Controled-release VEGF Plasmid Loaded PLGA Nanoparticles Therapy For Dopaminergic Neurons In Parkinson's Rats

ObjectiveTo observe the effect of vascular endothelial growth factor(VEGF)plasmid loaded poly-lactide-coglycolide(PLGA)nanoparticles on dopaminergic neurons of Parkinson's rats and to explore the mechanism underlying this effect.Method(1)The preparation and evaluation of partial lateral PD rat modelBefore the surgical procedures,the rats should be carry through repeated behavior test to make sure have no spontaneous rotations.Then rats were deeply anthetized with chloral hydrate(10%)and placed on a Korf stereotaxic instrument.A middle skin incision was made in the skull,stripped the periosteum in order to exposure the bregma and posterior.With reference to the rats stereotaxic atlas which write by Xin-min Bao,the coordinates of substantia nigra compacta(SNC)and ventral tegmental area(VTA)were determined:AP+4.8mm,ML+1.9mm,DV-7.5mm;AP+4.8 mm,ML+1.1mm,DV-8.0mm.Accroding to the above coordinates,two burr holes were drilled.8ul 6-OHDA were injected into the SNC and VTA,the injection rate was 1ul/min,and the syringe was left in place for additional 10 min before retracted slowly(1mm/min).Three weeks after the 6-OHDA surgery,the rotational behavior was tested with amphetamine administration.Only rats who rotated more than 7 turns/min were included in this study.(2)The preparation and evaluation of controled-release VEGF plasmid loaded PLGA nanoparticlesWe using the double emulsion-solvent evaporation technique to make the controledrelease VEGF plasmid loaded PLGA nanoparticles.Vacuum freeze-drying for 24 hours and stored in the cryogenic refrigerator.Using the scanning electron microscopy(SEM)to obse rve the nanoparticles morphology;using Malvern instruent to detected the mean particle,size distribution and zeta potential;the encapsulation efficiency was determined by UV detector.(3)Nanoparticles transplantionThe PD rats were randomly divided into five groups,which include the vehicle group;1ng/ml tail vein group;10ng/ml tail vein group;100ng/ml tail vein group;stereotaxic group.The vehicle group achieve 0.1M PBS through tail intravenous injection.The 2-4 groups achieve the different concentration of nanoparticles also through tail intravenous injection.The 1-4 groups achieve 4ul every two days of total ten days.The stereotaxic group achieve 20 ul nanoparticles for only one time through the intracranial injection according the above coordinates.(4)Testing methodThe rotational behavior was tested with amphetamine administration when the day after NS transplantion for 7?14?28days.The VEGF protein level of each group were tested by ELISA;the concentration of dopamine after transplantion were detected by High Performance Liquid Chromatography(HPLC);immune histochemical method was used to observe the expression of dopaminergic neurons.(5)Statistical analysisAll statistical analysis was performed with Graphpad Prism and SPSS 17.0.t-test was used to explore differences between groups.Significance was declared when p<0.05.Results(1)Characterization of NSThe sizes of NS was around 220.1±23.2nm for VEGF NS;the zeta potential was around-18.6±4.2mv;the obtained encapsulation efficiency was 91%.(2)Behavioral TestReduction of amphetamine-induced rotations in number was observed in rats receiving the PLGA nanoparticles which packaged the VEGF.Rats who were received tail intravenous injection 10ng/ml ? 100ng/ml ? 0.1M PBS showed a slight reduction in rotations at the end of the study(week 4).The stereotaxic group and 1ng/ml group showed a statistical reduction in the number of rotation at the end of this research when compared with the other groups(p<0.05).(3)ELISA analysis and HPLCThe 1ng/ml tail vein group and stereotaxic group showed a significant change when compared with the blank group(p<0.05).Though the 10ng/ml and 100ng/ml tail vein group have certain change,they don't have statistical influence.(4)TH immunofluorescenceAfter transplantion,the density of TH-positive fibers in the striatum which lesioned by 6-OHDA were increased except for the blank group.The 1ng/ml tail vein group and stereotaxic group played the obvious effect than others when compared with the blank group(p<0.05).Conclusion(1)Controled-release VEGF plasmid loaded PLGA nanoparticles displayed neuropr otective effects on DA fibers in the striatum in a rat model of PD.(2)Low-dose administration of VEGF displayed more neuroprotective effects on DA fibers in the striatum in a rat model of PD than high-dose.(3)After compared the effect of dopaminergic neuron protection between the tail vein and stereotaxic group,we confirmed that the controled-release VEGF plasmid loaded PLGA nanoparticles can cross the blood brain barrier.(4)Compared with the stereotaxic,transplant the controled-release VEGF plasmid loaded PLGA nanoparticles through tail vein was safe and convenient,which developing a new way in the treatment of Parkinson's disease.