【作者】 宋远见；

【导师】 宗志敏；

【作者基本信息】 中国矿业大学 ， 化学工艺， 2012， 博士

【摘要】 药物能够高效转染神经细胞，且可透过血脑屏障（BBB）入脑，对脑部疾病的基础研究和临床治疗具有重要意义。本文的研究目标是开发出能够携带药物高效转染神经细胞和透过BBB入脑的纳米药物载体。以聚（乳酸-羟基乙酸）共聚物（PLGA）、聚乙二醇（PEG）、乳铁蛋白（Lf）和二油酰磷脂酰乙醇胺（DOPE）等为主要原料，采用水/油/水（W/O/W）复乳法制得了Lf-PEG-PLGA/DOPE纳米载体，借助FTIR、¹H-NMR、XRD、TGA、UV-vis、DLLS和TEM等分析手段对该载体进行了鉴定和表征。以血红素加氧酶-1（HO-1）质粒为模型基因药物，考察了载体对药物的荷载效果，并将其在神经细胞转染和透过BBB入脑方面进行了初步应用。结果表明：Lf通过PEG以共价键与PLGA成功接枝；Lf-PEG-PLGA/DOPE纳米载体呈无定型结构，热稳定性较好，平均粒径为142.2nm，颗粒分布均匀，Zeta电位为+16.4mV，能够通过静电吸附作用与带负电的HO-1质粒结合；可以转染海马神经细胞，但其透过BBB的能力稍差，原因可能在于带正电荷的DOPE仅以嵌入的形式掺杂于纳米载体中，与载体主体的结合强度不够而易脱落。为使纳米载体中带正电荷的部分更加稳定，本文选用甲基丙烯酰氧乙基三甲基氯化铵（DMC）和十四烷酸（MA）通过共价键共同接枝于壳聚糖（CS）上，制得季铵盐-十四烷酸-壳聚糖（QMC）。再以PEG-醛为媒介将Lf接枝到QMC上，制得Lf-PEG-QMC，通过自组装法制得纳米载体。对载体进行了鉴定、表征和性能评价，并将其初步应用于神经细胞转染和透过BBB入脑实验。结果表明：阳离子成份DMC以共价键接枝到了CS上，MA和Lf也接枝到了CS上；该载体呈晶型结构，热稳定性很好，平均粒径为244.7nm，且分布比较均匀，Zeta电位为+27.9mV，能够荷载基因；还可荷载难溶性药物紫杉醇（PTX），包封率可达80%以上，具有较好的缓释效果；能够转染海马神经细胞并可透过BBB入脑；通过毒性实验，发现除QMC对离体海马神经细胞稍有毒性外，PEG-QMC和Lf-PEG-QMC对海马神经细胞及小鼠的心、肝、脾、肺和肾等均无明显毒性，但在尾静脉注射载体时多数小鼠出现短暂的抽搐，约10%死亡。为提高纳米载体的静脉注射安全性，本文换用生物相容性更好的八聚精氨酸（R8）作为阳离子成份与MA共接枝于CS，制得八聚精氨酸-十四烷酸-壳聚糖（RMC），以PEG-醛为媒介将Lf接枝到RMC上，制得Lf-PEG-RMC，通过自组装法制得纳米载体。对载体进行了鉴定、表征和性能评价，并将其初步应用于神经细胞转染和透过BBB入脑实验。结果表明：带正电荷的R8和MA共同接枝到了CS上，Lf接枝到了RMC上；该载体呈晶型结构，热稳定性很好，平均粒径为226.7nm，分布均匀，Zeta电位为+18.7mV且能够荷载基因；对PTX的包封率亦超过80%，可较好地控制药物缓慢释放；能够高效转染海马神经细胞并可透过BBB入脑；对离体海马神经细胞及小鼠心、肝、脾、肺和肾等均无明显毒性，尾静脉注射时小鼠未出现异常。为进一步增强纳米载体的靶向递药能力，本文试用油酸修饰的Fe₃O₄为超顺磁性材料，对应用前景较好的QMC和RMC及两者的衍生物纳米载体磁性化修饰，制得了相应系列的磁性纳米载体（MNC）。对载体进行了表征，通过VSM方法对其磁性能进行了初步分析，并将其初步应用于神经细胞摄取实验。结果表明：Fe₃O₄成功包入了QMC和RMC系列的纳米载体中，MNC的晶型与包入Fe₃O₄前相应纳米载体的晶型一致，具有较好的热稳定性和超顺磁性，且能够顺利进入神经细胞。以大鼠缺血性脑中风模型为基础，将荷载HO-1基因的Lf-PEG-QMC、Lf-PEG-RMC、磁性Lf-PEG-QMC和磁性Lf-PEG-RMC纳米载体初步应用于缺血性脑中风的治疗。结果表明：该4种荷载HO-1基因的纳米载体能够对缺血性脑中风诱导的海马神经元损伤起到明显的抑制作用，其中后两者的效果相对更好。更多还原

【Abstract】 It is important for the basic research and clinical treatment of brain diseases thatdrugs transfect neurons and enter brain through the blood-brain-barrier （BBB）. Thegoal of the project is to prepare nano carriers loading drugs which are able to transfectneurons and enter brain through the BBB.Lf-PEG-PLGA/DOPE nano carriers were made from Poly （lactic acid-glycolicacid） copolymer （PLGA）, polyethylene glycol （PEG）, lactoferrin （Lf） anddioleoylphosphatidyl ethanolamine （DOPE） by water/oil/water （W/O/W） doubleemulsion method. The Lf-PEG-PLGA/DOPE nano carriers were identificated andexosyndromed by means of FTIR,¹H-NMR, XRD, TGA, UV-vis, DLLS and TEM.We studied the nano carriers’ ability of loading drugs, transfecting hippocampalneurons and carring them into brain through the BBB using the model gene drugsHeme oxygenase-1（HO-1） plasmid. The results showed that Lf was successfullylinked to PLGA through covalent bond, the prepared nano carriers had amorphousstructure and good thermal stability with an average particle size of142.2nm,uniform particle size distribution and Zeta potential of+16.4mV. The nano carrierscould bind plasmid with negatively charge through electrostatic adsorption andtransfect the hippocampal neurons, but they had temperate ability to enter brainthrough the BBB with the probalble reason that the binding between nano carriers andDOPE with positive charge was too slender because of the only imbedded form ofDOPE in the nano carriers.To stabilize the positively charged groups in the nano carriers, QMC wereprepared by grafting Methacryloxyethyltrimethyl ammonium chloride （DMC） andmyristic acid （MA） to chitosan （CS） through covalent bond. Lf was grafted to QMCthrough PEG-aldehyde and then Lf-PEG-QMC nano carriers were prepared by meansof self-assembly. The nano carriers were identificated, exosyndromed and functionevaluated. Their abilities of transfecting neurons and entering brain through BBBwere studied. The results showed that DMC with positive charge was grafted to CS,MA and Lf were also grafted to CS. Lf-PEG-QMC nano carriers had the crystalstructure, good thermal stability with an average diameter of244.7nm, more evenlydistributed, and the Zeta potential of+27.9mV. The nano carriers could load gene andpackage insoluble paclitaxel with high encapsulation efficiency of more than80%andgood slow-releasing effect. They could efficiently transfect the hippocampal neurons and enter brain through the BBB without significant toxicity for the isolatedhippocampal neurons and heart, liver, spleen, lung and kidney of mice except forQMC. But brief convulsions were found in the majority of mice and approximately10%of them died after the tail vein injection with Lf-PEG-QMC nano carriers.To improve the security of the intravenous injection of nano carriers,8poly-arginine （R8） with positive charge and MA were grafted to CS to prepare RMC.Lf was grafted to RMC through PEG-aldehyde and then Lf-PEG-RMC nano carrierswere prepared by means of self-assembly. The nano carriers were identificated,exosyndromed and function evaluated. Their abilities of transfecting neurons andentering brain through the BBB were studied. The results showed that R8and MAwas successfully grafted to CS, Lf was grafted to the RMC and Lf-PEG-RMC nanocarriers were prepared with the crystal structure, good thermal stability, averageparticle size of226.7nm, uniform distribution and Zeta potential of+18.7mV. Thenano carriers could load gene and package paclitaxel with high encapsulationefficiency of more than80%and good slow-releasing effect. They could efficientlytransfect the hippocampal neurons and enter brain through the BBB withoutsignificant toxicity for the isolated hippocampal neurons, heart, liver, spleen, lung andkidney and abnormity after the tail vein injection with the nano carriers in mice.To enhance the potential applications of nano carriers, oleic acid modified Fe₃O₄magnetic materials were used to modify QMC, RMC together with their derivativesand prepared corresponding series of magnetic nano carriers （MNC）. They wereexosyndromed and performed by VSM analysis for their magnetic properties. Theresults showed that Fe₃O₄were successfully packed into QMC, RMC together withtheir derivatives. MNC had good thermal stability and magnetic properties withcrystal form consistent with that of the corresponding nano carriers without Fe₃O₄,and can easily enter the neurons.Lf-PEG-QMC, Lf-PEG-RMC, magnetic Lf-PEG-QMC and magneticLf-PEG-RMC nano carriers loading HO-1gene were initially applied to the braindiseases based on the ischemic stroke model of rats. The results showed that the4kinds of nano carriers loading HO-1especially the latter two kinds could significantlyinhibit the injury of hippocampal neurons induced by ischemic stroke.更多还原