Abstract 446: Cellular Uptake of Glycol Chitosan Nanoparticles in Idiopathic Pulmonary Fibrosis Fibroblasts in Response to the Collagen-Rich Microenvironment

Abstract

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by the presence of persistent fibrotic fibroblasts which have apoptosis-resistant properties in response to cell death inducing conditions including type I collagen rich matrix. Although selective targeting of fibrotic fibroblasts is a feasible concept for the effective therapy of IPF, no reliable drug carriers for targeting IPF fibroblasts have been reported. To address this limitation, biodegradable glycol chitosan nanoparticles (CNPs) were utilized as a carrier system for selective targeting of IPF patients-derived fibroblasts. Primary human lung fibroblasts from non-IPF and IPF patients (n = 6/group) were treated with various doses (50–450 μg/ml) of CNPs, and the subcellular localization of CNPs was examined as a function of time (3, 6, and 30 h) with a confocal microscope. CNPs were found in the cytoplasm of fibroblasts at 30 h post-treatment on tissue culture plates in the absence of collagen while the intracellular CNPs were clearly observed at 3 h post-treatment. Importantly, the cellular uptake of the CNPs was significantly increased at 3, 6, and 30 h post-treatment, when control and IPF fibroblasts were cultured on collagen, suggesting that fibroblast and collagen interaction promotes the CNP’s cellular internalization process. Clathrin-mediated endocytosis, caveolae-mediated endocytosis, and macropinocytosis were then suppressed by chlorpromazine (30 μM), genistein (100 μM), or amiloride (50 μM), respectively to elucidate the underlying mechanism of cellular uptake of CNPs in fibroblasts on the collagen matrix. Amiloride pre-treatment remarkably reduced the cellular internalization of CNPs in fibroblasts cultured on the collagen matrix. Furthermore, additional results also showed that sodium-hydrogen exchanger 1 (NHE-1) and PI3K play important roles in the increased cellular uptake of CNPs in the lung fibroblasts on the collagen matrix, suggesting that the subcellular localization of CNPs is largely due to the macropinocytosis. We propose that our approach can be a promising tactic for targeted delivery of therapeutics to the fibrotic fibroblasts residing in collagen matrix, which may lead to the improved treatment outcomes of this deadly disease.