Studies On Preparation Of Diphenylhydantoin Sodium Polybutylcyanoacrylate Nanoparticles And Its Pharmacodynamics For The Treatment Of Epilepsy

Objective:(1) To prepare Diphenylhydantoin sodium polybutylcyanoacrylatenanoparticles (DPH-PBCA-NPs) with three methods, then optimize theway of preparation and therefore to establish the best one.(2) To study the effect of the modification of DPH-PBCA-NPs bycoating with Tween-80 and the release characteristics ofdiphenylhydantoin sodium from the DPH-PBCA-NPs andsurface-modified DPH-PBCA-NPs with Tween-80 in vitro.(3) To evaluate the effects of DPH-PBCA-NPs and modifiedDPH-PBCA-NPs with Tween-80 in the epilepsy rat model and investigatethe advantages of nanoparticles drug delivery system.Methods:(1) Diphenylhydantoin sodium (DPH) was taken as the model drugand loaded by polybutylcyanoacrylate (PBCA) to prepareDPH-PBCA-NPs with the three methods of emulsion polymerizationincorporation, emulsion polymerization adsorption and interfacialpolymerization, respectively. The size and shape, drug loading, encapsulation ratio and Zeta potential of the DPH-PBCA-NPs wereevaluated to choose the better one.(2) One way experiment and orthogonal experiment were used tooptimize the methods and preparation condition.(3) DPH-PBCA-NPs were modified with Tween-80 and the differentcharacteristics between the DPH-PBCA-NPs and modifiedDPH-PBCA-NPs were compared.(4) The study of drug release behavior of the DPH-PBCA-NPs andmodified DPH-PBCA-NPs with Tween-80 in vitro were performed bydialysis method.(5) The lithium pilocarpine induced rat model of acute epilepsy wasestablished to investigate the changes of its behavior andelectroencephalogram (EEG) by using the Video-EEG monitoring, andtherefore to evaluate the effects of DPH-PBCA-NPs and modifiedDPH-PBCA-NPs with Tween-80 in controlling epilepsy.(6) Statistical treatment: The data was dealt with the StatisticalPackage for Social Sciences (SPSS 12.0 for Windows). The measurementdata were expressed as (?)±s, and t-test and ANOVA were used to assessthe differences between the groups, the x~2 test was used to assess thenumeration data, and P values below 0.05 were considered to bestatistically significant.Results: (1)A lot of floss and sediment could be were seen in theDPH-PBCA-NPs suspensions achieved by emulsion polymerizationincorporation. Transmission electron microscope (TEM) showed thatDPH-PBCA-NPs were adhered with each other, and nanoparticle sizeanalyzer displayed that the distribution of DPH-PBCA-NPs was wide. Onthe contrary, the DPH-PBCA-NPs made by emulsion polymerizationadsorption and interracial polymerization were spherical in shape andnarrow in distribution. Therefore, both the emulsion polymerizationadsorption and interfacial polymerization ought to be explored.(2) The pH, the amount of DPH, the stirring speed, the stirring timeand the reaction volume ratio (PBCA-NPs/DPH) were investigated asfactors influenced on the size and shape of the DPH-PBCA-NPs, the drugloading and encapsulation ratio by using one way experiment in theemulsion polymerization adsorption method. As a result, pH=7 and 2hours for stirring were chosen as the prepared condition. The amount ofDPH and PBCA-NPs, the stirring speed were optimized by theorthogonal-design experiment. We determined that the optimumconditions were: DPH, 75mg; PBCA-NPs, 35 ml and stirring speed,1,000rpm.(3) When reaction volume were 40ml, the optimized emulsionpolymerization adsorption method was as follows:①DPH, 75mg;②PBCA-NPs, 35ml;③stirring speed, 1,000rpm;④PH, 7;⑤temperature, 25℃;⑥stirring time, 2hours. In this condition, the DPH-PBCA-NPswere spherical in shape and narrow in distribution. The average size ofthe DPH-PBCA-NPs was about 120.40±5.32nm with a span of 0.57. Thedrug loading, encapsulation ratio was 22.1±0.41% and 82.51±1.53%,respectively, and the Zeta potential was-17.8mV.(4) The pH, the amount of DPH and olein, the phase volume ratio ofwater and oil (W/O) and the stirring speed were investigated on theparticle size and shape of the DPH-PBCA-NPs, the drug loading andencapsulation ratio by using one way experiment in interfacialpolymerization. As a result, the pH=7 and 800rpm of the stirring speedwere chosen as the prepared condition. The amount of DPH and olein andthe phase volume ratio of W/O were optimized by the orthogonal-designexperiment. We determined that the optimum conditions were: DPH,125mg; olein, 0.15ml; phase volume ratio of W/O, 1:1.(5) When reaction volume were 30ml, The optimized emulsioninterfacial polymerization method was as follows:①DPH,125mg;②phase volume ratio of W/O, 1:1;③olein, 0.15ml;④pH, 7;⑤stirringspeed, 800rpm;⑥Dextran-70, 1%(w/v);⑦α-BCA, 1%(v/v);⑧temperature, 25℃;⑨stirring time, 3hours. In this condition, theDPH-PBCA-NPs were spherical in shape and narrow in distribution. Theaverage grain size of the DPH-PBCA-NPs was about 189.80±3.45nmwith a span of 0.47. The drug loading and encapsulation ratio were 39.82±0.56% and 95.56±1.35%, respectively, and the Zeta potential was-34.8mV.(6) The particle size of modified DPH-PBCA-NPs withTween-80 was smaller than DPH-PBCA-NPs, there was significantdifference between them (t=3.813, P=0.001). The Zeta potential washigher after modified with Tween-80. The study of in vitro drug releasebehavior demonstrated that DPH-PBCA-NPs made by either the emulsionpolymerization adsorption or the interfacial polymerization and modifiedDPH-PBCA-NPs with Tween-80 show certain sustained releasecharacteristics.(7) The lithium pilocarpine induced rat model of acute epilepsy wassuccessfully established and its status epilepticus was confirmed by thenature of attack and EEG. The effects of DPH-PBCA-NPs and modifiedDPH-PBCA-NPs with Tween-80 to control status epilepticus were alsoinvestigated by the two indicators. The result showed that the effectiveratio of modified DPH-PBCA-NPs with Tween-80 and DPH-PBCA-NPswas 91.67% and 54.55%, respectively. The efficacy of modifiedDPH-PBCA-NPs with Tween-80 was better than DPH-PBCA-NPs(x~2=3.923, P=0.048) and DPH (x~2=4.557, P=0.033). The efficacy ofDPH-PBCA-NPs was higher than DPH (50%), there was no significantdifference between the two groups (x~2=0.043, P=0.835).Conelusion: (1) DPH-PBCA-NPs can be achieved by the emulsionpolymerization adsorption and interracial polymerization. DPH-PBCA-NPs possess suitable particles size, narrow distribution and high drugloading and encapsulation ratio, the stabilization of DPH-PBCA-NPsmade by interfacial polymerization is better than the emulsionpolymerization adsorption.(2) The particle size of modified DPH-PBCA-NPs with Tween-80are smaller than the DPH-PBCA-NPs, and the Zeta potential ofDPH-PBCA-NPs is higher after modified with Tween-80. It is easy topermeate into the BBB. The DPH-PBCA-NPs and modifiedDPH-PBCA-NPs with Tween-80 show certain sustained releasecharacteristics by the study the release characteristics ofdiphenylhydantoin sodium from the DPH-PBCA-NPs in vitro.(3) DPH-PBCA-NPs and modified DPH-PBCA-NPs withTween-80 can be used to improve the behavior and EEG of acute epilepsyrat model. The efficacy of modified DPH-PBCA-NPs with Tween-80 isbetter than DPH-PBCA-NPs and DPH.