RECEPTOR-MEDIATED PEPTIDE DELIVERY IN PULMONARY EPITHELIAL MONOLAYERS

The present study investigated the feasibility of utilizing receptor-m ediated endocytosis as a means to enhance peptide delivery to the pulm onary epithelium. The strategy employs a molecular conjugate consistin g 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 s imulate the conditions of the pulmonary epithelium and to allow accura te 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 ob served upon its conjugation with TF The effect was found to be concent ration-dependent, being more pronounced at low concentrations, i.e., 3 .9- and 1.2-fold increase over unconjugated HRP controls at the concen tration levels of 0.05 and 1.50 U/ml respectively. Effective peroxidas e uptake was shown to require the TF cognate moiety for the cell surfa ce receptor. Specific internalization of the conjugate by the TF endoc ytic pathway was verified by competition for the TF receptor. Conjugat e internalization was not followed by a proportional increase in trans cytosis, i.e., at 0.05 U/ml conjugate level, a 1.7-fold increase in tr anscytosis was observed as compared to 3.9-fold for endocytosis. Effec tive enhancement of transcytosis was achieved by treating the monolaye rs with brefeldin A (BFA), a compound known to affect intracellular tr ansport of TF receptor complexes. At 1.6 mu/ml concentration level, BF A promoted a >20-fold increase in the rate of transcytosis of the conj ugate in both the apical-to-basal and basal-to-apical directions. This effect was not associated with membrane leakage since BFA-treated mon olayers maintained tight barrier to transport of the paracellular perm eability solute C-14 mannitol. In addition, BFA had no significant eff ect on the transport of free HRP. Instead, the effect of BFA on conjug ate transport was mediated by TF receptors since excess free TF compet itively inhibited transcytosis of the conjugate. Thus, our results are consistent with the TF receptor-mediated transport of the conjugate a nd its enhancement through the intracellular rerouting of the conjugat e by BFA. The findings in this study may potentially be relevant to th e design of drug delivery systems that can enhance intra- or transcell ular uptake of therapeutic peptides in the pulmonary epithelium.