Multifunctional nanoparticles targeting cancer cells and capable of modulating the tumor immune microenvironment, and preparation method and application thereof

Abstract

The application provides a multifunctional nanoparticle targeting cancer cells and capable of regulating tumor immune microenvironment and a preparation method and application thereof; the preparation method comprises the following steps: a) mixing K3[Fe(CN)6], a Mo source, water and polyvinylpyrrolidone, and performing a hydrothermal reaction in the presence of a hydrochloric acid solution to obtain a reaction mixture; then the solid in the reaction mixture is separated, and then is sequentially subjected to washing and drying to obtain molybdenum-doped prussian blue nanoparticles; b) mixing the molybdenum-doped prussian blue nanoparticles with a cancer cell membrane, and performing coating to obtain a composite; and then the composite is washed with water to obtain the multifunctional nanoparticle. The product not only significantly improves the efficiency of photothermal therapy, but also generates active oxygen and consumes antioxidant glutathione in the tumor microenvironment, thereby enhancing chemical dynamic therapy and promoting the presentation of tumor-associated antigens; the coating of the cancer cell membrane as a warehouse of tumor-associated antigens further improves the immunotherapy potential of the product.

Description

Technical Field

[0001] This invention relates to the field of nanomaterials technology for cancer treatment, and more specifically, to a multifunctional nanoparticle that targets cancer cells and can regulate the tumor immune microenvironment, and a method for preparing the same. Background Technology

[0002] Tumor immunotherapy is a treatment strategy that utilizes a patient's own immune system to recognize and eliminate cancer cells. This approach aims to activate or enhance the natural functions of the immune system, enabling it to effectively recognize and attack tumor cells. Tumor immunotherapy includes various methods, such as immune checkpoint inhibitors, CAR-T cell therapy, cancer vaccines, and other immunomodulatory therapies. The main advantage of cancer immunotherapy lies in its ability to elicit a durable immune response, effectively inhibiting tumor recurrence and metastasis. Despite its promising therapeutic potential, challenges remain, including low response rates and immune escape. To enhance immunogenicity, one strategy is to induce apoptosis in tumor cells or directly deliver tumor-associated antigens (TAAs) to strengthen the immune response. Furthermore, immune checkpoints, such as programmed cell death protein 1 (PD-1) and its signaling pathway, play a crucial role in immune escape mechanisms. The use of immune checkpoint inhibitors can significantly enhance the immune system's ability to recognize tumors. Currently, researchers are actively exploring new strategies to improve the efficacy of immunotherapy and address the challenges in its application.

[0003] Enhancing the immune response to tumors is an important area of ​​current cancer research. Here are some key strategies for improving the tumor immune response: (1) using immune checkpoint inhibitors, such as antibodies against programmed death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4); (2) developing vaccines targeting specific tumor antigens; (3) employing cell therapy, such as CAR-T therapy; (4) tumor immunometabolic modulation therapy; and (5) combination therapies such as chemotherapy and radiotherapy. Treatments such as photothermal therapy (PTT) or chemodynamic therapy (CDT) are applied at the primary lesion site to induce tumor cell death and release immunogenic tumor-associated antigens. These antigens then activate dendritic cells, thereby triggering a T-cell-mediated anti-cancer immune response. The synergistic effect of these two therapies not only effectively promotes direct tumor ablation but also stimulates antigen presentation, further activating the immune response. This combined strategy is expected to overcome the limitations of single therapies and provide a comprehensive cancer treatment option. PTT and CDT have attracted widespread attention due to their ability to induce local hyperthermia and generate reactive oxygen species (ROS), respectively. ROS are a series of highly reactive molecules and free radicals produced from the partial reduction of oxygen. In organisms, ROS are typically generated by processes such as the mitochondrial respiratory chain and inflammatory responses of immune cells. While adequate amounts of ROS are essential for cell signaling and physiological functions, excessive ROS can lead to damage to cell structure and function. In cancer treatment, artificially increasing ROS levels in tumor cells is a common strategy to induce apoptosis or necrosis of cancer cells.

[0004] However, in the tumor microenvironment, the overexpression of the antioxidant glutathione (GSH) may reduce the efficacy of CDT by reacting with cytotoxic hydroxyl radicals. To address this challenge, the study introduced multivalent metal ions such as iridium (Ir) 3+ / Ir 4+ ), copper (Cu) + / Cu 2+ ) and manganese (Mn 2+ / Mn 4+ Elements such as molybdenum ions (Mo) are incorporated into the synthesized anticancer nanoparticles. 6+ / Mo 4+This substance not only effectively scavenges GSH but also participates in the generation of ROS through Fenton-like reactions (Angew. Chem. Int. Ed. 2019, 58, 946; Adv. Mater. 2019, 31, 1905271; Adv. Mater. 2020, 32, 1906024). The valence transitions of these metal ions can simultaneously achieve CDT effects and GSH scavenging, significantly enhancing anti-tumor efficacy. Furthermore, strategies for directly delivering tumor-associated antigens have also been used to enhance antigen presentation, stimulating tumor-specific immune responses through cancer nanovaccines and improving the efficacy of immunotherapy.

[0005] Prussian blue (PB) is a pharmaceutical formulation approved by the U.S. Food and Drug Administration (FDA) and is widely used in PTT (photothermal therapy) for cancer treatment due to its excellent photothermal conversion efficiency. PB's versatility stems from its ability to be doped with different metal ions to form so-called Prussian blue analogues (PBAs), which exhibit diverse therapeutic properties. In the study by Shi et al., they first synthesized PB nanoparticles and then formed molybdenum (Mo)-containing nanoparticles, such as molybdenum disulfide (MoS2), on their surface via chemical growth (references: Adv. Mater., DOI:10.1002 / adma.201503381; ACS Appl. Mater. Interfaces 2017, 9, 14, 12773; CN113577273A; CN114836768A; CN114214662A; Angew. Chem. Int. Ed. 2023, 62, e2022187), or added molybdenum using electrochemical deposition (reference: Int J Energy Res. 2022, 46, 17220). Although these methods achieved Mo doping, they made the entire process quite complex. Furthermore, these composite materials exhibit poor stability, particularly after injection into animal blood, where they easily detach from the nanoparticles, hindering their effective delivery to the tumor site and reducing their bioavailability. In some composite structures, Mo exists in a tetravalent form, failing to oxidize excess glutathione within the tumor and thus unable to disrupt the defensive microenvironment established by tumor cells to resist cytotoxic antioxidants. This limitation reduces the tumor-killing efficacy of ROS-generating therapeutic strategies.

[0006] In summary, the existing technologies still have the following drawbacks: (1) Due to the influence of cancer immune escape and tumor immunosuppressive microenvironment, the effects of most current immunotherapies are not satisfactory; (2) Although some nanomaterials have therapeutic potential, they lack imaging diagnostic functions, which limits their ability to carry out precise treatment under imaging guidance, thus affecting the maximization of treatment effects; (3) Most nanomaterials have failed to effectively improve the tumor microimmune environment, which limits the effective implementation and effect improvement of immunotherapy strategies. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a multifunctional nanoparticle that targets cancer cells and can regulate the tumor immune microenvironment, as well as its preparation method and application. Mo-doped Prussian blue (PMo) nanoparticles are synthesized in one step, and these particles are further encapsulated by cancer cell membranes (CCMs) to prepare PMo@CCM. Mo doping not only significantly improves the efficiency of PTT (phosphorus transduction) but also enhances CDT (cancer cell death response) by generating reactive oxygen species and consuming GSH. Simultaneously, the combined application of PTT and CDT effectively induces cell death and promotes the presentation of tumor-associated antigens. The coating of the cancer cell membrane serves as a repository for tumor-associated antigens, further enhancing the immunotherapeutic potential of PMo@CCM.

[0008] This invention provides a method for preparing multifunctional nanoparticles that target cancer cells and regulate the tumor immune microenvironment, comprising the following steps:

[0009] a) K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone were mixed and subjected to a hydrothermal reaction in the presence of hydrochloric acid solution to obtain a reaction mixture; the solid in the above reaction mixture was then separated, washed and dried sequentially to obtain molybdenum-doped Prussian blue nanoparticles.

[0010] b) The molybdenum-doped Prussian blue nanoparticles obtained in step a) are mixed with cancer cell membranes for coating to obtain a complex; the complex is then washed with water to obtain multifunctional nanoparticles that target cancer cells and can regulate the tumor immune microenvironment.

[0011] Preferably, the process of mixing K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone in step a) specifically involves:

[0012] a1) Dissolve K3[Fe(CN)6] in water, stir at 200 rpm to 400 rpm, and add polyvinylpyrrolidone; let the above mixture stand with stirring for later use to obtain solution A;

[0013] a2) Add the Mo source and polyvinylpyrrolidone to water and mix to obtain solution B;

[0014] a3) Add solution B dropwise to solution A and stir for 5 min to 15 min to obtain a mixture;

[0015] There is no order restriction between steps a1) and a2).

[0016] Preferably, the ratio of K3[Fe(CN)6], water and polyvinylpyrrolidone used in step a1) is (0.01mmol~0.1mmol):40mL:(2g~5g);

[0017] The ratio of Mo source, polyvinylpyrrolidone and water used in step a2) is (0.01mmol~0.1mmol): (2g~5g): 40mL.

[0018] Preferably, the Mo source in step a) is MoO3; the concentration of the hydrochloric acid solution is 0.01M to 0.02M; and the volume ratio of the hydrochloric acid solution to water is (0.5 to 1.5):1.

[0019] Preferably, the hydrothermal reaction process in the presence of hydrochloric acid solution described in step a) specifically includes:

[0020] Hydrochloric acid solution was added to a mixture of K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone and stirred for 5 min to 15 min. The mixture was then transferred to a Teflon-lined stainless steel autoclave for hydrothermal reaction to obtain the reaction mixture.

[0021] Preferably, the temperature of the hydrothermal reaction in step a) is 80℃~100℃ and the time is 15h~25h.

[0022] Preferably, the separation method in step a) is centrifugation; the centrifugation speed is 10,000 rpm to 20,000 rpm, and the time is 5 min to 15 min;

[0023] The washing method involves washing twice with deionized water and twice with anhydrous ethanol.

[0024] Preferably, in step b), the mass ratio of molybdenum-doped Prussian blue nanoparticles to cancer cell membrane is 1:(1-5); the coating method is to stir with a mixer at 20℃-30℃ for 2-4 hours; the number of water washings is 1-3 times, and the product is stored at 2℃-5℃ after washing.

[0025] This invention also provides a multifunctional nanoparticle that targets cancer cells and can regulate the tumor immune microenvironment, prepared using the preparation method described above.

[0026] This invention also provides an application of composite nanomaterials in the preparation of tumor immunotherapy drugs, wherein the composite nanomaterials are the multifunctional nanoparticles described in the above technical solution that target cancer cells and can regulate the tumor immune microenvironment.

[0027] This invention provides a multifunctional nanoparticle that targets cancer cells and regulates the tumor immune microenvironment, as well as its preparation method and application. The preparation method includes the following steps: a) mixing K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone, and carrying out a hydrothermal reaction in the presence of hydrochloric acid solution to obtain a reaction mixture; then separating the solid in the above reaction mixture, and washing and drying it sequentially to obtain molybdenum-doped Prussian blue nanoparticles; b) mixing the molybdenum-doped Prussian blue nanoparticles obtained in step a) with a cancer cell membrane for coating to obtain a complex; then washing the above complex with water to obtain multifunctional nanoparticles that target cancer cells and regulate the tumor immune microenvironment. Compared with existing technologies, this invention uses a one-step synthesis of Mo-doped Prussian blue (PMo) nanoparticles, which are further encapsulated by cancer cell membranes (CCMs) to prepare PMo@CCMs. Mo doping not only significantly improves the efficiency of photothermal therapy (PTT), but also enhances chemodynamic therapy (CDT) by generating ROS and consuming the antioxidant glutathione (GSH) in the tumor microenvironment. At the same time, the combined application of PTT and CDT effectively induces cell death and promotes the presentation of tumor-associated antigens. The coating of the cancer cell membrane serves as a repository for tumor-associated antigens, further enhancing the immunotherapeutic potential of PMo@CCMs.

[0028] Furthermore, this invention enhances the efficacy of immunotherapy by using a programmed cell death protein 1 antibody (anti-PD-1); the synergistic effect of PTT, CDT, and immunotherapy not only shows significant effects in treating primary tumors but also effectively inhibits the growth of distant tumors.

[0029] Furthermore, PMo@CCM also demonstrates superior performance in magnetic resonance imaging (MRI) and photothermal imaging (PTI), highlighting its versatility as a diagnostic tool; as a multifunctional nanoplatform, PMo@CCM shows potential for cancer diagnosis and treatment applications and provides new strategies for synergistic cancer therapy.

[0030] In addition, PMo@CCM significantly improves the tumor immune microenvironment (TIME) and effectively enhances the immunotherapy effect of cancer through the combination of PTT and CDT. Attached Figure Description

[0031] Figure 1 Characterization results of PMo and its CCM-coated PMo@CCM;

[0032] Figure 2Photothermal performance evaluation of PMo@CCM;

[0033] Figure 3 XPS spectrum of Mo in PMo@CCM;

[0034] Figure 4 For cell viability testing;

[0035] Figure 5 For photothermal (PTI) and magnetic resonance imaging (MRI) analysis;

[0036] Figure 6 For the evaluation of the therapeutic effect on proximal tumors in vivo;

[0037] Figure 7 An analysis of the efficacy of in vivo immunotherapy against distant tumors;

[0038] Figure 8 To analyze the percentages of helper T cells and cytotoxic T cells in (a) the spleen and (b) the distal tumor draining lymph nodes in different treatment groups by flow cytometry;

[0039] Figure 9 The concentration of the inflammatory factor interferon-γ (IFN-γ) in the serum of mice under different treatments was determined by ELISA.

[0040] Figure 10 Serum biochemical analysis results of mice after different treatments;

[0041] Figure 11 Representative H&E immunostaining micrographs of major organs of mice in different treatment groups after 15 days of treatment. Detailed Implementation

[0042] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] The following is a description of some of the terms used in this invention:

[0044] Tumor immunotherapy: Tumor immunotherapy is a treatment strategy that utilizes a patient's own immune system to recognize and eliminate cancer cells. This treatment aims to activate or enhance the natural functions of the immune system, enabling it to effectively recognize and attack tumor cells. Tumor immunotherapy includes various methods, such as immune checkpoint inhibitors, CAR-T cell therapy, cancer vaccines, and other immunomodulatory therapies.

[0045] Tumor Immune Microenvironment (TIME): The tumor immunosuppressive microenvironment refers to a special local environment surrounding a tumor, in which tumor cells actively suppress or disrupt the normal response of the immune system through various mechanisms, thereby evading immune surveillance and resistance.

[0046] Photothermal therapy (PTT) is a medical technology that uses light energy to convert into heat energy to treat diseases, and it has been widely used, particularly in the field of cancer treatment. This treatment method mainly involves irradiating targeted tumor tissue with light of a specific wavelength (usually near-infrared light), through which light absorbers (such as certain nanomaterials or dyes) absorb the light energy and convert it into heat energy. This locally generated high temperature can cause thermal death of tumor cells without affecting surrounding normal tissue.

[0047] Chemodynamic Therapy (CDT): Chemodynamic therapy is an emerging cancer treatment strategy that utilizes chemical reactions within the body to generate reactive oxygen species (ROS) sufficient to kill cancer cells. This treatment method primarily relies on the reaction between transition metal ions and specific chemicals in the tumor microenvironment (such as hydrogen peroxide) to produce high levels of ROS through the Fenton reaction or similar processes.

[0048] Reactive oxygen species (ROS) are a class of oxygen-containing chemical substances that play important roles in many physiological and pathological processes in organisms. ROS include both free radical and non-free radical forms, such as superoxide anion (O3). 2. - ), hydrogen peroxide (H2O2), singlet oxygen ( 1 O2) and hydroxyl radicals (OH·), etc.

[0049] The problems in the prior art that this invention aims to solve include: (1) In order to reshape the tumor immune microenvironment and enhance the effect of immunotherapy, this invention has developed multifunctional or composite nanomaterials. These materials significantly improve the effect of cancer treatment by combining the synergistic effects of chemokinetics, photothermal therapy and immunotherapy; (2) In view of the limitation of existing materials that only have therapeutic functions but lack imaging diagnostic capabilities, this invention has developed nanomaterials with dual functions of therapy and imaging, realizing real-time monitoring and precise guidance of the treatment process; (3) In order to solve the problem that most nanomaterials cannot improve the tumor microimmune environment, this invention has developed nanomaterials that can regulate the tumor microenvironment and activate the immune response, thereby improving the overall effect of immunotherapy.

[0050] Based on this, the present invention provides a method for preparing multifunctional nanoparticles that target cancer cells and can regulate the tumor immune microenvironment, comprising the following steps:

[0051] a) K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone were mixed and subjected to a hydrothermal reaction in the presence of hydrochloric acid solution to obtain a reaction mixture; the solid in the above reaction mixture was then separated, washed and dried sequentially to obtain molybdenum-doped Prussian blue nanoparticles.

[0052] b) The molybdenum-doped Prussian blue nanoparticles obtained in step a) are mixed with cancer cell membranes for coating to obtain a complex; the complex is then washed with water to obtain multifunctional nanoparticles that target cancer cells and can regulate the tumor immune microenvironment.

[0053] In this invention, K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone are first mixed and subjected to a hydrothermal reaction in the presence of hydrochloric acid solution to obtain a reaction mixture. The solid in the above reaction mixture is then separated, washed and dried sequentially to obtain molybdenum-doped Prussian blue nanoparticles (synthesis of PMo).

[0054] The present invention does not impose any special restrictions on the sources of K3[Fe(CN)6], Mo source, polyvinylpyrrolidone (PVP), and hydrochloric acid solution; commercially available products well known to those skilled in the art can be used. The water used can be deionized water well known to those skilled in the art.

[0055] In this invention, the process of mixing K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone is preferably as follows:

[0056] a1) Dissolve K3[Fe(CN)6] in water, stir at 200 rpm to 400 rpm, and add polyvinylpyrrolidone; let the above mixture stand with stirring for later use to obtain solution A;

[0057] a2) Add the Mo source and polyvinylpyrrolidone to water and mix to obtain solution B;

[0058] a3) Add solution B dropwise to solution A and stir for 5 min to 15 min to obtain a mixture;

[0059] There is no order restriction between steps a1) and a2).

[0060] In this invention, the ratio of K3[Fe(CN)6], water and polyvinylpyrrolidone in step a1) is (0.01mmol~0.1mmol):40mL:(2g~5g); the ratio of Mo source, polyvinylpyrrolidone and water in step a2) is (0.01mmol~0.1mmol):(2g~5g):40mL.

[0061] In this invention, the Mo source is preferably MoO3; the concentration of the hydrochloric acid solution is preferably 0.01M to 0.02M; and the volume ratio of the hydrochloric acid solution to water is preferably (0.5 to 1.5):1.

[0062] In this invention, the hydrothermal reaction process in the presence of hydrochloric acid solution is preferably as follows:

[0063] Hydrochloric acid solution was added to a mixture of K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone and stirred for 5 min to 15 min. The mixture was then transferred to a Teflon-lined stainless steel autoclave for hydrothermal reaction to obtain the reaction mixture.

[0064] In this invention, the temperature of the hydrothermal reaction is preferably 80℃~100℃, and the time is preferably 15h~25h.

[0065] After the above heat treatment is completed, the solids in the reaction mixture are separated. The separation method is preferably centrifugation. The centrifugation speed is preferably 10,000 rpm to 20,000 rpm, and the centrifugation time is preferably 5 min to 15 min.

[0066] In this invention, the washing method is preferably to wash twice with deionized water and twice with anhydrous ethanol; the drying method can be the room temperature drying method in a vacuum dryer, which is well known to those skilled in the art.

[0067] After obtaining the molybdenum-doped Prussian blue nanoparticles, the present invention mixes the obtained molybdenum-doped Prussian blue nanoparticles with a cancer cell membrane for coating to obtain a complex; then the above complex is washed with water to obtain multifunctional nanoparticles that target cancer cells and can regulate the tumor immune microenvironment (Preparation of PMo@CCM).

[0068] This invention does not impose any special restrictions on the source of the cancer cell membrane, and it is obtained using extraction methods for cancer cell membranes well known to those skilled in the art. In a preferred embodiment of this invention, the extraction process of the cancer cell membrane includes the following steps:

[0069] First, cancer cells were seeded into culture dishes containing 5%–15% fetal bovine serum (FBS) and cultured overnight; subsequently, 1×10⁶ cells were collected. 7 ~1×109 Cells were washed 2-4 times with phosphate-buffered saline (PBS), followed by centrifugation at 1000-1500g to collect the cell pellet. The collected cells were then resuspended in hypotonic lysis buffer (containing 15mM-25mM Tris-HCl, 5mM-15mM KCl, 1mM-4mM MgCl2, and protease inhibitors, pH 7-8) and homogenized using a Dounce homogenizer. The mixture was then centrifuged at 3000-3500 rpm to collect the supernatant. The supernatant was further centrifuged at 18000-22000g for 3-8 minutes. Finally, the supernatant was centrifuged at 2℃-6℃ at 55000g-65000g for 1-1.5 hours to precipitate the cancer cell membrane (CCM). The collected pellet was then treated with 5mM-15mM PBS. Wash with HEPES buffer (pH 7-8), and finally use the BCA method to determine the protein concentration of CCM.

[0070] In this invention, the mass ratio of the molybdenum-doped Prussian blue nanoparticles to the cancer cell membrane is preferably 1:(1-5), and can be 1:1, 1:2, 1:3, 1:4, or 1:5.

[0071] In this invention, the coating method is preferably to use a mixer to stir for 2h to 4h at 20℃ to 30℃; the number of water washings is preferably 1 to 3 times to remove unreacted residues, and after water washing, the product (the prepared PMo@CCM complex) is preferably stored at 2℃ to 5℃.

[0072] This invention also provides a multifunctional nanoparticle that targets cancer cells and can regulate the tumor immune microenvironment, prepared using the preparation method described above.

[0073] In previous studies, researchers first synthesized PB nanoparticles and then formed molybdenum (Mo)-containing nanostructures, such as molybdenum disulfide (MoS2), on their surface using a chemical growth method. While this method achieved Mo doping, the process was relatively complex. Furthermore, the composite materials exhibited poor stability, especially after injection into animal blood, where they easily detached from the nanoparticles, preventing them from effectively reaching the tumor site and exerting their effects. This detachment also reduced the material's bioavailability. Moreover, in the aforementioned composite structures, Mo predominantly exists in a tetravalent form, which prevents it from oxidizing excess glutathione within the tumor. Therefore, Mo cannot disrupt the defensive microenvironment established by tumor cells to resist cytotoxic antioxidants, a limitation that reduces the tumor-killing effectiveness of therapeutic strategies that generate reactive oxygen species.

[0074] This invention synthesizes molybdenum-doped Prussian blue nanoparticles using a simple method. These nanoparticles possess near-infrared absorption properties and can be used for photothermal therapy. In PMo@CCM nanoparticles, molybdenum plays a dual role: on the one hand, it consumes the antioxidant glutathione (GSH) in the tumor microenvironment, thereby reshaping the tumor immunosuppressive microenvironment and disrupting the tumor's self-protective mechanisms; on the other hand, molybdenum catalyzes the generation of reactive oxygen species (ROS), directly enhancing the killing effect on tumor cells. Furthermore, after modifying the surface of the nanoparticles with cancer cell membranes, these materials can specifically recognize the same type of cancer cells. Under laser irradiation, these cancer cell-carrying nanoparticles can further promote the release of cancer-associated antigens, thereby enhancing the effect of immunotherapy. Simultaneously, the synthesized PMo@CCM nanoparticles possess photothermal (PTI) and magnetic resonance (MRI) imaging capabilities, providing precise diagnostic guidance for treatment; combined with immune checkpoint inhibitors, such as anti-PD-1 antibodies, photothermal therapy (PTT), chemokinetic therapy (CDT), and enhanced immunotherapy can be achieved.

[0075] This invention also provides an application of composite nanomaterials in the preparation of tumor immunotherapy drugs, wherein the composite nanomaterials are the multifunctional nanoparticles described in the above technical solution that target cancer cells and can regulate the tumor immune microenvironment.

[0076] This invention provides an innovative molybdenum-doped Prussian blue nanoparticle (PMo@CCM) coated with a cancer cell membrane. The synthesis method for this nanoparticle is simple and easy to operate, demonstrating significant advantages in imaging-guided cancer therapy. PMo@CCM is not only suitable for photothermal therapy guided by MRI and photothermal imaging, but can also effectively integrate chemodynamic therapy and immunotherapy to achieve synergistic cancer treatment. The core advantage of PMo@CCM lies in its unique dual function. Molybdenum plays a crucial role in these nanoparticles: on the one hand, it effectively depletes the antioxidant glutathione in the tumor microenvironment, increasing the sensitivity of tumor cells to oxidative stress and immunotherapy, thereby disrupting the tumor's self-protective mechanisms; on the other hand, it catalyzes the generation of reactive oxygen species (ROS), significantly enhancing the tumor microenvironment's stress on tumor cell growth and attacking cancer cells. Furthermore, the synergistic effect of the photothermal therapy (PTT), chemodynamic therapy (CDT), and cancer cell membrane (CCM) coating of PMo@CCM greatly promotes the exposure and presentation of tumor-associated antigens. This mechanism effectively addresses the antigen scarcity problem commonly encountered in immunotherapy, while simultaneously promoting the activation of helper T cells and cytotoxic T cells, thus enhancing the overall immune response. To further enhance the immune response, we used intraperitoneal injection of anti-PD-1 antibodies to improve the interaction between T cells and tumor cells, thereby enhancing the immune system's ability to attack tumors. This multimodal therapeutic strategy produced a synergistic effect in animal models, significantly inhibiting the growth of primary tumors in mice and effectively limiting the progression of distant tumors.

[0077] In summary, this invention not only provides an effective means to combat recurrent tumors and promote antigen presentation, but also demonstrates significant efficacy in treating recurrent tumors. This approach, combining PTT, CDT, and immunotherapy, showcases its enormous clinical application potential in future cancer treatment, offering a novel and highly effective treatment strategy.

[0078] This invention provides a multifunctional nanoparticle that targets cancer cells and regulates the tumor immune microenvironment, as well as its preparation method and application. The preparation method includes the following steps: a) mixing K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone, and carrying out a hydrothermal reaction in the presence of hydrochloric acid solution to obtain a reaction mixture; then separating the solid in the above reaction mixture, and washing and drying it sequentially to obtain molybdenum-doped Prussian blue nanoparticles; b) mixing the molybdenum-doped Prussian blue nanoparticles obtained in step a) with a cancer cell membrane for coating to obtain a complex; then washing the above complex with water to obtain multifunctional nanoparticles that target cancer cells and regulate the tumor immune microenvironment. Compared with existing technologies, this invention uses a one-step synthesis of Mo-doped Prussian blue (PMo) nanoparticles, which are further encapsulated by cancer cell membranes (CCMs) to prepare PMo@CCMs. Mo doping not only significantly improves the efficiency of photothermal therapy (PTT), but also enhances chemodynamic therapy (CDT) by generating reactive oxygen species and consuming the antioxidant glutathione (GSH) in the tumor microenvironment. At the same time, the combined application of PTT and CDT effectively induces cell death and promotes the presentation of tumor-associated antigens. The coating of the cancer cell membrane serves as a repository for tumor-associated antigens, further enhancing the immunotherapeutic potential of PMo@CCMs.

[0079] Furthermore, this invention enhances the efficacy of immunotherapy by using a programmed cell death protein 1 antibody (anti-PD-1); the synergistic effect of PTT, CDT, and immunotherapy not only shows significant effects in treating primary tumors but also effectively inhibits the growth of distant tumors.

[0080] Furthermore, PMo@CCM also demonstrates superior performance in magnetic resonance imaging (MRI) and photothermal imaging (PTI), highlighting its versatility as a diagnostic tool; as a multifunctional nanoplatform, PMo@CCM shows potential for cancer diagnosis and treatment applications and provides new strategies for synergistic cancer therapy.

[0081] In addition, PMo@CCM significantly improves the tumor immune microenvironment (TIME) and effectively enhances the immunotherapy effect of cancer through the combination of PTT and CDT.

[0082] To further illustrate the present invention, the following embodiments will be described in detail.

[0083] Example

[0084] (1) Synthesis of PMo nanoparticles:

[0085] Preparation of solution A: Dissolve 0.05 mmol of K3[Fe(CN)6] in 40 mL of deionized water. Stir at 300 rpm and add 3 g of PVP. Let the mixture stand with magnetic stirring for later use.

[0086] Preparation of solution B: Add 0.05 mmol MoO3 and 3 g PVP to 40 mL of deionized water and mix thoroughly.

[0087] Mixing and Reaction: Slowly add solution B dropwise to solution A and stir for 10 minutes. Add 80 mL of 0.015 M hydrochloric acid solution and continue stirring for 10 minutes. Transfer the mixture to a Teflon-lined stainless steel autoclave and heat at 90°C for 20 hours.

[0088] Separation and drying: After heat treatment, centrifuge at 14,000 rpm for 10 minutes. Wash twice each with deionized water and anhydrous ethanol. Dry at room temperature in a vacuum dryer to obtain the PMo nanoparticle product.

[0089] (2) Extraction of cancer cell membranes:

[0090] First, cancer cells were seeded into culture dishes containing 10% fetal bovine serum (FBS) and cultured overnight. Then, approximately 1 × 10⁶ cells were collected. 8 Cells were washed three times with phosphate-buffered saline (PBS), followed by centrifugation at 1200g to collect the cell pellet. The collected cells were resuspended in hypotonic lysis buffer (containing 20mM Tris-HCl, 10mM KCl, 2mM MgCl2, and a protease inhibitor, pH 7.5) and homogenized using a Dounce homogenizer. The mixture was then centrifuged at 3200 rpm, and the supernatant was collected. The supernatant was further centrifuged at 20000g for 5 minutes. Finally, the supernatant was centrifuged at 4°C and 60000g for 1.2 hours to precipitate the cancer cell membrane (CCM). The collected pellet was washed with 10mM HEPES buffer (pH 7.5), and the protein concentration of the CCM was determined using the BCA method.

[0091] (3) Preparation of PMo@CCM:

[0092] First, freshly extracted CCM and PMo were mixed at a mass ratio ranging from 5:1 to 1:1 (3:1 in this example). The mixture was stirred for 3 hours at room temperature using a mixer to prepare the PMo@CCM complex. Afterward, the mixture was washed twice with deionized water to remove unreacted residues. Finally, the prepared PMo@CCM was stored at 4°C.

[0093] PMo and PMo@CCM were characterized and analyzed by transmission electron microscopy (TEM), particle size (CLS), zeta potential, inductively coupled plasma mass spectrometry (ICP-MS), and X-ray energy dispersive spectroscopy (XPS).

[0094] 1) In vitro photothermal performance measurement:

[0095] To evaluate the photothermal properties of PMo@CCM, 1 mL of PMo@CCM with a concentration of 50 μg / mL was first placed in a quartz test tube and subjected to different power densities (0.1, 0.3, 0.5, 0.8, 1.0 W / cm²). 2 The sample was irradiated with an 808 nm laser for 10 minutes. Then, it was irradiated with different concentrations (0, 5, 10, 20, 50 μg / mL) of PMo@CCM solution for 10 minutes each. The laser was applied at a power density of 0.3 W / cm². 2 Irradiation with an 808nm laser. 1 mL of a 50 μg / mL PMo@CCM solution was exposed to a power density of 0.5 W / cm². 2 The photothermal stability of PMo@CCM was evaluated by performing six on-off cycles under an 808 nm laser. Temperature was monitored using a digital thermometer. Finally, the photothermal conversion efficiency (η) of PMo@CCM was calculated using the flow equation.

[0096] 2) Glutathione consumption test:

[0097] First, freshly prepared PMo@CCM solutions were mixed with 80 μM glutathione solution at different concentrations (0, 50, 100, 200, 500, 1000 μg / mL) and reacted for 10 minutes. Then, the precipitate was removed by centrifugation, and the supernatant was collected. 10 μL of 0.1 M DTNB solution was added, and incubation continued for another 10 minutes. Afterward, the absorbance peak was measured at 412 nm using a UV-Vis spectrometer, and the analytical results were recorded.

[0098] 3) Determination of reactive oxygen species (ROS) generation:

[0099] 100 μM TMB solution was added to a solution containing 20 μM H2O2 and 50 μg / mL PMo@CCM. Absorbance at 652 nm was monitored every minute using a UV-Vis spectrophotometer. Additionally, intracellular ROS levels after different treatments were assessed using a fluorophore (HPF) probe. 4T1 cells were seeded at a density of 1 × 10⁴ cells / well in 96-well plates and incubated for 12 hours. Subsequently, cells were treated with PBS or 300 μg / mL PMo@CCM, followed by laser treatment at 808 nm (0.3 W / cm²). 2 Irradiate for 10 minutes. After 4 hours of incubation, wash the cells three times with PBS, then treat with a 5M high-pass filter for 30 minutes. Finally, wash twice with PBS, and acquire fluorescence images of the cells using a confocal laser scanning microscope at an excitation wavelength of 488nm and an emission wavelength of 530nm.

[0100] 4) Evaluation of cell viability after different treatments:

[0101] First, the cancer cells were divided into 1×10 4 Cells were seeded at a density of 10% fetal bovine serum in 96-well plates and incubated at 37°C for 12 hours. Afterwards, 3 μL of PBS or PMo@CCM (final concentration 10 mg / mL) was added to the cells, and incubation was continued for 3 hours to promote nanomaterial absorption. The laser-treated group was then treated with 0.3 W / cm² laser. 2 Cells were irradiated with an 808 nm laser for 10 minutes. After laser treatment, cells were cultured at 37°C for 24 or 48 hours. Then, 100 μL of fresh culture medium was added, along with 10 μL of CCK-8 cell viability assay solution, and the cells were incubated for another 30 minutes. Finally, cell viability was assessed by measuring the absorbance of each group at 450 nm using a microplate reader. As a control, cells treated with PBS but not laser irradiated were included.

[0102] 5) In vivo anti-cancer therapy:

[0103] All animal experiments were conducted in accordance with animal welfare and ethical review guidelines and were overseen by the Animal Care and Use Committee (IACUC) of the Changchun Institute of Applied Chemistry, Chinese Academy of Sciences (Approval No.: CIAC 2023.0174). In the experiments, 2 × 10⁶ animals were used... 6 One cancer cell (100 μL) was subcutaneously injected into the left hind limb of 5-7 week old female BALB / c mice. When the tumor volume reached approximately 100 mm², the tumor was inoculated. 3 Mice were randomly divided into seven groups and received the following treatments: (1) PBS, (2) PBS-L, (3) PBS + anti-PD-1, (4) PMo@CCM, (5) PMo@CCM-L, (6) PMo@CCM + anti-PD-1, and (7) PMo@CCM-L + anti-PD-1. Each group of mice received 200 μL (5 mg / mL) of the corresponding solution intravenously. For the laser treatment groups (2, 5, and 7), 12 hours after injection, mice received 1.2 W / cm² of anti-PD-1. 2 The primary tumor site was irradiated with an 808nm laser for 10 minutes. Additionally, 4T1 tumor cells were subcutaneously inoculated into the right hind limb of mice as distant tumors (day 0). On days 1, 3, 5, 7, 9, 11, and 13, mice in groups (3), (6), and (7) received intraperitoneal injections of anti-PD-1 at a dose of 8 μg / g. The volumes of the primary and distant tumors were measured every 2 days and daily, respectively, using the formula: Volume = Length × Width. 2 / 2. On day 15, the mice were sacrificed, photographed, and samples of primary and distant tumors were collected for hematoxylin-eosin (H&E), Ki-67, CD31, TUNEL staining, and histological analysis.

[0104] 6) In vivo immune activation assessment:

[0105] Flow cytometry was used to assess the activation of cytotoxic T lymphocytes (CTLs). First, spleen and tumor-draining lymph nodes were homogenized through a 200-mesh filter and transferred to 10 mL centrifuge tubes. Red blood cells were removed from spleen samples using erythrocyte lysis buffer. The treated cell suspension was incubated with anti-FVD-eF780 antibody at 4°C for 30 min, followed by washing twice with PBS containing 3% FBS and centrifuging at 4°C, 2000 rpm for 5 min. Next, the cells were co-incubated with CD45-eF506, CD3-FITC, CD4-APC, and CD8-PE antibodies at 4°C for 40 min. After incubation, the cells were washed once more with FACS buffer, centrifuged at 4°C, 2000 rpm for 5 min, and finally fixed with 4% paraformaldehyde. CD4+ in rat spleen and lymph nodes was analyzed by flow cytometry. + or CD8 + T cell count. In addition, serum interferon-γ (IFN-γ) levels were quantitatively measured using an ELISA kit to further assess immune activation status.

[0106] 7) In vivo biochemical analysis and hematological assessment:

[0107] Blood was collected from mice in all experimental groups, and serum was separated by centrifugation at 3000 rpm for 15 minutes. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN), and creatine kinase (CK) to assess liver and kidney function. Changes in these hematological parameters can provide important information about treatment-related effects, particularly on the immune and hematopoietic systems.

[0108] Test results and analysis:

[0109] ①Characteristics of PMo and PMo@CCM:

[0110] This invention is the first to synthesize a molybdenum-doped Prussian blue analogue (PMo) using a one-step method, achieved by mixing MoO3 and K3[Fe(CN)6]. Figure 1 a). Subsequently, PMo was mixed with CCM and stirred at room temperature for 1 hour to obtain the PMo@CCM complex ( Figure 1 b). PMo exhibits a regular cubic structure; however, after CCM coating, the surface of PMo@CCM becomes blurred, and new structural layers appear on the surface. Detailed observation of the elemental distribution using high-resolution transmission electron microscopy confirmed the presence of Mo doping. Figure 1c) The distributions of Mo and Fe elements significantly overlap, providing direct evidence for the presence of molybdenum. Furthermore, this invention also used inductively coupled plasma mass spectrometry (ICP-MS) to quantitatively analyze the Fe and Mo contents in PMo@CCM, showing that the Fe and Mo contents were 237.05 ppm and 7.73 ppm, respectively. Figure 1 d). The UV-Vis spectra of PMo and PMo@CCM show a high absorption peak near 808 nm (see...). Figure 1 e), indicating that these materials have the potential for application as photothermal agents and in photothermal therapy. CCM coating increased the particle size from 125.43±3.48 nm to 164.41±1.86 nm. Figure 1 f), while the zeta potential decreased from -17.7 ± 0.2 mV to -18.4 ± 0.3 mV (see f). Figure 1 g), these changes indicate that CCM was successfully coated onto the surface of PMo nanoparticles.

[0111] See Figure 1 As shown, Figure 1 Characterization results of PMo and its CCM-coated PMo@CCM are shown below; (a) transmission electron microscopy (TEM) image of PMo; (b) TEM image of PMo@CCM, with a scale bar (black) of 400 nm; (c) high-resolution TEM image of PMo@CCM revealing the distribution of Fe and Mo elements, with a scale bar (white) of 100 nm; (d) Fe and Mo content in PMo@CCM determined by inductively coupled plasma mass spectrometry (ICP-MS); (e) UV-Vis absorption spectra of PMo and PMo@CCM; (f) particle size analysis of PMo and PMo@CCM; and (g) zeta potential measurement results of PMo, PMo@CCM, and CCM.

[0112] ②Photothermal property characterization of PMo@CCM:

[0113] Due to the high absorption rate of PMo@CCM at 808 nm, the present invention then maintains the laser power at 0.3 W / cm². 2 The concentration of PMo@CCM was varied, and temperature changes were monitored over 10 minutes under laser irradiation. Figure 2 As shown in Figure a, the solution temperature increased with concentrations of 0, 5, 10, 20, and 50 μg / mL, indicating that the photothermal conversion effect of PMo@CCM is concentration-dependent. Furthermore, as... Figure 2As shown in b, when the PMo@CCM concentration is 50 μg / mL, the rate of temperature rise increases with increasing laser power density, indicating that the photothermal conversion effect of PMo@CCM is dependent on laser power density. The present invention then uses a 50 μg / mL PMo@CCM solution at 0.5 W / cm². 2 The laser was irradiated at a speed of 10 minutes, and then allowed to cool naturally at room temperature. The relationship between time and -ln(θ) was obtained from the cooling curve. Figure 2 c). To evaluate photothermal stability, PMo@CCM underwent six laser switching cycles. (e.g.) Figure 2 As shown in Figure d, the maximum temperature remained relatively stable, indicating that PMo@CCM possesses excellent photothermal stability at high temperatures. The above experiments demonstrate that PMo@CCM exhibits good photothermal properties and has potential for photothermal therapy.

[0114] ③ Extracellular GSH consumption and reactive oxygen species (ROS) generation:

[0115] The extracellular clearance capacity of GSH was assessed using 5,5'-dimercapto(2-nitrobenzoic acid) (DTNB). Figure 2 As can be seen, at 412 nm, the absorbance of DTNB gradually decreases with increasing PMo@CCM concentration, especially at a concentration of 1000 μg / mL where the absorbance (OD412) approaches zero. This indicates that PMo@CCM has a significant GSH-consuming capacity. To further elucidate its mechanism of action, this invention uses X-ray photoelectron spectroscopy (XPS) to analyze the valence state changes of Mo in PMo@CCM during GSH depletion. Figure 3 As shown in a and 3b, after GSH consumption, the percentage of hexavalent Mo in PMo@CCM decreased from 34.48% to 14.79%. This result indicates that hexavalent Mo can oxidize GSH, potentially making cancer cells more susceptible to the killing effects of ROS generated by CDT and PTT, thereby improving the therapeutic effect of cancer.

[0116] To further explore the application of PMo@CCM in chemokinetic therapy (CDT), this invention investigated its ability to generate reactive oxygen species (ROS). The molybdenum (Mo) element in PMo@CCM may react with intracellular H₂O₂ to generate ROS. To quantify ROS generation, 3,3',5,5'-tetramethylbenzidine (TMB) was used as an indicator. Figure 2 As shown in f, after incubation with PMo@CCM, the absorbance of TMB at 652 nm increased significantly over time, indicating that PMo@CCM effectively catalyzed the continuous generation of ROS. These results suggest that PMo@CCM has potential anti-tumor CDT effects.

[0117] See Figures 2-3 As shown, Figure 2 Photothermal performance evaluation of PMo@CCM; where (a) at 0.3 W / cm 2 (a) Temperature change (ΔT) curves of PMo@CCM solutions with concentrations of 0, 5, 10, 20, and 50 μg / mL under 808 nm laser irradiation; (b) Temperature change (ΔT) curves of PMo@CCM solutions with concentrations of 0, 5, 10, 20, and 50 μg / mL under 808 nm laser irradiation; 2 Temperature change curves of PMo@CCM solution with a concentration of 50 μg / mL under laser power density irradiation; (c) at 0.5 W / cm 2 After 10 minutes of laser irradiation, the laser was turned off, and the real-time temperature changes of PMo@CCM and pure water samples as they cooled to room temperature were recorded. A linear fitting curve of time versus -ln(θ) was obtained from the cooling data of the PMo@CCM solution. (d) The photothermal stability of PMo@CCM was tested through six laser on / off cycles. (e) The UV-Vis spectra of PMo@CCM at concentrations of 0, 100, 200, 500, and 1000 μg / mL after incubation with DTNB and GSH solutions for 10 minutes. (f) The change in absorbance at 652 nm within 30 minutes after mixing TMB with H2O2 or H2O2 + PMo@CCM. 652 ).

[0118] Figure 3 The XPS spectra of Mo in PMo@CCM show the changes in the content of each valence state of Mo before and after the reaction with GSH; among them, (a) before GSH treatment, Mo 6+ (purple line) The proportion is 34.48%; (b) After GSH treatment, Mo 6+ The proportion decreased to 14.79%.

[0119] ④ In vitro cancer cell synergistic therapy research:

[0120] This invention uses the CCK-8 method to evaluate PMo@CCM used alone. Figure 4 a) and its effect on the viability of 4T1 cancer cells when used in conjunction with laser irradiation, thereby validating its potential efficacy in chemokinetic therapy (CDT) and in synergy with photothermal therapy (PTT). Figure 4 As shown in b, there was no significant difference between the group treated with PBS only (control group) and the group irradiated with laser only (PBS-L group), indicating that the 808nm wavelength alone, 0.3W / cm², was the only effective treatment. 2Laser irradiation (L) had almost no effect on cell viability. After 24 hours, the cell viability of the PMo@CCM combined with laser irradiation (PMo@CCM-L group) significantly decreased to 33.9%, lower than the 48.9% of the PMo@CCM-only treatment group (*P<0.05). After 48 hours, the survival rate of the PMo@CCM-L group further decreased to 19.9%, significantly lower than the 37.6% of the PMo@CCM group (***P<0.001). Through these tests, the present invention demonstrates that the synergistic effect of CDT and PTT has a more significant effect in inducing cell death.

[0121] See Figure 4 As shown, Figure 4 Cell viability was assessed. (a) Cell viability of 4T1 cancer cells was measured for 24 and 48 hours after treatment with different concentrations (0, 10, 20, 50, 100, 200, 300, 400 μg / mL) of PMo@CCM. (b) Cell viability of 4T1 cells was measured for 24 and 48 hours after different treatments (PBS, PBS-L, PMo@CCM, PMo@CCM-L). Each experiment was repeated three times (n=3). "L" indicates the use of an 808 nm wavelength and 0.3 W / cm² filter. 2 Laser irradiation with varying power. Statistical significance was marked as *P < 0.05 and ***P < 0.001 to assess the effect of different treatments on cell viability.

[0122] ⑤ Photothermal imaging (PTI) and magnetic resonance imaging (MRI):

[0123] First, this invention evaluated the photothermal imaging (PTI) capability of the PMo@CCM composite material. After intravenous injection of PMo@CCM at different time points (0, 6, 12, 24, and 48 hours), this invention utilized 808 nm and 1.2 W / cm² thermal conductivity. 2 The tumor site was irradiated with laser, and its temperature change was monitored using an infrared thermal imager. The results showed that 12 hours after intravenous injection, the temperature of the tumor tissue significantly increased, reaching a peak of 58.6℃ (e.g., Figure 5 (as shown in a). Furthermore... Figure 5 b demonstrates the effect of PMo@CCM in photothermal imaging, with the best photothermal imaging effect observed 12 hours after injection, consistent with the highest accumulation of PMo@CCM in tumors.

[0124] Next, the present invention evaluated the in vitro and in vivo magnetic resonance imaging (MRI) capabilities of PMo@CCM. Figure 5c shows a standard curve illustrating the linear increase in T1-weighted MRI signal with PMo@CCM concentration in in vitro experiments. Subsequently, in vivo experiments conducted on a 1.0T small animal MRI scanner showed that 12 hours after intravenous injection of PMo@CCM, the T1-weighted MRI signal at the tumor sites in the coronal and axial planes was significantly enhanced compared to the control group (e.g., ...). Figure 5 (As shown in d). Furthermore, T1-weighted MRI signal intensity was significantly stronger after intratumoral injection of PMo@CCM solution. These results not only confirm the excellent accumulation properties of PMo@CCM in tumors but also highlight its potential application value in tumor diagnosis and image-guided therapy.

[0125] See Figure 5 As shown, Figure 5 Photothermal (PTI) and magnetic resonance imaging (MRI) analyses were performed; (a) a curve showing the change in tumor temperature over time; and (b) the results of intravenous injection of PMo@CCM at different time points (0, 6, 12, 24, and 48 hours) using 808nm, 1.2W / cm² thermometers. 2 (c) Photothermal imaging of tumor sites irradiated by laser (sample size n=3); (d) Linear relationship between T1-weighted MR signal and PMo@CCM concentration in in vitro experiments. The inset shows T1-weighted MRI images of different concentrations (0, 0.075, 0.15, 0.3, 0.6, 1.2 mg / mL) of PMo@CCM; (e) T1-weighted MRI images of mice before and 12 hours after intravenous injection of PMo@CCM, including MRI images of direct injection at the tumor site.

[0126] ⑥ Evaluation of in vivo anti-tumor effects:

[0127] This invention further investigated the combined therapeutic effect of PMo@CCM in mice. Primary tumors with a volume of approximately 100 mm² were injected into the left hind leg of each mouse. 3 Mice were then randomly divided into seven groups and received the following treatments: (1) PBS; (2) PBS-1; (3) PBS + anti-PD-1; (4) PMo@CCM; (5) PMo@CCM-L; (6) PMo@CCM + anti-PD-1; (7) PMo@CCM-L + anti-PD-1. Subsequently, mice received 200 μL of 5 mg / mL PMo@CCM or PBS solution intravenously. Twelve hours after injection, groups (2), (5), and (7) received 1.2 W / cm² of PMo@CCM at the tumor site. 2The mice were irradiated with an 808nm laser for 10 minutes. After this photothermal treatment, tumor cells were inoculated into the right hind limb of the mice to simulate tumor metastasis. On days 1, 3, 5, 7, 9, 11, and 13, groups (3), (6), and (7) were intraperitoneally injected with anti-PD-1 at a dose of 8μg / g. After 14 days of follow-up, the tumor volume and body weight of the mice in each group were monitored, and representative images were collected.

[0128] like Figure 6 As shown in figure a, the tumor volume in the PBS group increased rapidly, reaching the largest size. Conversely, the tumor growth in the PMo@CCM-L+ anti-PD-1 group was significantly inhibited, with the smallest volume and weight. Figure 6 b). This result highlights the potential of combined photothermal therapy (PTT), chemodynamic therapy (CDT), and immunotherapy in effectively eliminating tumor cells. The PMo@CCM-L treatment group had the second smallest tumor volume, indicating that PTT and CDT alone also have significant effects. Furthermore, compared to the PBS group, there was no significant change in tumor volume and weight in mice treated with PBS + anti-PD-1. The tumor volume in the PMo@CCM-L + anti-PD-1 group was significantly smaller than that in the PMo@CCM-L group; this enhanced effect may be due to PMo@CCM promoting antigen presentation and the simultaneous elimination of tumor cells through PTT and CDT. Immunostaining evaluation on day 15 showed ( Figure 6 c) Following PMo@CCM-L + anti-PD-1 treatment, the expression of the cell proliferation marker Ki-67 in tumor tissue was significantly reduced, indicating that this combination therapy has strong anti-cancer potential. In the PMo@CCM-L + anti-PD-1 group, the expression of the vascular endothelial cell marker CD31 was significantly reduced, indicating a strong inhibitory effect on tumor angiogenesis. Furthermore, TUNEL staining of the tumor tissue in this group showed strong green fluorescence, suggesting a high level of apoptosis. Finally, H&E staining showed increased intercellular spaces and significant signs of apoptosis in the primary tumor tissue of this group, confirming widespread cell death.

[0129] See Figure 6 As shown, Figure 6For in vivo evaluation of the therapeutic effect on proximal tumors: (1) PBS, (2) PBS-1, (3) PBS + anti-PD-1, (4) PMo@CCM, (5) PMo@CCM-L, (6) PMo@CCM + anti-PD-1, (7) PMo@CCM-L + anti-PD-1. Among them, (a) the curve of primary tumor volume change 14 days after different treatments. (b) the weight measurement results of primary tumors in mice in each treatment group on day 15. Data are expressed as mean ± standard deviation (mean ± SD). There are 4 mice in each group (n = 4). The statistical significance levels are *P < 0.05; **P < 0.01; ***P < 0.001. (c) Ki-67, CD31, TUNEL, and H&E immunostaining microscopic images of primary tumors in different treatment groups (Ki-67 labeled tumor cell proliferation, CD31 labeled angiogenesis, TUNEL labeled apoptosis, and H&E staining observed tumor tissue structure). Scale bar: 100μm.

[0130] To investigate the effectiveness of immunotherapy in cancer treatment, this invention analyzed the growth of distant tumors. The growth trajectory and final weight of the tumor were measured. Figure 7 a and Figure 7 As shown in b. Compared with the control group using PBS alone, the PBS + anti-PD-1 group did not show a significant reduction in tumor volume and weight, suggesting limited efficacy of anti-PD-1 therapy, possibly due to insufficient antigen presentation. Conversely, the PMo@CCM + anti-PD-1 group showed a significant reduction in both tumor volume and weight compared to the PBS group, primarily attributed to CDT-induced primary tumor necrosis and apoptosis, which promoted antigen release. Furthermore, the 4T1 tumor cell membranes coated on PMo@CCM, rich in tumor-associated antigens (TAAs), significantly improved the efficacy of immunotherapy by enhancing antigen presentation. Notably, compared with the PMo@CCM + anti-PD-1 group, the tumor volume in the PMo@CCM-L + anti-PD-1 group was consistently suppressed throughout the treatment period, with a final significant reduction in both volume and weight (*P < 0.05). This difference may be due to enhanced antigen supply triggered by PTT. Figure 7c presents the immunostaining results of distal tumors in each group, including Ki-67, CD31, TUNEL, and H&E staining. Ki-67 staining showed a significant reduction in tumor cell proliferation in the PMo@CCM-L + anti-PD-1 group, indicating the most significant immunotherapy effect in this group. CD31 staining showed that angiogenesis was significantly inhibited after PMo@CCM-L + anti-PD-1 treatment, exhibiting minimal green fluorescence. TUNEL staining indicated an increased apoptosis rate in this treatment group. H&E staining revealed increased intercellular spaces and the highest level of apoptosis in this group. These results demonstrate that, compared to other groups, the PMo@CCM-L + anti-PD-1 group showed a significant increase in apoptosis or necrosis, while angiogenesis and tumor replication were significantly inhibited, highlighting the significant efficacy of the combined immunotherapy strategy.

[0131] See Figure 7 As shown, Figure 7 For the efficacy analysis of in vivo immunotherapy on distant tumors: (1) PBS, (2) PBS-1, (3) PBS + anti-PD-1, (4) PMo@CCM, (5) PMo@CCM-L, (6) PMo@CCM + anti-PD-1, (7) PMo@CCM-L + anti-PD-1. Among them, (a) the curve of distant tumor volume change, including 4 mice in each group (n=4), with statistical significance levels of *P<0.05; **P<0.01; ***P<0.001. (b) Distant tumor weight on day 15 (n=4). (d) Immunostaining microscopic images of distant tumors: Ki-67, CD31, TUNEL and H&E staining were used to assess cell proliferation, angiogenesis, apoptosis and tissue structure changes of distant tumors in different treatment groups. The scale bar is 100 μm.

[0132] The above findings demonstrate that PMo@CCM-L + anti-PD-1 therapy exhibits significant efficacy in both primary and distant tumors, highlighting the synergistic effect between PTT (photothermal therapy), CDT (chemodynamic therapy), and immunotherapy. Particularly noteworthy is the effective stimulation of a comprehensive systemic anti-tumor immune response through multi-channel antigen presentation combined with the use of an immune checkpoint inhibitor (anti-PD-1). The PMo@CCM-L + anti-PD-1 treatment group not only significantly delayed tumor progression but also demonstrated the potential to prevent tumor recurrence. This finding strengthens the theoretical basis for advanced tumor immunotherapy using multimodal treatment strategies and provides strong scientific support for future treatment strategies.

[0133] ⑦ Anti-tumor immune response:

[0134] The findings of this invention highlight the significant immunomodulatory effects of the PMo@CCM formulation, particularly when used in combination with anti-PD-1 therapy. Flow cytometry analysis of CD4-positive T cells (CD4+) was performed. + Helper T cells) and CD8-positive T cells (CD8+) + The proportion of cytotoxic T cells (CTCs) allows for a deeper understanding of how these treatments affect the immune environment in mice. In the spleen, a key immune organ, particularly in the PMo@CCM-L, PMo@CCM+anti-PD-1, and PMo@CCM-L+anti-PD-1 treatment groups, CD4+ was observed. + The significantly increased proportion of T cells indicates a strong activation of helper T cell-mediated immune responses. Figure 8 As shown in figure a, CD4 in the PMo@CCM-L+ anti-PD-1 group + T cell count increased to 16.3%, significantly higher than the control group (8.34%), highlighting the effective immune activation effect of PMo@CCM combined with anti-PD-1 therapy. Simultaneously, CD8... + The significant increase in T cells, especially in the PMo@CCM-L+ anti-PD-1 group, with a percentage of 11.2% in the spleen, was significantly higher than that in the control group (4.08%), highlighting the enhanced cytotoxic response against tumor cells.

[0135] Analysis of the T-cell population in the distal tumor-draining lymph nodes further confirmed the systemic immune response induced by these treatments. Figure 8 In b, CD4 in the PMo@CCM-L+ anti-PD-1 group + T cells and CD8 + The T cell levels were 47.6% and 29.0%, respectively, significantly higher than other groups and significantly higher than the PBS control group, which were 29.4% and 14.6%, respectively. This not only demonstrates the direct local therapeutic effect of the treatment on the primary tumor site but also indicates the impact of PMo@CCM treatment on the systemic immune system; the enhanced immune response in distal lymph nodes is clear evidence of this systemic effect. As previous studies have shown, the proximity of immune organs to tumor lesions plays a crucial role in the immune system's ability to respond to tumors. In mice treated with PMo@CCM-L+ anti-PD-1, the populations of helper T cells and cytotoxic T cells in the spleen and distant lymph nodes were significantly increased, indicating a comprehensive and effective anti-tumor immune response.

[0136] See Figure 8 As shown, Figure 8 To analyze the helper T cells (CD4+) in (a) the spleen and (b) distal tumor draining lymph nodes by flow cytometry under different treatment groups (PBS, PBS + anti-PD-1, PMo@CCM-L and PMo@CCM-L + anti-PD-1).+ / CD3 + ) and cytotoxic T cells (CD8) + / CD3 + The percentage of ). Here, "L" represents an 808 nm wavelength laser with a power density of 1.2 W / cm². 2 .

[0137] Besides the involvement of immune cells, inflammatory mediators also significantly affect the efficacy of immunotherapy. Interferon-γ (IFN-γ), mainly composed of CD8... + T cell production is a key component in modifying overall tumor tissue and plays a crucial role in the tumor microenvironment (TME). Notably, ELISA assays showed that the PMo@CCM-L + anti-PD-1 group exhibited the highest levels of IFN-γ (…). Figure 9 This finding validates the hypothesis that PMo@CCM can counteract the immunosuppression of the tumor microenvironment (TME) during anti-PD-L1 therapy. Overall, these data demonstrate that PMo@CCM significantly enhances immune responses and anti-tumor activity by repairing the TME, modulating immune effector molecules and cellular components. This comprehensive immune activation suggests that the binding of PMo@CCM to anti-PD-1 not only directly targets tumors but also potentially modulates the immune system to prevent tumor recurrence and metastasis, providing a novel avenue for future cancer immunotherapy strategies.

[0138] See Figure 9 As shown, Figure 9 The concentrations of the inflammatory factor interferon-γ (IFN-γ) in the serum of mice under different treatments (PBS, PBS-L, PBS + anti-PD-1, PMo@CCM, PMo@CCM-L, PMo@CCM + anti-PD-1, and PMo@CCM-L + anti-PD-1) were measured by ELISA (sample size n = 3). Here, "L" indicates a power density of 1.2 W / cm². 2 808nm laser.

[0139] ⑧ In vivo toxicity analysis of PMo@CCM:

[0140] This invention first involves serum biochemical analysis of mice in different treatment groups. Following PMo@CCM-L+ anti-PD-1 treatment, serum aspartate aminotransferase (AST) levels in the treatment groups remained within the normal range (36.31-235.48 U / L), see [link to relevant documentation]. Figure 10a. Supporting information. This contrasts sharply with other groups of mice, which showed elevated AST levels exceeding the normal range, indicating potential liver damage. Furthermore, alanine aminotransferase (ALT) levels in all groups were within the normal range (10.06-96.47 U / L), suggesting that PMo@CCM-L + anti-PD-1 treatment had minimal adverse effects on liver function. Figure 10 b, Supporting Information). In addition, blood urea nitrogen (BUN) and creatine kinase (CK) levels ( Figure 10 c) and d) were also within the normal range, indicating that PMo@CCM-L+ anti-pd-1 treatment did not significantly impair kidney and cardiac function. Overall, these results highlight the beneficial characteristics of this therapy in terms of liver function, with negligible effects on kidney and cardiac function in mice, emphasizing its potential as a safe treatment option.

[0141] See Figure 10 As shown, Figure 10 Serum biochemical analysis results of mice after different treatments (PBS, PBS-L, PBS + anti-PD-1, PMo@CCM, PMo@CCM-L, PMo@CCM + anti-PD-1, PMo@CCM-L + anti-PD-1). This includes the concentrations of: (a) aspartate aminotransferase (AST, normal range: 36.31–235.48 U / L), (b) alanine aminotransferase (ALT, normal range: 10.06–96.47 U / L), (c) blood urea nitrogen (BUN, normal range: 10.81–34.74 U / L), and (d) creatine kinase (CK, normal range: 0–2070.55 U / L). Data are expressed as mean ± standard deviation (mean ± SD), with a sample size of n = 3. "L" indicates the use of an 808 nm laser with a power density of 1.2 W / cm². 2 .

[0142] In addition, H&E immunostaining micrographs of major organs (heart, liver, spleen, lungs, and kidneys) were also included. Figure 11 The results showed that no significant necrosis or damage was observed in any of the treatment groups. This result highlights the extremely low toxicity and excellent in vivo biocompatibility of PMo@CCM, further confirming its potential as a safe therapeutic agent.

[0143] See Figure 11 As shown, Figure 11Representative H&E immunostaining micrographs of major organs (heart, liver, spleen, lung, and kidney) in mice from different treatment groups (PBS, PBS-L, PBS + anti-PD-1, PMo@CCM, PMo@CCM-L, PMo@CCM + anti-PD-1, PMo@CCM-L + anti-PD-1) after 15 days of treatment. Scale bar: 100 μm.

[0144] In summary, this invention provides molybdenum-doped Prussian blue nanoparticles (PMo@CCM) encapsulated in cancer cell membranes, and has been successfully applied to synergistic cancer treatment using photothermal therapy, chemodynamic therapy, and immunotherapy guided by MRI and photothermal imaging. The molybdenum element in PMo@CCM has a dual function: on the one hand, it consumes the antioxidant glutathione in the tumor microenvironment, disrupting the tumor's protective mechanisms; on the other hand, it catalyzes the generation of ROS, enhancing toxicity to tumor cells. The synergistic effect of PTT, CDT, and the CCM coating promotes the exposure and presentation of tumor-associated antigens, effectively addressing the problem of antigen scarcity in immunotherapy, and promoting the activation of helper T cells and cytotoxic T cells. To enhance the immune response, this invention improves the interaction between T cells and tumor cells through intraperitoneal injection of anti-PD-1 antibodies. This multimodal treatment strategy produced a synergistic effect, significantly inhibiting the growth of primary tumors in mice and limiting the progression of distant tumors. This method combining PTT / CDT and immunotherapy not only effectively targets primary tumors and promotes antigen presentation but also shows significant efficacy against recurrent tumors, demonstrating its potential for future clinical application in cancer treatment.

[0145] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a multifunctional nanoparticle targeting cancer cells and capable of modulating the tumor immune microenvironment, characterized in that, Includes the following steps: a) K3[Fe(CN)6], Mo source, water, and polyvinylpyrrolidone are mixed and subjected to a hydrothermal reaction in the presence of hydrochloric acid solution to obtain a reaction mixture; the solid in the above reaction mixture is then separated, washed, and dried sequentially to obtain molybdenum-doped Prussian blue nanoparticles; the Mo source is MoO3; the concentration of the hydrochloric acid solution is 0.01M~0.02M; the volume ratio of the hydrochloric acid solution to water is (0.5~1.5):1; b) The molybdenum-doped Prussian blue nanoparticles obtained in step a) are mixed with cancer cell membranes for coating to obtain a complex; the complex is then washed with water to obtain multifunctional nanoparticles that target cancer cells and can regulate the tumor immune microenvironment.

2. The production method according to claim 1, characterized by, The process of mixing K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone as described in step a) is as follows: a1) Dissolve K3[Fe(CN)6] in water, stir at 200 rpm to 400 rpm, and add polyvinylpyrrolidone; let the above mixture stand with stirring for later use to obtain solution A; a2) Add the Mo source and polyvinylpyrrolidone to water and mix to obtain solution B; a3) Add solution B dropwise to solution A and stir for 5 min to 15 min to obtain a mixture; There is no order restriction between steps a1) and a2).

3. The preparation method according to claim 2, characterized in that, The ratio of K3[Fe(CN)6], water and polyvinylpyrrolidone used in step a1) is (0.01mmol~0.1mmol):40mL:(2g~5g); The ratio of Mo source, polyvinylpyrrolidone and water used in step a2) is (0.01mmol~0.1mmol):(2g~5g):40mL.

4. The method of claim 1, wherein, The hydrothermal reaction process described in step a) in the presence of hydrochloric acid solution is as follows: Hydrochloric acid solution was added to a mixture of K3[Fe(CN)6], Mo source, water and polyvinylpyrrolidone and stirred for 5 min to 15 min. The mixture was then transferred to a Teflon-lined stainless steel autoclave for hydrothermal reaction to obtain the reaction mixture.

5. The preparation method according to claim 1, characterized in that, The hydrothermal reaction in step a) is carried out at a temperature of 80℃~100℃ for 15h~25h.

6. The method of claim 1, wherein, The separation method described in step a) is centrifugation; the centrifugation speed is 10,000 rpm to 20,000 rpm, and the time is 5 min to 15 min; The washing method involves washing twice with deionized water and twice with anhydrous ethanol.

7. The preparation method according to claim 1, characterized in that, In step b), the mass ratio of molybdenum-doped Prussian blue nanoparticles to cancer cell membranes is 1:(1~5); the coating method is to stir with a mixer at 20℃~30℃ for 2h~4h; the number of water washings is 1~3 times, and the product is stored at 2℃~5℃ after washing.

8. A multifunctional nanoparticle targeting cancer cells and capable of modulating the tumor immune microenvironment, characterized in that, It is prepared by the preparation method described in any one of claims 1 to 7.

9. The use of a composite nanomaterial in the preparation of a tumor immunotherapy drug, characterized in that, The composite nanomaterial is the multifunctional nanoparticle described in claim 8 that targets cancer cells and can regulate the tumor immune microenvironment.

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