Targeting M2 macrophage delivery system of chidamide as well as preparation method and application of targeting M2 macrophage delivery system

Abstract

The invention provides a responsive hydrogel of chidamide extracellular vesicles and a preparation method thereof, engineered EVs loaded chidamide, and a TSPBA / TVA hydrogel with a pH responsive characteristic is prepared, the preparation can realize sustained and controlled release of EVs under an acidic condition, effectively overcome the defect of direct injection, establish a linkage regulation mechanism of a microenvironment pH value and disease progression, and improve the treatment effect of the chidamide extracellular vesicles. According to the mechanism, the administration dosage is reduced, the action time is prolonged, tumor microenvironment targeted drug delivery is realized, a new thought is provided for tumor treatment, and the mechanism has important clinical transformation value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a chidamide targeted M2 macrophage delivery system, a preparation method and an application thereof. Background Art

[0002] Non-Hodgkin's lymphoma (NHL) is a group of malignant tumors that originate in lymphoid tissue. Based on the cell of origin, it can be divided into B-cell (accounting for 85-90%), T-cell, and natural killer (NK) cell types. B-cell lymphoma can occur at any stage of normal B-cell development, with an annual incidence of approximately 20 per 100,000. Common subtypes include Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), and follicular lymphoma, with DLBCL being the most common, accounting for approximately 30-40% of all cases. Currently, the R-CHOP regimen, as a first-line treatment for DLBCL, can achieve a 5-year survival rate of 50%-60%. However, 30%-40% of patients still experience relapse and have a poor prognosis.

[0003] The key role of M2 macrophages in the tumor microenvironment (TME) in the progression of B-cell lymphoma. The proportion of M2 macrophages in the B lymphoma microenvironment is significantly increased, and their accumulation is closely associated with poor prognosis in patients. M2 macrophages can inhibit the cytotoxic effect of chimeric antigen receptor T cells (CAR-T). M2 macrophages mediate the drug resistance process of B lymphoma, and their enrichment level is significantly positively correlated with lymphoma recurrence. However, the relevant molecular mechanisms have not been fully elucidated. These clinical challenges highlight the urgency of in-depth analysis of the mechanism of action of the B lymphoma microenvironment and the development of new treatment strategies.

[0004] Chidamide, a selective histone deacetylase (HDAC) inhibitor, primarily targets class I (HDAC1 / 2 / 3) and class IIb (HDAC10) HDACs and has been approved for the treatment of peripheral T-cell lymphoma. The drug has also shown efficacy in B-cell lymphoma, leukemia, and breast cancer, but its mechanism of action remains unclear. Existing studies suggest that high-dose chidamide exerts direct antitumor effects by arresting the tumor cell cycle and inducing apoptosis, while low-dose chidamide primarily modulates the immune response. Notably, HDAC inhibitors can inhibit M2 macrophage polarization. Although high-dose regimens offer superior clinical efficacy, they can cause serious adverse reactions such as bone marrow suppression, liver and kidney damage, and electrolyte imbalances. Low-dose regimens, while offering immunomodulatory advantages, are limited in their effective application in tumor treatment due to their limited targeting. Therefore, optimizing drug delivery systems to enhance their targeting has become a key scientific question.

[0005] Extracellular vesicles (EVs) are lipid bilayer vesicles (30-150 nm) actively secreted by living cells. They can be transported through body fluids to transmit biological signals. Recent studies have confirmed that engineered EVs can serve as novel drug carriers, with progress already made in gene therapy, cardiac repair, and immunomodulation, but this remains a niche area in the field of lymphoma. When used as a delivery system, EVs' lipid bilayers can accommodate hydrophobic drugs, while their inner lumen is suitable for hydrophilic drugs. With advantages such as strong penetrability, good biocompatibility, and widespread biodistribution, EVs hold promise as ideal natural carriers.

[0006] This study aimed to construct a chidamide-EVs complex system to reduce drug toxicity and enhance immunomodulatory effects. To address the issues of burst release and uncontrolled dosage associated with direct EV injection, we introduced a hydrogel delivery system. Hydrogels are three-dimensional networks constructed from hydrophilic polymers, exhibiting four key properties: biocompatibility, biodegradability, drug loading, and controlled release. Given the acidic tumor microenvironment caused by lactic acid accumulation, we developed a pH-responsive TSPBA / TVA (TP) hydrogel. This system enables sustained and controlled release of EVs under acidic conditions, effectively overcoming the limitations of direct injection and establishing a mechanism for the coordinated regulation of microenvironmental pH and disease progression.

[0007] HDACs can regulate macrophage reprogramming by modifying STAT3, and the tumor-promoting role of M2 macrophages in lymphoma progression has been confirmed. Although cedabendine has immunomodulatory functions, its regulatory mechanism on the immune phenotype of macrophages is still unclear. This study found that STAT3 acetylation modification plays a key role in macrophage polarization in the DLBCL microenvironment. Based on this, we constructed a pH-responsive precision drug delivery platform: cedabendine was loaded into engineered EVs and sustained drug release was achieved with the help of smart hydrogels. This strategy not only reduces the dosage and prolongs the duration of action, but also achieves targeted drug delivery in the tumor microenvironment, providing a new approach for the treatment of high-grade or relapsed / refractory DLBCL, and has important clinical translational value. Summary of the Invention

[0008] In order to overcome the defects of the prior art, the present invention provides the following technical solutions:

[0009] The first aspect of the present invention provides a method for preparing a chidamide targeted M2 macrophage delivery system, comprising the following steps:

[0010] S1: Dissolve distearoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG2000-Mal) in methanol to prepare a 5-20 mg / mL solution;

[0011] S2: M2 macrophage targeting peptide (M2pep) was dissolved in PBS-ACN buffer at pH 6-7 to prepare a solution with a concentration of 0.5-2 mmol / L;

[0012] S3: Add the M2pep solution and tris(2-carbonylethyl)phosphine hydrochloride (TCEP) solution prepared in S2 to the DSPE-PEG2000-Mal methanol solution prepared in S1, and stir at room temperature for 40-50 h to mix uniformly to form a suspension;

[0013] S4: dialyzing the suspension prepared in S3 in distilled water for 40-50 hours to remove free peptides, obtaining a synthetic product, distearoylphosphatidylethanolamine-polyethylene glycol-M2 macrophage targeting peptide (DSPE-PEG-M2pep), which was dissolved in D2O for later use; further, the dialysis membrane had a molecular weight cutoff of ≥3500 Da;

[0014] S5: DSPE-PEG-M2pep was integrated into the membrane of extracellular vesicles (EVs), loaded with Chid, and incubated at 35-38°C for 2.5-4 hours. Subsequently, the mixture was ultracentrifuged at 0-4°C to remove unbound DSPE-PEG-M2pep to obtain Chid-loaded M2 macrophage-targeting peptide-modified extracellular vesicles (i.e., Chid@M2pep-EVs);

[0015] S6: First, the Chid@M2pep-EVs prepared in S5 were mixed with polyvinyl alcohol (PVA) solution at a volume ratio of 1.8 to 2.2:1, and then an equal volume of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA) solution was added to the PVA solution, and the pH value was adjusted to 9 to 10. The mixture was mixed and the pH value was adjusted to 9 to 10 to prepare Chid@M2pep-EVs / TP hydrogel.

[0016] Alternatively, the Chid@M2pep-EVs prepared in S5 were first mixed with the PVA solution at a volume ratio of 1.8 to 2.2:1, and then an equal volume of 3-aminophenylboronic acid-hyaluronic acid copolymer (HA-PBA) solution was added to the PVA solution, and the pH value was adjusted to 9 to 10, and the mixture was mixed to prepare the Chid@M2pep-EVs / HP hydrogel.

[0017] Furthermore, in S3, the volume ratio of the M2pep solution: DSPE-PEG2000-Mal solution: TCEP solution is 1:1:0.03-0.05; further preferably, the volume ratio of the M2pep solution: DSPE-PEG2000-Mal solution: TCEP solution is 1:1:0.04;

[0018] Furthermore, in S5, the mass ratio of DSPE-PEG-M2pep:EVs:Chid is 1:1:1-5;

[0019] Furthermore, in S5, the centrifugal speed is 100,000 to 150,000 × g, and the centrifugation time is 60 to 80 minutes;

[0020] Furthermore, in S6, the concentration of the PVA is 8 to 12 m / v%;

[0021] Furthermore, in S6, the concentration of TSPBA is 4 to 6 m / v%;

[0022] The second aspect of the present invention provides a targeted M2 macrophage delivery system of chidamide, which is prepared by any of the above methods;

[0023] The third aspect of the present invention provides the use of any of the above-mentioned M2 macrophage-targeted delivery systems of chidamide in the preparation of anti-tumor drugs;

[0024] Furthermore, the anti-tumor use is selected from any one or more of lymphoma, leukemia, and breast cancer; and further, the lymphoma is diffuse large B-cell lymphoma;

[0025] Furthermore, the anti-tumor effect is to reduce the dosage and / or prolong the duration of drug action; beneficial effects

[0026] The present invention achieves targeted delivery to M2 macrophages by engineering M2pep-EVs to load Chidamide, and loads it into TSPBA / TVA hydrogel with pH-responsive properties. This system can achieve controlled release of Chid@M2pep-EVs under acidic conditions, effectively overcoming drug side effects and establishing a linkage regulation mechanism between microenvironment pH and disease progression. This mechanism not only reduces the dosage and prolongs the duration of action, but also achieves targeted drug delivery in the tumor microenvironment, providing new ideas for tumor treatment, especially the treatment of high-grade or recurrent / refractory DLBCL, and has important clinical translational value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Figure 1: HDACs are highly expressed in macrophages and regulate the generation of M2 phenotype macrophages, including (A) visualization of cell populations in lymph nodes (LNs) and diffuse large B-cell lymphoma (DLBCL) by UMAP, (B) dot plots of different cell type markers; (C) number of interactions and interaction weight / strength of intercellular communication, (D) visualization of macrophage subtypes in LNs and DLBCL by UMAP, (E) dot plots of different cell type markers; (F) proportions of various cell subpopulations in macrophages, (G) heat map showing the relationship between macrophage subtypes and HDA Cs-related functional gene expression levels, (H) UMAP visualization of spatiotemporal differentiation trajectories of macrophage subtypes, (I) Expression dynamics of HDAC1, HDAC2, HDAC3, and STAT3 on pseudo-time trajectories, (J) Heat map showing gene expression dynamics on pseudo-time trajectories; (K) GSEA showing enrichment of HDAC target genes and JAK-STAT signaling pathway in the MRC1+ macrophage group, (L) Heat map showing DoRothEA analysis of the transcription factor activation landscape in different cell subpopulations, (M) Gene expression levels of HDACs in different cell types;

[0028] Figure 2 Chidamide induces the reprogramming of M2 macrophages into M1 macrophages, exerting inflammatory and lymphoma cytotoxic effects. (A) Effect of chidamide on RAW264.7 cell viability. Data are the IC of chidamide. 50 Summary of values, (B) Fluorescence images of macrophage morphology after chidamide treatment, (CD) Fluorescence images and ratios of macrophages of different phenotypes (M1 and M2) after chidamide treatment, (EH) Chidamide-induced macrophage polarization and the expression of CD86, CD206, TNFα, and IL10 were evaluated by flow cytometry, (IJ) TNFα and IL10 production by M1 and M2 macrophages were detected by ELISA, (KL) Transcription levels of M1 and M2 markers CD86 and CD206 were detected by qPCR, (M) Western blot analysis of HDACs, STAT3, and Ac-STAT3 expression in macrophages after treatment. (NO) ChIP analysis of STAT3 transcriptional activity using STAT3 or IgG antibodies, and quantification of DNA in the CD86 or CD206 promoter region by qPCR. (P) Fluorescence images of HDAC expression in macrophages after chidamide treatment. (Q) Co-culture experiments verifying the cytotoxic effect of chidamide-treated M1 macrophages reprogrammed against the B lymphoma cell line A20.

[0029] Figure 3For the construction and verification of Chid@M2pep-EVs, (A) the synthesis process of Chid@M2pep-EVs, (B) the DSPE-PEG-M2pep 1 H-NMR spectra, (C) TEM detection of the morphology of EVs and M2pep-EVs, (D-E) particle size and zeta potential of EVs and M2pep-EVs, (F) Western blot detection of EV biomarkers in BMSCs, EVs, and M2pep-EVs, (G) fluorescence images and cellular uptake of EVs and M2pep-EVs by different subtypes (M0, M1, and M2) of macrophages, (H) flow cytometry analysis of the uptake of M2pep-EVs by different subtypes of macrophages;

[0030] Figure 4 Construction and validation of TSPBA / PVA hydrogels. (A) A BALB / c mouse subcutaneous tumor model was used to measure the pH values ​​of serum and tumor interstitial fluid. (B) Synthesis process of TP hydrogel. (C) FTIR spectrum of TP hydrogel. (D) Hydrogel formed from a mixture of TSPBA and PVA and treated with PBS at pH 6.5. (E) SEM images of TP and Chid@M2pep-EVs / TP hydrogels. (F) Porosity of TP and Chid@M2pep-EVs / TP hydrogels. (G) Rheological properties of TP and Chid@M2pep-EVs / TP hydrogels. G': storage modulus, G”: loss modulus. (HI) EV release and weight residual levels of TP and Chid@M2pep-EVs / TP hydrogels at different pH conditions. (JK) Water absorption and water content of TP and Chid@M2pep-EVs / TP hydrogels.

[0031] Figure 5Chid@M2pep-EVs / TP regulates the reprogramming of M2 macrophages to the M1 phenotype; (A) Schematic diagram of the workflow for functional validation of Chid@M2pep-EVs / TP, (BD) Fluorescence images and ratios of markers of different macrophage phenotypes (M1 and M2) after treatment, (EH) Flow cytometry evaluation of the expression of CD86, CD206, TNFα, and IL10 after designated treatment to verify macrophage reprogramming, (IJ) qPCR detection of M (KN) ELISA was used to detect the expression of cytokines TNFα, IL6, IL10, and TGFβ in M1 and M2 macrophages. (O) Fluorescence images of HDACs expression in macrophages after treatment were shown. (5P-S) Relative mRNA levels of different macrophage cytokines after different treatments: (5P) IL6 level, (5Q) IL10 level, (5R) TNFα level, and (5S) TGFβ level.

[0032] Figure 6 Chid@M2pep-EVs / TP mediates macrophage cytotoxicity to eliminate lymphoma cells. (A) Schematic diagram of the interaction between treated macrophages and lymphoma cells. (B) After treatment, conditioned medium (CM) induced apoptosis in lymphoma cells, as assessed by flow cytometry. (C-D) Fluorescent images and proportions of apoptotic cells after treatment. (E) Ki67 expression after indicated treatments was assessed by flow cytometry to evaluate the lymphoma inhibitory effect. (F-H) CCK8 assay was used to detect the proliferation level of A20 lymphoma cells at different time points after indicated treatments.

[0033] Figure 7 Chid@M2pep-EVs / TP promotes macrophage reprogramming to suppress lymphoma in vivo. (A) Strategy to investigate the anti-lymphoma effect of Chid@M2pep-EVs / TP hydrogel in a BALB / c mouse subcutaneous tumor model. (B) Effects of corresponding treatments on lymphoma growth. (C) Representative images of lymphomas. (D) H&E staining of lymphoma tissues after treatment. (E) Immunofluorescence staining of macrophage markers in lymphoma tissues. (F) Evaluation of the degree of apoptosis of lymphoma cells after the indicated treatments by flow cytometry. (G) After treatment, the expression of macrophage reprogramming markers was evaluated by flow cytometry.

[0034] Figure 8Chid@M2pep-EVs / TP induces the reprogramming of M2 macrophages into M1 macrophages, exerting inflammatory and lymphoma-killing effects. (AB) Heatmaps and volcano plots of differentially expressed genes (DEGs) between the vehicle group and the Chid@M2pep-EVs / TP group. (C) Functional annotation of DEGs using GSEA enrichment. (D) Normalized FPKM values ​​of HDAC1, HDAC2, HDAC3, CD86, TNFα, and STAT3. (E) Quantitative PCR verification of the expression levels of HDAC1, HDAC2, HDAC3, CD86, TNFα, and STAT3 in animal model lymphoma tissues. (F) Western blot analysis of the expression of HDAC1, HDAC2, HDAC3, STAT3, and Ac-STAT3 in animal model lymphoma tissues. (GJ) Flow cytometry analysis of the expression levels of HDAC1, HDAC2, HDAC3, and STAT3 in macrophages from animal model lymphoma tissues.

[0035] Figure 9 Schematic diagram of the preparation process and related mechanism of the present invention. DETAILED DESCRIPTION

[0036] The present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0037] (1) Embodiment

[0038] Example 1: Synthesis and Characterization of DSPE-PEG-M2pep

[0039] First, distearoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG2000-Mal) was dissolved in methanol (10 mg / mL). Then, 1 mM of M2 macrophage targeting peptide (M2pep) (M2 macrophage targeting peptide (M2pep), whose amino acid sequence is YEQDPWGVKWWY, is a polypeptide with specific targeting function of M2 macrophages, and its molecular formula is C 83 H 101 N 17 O 20, with a molecular weight of 1656.79. Its structure consists of 12 amino acids, and the three-letter sequence is Tyr-Glu-Gln-Asp-Pro-Trp-Gly-Val-Lys-Trp-Trp-Tyr) solution (PBS / ACN at pH 6.5, 1:1 v / v) and 0.04 equivalents of tris(2-carbonylethyl)phosphine hydrochloride (TCEP) (10 mg / mL, dissolved in double deionized water) were added to the DSPE-PEG2000-Mal solution, and the solution was then stirred at room temperature for 48 hours. Subsequently, the above mixture was dialyzed in distilled water for 48 hours (molecular weight 3500 Da) to remove free peptide. The synthesized product was dissolved in deuterated water (D2O) and characterized by hydrogen nuclear magnetic resonance spectroscopy ( 1 H NMR) analysis was performed to confirm successful coupling.

[0040] Example 2: Preparation of Chid-loaded M2pep-modified EVs

[0041] Chidamide (Chid)-loaded M2pep-EVs (Chid@M2pep-EVs) were generated by self-assembly. Distearoylphosphatidylethanolamine-polyethylene glycol-M2 macrophage-targeting peptide (DSPE-PEG-M2pep) was incorporated into the membrane of extracellular vesicles (EVs) and loaded with Chid at a mass ratio of 1:1:2. The cells were incubated at 37°C for 2 hours. Subsequently, the mixture was ultracentrifuged at 120,000 × g for 70 minutes at 4°C to remove unbound DSPE-PEG-M2pep, resulting in the purified Chid-loaded M2 macrophage-targeted delivery system (Chid@M2pep-EVs).

[0042] Example 3: Preparation and characterization of TSPBA / PVA hydrogel

[0043] First, 200 μL of Chid@M2pep-EVs prepared in Example 2 was mixed with 100 μL of a polyvinyl alcohol (PVA) solution having a concentration of 8-12 m / v%. Then, an equal volume of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (TSPBA) solution having a concentration of 4-6 m / v% was added to the PVA solution. The pH was adjusted to 9.5, and the mixture was mixed to prepare a Chid@M2pep-EVs / TP hydrogel.

[0044] The hydrogels were confirmed using Fourier transform infrared spectroscopy (FTIR). After freeze-drying, the morphology of the TSPBA / PVA hydrogel (Chid@M2pep-EVs / TP hydrogel) was imaged by scanning electron microscopy (SEM). For in vivo experiments, administration was performed using a dual-syringe syringe (EFL, China).

[0045] Example 4: Preparation and characterization of HA-PBA / PVA hydrogel

[0046] First, 200 μL of Chid@M2pep-EVs prepared in Example 2 was mixed with 100 μL of a polyvinyl alcohol (PVA) solution having a concentration of 8-12 m / v%. Then, an equal volume of a 3-aminophenylboronic acid-hyaluronic acid copolymer (HA-PBA) solution having a concentration of 2-5 m / v% was added to the PVA solution. The pH was adjusted to 9.5 and mixed to prepare a Chid@M2pep-EVs / HP hydrogel.

[0047] The hydrogels were confirmed using Fourier transform infrared spectroscopy (FTIR). After freeze-drying, the morphology of the HA-PBA / PVA hydrogels (Chid@M2pep-EVs / HP hydrogels) was imaged by scanning electron microscopy (SEM). For in vivo experiments, administration was performed using a dual-syringe syringe (EFL, China).

[0048] (2) Experimental examples

[0049] 2.1 Overview

[0050] To elucidate differences in cellular composition and gene expression within the tumor microenvironment, we defined data from patients with diffuse large B-cell lymphoma (DLBCL) as the tumor group and data from tumor-free lymph nodes as the normal control group. Single-cell sequencing data from patients with DLBCL and tumor-free lymph nodes were collected from the Gene Expression Omnibus (GEO) database for single-cell sequencing analysis. In RNA-seq analysis, differentially expressed genes (DEGs) were identified based on thresholds (fold change >2 or <0.5 and p-value <0.05). Data from both groups were visualized using volcano plots and heat maps. Furthermore, gene set enrichment analysis (GSEA) was performed to elucidate the functions of the DEGs. These RNA-seq analyses were performed using the Sangerbox platform (http: / / sangerbox.com / Tool).

[0051] 2.2 Isolation and identification of extracellular vesicles (EVs) derived from bone marrow mesenchymal stem cells (BMSCs)

[0052] BMSC-derived EVs were extracted by gradient ultracentrifugation according to the guidelines provided by the International Society for Extracellular Vesicles. When the BMSC density reached 70–80% under microscopy, the culture medium was replaced with EV-free medium, and the supernatant was collected 48 hours later. The supernatant was then centrifuged at 2000 × g for 20 minutes to remove dead cells and at 10,000 × g for 30 minutes at 4°C to remove cellular debris. The supernatant was then transferred to an ultracentrifuge tube and ultracentrifuged at 100,000 × g for 70 minutes. After removal of the supernatant, the EVs pelleted at the bottom of the tube were resuspended in phosphate-buffered saline (PBS). To characterize the morphology and biological properties of the EVs, the extracted EVs were fixed in glutaraldehyde and negatively stained with phosphotungstic acid on copper grids. The EV morphology was then observed using transmission electron microscopy (TEM, Talos F200X G2, Thermo Fisher). Western blotting was used to identify EV biomarkers, including TSG101, Alix, CD9, and Calnexin. The diameter and zeta potential of EVs were measured by dynamic light scattering (DLS).

[0053] 2.3 Cellular Uptake of Chid@M2pep-EVs

[0054] RAW 264.7 cells were seeded in 24-well plates at a density of 100,000 cells per well 24 hours in advance and then divided into three groups: M0 subtype group (untreated), M1 subtype group (induced with 1 μg / mL LPS for 24 hours), and M2 subtype group (induced with a combination of 20 ng / mL IL-4 and 10 ng / mL IL-13 for 24 hours). Subsequently, 10 μg of Chid@M2pep-EVs (extracellular vesicles encapsulating chidamide and modified with an M2 macrophage-targeting peptide) or Chid@EVs (extracellular vesicles encapsulating chidamide) labeled with the cell membrane green fluorescent probe Dio were added to each group and incubated for 6 hours. The culture medium was then discarded, the cells were washed with PBS, and stained with TRITC-phalloidin. Finally, EV uptake by RAW264.7 cells was assessed by immunofluorescence microscopy and flow cytometry.

[0055] 2.4 Release and gel degradation kinetics of Chid@M2pep-EVs / TP hydrogel

[0056] To test the controlled-release properties of the hydrogel, Chid@M2pep-EVs / TP hydrogels (containing extracellular vesicles encapsulated with chidamide and modified with an M2 macrophage-targeting peptide) were transferred to a test tube containing 20 mL of PBS and placed at 37°C. At different time points (0, 6, 12, 24, 48, 72, 96, 120, 144, and 168 hours), 1 mL of supernatant was obtained and supplemented with an equal volume of PBS solution. Since Chid@M2pep-EVs were labeled with DIO, the concentration of released Chid@M2pep-EVs was measured based on fluorescence. Furthermore, after the hydrogel equilibrated, it was removed and weighed. The hydrogel was then removed and weighed at each time point, and the degradation rate of the hydrogel weight was calculated.

[0057] 2.5 Cell culture

[0058] Briefly, vector, TP, Chid@EVs / TP, and Chid@M2pep-EVs / TP were evenly applied to the upper Transwell chamber, while RAW264.7 cells were cultured in the lower Transwell chamber using EV-free medium at pH 6.5. To mimic the in vivo tumor microenvironment, when cell density reached 70%, 20 ng / mL IL-4 and 10 ng / mL IL-13 were added to the wells and incubated for 24 hours. The Transwells were then removed, the supernatant discarded, and replaced with fresh medium. After 24 hours, the conditioned medium was aspirated onto A20 cells to assay cell-to-cell communication.

[0059] 2.6 Mouse Model

[0060] For the A20 lymphoma model, BALB / c mice were subcutaneously inoculated with 1×10⁶ cells / mouse. One week after transplantation, the mice were randomly divided into four groups: control, TP, Chid@EVs / TP, and Chid@M2pep-EVs / TP. Tumors were measured with a vernier caliper every other day. Tumor volume = 0.5 × L × S 2 , where L represents the longest diameter and S represents the shortest diameter. After treatment, lymphoma samples were collected for subsequent experiments. All animal experiments were approved by the Animal Experimentation Ethics Committee of Fudan University.

[0061] 2.7 Tumor stromal fluid extraction and pH measurement

[0062] Gently wipe the tumor tissue with a paper towel to remove excess PBS, mince it, and transfer it to a 2 mL centrifuge filter tube. Then, centrifuge at 10,000 × g for 20 minutes at 4°C. The tumor stromal fluid will accumulate at the bottom of the tube. Measure the pH of the tumor stromal fluid using a micro-pH meter.

[0063] 2.8 Immunofluorescence staining and HE staining

[0064] Tissues or cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.5% Triton X-100 for 15 minutes, and blocked with 3% BSA for 30 minutes at 37°C. Specific binding of primary antibodies (CD68, CD86, CD206, HDAC1, HDAC2, HDAC3, and STAT3) was detected using fluorescent secondary antibodies. Observation was performed using a THUNDER Imager (Leica).

[0065] 2.9 Real-time quantitative PCR (qPCR)

[0066] Total RNA was extracted and reverse transcribed into cDNA. The reaction system was then prepared according to the kit instructions and run on an ABI PRISM 7500 qPCR instrument. PCR product accumulation was recorded in real time using the SYBR Green assay, and Ct values ​​were obtained. Relative mRNA expression was estimated using the 2-ΔΔCt method, using GAPDH as an internal control.

[0067] 2.10 ChIP-qPCR

[0068] ChIP experiments were performed according to the kit instructions. Plus Ultrasonic Chromatin IP Kit was used. Briefly, cells were fixed with 1wt% formaldehyde, incubated with glycine (final concentration 50mM), and washed three times with PBS. After cell lysis and chromatin extraction, chromatin was sonicated to 100-500bp. Subsequently, the lysate was incubated with ChIP-grade STAT3 and IgG-specific antibodies at 4°C overnight and then coupled to magnetic beads. The precipitate (input chromatin as a control) was eluted, the cross-linking was reversed, and the DNA was purified by chloroform / phenol extraction and resuspended in DNA elution buffer. The eluted DNA was analyzed by qPCR.

[0069] 2.11ELISA

[0070] Tumor tissue and cell supernatant were collected. IL-6, IL-10, TNF-α, and TGF-β concentrations were measured using ELISA kits according to the kit instructions. The total protein concentration of each sample was quantified by BCA and adjusted to a consistent level. A standard calibration curve was constructed using standard samples. The optical density of each sample was recorded using a microplate reader, and the concentration was calculated based on the standard curve.

[0071] 2.12 Western blotting

[0072] Protein was extracted from cells or tissues using a total protein extraction kit. Protein concentration was determined using a BCA protein assay kit. Protein lysates were separated by SDS-PAGE and subsequently transferred to a PVDF membrane. The membrane was probed with primary and secondary antibodies. Western blot images were captured using an Amersham Imager 600.

[0073] 2.13 Flow cytometry

[0074] Surface antigens in the cell suspension were stained with fluorescently labeled antibodies for 30 minutes at room temperature. For intracellular staining, cells were stimulated with 30 nM PMA and 1 μM ionomycin for 4–6 hours at 37°C. Following surface staining, cells were fixed with fixation buffer and subjected to intracellular staining using 1x permeabilization buffer for 30 minutes at room temperature. Cells were then analyzed using a CytoFLEX S flow cytometer.

[0075] 2.14 CCK-8 assay

[0076] RAW264.7 or A20 cells were seeded at a density of 10,000 cells per well in a 96-well plate. After treatment, 10 μl of CCK-8 solution was added to each well and incubated at 37°C for 2 hours. Finally, absorbance was measured at 450 nm.

[0077] 2.15 Dead / alive cell assay

[0078] A20 cells were seeded at a density of 100,000 cells per well in a 24-well plate. Conditioned medium from RAW264.7 cells treated with vehicle, TP, Chid@EVs / TP, or Chid@M2pep-EVs / TP was added to the A20 cells and incubated for 24 hours. Then, 250 μl of Calcein AM / PI solution was added to each well and incubated in the dark at 37°C for 30 minutes. After incubation, cell viability was assessed using a THUNDER Imager (Leica).

[0079] 2.16 Statistical Analysis

[0080] Analyses were performed using GraphPad Prism 8 software and R 4.3.0. All experiments were performed with n ≥ 3 biological replicates. P < 0.05 was considered statistically significant. Multiple group comparisons were performed using one-way analysis of variance (ANOVA) followed by the Tukey test, and two-group comparisons were performed using an unpaired two-tailed t-test. Data are expressed as mean ± standard deviation (SD).

[0081] (3) Experimental results

[0082] 3.1 Single-cell sequencing analysis reveals an increased proportion of M2 phenotype macrophages in DLBCL To explore potential therapeutic targets for DLBCL, we analyzed and compared the cell composition and differentially expressed genes between DLBCL and normal lymph nodes (LNs). We analyzed data from four DLBCL patients (GSE182434) and 10 normal lymph nodes (GSE131907). Cells were visualized using UMAP ( Figure 1 A). Based on the expression levels of classic marker genes, five major cell types were identified, including B cells, T cells, NK cells, macrophages, and other cells ( Figure 1 A, B). Based on this, we found that HDAC1 is highly expressed in B cells, while HDAC1, HDAC2, and HDAC3 are highly expressed in macrophages ( Figure 1 M). Cell communication analysis showed that macrophages occupy a significant number and interaction weight in the tumor microenvironment ( Figure 1 C). However, when the macrophage population was further subdivided and visualized by UMAP, we identified five cell types based on the expression levels of marker genes, including MRC1+ macrophages, APOC1+ macrophages, CD40+ macrophages, CCL5+ macrophages, and HES1+ macrophages ( Figure 1 D, E), and the proportion of MRC1+ macrophages (M2 phenotype macrophages) in the DLBCL group was significantly higher than that in the LN group ( Figure 1 F). In MRC1+ macrophages, HDAC family-related genes are significantly expressed ( Figure 1 G). We then performed pseudo-time series analysis to assess the temporal variability of macrophage gene expression ( Figure 1 H). HDAC family genes are expressed along pseudo-time trajectory in MRC1+ macrophages ( Figure 1 I, J, S6). In addition, we applied gene set enrichment analysis (GSEA) and found that HDAC target genes and JAK-STAT3 signaling pathway were significantly enriched ( Figure 1 K). Transcription factor activity DoRothEA analysis showed that the activity of STAT family protein STAT3 was significantly increased in _MRC1_+ macrophage clusters ( Figure 1 L).

[0083] In this study, we characterized a single-cell landscape of DLBCL. We found that within the DLBCL tumor microenvironment, STAT3, under the regulation of HDACs, promotes the generation of M2 macrophages. Abundant M2 macrophages suppress tumor immunity through the LGALS9-CD44 pair while promoting tumor cell proliferation through MIF-(CD74+CXCR4) and other pathways. HDACs have been reported to influence the reprogramming of M2 macrophages. Based on these data, we hypothesized that HDACs may serve as potential therapeutic targets for DLBCL.

[0084] 3.2Chid promotes M1 reprogramming to exert therapeutic effects

[0085] Chid is a small molecule targeted drug that targets class I HDACs (HDAC1, HDAC2, and HDAC3) and class IIb HDACs (HDAC10). To determine the IC50 of Chid against M2 macrophages, we induced RAW264.7 cells to transform into an M2 phenotype with IL4 and IL13, and then performed CCK-8 assay. The IC50 value of Chid against M2 phenotype RAW264.7 cells was 1.088 μM ( Figure 2 A). Immunofluorescence staining showed that the treated M2 phenotype RAW264.7 cells underwent a morphological transformation similar to that of M1 cells ( Figure 2 B). In addition to morphological changes, we confirmed the phenotypic switching of cells by staining for M1 and M2 cell-specific markers (CD86 and CD206). Figure 2 C, D). In addition, flow cytometry analysis showed that after treatment, the expression of M1 macrophage-related marker CD86 increased significantly, and the proportion of M1 cells secreting TNFα was higher. In contrast, the expression of M2-related marker CD206 decreased significantly, and the proportion of M2 cells secreting IL10 also decreased significantly ( Figure 2 EH). In addition, we measured the secretion levels of M1- and M2-related cytokines, including IL-6, IL-10, TNF-α, and TGF-β, using ELISA ( Figure 2 I, J). In addition, the relative mRNA levels of different macrophage markers (CD86 and CD206) and cytokines (IL-6, IL-10, TNF-α, and TGF-β) were measured by qPCR ( Figure 2K, L). The results showed that the proportion of M1 macrophages and the secretion of M1-related cytokines increased significantly after Chid treatment. Single-cell sequencing showed that STAT3 may be a key factor in HDACs regulating macrophage reprogramming. Western blotting showed that the expression of HDAC1, HDAC2 and HDAC3 increased in M2 macrophages, and the acetylation level of STAT3 increased significantly after Chid inhibited HDAC ( Figure 2 M). In addition, after Chid treatment, the transcriptional activity of STAT3 on CD86 was significantly increased, while the transcriptional activity on CD206 was significantly decreased ( Figure 2 N, O). In addition, immunofluorescence staining and Western blotting confirmed that HDACs were inhibited by Chid ( Figure 2 M, P). According to some studies, the effects of Chid concentration on different cell types are inconsistent. Therefore, we also studied the effect of Chid on lymphoma cells. When A20 cells were treated with Chid, the proportion of apoptotic cells increased, but the difference was not statistically significant ( Figure 2 Q). In addition, when A20 cells were co-cultured with M2 macrophages, Chid treatment significantly increased the apoptotic rate of A20 cells ( Figure 2 Q). To determine the cause of increased apoptosis in A20 cells, we added the conditioned medium (CM) of Chid-treated M2 macrophages to the culture medium of A20 cells, and the apoptotic rate of A20 cells also increased significantly.

[0086] Recently, researchers have been working to expand the indications of Chid, believing it has the potential to treat DLBCL. However, its underlying therapeutic mechanism has not yet been reported. Several studies have identified the immunomodulatory effects of Chid, but its regulatory effects on macrophages remain unclear. In our study, targeting M2 macrophages to promote their reprogramming to an M1 phenotype, thereby enhancing their tumor-killing effects, may provide a promising therapeutic strategy for DLBCL.

[0087] 3.3 Construction and Verification of M2pep-EVs

[0088] BMSCs were isolated from the bone marrow and EVs were extracted from BMSCs by ultracentrifugation. DSPE-PEG-M2pep can be specifically recognized by M2 macrophages. To effectively improve the uptake rate of Chid by M2 macrophages, M2pep-modified EVs (M2pep-EVs) were constructed as described in the experimental section ( Figure 3 A). DSPE-PEG-M2pep has excellent affinity for the phospholipid bilayer of EVs and is used to modify EVs via self-assembly. The self-assembly method relies on non-covalent forces and is a bio-friendly strategy that does not interfere with the bioactivity of engineered EVs.1 H NMR ( 1 The purified DSPE-PEG-M2pep, DSPE-PEG-NHS and M2-pep products were characterized by H NMR spectroscopy ( Figure 3 B). Subsequently, the morphology of EVs and M2pep-EVs was evaluated by transmission electron microscopy (TEM) ( Figure 3 C), the results showed typical double-layer membrane vesicles. In addition, the particle size and zeta potential of EVs and M2pep-EVs were analyzed by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS90. The particle size histogram showed that the average particle size of EVs and M2pep-EVs was 204.1 and 223.8 nm, respectively ( Figure 3 D). The zeta potentials of EVs and M2pep-EVs were 22.15±2.29 and 23.67±3.28 mV, respectively, showing negative surface charges ( Figure 3 E). In addition, Western blot analysis confirmed that EV markers (CD9, Alix, and TSG101) were enriched in EVs and M2pep-EVs, while non-EV markers (Calnexin) were not detected ( Figure 3 F). In addition, to confirm the ability of M2pep-EVs to target M2 macrophages, we added DIO-labeled EVs and M2pep-EVs to different subtypes of macrophages (M0, M1, and M2 subtypes). The results showed that M2 macrophages internalized more EVs, indicating that M2pep-modified EVs have enhanced targeting ability ( Figure 3 G). In addition, flow cytometry analysis further confirmed the targeting ability of M2pep-EVs ( Figure 3 H).

[0089] Currently, engineered EVs are being developed using surface modification technologies for the treatment of various diseases, including gene therapy for intrauterine adhesions and tumor microenvironment regulation. However, the application of engineered EVs in lymphoma treatment is less well-researched. In this study, Chid-loaded EVs were modified with M2pep to achieve targeted recognition and precise intervention of M2 macrophages. This EV-based, M2pep-modified targeted delivery system holds promise as a therapeutic strategy for B-cell lymphoma.

[0090] 3.4 Construction and detection of Chid@M2pep-EVs / TP hydrogel

[0091] Chid is degraded immediately after absorption, with a terminal elimination half-life of 17 hours. To overcome this shortcoming, a pH analysis of the tumor interstitial fluid of lymphoma-bearing mice was conducted ( Figure 4A), we synthesized a pH-responsive hydrogel for the delivery of Chid@M2pep-EVs. PVA is a polyol that reacts with phenylboronic acid to form pH-sensitive boronate ester bonds. PVA can be further cross-linked with N1-(4-borobenzyl)-N3-(4-boronophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diammonium (TSPBA) to form a stable hydrogel ( Figure 4 B). TSPBA linkers containing quaternary ammonium groups exhibit water solubility and can form gels in aqueous environments. The pH-responsive hydrogels ( Figure 4 C). When PVA was mixed with TSPBA, a hydrogel was formed and gradually dissolved in PBS at pH 6.5 ( Figure 4 D). The morphology and structure of TP and Chid@M2pep-EVs / TP hydrogels were examined by scanning electron microscopy (SEM). SEM images showed a porous cross-linked network structure. There was no significant difference in the porosity of the two hydrogels ( Figure 4 E, F). Rheological measurements were used to characterize the dynamic mechanical properties of the hydrogels. After hydrogel formation, there was no difference in the storage modulus (G') and loss modulus (G") between TSPVA / PBA(TP) hydrogel and Chid@M2pep-EVs / TP ( Figure 4 G). We then developed a sustained release curve of Dio-labeled Chid@M2pep-EVs from the hydrogel. The release curve showed that Chid@M2pep-EVs had an initial burst release in the first 2 days, followed by a sustained release phase over the next 5 days, with a cumulative release rate of 85.23±3.17% ( Figure 4 H). The residual weight curve of the hydrogel related to the release of Chid@M2pep-EVs is shown in Figure 2. Figure 4 I. In addition, we evaluated the water absorption and water retention of these hydrogels and found that the water absorption and water retention properties of Chid@M2pep-EVs / TP and TP hydrogels remained unchanged ( Figure 4 J, K). In summary, we successfully developed a pH-responsive TP hydrogel loaded with Chid@M2pep-EVs without affecting the performance of the hydrogel.

[0092] In our study, we utilized TP hydrogels to achieve sustained release and used M2pep-EVs to target M2 macrophages, while Chid loaded in the hydrogels acted on HDAC family proteins, thereby extending the therapeutic duration of Chid and enhancing its targeting effect.

[0093] 3.5Chid@M2pep-EVs / TP regulates the reprogramming of M2 macrophages to M1 phenotype

[0094] The recruitment of M2 macrophages in lymphoma plays an important role in suppressing immunity, participating in lymphoma angiogenesis, and promoting lymphoma cell proliferation and metastasis. To study the reprogramming effect of Chid@M2pep-EVs / TP on macrophages, we seeded RAW264.7 cells in the lower chamber of the Transwell system and added IL4 and IL13 to stimulate M2 macrophage polarization. The cells were divided into four groups: vehicle, TP, Chid@EVs / TP or Chid@M2pep-EVs / TP, and then added with pH 6.5 culture medium ( Figure 5 A). After treatment, the differences in M1 and M2 macrophage phenotypes were assessed by immunofluorescence staining for CD68, CD86, and CD206. In the vehicle and TP groups, the proportion of CD206-positive M2 macrophages was higher, while the proportion of CD86-positive M1 macrophages was lower. In the Chid@EVs / TP and Chid@M2pep-EVs / TP groups, the proportion of CD86-positive M1 macrophages increased significantly, while the proportion of CD206-positive M2 macrophages decreased. In addition, in the absence of M2-pep, the proportion of M1 macrophages in the Chid@EVs / TP group was significantly lower than that in the Chid@M2pep-EVs / TP group ( Figure 5 BD). Flow cytometry analysis showed that the proportion of M2 macrophages was higher in the vehicle and TP groups, and they mainly secreted the M2-related cytokine IL-10 ( Figure 5 EH). However, the proportion of M1 macrophages in the Chid@EVs / TP and Chid@M2pep-EVs / TP groups increased significantly, and the proportion of TNFα-positive cells associated with the M1 phenotype increased significantly ( Figure 5 EH). Figure 5 I, J and Figure 5As shown in Figure 2, we analyzed the expression levels of macrophage-related markers and cytokines by qPCR. The results showed that treatment with Chid@EVs / TP or Chid@M2pep-EVs / TP significantly increased the mRNA expression levels of M1 macrophage-related genes CD86, IL6, and TNFα, while significantly decreased the expression of M2 macrophage-related genes CD206, IL10, and TGFβ. The targeting effect of M2-pep further increased the expression of CD86 and TNFα and decreased the expression of TGFβ. In contrast, macrophages in the vehicle and TP groups were primarily M2 macrophages, with CD206, IL10, and TGFβ maintained at high levels, with no significant differences between the two groups. Cytokine secretion is a key mechanism by which macrophages exert their biological functions. To investigate the potential mechanisms of tumor cell apoptosis following macrophage polarization, we assayed the production of IL-6, TNF-α, IL-10, and TGF-β in cell supernatants by ELISA. Compared with the vehicle and TP groups, the secretion of IL-6 and TNFα in the Chid@EVs / TP and Chid@M2pep-EVs / TP groups was significantly increased, and the presence of M2pep further increased the secretion of TNF-α ( Figure 5 KN). At the same time, we verified the targeted inhibitory effect of Chid@M2pep-EVs / TP by immunofluorescence staining. The results showed that the expression of HDAC1, HDAC2, and HDAC3 in the Chid@EVs / TP and Chid@M2pep-EVs / TP groups was significantly decreased, and the decrease was more obvious in the Chid@M2pep-EVs / TP group ( Figure 5 O). M2 macrophages are recruited in lymphomas, but no studies have yet investigated precisely targeting these genes. In this study, by combining EVs, hydrogels, and Chid, we demonstrated for the first time the feasibility of targeting M2 macrophages to promote their reprogramming and kill lymphoma cells. We believe this therapeutic approach has significant potential for future clinical treatment.

[0095] 3.6Chid@M2pep-EVs / TP regulates the generation of M1 macrophages, inhibits lymphoma cell proliferation and promotes apoptosis

[0096] We confirmed that Chid@M2pep-EVs / TP promoted macrophage reprogramming and, based on this, began to explore the effects of Chid@M2pep-EVs / TP on lymphoma cells. As described above, after macrophages were treated with vehicle, TP, Chid@EVs / TP, and Chid@M2pep-EVs / TP groups ( Figure 5A), we removed the Transwell chamber and replaced the macrophage culture medium. After 24 hours, we collected the conditioned medium and added it to the A20 cell culture medium ( Figure 6 A). Flow cytometry analysis showed that macrophage conditioned medium treated with Chid@EVs / TP or Chid@M2pep-EVs / TP had a significant cytotoxic effect on lymphoma cells, with a significant increase in the proportion of apoptotic lymphoma cells. In contrast, lymphoma cells in the vehicle and TP groups did not undergo significant apoptosis. In addition, after M2pep modification, lymphoma cell apoptosis was further increased ( Figure 6 B). In addition to flow cytometry, immunofluorescence Calcein / PI staining showed no significant increase in lymphoma cell apoptosis in the vehicle and TP groups, while the proportion of apoptotic lymphoma cells increased in the Chid@EVs / TP group, and the effect was more significant in the Chid@M2pep-EVs / TP group ( Figure 6 In addition to apoptosis, we also evaluated the proliferation marker Ki67 in lymphoma cells by flow cytometry. The results showed that the proliferation activity of lymphoma cells was higher in the vehicle and TP groups, while the proportion of Ki67-positive cells was significantly reduced in the Chid@EVs / TP group, and the reduction was more obvious in the Chid@M2pep-EVs / TP group ( Figure 6 E). In addition, we also evaluated the cell viability of lymphoma cells by CCK8 assay. The three-day CCK8 assay showed no significant difference in cell viability between the vehicle and TP groups, while the cell viability of the Chid@EVs / TP and Chid@M2pep-EVs / TP groups decreased significantly from the first day, and on the third day, the cell viability of the Chid@M2pep-EVs / TP group was significantly lower than that of the Chid@EVs / TP group ( Figure 6 FH).

[0097] Our in vitro experiments showed that Chid@M2pep-EVs / TP can effectively reprogram M2 macrophages into M1 macrophages, promote the secretion of cytokine TNFα, and thus induce apoptosis of lymphoma cells, providing good effects for the treatment of lymphoma.

[0098] 3.7Chid@M2pep-EVs / TP achieves lymphoma therapeutic effects in vivo by regulating macrophage reprogramming

[0099] To further validate the therapeutic efficacy of Chid@M2pep-EVs / TP against lymphoma, we established a cell line-derived xenograft mouse model. On day -7, we subcutaneously injected 1×106 A20 cells into BALB / c mice. On day 0, the tumor volume reached approximately 100 mm. 3At this time, the mice were randomly divided into four groups: vehicle, TP, Chid@EVs / TP, and Chid@M2pep-EVs / TP, and were dosed once a week. Starting from day 0, tumor size was measured every other day. After four weeks, the mice were sacrificed and tissue samples were collected for subsequent analysis ( Figure 7 A). Compared with the vehicle and TP groups, the tumor growth rates of the Chid@EVs / TP and Chid@M2pep-EVs / TP treatment groups were significantly reduced, with the Chid@M2pep-EVs / TP group showing the slowest growth rate ( Figure 7 B). In addition, tumor volume assessment further confirmed that both Chid@EVs / TP and Chid@M2pep-EVs / TP treatments significantly inhibited lymphoma growth, with Chid@M2pep-EVs / TP showing a stronger inhibitory effect ( Figure 7 C). HE staining showed the pathological characteristics of lymphoma in the four treatment groups ( Figure 7 D). Immunofluorescence staining of pathological sections showed that in the vehicle and TP groups, the majority of macrophages were CD206-positive M2 macrophages, while the proportion of CD86-positive M1 macrophages increased significantly in the Chid@EVs / TP and Chid@M2pep-EVs / TP groups, with the Chid@M2pep-EVs / TP group having the highest proportion of M1 macrophages ( Figure 7 E). Flow cytometry analysis was used to evaluate the degree of lymphoma cell apoptosis under different treatments. The results showed that lymphoma cell apoptosis was significantly increased in the Chid@EVs / TP and Chid@M2pep-EVs / TP groups, and the apoptosis level in the Chid@M2pep-EVs / TP group was significantly higher than that in the Chid@EVs / TP group ( Figure 7 F). In addition, flow cytometry further confirmed that Chid@EVs / TP and Chid@M2pep-EVs / TP treatment induced the reprogramming of M2 macrophages to the M1 phenotype. Notably, the Chid@M2pep-EVs / TP treatment group had a higher proportion of M1 macrophages and increased TNFα secretion compared with the Chid@EVs / TP group ( Figure 7GI). In addition, we established a vehicle group and an EVs / TP group to exclude potential effects that EVs may cause in vivo. The experiments verified the therapeutic effects of Chid@M2pep-EVs / TP in vitro and in vivo. Previous studies have reported the therapeutic effects of Chid on DLBCL, but their detailed molecular mechanisms have not been explored. Although the immunomodulatory effects of Chid have been documented, its role in regulating macrophages is still unclear. Our study not only demonstrated that Chid exerts its anti-lymphoma effect by reprogramming macrophages and enhancing the cytotoxicity of M1 macrophages against lymphoma cells, but also achieved better therapeutic effects by utilizing engineered EVs and pH-responsive hydrogels. The Chid@M2pep-EVs / TP system we developed is expected to improve the clinical management of DLBCL in the future.

[0100] 3.8Chid@M2pep-EVs / TP promotes macrophage reprogramming by regulating STAT3 acetylation modification

[0101] We found that the therapeutic effect of Chid@M2pep-EVs / TP was attributed to its ability to promote the reprogramming of macrophages to M1 macrophages, thereby exerting cytotoxic effects on lymphoma cells. However, the molecular mechanism of Chid treatment is still unclear. Through single-cell sequencing analysis and in vitro experiments, we concluded that acetylation of STAT3 plays a regulatory role in the reprogramming of macrophages to M1 and M2 phenotypes. It has been reported that deacetylation of STAT3 promotes the generation of M2 macrophages. In order to study the significance of STAT3 in this process, we also verified it through animal experiments. Using the samples obtained from the above animal experiments, we performed RNA-seq on 5 samples each from the vehicle group and the Chid@M2pep-EVs / TP group. Compared with the vehicle group, 5632 upregulated genes and 3928 downregulated genes were identified in the Chid@M2pep-EVs / TP group through heat map and volcano map analysis ( Figure 8 To explore the biological functions of DEGs, GSEA showed that these genes were mainly associated with the downregulation of TGFβ signaling, JAK-STAT3 signaling, and HDAC pathways, and the upregulation of TNFα signaling ( Figure 8 C). In addition, the expression of M2-related genes was significantly decreased, while the expression of M1-related genes was significantly increased in the Chid@M2pep-EVs / TP group ( Figure 8 D). We also verified the expression levels of these genes by qPCR ( Figure 8E). The molecular mechanisms identified by single-cell sequencing, RNA-seq analysis, and in vitro experiments were further validated in animal experimental samples. Western blot analysis showed that the acetylation level of STAT3 protein was significantly increased in the Chid@M2pep-EVs / TP group ( Figure 8 F). Flow cytometry analysis confirmed that the expression of HDAC1, HDAC2, and HDAC3 decreased, while the expression of STAT3 increased in macrophages after Chid@M2pep-EVs / TP hydrogel treatment ( Figure 8 GJ).

[0102] The molecular mechanism verified in vivo is consistent with our experimental results, indicating that under the action of Chid, STAT3 acetylation levels are increased, thereby promoting the expression of M1 macrophage-related genes. The increase in the proportion of M1 macrophages contributes to the therapeutic effect of lymphoma.

[0103] In this study, we found that STAT3 acetylation regulates macrophage polarization in the tumor microenvironment of DLBCL. At the same time, based on the acidic microenvironment of tumors, we developed a pH-responsive drug delivery system for precise drug delivery. By delivering engineered extracellular vesicles (EVs) loaded with Chid via pH-responsive hydrogels, we achieved sustained drug release, reduced drug dosage, prolonged drug efficacy, and achieved the goal of precise drug delivery ( Figure 9 This strategy is expected to improve the treatment outcomes for patients with high-grade or relapsed / refractory DLBCL and lay the foundation for future clinical applications.

[0104] Although the present invention has been described above with reference to embodiments, various modifications may be made thereto and equivalent components may be substituted without departing from the scope of the present invention. In particular, as long as there are no structural conflicts, the various features of the embodiments disclosed herein may be combined with each other in any manner, and the omission of an exhaustive description of such combinations in this specification is solely for the sake of space and resource conservation. Therefore, the present invention is not limited to the specific embodiments disclosed herein, but includes all technical solutions falling within the scope of the claims.

Claims

1. A method for preparing a chidamide targeted M2 macrophage delivery system, characterized in that: The following steps are involved: S1: Dissolve distearoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG2000-Mal) in methanol to prepare a 5-20 mg / mL solution; S2: M2 macrophage targeting peptide (M2pep) was dissolved in PBS-ACN buffer at pH 6-7 to prepare a solution with a concentration of 0.5-2 mmol / L; S3: Add the M2pep solution and tris(2-carbonylethyl)phosphine hydrochloride (TCEP) solution prepared in S2 to the DSPE-PEG2000-Mal methanol solution prepared in S1, and stir at room temperature for 40-50 h to mix uniformly to form a suspension; S4: dialyzing the suspension prepared in S3 in distilled water for 40-50 hours to remove free peptides, obtaining a synthetic product, distearoylphosphatidylethanolamine-polyethylene glycol-M2 macrophage targeting peptide (DSPE-PEG-M2pep), which was dissolved in D2O for later use; further, the dialysis membrane had a molecular weight cutoff of ≥3500 Da; S5: DSPE-PEG-M2pep was integrated into the membrane of extracellular vesicles (i.e., EVs), loaded with chidamide (i.e., Chid), and incubated at 35-38°C for 2.5-4 hours. Subsequently, the mixture was ultracentrifuged at 0-4°C to remove unbound DSPE-PEG-M2pep to obtain Chid-loaded M2 macrophage-targeting peptide-modified extracellular vesicles (i.e., Chid@M2pep-EVs); S6: First, the Chid@M2pep-EVs prepared in S5 were mixed with polyvinyl alcohol (i.e., PVA) solution at a volume ratio of 1.8 to 2.2:1, and then an equal volume of N1-(4-bromobenzyl)-N3-(4-bromophenyl)-N1,N1,N3,N3-tetramethylpropane-1,3-diamine (i.e., TSPBA) solution was added to the mixture, and the pH value was adjusted to 9 to 10. The mixture was mixed and the pH value was adjusted to 9 to 10 to prepare Chid@M2pep-EVs / TP hydrogel. Alternatively, the Chid@M2pep-EVs prepared in S5 are first mixed with the PVA solution in a volume ratio of 1.8 to 2.2:1, and then a 3-aminophenylboronic acid-hyaluronic acid copolymer (i.e., HA-PBA) solution with an equal volume to the PVA solution is added, the pH value is adjusted to 9 to 10, and the mixture is mixed to prepare the Chid@M2pep-EVs / HP hydrogel.

2. The method for preparing the M2 macrophage-targeted delivery system of chidamide according to claim 1, characterized in that: In S3, the volume ratio of the M2pep solution: DSPE-PEG2000-Mal solution: TCEP solution is 1:1:0.03-0.05; further preferably, the volume ratio of the M2pep solution: DSPE-PEG2000-Mal solution: TCEP solution is 1:1:0.

04.

3. The method for preparing the M2 macrophage-targeted delivery system of chidamide according to claim 1, characterized in that: In S5, the mass ratio of DSPE-PEG-M2pep:EVs:Chid is 1:1:1~5.

4. The method for preparing the M2 macrophage-targeted delivery system of chidamide according to claim 1, characterized in that: In S5, the centrifugal speed is 100,000 to 150,000×g, and the centrifugal time is 60 to 80 minutes.

5. The method for preparing the M2 macrophage-targeted delivery system of chidamide according to claim 1, characterized in that: In S6, the concentration of the PVA is 8 to 12 m / v%.

6. The method for preparing the M2 macrophage-targeted delivery system of chidamide according to claim 1, characterized in that: In S6, the concentration of TSPBA is 4 to 6 m / v%.

7. A targeted M2 macrophage delivery system of chidamide, characterized in that: The product is prepared by the method according to any one of claims 1 to 6.

8. Use of the M2 macrophage-targeted delivery system of chidamide according to claim 7 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that The anti-tumor use is selected from any one or more of lymphoma, leukemia, and breast cancer; further, the lymphoma is diffuse large B-cell lymphoma.

10. The use according to claim 8 or 9, characterized in that The anti-tumor effect is to reduce the dosage and / or prolong the drug action time.

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