Engineered drug-loaded macrophage for resisting transplant rejection

Abstract

The invention relates to the technical field of medicines, and particularly discloses an engineered drug-loaded macrophage for resisting transplant rejection. PD-L1 is overexpressed on the surface of a cell membrane of the engineered drug-loaded macrophage, and the engineered drug-loaded macrophage internally contains nanoparticles encapsulated with rapamycin. Due to the biological characteristic that the macrophages can respond to inflammation signals, the engineered drug-loaded macrophages can be efficiently recruited to rejection reaction parts by chemotactic factors of transplantation parts, and the engineered drug-loaded macrophages can penetrate a tissue barrier as an active carrier, respond to external stimulation to exocytosis and release nanoparticles encapsulated with rapamycin after reaching a target position, and can be used for preparing a drug carrier. Local precise delivery of PD-L1 and rapamycin is realized, and immunosuppression toxicity of systemic administration is reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to an engineered drug-loaded macrophage for treating transplant rejection. Background Technology

[0002] Organ transplantation is the most effective treatment for end-stage organ failure, but transplant rejection remains a major obstacle to long-term graft survival and patients' quality of life. While widely used immunosuppressants (such as tacrolimus and cyclosporine A) can effectively reduce acute rejection, their limitations are becoming increasingly apparent: they cannot completely suppress long-term immune activation and are often accompanied by serious side effects such as infection, malignancy, and nephrotoxicity. More importantly, these drugs interfere with the function and stability of regulatory T cells, which are crucial for maintaining immune tolerance. To overcome these limitations, clinical practice has begun to combine rapamycin, an mTOR inhibitor, with drugs that have less impact on Tregs and may even promote their function. This drug aims to suppress rejection while helping to establish long-term immune tolerance. However, rapamycin's broad target range can cause serious adverse reactions such as bone marrow suppression, limiting its application. Studies have shown that a PD-L1-Ig fusion protein that enhances the PD-1 / PD-L1 immunosuppressive axis has shown potential for synergistic prevention of transplant rejection when used in combination with rapamycin. This suggests that the combination of rapamycin and a PD-L1 inhibitor may offer a better option for patients by improving efficacy while reducing side effects. However, currently used soluble PD-L1-Ig formulations suffer from low receptor clustering efficiency and lower immunosuppressive efficacy compared to membrane-bound forms. More importantly, systemic administration may induce widespread immunosuppression, posing toxicity risks. Therefore, achieving localized and precise synergistic delivery of the two drugs is a core challenge that translational research urgently needs to address. Summary of the Invention

[0003] This invention provides engineered drug-loaded macrophages for treating transplant rejection. These engineered cells overexpress PD-L1 on their surface and contain encapsulated rapamycin nanoparticles. Due to their biological characteristics of responding to inflammatory signals, these engineered drug-loaded macrophages can be efficiently recruited to the rejection site by chemokines at the transplant site. As an active carrier, they can penetrate tissue barriers and, upon reaching the target site, release the encapsulated rapamycin nanoparticles via exocytosis in response to external stimuli. This achieves precise local delivery of PD-L1 and rapamycin, reducing the immunosuppressive toxicity of systemic administration.

[0004] The technical solution provided by this invention is as follows: In a first aspect, the present invention provides an engineered drug-loaded macrophage for resisting transplant rejection, wherein the engineered drug-loaded macrophage overexpresses PD-L1 on its cell membrane surface and contains nanoparticles encapsulating rapamycin inside.

[0005] In conjunction with the first aspect of the invention, in some embodiments, the nanoparticles have a core-shell structure, wherein the core contains rapamycin and the shell is a biodegradable polymer material.

[0006] In conjunction with the first aspect of the invention, in some embodiments, the engineered drug-loaded macrophages are further modified with azide groups on their surface.

[0007] Secondly, the present invention provides a method for preparing engineered drug-loaded macrophages, comprising: Inducing macrophages to overexpress the immunosuppressive protein PD-L1; The induced macrophages engulfed nanoparticles containing rapamycin, forming engineered drug-loaded macrophages.

[0008] In conjunction with a second aspect of the invention, in some embodiments, the induction of macrophage overexpression of the immunosuppressive protein PD-L1 includes: co-incubating macrophages with IFN-γ.

[0009] In conjunction with a second aspect of the invention, in some embodiments, the step of causing induced macrophages to phagocytose nanoparticles encapsulated with rapamycin includes: co-incubating induced macrophages with nanoparticles encapsulated with rapamycin.

[0010] In conjunction with the second aspect of the present invention, in some embodiments, the preparation steps of the nanoparticles include: providing a precursor liquid containing an organic solvent and rapamycin and a biodegradable polymer dissolved therein; adding the precursor liquid to an aqueous solution containing an emulsifier, emulsifying to form an oil / water emulsion; and evaporating the organic solvent under stirring conditions to obtain nanoparticles.

[0011] In conjunction with the second aspect of the present invention, in some embodiments, the modification step of the azide group includes: co-incubating azidomannose with induced macrophages.

[0012] Thirdly, the present invention provides the application of the above-mentioned engineered drug-loaded macrophages in the preparation of drugs for treating transplant rejection diseases.

[0013] In conjunction with a third aspect of the invention, in some embodiments, the transplant rejection disease is an acute rejection reaction caused by skin transplantation.

[0014] Compared with the prior art, the present invention has at least the following beneficial effects: 1. Addressing the challenge of precise local delivery of PD-L1 and rapamycin, this invention provides engineered drug-loaded macrophages for combating transplant rejection. These macrophages are engineered to overexpress PD-L1 on their cell membrane surface and contain nanoparticles encapsulated with rapamycin. Based on the unique inflammatory signaling ability of macrophages, inflammatory chemokines produced at the transplant site can efficiently recruit engineered macrophages to the rejection site. These cells, acting as active carriers, can actively penetrate tissue barriers for precise targeting. Upon reaching the target site, they can respond to specific external stimuli and release large quantities of the encapsulated rapamycin nanoparticles through exocytosis.

[0015] 2. The PD-L1 overexpressed on the engineered macrophage membrane is a natural membrane protein that is translated and expressed by the cell itself after stimulation by IFN-γ. Its structure, orientation and function are superior to those of the soluble PD-L1-Ig fusion protein, thus exhibiting higher stability and biological activity. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This study outlines the design, preparation, and characterization of engineered macrophage TTMs. A shows a schematic diagram of the engineered macrophage TTM preparation process. B-C demonstrate immunofluorescence assays of the expression level and quantification of endogenous PD-L1 in RAW264.7 cells after 48 hours of treatment with different concentrations of PD-L1 (** p < 0.01, *** p < 0.001, **** p < 0.0001). D shows confocal microscopy analysis of macrophage surface modification with azide labeling. E demonstrates confocal microscopy analysis of macrophages exhibiting both PD-L1 overexpression and azide labeling. F shows flow cytometry analysis of macrophages overexpressing PD-L1 and labeled with azide (Mφ). PD-L1@N3 The uptake capacity of ) was not significant in ns; G was the value of Mφ compared by flow cytometry. PD-L1@N3 Compared with the nanoparticle uptake capacity of ordinary RAW264.7 cells; H is the confocal verification of Mφ PD-L1@N3 The uptake capacity was comparable to that of ordinary RAW264.7 cells; I shows the intracellular uptake of RAPA NPs by TMM using transmission electron microscopy (TEM); J shows the quantitative analysis of Mφ by HPLC. PD-L1@N3The loading efficiency of RAPA NPs was not significant in ns; K represents the expression level of PD-L1 in TTM detected by qRT-PCR, ** p < 0.01, *** p < 0.001, **** p < 0.0001; L represents the expression levels of CD68 and PD-L1 in TTM detected by Western blot.

[0018] Figure 2 The data represents the release of rapamycin; where: A is the standard curve of rapamycin concentration, and B is the drug release curve of rapamycin nanoparticles.

[0019] Figure 3 This study aims to evaluate the bioactivity and biosafety of TTM cells in vitro. A represents the CCK-8 assay used to assess the cell viability of TTM cells treated with IFN-γ and mannose azide (AC4ManNAz). B-C show schematic diagrams and quantitative analyses of the chemotactic activity of TTM and standard RAW264.7 cells under MCP-1 induction using Transwell assays. D shows the detection of TTM drug release in the LPS inflammation model by high performance liquid chromatography; E shows the assessment of TTM cytotoxicity by Calcein-AM / PI double staining; F shows the fluorescence spectrum of TTM ROS level measured by DCFH-DA fluorescence method; G shows the flowchart of TTM ROS level measurement by DCFH-DA fluorescence method; H shows the quantitative analysis of TTM ROS level measured by DCFH-DA fluorescence method, with no significant difference in ns; I shows the IL-1 secretion level in TTM detected by ELISA; J shows the IL-2 secretion level in TTM detected by ELISA; K shows the IL-6 secretion level in TTM detected by ELISA; L shows the TNF-α secretion level in TTM detected by ELISA, with no significant difference in ns.

[0020] Figure 4This document outlines the blood compatibility assessment and in vivo biosafety evaluation of TTM. Specifically: A shows the hemolysis assay for TTM blood compatibility; B is a schematic diagram illustrating the procedure for three intravenous injections of PBS, RAW264.7 cells, or TTM into normal mice within a 48-hour interval; C shows the ELISA assay for IgG concentrations after PBS, RAW264.7 cell, or TTM treatment; D shows the ELISA assay for IgM concentrations after PBS, RAW264.7 cell, or TTM treatment; and E-I show the serum biochemical and hematological assays for liver function indicators (ALT, AST). Quantitative analysis of ALB, TBIL, and CHO levels; quantitative analysis of serum biochemical and hematological test results for renal function indicators (BUN, CREA, UA); quantitative analysis of complete blood cell count including white blood cell subsets (WBC), lymphocytes (Lymph), neutrophils (Gran), monocytes (MON), red blood cells (RBC), platelets (PLT), mean corpuscular volume (MCV), and hemoglobin (HGB); and H&E staining analysis of the morphological characteristics of the heart, liver, spleen, lungs, and kidneys.

[0021] Figure 5 This study investigated the in vivo graft targeting performance of TTM in a mouse skin graft model. Specifically: A) in vivo imaging detection of the fluorescence intensity of DBCO-Cy5 in mice treated with PBS, RAW264.7 cells, or TTM; B) quantitative analysis of the fluorescence intensity of DBCO-Cy5 in mice treated with PBS, RAW264.7 cells, or TTM; and C) immunofluorescence staining verification of the interaction between DBCO-Cy5 and CD68 in the graft tissue. + Co-localization of macrophages; D shows in vivo imaging detection of local fluorescence signals in the graft at different time points after skin grafting surgery with probe application; E shows quantitative analysis of DBCO-Cy5 fluorescence intensity after TTM injection at different time points after skin grafting surgery, ** p < 0.01, *** p < 0.001, **** p < 0.0001; F shows Western Blot detection of protein expression levels of macrophage biomarkers CSF-1, MCP-1, and CD68; G shows quantitative analysis of protein expression levels of macrophage biomarkers CSF-1, MCP-1, and CD68 detected by Western Blot, ** p < 0.01, *** p < 0.001, **** p < 0.0001; H shows qRT-PCR detection of mRNA expression levels of macrophage biomarkers CSF-1, MCP-1, and CD68, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0022] Figure 6This study evaluates the efficacy of TTM (Thin-Methoxygen Transplantation) in treating mouse skin grafts. A shows a schematic diagram of the drug administration process after establishing the mouse skin graft model; B shows the Kaplan-Meier survival analysis of PBS, RAW264.7 cells, and macrophages overexpressing PD-L1 (Mφ). PD-L1 The effects of rapamycin nanoparticles (RAPA NPs) and TTM treatment on skin graft survival; C represents the H&E staining assay for PBS, RAW264.7 cells, and Mg2+. PD-L1 The effects of RAPA NPs and TTM treatment on skin graft structure; D represents immunohistochemical detection of PBS, RAW264.7 cells, and Mg2+. PD-L1 The expression levels of CD3 in RAPA NPs and TTM-treated skin graft tissues; E represents the immunohistochemical detection of PBS, RAW264.7 cells, and Mg2+. PD-L1 The expression levels of Ki67 in RAPA NPs and TTM-treated skin grafts were measured; F represents the immunohistochemical detection of PBS, RAW264.7 cells, and Mg2+. PD-L1 The expression levels of Granzyme B in RAPA NPs and TTM-treated skin graft tissues were determined.

[0023] Figure 7 TTM's responsiveness to allogeneic Th1 and CD8 + T cell activation and proliferation inhibition efficacy assay; where: A is flow cytometry evaluation of TTM's effect on CD4. + The regulatory effect of T cell proliferation; B represents the effect of TTM on CD4. + Quantitative analysis of the effect on T cell proliferation, ** p < 0.01, *** p < 0.001, **** p < 0.0001; C~F show the expression of cytokines (IL-2, IFN-γ, IL-4, IL-10) in the supernatant of TTM co-culture with allogeneic reactive T cells, ** p < 0.01, *** p < 0.001, **** p < 0.0001; G shows the flow cytometry analysis of PBS, RAW264.7 cells, and Mg2+ cells. PD-L1 RAPA NPs and TTM group on CD8 in spleen + The effect of T cell frequency; H represents flow cytometry analysis of PBS, RAW264.7 cells, and Mφ. PD-L1 RAPA NPs and TTM group on CD8 in spleen + Quantitative analysis of T cell frequency, ** p < 0.01, *** p < 0.001, **** p < 0.0001; I represents flow cytometry analysis of PBS, RAW264.7 cells, and Mg2+ cells. PD-L1 RAPA NPs and TTM group on CD8 in lymph nodes+ The effect of T cell frequency; J represents flow cytometry analysis of PBS, RAW264.7 cells, and Mφ. PD-L1 RAPA NPs and TTM group on CD8 in lymph nodes + Quantitative analysis of T cell frequency, ** p < 0.01, *** p < 0.001, **** p < 0.0001; K represents flow cytometry analysis of PBS, RAW264.7 cells, and Mg2+ cells. PD-L1 RAPA NPs and TTM group showed CD8 levels in lymph nodes infiltrated by transplanted tissue. + The effect of T cell frequency, L represents flow cytometry analysis of PBS, RAW264.7 cells, and Mφ. PD-L1 RAPA NPs and TTM group showed CD8 levels in lymph nodes infiltrated by transplanted tissue. + Quantitative analysis of T cell frequency, ** p<0.01, *** p<0.001, **** p<0.0001.

[0024] Figure 8 Transcriptome sequencing analysis of complement activation levels of C3, C5, C3ar, C5ar, CCR5, and CCL5 in grafts using TTM, and qPCR detection of related mRNA expression levels; where: A represents transcriptome analysis using PBS, RAW264.7 cells, and Mg2+ cells. PD-L1 RAPNPs and TTM group showed the complement activation levels of C3, C5, C3ar, C5ar, CCR5, and CCL5; B~G were quantitative analyses of the expression levels of C3, C5, C3ar, C5ar, CCR5, and CCL5-related mRNAs detected by qPCR. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0025] Figure 9 The effect of TTM on the differentiation and function of regulatory T cells (Tregs); where: A~D represent the effect of flow cytometry on CD25 in the spleen and lymph nodes. + FoxP3 + Frequency and quantification of regulatory T cells (Tregs), ** p < 0.01, *** p < 0.001, **** p < 0.0001. E-F show flow cytometry detection of CD25 in single-cell suspensions prepared from allogeneic skin tissue. + FoxP3 + Frequency and quantification of regulatory T cells (Tregs), ** p < 0.01, *** p < 0.001, **** p < 0.0001: G~H represent Foxp3 detected by flow cytometry. + IL-10 +Frequency and quantification of regulatory T cells (Tregs), ** p < 0.01, *** p < 0.001, **** p < 0.0001. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0027] In transplant rejection treatment, rapamycin is often used as an adjunct therapy to alleviate side effects such as infection, malignancy, and nephrotoxicity caused by calcineurin inhibitors like cyclosporine A and tacrolimus. However, due to its broad target range, systemic administration can easily lead to excessive immunosuppression, making precise delivery of rapamycin to the rejection site a key challenge. Studies have shown that rapamycin combined with programmed death-ligand-1 (PD-L1) has a synergistic immunosuppressive effect. PD-1 / PD-L1 are important immune checkpoints; their binding can inhibit effector T cell activity and promote regulatory T cell function, thereby regulating peripheral tolerance. However, soluble PD-L1-Ig suffers from low receptor clustering efficiency, weak potency, and the potential to induce systemic immunosuppressive toxicity. To overcome these limitations, research has shifted towards cell-based delivery strategies. By modifying human embryonic stem cells, mesenchymal stem cells, and dendritic cells to highly express membrane-bound PD-L1, these platforms can continuously inhibit local T cell activation and promote immune tolerance. However, they still have shortcomings in terms of targeting, precision of regulation, and real-time tracking.

[0028] This invention provides engineered drug-loaded macrophages for combating transplant rejection. These engineered macrophages overexpress PD-L1 on their cell membrane surface and contain nanoparticles encapsulated with rapamycin. Compared to human embryonic stem cells, mesenchymal stem cells, and dendritic cells, macrophages possess inflammatory chemotaxis capabilities. Inflammatory factors released at the transplant rejection site can directionally recruit these engineered macrophages to the lesion. As active carriers, macrophages, leveraging their natural migratory abilities, can cross physiological barriers to precisely reach the target area. Upon receiving specific stimuli, they release large quantities of their loaded rapamycin nanoparticles via exocytosis. This effectively solves the problem of precise in vivo delivery of the two aforementioned immunosuppressive signals (rapamycin and membrane-bound PD-L1).

[0029] In some embodiments, the nanoparticles have a core-shell structure, with the core containing rapamycin and the shell being a biodegradable polymeric material. The core provides a stable storage microenvironment for the drug; the shell acts as a physical and chemical barrier, effectively preventing drug leakage or premature degradation before reaching the transplant rejection site, significantly improving drug loading stability and in vivo bioavailability. The biodegradable polymeric material is one or more of the following substances: (1) Polylactic acid (PLA); (2) Polycaprolactone (PCL); (3) Polylactic acid-glycolic acid copolymer (PLGA); (4) Chitosan; (5) Gelatin; (6) 3-hydroxybutyric acid-3-hydroxyhexanoic acid copolyester (PHBHHx); (7) Silk fibroin (SF).

[0030] Preferably, the biodegradable polymer material is PLGA. PLGA achieves long-term release of rapamycin through slow hydrolysis in vivo, effectively avoiding the risk of excessively high local concentrations caused by burst drug release, significantly reducing dosing frequency, and maintaining a stable and effective drug concentration at the lesion site over a long period. Furthermore, due to the hydrophobic nature of rapamycin, PLGA encapsulation technology effectively protects the drug from rapid metabolism and clearance in the body, prolonging drug circulation time; it also promotes selective accumulation of the drug at the target site, reduces non-specific distribution throughout the body, successfully achieving targeted delivery of local drug administration, significantly improving drug bioavailability and reducing systemic toxicity.

[0031] In some embodiments, the engineered drug-loaded macrophages are further modified with azide groups on their surface. These modified azide groups can be used for orthogonal labeling. Specifically, the azide reaction probe DBCO-Cy5 can specifically bind to the azide groups modified on the macrophage surface via a copper-free click reaction. The Cy5 fluorescent group carried on the probe emits bright red fluorescence upon excitation at a specific wavelength. Because this reaction is highly specific, it ensures precise labeling to the target cells, greatly reducing background signal interference, thus enabling real-time labeling and tracking during the application of engineered macrophages.

[0032] This invention also provides a method for preparing engineered drug-loaded macrophages, comprising the following steps: inducing macrophages to overexpress the immunosuppressive protein PD-L1; and phagocytosing the induced macrophages with nanoparticles encapsulated with rapamycin to form engineered drug-loaded macrophages. Stable expression of PD-L1 on the cell membrane provides immediate immune checkpoint inhibition signals, while intracellular nanoparticles respond to the microenvironment and provide long-lasting sustained release of rapamycin; both can synergistically enhance local immunosuppression. Simultaneously, relying on the macrophages' natural chemotaxis towards inflammatory sites, these engineered macrophages can achieve targeted delivery, precisely enriching the dual therapeutic effects at the graft site, thereby improving the efficacy against transplant rejection while minimizing the widespread immunosuppression and toxic side effects caused by traditional systemic drug administration. The preparation steps are mainly divided into two parts and are relatively simple. The phagocytosis step only requires conventional cell culture and incubation equipment, without the need for special instruments, which is conducive to large-scale production.

[0033] In some embodiments, inducing macrophage overexpression of the immunosuppressive protein PD-L1 includes co-incubating macrophages with IFN-γ. The advantage of this invention, which induces macrophage overexpression of the immunosuppressive protein PD-L1 through co-incubation with IFN-γ, lies in its ability to highly mimic the pathophysiological state in vivo, controllably and efficiently activating the classic JAK-STAT1 signaling pathway in macrophages and initiating the transcriptional program of the PD-L1 gene. Compared to existing soluble PD-L1-Ig fusion proteins used alone, PD-L1 expressed by macrophages through their own transcription and translation exhibits superior stability and biological activity.

[0034] In some embodiments, the process of inducing induced macrophages to phagocytose nanoparticles encapsulated with rapamycin includes co-incubating the induced macrophages with the nanoparticles encapsulated with rapamycin. This method utilizes the inherent phagocytic function of macrophages to actively take up and internalize nanoparticles encapsulated with rapamycin from the surrounding environment, thereby loading rapamycin into the cells. Compared with common viral vector methods, chemical or physical delivery methods, this loading strategy based on natural cell function not only significantly increases the drug loading capacity per cell but also has advantages such as ease of operation, lower cost, and higher safety.

[0035] In some embodiments, the preparation steps of the nanoparticles include: providing a precursor fluid containing an organic solvent and rapamycin dissolved therein, and a biodegradable polymer; adding the precursor fluid to an aqueous solution containing an emulsifier, emulsifying to form an oil / water emulsion; and evaporating the organic solvent under stirring conditions to obtain the nanoparticles. The preparation of rapamycin nanoparticles employs a single emulsification method, which combines ease of operation with process stability. This method is simple, efficient, and reproducible. In terms of application, the single emulsification method is suitable for encapsulating rapamycin, a hydrophobic drug, achieving high drug loading and encapsulation efficiency. The resulting nanoparticles exhibit good sustained-release properties, which is beneficial for achieving sustained release of rapamycin. In terms of production, this process is relatively straightforward, has mature process parameters, and requires relatively low-level experimental equipment, which is conducive to technology popularization and process scale-up.

[0036] In some embodiments, the modification step of the azido group includes co-incubating azidomannose with induced macrophages. Due to its acetyl group, azidomannose easily penetrates the cell membrane, is internalized by macrophages, and then hydrolyzed by esterases within the macrophages, releasing the core structure ManNAz. The cell's own sialic acid synthase system misidentifies ManNAz as a natural substrate, thereby integrating it into the metabolic pathway and ultimately activating it into CMP-azidosialic acid. Subsequently, glycosyltransferases in the Golgi apparatus add CMP-azidosialic acid to the glycan ends of newly synthesized glycoproteins, glycolipids, and other molecules, and transport them to the cell membrane surface. This process results in the outer cell membrane being labeled with the azido group, thereby providing a target for subsequent click chemistry-based specific recognition.

[0037] This invention also provides the application of engineered drug-loaded macrophages in the preparation of drugs for treating transplant rejection. Transplant rejection is a specific immune response launched by the recipient's immune system against the donor graft after allogeneic organ, tissue, or cell transplantation, leading to graft dysfunction or even loss. Common types include bone marrow transplant rejection, heart transplant rejection, corneal transplant rejection, intestinal transplant rejection, liver transplant rejection, lung transplant rejection, pancreas transplant rejection, kidney transplant rejection, and skin transplant rejection. The engineered drug-loaded macrophages provided by this invention can precisely deliver PD-L1 and rapamycin to the rejection site, thereby inhibiting the rejection reaction.

[0038] In some embodiments, the transplant rejection disease is an acute rejection reaction caused by skin grafts. Symptoms of rejection caused by skin grafts include one or more of the following: (1) Symptoms similar to influenza, such as fever, chills, fatigue, loss of appetite and muscle and joint pain; (2) Abnormal color, obvious swelling and hardening, persistent pain and severe itching appear at the transplanted skin site; (3) Red macules, papules or patches appear on the surface of the transplanted skin. In severe cases, blisters and blood blisters may appear. After the rupture, erosion, exudation or crusting may form, with pinpoint or patchy bleeding, which may then develop into deep ulcers. (4) The transplanted skin necroses, forms a thick scab, and eventually falls off completely, exposing the underlying tissue.

[0039] The technical solution provided by the present invention will be described in detail below with reference to the embodiments.

[0040] Example 1: Preparation of engineered macrophage TTM (1) IFN-γ-induced construction of macrophages overexpressing PD-L1 (Mφ) PD-L1 Optimal concentration screening IFN-γ was dissolved in deionized water to obtain IFN-γ solution; the IFN-γ solution was diluted with complete culture medium containing 10% FBS to prepare working solutions with concentrations of 30, 50, 100, and 150 ng / mL. 1 mL of each working solution was incubated with the mouse macrophage cell line RAW264.7 for 48 h, and then the expression of PD-L1 was verified by immunofluorescence. Figure 1 (B~C), and finally determined to construct PD-L1 overexpressing macrophages Mφ PD-L1 The optimal IFN-γ induction concentration was 100 ng / mL, ** p<0.01, *** p<0.001, **** p<0.0001.

[0041] (2) Constructing macrophages Mφ with azide-labeled cell membrane surface using azidomannose N3 Optimal concentration screening RAW264.7 cells metabolize azidomannose (AC4ManNAz) via the sialic acid pathway, successfully labeling its azide group onto the cell membrane surface. DBCO-Cy5 can undergo a click chemical reaction with this azide, thereby coupling with the Cy5 fluorescent group to achieve fluorescent labeling of RAW264.7 cells. The specific implementation method is as follows: First, weigh an appropriate amount of AC4ManNAz powder and dissolve it in complete culture medium to prepare working solutions of 30μM, 50μM, and 60μM. Take 1mL of each concentration of working solution and co-incubate with RAW264.7 cells for 48 h. Wash three times with PBS, add serum-free culture medium containing 30μM DBCO-Cy5, and react at 37℃ in the dark for 0.5 h. Then wash three times with PBS to remove free DBCO-Cy5, fix with 4% paraformaldehyde at room temperature for 10 min, and further label the cell nuclei with DAPI staining. Finally, laser confocal microscopy revealed significant enrichment of DBCO-Cy5 fluorescence signal on the cell membrane, confirming that the optimal concentration of AC4ManNAz for cell labeling was 50 μM. Furthermore, the cell membranes of RAW264.7 cells were labeled with a 5 μM Cell Mask probe to further investigate the modification efficacy of the azide group. The results showed clear co-localization between the DBCO-Cy5-labeled azide group and the Cell Mask-labeled RAW264.7 cell membrane, confirming the effectiveness of azide modification on the RAW264.7 cell membrane surface. Figure 1 D).

[0042] (3) Preparation of engineered macrophages Mφ by IFN-γ induction combined with glucose metabolism labeling PDL1@ N3 The incubation solution was prepared according to the optimal concentrations (IFN-γ: 100 ng / mL; AC4ManNAz: 50 μM) determined in steps (1) and (2). RAW264.7 cells were incubated in this solution for 48 h. The total volume of the incubation system was 1 mL. This yielded dual-modified macrophages Mφ that overexpressed PD-L1 and had azide-labeled surfaces. PD-L1@N3 Subsequently, the results were verified by immunofluorescence and DBCO-Cy5 reaction, indicating that Mφ PD-L1@N3 It exhibits PD-L1 overexpression and azide labeling characteristics. Confocal microscopy shows that PD-L1 and azide groups are located in different membrane regions without overlap. Figure 1 E), indicating that the dual modification does not cause steric hindrance interference. This steric separation may help preserve the function of PD-L1 for immune checkpoint binding.

[0043] (4) Screening of drug loading time points on macrophages To assess whether surface engineering interferes with the core phagocytic function of macrophages, DiI-labeled PLGA nanoparticles were used, and the uptake kinetics were analyzed by flow cytometry. The results showed that the internalization of the nanoparticles exceeded 90% within 8 hours, and no further increase was observed thereafter. Figure 1 F), ns showed no significant difference. Therefore, 8 hours was chosen as the optimal incubation time. Furthermore, Mφ PD-L1@N The uptake capacity of 3 is comparable to that of ordinary RAW264.7 cells, as confirmed by flow cytometry and confocal microscopy. Figure 1 G~H), the above results indicate that Mφ PD-L1@N3 The phagocytic function of macrophages is preserved.

[0044] (5) Rapamycin nanoparticles (RAPA NPs) were prepared by a single emulsion method. First, weigh 2 mg of RAPA and 10 mg of PLGA and dissolve them separately in 0.5 mL of dichloromethane. Next, pipette 0.1 mL of RAPA into the PLGA dispersion and mix thoroughly. Then, add 3 mL of 0.5% PVA solution (w / v) and further treat the mixture using an ultrasonic homogenizer at 20% power for 5 min. Next, add 10 mL of ultrapure water and stir on a magnetic stirrer at room temperature for 3 h to facilitate the volatilization of dichloromethane. Finally, centrifuge the mixture at 22000 g for 20 min, collect the precipitate, resuspend the precipitate in 4 mL of ultrapure water to obtain the final product, RAPA NPs, which are then lyophilized for subsequent experiments.

[0045] (6) Mφ PDL1@ N3 Engineered macrophages TTM prepared by phagocytosis of rapamycin-loaded nanoparticles RAW264.7 cells (5 × 10⁻⁶) 4 (cells / mL) were seeded into a confocal dish. After cell adhesion, 50 μM MAC4ManNAz and 100 ng / mL IFN-γ were added and the cells were incubated for 48 h. The total incubation volume was 1 mL. Engineered macrophages (Mφ) were thus prepared. PD-L1@N3 Subsequently, the RAPA NPs from Example 5 were added to a culture dish at a concentration of 25 μg / mL and cultured at 37°C for 8 h. Free RAPA NPs were then removed by rinsing with phosphate-buffered saline (PBS), thus constructing engineered drug-loaded macrophage TTM cells. Figure 1 A). Intracellular uptake of RAPA NPs by TMMs was confirmed by transmission electron microscopy (TEM), showing intact nanoparticles with occasional deformation, consistent with endocytosis. Figure 1 I). HPLC quantitative analysis showed high loading efficiency, with approximately 94% of rapamycin internalized at 8 hours and approximately 95% internalized at 24 hours. Figure 1 J). Western blot and qRT-PCR analyses showed that nanoparticle loading did not alter the expression of CD68 or PD-L1, TTM maintained stable CD68 levels, and PD-L1 expression increased 3.2-fold compared to ordinary RAW264.7 cells. Figure 1 K~L). Furthermore, a standard curve for RAPA was established using high-performance liquid chromatography (HPLC). Subsequently, RAPA NPs were dialyzed, with 0.5 mL samples collected continuously for 72 hours for analysis. The results showed that after 72 hours of dialysis, the drug release rate of RAPA NPs was only 15%, while the release rate of RAPA without PLGA encapsulation was as high as 92% after 72 hours. These results confirm that PLGA encapsulation of RAPA can prevent drug leakage. Figure 2 (A~B).

[0046] Example 2: Evaluation of TTM chemotactic function and inflammatory response drug release The CCK-8 assay was used to evaluate the control group (normal RAW264.7 cells) and Mφ. N3 Mφ PD-L1 Cell viability was assessed using TTM cells, and the results showed that the viability of treated TTM cells exceeded 95%, with no significant difference compared to the Control group and all other groups. Figure 3 A). Furthermore, the chemotactic capacity of the TTM and Control groups (standard RAW264.7 cells) under MCP-1 induction was evaluated using Transwell assays. The results showed that the TTM and Control groups exhibited similar migration abilities under MCP-1 induction. Figure 3 (B~C), indicating that surface modification had no significant effect on the chemotactic function of macrophages. High-performance liquid chromatography (HPLC) analysis further confirmed that TTM possessed inflammatory microenvironment-responsive drug release properties. Results showed that TTM released 10.8±2.5% of encapsulated rapamycin within 36 hours. Furthermore, under LPS stimulation, the drug release from TTM increased to 24.8±2.0% within 24 hours, a 2.3-fold increase compared to the release under LPS-free conditions. Figure 3 D).

[0047] Example 3: In vitro safety assessment of TTM To assess the cytotoxicity and oxidative stress of engineered macrophages, PI staining was performed to reveal that most cells were viable, with only a very small number showing PI-positive signals. Figure 3 E). Subsequently, ROS levels, as measured by DCFH-DA fluorescence assay, did not change significantly after modification, consistent with the Control group (ordinary RAW264.7 cells). Figure 3(F~H), indicating that surface labeling and nanoparticle loading did not induce oxidative stress. Furthermore, ELISA was used to detect cytokine secretion, and the results showed that TTM and Mφ... N3 Mφ PD-L1 No significant differences were observed in the secretion levels of IL-1, IL-2, IL-6, or TNF-α between the control (normal RAW264.7 cells) group and the control group. Figure 3 I–L) further supports the immune safety of TTM in vitro.

[0048] Example 4: In vivo safety assessment of TTM All cell types tested included TTM, RAW264.7 cells, and Mφ. PD-L1@N3 The hemolysis caused by the PBS group was similar to that in the PBS group, and extremely mild, while Triton X-100 caused complete hemolysis. Figure 4 A) demonstrates that TTM has good blood compatibility and is suitable for systemic administration.

[0049] To investigate the effect of TTM on immunogenicity in mice, 6-8 week old male C57BL / 6J mice were selected as the research subjects. They were cultured in an SPF environment with a standard diet for one week, and their body weight was approximately 20g. The mice were then randomly divided into groups of three and treated via tail vein injection. Each mouse received three intravenous injections of PBS (5×10⁻⁶ mg / L) at 48-hour intervals. 6 RAW264.7 cells or 5 × 10⁶ cells 6 TTM ( Figure 4 B), Seven days after drug administration, samples were collected from mice for analysis of immunogenicity, physiological indicators, and organ pathological changes. Specific experimental groups were as follows: (1) PBS group (PBS, 200 μL / animal, once every 48 h, for a total of 3 times); (2) RAW264.7 group (5×10 6 RAW264.7 cells, 200 μL / bird, every 48 hours, for a total of 3 times); (3) TTM group (5×10 6 One TTM cell, 200 μL / animal, once every 48 hours, for a total of 3 times.

[0050] ELISA results showed that the concentrations of IgG and IgM in the TTM group were comparable to those in the PBS group, indicating that TTM has low immunogenicity. Figure 4 C~D). Serum biochemistry and hematology tests assessed organ safety 48 hours after the last administration. Liver (ALT, AST, ALB, TBIL, CHO) and kidney (BUN, CREA, UA) parameters remained within the normal range, and there were no significant differences between groups. Figure 4 E~L). In addition, the complete blood cell count, including white blood cell subsets, red blood cells, platelets, MCV, and hemoglobin, did not change significantly. Figure 4 M~V). Finally, histological analysis of the heart, liver, spleen, lungs, and kidneys showed that the organ structures were intact, with no evidence of inflammatory infiltration, necrosis, or tissue damage. Figure 4 The above results indicate that, under the test conditions, multiple systemic administrations of TTM were well tolerable.

[0051] Example 5: Monitoring TTM Targeting Migration in a Mouse Skin Transplant Model via In Vivo Orthogonal Bioreaction To further evaluate the targeting migration of TTM in a mouse skin graft model, a mouse skin graft model was first constructed using 6-8 week old male BALB / c and C57BL / 6J mice as experimental subjects. These mice were acclimatized for one week in an SPF environment with a standard diet, and their weight was approximately 20g. BALB / c mice served as donors, and C57BL / 6J mice as recipients. Both donor and recipient mice were safely anesthetized. Under sterile conditions, a whole piece of skin was harvested from the back of the donor mouse and placed in cold, sterile saline. Subcutaneous fat and connective tissue were carefully removed to create a full-thickness skin graft. The prepared skin graft was trimmed to a standard size. Subsequently, in the recipient mouse's prepared area, a piece of skin identical in shape and size to the donor skin graft was cut with fine scissors, exposing the underlying vascularized granulation tissue bed. The prepared donor skin graft was then precisely placed on the recipient wound, ensuring edge alignment. Finally, the skin graft was fixed and bandaged. On the 4th day after surgery, the constructed skin graft model mice were randomly divided into 5 mice per group and treated with the drug via tail vein injection. The specific experimental groups were as follows: (1) Control group (PBS, 200 μL / animal, injected once on the 4th day after surgery); (2) RAW264.7 group (5×10 6 200 μL of RAW264.7 cells per animal, injected once on the 4th day after surgery. (3) TTM group (5×10 6 One TTM cell, 200 μL / animal, injected once on the 4th day after surgery.

[0052] 24 hours later, DBCO-Cy5 was injected, and imaging was performed 6 hours later. Only the TTM group showed strong localized graft fluorescence, while the Control and RAW264.7 groups showed weaker signals. Figure 5 A). Quantitative analysis showed that the graft fluorescence intensity in the TTM group was approximately 1.6 times that of the other groups ( Figure 5 B). Immunofluorescence staining confirmed the interaction between DBCO-Cy5 and CD68 in graft tissue.+ Colocalization of macrophages ( Figure 5 C).

[0053] To investigate inflammatory adaptive migration, on postoperative day 2, the constructed skin graft model mice were randomly divided into groups of 5. TTM was administered via tail vein injection on postoperative days 2, 4, and 6. The specific experimental groups were as follows: (1) POD3 group (5×10 6 One TTM cell, 200 μL / animal, administered once on the second day after surgery); (2) POD5 group (5×10 6 One TTM cell, 200 μL / animal, administered once on the 4th day after surgery); (3) POD7 group (5×10 6 One TTM cell, 200 μL / animal, administered once on day 6 post-surgery.

[0054] DBCO-Cy5 was injected 24 hours after drug administration, and in vivo imaging was performed 6 hours later. Local fluorescence of the graft was observed at all time points, with the maximum fluorescence intensity occurring on postoperative day 5, coinciding with the peak of inflammatory activity. Figure 5 D). Quantitative analysis showed a strong correlation between fluorescence intensity and repulsion level. Figure 5 E).

[0055] In addition, for untreated skin graft model mice, skin graft tissue was collected on days 3 and 7 post-transplantation. Western blotting was used to detect the expression of key molecules that recognize and regulate macrophages. The results showed that the expression of CSF-1, MCP-1, and CD68 in the transplanted tissue gradually increased with time after transplantation, upregulating by approximately 2.65-fold, 2.02-fold, and 1.54-fold, respectively, on day 7. Figure 5 FG). Similarly, qRT-PCR confirmed elevated expression of CSF-1 and MCP-1 (FG). Figure 5 This supports its recruitment role of TTM during the transplant rejection phase. These results indicate that TTM can migrate in inflammatory grafts in response to chemokine (MCP-1, CSF-1) signals, and selective visualization can be achieved using bioorthogonal calibration.

[0056] Example 6: TTM significantly prolongs graft survival in skin-transplanted mice To evaluate the therapeutic effect of TTM in a mouse skin graft model, mice were randomly divided into groups of 5 on postoperative day 3. PBS, RAW264.7 cells, and Mg2+ were injected via tail vein on postoperative days 3, 5, and 7.PD-L1 The mouse skin transplantation model was constructed using RAPA NPs or TTM, following the same procedure as in Example 5. Specific experimental groups were as follows: (1) PBS group (PBS, 200 μL / animal, once every 48 h, for a total of 3 times); (2) RAW264.7 group (5×10 6 RAW264.7 cells, 200 μL / cell, every 48 hours, for a total of 3 times); (3) Mφ PD-L1 Group (5×10) 6 Mφ PD-L1 Cells, 200 μL / cell, every 48 hours, for a total of 3 times); (4) RAPA NPs group (2 mg / kg RAPA NPs nanoparticles, 40 μg RAPA / animal, once every 48 hours, for a total of 3 times); (5) TTM group (5×10 6 One TTM cell, 200 μL / animal, once every 48 hours, for a total of 3 times.

[0057] Grafts and serum were collected on the ninth day post-surgery, and graft survival was monitored daily until day 35. Figure 6 A). Kaplan-Meier survival analysis showed that TTM treatment significantly prolonged graft survival compared to all other groups. Grafts in the PBS and RAW264.7 cell groups were rejected within 15 days, while TTM extended survival to 35 days, significantly longer than Mφ. PD-L1 (P<0.01) and RAPA NPs (P<0.05) Figure 6 B). Histological analysis showed that the grafts in the PBS group and the RAW264.7 group exhibited severe necrosis, dense inflammatory cell infiltration, and tissue destruction, consistent with the manifestations of acute rejection. Figure 6 C). Mφ PD-L1 The RAPA NPs group showed partial preservation of tissue integrity, but inflammatory changes were still present. In contrast, the grafts in the TTM group largely preserved structure and showed minimal inflammatory cell infiltration. CD3 staining revealed abundant T cell infiltration and Mφ in the grafts of the PBS and RAW264.7 groups. PD-L1 Infiltration was reduced in the TTM-treated and RAPA NPs groups, while almost no CD3 was observed in the grafts of the TTM-treated groups. + cell( Figure 6 D). Ki67 staining, as an indicator of proliferation activity, showed a similar trend; compared to all other groups, the TTM group had the lowest proliferation activity. Figure 6E). Granzyme B expression reflects the activity of cytotoxic T cells, and the TTM treatment group significantly inhibited Granzyme B expression compared to all other groups. Figure 6 F).

[0058] Example 7: TTM inhibits allogeneic reactive T cell response To evaluate TTM for CD4 + The regulatory role of T cells was first investigated by extracting and purifying CD4 from the spleen of C57BL / 6J mice using a magnetic bead separation method. + T cells were resuspended in complete culture medium, and CD4+ cells were counted. + T cells were randomly divided into 5 groups, with 4 × 10⁴ cells per group. 5 CD4 cells / mL + T cells were then used in a direct co-culture method, where the constructed co-culture system was incubated at 37°C with 5% CO2 for 72 hours. The specific co-culture groups were as follows: (1) PBS group (100μL PBS + 100μL 4×10 5 CD4 + T cells); (2) RAW264.7 group (100μL 1×10 5 RAW264.7 cell suspension + 100 μL 4 × 10 5 CD4 + T cell suspension); (3) Mφ PD-L1 Group (100μL 1×10) 5 Mφ PD-L1 Cell suspension + 100 μL 4 × 10 5 CD4 + T cell suspension); (4) RAPA NPs group (100 μL 100 nM RAPA NPs + 100 μL 4 × 10 5 CD4 + T cell suspension); (5) TTM group (100μL 1×10 5 100 μL of TTM cell suspension + 4 × 10 5 CD4 + T cell suspension).

[0059] Evaluation of TTM's effect on CD4 by flow cytometry + The regulatory effect of T cell proliferation: The results showed that after in vitro co-culture experiments, TTM had an effect on CD4+. + The inhibitory effect on T cell proliferation was significantly stronger than that on Mφ. PD-L1The group (P<0.001) and the RAPA NPs group (P<0.01) Figure 7 A~B). Cytokine analysis of the co-culture supernatant showed that TTM reduced the production of IL-2 and IFN-γ, while increasing the levels of IL-4 and IL-10 (A~B). Figure 7 C~F), consistent with the shift towards anti-inflammatory cytokine lineages. Furthermore, flow cytometry analysis of the spleen showed that the TTM group had CD8... + T cell frequency decreased to 24.3%, while Mφ PD-L1 The percentage was 35.7% in the RAPA NPs group and 28.6% in the RAPA NPs group. Figure 7 G~H). A consistent reduction was also observed in lymph nodes, CD8 in the TTM group. + The T cell frequency was the lowest in all groups. Figure 7 I~J). Further analysis of the lymphocytes infiltrating the transplanted tissue revealed that, although the proportion of lymphocytes in the skin tissue was lower than in the spleen or lymph nodes, flow cytometry showed that CD8+ cells were significantly increased after TTM treatment. + T cell infiltration remains reduced. Figure 7 K). Quantitative analysis showed that, compared with Mφ PD-L1 Compared with the RAPA NPs group, CD8 in TTM + T cell infiltration decreased by 54.22% and 53.91%, respectively. Figure 7 The results (L) were consistent with those in secondary lymphoid organs. These results indicate that TTM can significantly suppress the allogeneic reactive T cell response during rejection.

[0060] Example 9: TTM downregulation of complement activation enhances rapamycin-mediated mTOR inhibition It is known that IFN-γ promotes complement activation by upregulating the expression of C3 and C5. Therefore, transcriptome sequencing and qPCR analysis were used to detect factors related to the complement signaling pathway. Compared with the PBS group and the RAPA NPs group, the expression of C3, C5, C3ar, and C5ar in the transplanted tissues of the TTM group was significantly downregulated. Figure 8 (A~E), suggesting that TTM treatment can alleviate complement-mediated damage. Next, the effect of TTM treatment on immune cell migration was verified by evaluating the chemokine CCL5 and its receptor CCR5. qPCR analysis of transplanted tissues collected on day 9 post-surgery showed that, compared with the PBS and RAPA NPs groups, the expression of both CCL5 and CCR5 was reduced in TTM-treated mice, and compared with RAPA NPs, the expression of CCL5 and CCR5 in the TTM group was reduced by 78.6% and 59.5%, respectively. Figure 8(F~G). The above findings suggest that TTM attenuates chemokine signaling involved in T cell recruitment, which may correspondingly reduce T cell activation and proliferation in transplanted tissues.

[0061] Example 10: TTM promotes Treg differentiation and establishes a transplant tolerance microenvironment. To further examine the role of TTM in promoting transplant tolerance, flow cytometry analysis of the spleen and lymph nodes assessed the effects of TTM on regulatory T cell (Treg) differentiation and function. The results showed that, compared with RAPA NPs, TTM-treated mice had significantly higher CD25 levels. + FoxP3 + Increased frequency of regulatory T cells ( Figure 9 A~D). Single-cell suspensions prepared from allogeneic skin tissue confirmed that, compared to RAPA NPs, the TTM group showed a 1.79-fold increase in regulatory T cells (Tregs). Figure 9 E~F). These observations are consistent with the known ability of rapamycin to promote Foxp3 induction and the role of PD-L1 in stabilizing regulatory T cells (Tregs) under inflammatory conditions. Finally, regarding Foxp3... + IL-10 + Functional evaluation of Treg shows that, compared to Mφ PD-L1 In comparison, TTM enables IL-10 + The proportion of Tregs increased by 2.3 times, and by 1.5 times compared to RAPA. Figure 9 (G~H), indicating enhanced inhibitory capacity of regulatory T cells (Tregs). Overall, these results suggest that TTMs induce Foxp3 through a combined effect of PD-L1 signaling and rapamycin-mediated mTOR inhibition. + Treg differentiation enhances their immunosuppressive function, thereby establishing a tolerable transplant microenvironment.

[0062] In the description of this specification, the references to terms such as "one embodiment / mode," "some embodiments / modes," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment / mode or example is included in at least one embodiment / mode or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment / mode or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments / modes or examples. Furthermore, without contradiction, those skilled in the art can combine and integrate the different embodiments / modes or examples described in this specification, as well as the features of different embodiments / modes or examples.

[0063] It should be noted that in this invention, relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly specified.

[0064] The above description is merely a specific embodiment of the present invention, enabling those skilled in the art to understand or implement the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. An engineered drug-loaded macrophage for resisting transplant rejection, characterized in that: The engineered drug-loaded macrophages overexpress PD-L1 on their cell membrane surface and contain nanoparticles encapsulating rapamycin inside.

2. The engineered drug-loaded macrophages according to claim 1, characterized in that: The nanoparticles have a core-shell structure, with the core containing rapamycin and the shell being a biodegradable polymer material.

3. The engineered drug-loaded macrophages according to claim 1, characterized in that: The engineered drug-loaded macrophages are also modified with azide groups on their surface.

4. A method for preparing engineered drug-loaded macrophages, characterized in that, include: Inducing macrophages to overexpress the immunosuppressive protein PD-L1; The induced macrophages engulfed nanoparticles containing rapamycin, forming engineered drug-loaded macrophages.

5. The method for preparing engineered drug-loaded macrophages according to claim 4, characterized in that: Inducing macrophage overexpression of the immunosuppressive protein PD-L1 involves co-incubating macrophages with IFN-γ.

6. The method for preparing engineered drug-loaded macrophages according to claim 4, characterized in that: Enabling induced macrophages to engulf nanoparticles encapsulated with rapamycin involves co-incubating induced macrophages with nanoparticles encapsulated with rapamycin.

7. The method for preparing engineered drug-loaded macrophages according to claim 4, characterized in that: The preparation method further includes a nanoparticle preparation step, which includes: A precursor fluid is provided; the precursor fluid contains an organic solvent and rapamycin and a biodegradable polymer dissolved therein; The precursor fluid is added to an aqueous solution containing an emulsifier and emulsified to form an oil / water emulsion. The organic solvent was evaporated under stirring to obtain nanoparticles.

8. The method for preparing engineered drug-loaded macrophages according to claim 4, characterized in that: The preparation method further includes an azide group modification step, wherein the azide group modification step includes: Azide-mannose was co-incubated with induced macrophages.

9. The use of the engineered drug-loaded macrophages according to any one of claims 1 to 3 in the preparation of a medicament for treating transplant rejection.

10. The application according to claim 9, characterized in that: The transplant rejection disease mentioned refers to an acute rejection reaction caused by skin grafts.

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