| 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... |
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