Preparation of all-trans retinoic acid and irinotecan co-loaded liposomes and their in vitro evaluation against anaplastic thyroid cancer

ObjectiveTo prepare all-trans retinoic acid (ATRA) and irinotecan (IRI) co‑loaded liposomes (ATRA-IRI@Lip) and evaluate their in vitro therapeutic effects against anaplastic thyroid carcinoma (ATC) and their ability to eliminate cancer stem cells (CSCs).MethodsATRA-IRI@Lip was obtained through the co-encapsulation of ATRA and IRI within liposomes, using the thin-film hydration method. The particle size, Zeta potential, encapsulation efficiency, and in vitro release profiles of ATRA-IRI@Lip were characterized. Hemolysis assay was conducted to evaluate blood compatibility. Cellular uptake of FITC-labeled liposomes was assessed. The in vitro anti-ATC effects were evaluated by cell counting kit-8 (CCK‑8), Calcein‑AM/PI staining, and Transwell assays. The synergistic effect of ATRA and IRI was analyzed using the combination index, while flow cytometry was performed to detect the proportions of aldehyde dehydrogenase-positive cells and CD133-positive cells.ResultsThe prepared ATRA-IRI@Lip had an average particle size of (210.3±5.2) nm, a polydispersity index of 0.254, and an average surface potential of －(13.7±2.3) mV. The encapsulation efficiencies of ATRA and IRI were (88.1±2.9)% and (86.8±1.6)%, respectively. In vitro release studies showed that ATRA-IRI@Lip significantly delayed the release of both ATRA and IRI, with 48-h cumulative release rates of (66.2±3.6)% and (77.7±4.1)%, respectively. Hemolysis assays demonstrated that all tested concentrations of ATRA-IRI@Lip exhibited hemolysis rates below 3%, indicating good hemocompatibility. CCK-8 and Calcein-AM/PI staining demonstrated that ATRA-IRI@Lip significantly inhibited the viability of ATC cells and promoted cell death, with an IC₅₀ significantly lower than that of the free IRI group and the free ATRA+IRI group (both P<0.01). Combination index analysis demonstrated that both the free ATRA+IRI group and the ATRA-IRI@Lip group exhibited synergistic effects (combination index <1), with stronger synergism observed in the ATRA-IRI@Lip group. Flow cytometry results showed that ATRA-IRI@Lip significantly decreased the proportions of aldehyde dehydrogenase-positive cells and CD133-positive cells (both P<0.01).ConclusionATRA-IRI@Lip was successfully prepared, achieving synergistic effects of differentiation therapy and chemotherapy of ATRA and IRI, thereby effectively eliminating ATC cells and CSCs.