Nanometer battery engineering-based adoptive macrophage and preparation method and application thereof

Abstract

The invention discloses an engineered adoptive macrophage based on a nano battery as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The preparation method disclosed by the invention comprises the following steps: taking an immunologic adjuvant working solution as a raw material, and obtaining nanoparticles with high drug loading capacity through an assembly effect; as the main energy of ChARM comes from an immunologic adjuvant, the high-drug-loading nano-particle is also called a high-energy-density nano-battery. Diluting the obtained nano particles with a cell culture medium, and co-incubating the diluted nano particles and macrophages to obtain engineered macrophages loaded with the anti-tumor or anti-inflammatory nano battery; the assembly mode is any one of supramolecular assembly, prodrug assembly or metal coordination assembly; the high-density drug-loaded nano battery can endow engineered cells with excellent biological functions. The method solves the technical problems that the preparation of the existing CAR-M is expensive, the gene modification is complex, and the nano-drug delivery system is unstable.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an engineered adoptive macrophage based on nanobatteries, its preparation method, and its application. Background Technology

[0002] In recent years, cancer immunotherapy has made groundbreaking progress. Among them, chimeric antigen receptor T-cell (CAR-T) therapy has achieved breakthrough success in the treatment of hematologic malignancies. However, CAR-T technology faces significant challenges in the treatment of solid tumors, mainly due to factors such as the high heterogeneity of tumors, their dense stromal fibrosis structure, and the presence of immunosuppressive factors (such as TGF-β and PD-L1) in the tumor immune microenvironment (TIME). The TIME of solid tumors is characterized by the complex coexistence of immunosuppressive cells and molecular patterns, posing multiple obstacles to the efficacy of immunotherapy. Tumor-associated macrophages (TAMs) are a core component of this immunosuppressive environment. They typically exhibit a pro-tumorigenic M2 phenotype, secreting immunosuppressive factors such as IL-10 and TGF-β, promoting angiogenesis, tumor invasion, and metastasis, thereby significantly weakening the anti-tumor immune response.

[0003] Given this complexity, macrophage-mediated immunomodulation has emerged as a potential strategy for addressing the challenges of solid tumor treatment. As a key component of the innate immune system, macrophages possess several therapeutic advantages: i) proven tumor microenvironment tropism, enabling them to penetrate the dense stromal barrier; ii) inherent antigen-presenting capabilities, promoting cross-initiation of adaptive immune responses; and iii) significant phenotypic plasticity, allowing dynamic switching between pro-tumor (M2) and anti-tumor (M1) polarization states. In recent years, adoptive macrophages have emerged as a novel strategy for solid tumor immunotherapy, including the in vitro engineering of macrophages to possess specific anti-tumor functions, such as chimeric antigen receptor macrophages (CAR-M), drug-loaded macrophages, and macrophage backpack delivery systems. Preclinical studies have confirmed that CAR-M can mediate a potent anti-tumor effect through antigen-specific recognition mechanisms, but it still faces three major challenges: i) the genetic engineering process is complex and time-consuming; ii) infused macrophages are easily reversed to the M2 phenotype by immunosuppressive factors; and iii) the limited reprogramming ability of surrounding TAMs restricts the expansion of its anti-tumor effect. In contrast, drug-loaded macrophage strategies mainly utilize living macrophages as target carriers to deliver drugs to the tumor site, failing to fully utilize the anti-tumor function of macrophages themselves; while macrophage backpack technology can synergistically utilize the dual functions of backpack drugs and macrophages, the backpack may detach prematurely in systemic circulation, leading to off-target toxicity. Summary of the Invention

[0004] The purpose of this invention is to provide an engineered adoptive macrophage based on nanobatteries, its preparation method, and its application, in order to solve the technical problems of the high cost and complex genetic modification of existing CAR-M preparations, as well as the instability of nanodrug delivery systems.

[0005] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a method for preparing engineered adoptive macrophages based on nanobatteries, comprising the following steps: S1: High-drug-load nanoparticles were obtained through assembly using an immunoadjuvant working solution as a raw material; given ChAR... M's main energy comes from immune adjuvants, and these high-drug-load nanoparticles are also known as high-energy-density nanobatteries. S2: After diluting the obtained nanoparticles with cell culture medium, they were co-incubated with macrophages to obtain engineered adoptive macrophages based on nanobatteries, that is, engineered macrophages that can be loaded with anti-tumor or anti-inflammatory nanobatteries. In S1, the assembly method is any one of supramolecular assembly, prodrug assembly, or metal coordination assembly.

[0006] Furthermore, in S1, the working solution of the immune adjuvant is obtained by mixing a small molecule immunodrug with a dimethyl sulfoxide solution.

[0007] Furthermore, in S1, when the assembly method is supramolecular assembly, the specific steps are as follows: After stirring and mixing the small molecule immunodrug and dimethyl sulfoxide solution, an immunoadjuvant working solution is obtained; Subsequently, an aqueous solution was added to the immunoadjuvant working solution, and nanoparticles were formed through self-assembly under ultrasonic conditions.

[0008] Furthermore, the small molecule immunotherapy drug is the Toll-like receptor 7 / 8 agonist R848; The volume ratio of the working solution to the aqueous solution of the immunoadjuvant is 5%.

[0009] Furthermore, the stirring and mixing are carried out at room temperature; The concentration of the nanoparticles is 10 µM.

[0010] Furthermore, the macrophages are M1 type macrophages or M2 type macrophages; The ratio of nanoparticles to macrophages is 17 µg: 10^6 cells.

[0011] Furthermore, the co-incubation culture system is cultured under aseptic conditions at 37 °C and 5% CO2.

[0012] The present invention also discloses adoptive macrophages based on nanobatteries prepared by the above preparation method, wherein the drug loading of the adoptive macrophages based on nanobatteries is 0.5 ng / cell.

[0013] This invention also discloses the application of the above-mentioned nano-battery-based engineered adoptive macrophages in the preparation of disease treatment systems, wherein the nano-battery-based engineered adoptive macrophages are used to prepare disease treatment systems by loading small molecule drugs; The small molecule drug is either a small molecule drug that can induce M2 macrophages to become M1 type or a small molecule drug that can induce M1 macrophages to become M2 type polarized. When the small molecule drug is one that can induce M2 macrophages to become M1, it can promote immune activation of the tumor microenvironment. When the small molecule drug is one that can induce M1 macrophages to polarize to M2, it can suppress excessive inflammation or promote tissue repair.

[0014] Furthermore, when the small molecule drug is a small molecule drug that can induce M2 macrophages to reverse polarize into M1, the small molecule drug is an immune agonist small molecule drug, a metabolic small molecule drug, or a small molecule drug that reverses M2 polarization into M1. The immune-stimulating small molecule drug is one or more of the following: STING agonist CDN, TLR agonist and AMPK activator; The metabolic small molecule drug is one or both of the aryl hydrocarbon receptor AhR inhibitor and glutamine transporter inhibitor; The small molecule drug with reverse polarization M2 being M1 is one or both of histamine receptor inhibitors and aspirin; When the small molecule drug is a small molecule drug that can induce M1 macrophages to polarize to M2, the small molecule drug is a flavonoid or a polyphenol.

[0015] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses a method for preparing engineered adoptive macrophages based on nanobatteries. The method uses a small molecule drug, an immunoadjuvant (R848), as a guiding molecule to self-assemble nanoparticles, namely high-energy-density nanobatteries (RR NPs). These anti-tumor "nanobatteries" are then loaded into macrophages to form engineered adoptive macrophages (ChAR-M). Within the ChAR-M system, RR NPs, acting as long-lasting nanobatteries with high energy density, continuously provide anti-tumor power, maintaining M1 phenotype stability and resisting TAM repolarization in the harsh microenvironment of sepsis. ChAR-M exerts its therapeutic effect through three main mechanisms: (i) long-lasting M1 maintenance; (ii) anti-M2 polarization; and (iii) paracrine regulation. The sustained release of R848 not only maintains M1 polarization but also endows ChAR-M with the ability to inhibit M2 polarization, forming a persistent pro-inflammatory state. All three mechanisms mentioned above rely on nanoparticles with high drug loading (>90%) obtained through drug self-assembly to ensure high energy density, thereby better achieving long-lasting, anti-polarization, and paracrine effects. In contrast, traditional nanoparticles such as liposomes and micelles with low drug loading capacity cannot achieve these three mechanisms. Pro-inflammatory cytokines secreted by ChAR-M and exosomes containing R848 are internalized by surrounding M2 macrophages. By activating the TLR / NF-κB pathway, they downregulate M2 markers (such as Cd206) and upregulate M1 markers (such as Nos2 and Il6), triggering the M2 to M1 phenotype conversion and producing a cascade polarization effect. This synergistic effect of M1 maintenance, M2 inhibition, and paracrine-driven M1 polarization jointly constructs a pro-inflammatory microenvironment, enhances antigen presentation and co-stimulatory signals (CD80 / CD86), activates CD8+ and CD4+ T cells, reduces Treg cells, and inhibits angiogenesis. The remodeled tumor microenvironment, combined with the direct killing effects of M1 macrophages and T cells, synergistically enhances anti-tumor immunity by inducing tumor cell apoptosis. This multi-effect strategy addresses key limitations of existing macrophage therapies and shows significant promise in the field of solid tumor immunotherapy. It significantly overcomes the technical challenges of the high cost and genetic complexity of existing CAR-M preparations, as well as the instability of nanomedicine delivery systems.

[0016] The present invention also discloses that the adoptive macrophages based on nanobatteries prepared by the above preparation method can achieve targeted regulation of diseases by maximizing the loading of nanomedicines without affecting the biological function of the macrophages themselves.

[0017] This invention also discloses the application of the above-mentioned engineered adoptive macrophages based on nanobatteries in the preparation of disease treatment systems. According to relevant experimental results, the engineered adoptive macrophages of this invention can deliver drugs efficiently and accurately, without premature leakage under physiological conditions, and are not easily affected by opsonin effects, exhibiting good stability and good biosafety. The assembly method in this invention will provide a new model for developing easy-to-manufacture and multifunctional live cell delivery systems, and guide a wider range of immune microenvironment regulation strategies to improve the efficacy of immunotherapy. Attached Figure Description

[0018] Figure 1 Characterization results of the RR NBs nanocells in Example 1 Where: A represents particle size and polydispersity index; B represents the morphology results from transmission electron microscopy; and C represents the ultraviolet absorption spectrum.

[0019] Figure 2 This is a schematic diagram of the kinetic simulation of the RR NBs nanocells obtained in Example 1; Figure 3 The stability of the nano-batteries RR NBs obtained in Example 1; Where: A represents the stability of RR NBs in aqueous solution; B represents the stability of RR NBs in cell culture medium; Figure 4 The drug release curve of the nanobatteries RR NBs obtained in Example 1; Figure 5 Cellular uptake and quantification of the nanobatteries RR NBs obtained in Example 1; Figure 6 Flow cytometry detection and quantification of RR NBs nanobatteries obtained in Example 1 for cellular uptake; Where: A is; B is Figure 7 Cytotoxicity of the nanobatteries RR NBs obtained in Example 1; Figure 8 Functional evaluation of the ChAR-M obtained in Example 3; Where: A represents the migration ability and quantification of ChAR-M; B represents the chemotactic ability and quantification of ChAR-M.

[0020] Figure 9 Mechanism evaluation of ChAR-M obtained in Example 3; Where: A represents the polarization capacity of ChAR-M; B represents the long-term polarization stability of ChAR-M; C represents the anti-M2 capacity of ChAR-M; and D represents the paracrine verification of ChAR-M.

[0021] Figure 10This serves as PCR verification of ChAR-M paracrine secretion in Example 3; Where: A represents the relative expression of the Nos2 gene; B represents the relative expression of the Cd206 gene; C represents the relative expression of the Tnfa gene; and D represents the relative expression of the Il1a gene.

[0022] Figure 11 Biodistribution assessment of ChAR-M obtained in Example 3; Where: A represents the tissue distribution of ChAR-M; B represents the quantitative distribution of ChAR-M tissue.

[0023] Figure 12 Evaluation of the antitumor effect of ChAR-M in Example 3; Where: A is the tumor size at the experimental endpoint; B is the tumor size at the experimental endpoint; C is the tumor growth curve.

[0024] Figure 13 Pathological evaluation of the antitumor effect of ChAR-M in Example 3; Figure 14 Analysis of the remodeling of the tumor immune microenvironment of ChAR-M in Example 3; Where: A represents CD80 after treatment. + The proportion of macrophages; B represents CD206 after treatment. + The proportion of macrophages; C represents CD8+ after treatment. + The proportion of T cells; D represents CD4+ after treatment. + The proportion of T cells; E represents the Foxp3 level after treatment. + The proportion of Treg cells.

[0025] Figure 15 The diagram shows a process schematic of the ChAR-M preparation method disclosed in this invention. Detailed Implementation

[0026] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0027] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0028] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0029] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0030] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0031] This invention provides a method for preparing engineered adoptive macrophages based on nanobatteries, which can be used to regulate the tumor immunosuppressive microenvironment and improve the efficacy of immunotherapy. This method can broadly guide the development of live cell-based functional carriers. The method is as follows: Figure 15 As shown, drug A is an immune adjuvant, including benzene ring-containing immune adjuvant biomolecules with a large π structure, such as imiquimod and remiquimod; drug B is a small molecule drug that can assemble with drug A, or a metal ion that can coordinate with it; the macrophages are M1 or M2 type macrophages, which can use their strong phagocytic ability to engulf nanoparticles; and the nanoparticles are co-incubated at a certain ratio and time to form nanobattery-engineered adoptive macrophages (ChAR-M).

[0032] The main steps include: The main approach involves using immune adjuvant biomolecules with benzene rings and large π structures to form prodrugs through physical mixing or simple linking. These prodrugs are then directly assembled into nanoparticles under the influence of gold molecules. Furthermore, the phagocytic activity of macrophages is used to efficiently load the nanoparticles, preparing them into nano-battery-engineered adoptive macrophages. Finally, the disease targeting of macrophages is used to achieve precise regulation of diseases.

[0033] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0034] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0035] Example 1 A method for preparing engineered adoptive-charged macrophages based on nanoparticles includes the following steps: S1: Dissolve small molecule drugs of immune adjuvants in DMSO in an appropriate proportion, and stir at room temperature to obtain working solution; S2: Add an appropriate amount of aqueous solution and form nanoparticles through self-assembly under ultrasonic conditions; S3: The nanoparticles obtained in S2 were diluted with cell culture medium and co-incubated with M1 macrophages to obtain engineered macrophages loaded with anti-tumor nanoparticles. In S1, the immune adjuvant small molecules form nanoparticles through molecular self-assembly. The small molecule immunotherapy drug is R848, a Toll-like receptor 7 / 8 (TLR) agonist. The volume ratio of the working solution to the aqueous solution of the immunoadjuvant is 5%; The concentration of the nanoparticles is 10 µM; The ratio of nanoparticles to macrophages is 17 µg: 10^6 cells.

[0036] The co-incubation culture system was cultured under aseptic conditions at 37 ℃ and 5% CO2.

[0037] Example 2 Unlike Example 1, the assembly methods of the rice cell include, but are not limited to, supramolecular assembly, prodrug assembly, and metal coordination assembly.

[0038] Example 3 Unlike Example 1, in this example, the drug can be polarized towards M1, and the active ingredient is an immune agonist small molecule drug, a metabolic small molecule drug, or other small molecule drugs that have reverse polarization M2 to M1.

[0039] The prepared nanobatteries are anti-tumor type, and the cells used in this process are M1 type macrophages.

[0040] Example 4 Unlike Example 1, in this example, the drug can be a drug polarized towards M2, such as a flavonoid or polyphenol small molecule drug.

[0041] The prepared nanobatteries are anti-inflammatory, and the cells used are M2 macrophages.

[0042] Example 5 Unlike Example 1, the concentrations of the cells (nanoparticles) incubated by the nanobatteries were 0.1, 0.25, 0.5, 1, 2.5, 5, 10, and 25 µM, respectively.

[0043] Example 6 Unlike Example 1, the incubation times for the nanobatteries (nanoparticles) were 0.5, 1, 2, 4, and 6 hours, respectively.

[0044] Figure 1 The following are statistical diagrams of the particle size and polydispersity index of the coordination-assembled nanoparticles RR NBs obtained in Examples 1 to 4, as shown in the figure. Figure 1 As shown in Figure A, the hydrodynamic diameter distribution of RR NBs is approximately 170 nm, and the polydispersity index (PDI) is 0.21, indicating that RR NBs have good dispersibility. Furthermore, the morphology of the RR NBs in Example 2 was characterized by TEM, as shown... Figure 1 As shown in Figure B, the RR NBs exhibit a uniform morphology and a regular spherical structure. The actual size of the nanoparticles is approximately 130-140 nm, with good dispersion and no aggregation. Further calculations revealed that the drug loading of R848 in the RR NBs was 75.1%. The drug loading of the RR NBs significantly exceeds that of traditional nanomedicines, indicating that the nanoparticles of this application can deliver drugs more efficiently.

[0045] further, Figure 1 The violet-visible spectral results in C show a significant blue shift in the violet-visible spectrum after molecular aggregation to form nanostructures, confirming the formation of these nanostructures. To elucidate the self-assembly mechanism of RssR NB, the researchers conducted a 200-nanosecond molecular dynamics (MD) simulation. The simulation results ( Figure 2 This visually demonstrates the process by which nanoparticles dynamically form stable assemblies from their initial self-structure (0 ns), with the π-π stacking interaction between imidazoquinoline structures identified as the key driving force for self-assembly. Dynamic light scattering (DLS) stability monitoring over 7 days showed... Figure 3 A- Figure 3The particle size and polydispersity index (PDI) of RR NB remained stable in both aqueous solution and DMEM culture environments, confirming its excellent stability. This nanoparticle design is responsive to reducing factors (such as GILT) in the endosome-lysosomal microenvironment. Stimulation-responsive release kinetics experiments further confirmed that the release rate of R848 in PBS was <10%, while sustained release was achieved upon GILT stimulation. Figure 4 This highlights the long-acting drug delivery characteristics of RR NBs.

[0046] Based on the above results, this application conducted experiments and verifications on the application effects of the obtained adoptive macrophages engineered based on nanobatteries: Construction of Antitumor-Induced ChAR-Ms: This application further constructs engineered adoptive macrophages (ChAR-Ms) based on nanobatteries. A key challenge in constructing ChAR-Ms was maximizing nanoparticle loading efficiency while minimizing cytotoxicity. To optimize the incubation time of R848 nanobatteries in ChAR-Ms, this application used 6-FAM-labeled RR NBs for time-dependent cell uptake experiments. Confocal laser scanning microscopy (CLSM) analysis revealed a unique intracellular distribution (…). Figure 5 Within 1-2 hours, nanoparticles were initially localized in lysosomes, and after 4-6 hours, they redistributed to the cytoplasm. The distribution of green fluorescence of 6-FAM-labeled NPs in the cytoplasm increased in a time-dependent manner. Notably, the fluorescence intensity plateaued after 4 hours, indicating an upper time limit for nanoparticle accumulation. Flow cytometry quantification results ( Figure 6 This further confirmed that the internalization of nanoparticles was time-dependent within 4 hours, and that uptake reached saturation at 4 hours. Based on these results, 4 hours was ultimately chosen as the incubation time for ChAR-M preparation. Uninternalized RR NBs were removed by changing the culture medium, thereby ensuring optimal loading efficiency while minimizing adverse effects on macrophage function.

[0047] Cytotoxicity and functional evaluation of antitumor ChAR-M: First, the biocompatibility of RR NBs was assessed to ensure that the loading of nanobatteries would not induce cytotoxicity in macrophages. This was determined through an MTT assay (…). Figure 7 This study demonstrated that RR NBs exhibited excellent cell tolerance at all tested concentrations, with macrophage survival rates exceeding 90%. These results confirm the good biocompatibility of RR NBs with RAW264.7 cells. Subsequently, this application investigated whether incubation with 10 µM RR NBs for 4 hours affected normal macrophage function. Figure 8As shown in Figure A, the migration ability of ChAR-M is comparable to that of ordinary M1 macrophages, indicating that it retains the inherent migration characteristics crucial for in vivo therapeutic applications. Furthermore, this application evaluated the chemotactic ability of ChAR-M using a Transwell assay. Figure 8 (B) The results showed that ChAR-M cells had the same chemotactic capacity as normal M1 macrophages. These data collectively confirm that loading RR NBs does not impair macrophage survival and function.

[0048] Assessment of Macrophage Polarization and Durability of Antitumor ChAR-M: This application analyzed the ability of nanobatteries to reprogram macrophage phenotypes using flow cytometry. Macrophages were co-incubated with RR NBs for 4 hours, then cultured for another 24 hours with fresh medium, using untreated M0 macrophages as a negative control. Results showed that RR NBs significantly promoted M2 polarization: compared with IL-4-treated M2 controls, their CD80... + CD86 + The proportion of cells was significantly increased and comparable to that of the LPS-stimulated M1 macrophage group. Figure 9 A). Notably, the M1 polarization ability of RR NBs is significantly better than that of free R848 drug, which may be attributed to the stronger cellular uptake efficiency of the nanoparticles.

[0049] To evaluate the persistence of antitumor ChAR-M cells in maintaining the M1 phenotype, this application used LPS-stimulated M1 macrophages as a control and detected CD80 levels. + CD86 + Cell ratio. For example... Figure 9 As shown in B, ChAR-M maintained a significantly higher CD80 level than the control group even after four consecutive passages. + CD86 + The expression was enhanced, while the control group M2 macrophages lost CD80 after only two passages. + CD86 + Expression was restored to the unpolarized M0 phenotype. This indicates that RR NBs can effectively maintain the long-term stability of the M1 state. Furthermore, the phenotypic stability of ChAR-M cells was detected by flow cytometry under M2 cell co-culture stimulation. Results ( Figure 9 C) shows that ChAR-Ms maintain their phenotype for a longer period compared to unmodified M1 macrophages. These findings confirm that nanobattery-driven ChAR-Ms possess enhanced phenotypic stability and can continue to exert immunomodulatory effects in the tumor microenvironment.

[0050] Paracrine effect of antitumor ChAR-M: Macrophages can regulate surrounding cells by secreting cytokines and exosomes. To investigate the paracrine effect of ChAR-M, M2 macrophages were co-cultured with ChAR-M conditioned medium (CM) for 24 hours and then analyzed by flow cytometry. The results showed ( Figure 9 D), ChAR-M's CM can effectively induce M2 macrophages to transform into the M1 phenotype, CD80 + CD86 + The proportion of cells was significantly higher than that of the untreated control group.

[0051] Validation of ChAR-M-mediated macrophage phenotypic remodeling by PCR: This application used real-time quantitative PCR to characterize macrophage polarization status, with untreated M0 macrophages serving as a blank control. Analysis of macrophages treated with ChAR-M conditioned medium (CM) showed that M1 markers... iNos Significantly upward, while M2 markers Cd206 Significantly lowered ( Figure 10 A- Figure 10 B). Simultaneous detection revealed pro-inflammatory cytokines. Tnfα and Il1a Significantly increased ( Figure 10 C- Figure 10 D). Compared to the free drug treatment group, ChAR-M exhibited stronger M1-promoting and M2-inhibiting effects. This synergistic effect stems from the continuous supply of antitumor active substances from the nanobatteries, which can maintain strong M1 polarization in the tumor microenvironment.

[0052] In vivo distribution characteristics of ChAR-M: To analyze the biodistribution characteristics of ChAR-M in a 4T1 tumor model, this application used DiD fluorescent dye to label ChAR-M, and used LPS-induced M1 macrophages as a control. Intravenous infusion of 3×10 6 Six hours after cell collection, major organs (heart, lung, liver, spleen, kidney, and tumor) were harvested for in vitro fluorescence imaging. Results showed that ChAR-M was significantly enriched in the tumor. Figure 11 A- Figure 11 (B) Notably, the distribution pattern of ChAR-M is highly consistent with that of natural M1 macrophages, confirming that the loading of nanobatteries did not affect the inherent chemotactic migration ability and lesion-targeting properties of macrophages. Comparative analysis shows that ChAR-M loaded with nanoparticles fully retains the core functions of macrophages, ensuring precise targeting of tumor tissue.

[0053] Evaluation of the antitumor efficacy of ChAR-M in a 4T1 breast tumor model: This application evaluated the antitumor therapeutic effect of ChAR-M in a 4T1 tumor-bearing mouse model. Mice were randomly divided into four groups: PBS group, M0 macrophage group, M1 macrophage group, and ChAR-M group. The PBS group served as a negative control to assess natural tumor progression without intervention, the M0 macrophage group was used to highlight the necessity of M1 polarization, the M1 macrophage group served as a control to clarify the role of nanobatteries (NB), and the ChAR-M group validated the efficacy of NB-loaded M1 macrophages. When the tumor volume reached approximately 50 mm³, each group of mice received intravenous injection (4 × 10⁻⁶) every 4 days. 6 Cells / animal). Tumor volume monitoring results showed ( Figure 12 A~ Figure 12 C), the M0 macrophage group was similar to the PBS group, showing no antitumor activity; the M1 macrophage group exhibited moderate tumor growth inhibition, while the ChAR-M group showed the most significant inhibitory effect, with the smallest final tumor burden and volume. Notably, the efficacy of ChAR-M was significantly superior to the M1 macrophage group, highlighting the crucial role of NB in ​​enhancing therapeutic efficacy. Histopathological analysis ( Figure 13 The results showed that tumor tissue in the ChAR-M group exhibited extensive necrosis and disordered nuclear structure, consistent with its significant growth inhibition. Furthermore, compared to other groups, no micrometastases were detected in the lungs and livers of mice treated with ChAR-M, which may be attributed to sustained immune activation blocking metastatic spread. These results demonstrate that ChAR-M achieves local tumor clearance through direct cytotoxicity and tumor microenvironment (TME) remodeling, while simultaneously inhibiting distant metastasis through systemic immune activation, showcasing comprehensive therapeutic potential.

[0054] Flow cytometry analysis showed that ( Figure 14 Compared to the PBS control group, ChAR-M treatment significantly increased the CD80 marker of M1 macrophages in tumor tissue. + Expression of [a specific substance] was reduced (22.9% vs 5.7%), while the expression of the M2 biomarker CD206 was significantly reduced. + The proportion of tumor-associated macrophages polarized and reprogrammed towards an anti-tumor phenotype was successfully achieved (5.3% vs 50.9%). Regarding adaptive immunity, CD8+ cells with anti-tumor activity were observed in the ChAR-M treatment group. + T cells (33.5% vs 8.2%) and CD4 +T cell infiltration was significantly increased (41.6% vs 11.6%), while the proportion of immunosuppressive Treg cells was significantly decreased (11.4% vs 31.8%). This optimized immune microenvironment was significantly superior to the traditional M1 macrophage therapy group, demonstrating that the RssR nanobatteries synergistically activate innate and adaptive immune responses by enhancing the antigen-presenting function and immunomodulatory capacity of macrophages, thereby constructing a more efficient anti-tumor immune microenvironment.

[0055] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing engineered adoptive macrophages based on nanobatteries, characterized in that, Includes the following steps: S1: Nanoparticles are obtained through assembly using an immune adjuvant working solution as raw material; S2: The obtained nanoparticles were diluted with cell culture medium and co-incubated with macrophages to obtain engineered macrophages loaded with anti-tumor nanoparticles; In S1, the assembly method is any one of supramolecular assembly, prodrug assembly, or metal coordination assembly.

2. The method for preparing engineered adoptive macrophages based on nanobatteries according to claim 1, characterized in that, In S1, the working solution of the immune adjuvant is obtained by mixing a small molecule immunodrug with a dimethyl sulfoxide solution.

3. The method for preparing engineered adoptive macrophages based on nanobatteries according to claim 1, characterized in that, In S1, when the assembly method is supramolecular assembly, the specific steps are as follows: After stirring and mixing the small molecule immunodrug and dimethyl sulfoxide solution, an immunoadjuvant working solution is obtained; Subsequently, an aqueous solution was added to the immunoadjuvant working solution, and nanoparticles were formed through self-assembly under ultrasonic conditions.

4. The method for preparing engineered adoptive macrophages based on nanobatteries according to claim 3, characterized in that, The small molecule immunotherapy drug is R848, a Toll-like receptor 7 / 8 agonist. The volume ratio of the working solution to the aqueous solution of the immunoadjuvant is 5%.

5. The method for preparing engineered adoptive macrophages based on nanobatteries according to claim 3, characterized in that, The stirring and mixing are carried out at room temperature; The concentration of the nanoparticles is 10 µM.

6. The method for preparing engineered adoptive macrophages based on nanobatteries according to claim 1, characterized in that, The macrophages are either M1 or M2 type macrophages; The ratio of nanoparticles to macrophages is 17 µg: 10^6 cells.

7. A method for preparing engineered adoptive macrophages based on nanobatteries according to claim 1, characterized in that, The co-incubation culture system was cultured under aseptic conditions at 37 ℃ and 5% CO2.

8. A type of adoptive macrophage engineered based on nanobatteries, characterized in that, The drug-loaded bioengineered adoptive macrophages based on nanobatteries are prepared by any one of the preparation methods described in claims 1 to 7, and the drug loading capacity is 0.5 ng / cell.

9. The application of the nano-battery-based engineered adoptive macrophages in the preparation of a disease treatment system as described in claim 8, characterized in that... The aforementioned disease treatment system is prepared by loading small molecule drugs onto engineered adoptive macrophages based on nanobatteries. The small molecule drug is either a small molecule drug that can induce M2 macrophages to become M1 type or a small molecule drug that can induce M1 macrophages to become M2 type polarized. When the small molecule drug is one that can induce M2 macrophages to become M1, it can promote immune activation of the tumor microenvironment. When the small molecule drug is one that can induce M1 macrophages to polarize to M2, it can suppress excessive inflammation or promote tissue repair.

10. The application of nano-battery-engineered adoptive macrophages in the preparation of a disease treatment system according to claim 9, characterized in that, When the small molecule drug is a small molecule drug that can induce M2 macrophages to reverse polarize into M1, the small molecule drug is an immune agonist small molecule drug, a metabolic small molecule drug, or a small molecule drug that reverses polarization from M2 to M1. The immune-stimulating small molecule drug is one or more of the following: STING agonist CDN, TLR agonist and AMPK activator; The metabolic small molecule drug is one or both of the aryl hydrocarbon receptor AhR inhibitor and glutamine transporter inhibitor; The small molecule drug with reverse polarization M2 being M1 is one or both of histamine receptor inhibitors and aspirin; When the small molecule drug is a small molecule drug that can induce M1 macrophages to polarize to M2, the small molecule drug is a flavonoid or a polyphenol.

Images