Remote Co-Loading of Doxorubicin and Hydralazine into PEGylated Liposomes: In Vitro Anti-Proliferative Effect Against Breast Cancer

Doxorubicin (DOX), an anthracycline chemotherapeutic agent, demonstrates efficacy against various types of cancer. Combining DOX with the antihypertensive drug hydralazine (HDZ) has been proposed as cardioprotective combination therapy, allowing for the use of a reduced DOX dose. The current study describes the remote co-loading of DOX and HDZ into PEGylated liposomes using, for the first time, a simultaneous pH gradient technique. First, PEGylated liposomes were prepared using an ethanol injection method and remotely loaded with DOX and HDZ. Then, DOX- and HDZ-loaded liposomes (Lip-DOX-HDZ) were characterized using DLS, TEM, FTIR, thermal analysis, drug leakage, and stability. Furthermore, the cellular uptake and cytotoxicity were evaluated in two human breast cancer cell lines (MCF7 and MDA-MB-231) and two normal cell lines (human dermal fibroblasts (HDFs) and rat cardiac cells (H9C2)). The results revealed that Lip-DOX-HDZ had a particle size of 158 ± 18 nm, PDI of 0.22 ± 0.08, and zeta potential of -22 ± 5 mV. The encapsulation efficiency of DOX and HDZ was 90% and 30%, respectively. Moreover, the IC₅₀ values of Lip-DOX-HDZ showed higher cytotoxicity against the MDA-MB-231 (5.5 ± 0.4 µM) and MCF7 (6.25 ± 0.9 µM) breast cancer cell lines compared to normal cells: HDF cells (20 ± 3.0 µM) and H9C2 cardiac cells (19.37 ± 2.0 µM). Our study found that remotely loaded Lip-DOX-HDZ showed a ~4-fold lower toxicity and selectivity for normal cells (HDFs and H9C2), compared to breast cancer cells. This suggests that Lip-DOX-HDZ is a promising nanocarrier for both DOX and HDZ, clinically potent molecules.