An engineered apoptosome based on apoptin and liposome, and a preparation method and application thereof

Abstract

The application discloses a kind of bioengineered apoptotic bodies based on apoptotic body membrane protein and liposome and preparation method and application thereof.The bioengineered apoptotic bodies are formed by the chimerization of apoptotic body membrane protein and liposome loaded with gastrodin and Cy5.The bioengineered apoptotic bodies simulating the natural apoptosis process of mesenchymal stem cells can promote the polarization of microglial cells to M2 type, release anti-inflammatory factors and neurotrophic factors, promote the proliferation of neurons, improve the antioxidant capacity of cells, improve the nervous motor function, relieve brain edema, and thus effectively treat traumatic brain injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a kind of biological engineering apoptotic body based on apoptotic body membrane protein and liposome and its preparation method and application. BACKGROUND

[0002] Traumatic brain injury (TBI) causes brain tissue destruction and significant neurological impairment, and most patients have poor prognosis. Current clinical treatment strategies for TBI include surgery, hyperbaric oxygen therapy, functional electrical stimulation, and rehabilitation therapy. However, these treatment strategies are mostly focused on relieving symptoms and maintaining patient life. In contrast, the recovery of brain function in TBI patients mainly relies on the body's autonomous repair mechanism. When TBI occurs, damage to the cerebral vascular system disrupts the blood-brain barrier, allowing immune cells including macrophages, neutrophils, and lymphocytes to infiltrate into the brain parenchyma. Damaged nerve cells cannot maintain oxidative stress homeostasis, resulting in the production of reactive oxygen species. This activates immune cells, which in turn cause further damage to nerve cells. Ultimately, the inflammatory cascade in damaged brain tissue intensifies, indirectly causing neuronal death and neurodegeneration. Timely response to neuroinflammation triggered by TBI is crucial to prevent the persistence of chronic inflammatory cascade and damage to brain function. Therefore, the key challenge of TBI treatment is to control the development of neuroinflammation and maximize the inhibition of secondary damage.

[0003] Microglia are important cells in the central nervous system and play an important role in maintaining the homeostasis of the brain microenvironment. When TBI occurs, microglia are rapidly activated and transformed into different reactive phenotypes. Similar to peripheral macrophages, microglia are generally divided into two categories: classically activated M1 microglia and alternatively activated M2 microglia. As an important participant in neuroinflammation after TBI, microglia are rapidly activated to M1 and secrete a large amount of pro-inflammatory cytokines and chemokines, driving the progression of neuroinflammatory response. In contrast, M2 microglia are mainly responsible for phagocytosis and clearance of cellular waste in the body. In addition, they also have anti-inflammatory and neuroprotective effects. Therefore, developing TBI therapies targeting the regulation of microglial phenotype not only can reduce neuroinflammation, but also can promote nerve repair and improve neurological function.

[0004] Mesenchymal stem cells (MSCs) promote the polarization of M2 microglia through paracrine cytokines and metabolites, including TGF-β, IL-10, PGE2 and IDO. The survival rate of transplanted MSCs is low, and most of the transplanted MSCs will undergo apoptosis in vivo. After MSCs apoptosis, double-layer lipid membrane vesicles, i.e. apoptotic bodies, are released, which encapsulate organelle fragments and genetic material. At the same time, the "eat-me" signal (such as calreticulin) is flipped from the inner layer to the outer layer of the cell membrane and anchored on the surface of the apoptotic cell membrane and the apoptotic body, attracting the specific recognition and phagocytosis of macrophages. The process of macrophages phagocytizing and removing apoptotic cell products (apoptotic bodies) is called efferocytosis. During efferocytosis, macrophages are polarized to M2 type, thereby protecting surrounding healthy cells while removing apoptotic cells.

[0005] Recently, different types of bioengineered apoptotic bodies have been developed for the treatment of myocardial infarction and Parkinson's disease, which play a role by enhancing the cytotoxicity and anti-inflammatory effects of macrophages. However, to the best of our knowledge, no study has evaluated the therapeutic effect of MSC-derived apoptotic bodies on TBI and its biological mechanism. SUMMARY

[0006] In view of the above technical problems to be solved, the purpose of the present application is to provide a drug capable of effectively treating traumatic brain injury and promoting nerve recovery.

[0007] In order to achieve the above purpose of the application, the present application provides a bioengineered apoptotic body based on apoptotic body membrane protein and liposome, which is obtained 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 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 membrane protein.

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

[0011] According to a preferred embodiment, the molar ratio of DSPC, DSPE-TK-PEG2000, 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 lipid membrane mass, respectively, and more preferably 2% and 0.05%, respectively.

[0013] According to a preferred embodiment, the mass ratio of the apoptotic body membrane protein to the liposomes 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 liposomes, which includes: 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 application of the bioengineered apoptotic bodies based on apoptotic membrane proteins and liposomes in the preparation of drugs for treating traumatic brain injury.

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

[0017] This invention constructs a bioengineered apoptotic body (ALP) that mimics the natural apoptosis process of MSCs, regulating the release of anti-inflammatory factors from microglia and promoting neuronal proliferation, for the purpose of polarizing M2-type microglia to treat TBI. ALP is prepared by chimerating an apoptotic body membrane protein (ApoM) with blood-brain barrier penetration capability and targeting ability with liposomes loaded with gastrodin and Cy5. Gastrodin (GAS) is a glucosinolate analog extracted from the rhizome of Asparagus cochinchinensis, which can stimulate neuronal proliferation and inhibit microglia activation in inflammatory environments, and is released in highly reactive oxygen species (ROS) microenvironments to stimulate neuronal proliferation. ApoM, on the other hand, can induce microglia to polarize to the M2 type, releasing anti-inflammatory factors and neurotrophic factors. In this invention, gastrodin and ApoM can be released responsively in highly reactive oxygen species (ROS) microenvironments, exerting therapeutic and regulatory functions. Furthermore, both gastrodin and ApoM have the ability to inhibit the activation of the NF-κB signaling pathway in microglia. In vivo studies have shown that ALP effectively alleviates neuroinflammation and improves motor function and blood perfusion in TBI model mice by activating M2 polarization of microglia in the brain. This invention proposes a novel strategy for repairing brain injury by effectively activating the immune response of microglia. Attached Figure Description

[0018] Figure 1 A schematic diagram of ALP treatment for TBI is shown. (a) Preparation of ROS-responsive bioengineered apoptotic bodies (ALPs); (b) Brain microenvironment in TBI mice: Elevated ROS at the site of injury not only leads to neuronal apoptosis but also activates glial cells to transform into M1 type, releasing pro-inflammatory factors to amplify the damage and further activating the inflammation-related NF-κB signaling pathway, exacerbating the inflammatory response; (c) Mechanism of action of ALP in TBI treatment: Gastrodin (GAS) can reduce ROS levels and stimulate neuronal proliferation; ApoM can regulate the transformation of microglia into M2 type, releasing anti-inflammatory factors and neurotrophic factors to promote repair; GAS and ApoM can also inhibit the activation of the NF-κB signaling pathway to alleviate inflammation.

[0019] Figure 2 Biochemical characterization of ALP is shown. (a) Schematic diagram of MSC-derived apoptotic bodies (Apo); (b) Morphological features of Apo under an optical microscope (scale bar = 4 μm); (c) Expression of CRT protein (green) on the surface of apoptotic MSCs and Apo (enlarged view); (d) Transmission electron microscopy images of LP and ALP (scale bar = 50 nm); (e) CRT and Na+ in LP, ALP, and ApoM. + -K +(f) Western blotting analysis of ATP protein; (g) SDS-PAGE analysis of LP, ALP, ApoM, Apo and MSCM proteins; (h) 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 This study demonstrates that ALP promotes neuronal proliferation in vitro. (ab) 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 in crossing the blood-brain barrier in vitro (left: schematic diagram of the in vitro BBB model); (d) Scratch 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) ROS-damaged H in N2a cells (g) Flow cytometry analysis of the effects of different treatment groups on CD206 expression 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 of 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 The in vivo therapeutic effect of ALP on TBI mice is shown. (ab) Metabolic distribution of MSCs, LP and ALP at different time points in TBI mice (a) and semi-quantitative fluorescence analysis (b); (c) Schematic diagram of ALP in vivo treatment; (d) H&E staining and Nissl staining of brain tissue sections of TBI mice on day 10 after different treatments (scale bar = 250 μm); (ef) OCT imaging observation of blood flow changes at the injury site in TBI mice (e) and quantitative analysis of vascular density (f) (scale bar = 1 mm); (g) Measurement of brain tissue water content of TBI mice on day 5 after different treatments; (hi) SOD enzyme activity (h) and MDA level (j) in brain tissue of TBI mice on day 10.

[0022] Figure 5The behavioral tests of TBI mice by ALP are shown. (a) Schematic diagram of the behavioral test timeline; (b) mNSS assessment of changes in motor, sensory, balance and reflex functions of TBI mice; (cf) OFT test assessment of TBI mice's voluntary movement ability and emotional behavior (c), including average speed (d), central area movement distance (e) and total movement distance (f); (gk) Catwalk system assessment of TBI mice's motor function, mainly analyzing gait pattern changes (g) and four dynamic indicators: support base 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 and semi-quantitative fluorescence in the brain tissue of TBI mice (CD206, a marker of M2 microglia, and quantitative analysis (b); Ly6G, a marker of neutrophils, and quantitative analysis (d); FoxP3, a marker of Treg cells, and quantitative analysis (f); (g) Immunofluorescence staining of the injury sites in 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 the levels of inflammation-related cytokines (TGFβ (h), IL-6 (i), TNF-α (j)) and neurotrophic factors (NGF (k), GDNF (l), BDNF (m)) in brain tissue.

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

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

[0026] 1 Method

[0027] 1.1 Extraction of apoptotic bodies (Apo) and membrane proteins (ApoM)

[0028] Apoptotic bodies (Apo) derived from mesenchymal stem cells (MSCs) were obtained through induction with asteroidin (50606ES10, Yisheng Biotechnology). After MSCs were co-incubated with 0.5 μM asteroidin for 6 hours, the serum-free culture supernatant was collected. Cell debris was removed by centrifugation at 50 g, 4 °C for 5 minutes, followed by centrifugation at 1000 g, 4 °C for 15 minutes to collect the apo bodies (Apo). The Apo suspension was smeared and its morphology was observed under a light microscope.

[0029] For Apo membrane protein (ApoM) extraction, 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, 4°C for 10 minutes, followed by centrifugation at 10000g, 4°C for 10 minutes to collect the precipitate. The precipitate was resuspended in ddH2O and stored at -80°C. The ApoM concentration was determined using a BCA kit (P0010, Beyotime).

[0030] 1.2 CRT Detection of Apo Surface

[0031] The expression of calreticulin (CRT) on the surface of Apo cells was observed by immunofluorescence staining. MSCs were treated with astrocytocin for different durations and then fixed with 4% paraformaldehyde (P0099, Beyotime) for 20 minutes. After blocking for 1 hour, 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-labeled secondary antibody (A0423, Beyotime) at room temperature in the dark for 1 hour. After washing three times with PBS, DAPI (C1006, Beyotime) was used for counterstaining. After washing with PBS, CRT expression on the Apo surface was detected under a fluorescence microscope.

[0032] 1.3 Preparation and Characterization of ALP

[0033] Preparation: ROS-responsive liposomes (LPs) were prepared using the Bangham method and freeze-thaw method. DSPC (distearylphosphatidylcholine, 850365C, Avanti), DSPE-TK-PEG2000 (distearylphosphatidylethanolamine-ketothiol-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 solution was dried under nitrogen to form a uniform film, and then vacuum dried for 3 hours to obtain the lipid membrane. 2 mL of PBS solution containing 200 μg / ml gastrodin (PHL89324, Sigma) and 5 μg / ml Cy5 (146368-11-8, MCE) was added. The mass percentages of gastrodin and Cy5 were 2% and 0.05% of the lipid membrane mass, respectively. After five freeze-thaw cycles, LPs with uniform particle size were obtained using a liposome extruder. ApoM-coated LP (ALP) was prepared at a mass ratio of LP to ApoM of 100:1. Utilizing the flowability of the liposome phospholipid bilayer, ApoM was embedded 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.).

[0034] Characterization: The zeta potential and particle size distribution of nanoparticles (NPs) were detected using a dynamic light scattering instrument (Nano ZS90, Malvern). The morphology of NPs was observed by transmission electron microscopy (TEM) after negative staining with 2% phosphotungstic acid (P4006, Sigma). SDS-PAGE and Western blotting were used to detect protein expression on the NP surface. The absorption and emission spectra of ALP were measured using a UV-Vis spectrophotometer and a fluorescence spectrophotometer, respectively, and an ALP drug release curve was plotted based on Cy5 absorbance.

[0035] 1.4 Cell Culture

[0036] C57BL / 6J mouse bone marrow mesenchymal stem cells (MSCs) were purchased from Cyagen Biosciences. Cells were expanded in an incubator using DMEM / F12 medium (Gibco) containing 10% fetal bovine serum (FBS). The MSCs used in this invention were within the 15th passage. 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 medium (Gibco) containing 10% FBS.

[0037] 1.5 Cytotoxicity Detection

[0038] All cell viability assays were performed using the Cell Counting Kit-8 (CCK-8) (C0041, Beyotime).

[0039] Establishment of an 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.

[0040] Assay for promoting N2a cell proliferation with gastrodin (Gas): 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 another 12 hours. The supernatant was discarded, and the cells were replaced with serum-free medium containing different concentrations of gastrodin and incubated for another 12 hours.

[0041] Assay for promoting N2a cell proliferation by M2 microglia: BV2 cells pre-cultured for 24 hours were treated with different concentrations of IL-4 (SRP3211, Sigma) instead of gastrodin. A control group was prepared by co-culturing the same number of blank N2a cells with a corresponding proportion of pre-treated BV2 cells.

[0042] ALP biocompatibility assay: N2a cells and bEnd.3 cells were seeded in 96-well plates and cultured for 12 hours. The medium was then replaced with serum-free medium and co-incubated with different concentrations of ALP for 12 hours. Cell viability was then detected using the CCK8 assay kit.

[0043] 1.6 ALP cell uptake assay

[0044] Flow cytometry analysis: N2a and BV2 cells were seeded in 24-well plates and cultured for 12 hours. The medium was then replaced with serum-free medium containing LP and ALP. Cells were washed twice with PBS at different time points, digested, and collected. Fluorescence intensity was detected by flow cytometry. Flow cytometry data were analyzed using FlowJo software.

[0045] Confocal laser scanning microscopy (CLSM) observation: As described above, replace the 24-well plate with an 8-well chamber slide (154534, Thermo) suitable for CLSM.

[0046] 1.7 ALP in vitro blood-brain barrier (BBB) ​​crossing experiment

[0047] An in vitro BBB model was constructed 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%, the medium was replaced with serum-free medium containing LP or ALP. The fluorescence intensity of the lower layer cells was detected by flow cytometry to analyze the ability of LP and ALP to cross the BBB in vitro.

[0048] 1.8 Cell Scratch Assay

[0049] The scratch technique was used to simulate mechanical cell damage, and the efficacy of drugs was assessed by the wound area. N2a cells were seeded in 24-well plates and cultured for 12 hours. Scratches were created using a scratcher (201925, SPL), and images were acquired under an optical microscope. Subsequently, cells were co-incubated for 24 hours with 5 μg / ml gastrodin (Gas), 100 ng / ml apoptotic bodies (Apo), and LP and ALP at concentrations equivalent to free gastrodin, respectively, and images were acquired again. Untreated scratched cells served as a control group. ImageJ software was used to measure the wound healing area to assess the ability of ALP to promote N2a cell proliferation.

[0050] 1.9 Evaluation of ALP's ROS removal capability

[0051] N2a cells were co-incubated with H2O2 (0.03 μM) and drugs (gastrodin, apoptotic bodies, LP, and ALP) for 12 hours, with untreated cells serving as a control. Cells were then incubated with 10 μM DCFH-DA (S0033M, Beyotime) for 30 minutes, washed twice with PBS, and fluorescence intensity was assessed by flow cytometry to evaluate the level of ROS scavenging by ALP.

[0052] Mitochondrial membrane potential detection (JC-1, C2003S, Beyotime): Mitochondrial membrane potential can reflect the early apoptotic state of cells. N2a cells were stained with JC-1 for 20 minutes, and then oxidative damage was observed using confocal laser scanning microscopy (CLSM).

[0053] 1.10 Western blotting

[0054] Cells were treated on ice with lysis buffer (P0037, Beyotime) containing 1% PMSF (ST506, Beyotime) for 1 minute to collect proteins. Tissue samples were ground before protein extraction. Sample concentrations were standardized according to the BCA kit results, and proteins were separated by SDS-PAGE electrophoresis (Bio-Rad). Proteins were transferred to PVDF membranes using a transfer apparatus (Bio-Rad) and blocked at room temperature for 1 hour. Primary antibody was added and incubated overnight at 4°C, followed by three TBST washes (5 minutes each). Secondary antibody (170-6515, Bio-Rad) was added and incubated at room temperature for 2 hours, followed by three more TBST washes. ECL chemiluminescent substrate (1705060, Bio-Rad) was added for development, and images were acquired using the Bio-Rad imaging system. Quantitative analysis was performed using ImageJ software.

[0055] 1.11 BV2 cell polarization detection

[0056] BV2 cells were seeded in 24-well plates and cultured for 12 hours. The 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 dosages) and cultured for another 24 hours. After cell collection, Fc inhibitors were added, and cells were stained with CD206 antibody (Supplementary Table 3) and analyzed by flow cytometry.

[0057] 1.12 Real-time quantitative PCR (qRT-PCR)

[0058] 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 the SYBR Green SuperPremix kit (64477446, Bio-Rad) on a real-time quantitative PCR system (CFX96, Bio-Rad), with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control gene. Two... -ΔΔCT The relative expression levels of each gene were calculated using a method. All primers were synthesized by BGI Genomics Co., Ltd., and their sequences are detailed in Supplementary Table 1.

[0059] Table 1. Primer sequences

[0060]

[0061] 1.13 Laboratory Animals

[0062] Male C57BL / 6 mice (8-10 weeks old, 26-30 g) were purchased from the Animal Experimentation Centre of the Faculty of Health Sciences, University of Macau. All experimental procedures were in accordance with animal welfare standards and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Macau.

[0063] Establishment of a moderate traumatic brain injury (TBI) model: Mice were anesthetized with 1.5% isoflurane gas, and the skull was exposed. A circular window with a diameter of 3 mm was drilled using a skull drill (78001, RWD) without damaging the dura mater. A moderate TBI model was established using a precision impactor (68099Ⅱ, RWD) with the following standard parameters: impact velocity = 4 m / s, depth = 1.0 mm, and residence time = 0.4 s. The degree of blood-brain barrier damage was assessed by observing the penetration of 2% Evans blue into the brain injury site after intravenous injection via the tail vein.

[0064] 1.14 Biodistribution of ALP in vivo

[0065] Nine TBI mice were randomly divided into three groups. Two hours after model establishment, each group was injected via tail vein with LP, ALP, or MSCs. The biodistribution of these MSCs was observed at different time points using a small animal in vivo imaging system.

[0066] 1.15 ALP in vivo therapeutic effect

[0067] Forty TBI mice were randomly divided into five groups (control group, Gas group, Apo group, LP group, and ALP group), and eight untreated mice were used as the normal control group. Treatment was administered via tail vein on days 1, 3, and 5 after model establishment. On day 10, mice were sacrificed, and brain tissue was collected and embedded in paraffin. The treatment effect was assessed by H&E staining and Nissl staining.

[0068] 1.16 Optical coherence tomography

[0069] Vascular signals at the injury site were acquired using the previously reported optical coherence tomography technique. As mentioned above, blood flow signals were acquired after skull incision (labeled "Pre") and on days 1, 5, and 10 after modeling, and cerebral vascular density was quantified using MATLAB software.

[0070] 1.17 Cerebral edema detection

[0071] Five days after treatment, mice (n=3) were sacrificed, and brain tissue was collected to determine water content. First, the fresh brain tissue was weighed, and then the dehydrated brain tissue was dried at 65°C for 24 hours and the weight was recorded.

[0072] The formula for calculating brain water content is as follows:

[0073] .

[0074] 1.18 Detection of Superoxide Dismutase (SOD) Activity and Malondialdehyde (MDA) Levels

[0075] On day 10 post-treatment, TBI mice were sacrificed, and the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in brain tissue were measured. Brain tissue was mixed with SOD sample preparation solution and homogenized on ice. The mixture was then centrifuged at 12000g, 4℃ for 5 minutes, and the supernatant was used to determine SOD activity in the brain tissue using the WST-8 method (S0101M, Beyotime). MDA level detection (S0131S, Beyotime) was performed according to the kit instructions.

[0076] 1.19 Behavioral Test

[0077] Another 40 TBI mice were randomly divided into 5 groups (control group, Gas group, Apo group, LP group, and ALP group) for behavioral testing. Behavioral experiments were performed on TBI mice on days 3, 10, and 20 after different treatments.

[0078] mNSS score: The behavior of TBI mice was scored using a randomized double-blind method based on a standardized scale.

[0079] Open field test: The experiment was conducted in an open field chamber measuring 50cm×50cm×40cm. The movement trajectory of TBI mice was recorded over 8 minutes using a digital camera. Data such as total distance, average speed, and movement distance in the central area were analyzed using Smart 3.0 software.

[0080] Catwalk gait analysis: Mice underwent daily training for three consecutive days prior to the experiment. Before each formal experiment, TBI mice were allowed to acclimatize in the dark for one hour. Gait data were collected using the Noldus Catwalk system, and four dynamic indicators (average distance between forelimbs and hindlimbs, average stride length between left and right limbs, swing speed, and stride length) were analyzed based on mouse gait behavior.

[0081] 1.20 Brain Tissue Cytokine Detection

[0082] On day 10 post-treatment, TBI mice were sacrificed and brain tissue was collected. Damaged areas were excised, trimmed, and digested with collagenase to form a single-cell suspension. The supernatant was collected by centrifugation, and cytokines (TGFβ, TNFα, and IL-6) and neurotrophic factors (NGF, GDNF, and BDNF) were detected using an ELISA kit. The precipitated cells were used for flow cytometry analysis. A blocking agent was added to the precipitated single-cell suspension, and flow cytometry staining was performed using antibodies. Foxp3 antibody staining requires membrane permeabilization (00-5523-00, Thermo).

[0083] 1.21 Immunofluorescence staining

[0084] As described in the "ALP In vivo therapeutic effect" section above, immunofluorescence staining was performed on paraffin sections. First, the apoptosis status in brain tissue sections was analyzed using the One Step TUNEL apoptosis detection kit. Subsequently, IF staining was performed: paraffin sections were dewaxed and antigen retrieval was performed, followed by serum blocking for 1 hour. Iba1, TNFα, and CD206 primary antibodies were added, and the sections were incubated overnight at 4°C. After washing three times with TBS, fluorescently labeled secondary antibody was added and incubated for 1 hour. After counterstaining with DAPI, observation was performed using a fluorescence microscope.

[0085] 1.22 TBI mouse RNA sequencing

[0086] On day 5 after TBI treatment, brain tissue was harvested from mice after perfusion with saline and flash-frozen in liquid nitrogen for 5 seconds. Samples were sent to Beijing Novogene Technology Co., Ltd. for RNA-Seq library preparation, and data were analyzed via https: / / ap-magic.novogene.com / .

[0087] 1.23 Biocompatibility of ALP

[0088] Twelve male C57BL / 6 mice were randomly divided into two groups for in vivo biocompatibility testing, with six untreated mice serving as a control group. Blood samples were collected 14 days after tail vein injection of LP and ALP for complete blood count. The biocompatibility of the nanoparticles was observed using paraffin sections of major organs stained with H&E.

[0089] 1.24 Statistical Analysis

[0090] Statistical analysis was performed using Origin 2022. Differences between groups were assessed using independent samples t-tests (***P<0.001; **P<0.01; *P<0.05). All statistical analyses were performed using SPSS 26.0 software.

[0091] 2 Results and Discussion

[0092] 2.1 Preparation and Biochemical Characterization of ALP

[0093] First, primary mouse mesenchymal stem cells (MSCs) were treated with astrocytocin to induce apoptosis, and apoptotic bodies (Apo) were prepared. Figure 2 (a) Microscopic observation showed that the apoptotic bodies derived from MSCs exhibited 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 "phagocytic" signaling molecule, calreticulin (CRT) is exposed on the outer surface of the cell membrane during apoptosis, attracting macrophages for phagocytosis and promoting cell burial. Immunofluorescence imaging showed that Apo highly expressed CRT ( Figure 2(c in the text)

[0094] This invention utilizes a mechanical extrusion method to fuse Apo membrane protein (ApoM) with liposomes (LP) loaded with gastrodin (Gas) and Cy5, thereby preparing engineered apoptotic bodies (ALPs). Transmission electron microscopy (TEM) observation shows that both LPs and ALPs exhibit uniform spherical structures. Figure 2 (d) Western blot and SDS-PAGE analyses showed that ALP expresses multiple ApoM proteins, including CRT and Na. + -K + ATPase ( Figure 2 The presence of ef in the data suggests that ALP may have similar biological functions to Apo.

[0095] 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 detection results confirmed that the membrane protein was successfully modified onto the liposome surface. Furthermore, fluorescence imaging monitoring of ALP distribution in vivo was achieved by loading the molecular dye Cy5. Characteristic peaks of Gas and Cy5 in ALP were detected by UV absorption and fluorescence emission spectroscopy, confirming successful drug loading.

[0096] The responsiveness of ALP to reactive oxygen species (ROS) was evaluated using drug release assays. In the presence of hydrogen peroxide (H₂O₂), ALP rapidly released the drug: the release efficiency reached 42.0 ± 1.6% at 1 hour and 91.7 ± 5.0% at 12 hours. Figure 2 (h in the text). Continuous observation for 7 days showed that the ALP particle size distribution remained stable, indicating its excellent stability. Figure 2 (i in the text).

[0097] 2.2 ALP promotes neuronal proliferation in vitro

[0098] Calreticulin (CRT) expressed on the apoptotic body membrane can bind to CD91 protein (low-density lipoprotein receptor-associated protein) on the surface of microglia and neurons, thereby enhancing ALP uptake by microglia and neurons. The uptake of ALP by neuronal N2α cells was first investigated. Figure 3 As shown in ab, ALP exhibits superior cellular uptake capacity compared to LP, indicating that ALP can actively target nerve cells.

[0099] Subsequently, an in vitro blood-brain barrier (BBB) ​​model was constructed using Transwell experiments to assess whether ALPs possess a similar ability to cross the BBB as MSCs. Figure 3(c) In the experiment, endothelial cells bEnd.3 were seeded in the upper chamber, and neuronal cells N2a were seeded in the lower chamber. After adding LP and ALP to the upper layer, the fluorescence intensity of the cells in the lower layer was measured. The results showed that the amount of ALP actively crossing the BBB was significantly higher than that of LP.

[0100] To simulate the high ROS microenvironment in damaged brain tissue, an in vitro ROS injury model was constructed by damaging neurons with H2O2. When the H2O2 concentration was 0.02 μM, the cell survival rate was 63.04 ± 4.37%, therefore this concentration was chosen for establishing the in vitro ROS injury model. Gastrodin (Gas), a glucosinolate extracted from the traditional Chinese medicine Asparagus cochinchinensis, possesses natural anti-inflammatory, antioxidant, and neuroprotective effects. Gas can improve the survival rate of ROS-damaged neurons; treatment with 5 μg / ml gastrodin increased the survival rate of damaged neurons by 23.86 ± 8.41%.

[0101] Mechanical damage to N2a cells was simulated using a scratch assay. For example... Figure 3 As shown in 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.

[0102] To verify the ROS scavenging effect of ALP, 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect intracellular ROS levels. The results showed that H2O2 treatment increased intracellular ROS levels in N2a cells, while ALP treatment significantly reduced intracellular ROS levels. Intracellular ROS levels are closely related to mitochondrial function: elevated ROS levels lead to decreased mitochondrial membrane potential and impaired mitochondrial function. The JC-1 fluorescent probe was used to assess 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 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.

[0103] To investigate the biological mechanism by which ALP resists ROS damage, the expression of antioxidant and anti-apoptotic proteins in neuronal cells was examined. Nuclear factor E2-related factor 2 (Nrf2) is a redox-sensitive transcription factor. After ROS damage, Nrf2, which is originally located in the cytoplasm, translocates to the nucleus and upregulates the expression of heme oxygenase-1 (HO-1), the product of which has antioxidant activity.

[0104] Furthermore, overexpression of B-lymphoma-2 (Bcl-2) can reduce the production of oxygen free radicals and the formation of lipid peroxides, thereby inhibiting apoptosis. Western blotting results showed that ALP treatment upregulated the protein expression of Nrf2, HO-1, and Bcl-2 in ROS-damaged neurons, 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 the middle.

[0105] Furthermore, ALP can indirectly promote neuronal proliferation by regulating microglia. Under physiological conditions, microglia have functions such as immune regulation, neurotrophic function, and homeostasis maintenance. ALP was found to actively target microglia because microglia also highly express CD91 on their surface to bind to CRT on apoptotic bodies. Elevated ROS levels in the brain after TBI promote microglia polarization towards the M1 type, exacerbating the inflammatory response and damaging neurons; while Apo can promote M2 microglia repolarization through cell burial to inhibit inflammation. Flow cytometry results showed that ALP treatment upregulated the expression of the M2 type marker CD206 in microglia, indicating that ALP can enhance the polarization of M2 microglia. Figure 3 g in (the middle part).

[0106] Further analysis using qRT-PCR investigated the effects of different treatments on the expression of phenotypic markers and the secretion levels of cytokines / neurotrophic factors in LPS-pretreated microglia. The results showed that ALP treatment significantly downregulated the expression of the M1 marker CD86, while upregulating the expression of the 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 decreased the release of pro-inflammatory cytokines (IL-6, IL-1β). Figure 3 (h in the middle).

[0107] The effect of M2 microglia on ROS-damaged neurons was analyzed using a CCK-8 assay kit. When the neuron-to-microglia ratio 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).

[0108] Nuclear factor-κB (NF-κB) is an important intracellular nuclear transcription factor involved in the regulation of inflammatory responses, immune responses, and apoptosis, and is a classic ROS-related signaling pathway. ALP may exert its anti-inflammatory effect by affecting the NF-κB signaling pathway. Western blot analysis showed that the expression of both P65 and P-P65 in microglia decreased after ALP treatment, indicating that the H2O2-activated NF-κB signaling pathway was inhibited; at the same time, IKβα expression increased, suggesting that more activated NF-κB regulates the return to the resting state through negative feedback. Figure 3 j in the middle.

[0109] 2.3 In vivo therapeutic effect of ALP on TBI mice

[0110] Further investigation was conducted into the biodistribution of ALP in vivo to assess its ability to penetrate the blood-brain barrier and enter the brain tissue of TBI mice. This invention used a precision striker to establish a moderate TBI mouse model. Mice were sacrificed after intravenous injection of Evans blue dye via the tail vein, and brain tissue was harvested to observe blood-brain barrier leakage. The results showed that Evans blue dye could penetrate to the site of brain injury, indicating that the blood-brain barrier in TBI mice had been disrupted.

[0111] Subsequently, in vivo imaging was used to assess the biodistribution of MSCs, LP, and ALP in TBI mice. Fluorescence imaging showed that MSCs were delivered to the brain 72 hours after intravenous injection; LP reached the brain region 12-24 hours after injection, possibly due to the enhanced permeability retention effect (EPR) at the injury site after blood-brain barrier damage; while ALP was rapidly delivered to the TBI injury site, reaching peak accumulation at 12 hours and remaining in the brain for a long time (approximately 96 hours). Notably, the peak fluorescence intensity of ALP was significantly stronger than that of MSCs and LP, indicating that apoptotic body membrane modification significantly enhanced the targeting of ALP to TBI. Figure 4 (ab in the text).

[0112] Based on the drug's biodistribution in vivo, TBI mice were treated with three doses, each 48 hours apart. The experimental procedure was as follows: Figure 4 As shown in c. Treatment efficacy was assessed by H&E staining and Nissl staining of brain tissue sections processed on day 10. Figure 4 As shown in d, H&E staining results revealed that, compared to the normal group, the cortical pyramidal cells in the TBI control group were scattered and shrunken, with numerous degenerated neurons and irregular nuclei; while in the ALP group, the cortical pyramidal cells were neatly arranged and morphologically normal, with significantly reduced neuronal necrosis. Nissl staining showed that the number of Nissl bodies in the brain of 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.

[0113] Further optical coherence tomography (OCT) was used to monitor changes in cerebral blood vessels in TBI mice during treatment. For example... Figure 4 As shown in the ef, the vascular density in the brain injury area of ​​mice treated with ALP increased significantly from 30.66±1.2% on the initial day to 143.44±0.8% on day 10, indicating that ALP can promote cerebral angiogenesis and thus improve the nutritional supply to damaged brain tissue.

[0114] In addition, brain tissue was harvested on day 5 post-treatment to measure water content and assess cerebral edema. The brain water contents of each group were as follows: control group 81.11±2.62%, Gas group 78.96±2.37%, Apo group 78.57±1.35%, LP group 78.36±1.66%, ALP group 77.81±0.877%, and normal group 77.89±0.426%. ALP significantly alleviated cerebral edema symptoms after TBI (transient brain injury). Figure 4 g in (the middle part).

[0115] Brain ROS levels were assessed by detecting superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels in brain tissue. Compared with the control group, the ALP group showed significantly increased SOD activity and significantly decreased MDA levels, indicating that ALP can effectively reduce ROS levels in brain tissue after TBI. Figure 4 hi in the middle.

[0116] 2.4 Assessment of Neurological Function Recovery in TBI Mice

[0117] The neurological function recovery of TBI mice after treatment was assessed using a modified neurological deficit score (mNSS), open field test (OFT), and Catwalk gait analysis system. Neurological function was assessed in TBI mice on days 3, 10, and 20 after model establishment. Figure 5 a). mNSS results showed that the neurological function scores of mice in the ALP-treated group were significantly lower than those in the control group ( Figure 5 (b) in the middle.

[0118] Subsequently, based on the mouse movement trajectory, OFT was used to assess the motor ability and emotional behavior of TBI mice. Figure 5 c). Compared with the control group, the ALP-treated TBI mice showed a higher average speed ( Figure 5 d) longer total distance of motion ( Figure 5 f) and the increased central area activity distance ( Figure 5 (e). These results indicate that ALP treatment can improve the motor ability and autonomous exploration behavior of TBI mice and promote the recovery of motor function.

[0119] The Catwalk system can analyze gait changes in TBI mice caused by movement disorders and pain during natural walking. This experiment focused on analyzing changes in gait patterns and four dynamic indicators (stance width, footprint position, swing speed, and stride length). Control group mice exhibited gait abnormalities due to decreased motor coordination. Figure 5 (g) is characterized by an increased distance between the forelimbs and hindlimbs. Figure 5 (h) The distance between the left and right footprints increased ( Figure 5 i) shortened stride length ( Figure 5 j) and the speed of oscillation slowed down ( Figure 6 (k in the text). In contrast, the gait pattern of the ALP-treated mice was almost identical to that of normal mice, and all dynamic indicators were better than those of the control group, suggesting that ALP may enhance the motor coordination ability of mice.

[0120] 2.5 Immune mechanism of ALP treatment

[0121] Microglia are intrinsic immune cells of the central nervous system. Previous studies have shown that regulating M2 microglia polarization can promote anti-inflammatory responses and neural repair. To elucidate 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, compared with the control group, ALP treatment significantly increased the number of M2 microglia (F4 / 80). + CD11b + CD206 + The proportion of M1 microglia (F4 / 80) was reduced, while the proportion of M1 microglia (F4 / 80) was also reduced. + CD11b + CD80 + The ratio indicates that ALP can promote microglial polarization from M1 to M2.

[0122] In the pathological state of TBI, the infiltration of large numbers of neutrophils into brain tissue exacerbates the inflammatory response and cerebral edema. Flow cytometry analysis showed that neutrophils (CD45+) were present in the brain tissue of the ALP group. + CD11b + Ly6G + The level of [unclear] was significantly lower than that in the control group, which helps to alleviate secondary damage from TBI. Figure 6 (cd in the middle). Regulatory T cells (Tregs) act as CD4. + A subset of T cells whose primary function is to suppress excessive immune responses in the inflammatory microenvironment. Compared to the control group, the ALP group showed a higher concentration of Treg cells (CD4+) in brain tissue. + FoxP3 + The increased proportion of ) helps suppress excessive inflammatory response at the site of TBI injury ( Figure 6 (ef in the text)

[0123] Immunofluorescence staining was used to further assess neuronal apoptosis and microglial activation. Figure 6 As shown in g, the green fluorescence intensity of TdT-mediated dUTP nick-end labeling (TUNEL) was reduced in brain tissue sections of the ALP group. Figure 6 (g in line 1) indicates a significant reduction in neuronal apoptosis in the damaged area. Furthermore, ALP treatment can inhibit microglial hyperactivation to alleviate early inflammatory damage: in the control group, microglial activation at the damaged site was enhanced, while ALP treatment reduced this hyperactivation. Figure 6 (g in line 2). More importantly, ALP treatment significantly reduced the staining intensity of M1 microglia (TNFα) in the brains of TBI mice, while significantly increasing the staining intensity of M2 microglia (CD206). Figure 6 (g in line 3-4).

[0124] Serum cytokine detection showed that ALP treatment significantly decreased the levels of pro-inflammatory cytokines (TNF-α) and significantly increased the levels of anti-inflammatory cytokines (TGFβ, IL-6) in TBI mice. Figure 7 (hj); at the same time, the levels of neurotrophic factors (NGF, GDNF, BDNF) also increased significantly ( Figure 7 (km in the text). These results indicate that ALP effectively inhibits the development of neuroinflammation after TBI by promoting M2 microglia polarization and improving the immune microenvironment.

[0125] 2.6 RNA sequencing analysis of brain tissue after ALP treatment

[0126] Neuroinflammation induced by total brain injury (TBI) is a key factor leading to poor patient prognosis. 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 function recovery by inducing microglia to M2 polarization. However, previous research has not explored the molecular mechanisms of apoptotic body therapy for TBI in depth; therefore, RNA sequencing (RNA-seq) technology was used to analyze the gene expression profile and signaling pathway activation in the brain tissue of TBI-affected mice.

[0127] Differential gene Wahn analysis showed that the ALP, LP, and TBI groups had 485, 408, and 420 specific expression genes, respectively, and 12,300 common expression genes. Figure 7 a) in the example. Figure 7 As shown in b, compared with the TBI group, ALP treatment resulted in significant downregulation of 292 genes and significant upregulation of 379 genes. In contrast, compared with the LP group, ALP treatment only led to downregulation of 257 genes. Figure 7(c) This difference in downregulated genes may stem from the therapeutic effect of apoptosis-inducing membrane proteins on the surface of ALP.

[0128] Cluster heatmap analysis of differentially expressed genes in the ALP, LP, and TBI groups revealed that the expression of apoptosis-related genes (CASP8, CASP9, FADD, and CASP12), Toll-like receptor signaling pathway-related genes (MYD88, TIRAP, and TICAM2), and NF-κB signaling pathway-related genes (P50, IKKα, CK2, and P65) were all significantly downregulated. Figure 7 (d in the text). The decrease in these gene expressions indicates that ALP treatment not only reduces apoptosis but also inhibits the inflammatory response activated by the Toll-like receptor signaling pathway and the NF-κB signaling pathway.

[0129] Western blot results showed that ALP treatment reduced the expression levels of NF-κB signaling pathway-related proteins (including P-P65, P-Ikkβ, and Ikβα) in the brain tissue of TBI mice. Figure 7 (f in the text). Furthermore, KEGG enrichment analysis showed that, compared to the TBI group, the differentially expressed genes in the ALP group were mainly involved in inflammation-related signaling pathways, protein metabolism, cell adhesion, and calcium homeostasis regulation. Among these, the TNF signaling pathway, NF-κB signaling pathway, NOD-like receptor signaling pathway, and Toll-like receptor signaling pathway are key regulatory pathways in the inflammatory process and immune response. Figure 1 (e in the text).

[0130] Gene ontology (GO) analysis showed that, compared with the TBI or LP groups, the differentially expressed genes in the ALP group mainly exhibited changes in receptor ligand activity, hormones, chemokines, and organic acid binding properties at the molecular function (MF) level; changes in receptor complexes, extracellular matrix, inflammation-related complexes, and synapse-related genes at the cellular component (CC) level; and significant effects on cellular secretion, leukocyte-mediated immune responses, immune system processes, calcium homeostasis, blood circulation, and neuropeptide signaling pathways at the biological process (BP) level. ​ The difference in treatment efficacy between the ALP and LP groups may stem from the fact that the modification of the apoptosis body membrane leads to different in vivo metabolic processes, antioxidant damage activity, immunomodulatory capabilities, and effects on inflammatory responses of the nanomedicine.

[0131] These results indicate that the most important mechanism by which ALP treats TBI is by inhibiting inflammation-related signaling pathways, including NF-κB, to maintain the homeostasis of the brain's immune system, thereby reducing secondary damage caused by the development of neuroinflammation after TBI.

[0132] 2.7 Biocompatibility of ALP

[0133] The biocompatibility of ALP was assessed at both the cellular and small animal levels. At the cellular level, cell viability was measured after co-incubating bEnd.3 and N2a cells with different concentrations of ALP (0–2 mg / ml) for 12 hours. The results showed that the viability of vascular endothelial cells and neurons did not decrease significantly, indicating that ALP has no significant toxic effect on normal cells.

[0134] Mice in each treatment group undergoing TBI administration were sacrificed on day 14, and blood samples were collected for complete blood count and histopathological examination of major organs using hematoxylin and eosin (H&E) staining. Results showed no statistically significant differences in any hematological parameters between the LP and ALP groups compared to the normal mouse group. Furthermore, no significant morphological changes were observed in the organ tissue sections of both the LP and ALP groups. Therefore, the ALP developed in this invention is a biocompatible nanomedicine suitable for TBI treatment.

[0135] This invention proposes an innovative strategy for treating triglyceride-associated brain injury (TBI). The developed bioengineered MSC-derived apoptotic bodies (ALPs) effectively promote nerve cell regeneration and regulate microglia polarization. On one hand, ALPs can scavenge ROS and directly stimulate nerve cell proliferation by releasing gastrodin in response to ROS; on the other hand, the apoptotic body membrane coating the surface of ALPs can promote M2 microglia polarization and release anti-inflammatory cytokines and neurotrophic factors through cytotoxicity. ​ A schematic diagram of ALP treatment for TBI is shown. (a) Preparation of ROS-responsive bioengineered apoptotic bodies (ALPs); (b) Brain microenvironment in TBI mice: Elevated ROS at the site of injury not only leads to neuronal apoptosis but also activates glial cells to transform into M1 type, releasing pro-inflammatory factors to amplify the damage and further activating the inflammation-related NF-κB signaling pathway, exacerbating the inflammatory response; (c) Mechanism of action of ALP in TBI treatment: Gastrodin (GAS) can reduce ROS levels and stimulate neuronal proliferation; ApoM can regulate the transformation of microglia into M2 type, releasing anti-inflammatory factors and neurotrophic factors to promote repair; GAS and ApoM can also inhibit the activation of the NF-κB signaling pathway to alleviate inflammation.

[0136] In vivo studies have shown that ALP can actively cross the blood-brain barrier and rapidly accumulate at the site of TBI injury. This phenomenon may stem from the fact that CRT, highly expressed on the surface of apoptotic bodies, can bind to CD91, which is highly expressed in microglia and neurons. ALP exhibited significant anti-inflammatory, antioxidant, nerve regeneration-promoting, and cerebrovascular repair effects in a TBI mouse model. Behavioral experiments showed that ALP treatment enhanced the recovery of neuromotor function in TBI mice.

[0137] More importantly, the biological mechanisms by which ALP treats TBI were explored in depth. ALP not only promotes the polarization of M2 microglia in the brain but also reduces neutrophil infiltration and recruits Treg cells, indicating that it can improve the brain's immune microenvironment. RNA sequencing results suggest that Apo may exert its therapeutic effect by influencing multiple inflammation-related signaling pathways, including NF-κB.

[0138] In summary, this invention systematically demonstrates the potential of bioengineered apoptotic bodies as a strategy for regulating microglia in the treatment of TBI. These findings provide a promising new direction for future TBI treatment research.

Claims

1. A bioengineered apoptotic body based on apoptotic membrane proteins and liposomes, characterized in that, The bioengineered apoptotic body is formed by chimerism of apoptotic body membrane proteins with liposomes loaded with gastrodin and Cy5; The apoptotic body membrane protein was obtained by inducing mesenchymal stem cells with astrocytocin and then extracting the membrane protein. The liposomes loaded with gastrodin and Cy5 were obtained by adding gastrodin and Cy5 to a lipid membrane made of DSPC, DSPE-TK-PEG2000 and cholesterol. The molar ratio of DSPC, DSPE-TK-PEG2000, and cholesterol is (50-70):(3-8):(30-40).

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

3. The bioengineered apoptotic body according to claim 1 or 2, characterized in that, The mass ratio of the apoptotic body membrane protein to the liposomes loaded with gastrodin and Cy5 is 1:50 to 1:

150.

4. A method for preparing bioengineered apoptotic bodies as described in any one of claims 1 to 3, characterized in that... include: The bioengineered apoptotic bodies were prepared by mechanically extruding apoptotic membrane proteins into liposomes loaded with gastrodin and Cy5.

5. The use of the bioengineered apoptotic body as described in any one of claims 1 to 3 in the preparation of a medicament for treating traumatic brain injury.

6. The use of the bioengineered apoptotic bodies as described in any one of claims 1 to 3 in the preparation of a medicament for promoting brain injury recovery, wherein promoting brain injury recovery includes any one or more of the following: reducing neuroinflammation, improving brain motor function, promoting neuronal proliferation, promoting nerve repair, inhibiting microglia activation, promoting M2 microglia polarization, improving cellular antioxidant capacity, reducing brain tissue ROS levels, reducing neuronal apoptosis and necrosis in the brain, and alleviating cerebral edema.

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