Abstract 3017: Combination effect of vinorelbine and doxorubicin in free solutions and liposomes in a multicellular spheroids model

Abstract Purpose: Combination drug therapy is a cornerstone approach to treat cancers, yet strategies of delivering drug combinations, including optimal drug ratios and drug formulations, remain challenging. Doxorubicin (DOX) and vinorelbine (VNR) are well established anticancer drugs that inhibit different phases of cell division, thus making them promising candidates for combination therapy. This study evaluated the combination effect of DOX and VNR in various formulations (free solution, pH-sensitive liposomes, and non-pH-sensitive liposomes) at various drug ratios against multicellular spheroids (MCSs) of A549 cells (NSCLC) by cell viability assay and mathematical modeling. Methods: Non-pH-sensitive liposomes (NonFlip) are composed of cholesterol, POPC and PEG-Ceramide. pH sensitive liposomes (Flip) are composed of POPC, PEG-Ceramide and lipids with a pH-sensitive conformational switch (flipids). A549 MCSs were constructed in 96-well ultra-low attachment microplates with RPMI-1640 and 0.3% collagen. MCSs (∼500 μm) were treated with free or formulated DOX, VNR, or DOX:VNR combinations at various ratios. Cell viability was assessed using CellTiter-Glo 3D assay. The effects of drug combination were evaluated by three mathematical models (Greco’s, unified theory, and Bliss independent) using the GraphPad Prism 10.6.0 software. Results: For individual drugs, free DOX, NonFlip DOX, and Flip DOX exhibited similar IC50 values around 5 μM. Free VNR and Flip VNR showed IC50 around 44 μM, while NonFlip VNR displayed IC50 exceeding 200 μM. For combinations, DOX:VNR ratios of 5:1, 1:1, and 1:5 yielded IC50 values of approximately 5, 8, and 16 μM, respectively, in free drug and Flip groups. NonFlip groups at 5:1 and 1:1 showed IC50 values of approximately 5 and 8 μM, but the IC50 of NonFlip DOX:VNR 1:5 exceeded 200 μM. Compared to individual drugs, most of the DOX:VNR combinations in Free drug solution, Flip formulations, and NonFlip formulations demonstrated synergism in Greco's and unified theory models (R2>0.95). To optimize the synergism, Greco's model favored the DOX:VNR ratio of 5:1 while unified theory favored 1:5. In contrast, the Bliss independent model fitted the cell viability data poorly (R2<0.9) and did not show synergistic effects. Conclusions: Greco's and unified theory modeling of the dose dependent cell viability data demonstrated the synergistic effect of combining DOX and VNR in various formulations. The poor fitting of the cell viability data by the Bliss independent model suggests DOX and VNR may not suppress the cancer cell growth independently. Further studies are needed to confirm these findings. Citation Format: Yong Zhu, Zizhao Xu, Yifan Lu, Shen Zhao, Md Delowar Hossain, Xin Guo. Combination effect of vinorelbine and doxorubicin in free solutions and liposomes in a multicellular spheroids model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3017.