Mangiferin Modified BMSC Drug Delivery System For Treatment Of Cerebral Ischemia-Reperfusion Injury

As a cutting-edge discipline in the field of regeneration,MSCs have shown potential value in disease treatment and drug delivery in recent years due to their multi-directional differentiation potential,powerful paracrine system,auto-homing properties and low immunity.Objective:In this study,MGF relies on CXCR4/SDF-1αaxis to promote the homing efficiency of bMSCs and improve their biological characteristics,and MSCs drug delivery platform is used to carry the neuroprotective agent MGF to construct a synergistic targeted therapy system combined with drugs and stem cells for the treatment of cerebral ischemia-reperfusion injury.Methods:（1）To examine the effects of MGF on bMSCs proliferation and migration in vitro,and select the optimal time.The expression of Cyclin D and CXCR4 is determined by WB（Western-blot）.The expression of CXCR4 is also verified by immunofluorescence staining and q PCR.（2）To verify the neuroprotective effect of MGF in vitro and its mechanism.The expression of NGF and BDNF in bMSCs and RA cells induced by MGF are determined by WB,Elisa and q PCR.At the same time,the regulation of pro-inflammatory factor TNF-αand anti-inflammatory factor IL-10 in RM cells is detected by WB and Elisa.（3）M-MGF is prepared by the thin film hydration method,and the morphology,size,stability,drug loading capacity,release behavior,and biological activity of micelles are verified.The MGF@M-bMSCs synergistic system is constructed through the uptake of M-MGF by M-bMSCs,and the optimal time and drug loading of the micelles are screened to verify the in vitro release ability of the system in vitro.The neuroprotective activity of the system is verified by acting on H₂O₂-damaged PC12 cells.（4）The migration ability and mechanism of M-bMSCs on H₂O₂-damaged PC12 were verified by Transwell experiment in vitro.The biological distribution and brain targeting ability of the synergistic system are investigated by imaging in vivo,and the brain homing efficiency of the synergistic system is evaluated by counting 5-bromodeoxyuridine positive cells.（5）A rat middle cerebral artery occlusion model（MCAO）is preparated,and SD rats are randomly divided into Sham group,Model group,Edaravone group,M-MGF group,MGF@bMSCs group and MGF@M-bMSC group.7 days after treatment,the brains are collected to investigate the volume of cerebral infarction,water content,neurological function in each group and histopathological staining（HE,Nissl,TUNEL）in each group.The experiments also verify the brain neurotrophic factors NGF and BDNF,the regulation of microglia phenotype and the expression of the factor TNF-α.Results:（1）bMSCs treated by 20μM MGF for 24 h can significantly promote cell proliferation and migration.They can also improve the expression of cyclin Cyclin D1 and CXCR4.This indicates that MGF can improve the basic biological properties of bMSCs in vitro.（2）MGF-induced bMSCs and RA cells for 24 h could increase the protein expression,concentration and m RNA degree of NGF and BDNF.In addition,MGF up-regulates the expression of IL-10 and down-regulates the expression of TNF-αin LPS-induced RM cells.It shows that MGF can promote the repair of nerve damage and inhibit the occurrence of neuroinflammation.（3）M-MGF is a uniform spherical shape of 30 nm,and the drug loading can reach94.78%with the following properties that relatively stable at normal temperature and physiological p H,it is nearly unchange in particle size and PDI in NS,PBS 7.4 and L-DMEM solutes,with constantly and slowly releasing speed,and good biological activity.The optimal time of M-bMSCs uptake of M-MGF is 6 h,and the drug loading is up to 178.17pg/cell.The system is released stably in vitro,and could improve the survival rate of H₂O₂-damaged PC12 cells.This indicates that MGF@M-bMSCs are successfully built with favourable performance.（4）Transwell chambers in vitro showed that M-bMSCs could migrate to H₂O₂-damaged PC12 cells and rely on the CXCR4/SDF-1αaxis.In vivo imaging find that the biological distribution of the system in vivo was obvious at 4 h,and the brain enrichment ability was strong.The positive cell markers in the brain of Brd U show that M-bMSCs have stronger targeting ability to brain injury than bMSCs.This indicates that the homing efficiency of the bMSCs drug-loading system modified by MGF is significantly improved.（5）MGF@M-bMSCs can facilitate neurological function,reduce cerebral infarct volume and brain water content,improve brain pathological tissue structure,and prevent neuronal damage.In-depth mechanism exploration find that the synergistic system up-regulates the expression of neurotrophic factors NGF and BDNF at the site of brain injury,regulates the phenotypic transformation of brain microglia,and inhibits the expression of TNF-α.It shows that the system can reduce the damage caused by cerebral ischemia-reperfusion through nerve function repair and remodeling,inhibition of neuroinflammation.Conclusion:MGF improves the proliferation and migration of bMSCs in vitro,and has neuroprotective activity by promoting the secretion of neurotrophic substances and anti-inflammatory effects.The synergistic system MGF@M-bMSCs has excellent drug-loading capacity,stable in vitro release performance and good biological activity.This system can also be efficiently enriched in the pathological microenvironment of the brain,and possess ideal therapeutic effect on cerebral ischemia-reperfusion injury.