HPMA based macromolecular therapeutics: internalization, intracellular pathway and cell death depend on the character of covalent bond between the drug and the peptidic spacer and also on spacer composition

Polymeric conjugates based on N-(2-hydroxypropyl)methacrylamide (HPMA) have been tested as potential carrier for anticancer drug - doxorubicin (Dox). Two types of conjugates were synthesized: (a) conjugates containing Dox bound through an amidic bond to an oligopeptidic side-chain (usually GFLG) and (b) hydrolytically cleavable conjugates wherein Dox is bound to the polymeric carrier through a pH sensitive bond. The mechanism of action of both conjugates is different and reflects the diverse way and intensity of their intracellular accumulation. All conjugates containing doxorubicin bound via an amidic bond directly penetrate the plasma membrane and are detectable in all associated cellular membranes, i.e. membranes of the endocytic compartment, a nuclear membrane as well as membranes of Golgi and endoplasmic reticulum. We have never been able to detect released doxorubicin inside the nuclei of the treated cells. The cytotoxicity of these conjugates seems to be primarily caused by the damage of cellular membranes. Necrosis is the main mechanism of the cell death. Conjugates containing hydrolytically bound doxorubicin are internalized by endocytosis and fluid phase pinocytosis and doxorubicin is cleaved from the polymeric carrier at low pH in late endosomes and lysosomes. An apoptosis is the main mechanism of the cell death. The spacer influences the rate of the intracellular release of the drug rather than the rate of internalization.