A nanosystem for regulating the acidic microenvironment of tumors and promoting the efficacy of photoimmunotherapy and its application

Abstract

The present invention relates to the field of environmental protection technology, and discloses a nanosystem and application for regulating the acidic microenvironment of tumors to promote the efficacy of photoimmunotherapy, including a nanosystem for regulating the acidic microenvironment of tumors to promote the efficacy of photoimmunotherapy, the nanosystem including a nanocarrier PEG-NH2 and nanomedicines Ce6-COOH and FX-11; wherein, the equivalent ratio of PEG-NH2 to Ce6-COOH is: 1:0.5-1.5; the mass ratio of PEG-NH2 to FX-11 is: 20-80:1. The present invention constructs the nanomaterial FX-11@PEG-Ce6, which, in addition to killing tumor cells through photodynamic therapy (PDT), can also induce tumor cell immunogenic death (ICD), promote the maturation of dendritic cells (DCs), and further promote the activation and infiltration of CD8+T cells; at the same time, FX-11@PEG-Ce6 can also restore the function of CD8+T cells by improving the acidic microenvironment; these two effects can synergistically enhance the efficacy of α-PD-1, exert a synergistic immunotherapy effect, and provide a new approach for photodynamic immunotherapy.

Description

Technical Field

[0001] The present invention relates to the field of environmental protection technology, and in particular to a nanosystem for regulating the acidic microenvironment of tumors to promote the efficacy of photoimmunotherapy and its application. Background Art

[0002] Gastric cancer (GC) is the fifth most common malignancy worldwide and the fourth leading cause of cancer death. In recent years, immunotherapy has transformed the landscape of cancer treatment, and GC is no exception. Treatment targeting programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has brought significant clinical benefits, but the widespread use of α-PD-1 drugs has been hampered by relatively low response rates. Due to the high heterogeneity and individual differences within tumors, single therapy can no longer meet clinical treatment needs. It is crucial to identify effective combination therapy modalities to enhance anti-tumor immune responses. Recent clinical trials have shown that several anti-PD-1 antibody (α-PD-1) drugs combined with standard chemotherapy or targeted therapy have encouraging effects on patients with GC.

[0003] Photodynamic therapy (PDT) can effectively deliver photosensitizers to tumor sites and, under laser irradiation, induce cytotoxic effects on tumor cells. Compared with traditional cancer therapies, PDT has the advantages of high selectivity and minimal invasiveness. PDT can induce immunogenic cell death (ICD), which helps eliminate tumor cells. During ICD, calreticulin (CRT) and high-mobility group protein B1 (HMGB1) promote the maturation of dendritic cells (DCs), thereby activating cytotoxic T lymphocytes (CTLs) and inducing effective antitumor immune responses. In recent years, due to the advantages of PDT, such as its non-invasiveness, high spatiotemporal selectivity, and low drug tolerance, an increasing number of studies have combined PDT with immunotherapy. Encouragingly, the combined use of PDT and immunotherapy can help eliminate residual tumor cells and achieve better therapeutic effects. Among the numerous photosensitizers, the second-generation photosensitizer hematoporphyrin derivative (Ce6) has been widely used in PDT.

[0004] However, conventional photodynamic therapy (PDT) faces several major obstacles, including the acidic, immunosuppressive tumor microenvironment (TME). Like almost all tumors, gastric cancer metabolizes glucose for energy through aerobic glycolysis (Warburg effect) and produces excess lactate, resulting in a malnourished, hypoxic, and low-pH TME. As a result, an immunosuppressive TME is established to gain immune evasion potential and regulate cancer growth. Several small molecule inhibitors have been developed to counteract the Warburg effect and alleviate the acidic TME. FX-11 is a specific inhibitor of lactate dehydrogenase A (LDHA). FX-11 has shown the ability to inhibit lactate production in prostate cancer cells, thereby inhibiting tumor growth. However, both FX-11 and Ce6 have exhibited short circulation half-lives, limited tumor accumulation, and significant side effects in clinical trials. Therefore, new drug delivery systems are urgently needed to enhance drug circulation, tumor accumulation, and bioavailability in vivo.

[0005] Nanoparticle (NP) platforms with intrinsic therapeutic properties, controlled release, and targeted delivery of multiple therapeutic agents have shown great application prospects in cancer treatment. Nanoparticle-based photodynamic therapy (PDT) can enable the destroyed cancer cells to serve as in situ vaccines and enhance cancer immunogenicity. It has been reported that the efficacy of the photosensitizer Ce6 is improved when used as part of a nanoparticle system due to its low water solubility. Polyethylene glycol (PEG) is a biodegradable functional polymer that is widely used in the preparation of nanoparticles due to its excellent biocompatibility. Therefore, in this study, we developed PEG-Ce6 encapsulated with FX-11, named FX-11@PEG-Ce6, and evaluated the effect of FX-11@PEG-Ce6 in photoimmunotherapy of gastric cancer. Summary of the Invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides a nanosystem and application for regulating the acidic microenvironment of tumors to promote the efficacy of photoimmunotherapy.

[0007] The present invention is implemented by the following technical solutions: a nanosystem for regulating the acidic microenvironment of tumors to promote the efficacy of photoimmunotherapy, the nanosystem comprising a nanocarrier PEG-NH2 and nanomedicines Ce6-COOH and FX-11;

[0008] Among them, the equivalent ratio of PEG-NH2 to Ce6-COOH is: 1:0.5-1.5;

[0009] The mass ratio of PEG-NH2 to FX-11 is: 20-80:1;

[0010] The PEG is connected to Ce6 through an amide bond and assembled with FX-11 through hydrophobic interaction to form a nanosystem, namely FX-11@PEG-Ce6.

[0011] As a further improvement of the above scheme, the preparation steps are:

[0012] Step S1, PEG-Ce6 synthesis:

[0013] PEG-NH2 was dissolved in dichloromethane, and a DMF solution containing Ce6-COOH, EDC and DMAP was added. After the reaction was completely dissolved, the reaction solution was concentrated by vacuum distillation, precipitated with a large amount of ice ether, filtered and collected, and vacuum dried to obtain PEG-Ce6;

[0014] Step S2, synthesis of FX-11@PEG-Ce6:

[0015] PEG-Ce6 was dissolved in water, and then a DMSO solution containing FX-11 was added. After stirring and mixing, the mixture was centrifuged to remove free FX-11 and collect the precipitate to obtain FX-11@PEG-Ce6.

[0016] As a further improvement of the above scheme, the FX-11@PEG-Ce6 kills tumor cells through photodynamic therapy.

[0017] As a further improvement of the above scheme, the FX-11@PEG-Ce6 induces the immunogenic death of tumor cells, promotes the maturation of dendritic cells, promotes the activation and infiltration of CD8+T cells, restores the function of CD8+T cells by improving the acidic microenvironment, and synergistically enhances the efficacy of α-PD-1.

[0018] A nanosystem is used for preparing biological products.

[0019] As a further improvement of the above solution, the biological product includes: reagents, kits, and chips.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The present invention constructs a nanomaterial, FX-11@PEG-Ce6, which, in addition to killing tumor cells through photodynamic therapy (PDT), can also induce immunogenic cell death (ICD) of tumor cells, promote the maturation of dendritic cells (DCs), and further promote the activation and infiltration of CD8+ T cells. Simultaneously, FX-11@PEG-Ce6 can also restore the function of CD8+ T cells by improving the acidic microenvironment. These two effects can synergistically enhance the efficacy of α-PD-1, exert a synergistic immunotherapy effect, and provide a new approach for photodynamic immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Analytical spectrum of FX-11@PEG-Ce6, where:

[0023] Figure 1 A is a representative transmission electron microscopy image of FX-11@PEG-Ce6;

[0024] Figure 1 B is the particle size distribution of FX-11@PEG-Ce6 using dynamic light scattering method;

[0025] Figure 1 C is the detection diagram of the Zeta point of FX-11@PEG-Ce6;

[0026] Figure 1 D is the statistical graph of the particle size and polydispersity index of FX-11@PEG-Ce6 in water over 8 days;

[0027] Figure 1 E is the statistical graph of the particle size and PDI of FX-11@PEG-Ce6 in phosphate-buffered saline (PBS) within 8 days;

[0028] Figure 1 F is the statistical graph of particle size and PDI of FX-11@PEG-Ce6 in 10% fetal bovine serum (FBS) within 8 days;

[0029] Figure 1 G is a graph showing the drug release curve of FX-11 from FX-11@PEG-Ce6 in PBS buffer at different pH values;

[0030] Figure 1 H is an expression graph showing the in vitro cellular uptake of FX-11@PEG-Ce6 when MKN45(H) cells were co-incubated with free Ce6 and FX-11@PEG-Ce6;

[0031] Figure 1 I is an expression graph showing the in vitro cellular uptake of FX-11@PEG-Ce6 when MKN28(I) cells were co-incubated with free Ce6 and FX-11@PEG-Ce6;

[0032] Figure 2 The analytical profile of FX-11@PEG-Ce6's lysosomal escape and cellular internalization is shown below:

[0033] Figure 2 A is the expression diagram of lysosomal escape of FX-11@PEG-Ce6 in MKN45 (A) cells;

[0034] Figure 2 B is the expression diagram of lysosomal escape of FX-11@PEG-Ce6 in MKN28 (B) cells;

[0035] Figure 2C is the expression graph of FX-11@PEG-Ce6 incubated with MKN45 and MKN28 cells for 4 hours or 24 hours, and the cellular internalization of FX-11@PEG-Ce6 was analyzed by flow cytometry;

[0036] Figure 2 D is the expression graph of the cell internalization of FX-11@PEG-Ce6 after incubation with MKN45 and MKN28 cells at 4°C or 37°C;

[0037] Figure 2 E is the expression graph of FX-11@PEG-Ce6 internalization in MKN45 and MKN28 cells treated with CPZ;

[0038] Figure 2 F is the expression graph of cellular internalization of FX-11@PEG-Ce6 in MKN45 and MKN28 cells treated with Mβ-CD;

[0039] Figure 2 G is the expression graph of FX-11@PEG-Ce6 internalization in MKN45 and MKN28 cells treated with Dynasore;

[0040] Figure 3 The analytical profile of FX-11@PEG-Ce6-induced immune activation in vitro; wherein:

[0041] Figure 3 A is the CD3 in T cells after incubation at pH 6.5 and 7.4 + CD8 + Distribution diagram of cell proportions;

[0042] Figure 3 B is the expression diagram of the relative levels of lactate in MKN45 cells and MKN28 cells after different treatments;

[0043] Figure 3 C is the expression graph of calreticulin (CRT) by immunofluorescence staining in MKN45 cells treated with Ce6 or FX-11@PEG-Ce6 with or without illumination;

[0044] Figure 3 D is the expression of calreticulin (CRT) by immunofluorescence staining in MKN28 cells treated with Ce6 or FX-11@PEG-Ce6 with or without illumination;

[0045] Figure 3 E is the expression graph of high mobility group protein B1 (HMGB1) by immunofluorescence staining in MKN45 cells treated with Ce6 or FX-11@PEG-Ce6 with or without illumination;

[0046] Figure 3 F is the expression graph of high mobility group protein B1 (HMGB1) by immunofluorescence staining in MKN28 cells treated with Ce6 or FX-11@PEG-Ce6 with or without illumination;

[0047] Figure 3 G is CD80 after treatment with Ce6 or FX-11@PEG-Ce6 with or without illumination + CD86 + Expression graph of the proportion of dendritic cells (DCs);

[0048] Figure 4 The analytical profile of FX-11@PEG-Ce6 showed that it enhanced T cell-mediated cytotoxicity and promoted the therapeutic effect of α-PD-1 in vitro;

[0049] Figure 4 A is the expression diagram of the ratio of TNF-α, IFN-γ and granzyme B in MKN45 cells in different treatment groups;

[0050] Figure 4 B is the expression graph of the ratio of TNF-α, IFN-γ and granzyme B in MKN28 cells in different treatment groups;

[0051] Figure 4 C is the expression graph of apoptosis rate of MKN45 cells in different treatment groups;

[0052] Figure 4 D is the expression graph of apoptosis rate of MKN28 cells in different treatment groups;

[0053] Figure 5 Analytical spectrum of FX-11@PEG-Ce6 enhancing the in vivo photoimmunotherapy effect; wherein:

[0054] Figure 5 A is an expression diagram of the distribution of free Ce6 and FX-11@PEG-Ce6 in MFC tumor-bearing mice evaluated at 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours using a small animal imaging system;

[0055] Figure 5 B is an in vitro fluorescence imaging of the main organs and tumors of MFC tumor-bearing mice treated with free Ce6 or FX-11@PEG-Ce6;

[0056] Figure 5 C is an in vitro fluorescence quantitative analysis of the expression of major organs and tumors in MFC tumor-bearing mice treated with free Ce6 or FX-11@PEG-Ce6;

[0057] Figure 5D is a photograph of tumors in MFC-bearing mice treated with control group, control group (+), α-PD-1, PEG-Ce6 (+), FX-11@PEG-Ce6, FX-11@PEG-Ce6+α-PD-1, FX-11@PEG-Ce6 (+), and FX-11@PEG-Ce6+α-PD-1 (+), respectively;

[0058] Figure 5 E is a statistical graph of the tumor weight of MFC tumor-bearing mice under different treatments;

[0059] Figure 5 F is a statistical graph of the tumor growth curves of MFC tumor-bearing mice under different treatments;

[0060] Figure 5 G is the expression graph of Ki-67 and TUNEL staining of tumors in MFC tumor-bearing mice that received different treatments;

[0061] Figure 6 FX-11@PEG-Ce6 enhanced CD8 + Analytical spectrum of T cell function; including:

[0062] Figure 6 A is the expression graph of CD8 staining in tumors of MFC tumor-bearing mice treated with control group, control group (+), α-PD-1, PEG-Ce6 (+), FX-11@PEG-Ce6, FX-11@PEG-Ce6+α-PD-1, FX-11@PEG-Ce6 (+), and FX-11@PEG-Ce6+α-PD-1 (+);

[0063] Figure 6 B is flow cytometry analysis showing the CD3 + CD8 + Expression plots of cell proportions;

[0064] Figure 6 C is the TNF-α expression in the tumors of MFC-bearing mice receiving different treatments + IFN-γ + and granzymeB + CD8 + Expression graphs of representative flow cytometry results of TILs;

[0065] Figure 7 Analytical profile demonstrating that FX-11@PEG-Ce6 enhances the efficacy of α-PD-1 in patient-derived xenograft (PDX) models;

[0066] Figure 7A is a schematic diagram of the establishment of a humanized gastric cancer PDX mouse model;

[0067] Figure 7 B is the biodistribution of free Ce6 and FX-11@PEG-Ce6 in vivo observed by a small animal in vivo imaging system;

[0068] Figure 7 C is the in vitro fluorescence imaging of tumors and major organs after tail vein injection of free Ce6 and FX-11@PEG-Ce6;

[0069] Figure 7 D is the in vitro fluorescence quantitative statistical graph of tumors and major organs after tail vein injection of free Ce6 and FX-11@PEG-Ce6;

[0070] Figure 7 E is a photo of tumors in PDX mouse models treated with control group, control group (+), α-PD-1, PEG-Ce6 (+), FX-11@PEG-Ce6, FX-11@PEG-Ce6+α-PD-1, FX-11@PEG-Ce6 (+), and FX-11@PEG-Ce6+α-PD-1 (+);

[0071] Figure 7 F is the tumor growth curve of PDX mouse models with different treatments;

[0072] Figure 7 G is the tumor weight of PDX mouse models with different treatments;

[0073] Figure 7 H is the body weight of PDX mouse models with different treatments;

[0074] Figure 7 I is the expression diagram of Ki-67 and TUNEL staining of PDX mouse model tumors with different treatments;

[0075] Figure 8 Analytical profile for in vivo safety assessment of FX-11@PEG-Ce6; wherein:

[0076] Figure 8 A is the alanine aminotransferase (ALT) level in MFC tumor-bearing mice treated with control group, control group (+), α-PD-1, PEG-Ce6 (+), FX-11@PEG-Ce6, FX-11@PEG-Ce6+α-PD-1, FX-11@PEG-Ce6 (+), and FX-11@PEG-Ce6+α-PD-1 (+);

[0077] Figure 8 B is the aspartate aminotransferase (AST) level in MFC tumor-bearing mice with different treatments;

[0078] Figure 8 C is the blood urea nitrogen (BUN) level of MFC tumor-bearing mice with different treatments;

[0079] Figure 8 D is the serum creatinine (CR) level of MFC tumor-bearing mice with different treatments;

[0080] Figure 8 E is a representative image of H&E staining of the heart, liver, spleen, lung, and kidney of MFC tumor-bearing mice with different treatments;

[0081] Figure 9 The expression diagram of UV-visible absorption spectra of FX-11, PEG-Ce6 and FX-11@PEG-Ce6;

[0082] Figure 10 The drug release curves of Ce6 in FX-11@PEG-Ce6 in PBS buffer with different pH values;

[0083] Figure 11 The pH value of the culture medium of MKN45 and MKN28 cells treated with PEG-Ce6, FX-11, and FX-11@PEG-Ce6 is detected; wherein:

[0084] Figure 11 a is the pH value of the culture medium of MKN45 cells treated with PEG-Ce6, FX-11, and FX-11@PEG-Ce6;

[0085] Figure 11 b pH value of the culture medium of MKN28 cells treated with PEG-Ce6, FX-11, and FX-11@PEG-Ce6;

[0086] Figure 12 Analytical profiles for quantitative analysis of the immunofluorescence intensity of calreticulin (CRT) in MKN45 and MKN28 cells treated with Ce6 or FX-11@PEG-Ce6 with or without light irradiation; wherein:

[0087] Figure 12 a is a quantitative analysis of the immunofluorescence intensity of calreticulin (CRT) in MKN45 cells treated with Ce6 or FX-11@PEG-Ce6 with or without light irradiation;

[0088] Figure 12 b is a quantitative analysis of the immunofluorescence intensity of calreticulin (CRT) in MKN28 cells treated with Ce6 or FX-11@PEG-Ce6 with or without light irradiation;

[0089] Figure 13The analytical spectrum is for quantitative analysis of the immunofluorescence intensity of high mobility group protein B1 (HMGB1) in MKN45 and MKN28 cells irradiated with Ce6 or FX-11@PEG-Ce6 or not irradiated with light; wherein:

[0090] Figure 13 a is a statistical graph showing the quantitative analysis of the immunofluorescence intensity of high mobility group protein B1 (HMGB1) in MKN45 cells irradiated with Ce6 or FX-11@PEG-Ce6 or without light irradiation;

[0091] Figure 13 b is a statistical graph showing the quantitative analysis of the immunofluorescence intensity of high mobility group protein B1 (HMGB1) in MKN28 cells irradiated with Ce6 or FX-11@PEG-Ce6 or without light irradiation;

[0092] Figure 14 Analytical profiles of cell viability of MKN45 cells after treatment with PEG-Ce6, free FX-11, and FX-11@PEG-Ce6, respectively;

[0093] Figure 15 Analytical profiles of cell viability of MKN28 cells after treatment with PEG-Ce6, free FX-11, and FX-11@PEG-Ce6, respectively;

[0094] Figure 16 The cell viability analysis spectrum of MKN45 and MKN28 after treatment with different drug doses; wherein:

[0095] Figure 16 a is a statistical graph of cell viability of MKN45 cells after treatment with different drug doses;

[0096] Figure 16 b is a statistical graph of cell viability of MKN28 cells after treatment with different drug doses;

[0097] Figure 17 Analytical profiles of relative lactate levels in tumors of MFC-bearing mice treated with control, control (+), α-PD-1, PEG-Ce6 (+), FX-11@PEG-Ce6, FX-11@PEG-Ce6+α-PD-1, FX-11@PEG-Ce6 (+), and FX-11@PEG-Ce6+α-PD-1 (+);

[0098] Figure 18 The relative lactate levels of tumors in each group were analyzed in the PDX mouse model.

[0099] Figure 19Analytical profiles of CD8 staining in patient-derived xenograft (PDX) mouse models treated with control, control (+), α-PD-1, PEG-PEG-Ce6 (+), FX-11, FX-11@PEG-Ce6+α-PD-1, FX-11-11@PEG-Ce6 (+), and FEG-Ce61 (+);

[0100] Figure 20 Analytical profile for in vivo safety assessment of FX-11@PEG-Ce6; wherein:

[0101] Figure 20 aAlanine aminotransferase (ALT) levels in patient-derived xenograft (PDX) mouse models treated with control, control (+), α-PD-1, PEG-Ce6 (+), FX-11, PEG-Ce6, FX-11, PEG-Ce6 + α-PD-1, FX-11, and PEG-Ce6 + α-PD-1 (+);

[0102] Figure 20 b Aspartate aminotransferase (AST) levels in PDX mouse models with different treatments (B);

[0103] Figure 20 c is the expression of blood urea nitrogen (BUN) in PDX mouse models after different treatments;

[0104] Figure 20 d is the expression diagram of serum creatinine (CR) in PDX mouse model;

[0105] Figure 20 e are representative images of H&E staining of the heart, liver, spleen, lung, and kidney of PDX mouse models after different treatments. DETAILED DESCRIPTION

[0106] The present invention will be further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments.

[0107] Example 1:

[0108] The present embodiment provides a nanosystem for regulating the acidic microenvironment of tumors to promote the efficacy of photoimmunotherapy. The nanosystem includes a nanocarrier PEG-NH2 and nanomedicines Ce6-COOH and FX-11, wherein FX-11 is LDHA Inhibitor FX11; Ce6 is dihydrochlorin e6;

[0109] Among them, the equivalent ratio of PEG-NH2 to Ce6-COOH is: 1:0.5-1.5;

[0110] The mass ratio of PEG-NH2 to FX-11 is: 20-80:1;

[0111] PEG and Ce6 are connected through amide bonds and assembled with FX-11 through hydrophobic interaction to form a nanosystem, namely FX-11@PEG-Ce6.

[0112] The preparation steps are:

[0113] Step S1, PEG-Ce6 synthesis:

[0114] PEG-NH2 was dissolved in dichloromethane, and a DMF solution containing Ce6-COOH, EDC and DMAP was added. After the reaction was completely dissolved, the reaction solution was concentrated by vacuum distillation, precipitated with a large amount of ice ether, filtered and collected, and vacuum dried to obtain PEG-Ce6;

[0115] Step S2, synthesis of FX-11@PEG-Ce6:

[0116] PEG-Ce6 was dissolved in water, and then a DMSO solution containing FX-11 was added. After stirring and mixing, the mixture was centrifuged to remove free FX-11 and collect the precipitate to obtain FX-11@PEG-Ce6.

[0117] FX-11@PEG-Ce6 kills tumor cells through photodynamic therapy;

[0118] FX-11@PEG-Ce6 induces immunogenic death of tumor cells, promotes dendritic cell maturation, and promotes the activation and infiltration of CD8+T cells. It restores the function of CD8+T cells by improving the acidic microenvironment and synergistically enhances the efficacy of α-PD-1.

[0119] A nanosystem is used to prepare biological products; the biological products include reagents, test kits, and chips.

[0120] Example 2:

[0121] FX-11@PEG-Ce6 was prepared by the following steps:

[0122] 1. Synthesis of PEG-Ce6:

[0123] Weigh 50 mg of PEG-NH2 and dissolve it in 5 mL of dichloromethane. Add 2 mL of a DMF solution containing Ce6-COOH, EDC, and DMAP (the equivalent ratio of Ce6-COOH, EDC, and DMAP is 1:2:1). Once completely dissolved, react at 50°C for 1 hour. Concentrate the reaction solution by vacuum distillation. Precipitate with a large amount of icy ether, filter, collect, and vacuum dry to obtain PEG-Ce6.

[0124] 2. Synthesis of FX-11@PEG-Ce6:

[0125] 20 mg of PEG-Ce6 was dissolved in 10 ml of water, and then 200 μl of DMSO containing 1 mg of FX-11 was added. After stirring for 24 hours, the mixture was centrifuged at 13,000 rpm / min to remove free FX-11 and collect the precipitate.

[0126] Example 3:

[0127] 1. Synthesis and characterization of FX-11@PEG-Ce6:

[0128] PEG was chosen as the shell of the nanoparticles, and FX-11 and Ce6 were used as the core due to their hydrophobic nature. PEG and Ce6 are linked by amide bonds and can self-assemble into nanoparticles with FX-11 through hydrophobic interactions.

[0129] The morphology of FX-11@PEG-Ce6 was examined by transmission electron microscopy (TEM). Figure 1 As shown in Figure A, the particle size of FX-11@PEG-Ce6 is uniform, regular spherical, evenly dispersed, and there is no obvious aggregation. Dynamic light scattering (DLS) data show that the average particle size of FX-11@PEG-Ce6 is 132±4.10 nm. Figure 1 As shown in B. The Zeta potential of FX-11@PEG-Ce6 is -5.5±0.4 mV. Figure 1 C. In addition, the UV-Vis spectrum showed that FX-11@PEG-Ce6 had the same absorption peaks as FX-11 and PEG-Ce6, as shown in Figure 9 To evaluate the stability of FX-11@PEG-Ce6, the particle size and polydispersity index (PDI) of the nanoparticles were measured in water, phosphate buffered saline (PBS), and 10% fetal bovine serum (FBS) over eight days. Figure 1 Figure DF shows that the particle size and PDI of FX-11@PEG-Ce6 in water and PBS are relatively stable, indicating that FX-11@PEG-Ce6 has good stability in vitro. In 10% FBS, the diameter of FX-11@PEG-Ce6 increases slightly, which may be due to the adsorption of serum proteins on its surface.

[0130] Next, the drug release behavior of FX-11@PEG-Ce6 at different pH values ​​was studied. The results showed that nanoencapsulated FX-11 and Ce6 had a good sustained release effect. At the same time, the drug release was faster in acidic solution, which suggests that FX-11@PEG-Ce6 may have better efficacy in acidic tumor microenvironment, such as Figure 1 G and Figure 10 As shown. The drug loading efficiency of FX-11@PEG-Ce6 was 2.43% ± 0.26%. To evaluate the in vitro uptake of nanoparticles, gastric cancer cell lines were treated with free Ce6 and Ce6-loaded FX-11@PEG-Ce6. Figure 1 As shown in Figures H and I, significant red fluorescence was observed in gastric cancer cells treated with Ce6-loaded FX-11@PEG-Ce6. This indicates that FX-11@PEG-Ce6 is better taken up by tumor cells, which may be due to the enhanced permeability and retention (EPR) effect.

[0131] 2. Lysosomal escape and cellular internalization of FX-11@PEG-Ce6 by gastric cancer cells:

[0132] To evaluate the lysosomal escape ability of FX-11@PEG-Ce6, gastric cancer cell lines were treated with FX-11@PEG-Ce6 for 4 h. The results showed that there was no obvious colocalization between FX-11@PEG-Ce6 and lysosomes, indicating that FX-11@PEG-Ce6 was able to escape from lysosomes. Figure 2 As shown in A and B. Figure 2 As shown in C, the internalization of FX-11@PEG-Ce6 mainly occurred within the first 4 hours, as there was no significant change in the intracellular red fluorescence intensity of FX-11@PEG-Ce6 at 4 and 24 hours. MKN45 and MKN28 cells were treated with FX-11@PEG-Ce6 for 4 hours at 4°C and 37°C, respectively. The results showed that the cellular uptake of FX-11@PEG-Ce6 at 37°C was much higher than that at 4°C, as shown in Figure 3. Figure 2 As shown in D, this indicates that the process of cellular internalization of FX-11@PEG-Ce6 is energy-consuming. Then, different endocytosis inhibitors were used to elucidate the specific endocytic pathways involved in the uptake of FX-11@PEG-Ce6. Chlorpromazine (CPZ) can inhibit clathrin-mediated endocytosis. Gastric cancer cell lines were treated with CPZ for 4 hours before adding FX-11@PEG-Ce6. The results showed that CPZ had no significant effect on the intracellular mean fluorescence intensity, as shown in Figure 5. Figure 2 As shown in Figure E, this indicates that clathrin-mediated endocytosis is not the cause of FX-11@PEG-Ce6 cellular internalization. Methyl-β-cyclodextrin (Mβ-CD) can inhibit caveolin-dependent endocytosis. Figure 2As shown in Figure F, after co-incubation with Mβ-CD, the mean fluorescence intensity of FX-11@PEG-Ce6 was significantly reduced, indicating that the endocytosis of FX-11@PEG-Ce6 is related to caveolin. Dynasore is a drug that inhibits dynein, which is required for clathrin- and caveolin-dependent endocytosis. Similarly, after treatment with Dynasore, the mean fluorescence intensity of FX-11@PEG-Ce6 was slightly reduced, as shown in Figure 5. Figure 2 G. Therefore, the above results indicate that FX-11@PEG-Ce6 can escape from lysosomes and the endocytosis of FX-11@PEG-Ce6 is mediated by caveolin.

[0133] 3. FX-11@PEG-Ce6 induces immune activation in vitro:

[0134] The pH value of the culture medium was 6.5 to simulate the acidic tumor microenvironment, and the pH value of the culture medium was 7.4 to simulate the physiological environment. + The proportion of T cells decreased significantly, such as Figure 3 This suggests that the acidic microenvironment may hinder the proliferation of cytotoxic T cells and lead to an immunosuppressive microenvironment. Subsequently, the lactate levels and culture medium pH values ​​of MKN45 and MKN28 cells treated with PEG-Ce6, FX-11, and FX-11@PEG-Ce6 were compared. Figure 3 B and Figure 11 It was found that the FX-11@PEG-Ce6 group significantly reduced lactate production and the acidity of the extracellular medium compared with the free FX-11 group, indicating that the nanoparticles enhanced the cellular uptake of FX-11. This suggests that FX-11@PEG-Ce6 may have the potential to reverse the acidic immunosuppressive microenvironment.

[0135] Photodynamic therapy-induced immunogenic cell death contributes to the maturation of dendritic cells, which is also crucial for immune activation. To evaluate the photodynamic therapy (PDT) effect of FX-11@PEG-Ce6, MKN45 and MKN28 cells were treated, and confocal laser scanning microscopy (CLSM) was used to evaluate immunogenic cell death (ICD) biomarkers including cell surface-exposed calreticulin (CRT) and high-mobility group protein B1 (HMGB1) release. Figure 3 C, D and Figure 12 As shown in the figure, CRT expression increased significantly after light irradiation. In addition, FX-11@PEG-Ce6 showed stronger green fluorescence than the free Ce6 group, indicating that the nanoformulation Ce6 can significantly accumulate in gastric cancer cells. Figure 3 E, F, and Figure 13As shown in Figure 3, the green fluorescence of HMGB1 in cells treated with photodynamic therapy is very weak, indicating that HMGB1 can be effectively released after irradiation. These findings indicate that FX-11@PEG-Ce6 can induce a strong ICD response under light irradiation.

[0136] As the most effective antigen-presenting cells, dendritic cells (DCs) play a key role in the activation of antitumor immunity during ICD. To evaluate the DC maturation effect triggered by FX-11@PEG-Ce6, the mouse gastric cancer cell line MFC was co-cultured with bone marrow-derived DCs, and the maturation of DCs was evaluated by flow cytometry. The proportion of mature DCs increased significantly after light irradiation, and the largest proportion of mature DCs was observed in the FX-11@PEG-Ce6 light-irradiated group, indicating that FX-11 can also promote the maturation of DCs, which may be due to the reduction of lactate production, such as Figure 3 G. In summary, FX-11@PEG-Ce6 combined with light irradiation can significantly induce ICD response, promote DCs maturation, and reverse the acidic immunosuppressive microenvironment.

[0137] FX-11@PEG-Ce6 enhances T cell-mediated cytotoxicity and promotes the efficacy of α-PD-1 combined immunotherapy in vitro

[0138] After verifying the ability of FX-11@PEG-Ce6 to activate immune cells, we attempted to verify the cytotoxicity of FX-11@PEG-Ce6 itself and the effect of combined immunotherapy. First, the anti-tumor effect of FX-11@PEG-Ce6 itself was evaluated using CCK-8 experiments. The data showed that both FX-11 and PEG-Ce6 have certain anti-tumor effects, such as Figure 14 and Figure 15 As shown. After light irradiation, the PEG-Ce6 group and the FX-11@PEG-Ce6 group showed stronger anti-tumor ability, which indicates that the photosensitizer can exert obvious photodynamic therapy effect after light irradiation, but FX-11@PEG-Ce6 can achieve the strongest anti-tumor ability under light irradiation, which is mainly attributed to the photodynamic therapy induced by FX-11@PEG-Ce6 and the inhibition of cell glycolysis. At the same time, it was found that regardless of the presence or absence of light, FX-11@PEG-Ce6 always showed dose-dependent cytotoxicity. In addition, at the same drug dose, the cytotoxicity of the light-irradiated group was always stronger, as shown in Figure 2. Figure 16 As shown, this also confirms the previous view. Next, we began to verify the effect of FX-11@PEG-Ce6 on immune cells. First, gastric cancer cells were co-cultured with activated peripheral blood mononuclear cells (PBMCs), and the co-cultured cells were treated differently. Figure 4As shown in A and B, the expression levels of TNF-α, IFN-γ and granzyme B in the PEG-Ce6(+) group were higher than those in the control group, indicating that the immunogenic cell death induced by PEG-Ce6 can promote the function of cytotoxic T lymphocytes (CTLs). At the same time, the expression levels of TNF-α, IFN-γ and granzyme B in the FX-11@PEG-Ce6 group were higher than those in the PEG-Ce6 group, indicating that loading FX-11 can effectively enhance the function of cytotoxic T lymphocytes (CTLs). This shows that both PEG-Ce6 and FX-11 can stimulate CTLs to secrete cytokines. In addition, the combined use of FX-11@PEG-Ce6, light and α-PD-1 triggered the highest apoptosis rate of GC cells, as shown in Figure 5. Figure 4 C and D. Therefore, these data indicate that FX-11@PEG-Ce6 can enhance CD8+ T cell-mediated cytotoxicity and enhance the therapeutic effect of α-PD-1 in vitro. V. FX-11@PEG-Ce6 enhances the effect of photoimmunotherapy in vivo:

[0139] In order to study the therapeutic effect of FX-11@PEG-Ce6 in vivo, the biodistribution of FX-11@PEG-Ce6 in MFC tumor-bearing mice was first evaluated. The small animal imaging system was first used to monitor the real-time distribution of Ce6 and FX-11@PEG-Ce6 in the body after tail vein injection. The results showed that compared with FX-11@PEG-Ce6, free Ce6 almost completely disappeared 24 hours after administration, indicating that the retention ability of free Ce6 in the body was poor. At the same time, within 24 hours, there was almost no fluorescence aggregation in the tumor area of ​​the free Ce6 group, while significant fluorescence aggregation was observed in the tumor area of ​​the FX-11@PEG-Ce6 group, indicating that FX-11@PEG-Ce6 has a better targeting effect on tumor tissue in vivo, such as Figure 5 As shown in A. This may be attributed to the EPR effect. In vitro fluorescence imaging of major organs and tumors verified the tumor targeting properties of FX-11@PEG-Ce6, as shown in Figure 5 As shown in B and C. Figure 5 As shown in DF, the FX-11@PEG-Ce6+α-PD-1(+) group showed the most significant therapeutic effect, which is consistent with the in vitro data. In addition, the relative levels of intratumoral lactate in each group after treatment were measured. The results showed that FX-11@PEG-Ce6 treatment can improve the acidic environment in the tumor, such as Figure 17 In addition, Ki-67 and TUNEL staining were performed on tumor tissues. The FX-11@PEG-Ce6+α-PD-1(+) group showed the lowest cell proliferation level and the highest cell apoptosis level, as shown in Figure 2. Figure 5G. In summary, FX-11@PEG-Ce6 combined with α-PD-1 antibody treatment demonstrated significant antitumor efficacy in vivo, consistent with in vitro experimental results. Taken together, these results indicate that the FX-11@PEG-Ce6+α-PD-1(+) group possessed the strongest tumor-killing ability and effectively alleviated the acidic tumor microenvironment.

[0140] 6. FX-11@PEG-Ce6 enhances CD8+ T cell function in vivo:

[0141] In order to evaluate the effect of FX-11@PEG-Ce6 on CD8+ T cells in vivo, immunofluorescence staining and flow cytometry analysis were performed on tumor tissues, and the expression levels of CD8 in each group after treatment were detected. The results showed that the CD8 + The level of T cell infiltration was higher than that in the PEG-Ce6(+) group, which indicated that FX-11 loading could effectively reverse the immunosuppressive microenvironment. + The T cell infiltration level was also higher than that in the FX-11@PEG-Ce6 group, indicating that FX-11@PEG-Ce6-induced immune cell death can effectively promote dendritic cell maturation and activate CD8 + At the same time, CD8 + The highest level of T cell infiltration was observed, which confirmed that FX-11@PEG-Ce6 could exert a strong photoimmunotherapy effect, e.g. Figure 6 As shown in A and B. In addition, the expression levels of TNF-α, IFN-γ and granzyme B in the FX-11@PEG-Ce6+α-PD-1(+) group were also the highest, as shown in Figure 6 C. These results indicate that FX-11@PEG-Ce6 can enhance the tumor-killing ability of CD8+ T cells in vivo by improving the acidic microenvironment and promoting DC cell maturation.

[0142] 7. FX-11@PEG-Ce6 enhances the efficacy of α-PD-1 in patient-derived xenograft (PDX) models:

[0143] To further evaluate the effect of FX-11@PEG-Ce6 on the efficacy of α-PD-1, a humanized gastric cancer PDX mouse model was established. Figure 7 As shown in A. The tumor-infiltrating CD8 +T cells were adoptively transferred into immunodeficient mice bearing gastric cancer xenografts. Next, a small animal in vivo imaging system was used to observe the real-time distribution of FX-11@PEG-Ce6 in vivo. After tail vein injection of FX-11@PEG-Ce6, the subcutaneous tumor area in the right groin of the mouse showed a stronger fluorescence signal, indicating that FX-11@PEG-Ce6 has a stronger tumor targeting ability, which is consistent with previous results, such as Figure 7 As shown in B. At the same time, the in vitro fluorescence imaging results of tumors and major organs also support this conclusion, as shown in Figure 7 As shown in C and D. Consistently, it was found that the xenograft tumor volume in the FX-11@PEG-Ce6+α-PD-1(+) group was the smallest, as shown in Figure 7 The results of the measurement of intratumoral lactate levels in each group showed that FX-11@PEG-Ce6 could increase lactate production, thereby improving the acidic microenvironment. Figure 18 There was no significant difference in body weight among the groups, indicating that FX-11@PEG-Ce6 had no obvious toxicity. Figure 7 H. In addition, the fluorescence of Ki-67, TUNEL, and CD8 in tumor tissues extracted from PDX models was also detected. Consistent with previous findings, the FX-11@PEG-Ce6+α-PD-1(+) group showed the highest level of cytotoxic T cell infiltration and GC cell apoptosis rate, and the lowest level of GC cell proliferation, as shown in Figure 3. Figure 7 I. Figure 19 Taken together, these data confirm that FX-11@PEG-Ce6 can enhance the efficacy of α-PD-1 therapy in PDX mouse models.

[0144] 8. In vivo safety assessment of FX-11@PEG-Ce6:

[0145] To examine the biosafety of FX-11@PEG-Ce6, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and serum creatinine (CR) in MFC tumor-bearing mice were measured, as shown in Figure 3. Figure 8 As shown in AD. Figure 8 As shown in Figure E, H&E staining of the heart, liver, spleen, lungs, and kidneys showed no significant toxicity to major organs. Similarly, the data from the PDX model also confirmed the biosafety of FX-11@PEG-Ce6 in vivo. Figure 20 Taken together, these results indicate that FX-11@PEG-Ce6 has no obvious systemic toxicity in vivo.

[0146] In summary, the present invention has constructed a new type of nanomaterial (FX-11@PEG-Ce6). In addition to killing tumor cells through photodynamic therapy (PDT), it can also induce tumor cell immunogenic death (ICD), promote the maturation of dendritic cells (DCs), and then promote the activation and infiltration of CD8+T cells. At the same time, FX-11@PEG-Ce6 can also restore the function of CD8+T cells by improving the acidic microenvironment. These two effects can synergistically enhance the efficacy of α-PD-1, exert a synergistic immunotherapy effect, and provide a new approach for photodynamic immunotherapy.

[0147] The above embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and replacements made by technicians in this field on the basis of the present invention fall within the scope of protection required by the present invention.

Claims

1. A nanomaterial for regulating the acidic microenvironment of tumors to enhance the efficacy of photoimmunotherapy, characterized in that: The nanomaterial includes a nanocarrier PEG-NH2, a nanomedicine Ce6-COOH and a lactate dehydrogenase A inhibitor FX-11; Among them, the equivalent ratio of PEG-NH2 to Ce6-COOH is: 1:0.5-1.5; The mass ratio of PEG-NH2 to FX-11 is: 20-80:1; The PEG is connected to Ce6 through an amide bond and assembled with FX-11 through hydrophobic interaction to form a nanomaterial, namely FX-11@PEG-Ce6.

2. The nanomaterial for regulating the acidic microenvironment of tumors and enhancing the efficacy of photoimmunotherapy according to claim 1, characterized in that: The preparation steps are: Step S1, PEG-Ce6 synthesis: PEG-NH2 was dissolved in dichloromethane, and a DMF solution containing Ce6-COOH, EDC and DMAP was added. After the reaction was completely dissolved, the reaction solution was concentrated by vacuum distillation, precipitated with a large amount of ice ether, filtered and collected, and vacuum dried to obtain PEG-Ce6; Step S2, synthesis of FX-11@PEG-Ce6: PEG-Ce6 was dissolved in water, and then a DMSO solution containing FX-11 was added. After stirring and mixing, the mixture was centrifuged to remove free FX-11 and collect the precipitate to obtain FX-11@PEG-Ce6.

3. The nanomaterial for regulating the acidic microenvironment of tumors and enhancing the efficacy of photoimmunotherapy according to claim 1, characterized in that: The FX-11@PEG-Ce6 kills tumor cells through photodynamic therapy.

4. The nanomaterial for regulating the acidic microenvironment of tumors and enhancing the efficacy of photoimmunotherapy according to claim 1, characterized in that: The FX-11@PEG-Ce6 induces immunogenic death of tumor cells, promotes maturation of dendritic cells, promotes activation and infiltration of CD8+T cells, restores the function of CD8+T cells by improving the acidic microenvironment, and synergistically enhances the efficacy of α-PD-1.

5. The nanomaterial according to any one of claims 1 to 4, characterized in that The nanomaterial is used for preparing biological products.

6. The nanomaterial according to claim 5, wherein The biological product is a reagent or a kit.

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