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 (∖G 2500 Ωcm²) 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 μg/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 μg/mL of nonradiolabeled Tf. Basolateral samples (500 μL) 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 μg/mL.

Results. BFA treatment resulted in a small significant reduction in TfR at the basolateral surface of type II cell-like monolayers, while it had no effect on TfR distribution in type I cell-like monolayers. In contrast, BFA treatment significantly altered the endocytosis of TfR, reducing the basolateral uptake of ⁵⁹Fe-Tf while greatly increasing the apical uptake of ⁵⁹Fe-Tf. BFA treatment, however, did not affect the TfR-specific uptake of ⁵⁹Fe-Tf in type I cell-like monolayers. TfR-specific apical-to-basolateral transcytosis of ¹²⁵I-Tf and ¹²⁶I-GCSF-Tf conjugates was significantly enhanced in the presence of BFA in type II cell-like monolayers, whereas it had no effect on apical-to-basolateral transport of ¹²⁵I-GCSF. BFA-enhanced transport of ¹²⁵I-GCSF-Tf was approximately 3-fold higher than that of ¹²⁵I-GCSF in the presence or absence of BFA. Moreover, ¹²⁵I-GCSF transport in the presence of BFA was not significantly different from non-specific ¹²⁵I-GCSF-Tf transport. Chromatographic analyses and bio-assays revealed that GCSF-Tf was not degraded during transport via TfR-specific processes, and that GCSF retained biologic activity when liberated from the conjugate via dithiothreitol reduction.

Conclusion. This study suggests the possibility of using TfR-mediated transcytosis for systemic delivery of therapeutic proteins via the alveolar epithelium.