Soluble, crosslinked N-(2-hydroxypropyl)methacrylamide copolymers as potential drug carriers: Pinocytosis by rat visceral yolk sacs and rat intestine cultured in vitro. Effect of molecular weight on uptake and intracellular degradation

Abstract

N- (2-Hydroxypropyl)methacrylamide (HPMA) copolymers containing oligopeptide side-chains terminating in a p- nitrophenyl ester were crosslinked with dityrosylhexamethylene-diamine to a level below the gel point. The resultant soluble crosslinked HPMA copolymer sample was then fractionated to produce five samples of mean molecular weights (M̄ w) between 34,000 and >400,000. These fractions were radiolabelled with [ 125I] iodide and used to study uptake by rat visceral yolk sacs and adult rat small intestine cultured in vitro. Rat visceral yolk sacs captured these HPMA copolymers at a rate consistent with uptake by fluid-phase pinocytosis. The oligopeptide crosslinks of the internalized copolymers were degraded intracellularly, resulting in the release of [ 125I] iodotyrosine back into the culture medium. The rate of uptake of copolymer by the yolk sac decreased markedly with increasing molecular weight, but molecular weight had no effect on the extent of intracellular degradation of the different HPMA copolymer fractions. The effect of 2,4-dinitrophenol (a known inhibitor of pinocytosis) and leupeptin (a lysosomal thiol-pro-teinase inhibitor) confirmed that accumulation of copolymers was by pinocytosis and indicated the importance of lysosomal thiol-proteinases in oligopeptide crosslink degradation. Incubation of copolymer with purified cathepsin B confirmed that this enzyme is able to cleave oligopeptide crosslinks. Crosslinked HPMA copolymers were also found to be pinocytosed by rat intestine and also translocated across to the serosal side. Uptake and translocation were both inhibited by low temperature (15°C) and once again pinocytosis was shown to be size-dependent. However, in this case the rate of uptake increased with increasing polymer size .