Transcytosis of GCSF-transferrin across rat alveolar epithelial cell monolayers.
OBJECTIVE
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 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.


CONCLUSIONS
This study suggests the possibility of using TfR-mediated transcytosis for systemic delivery of therapeutic proteins via the alveolar epithelium.
Widera
Adam
A
Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.
Kim
Kwang-Jin J
KJ
Crandall
Edward D
ED
Shen
Wei-Chiang
WC
D000818:Animals; D001692:Biological Transport; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D004705:Endocytosis; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D004848:Epithelium / Q000378:metabolism; D000069585:Filgrastim; D005712:Galvanic Skin Response; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000494:pharmacology*; D066298:In Vitro Techniques; D008297:Male; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011990:Receptors, Transferrin / Q000187:drug effects / Q000378:metabolism; D011994:Recombinant Proteins; D013997:Time Factors; D014168:Transferrin / Q000737:chemistry / Q000494:pharmacology*
D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D016179:Granulocyte Colony-Stimulating Factor; D020126:Brefeldin A; D000069585:Filgrastim