Nanoparticles Loaded With Melatonin Protect Adipose Derived Stem Cells From Injury Induced By Hypoxia/Reoxygenation

Objective: Mesenchymal stem cells (MSCs) transplantation is a promising therapeutic strategy for heart failure after myocardial infarction. However, those cells faced low survival rate due to oxidative stress and inflammatory microenvironment in ischemic heart tissue. Melatonin, as a powerful endogenous antioxidant, has been used to protect cells from oxidative injury. However, the effects of melatonin were limited due to short half-life and the unclear molecular mechanisms by which melatonin protects cells from apoptosis and necrosis. Nano drug delivery carriers protect loaded drugs from degradation in physiological environment and release them in a controlled manner, which results in longer drug effects and fewer side-effects.Therefore, we constructed poly(lactide-co-glycolide)- monomethoxy-poly-(polyethylene glycol) (PLGA-mPEG) nanoparticles loaded with melatonin and observed whether their protective effect to adipose derived mesenchymal stem cells(ADSCs) is better than melatonin alone both in vitro and in vivo.Method: The ADSCs, isolated from rats, were identified by flow cytometry and differentiation; Melatonin loaded PLGA-mPEG nanoparticles are prepared by a thin film evaporation-sonication method. Different combinations of mixing ratio of melatonin and PLGA-mPEG were considered to find the optimum condition for the formation of drug delivery system; The apoptotic and necrotic rates of ADSCs pretreated by melatonin or melatonin nanoparticles were detected by a flow cytometry method; The effect that melatonin protect the mitochondrial function of ADSCs from oxidative injury was measured by both Western Blot and immunofluorescent and the mechanism of it was investigated mainly focused on mitochondrial p53-CypD signal pathway; In in vivo study, the survival rate of ADSCs in myocardial infection area was detected in a in vivo Imaging System.Results: 1. ADSCs were positive for CD90 and CD29 negative for CD34, CD45,which is in line with the phenotypical characteristics of mesenchymal stem cells. 2.Melatonin from PLGA-mPEG nanoparticles (wPLGA-mPEG/wmelatonin= 5:1) is released in a controlled manner and 62% was released within 48 hours. 3. Both melatonin nanoparticles and melatonin along reduce the generation of ROS, inhibit the activation of apoptotic protein and decrease the death rate of ADSCs from 39.2 ±3.4% to 7.6±3.8% and 14.2±2.8% separately. 4. The melatonin pretreatment inhibits the translocation of p53 from cytoplasm to mitochondria and decreases the formation of p53-CypD complex. 5. The in vivo imaging system shows that more ADSCs survive in the infarcted heat areas after pretreated by melatonin nanoparticles.Conclusions: 1. Melatonin nanoparticles have a better cell protective effect against H/R than melatonin alone. 2. Melatonin protects ADSCs from oxidative injury by mitochondrial p53-CypD signal pathway that controls the opening of mitochondrial permeability transition pore (mPTP). 3. ADSCs, pretreated by melatonin, get better survival ability in the microenvironment of myocardial infarction.