Synthesis of HPMA Copolymers Containing Doxorubicin Bound via a Hydrazone Linkage. Effect of Spacer on Drug Release and in vitro Cytotoxicity

Abstract

The aim of this study was the synthesis, physico-chemical characterization and preliminary evaluation of biological activity of novel polymer drugs based on conjugates of anti-cancer drug doxorubicin (Dox) with water-soluble polymer drug carriers based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In the conjugates, Dox is attached to the polymer via a pH-sensitive linkage susceptible to hydrolysis at pH ≈ 5–6, thus enabling intracellular Dox release. Seven Dox-containing polymer conjugates differing in the length and structure of the single-amino-acid or oligopeptide spacer were synthesized (Gly, β-Ala, 6-aminohexanoyl, 4-aminobenzoyl, GlyGly, GlyLeuGly, Gly-DL-PheLeuGly) and the relationship between the spacer structure and the rate of in vitro Dox release was studied at various pH. The rate of Dox release at pH 5 (close to lysosomal pH) ranged from 70 to 96% of total Dox/48 h, depending on the spacer structure and being the highest for the conjugate containing the 6-aminohexanoyl spacer. The rate of Dox release from most conjugates incubated at pH 7.4 (blood pH) was more than 10 times slower, ca. 4–10% of total Dox/48 h. Molecular weight of the polymer (25 000–115 000 g/mol) did not significantly influence the rate. The presence of lysosomal enzyme cathepsin B in incubation media increased the rate of Dox release from the conjugates with oligopeptide GlyLeuGly and Gly-DL-PheLeuGly spacers by 15–30%, whereas the release from conjugates with other spacers remained unchanged. Cytotoxicity of all hydrazone conjugates for mouse EL-4 T cell lymphoma cells was much higher and close to that of free Dox (IC50 ≈ 0.1–0.34 μg Dox/mL), in contrast to cytotoxicity of similar classic conjugates bearing Dox attached via an amide bond (IC50 ≈ 19 μg Dox/mL).