Preparation Of Plga-peg-EGCG Sustained-release Nanoparticles And Synergetic Effect With Nimodipine In The Treatment Of Subarachnoid Hemorrhage

The biological activities of EGCG largely depended on its anti-oxidative,anti-inflammatory and anti-virus activities.In addition,EGCG regulated variety signaling pathways,which involved in autophagy and apoptosis,and eventually affected cell survival.Therefore,EGCG exhibited potential clinical application values in the prevention and treatment of cancer,cardiovascular,and neurodegenerative diseases.However,the low bioavailability of EGCG limited the therapeutic effects.Sustained-released drug nanoparticles can improve the stability,cell uptake and prolonged the elimination of drugs in vitro.Nimodipine is the only clinical drug for white matter damage,and its long-term use can lead to reduced blood pressure.In the present study,we prepared PEG-PLGA-EGCG nanoparticles based on the process at optimum conditions.Subsequently,the synergetic effect of EGCG and nimodipine was assessed in the treatment of subarachnoid hemorrhage（SAH）.Methods and resultsPrepared the sustained released EGCG nanoparticlesEGCG was encapsulated in polyethylene glycol（PEG）and poly（lactic-co-glycolic acid）（PLGA）by using double emulsion-solvent evaporation method.Firstly,the dosages of PEG-PLGA,EGCG,and Tween-80 were analyzed.Particle size,drug loading rate,and encapsulation efficiency were investigated by using nanoparticle potentiometer,laser particle sizer,and HPLC.Based on the obtained average particle size in the range of 106-256 nm,Zeta potential in the range of 78-86 %,and entrapment efficiency from 78 % to 86 %,response surface methodology with central composite factorial design was used to investigate the optimization of sustained released-system components,which will select the optimal formulation requiring a minimum of experiments.The optimization was 12 mg/ml PEG-PLGA,7mg/ml EGCG and 1.5% Tween 80,and the property of obtained nanoparticle was particle size 168 nm,Zeta potential of-22.8 m V,and entrapment efficiency of 86 %.The morphological characterization of the nanoparticle was observed by using transmission electron microscope,which are distributed evenly round in shape and less aggregation.In vitro release profile and stability of EGCG nanoparticlesTwo different release mediums were used to evaluate in vitro release profile.The first one was obtained an acid buffer solution（p H 3.0）with ascorbic acid 0.25%,and the second one was 0.1 M PBS buffer solution（p H 7.4）.The in vitro cumulative drug release profile revealed a sequential and controlled release of EGCG.Drug release of EGCG nanoparticles in acid and PBS buffer solution was 9.26 % and10.35 % in 24 h,and 15.94 % and 26.98 % in 7d,respectively.However,drug release of EGCG in acid and PBS buffer solution was 41 %and 100 % in 24 h,respectively.No detection was found in EGCG group at 7 d.The particle size of EGCG nanoparticles was decreased in 6 months.The synergetic effect of EGCG nanoparticles with nimodipine in the inhibition of neuronal cell damage after SAHUsing SAH mouse model,the synergetic effect of EGCG nanoparticles with nimodipine was evaluated after SAH.MDA,SOD,GSH-Px,and T-AOC were used to detect oxidative stress;LDH and Nissl staining were used to evaluate the damage of neuronal cells;the expression of Ca MKII,Atg5,Beclin-1,and Caspase-3 were used to analyze autophagic and apoptotic activity.The results showed that there are significantly decreased MDA level and increased SOD GSH-Px,and T-AOC levels to block the oxidative stress after SAH in the EGCG nanoparticle and EGCG nanoparticle combined with nimodipine groups.In addition,the down-expression of Ca MKII-induced autophagy and apoptosis was also detected in these two groups.As a result,the damage of neuronal cells was restored in the EGCG nanoparticle and EGCG nanoparticle combined with nimodipine groups after SAH.Conclusion: EGCG PEGylated-PLGA nanoparticles was stably and evenly round in shape with 168 nm in particle size.These nanoparticles have sustained release profile,which improve the bioavailability of EGCG.EGCG nanoparticle combined with nimodipine had a significantly synergetic effect in the treatment of SAH.Therefore,EGCG nanoparticle could be a stable,safety,and effective potential drug release strategy for the treatment of SAH.