Receptor-mediated peptide delivery in pulmonary epithelial monolayers.
The present study investigated the feasibility of utilizing receptor-mediated endocytosis as a means to enhance peptide delivery to the pulmonary epithelium. The strategy employs a molecular conjugate consisting of a cognate moiety, transferrin (TF), covalently-linked to a model polypeptide, horseradish peroxidase (HRP), via a reversible disulfide linkage. A cultured alveolar epithelial monolayer system was used to simulate the conditions of the pulmonary epithelium and to allow accurate quantitation of intra- and transcellular peroxidase transport. The alveolar cells were isolated from rat lungs by enzymatic digestion and grown on microporous tissue culture-treated polycarbonate filters. A significant increase in the uptake of HRP by the cell monolayer was observed upon its conjugation with TF. The effect was found to be concentration-dependent, being more pronounced at low concentrations, i.e., 3.9- and 1.2-fold increase over unconjugated HRP controls at the concentration levels of 0.05 and 1.50 U/ml respectively. Effective peroxidase uptake was shown to require the TF cognate moiety for the cell surface receptor. Specific internalization of the conjugate by the TF endocytic pathway was verified by competition for the TF receptor. Conjugate internalization was not followed by a proportional increase in transcytosis, i.e., at 0.05 U/ml conjugate level, a 1.7-fold increase in transcytosis was observed as compared to 3.9-fold for endocytosis. Effective enhancement of transcytosis was achieved by treating the monolayers with brefeldin A (BFA), a compound known to affect intracellular transport of TF receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)
Deshpande
D
D
Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown 26506.
Toledo-Velasquez
D
D
Wang
L Y
LY
Malanga
C J
CJ
Ma
J K
JK
Rojanasakul
Y
Y
D000818:Animals; D020126:Brefeldin A; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000008:administration & dosage / Q000493:pharmacokinetics; D004705:Endocytosis / Q000502:physiology; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006735:Horseradish Peroxidase; D018796:Immunoconjugates; D008168:Lung / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010455:Peptides / Q000008:administration & dosage*; D011500:Protein Synthesis Inhibitors / Q000008:administration & dosage / Q000493:pharmacokinetics; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011956:Receptors, Cell Surface / Q000378:metabolism*; D014168:Transferrin / Q000008:administration & dosage
D003517:Cyclopentanes; D018796:Immunoconjugates; D010455:Peptides; D011500:Protein Synthesis Inhibitors; D011956:Receptors, Cell Surface; D014168:Transferrin; D020126:Brefeldin A; D006735:Horseradish Peroxidase