Novel Doxorubicin Derivatives: Synthesis and Cytotoxicity Study in 2D and 3D in Vitro Models

Multidrug resistance (MDR) of tumors to chemotherapeutics often leads to failure of cancer treatment. The aim of the study was to prepare novel MDR-overcoming chemotherapeutics based on doxorubicin (DOX) derivatives and to evaluate their efficacy in 2D and 3D in vitro models. To overcome MDR, we synthesized five DOX derivatives, and then obtained non-covalent complexes with human serum albumin (HSA). Drug efficacy was evaluated for two tumor cell lines, namely human breast adenocarcinoma MCF-7 cells and DOX resistant MCF-7/ADR cells. Additionally, MCF-7 cells were entrapped in alginate-oligochitosan microcapsules, and generated tumor spheroids were used as a 3D in vitro model to study cytotoxicity of the DOX derivatives. Due to 3D structure, the tumor spheroids were more resistant to chemotherapy compared to monolayer culture. DOX covalently attached to palmitic acid through hydrazone linkage (DOX-N₂H-Palm conjugate) was found to be the most promising derivative. Its accumulation levels within MCF-7/ADR cells was 4- and 10-fold higher than those of native DOX when the conjugate was added to cultivation medium without serum and to medium supplemented with 10% fetal bovine serum, respectively. Non-covalent complex of the conjugate with HSA was found to reduce the IC₅₀ value from 32.9 µM (for free DOX-N₂H-Palm) to 16.8 µM (for HSA-DOX-N₂H-Palm) after 72 h incubation with MCF-7/ADR cells. Palm-N₂H-DOX conjugate was found to be the most promising DOX derivative in this research. The formation of non-covalent complex of Palm-N₂H-DOX conjugate with HSA allowed improving its anti-proliferative activity against both MCF-7 and MCF-7/ADR cells.