2 were degraded to produce Mn 2+ and O2, which decomposed the HSA nanoparticles formed by the biomineralization method, promoting the release of the drug. At the same time, the PD-L1 antibody was released, allowing it to bind to the PD-L1 on the cell surface and block the immunosuppressive effect. After 24 hours of incubation, Ce6, Mn 2+ The release rates of PD-L1 antibody were 72.3%, 13.14% and 95.22% respectively, which showed that the nanoparticles HMC@anti-PD-L1 can be specifically released in the tumor microenvironment and exert therapeutic effects.

[0074] A dissolved oxygen meter was used to measure the amount of O2 produced by nanoparticles HMC@anti-PD-L1 under normal physiological conditions and in weakly acidic / H2O2 solutions. Fig.11 As shown, the nanoparticles HMC@anti-PD-L1 were placed in a PBS solution with a pH of 7.4 and a buffer solution with a pH of 6.5 / H2O2. In the weak acid / H2O2 solution, the dissolved oxygen meter probe showed a significant increase in value after 1 minute. After 10 minutes of detection, the oxygen content was 16.3 mg / L, while there was no significant change in the oxygen content in the pH 7.4 solution. Fig.12 As shown in the figure, after the HMC@anti-PD-L1 nanoparticles loaded with MnO2 reacted with the weak acid / H2O2 solution, bubbles appeared on the tube wall. This shows that the HMC@anti-PD-L1 nanoparticles can continuously release O2 in the tumor microenvironment.

[0075] Therefore, our next step is to verify whether the nanoparticles can enhance the sonodynamic therapy to produce more ROS. Fig.13 As shown, nanoparticles HMC@anti-PD-L1 were irradiated with ultrasound (1W / cm 2 ), and the DPBF co-incubated with it showed obvious fluorescence quenching. Among them, the fluorescence quenching was more obvious in the weak acid / H2O2 solution. And with the passage of time, the singlet oxygen production gradually increased, indicating that the nanoparticle HMC@anti-PD-L1 has a good singlet oxygen production, produces O2 under dual response, and can further increase ROS production, laying an experimental foundation for subsequent cell and in vivo sonodynamic therapy efficacy experiments.

[0076] Combination Fig.14 As shown in the figure, since the nanoparticle HMC@anti-PD-L1 itself has no toxicity to cells, it does not affect cell survival. When the drug Ce6 concentration is 200μg / ml, the cell survival rate is still greater than 90%.

[0077] The main organs were stained to study the potential toxicity of the nanoparticle HMC@anti-PD-L1, and the staining results did not reveal any histopathological abnormalities, lesions or degeneration. Then, the biosafety test results showed that there was no significant difference in AST / ALT (liver function index) and BUN / CRE (kidney function index) between the different treatment groups, proving that the nanoparticle HMC@anti-PD-L1 treatment had no effect on the liver and kidney function of mice.

[0078] HSA is the most abundant protein in plasma. It has high biocompatibility, good biodegradability and non-immunogenicity. It can target secreted proteinacidic and rich incysteine ​​(SPARC) in tumor tissue. SPARC can specifically adsorb albumin containing drugs and aggregate on tumor cells, thereby increasing local drug concentration and enhancing tumor killing ability. Compared with other nano drug delivery systems, HSA nanoparticles encapsulate or adsorb drugs through intermolecular disulfide bonds, and have better controllable drug release performance, better drug encapsulation capacity, better storage stability and tumor passive targeting ability. HSA nanoparticles can pass through tumor blood vessels through the EPR effect and reach the tumor tissue gap. After responding to glutathione in the tumor, they are cleaved into drug-loaded small molecule complexes, which can better penetrate into the tumor tissue gap. Therefore, the effective EPR-derived tumor accumulation of macromolecular drug delivery systems and the strong penetration of small molecule drugs in the tumor tissue gap can be achieved at the same time. This "from large to small" drug loading strategy may be an important solution to improve the effect of cancer treatment.

[0079] In addition, HSA nanoparticles can isolate inorganic ions through biomineralization under alkaline conditions to form protein-coated metal oxide nanoclusters. In recent years, manganese dioxide nanoparticles (MnO2) have attracted extensive attention as a unique TME-responsive nanomaterial. MnO2 can not only interact with H + Or glutathione (GSH) decomposes to generate Mn 2+ ions, which can significantly enhance T1-magnetic resonance (MR) imaging contrast for tumor-specific imaging and detection, and can also trigger the decomposition of H2O2 present in the TME into water and oxygen, thereby alleviating the tumor hypoxic microenvironment, an effect that can enhance many therapeutic modalities such as sonodynamic therapy.

[0080] The present application is further described below through a study of the drug delivery and permeability properties of nanoparticles HMC@anti-PD-L1 combined with sonodynamic therapy in vivo and in vitro.

[0081] Tumors are not just composed of tumor cells, their structure is more like a mutated organ, so they have a complex structure. Since the multicellular spheroid model is closer to solid tumors in terms of biological morphology, internal environment, and three-dimensional morphology, it is regarded as a relatively valuable research method in the study of tumor treatment. In order to simulate the penetration ability of nanoparticles HMC@anti-PD-L1 in tumor tissue, a 4T1 breast cancer 3D cell spheroid model was constructed, and free Ce6 and nanoparticles HMC@anti-PD-L1 were used to explore the in-depth delivery effect of nanoparticles HMC@anti-PD-L1 in 3D cell spheroids under simulated tumor microenvironment conditions.

[0082] The specific construction steps are as follows: (1) Mouse breast cancer 4T1 cells were cultured at 1×10 4 The cells were plated on laser confocal culture dishes, and nanoparticles HMC@anti-PD-L1 and free Ce6 were added and incubated for different time periods. The cells were washed with PBS three times and divided into ultrasound irradiation group and non-ultrasound irradiation group. The ultrasound irradiation group was irradiated with 1 W / cm 2 The cells were irradiated for 1 minute, and the non-ultrasound irradiation group was used as the control group. Laser confocal microscopy and flow cytometry were used to detect the intracellular uptake of the drugs in the above-mentioned different treatment groups at different time periods.

[0083] (2) 3D tumor cell spheroids were prepared to simulate the multilayered cell morphology of tumors in vitro. Nanoparticles HMC@anti-PD-L1 were added to pH 6.5, pH 7.4, and H2O2 buffers and incubated with tumor spheroids for different time periods. The spheroids were divided into ultrasound irradiation group and non-ultrasound irradiation group. The ultrasound irradiation group was irradiated at 1 W / cm 2 The cells were irradiated for 1 minute, and the non-ultrasound irradiation group was used as the control group. After treatment, a laser confocal microscope was used to scan them in three dimensions to detect the drug release and penetration of the nanoparticles HMC@anti-PD-L1 in a weak acid / H2O2 environment.

[0084] Combination Fig.15 As shown in the figure, free Ce6 can evenly penetrate into the tumor cell sphere, and the nanoparticle HMC@anti-PD-L1 can also be evenly distributed in the 3D cell sphere under weak acid / H2O2 conditions, which means that the nanoparticle "changes from large to small" after the response decomposition, and small molecule drugs can more easily penetrate into the tumor cell sphere, which is the same as the penetration effect of free Ce6. Therefore, it is further demonstrated that the change in size can effectively improve the deep penetration ability of chemotherapy drugs into solid tumors. Here, at the level of 4T1 3D cell spheres, it was verified that the dual-responsive nanoparticle HMC@anti-PD-L1 released small molecule compound drugs due to the change in size after being treated in a simulated tumor microenvironment, making it easier to penetrate into the tumor cell sphere, significantly enhancing its inward penetration ability and the ability to be taken up by cells.

[0085] In order to detect the ability of drug release in biological tissues, a Balb / c mouse model of breast cancer was established, and the nanoparticle HMC@anti-PD-L1 solution was injected intratumorally and intravenously into tumor-bearing mice. The in vivo distribution process of the nanoparticles at different time points was dynamically observed using small animal fluorescence in vivo imaging; then, the main organs and tumor tissues were removed, and the distribution of the nanoparticles HMC@PD-L1 in the ex vivo tissues was detected by fluorescence imaging, and quantitative analysis was performed using flow cytometry; according to the drug distribution time point, the time point with the highest amount of drug in the tumor was selected and divided into the ultrasound irradiation group and the non-ultrasound irradiation group. The ultrasound irradiation group was irradiated at 2W / cm 2 The mouse tumor was locally acted on, the irradiation time was 1 minute, and the non-ultrasound irradiation group was used as the control group. Then, after pathological sectioning, the distribution of nanoparticles in different tissues and cells of the animals was observed using a laser confocal microscope to study the tissue distribution of nanoparticles in vivo and the tumor penetration behavior.

[0086] Combination Fig.16A As shown, after intratumoral injection, the delivery and distribution of nanoparticles HMC@anti-PD-L1 in tumor tissue were similar to those of free Ce6, and the delivery penetration was increased after ultrasound irradiation; combined with Fig. 16B As shown, after intravenous injection, the nanoparticle HMC@anti-PD-L1 was deeply delivered and evenly distributed in the tumor tissue. After ultrasound irradiation, the drug delivery effect was enhanced. In the non-ultrasound irradiation group, whether it was intratumoral injection or intravenous injection of nanoparticle HMC@anti-PD-L1, red fluorescent Ce6 was evenly distributed in the tumor tissue, which was the same as the injection of free Ce6. It further shows that the nanoparticle HMC@anti-PD-L1 can dual-respond to degradation and release small molecule drugs in the tumor microenvironment in vivo, promoting the penetration of drugs in highly heterogeneous tumors. Under ultrasound irradiation, the distribution of Ce6 is compared with the non-ultrasound irradiation group, and the red color in the tumor tissue should be more obvious, indicating that ultrasound can stimulate and promote drug release. The reason is that due to the thermal effect, mechanical effect and cavitation effect of ultrasound itself, combined with the O2 generated by the response of the nanoparticle HMC@anti-PD-L1, the cavitation effect of ultrasound can be enhanced to promote the release of drugs to a distant place.

[0087] According to the above in vitro experiments, the nanoparticle HMC@anti-PD-L1 is a nanoparticle with dual-responsive drug release properties, which gives it significant potential for synergistic sonodynamic therapy of tumors.

[0088] Finally, the present application is further illustrated by the anti-tumor immune response effect of the nanoparticle HMC@anti-PD-L1.

[0089] (1) Anti-tumor inhibitory effect of nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy on distal tumors

[0090] Currently, the main cause of death in cancer patients is tumor metastasis. Traditional treatments such as surgical resection, chemotherapy and radiotherapy are rarely effective against tumor metastasis. Studies have shown that abscopal effects are attributed to the host immune response stimulated by cancer therapy, which is of great significance for evaluating the long-term efficacy of cancer therapy.

[0091] Therefore, the bilateral 4T1 subcutaneous tumor model was used as the research object, and the experimental design was as follows Fig.17A As shown, the experimental steps are:

[0092] a. Thirty 5-6 week old Balb / c female mice were inoculated with an orthotopic tumor on the left flank. Four days later, a distal tumor was inoculated on the right flank to establish an animal model.

[0093] b. When the left tumor volume reaches 100mm 3 At the same time, the mice were randomly divided into the Saline group (treated with saline), the HMC group (treated with HSA nanoparticles loaded with MnO2 and Ce6), the HMC@anti-PD-L1 group (treated with HSA nanoparticles loaded with MnO2, Ce6 and PD-L1 antibodies), the Saline+US group (treated with saline combined with sonodynamic therapy), the HMC+US group (treated with HSA nanoparticles loaded with MnO2 and Ce6 combined with sonodynamic therapy), and the HMC@anti-PD-L1+US group (treated with HSA nanoparticles loaded with MnO2, Ce6 and PD-L1 antibodies combined with sonodynamic therapy). 200 μl of HMC or HMC@anti-PD-L1 was injected into the tail vein of the mice in each group. Six hours later, the mice were treated with a power of 2.5 W / cm 2 The ultrasound machine irradiated only the left tumor of the mouse for 5 minutes.

[0094] c. The mice were given medication and ultrasound irradiated every 3 days for a total of 4 treatments. During the treatment, the bilateral tumor growth volume and mouse weight were measured every 3 days for a total of 15 days. At the same time, the death of mice was followed up for 45 days. According to the Animal Welfare Act and relevant experimental regulations, when the tumor volume reached 2000mm 3 The death of animals was recorded.

[0095] Combination Fig. 17B As shown, the HMC@anti-PD-L1+US group can effectively inhibit the in situ tumor, while the other treatment groups did not show obvious inhibitory effects and the tumor continued to grow rapidly. Fig. 17CAs shown in the figure, the HMC@anti-PD-L1+US group can effectively inhibit the in situ tumor and significantly delay the growth of the corresponding distal tumor. The growth of the distal tumor in other treatment groups was not effectively inhibited. Fig.17D As shown, the survival rate of tumor-bearing mice in the HMC@anti-PD-L1+US group was significantly higher than that in other treatment groups.

[0096] The above results show that the nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy can not only effectively inhibit local tumors but also has a significant inhibitory effect on untreated distal tumors, inducing a distant effect. According to the results, it can be speculated that after the nanoparticle HMC@anti-PD-L1 is combined with sonodynamic therapy for in situ tumors, on the one hand, it can activate T lymphocytes in the distant tumor microenvironment, so that it can enter the systemic circulation along the surface lymphatic vessels or blood vessels to the distant tumor site, thereby exerting an anti-tumor effect; on the other hand, after the nanoparticle HMC@anti-PD-L1 enters the systemic circulation through the tumor blood vessels, it reaches the distant tumor, activates T lymphocytes in the tumor microenvironment, blocks the recognition of PD-1 / PD-L1, avoids the immune escape of the tumor, and inhibits the growth of tumor volume. In addition, the nanoparticle HMC@anti-PD-L1 that enters the blood circulation can bind to PD-1 on T cells in the blood, activating it into toxic T lymphocytes with killing function, thereby improving the immune ability of the entire mouse body. In anti-tumor treatment, weight loss in mice is often used as an important indicator for judging the side effects or toxicity of treatment. Combined with Fig.17E As shown, during the process of mice receiving nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy, the weight of tumor-bearing mice increased slightly and then stabilized, proving that nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy is safe and has no toxic side effects.

[0097] The above results indicate that nanoparticle HMC@anti-PD-L1-mediated O2-enhanced sonodynamic therapy can not only inhibit local subcutaneous tumors, but also exert a distal effect to inhibit tumor metastasis.

[0098] (2) Nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy induces ICD in tumor cells in vivo

[0099] In order to further verify whether nanoparticle HMC@anti-PD-L1-mediated sonodynamic therapy can trigger ICD in tumors, activate DCs to trigger anti-tumor immune responses and produce distal anti-tumor effects. Previous studies have shown that CRT exposure is necessary to increase immunogenic killing and can be used as a representative biomarker for determining ICD.

[0100] The bilateral 4T1 subcutaneous tumor model was also used as the research object. The experimental steps were as follows:

[0101] a. Thirty 5-6 week old Balb / c female mice were inoculated with an orthotopic tumor on the left flank. Four days later, a distal tumor was inoculated on the right flank to establish an animal model.

[0102] b. When the left tumor volume reaches 100mm 3 The mice were randomly divided into the Control group (treated with saline), the US group (sonodynamic therapy), the HMC group (treated with HSA nanoparticles loaded with MnO2 and Ce6), the HMC+US group (treated with HSA nanoparticles loaded with MnO2 and Ce6 combined with sonodynamic therapy), the HMC@anti-PD-L1 group (treated with HSA nanoparticles loaded with MnO2, Ce6 and PD-L1 antibodies), and the HMC@anti-PD-L1+US group (treated with HSA nanoparticles loaded with MnO2, Ce6 and PD-L1 antibodies combined with sonodynamic therapy). 200 μl of HMC or HMC@anti-PD-L1 was injected into the tail vein of the mice in each group. Six hours later, the mice were treated with a power of 2.5 W / cm 2 The ultrasound machine irradiated only the left tumor of the mouse for 5 minutes.

[0103] c. The mice were medicated and irradiated with ultrasound every 3 days for a total of 4 treatments. During the treatment, the bilateral tumor growth volume and the weight of the mice were measured every 3 days for a total of 15 days.

[0104] d. On the third day after treatment, the mice were killed and the tumor was completely removed and made into paraffin tumor blocks for subsequent use.

[0105] e. Cut 5 μm thin sections from the paraffin tumor block and place them on a slide. After dewaxing, washing with PBS and fixing with 4% paraformaldehyde, incubate with anti-CRT antibody at 4°C overnight.

[0106] f. After washing with PBS, the cells were stained with Alexa Fluor 594 fluorescently labeled secondary antibody for 30 minutes, and then the nuclei were stained with DAPI for 10 minutes after washing with PBS.

[0107] g. After washing with PBS for several times, add an appropriate amount of anti-fluorescence quenching sealing solution on the slide and cover it with a coverslip for CLSM observation.

[0108] Combination Fig.18As shown in the figure, the groups not treated with ultrasound (i.e., the Control group, HMC group, and HMC@anti-PD-L1 group) could not induce CRT exposure, while the HMC+US group could induce a small amount of CRT exposure. However, a large amount of red fluorescence appeared in the tumor sections of the HMC@anti-PD-L1+US group, proving that nanoparticles HMC@anti-PD-L1 combined with sonodynamic therapy can increase CRT exposure and have a strong ICD induction effect in vivo.

[0109] (3) Detection of tumor-infiltrating T lymphocytes by flow cytometry

[0110] The bilateral 4T1 subcutaneous tumor model was also used as the research object. The experimental steps were as follows:

[0111] a. Thirty 5-6 week old Balb / c female mice were inoculated with an orthotopic tumor on the left flank. Four days later, a distal tumor was inoculated on the right flank to establish an animal model.

[0112] b. When the left tumor volume reaches 100mm 3 The mice were randomly divided into the Control group (treated with saline), the US group (sonodynamic therapy), the HMC group (treated with HSA nanoparticles loaded with MnO2 and Ce6), the HMC+US group (treated with HSA nanoparticles loaded with MnO2 and Ce6 combined with sonodynamic therapy), the HMC@anti-PD-L1 group (treated with HSA nanoparticles loaded with MnO2, Ce6 and PD-L1 antibodies), and the HMC@anti-PD-L1+US group (treated with HSA nanoparticles loaded with MnO2, Ce6 and PD-L1 antibodies combined with sonodynamic therapy). 200 μl of HMC or HMC@anti-PD-L1 was injected into the tail vein of the mice in each group. Six hours later, the mice were treated with a power of 2.5 W / cm 2 The ultrasound machine irradiated only the left tumor of the mouse for 5 minutes.

[0113] c. The mice were medicated and irradiated with ultrasound every 3 days for a total of 4 treatments. During the treatment, the bilateral tumor growth volume and the weight of the mice were measured every 3 days for a total of 15 days.

[0114] d. On the third day after treatment, the mice were killed and the tumor masses were completely removed and weighed. The tumor masses were minced and ground in 4°C PBS, and filtered through a cell sieve (40 μm) to prepare a tumor tissue cell suspension.

[0115] e. Stain with FITC-CD3, APC / Cy7-CD45, PerCP-CD4 and APC-CD8a antibodies and incubate at room temperature in the dark for 30 minutes.

[0116] f. Centrifuge the sample (350×g, 5 minutes), resuspend the cell pellet in PBS, and analyze the sample by flow cytometry.

[0117] g. Take out 100 μl of the collected T lymphocytes, stain them with labeled anti-CD25-APC, anti-CD4-FITC and anti-FOXP3-PE, and then detect them using a flow cytometer.

[0118] The infiltration of immune effector cells into tumors is also an important step in achieving tumor suppression and distant anti-tumor effects. Using distant tumors as the target of immunotherapy, the proportions of activated CD8+T and CD4+T cells in tumors were detected. Fig.19 As shown, the proportion of CD8+T in the tumor of the control group was less than 5%, and CD4+T was only 17.8%. However, the HMC@anti-PD-L1+US group showed the highest percentage of activated effector cells in the tumor, with CD8+T accounting for 21.4% and CD4+T accounting for 27.4%. It is known that CD8+T cells are highly cytotoxic to tumor cells, while CD4+T cells are considered to be helper cells that can recognize tumor cells and then interact with CD8+T cells to inhibit tumors. This shows that nanoparticle HMC@anti-PD-L1-mediated sonodynamic therapy has a strong effect on activating effector cells.

[0119] In contrast, regulatory T lymphocytes are a type of T cell subset with negative regulatory effects that maintain self-stability and prevent autoimmune diseases. They are characterized by immune anergy and immunosuppression and are the main helpers of tumor immune escape. The effect of nanoparticle HMC@anti-PD-L1-mediated sonodynamic therapy on regulatory T lymphocytes in tumor tissue. After treatment with nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy, tumor cells were stained with anti-CD25-APC, anti-CD4-FITC, and anti-FOXP3-PE. Flow cytometry results showed that compared with the mouse tumor tissues in the Control group, the distal tumor tissues in the HMC@anti-PD-L1+US group showed a decrease in regulatory T lymphocytes.

[0120] Combination Fig. 20 As shown in the figure, the detection of different immune factors in tumor tissues further illustrates that immune cells play a major role in suppressing tumors in distant tumor tissues. Among them, immune factors such as TNF-α, IL-6, and IFN-γ that can inhibit tumor proliferation and growth have relatively high concentrations in the treatment group. This shows that after treatment, the immune microenvironment of mouse tumors has been greatly improved, from the original immunosuppressive state to the immune-promoting state, further proving the effectiveness of the designed immunotherapy strategy.

[0121] (4) Anti-tumor recurrence by memory T cells

[0122] In order to further verify the tumor immune memory effect of nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy, a tumor re-implantation model after tumor treatment was constructed.

[0123] Combination Fig.21A As shown in the figure, the specific steps of the experiment on distant tumor recurrence after nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy are as follows:

[0124] a. A unilateral 4T1 tumor-bearing mouse model was established, and the tumor-bearing mice were randomly divided into 2 groups (6 mice / group), a control group (treated with normal saline) and a HMC@anti-PD-L1+US group (nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy). Six hours after administration, the power was 2.5 W / cm 2 The ultrasound machine was used to irradiate the right tumor of the mouse for 5 minutes. The mice were given medication and ultrasound irradiation every 3 days for a total of 4 treatments.

[0125] b. After 18 days, inject 100 μl of 1×10 6 The 4T1 cell suspension was added to the mouse body and the left tumor growth volume and mouse body weight were measured every 3 days for 30 days. The death of mice was also observed for 50 days. According to the Animal Welfare Act and relevant experimental regulations, when the tumor volume reached 2000mm 3 The death of animals was recorded.

[0126] Combination Figures 21B-21C As shown in the figure, compared with the control group, the tumor growth rate of mice in the HMC@anti-PD-L1+US group was the slowest. This is because sonodynamic therapy can improve the immunogenicity of tumor cells. Subsequently, the tumor growth rate of mice in the HMC@anti-PD-L1+US group was slower, and their survival status was also the most ideal. Two weeks after tumor implantation, combined with Fig.21D As shown in the figure, the survival rate of the HMC@anti-PD-L1+US group was significantly higher than that of the control group.

[0127] Memory T cells can produce a stronger immune response faster than the first exposure to the antigen, which is also one of the important indicators for measuring tumor immunotherapy. Memory T cells are divided into central memory T cells (TCM) and effector memory T cells (TEM). The TEM of the treated mice was tested, and the specific test steps are as follows:

[0128] a. A unilateral 4T1 tumor-bearing mouse model was established, and the tumor-bearing mice were randomly divided into 2 groups (6 mice / group), a control group (treated with normal saline) and a HMC@anti-PD-L1+US group (nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy). Six hours after administration, the power was 2.5 W / cm 2 The ultrasound machine was used to irradiate the right tumor of the mouse for 5 minutes. The mice were given medication and ultrasound irradiation every 3 days for a total of 4 treatments.

[0129] b. Euthanize the mice, dissect and collect the spleen of each group of mice, cut them into pieces, grind them, rinse them with PBS, filter the filtrate with a 40μm filter, centrifuge, add red blood cell lysis buffer, shake and centrifuge to remove the supernatant. Repeat centrifugation twice with PBS, then take out 100μl, add anti-CD3-FITC, anti-CD8-PerCP, anti-CD44-PE and anti-CD62L-APC antibodies to detect the proportion of effector memory T cells. Refer to the instruction manual for specific staining methods and antibody concentrations.

[0130] The results are combined as Fig. 22 As shown in the figure, compared with the Control group, the proportion of effector memory CD8+T cells in the CD8+T cells of the mice in the HMC@anti-PD-L1+US group was 36.1%, which was significantly increased. This shows that after the nanoparticle HMC@anti-PD-L1 combined with sonodynamic therapy, the immune system of the mice showed a certain memory.

[0131] In view of this, the drug delivery system prepared by the present invention can not only utilize TME response to improve drug release rate, but also regulate unfavorable TME, and further optimize the efficacy of sonodynamic therapy and its induced tumor immune response therapy.

[0132] The above implementation modes are merely descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary engineering and technical personnel in the field shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing a tumor microenvironment responsive nano drug delivery system, characterized in that: The nano drug delivery system is a conjugate of PD-L1 antibody, MnO2 and Ce6 coupled to HSA nanoparticles. The preparation method of the nano drug delivery system comprises the following steps: (1) First, HSA nanoparticles were dissolved in deionized water to prepare a HSA solution with a concentration of 10 mg / mL, KMnO4 solution was added to the HSA solution, and the reaction was continued in a water bath at 25°C for 4 hours, and the obtained HSA-manganese particles were dialyzed and ultrafiltered for later use; (2) reacting HSA-manganese particles and DBCO-NHS at room temperature overnight; then removing free DBCO-NHS by dialysis ultrafiltration to obtain DBCO-modified albumin-manganese particles, i.e., HSA-manganese particles-DBCO; (3) Weigh 15 mg of Ce6 powder, 9.6 mg of EDC, and 10.9 mg of NHS, take 1 mL of DMSO to completely dissolve the three, and stir for 4 hours in a dark place to obtain a Ce6 activated sample; take HSA-manganese particle-DBCO and 0.2 mL of Ce6 activated sample and stir overnight, then remove free Ce6 by dialysis ultrafiltration to obtain HSA-manganese particle-Ce6 nanoparticle surface modified with DBCO, i.e. DBCO-HMC; (4) activating the thiol group of the PD-L1 antibody using Traut's reagent, and then stirring with N3-PEG-Mal to obtain N3-PEG-PD-L1 antibody; (5) The N3-PEG-PD-L1 antibody was subjected to a copper-free click reaction with DBCO-HMC to couple the PD-L1 antibody to the HSA nanoparticles, thereby obtaining the nanoparticles HMC@anti-PD-L1.

2. The preparation method according to claim 1, characterized in that: In step (1), 150 mg of HSA nanoparticles were dissolved in 15 mL of deionized water to prepare an HSA solution with a concentration of 10 mg / mL, and 3 mL of a 20 mM KMnO4 solution was added to the HSA solution.

3. The preparation method according to claim 1, characterized in that: The obtained nanoparticles HMC@anti-PD-L1 were tested to determine whether the HSA nanoparticles successfully carried MnO2, Ce6 and PD-L1 antibodies; the test content included the morphological structure, particle size, potential, optical properties, drug encapsulation rate of the nanoparticles HMC@anti-PD-L1 and biosafety testing in cells and in vivo.

4. The preparation method according to claim 3, characterized in that: The particle size of nanoparticles HMC@anti-PD-L1 was measured by DLS method and was 409.08~508.32nm.

5. The preparation method according to claim 3, characterized in that: TEM observation showed that the nanoparticles HMC@anti-PD-L1 were spherical in morphology.

6. The preparation method according to claim 3, characterized in that: The results of X-ray photoelectron spectroscopy analysis showed that the peaks of Mn(2p1 / 2) and Mn(2p3 / 2) were detected at binding energies of around 652.9 eV and 641.8 eV, indicating that the Mn element in the synthesized MnO2 was in valence state IV.

7. The preparation method according to claim 3, characterized in that: The encapsulation efficiency and drug loading of MnO2 in the nanoparticles HMC@anti-PD-L1 were measured by ICP-OES to be 63% and 1.21%, respectively; The ELISA experiment measured the encapsulation efficiency and drug loading of PD-L1 antibody in the nanoparticles HMC@anti-PD-L1 to be 78.98% and 1.46%, respectively. The UV-visible absorption spectrum analysis showed that the encapsulation efficiency and drug loading of Ce6 in the nanoparticles HMC@anti-PD-L1 were 48% and 3.1%, respectively.

8. The preparation method according to claim 3, characterized in that: HE staining was performed on major organs to study the potential toxicity of nanoparticles HMC@anti-PD-L1, and no histopathological abnormalities, lesions, or degeneration were found in the HE staining results.

9. The preparation method according to claim 1, characterized in that: The changes in the structure, particle size, potential and other physical and chemical properties of nanoparticles HMC@antiPD-L1 in the simulated tumor micro-acid environment and H2O2 environment in vitro, as well as the Mn 2+ , qualitative and quantitative analysis of O2 release is used to determine whether the nanoparticles HMC@anti-PD-L1 have the ability of responsive release in the micro-acidic environment of the tumor.

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