靶向性紫杉醇脂质体的制备及药效的初步研究

硕士学位论文

摘要

脂质体作为药物载体具有许多明显的优势，已引起越来越多的关注与研究。本论文瞄准脂质体在药物靶向传输中的研究与应用这一前沿方向，在靶向脂质体载药纳米颗粒的制备及其体外体内靶向药效研究方面开展了如下三个方面的工作:

（1）紫杉醇脂质体纳米药物的制备及其质量评价

针对紫杉醇药物在临床应用中难溶于水、毒副作用大以及生物利用率低等缺陷，以大豆磷脂、胆固醇为药物载体材料，采用超声薄膜法与逆相蒸发法两种不同的方法制备了紫杉醇脂质体纳米药物，并比较了这两种不同的制备方法对所制备的纳米药物的粒径大小、分散性、包封率以及物理稳定性的影响。结合实验结果与后续实验的需要，得到如下结论：与逆相蒸发法相比，超声薄膜法制备的紫杉醇脂质体纳米药物粒径在200nm左右、分散性好，更能满足实验需要。同时，还比较了药物投药量与分散体系对所制备的紫杉醇脂质体纳米药物粒径与稳定性的影响。

（2）脂质体纳米颗粒的生物修饰及其在细胞成像中的应用

以大豆磷脂、胆固醇、对硝基苯碳酸酯-聚乙二醇-二棕榈酰磷脂酰乙醇胺（DPPE-PEG-pNP）为原料，制备了一种新型的表面易修饰带氨基活性分子的脂质体纳米颗粒，并在脂质体纳米颗粒表面修饰了抗-CK19抗体，开展了MGC细胞的识别与成像研究。我们制备的这种脂质体纳米颗粒在进行生物修饰时颗粒展现出了如下几个方面的优势：反应的条件温和，溶液的pH值控制在8.0即可；连接的效率高、速度快，反应时间只需4h；连接上去的活性分子还能保持很高的活性。这种修饰了抗-CK19抗体的脂质体纳米颗粒在MGC细胞的识别与成像中时中表现出很好的特异性。

（3）修饰了TMP1肽的紫杉醇脂质体纳米药物的制备及其体外与体内靶向药效研究

在前面两个部分工作的基础上，本章对脂质体纳米颗粒在紫杉醇的主动靶向运输方面的应用做了进一步的研究。利用上一章颗粒表面生物修饰的方法在紫杉醇脂质体纳米颗粒表面修饰了同济医科大学最近发现的TMP1肽，并考察了这种TMP1肽修饰的紫杉醇脂质体颗粒的体外与体内的靶向药效。结果表明这种紫杉醇脂质体纳米药物在体外显示出了对PE-3M-1E8前列腺癌细胞的靶向抑制作用。在此基础上，还初步开展了这种修饰了TMP1肽的紫杉醇脂质体的体内药效实验，结果显示TMP1修饰的紫杉醇脂质体对肿瘤的抑制效果跟紫杉醇脂质体的效果相当，但这两者的体内抑瘤效果明显强于游离紫杉醇药物。

关键词：脂质体；紫杉醇；靶向性；抗-CK19抗体；TMP1肽链

靶向性紫杉醇脂质体的制备及其药效的初步研究

Abstract

Liposomes as drug carrier have caught more and more attention and research because of its obvious advantages. Aimed to the application of liposomes in targeted drug delivery, paclitaxel-containing liposomes were prepared and their drug effect were studied in vitro and in vivo, including the following three aspects:

(1)Preparation and quality evaluation of paclitaxel-containing liposomes

Paclitaxel-containing liposomes were prepared through film-sonic method and reverse phase evaporation method by using soy bean phospholipids and cholesterol. The influence of different preparation methods on size, dispersion, entrapment efficiency, as well as physical stability of paclitaxel-containing liposomes were investigated. The results showed that film-sonic method was more feasible to prepare paclitaxel-containing liposomes than reverse phase evaporation method. The as-prepared liposomes have overcome the disadvantages of paclitaxel in clinic, such as poor hydrophilicity, side effect and low bioavailability. The influence of amount of drug used in liposome preparation and dispersion buffer on size and stability of paclitaxel-containing liposomes was also studied.

(2)Biomodification of liposomes and their application in cell imaging

A novel liposome which could be easily surface-modified with bio-molecules

groups was prepared by using soy bean phospholipids, cholesterol and containing NH

2

DPPE-PEG-pNP. Anti-cytokeratins 19 antibody was connected to the surface of the liposome nanoparticles. After incubation with MGC cells and COS-7 cells, MGC cells were selectively recognized by anti-cytokeratins 19 antibody modified liposome nanoparticles and well imaged. This kind of liposomes has following advantages: surface modification can be performed efficiently and quickly under mild conditions; bio-molecules could remain high activity after modification.

(3) Preparation of TMP1 peptide modified paclitaxel-containing liposome and study of targeted drug delivery in vitro and in vivo

Based on the above work, applications of liposomes in targeted drug delivery were studied further. TMP1 peptide was combined to the surface of paclitaxel-containing liposomes and its targeted drug delivery was studied in vitro and in vivo. The in vitro results indicated that compared with paclitaxel-containing liposome, the viability of PE-3M-1E8 cells decreased after incubation with TMP1 modified paclitaxel-containing liposome. Also, the primary in vivo experiment results revealed