(T1330-09-52) Co-Formulation of Vinorelbine and Doxorubicin into Liposomes for the Treatment of Non-Small Cell Lung Cancer

Purpose: While drug combinations are standard practice for treating non-small cell lung cancer (NSCLC) in clinic, the different pharmacokinetic profiles of the individual drugs may compromise their synchronized exposure and synergistic effects against solid tumors. Nano-formulations such as liposomes offer the opportunity to address this issue by combining multiple drugs into a single formulation. Previously, we reported pH-sensitive liposomes based on the protonation-induced conformational switch of flipids in efforts to enhance the drug release at the acidic microenvironment inside solid tumors[1]. In this study, we aim to 1) construct liposomes that are loaded with doxorubicin (DOX), vinorelbine (VNR), or both of the drugs, and 2) to characterize their physicochemical properties and drug release. Some of the liposomes will contain flipids to enhance their drug release at low pH and are called fliposomes hereinafter.

Methods: Liposome formulations were prepared by thin-film hydration, freeze-thawing, extrusion, size-exclusion chromatography, ion-driven remote drug-loading and removal of free drug by cation-exchange resin. The size and polydispersity index (PDI) were measured using dynamic light scattering on a Zetasizer (Malvern, UK). The drug encapsulation efficiency (EE) and drug release were determined by HPLC. For drug release study, a buffer-diluted sample of a liposome formulation was mixed with cation-exchange resin in a glass vial and incubated at 37 ℃ on an orbital shaker at 100 rpm for 50 hours, during which the diluted formulation was aliquoted from the mixture and the concentration of the remaining drugs in the aliquots was quantified by HPLC [Waters 2690, C18 column (4.6 x 250 mm, 5μm, Phenomenex®), UV at 270 nm]. HPLC gradient solvents: A, water with 0.1% trifluoroacetic acid (TFA); B, acetonitrile/methanol (4/1) with 0.1% TFA. The column was eluted at a flow rate of 1 mL/min, 35 ℃ using a linear gradient from 10% solvent B to 75% solvent B over 15 min followed by re-equilibrating the column under initial conditions for 2 min.

Results: The liposome formulations of VNR were prepared using either DPPC, POPC, or a mixture of DPPC and POPC, together with PEG-Ceramide. Some of the liposomes also contained a flipid. The liposomes showed hydrodynamic diameters smaller than 150 nm and high homogeneity. Liposomes without the flipid all achieved high encapsulation efficiency of VNR ( >70%). For fliposomes of VNR, only those containing POPC achieved high encapsulation efficiency (Table 1A). As shown in Table 1B, liposomes with or without the pH-sensitive flipid were successfully loaded with DOX, VNR, and both drugs (encapsulation efficiency >75%). Such liposomes carried hydrodynamic diameters ~130 nm, low PDI ( < 0.02), and nearly neutral pH. DOX-loaded liposomes exhibited a cumulative release of approximately 20% to 30% over 50 hours at pH 7.4. Surprisingly, at pH 5.5, the DOX-loaded fliposome did not demonstrate enhanced drug release compared to the DOX-loaded liposome without the flipid. In contrast, VNR-loaded fliposomes showed higher release compared to their liposome counterparts without the flipid at both pH 7.4 and 5.5. Liposomes without the flipid released approximately 20% of VNR, whether single-loaded or dual-loaded, whereas fliposome formulations released 2- to 3-fold more VNR (Figure 2).

Conclusion: Liposomes loaded with DOX, VNR, or both drugs were developed with size ~ 130 nm, low PDI, mostly high encapsulation efficiency and nearly neutral pH. The fliposome formulation containing VNR showed higher drug release compared to its counterpart without the flipid. However, this effect was not observed in the DOX-loaded fliposome formulation. In the future, we will evaluate the anticancer activities of these formulations in multicellular spheroids of human lung cancer cells[2].

References: [1] Zhao, Shen (2018). Design and in vitro characterization of lipids with a pH-sensitive conformational switch and their liposomes for anticancer drug delivery.
[2] Xu, Z et al. (2024). Construction of An Orthotopic Xenograft Model of Non-small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities. Journal of Visualized Experiments: Jove, (207).

Acknowledgements: This work was supported by Seed Grant of JieDu Center of Drug Development, University of the Pacific.

Figure 1. Perturbation of lipid bilayer by a protonation-induced conformational switch[1].

Table 1. Physicochemical properties of liposome formulations loaded with DOX, VNR, or both drugs.

Figure 2. Drug release from liposomes loaded with DOX, VNR, or both drugs at pHs 7.4, and 5.5 for 50 hours.