Nanometer sponge immune medicament as well as preparation method and anti-tumor application thereof

Abstract

The invention provides a nano sponge immune medicament as well as a preparation method and anti-tumor application thereof. A novel cellular nano sponge is designed, a low-inflammation photothermal therapy and a metabolic regulation function are combined, and metastasis and relapse of tumors are systematically inhibited by neutralizing and combining lactic acid metabolism interference through inflammatory cytokines. The nano sponge is modified by surface positive charges of photo-thermal core mesoporous polydopamine and then carries siRNA and M1 type macrophage membrane fragments, so that targeted therapy and multi-functional intervention on inflammatory lesions can be realized. A nano sponge treatment mode is innovatively applied to the anti-tumor field, and low-inflammation photo-thermal treatment is realized by utilizing a strategy of neutralizing cytokines by a biological membrane surface receptor. In-vivo and in-vitro experiments prove that the nano sponge has the characteristic of inflammation homing, inhibits tumor growth, prevents metastasis and recurrence, can effectively regulate the primary tumor microenvironment and the inflammation microenvironment of the ecological niche (PMN) before tumor metastasis at the secondary part, and brings new breakthrough and possibility to the field of tumor treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of tumor drugs, in particular to a nano sponge immune agent and a preparation method and anti-tumor application thereof. Background Art

[0002] Tumor metastasis and recurrence are hallmarks of most tumors and lead to the death of approximately 90% of cancer patients. Current clinical interventions, such as tumor resection, chemotherapy, and radiotherapy, can eliminate some primary tumors, but often fail to prevent tumor recurrence and may even promote metastasis. In recent years, the spatiotemporal selectivity of photothermal therapy (PTT) has provided another approach for the treatment of solid tumors. PTT has the ability of non-invasive and local precision treatment. Appropriate PTT destroys the integrity of tumor cells and releases tumor-associated antigens, inducing immunogenic cell death (ICD) to initiate a controlled immune anti-tumor response, and inhibits tumor metastasis and recurrence through immune memory effects and specificity.

[0003] However, recent studies have shown that excessive PTT will trigger an uncontrolled inflammatory response, which may overwhelm the immunogenicity triggered by PTT and lead to skin abscesses, permanent tissue damage, metastasis, and promotion of tumor regeneration. Worse still, the abnormal metabolite of tumors, lactate (LA), will act as an amplifier of inflammation, and there will be signal crosstalk with various immune cells such as macrophages and lymphocytes, directly or indirectly exacerbating the formation of the tumor immunosuppressive microenvironment (TME). Specifically, inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) initiate epithelial-mesenchymal transition (EMT) through the NF-κB pathway, enhancing the stemness and migration behavior of cancer cells. On the other hand, the pre-metastatic niche (PMN) is quietly established during primary tumor formation and is exacerbated by excessive PTT inflammatory response. Various inflammatory mediators will reshape the lung parenchyma and provide "soil" for circulating tumor cells (CTCs). Along with the PTT-related inflammatory response, immunosuppressive cells flood into tumor lesions and are nourished in an environment with high tumor metabolite lactate, becoming accomplices in tumor recurrence and metastasis. This metabolic imbalance within the tumor will drive the PTT inflammatory response, and its metabolite lactate is known as the mastermind of immune chaos.

[0004] To alleviate the inflammatory microenvironment in tumors and PMNs, researchers have discovered nanoparticles that wrap around leukocyte membranes (such as neutrophil membranes and macrophage membranes). These nanoparticles not only have inflammatory "homing" properties, but can also act as bait to adsorb inflammatory molecules. They are called nanosponges. They have been shown to be able to provide anti-inflammatory treatments for diseases such as arthritis and sepsis, but have not yet been used in tumor diseases, and an integrated treatment plan to address the crosstalk between treatment-related inflammation and tumor metabolism has not yet been reported.

[0005] Based on the above background, we studied a cell-like nanosponge (PL@M1M) as a new generation of optical immunoreagents, aiming to systematically target primary tumors and inhibit tumor metastasis and recurrence. Mesoporous dopamine (mPDA) was used as the PTT core to adsorb siRNA of lactate dehydrogenase A (siLDHA) and wrap the membrane of M1 macrophages. Due to the multiple receptors on the surface of the inflammatory cell membrane of PL@M1M, it not only accumulates in the inflammatory lesions, but also neutralizes a large number of inflammatory cytokines. Therefore, PL@M1M can avoid the inflammatory cascade caused by PTT. On the other hand, PL@M1M will cut off the "nutrition" supply of PMN, which is the "soil" for the breeding of circulating tumor cells (CTCs), making PMN unsuitable for their survival, thereby further preventing metastasis. At the same time, siLDHA will interfere with the production of lactate, cut off the positive feedback communication between inflammation and lactate, and reorganize the overall immune microenvironment of the tumor. In conclusion, PL@M1M improves the deficiencies of traditional PTT anti-tumor therapy, systematically inhibits tumor metastasis and recurrence, and provides a paradigm for the development of a new generation of photothermal nanomedicines. Summary of the invention

[0006] In view of the needs of the prior art, the present invention constructs a cell-like nanosponge PL@M1M system, in which the surface of mesoporous polydopamine (mPDA) is modified with tertiary amines by N,N-dimethylethylenediamine (DMEA) to promote the loading of siLDHA and serve as a PTT agent and lactate metabolism regulator (i.e., PL). Finally, PL is encapsulated by newly collected M1 macrophage (M1M) membrane fragments (i.e., PL@M1M). Ultimately, PL@M1M can inhibit tumor metastasis and recurrence in multiple steps. First, based on the "homing" inflammatory ability of M1M, PL@M1M can significantly target and destroy primary tumors through photoimmunotherapy. Subsequently, PL@M1M spontaneously neutralizes PTT inflammatory cytokines through membrane surface receptors, inhibiting the EMT process of primary tumors. PL@M1M "homes" to PMNs, performs anti-inflammatory actions by neutralizing cytokines, and prevents CTCs from colonizing in PMNs. Finally, siLDHA could further prevent tumor metastasis and recurrence by blocking tumor inflammation-lactate metabolism crosstalk, reducing immunosuppressive cell tolerance, and revitalizing the immunosuppressive tumor microenvironment.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A nanosponge immune agent, wherein the surface of a nanophotothermal agent is modified by a modifier, and then a lactic acid LA metabolism disruptor is loaded by electrostatic adsorption, and finally the surface of the nanophotothermal agent is wrapped with a cell membrane bionic coat, and finally a cell-like nanosponge immune agent is constructed;

[0009] The nano-photothermal agent is selected from organic photothermal materials; the organic photothermal materials are selected from small molecule organic dyes, supramolecular complexes, and conjugated polymers;

[0010] Small molecule organic dyes are preferably: indocyanine green (ICG), rhodamine B;

[0011] Preferred supramolecular complexes are: dye-metal complexes, MOF-dye complexes;

[0012] Preferred conjugated polymers include polythiophene and polystyrene.

[0013] The modifier is selected from: an amine compound or a cationic polymer;

[0014] The lactate metabolism disruptor: regulates the production of lactate by inhibiting pyruvate dehydrogenase kinase 1 and lactate dehydrogenase A subunit LDHA target, regulates the transport of lactate through lactate transporters including monocarboxylic acid transporter 1 and monocarboxylic acid transporter 4, and regulates the degradation of lactate through lactate oxidase;

[0015] The cell membrane bionic coat is selected from macrophage membrane, macrophage exosome membrane and neutrophil membrane.

[0016] As a preferred embodiment, when the nanophotothermal agent is a conjugated polymer, it is mesoporous polydopamine mPDA.

[0017] As a preferred embodiment, when the modifier is an amine compound, it is selected from DMEA or triethylenetetramine, preferably DMEA;

[0018] When the modifier is a cationic polymer, it is selected from polylysine, polyacrylamine PAM, polyamine, and polyvinylamine.

[0019] As a preferred embodiment, the lactic acid LA metabolism disruptor is preferably siLDHA to regulate the production of lactic acid LA.

[0020] As a preferred embodiment, the cell membrane bionic coat is preferably an M1 macrophage membrane.

[0021] As a preferred embodiment, the method for preparing the nanosponge immune agent of the anti-tumor drug comprises the following steps:

[0022] The first step is to synthesize PL@M1M:

[0023] The polyoxyethylene polyoxypropylene ether block copolymer and 1,2,4-trimethylbenzene are dissolved in a water-alcohol mixture, and tris (hydroxymethyl)aminomethane (Tris) and dopamine aqueous solution are added, stirred at room temperature for 24 hours, centrifuged, and the template agent polyoxyethylene polyoxypropylene ether block copolymer and 1,2,4-trimethylbenzene are removed by ultrasonication to obtain mPDA suspended in ethanol;

[0024] Modification of mPDA: mPDA was suspended in Tris buffer, DMEA was added, stirred at room temperature for 2 hours, collected by centrifugation, and the surface charge was detected by dynamic light scattering technology DLS and Zeta potential analysis. The siLDHA loading capacity was tested by gel retention experiment. After mixing and stirring, the PL complex was obtained by centrifugation;

[0025] Step 2: Synthesis of PL@M1M nanosponges: Lipopolysaccharide LPS induces M0 macrophages to polarize into M1 macrophages, extracts M1M, mixes with PL ultrasonically, extrude and centrifuge to obtain PL@M1M; the morphology, size, surface potential and protein expression are analyzed by transmission electron microscopy TEM, dynamic light scattering technology DLS, agarose gel electrophoresis SDS-PAGE and flow cytometry, and the stability and hemolysis are evaluated.

[0026] As a preferred embodiment, the method for preparing the nanosponge immune agent of the anti-tumor drug of the present invention comprises the following steps:

[0027] The first step is to synthesize PL@M1M

[0028] First, 0.36 g of polyoxyethylene polyoxypropylene ether block copolymer and 0.36 g of 1,2,4-trimethylbenzene were dissolved in a mixture of 65 mL of water and 60 mL of ethanol; after stirring for 30 min, 5 mL of 18 mg / mL Tris aqueous solution was added to the mixture, and then 5 mL of 12 mg / mL dopamine aqueous solution was added; the reaction mixture was stirred at room temperature for 24 h; then the particles were separated by centrifugation at 13000 rpm for 10 min; the template was removed by repeated ultrasound, that is, the sample was first ultrasound-treated in ethanol for 30 min, centrifuged, and dispersed in a mixed solution of ethanol and acetone in a volume ratio of 2:1 for ultrasound treatment; the final product was suspended in ethanol for further use;

[0029] 1 mg of mPDA was suspended in 2 mL of Tris buffer, and then different concentrations of DMEA were added and stirred at room temperature for 2 hours; DMEA-modified mPDAD was collected by centrifugation; the surface charge of mPDAD at different DMEA concentrations was detected by DLS; the loading capacity of siLDHA was tested by gel retention experiment, and siLDHA and mPDAD were mixed at a mass ratio of siLDHA:mPDAD=1:0-1:6, and after stirring for 4 hours, the mPDAD and siLDHA complex PL was obtained by centrifugation;

[0030] Step 2: Synthesis of PL@M1M nanosponge

[0031] LPS was used to induce M0 macrophages to polarize into M1 macrophages, and M1M was extracted using a membrane protein extraction kit. The total protein content was analyzed by BCA protein assay. The extracted M1M and PL were sonicated, extruded through a polycarbonate membrane, and centrifuged to obtain PL@M1M. The morphology of mPDA and PL@M1M was observed by TEM, and their hydrodynamic diameters and surface potentials were analyzed by DLS. The protein expressions of M1M and PL@M1M were analyzed by SDS-PAGE and flow cytometry. The spatial localization of M1M and PL was determined by observing the overlap of Cy5 fluorescence and the cell membrane dye DiO. Finally, the stability of PL@M1M in PBS and its hemolytic activity were evaluated.

[0032] The present invention also provides an anti-tumor drug composition, comprising the above-mentioned nano sponge immune agent.

[0033] The present invention also provides an application of a nanosponge immunoagent in the preparation of an anti-tumor drug, wherein the nanosponge immunoagent PL@M1M overcomes the side effects of photothermal anti-tumor drugs, such as systemic inflammatory cascade tumor-promoting reactions; the nanosponge immunoagent PL@M1M utilizes the inflammation targeting ability of M1M to ablate primary tumors through photoimmunotherapy, and neutralizes inflammatory factors through membrane surface receptors to inhibit the epithelial-mesenchymal transition (EMT) process of the tumor; PL@M1M "homes" to PMN to prevent the colonization of circulating tumor cells (CTCs); siLDHA blocks the interaction between tumor inflammation and lactate metabolism, reduces immunosuppression, activates the tumor microenvironment, and prevents tumor metastasis and recurrence; the nanosponge immunoagent PL@M1M can achieve comprehensive inhibition of tumor metastasis and recurrence through the multiple mechanisms of M1M's inflammation targeting, neutralization of cytokines, and activation of the tumor microenvironment.

[0034] As a preferred embodiment, the nanosponge immune agent of the present invention is used as a drug for treating the following cancers: including but not limited to various solid tumors appearing on the surface or inside of organs, including skin cancer, malignant melanoma, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, breast cancer, laryngeal cancer, thyroid cancer, tongue cancer, prostate cancer, penile cancer, testicular tumors, vaginal malignancies, and vulvar malignancies.

[0035] The mechanism of the present invention is as follows:

[0036] The present invention constructs a cellular nanosponge PL@M1M system, in which the surface of mesoporous polydopamine (mPDA) is modified with tertiary amines by N,N-dimethylethylenediamine (DMEA) to promote the loading of siLDHA and serve as a PTT agent and lactate metabolism regulator (i.e., PL). Finally, PL is encapsulated by newly collected M1 macrophage (M1M) membrane fragments (i.e., PL@M1M). Ultimately, the nanosponge immunopharmaceutical PL@M1M can inhibit tumor metastasis and recurrence in multiple steps. First, based on the "homing" inflammatory ability of M1M, the nanosponge immunopharmaceutical PL@M1M can significantly target and destroy primary tumors through photoimmunotherapy. Subsequently, PL@M1M spontaneously neutralizes PTT inflammatory cytokines through membrane surface receptors, inhibiting the EMT process of primary tumors. The nanosponge immunopharmaceutical PL@M1M "homes" to PMN, performs anti-inflammatory actions by neutralizing cytokines, and prevents CTCs from colonizing in PMN. Finally, siLDHA could further prevent tumor metastasis and recurrence by blocking tumor inflammation-lactate metabolism crosstalk, reducing immunosuppressive cell tolerance, and reinvigorating the immunosuppressive tumor microenvironment (TME).

[0037] The first aspect of the present invention provides a method for preparing the cellular nanosponge PL@M1M according to the first aspect of the present invention, the method comprising the following steps:

[0038] 1. Synthesis of mPDA loaded with siLDHA: mPDA is an excellent photothermal agent with high biocompatibility, high photothermal conversion efficiency, simple preparation process, and rich surface functional groups. It has broad application prospects in the biomedical field, especially in drug delivery systems and cancer treatment applications such as photothermal therapy. On this basis, we used DMEA modification to form a stable chemical bond with the aldehyde group of the mPDA functional group through chemical reactions such as Michael addition / Schiff base reaction, so it has good stability in the intracellular environment (mPDAD), which helps to protect siRNA and improve delivery efficiency. After surface modification, a positive charge is obtained to obtain mPDAD / siLDHA (PL) by electrostatic adsorption of siLDHA.

[0039] 2. Construction of cellular nanosponge PL@M1M: PL was biomimetically coated with M1 macrophage membrane to achieve inflammatory homing and inflammatory cytokine neutralization ability. First, the M1 macrophage membrane was collected and LPS (100pg / mL) was used to induce the polarization of M0 macrophages into M1 macrophages. M1 macrophage membrane fragments (M1M) were obtained using a membrane protein extraction kit, and the total protein content in the obtained M1M was analyzed by BCA protein assay. The extracted M1M was first treated with PL ultrasound for 5 minutes, then repeatedly pushed with a micro extruder, and finally the suspension was centrifuged to obtain PL@M1M.

[0040] The nanosponge PL@M1M system of the present invention can be directly administered intratumorally or intravenously. PL@M1M ablates the primary tumor and inhibits its EMT process through inflammation "homing" low-inflammation PPT, and reorganizes TME in combination with metabolic interference. More importantly, PL@M1M also relieves the pressure on PMN, preventing it from providing "nutrients" as "soil" for the proliferation of circulating tumor cells (CTCs). PL@M1M overcomes the inflammation-related side effects of traditional photothermal agents, cuts off the connection between PTT inflammation and tumor metabolic communication, and inhibits and eliminates the primary tumor to the greatest extent. The results of anti-spontaneous metastatic tumors also prove that after treatment with nanosponge PL@M1M, the number of metastatic nodules in the mouse lungs was significantly reduced, and the inflammatory state of PMN in the lungs was significantly changed. A satisfactory structure was also achieved in the challenge model, which prolonged the survival rate of mice. Finally, the results of blood routine, liver function, and kidney function tests on mice showed that the nanosponge PL@M1M has good biosafety.

[0041] The present invention has the following advantages:

[0042] (1) We designed a novel cell-like nanosponge (PL@M1M) that combines low-inflammatory photothermal therapy and metabolic regulation functions to systemically inhibit tumor metastasis and recurrence through neutralization of inflammatory cytokines and interference with lactate metabolism.

[0043] (2) The nanosponges have a unique structure, including mesoporous polydopamine surface modified with positive charges as the photothermal core, carrying siRNA and M1 macrophage membrane fragments, which can achieve targeted treatment and multi-functional intervention of inflammatory foci. For the first time, the nanosponges treatment method was applied to the anti-tumor field, and the strategy of neutralizing cytokines by biomembrane surface receptors was cleverly used to achieve low-inflammatory photothermal therapy.

[0044] (3) In vitro experiments have shown that nanosponges have inflammatory homing properties and exhibit significant anti-tumor effects, including inhibiting tumor growth, preventing metastasis and recurrence, and can effectively regulate the primary tumor microenvironment and the inflammatory microenvironment of PMNs in secondary sites, bringing new possibilities to the field of tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is a schematic diagram of the PL synthesis characterization of mPDA of the present invention.

[0046] Figure 2 It is a schematic diagram of the synthesis characterization of PL@M1M of the present invention.

[0047] Figure 3 It is a schematic diagram of characterizing the photothermal and siLDHA controlled release performance of the present invention.

[0048] Figure 4This is a schematic diagram of the cytokine neutralization ability test of PL@M1M.

[0049] Figure 5 This is a schematic diagram of the targeted metabolic regulation and downregulation of PD-L1 mediated by PL@M1M of the present invention.

[0050] Figure 6 This is a schematic diagram of the low-inflammation photothermal anti-tumor experiment mediated by PL@M1M of the present invention.

[0051] Figure 7 This is a schematic diagram of the in vivo inflammation homing ability of PL@M1M of the present invention.

[0052] Figure 8 This is a schematic diagram of the in vivo nanosponge PL@M1M combined anti-tumor effect of the present invention.

[0053] Fig. 9 This is a schematic diagram of PL@M1M of the present invention enhancing the anti-tumor immune response in vivo.

[0054] Fig.10 This is a schematic diagram showing that the present invention can alleviate tumor-related inflammatory response after PTT.

[0055] Fig.11 Schematic diagram of PL@M1M of the present invention inhibiting spontaneous metastasis.

[0056] Fig.12 This is a schematic diagram of the anti-tumor recurrence immune memory effect induced by PL@M1M of the present invention. DETAILED DESCRIPTION

[0057] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0058] The specific implementation example is provided to further illustrate the present invention and is not intended to limit the protection scope of the present invention.

[0059] The present invention provides a nanosponge immune agent, which uses a modifier to modify the surface of photothermal nanoparticles, then carries a lactic acid LA metabolism disruptor through electrostatic adsorption, and finally wraps the surface of the photothermal nanoparticles with a cell membrane bionic coat, and finally constructs a cell-like nanosponge immune agent PL@M1M;

[0060] The nano-photothermal agent is selected from organic photothermal materials; the organic photothermal materials are selected from small molecule organic dyes, supramolecular complexes, and conjugated polymers;

[0061] Small molecule organic dyes are preferably: indocyanine green (ICG), rhodamine B;

[0062] Preferred supramolecular complexes are: dye-metal complexes, MOF-dye complexes;

[0063] Preferred conjugated polymers include polythiophene and polystyrene.

[0064] The modifier is selected from: an amine compound or a cationic polymer;

[0065] The lactic acid LA metabolism disruptor: regulates the production of lactic acid LA by inhibiting PKD1 and LDHA targets, regulates the transport of lactic acid LA through lactate transporters including MCT1 and MCT4, and regulates the degradation of lactic acid LA through lactate oxidase;

[0066] The cell membrane bionic coat is selected from macrophage membrane, macrophage exosome membrane and neutrophil membrane.

[0067] In some embodiments, the photothermal nanoparticles are conjugated polymers mPDA.

[0068] In some embodiments, when the nanophotothermal agent is a conjugated polymer, it is mesoporous polydopamine mPDA.

[0069] In some embodiments, when the modifier is an amine compound, it is selected from DMEA or triethylenetetramine, preferably DMEA;

[0070] When the modifier is a cationic polymer, it is selected from polylysine, polyacrylamine PAM, polyamine, and polyvinylamine PEI.

[0071] In some embodiments, the lactate LA metabolism disruptor, preferably siLDHA, is used to regulate the production of lactate LA.

[0072] In some embodiments, the cell membrane bionic coat is preferably an M1 macrophage membrane.

[0073] In some embodiments, a method for preparing the nanosponge immunotherapy agent comprises the following steps:

[0074] The first step is to synthesize PL@M1M

[0075] First, 0.36 g of polyoxyethylene polyoxypropylene ether block copolymer (F127) and 0.36 g of 1,3,5-trimethylbenzene (TMB) were dissolved in a mixture of 65 mL of water and 60 mL of ethanol; after stirring for 30 min, 5 mL of 18 mg / mL Tris aqueous solution was added to the mixture, followed by 5 mL of 12 mg / mL dopamine aqueous solution; the reaction mixture was stirred at room temperature for 24 h; then the particles were separated by centrifugation at 13000 rpm for 10 min. The template TMB and F127 were removed by repeated ultrasound, i.e., the sample was first ultrasound-treated in ethanol for 30 min, centrifuged, and dispersed in a mixed solution of ethanol and acetone in a volume ratio of 2:1 for ultrasound treatment; the final product was suspended in ethanol for further use;

[0076] 1 mg of mPDA was suspended in 2 mL of Tris buffer, and then different concentrations of DMEA were added and stirred at room temperature for 2 hours; DMEA-modified mPDAD was collected by centrifugation; the surface charge of mPDAD at different DMEA concentrations was detected by dynamic light scattering (DLS); the loading capacity of siLDHA was tested by gel retention experiment, and siLDHA and mPDAD were mixed at a mass ratio of siLDHA:mPDAD=1:0-1:6, and after stirring for 4 hours, the mPDAD and siLDHA complex PL was obtained by centrifugation;

[0077] Step 2: Synthesis of PL@M1M nanosponge

[0078] M0 macrophages were polarized into M1 macrophages induced by lipopolysaccharide (LPS). M1M was extracted using a membrane protein extraction kit, and the total protein content was analyzed by BCA protein assay. The extracted M1M and PL were sonicated, extruded through a polycarbonate membrane, and centrifuged to obtain PL@M1M. The morphology of mPDA and PL@M1M was observed by TEM, and their hydrodynamic diameters and surface potentials were analyzed by dynamic light scattering (DLS). The protein expressions of M1M and PL@M1M were analyzed by SDS-PAGE and flow cytometry. The spatial localization of M1M and PL was determined by observing the overlap of Cy5 fluorescence and the cell membrane dye DiO. Finally, the stability of PL@M1M in PBS and its hemolytic activity were evaluated.

[0079] This embodiment also provides an anti-tumor drug composition, including the above-mentioned nanosponge immune agent.

[0080] The embodiment also provides an application of a nanosponge immunoagent in the preparation of an anti-tumor drug, wherein the nanosponge immunoagent PL@M1M overcomes the side effects of photothermal anti-tumor drugs, such as systemic inflammatory cascade tumor-promoting reactions; the nanosponge immunoagent PL@M1M utilizes the inflammation targeting ability of M1M to ablate primary tumors through photoimmunotherapy, and neutralizes inflammatory factors through membrane surface receptors to inhibit the epithelial-mesenchymal transition (EMT) process of the tumor; PL@M1M "homes" to PMN to prevent the colonization of circulating tumor cells (CTCs); siLDHA blocks the interaction between tumor inflammation and lactate metabolism, reduces immunosuppression, activates the tumor microenvironment, and prevents tumor metastasis and recurrence; the nanosponge immunoagent PL@M1M can achieve comprehensive inhibition of tumor metastasis and recurrence through the multiple mechanisms of M1M's inflammation targeting, neutralization of cytokines, and activation of the tumor microenvironment.

[0081] The nanosponge PL@M1M drug of the present invention is a drug for treating the following cancers: including but not limited to various solid tumors appearing on the surface or inside of organs, including skin cancer, malignant melanoma, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, breast cancer, laryngeal cancer, thyroid cancer, tongue cancer, prostate cancer, penile cancer, testicular tumors, vaginal malignancies, and vulvar malignancies.

[0082] Example 1: Preparation of Nanosponge Immunotherapy Agent

[0083] The cell-like nanosponge PL@M1M with inflammation homing ability constructed in this example includes a mesoporous polydopamine nanophotothermal core carrying siLDHA and a bionic cell membrane shell. The preparation method of the nanodiagnosis and treatment system includes the following three steps:

[0084] The first step is to synthesize PL@M1M

[0085] First, 0.36 g of polyoxyethylene polyoxypropylene ether block copolymer F127 and 0.36 g of 1,2,4-trimethylbenzene (TMB) were dissolved in a mixture of water (65 mL) and ethanol (60 mL). After stirring for 30 min, an aqueous solution of tris (hydroxymethyl) aminomethyl (Tris) (18 mg / mL, 5 mL) was added to the mixture, followed by an aqueous solution of dopamine (12 mg / mL, 5 mL). The reaction mixture was stirred at room temperature for 24 h; then the particles were separated by centrifugation at 13000 rpm for 10 min. The template TMB and F127 were removed by repeated ultrasound (3 times), i.e., the sample was first ultrasound-treated in ethanol for 30 min, centrifuged, and dispersed in a mixed solution of ethanol and acetone (2 / 1, v / v) for ultrasound treatment. The final product was suspended in ethanol for further use.

[0086] And modified by DMEA to load siLDHA. 1 mg mPDA was suspended in 2 mL Tris buffer (pH 8.5), and then different concentrations of DMEA (0-10 mg / mL) were added and stirred at room temperature for 2 hours. DMEA-modified mPDAD was collected by centrifugation (13000 rpm, 10 minutes). The surface charge of mPDAD at different DMEA concentrations was detected by dynamic light scattering technology (DLS). The loading capacity of siLDHA was tested by gel retention experiment. siLDHA and mPDAD were mixed in different mass ratios (siLDHA: mPDAD = 1: 0-1: 6). After stirring for 4 hours, the mPDAD and siLDHA complex (PL) was obtained by centrifugation (13000 rpm, 10 minutes).

[0087] Results As shown by transmission electron microscopy (TEM), mPDA exhibits spherical mesopores ( Figure 1 A), and its surface zeta potential is -28.3 mV ( Figure 1 B). DMEA enriches the surface of mPDA with tertiary amine groups and imparts positive charges to it through Michael addition / Schiff base reaction. The final zeta potential of mPDAD after DMEA modification is +8.0 mV ( Figure 1 B), which is conducive to nucleic acid adsorption. In order to study the loading capacity of DMEA-modified mPDA as siLDHA nanocarrier (mPDAD), agarose gel electrophoresis delay test showed ( Figure 1 C), when the mass ratio of siLDHA to mPDAD is 1:6, siLDHA can be completely adsorbed by mPDAD.

[0088] Step 2: Synthesis of PL@M1M nanosponge immunotherapy agents

[0089] M0 macrophages were polarized to M1 macrophages by LPS (100 pg / mL), and M1M was extracted using a membrane protein extraction kit. The total protein content was analyzed by BCA protein assay. The extracted M1M and PL were sonicated for 5 minutes, extruded through a 400 nm polycarbonate membrane 13 times, and centrifuged to obtain PL@M1M (4°C, 13000 rpm, 10 minutes). The morphology of mPDA and PL@M1M was observed by TEM, and their hydrodynamic diameter, surface potential, and protein expression of M1M and PL@M1M were analyzed by DLS. The spatial localization of M1M and PL was determined by observing the overlap of Cy5 fluorescence and cell membrane dye DiO; finally, the stability of PL@M1M in PBS and its hemolytic activity were evaluated.

[0090] The results are as follows Figure 2 As shown in A, the thickness of the bionic film on the PL@M1M surface is about 10 nm. Figure 2As shown in B and C, PL@M1M retains the inflammatory cytokine receptors on the surface of M1 macrophage membrane. Figure 2 As shown in Figure C, polyacrylamide gel electrophoresis demonstrated that the characteristic membrane proteins of M1M were retained in PL@M1M.

[0091] Example 2: Performance test of nanosponge immune agent PL@M1M

[0092] The performance of the cellular nanosponge prepared by the present invention is mainly evaluated by the following methods:

[0093] (1) Characterization of photothermal and siLDHA controlled release performance

[0094] 200 μg / mL of PL@M1M was exposed to laser (808 nm, 1.0 W / cm 2 , 10min), the temperature rapidly increased to ~50℃ ( Figure 3 A), and has the ability to repeatedly stabilize the photothermal effect ( Figure 3 B).

[0095] The photothermal / pH dual-responsive siLDHA release ability of PL@M1M was then tested. We established two buffer systems (pH 5.5 or pH 7.4) and controlled laser irradiation, and calculated the siLDHA release amount of PL@M1M at different time points by Cy5 fluorescence intensity detection. When the pH changed from 7.4 to 5.5, the permeability of the biomembrane increased, and the release amount of siLDHA increased by 2.2 times accordingly. When laser irradiation was added (pH 5.5vs pH 5.5+L), the release amount of siLDHA increased by 2.7 times, and about 81.7% of siLDHA was released from PL@M1M ( Figure 3 C). Therefore, PL@M1M has the photothermal / pH dual-responsive siLDHA release ability.

[0096] (2) Detection of PL@M1M cytokine neutralization ability

[0097] Next, we studied the sponging ability of PL@M1M on cytokines. The workflow is as follows: Figure 4 As shown in A. Previously, a similar method was used to construct M0 macrophage membrane-encapsulated PL@M0 and erythrocyte membrane (RM)-encapsulated PL@RM. When 500pg TNF-α or IL-6 were incubated with different nanoreactors in 1mL deionized water, 200μg PL@M1M could neutralize TNF-α and IL-6 with efficiencies of 54.4% and 65.6%, respectively. This was significantly higher than mPDA, PL@RM and PL@M0 ( Figure 4B and C). Next, TNF-α or IL-6 were incubated with different concentrations of PL@M1M (0-400μg / mL) at 500pg / mL. As the amount of PL@M1M increased, the residual cytokines decreased. When 400μg / mL of PL@M1M was added, TNF-α and IL-6 were cleared by 66.9% and 79.4%, respectively ( Figure 4 D and E). In summary, we obtained an easily prepared cellular nanosponge PL@M1M, which retains the functions of M1M and can serve as a nucleic acid carrier, photothermal agent, and multiple cytokine blocker.

[0098] Example 3: Study on PL@M1M-mediated targeted metabolic regulation and downregulation of PD-L1

[0099] The PL@M1M mouse breast cancer cell line 4T1 prepared according to the preparation method provided in Example 1 was purchased from ATCC, USA. The mouse monocyte macrophage cell line RAW264.7 was purchased from the cell bank of the Chinese Academy of Sciences. 4T1 cells were cultured in 1640 medium and RAW264.7 cells were cultured in DMEM.

[0100] (1) Targeted endocytosis ability of PL@M1M

[0101] PL@M1M can effectively avoid the unnecessary release and degradation of siLDHA in vitro. Flow cytometry quantification ( Figure 5 A) shows that free siLDHA is negatively charged and easily degraded, and it is not easy to cross the cell membrane (endocytosis efficiency <1%). The endocytosis efficiency of nanoscale PL in 4T1 cells immediately increased to 10%, while PL@M1M (55.5%) was 5.5 times higher than PL. This may be due to the fact that M1M binds to tumor cells more effectively through receptor-ligand interactions. To verify the camouflage ability of M1M, the phagocytosis of PL@M1M by M2 macrophages was shown. The internalization efficiency of siLDHA is negligible, but the efficiency of M2 engulfing PL (33.3%) is significantly higher than PL@M1M (2.17%), about 15 times, because M1M gives PL@M1M camouflage ability to escape M2 phagocytosis ( Figure 5 B).

[0102] (2) PL@M1M silencing siLDHA inhibits LA production and PL-L1 expression

[0103] To detect the gene silencing efficiency of PL@M1M, the LDHA gene in cells incubated with Lipo-siL (Lipofectamine 3000) or PL@M1M was expressed as follows: Figure 5 C) and protein ( Figure 5D) expression was significantly inhibited. After incubation with Lipo-siL or PL@M1M, the LA concentration of tumor cells was significantly downregulated ( Figure 5 E).LA plays a crucial role in driving TME. Recent studies have reported that LA concentration is positively correlated with PD-L1 expression in tumors, which will help tumor cells escape immune surveillance. We used WB ( Figure 5 F) The experiment reached a similar conclusion, that is, in 4T1 cells, reprogramming LA metabolism will reduce the expression of PL-L1 and is expected to relieve the immune blockade.

[0104] Example 4: PL@M1M-mediated low-inflammation photoimmunotherapy experiment

[0105] (1) Low-inflammation photothermal anti-tumor and ICD activation experiments of PL@M1M

[0106] Initially, CCK-8 assays confirmed that mPDA, PL, and PL@M1M had no obvious “dark” cytotoxicity in 4T1 cells ( Figure 6 A). However, under laser irradiation (808 nm, 1.0 W / cm2, 10 min), mPDA, PL, and PL@M1M exhibited concentration-dependent cytotoxic effects ( Figure 6 B). Subsequently, the ICD marker high mobility group box 1 protein (HMGB1) was detected ( Figure 6 C), PL@M1M+L group can induce strong ER stress and promote the release of DAMPs, thereby enhancing ICD.

[0107] Then, the pretreated 4T1 cells were co-cultured with DC2.4 cells. After 24 hours of co-stimulation, the nanosponge PL@M1M+L activated about 19.2% of DC cells, which was about 2 times higher than that of the PBS group (9.4%). Figure 6 D). In order to simulate the effect of inflammatory factor neutralization on DC cell maturation after multiple administration in vivo. In the co-culture system, we added an equal amount of PL@M1M to the PL@M1M+L group and found that the maturity of DC cells (17.4%) was not significantly different from that of the mPDA+L group and the PL@M1M+L group. The concentrations of TNF-α and IL-6 in the cell culture supernatant of each group were then detected ( Figure 6 E, F). The mPDA+L group released a large amount of inflammatory cytokines, while PL@M1M+L was able to neutralize some of the cytokines. The PL@M1M+L+(PL@M1M) group had the best neutralization ability for TNF-α and IL-6 concentrations.

[0108] (2) PL@M1M low-inflammation PTT to prevent tumor cell EMT experiment

[0109] Next, to investigate the hypoinflammatory PTT effect of PL@M1M, RAW 264.7 cells were co-cultured with different 4T1 cell samples. mPDA+L induced macrophages to release a large amount of proinflammatory cytokines (TNF-α and IL-6), which may lead to the activation of NF-kB inflammation-related pathways: such as cell migration and proliferation. The cytokine concentration detected in the supernatant of the PL@M1M+L+(PL@M1M) group was the lowest among the experimental groups, as confirmed by WB experiments ( Figure 6 G), the nanosponge PL@M1M+L+(PL@M1M) group inhibited the activation of NF-κB signaling pathway and the expression of COX-2 protein induced by PTT.

[0110] like Figure 6 As shown in H, after TNF-α treatment, 4T1 cells showed reduced expression of the cell adhesion protein E-Ca and enhanced expression of the cell motility protein vimentin (Vim), with EMT-like characteristics. PL@M1M treatment significantly inhibited the EMT process and reduced the invasive ability of 4T1 cells. Subsequently, the scratch wound healing experiment confirmed that after 24h, the migration ability of 4T1 cells in the presence of TNF-α (10ng / mL) was 2.5 times (64.5%) compared with the control group (25.5%). When we blocked TNF-α using TNF-α antibody (Infliximab 10ng / mL) or nanosponge PL@M1M (200μg / mL), the cell migration ability was reduced to 38.6% or 28.7% ( Figure 6 I). In summary, PL@M1M meets the requirements of low-inflammatory photoimmunotherapy and can not only activate ICD but also inhibit the PTT-inflammatory cascade.

[0111] Example 5: In vivo experimental study of the homing ability of the nanosponge PL@M1M by in vivo fluorescence imaging

[0112] In order to more intuitively observe the accumulation of cellular nanosponges in tumor sites and various organs, the biodistribution of PL@M1M in vivo was studied by using Cy5-siLDHA as a fluorescent indicator. The in vitro fluorescence semi-quantitative results 48 hours after intravenous injection showed that the tumor accumulation of PL@M1M was 26.2 times and 4.6 times higher than that of Cy5-siLDHA and PL@RM, respectively ( Figure 7 A). In PMNs, semi-quantitative analysis of the lungs in vitro at 48 h showed that the accumulation of PL@M1M was 5.1-fold and 3.9-fold higher than that of Cy5-siLDHA and PL@RM, respectively ( Figure 7 B). These data suggest that PL@M1M has good inflammatory “homing” properties, not only accumulating at the primary tumor site, but also effectively targeting PMNs in vivo. We also measured the concentrations of inflammatory cytokines in serum at different times after tumor inoculation ( Figure 7C), TNF-α and IL-6 concentrations in 14-day tumor-bearing mice increased 2.6-fold and 3.9-fold, respectively, compared with healthy mice, in a time-dependent manner. When we exposed 14-day tumor-bearing mice to PTT, TNF-α and IL-6 concentrations increased again.

[0113] Example 6: Application of cell-like nanosponge PL@M1M in anti-tumor drugs

[0114] (1) Monitoring the anti-tumor effect of nanosponge immunotherapy in vivo

[0115] A bilateral 4T1 tumor-bearing mouse model was established and the tumor-bearing mice were randomly divided into 5 groups. G1: saline, G2: mPDA+, G3: PL@RM+L, G4: P@M1+L, G5: PL@M1M+L were injected intravenously 3 times every 3 days. Among them, G4 and G5 mice were injected with P@M1 or PL@M1M 1 day after the first administration to exert a stronger cytokine neutralization effect. The specific workflow is shown in Fig.10 A. 8 hours after the first administration, laser irradiation (808 nm, 1.0 W / cm 2 , 10min). During the whole period, the volume of primary and distal tumors and the weight of mice were monitored every three days. Immunological indicators were detected on the 8th day, and the remaining mice were killed on the 21st day to collect the main organs and primary tumors. First, in the mice treated with PL@M1M, the tumor area showed a strong photothermal heating ability ( Figure 8 A). Compared with other treatment groups, PL@M1M+L even completely inhibited tumor growth ( Figure 8 B). In addition, tumor tissue was collected and photographed on day 21 ( Figure 8 C) and weighing ( Figure 8 D), the smallest and lightest tumor of PL@M1M+L had the strongest antitumor efficacy (89.5%) ( Figure 8 E).

[0116] On the other hand, LA metabolic disorder is an important culprit for the proliferation of malignant tumors. Due to its excellent "homing" ability, the gene silencing ability of siLDHA in the PL@M1M+L group was more obvious, and the LA concentration in the tumor of the PL@M1M+L group was as low as 4.5μmol / g ( Figure 8 F).

[0117] (2) Nanosponge immunotherapy PL@M1M enhances anti-tumor immune response in vivo

[0118] First, considering that PTT-induced antigen release contributes to the maturation of DCs and induces systemic immune cascade reactions. On the 8th day of treatment, we detected that the number of activated DCs in the single PTT group (mPDA+L: 15.3%) was only 1.3 times that of the saline group (11.7%). Fig. 9 A). In contrast, changes in the LA microenvironment enhanced the differentiation and maturation of monocytes into dendritic cells, and the maturation rate of dendritic cells in the nanosponge PL@M1M+L (23.9%) was more than twice that of saline.

[0119] At the 21st day of treatment endpoint, tumor tissues were dissected from mice in each group, and anti-tumor immune cells (CTL, Th) and tumor immunosuppressive cells (Treg, MDSCs, TAMs) were analyzed using flow cytometry. LA metabolic interference significantly improved the enrichment of CTL in the tumor area, and the CTL in the PL@RM+L (7.3%) group and the PL@M1M+L (10.2%) group increased by 4.4 times and 6.1 times, respectively, compared with the saline group ( Fig. 9 B). In addition, Th cells (CD3 + CD4 + )(15.1%) was 7.4 times that of the saline group (2.0%) Fig. 9 C). We then further analyzed the immunosuppressive cells in the tumor, Treg ( Fig. 9 D), MDSCs( Fig. 9 E), and TAMs ( Fig. 9 F) cells, PL@M1M treatment can maximally inhibit the infiltration of immunosuppressive cells in the tumor area. The above results show that the anti-tumor nanosponge immunoagent PL@M1M combined with siLDHA to interfere with LA metabolism and low-inflammatory PTT can effectively reprogram TME and has great potential in treating cancer metastasis and recurrence.

[0120] (3) The nanosponge immunoagent PL@M1M can alleviate tumor-related inflammatory response after PTT.

[0121] PTT has been shown to promote the infiltration of inflammatory cells within tumors. We next evaluated the anti-inflammatory effects of PL@M1M in tumor-bearing mice. First, serum pro-inflammatory cytokines such as Fig.10 As shown in A, compared with the saline group, the PTT of mPDA increased the levels of TNF-α and IL-6 by about 5.2 and 4.3 times, respectively, while the levels of P@M1 and PL@M1M in the laser group did not increase significantly. On the other hand, tumor-bearing mice tended to develop splenic swelling due to the presence of tumor-related inflammation. The spleen weight of the saline group (0.48 g) was 1.9 times greater than that of healthy mice (0.26 g). Fig.10 B and C). In contrast, mPDA+L induced more severe spleen swelling (0.76 g). However, PL@M1M+L treatment restored the spleen weight (0.24 g) of tumor-bearing mice to healthy levels, likely due to reduced systemic inflammation, which is important for the safe use of nanomedicines. The above results indicate that the anti-tumor nanosponge immunoagent PL@M1M is able to avoid the PTT-induced residual tumor EMT process and successfully reduce systemic inflammatory responses.

[0122] (4) Nanosponge immunotherapy PL@M1M can alleviate PMN inflammatory microenvironment and inhibit spontaneous metastasis

[0123] We found through lung HE staining that very serious spontaneous lung metastasis was observed on day 21 in the saline group. Fig.11 A). Although the mPDA+L and PL@RM+L groups had fewer lung metastatic nodules, the interstitial space of the lung tissue was dense, which may be related to the lung inflammatory response induced by TDSFs or other mediators. The PL@M1M+L group had normal lung histology and no metastatic nodules. Therefore, the above research results systematically confirmed that the anti-tumor nanosponge immunoagent PL@M1M can inhibit the PTT-inflammatory cascade in PMNs, deprive PMNs of "nutrients" as CTC "soil", and effectively inhibit the occurrence of spontaneous metastasis.

[0124] (5) Immune memory induced by nanosponge immunotherapy PL@M1M against tumor recurrence

[0125] Immune memory provides long-term protection against a second wave of pathogen infection. 4T1 cells were first inoculated into the left mammary pad, and 7 days later, PL@M1M+L was used for multiple times. On the 8th day of treatment, mice that were completely cured by PL@M1M were selected as the experimental group, and the saline group with surgical removal of the primary tumor was used as the control group. Tumor-specific central memory T cells (Tcm, CD3 + CD8 + CD44 + CD62L + ) and effector memory T cells (Tem, CD3 + CD8 + CD44 + CD62L - ).like Fig.12As shown in A and B, compared with the saline group, the Tcm and Tem of the PL@M1M+L group were enhanced by 5.5 and 2.1 times, respectively, which largely contributed to its resistance to tumor recurrence. Therefore, on the 13th day after the resection of the primary tumor (day 21), a re-challenge tumor model was established by injecting 4T1 cells into the opposite mammary pad of mice. Rapid tumor growth was observed in the control group after re-modeling, growing to 1200mm within 15 days. 3 , the tumor grew twice as fast as the initial inoculation ( Fig.12 C). However, the PL@M1M+L group greatly inhibited tumor growth, and its growth rate was only about 1 / 4 of that of the control group. The survival rate of PL@M1M+L within 38 days was 100%, significantly higher than the control group's 0%, and 60% of the mice survived on day 56 ( Fig.12 D). The above results suggest that PL@M1M-based photoimmunotherapy can not only inhibit the growth of established tumors but also prevent tumor recurrence.

[0126] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the technology can modify or change the above embodiments without violating the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the relevant technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A nanosponge immune agent, characterized in that: The surface of the nanophotothermal agent is modified by a modifier, and then the lactic acid metabolism disruptor is loaded by electrostatic adsorption. Finally, the surface of the nanophotothermal agent is wrapped with a cell membrane bionic coat, and finally a cell-like nanosponge immune agent is constructed. The nano-photothermal agent is selected from organic photothermal materials; the organic photothermal materials are selected from small molecule organic dyes, supramolecular complexes, and conjugated polymers; The modifier is selected from: an amine compound or a cationic polymer; The lactate metabolism disruptor: regulates the production of lactate by inhibiting pyruvate dehydrogenase kinase 1 and lactate dehydrogenase A subunit LDHA target, regulates the transport of lactate through lactate transporters including monocarboxylic acid transporter 1 and monocarboxylic acid transporter 4, and regulates the degradation of lactate through lactate oxidase; The cell membrane bionic coat is selected from macrophage membrane, macrophage exosome membrane and neutrophil membrane.

2. The nanosponge immunomodulator according to claim 1, characterized in that: When the nanophotothermal agent is a conjugated polymer, it is mesoporous polydopamine mPDA.

3. The nanosponge immunomodulator according to claim 1, characterized in that: When the modifier is an amine compound, it is selected from dimethylethanolamine DMEA or triethylenetetramine, preferably DMEA; When the modifier is a cationic polymer, it is selected from polylysine, polyacrylamine, polyamine, and polyvinylamine.

4. The nanosponge immunomodulator according to claim 1, characterized in that: The lactate metabolism disruptor is preferably a siRNA of lactate dehydrogenase A to regulate the production of lactate.

5. The nanosponge immunomodulator according to claim 1, characterized in that: The cell membrane bionic coat is preferably an M1 macrophage membrane.

6. The method for preparing the nanosponge immunopharmaceutical according to any one of claims 1 to 5, characterized in that: The steps include: The first step is to synthesize PL@M1M: The polyoxyethylene polyoxypropylene ether block copolymer and 1,2,4-trimethylbenzene are dissolved in a water-alcohol mixture, and tris (hydroxymethyl)aminomethane (Tris) and dopamine aqueous solution are added, stirred at room temperature for 24 hours, centrifuged, and the template agent polyoxyethylene polyoxypropylene ether block copolymer and 1,2,4-trimethylbenzene are removed by ultrasonication to obtain mPDA suspended in ethanol; Modification of mPDA: mPDA was suspended in Tris buffer, DMEA was added, stirred at room temperature for 2 hours, collected by centrifugation, and the surface charge was detected by dynamic light scattering technology DLS and Zeta potential analyzer. The siLDHA loading capacity was tested by gel retention experiment. After mixing and stirring, the PL complex was obtained by centrifugation; Step 2: Synthesis of PL@M1M nanosponges: Lipopolysaccharide LPS induces M0 macrophages to polarize into M1 macrophages, extracts M1M, mixes with PL ultrasonically, extrude and centrifuge to obtain PL@M1M; the morphology, size, surface potential and protein expression are analyzed by transmission electron microscopy TEM, dynamic light scattering technology DLS, agarose gel electrophoresis SDS-PAGE and flow cytometry, and the stability and hemolysis are evaluated.

7. The method for preparing the nanosponge immunomodulator according to claim 6, characterized in that The steps include: The first step is to synthesize PL@M1M First, 0.36 g of polyoxyethylene polyoxypropylene ether block copolymer and 0.36 g of 1,2,4-trimethylbenzene were dissolved in a mixture of 65 mL of water and 60 mL of ethanol; after stirring for 30 min, 5 mL of 18 mg / mL Tris aqueous solution was added to the mixture, and then 5 mL of 12 mg / mL dopamine aqueous solution was added; the reaction mixture was stirred at room temperature for 24 h; then the particles were separated by centrifugation at 13000 rpm for 10 min; the template was removed by repeated ultrasound, that is, the sample was first ultrasound-treated in ethanol for 30 min, centrifuged, and dispersed in a mixed solution of ethanol and acetone in a volume ratio of 2:1 for ultrasound treatment; the final product was suspended in ethanol for further use; 1 mg of mPDA was suspended in 2 mL of Tris buffer, and then different concentrations of DMEA were added and stirred at room temperature for 2 hours; DMEA-modified mPDAD was collected by centrifugation; the surface charge of mPDAD at different DMEA concentrations was detected by DLS; the loading capacity of siLDHA was tested by gel retention experiment, and siLDHA and mPDAD were mixed at a mass ratio of siLDHA:mPDAD=1:0-1:6, and after stirring for 4 hours, the mPDAD and siLDHA complex PL was obtained by centrifugation; Step 2: Synthesis of PL@M1M nanosponge LPS was used to induce M0 macrophages to polarize into M1 macrophages, and M1M was extracted using a membrane protein extraction kit. The total protein content was analyzed by BCA protein assay. The extracted M1M and PL were sonicated, extruded through a polycarbonate membrane, and centrifuged to obtain PL@M1M. The morphology of mPDA and PL@M1M was observed by TEM, and their hydrodynamic diameters and surface potentials were analyzed by DLS. The membrane proteins of M1M and PL@M1M were analyzed by SDS-PAGE and flow cytometry. The spatial localization of M1M and PL was determined by observing the overlap of Cy5 fluorescence and the cell membrane dye DiO. Finally, the stability of PL@M1M in PBS and its hemolytic activity were evaluated.

8. An anti-tumor pharmaceutical composition, characterized in that: The nanosponge immunomodulator comprises the nanosponge immunomodulator according to any one of claims 1 to 5.

9. Use of a nanosponge immunopharmaceutical according to any one of claims 1 to 5 in the preparation of anti-tumor drugs, characterized in that: The nanosponge immunoagent overcomes the side effects of photothermal anti-tumor drugs, such as systemic inflammatory cascade-promoting tumor reactions; the nanosponge immunoagent utilizes the inflammation targeting ability of M1M to ablate primary tumors through photoimmunotherapy, and neutralizes inflammatory factors through membrane surface receptors to inhibit the epithelial-mesenchymal transition (EMT) process of tumors; PL@M1M "homes" to PMN to prevent the colonization of circulating tumor cells (CTCs); siLDHA blocks the interaction between tumor inflammation and lactate metabolism, reduces immunosuppression, activates the tumor microenvironment, and prevents tumor metastasis and recurrence; the nanosponge immunoagent PL@M1M can achieve comprehensive inhibition of tumor metastasis and recurrence through multiple mechanisms of M1M's inflammation targeting, neutralization of cytokines, and activation of the tumor microenvironment.

10. The use of the nanosponge immune agent according to claim 9 in the preparation of anti-tumor drugs, characterized in that: The tumors include but are not limited to various solid tumors appearing on the surface or inside of organs, including skin cancer, malignant melanoma, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, breast cancer, laryngeal cancer, thyroid cancer, tongue cancer, prostate cancer, penile cancer, testicular tumors, vaginal malignancies, and vulvar malignancies.

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