Bioengineering apoptotic body based on apoptotic body membrane protein and liposome as well as preparation method and application of bioengineering apoptotic body

Abstract

The invention discloses a bioengineering apoptotic body based on apoptotic body membrane protein and lipidosome as well as a preparation method and application of the bioengineering apoptotic body. The bioengineered apoptotic body is formed by chimeric combination of apoptotic body membrane protein and liposome loaded with gastrodin and Cy5. The bioengineered apoptotic body for simulating the natural apoptosis process of the mesenchymal stem cells is constructed, microglial cells can be promoted to be polarized to an M2 type, anti-inflammatory factors and neurotrophic factors are released, neuronal proliferation is promoted, the cell oxidation resistance is improved, the neuromotor function is improved, and encephaledema is relieved, so that traumatic brain injury is effectively treated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a bioengineered apoptotic body based on apoptotic body membrane protein and liposome, and a preparation method and application thereof. Background Art

[0002] Traumatic brain injury (TBI) causes brain tissue destruction and significant neurological impairment, resulting in a poor prognosis for most patients. Current clinical treatment strategies for TBI include surgery, hyperbaric oxygen therapy, functional electrical stimulation, and rehabilitation. However, these strategies primarily focus on symptom relief and survival. In contrast, the recovery of brain function in TBI patients relies primarily on the body's own repair mechanisms. During TBI, damage to the cerebrovascular system disrupts the blood-brain barrier, allowing immune cells, including macrophages, neutrophils, and lymphocytes, to infiltrate the brain parenchyma. Damaged neurons are unable to maintain oxidative stress homeostasis, resulting in the production of reactive oxygen species (ROS). This activates immune cells, further damaging neurons. Ultimately, the inflammatory cascade in the damaged brain tissue intensifies, indirectly leading to neuronal death and neurodegeneration. Promptly addressing TBI-induced neuroinflammation is crucial to prevent the persistence of a chronic inflammatory cascade and the resulting impairment of brain function. Therefore, a key challenge in TBI treatment lies in controlling the progression of neuroinflammation and minimizing secondary injury.

[0003] Microglia are crucial cells in the central nervous system, playing a crucial role in maintaining homeostasis in the brain's microenvironment. Upon TBI, microglia rapidly activate and transform into distinct reactive phenotypes. Similar to peripheral macrophages, microglia are generally classified into two types: classically activated M1 microglia and alternatively activated M2 microglia. As key players in neuroinflammation after TBI, microglia rapidly activate into the M1 type and secrete numerous proinflammatory cytokines and chemokines, driving the progression of the neuroinflammatory response. In contrast, M2 microglia primarily phagocytose and clear cellular waste products. Furthermore, they exhibit anti-inflammatory and neuroprotective properties. Therefore, developing TBI therapies that modulate microglial phenotypes could not only alleviate neuroinflammation but also promote neurorepair and improve neurological function.

[0004] Mesenchymal stem cells (MSCs) promote the polarization of M2 microglia by paracrine secretion of cytokines and metabolites, including TGF-β, IL-10, PGE2, and IDO. Transplanted MSCs have a low survival rate, and most undergo apoptosis in vivo. Following MSC apoptosis, they release bilayer lipid membrane vesicles, known as apoptotic bodies, which encapsulate organelle fragments and genetic material. Simultaneously, "eat-me" signals (such as calreticulin) flip from the inner to the outer layer of the cell membrane and become anchored to the apoptotic cell membrane and the surface of apoptotic bodies, attracting specific recognition and phagocytosis by macrophages. The process by which macrophages engulf and eliminate apoptotic cell products (apoptotic bodies) is called efferocytosis. During efferocytosis, macrophages polarize toward the M2 microglia, thereby clearing apoptotic cells while protecting surrounding healthy cells.

[0005] Recently, different types of bioengineered apoptotic bodies have been developed for the treatment of myocardial infarction and Parkinson's disease by enhancing the cytotoxic and anti-inflammatory effects of macrophages. However, to our knowledge, no studies have evaluated the therapeutic effects and biological mechanisms of MSC-derived apoptotic bodies in TBI. Summary of the Invention

[0006] In view of the above technical problems to be solved, the purpose of the present invention is to provide a drug that can effectively treat traumatic brain injury and promote neurological recovery.

[0007] In order to achieve the above object of the invention, the present invention provides a bioengineered apoptotic body based on apoptotic body membrane protein and liposome, which is formed by chimerizing apoptotic body membrane protein with liposome loaded with gastrodin and Cy5.

[0008] According to a preferred embodiment, the apoptotic body membrane protein is derived from mesenchymal stem cells.

[0009] According to a preferred embodiment, the apoptotic body membrane protein is obtained by inducing mesenchymal stem cells with staurosporine and extracting the membrane protein.

[0010] According to a preferred embodiment, the liposome loaded with gastrodin and Cy5 is obtained by adding gastrodin and Cy5 to a lipid membrane made of DSPC, DSPE-TK-PEG2000 and cholesterol.

[0011] According to a preferred embodiment, the molar ratio of DSPC, DSPE-TK-PEG2000 and cholesterol is (50-70):(3-8):(30-40), more preferably 60:5:35.

[0012] According to a preferred embodiment, the mass of gastrodin and Cy5 accounts for 1-3% and 0.02-0.08% of the mass of the lipid membrane, respectively, more preferably 2% and 0.05%, respectively.

[0013] According to a preferred embodiment, it is characterized in that the mass ratio of the apoptotic body membrane protein to the liposome loaded with gastrodin and Cy5 is 1:50 to 1:150, more preferably 1:100.

[0014] On the other hand, the present invention also provides a method for preparing the bioengineered apoptotic body based on apoptotic body membrane protein and liposome, which comprises: embedding the apoptotic body membrane protein into liposomes loaded with gastrodin and Cy5 by mechanical extrusion to obtain the bioengineered apoptotic body.

[0015] On the other hand, the present invention also provides the use of the bioengineered apoptotic body based on apoptotic body membrane protein and liposome in the preparation of a drug for treating traumatic brain injury.

[0016] On the other hand, the present invention also provides the use of the bioengineered apoptotic bodies based on apoptotic body membrane proteins and liposomes in the preparation of drugs for promoting brain injury recovery, wherein the promotion of brain injury recovery includes any one or more of the following: reducing neuroinflammation, improving brain motor function, promoting neuronal proliferation, promoting neural repair, inhibiting microglial activation, promoting M2 microglial polarization, improving cell antioxidant capacity, reducing brain tissue ROS levels, reducing neuronal apoptosis and necrosis in the brain, and alleviating brain edema.

[0017] This invention constructs a bioengineered apoptotic polysome (ALP) that mimics the natural apoptosis process of MSCs. It can regulate the release of anti-inflammatory factors from microglia and promote neuronal proliferation, potentially enabling the polarization of M2 microglia to treat TBI. ALP is prepared by conjugating the blood-brain barrier-penetrating and targeted apoptotic polysome membrane protein (ApoM) with liposomes loaded with gastrodin and Cy5. Gastrodin (GAS), a glucoside analogue extracted from the rhizome of Asparagus cochinchinensis, stimulates neuronal proliferation and inhibits microglial activation in inflammatory environments. Its release in microenvironments high in reactive oxygen species (ROS) stimulates neuronal proliferation, while ApoM promotes microglial polarization toward the M2 phenotype, releasing anti-inflammatory and neurotrophic factors. In this invention, gastrodin and ApoM are responsively released in microenvironments high in reactive oxygen species (ROS), exerting therapeutic and regulatory functions. Furthermore, both gastrodin and ApoM inhibit the activation of the NF-κB signaling pathway in microglia. In vivo studies have shown that ALP effectively reduces neuroinflammation and improves motor function and blood perfusion in TBI model mice by activating the M2 polarization of microglia in the brain. This study proposes a new strategy to repair brain damage by effectively activating the immune response of microglia. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of ALP treatment for TBI. (a) Preparation of ROS-responsive bioengineered apoptotic bodies (ALP); (b) Microenvironment of the brain of a TBI mouse: Elevated ROS at the injury site not only leads to neuronal apoptosis but also activates glial cells to transform into the M1 phenotype, releasing proinflammatory cytokines to amplify the injury, further activating the inflammation-related NF-κB signaling pathway and exacerbating the inflammatory response; (c) Mechanism of action of ALP treatment for TBI: Gastrodin (GAS) reduces ROS levels and stimulates neuronal proliferation; ApoM regulates the transformation of microglia into the M2 phenotype, releasing anti-inflammatory and neurotrophic factors to promote repair; GAS and ApoM also inhibit activation of the NF-κB signaling pathway to alleviate inflammation.

[0019] Figure 2 Figure 2 shows the biochemical characterization of ALP. (a) Schematic diagram of the collection of MSC-derived apoptotic bodies (Apo); (b) Apo morphology under optical microscopy (scale bar = 4 μm); (c) Expression of CRT protein (green) on the surface of apoptotic MSCs and Apo (enlarged image); (d) Transmission electron microscopy images of LP and ALP (scale bar = 50 nm); (e) CRT and Na in LP, ALP, and ApoM. + -K +Western blotting analysis of ATP protein; (f) Protein SDS-PAGE analysis of LP, ALP, ApoM, Apo, and MSCM; (g) Particle size distribution and zeta potential of LP and ALP (inset); (h) Drug release characteristics of ALP to ROS; (i) Particle size stability of LP and ALP over 7 days.

[0020] Figure 3 ALP promotes neuronal proliferation in vitro. (a, b) Flow cytometry (a) and fluorescence microscopy (b) were used to detect the uptake of LP and ALP by N2a cells at different time points (scale bar = 10 μm); (c) Transwell assay was used to analyze the efficiency of LP and ALP crossing the blood-brain barrier in vitro (left: schematic diagram of the in vitro BBB model); (d) Wound wound assay was used to analyze the proliferation efficiency of N2a cells after different treatments; (e) JC-1 fluorescence staining of N2a cells in each group (scale bar = 20 μm); (f) H-induced cytotoxicity in ROS-damaged N2a cells. (g) Flow cytometry analysis of the effects of different treatment groups on the expression of CD206 in BV2 microglia; (h) qRT-PCR quantitative analysis of the expression of various proteins in LPS-induced BV2 cells after different treatments; (i) Survival rate of ROS-damaged N2a cells after co-culture with M2-type BV2 cells; (j) Expression of NF-κB signaling pathway-related proteins P-P65, P65, and IKβα in BV2 cells after different treatments.

[0021] Figure 4 Figure 3 shows the in vivo therapeutic effect of ALP on TBI mice. (a) Metabolic distribution of MSCs, LP, and ALP at different time points in TBI mice (a) and fluorescence semi-quantitative analysis (b); (c) Schematic diagram of ALP treatment in vivo; (d) H&E and Nissl staining of brain tissue sections from TBI mice on day 10 after different treatments (scale bar = 250 μm); (ef) OCT imaging of blood flow changes at the injury site in TBI mice (e) and quantitative analysis of vascular density (f) (scale bar = 1 mm); (g) Water content in brain tissue of TBI mice on day 5 after different treatments; (hi) SOD activity (h) and MDA levels (j) in brain tissue of TBI mice on day 10.

[0022] Figure 5Figure 2 shows behavioral testing of TBI mice using ALP. (a) Schematic diagram of the behavioral testing timeline; (b) mNSS assesses changes in motor, sensory, balance, and reflex function in TBI mice; (cf) OFT assesses locomotor ability and emotional behavior in TBI mice (c), including average speed (d), center-zone distance traveled (e), and total distance traveled (f); (gk) Catwalk system assesses motor function in TBI mice, primarily analyzing changes in gait pattern (g) and four dynamic metrics: base of support width (h), footprint position (i), swing speed (k), and stride length (j).

[0023] Figure 6 The immune mechanism of ALP treatment for TBI is shown. (af) Effects of different drugs on the distribution of immune cells in the brain tissue of TBI mice and their fluorescence semi-quantification (M2 microglia marker CD206 (a) and quantitative analysis (b); neutrophil marker Ly6G (c) and quantitative analysis (d); Treg cell marker FoxP3 (e) and quantitative analysis (f); (g) Immunofluorescence staining of the injury site of TBI mice after different treatments (apoptosis (TUNEL), glial cells (Iba1), M1 microglia (TNFα), and M2 microglia (CD206); scale bar = 50 μm); (hm) ELISA detection of inflammatory cytokines (TGFβ (h), IL-6 (i), TNF-α (j)) and neurotrophic factors (NGF (k), GDNF (l), BDNF (m)) in brain tissue.

[0024] Figure 7 Figure 2 shows RNA sequencing analysis of ALP-treated TBI mice. (a) Venn diagram of abnormally expressed genes in the ALP, LP, and TBI groups; (b) Volcano plot of abnormally expressed genes in the ALP and TBI groups; (c) Volcano plot of abnormally expressed genes in the ALP and LP groups; (d) Gene heatmap of the ALP, LP, and TBI groups; (e) KEGG enrichment analysis of abnormally expressed genes in the ALP and TBI groups; (f) Western blot analysis of NF-κB signaling pathway proteins in the ALP and TBI groups; (g) GO analysis of abnormally expressed genes in the ALP and TBI groups; (h) GO analysis of abnormally expressed genes in the ALP and LP groups. DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0026] 1 Method 1.1 Extraction of apoptotic bodies (Apo) and membrane proteins (ApoM) Apoptotic bodies (Apo) derived from mesenchymal stem cells (MSCs) were obtained by induction with staurosporine (50606ES10, Yisheng Biotechnology). MSCs were incubated with 0.5 μM staurosporine for 6 hours, and the serum-free culture supernatant was collected. Cell debris was removed by centrifugation at 50g and 4°C for 5 minutes, followed by centrifugation at 1000g and 4°C for 15 minutes to collect the apoptotic bodies (Apo). The Apo suspension was smeared and its morphology was observed under a light microscope.

[0027] To extract apo membrane protein (ApoM), Apo was lysed on ice for 1 hour in TM buffer (0.01M Tris and 0.001M MgCl2) containing protease inhibitors. The supernatant was collected by centrifugation at 100g and 4°C for 10 minutes, followed by centrifugation at 10,000g and 4°C for 10 minutes to collect the precipitate. The precipitate was resuspended in ddH2O and stored at -80°C. ApoM concentration was determined using a BCA assay kit (P0010, Beyotime).

[0028] 1.2 Apo surface CRT detection Immunofluorescence staining was used to investigate calreticulin (CRT) expression on the surface of apo mesenchymal stem cells. MSCs were treated with staurosporine for various durations and then fixed with 4% paraformaldehyde (P0099, Beyotime) for 20 minutes. After 1 hour of blocking buffer treatment, anti-CRT antibody (27298-1-AP, Proteintech, 1:200 dilution) was added and incubated overnight at 4°C. After washing twice with PBS, the cells were incubated with Alexa Fluor 488-conjugated secondary antibody (A0423, Beyotime) for 1 hour at room temperature in the dark. After three washes with PBS, the cells were counterstained with DAPI (C1006, Beyotime). After washing with PBS, CRT expression on the surface of apo mesenchymal stem cells was examined under a fluorescence microscope.

[0029] 1.3 Preparation and characterization of ALP Preparation: ROS-responsive liposomes (LPs) were prepared using the Bangham and freeze-thaw methods. DSPC (distearylphosphatidylcholine, 850365C, Avanti), DSPE-TK-PEG2000 (distearylphosphatidylethanolamine-thioketal-polyethylene glycol 2000, R-D526, Xi'an Ruixi Biotechnology), and cholesterol (C8667, Sigma) were dissolved in chloroform at a molar ratio of 60:5:35. The mixture was dried under nitrogen to form a uniform film and then vacuum-dried for 3 hours to obtain a lipid membrane. A 2 mL PBS solution containing 200 μg / mL gastrodin (PHL89324, Sigma) and 5 μg / mL Cy5 (146368-11-8, MCE) was added, with the mass of gastrodin and Cy5 accounting for 2% and 0.05% of the lipid membrane mass, respectively. After five freeze-thaw cycles, LPs with uniform particle size were obtained by liposome extrusion. ApoM-coated LP (ALP) was prepared with a mass ratio of LP to ApoM of 100:1. The fluidity of the liposomal phospholipid bilayer was utilized to embed ApoM into the mobile phase using an extruder (610000, Avanti) (Reference: Xiaorui Geng et al., Active-Targeting NIR-II Phototheranostics in Multiple Tumor Models Using Platelet-Camouflaged Nanoprobes, ACS Appl Mater Interfaces . 2020, 12(50):55624-55637.).

[0030] Characterization: The zeta potential and size distribution of the nanoparticles (NPs) were determined using a dynamic light scattering instrument (Nano ZS90, Malvern). The morphology of the NPs was observed by transmission electron microscopy (TEM) after negative staining with 2% phosphotungstic acid (P4006, Sigma). Surface protein expression on the NPs was analyzed by SDS-PAGE and western blotting. The absorption and emission spectra of ALP were measured by UV-visible spectrophotometry and fluorescence spectrophotometry, respectively. The ALP drug release curve was plotted based on Cy5 absorbance.

[0031] 1.4 Cell culture C57BL / 6J mouse bone marrow mesenchymal stem cells (MSCs) were purchased from Saiye Biotechnology. The cells were expanded in an incubator using DMEM / F12 medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco). The MSCs used in this study were within passage 15. Neuro-2a (N2a) cells and bEnd.3 cells were purchased from ATCC, and BV2 cells were purchased from the Shanghai Institutes for Biological Sciences. All three cell lines were expanded using DMEM supplemented with 10% FBS (Gibco).

[0032] 1.5 Cytotoxicity assay All cell viability experiments were performed using the Cell Counting Kit-8 (CCK-8) (C0041, Beyotime).

[0033] Establishment of in vitro reactive oxygen species (ROS) damage model: N2a cells were seeded in 96-well plates and cultured for 12 hours. The culture medium was then replaced with serum-free medium and incubated with different concentrations of H2O2 (H1009, Sigma) for 12 hours.

[0034] Gastrodin (Gas)-promoting N2a cell proliferation assay: N2a cells were seeded in 96-well plates and cultured for 12 hours. The culture medium was then replaced with serum-free medium and incubated with 0.02 μM H₂O₂ for 12 hours. The supernatant was discarded and the cells were replaced with serum-free medium containing various concentrations of gastrodin and incubated for another 12 hours.

[0035] M2 microglia stimulate N2a cell proliferation: As described above, BV2 cells pre-cultured for 24 hours were treated with varying concentrations of IL-4 (SRP3211, Sigma) instead of gastrodin. For the control group, an equal number of blank N2a cells were co-cultured with the corresponding proportions of pre-treated BV2 cells.

[0036] ALP biocompatibility test: N2a cells and bEnd.3 cells were seeded in 96-well plates and cultured for 12 hours. The culture medium was then replaced with serum-free medium and incubated with different concentrations of ALP for 12 hours. The cell viability was detected using the CCK8 kit.

[0037] 1.6 ALP cellular uptake experiment Flow cytometry: N2a and BV2 cells were seeded in 24-well plates and cultured for 12 hours. The culture medium was then replaced with serum-free medium containing LP and ALP. At different time points, the cells were washed twice with PBS and then digested and harvested. Fluorescence intensity was measured by flow cytometry. Flow cytometric data were analyzed using FlowJo software.

[0038] Confocal laser scanning microscopy (CLSM) observation: As described above, the 24-well plate was replaced with an 8-well chamber slide suitable for CLSM (154534, Thermo).

[0039] 1.7 ALP in vitro blood-brain barrier (BBB) ​​translocation assay An in vitro BBB model was established using Transwell chambers (354541, Corning). bEnd.3 cells were cultured in the upper layer and N2a cells in the lower layer. When the cell density in the upper layer reached 95%, serum-free culture medium containing LP or ALP was replaced. Flow cytometry was used to measure the fluorescence intensity of cells in the lower layer to analyze the ability of LP and ALP to cross the BBB in vitro.

[0040] 1.8 Cell scratch assay A wound wound assay was used to simulate mechanical cell injury, and drug efficacy was assessed by wound area. N2a cells were seeded in 24-well plates and cultured for 12 hours. Wounds were then created using a scratcher (201925, SPL), and images were acquired under a light microscope. Subsequently, 5 μg / ml gastrodin (Gas), 100 ng / ml apoptotic bodies (Apo), and LP and ALP at concentrations equivalent to free gastrodin were added, and the cells were incubated for 24 hours. Images were then acquired again. Untreated scratched cells served as a control group, and wound healing area was measured using Image J software to assess the ability of ALP to promote N2a cell proliferation.

[0041] 1.9 Evaluation of ALP's ROS scavenging ability N2a cells were incubated with H2O2 (0.03 μM) and drugs (gastrodin, apoptotic bodies, LP, and ALP) for 12 hours. Untreated cells served as controls. They were then incubated with 10 μM DCFH-DA (S0033M, Beyotime) for 30 minutes, washed twice with PBS, and the level of ROS scavenging by ALP was assessed by flow cytometry using fluorescence intensity.

[0042] Mitochondrial membrane potential assay (JC-1, C2003S, Beyotime): Mitochondrial membrane potential can reflect the early stages of cell apoptosis. N2a cells were stained with JC-1 for 20 minutes, and then confocal laser scanning microscopy (CLSM) was used to observe cellular oxidative damage.

[0043] 1.10 Western blotting Cells were treated on ice for 1 minute with lysis buffer (P0037, Beyotime) containing 1% PMSF (ST506, Beyotime) to collect proteins. Tissue samples were ground and then protein extracted. Sample loading concentrations were standardized based on the results of the BCA assay kit, and proteins were separated by SDS-PAGE electrophoresis (Bio-Rad). Proteins were transferred to a PVDF membrane using a transfer system (Bio-Rad) and blocked for 1 hour at room temperature. Primary antibodies were added for overnight incubation at 4°C, followed by three 5-minute washes in TBST. Secondary antibodies (170-6515, Bio-Rad) were incubated for 2 hours at room temperature, and the membranes were washed again three times in TBST. ECL chemiluminescent substrate (1705060, Bio-Rad) was added for development. Images were acquired using a Bio-Rad imaging system, and quantitative analysis was performed using Image J software.

[0044] 1.11 BV2 cell polarization assay BV2 cells were seeded in 24-well plates and cultured for 12 hours. The culture medium was then replaced with serum-free medium containing 100 ng / ml LPS (L2630, Sigma) and drugs (gastrodin, apoptotic bodies, LP, and ALP, at the same doses as above) for an additional 24 hours. Cells were harvested, treated with Fc blockers, stained with a CD206 antibody (Supplementary Table 3), and analyzed by flow cytometry.

[0045] 1.12 Real-time quantitative PCR (qRT-PCR) Total RNA was extracted from BV2 cells using Trizol reagent (15596018, Invitrogen), and cDNA was prepared using the iScript™ cDNA Synthesis Kit (1708891, Bio-Rad). Amplification was performed using a SYBR Green SuperPremix Kit (64477446, Bio-Rad) on a real-time fluorescence quantitative PCR instrument (CFX96, Bio-Rad), using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal reference gene. -ΔΔCT The relative expression of each gene was calculated using the PCR method. All primers were synthesized by BGI, and their sequences are shown in Supplementary Table 1. Table 1. Primer sequences

[0046] 1.13 Experimental Animals Male C57BL / 6 mice (8–10 weeks old, 26–30 g) were purchased from the Animal Experimentation Center, Faculty of Health Sciences, University of Macau. All experimental procedures complied with animal welfare standards and were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Macau.

[0047] A moderate traumatic brain injury (TBI) model was established: Mice were anesthetized with 1.5% isoflurane gas, and the skull was exposed. A 3 mm diameter circular window was drilled through the dura mater without damaging it using a skull drill (78001, RWD). A moderate TBI model was established using a precision impactor (68099II, RWD) with standard parameters: impact velocity = 4 m / s, depth = 1.0 mm, and dwell time = 0.4 s. After injection of 2% Evans blue via the tail vein, the extent of blood-brain barrier damage was assessed by observing brain penetration at the injury site.

[0048] 1.14 Biodistribution of ALP in vivo Nine TBI mice were randomly divided into three groups and injected with LP, ALP, or MSCs via the tail vein 2 hours after model establishment. The biodistribution of these MSCs was observed at different time points using a small animal in vivo imaging system.

[0049] 1.15 ALP therapeutic effect in vivo Forty mice with TBI were randomly divided into five groups (control, Gas, Apo, LP, and ALP). Eight untreated mice served as a normal control group. Drugs were administered via the tail vein on days 1, 3, and 5 after model establishment. Mice were sacrificed on day 10 and brain tissue was paraffin-embedded. The therapeutic effects were assessed by H&E and Nissl staining.

[0050] 1.16 Optical Coherence Tomography Optical coherence tomography (OCT) was used to acquire vascular signals at the injury site. As previously described, blood flow signals were collected after craniotomy (labeled "Pre") and on days 1, 5, and 10 after modeling. Cerebral vascular density was quantified using MATLAB software.

[0051] 1.17 Brain Edema Detection On day 5 after treatment, mice (n = 3) were sacrificed and brain tissue was removed for water content determination. Fresh brain tissue was weighed first, followed by dehydrated brain weight after drying at 65°C for 24 hours.

[0052] The formula for calculating brain water content is as follows: .

[0053] 1.18 Superoxide dismutase (SOD) activity and malondialdehyde (MDA) level detection Ten days after treatment, TBI mice were sacrificed, and brain tissue superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels were measured. Brain tissue was mixed with SOD sample preparation solution and homogenized on ice. The mixture was then centrifuged at 12,000 g at 4°C for 5 minutes. The supernatant was collected and assayed for SOD activity using the WST-8 assay (S0101M, Beyotime). MDA levels were assayed (S0131S, Beyotime) according to the kit instructions.

[0054] 1.19 Behavioral Testing Another 40 TBI mice were randomly divided into five groups (control, Gas, Apo, LP, and ALP) for behavioral testing. Behavioral experiments were performed on the TBI mice on days 3, 10, and 20 after different treatments.

[0055] mNSS scoring: The behavior of TBI mice was scored using a randomized double-blind method according to a standardized scale.

[0056] Open field test: The experiment was conducted in a 50 cm × 50 cm × 40 cm open field chamber. The activity trajectory of TBI mice was recorded using a digital video camera over an 8-minute period. Data such as total distance traveled, average speed, and central area activity distance were analyzed using Smart 3.0 software.

[0057] Catwalk gait analysis: Mice were trained daily for three consecutive days before the experiment. TBI mice were acclimated to a dark environment for 1 hour before each experiment. Gait data were collected using the Noldus Catwalk system, and four dynamic metrics (average distance between forelimbs and hindlimbs, average span between left and right limbs, swing velocity, and stride length) were analyzed based on the mice's gait behavior.

[0058] 1.20 Brain Tissue Cytokine Detection Ten days after treatment, TBI mice were sacrificed and brain tissue was collected. The injured area was excised, trimmed, and digested with collagenase to obtain a single-cell suspension. The supernatant was collected by centrifugation and assayed for cytokines (TGFβ, TNFα, and IL-6) and neurotrophic factors (NGF, GDNF, and BDNF) using ELISA kits. The pelleted cells were used for flow cytometry analysis. Blocking agent was added to the pelleted single-cell suspension, and flow cytometry staining with antibodies was performed. Staining with the Foxp3 antibody required membrane permeabilization (00-5523-00, Thermo).

[0059] 1.21 Immunofluorescence staining As described in the "In Vivo Effects of ALP Treatment" section above, immunofluorescence staining was performed on paraffin sections. First, apoptosis in brain tissue sections was analyzed using the One Step TUNEL Apoptosis Detection Kit. Subsequently, IF staining was performed: paraffin sections were deparaffinized and antigen retrieval performed, followed by serum blocking for 1 hour. Primary antibodies against Iba1, TNFα, and CD206 were added, and the sections were incubated overnight at 4°C. After washing three times with TBS, fluorescently labeled secondary antibodies were added and incubated for 1 hour. Following counterstaining with DAPI, sections were observed under a fluorescence microscope.

[0060] 1.22 RNA sequencing of TBI mice On day 5 after TBI, brain tissue was harvested from mice after perfusion with normal saline and snap-frozen in liquid nitrogen for 5 seconds. The samples were sent to Beijing Novogene Technology Co., Ltd. for RNA-Seq library preparation, and the data were analyzed using https: / / ap-magic.novogene.com / .

[0061] 1.23 Biocompatibility of ALP Twelve male C57BL / 6 mice were randomly divided into two groups for in vivo biosafety testing. Six untreated mice served as a control group. Blood samples were collected 14 days after tail vein injection of LP and ALP for routine blood tests. The biocompatibility of the nanoparticles was assessed using paraffin sections of major organs stained with H&E.

[0062] 1.24 Statistical Analysis Statistical analyses were performed using Origin 2022. Differences between groups were assessed using independent-sample t-tests (***P < 0.001; **P < 0.01; *P < 0.05). All statistical analyses were performed using SPSS 26.0 software.

[0063] 2 Results and Discussion 2.1 Preparation and biochemical characterization of ALP First, primary mouse mesenchymal stem cells (MSCs) were treated with staurosporine to induce apoptosis, and apoptotic bodies (Apo) were prepared ( Figure 2 (a) Microscopic observation showed that MSCs-derived apoptotic bodies presented irregular spherical structures ( Figure 2 (b) Dynamic light scattering (DLS) results showed that the particle size distribution of Apo was 1003 ± 22.16 nm. As a typical "eat me" signaling molecule, calreticulin (CRT) is exposed on the outer surface of the cell membrane during apoptosis, attracting macrophages to engulf and promote efferocytosis. Immunofluorescence imaging showed that CRT was highly expressed on the surface of Apo ( Figure 2 c) in the above example.

[0064] The present invention fuses Apo membrane protein (ApoM) with liposomes (LP) loaded with gastrodin (Gas) and Cy5 by mechanical extrusion to prepare engineered apoptotic liposomes (ALP). Transmission electron microscopy (TEM) observations show that both LP and ALP exhibit uniform spherical structures ( Figure 2 Western blotting and SDS-PAGE analysis showed that ALP expressed multiple ApoM proteins, including CRT and Na + -K + ATPase ( Figure 2 ef in ), suggesting that ALP may have similar biological functions to Apo.

[0065] like Figure 2 As shown in Figure g, the particle size distributions of LP and ALP were 169.0 ± 6.0 nm and 163.2 ± 3.1 nm, respectively, with no significant difference in particle size after membrane protein modification. Zeta potential measurements confirmed the successful surface modification of the membrane protein onto the liposomes. Furthermore, loading the liposomes with the molecular dye Cy5 enabled fluorescence imaging of the in vivo distribution of ALP. UV absorption and fluorescence emission spectra detected characteristic peaks of Gas and Cy5 in ALP, confirming successful drug loading.

[0066] The responsiveness of ALP to reactive oxygen species (ROS) was evaluated by drug release experiments. In the presence of hydrogen peroxide (H2O2), ALP was able to rapidly release the drug: the release efficiency reached 42.0±1.6% in 1 hour and 91.7±5.0% in 12 hours ( Figure 2h in the figure). Observation for 7 consecutive days showed that the particle size distribution of ALP remained stable, indicating its excellent stability ( Figure 2 (i in the text).

[0067] 2.2 ALP promotes neuronal proliferation in vitro Calreticulin (CRT) expressed on the membrane of apoptotic bodies can bind to CD91 protein (low-density lipoprotein receptor-related protein) on the surface of microglia and neurons, thereby enhancing the uptake of ALP by microglia and neurons. First, the uptake of ALP by neuronal cells N2a was studied. Figure 3 As shown in Figures ab, ALP exhibited superior cellular uptake ability compared with LP, indicating that ALP can actively target neural cells.

[0068] Subsequently, an in vitro blood-brain barrier (BBB) ​​model was constructed by Transwell assay to evaluate whether ALP has the ability to cross the BBB similar to MSCs ( Figure 3 (c) In this experiment, endothelial cells (bEnd.3) were seeded in the upper chamber, and neuronal cells (N2a) were seeded in the lower chamber. LP and ALP were added to the upper chamber, and the fluorescence intensity of the cells in the lower chamber was measured. The results showed that ALP actively crossed the BBB at a significantly higher rate than LP.

[0069] To simulate the high ROS microenvironment in damaged brain tissue, an in vitro ROS injury model was established using H2O2-induced neuronal injury. At a H2O2 concentration of 0.02 μM, the cell survival rate was 63.04±4.37%, and this concentration was therefore selected to establish the in vitro ROS injury model. Gastrodin (Gas), a glucoside extracted from the traditional Chinese medicine Asparagus cochinchinensis, possesses natural anti-inflammatory, antioxidant, and neuroprotective properties. Gas can enhance the survival rate of ROS-injured neurons; treatment with 5 μg / ml of gastrodin increased the survival rate of injured neurons by 23.86±8.41%.

[0070] The mechanical damage of N2a cells was simulated by scratch test. Figure 3 As shown in Figure d, compared with the control group (4.95±0.08%), the survival rate of N2a cells increased to 60.47±1.56% after ALP treatment, indicating that ALP can significantly promote neuronal cell proliferation.

[0071] In order to verify the ROS scavenging effect of ALP, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect the intracellular ROS level. The results showed that the intracellular ROS level of N2a cells increased after H2O2 treatment, while ALP treatment could significantly reduce the intracellular ROS level. The intracellular ROS level is closely related to mitochondrial function: increased ROS levels will lead to a decrease in mitochondrial membrane potential and impaired mitochondrial function. The JC-1 fluorescent probe was used to evaluate the mitochondrial membrane potential: under normal mitochondrial membrane potential, JC-1 forms aggregates in the matrix and emits red fluorescence; when the membrane potential decreases, free JC-1 exhibits green fluorescence. Figure 3 As shown in Figure e, ALP treatment can restore the mitochondrial membrane potential of ROS-damaged N2a cells, indicating that ALP can play a role by inhibiting mitochondrial damage caused by high cellular ROS levels.

[0072] To explore the biological mechanism by which ALP protects against ROS damage, we examined the expression of antioxidant and anti-apoptotic proteins in neuronal cells. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor. After cells are damaged by ROS, Nrf2, originally located in the cytoplasm, translocates to the nucleus, upregulating the expression of heme oxygenase-1 (HO-1), the product of which has antioxidant activity.

[0073] In addition, overexpression of B lymphocytoma-2 (Bcl-2) can reduce the generation of oxygen free radicals and the formation of lipid peroxides, thereby inhibiting cell apoptosis. Western blotting results showed that ALP treatment can upregulate the protein expression of Nrf2, HO-1, and Bcl-2 in ROS-damaged neuronal cells, indicating that ALP not only upregulates HO-1 expression by activating Nrf2, but also induces Bcl-2 overexpression to protect cells from oxidative damage-induced apoptosis ( Figure 3 f in ).

[0074] In addition, ALP can also indirectly promote neuronal proliferation by regulating microglia. Under physiological conditions, microglia have functions such as immune regulation, neurotrophic and maintaining homeostasis. It was found that ALP can also actively target microglia, because CD91 is also highly expressed on the surface of microglia to bind to CRT on apoptotic bodies. The elevated ROS level in the brain after TBI will cause microglia to polarize to the M1 type, aggravating the inflammatory response and damaging neurons; while Apo can promote the repolarization of M2 microglia through efferocytosis to inhibit inflammation. Flow cytometry results showed that ALP treatment can upregulate the expression of the M2 marker CD206 in microglia, indicating that ALP can enhance the polarization of M2 microglia ( Figure 3 g in the .

[0075] The effects of different treatments on the expression of phenotypic markers and cytokine / neurotrophic factor secretion levels of LPS-pretreated microglia were further analyzed by qRT-PCR. The results showed that ALP treatment significantly downregulated the expression of M1 marker CD86, while upregulated the expression of M2 markers CD163 and CD206. More importantly, ALP treatment significantly increased the secretion of anti-inflammatory cytokines (IL-10, TGF-β) and neurotrophic factors (NGF, GDNF, BDNF), and reduced the release of pro-inflammatory cytokines (IL-6, IL-1β). Figure 3 h in the text).

[0076] The CCK-8 kit was used to analyze the effect of M2 microglia on ROS-damaged neurons. When the ratio of neurons to microglia was 2:1, the survival rate of damaged neurons increased from 65.78±3.22% to 86.71±7.56%. This neuroregenerative effect may be attributed to the neurotrophic and anti-inflammatory factors secreted by M2 microglia ( Figure 3 (i in the text).

[0077] Nuclear factor κB (NF-κB) is an important nuclear transcription factor in cells that participates in the body's inflammatory response, immune response, and apoptosis regulation. It is a classic ROS-related signaling pathway. ALP may exert its anti-inflammatory effects by affecting the NF-κB signaling pathway. Protein immunoblotting analysis showed that the expression of P65 and P-P65 in microglia decreased after ALP treatment, indicating that the NF-κB signaling pathway activated by H2O2 was inhibited; at the same time, the expression of IKβα increased, suggesting that more activated NF-κB returns to a resting state through negative feedback regulation ( Figure 3 in the j).

[0078] 2.3 In vivo therapeutic effect of ALP on TBI mice The biodistribution of ALP in vivo was further investigated to assess its ability to penetrate the blood-brain barrier and enter the brain tissue of TBI mice. A moderate TBI mouse model was established using a precision impactor. Following tail vein injection of Evans blue dye, the mice were sacrificed and brain tissue was removed to assess blood-brain barrier leakage. The results showed that Evans blue dye could penetrate the brain injury site, indicating that the blood-brain barrier of TBI mice was disrupted.

[0079] Subsequently, an in vivo imaging system was used to evaluate the biodistribution of MSCs, LP, and ALP in TBI mice. Fluorescence imaging showed that MSCs could be delivered to the brain 72 hours after intravenous injection; LP could reach the brain region 12-24 hours after injection, which may be due to the enrichment of ALP at the injury site through the enhanced permeability retention effect (EPR effect) after blood-brain barrier damage; and ALP could be rapidly delivered to the TBI injury site, reaching peak accumulation at 12 hours and remaining in the brain for a long time (about 96 hours). It is worth noting that the peak fluorescence intensity of ALP was significantly stronger than that of MSCs and LP, indicating that the modification of the apoptotic body membrane significantly enhanced the targeting of ALP to TBI ( Figure 4 ab in).

[0080] Based on the results of drug biodistribution in vivo, TBI mice were treated with three doses, each with an interval of 48 hours. Figure 4 As shown in c. The therapeutic effect was evaluated by H&E staining and Nissl staining of brain tissue sections on day 10. Figure 4 As shown in (d), H&E staining revealed that compared with the normal control group, the cortical pyramidal cells in the TBI control group were scattered and reduced in size, with numerous degenerated neurons and irregular nuclei. In contrast, the cortical pyramidal cells in the ALP group were neatly arranged and morphologically normal, with significantly less neuronal necrosis. Nissl staining revealed that the number of Nissl bodies in the ALP group was significantly higher than that in the TBI control group, suggesting that ALP can effectively reduce neuronal apoptosis and necrosis in the brains of TBI mice.

[0081] Optical coherence tomography (OCT) was further used to monitor the changes in cerebral blood vessels in TBI mice during treatment. Figure 4 As shown in Figure ef, the vascular density in the brain injury area of ​​mice in the ALP treatment group increased significantly from 30.66±1.2% on the initial day to 143.44±0.8% on the 10th day, indicating that ALP can promote cerebral angiogenesis, thereby improving the nutritional supply of damaged brain tissue.

[0082] In addition, brain tissue was taken on the fifth day after treatment to measure water content to assess brain edema. The brain water content of each group was: 81.11±2.62% in the control group, 78.96±2.37% in the Gas group, 78.57±1.35% in the Apo group, 78.36±1.66% in the LP group, 77.81±0.877% in the ALP group, and 77.89±0.426% in the normal group. ALP can significantly alleviate the symptoms of brain edema after TBI ( Figure 4 g in the .

[0083] The level of ROS in the brain was assessed by detecting the activity of superoxide dismutase (SOD) and malondialdehyde (MDA) in brain tissue. Compared with the control group, the ALP group showed a significant increase in SOD enzyme activity and a significant decrease in MDA level, indicating that ALP can effectively reduce the level of ROS in brain tissue after TBI ( Figure 4 in the text).

[0084] 2.4 Assessment of neurological function recovery in TBI mice The neurological function recovery of TBI mice after treatment was evaluated by modified neurological deficit score (mNSS), open field test (OFT) and Catwalk gait analysis system. Neurological function of TBI mice was evaluated on the 3rd, 10th and 20th days after model establishment ( Figure 5 mNSS results showed that the neurological function scores of mice in the ALP treatment group were significantly lower than those in the control group ( Figure 5 b) in the above example.

[0085] Then, based on the movement trajectory of the mice, OFT was used to evaluate the motor ability and emotional behavior of TBI mice ( Figure 5 Compared with the control group, the TBI mice in the ALP-treated group showed a higher average speed ( Figure 5 d in), longer total movement distance ( Figure 5 f) and the increased central region activity distance ( Figure 5 These results indicate that ALP treatment can improve the motor ability and autonomous exploratory behavior of TBI mice and promote the recovery of motor function.

[0086] The Catwalk system can analyze gait changes caused by motor impairment and pain in TBI mice during natural walking. This experiment focused on analyzing changes in gait patterns and four dynamic indicators (base of support width, footprint position, swing speed, and stride length). The control group mice showed gait abnormalities due to reduced motor coordination ( Figure 5 g in the figure), characterized by a widening of the distance between the front and rear limbs ( Figure 5 h in), the distance between the left and right footprints increases ( Figure 5 i in), shortened stride length ( Figure 5 j in) and the slowing of the swing speed ( Figure 5 In contrast, the gait pattern of the ALP-treated mice was almost the same as that of the normal mice, and all dynamic indices were better than those of the control group, suggesting that ALP may enhance the motor coordination ability of the mice.

[0087] 2.5 Immune Mechanisms of ALP Therapy Microglia are immune cells that are intrinsic to the central nervous system. Studies have shown that regulating the polarization of M2 microglia can promote anti-inflammatory responses and neural repair. To clarify the mechanism of action of ALP, the levels of immune cells in the brains of TBI mice were quantitatively analyzed by flow cytometry. Figure 6 As shown in ab, ALP treatment significantly increased the number of M2 microglia (F4 / 80 + CD11b + CD206 + ) ratio, while reducing the M1 microglia (F4 / 80 + CD11b + CD80 + ) ratio, indicating that ALP can promote the polarization of microglia from M1 to M2.

[0088] Under the pathological state of TBI, a large number of neutrophils infiltrating the brain tissue will aggravate the inflammatory response and brain edema. Flow cytometry analysis showed that the neutrophils (CD45 + CD11b + Ly6G + ) levels were significantly lower than those in the control group, which helps to reduce the secondary injury of TBI ( Figure 6 Regulatory T cells (Treg) are CD4 + A subset of T cells whose main function is to suppress excessive immune response in the inflammatory microenvironment. Compared with the control group, the Treg cells (CD4 + Foxp3 + ) ratio, which helps to inhibit excessive inflammatory response at the TBI injury site ( Figure 6 ef in).

[0089] Immunofluorescence staining was used to further evaluate neuronal apoptosis and microglial activation. Figure 6 As shown in g, the green fluorescence intensity of TdT-mediated dUTP nick end labeling (TUNEL) in brain tissue sections of the ALP group decreased ( Figure 6 g, row 1), indicating that neuronal apoptosis in the injured area was significantly reduced. In addition, ALP treatment can inhibit the excessive activation of microglia to alleviate early inflammatory damage: microglial activation was enhanced in the injured area of ​​the control group, while ALP treatment can alleviate its excessive activation state ( Figure 6 More importantly, ALP treatment significantly reduced the staining intensity of M1 microglia (TNFα) and significantly increased the staining intensity of M2 microglia (CD206) in the brains of TBI mice ( Figure 6 g in , lines 3-4).

[0090] Serum cytokine detection showed that the level of pro-inflammatory cytokines (TNF-α) in TBI mice was significantly reduced, while the levels of anti-inflammatory cytokines (TGFβ, IL-6) were significantly increased after ALP treatment ( Figure 6 hj in); At the same time, the levels of neurotrophic factors (NGF, GDNF, BDNF) also increased significantly ( Figure 6 These results suggest that ALP effectively inhibits the development of neuroinflammation after TBI by promoting M2 microglial polarization and improving the immune microenvironment.

[0091] 2.6 RNA sequencing analysis of brain tissue after ALP treatment Neuroinflammation triggered by TBI is a key factor contributing to poor patient outcomes. Microglia-mediated immune cascades play a crucial role in the pathogenesis of neuroinflammation following TBI. Experimental studies have shown that ALP exerts anti-inflammatory effects and promotes neurological recovery by inducing microglial polarization toward the M2 type. However, no previous studies have explored the molecular mechanisms of apoptotic bodies in treating TBI. Therefore, RNA sequencing (RNA-seq) was used to analyze gene expression profiles and signaling pathway activation in brain tissue from mice with TBI.

[0092] Wayne analysis of differentially expressed genes showed that there were 485, 408, and 420 specific expressed genes in the ALP, LP, and TBI groups, respectively, and 12,300 common expressed genes ( Figure 7 a in ). Figure 7 As shown in (b), compared with the TBI group, 292 genes were significantly down-regulated and 379 genes were significantly up-regulated after ALP treatment. However, compared with the LP group, ALP treatment only resulted in the down-regulation of 257 genes ( Figure 7 (c) The difference in down-regulated genes may be due to the therapeutic effect of apoptotic body membrane proteins on the surface of ALP.

[0093] The differentially expressed genes in the ALP, LP, and TBI groups were analyzed using cluster heat maps. It was found that the expression of genes related to apoptosis (CASP8, CASP9, FADD, and CASP12), genes related to the Toll-like receptor signaling pathway (MYD88, TIRAP, and TICAM2), and genes related to the NF-κB signaling pathway (P50, IKKα, CK2, and P65) were significantly downregulated ( Figure 7 The decreased expression of these genes indicated that ALP treatment not only reduced cell apoptosis but also inhibited the inflammatory response through activation of the Toll-like receptor signaling pathway and the NF-κB signaling pathway.

[0094] Western blot results showed that the expression levels of NF-κB signaling pathway-related proteins (including P-P65, P-Ikkβ, and Ikβα) in the brain tissue of TBI mice were reduced after ALP treatment ( Figure 7 In addition, KEGG enrichment analysis showed that compared with the TBI group, the differentially expressed genes in the ALP group were mainly involved in inflammatory response-related signaling pathways, protein metabolism, cell adhesion, and calcium homeostasis regulation. Among them, the TNF signaling pathway, NF-κB signaling pathway, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway are key regulatory pathways for the inflammatory process and immune response ( Figure 7 e in the above example).

[0095] Gene ontology (GO) analysis showed that compared with the TBI or LP groups, the differentially expressed genes in the ALP group were mainly characterized by changes in receptor ligand activity, hormone, chemokine, and organic acid binding properties in terms of molecular function (MF); changes in receptor complexes, extracellular matrix, inflammation-related complexes, and synapse-related genes in terms of cellular components (CC); and significant changes in cell secretion, leukocyte-mediated immune response, immune system processes, calcium homeostasis, blood circulation, and neuropeptide signaling pathways in terms of biological processes (BP). Figure 7 The difference in therapeutic effects between the ALP and LP groups may be due to the fact that the modification of the apoptotic body membrane leads to different in vivo metabolic processes, antioxidant damage activities, immunomodulatory abilities, and effects on the inflammatory response process of the nanomedicine.

[0096] These results indicate that the most important mechanism of ALP in treating TBI is to maintain the homeostasis of the brain immune system by inhibiting inflammation-related signaling pathways including NF-κB, thereby alleviating secondary damage caused by the development of neuroinflammation after TBI.

[0097] 2.7 Biocompatibility of ALP The biocompatibility of ALP was evaluated at both the cellular and animal levels. At the cellular level, bEnd.3 and N2a cells were incubated with varying concentrations of ALP (0-2 mg / ml) for 12 hours and then their viability was measured. The results showed no significant decrease in the viability of either endothelial cells or neurons, indicating that ALP has no significant toxic effects on normal cells.

[0098] TBI mice in each treatment group were sacrificed 14 days after dosing. Blood was collected for complete blood counts, and major organs were harvested for histopathological examination using hematoxylin and eosin (H&E) staining. The results showed no statistically significant differences in hematological parameters between the LP and ALP groups compared with the normal mice. Furthermore, no significant morphological changes were observed in organ tissue sections from the LP and ALP groups. Therefore, the ALP developed in this invention is a biocompatible nanodrug suitable for the treatment of TBI.

[0099] This study proposes an innovative strategy for treating TBI. The bioengineered MSC-derived apoptotic bodies (ALPs) developed here effectively promote neuronal regeneration and regulate microglial polarization. ALPs scavenge ROS through the ROS-responsive release of gastrodin, directly stimulating neuronal proliferation. Furthermore, the apoptotic body membrane coated on the ALP surface promotes M2 microglial polarization and releases anti-inflammatory cytokines and neurotrophic factors through efferocytosis. Figure 1 Schematic diagram of ALP treatment for TBI. (a) Preparation of ROS-responsive bioengineered apoptotic bodies (ALP); (b) Microenvironment of the brain of a TBI mouse: Elevated ROS at the injury site not only leads to neuronal apoptosis but also activates glial cells to transform into the M1 phenotype, releasing proinflammatory cytokines to amplify the injury, further activating the inflammation-related NF-κB signaling pathway and exacerbating the inflammatory response; (c) Mechanism of action of ALP treatment for TBI: Gastrodin (GAS) reduces ROS levels and stimulates neuronal proliferation; ApoM regulates the transformation of microglia into the M2 phenotype, releasing anti-inflammatory and neurotrophic factors to promote repair; GAS and ApoM also inhibit activation of the NF-κB signaling pathway to alleviate inflammation.

[0100] In vivo studies have demonstrated that ALP can actively penetrate the blood-brain barrier and rapidly accumulate at the site of TBI injury. This phenomenon may be due to the ability of CRT, highly expressed on the membrane surface of apoptotic bodies, to bind to CD91, which is highly expressed on microglia and neurons. ALP exhibits significant anti-inflammatory, antioxidant, neuroregenerative, and cerebrovascular repair effects in a TBI mouse model. Behavioral studies have shown that ALP treatment enhances the recovery of neuromotor function in TBI mice.

[0101] More importantly, the biological mechanisms of ALP in treating TBI were further explored. ALP not only promotes the polarization of M2 microglia in the brain but also reduces neutrophil infiltration and recruits Treg cells, suggesting that it can improve the brain's immune microenvironment. RNA sequencing results suggest that ALP may exert its therapeutic effects by affecting multiple inflammatory signaling pathways, including NF-κB.

[0102] In summary, this study systematically demonstrates the potential of bioengineered apoptotic bodies as a microglial-modulating strategy for treating TBI. These findings provide a promising new direction for future TBI treatment research.

Claims

1. A bioengineered apoptotic body based on apoptotic body membrane protein and liposome, characterized in that: The bioengineered apoptotic body is formed by chimerizing apoptotic body membrane protein with liposomes loaded with gastrodin and Cy5.

2. The bioengineered apoptotic body according to claim 1, characterized in that The apoptotic body membrane protein is derived from mesenchymal stem cells.

3. The bioengineered apoptotic body according to claim 1, characterized in that The apoptotic body membrane protein is obtained by inducing mesenchymal stem cells with staurosporine and extracting the membrane protein.

4. The bioengineered apoptotic body according to claim 1, wherein The liposome loaded with gastrodin and Cy5 is obtained by adding gastrodin and Cy5 to a lipid membrane prepared from DSPC, DSPE-TK-PEG2000 and cholesterol.

5. The bioengineered apoptotic body according to claim 4, characterized in that The molar ratio of DSPC, DSPE-TK-PEG2000, and cholesterol is (50-70):(3-8):(30-40).

6. The bioengineered apoptotic body according to claim 4, characterized in that The mass of gastrodin and Cy5 account for 1-3% and 0.02-0.08% of the lipid membrane mass, respectively.

7. The bioengineered apoptotic body according to any one of claims 1 to 6, characterized in that The mass ratio of the apoptotic body membrane protein to the liposome loaded with gastrodin and Cy5 is 1:50 to 1:

150.

8. A method for preparing the bioengineered apoptotic body according to any one of claims 1 to 7, characterized in that include: The bioengineered apoptotic body is prepared by embedding the apoptotic body membrane protein into the liposome loaded with gastrodin and Cy5 through a mechanical extrusion method.

9. Use of the bioengineered apoptotic body according to any one of claims 1 to 7 in the preparation of a medicament for treating traumatic brain injury.

10. Use of the bioengineered apoptotic bodies according to any one of claims 1 to 7 in the preparation of a medicament for promoting recovery from brain injury, wherein the promoting recovery from brain injury comprises any one or more of the following: alleviating neuroinflammation, improving brain motor function, promoting neuronal proliferation, promoting neural repair, inhibiting microglial activation, promoting M2 microglial polarization, improving cellular antioxidant capacity, reducing ROS levels in brain tissue, reducing neuronal apoptosis and necrosis in the brain, and alleviating cerebral edema.

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