免疫靶向脂质体包载BDNF经面部皮内给药沿三叉神经入脑治疗阿尔茨海默病研究

Our Pre-experimental data showed Evans blue entered the trigeminal nerve via the intradermal injection in skin beside the nose, and several previous studies revealed that the trigeminal nerve was the main pathway for drug delivery after the intranasal administration of some proteins. Meanwhile The trigeminal nerve and its nerve ending distributed in facial skin extensively. Thus we proposed that the immuno-liposomes, which targeted to nerve ending via the myelin associated glycoproteins, might enter the brain via trigeminal nerve. The huge weight bio-molecules showed promising therapeutic effects on some diseases in central nervous system, for example, the BDNF was studied extensively and showed potential pharmacological effects on stroke, AD, PD and others. Therefore We will take the BDNF as model drug for huge weight bio-molecules, and construct immuno-liposomes to carry BDNF for subcantaneous administration in facial skin and deliver the BDNF to brain. we'll execute the plans: (1) the preferred administration route for the immuno-liposomes will be selected; (2) immuno-liposomes will be constructed and the characteristics of immuno-liposomes, including the size，shape, Entrapment rate be evaluated;（3）the preferred pathway for immuno-liposomes delivery and the mechanisms of BDNF entry into nerve system of immuno-liposomes were selcected in vivo; and the targeting efficiency of the immuno-liposomes delivery to the nerve ending in skin, trigeminal nerve and brain will also be evaluated with immunohistochemisty , westernblot and the fluorescence image techniques. (4) the pharmacodynamic and toxicity of immuno-liposomes will be evaluated . After above plans being finished, we will establish a novel safe administration route for targeted drug delivery to brain and construct an stable immuno-liposomes to targeted the nerve ending in facial skin;meanwhile,we will get some characteristics of immuno-liposomes,including the pharmacokinetics and the possible therapeutic effects on AD. All these results will be used to evaluate the feasibility of targeted drug delivery to brain of immuno-liposomes via trigeminal nerve and the therapeutic effects of encapsulated BDNF.

本项目前期在动物的鼻侧皮下和皮内给药可由三叉神经进入脑内，利用三叉神经在头面部的分布特点，并结合以往研究结果的基础上，提出构建针对皮肤神经末梢的靶向性制剂实现经头面皮肤内给药由三叉神经递送靶向入脑的假设。鉴于未来生物大分子是研究和开发的重点，课题拟将以研究较为确切的BDNF作为工具药，利用免疫靶向技术构建针对神经细胞膜成分髓磷脂相关糖蛋白的主动靶向脂质体，以此包载BDNF。在研究其表征的基础上，采用皮内注射免疫靶向脂质体后进入皮肤和入脑的靶向效果；采用小动物荧光成像技术、BDNF免疫靶向脂质体、分子生物学、病理学等技术相结合的手段研究在头面部皮肤给药后递送途径及可能机理及进入脑内的动力学过程和药效学评价。通过上述研究，希望了解BDNF的免疫靶向脂质体经头面皮肤表面给药是否选择性经三叉神经递送入脑，并了解经此途径和方式给药是否具有治疗阿尔茨海默病的效果

项目研究的主要目标在研究头面部（三叉神经支配区）皮肤皮内给药递送入脑，构建几种制剂并研究其在头面部皮肤给药入脑的效率、入脑途径。 通过项目组成员共同努力主要完成以下：（1）在以往研究的基础上，项目组成员对头面部皮内给药绕开血脑屏障入脑的可行星进行研究，在大鼠实验获得较好的效果。利用hBDNF（40kDa）、猪胰岛素、FITC-Dextrans（40kDa）和伊文思蓝（960Da）作为工具药，发现大鼠触须垫内给药可以绕开血脑屏障进入脑内，其递送途径主要示三叉伸经及其附属结构；皮内给药的入脑效率高于鼻腔给药。（2）对头面部皮内给药绕开血脑屏障入脑的机制进行了研究，获得较为详细递送通路，主要包括通过三叉神经的神经细胞及其轴突、神经外膜和束膜、神经血管外周隙等结构参与；同时也通过淋巴管进入鼻黏膜，经鼻腔-脑通路，包括嗅区进入脑内；淋巴管或淋巴系统在皮内给药入脑的递送过程起到不同的作用。（3）构建几种制剂包脂质体体、PEG脂质体和MBP抗体配接的脂质体。结果显示皮内给药后，脂质体和PEG脂质体可有效提高hBDNF入脑，并且获得较好的疗效，但是对MBP抗体配接的脂质体则提升效果不佳，可能被淋巴系统吞噬有关。这些结果提示，在大鼠体内详细的观察了头面部皮内注射给药绕开血脑屏障入脑治疗脑部疾病的可能性。在人体，利用皮内注射给药已经被用于治疗三叉神经痛和热痛觉过敏。因此，头面给药具有潜在的中枢神经系统和颅神经的疾病治疗作用，这是一种潜在的绕过血脑屏障的给药方式，制剂可影响该给药方式的效率。