Evaluation of protein-N-(2-hydroxypropyl)methacrylamide copolymer conjugates as targetable drug carriers. 1. Binding, pinocytic uptake and intracellular distribution of transferrin and anti-transferrin receptor antibody conjugates

The transferrin receptor of human skin fibroblasts was studied as an in vitro model target antigen receptor for interaction with protein-polymer conjugates having potential for targeted drug delivery. Pinocytic uptake of 125I-labelled N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugated to monoclonal antibody B3/25 (specific for the transferrin receptor) or transferrin was up to 9-fold greater than uptake of the parent HPMA copolymer. The ability of these conjugates to bind specifically was confirmed by Scatchard analysis. Pinocytic internalisation was dependent on the molecular mass of the conjugate. Intracellular routing following internalisation was evaluated using density-gradient centrifugation. Unmodified HPMA copolymer was transferred via the endosomal compartment into secondary lysosomes, where, being resistant to degradation, it accumulated. Although the majority of endocytosed transferrin is recycled via the endosome, it was shown that any transferrin reaching the lysosomes was rapidly degraded and low-molecular-weight degradation products were released. Monoclonal antibody B3/25 showed a subcellular distribution consistent with prolongation on the cell surface, followed by internalisation and subcellular trafficking, via endosomes, into the lysosomal compartment, with subsequent degradation. Conjugation of protein to HPMA copolymer increased lysosomal accumulation of polymer up to 9-fold, with no detectable degradation of conjugate. The data presented here have implications regarding clinical potential of protein-HPMA copolymer conjugates designed for lysosomotropic drug delivery.