A mesenchymal stem cell apoptosis vesicle release system and its construction method and application

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, specifically a mesenchymal stem cell apoptotic vesicle delivery system and its construction method and application. The system of the present invention is co-incubated with α-mangostin and mesenchymal stem cell huMSC, α-M induces huMSC apoptosis, and while producing apoptotic vesicles, apoptotic vesicles α-M / AB loaded with α-M are obtained by huMSC's uptake of α-M and the hydrophobic interaction of α-M and apoptotic vesicle lipid membrane; α-M / AB is co-incubated with cell-penetrating peptide MAP activated by matrix metalloproteinase MMP9 responsiveness, to obtain α-M / AB-MAP. The system can target the lesion site, promote the survival of neurons in the cerebral ischemic area, and promote microglia to polarize from M1 to M2 phenotype, regulate the inflammatory immune microenvironment, promote the proliferation of vascular endothelial cells, promote angiogenesis to remove reactive oxygen species, have a neuroprotective effect on damaged neurons, can be used for the treatment of ischemic stroke patients, effectively reduce infarct area, improve neurological function, and have high safety.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a mesenchymal stem cell apoptosis vesicle release system and a construction method and application thereof. Background Art

[0002] Ischemic stroke refers to a severe acute cerebrovascular disease caused by cerebral ischemia due to occlusion of the cerebral artery, accounting for more than 80% of strokes. It is characterized by high morbidity, high disability rate, and high mortality rate. It is currently one of the main causes of disability in adults. After ischemic stroke occurs, acute ischemia leads to neuroexcitotoxicity and excessive reactive oxygen species (ROS) production, and a large number of neurons in the infarct area die. Necrotic neurons release damage-associated pattern molecules (DAMPs) to activate microglia to polarize to the proinflammatory M1 phenotype, promote the massive production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), further destroy neurons, and cause secondary damage. In addition, vascular endothelial cells and microglia secrete matrix metalloproteinases (MMPs) such as MMP2 and MMP9 to participate in the hydrolysis of extracellular matrix and basement membrane, further destroy the blood-brain barrier (BBB), and can also lead to the occurrence of hematogenous cerebral edema.

[0003] Intravenous infusion of recombinant tissue plasminogen activator (rt-PA) to achieve reperfusion is currently the only FDA-approved therapy. However, rt-PA has a narrow therapeutic window and must be treated within 4.5 hours after the onset of stroke to achieve substantial therapeutic effects. In addition, rt-PA has a short half-life, and reperfusion injury caused by blood flow restoration after treatment can aggravate neuronal damage, which greatly limits the clinical benefits of rt-PA thrombolytic therapy. Therefore, it is urgent to explore more effective treatment methods. After blood flow is restored, while clearing excess ROS and inducing microglia to polarize to the reparative M2 phenotype, trying to promote the survival of damaged neurons and promote microvascular regeneration in the lesion area to improve microcirculation will be beneficial to maximize tissue repair, restore neurological function, and achieve better therapeutic effects. In addition, by utilizing MMPs highly expressed in the damaged tissue area, responsive targeting of nanoformulations to ischemic reperfusion lesion tissues can be achieved.

[0004] At present, a large number of studies have shown that extracellular vesicles derived from mesenchymal stem cells contain biologically active substances of stem cells, which can replace stem cells to play important biological functions such as organ and tissue damage repair. Exosomes derived from mesenchymal stem cells have good cell regeneration and repair-promoting abilities, and have been shown to promote neurological recovery and brain remodeling after focal cerebral ischemia in rats and mice. However, the complex separation method and low yield make exosomes still difficult to apply in practice. Therefore, it is urgent to develop a new drug delivery therapy system based on stem cell vesicles that is easy to obtain, has a high yield, and integrates multiple therapeutic effects to effectively improve the therapeutic effect of ischemic stroke.

[0005] Apoptotic vesicles are vesicles produced by cell disintegration during apoptosis. The apoptotic process is controlled by a unique and precise signaling pathway. Certain metabolic pathways remain active during apoptosis, so that apoptotic vesicles contain a unique set of apoptotic metabolites. These microRNAs, mRNAs, DNA, proteins, and lipids can act as messengers to mediate intercellular communication and produce unique biological functions. More and more studies have shown that after phagocytic cells engulf apoptotic vesicles, they can initiate and promote tissue repair and induce phagocytes to transform into an M2 anti-inflammatory phenotype. Therefore, apoptotic vesicles are particularly suitable as therapeutic carriers for regulating inflammatory responses. In addition, studies have reported that stem cell-derived apoptotic vesicles can improve myocardial infarction by regulating receptor cell autophagy, regulate liver macrophage homeostasis to treat type 2 diabetes, mediate stem cell proliferation during epithelial tissue repair, promote endometrial regeneration and restoration of fertility in an intrauterine adhesion model, and induce angiogenesis to promote dental pulp regeneration, indicating that apoptotic vesicles have great application potential in the field of tissue repair. Compared with cell-based therapies, the characteristics of easy large-scale production, good stability, and easy availability give apoptotic vesicles obvious advantages in clinical treatment.

[0006] The present invention constructs a multifunctional mesenchymal stem cell apoptotic vesicle delivery system α-M / AB-MAP based on mesenchymal stem cell apoptotic vesicles derived from human umbilical cord. The natural product α-mangostin is loaded in the apoptotic vesicles to play a comprehensive neuroprotective effect, and the cell-penetrating peptide MAP activated by MMP9 responsiveness is modified on the surface of the apoptotic vesicle membrane to promote the accumulation of apoptotic vesicles in the ischemic reperfusion lesion area of ​​cerebral stroke, so that the entire multifunctional mesenchymal stem cell apoptotic vesicle treatment system can play a better therapeutic effect. Summary of the invention

[0007] The purpose of the present invention is to provide a multifunctional mesenchymal stem cell apoptotic vesicle delivery system with high safety, which can effectively reduce the infarct area and improve the neural function for patients with ischemic stroke, as well as a construction method and application thereof.

[0008] The multifunctional mesenchymal stem cell apoptosis vesicle release system provided by the present invention is constructed by the following steps:

[0009] (1) A certain concentration of α-mangostin (denoted as α-M) was co-incubated with human umbilical cord-derived mesenchymal stem cells (huMSCs). α-M induced huMSC apoptosis and produced apoptotic vesicles. At the same time, apoptotic vesicles loaded with α-M were obtained through the uptake of α-M by huMSCs and the hydrophobic interaction between α-M and the lipid membrane of apoptotic vesicles, denoted as α-M / AB.

[0010] (2) Subsequently, α-M / AB was co-incubated with MMP9-responsive activated cell-penetrating peptide (MAP) to prepare a multifunctional mesenchymal stem cell apoptotic vesicle delivery system, which was designated as α-M / AB-MAP.

[0011] The sequence of the cell-penetrating peptide MAP activated by MMP9 responsiveness in step (2) is:

[0012] Ac-FAEKFKEAVKDYFAKFWD-GSG-RRRRRRRRRR-PVGLIG-EGGEGGEGG (SEQ. ID. NO1).

[0013] The peptide is connected to the phospholipid bilayer of α-M / AB by hydrophobic interaction through the front α-helix (Ac-FAEKFKEAVKDYFAKFWD); followed by an arginine-rich transmembrane peptide (RRRRRRRRR) composed of 9 arginines, and a MMP-2 / 9 sensitive peptide (PVGLIG) is designed to be connected after the transmembrane peptide, aiming to make it specifically hydrolyzed and cleaved by MMP-2 / 9 with increased expression levels in the brain injury site and expose the arginine-rich transmembrane peptide, thereby bringing apoptotic vesicles into the cells at the injury site, making the MAP transmembrane peptide targeted to the brain injury area; finally, a polyanionic inhibitory peptide (EGGEGGEGG) is connected, and through the interaction of positive and negative charges, the positive charge of the arginine oligopeptide remains shielded during the circulation process.

[0014] Furthermore, the specific process of step (1) is as follows:

[0015] 10-15 ml of DMEM low-glucose serum-free culture medium containing 5 μg / ml α-M was mixed with 0.5-1.5×10 6 The huMSCs were incubated for 3-12 hours, the culture medium was collected, and the supernatant was taken and centrifuged at 600-1000g for 2-3 times, each time for 8-12 minutes. The supernatant was taken and centrifuged at 10000-12500g for 15-30 minutes. The precipitate was resuspended in phosphate buffer to obtain α-M / AB.

[0016] Furthermore, the specific process of step (2) is as follows:

[0017] MAP and α-M / AB were co-incubated in phosphate buffered saline (PBS) at a protein ratio of 1:100 to 2:100 for 2-4 hours. MAP was modified into the phospholipid bilayer of α-M / AB by hydrophobic insertion. The co-incubated PBS was centrifuged at 600-1000g for 2-3 times, each time for 8-12 minutes, the supernatant was taken, and the supernatant was centrifuged at 10000-12500g for 15-30 minutes. The precipitate was resuspended in phosphate buffer to obtain α-M / AB-MAP.

[0018] In the present invention, the α-M is a xanthone compound, which has outstanding antioxidant and anti-inflammatory abilities, can effectively remove high levels of ROS in the infarct area, and can play an anti-inflammatory role.

[0019] In the present invention, the mesenchymal stem cell apoptotic vesicles α-M / AB loaded with α-M have the effects of anti-inflammation, anti-apoptosis, scavenging ROS and promoting angiogenesis at the cellular level.

[0020] The α-M / AB-MAP constructed by the present invention can be enriched in the infarct area of ​​the ischemic brain area of ​​MCAO (middle cerebral artery occlusion) rats after administration through the tail vein, and can effectively reduce the area of ​​cerebral infarction and improve neurological function; at the same time, α-M / AB-MAP has good safety. Therefore, it can be used to treat patients with ischemic stroke.

[0021] In this system, α-M plays an anti-inflammatory role, promotes the polarization of microglia to the M2 phenotype, and clears ROS; AB plays an anti-inflammatory role, promotes the polarization of microglia to the M2 phenotype, and promotes the survival and angiogenesis of neurons in the injured area; MAP plays a cell-penetrating peptide function by responding to the high level of MMP9 in the lesion area, and delivers α-M / AB to the lesion area of ​​cerebral ischemia-reperfusion. A systematic evaluation showed that the multifunctional apoptotic vesicle α-M / AB promotes the polarization of microglia to the M2 phenotype, promotes the migration and tube formation of vascular endothelial cells, and also clears ROS, promotes the survival of neurons and improves cell viability; through MAP modification, α-M / AB-MAP can effectively target the brain injury area and reduce the retention of apoptotic vesicles in the liver and lungs, thereby enhancing the treatment of ischemic stroke, reducing the infarct area, and improving neurological function, which provides a new strategy for stem cell-based regenerative medicine therapy in the treatment of ischemic stroke.

[0022] This delivery system can respond to the MMP released from the ischemic brain injury area during ischemia-reperfusion injury, thereby promoting its retention and enrichment in the ischemic injury area. Through the synergistic effect of huMSC apoptotic vesicles and α-M, it promotes neuronal survival, promotes angiogenesis, regulates the inflammatory immune microenvironment, clears ROS, and exerts a neuroprotective effect on damaged neurons.

[0023] In the present invention, the selected therapeutic drug is α-M, which is loaded into the stem cell apoptotic vesicles by co-incubation with huMSCs.

[0024] In the present invention, lipopolysaccharide (LPS) was used to stimulate BV2 microglia to be in an inflammatory activation state, and then the cells were co-incubated with α-M / AB to detect the secretion of inflammatory factors to investigate the anti-inflammatory effect of apoptotic vesicles.

[0025] In the present invention, PC-12 cells treated with oxygen glucose deprivation / reoxygenation model (OGD / R) were co-incubated with α-M / AB to study the effects of apoptotic vesicles in scavenging ROS, promoting cell survival, and resisting apoptosis. PC-12 cells are rat adrenal pheochromocytoma cells that can differentiate into neurons and glial cells and are commonly used neural cell lines in the field of brain disease research.

[0026] In the present invention, BCEC rat brain capillary endothelial cells were co-cultured with α-M / AB to detect the effects of apoptotic vesicles on promoting BCEC tube formation and migration and the effects on BCEC cell viability.

[0027] The animals used in the present invention are SD male rats weighing between 240 and 260 g, which are common rat species used for middle cerebral artery occlusion (MCAO) models.

[0028] The present invention describes the preparation process and related characterization of a multifunctional mesenchymal stem cell apoptotic vesicle delivery system α-M / AB-MAP, and provides evaluation results of its neuroprotective effects at the cellular and animal levels.

[0029] The present invention proves that the delivery system has significant anti-inflammatory effects at the cellular level through enzyme-linked immunosorbent assay (ELISA) and other technologies, and can reduce the secretion level of pro-inflammatory cytokines of inflammatory-activated microglia and increase the secretion level of anti-inflammatory cytokines; through fluorescence imaging and other technologies, it is proved that the delivery system can significantly reduce the level of cellular ROS; through MTT and other experiments, it is confirmed that the delivery system can significantly improve the cell viability, migration, tube formation and significant neuroprotective effect of vascular endothelial cells.

[0030] The present invention proves at the animal level through fluorescence imaging technology that the system can significantly improve the targeted accumulation in the cerebral ischemia-reperfusion injury area through the modification of MAP.

[0031] The present invention confirms that α-M / AB-MAP can reduce infarct area and improve neurological deficits in the MCAO rat model. At the same time, α-M / AB-MAP also has good safety. The multifunctional mesenchymal stem cell apoptotic vesicles provide a promising treatment strategy for ischemic stroke. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1-Figure 8 It is the characterization of α-M / AB-MAP.

[0033] Figure 1 This is a structural model diagram of the apoptotic vesicle α-M / AB-MAP release system of multifunctional mesenchymal stem cells.

[0034] Figure 2 For huMSCs, add 5 μg / ml α-M or 1 mM HO 2 O 2 The morphological changes after 3 hours of incubation showed that huMSC cells shrank and cell membrane blebbled, showing typical morphological characteristics of apoptosis. Among them, (A) is the light microscopic morphology of huMSC under normal culture conditions; (B) is the light microscopic morphology of huMSC after adding 5μg / ml α-M and incubating for 3 hours; (C) is the light microscopic morphology of huMSC after adding 1mM H 2 O 2 Light microscopic morphology of huMSCs after 3 h of incubation.

[0035] Figure 3 (5μg / mlα-M forms apoptotic vesicles as α-M / AB, 1mM H 2 O 2 The apoptotic vesicles formed are OX-AB. Phosphatidylserine (PtdSer) signals can be detected on the apoptotic vesicles α-M / AB and OX-AB. (A) is the fluorescence image of α-M / AB combined with AnnexinV-FITC; (B) is the fluorescence image of OX-AB combined with AnnexinV-FITC.

[0036] Figure 4 More than 90% of the extracted apoptotic vesicles can detect positive PtdSer externalization signals, indicating that the extracted apoptotic vesicles have a high purity. Among them, (A) is the positive detection ratio of PtdSer externalization signals of α-M / AB; (B) is the positive detection ratio of PtdSer externalization signals of OX-AB.

[0037] Figure 5 The BCA method was used to compare the α-M induction and H 2 O 2 The yield of extracellular vesicles secreted into the culture medium by induced apoptotic vesicles and uninduced huMSCs showed that apoptotic vesicles had an advantage in yield (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0038] Figure 6 Transmission electron microscopy images of (A) OX-AB, (B) α-M / AB and (C) α-M / AB-MAP.

[0039] Figure 7 Western Blot quantification of huMSC, OX-AB and α-M / AB Cleaved Caspase 3 levels.

[0040] Figure 8 The particle size distribution of OX-AB, α-M / AB and α-M / AB-MAP after incubation in HBSS or HBSS containing 10% FBS at room temperature for 24 hours. Among them, (A) is the particle size distribution of OX-AB after incubation in HBSS for 24 hours; (B) is the particle size distribution of OX-AB after incubation in HBSS containing 10% FBS for 24 hours; (C) is the particle size distribution of α-M / AB after incubation in HBSS for 24 hours; (D) is the particle size distribution of α-M / AB after incubation in HBSS containing 10% FBS for 24 hours; (E) is the particle size distribution of α-M / AB-MAP after incubation in HBSS for 24 hours; (F) is the particle size distribution of α-M / AB-MAP after incubation in HBSS containing 10% FBS for 24 hours

[0041] Figure 9-Figure 19 :Anti-inflammatory, neuroprotective and angiogenic effects of α-M / AB at the cellular level.

[0042] Fig. 9 The iNOS and Arg-1 levels of BV2 cells treated with LPS after incubation with PBS, α-M, OX-AB, and α-M / AB for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001. (A) is the relative iNOS level; (B) is the Arg-1 level.

[0043] Fig.10 The levels of IL-1β, IL-6 and TNF-α in BV2 cells treated with LPS after co-incubation with PBS, α-M, OX-AB, and α-M / AB for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001. Among them, (A) is the level of IL-1β; (B) is the level of IL-6; (C) is the level of TNF-α.

[0044] Fig.11 The levels of IL-4, IL-10 and TGF-β in BV2 cells treated with LPS after co-incubation with PBS, α-M, OX-AB, and α-M / AB for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001. Among them, (A) is the level of IL-10; (B) is the level of TGF-β; (C) is the level of IL-4.

[0045] Fig.12 Cell viability of PC-12 cells treated with OGD / R after incubation with PBS, α-M, OX-AB, and α-M / AB for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0046] Fig.13 The Bcl-2 / Bax levels of PC-12 cells treated with OGD / R after incubation with PBS, α-M, OX-AB, and α-M / AB for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0047] Fig.14 The apoptosis rate of PC-12 cells treated with OGD / R after incubation with (B) PBS, (C) α-M, (D) OX-AB, and (E) α-M / AB for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001. Among them, (A) is the apoptosis rate of PC-12 cells not treated with OGD / R; (F) is the quantitative results of apoptosis rate of each group.

[0048] Fig.15 , 16 The ROS levels of PC-12 cells treated with OGD / R after incubation with PBS, α-M, OX-AB, and α-M / AB for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0049] Fig.17 Cell viability of BCEC cells after incubation with PBS, α-M, OX-AB, and α-M / AB for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0050] Fig.18 The wound healing of BCEC cells treated with scratches and then incubated with PBS, α-M, OX-AB, and α-M / AB for 48 hours (n=3). (A) is a light microscope image; (B) is a quantitative result.

[0051] Fig.19The tube formation of BCEC cells after being plated on Matrigel and incubated with PBS, α-M, OX-AB, and α-M / AB for 8 hours (n=3). (A) is a light microscopy image; (B) is the quantitative result of the relative total tubule length; (C) is the quantitative result of the relative branch length.

[0052] Figure 20-Figure 29 :Investigation on the targeting ability of α-M / AB-MAP in the cerebral ischemia-reperfusion injury area.

[0053] Fig. 20 (A) is the content of MMP9 in the supernatant of BV2 cells treated with different concentrations of LPS for 24 hours (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001. Fig. 20 (B) The content of MMP9 in the supernatant of BCEC cells at different time points after OGD / R treatment (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001. Fig. 20 (C) is the content of MMP9 in the homogenate of the affected and normal sides of the brain of MCAO rats 24 hours after surgery (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0054] Fig.21 , Fig. 22 α-M / AB, α-M / AB-MAP, α-M / AB and MMP inhibitor GM6001 were added to BCEC cells after OGD / R treatment for 12 hours, and the uptake of apoptotic vesicles by BCEC was observed 16 hours later (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0055] Fig.23 , Fig.24 α-M / AB, α-M / AB-MAP, α-M / AB and MMP inhibitor GM6001 were added to BV2 cells treated with 500 ng / ml LPS for 24 h, and the uptake of apoptotic vesicles by BCECs 16 h later (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0056] Fig.25 These are in vitro live imaging images of the brain of MCAO rats 4h, 12h, 24h, and 48h after intravenous injection of DiR, DiR-labeled α-M / AB, and DiR-labeled α-M / AB-MAP.

[0057] Fig.26 Quantification of relative fluorescence intensity of the affected side of the brain in MCAO rats after intravenous injection of DiR, DiR-labeled α-M / AB and DiR-labeled α-M / AB-MAP 4h, 12h, 24h, 48h (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0058] Fig. 27 The ratio of fluorescence intensity between the affected side (Ipsi) and the normal side (Contra) of the brain in MCAO rats 4h, 12h, 24h, and 48h after intravenous injection of DiR, DiR-labeled α-M / AB, and DiR-labeled α-M / AB-MAP (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0059] Fig.28 , 29 Relative fluorescence intensity of major organs of MCAO rats 24 h after intravenous injection of DiR, DiR-labeled α-M / AB and DiR-labeled α-M / AB-MAP (n=3). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0060] Figure 30-Figure 38 :Investigation of the neuroprotective effect of α-M / AB-MAP at the animal level.

[0061] Fig.30 TTC staining images of brain sections collected from MCAO rats 48 hours after intravenous injection of different preparations.

[0062] Fig.31 For quantification of infarct area (n=6). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0063] Fig.32 The ROS levels in the lesion area of ​​MCAO rats 24 hours after intravenous injection of different preparations (n=6). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0064] Fig.33 The body weight changes of MCAO rats within 7 days after intravenous injection of different preparations (n=6). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0065] Fig.34The neurological function scores of MCAO rats 72 hours after intravenous injection of different preparations (n=6). *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0066] Figure 35-38 Behavioral evaluation of MCAO rats 72 hours after intravenous injection of different preparations (n=6). Fig.35 Score forelimb placement, Fig.36 is the time for adhesive removal, Fig.37 is the adhesion contact reaction time, Fig.38 Forelimb touch asymmetry rate. *P<0.05, **P<0.01, or ***P<0.001, ****P<0.0001.

[0067] Fig.39 :The preparation process and functional model diagram of α-M / AB-MAP. Among them: (A) The preparation process of α-M / AB-MAP. (B) The functional model diagram of α-M / AB-MAP. DETAILED DESCRIPTION

[0068] The present invention will be further described below through specific embodiments.

[0069] Example 1: Preparation and characterization of α-M / AB-MAP

[0070] Prepare 1 mM HO 2 O 2 Induced apoptotic vesicles (OX-AB), α-M / AB and α-M / AB-MAP. For the preparation of OX-AB, 10 ml of 1 mM H 2 O 2 DMEM low glucose serum-free culture medium with 1×10 6 The cells were incubated with huMSCs for 3 h, and the culture medium was collected and centrifuged twice at 600 g for 10 min each time. The supernatant was taken and centrifuged at 12500 g for 20 min. The precipitate was resuspended in phosphate buffer to obtain the apoptotic vesicles OX-AB. For the preparation of α-M / AB, 10 ml of DMEM low-glucose serum-free culture medium containing 5 μg / ml α-M was mixed with 1×10 6The huMSCs were incubated for 3 hours, the culture medium was collected, centrifuged twice at 600g for 10 minutes each time, the supernatant was taken, and the mixture was centrifuged at 12500g for 20 minutes. The precipitate was resuspended in phosphate buffer to obtain α-M / AB. For the preparation of α-M / AB-MAP, MAP and α-M / AB were incubated in phosphate buffer (PBS) at a protein ratio of 1:100 to 2:100 for 3 hours, centrifuged twice at 600g for 10 minutes each time, the supernatant was taken, and the mixture was centrifuged at 12500g for 20 minutes. The precipitate was resuspended in phosphate buffer to obtain α-M / AB-MAP.

[0071] The particle size was measured using a nanoparticle tracking analyzer, and its morphology and size were observed under a transmission electron microscope (TEM).

[0072] Morphological observation of huMSC apoptosis process: huMSCs were treated with 5 μg / ml α-M or 1 mM H 2 O 2 After incubation for 3 h, the morphological changes of MSCs were observed under an optical microscope.

[0073] Investigation of apoptotic vesicle markers: (1) Investigation of phosphatidylserine (PtdSer) externalization on the membrane of apoptotic vesicles: According to the instructions of the cell apoptosis staining kit, Annexin V reagent was mixed with OX-AB or α-M / AB in phosphate buffer and incubated for 5 min. The cells were observed and photographed under an inverted fluorescence microscope, and the proportion of AnnexinV+ vesicles was analyzed by flow cytometry; (2) Investigation of the expression of Cleaved Caspase 3 in apoptotic vesicles: The relative contents of Cleaved Caspase 3 in huMSC, OX-AB, and α-M / AB were compared by Western Blot.

[0074] Investigation of apoptotic vesicle yield: 2×10 6 huMSCs were incubated with 5 μg / ml α-M or 1 mM HO 2 O 2 After 3 h of incubation, the culture medium was collected and centrifuged twice at 600 g for 10 min each time. The supernatant was centrifuged at 12500 g for 20 min and the precipitate was used to detect the protein content by BCA method. 2 O 2 Treated huMSCs served as controls.

[0075] Stability study: OX-AB and α-M / AB were placed in HBSS or Hank's balanced salt solution (HBSS) containing 10% fetal bovine serum (FBS) for 24 h, and the changes in the size of apoptotic vesicles were examined using a nanoparticle tracking analyzer.

[0076] The results show that: Figure 2 When huMSCs were treated with 5 μg / ml α-M or 1 mM H 2 O 2 After 3 hours of incubation, cell shrinkage and cell membrane blebbing were observed, which are typical morphological features of apoptosis, and apoptotic vesicles were produced. Figure 3 As shown in the figure, the green fluorescence signal of AnnexinV-FITC can be detected in the extracted apoptotic vesicles, indicating that the apoptotic vesicles have externalized PtdSer, which is also a marker of apoptotic vesicles. Figure 4 As shown in Figure 2, flow cytometry analysis showed that the positive rate of PtdSer externalization signals in the extracted apoptotic vesicles reached more than 90%, indicating that the purity of the extracted apoptotic vesicles was high. Figure 5 As shown, the BCA method compared the apoptotic vesicles induced by α-M, α-M / AB, and H 2 O 2 The yield of induced apoptotic vesicles and OX-AB uninduced extracellular vesicles secreted into the supernatant showed that apoptotic vesicles had an advantage in yield. Figure 6 As shown in Figure 2, all three apoptotic vesicles present a saucer-like vesicle structure with a particle size of approximately 120 nm. Figure 7 As shown in Figure 3, the apoptotic marker Cleaved Caspase 3 can be detected in the extracted apoptotic vesicles, indicating that huMSCs have undergone apoptosis. Figure 8 As shown, the particle sizes of the three apoptotic vesicles can remain relatively stable in HBSS and HBSS with 10% FBS at room temperature for 24 hours.

[0077] Example 2: Cellular investigation of the anti-inflammatory, neuroprotective and angiogenic effects of α-M / AB

[0078] Investigation of anti-inflammatory effect: In serum-free culture medium, BV2 cells were stimulated with 100 ng / ml LPS for 24 h, and then 20 μg / ml of apoptotic vesicles or an equivalent amount of α-M (1.1 μg / ml) were added and incubated for 24 h. The relative level of intracellular iNOS and the cytokine level in the cell culture supernatant were detected using an inducible nitric oxide synthase (iNOS) detection kit and an ELISA kit.

[0079] Investigation of neuroprotective effect: PC12 cells were cultured in sugar-free DMEM medium and placed in an anaerobic box for 10 hours. The cells were taken out and cultured in high-glucose DMEM medium for 2 hours. The medium was then replaced with high-glucose DMEM medium containing 20 μg / ml apoptotic vesicles or an equivalent amount of α-M (1.1 μg / ml) and cultured for 24 hours. The cell viability was detected by MTT method. At the same time, the culture supernatant was collected and the Bcl-2 / Bax level was detected by ELISA kit. The cells were collected and apoptosis was detected by Annexin V / PI cell apoptosis staining kit.

[0080] ROS investigation: PC12 cells were cultured in sugar-free DMEM medium, placed in an anaerobic box for 10 hours, the cells were removed, high-glucose DMEM medium was added and cultured for 2 hours, and then the medium was replaced with high-glucose DMEM medium containing 20μg / ml apoptotic vesicles or an equivalent amount of α-M (1.1μg / ml) and cultured for 24 hours. The cells were then incubated with high-glucose DMEM medium containing DCFH-DA for 60 minutes, and fixed with 4% paraformaldehyde for 20 minutes. After staining the cell nucleus with Hoechst, the cells were observed under an inverted fluorescence microscope and photographed. Image J was used to semi-quantify the fluorescence intensity.

[0081] Study on the promotion of endothelial cell migration, tube formation and cell viability: After incubation with 10 μg / ml apoptotic vesicles or an equivalent amount of α-M (0.55 μg / ml) in serum-free medium for a period of time, MTT (24h) experiment, scratch migration experiment (48h), and matrix gel tube formation experiment (8h) were performed.

[0082] The results show that: Figure 9-11 As shown in the figure, α-M / AB can significantly reduce the M1 marker iNOS of BV2 cells stimulated by LPS, and significantly reduce the secretion of pro-inflammatory cytokines IL-1β, IL-6 and TNF-α; at the same time, it significantly increases the M2 marker arginase-1 (Arg-1), and significantly increases the levels of anti-inflammatory cytokines interleukin-4 (IL-4), interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). Its effect is better than OX-AB without α-M and free α-M, and can exert the best anti-inflammatory effect. Figure 12-14 As shown in the figure, α-M / AB significantly increased the cell viability of PC12 cells treated with OGD / R and increased the level of Bcl-2 / Bax, which indicates that α-M / AB improved its anti-apoptotic ability and reduced its apoptotic rate, and had a good neuroprotective effect. Overall, the neuroprotective effect of α-M / AB was better than that of OX-AB and free α-M. Fig.15 , 16 As shown in the figure, α-M / AB significantly reduced the ROS level of PC12 cells treated with OGD / R, which was due to the loading of α-M with antioxidant activity; OX-AB could also reduce ROS to a certain extent, but the effect was not as good as α-M / AB. Figure 17-19As shown in the results, α-M / AB and OX-AB can significantly improve the cell viability, migration, and tube formation ability of BCEC cells, and increase the total length of tubules and the number of nodules, while free α-M has no such effect, which indicates that stem cell apoptotic vesicles have the ability to promote BCEC to form vascular structures. In summary, using stem cell apoptotic vesicles as the carrier of α-M, α-M / AB combines the multiple therapeutic effects of α-M and apoptotic vesicles, including anti-inflammation, anti-apoptosis, scavenging ROS, and promoting angiogenesis, and can play a better role in promoting tissue repair and achieve the best therapeutic effect.

[0083] Example 3: Investigation of the targeting ability of α-M / AB-MAP in the cerebral ischemia-reperfusion injury area

[0084] Investigation of MMP: ELISA was used to detect (1) the culture supernatant of BV2 cells stimulated with different concentrations of LPS for 24 h; (2) the culture supernatant of BCEC cells cultured in sugar-free DMEM medium in an anaerobic box for 10 h, then replaced with high-glucose DMEM medium and cultured normally for 24 h and 48 h; (3) the level of MMP9 in brain tissue homogenate of MCAO rats 24 h after surgery.

[0085] The ability of α-M / AB-MAP to respond to MMP9 was investigated: (1) For BCEC cells, BCEC were cultured in an anaerobic box with sugar-free DMEM medium for 10 h, and the medium was replaced with DMEM high-glucose medium or high-glucose medium supplemented with the broad-spectrum MMP inhibitor GM6001 to 2 μM for 12 h. DiI-labeled α-M / AB or α-M / AB-MAP was then added to a concentration of 10 μg / ml. After 16 h, the medium was discarded and the cells were fixed with 4% paraformaldehyde for 20 min. After staining the cell nucleus with Hoechst, the cells were observed under an inverted fluorescence microscope and photographed. Image J was used to semi-quantify the fluorescence intensity; (2) BV2 cells were cultured with a culture medium containing 500 ng / ml LPS or a culture medium containing 500 ng / ml LPS and 2 μM GM6001 for 24 h, and then DiI-labeled α-M / AB or α-M / AB-MAP was added to a concentration of 20 μg / ml. After 16 h, the culture medium was discarded and the cells were fixed with 4% paraformaldehyde for 20 min. After staining the cell nucleus with Hoechst, the cells were observed under an inverted fluorescence microscope and photographed. Image J was used to semi-quantify the fluorescence intensity.

[0086] In vivo targeting study of α-M / AB-MAP: SD male healthy rats weighing 240-260g were selected for MCAO surgery and injected with DiR-labeled α-M / AB or α-M / AB-MAP through the tail vein. They were anesthetized and killed at the pre-set time point after injection, and the complete brain tissue and major organs were collected, and images were collected using a small animal in vivo imager (IVIS). The fluorescence intensity of the samples was semi-quantitated using ROI analysis (Region-of-Interest Analysis).

[0087] The results show that: Fig. 20 As shown in (A)-(C), after LPS stimulation, the level of MMP9 secreted by BV2 cells increased significantly and was dose-dependent; after OGD / R treatment, the level of MMP9 secreted by BCEC cells increased significantly and was time-dependent; after MCAO surgery, the MMP9 level in the brain tissue on the side of the injury area (Ipsi) was significantly increased, while the MMP9 level in the brain tissue on the side of the non-infarct area (contra) was not significantly different from that in the sham operation group. In fact, in the stroke injury area, inflammatory-activated microglia and endothelial cells stimulated by ischemia-reperfusion oxidative stress secrete MMPs, which are important sources of MMPs in the injury area.

[0088] Investigation of the ability of α-M / AB-MAP to respond to MMP9: Fig.21 , 22 As shown in Figure 3 , MAP modification significantly promoted BCEC uptake of AB due to the increase in MMP9 after OGD / R, while the MMP inhibitor GM6001 could inhibit this effect; Fig.23 , 24 As shown in the figure, due to the increase of MMP9 after LPS stimulation, the modification of MAP significantly promoted the uptake of AB by BV2, while the MMP inhibitor GM6001 could inhibit this effect. This suggests that MAP can promote the uptake of apoptotic vesicles by cells in the area with high MMP9 levels by responding to MMP9.

[0089] In vivo targeting study of α-M / AB-MAP: The distribution of apoptotic vesicles in the brain Figure 25-27 As shown in the figure, 12 hours after injection, the accumulation of α-M / AB-MAP in the affected hemisphere was significantly higher than that of α-M / AB, and this significant difference lasted until 48 hours, at which time the accumulation of α-M / AB-MAP in the affected hemisphere was 2.0 times that of α-M / AB; the ratio of fluorescence intensity between the affected hemisphere and the normal hemisphere showed that the ability of α-M / AB-MAP to selectively accumulate in the affected hemisphere was stronger than that of α-M / AB, and at 48 hours, this value was 1.9 times that of α-M / AB; in addition, as Fig.28 , 29As shown in the figure, the quantitative results of fluorescence intensity of major organs showed that apoptotic vesicles were mainly retained in the liver, spleen, and lungs, but MAP modification reduced the retention in the liver and lungs, which was conducive to the long circulation of apoptotic vesicles and increased uptake in the brain injury area. This may also be attributed to the fact that MAP modification has a certain degree of shielding effect on the "eat me" signal on apoptotic vesicles.

[0090] Example 4: Animal level study on the neuroprotective effect of α-M / AB-MAP

[0091] Evaluation of cerebral infarction area: SD male rats weighing between 240 and 260 g were selected for MCAO surgery, and apoptotic vesicles or an equivalent amount of α-M (88 μg / kg) were injected through the tail vein at a dose of 1.6 mg / kg (protein equivalent). Each group of rats was given the drug within half an hour after the MCAO model was established. The brain was anesthetized and removed 48 hours later, and TTC staining was performed after frozen sections. After staining, the brain slices were placed on black cardboard and recorded and photographed with a digital camera. Then the Image Tool software was used to calculate the percentage of infarction area of ​​each brain slice. The calculation formula for the percentage of infarction area is: percentage of infarction area = (infarction area / hemi-cerebral area where the infarction area is located) × 100%.

[0092] Evaluation of neurological deficits: Each group of rats was given medication within half an hour after the MCAO model was established. After 72 hours, the neurological impairment of each group was evaluated according to the five-level four-point method established by Longa, and the neurological function was evaluated by forelimb placement score, adhesion removal time, adhesion touch reaction time, and forelimb touch asymmetry rate.

[0093] Evaluation of ROS level in infarct area: each group of rats was given medication within half an hour after the MCAO model was established. After 24 hours, the brain was cut into ischemic hemispheres by anesthesia. The fragments of the ischemic hemisphere were soaked in pancreatic cell digestion liquid and the cells were extracted. The intracellular ROS level was detected using a reactive oxygen species detection kit.

[0094] Body weight examination: Each group of rats was given medication within half an hour after the MCAO model was established, and the body weight of each rat was recorded for 7 consecutive days.

[0095] The results showed that TTC staining results were as follows: Fig.30 Compared with the Sham group, the Model group showed very significant cerebral infarction, and the quantitative results ( Fig.31 ) showed that the infarct area percentage of the Model group was as high as 73.8%, while α-M / AB-MAP reduced the infarct area percentage to 36.6% after 48 hours of administration, preliminarily confirming that α-M / AB-MAP has a neuroprotective effect. α-M / AB can also slightly reduce the infarct area, but the effect is not as good as α-M / AB-MAP. The results of ROS content in the ischemic hemisphere are as follows Fig.32Both α-M / AB-MAP and free α-M can significantly reduce the level of ROS in the ischemic hemisphere, while α-M / AB cannot significantly reduce the level of ROS in the ischemic hemisphere.

[0096] The neurological deficits of each treatment group were evaluated according to the five-level four-point method. Fig.34 As shown. The higher the score, the more severe the neurological damage. The rats in the Model group scored 3.5 points. However, the α-M / AB-MAP treatment group showed improved neurological deficits in MCAO rats 72 hours after administration, with a score of 1.5 points, indicating that α-M / AB-MAP can significantly improve the neurological damage in MCAO rats, and its effect is better than α-M / AB, while there is no significant difference between free α-M and the Model group. Body weight analysis ( Fig.33 ) and behavioral analysis ( Figure 35-38 ) also obtained similar results.

[0097] In summary, α-M / AB-MAP can exert the best neuroprotective effect, significantly reduce the infarct area of ​​the ischemic hemisphere, reduce ROS, promote weight recovery and behavioral recovery, and its effect is better than α-M / AB; while α-M can only clear ROS and has limited therapeutic effect. This may be due to the limited accumulation of α-M / AB in the lesion area, while α-M does not have the function of promoting cell survival and tissue repair like apoptotic vesicles. On the contrary, after modification by MAP, α-M / AB-MAP can better target the ischemic hemisphere infarct lesion area, and achieve the best therapeutic effect through the synergistic effect of α-M and stem cell apoptotic vesicles.

Claims

1. A method for constructing a mesenchymal stem cell apoptosis vesicle release system. It is characterized in that The specific steps are as follows: (1) α-mangostin was co-cultured with human umbilical cord-derived mesenchymal stem cells (huMSCs). α-mangostin induced huMSC apoptosis and produced apoptotic vesicles. At the same time, apoptotic vesicles loaded with α-mangostin were obtained through the uptake of α-mangostin by huMSCs and the hydrophobic interaction between α-mangostin and the lipid membrane of apoptotic vesicles, which were recorded as α-mangostin / AB. (2) Subsequently, α-mangostin / AB was co-incubated with MMP9-responsive cell-penetrating peptide MAP to prepare a multifunctional mesenchymal stem cell apoptotic vesicle delivery system, which was denoted as α-mangostin / AB-MAP; The sequence of the MAP is shown as SEQ.ID.NO 1.

2. The method for constructing the mesenchymal stem cell apoptosis vesicle delivery system according to claim 1, Features: The specific process of step (1) is as follows: 10-15 ml of DMEM low-glucose serum-free culture medium containing 5 μg / ml α-mangostin was mixed with 0.5-1.5×10 6 The huMSCs were incubated for 3-12 hours, the culture medium was collected, centrifuged at 600-1000 g for 2-3 times, each time for 8-12 minutes, the supernatant was taken, and centrifuged at 10000-12500 g for 15-30 minutes. The precipitate was resuspended in phosphate buffer to obtain α-mangostin / AB; The specific process of step (2) is as follows: MAP and α-mangostin / AB were co-incubated in phosphate buffered saline (PBS) at a protein ratio of 1:100 to 2:100 for 2-4 hours. MAP was modified into the phospholipid bilayer of α-mangostin / AB by hydrophobic insertion. The co-incubated PBS was centrifuged at 600-1000g for 2-3 times, each time for 8-12 minutes, the supernatant was taken, and the supernatant was centrifuged at 10000-12500g for 15-30 minutes. The precipitate was resuspended in phosphate buffer to obtain α-mangostin / AB-MAP.

3. The mesenchymal stem cell apoptotic vesicle release system obtained by the construction method of claim 1 or 2 is denoted as α-mangostin / AB-MAP.

4. Use of the mesenchymal stem cell apoptotic vesicle delivery system obtained by the construction method as described in claim 3 in the preparation of a drug system for treating ischemic stroke patients.

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