Molecular Imaging Guided Research On Therapy Efficacy Of MSCs For Ischemic Stroke After PHD2 Silencing

ObjectivesStroke is an acute blood circulation disorder that seriously jeopardizes the safety and health of human life.Stroke has a "three high" characteristic of high morbidity,high disability,and high mortality.At present,Stroke is the second most common cause of death and a major cause of disability worldwide,of which more than 70% are ischemic stroke.Although many drugs have been shown to have therapeutic effects on stroke in animal experiments.Currently,the only drug approved by the FDA for the treatment of acute ischemic stroke is the thrombolytic drug recombinant tissue plasminogen activator(r-t PA).Due to limited time window,risk of intracranial hemorrhage,and reperfusion injury,only less than 10% of patients with acute ischemic stroke have indications for revascularization therapy;Moreover,approximately half of the patients who do receive t PA for acute ischemic stroke have a significant long-term disability.Therefore,restorative therapeutics after stroke is extremely important.Mesenchymal stem cells(MSCs)are pluripotent stem cells that can self-renewal and have multi-directional differentiation potential,such as bone,fat,cartilage,and bone marrow stromal cells.Under specific conditions,MSCs can differentiate into neurons,glia and endothelial cells.MSCs have received considerable study worldwide.Although the safety of MSCs has been affirmed in clinical applications,there has been some controversy in its efficacy.This may be related to its lower migration and lower survival ability after transplantation.In this study,MRI visible si RNA nanoparticles were designed to deliver si RNA targeting PHD2 of MSCs,thereby enhancing the migration and survival of MSCs after PHD2 silencing,and thus enhancing the therapy efficacy of MSCs for ischemic stroke.At the same time,Combining BLI with MRI,homing/migration and survival of MSCs were dynamic,noninvasive and continuous observed in vivo.Part Ⅰ Construction and characterization of MRI-visible si RNA delivery nanoparticlesObjective: The research was focusing on constructing the magnetic nanoparticles combining si RNA delivery and MSCs tracking with MRI together.The si RNA condenses,release,protection,delivery and MRI imaging charactors of the nanoparticles were identified.Methods: Alkyl-PEI was synthesized using hyperbranched amphiphilic low molecular weight polyethylenimine.The SPIO nanoparticles and Alkyl-PEI were used to obtain the Alkyl-PEI/SPIO nanoparticles.The morphology and size of the nanoparticles were characterized by transmission electron microscopy(TEM),dynamic light scattering(DLS)and zeta-potential.si RNA biding affinity,release and protection charactors of the nanoparticles were characterized by agarose gel electrophoresis analysis.The T2 relaxivity of the nanoparticles was measured on a clinical 1.5T MR scanner.FITC-labeled si RNA was delivered by the nanoparticles into MSCs,immunofluorescence imaging and Prussian blue staining were carried out to detect the transfection efficacy of the nanoparticles.Cell Counting Kit-8(CCK-8)test was carried out to detected the cytotoxicity of the nanoparticle after incubation with MSCs at different time points.Results: DLS results showed that the sizes of the Alkyl-PEI/SPIO nanoparticles in water were approximately 80-120 nm.TEM images showed the monodispersed structure of the nanoparticles,which contained iron oxide clusters inside the core.The zeta potential of the Alkyl-SPIO/si RNA nanocomplexes increased with the increase of N/P ratio,indicated successful loading of si RNA on the nanoparticles.This unique structure resulted in a significantly high r2 value with 549.7 m M-1S-1,and T2-weighted signal intensity decreased gradually with the increased iron concentration of the nanoparticles.An electrophoretic retardation assay was employed to investigate the binding ability of the Alkyl-PEI/SPIO nanoparticles to the negatively charged si RNA.The free si RNA was completely retarded when the nitrogen/nucleic acid phosphate(N/P)ratio was 20.To test whether the si RNA was released when the nanocomplexes entered a biological environment,the prepared Alkyl-SPIO/si RNA nanocomplexes were subjected to a heparin decomplexation assay.The si RNA was successfully released from the Alkyl-PEI/SPIO nanoparticles when incubated with a certain amount of heparin.Additionally,the serum stability assay showed that Alkyl-PEI/SPIO effectively protected the si RNA from degradation.With the CCK-8 test,the incubation of the MSCs with the Alkyl-SPIO/si RNA nanocomplexes showed no obvious cytotoxicity compared with the control group even 72 hours later.Conclusions: MRI-visible delivery system with low cytotoxicity and high transfection efficacy were constructed in this section.The nanoparticles could efficiently condense,releasePart Ⅱ In vitro research on therapy efficacy of MSCs after PHD2 SilencingObjective: The research was to explore the migration,survival ability of MSCs after inhibiting the hif-prolyl hydroxylase 2(PHD2)expression of MSCs with si RNA delivered by MRI-visible nanoparticles,and also to reveal the underlying mechanism.Methods: si RNA(50 n M,100 pmol per well)and an appropriate amount of Alkyl-PEI/SPIO(N/P=20)were mixed and added into the culture medium of MSCs seeded on six-well plates at a density of 5×10^5 cells/well,after incubation for 6h,the fresh medium was changed and MSCs were culture to 48 h.Western blot was carried out to detect the expression of PHD2,C-X-C chemokine receptor type 4(CXCR4)and hypoxia-inducible factor 1α(HIF-1α).Transwell assay was used to detect the migration ability of MSCs after PHD2 silencing.And H2O2 apoptosis assay was carried out to detect the survival ability of MSCs in the oxidative stress environment after PHD2 silencing.Results: Western blot results showed that si PHD2(si RNA targeting PHD2)delivery by Alkyl-PEI/SPIO nanoparticles significantly decreased PHD2 expression in MSCs.PHD2 silencing in MSCs significantly increased the expression of CXCR4,a receptor expressed on stem cells that is critical for stem cell migration.And also,the expression of HIF-1α,related to the survival ability of cells,increased significantly after PHD2 silencing.Transwell assay showed more si PHD2-MSCs migrated to the lower chamber than si CON-MSCs,indicating enhanced migration ability of si PHD2-EPCs after PHD2 silencing.H2O2 apoptosis assay results showed that BLI intensity was significantly higher in the si PHD2-MSCs group,indicating that more MSCs survived in the oxidative stress environment after PHD2 silencing.Conclusions: The in vitro experiments showed expression of PHD2 in MSCs was efficiently inhibited with si PHD2 delivered by the nanoparticles.PHD2 silencing significantly increased the migration and survival ability of MSCs via enhanced expression of CXCR4 and HIF-1α.Part Ⅲ In vivo research on therapy efficacy of MSCs after PHD2 SilencingObjective: Labeling MSCs with lentiviral vector carrying dual reporter gene and MRI visible nanoparticles.Detecting the dynamic of MSCs after transplantation with the muti-modal imaging combined BLI with MRI.And also exploring the therapy efficacy of MSCs for ischemic stroke after PHD2 silencing,offering new methods and ideas to the ischemic stroke therapy with MSCs.Methods: MSCs were co-cultured with adequate luc2–e GFP lentiviral vector plus 5 mg/m L polybrene,six hours later,fresh complete medium was changed,and 72 hour later,the MSCs were passaged.MSCs of different density were planted into the 12 well plants to detect the correlation between the cell number and luminescence intensity using BLI.1 day after the photothrombotic ischemic stroke model was established,1×10^6 MSCs were intracardially injected into the left ventricular of the mice.Mice were sent for BLI and MRI scanning at different time points to detect the homing/migration and survival of MSCs in vivo.7 days after MSCs transplantation,infarct volume was evaluated by T2WI;Western blot was used to detect the expression of BDNF and VEGF;Immunohistochemical staining was used to evaluate the angiogenesis and neurogenesis with the specific marker,CD31 and doublecortin(DCX);Alao Prussian blue staining was used to make sure that MSCs existed in the peri-infarct area even 7 days after MSCs transplantation.14 days after MSCs transplantation,fractional anisotropy(FA)value and fiber counts were evaluated by DTI scaning;Neural plasticity and white matter recovery were evaluated by Neu N and myelin basic protein(MBP)staining respectively.m NSS and foot-faults test were carried out at different time points to detect the functional recovery after ischemic stroke.Results:As the results showed,BLI intensity was in high linear correlation with the MSCs number.1 day after MSCs transplantation,the T2*WI results showed that transplanted MSCs migrated to the peri-infart area of the brain.3 days and 7 days after transplantation,MSCs were observed mainly in the peri-infarct area.Significantly higher BLI signal intensity in the si PHD2-MSCs group 1 day after MSCs transplantation was observed,and 3 days and 7 days after transplantation,the BLI signal in the si PHD2-MSCs group was still higher than the control group.Prussian blue staining confirmed the exitance of MSCs in the peri-infarct area 7 days after transplantation.A higher level of BDNF was observed in the si PHD2-MSCs group,however,no significant differences were found in the expression of VEGF 7 days after MSCs transplantation.si PHD2-MSCs treatment significantly reduced the infarct volume,as the T2 WI results showed.A significantly increased expression of DCX and CD 31,was observed in the subventricular zone(SVZ)of the mice that received si PHD2-MSCs.14 days after MSCs transplantation,with the in vivo DTI analysis,increased FA value and fiber counts in the ipsilesional corpus callosum were observed in the si PHD2-MSCs group.A higher level of MBP and Neu N was observed in the si PHD2-MSCs group.Conclusions: The effective combination of BLI with MRI methods could be used to observe the dynamics of MSCs in vivo,guiding the transplantation of MSCs for the treatment of ischemic stroke.PHD2 silencing enhanced the migration and survival ability of MSCs,thereby improving the therapeutic efficacy of MSCs for ischemic stroke.