Synthesis, structural characterization, thermal behavior and cytotoxic/antiproliferative activity assessments of poly(maleic anhydride-alt-acrylic acid)/hydroxyurea polymer/drug conjugate

Abstract

As drug carrier, poly(maleic anhydride-alt-acrylic acid) copolymer [poly(MA-alt-AA)], was prepared traditionally by radical initiated (benzoyl peroxide (BPO)), complex-radical polymerization technique via charge transfer complex (CTC) (50/50 in p-dioxane, at 70 °C under a nitrogen atmosphere). The pure alternating carrier was then conjugated as poly[MA-alt-AA]/HX (or MAAA/HX) copolymer/drug couple by ring opening reaction with an antineoplastic agent hydroxyurea (HX) for 48 h at 75 ± 0.1 °C in dimethylformamide (DMF), presence of triethylamine (Et3N) catalytic base. Chemical structure of the copolymer, antineoplastic agent and conjugate was characterized by Fourier Transform Infrared (ATR-FTIR) and also Nuclear Magnetic Resonance (proton and carbon NMR). Physical characterizations were also carried out by Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). To verify the elemental composition and also conjugation reaction of conjugated product, quantitative elemental analysis was especially carried out. Cytotoxicity was determined with Lactate Dehydrogenase (LDH) leakage assay using LDH cytotoxicity detection kit provided by Roche Diagnostics GMBH (Mannheim, Germany) based on the protocol in the user’s manual. Compared with 5-FU, toxicity values are as follows respectively: HX > MAAA > 5-FU ̴ MAAA/HX. Furthermore, antiproliferative activity studies was examined on HeLa (human cervical cancer) and (C6) rat brain tumor cells using proliferation BrdU ELISA assay. Conjugated-drug was identified to have the higher antiproliferation than the copolymer and free-drug (Hydroxyurea) on all studied concentrations and cellular potency of inhibitions were found as MAAA/HX > HX > MAAA at the highest concentration, 100 μg/mL. Toxic drug (HX) was modified as nontoxic MAAA/HX conjugate with good properties.