Contrastive Studies of Cytarabine/Daunorubicin Dual-Loaded Liposomes Prepared by pH Gradient and Cu²⁺ Gradient Method

Conventional combination chemotherapy often leads to unsatisfactory clinical outcomes due to the different distribution characteristics in vivo and the superimposed systemic toxicity of the drug cocktail. Co-encapsulated nano preparations have been gradually developed in recent years. In this work, cytarabine (Ara-C)/daunorubicin (DNR) liposomes were prepared by the pH gradient (ADL-pH) and Cu²⁺ gradient (ADL-Cu) methods. Ara-C did not show significant release from either ADL-Cu or ADL-pH in vitro during 168 h, which related to its logP_oct. Different drug-loading patterns showed different release characteristics of DNR due to the different existence forms, ADL-pH contains the citrate form, while in ADL-Cu, there is the Cu²⁺ complex. To evaluate the release behavior, daunorubicin liposome (DL) and daunorubicin-Cu²⁺ complex (DNR-Cu) were prepared. The addition of EDTA in the release medium significantly increased the release rate of DNR from DL-Cu, while lower pH accelerated DNR release from both DL-pH and DL-Cu. The PK confirmed that ADL-Cu and ADL-pH could prolong the drug circulation time, and ADL-Cu had a mean retention time 1.5 times that of ADL-pH. Furthermore, both liposomes allowed the two drugs to maintain a relatively constant plasma concentration ratio for a prolonged time. Cytotoxicity assays showed that Ara-C/DNR with a molar ratio of 5:1 and 3:1 exhibited an excellent synergistic effect, which was more obvious at 5:1. In vitro antitumor results revealed that ADL-Cu exhibited more cytotoxicity than ADL-pH. All factors tested in this work suggest the considerable potential of ADL-Cu and ADL-pH for anticancer treatment.