Transcytosis of GCSF-transferrin across rat alveolar epithelial cell monolayers
Abstract
PURPOSE: The purpose of this study was to use primary cultured rat alveolar epithelial cell monolayers to examine the potential of using transferrin receptor (TfR)-mediated transcytosis for noninvasive systemic protein drug delivery via the pulmonary route.
METHODS: Freshly isolated rat type II pneumocytes were plated onto tissue culture-treated polycarbonate 12-mm Transwells. AEC monolayers (> or = 2500 omega(cm2)) were treated with keratinocyte growth factor (10 ng/mL) for maintenance of type II cell-like characteristics. Filgrastim (GCSF)-Tf conjugates were prepared using the linkers SPDP and DPDPB. TfR-specific binding and uptake were determined using 125I-Tf and 59Fe-Tf treatment, respectively. Apical-to-basolateral (A-to-B) transferrin receptor (TfR)-mediated transcytosis was determined by dosing the apical compartment with 1.5 microg/mL of 125I-Tf or 125I-GCSF-Tf. Nonspecific TfR-independent transport of 125I-Tf and 125I-GCSF-Tf was determined in parallel by including 150 microg/mL of nonradiolabeled Tf. Basolateral samples (500 microL) were taken at 2, 4, and 6 h post-dosing, subjected to 15% trichloroacetic acid precipitation, and assayed in a Packard gamma counter. TfR-specific transport was determined as the difference between total and nonspecifc transport. The effects of brefeldin-A (BFA) on TfR distribution and (A-to-B) transport of 125I-Tf, 125I-GCSF and 125I-GCSF-Tf was studied by including the agent in the apical fluid at 1 microg/mL.
RESULTS: BFA treatment…
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