title: Transcytosis of GCSF-transferrin across rat alveolar epithelial cell monolayers.
abstract: 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.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California 90033, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Widera
authors.forename: Kwang-Jin J
authors.initials: KJ
authors.lastname: Kim
authors.forename: Edward D
authors.initials: ED
authors.lastname: Crandall
authors.forename: Wei-Chiang
authors.initials: WC
authors.lastname: Shen
mesh_terms: 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*
chemical_list: D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D014168:Transferrin; D016179:Granulocyte Colony-Stimulating Factor; D020126:Brefeldin A; D000069585:Filgrastim