Browsing Theses and Dissertations School of Pharmacy by Author "Kitchens, Kelly Marie"
Poly (amidoamine) dendrimers as oral drug carriersKitchens, Kelly Marie; Ghandehari, Hamidreza (2007)The objective of the research described in this dissertation was to evaluate the influence of the physicochemical properties of poly (amidoamine) (PAMAM) dendrimers on their cytotoxicity, permeability and mechanisms of transport across Caco-2 cell monolayers as models of the intestinal epithelial barrier. The influences of structural properties including surface charge, molecular size, and model drug loading on PAMAM dendrimer permeability, tight junction modulation, internalization and intracellular trafficking were evaluated. Cationic PAMAM-NH2 (generations 2 (G2) and 4 (G4)), neutral PAMAM-OH (G2), and anionic PAMAM-COOH (G1.5-G3.5) dendrimers were conjugated to fluorescein isothiocyanate (FITC), as a model drug compound. The permeability of fluorescently labeled PAMAM dendrimers, and of 14C-mannitol in the presence of unlabeled and FITC labeled PAMAM dendrimers, was measured in the apical-to-basolateral direction. Caco-2 cells were incubated with the dendrimers followed by mouse anti-occludin and rhodamine phalloidin for tight junctions, or anti-clathrin, anti-early endosomal antigen 1 (EEA-1), and anti-lysosome associated membrane protein 1 (LAMP-1) for endocytosis markers, then visualized using confocal laser scanning microscopy. Transmission electron microscopy (TEM) was employed to monitor changes in Caco-2 cell monolayer morphology upon incubation with PAMAM dendrimers. The rate of G4NH2 absorption and permeability across Caco-2 cells was measured in the absence and presence of endocytosis inhibitors: brefeldin A; colchicine; filipin; and sucrose. The overall rank order of PAMAM permeability was G3.5COOH > G2NH 2 > G2.5COOH > G1.5COOH > G2OH. 14C-Mannitol permeability significantly increased in the presence of labeled cationic and anionic PAMAM dendrimers compared to unlabeled dendrimers. PAMAM dendrimers caused disruption in occludin and rhodamine phalloidin staining patterns, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. TEM analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. The rate of G4NH2 uptake and permeability significantly declined in the presence of endocytosis inhibitors. These studies demonstrate a size and charge window for PAMAM dendrimer cytotoxicity and permeability. Confocal studies suggested PAMAM dendrimers are transported via a combination of the paracellular pathway by opening the tight junctions and endocytosis mechanism(s). The contribution of endocytosis to G4NH 2 transport was further confirmed by endocytic inhibition of G4NH 2 absorption and transport. These studies collectively demonstrate the potential of PAMAM dendrimers in the oral delivery of bioactive agents for systemic absorption.