Cyclodextrin-based nanosponge co-delivery of doxorubicin and EMD: synergistic anticancer activity with improved selectivity toward cancer cells

Abstract

Combination therapy is a promising strategy in cancer treatment aiming at improving therapeutic efficacy and overcoming tumour resistance. Cyclodextrin-based nanosponges (EpCN) were developed here for the co-delivery of doxorubicin (DX), a hydrophilic chemotherapeutic agent, alongside N,N-bis (5-ethyl-2-hydroxybenzyl) methylamine (EMD), a hydrophobic compound targeting c-Myc. EpCNs were synthesized by crosslinking β-cyclodextrin with epichlorohydrin, then DX and EMD were loaded either separately or together into the nanosponge. The nanosponges were extensively characterized combining Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM) and Nuclear Magnetic Resonance (NMR). The dual-loaded nanosponges with DX and EMD (DX/EMD-EpCN) displayed uniform sizes (30 ± 13 nm), high encapsulation efficiency (>98%), a zeta potential of +23 ± 4 mV, and a pH-responsive drug release, with faster release at acidic pH mimicking tumour conditions. In vitro studies were carried out on cancerous (A549 and MCF-7) and non-cancerous (WI-38) cells to explore the therapeutic potential of the drug-loaded EpCNs. Cytotoxicity results demonstrated that DX/EMD-EpCNs significantly reduced cell viability, more than free drugs or single drug-loaded EpCNs in both cancerous cell lines. The therapeutic potential of combining DX and EMD was improved by the encapsulation into EpCN, as indicated by a strong synergism (combination index: <0.6), with a reduced effective dose, and improved drug uptake in cancer cells while sparing normal cells. Cell cycle analysis reveals that DX/EMD-EpCNs induced multi-phase arrest at the G0/G1 and G2/M phases, leading to a superior apoptotic induction as confirmed by the annexin V/Zombie UV staining. Western blot analysis demonstrated that the DX/EMD-EpCN significantly suppressed c-Myc and Bcl-2 expression while increased cleaved-PARP expression in both cancer cell lines, indicating the activation of caspase-dependent apoptosis. In contrast, the downregulation of c-Myc and Bcl-2 by single drug-loaded EpCNs altered cell cycle progression but did not significantly induced apoptosis. The co-delivery of DX and EMD by EpCNs enhanced therapeutic potency through various mechanisms. These findings highlight the potential of cyclodextrin-based nanosponges as a versatile drug delivery platform for combination chemotherapy profiting from their capability of simultaneously encapsulating both hydrophilic and hydrophobic drugs.