【作者】 胡凯莉；

【导师】 蒋新国； 冯林音； 张奇志； 陈钧；

【作者基本信息】 复旦大学 ， 药剂学， 2009， 博士

【摘要】 血脑屏障（Blood-brain barrier，BBB）在防止脑内环境受到外界毒物侵害的同时也很大程度上限制了药物的脑内转运及脑部疾病的治疗。以非侵袭性给药途径提高药物脑内递送的研究，因其能够克服外科手术所带来的创伤和危险，已成为国内外脑内靶向给药的研究重点。其中尤以通过受体介导转运入脑的微粒递药系统的研究最为成功，转铁蛋白受体的单克隆抗体OX26连接的空间稳定免疫脂质体已被证明可以通过血脑屏障上的特异受体，成功将小分子药物、蛋白及基因药物递送入脑，但OX26为小鼠抗大鼠转铁蛋白受体的单克隆抗体，具有很强的种属特异性，只能与大鼠转铁蛋白受体特异性结合，还存在严重的免疫原性。此外，载药系统脂质体的体内外稳定性均较差，若载药系统在未到达脑微血管的吸收部位前解体即会严重影响药物的脑内递送效果。鉴于此，有必要而且也亟需探寻新型的靶向功能分子和载药系统，构建更为合理、有效的脑内靶向递药系统。为了克服以上缺陷，本课题构建两种新型的生物可降解脑内递药系统—乳铁蛋白修饰聚乳酸—聚羟基乙酸纳米粒载药系统(Lf-NP_PLGA)和乳铁蛋白修饰聚乳酸纳米粒载药系统(Lf-NP_PLA)。乳铁蛋白（Lactoferrin，Lf）是一种存在于哺乳动物体内的阳离子糖蛋白，已有研究证实Lf可以通过受体介导的胞吞转运入脑，且其入脑量显著高于转铁蛋白和OX26。Lf-NP_PLGA和Lf-NP_PLA可借助表面Lf以受体介导胞吞转运方式增加其脑内转运；将生物可降解的纳米粒作为药物的载体，不仅安全性好，还能显著提高药物的稳定性和输送能力；纳米粒采用复乳／溶媒蒸发法制备，适合于包载水溶性大分子药物，如蛋白多肽和基因药物，可以掩盖其自身的理化性质，代之以纳米粒的特性；纳米粒表面经PEG修饰，可避免单核巨噬系统的吞噬，延长在血浆中的半衰期，提高血浆药时曲线下面积（Area under curve，AUC），具有长循环作用。帕金森病（Parkinson’s disease，PD）是一种常见的神经系统退行性疾病，在50岁以上的人群中发病率达1～2％，目前尚缺乏能够根治的药物和手段，临床仍以对症治疗为主。近年来，神经保护药物的开发为PD的治疗带来了新的希望。脑内注射实验证明，促肾上腺皮质激素释放激素相关肽Urocortin（UCN）对大鼠PD模型具有很好的治疗效果。但UCN静脉注射后不能入脑，极大限制了其应用。鉴于此，我们采用新型生物可降解脑靶向纳米载药系统Lf-NP_PLGA包载UCN进行脑内递送，以期提高对PD的治疗效果。本文第一部分为Lf-NP_PLGA的脑内递药研究。第一章首先由复乳／溶媒蒸发法制备马来酰亚胺—聚乙二醇（Maleimide-PEG，MAL-PEG）和甲氧基聚乙二醇（MPEG）表面共修饰的聚乳酸-聚羟基乙酸（PLGA）纳米粒，然后通过其表面MAL-PEG与巯基化Lf共价连接而制备Lf-NP_PLGA。该纳米粒的平均粒径在100nm以下，Zeta电位在-20 mV以下。免疫电镜观察证实纳米粒表面存在具有活性的Lf，Lf-NP_PLGA可由表面的Lf通过受体介导的胞吞转运入脑。利用ELISA法测得每个Lf-NP_PLGA表面连接的Lf约为42个。以粒径为指标，最终筛选的优化制备条件为：聚合物总量25mg，巯基化试剂2-IT∶Lf摩尔比为40∶1；外水相为1％的胆酸钠溶液。第一部分第二章采用香豆素-6作为纳米粒的荧光探针，bEnd．3作为体外BBB模型，以未连接Lf的普通纳米粒NP_PLGA为对照，对Lf-NP_PLGA的脑内递药特性进行了体外评价。在pH 7.0和4.0的条件下，香豆素-6在NP_PLGA和Lf-NP_PLGA中24 h的累积泄漏量均小于3.5％，证实香豆素-6是一种较为理想的纳米粒探针，能用以较为准确的示踪NP_PLGA和Lf-NP_PLGA体内外生物学行为。细胞摄取实验结果发现，bEnd．3在15，30和60 min时，对Lf-NP_PLGA的摄取均显著高于NP_PLGA。摄取抑制实验的结果证实，Lf-NP_PLGA是由LfR转运入脑，通过穴样凹陷，包被凹陷和巨胞饮途径内吞，为高尔基体和溶酶体参与的能量依赖过程。细胞活力测定结果显示，Lf，NP_PLGA和Lf-NP_PLGA浓度高达3 mg／mL时均对细胞活力没有显著影响。以上体外实验证明，Lf-NP_PLGA是一种具有脑内递药特性且毒性较低的药物传递系统。第一部分第三章以香豆素-6作为Lf-NP_PLGA的荧光探针，NP_PLGA作为对照，采用药动学方法考察了Lf-NP_PLGA小鼠脑内递送特性。结果显示，Lf-NP_PLGA在脑中的AUC是NP_PLGA的2.49倍，在血中AUC为NP_PLGA的1.14倍，与NP_PLGA相比较其脑靶向指数为2.19。组织分布结果表明，两种纳米粒主要在肝脾聚集，与NP_PLGA相比，Lf-NP_PLGA在心和脾的分布增多，但在肾的分布减少。小鼠尾静脉注射60 mg／kg载香豆素-6的Lf-NP_PLGA和NP_PLGA 1 h后，脑组织切片显示，Lf-NP_PLGA在黑质区，纹状体区及大脑皮质区的绿色荧光颗粒分布均显著高于NP_PLGA。单核—巨噬细胞特异性染色结果显示，高剂量Lf-NP_PLGA不引起BALB／c鼠大脑、小脑、心和肺的巨噬细胞增多，仅对肝、脾和肾产生一过性的轻微毒性。以上体内评价结果证实，Lf-NP_PLGA具有一定的脑内递药特性，并且是一种毒性较低的脑内递药系统。第一部分第四章建立了单侧损毁的急性6-OHDA PD大鼠模型及慢性鱼藤酮PD大鼠模型。采用Lf-NP_PLGA包载UCN多肽，通过行为学、免疫组织化学、神经递质的代谢产物三方面指标考察了Lf-NP_PLGA-NUC对于两种PD大鼠模型的治疗作用。结果表明，高剂量的Lf-NP_PLGA-NUC对于6-OHDA和鱼藤酮PD模型大鼠的行为都有显著的改善作用，并能提高纹状体内TH和DA的含量，显著优于同等剂量的未修饰纳米粒NP_PLGA-NUC。以上结果充分证实Lf-NP_PLGA是一种有效的脑内递药系统，为具有脑内治疗活性药物，特别是稳定性差、难以通过血脑屏障的蛋白多肽药物脑内递送提供了有效手段。本文第二部分为Lf-NP_PLA的脑内递药研究。第一章设计并制备了Lf-NP_PLA脑靶向纳米载药系统。Lf-NP_PLA的平均粒径在150 nm以下，Zeta电位为-20 mV左右。X射线光电子能谱对Lf-NP_PLA的表面元素分析结果显示，Lf-NP_PLA表面的N元素来自于Lf，证实NP_PLA与Lf共价连接的存在。免疫电镜观察结果进一步证实Lf-NP_PLA表面存在Lf，并从另一个角度证实Lf-NP_PLA可由表面的Lf通过受体介导的胞吞转运入脑。利用ELISA法测得MAL-PEG-PLA∶MPEG-PLA和Lf-SH∶MAL-PEG-PLA为不同比例时，Lf-NP_PLA表面连接的Lf的量。以粒径和表面Lf的量为指标，最终筛选的优化制备条件为：MPEG-PLA∶MAL-PEG-PLA质量比为9∶1；巯基化试剂2-IT∶Lf摩尔比为40∶1；MAL-PEG-PLA∶巯基化蛋白摩尔比为3∶2；反应时间为9 h。第二部分第二章以香豆素-6作为荧光探针，对Lf-NP_PLA的脑内递药特性进行了体外评价。结果显示，pH 7.0和4.0的条件下，香豆素-6在NP_PLA和Lf-NP_PLA中24 h的累积泄漏量均小于6％，证实香豆素-6能用以较为准确的示踪其体内外的生物学行为。小鼠脑毛细血管内皮细胞bEnd．3摄取载香豆素-6的NP_PLA和Lf-NP_PLA的定性观察结果显示，bEnd．3在30，60和120 min时，对Lf-NP_PLA的摄取均显著高于NP_PLA。摄取抑制试验的结果证实了Lf-NP_PLA可以通过Lf介导的胞吞转运过程被摄取，通过包被凹陷和巨胞饮途径内吞，为高尔基体参与的能量依赖过程。细胞活力测定结果显示，3 mg／mL的Lf，NP_PLA和Lf-NP_PLA对细胞活力均没有显著影响。以上体外实验证明，Lf-NP_PLA可以借助Lf经由受体介导的胞吞转运入脑，是一种具有脑内递药特性且毒性较低的药物传递系统。第二部分第三章以香豆素-6作为Lf-NP_PLA的荧光探针，以NP_PLA作为对照，采用药动学方法考察了Lf-NP_PLA小鼠脑内递药的特性。小鼠尾静脉注射60 mg／kg载香豆素-6的Lf-NP_PLA和NP_PLA 1h后，脑组织切片显示，Lf-NP_PLA第三脑室室周区，纹状体区及大脑皮质区的绿色荧光颗粒分布均显著高于NP_PLA。Lf-NP_PLA在脑中的AUC是NP_PLA的2.98倍，在血中AUC为NP_PLA的0.92倍，Lf-NP_PLA的脑靶向指数为3.22。组织分布结果表明，两种纳米粒主要在肝脾聚集，与NP_PLA相比，Lf-NP_PLA在肝、脾和肾的分布显著增多。单核—巨噬细胞特异性染色结果显示，高剂量Lf-NP_PLA对BALB／c鼠不引起大脑、小脑、心、肾和肺的巨噬细胞增多，仅对肝和脾产生轻微的一过性毒性。本文第三部分制备了包载牛血清白蛋白（BSA）的Lf-NP_PLGA和Lf-NP_PLA，比较了两种纳米载药系统在体外的释放和降解情况，并对它们的脑内递药性能进行了比较。体外释放试验结果表明，在37℃的PBS两者释放行为相似，均符合Weibull分布，15天的累积释放百分率在45％左右。在含有血浆的PBS中，Lf-NP_PLGA和Lf-NP_PLA的释放均有所加快，15天的累积释放百分率分别达到88％和61％，Lf-NP_PLGA的释放已比较完全。以Lf作为靶向功能分子构建的Lf-NP_PLGA和Lf-NP_PLA均能显著提高药物的入脑。相比较而言，Lf-NP_PLA在具有较高脑靶向效率的同时，被网状内皮系统摄取也更为显著。Lf-NP_PLGA虽然脑靶向性能稍逊，但可能具有更好的安全性。更多还原

【Abstract】 Blood-brain barrier（BBB） protects the internal environment of brain fromoutside toxicities, but meanwhile, it restricts entry of most drugs for brain diseasetherapy. Therefore, the research of enhancement of brain drug delivery throughnoninvasive means, especially development of novel drug delivery system for braindrug targeting, is on focusing in pharmaceutics. Among them, the most successfulstrategy is nanoparticle delivery systems targeting to the brain through receptormediated transcytosis. A murine anti-rat transferrin receptor monoclonal antibody,OX26, was used as brain targeting molecule and coupled with liposome, the formedimmunoliposome was proved delivery successfully small molecule drugs, proteins, aswell as gene therapies into the brain via receptor-mediated transcytosis. Nevertheless,the OX26 is only effective in rats, and it also possesses strong immunogenicity. Inaddition to that, liposome has poor stability both in vitro and in vivo, if itdisintegrated before arriving at the brain microvessels, the brain delivery capacity ofthe system will be largely impaired. In view of these, there is an urgent need toexplore new brain targeting molecule as well as drug loading vehicle to constructmore reasonable and effective brain drug delivery systems.To solve this problem, two novel biodegradable brain drug targeting systems,lactoferrin conjugated polyethylene glycol-polylactide-polyglycolide nanoparticlesystem(Lf-NP_PLGA) and lactoferrin conjugated polyethylene glycol-polylactidenanoparticle system(Lf-NP_PLA) were developed. Lactoferrin is a mammalian cationiciron-binding glycoprotein which has been demonstrated to cross the BBB viareceptor-mediated transcytosis. Markedly high brain uptake was observed forlactoferrin relative to transferrin and OX26 in vivo. The surface modified lactoferrincan enhance the nanoparticles’ brain entrance via receptor-mediated transcytosis; thebiodegradable nanoparticle served as drug carrier, which can endow the drug deliverysystem with good safety and increase its drug loading capacity; the double-emulsionand solvent evaporation preparation method facilitated encapsulation ofmacromolecules such as proteins, peptides and genes, and can mask theirphysico-chemical characteristics instead of nanoparticle properties; the stealthy pegylated nanoparticle can avoid the uptake by reticuloendothelial system, prolongtheir plasma half-life, and enhance area under the concentration-time curve（AUC）.Parkinson’s disease is a devastating degenerative neurological illness affecting1-2% of the ’over 50’ population. There are no effective therapies for PD at presentand clinical available treatments are all for symptom relief. Nowadays, thedevelopment of neuroprotective agents throws some new light on PD therapy.Following intracerebral injection, a corticotrophin releasing hormone related peptide,urocortin（UCN） significantly improved the behavior of PD model rats. But thepeptide does not cross the BBB after intravenous injection, which greatly restrains itsutilization. Therefore, we encapsulated it into the novel biodegradable brain drugtargeting system Lf-NP_PLGA, in order to enhance its therapeutic effect for PD.The first part described the research of Lf-NP_PLGA brain delivery system. Thefirst chapter is construction and characterization of Lf-NP_PLGA. The pegylated polylactide-poly glycolide nanoparticle, whose surface was co-modified withmaleimide-polyethylene glycol（MAL-PEG） and methoxy-polyethylene glycol（MPEG）, was made by double-emulsion and solvent evaporation method andcovalently conjugated with thiolated lactoferrin（Lf） via its maleimide functionalgroup. The average particle size of Lf-NP_PLGA was below 100 nm, and its zetapotential was around -20 mV. Immuno-gold staining result showed that bioactive Lfwas covalently coupled to the nanoparticle’s surface. ELISA detected the surface Lfnumber per nanoparticle was around 42. The optimized preparation conditions are:total polymer amount is 25 mg; 2-IT: Lf when thiolation is 40: 1; and the outer waterphase is 1% sodium cholate solution.In the second chapter of part one, a lipophilic fluorescent dye, coumarin-6,serving as a nanoparticle probe, was incorporated in Lf-NP_PLGA to investigate its braindrug delivery characteristics in a bEnd.3 BBB model with unconjugated nanoparticle(NP_PLGA) as control. The uptake results demonstrated that the uptake amount ofLf-NP_PLGA was significantly higher than that of NP_PLGA at 15, 30 and 60 min. MTTresults illustrated that after incubation with Lf-NP_PLGA, NP_PLGA and Lf respectively,the cell viability was always above 80% even at a relative high concentration of 3mg/mL. The uptake inhibition experiment results showed that Lf-NP_PLGA wastransport into brain by an energy-dependent process involving LfR, caveolae, clathrin,golgi apparatus, lysosome and macropinocytosis. All the in vitro results showed thatLf-NP_PLGA is a good brain delivery system with very low toxicity. Besides, the release of coumarin-6 from Lf-NP_PLGA and NP_PLGA in pH 7.0 and pH 4.0 PBS after 24 h wereall below 3.5% which proved that coumarin-6 is an ideal nanoparticle probe that canindicate accurately the in vitro and in vivo behavior of Lf-NP_PLGA and NP_PLGA.In the third chapter, the brain delivery property of Lf-NP_PLGA was evaluated bypharmacokinetics studies with NP_PLGA as control. The biodistribution result in micerevealed the AUC of Lf-NP_PLGA in cerebrum was 2.49 times of that of NP_PLGA, andthe brain drug targeting index of Lf-NP_PLGA was 2.19 compared with NP_PLGA. The twokinds of nanoparticles were both mainly distributed to the liver and spleen. Whilecompared to NP_PLGA, Lf-NP_PLGA distributed more to heart and spleen but less tokidney. One hour after a dose of 60 mg/kg Lf-NP_PLGA or NP_PLGA injection in micecaudal vein, fluorescent microscopy of brain coronal sections revealed a higheraccumulation of Lf-NP_PLGA in the substantia nigra, striatum and cerebral cortex regionthan that of NP_PLGA. Immunostaining of monocyte-macrophage demonstrated thathigh dose of Lf-NP_PLGA cannot induce the increase amount of macrophage incerebrum, cerebellum, heart and lung in BALB/c mice, only had light toxicity to liver,spleen and kidney, and its acute toxicity was transient. These results proved the braindelivery property and safety of Lf-NP_PLGA in vivo.In the fourth chapter, the urocortin（UCN） peptide was encapsulated inLf-NP_PLGA and NP_PLGA and their pharmacodynamics studies were carried out inunilateral 6-OHDA lesioned acute rat Parkinson’s disease（PD） model and chronicrotenone rat PD model with evaluation of behavioral, immunohistochemical andneurotransmitters’ changes. The results illustrated that the high dose Lf-NP_PLGA-UCNimproved the behavior of the rats in both PD model with increase of their striatumtyrosine hydroxylase and domapine levels, which is much better than the same dose ofunconjugated NP_PLGA-UCN. The above results proved that Lf-NP_PLGA is an effectivebrain delivery system which is promise in delivery brain active drugs, especiallyprotein and peptides with poor stability and low BBB transport.The second part describes the research of Lf-NP_PLA brain delivery system. In thefirst chapter we constructed and characterized Lf-NP_PLA. The average particle size ofLf-NP_PLA was below 150 nm, and its zeta potential was around -20 mV. X-rayphotoelectron spectroscopy and immuno-gold staining result showed that bioactive Lfwas covalently coupled to the nanoparticle’s surface. ELISA detected the surface Lfnumber per nanoparticle constructed with different ratios of MAL-PEG-PLA toMPEG-PLA and Lf-SH to NP_PLA. The optimized preparation conditions are: MAL-PEG-PLA: MPEG-PLA weight ratio is 1: 9; Lf-SH: MAL-PEG-PLA is 2: 3;2-IT: Lf when thiolation is 40: 1; and the reacting time is 9 h.In the second chapter of part two, the brain delivery property of Lf-NP_PLA wascompared with unconjugated nanoparticle(NP_PLA) in a bEnd.3 BBB model withcoumarin-6 incorporated in them as nanoparticle probe. The results showed that thecoumarin-6 release from Lf-NP_PLA and NP_PLA in pH 7.0 and pH 4.0 PBS after 24 hwere all below 6% which proved that coumarin-6 can indicate accurately the in vitroand in vivo behavior of Lf-NP_PLA and NP_PLA. The bEnd.3 cells uptake resultsdemonstrated that the uptake amount of Lf-NP_PLA was significantly higher than that ofNP_PLA at 30, 60 and 120 min. The uptake inhibition test showed that Lf-NP_PLGA wasuptake by an Lf mediated energy-dependent process involving clathrin, golgiapparatus and macropinocytosis. MTT results showed that none of Lf-NP_PLA, NP_PLAor Lf has significant toxicity on the cells. All the in vitro results showed that Lf-NP_PLAcan be delivery into the brain through Lf receptor mediated process and Lf-NP_PLA is agood brain delivery system with low toxicity.In the third chapter of part two, the brain delivery property of Lf-NP_PLA wasevaluated by pharmacokinetics studies with NP_PLA as control. One hour after a dose of60 mg/kg coumarin-6 loaded Lf-NP_PLA or NP_PLA injection in mice caudal vein,fluorescent microscopy of brain coronal sections revealed a higher accumulation ofLf-NP_PLA in third ventricle region, striatum and cerebral cortex region than that ofNP_PLA. The biodistribution result in mice revealed the AUC of Lf-NP_PLA in cerebrumwas 2.98 times of that of NP_PLA, and the brain drug targeting index of Lf-NP_PLA was3.22 compared with NP_PLA. The two kinds of nanoparticles were mainly distributed tothe liver and spleen. While compared to NP_PLA, Lf-NP_PLA distributed more to liver,spleen and kidney. Immunostaining of monocyte-macrophage demonstrated that highdose of Lf-NP_PLA cannot induce the increase amount of macrophage in cerebrum,cerebellum, heart kidney and lung in BALB/c mice, only had light toxicity to liver,and spleen, and its acute toxicity was transient.In the third part, we prepared BSA loaded Lf-NP_PLGA and Lf-NP_PLA, comparedthe releasing and degradation behavior of them as well as its brain delivery properties.The release results showed that, the two kinds of nanoparticles possess similar releasebehavior in PBS at 37℃, which all fit the Weibull distribution, with a 45% release ofBSA during the 15 days period. While in PBS containing 5% plasma, the release ofthe two kinds of nanoparticles was more complete, with an 88% release for Lf-NP_PLGA and 61% release for Lf-NP_PLA. Using Lf as a targeting molecule, bothLf-NP_PLGA and Lf-NP_PLA can enhance brain delivery. Lf-NP_PLA has a better braintargeting property but its uptake by the reticuloendothelial system was moreprominent. In comparison, Lf-NP_PLGA has a moderate brain targeting property withgood safety.更多还原