转铁蛋白受体介导的聚合物泡囊递药系统的构建及其脑内递药特性研究
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摘要
随着人类社会的老龄化,脑部疾患的发病率正呈逐年上升的趋势,已经成为危害人类生命和健康的主要疾病之一。血脑屏障(BBB)的存在严重影响了脑部疾病的治疗,而脑内靶向递药系统的研究为其带来新的希望,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,代表性的研究为转铁蛋白受体的单克隆抗体OX26介导入脑的空间稳定免疫脂质体。其具有诸多优点,但也存在某些不足,如载药系统脂质体的体内外稳定性相对较差,往往尚未到达脑微血管的吸收部位,载药系统即已解体,严重影响了药物的脑内递释效果。此外,脂质体的性质如包封性、膜稳定性、降解性等不易调控,也降低了药物到达靶部位的可能性。鉴于此,有必要探寻新型的载药系统,构建更为合理、有效的脑内靶向递药系统。
聚合物泡囊是一种新型的药物载体。它具有脂质体和纳米粒等微粒载药系统的主要优点,而且其体内外的稳定性显著优于脂质体;其水溶性的内腔结构对水溶性药物如蛋白多肽类药物的包封量可望优于纳米粒;其膜层较厚,有利于包封脂溶性药物,可与水溶性内腔的水溶性药物共同发挥“鸡尾酒疗法”作用;此外,通过聚合物分子的种类、分子量、嵌段比例等选择,所构建泡囊载药系统的理化特性如粒径、膜厚、渗透性、载药量、表面PEG修饰、靶向功能基修饰等乃至体内行为更具有主观的可调控性。因此以聚合物泡囊为药物载体可望获得更佳的脑内靶向递药特性。
本文以可生物降解的聚合物泡囊为基础,构建了一类新型的转铁蛋白(Tf)受体介导的脑内递药系统—OX26-聚合物泡囊系统(OX26-PO)和Tf-聚合物泡囊系统(Tf-PO)。
本文第一部分为聚合物泡囊的制备与表征。选择可生物降解的聚乙二醇—聚己内酯(PEG-PCL)制备聚合物泡囊,探讨PEG-PCL形成泡囊的条件,通过考察其理化性质如粒径、膜厚、稳定性、渗透性、药物释放机制等,探讨其作为脑内靶向递药载体的可行性。采用开环聚合法合成Maleimide-PEG-PCL和MPEG-PCL。注入法制备聚合物泡囊,发现当PEG-PCL中PEG的体积百分比(f_(hydrophilic))位于0.16～0.32之间时可注入法形成泡囊。泡囊的粒径及膜厚与PEG-PCL分子量相关。其膜层厚度(≥10 nm)大于脂质体(3～4 nm),其体外粒径稳定性显著优于脂质体。渗透性研究表明,PEG-PCL泡囊的渗透性比脂质体低20倍,而且这种渗透性可采用二噁烷进行调节。常温下PEG-PCL泡囊内药物的释放符合Ficks第一定律,通过调节泡囊的膜厚可调节药物的释放速度。PEG-PCL泡囊内药物的释放受到温度、pH及血清成分的影响。在37℃,pH 4或血清的条件下药物的释放为扩散和骨架溶蚀的混合机制,释放前期的释药规律主要符合Ficks扩散,释放后期的释药规律主要为溶蚀机制。上述实验结果表明,PEG-PCL泡囊的体外性能良好,可以作为脑内递药系统的药物载体。
第二部分为脑内靶向递药系统OX26-PO的构建及其脑内递药特性评价。将Maleimide-PEG-PCL和MPEG-PCL以一定比例混合,注入法制得聚合物泡囊,将PEG-PCL聚合物泡囊与巯基化的OX26通过Maleimide相连,构建了一种新型的通过转铁蛋白受体介导的脑内靶向递药系统OX26-PO。X射线光电子能谱和免疫透射电镜实验证实泡囊表面连接有OX26。以6-香豆素为荧光探针,考察各种表面OX26密度的OX26-PO在大鼠体内的药动学及脑内递药特性。结果表明,OX26-PO具有一定的脑内靶向递药特性;随着OX26-PO表面OX26密度的增加,血浆中药时曲线下面积减少;当每个PO表面OX26数目达到34时,OX26-PO在2h的大脑渗透表面积(PS)值及摄取量(%ID/g)均达到最大值。与普通泡囊相比,OX26_(34)-PO的%ID/g是PO的2.62倍,PS值是PO的3.48倍。大鼠尾静脉注射50 mg/kg载6-香豆素的OX26_(34)-PO和PO 30 min后,脑组织冰冻切片显示,OX26-PO在大脑皮层、侧脑室脉络丛和第三脑室室周区的绿色荧光颗粒分布均显著高于PO,进一步验证了上述结果。单核一巨噬细胞特异性染色结果显示,高剂量的OX26-PO对SD大鼠不引起大脑、心和肝的巨噬细胞增多,仅对脾、肺和肾产生轻微毒性,且对肺和肾产生的急性毒性为一过性。上述实验结果证明,OX26_(34)-PO可望成为一种具有脑内递药特性、毒性较低的新型药物传递系统。
第三部分以AVP_(4-9)类似物NC-1900为模型多肽药物,将NC-1900包载于OX26_(34)-PO中,构建一种肽类药物脑内递药系统,并通过药效学考察其脑内递药特性。通过考察NC-1900的理化性质发现NC-1900是一种高水溶性的强碱性多肽,其log P低于—4,在中性环境下主要以阳离子形式存在,高低浓度的NC-1900溶液均能诱导离子敏感型的去乙酰结冷胶发生胶凝。根据NC-1900的理化性质,采用泡囊包裹即型凝胶的方式载NC-1900,可大大提高NC-1900的包封率。采用星点设计-效应面优化法对NC-1900的载药工艺进行了优化。根据优化的处方,载NC-1900泡囊的载药量约为1.2%,包封率约为30%,符合实验预期要求。NC-1900-PO的体外释放结果表明,NC-1900从凝胶中的释放是整个释放过程的限速过程,而且NC-1900从凝胶中的释放符合Ficks扩散机制。通过药效学考察NC-1900-OX26-PO的脑内递药性能,结果显示,NC-1900-OX26-PO对东莨菪碱诱导的大鼠学习和记忆障碍有明显的改善作用,且作用效果呈剂量依赖性。根据量效关系方程,NC-1900-OX26-PO对大鼠学习记忆障碍改善作用的半数有效剂量为0.82 ng/kg。上述结果表明,OX26-PO可为肽类或蛋白类药物的脑内递药提供新的载药系统。
第四部分以阿霉素为模型药物,构建了一种新型的小分子化疗药物的脑内递药系统—载阿霉素Tf-PO。采用主动载药法制备载阿霉素Tf-PO。对C6细胞的抑制结果(IC_(50))显示,载阿霉素Tf-PO对C6的抑制效果是阿霉素溶液的8.62倍,是载药PO的3.75倍,证实Tf-PO能够显著增加阿霉素对C6的体外抑制效果。药动学与脑内组织分布结果表明,载阿霉素Tf-PO可显著增加阿霉素的大脑组织摄取量,尤其是脑内肿瘤部位的摄取量。尾静脉注射载阿霉素Tf-PO 24 h后,其在大脑皮层、左侧纹状体、肿瘤部位的ID%/g值分别是载阿霉素PO的3.30,3.54,3.60倍,分别是阿霉素溶液的3.47,3.16,6.79倍。尾静脉注射载阿霉素Tf-PO 4 h后,其在大脑皮层、左侧纹状体、肿瘤部位的ID%/g值分别是阿霉素溶液的2.08,2.59,6.92倍,分别是载阿霉素PO的2.38,2.23,2.72倍。该结果为提高阿霉素对脑胶质瘤的治疗效果提供了有价值的实验资料。
第五部分构建了三种泡囊载药系统,并对其药动学及组织分布进行了比较,以期比较评价OX26和Tf两种靶向功能基与泡囊相连后的脑内递药特性。结果表明,聚合物泡囊在血浆中的清除速率及组织对泡囊的摄取与泡囊表面蛋白的种类和密度密切相关。泡囊与Tf相连后,其肝、脾、大脑的PS值及摄取量显著性增加,但肺的PS值及摄取量显著性减少。泡囊与OX26相连后各器官对OX26-PO的摄取与PO表面OX26密度相关:与表面蛋白密度相近的Tf-PO相比,OX26_(34)-PO的心、肝、脾、肺、肾、大脑PS值及摄取量均无显著性差异。
With population aging,brain diseases are rapidly increasing and threatening human health.However,the delivery of therapeutic agents for brain diseases is far from efficient,due to the blood-brain barrier(BBB).The BBB drug targeting technology designed as molecular "Trojan horse" could bring hope to these molecules. One of the most effective "Trojan horse" BBB delivery systems is liposome-based "Trojan horse",composed of pegylated liposomes as drug carriers and surface-conjugated targetors such as the transferrin(Tf) receptor monoclonal antibody (TFRmAb) OX26 which could initiate the endogenous receptor-mediated transcytosis (RMT) to cross the BBB.However,liposome seems not perfect as a drug carrier because of its unstability.Before liposomes reach the brain capillary,they will rupture in blood and decrease the brain drug delivery.Moreover,many lipid vesicles properties such as encapsulant retention,membrane stability,and degradation are not particularly well-controlled and minimize the likelihood of delivering drugs to an intended target site.Therefore,new drug carriers with better vesicles properties such as good stability and low permeability are necessary for the construction of the BBB targeting delivery system with more rational and effective brain delivery property.
As a new class of synthetic thin-shelled capsules based on block copolymer chemistry,polymersomes are self-assembled vesicles of amphiphilic block copolymers with thicker and tougher membrane than lipids.Polymersomes have the same advantages as liposomes and nanoparticles.Compared with liposomes, polymersomes are more stable.The internal aqueous cavity of the polymersome is expected to load more hydrophilic drugs including peptides and protein than nanoparticles.The combination of a thick wall for a hydrophobic drug and a vesicular lumen for a hydrophilic drug will lead to synergistic effect like cocktails.Moreover, the physical and chemical properties of polymersomes including particles size, membrane thickness,permeability,drug loading,surface modification,and even in vivo behavior may be broadly tunable through rich diversity of block copolymer chemistries(block fraction,block architecture).Therefore,polymersomes are good candidates for drug delivery carriers,which are expected to have better brain delivery property.
The present study focused on the preparation and evaluation of brain delivery property of polymersome-based molecular "Trojan horse",where OX26 or transferrin, was conjugated to the polymersomes' surface(OX26-PO or Tf-PO).
The first part described the preparation and characterization of polymersomes. Block copolymer poly(ethyleneglycol)-poly(ε-caprolactone)(PEG-PCL) was selected for preparing polymersomes due to its biodegradability and commercial availability. The feasibility of PEG-PCL polymersomes as drug carriers were evaluated through the investigation of the formation of polymersomes and the physico-chemical properties such as particles size,membrane thickness,stability,permeability and drug releasing characteristics.The MPEG-PCL or Maleimide-PEG-PCL diblock copolymers were synthesized by ring-opening polymerization ofε-CL in dry toluene under moisture-free high purity nitrogen atmosphere,using MPEG or Maleimide-PEG-PCL as the initiator.It was found that PEG-PCL copolymers self-assembling into polymersomes possess a hydrophilic volume fraction(f_(hydrophilic)) of 0.16～0.32 via a solvent injection technique.The vesicle size and membrane thickness correlated with the molecular weight of copolymers.The membrane thickness of polymersome was above 10 nm,much thicker than that of liposome.The in vitro stability tests of vesicle size showed that polymersomes were more stable than liposomes.It was shown that PEG-PCL polymersomes were 20 times less permeable than liposomes,but their permeability could be tunable by dioxane.The release of drugs from polymersomes at room temperature was consistent with Fick's first law and the flux of the drug across the vesicle wall could be tunable by the adjustment of the molecular weight.High temperature,low pH value and the existence of serum accelerated the release of drugs from polymersomes.The release of drugs from polymersomes was expressed as the combination of Fick's diffusion and erosion mechanism at 37℃,pH 4 or in the existence of serum.During the initial release process,it was mainly attributed to Fick's diffusion,followed by erosion mechanism mainly in the following release process.
The second part described the preparation and evaluation of brain delivery property of OX26-PO.Activated PEG-PCL polymersomes were made with a blend of MPEG-PCL and Maleimide-PEG-PCL.OX26 was thiolated and conjugated to the polymersomes' surface through the maleimide function.Therefore,a novel drug carrier for brain delivery through transferrin receptor mediated transcytosis,PEG-PCL polymersomes conjugated with OX26(OX26-PO),was developed.Coupling of OX26 with PO was confirmed by immuno-gold labeling of OX26 visualized under the TEM and X-ray photoelectron spectroscopy test.The results of pharmacokinetics and brain delivery of 6-coumarin labeled OX26-PO in rats proved that the increase of surface OX26 density of OX26-PO decreased blood AUC.The optimized OX26 number conjugated per polymersome was 34,which can acquire the greatest blood-brain barrier(BBB) permeability surface area(PS) product and percentage of injected dose per gram brain(%ID/g brain).Compared with PO,the average%ID/g brain and PS product for OX26_(34)-PO were 2.62-fold and 3.48-fold of those of PO,respectively. The microscopic examination of coronal sections of the rat brain after i.v.injection of fluorescent polymersomes showed that coumarin-6 labeled OX26_(34)-PO were localized in the cerebral cortex,the peri-ventricular region of the lateral ventricle and the third ventricle,where OX26_(34)-PO exhibited a higher accumulation than PO,validating the results of brain delivery property of OX26_(34)-PO.Immunostaining of monocyte macrophage demonstrated that high dose of OX26-PO could not induce the increase amount of macrophage in cerebrum,heart and liver in Sprague-Dawley rats,only had light toxicity to lung,spleen and kidney,and its acute toxicity to lung and kidney was transient.Derived from the above experimental results,we could conclude that OX26-PO was a novel brain drug delivery system with low toxicity.
The third part described the preparation and evaluation of brain delivery property of peptides loaded OX26_(34)-PO.NC-1900,a vasopressin fragment AVP_(4-9) analog,was encapsulated into OX26-PO as a model peptide and the enhanced brain delivery of NC-1900 was evaluated with pharmacodynamic experiments.It was found that NC-1900 was a highly hydrophilic peptide with strong base property.Its logP was below -4.NC-1900 was a cationic peptide in neutral environment,inducing the gelatination of gellan gum at a high or low concentration.According the physical and chemical properties of NC-1900,a new method for the encapsulation of NC-1900 was developed.The gellan gum trapped into the polymersomes was used as a reservoir to capture NC-1900 permeating into inner aqueous cavity of polymersomes,which highly increased the encapsulation efficiency of NC-1900.The preparation of NC-1900 loaded OX26-PO was optimized by central composite design and response surface method.According to the optimized preparation,the drug loading capability of NC-1900 loaded OX26-PO was 1.20%and a relative high encapsulation efficiency of 30%.of NC-1900 was encapsulated into OX26-PO,which reached the expected objective.In vitro release tests showed that the release of NC-1900 from the gel was a rate-limiting step of the release ofNC-1900 from OX26-PO and it could be expressed as Fick's diffusion.It was shown that OX26-PO significantly enhanced the brain delivery of NC-1900 with ameliorating the scopolamine-induced learning and memory impairments in a dose-dependent manner.The dose-response relation of NC-1900 loaded OX26-PO revealed that the median effective dose of NC-1900 was 0.82 ng/kg.The above experiments results indicated that OX26-PO might serve as a potential brain delivery system especially for large-molecule drugs such as peptides and proteins.
The fourth part described the preparation and evaluation of brain delivery property of Tf-PO loaded with small molecule chemotherapeutics.Doxorubicin,as a model drug,was encapsulated into Tf-PO via a pH gradient.From IC_(50),the C6 inhibition efficiency of doxorubicin loaded Tf-PO was 8.62 times higher than that of doxorubicin solution,and 3.75 times higher than that of doxorubicin loaded PO, which revealed that doxorubicin loaded Tf-PO can enhance the drug inhibition effect on C6 glioma cells.It was shown in pharmacokinetic and brain distribution experiments that Tf-PO significantly enhanced brain delivery of doxorubicin, especially the delivery of doxorubicin into brain tumor.At 24h after i.v.injection of doxorubicin loaded Tf-PO in C6 glioma transplanted rats,%ID/g of brain cortex,left striaturn,and tumor in Tf-PO group was 3.30,3.54,3.60 times higher than that in PO group,respectively and 3.47,3.16,6.79 times higher than that in doxorubicin solution group,respectively.At 4h after i.v.injection of doxorubicin loaded Tf-PO in C6 glioma transplanted rats,%ID/g of brain cortex,left striatum,and tumor in Tf-PO group was 2.38,2.23,2.72 times higher than that in PO group,respectively and 2.08, 2.59,6.92 times higher than that in doxorubicin solution group,respectively.The above experiments results indicated that Tf-PO might enhance the chemotherapeutic effect of doxorubicin for brain glioma.
The fifth part described the preparation and comparison of pharmacokinetics and tissue distribution of three brain drug delivery systems based on polymersomes.It was shown that the clearance of polymersomes in blood and tissue uptake of polymersomes correlated to the type and density of surface protein conjugated to polymersomes.Following the conjugation with tranferrin,the organ PS product and uptake of polymersomes for liver,brain and spleen were significantly increased while those for lung were significantly decreased.The organ uptake of OX26-PO correlated with surface OX26 density.There was no significant difference of organ PS product and uptake between OX26_(34)-PO and Tf-PO with the similar surface protein density.
聚合物泡囊是一种新型的药物载体。它具有脂质体和纳米粒等微粒载药系统的主要优点,而且其体内外的稳定性显著优于脂质体;其水溶性的内腔结构对水溶性药物如蛋白多肽类药物的包封量可望优于纳米粒;其膜层较厚,有利于包封脂溶性药物,可与水溶性内腔的水溶性药物共同发挥“鸡尾酒疗法”作用;此外,通过聚合物分子的种类、分子量、嵌段比例等选择,所构建泡囊载药系统的理化特性如粒径、膜厚、渗透性、载药量、表面PEG修饰、靶向功能基修饰等乃至体内行为更具有主观的可调控性。因此以聚合物泡囊为药物载体可望获得更佳的脑内靶向递药特性。
本文以可生物降解的聚合物泡囊为基础,构建了一类新型的转铁蛋白(Tf)受体介导的脑内递药系统—OX26-聚合物泡囊系统(OX26-PO)和Tf-聚合物泡囊系统(Tf-PO)。
本文第一部分为聚合物泡囊的制备与表征。选择可生物降解的聚乙二醇—聚己内酯(PEG-PCL)制备聚合物泡囊,探讨PEG-PCL形成泡囊的条件,通过考察其理化性质如粒径、膜厚、稳定性、渗透性、药物释放机制等,探讨其作为脑内靶向递药载体的可行性。采用开环聚合法合成Maleimide-PEG-PCL和MPEG-PCL。注入法制备聚合物泡囊,发现当PEG-PCL中PEG的体积百分比(f_(hydrophilic))位于0.16～0.32之间时可注入法形成泡囊。泡囊的粒径及膜厚与PEG-PCL分子量相关。其膜层厚度(≥10 nm)大于脂质体(3～4 nm),其体外粒径稳定性显著优于脂质体。渗透性研究表明,PEG-PCL泡囊的渗透性比脂质体低20倍,而且这种渗透性可采用二噁烷进行调节。常温下PEG-PCL泡囊内药物的释放符合Ficks第一定律,通过调节泡囊的膜厚可调节药物的释放速度。PEG-PCL泡囊内药物的释放受到温度、pH及血清成分的影响。在37℃,pH 4或血清的条件下药物的释放为扩散和骨架溶蚀的混合机制,释放前期的释药规律主要符合Ficks扩散,释放后期的释药规律主要为溶蚀机制。上述实验结果表明,PEG-PCL泡囊的体外性能良好,可以作为脑内递药系统的药物载体。
第二部分为脑内靶向递药系统OX26-PO的构建及其脑内递药特性评价。将Maleimide-PEG-PCL和MPEG-PCL以一定比例混合,注入法制得聚合物泡囊,将PEG-PCL聚合物泡囊与巯基化的OX26通过Maleimide相连,构建了一种新型的通过转铁蛋白受体介导的脑内靶向递药系统OX26-PO。X射线光电子能谱和免疫透射电镜实验证实泡囊表面连接有OX26。以6-香豆素为荧光探针,考察各种表面OX26密度的OX26-PO在大鼠体内的药动学及脑内递药特性。结果表明,OX26-PO具有一定的脑内靶向递药特性;随着OX26-PO表面OX26密度的增加,血浆中药时曲线下面积减少;当每个PO表面OX26数目达到34时,OX26-PO在2h的大脑渗透表面积(PS)值及摄取量(%ID/g)均达到最大值。与普通泡囊相比,OX26_(34)-PO的%ID/g是PO的2.62倍,PS值是PO的3.48倍。大鼠尾静脉注射50 mg/kg载6-香豆素的OX26_(34)-PO和PO 30 min后,脑组织冰冻切片显示,OX26-PO在大脑皮层、侧脑室脉络丛和第三脑室室周区的绿色荧光颗粒分布均显著高于PO,进一步验证了上述结果。单核一巨噬细胞特异性染色结果显示,高剂量的OX26-PO对SD大鼠不引起大脑、心和肝的巨噬细胞增多,仅对脾、肺和肾产生轻微毒性,且对肺和肾产生的急性毒性为一过性。上述实验结果证明,OX26_(34)-PO可望成为一种具有脑内递药特性、毒性较低的新型药物传递系统。
第三部分以AVP_(4-9)类似物NC-1900为模型多肽药物,将NC-1900包载于OX26_(34)-PO中,构建一种肽类药物脑内递药系统,并通过药效学考察其脑内递药特性。通过考察NC-1900的理化性质发现NC-1900是一种高水溶性的强碱性多肽,其log P低于—4,在中性环境下主要以阳离子形式存在,高低浓度的NC-1900溶液均能诱导离子敏感型的去乙酰结冷胶发生胶凝。根据NC-1900的理化性质,采用泡囊包裹即型凝胶的方式载NC-1900,可大大提高NC-1900的包封率。采用星点设计-效应面优化法对NC-1900的载药工艺进行了优化。根据优化的处方,载NC-1900泡囊的载药量约为1.2%,包封率约为30%,符合实验预期要求。NC-1900-PO的体外释放结果表明,NC-1900从凝胶中的释放是整个释放过程的限速过程,而且NC-1900从凝胶中的释放符合Ficks扩散机制。通过药效学考察NC-1900-OX26-PO的脑内递药性能,结果显示,NC-1900-OX26-PO对东莨菪碱诱导的大鼠学习和记忆障碍有明显的改善作用,且作用效果呈剂量依赖性。根据量效关系方程,NC-1900-OX26-PO对大鼠学习记忆障碍改善作用的半数有效剂量为0.82 ng/kg。上述结果表明,OX26-PO可为肽类或蛋白类药物的脑内递药提供新的载药系统。
第四部分以阿霉素为模型药物,构建了一种新型的小分子化疗药物的脑内递药系统—载阿霉素Tf-PO。采用主动载药法制备载阿霉素Tf-PO。对C6细胞的抑制结果(IC_(50))显示,载阿霉素Tf-PO对C6的抑制效果是阿霉素溶液的8.62倍,是载药PO的3.75倍,证实Tf-PO能够显著增加阿霉素对C6的体外抑制效果。药动学与脑内组织分布结果表明,载阿霉素Tf-PO可显著增加阿霉素的大脑组织摄取量,尤其是脑内肿瘤部位的摄取量。尾静脉注射载阿霉素Tf-PO 24 h后,其在大脑皮层、左侧纹状体、肿瘤部位的ID%/g值分别是载阿霉素PO的3.30,3.54,3.60倍,分别是阿霉素溶液的3.47,3.16,6.79倍。尾静脉注射载阿霉素Tf-PO 4 h后,其在大脑皮层、左侧纹状体、肿瘤部位的ID%/g值分别是阿霉素溶液的2.08,2.59,6.92倍,分别是载阿霉素PO的2.38,2.23,2.72倍。该结果为提高阿霉素对脑胶质瘤的治疗效果提供了有价值的实验资料。
第五部分构建了三种泡囊载药系统,并对其药动学及组织分布进行了比较,以期比较评价OX26和Tf两种靶向功能基与泡囊相连后的脑内递药特性。结果表明,聚合物泡囊在血浆中的清除速率及组织对泡囊的摄取与泡囊表面蛋白的种类和密度密切相关。泡囊与Tf相连后,其肝、脾、大脑的PS值及摄取量显著性增加,但肺的PS值及摄取量显著性减少。泡囊与OX26相连后各器官对OX26-PO的摄取与PO表面OX26密度相关:与表面蛋白密度相近的Tf-PO相比,OX26_(34)-PO的心、肝、脾、肺、肾、大脑PS值及摄取量均无显著性差异。
With population aging,brain diseases are rapidly increasing and threatening human health.However,the delivery of therapeutic agents for brain diseases is far from efficient,due to the blood-brain barrier(BBB).The BBB drug targeting technology designed as molecular "Trojan horse" could bring hope to these molecules. One of the most effective "Trojan horse" BBB delivery systems is liposome-based "Trojan horse",composed of pegylated liposomes as drug carriers and surface-conjugated targetors such as the transferrin(Tf) receptor monoclonal antibody (TFRmAb) OX26 which could initiate the endogenous receptor-mediated transcytosis (RMT) to cross the BBB.However,liposome seems not perfect as a drug carrier because of its unstability.Before liposomes reach the brain capillary,they will rupture in blood and decrease the brain drug delivery.Moreover,many lipid vesicles properties such as encapsulant retention,membrane stability,and degradation are not particularly well-controlled and minimize the likelihood of delivering drugs to an intended target site.Therefore,new drug carriers with better vesicles properties such as good stability and low permeability are necessary for the construction of the BBB targeting delivery system with more rational and effective brain delivery property.
As a new class of synthetic thin-shelled capsules based on block copolymer chemistry,polymersomes are self-assembled vesicles of amphiphilic block copolymers with thicker and tougher membrane than lipids.Polymersomes have the same advantages as liposomes and nanoparticles.Compared with liposomes, polymersomes are more stable.The internal aqueous cavity of the polymersome is expected to load more hydrophilic drugs including peptides and protein than nanoparticles.The combination of a thick wall for a hydrophobic drug and a vesicular lumen for a hydrophilic drug will lead to synergistic effect like cocktails.Moreover, the physical and chemical properties of polymersomes including particles size, membrane thickness,permeability,drug loading,surface modification,and even in vivo behavior may be broadly tunable through rich diversity of block copolymer chemistries(block fraction,block architecture).Therefore,polymersomes are good candidates for drug delivery carriers,which are expected to have better brain delivery property.
The present study focused on the preparation and evaluation of brain delivery property of polymersome-based molecular "Trojan horse",where OX26 or transferrin, was conjugated to the polymersomes' surface(OX26-PO or Tf-PO).
The first part described the preparation and characterization of polymersomes. Block copolymer poly(ethyleneglycol)-poly(ε-caprolactone)(PEG-PCL) was selected for preparing polymersomes due to its biodegradability and commercial availability. The feasibility of PEG-PCL polymersomes as drug carriers were evaluated through the investigation of the formation of polymersomes and the physico-chemical properties such as particles size,membrane thickness,stability,permeability and drug releasing characteristics.The MPEG-PCL or Maleimide-PEG-PCL diblock copolymers were synthesized by ring-opening polymerization ofε-CL in dry toluene under moisture-free high purity nitrogen atmosphere,using MPEG or Maleimide-PEG-PCL as the initiator.It was found that PEG-PCL copolymers self-assembling into polymersomes possess a hydrophilic volume fraction(f_(hydrophilic)) of 0.16～0.32 via a solvent injection technique.The vesicle size and membrane thickness correlated with the molecular weight of copolymers.The membrane thickness of polymersome was above 10 nm,much thicker than that of liposome.The in vitro stability tests of vesicle size showed that polymersomes were more stable than liposomes.It was shown that PEG-PCL polymersomes were 20 times less permeable than liposomes,but their permeability could be tunable by dioxane.The release of drugs from polymersomes at room temperature was consistent with Fick's first law and the flux of the drug across the vesicle wall could be tunable by the adjustment of the molecular weight.High temperature,low pH value and the existence of serum accelerated the release of drugs from polymersomes.The release of drugs from polymersomes was expressed as the combination of Fick's diffusion and erosion mechanism at 37℃,pH 4 or in the existence of serum.During the initial release process,it was mainly attributed to Fick's diffusion,followed by erosion mechanism mainly in the following release process.
The second part described the preparation and evaluation of brain delivery property of OX26-PO.Activated PEG-PCL polymersomes were made with a blend of MPEG-PCL and Maleimide-PEG-PCL.OX26 was thiolated and conjugated to the polymersomes' surface through the maleimide function.Therefore,a novel drug carrier for brain delivery through transferrin receptor mediated transcytosis,PEG-PCL polymersomes conjugated with OX26(OX26-PO),was developed.Coupling of OX26 with PO was confirmed by immuno-gold labeling of OX26 visualized under the TEM and X-ray photoelectron spectroscopy test.The results of pharmacokinetics and brain delivery of 6-coumarin labeled OX26-PO in rats proved that the increase of surface OX26 density of OX26-PO decreased blood AUC.The optimized OX26 number conjugated per polymersome was 34,which can acquire the greatest blood-brain barrier(BBB) permeability surface area(PS) product and percentage of injected dose per gram brain(%ID/g brain).Compared with PO,the average%ID/g brain and PS product for OX26_(34)-PO were 2.62-fold and 3.48-fold of those of PO,respectively. The microscopic examination of coronal sections of the rat brain after i.v.injection of fluorescent polymersomes showed that coumarin-6 labeled OX26_(34)-PO were localized in the cerebral cortex,the peri-ventricular region of the lateral ventricle and the third ventricle,where OX26_(34)-PO exhibited a higher accumulation than PO,validating the results of brain delivery property of OX26_(34)-PO.Immunostaining of monocyte macrophage demonstrated that high dose of OX26-PO could not induce the increase amount of macrophage in cerebrum,heart and liver in Sprague-Dawley rats,only had light toxicity to lung,spleen and kidney,and its acute toxicity to lung and kidney was transient.Derived from the above experimental results,we could conclude that OX26-PO was a novel brain drug delivery system with low toxicity.
The third part described the preparation and evaluation of brain delivery property of peptides loaded OX26_(34)-PO.NC-1900,a vasopressin fragment AVP_(4-9) analog,was encapsulated into OX26-PO as a model peptide and the enhanced brain delivery of NC-1900 was evaluated with pharmacodynamic experiments.It was found that NC-1900 was a highly hydrophilic peptide with strong base property.Its logP was below -4.NC-1900 was a cationic peptide in neutral environment,inducing the gelatination of gellan gum at a high or low concentration.According the physical and chemical properties of NC-1900,a new method for the encapsulation of NC-1900 was developed.The gellan gum trapped into the polymersomes was used as a reservoir to capture NC-1900 permeating into inner aqueous cavity of polymersomes,which highly increased the encapsulation efficiency of NC-1900.The preparation of NC-1900 loaded OX26-PO was optimized by central composite design and response surface method.According to the optimized preparation,the drug loading capability of NC-1900 loaded OX26-PO was 1.20%and a relative high encapsulation efficiency of 30%.of NC-1900 was encapsulated into OX26-PO,which reached the expected objective.In vitro release tests showed that the release of NC-1900 from the gel was a rate-limiting step of the release ofNC-1900 from OX26-PO and it could be expressed as Fick's diffusion.It was shown that OX26-PO significantly enhanced the brain delivery of NC-1900 with ameliorating the scopolamine-induced learning and memory impairments in a dose-dependent manner.The dose-response relation of NC-1900 loaded OX26-PO revealed that the median effective dose of NC-1900 was 0.82 ng/kg.The above experiments results indicated that OX26-PO might serve as a potential brain delivery system especially for large-molecule drugs such as peptides and proteins.
The fourth part described the preparation and evaluation of brain delivery property of Tf-PO loaded with small molecule chemotherapeutics.Doxorubicin,as a model drug,was encapsulated into Tf-PO via a pH gradient.From IC_(50),the C6 inhibition efficiency of doxorubicin loaded Tf-PO was 8.62 times higher than that of doxorubicin solution,and 3.75 times higher than that of doxorubicin loaded PO, which revealed that doxorubicin loaded Tf-PO can enhance the drug inhibition effect on C6 glioma cells.It was shown in pharmacokinetic and brain distribution experiments that Tf-PO significantly enhanced brain delivery of doxorubicin, especially the delivery of doxorubicin into brain tumor.At 24h after i.v.injection of doxorubicin loaded Tf-PO in C6 glioma transplanted rats,%ID/g of brain cortex,left striaturn,and tumor in Tf-PO group was 3.30,3.54,3.60 times higher than that in PO group,respectively and 3.47,3.16,6.79 times higher than that in doxorubicin solution group,respectively.At 4h after i.v.injection of doxorubicin loaded Tf-PO in C6 glioma transplanted rats,%ID/g of brain cortex,left striatum,and tumor in Tf-PO group was 2.38,2.23,2.72 times higher than that in PO group,respectively and 2.08, 2.59,6.92 times higher than that in doxorubicin solution group,respectively.The above experiments results indicated that Tf-PO might enhance the chemotherapeutic effect of doxorubicin for brain glioma.
The fifth part described the preparation and comparison of pharmacokinetics and tissue distribution of three brain drug delivery systems based on polymersomes.It was shown that the clearance of polymersomes in blood and tissue uptake of polymersomes correlated to the type and density of surface protein conjugated to polymersomes.Following the conjugation with tranferrin,the organ PS product and uptake of polymersomes for liver,brain and spleen were significantly increased while those for lung were significantly decreased.The organ uptake of OX26-PO correlated with surface OX26 density.There was no significant difference of organ PS product and uptake between OX26_(34)-PO and Tf-PO with the similar surface protein density.
引文
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[8] Olivier JC, Huertas R, Lee HJ, Calon F, Pardridge WM. Synthesis of Pegylated immunonanoparticles[J]. Pharm Res, 2002, 19 (8): 1137-1143.
[9] Ahmed F, Discher DE. Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis triggered controlled release vesicles[J]. J Control Release, 2004, 96: 37-53.
[10] Kang YS, Bickel U, Pardridge WM. Pharmacokinetics and saturable blood-brain barrier transport of biotin bound to a conjugate of avidin and a monoclonal antibody to the transferring receptor[J]. Drug Metab Dispos, 1994, 22(1): 99-105.
[11] Calvo P, Gouritin B, Chacun H, Desmaele D, D'Angelo J, Noel JP, Georgin D, Fattal E, Andreux JP, Couvreur P. Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery [J]. Pharm Res, 2001,18(8): 1157-1166.
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[13] Thorn SR, Bhopale VM, Fisher D, Zhang J, Gimotty P. Delayed neuropathology after carbon monoxide poisoning is immune-mediated[J]. Proc Natl Acad Sci, 2004,101 (37): 13660-13665.
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[22] Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses[J]. Nat Rev Drug Discov, 2002, 1: 131-139.
[23] Qian Z, Li H, Sun H, Ho K. Targeted Drug Delivery via the transferrin receptor-mediated endocytosis pathway[J]. Pharmacol Rev, 2002, 54: 561-587.
[24] Photos PJ, Bacakova L, Discher B, Bates FS, Discher DE. Polymer vesicles in vivo: correlations with PEG molecular weight[J]. J Control Release, 2003, 90: 323-334.
[1]Mishima K,Tsukikawa H,Miura I,Inada K,Abe K,Matsumoto Y,Egashira N,Iwasaki K,Fujiwara M.Ameliorative effect of NC-1900,a new AVP_(4-9)analog,through vasopressin V_(1A) receptor on scopolamine-induced impairments of spatial memory in the eight-arm radial maze[J].Neuropharmacology,2003,44:541-552.
[2]Xie Y,Lu W,Jiang X.Improvement ofcationic albumin conjugated pegylated nanoparticles holding NC-1900,a vasopressin fragment analog,in memory deficits induced by scopolamine in mice[J].Behav Brain Res,2006,173:76-84.
[3]Carfors J,Edsman K,Petersson R,et al.Rheological evaluation of Gelrite in situ gels for ophthalmic use[J]. Eur J Pharm Sci, 1998, 6(2): 113-119.
[4] Dian R. Arifin, Andre F. Palmer. Polymersome encapsulated hemoglobin: a Novel type of oxygen carrier[J]. Biomacromolecules, 2005, 6: 2172-2181
[5] Bickel U, Yoshikawa T, Pardridge WM. Delivery of peptides and proteins through the blood-brain barrier[J]. Adv Drug Deliv Rev, 2001, 46 (1-3): 247-279.
[6] Pardridge WM, Buciak JL, Friden PM. Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo[J]. J Pharmacol Exp Ther, 1991, 259: 66-70.
[1]Brain G,Spyridoy V,Melpomeni P,Michalis K,Panayiotis D,Nicolaos P,Haralabos P K.Intratumoral doxorubicin in patients with malignant brain gliomas[J].Am J Clin Oncol(CCT),2002,25(1):60-64.
[2]Klaus F,Jorg D,Peter H,Caecilie W,Beate W,Saskia P,Andreas S,Winfried U,Ulrich B.Long-term stabilization in patients with malignant glioma after treatment with liposomal doxorubicin[J].CANCER,2001,92(7):1936-1942.
[3]Qian Z,Li H,Sun H,Ho K.Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway[J].Pharmacol Rev,2002,54:561-587.
[1]Pardridge WM.Blood-brain barrier delivery[J].Drug Discovery Today,2007,12:54-61.
[2]Qian Z,Li H,Sun H,Ho K.Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway[J].Pharmacol Rev,2002,54:561-587.
[3]Huang R,Qu Y,Ke W,Zhu J,Pei Y,Jiang C.Efficient gene delivery targeted to the brain using a transferrin-conjugated polyethyleneglycol-modified polyamidoamine dendrimer[J].FASEB J,2007,21:1117-1125.
[4]Bickel U,Yoshikawa T,Pardridge WM.Delivery of peptides and proteins through the blood-brain barrier[J].Adv Drug Deliv Rev,2001,46(1-3):247-279.
[5]Pardridge WM,Buciak JL,Friden PM.Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo[J].J Pharmacol Exp Ther,1991,259:66-70.
[6]Bin J,Jun M,Makoto H,Kaori I,Hidetaka A,Hideyoshi H,Tetsuya S.Pharmacokinetics and brain uptake of lactoferrin in rats.Life Sciences,2006,78:851-855.
[1]Discher BM,Won YY,Ege DS,Lee J,Bates FS,Discher DE,D.A.Hammer.Polymersomes:tough vesicles made from diblock copolymers[J].Science,1999,284:1143-1146.
[2]Photos PJ,Bacakova L,Discher B,Bates FS,Discher DE.Polymer vesicles in vivo:correlations with PEG molecular weight[J].J Control Release,2003,90:323-334.
[3]Discher DE,Eisenberg A.Polymer vesicles[J].Science,2002,297:967-973.
[4]Fenghua Meng,Christine Hiemstra,Gerard H.M.Engbers,Jan Feijen.Biodegradable polymersomes[J].Macromolecules,2003,36,3004-3006.
[5]Kukula H,Schlaad H,Antonietti M,Forster S.The formation of polymer vesicles or "peptosomes" by polybutadiene-block-poly(L-glutamate)s in dilute aqueous solution[J].J AM CHEM SOC,2002,124(8):1658-1663.
[6] Brown MD, Schatzlein A, Brownlie A, Jack V, Wang W, Tetley L, Gray A, Uchegbu IF. Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents[J]. Bioconjugate Chem, 2000, 11: 880-891.
[7] Bermudez H, Brannan AK, Hammer DA, Bates FS, Discher DE. Molecular weight dependence of polymersome membrane structure, elasticity, and stability[J]. Macromolecules, 2002, 35: 8203-8208.
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[24] Discher DE, Ortiz V, Srinivas G, Klein ML, Kim Y, Christian D, Cai S, Photos P, Ahmed F. Emerging applications of polymersomes in delivery: From molecular dynamics to shrinkage of tumors[J]. Prog. Polym. Sci, 2007, 32: 838-857.
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[2] Pardridge WM. Brain drug targeting [M]. Cambridge: Cambridge University Press, 1998: 82.
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[7] Photos PJ, Bacakova L, Discher B, Bates FS, Discher DE. Polymer vesicles in vivo: correlations with PEG molecular weight[J]. J Control Release, 2003, 90: 323-334.
[8] Ahmed F, Pakunlu R, Brannan A, Bates F, Minko T, Discher DE. Biodegradable polymersomes loaded with both paclitaxel and doxorubicin permeate and shrink tumors, inducing apoptosis in proportion to accumulated drug[J]. J Control Release, 2006, 116: 150-158
[9] Bermudez H, Brannan AK, Hammer DA, Bates FS, Discher DE. Molecular weight dependence of polymersome membrane structure, elasticity, and stability[J]. Macromolecules, 2002, 35: 8203-8208.
[10] Ahmed F, Discher DE. Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis triggered controlled release vesicles[J]. J Control Release, 2004, 96: 37-53.
[11] Meng F, Engbers G, Feijen J. Biodegradable polymersomes as a basis for artificial cells: encapsulation, release and targeting[J]. J Control Release, 2005, 101: 187-198.
[12] Flaten G, Bunjes H, Luthman K, Brandl M. Drug permeability across a phospholipids vesicle-based barrier 2. Characterization of barrier structure, storage stability and stability towards pH changes[J]. Eur J Pharm Sci, 2006, 28: 336-343
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[15] Mishima K, Tsukikawa H, Miura I, Inada K, Abe K, Matsumoto Y, Egashira N, Iwasaki K, Fujiwara M. Ameliorative effect of NC-1900, a new AVP4.9 analog, through vasopressin V_(1A) receptor on scopolamine-induced impairments of spatial memory in the eight-arm radial maze[J]. Neuropharmacology, 2003, 44: 541-552.
[16] Xie Y, Lu W, Jiang X. Improvement of cationic albumin conjugated pegylated nanoparticles holding NC-1900, a vasopressin fragment analog, in memory deficits induced by scopolamine in mice[J]. Behav Brain Res, 2006, 173: 76-84.
[17] Brain G, Spyridoy V, Melpomeni P, Michalis K, Panayiotis D, Nicolaos P, Haralabos P K. Intratumoral doxorubicin in patients with malignant brain gliomas[J]. Am J Clin Oncol (CCT), 2002, 25(1): 60-64.
[18] Klaus F, Jorg D, Peter H, Caecilie W, Beate W, Saskia P, Andreas S, Winfried U, Ulrich B. Long-term stabilization in patients with malignant glioma after treatment with liposomal doxorubicin[J]. CANCER, 2001, 92 (7): 1936-1942.
[19] Qian Z, Li H, Sun H, Ho K. Targeted drug delivery via the transferrin receptor-mediated endocytosis pathway[J]. Pharmacol Rev, 2002, 54: 561-587.
[1]Discher BM,Won YY,Ege DS,Lee J,Bates FS,Discher DE,D.A.Hammer.Polymersomes:tough vesicles made from diblock copolymers[J].Science,1999,284:1143-1146.
[2]Discher DE,Eisenberg A.Polymer vesicles[J].Science,2002,297:967-973.
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[6]Kukula H,Schlaad H,Antonietti M,Forster S.The formation of polymer vesicles or "peptosomes" by polybutadiene- block-poly(L-glutamate)s in dilute aqueous solution[J].J AM CHEM SOC,2002,124(8):1658-1663.
[7] Brown MD, Schatzlein A, Brownlie A, Jack V, Wang W, Tetley L, Gray A, Uchegbu IF. Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents[J]. Bioconjugate Chem, 2000, 11: 880-891
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[9] Bermudez H, Brannan AK, Hammer DA, Bates FS, Discher DE. Molecular weight dependence of polymersome membrane structure, elasticity, and stability[J]. Macromolecules, 2002, 35: 8203-8208.
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[11] Napoli A, Boerakker M, Tirelli N, Nolte R, Sommerdijk N, Hubbell J. Glucose-oxidase based self-destructing polymeric vesicles[J]. Langmuir, 2004, 20: 3487-3491.
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[19] Flaten G, Bunjes H, Luthman K, Brandl M. Drug permeability across a phospholipids vesicle-based barrier 2. Characterization of barrier structure, storage stability and stability towards pH changes[J]. Eur J Pharm Sci, 2006, 28: 336-343
[20] Hara M, Yuan H, Yang Q, Hoshino T, Yokoyama A, Miyake J. Stabilization of liposomal membranes by thermozeaxanthins: carotenoid-glucoside esters[J]. Biochim Biophys Acta, 1999, 1461: 147-154
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[1]Bickel U,Yoshikawa T,Pardridge WM.Delivery of peptides and proteins through the blood-brain barrier[J].Adv Drug Deliv Rev,2001,46(1-3):247-279.
[2]Tsuji A,Tamai Ⅱ.Carrier-mediated or specialized transport of drugs across the blood-brain barrier[J].Adv Drug Deliv Rev,1999,36(2-3):277-290.
[3]Huwyler J,Wu D,Pardridge WM.Brain drug delivery of small molecules using immunoliposomes[J].Proc Natl Acad Sci 1996,93(24):14164-14169.
[4]Pardridge WM,Buciak JL,Friden PM.Selective transport of an anti-transferrin receptor antibody through the blood-brain barrier in vivo[J].J Pharmacol Exp Ther,1991,259:66-70.
[5]Huwyler J,Wu D,Pardridge WM.Brain drug delivery of small molecules using immunoliposomes[J].Proc Natl Acad Sci,1996,93(24):14164-14169.
[6]Ellmann GL.Tissue sulfhydryl groups[J].Arch Biochem Biophys,1959,82 (1): 70-77.
[7] Lu W, Zhang Y, Tan Y, Hu K, Jiang X, Fu S. Cationic albumin-conjugated Pegylated nanoparticles as novel drug carrier for brain delivery[J]. J Control Release, 2005, 107:428-448.
[8] Olivier JC, Huertas R, Lee HJ, Calon F, Pardridge WM. Synthesis of Pegylated immunonanoparticles[J]. Pharm Res, 2002, 19 (8): 1137-1143.
[9] Ahmed F, Discher DE. Self-porating polymersomes of PEG-PLA and PEG-PCL: hydrolysis triggered controlled release vesicles[J]. J Control Release, 2004, 96: 37-53.
[10] Kang YS, Bickel U, Pardridge WM. Pharmacokinetics and saturable blood-brain barrier transport of biotin bound to a conjugate of avidin and a monoclonal antibody to the transferring receptor[J]. Drug Metab Dispos, 1994, 22(1): 99-105.
[11] Calvo P, Gouritin B, Chacun H, Desmaele D, D'Angelo J, Noel JP, Georgin D, Fattal E, Andreux JP, Couvreur P. Long-circulating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery [J]. Pharm Res, 2001,18(8): 1157-1166.
[12] Janeway CA, Travers P, Walport M, Shlomchik MJ. Immunobiology[M]. New York: Garland publishing, 2001: 665.
[13] Thorn SR, Bhopale VM, Fisher D, Zhang J, Gimotty P. Delayed neuropathology after carbon monoxide poisoning is immune-mediated[J]. Proc Natl Acad Sci, 2004,101 (37): 13660-13665.
[14] Alric L, Orfila C, Carrere N, Beraud M, Carrera G, Lepert JC, Duffaut M, Pipy B, Vinel JP. Reactive oxygen intermediates and eicosanoid production by kupffer cells and infiltrated macrophages in acute and chronic liver injury induced in rats by CCl_4[J]. Inflamm Res, 2000,49 (12): 700-707.
[15] Smetana K Jr, Petrovicky P, Zach P, Nemcova V, Jelinkova M, Vacik J, Gabius HJ. Brain lesion-induced alteration of selected phenotypic properties of spleen macrophages and their partial restoration in the course of foreign body reaction against intraperitoneally implanted polymers[J]. J Mater Sci Mater Med, 1999,10(7): 425-429.
[16] Peracchia MT, Gref R, Minamitake Y, Domb A, Lotan N, Langer R, PEG-coated nanospheres from amphiphilic diblock and multiblock copolymers: investigations of their drug encapsulation and release characteristics[J]. J Control Release, 1997,46: 223-231.
[17] Meng F, Engbers G, Feijen J. Biodegradable polymersomes as a basis for artificial cells: encapsulation, release and targeting[J]. J Control Release, 2005, 101: 187-198.
[18] Weissenbock A, Wirth M, Gabor F. WGA-grafted PLGA-nanospheres: preparation and association with Caco-2 single cells[J]. J Control Release, 2004, 99(3): 383-392.
[19] Fleiner M., Benzinger P, Fichert T, Massing U. Studies on protein-liposome coupling using novel thiol-reactive coupling lipids: influence of spacer length and polarity[J]. Bioconjugate Chem, 2001, 12: 470-475.
[20] Mercadal M, Domingo JC Petriz J, Garcia J, Madariaga MA. A novel strategy affords high-yield coupling of antibody to extremities of liposomal surface-grafted PEG chains[J]. Biochim Biophys Acta, 1999, 1418: 232-238.
[21] Pardridge WM. Brain drug targeting [M]. Cambridge: Cambridge University Press, 1998: 82.
[22] Pardridge WM. Drug and gene targeting to the brain with molecular Trojan horses[J]. Nat Rev Drug Discov, 2002, 1: 131-139.
[23] Qian Z, Li H, Sun H, Ho K. Targeted Drug Delivery via the transferrin receptor-mediated endocytosis pathway[J]. Pharmacol Rev, 2002, 54: 561-587.
[24] Photos PJ, Bacakova L, Discher B, Bates FS, Discher DE. Polymer vesicles in vivo: correlations with PEG molecular weight[J]. J Control Release, 2003, 90: 323-334.
[1]Mishima K,Tsukikawa H,Miura I,Inada K,Abe K,Matsumoto Y,Egashira N,Iwasaki K,Fujiwara M.Ameliorative effect of NC-1900,a new AVP_(4-9)analog,through vasopressin V_(1A) receptor on scopolamine-induced impairments of spatial memory in the eight-arm radial maze[J].Neuropharmacology,2003,44:541-552.
[2]Xie Y,Lu W,Jiang X.Improvement ofcationic albumin conjugated pegylated nanoparticles holding NC-1900,a vasopressin fragment analog,in memory deficits induced by scopolamine in mice[J].Behav Brain Res,2006,173:76-84.
[3]Carfors J,Edsman K,Petersson R,et al.Rheological evaluation of Gelrite in situ gels for ophthalmic use[J]. Eur J Pharm Sci, 1998, 6(2): 113-119.
[4] Dian R. Arifin, Andre F. Palmer. Polymersome encapsulated hemoglobin: a Novel type of oxygen carrier[J]. Biomacromolecules, 2005, 6: 2172-2181
[5] Bickel U, Yoshikawa T, Pardridge WM. Delivery of peptides and proteins through the blood-brain barrier[J]. Adv Drug Deliv Rev, 2001, 46 (1-3): 247-279.
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