【作者】 王夏红；

【导师】 肖波；

【作者基本信息】 中南大学 ， 神经病学， 2007， 博士

【摘要】 目的：(1)初选苯妥英钠聚氰基丙烯酸正丁酯纳米微粒(DPH-PBCA-NPs)的制备方法，并对制备工艺进行优化，建立DPH-PBCA-NPs最佳的制备方法和工艺。(2)利用Tween-80对DPH-PBCA-NPs进行表面修饰，探讨Tween-80对DPH-PBCA-NPs进行表面修饰的可行性。考察DPH-PBCA-NPs和Tween-80修饰的DPH-PBCA-NPs体外释药规律，探讨其缓释效应。(3)利用癫痫动物模型评价DPH-PBCA-NPs和Tween-80修饰的DPH-PBCA-NPs的抗癫痫疗效，探讨纳米载药系统的优越性。方法：(1)选择α-氰基丙烯酸正丁酯(α-butylcyanoacrylate，α-BCA)为载体，应用乳化聚合包入法、乳化聚合吸附法和界面聚合法制备DPH-PBCA-NPs，考察DPH-PBCA-NPs的粒径、形状、载药量、包封率和Zeta电位等指标，初选出制备方法。(2)通过单因素试验结合正交试验对初选DPH-PBCA-NPs的制备方法进行工艺优化，确立DPH-PBCA-NPs最佳的制备方法和工艺。(3)利用Tween-80作为表面活性剂对DPH-PBCA-NPs进行表面修饰，比较Tween-80修饰前后纳米微粒的变化。(4)采用动态透析法进行体外释药，对DPH-PBCA-NPs和Tween-80修饰的DPH-PBCA-NPs体外释药规律进行研究。(5)构建氯化锂-匹罗卡品急性癫痫大鼠模型，利用视频脑电监测仪，观察脑电图动态变化过程和致痫大鼠的行为学改变，评估DPH-PBCA-NPs和Tween-80修饰的DPH-PBCA-NPs的抗癫痫疗效。(6)统计学处理：采用SPSS 12.0统计软件包对所有数据进行统计，计量资料以均数±标准差((?)±s)表示，用t检验和方差分析对各组数据进行比较；计数资料采用x~2检验。P＜0.05有统计学意义。结果：(1)乳化聚合包入法制备的DPH-PBCA-NPs，透射电子显微镜显示纳米微粒相互粘连，形状不匀称，纳米粒度分析仪显示粒径分布不均匀，纳米胶体溶液中出现絮状物和沉淀。而乳化聚合吸附法和界面聚合法制备的DPH-PBCA-NPs，纳米微粒形状呈均匀球形，分散良好，呈现单一分布峰，分布集中。因此，初选出乳化聚合吸附法和界面聚合法，并对其制备工艺进行研究。(2)通过单因素试验考察了pH值、苯妥英钠用量、搅拌速度、搅拌时间以及反应物体积比(PBCA-NPs与苯妥英钠溶液)对乳化聚合吸附法制备的DPH-PBCA-NPs的粒径、载药量和包封率的影响，确定pH值为7、搅拌时间2h作为基本的制备条件。对苯妥英钠用量、PBCA-NPs胶体溶液体积和搅拌速度设计正交试验，确定苯妥英钠75mg、PBCA-NPs体积35ml、搅拌速度1，000rpm为最佳的制备条件。(3)优化后DPH-PBCA-NPs乳化聚合吸附法的制备工艺如下：当反应体积为40ml时，苯妥英钠用量75mg，PBCA-NPs用量35ml，搅拌速度1.000 rpm，反应体系的pH值为7，温度25℃，搅拌时间2h。该工艺条件制备的DPH-PBCA-NPs性状及各种指标如下：纳米微粒呈球形，形态均匀，分散性佳，平均粒径120.40±5.32nm，跨度0.57，载药量22.1+0.41％，包封率82.51±1.53％，Zeta电位-17.8mV。(4)通过单因素试验考察了pH值、苯妥英钠用量、三油酸甘油酯用量、水相与有机相体积比(W／O)以及搅拌速度对界面聚合法制备DPH-PBCA-NPs的粒径、载药量和包封率的影响，确定pH 7，搅拌速度800rpm作为基本的制备条件。对苯妥英钠用量、三油酸甘油酯用量以及水相与有机相体积比设计正交试验，确定苯妥英钠125mg、三油酸甘油酯0.15ml、水相与有机相体积比1：1为最佳的制备条件。(5)优化后DPH-PBCA-NPs界面聚合法制备工艺如下：当反应体积为30ml时，苯妥英钠量125mg，水相与有机相体积比(W／O)1：1，三油酸甘油酯0.15ml，反应体系pH值为7，搅拌速度800 rpm，1％(w／v)的Dextran-70，α-BCA为1％(v／v)，温度25℃，搅拌时间3h。该工艺条件制备的DPH-PBCA-NPs性状及各种指标如下：纳米粒的形态均匀，呈球形，分散性佳。平均粒径189.80±3.45 nm，跨度0.47，载药量39.82±0.56％，包封率95.56±1.35％，Zeta电位-34.8mV。(6)Tween-80修饰后DPH-PBCA-NPs的粒径减小，与未修饰的DPH-PBCA-NPs相比具有显著性差异(t=3.813，P=0.001)，Zeta电位也有明显的升高。体外释药结果表明乳化聚合吸附法和界面聚合法制备的DPH-PBCA-NPs及Tween-80修饰的DPH-PBCA-NPs均具有缓释性。(7)成功构建氯化锂-匹罗卡品急性癫痫大鼠模型，致痫大鼠在行为和脑电图上均表现出了癫痫持续状态。行为学结合脑电图考察DPH-PBCA-NPs和Tween-80修饰的DPH-PBCA-NPs对致痫大鼠的抗癫痫疗效，Tween-80修饰的DPH-PBCA-NPs组有效率为91.67％，DPH-PBCA-NPs组有效率为54.55％，Tween-80修饰的DPH-PBCA-NPs疗效优于DPH-PBCA-NPs(x~2=3.923，P=0.048)和苯妥英钠(x~2=4.557，P=0.033)，DPH-PBCA-NPs的有效率高于苯妥英钠普通剂型(50％)，二者相比无显著性差异(x~2=0.043，P=0.835)。结论(1)乳化聚合吸附法和界面聚合法均可以制备出粒径合适、形态均匀、载药量和包封率良好的DPH-PBCA-NPs，界面聚合法制备的DPH-PBCA-NPs稳定性优于乳化聚合吸附法。(2)Tween-80修饰后的DPH-PBCA-NPs粒径减小，Zeta电位升高，有利于透过血脑屏障。体外释药显示乳化聚合法吸附法和界面聚合法制备的DPH-PBCA-NPs和Tween-80修饰的DPH-PBCA-NPs具有较好的缓释性。(3)DPH-PBCA-NPs和Tween-80修饰的DPH-PBCA-NPs能够改善致痫大鼠的行为学和脑电图，具有抗癫痫疗效。Tween-80修饰的DPH-PBCA-NPs疗效优于DPH-PBCA-NPs和苯妥英钠普通剂型。更多还原

【Abstract】 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.更多还原