Preparation method and application of apoptotic neutrophile granulocyte membrane fused HG106 liposome

Abstract

The invention discloses a preparation method and application of an apoptotic neutrophile granulocyte membrane fused HG106 liposome, and relates to the technical field of pharmaceutical preparations, and the technical key points are as follows: an apoptotic neutrophile granulocyte membrane (ANM) is fused with the HG106 liposome (HG106-ANM at Lip), nanoparticles are disguised into apoptotic bodies through the apoptotic neutrophile granulocyte membrane, and the apoptotic neutrophile granulocyte membrane is fused with the HG106 liposome (HG106-ANM at Lip). An eat me signal on a cell membrane of the apoptotic body enables the nanoparticles to be more easily taken by the dendritic cells; the HG106-ANM-coated Lip promotes the interment effect of the dendritic cells through simulation of apoptotic bodies and targeted inhibition of the SLC7A11, and an important theoretical basis is laid for developing a safe and efficient new scheme for wound treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to a preparation method and application of apoptotic neutrophil membrane-fused HG106 liposome. Background Art

[0002] Chronic wounds and large wounds pose significant clinical challenges due to their complex pathophysiology and poor healing outcomes. The excessive inflammatory response caused by a large number of cell apoptosis during wound repair is the key issue that makes these wounds difficult to heal or even fail to heal. Effeminate is the process by which professional phagocytes such as dendritic cells and macrophages engulf and internalize apoptotic cells. It is considered a key link in the wound healing process. The process of phagocyte clearance of apoptotic cells is crucial for alleviating inflammatory responses and promoting tissue repair. Unfortunately, in chronic wounds and large wounds, the effeminate function of phagocytes is impaired. Previous reports have suggested that the reason may be that excessive inflammatory response limits the effeminate function of phagocytes. Apoptotic cells that are not cleared in time eventually trigger a strong inflammatory response. Therefore, strengthening the clearance of apoptotic cells may break this vicious cycle and promote wound repair.

[0003] Dendritic cells are not only key antigen-presenting cells but also play a crucial role in the efferocytosis process. Under normal physiological conditions, the body continuously produces apoptotic cells, which contain a large number of self-antigens. Dendritic cells eliminate these apoptotic cells through efferocytosis, preventing the release and accumulation of self-antigens, maintaining autoimmune tolerance, and preventing the occurrence of autoimmune diseases. Recent studies have emphasized the role of dendritic cells in wound healing, particularly their ability to regulate immune homeostasis and promote tissue regeneration. The efferocytosis process of dendritic cells accelerates wound repair by regulating the inflammatory environment, releasing repair factors, and activating regenerative pathways. Therefore, enhancing the efferocytosis ability of DCs may be a promising therapeutic strategy for chronic wound healing.

[0004] Studies have reported that SLC7A11 acts as a molecular brake on dendritic cell efferocytosis, and that drug inhibition or genetic knockout of SLC7A11 can enhance dendritic cell efferocytosis. Furthermore, SLC7A11 is abnormally overexpressed in diabetic mouse wound models, and targeted inhibition of SLC7A11 can effectively reduce excessive accumulation of apoptotic cells in wounds and accelerate wound healing. However, topical application of existing small molecule inhibitors of SLC7A11, such as erastin and sulfasalazine, may affect protein synthesis and redox balance in normal cells or tissues.

[0005] Therefore, the present invention aims to provide a method for preparing apoptotic neutrophil membrane-fused HG106 liposomes and its application to solve the above problems. Summary of the Invention

[0006] The purpose of the present invention is to solve the above problems and provide a preparation method and application of apoptotic neutrophil membrane-fused HG106 liposomes. HG106-ANM@Lip promotes dendritic cell efferocytosis by mimicking apoptotic bodies and targeted inhibition of SLC7A11, thereby reshaping the inflammatory microenvironment and enhancing the efficiency of apoptotic cell clearance, laying an important theoretical foundation for the development of new safe and efficient wound treatment solutions.

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

[0008] The present invention provides a method for preparing apoptotic neutrophil membrane-fused HG106 liposomes, the method comprising the following steps:

[0009] S1. Mouse bone marrow neutrophils were isolated from the bone marrow of male C57BL / 6 mice;

[0010] S2. The isolated neutrophils were used to obtain neutrophil membranes using a cell membrane extraction kit; first, 5 μM staurosporine was used to co-incubate mouse bone marrow neutrophils for 3 hours to induce neutrophil apoptosis. After collecting the suspended cells, the cell suspension was transferred to an appropriately sized ice-bath pre-cooled glass homogenizer and homogenized for about 30-50 s until 70-80% of the cells had no perinuclear halo and intact cell morphology, indicating that the cells had been fully broken. Subsequently, the cell nuclei and unbroken cells were removed by centrifugation at 700 g for 10 minutes at 4°C, and the supernatant was collected. The cell membrane fragments were precipitated by centrifugation at 14,000 g for 30 minutes at 4°C to resuspend and collect the cell membrane protein concentration.

[0011] S3. Dissolve 30 mg of soybean lecithin, 10 mg of cholesterol, and 10 mg of DSPE-PEG-2000 in 5 ml of chloroform to prepare solution 1. Then dissolve 5 mg of HG106 in 10 mL of tetrahydrofuran to prepare solution 2. Take 2 ml of each of the above two solutions and 4 ml of chloroform and add them to a round flask. Rotary evaporate at 40°C to form a thin film. Then add 1 ml of sterile PBS to the flask and sonicate in an ice-water bath for 20 minutes to obtain HG106 liposomes (HG106@Lip).

[0012] S4. Cell membranes were mixed with HG106@Lip at a mass ratio of 1:10. The mixed liposomes were then passed through 200 nm and 100 nm polycarbonate porous membrane filters 11 times using an extruder. Free drug not encapsulated by the liposomes was then removed using dialysis tubing (MWCO = 3500 Da) to synthesize HG106-ANM@Lip.

[0013] In S1, neutrophils from male C57BL / 6 mice were isolated and verified by flow cytometry. In S3, HG106-ANM@Lip was prepared by thin film hydration method.

[0014] An application of HG106 liposomes fused with apoptotic neutrophil membranes. The HG106-ANM@Lip can be used to prepare a drug for treating chronic wounds and large-area wound healing. The "eat me" signal on the HG106-ANM@Lip promotes the recognition and uptake of HG106-ANM@Lip by dendritic cells, enhancing the ability of BMDCs to phagocytose apoptotic cells in vitro, regulating the inflammatory environment, releasing repair factors, and activating regenerative pathways to accelerate wound repair.

[0015] Compared with the existing technology, this solution has the following beneficial effects:

[0016] This invention utilizes a novel inhibitor, HG106, for targeted intervention, demonstrating superior biosafety compared to traditional drugs. Leveraging the unique advantages of apoptotic neutrophil membrane (ANM) biomimetic nanocarriers, the system integrates the dual mechanisms of PS-mediated phagocytosis enhancement and CXCR1 / 2 inflammation targeting to create a precise delivery system. By targeting SLC7A11 and synergizing with the biomimetic carrier, this strategy not only mitigates the risk of exogenous apoptotic cells increasing the efferocytosis burden, but also reshapes the inflammatory microenvironment and enhances apoptotic cell clearance, providing a dual intervention pathway for reversing the "inflammation-repair imbalance" in chronic wounds. This organic combination of molecular regulation and biomimetic delivery lays an important theoretical foundation for the development of safe and effective new wound treatment options. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the preparation method in an embodiment of the present invention;

[0018] Figure 2 1. It is a material preparation flow chart and mechanism diagram in an embodiment of the present invention;

[0019] Figure 3 Schematic diagram of the characterization and biosafety of HG106-ANM@Lip in an embodiment of the present invention;

[0020] Figure 4 Schematic diagram of the cellular uptake efficiency of HG106-ANM@Lip in an embodiment of the present invention;

[0021] Figure 5 Schematic diagram of the in vitro cell-stimulating effect of HG106-ANM@Lip in an embodiment of the present invention;

[0022] Figure 6 This is a schematic diagram of the changes in BMDCs after HG106-ANM@Lip intervention in an embodiment of the present invention;

[0023] Figure 7 Schematic diagram of the characterization of HG106-ANM@Lip hydrogel and its effect on promoting wound healing in diabetic mice according to an embodiment of the present invention;

[0024] Figure 8 Schematic diagram of the histological manifestation of HG106-ANM@Lip promoting diabetic wound healing in mice according to the embodiment of the present invention;

[0025] Figure 9 This is a schematic diagram of the effect of HG106-ANM@Lip in promoting wound healing in Bama pigs in an embodiment of the present invention. DETAILED DESCRIPTION

[0026] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0027] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0028] Example:

[0029] 1. Experimental Results

[0030] Small molecule inhibitors and cell membrane screening:

[0031] In order to screen the best small molecule inhibitors that promote dendritic cell efferocytosis, three SLC7A11 small molecule inhibitors reported in the literature were selected, namely HG106, SAS, and Erastin. The cell model used immortalized bone marrow dendritic cells (MBDCs), which basically have the phagocytic function, cell phenotype, and morphological characteristics of primary bone marrow dendritic cells. HG106 and Erastin were respectively intervened in BMDCs in vitro at concentrations ranging from 0-10μM, and SAS was intervened in 0-1mM to screen the best intervention drugs and intervention concentrations. Flow cytometric analysis results showed that ( Figure 3AC), HG106 has the best effect of promoting BMDC efferocytosis in vitro, and the phagocytosis rate is the highest at a concentration of 5μM. Therefore, 5μM HG106 was used as the intervention condition in subsequent experiments. In addition, in order to further screen the best cell membrane for modifying liposomes, 5 commonly used apoptotic cell membranes of cells that play an important role in wound repair were screened, including apoptotic neutrophil membrane (ANM), apoptotic fibroblast membrane (AFM), apoptotic endothelial cell membrane (AEM), and apoptotic red blood cell membrane (ARBM). Flow cytometry analysis was used to compare the in vitro uptake effect of BMDC on liposomes modified with different apoptotic cell membranes. The results showed that ( Figure 3 DE), within two hours, the fluorescence intensity of the apoptotic neutrophil membrane-modified liposome group was the strongest, indicating that apoptotic neutrophils had the best effect in promoting BMDC uptake.

[0032] Physicochemical properties and safety of HG106-ANM@Lip

[0033] The preparation of HG106-ANM@Lip requires first promoting apoptosis of neutrophils and extracting neutrophil membranes containing surface functional proteins and efferocytosis signals, and then repeatedly extruding them through an extruder, such as Figure 2 As shown in A. First, neutrophils were isolated from the bone marrow of male C57BL / 6 mice, and the purity of the isolated neutrophils was verified to be over 90% by flow cytometry analysis. Neutrophil membranes were obtained using a cell membrane extraction kit, and the protein content in the cell membrane was quantitatively detected using the BCA method. The results showed that approximately 200 million cells could extract about 1 mg of cell membrane protein. HG106@Lip lipids were prepared by a thin film hydration method and fused with ANM by co-extrusion through an extruder. The fused liposomes passed through 200nm and 100nm polycarbonate filter membranes in turn, and the obtained HG106-ANM@Lip was detected by HPLC to have an encapsulation efficiency of 63.51% and a drug loading of 3.02%. Dynamic scattering (DLS) measurements were then used to confirm that the diameter of the fused liposomes remained at approximately 100nm and had a narrow particle size distribution ( Figure 3 H). At the same time, cryo-transmission electron microscopy (TEM) images showed that HG106-ANM@Lip appeared as uniform spheres ( Figure 3 F), and the particle size is consistent with the DLS measurement data. The Zeta potential of HG106@Lip and HG106-ANM@Lip in PBS solution is approximately -40mV and -30mV, respectively. Figure 3G), indicating that the Zeta potential of the liposomes after fusion with the cell membrane has changed to a certain extent, which indirectly verifies the fusion of the cell membrane and the liposomes. In order to further clarify the fusion efficiency of the liposomes and the cell membrane, ANM and HG106@Lip were stained with different fluorescent dyes before fusion, and the fusion efficiency was observed using a fluorescence confocal microscope ( Figure 3 I).

[0034] Finally, the in vitro biosafety of HG106-ANM@Lip was tested through the CKK-8 experiment, and it was found that at a high concentration of 400 μg / ml, BMDCs and L929 cells cultured in vitro still had high biological activity, indicating that HG106-ANM has good biocompatibility and cell safety, providing safety guarantees for subsequent further cell and animal studies.

[0035] In vitro cellular uptake of HG106-ANM@Lip

[0036] Dendritic cells are recruited to the wound surface by the find me signal released by early apoptotic cells in the wound. As apoptosis progresses, the eat me signal in the cell membrane is exposed, mediating dendritic cells to recognize and engulf apoptotic cells. This process is called efferocytosis. Inspired by the natural process of efferocytosis, biomimetic nanomedicines that inherit the eat me signal are more easily recognized and taken up by phagocytes, thereby improving the bioavailability of drugs. Lip, NM@Lip, and HG106@Lip used in in vitro cell uptake experiments were all labeled with the fluorescent dye PKH26. At the same time, Figure 4 As shown in Figure AE, the uptake rate of Lip and NM@Lip by BMDC was only about 50%, and there was no statistical difference between the two groups, while the uptake rate of ANM@Lip group was as high as 95%. In the nanoparticle uptake experiment, the fluorescence intensity of flow cytometry is also an important indicator for evaluating uptake. Figure 4 B Figure 4 In Figure 5, the ANM@Lip group had the highest fluorescence intensity, which was basically consistent with the uptake rate. In addition, representative liposome uptake images were captured using a fluorescence confocal microscope, which directly showed that BMDCs could uptake more HG106-ANM@Lip within a certain period of time. Flow cytometry analysis revealed that BMDC uptake of HG106-ANM@Lip was time-dependent, with the cell uptake rate reaching a peak at 2 hours ( Figure 4 C and Figure 4 G), the fluorescence intensity reached a high level at 2 h, and still increased slightly at 4 h. Figure 4 D and Figure 4 H. Based on the comprehensive results and the consideration of reducing drug cytotoxicity, 2h was finally selected as the intervention time for subsequent cell burial effect research.

[0037] HG106-ANM@Lip enhances dendritic cell efferocytosis in vitro

[0038] During wound repair, a large number of apoptotic cells, including neutrophils and fibroblasts, are produced. Efficiently clearing excessive apoptotic cells from the wound surface is crucial for promoting tissue repair and maintaining wound microenvironmental homeostasis. Diabetic wounds or large wounds produce a large number of apoptotic cells during healing, leading to a relative impairment of phagocyte function. Therefore, it was hypothesized that HG106-ANM@Lip could enhance the ability of BMDCs to phagocytose apoptotic cells in vitro. To verify the in vitro efferocytosis-promoting function of HG106-ANM@Lip, BMDCs were first labeled with dyes for differentiation. BMDCs were then pretreated with different nanoparticles. Subsequently, a 10-fold increase in the number of pretreated and stained Jurkat T cells was added to the BMDC-cultured cell culture plates. After incubation for 2 hours, efferocytosis was analyzed by flow cytometry. The results showed that both ANM@Lip- and HG106@Lip-treated BMDCs exhibited enhanced efferocytosis, as evidenced by an increased phagocytic rate. Compared with ANM@Lip and HG106@Lip, BMDCs treated with HG106-ANM@Lip showed a higher level of efferocytosis. In the same period of time, the phagocytosis rate of apoptotic cells could reach about 50% ( Figure 5 AC). Furthermore, considering that phagocytes can engulf multiple apoptotic cells, we added a method to evaluate phagocytosis by using mean fluorescence intensity, and the results were generally consistent with those described above. Furthermore, to more intuitively demonstrate efferocytosis, we used fluorescence confocal microscopy to observe fluorescence images of apoptotic cells phagocytosed by BMDCs under different treatments. Clearly, HG106-ANM@Lip-treated BMDCs engulfed more apoptotic cells, further confirming that HG106-ANM@Lip synergistically enhances BMDC efferocytosis in vitro.

[0039] Changes in dendritic cell phenotype and migration ability

[0040] By detecting the protein expression levels of MERTK and AXL during dendritic cell burial, we further elucidated the specific mechanism by which HG106-ANM@Lip intervention synergistically enhanced dendritic cell burial. MERTK and AXL are important receptors that play a role in the process of dendritic cell burial. MERTK and AXL mediate the recognition and uptake of apoptotic cells by phagocytes by recognizing phosphatidylserine (PS) exposed on the surface of apoptotic cells. Therefore, their expression levels are indeed closely related to the efficiency of burial. Figure 6As shown in Figure B, the SLC7A11 protein expression of BMDCs after HG106@Lip and HG106-ANM@Lip intervention showed a feedback upregulation, which is consistent with the application characteristics of small molecule channel protein inhibitors. With the functional inhibition of SLC7A11, the in vitro efferocytosis of dendritic cells was enhanced, which was manifested at the protein level as an increase in the expression of two important receptor proteins, MERTK and AXL, such as Figure 6 As shown in CD, both ANM@Lip and HG106@Lip enhance MERTK and AXL protein expression, consistent with predicted results. Furthermore, HG106-ANM@Lip combines the functions of apoptotic neutrophil membranes and small molecule inhibitors, synergistically promoting MERTK and AXL protein expression through apoptotic signaling and SLC7A11 inhibition, which is consistent with previous results on apoptotic cell phagocytosis.

[0041] In addition, upregulation of MERTK and AXL expression can activate the PI3K-Akt / STAT3 downstream pathway, leading to upregulation of IL-10 / TGF-β gene expression. In wound repair, the anti-inflammatory effect mediated by IL-10 / TGF-β can limit excessive inflammatory response, maintain wound inflammation homeostasis, and promote wound healing. Therefore, RT-qPCR was used to detect the gene changes of BMDCs after intervention to verify this hypothesis. Figure 6 As shown in EF, similar to the WB results, HG106-ANM@Lip promotes the upregulation of the anti-inflammatory factor IL-10 gene through a synergistic effect. In addition, HG106@Lip can also promote the upregulation of TGF-β genes, while ANM@Lip does not seem to have this effect. Interestingly, although ANM@Lip cannot promote the upregulation of BMDCs' genes IL-1 and IL-6, HG106-ANM@Lip still shows a synergistic effect between apoptotic neutrophil membranes and HG106, as shown by significantly higher IL-1 and IL-6 gene expression in BMDCs treated with HG106-ANM@Lip than in the HG106@Lip treatment group. This may be because the efferocytosis signal on the surface of apoptotic neutrophils initiates early efferocytosis and activates the expression of pro-inflammatory factors in order to recruit more phagocytes such as dendritic cells for efferocytosis.

[0042] Early apoptotic cells release find me signals (ATP / UTP) to attract dendritic cells to migrate to the site of injury. Therefore, in order to further explore whether apoptotic neutrophil membranes can still have the chemotactic function of dendritic cells, the changes in the in vitro migration function of BMDCs after intervention were explored through the trasnwell cell experiment. Figure 6As shown in Figure 1J, neutrophil membrane-modified liposomes have the effect of promoting BMDCs migration, which may be because their surface inherits neutrophil-related cell chemokines, such as CXCL1 and CX3CL1, which recruit more BMDCs. In addition, the neutrophil membrane after apoptosis treatment has increased apoptosis-related signaling molecules, such as eat-me signals, and may also retain some find-me signals such as ATP / UTP. These synergistic chemokines further enhance the BMDCs recruitment ability of nanoparticles.

[0043] Material characterization of hydrogels

[0044] In order to better apply to in vivo experiments, hydrogels have shown great potential in wound repair due to their high drug loading, controlled release and good biocompatibility. A PLGA-PEG-PLGA thermosensitive hydrogel was used to carry HG106-ANM@Lip. After loading HG106-ANM@Lip, the thermosensitive hydrogel can still automatically gel at 25°C and has good adhesion after gelation ( Figure 7 A), and then the morphological characteristics of the hydrogel before and after loading HG106-ANM@Lip were observed by scanning electron microscopy. There was no obvious change, and spherical HG106-ANM@Lip( Figure 7 B) Drug release curve was detected by HPLC. Figure 7 It can be observed in C that the drug in the hydrogel is released by nearly 40% within 3 hours, and the liposomes are quickly released into the wound surface, initiating endocytosis and recruiting dendritic cells. The release reaches about 70% within 24 hours, and then the remaining drug is slowly released over the next two days. Similar to the release curve, it was observed through in vivo fluorescence imaging of mice that the fluorescence intensity of the hydrogel loaded with stained HG106-ANM@Lip decreased by about 50% within 24 hours and slowly decreased over the next two days. Compared with the directly injected stained HG106-ANM@Lip, the fluorescence intensity of the hydrogel group was higher within 72 hours, and the fluorescence image showed that it was more concentrated in the wound surface and had higher bioavailability ( Figure 7 DE), the hydrogel was used in subsequent animal experiments to carry different groups of nanoparticles for group research.

[0045] In vivo study on promoting diabetic wound healing in mice

[0046] Based on the excellent performance of HG106-ANM@Lip in promoting cell burial in vitro, combined with safe and efficient hydrogel materials, a chronic wound model was established using diabetic mice. The specific wound membrane formation and treatment process is shown in Figure 7F. First, STZ was injected intraperitoneally to establish a diabetic mouse model. After the blood glucose level of the mice stabilized above 16.7 mmol / L, a circular full-thickness wound with a diameter of 6 mm was made on the back of the mice, and the intervention groups were divided into groups. Figure 7 G is the representative healing situation of each group within 14 days of membrane formation intervention, and the wound healing superposition diagram ( Figure 7 H) and wound area heat map ( Figure 7 I) It can be more intuitively seen that the group loaded with HG106-ANM@Lip hydrogel has a faster healing rate and is basically healed on the 14th day. Compared with the PBS group, Gel group, ANM@Lip group, and HG106@Lip group, the wound healing speed of mice in the HG106-ANM@Lip group is significantly faster, especially on the 7th day. This shows that HG106-ANM@Lip plays an important role in promoting diabetic wound healing ( Figure 7 J). In addition, the wound healing was analyzed by histological staining, and the tissue was stained with H&E on the 14th day after the operation ( Figure 8 A), the results showed that the wound length of the HG106-ANM@Lip group was the shortest compared with the PBS group, Gel group, ANM@Lip group, and HG106@Lip group ( Figure 8 B), and Masson staining was used to evaluate collagen deposition in wound tissue on day 14 ( Figure 8 C), the results showed that the relative collagen deposition rate of the wound surface in the HG106-ANM@Lip group was higher than that in the PBS group, Gel group, ANM@Lip group, and HG106@Lip group, and was closer to the collagen ratio of normal tissue ( Figure 8 D). In order to verify whether HG106-ANM@Lip accelerates wound healing by promoting cell burial in vivo, the efficiency of cell burial in wounds was explored by various methods. First, immunofluorescence staining was performed on the wound tissue on day 3 ( Figure 8 E) The number of apoptotic cells in the wound was assessed, and the apoptosis status of the wound was analyzed by the total fluorescence intensity of Cleaved Caspase-3. Cleaved Caspase-3 is a marker molecule for cell apoptosis, and its expression level indirectly reflects the degree of cell apoptosis in the wound. Statistical results showed that the total fluorescence intensity representing apoptosis signals in the wound of the HG106-ANM@Lip group was the lowest compared with the PBS group, Gel group, ANM@Lip group, and HG106@Lip group, indicating that the number of apoptotic cells in the wound was the lowest and the clearance rate of apoptotic cells by efferocytosis was the highest ( Figure 8 F). In addition, the in vivo burial effect was verified at the protein level by WB experiments ( Figure 8G), the results showed that HG106-ANM@Lip and HG106@Lip feedback upregulated the expression of SLC7A11 protein in the wound surface ( Figure 8 H), which proved that HG106-ANM@Lip and HG106@Lip had significant inhibitory effects on the SLC7A11-dominated pathway. The expression of Cleaved Caspase-3 protein showed that the expression of Cleaved Caspase-3 protein in the wound of HG106-ANM@Lip group was the lowest compared with that of PBS group, Gel group, ANM@Lip group and HG106@Lip group ( Figure 8 I). In addition, compared with other groups, the expression of MERTK protein in the HG106-ANM@Lip group was significantly increased, which also verified the significant increase in the efficiency of cell burial in the wound ( Figure 8 Finally, the biosafety of our nanoformulation was investigated by histological staining of mouse internal organs. The results showed that it did not cause significant toxicity or damage to important organs such as the liver and kidneys.

[0047] Enhance wound healing in Bama pigs

[0048] In order to further explore the in vivo application effect of HG106-ANM@Lip and lay a solid foundation for clinical research and application, HG106-ANM@Lip was applied to the wounds of Bama pigs. Since the structure of pig skin is similar to that of humans, and the wound healing process is basically the same as that of humans, studies have shown that the repair process of large-area wounds requires a longer time and more complex physiological processes, which are very similar to chronic wounds, and there is also an excessive load of apoptotic cells. Therefore, the pig large wound model is a good alternative model for chronic wound healing. Similarly, a full-thickness defect wound with a diameter of 3 cm was established on the back of the pig, and a 21-day observation was carried out after membrane formation ( Figure 9 A), and the experiment was divided into three groups: Control group, recombinant human epidermal growth factor group (RHEGF), and HG106-ANM@Lip loaded hydrogel group. First, the wound healing status was observed for 21 days and representative wound photos were recorded ( Figure 9 B) and color images of wound surfaces at multiple time points ( Figure 9 C), in addition, the heat map of wound area statistics can more clearly and intuitively show that the HG106-ANM@Lip group has a better effect in promoting wound healing ( Figure 9 E). Statistical analysis showed that ( Figure 9 D), at the observation time points of 7 days, 14 days and 21 days, the wound healing rate of the HG106-ANM@Lip group was significantly higher than that of the Control group and the RHEGF group. In addition, H&E staining was performed on the tissues on the 7th and 14th days ( Figure 9F), the growth rate of granulation tissue in the early stage of wound healing (day 7) was analyzed. The results showed that the thickness of granulation tissue in the wound of HG106-ANM@Lip group was significantly higher than that in the other two groups, almost consistent with the depth of the wound ( Figure 9 H). Statistical analysis of wound epidermal coverage was performed on H&E-stained sections 14 days later. The results showed that the wound surface of the HG106-ANM@Lip group was completely covered by regenerated epidermis, while the wound surface of the Control group and the RHEGF group still had a long area of skin loss ( Figure 9 I). Masson staining of wound tissue on day 21 was used to analyze the collagen deposition ratio of the new tissue. The statistical results showed that

[0049] The average collagen deposition rate of HG106-ANM@Lip relative to normal tissue reached about 80%, which was significantly higher than 60% of the Control group and 40% of the RHEGF group.

[0050] 2. Discussion

[0051] Wound healing is a complex and multi-stage biological process involving three key stages: inflammation, proliferation, and remodeling. Numerous studies have shown that the inflammatory response in wounds is inseparable from the repair process. Moderate inflammation helps to eliminate pathogens and damaged cells, but excessive or persistent inflammation can lead to tissue damage and delayed healing. In chronic wounds and large-area wounds, such as diabetic wounds, cell apoptosis generally increases, and the failure to clear apoptotic cells in a timely manner (i.e., impaired efferocytosis) further exacerbates local inflammation, causing the wound to fall into a vicious cycle of "inflammation-repair imbalance."

[0052] The present invention deeply analyzes this pathological mechanism, focusing on the key role of dendritic cells (DC) in the clearance of apoptotic cells. Under normal circumstances, DC not only clears apoptotic cells through burial, avoids the release of self-antigens and abnormal immune activation, but also regulates the local microenvironment and promotes tissue regeneration. However, in chronic or diabetic wounds, excessive apoptotic cells overload DC, and coupled with the interference of the local inflammatory environment, its burial function is significantly inhibited. In recent years, SLC7A11 has been identified as a "molecular brake" that regulates the DC burial process. Studies have shown that abnormally high expression of SLC7A11 in diabetic wounds will inhibit the effective phagocytosis of apoptotic cells by DC, further hindering wound repair.

[0053] To break this vicious cycle, the present invention utilizes a novel small molecule inhibitor, HG106, to precisely target SLC7A11 to deregulate the negative regulation of DC efferocytosis. Compared to traditional drugs (such as Erastin and SAS), HG106 at 5 μM not only exhibits a significant advantage in promoting efferocytosis but also exhibits superior biosafety, mitigating adverse effects on normal cellular protein synthesis and redox homeostasis. This molecular intervention strategy offers a novel approach for regulating local inflammation and promoting the clearance of apoptotic cells.

[0054] On the other hand, the present invention utilizes apoptotic neutrophil membranes (ANM) to modify liposomes and construct the HG106-ANM@Lip biomimetic nanodelivery system. ANM not only retains the inflammatory tropism of neutrophils (mainly mediated by CXCR1 / 2 receptors), but also promotes DC recognition and uptake of nanoparticles through the "eat me" signals (such as phosphatidylserine, PS) exposed on its surface. In vitro flow cytometry analysis and confocal imaging both showed that ANM modification significantly improved the uptake rate of nanoparticles in DC, and the combined effect of HG106 increased the ability of BMDCs to phagocytose apoptotic cells by about 50%, indicating that the two have a synergistic effect in promoting cell burial.

[0055] Furthermore, to achieve localized, sustained drug release and minimize systemic side effects, the present invention further incorporates HG106-ANM@Lip into a thermosensitive PLGA-PEG-PLGA hydrogel. This hydrogel rapidly gels at 25°C and exhibits excellent adhesion and biocompatibility. In vitro and in vivo drug release curves and in vivo fluorescence imaging data confirm that it maintains high concentrations of the nanoformulation locally at the wound surface, thereby optimizing the therapeutic effect.

[0056] In in vivo experiments, HG106-ANM@Lip demonstrated significant healing-promoting effects in both diabetic mouse models and Bama pig large wound models. Indicators such as wound healing rate, re-epithelialization, granulation tissue formation, and collagen deposition were significantly superior to those in the control group and single-treatment groups (using only ANM@Lip or HG106@Lip). Histological analysis showed that the expression of apoptosis markers (such as Cleaved Caspase-3) in the wounds of the HG106-ANM@Lip-treated group was lowest, while the expression of key receptor proteins (such as MERTK) was significantly upregulated, further validating the significant advantages of this nanosystem in enhancing DC efferocytosis and reshaping the local inflammatory microenvironment.

[0057] 3. Conclusion

[0058] This study, exploring the pathological mechanisms, reveals that limited SLC7A11-mediated dendritic cell efferocytosis in chronic wounds is a key factor contributing to the inflammation-repair imbalance. By targeting SLC7A11 with the novel small molecule inhibitor HG106 and constructing a biomimetic nanopharmaceutical using liposomes modified with apoptotic neutrophil membranes, this approach achieves a dual intervention: effectively relieving SLC7A11's negative regulation of efferocytosis and enhancing DC clearance of apoptotic cells; and further enhancing DC uptake by promoting neutrophil chemotaxis and "eat me" signaling. Combined with a PLGA-PEG-PLGA thermosensitive hydrogel-based localized delivery system, this approach achieves sustained, concentrated drug release at the wound site, significantly accelerating the healing process in diabetic and large-area wounds. Overall, this strategy not only provides an innovative approach for chronic wound treatment but also lays a solid theoretical and practical foundation for the future development of safe and effective wound repair solutions.

[0059] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for preparing apoptotic neutrophil membrane-fused HG106 liposomes, characterized by: The method comprises the following steps: S1. Mouse bone marrow neutrophils were isolated from the bone marrow of male C57BL / 6 mice; S2. Using a cell membrane extraction kit to obtain neutrophil membranes from the isolated neutrophils; S3. Dissolve 30 mg of soybean lecithin, 10 mg of cholesterol, and 10 mg of DSPE-PEG-2000 in 5 ml of chloroform to prepare solution 1. Dissolve 5 mg of HG106 in 10 ml of tetrahydrofuran to prepare solution 2. Take 2 ml of each of the above two solutions and 4 ml of chloroform and add them to a round flask. Rotary evaporate at 40°C to form a thin film. Then, add 1 ml of sterile PBS to the flask and sonicate in an ice-water bath for 20 minutes to obtain HG106@Lip. S4. Neutrophil membranes and HG106@Lip were mixed in a mass ratio of 1:10, and the mixed liposomes were passed through 200 nm and 100 nm polycarbonate porous membrane filters using an extruder. The above filtration operation was repeated several times. Subsequently, dialysis tubing was used to remove free drugs not encapsulated by liposomes to synthesize HG106-ANM@Lip.

2. The method for preparing apoptotic neutrophil membrane-fused HG106 liposomes according to claim 1, wherein: Neutrophils isolated from male C57BL / 6 mice were validated by flow cytometry analysis as described in S1.

3. The method for preparing apoptotic neutrophil membrane-fused HG106 liposomes according to claim 1, wherein: The specific steps of S2 are: incubating mouse bone marrow neutrophils with 5 μM staurosporine for 3 hours to induce neutrophil apoptosis; collecting the suspended neutrophils and transferring the cell suspension to a pre-cooled glass homogenizer in an ice bath, and homogenizing for about 30-50 seconds until 70-80% of the neutrophils have no perinuclear halo and intact cell morphology; Subsequently, the cells were centrifuged at 700 g for 10 minutes at 4°C to remove neutrophil nuclei and unbroken neutrophils, and the supernatant was collected. The cells were centrifuged at 14,000 g for 30 minutes at 4°C to precipitate neutrophil membrane fragments, which were resuspended in pure water and collected to measure the cell membrane protein concentration.

4. The method for preparing apoptotic neutrophil membrane-fused HG106 liposomes according to claim 1, wherein: In S3, HG106-ANM@Lip was prepared by a thin film hydration method.

5. The use of the apoptotic neutrophil membrane-fused HG106 liposome according to claim 1, characterized in that: The HG106-ANM@Lip can be used to prepare medicines for treating chronic wounds and healing large-area wounds.

6. The use of the apoptotic neutrophil membrane-fused HG106 liposome according to claim 5, characterized in that: The eat me signal on HG106-ANM@Lip can promote the recognition and uptake of HG106-ANM@Lip by dendritic cells, enhance the ability of BMDC to phagocytize apoptotic cells in vitro, regulate the inflammatory environment, release repair factors, activate regeneration pathways and accelerate wound repair.

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