纳米粒—多肽药物转运Caco-2细胞模型的机理研究

【作者】 李正荣；

【导师】 王文； 李朝军；

【作者基本信息】 南京师范大学 ， 水产养殖， 2003， 硕士

【摘要】 诸多因素影响口服药物的生物利用度，为探明药物吸收过程中物理和生化屏障的机理很有必要建立合适的药物体外吸收模型。实验中，我们建立Caco-2细胞模型以模拟小肠上皮细胞来研究这些主要的吸收屏障。Caco-2细胞系来源于人结肠癌细胞，在一定的培养条件下能自发形成有极性的、具微绒毛以及紧密连接等类似于小肠上皮细胞刷状缘侧的分化特征，因此这种细胞能模拟小肠上皮细胞，是体外研究药物转运机理、吸收特性的理想模型。另外，磷脂是哺乳动物上皮细胞膜上的主要脂质形式，可作为载体转运多种药物。一些由磷脂组成脂质体包裹的药物能降低药物的毒性以使药物作用于靶位点，增强药物的治疗效果。 本论文中建立、评估了Caco-2细胞模型，并应用此模型对壳聚糖及其衍生物N-三甲基壳聚糖(TMC)、EDTA螯合的壳聚糖(CEC)包裹的脂质体药物(胰岛素、醋酸亮丙瑞林等)的吸收特性进行了较详尽的研究。1、Caco-2细胞模型的建立与评估 本章实验中Caco-2细胞接种于涂有鼠尾胶原的Transwell中培养20—28天后，通过电子显微镜观察细胞的形态特征以及EVOM皮肤伏特计、碱性磷酸酶试剂盒对单层Caco-2细胞的定性测量。结果表明本实验室建立的Caco-2细胞模型具有微绒毛、紧密连接等结构特征；单层细胞的跨膜电阻值(TEER)在350~550Ω*cm~2，碱性磷酸酶主要集中在绒毛面，与小肠类似，可以作为研究小肠药物过程的体外模型。2、纳米粒药物在Caco-2细胞模型中吸收特性的研究 我们设计壳聚糖、TMC、CEC以及脂质体等纳米材料包裹一些固有吸收性较差的药物(胰岛素、醋酸亮丙瑞林)以提高其生物利用率。从实验通透结果：ⅰ)壳聚糖包裹的醋酸亮丙瑞林>壳聚糖、脂质体共包裹的醋酸亮丙瑞林>醋酸亮丙瑞林>脂质体包裹的醋酸亮丙瑞林；ⅱ)壳聚糖、脂质体共包裹的胰岛素>CEC、脂质体共包裹的胰岛素>TMC、脂质体共包裹的胰岛素>脂质体包裹的胰岛素>胰岛素，可得出：醋酸亮丙瑞林与胰岛素转运Caco-2单层细胞的机制不同；另外，脂质体可能有两种不同的功能：ⅰ)对多药耐药性(MDR)药物而言，脂质 硕士学位论文 中文摘要体可作为调节物以增加药物的吸收：h）对非＊＊R型药物，脂质体作为药物的贮存库延缓了药物的释放时间，同时避免胃肠中一些蛋白酶或多肽对药物的降解作用。壳聚糖能够促进胰岛素等药物的渗透量，TMC、CEC为壳聚糖的衍生物，推测它们有着类似于壳聚糖促吸收的功能。此外，纳米微粒会导致Caco－2细胞TEER的降低，然而这种作用是可逆的。3、纳米粒葡聚糖转运细胞后的实时监控 为了进一步证实细胞摄取多肽药物的机理，我们以葡聚糖作为药物的模型。葡聚糖是中性、固有吸收性较差的大分子物质 （1 kd），用罗丹明标志。实验中各种辅料包裹葡聚糖以及葡聚糖分别转运Caco－2细胞30min后，通过荧光显微镜检测葡聚糖的位置，结果唯有壳聚糖、脂质体共包裹的以及壳聚糖包裹的能增加葡聚精的吸收。 接着通过激光共聚焦扫描显微镜观察活细胞摄取葡聚精后的动态过程，得到了与通透试验一致的结果，即壳聚糖一高效生物或附性共聚体，是药物跨膜运输的促进吸收剂，推测主要通过胞旁转运、细胞内吞的方式提高药物的吸收。实验结束后Caco－2单层细胞用台酚蓝染色未见细胞内有这种物质的吸收，表明细胞未受到损坏。总之，壳聚精对细胞膜的作用是温和的、可逆的，因此壳聚糖可作为药物跨肠上皮细胞吸收的赋形剂。更多还原

【Abstract】 The multifaceted nature of the problems associated with poor oral bioavailability of drug molecules requires a systematic and reductionist approach to understand the underlying factors affecting the bioavailability and absorption of these molecules. Thus use of appropriate in vitro models is extremely useful in elucidating the role of various physical and biochemical barriers to drugs absorption. In this chapter, we have discussed the use of one such in vitro model - Caco-2 cells, in elucidating the roles of the physical and biochemical barriers to drug absorption posed by the intestinal epithelium. Caco-2 cell line, derived from a colon carcinoma, is able to differentiate spontaneously when grown in standard culture conditions. The differentiated cells polarized, formed microvilli and T-junctions associated with the duodenal enterocytes brush border. This cell line thus represents an appropriate model for the study of transport mechanisms related to the intestinal barrier and can be used to study the absorption of drugs. In addition, liposome is the major lipids in the plasma membrane of mammalian cells. Liposome have been used increasingly as carriers for the delivery of a variety of drugs. Some drugs encapsulated in liposomes made of phospholipid mixtures have shown reduced toxicity and enhanced therapeutic efficacy.In this research, Caco-2 cell monolayer model was established and evaluated. The absorptive characteristics of several drugs, such as insulin , leuprolide acetate(LA),which are encapsulated in chitosan , N-trimethyl chitosan(TMC) , chitosan+EDTA(CEC) coated or uncoated liposome, were also carefully studied in this model. 1 , The establishment and evaluation of Caco-2 cell monolayer modelIn this work, Caco-2 cells were seeded on Transwell, Which was coated with rat tail collagen I ,and grew for about 20-28 days for using; Electon microscope was used to monitor the cell morphological in experiment, the transepithelial electrical resistance(TEER) and alkali phosphatase were tested, the result shows that the Caco-2 cell monolayer in our laboratory posses microvillus and tight junction, the TEERvalues ranged from 350 to 550Q*cm2 and alkali phosphatase located in brush side under the cultivation condition.2, The study of absorptive characteristics of nano-particle drug in Caco-2 cell monolayer model.In my present experiments, the design of targeted oral liposome , chitosan and TMC , CEC is anticipated to improve the systemic delivery of poorly absorbed agents, such as insulin LA. From the results: i) encapsulated LA in Chitosan> encapsulated LA in Chitosan-coated liposome>LA> encapsulated LA in liposome; ii) encapsulated insulin in Chitosan-coated liposome Encapsulated insulin in CEC coated liposome > encapsulated insulin in TMC coated liposome > encapsulated insulin in liposome >insulin, we can conclude that the absorptive mechanism of LA is different from insulin; and that Liposome have two kinds of mechanisms: i) To multidrug resistance(MDR),it act as MDR modulators of the transport of drug in Caco-2 cell layers, ii) To LA, such liposome systems are advantageous in providing depots of drug, the reservoirs may also serve a protective functions preserving protein functionality in the presence of gastrointestinal(GI) proteases and bile salts. Chitosan is able to increase the uptake of insulin, LA; TMC, CEC are derivative of chitosan , it has been proposed that they most likely acts by the same mechanism as chitosan. Besides, the nano-particles reduces the TEER of Caco-2 cell, however, the effect is reversible.3 , Fluorescence and Confocal Microscopy analysis of movement of nano-particles Dextran.To confirm the involvement of cytosis for peptide drugs, we use the Dextran as a model for peptide drugs. Dextran, a poorly absorbed , neutral, hydrophilic and large molecular weight (Mw.10Kd) marker is labeled with Rhodanmine. Apparent permeabilities of Caco-2 cells to Dextran , uncoated Dextran liposome , chitosan coated Dextran and chitosan coated Dextran lipos更多还原