Construction method and application of MDSC exosome intelligent delivery system based on double tissue anchoring platelets

Abstract

The invention relates to a construction method and application of an MDSC exosome intelligent delivery system based on double tissue anchoring platelets. The invention develops an MDSC exosome (MDEVs) intelligent delivery system based on double tissue anchoring platelets to mediate space-time tissue retention of the MDEVs so as to maintain local immune reprogramming of the MDEVs to relieve chronic inflammation of periodontal tissues. By means of the overexpression of integrin alpha5 and the natural characteristics of platelets, the engineered platelets modified by MDEVs simultaneously anchor and degrade fibronectin and collagen in inflammatory tissues, reduce the non-specific uptake of macrophages to MDEVs, and serve as an intelligent storage cavern of MDEVs to realize on-demand release of MDEVs. These functions collectively promote retention of the modified MDEVs in a mouse periodontitis focus for at least 10 days.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for constructing and applying an MDSC exosome intelligent delivery system based on dual tissue-anchored platelets. Background Art

[0002] A series of immune-inflammatory diseases, such as autoimmune diseases, colitis, periodontitis and osteoarthritis, are often attributed to the vicious cycle caused by dysregulated immune activation and subsequent persistent inflammation, leading to irreversible tissue destruction and disease progression. [1] .

[0003] Chronic periodontitis is an immune inflammatory disease initiated by plaque, characterized by inflammation of periodontal tissues and destruction of periodontal supporting tissues. Periodontal microbial flora imbalance is the initiating factor of periodontitis, while host immune response imbalance is crucial to the destruction of periodontal tissues. + T cells are thought to play a key role in mediating bone loss caused by periodontitis [2] Therefore, treatment that regulates the local excessive immune response in periodontitis is a novel therapeutic strategy with great potential.

[0004] The persistent inflammatory state of tissues in chronic inflammatory diseases often requires sustained and effective local exposure to therapeutic agents to achieve optimal effects. However, current treatments, such as anti-inflammatory drugs and nonspecific immunosuppressive drugs, are often rapidly cleared from the target site even when administered locally. [3] , which results in short-term treatment effects and a series of side effects [4] Currently, long-term, frequent, and high-dose administration is often used to produce the desired therapeutic effect, but this measure may further aggravate the above-mentioned complications. [5] .

[0005] The development of drug delivery systems helps achieve local long-term retention of various preparations at the lesion site [6] According to recent literature, constructing a cargo reservoir in the extracellular space by transforming cargo into a form that can bind to extracellular matrix proteins is a relatively novel drug delivery strategy. [7] . Therefore, ECM proteins are ideal targets for delivery vectors, thereby achieving the accumulation of therapeutic drugs carried by the vector in the extracellular space. ECM is composed of various fibrous components such as collagen and fibronectin. The extracellular matrix (ECM) not only provides a physical scaffold for tissue cells, but also sends signals to cells through ECM receptors. ECM proteins interact with cells directly through cell surface integrin receptors or through intermediate molecules, and are then recognized by cell surface receptors [8] .

[0006] Collagen is one of the most abundant components of the ECM [9] However, it is worth noting that chronic inflammatory tissues usually express a large number of matrix metalloproteinases (MMPs). This family of proteins plays a key role in mediating the extensive degradation of collagen, thereby leading to inflammatory destruction of tissues.

[10] However, according to literature reports, fibronectin in ECM seems to be less affected by inflammatory degradation conditions.

[11] Therefore, delivery platforms based on a single targeted design are insufficient to achieve ideal ECM binding in inflammatory degradative tissues.

[0007] Due to their intrinsic biological functionality and ease of biological and chemical modification, living cells are increasingly being used for therapeutic purposes or as vehicles for delivering various drugs to target lesions.

[12] As a special type of cell, platelets are used as a delivery platform due to their various pathophysiological properties.

[13] It is a small, anucleate cell isolated from mature megakaryocytes that can directly bind to exposed subvascular collagen during vascular injury, acting as the main mediator of physiological hemostasis and pathological thrombosis.

[14] In particular, genetic engineering of their precursor cells can endow platelets with additional functions.

[15] Specifically, integrin α5β1, a specific transmembrane receptor for fibronectin, can be overexpressed in megakaryocytes.

[16] , thus giving its platelets the characteristic of integrin overexpression.

[0008] In recent years, the development of biomimetic immunomodulatory agents has gradually developed into a new type of anti-inflammatory biological therapeutic drug. It is worth noting that myeloid-derived suppressor cells (MDSCs) are also an important heterogeneous cell population with immunosuppressive effects in the human body.

[17] Despite the potential uncertainty regarding the therapeutic effects of MDSCs, it remains attractive to exploit the immunosuppressive potential of MDSC derivatives such as exosomes, also known as small extracellular vesicles (EVs), to alleviate inflammation.

[0009] EVs are lipid bilayer vesicles naturally released by cells into the extracellular space. They play a fundamental role in intercellular communication and are involved in regulating a range of biological activities.

[18] Exosomes (50-150nm in size) are rich in a variety of biologically active substances such as RNA, DNA fragments, lipids and proteins, and are therefore widely involved in the exchange of substances and information between cells. Based on this, MDEVs remain an attractive option for restoring immune balance in chronic inflammation. The treatment of EVs also faces the challenge of high clearance rate in the body. Previous work has mostly adopted frequent dosing to maintain the local concentration and function of EVs.

[19] . Summary of the Invention

[0010] The purpose of the present invention is to solve the problems of excessively fast drug clearance and short-term secondary therapeutic effects of anti-inflammatory or immunotherapy drugs in the prior art and to provide a method for constructing and applying an MDSC exosome intelligent delivery system based on dual tissue-anchored platelets.

[0011] The purpose of the present invention can be achieved by the following technical solutions:

[0012] A method for constructing an intelligent delivery system of MDSC exosomes based on dual tissue-anchored platelets, the specific steps are as follows:

[0013] S1, induction of myeloid-derived suppressor cells (MDSCs) in vitro;

[0014] S2, isolating the myeloid-derived suppressor cells (MDSCs) obtained in step S1 to obtain exosomes (MDEVs), and modifying the free thiol groups on the surface of the exosomes (MDEVs);

[0015] S3. Construction of a megakaryocyte cell line stably overexpressing mouse integrin α5, and introduction of azide groups onto the cell surface via the sialic acid biosynthesis pathway;

[0016] S4, inducing the cells obtained in step S3 to mature and performing cell metabolic labeling to generate platelets modified with azide groups (A5 / PLT);

[0017] S5, reacting the platelet modified with an azide group obtained in step S4 with a matrix metalloproteinase (MMPs) cleavable polypeptide linker (L) to obtain A5 / PLT-L;

[0018] S6. The exosomes modified with free thiol groups obtained in step S2 are mixed with the A5 / PLT-L obtained in step S5 to obtain an MDSC exosome intelligent delivery system based on dual tissue anchored platelets (A5 / PLT-MDEV).

[0019] Furthermore, in step S1, bone marrow cells (BMCs) are cultured with complete DMEM containing GM-CSF, IL-6 and PGE2 to induce differentiation and obtain myeloid-derived suppressor cells (MDSCs).

[0020] Furthermore, in step S3, the megakaryocyte cell line is L8057.

[0021] Furthermore, in step S3, the method for constructing a megakaryocyte cell line stably overexpressing mouse integrin α5 is as follows:

[0022] A lentiviral system carrying puromycin-resistant mouse integrin α5 (mITGA5) was packaged in HEK293T cells. The lentivirus was collected after transfection and used to infect the megakaryocyte cell line L8057 cells under the action of polybrene. Puromycin was added to screen for the megakaryocyte cell line L8057 that stably overexpressed mouse integrin α5.

[0023] Furthermore, in step S4, cells overexpressing mouse integrin α5 are cultured in a complete culture medium containing thrombopoietin (TPO) and Ac4ManNAz to induce maturation and perform cell labeling to obtain platelets modified with azide groups.

[0024] Furthermore, in step S6, the obtained MDEVs were modified onto A5 / PLT-L through a click reaction between the free thiol groups on MDEVs and the maleimide groups on the peptide linker on A5 / PLT-L to collect A5 / PLT-MDEVs.

[0025] In addition, the present invention also provides an application of an MDSC exosome intelligent delivery system based on dual tissue-anchored platelets in the preparation of drugs for treating inflammatory diseases.

[0026] Furthermore, the MDSC exosome intelligent delivery system based on dual tissue-anchored platelets is used in the preparation of drugs for the treatment of periodontitis.

[0027] Furthermore, the MDSC exosome intelligent delivery system based on dual tissue-anchored platelets is used in the preparation of drugs that synergistically anchor collagen and fibronectin in inflammatory tissues, reduce the nonspecific uptake of MDEVs by macrophages, and prolong the local retention time of MDEVs in various inflammatory tissues, thereby effectively improving the bioavailability in inflammatory tissues.

[0028] Furthermore, the above-mentioned dual tissue-anchored platelet-based MDSC exosome intelligent delivery system is used in the preparation of drugs that improve inflammatory tissues by upregulating the proportion of regulatory T cells, inducing T cell apoptosis, and reducing the proportion of pro-inflammatory T cells.

[0029] The principles of the present invention are as follows:

[0030] Chronic periodontitis is the result of a vicious cycle of dysregulated immune activation and persistent tissue inflammation [1] Chronic inflammation often requires continuous and effective local drug exposure for optimal results. However, current treatments often suffer from high drug clearance even when administered topically. [4]However, traditionally, long-term high-dose administration is usually used to achieve ideal therapeutic effects, but this may further aggravate off-target complications. Therefore, there is an urgent need to develop long-acting local immune preparations for the safe and effective treatment of chronic periodontitis.

[0031] In recent years, drug delivery strategies based on affinity for extracellular matrix (ECM) proteins can enhance tissue retention of growth factors or anti-inflammatory agents to improve therapeutic efficacy while reducing systemic side effects, thus becoming a promising strategy to improve drug bioavailability. [7a] . However, the extensive ECM degradation in chronic inflammatory tissue sites makes single tissue anchoring insufficient to achieve effective tissue adhesion. Therefore, the design of a dual ECM protein-targeted drug delivery platform is novel and important. Platelets, as a special type of cell, have a natural collagen targeting ability. The present invention cleverly utilizes this characteristic of platelets and simultaneously gives them another fibronectin targeting ability through genetic engineering, thereby developing them into a bionic delivery platform with dual tissue anchoring effects.

[0032] MDSC-derived exosomes (MDEVs) inherit multiple immunosuppressive molecules from their parent cells, thus possessing immunomodulatory potential. However, their in vivo application faces the same challenges as other drugs: high drug clearance at lesions and low bioavailability. This invention addresses this challenge by employing an engineered platelet delivery platform.

[0033] In summary, the present invention uses MDEVs as backpacks and modifies them on dual tissue-anchored platelets with overexpression of integrin α5 through MMP-cleavable peptides, in order to prolong the local retention time of MDEVs in various inflammatory tissues (named A5 / PLT-MDEV), thereby mediating its long-term immune reprogramming effect to effectively improve tissue inflammation.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] Based on the immunological pathogenesis of periodontitis, the present invention aims to focus on the upstream dysregulated immune tolerance and attempt to develop new biological agents to reprogram the immune microenvironment to rescue the chronic inflammation and tissue damage associated with immune dysregulation.

[0036] Dual targeting is an interesting and effective approach to achieve tissue accumulation mediated by ECM anchoring. This invention develops a novel delivery platform that can leverage multiple molecular recognition mechanisms within the damaged ECM to anchor multiple matrix proteins, such as collagen and fibronectin, thereby synergistically extending the local retention of the delivered therapeutic agent.

[0037] Combining the platelet's natural ability to recognize collagen and the genetically engineered ability to target fibronectin, the present invention further modifies platelets through a biointerface strategy to develop a delivery platform with dual tissue anchoring, thereby helping therapeutic drugs to be effectively retained in inflamed tissues to maintain local anti-inflammatory effects.

[0038] The present invention solves the problem of high in vivo clearance rate through a dual tissue-anchored platelet delivery platform of MDSC-derived exosomes (MDEVs).

[0039] The present invention utilizes dual tissue-anchored platelets as a delivery platform to explore MDEVs as a new type of immunomodulatory preparation, achieving the spatiotemporal retention of MDEVs in a variety of chronic inflammatory tissues, thereby maintaining their immunomodulatory activity, thereby effectively alleviating chronic inflammation and tissue destruction, and ultimately rescuing the progression of periodontitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Verification of integrin expression in A5 / PLT. (A, B) Representative flow cytometry images (A) and MFI quantification (B) of overexpressed integrin α5 in A5 / PLT. (C) Western blotting analysis of background expression of overexpressed integrin α5 and integrin β1 in A5 / PLT.

[0041] Figure 2 The phenotype of myeloid-derived suppressor cells (MDSCs) obtained in vitro was analyzed by flow cytometry. (A) CD11b in MDSCs + Gr-1 + The proportion of cells. (B) In CD11b + Gr-1 + The percentages of M-MDSCs and PMN-MDSCs in the cells.

[0042] Figure 3 Characterization of small extracellular vesicles (MDEVs) isolated from MDSCs. (A) Representative transmission electron microscopy (TEM) images of MDEVs stained with uranyl acetate. Scale bar: 100 nm. (B-C) Size distribution of MDEVs as determined by nanoflow cytometry (B) and nanoparticle tracking analysis (NTA) (C).

[0043] Figure 4Characterization of A5 / PLT-MDEV. (A) Representative transmission electron microscopy (TEM) image of A5 / PLT-MDEV. Scale bar: 200 nm. Orange arrows indicate MDEVs. Inset: Magnified view of a single MDEV. Scale bar: 50 nm. (B) Representative cryo-scanning electron microscopy (cryo-SEM) image of the surface morphology of A5 / PLT-MDE. Scale bar: 500 nm. Orange circles indicate MDEVs. (C) Representative confocal images of A5 / PLT-MDEVs. A5 / PLT and MDEVs were labeled with FITC-conjugated WGA (green) and PKH26 (red), respectively. Scale bar: 2 μm. (D) Surface potential of A5 / PLT-MDEVs detected by dynamic light scattering. (E-F) Representative flow cytometry images (E) and mean fluorescence intensity (MFI) analysis (F) of A5 / PLT-MDEVs synthesized by covalent conjugation of A5 / PLT-L with MDEVs labeled with free thiol-modified PKH26. (G) Characteristic protein analysis of each group by Western blotting. (H) Cumulative MDEV release curves from A5 / PLT-MDEVs under different conditions.

[0044] Figure 5 Validation of reduced macrophage uptake of platelets. (A, B) Representative flow cytometry images (A) and corresponding MFI analysis (B) of various PKH26-labeled agents (with equivalent fluorescent MDEV) uptake by Raw264.7 macrophages after 2, 6, 18, and 24 hours of co-incubation. (C, D) Representative confocal images of the intracellular accumulation of various agents in Raw264.7 cells after 2 hours (C) and 6 hours (D) of co-incubation. DAPI and PKH26 represent cell nuclei and MDEV, respectively. BF, bright field. Scale bar: 20 μm.

[0045] Figure 6 Retention of A5 / PLT-MDEV in periodontitis tissues of mice. (AB) Representative confocal images (A) and quantitative analysis (B) of the binding of various agents to PKH26-labeled MDEV on fibronectin-coated culture plates. Scale bar: 6 μm. (CD) Representative confocal images (C) and quantitative analysis (D) of the binding of various fluorescent samples on cryosections of gingival tissue from mice with periodontitis. Scale bar: 40 μm. (EF) Representative confocal images (E) and quantitative analysis (F) of the binding of various fluorescent samples on cryosections of inflamed gingival tissue from patients with periodontitis. Scale bar: 40 μm. (G) Experimental design to evaluate the retention of agents within periodontal lesions in mice. (HI) Representative ex vivo imaging (H) and quantitative analysis (I) of the retention of various fluorescent agents at inflammatory sites at different time points.

[0046] Figure 7To verify the immunosuppressive effect of MDEV released in vitro. (AB) Flow cytometric profiles (A) and corresponding apoptosis rates (B) of Annexin V-positive cells after mouse splenic lymphocytes were co-incubated with each group of samples for 24 hours. (CD) Flow cytometric profiles (C) and corresponding proliferation rates (D) of mouse splenic lymphocytes after 72 hours of co-incubation with each group of samples. (EF) Typical flow cytometric profiles (E) and CD4 + Foxp3 + The proportion of T cells (F).

[0047] Figure 8 To verify the long-term immune regulatory effect of A5 / PLT-MDEV in inflamed periodontal tissues. (A) Experimental design. (BC) CD4 + Foxp3 + Representative flow cytometry images (B) and ratios (C) of Treg cells. (D-E) CD4 + IL-17a + Representative flow cytometry images (D) and ratios (E) of cells. (FG) CD4 + IFN-γ + Representative flow cytometry images of cells (F) and ratios (G). (HI) CD4 + TNF-α + Representative flow cytometry images (H) and ratios (I) of cells. (JK) CD4 + CD69 + CD103 + Representative flow cytometry images of T cells (J) and ratios (K). (LM) TUNEL-positive CD3 + Representative confocal imaging (L) and quantitative analysis (M) of lymphocytes. Scale bar: 20 μm.

[0048] Figure 9The therapeutic effect of A5 / PLT-MDEV in a periodontitis mouse model. (A) H&E-stained tissue sections of periodontal tissues in different groups. Scale bar: 80 μm. G, gingiva; B, bone area; T, tooth; I, inflammatory cell infiltration. (B) Representative TNF-α immunohistochemical staining images of tissue sections in each group. Scale bar: 40 μm. (C) Typical IL-1β immunohistochemical staining tissue sections in each group. Scale bar: 40 μm. (D) Representative 3D micro-CT images of bone resorption around the maxillary molar area observed from the buccal and palatal sides, as shown by the distance between the cementum and the alveolar bone ridge (ABC). The upper and lower red dashed lines indicate the levels of CEJ and ABC, respectively. Scale bar: 500 μm. (E) Representative TRAP staining images of each group. Scale bar: 40 μm. G, gingiva; B, bone area. The red arrows represent TRAP. + Cell. (F) Figure 7 Corresponding quantitative analysis of TNF-α positive area in B. (G) Figure 7 Quantitative analysis of IL-1β positive area in C. (H) Quantitative analysis of alveolar bone loss. (I) Figure 7 TRAP in E + Corresponding quantitative analysis of osteoclast numbers.

[0049] Figure 10 (A) The preparation process of A5 / PLT-MDEV. (B) A5 / PLT-MDEV can simultaneously target and bind to extracellular fibronectin and collagen, effectively anchoring inflammatory and degradative tissues. A5 / PLT-MDEV mediates the localized, on-demand release of MDEVs, reducing nonspecific clearance of MDEVs by macrophages, thereby synergistically enhancing the spatiotemporal retention of MDEVs in inflamed tissues, maintaining immune reprogramming and effectively controlling inflammation in periodontal and joint areas. DETAILED DESCRIPTION

[0050] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0051] In the following examples, the sources of the reagents are as follows:

[0052] All flow cytometry antibodies were purchased from Biolegend. Immunohistochemistry and Western blotting antibodies were purchased from CST, Abcam, Santa Cruz, and Proteintech. Cytokines were purchased from Nearshore Biotechnology Co., Ltd. Chemical reagents were purchased from Aladdin. Serum and flow cytometry reagents were purchased from Thermo Fisher Scientific. Reagents such as Ac4ManNAz and PKH26 were purchased from Sigma.

[0053] In the following examples, the sources of the animals are as follows:

[0054] Female C57BL / 6 mice aged 6–8 weeks were housed in a SPF environment at the Animal Center of Renji Hospital. All animal experiments were conducted in accordance with ethical guidelines and approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.

[0055] In the following examples, the statistical analysis method is as follows:

[0056] All statistical data are expressed as mean ± standard error (SEM). Two-group comparisons were performed using an unpaired two-tailed Student's t-test using GraphPad software, and multiple group comparisons were performed using one-way analysis of variance (ANOVA) and Tukey's analysis. Differences were considered statistically significant when p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0057] Unless otherwise specified, the raw materials or processing techniques are conventional commercially available raw materials or conventional processing techniques in the art.

[0058] The following describes some embodiments of the present invention in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0059] Example 1

[0060] This embodiment provides a method for constructing an intelligent delivery system of MDSC exosomes based on dual tissue-anchored platelets. The specific steps are as follows:

[0061] S1. Cell culture

[0062] L8057 cells, Raw264.7 cells and mouse spleen cells were cultured in complete DMEM medium (supplemented with 10% FBS and 1% penicillin / streptomycin) at 37°C in a 5% CO2 humidified environment.

[0063] S2. Induction of MDSCs in vitro

[0064] Cells flushed from the tibia and femur of female C57BL / 6 mice were cultured in complete DMEM supplemented with 20 ng / ml GM-CSF, 20 ng / ml IL-6, and 2 μM PGE2 for 5 days to induce differentiation into MDSCs. The MDSC phenotype of the obtained cells was assessed by flow cytometry using antibodies against CD11b-FITC, Gr-1-PE, Ly6C-PerCP / Cy5.5, and Ly6G-APC.

[0065] S3. Collection and characterization of MDSC-derived EVs

[0066] MDSC-derived exosomes (MDEVs) were isolated and purified by ultraspeed gradient centrifugation at 4°C. Briefly, after collecting in vitro induced MDSCs, the cells were cultured in serum-free medium for 48 hours, and the supernatant was collected by centrifugation at 300g for 10 minutes. The supernatant was then centrifuged at 2,500g for 20 minutes to remove cell debris, and the supernatant was further centrifuged at 12,000g for 30 minutes to remove microvesicles. Next, the collected supernatant was centrifuged at 100,000g for 70 minutes, and the collected MDEV pellet was resuspended in PBS and subjected to another round of centrifugation at 100,000g for 70 minutes. Finally, the purified MDEV pellet was resuspended in PBS and stored in aliquots at -80°C for later use.

[0067] The morphology of the obtained MDEVs was observed using a transmission electron microscope (TEM, HT7800, HITACHI) after staining with 2 wt % uranyl acetate. The size distribution and concentration of MDEVs were determined using a nanoflow cytometer (NanoFCM, China) and nanoparticle tracking analysis (NTA, NS300, Malvern).

[0068] S4. Construction of Stable Overexpression Cell Lines and Cell Labeling

[0069] A lentiviral system carrying puromycin-resistant mouse integrin α5 (mITGA5) was packaged in HEK293T cells. Lentivirus was collected from the culture medium 48 hours after transfection. Subsequently, the megakaryocyte cell line L8057 cells were infected with the lentivirus in the presence of 6 μg / ml polybromide for 12 hours, and fresh culture medium was replaced. After 48 hours, 2.5 μg / ml puromycin was added to the culture medium of the infected cells to select cells that stably overexpressed mouse integrin α5 (ITGA5-L8057). ITGA5-L8057 cells were cultured in complete medium containing 50 μM Ac4ManNAz for 72 hours to introduce azide groups to the cell surface through the sialic acid biosynthesis pathway.

[0070] S5. Collection and Characterization of Engineered Platelets

[0071] ITGA5-L8057 cells or L8057 cells were cultured in complete medium containing 20 ng / ml thrombopoietin (TPO) and 50 μM Ac4ManNAz for 72 hours to induce maturation and label the cells, generating azide-modified platelets. The collected culture medium was then centrifuged at 1,400 rpm for 5 minutes to collect the supernatant, followed by centrifugation at 12,000 rpm for 15 minutes to collect the precipitated ITGA5 platelets (A5 / PLT) or platelets (PLT). The resulting platelets were gently resuspended in PBS containing 1 μM PGE1 or Tyrode's buffer. Surface markers of CD41 and P-Selectin were detected on A5 / PLTs by flow cytometry. Overexpression of integrin α5 on A5 / PLTs was confirmed by immunofluorescence, flow cytometry, and Western blotting. Expression of integrin β1 was detected by Western blotting.

[0072] Preparation of S6 and A5 / PLT-MDEV

[0073] First, A5 / PLT-L or PLT-L was synthesized by reacting an azide-containing A5 / PLT sample (or an equivalent amount of PLT sample) with an MMP-cleavable peptide linker (L) containing both an alkyne and a maleimide group. The azide group on A5 / PLT (or PLT) reacted with the alkyne group on the peptide linker via a click reaction. Specifically, the A5 / PLT or PLT precipitate collected by centrifugation was resuspended in 1 ml of a mixed solution containing 25 μM peptide linker (L), 50 μM copper(II)-TBTA complex, and 10 mM sodium ascorbate and stirred at room temperature overnight. Subsequently, the mixture was stirred with 50 μM mPEG(2K)-azide at room temperature for 15 minutes to quench the reaction between the peptide linker (L) and A5 / PLT (or PLT). The mixture was then centrifuged at 12,000 rpm for 15 minutes to collect the precipitated A5 / PLT-L or PLT-L. The resulting sample was washed three times with PBS.

[0074] Next, to modify free thiol groups on the surface of MDEVs, MDEVs were incubated with stirring in a PBS solution containing 2 mg / ml Traut's reagent. After incubation for 1 hour, the mixture was ultrafiltered (molecular weight cutoff = 10 kD) to remove excess reagent.

[0075] Finally, the resulting MDEVs were modified onto A5 / PLT-L or PLT-L via a click reaction between the free thiol groups on the MDEVs and the maleimide groups on the peptide linkers of equal amounts of A5 / PLT-L or PLT-L to collect A5 / PLT-MDEVs or PLT-MDEVs. Specifically, the obtained A5 / PLT-L or PLT-L sample and the thiolated MDEV sample were mixed in PBS and incubated with stirring at room temperature for 1 hour. The mixed solution was then centrifuged at 12,000 rpm for 15 minutes to remove free MDEVs from the supernatant. The collected A5 / PLT-MDEV or PLT-MDEV samples were washed three times with PBS for further use.

[0076] Example 2

[0077] This embodiment provides a method for characterizing A5 / PLT-MDEV, and the specific steps are as follows:

[0078] S1. To observe the morphology of the prepared A5 / PLT-MDEV, the sample was drop-cast onto a carbon-coated grid and immediately stained with 2 wt% uranyl acetate for 1 minute. The grid was imaged using an 80 kV transmission electron microscope (TEM, HT7800, HITACHI). To observe the surface morphology of the A5 / PLT-MDEV, the A5 / PLT-L and A5 / PLT-MDEV samples were cryo-scanning electron microscopy (cryo-SEM, Regulus822, HITACHI) in liquid nitrogen. To further observe the surface decoration of MDEVs on A5 / PLT, MDEVs and A5 / PLT-L were labeled with PKH26 and FITC-WGA dyes, respectively. Fluorescently labeled MDEVs and A5 / PLT-L were then used to synthesize A5 / PLT-MDEVs and imaged using a confocal microscope (Leica TSC SP8). The surface zeta potential of A5 / PLT-MDEVs, MDEVs, and A5 / PLTs was measured by dynamic light scattering (DLS, Zetasizer, Malvern). To further confirm the covalent binding of MDEVs to the A5 / PLT-L complex, samples of unmodified PKH26-labeled MDEVs or MDEVs modified with free thiol groups were prepared and used in the preparation of A5 / PLT-MDEVs. The resulting samples were then analyzed by flow cytometry to analyze the fluorescence intensity of the two A5 / PLT-MDEV samples compared to the A5 / PLT-L complex. To identify characteristic proteins on A5 / PLT-MDEVs, Western blotting was used to measure the protein levels of CD9, CD81, ALIX, TSG101, Calnexin, TGF-β, IL-10, Arg-1, integrin α5, and integrin β1 in A5 / PLTs, MDEVs, and A5 / PLT-MDEVs.

[0079] S2. Western blotting:

[0080] Western blotting was performed as follows: protein samples were collected and heated at 95°C for 10 minutes, and 20 μl of the sample was used for SDS-PAGE electrophoresis on a gradient precast polyacrylamide gel. The proteins on the gel were transferred to a PVDF membrane, which was then blocked in 5 wt% skim milk for 2 hours and incubated with various primary antibodies at the recommended dilutions overnight at 4°C. The membrane was washed three times with TBST for 5 minutes each and incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. After three TBST washes, the membrane was incubated with ECL substrate and imaged using an Odyssey infrared imaging system (LI-COR). β-actin was used as a control.

[0081] The experimental results of characterization of the dual tissue-anchored platelet delivery system are shown below:

[0082] By taking advantage of the inherent affinity of platelets for binding to collagen, this example also modified integrin α5β1 on megakaryocytes (MKs) by gene overexpression. Megakaryocytes are the precursors of platelets. The mouse megakaryocyte cell line L8057 was used to overexpress the gene of integrin α5 (named ITGA5-L8057). After the engineered megakaryocytes were subsequently treated with thrombopoietin to induce their maturation, the megakaryocytes were able to produce platelets. The results of flow cytometry, Western blotting, and immunofluorescence assays ( Figure 1 ) consistently verified that A5 / PLT also inherited the overexpression of integrin α5 and the expression of β1 subunit from ITGA5-L8057 cells.

[0083] MDEVs are derived from MDSCs, so this example first obtained MDSCs by inducing mouse bone marrow cell differentiation in vitro. Flow cytometry analysis confirmed their CD11b + Gr-1 + Phenotype ( Figure 2 A). Among them, M-MDSCs (expressing CD11b + Ly6G – Ly6C hi ) is the main subpopulation of MDSCs obtained in the present invention ( Figure 2 B). Transmission electron microscopy (TEM) imaging showed that the extracted MDEVs were hemispherical or disc-shaped ( Figure 3 A), which is the typical morphology of small EVs. Nanoflow cytometry ( Figure 3 B) and NTA( Figure 3 C) It was consistently confirmed that the particle size range of the obtained MDEVs was mainly 50-150 nm.

[0084] After treating azide-modified ITGA5-L8057 cells with thrombopoietin, azide-modified integrin α5 engineered platelets (A5 / PLT) were obtained. After co-incubation of A5 / PLT and MMP-cleavable peptide chains, a click reaction between the azide group on the surface of A5 / PLT and the alkyne group on one side of the peptide chain resulted in the A5 / PLT-L conjugate. Subsequently, the obtained A5 / PLT-L was reacted with PKH26-labeled MDEVs modified with free thiol groups to obtain A5 / PLT-MDEV. Its transmission electron microscopy (TEM) suggests that ( Figure 4 A), irregularly shaped micron-sized platelets were modified with some nanoparticles with a size of 50-150 nm. This result shows that MDEVs were successfully modified onto A5 / PLT. Cryo-scanning electron microscopy (cryo-SEM) image ( Figure 4B) Tips. Compared with the relatively smooth surface of the A5 / PLT-L group, the surface of A5 / PLT in the A5 / PLT-MDEV group was modified with nano-sized vesicles. In addition, confocal imaging of A5 / PLT-MDEV ( Figure 4 C) The extensive overlap of PKH26-labeled MDEVs and WGA-FITC-labeled A5 / PLT further confirmed that MDEVs were backpacking on the surface of A5 / PLT.

[0085] Surface zeta potential ( Figure 4 D) The presence of MDEVs on the surface of A5 / PLT was consistently verified. Fluorescently labeled MDEVs were used to prepare A5 / PLT-MDEVs, and flow cytometry images and mean fluorescence intensity (MFI) quantification were performed ( Figure 4 Figure 4E-4F shows that after the A5 / PLT-L conjugate was mixed with the modified MDEVs, the fluorescence intensity was significantly higher than that of the other groups, indicating that A5 / PLT-MDEV was effectively generated through the click reaction between the thiol-modified MDEVs and the maleimide groups on the A5 / PLT-L conjugate.

[0086] like Figure 4 Western blotting results in G show that the A5 / PLT-MDEV group was similar to the MDEV group, confirming the presence of MDEVs in the A5 / PLT-MDEV sample. In addition, A5 / PLT-MDEV also showed expression of integrin α5 and integrin β1, similar to the A5 / PLT group.

[0087] To verify the release of MMPs in response to MDEVs, A5 / PLT-MDEV samples were incubated under different MMP9-related conditions as follows. Figure 4 As shown in Figure ​H, upon MMP9 treatment, more than 60% of MDEVs were rapidly released from A5 / PLT-MDEV within 12 h, indicating that A5 / PLT-MDEV has the ability to release MDEVs on demand.

[0088] Example 3

[0089] This example provides an experimental method for macrophage uptake of A5 / PLT-MDEV. The specific steps are as follows: the Raw264.7 cell line was used for the experiment. After the cells were attached to the wall of a 96-well plate (2×10^ 4 cells), PKH26-labeled MDEV (5×10^ 8 / ml) and PLT-MDEV and A5 / PLT-MDEV samples with the same fluorescence intensity. After co-incubation in the incubator for 2, 6, 18 and 24 hours, the supernatant was removed and washed three times with PBS to completely remove the free sample. The cells were then washed, trypsinized, stained with F4 / 80-FITC antibody, and analyzed by flow cytometry to determine the intracellular fluorescence signals in different groups at predetermined time points. In order to further observe the macrophage uptake activity of A5 / PLT-MDEV, Raw264.7 cells were also seeded on 8-well chambers and co-incubated with similar fluorescent formulas as mentioned above. After 2 and 6 hours of co-culture, the cell samples were thoroughly rinsed, fixed with 4% PFA, washed and sealed with a mounting medium containing DAPI, and then imaged by confocal microscopy.

[0090] The experimental results of anti-phagocytic effect are shown below:

[0091] The present invention studies the effect of platelet-based backpack strategy on macrophage phagocytosis of MDEVs in vitro. As shown in the flow cytometry figure ( Figure 5 A) and quantitative MFI analysis ( Figure 5 B) shows that at each time point, the intracellular fluorescence intensity of the A5 / PLT-MDEV group and the PLT-MDEV group was significantly lower than that of the MDEV group, but there was no significant difference between the A5 / PLT-MDEV group and the PLT-MDEV group. In particular, the MDEV group showed the highest macrophage uptake rate within 6 hours, as shown by the steepest slope of the MFI increment from the 2nd to the 6th hour, while the MFI increment slope of the platelet-based group was relatively gentle ( Figure 5 B).

[0092] Confocal microscopy of macrophage uptake after 2 h co-incubation ( Figure 5 C) further revealed that free MDEVs were significantly internalized by cells compared with the platelet-based formulation, and the confocal microscopy results of cellular uptake at 6 h ( Figure 5 D) Consistently, the intracellular fluorescence signal of the MDEV group was significantly higher than that of the platelet-based groups. Therefore, these results consistently indicate that the strategy of modifying nanoscale MDEV onto micron-sized platelets effectively slows the rate of macrophage uptake of bound MDEV.

[0093] In addition, this example also provides an in vitro fibronectin binding experimental method for A5 / PLT-MDEV, the specific steps of which are as follows:

[0094] To evaluate the fibronectin binding ability of A5 / PLT-MDEV in vitro, the culture plates were first treated with 2 μg / cm 2 The fibronectin solution containing 1×107 Samples of PKH26-labeled MDEVs, PLT-MDEVs, and A5 / PLT-MDEVs were added to fibronectin-coated plates and incubated at 37°C for 2 hours. The plates were then washed three times with PBS under shaking, and residual fluorescent MDEVs were observed by confocal microscopy. Fluorescence intensity was quantified using Image J.

[0095] The experimental results of the fibronectin binding ability of A5 / PLT-MDEV are shown below:

[0096] like Figure 6 As shown in A-6B, confocal imaging and corresponding quantitative analysis showed that the A5 / PLT-MDEV group retained significantly more fluorescently labeled MDEVs on the plate compared to the other groups. The PLT-EV group had more residual fluorescence than the MDEV group, but there was no significant difference between the two groups.

[0097] Example 4

[0098] This example provides a method for establishing a periodontitis mouse model, and the specific steps are as follows:

[0099] A mouse model of periodontitis was established using the suture method. Briefly, 8-0 silk suture was placed around the maxillary first molar (M1) of 6-8-week-old female C57BL / 6 mice for 10 days. After the suture was removed, the mice with periodontitis were used for subsequent experiments. To confirm the expression of fibronectin in inflamed periodontal tissue, maxillary samples from each group were decalcified in 10% EDTA for 15 days, embedded, sectioned, and immunohistochemically stained for fibronectin.

[0100] Example 5

[0101] This example provides a method for evaluating the binding ability of A5 / PLT-MDEV to frozen sections of mouse and human periodontitis. The specific steps are as follows:

[0102] Mouse gingival cryosections were obtained from localized gingivae of mice with periodontitis established by the ligature method. Human gingival cryosections were obtained from consenting patients undergoing periodontitis surgery. The cryosectioning process was performed according to guidelines approved by the Ethics Review Committee of Renji Hospital, Shanghai Jiao Tong University School of Medicine. To evaluate the tissue-anchoring ability of A5 / PLT-MDEV in vitro, mouse and human periodontitis cryosections were blocked with 3% BSA at room temperature for 1 hour and then dripped with 1×10 7 PKH26-labeled MDEVs, PLT-MDEVs, and A5 / PLT-MDEVs were incubated at room temperature for 2 hours, washed, dried, and mounted. Finally, images were obtained using confocal microscopy and quantified using Image J.

[0103] Example 6

[0104] This example provides a method for determining the in vivo tissue retention of A5 / PLT-MDEV in a periodontitis mouse model. The specific steps are as follows:

[0105] After establishing a mouse periodontitis model, the silk thread was removed and used to investigate the local tissue retention of A5 / PLT-MDEVs. First, a single dose (20 μl) of different preparations containing equal amounts of PKH26-labeled MDEVs, including free MDEVs (1 × 10 8 ), PLT-MDEV, and A5 / PLT-MDEV were injected into the gingival tissue surrounding M1 and M2, respectively (n = 5 per group). The retention of fluorescent MDEVs in each group was assessed over 12 days. Mice in each group were sacrificed at predetermined time points, and the maxillae of the treated side were harvested for ex vivo imaging (PerkinElmer) to determine the retention of fluorescent signal within periodontitis lesions.

[0106] The experimental results of dual-anchored platelet-mediated tissue retention are shown below:

[0107] Each group of samples prepared with PKH26-labeled MDEVs were incubated with frozen sections, and after thorough washing to remove unbound samples, the residual fluorescent samples on frozen sections of mouse and human gingiva were detected. Figure 6 As shown in C-6D, the A5 / PLT-MDEV group exhibited significantly higher fluorescence signals on mouse cryosections compared to the other groups (p<0.0001). Frozen sections of mice treated with the free MDEV group showed limited fluorescence signals, while the fluorescence of the PLT-MDEV group was also significantly higher than that of the MDEV group (p<0.05), which was attributed to the inherent collagen binding ability of platelets.

[0108] Similar results were obtained on frozen sections of human gingiva, with the A5 / PLT-MDEV group showing significantly more bound fluorescent MDEVs than the other groups ( Figure 6 E-6F). Therefore, both the PLT-MDEV group and the A5 / PLT-MDEV group exhibited superior adhesion to inflammatory tissues, with the latter significantly superior to the former, confirming the tissue anchoring ability mediated by the dual targeting function conferred by the engineered platelet delivery system.

[0109] Depend on Figure 6 As shown in G, the maxilla treated with different samples was collected at different time points within 12 days and imaged in vitro. Figure 6 H) and the corresponding quantitative analysis ( Figure 6I) Consistently, the majority of free MDEVs in the MDEV group dissipated after 1 day, with their local fluorescence intensity significantly lower than that of the PLT-MDEV group (p < 0.01), while PLT-MDEV showed no significant inferiority compared to A5 / PLT-MDEV. Fluorescent MDEVs in the PLT-MDEV group persisted in inflamed gingiva until at least day 3, while A5 / PLT-MDEV displayed a significantly higher fluorescence signal (p < 0.01). Notably, the retention of A5 / PLT-MDEV in the gingiva was prolonged to at least 10 days.

[0110] Example 7

[0111] This example provides an in vitro immunosuppression experimental method, the specific steps are as follows:

[0112] A5 / PLT-MDEV samples were incubated with 4 μg / ml MMP9 in PBS to collect the supernatant containing released MDEVs. MDEVs in the supernatant were then isolated using the ExoQuick-TC isolation kit and resuspended in PBS for later use. Spleen cells were isolated from healthy C57BL / 6 mice and seeded in flat-bottom 96-well plates (1×10 cells per well). 6 The plates were coated with CD3 (1.5 μg / ml), soluble anti-CD28 (1 μg / ml) and IL-2 (200 U / ml) to activate spleen cells and perform subsequent experimental detection.

[0113] To examine the effects of released MDEVs on lymphocyte apoptosis, spleen cells were incubated with MDSCs (the number was 1 / 10 of the spleen cells) and 2×10 8 The cells were incubated with 100 μg / ml of released MDEVs for 24 hours. Cell samples were then collected, stained with mouse CD3-APC antibody, washed, and stained with Annexin V-FITC and propidium iodide solution for 15 minutes at room temperature before immediate flow cytometry analysis.

[0114] To determine the effect of released MDEVs on lymphocyte proliferation, spleen cells were incubated with MDSCs and released MDEVs for 72 h and treated with 50 μM EdU working solution for the last 20 h. Subsequently, cell samples were collected and stained with mouse CD3-APC antibody. EdU staining was performed and the expression of Alexa Fluor 488-EdU-positive CD3 + The percentage of cells.

[0115] To investigate the induction effect of released MDEVs on Tregs, spleen cells were incubated with MDSCs and MDEVs for 72 h and then collected for flow cytometry staining. Mouse antibodies CD3-PE, CD4-PerCP / Cy5.5, and Foxp3-APC were used to analyze the expression of CD4 in spleen. + CD4 T cells + Foxp3 + The proportion of Treg cells.

[0116] The experimental results of in vitro immunomodulatory effects are shown below:

[0117] Flow cytometry images ( Figure 7 A) and quantitative analysis of apoptosis rate ( Figure 7 B) showed that the proportion of Annexin V-positive lymphocytes induced by released MDEVs was significantly higher than that in the control group, indicating that A5 / PLT-MDEV effectively promoted the apoptosis of lymphocytes.

[0118] like Figure 7 As shown in C-7D, the flow cytometry profiles and proliferation rates indicated that the released MDEVs and MDSCs resulted in a significant decrease in the FITC-EdU positive percentage compared with the control group, confirming the anti-proliferative effect of MDEVs released by A5 / PLT-MDEV.

[0119] In addition, flow cytometry images ( Figure 7 E) and quantitative analysis ( Figure 7 F) showed that the MDEV group significantly increased CD4 + Foxp3 + The proportion of cells was significantly decreased (p<0.01), and there was no significant difference between the MDEV group and the MDSC group.

[0120] Example 8

[0121] This example provides an A5 / PLT-MDEV treatment method in a mouse periodontitis model. The specific steps are as follows:

[0122] To investigate the in vivo immunosuppressive capacity and therapeutic efficacy of A5 / PLT-MDEV, a mouse periodontitis model was established as described in Example 4, and various formulations were injected into the inflamed gingival tissue surrounding M1 and M2 according to the following treatment regimen (n = 5 per group). To determine the role of prolonged tissue retention on the immunotherapeutic efficacy of A5 / PLT-MDEV, mice received a single dose (20 μl) of PBS, free MDEVs (1 × 10^ 8), PLT-MDEV, and A5 / PLT-MDEV (containing equal amounts of MDEVs) were designated as the control, MDEV-1, PLT-MDEV, and A5 / PLT-MDEV groups, respectively. In addition, free MDEVs were injected three times on days 0, 3, and 6, designated the MDEV-3 group, to explore the dose-dependent effects of free MDEVs.

[0123] The experimental results of the long-term local immune regulation effect in mice with periodontitis are as follows:

[0124] At the end of the treatment study, gingival tissues and draining cervical lymph nodes (CLNs) from periodontitis lesions were collected for flow cytometric analysis to evaluate the immunomodulatory effects of A5 / PLT-MDEV ( Figure 8 A).

[0125] like Figure 8 As shown in B-8C, the gingival CD4 + Foxp3 + The proportion of Treg cells was significantly higher than that in the control group, while the proportion in the MDEV-1 group was almost unchanged. This result indicates that a single dose of PLT-MDEV preparation facilitates the local accumulation of MDEVs and effectively upregulates Treg cells compared to a single dose of free MDEVs.

[0126] In addition, if Figure 8 As shown in D-8E, gingival CD4 + IL-17a + The cell ratio was significantly lower than that in the MDEV-1 group (p < 0.001), while the effect in the A5 / PLT-MDEV group was comparable to that in the MDEV-3 group. Compared with free MDEVs, a single dose of A5 / PLT-MDEV produced a long-lasting Th17 inhibitory effect similar to that of three repeated doses in the MDEV-3 group.

[0127] like Figure 8 As shown in F-8G, through the expression of gingival CD4 + IFN-γ + Subpopulation analysis showed that A5 / PLT-MDEV treatment also led to the + IFN-γ + The proportion of CD4 T cells in the gingiva was significantly decreased compared with the control group (p<0.0001) and the MDEV-1 group (p<0.01). + TNF-α + Analysis of T cells showed that all groups had a significant reduction in the proportion of this subset, with A5 / PLT-MDEV being the most significant ( Figure 8 H-8I). Figure 8 As shown in J-8K, the A5 / PLT-MDEV group had CD4 + CD69 + CD103 - Tissue-resident memory T(T RM ) cells were significantly lower than those in the MDEV-1 group (p<0.001).

[0128] Example 9

[0129] This embodiment provides an in vivo flow cytometry analysis method, the specific steps are as follows:

[0130] In an in vivo study of periodontitis mice, mice were sacrificed, and gingival tissue surrounding M1 and M2 gingiva was isolated from the maxilla. The gingival tissue was cut into small pieces and enzymatically digested with a mixture of collagenase IV, DNase I, and Dispase II at 37°C for 3 hours, after which the suspension was filtered. Simultaneously, cervical draining lymph nodes (CLNs) from each group of mice were homogenized and filtered to collect single-cell suspensions. For intracellular cytokine analysis, gingival and CLN cell suspensions were incubated with a leukocyte activation cocktail for 5 hours. Next, cells were stained for viability using the fixable viability stain BV421 for 15 minutes at room temperature, followed by FcγR blocking using anti-CD16 / CD32 antibodies for 15 minutes at 4°C. Surface marker staining was performed using antibodies against CD45-FITC, CD3-BV650, CD4-PerCP / Cy5.5, CD103-APC-Cy7, and CD69-APC. For detection of intracellular markers, cells were fixed, permeabilized, and stained with antibodies against TNF-α-PE, IL-17a-APC, IFN-γ-PE-Cy7, and Foxp3-PE. Finally, samples were analyzed by flow cytometry.

[0131] In addition, CD3 and TUNEL immunofluorescence staining were performed on tissue sections of periodontitis mice to determine the CD3 + Apoptosis of lymphocytes. Figure 8 As shown in L-8M, compared with the MDEV-1 group, A5 / PLT-MDEV had a + The proportion of TUNEL-positive cells in the population was significantly increased (p<0.0001), and the MDEV-1 group also had a significant difference compared with the control group (p<0.05). This result indicates that the released MDEVs promoted the CD3 + The above results indicate that A5 / PLT-MDEV significantly enhances the bioavailability of MDEVs, enabling a single dose to maintain local immune reprogramming effects in inflamed periodontal tissues.

[0132] Example 10

[0133] This embodiment provides a micro-CT analysis method, the specific steps are as follows:

[0134] To assess the extent of alveolar bone loss in the mouse periodontitis model following various treatments, maxillary bones were collected and fixed with 4% PFA for 24 hours before micro-CT scanning (Bruker). Three-dimensional buccal and lingual views were generated using corresponding software. Quantitative analysis of alveolar bone loss was performed based on 3D imaging.

[0135] Example 11

[0136] This embodiment provides a histological analysis method for mouse periodontal tissue, and the specific steps are as follows:

[0137] After micro-CT analysis, the maxillary bone samples were decalcified in 10% EDTA for 15 days and then embedded in paraffin. Serial sections were generated along the mesial-distal direction. The sections were then stained with H&E to observe the tissue morphology of each sample. Trap staining was performed on each group of sections and quantified to analyze Trap. + Multinuclear cells were used to identify osteoclasts. In addition, immunohistochemical staining for TNF-α, IL-1β, and IL-6 was performed to assess tissue inflammation and quantitative analysis was performed using Image J. In addition, immunofluorescence dual-color staining for CD3 and TUNEL was performed to identify CD3 infiltrating periodontitis lesions. + The apoptosis of lymphocytes was then determined by Image J.

[0138] The therapeutic efficacy of periodontitis in mice was evaluated as follows:

[0139] This example further studies the effect of the long-lasting immunomodulatory function of A5 / PLT-MDEV on alleviating local inflammation of periodontitis in mice. Figure 9 As shown in the H&E-stained tissue sections in A, the control group displayed severe inflammatory cell infiltration, disorganized connective tissue fibers, and tissue destruction, while A5 / PLT-MDEV significantly ameliorated these pathological changes.

[0140] To further evaluate tissue inflammatory activity, immunohistochemical staining of TNF-α and IL-1β was performed on tissue sections. Compared with the MDEV-1 group, the TNF-α in the gingival tissue of the A5 / PLT-MDEV group was significantly higher than that of the MDEV-1 group. + The area was significantly reduced (p<0.01), and the MDEV-1 group also showed a significant difference compared with the control group ( Figure 9 B, 9F). Multiple doses of MDEV-3 further significantly reduced the TNF-α-positive area.

[0141] Similarly, the immunohistochemical results of IL-1β ( Figure 9 C, 9G) showed that A5 / PLT-MDEV also significantly reduced IL-1β production in inflamed gingiva relative to the control group (p < 0.0001) and was significantly better than the MDEV-1 group (p < 0.001).

[0142] Typical three-dimensional micro-CT images of the maxilla in each group showed that in the control group, there was significant alveolar bone resorption around the maxillary first molar (M1) and second molar (M2). Figure 9 D) and corresponding quantitative analysis ( Figure 9 H) showed that A5 / PLT-MDEV and MDEV-3 groups significantly reduced alveolar bone loss compared with the control group (p < 0.0001), while MDEV-1 group had no significant inhibitory effect on bone loss compared with the control group.

[0143] Typical Trap staining results ( Figure 9 E, 9I) showed that A5 / PLT-MDEV significantly reduced Trap + The number of osteoclasts (p < 0.0001 vs. control group; p < 0.01 vs. MDEV-1 group) confirmed the ability of A5 / PLT-MDEV to significantly attenuate bone resorption activity.

[0144] In summary, this example reports a dual-targeted platelet-anchored inflammatory tissue delivery system by modifying MDSCs exosomes (MDEVs) to achieve long-term retention of MDEVs in local inflammatory tissues, thereby persistently restoring immune homeostasis and achieving a potent inflammation relief effect. A5 / PLT-MDEV is prepared by coupling MDEVs to platelets overexpressing α5 integrin via MMP-cleavable peptides, and has been shown to carry typical immunosuppressive proteins. Due to the overexpression of integrin α5 and the inherent collagen targeting properties of platelets, A5 / PLT-MDEV can simultaneously target and bind to fibronectin and collagen in inflammatory degradative tissues, while helping to reduce the nonspecific uptake of MDEVs by macrophages. In addition, A5 / PLT-MDEV can also serve as an intelligent micro-reservoir for MDEVs, which can be released on demand through the action of MMPs rich in inflammatory tissues, thereby exerting multiple inhibitory effects on lymphocyte activity. A single injection of A5 / PLT-MDEV significantly prolonged the local retention of MDEVs in inflamed gums and knee joints for at least 10 days. Therefore, the optimized bioavailability of A5 / PLT-MDEV enables a single injection dose to effectively reprogram an overactive immune response in the microenvironment of periodontitis and arthritis in mice, including upregulating Treg cell infiltration, inducing T cell apoptosis, and inhibiting the secretion of pro-inflammatory cytokines by CD4 T cells.+ T cells, especially weakened CD4 + CD69 + CD103 - Tissue-resident memory T cells. Due to the effective restoration of immune balance, A5 / PLT-MDEV significantly reduced tissue inflammation and subsequent tissue destruction in mouse periodontitis and collagen-induced arthritis models, thereby rescuing disease progression. The synthesis method of this platform has been proven to be simple and easy to implement, and it can realize the preparation of delivery platforms of cell line-derived platelets and primary stem cell-derived platelets, both of which have inflammatory tissue anchoring efficacy. In view of the progress in the clinical translation of autologous induced pluripotent stem cell-derived platelets

[20] , the method of using stem cell-derived platelets in the present invention suggests its great potential as a personalized precision treatment platform.

[0145] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

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Claims

1. A method for constructing an intelligent delivery system of MDSC exosomes based on dual tissue-anchored platelets, characterized in that: The specific steps are as follows: S1, induction of myeloid-derived suppressor cells in vitro; S2. isolating the myeloid-derived suppressor cells obtained in step S1 to obtain exosomes, and modifying free thiol groups on the surface of the exosomes; S3. Construction of a megakaryocyte cell line stably overexpressing mouse integrin α5, and introduction of azide groups onto the cell surface via the sialic acid biosynthesis pathway; S4, inducing the cells obtained in step S3 to mature and performing cell metabolic labeling to generate platelets modified with azide groups; S5, reacting the platelet modified with an azide group obtained in step S4 with a matrix metalloproteinase-cleavable polypeptide linker to obtain A5 / PLT-L; S6. The exosomes modified with free thiol groups obtained in step S2 are mixed with A5 / PLT-L obtained in step S5 to obtain an MDSC exosome intelligent delivery system based on dual tissue-anchored platelets.

2. The method for constructing a MDSC exosome intelligent delivery system based on dual tissue-anchored platelets according to claim 1, characterized in that: In step S1, bone marrow cells are cultured in complete DMEM containing GM-CSF, IL-6, and PGE2 to induce differentiation and obtain myeloid-derived suppressor cells.

3. The method for constructing a MDSC exosome intelligent delivery system based on dual tissue-anchored platelets according to claim 1, characterized in that: In step S3, the megakaryocyte cell line is L8057.

4. The method for constructing a MDSC exosome intelligent delivery system based on dual tissue-anchored platelets according to claim 3, characterized in that: In step S3, the method for constructing a megakaryocyte cell line stably overexpressing mouse integrin α5 is as follows: The lentiviral system carrying puromycin-resistant mouse integrin α5 was packaged in HEK293T cells. The lentivirus was collected after transfection and used to infect the megakaryocyte cell line L8057 cells under the action of polybrene. Puromycin was added to screen the megakaryocyte cell line L8057 that stably overexpressed mouse integrin α5.

5. The method for constructing a MDSC exosome intelligent delivery system based on dual tissue-anchored platelets according to claim 1, characterized in that: In step S4, cells overexpressing mouse integrin α5 are cultured in a complete medium containing thrombopoietin and Ac4ManNAz to induce maturation and perform cell labeling to obtain platelets modified with azide groups.

6. The method for constructing a MDSC exosome intelligent delivery system based on dual tissue-anchored platelets according to claim 1, characterized in that: In step S6, the obtained MDEVs were modified onto A5 / PLT-L via a click reaction between the free thiol groups on MDEVs and the maleimide groups on the peptide linker on A5 / PLT-L to collect A5 / PLT-MDEVs.

7. Use of an intelligent delivery system of MDSC exosomes based on dual tissue-anchored platelets obtained by the construction method according to any one of claims 1 to 5 in the preparation of drugs for treating inflammatory diseases.

8. The use according to claim 7, characterized in that The application of the MDSC exosome intelligent delivery system based on dual tissue-anchored platelets in the preparation of drugs for treating periodontitis.

9. The use according to claim 7, characterized in that The dual tissue-anchored platelet-based MDSC exosome intelligent delivery system is used in the preparation of drugs that synergistically anchor collagen and fibronectin in inflammatory tissues, reduce the nonspecific uptake of MDEVs by macrophages, and prolong the local retention time of MDEVs in various inflammatory tissues, thereby effectively improving the bioavailability in inflammatory tissues.

10. The use according to claim 9, characterized in that The MDSC exosome intelligent delivery system based on dual tissue-anchored platelets is used in the preparation of drugs that improve inflammatory tissues by increasing the proportion of regulatory T cells, inducing T cell apoptosis, and reducing the proportion of pro-inflammatory T cells.

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