(T1230-01-01) Exploring Rifamycin Cytotoxic Potential in 2D and 3D Cancer Cell Models Using Liposomes as Delivery System

Purpose: Drug repurposing is a way to explore novel approaches for diseases treatment, including cancer. Rifamycin, a well-known antibiotic, inhibits RNA synthesis and probably can be explored as cytotoxic agent [1-3]. Thus, our goal was to investigate rifamycin cytotoxic activity towards cancer cell lines in monolayers and spheroids, using liposomes as delivery system.

Methods: Liposomes were produced by thin-film evaporation method using phosphatidylcholine, cholesterol and DOPE-PEG_5k (Figure 1). Non-encapsulated drug was removed using dialysis bags and encapsulated drug was directly determined by UV spectrophotometer. Liposomes were characterized regarding size and surface charge by dynamic light scattering, and stability at 4°C and 25°C. Formulation biocompatibility was evaluated by hemolysis assay. Finally, cytotoxicity of rifamycin SV and liposomes was evaluated in U87, SKOV3-TR and A549 cell lines using monolayer and spheroid culture [4].

Results: Unloaded and rifamycin-loaded liposomes showed size smaller than 100 nm and polydispersity around 0.2, with slightly negative zeta potential. Rifamycin drug loading in liposomes was 3mg of drug/10mg of lipid films. Both formulations were stable for at least 21 days when stored at 4°C, keeping above 60% of drug encapsulated. In the concentrations tested, liposomes formulation (unloaded and loaded) and free rifamycin were biocompatible and non-hemolytic. Blank liposomes were not cytotoxic in tested cell lines. Free rifamycin and rifamycin-loaded liposomes demonstrated cytotoxic activity towards tumor cell lines tested, without significant differences in 2D model (Figure 2). In spheroids model, after 24 h incubation the same behavior as monolayers was observed, however, after 48 h incubation, loaded liposomes presented higher cytotoxicity than free drug (Figure 3).

Conclusion: Rifamycin was successfully encapsulated in liposomes, leading to stable, biocompatible, and non-hemolytic formulation. The liposomal rifamycin exhibited cytotoxicity against cancer cells comparable to that of the free drug in both monolayer cell cultures and spheroid models. However, after 48 h incubation, loaded liposomes presented higher cytotoxicity, showing a better tumor permeability than free drug. Liposomes could be further functionalized with antibodies for targeted effect.

References: [1] Gao, Y., et al., Antibiotics for cancer treatment: A double-edged sword. Journal of Cancer, 2020. 11(17): p. 5135-5149.
[2] Courtois, A., et al., Inhibition of multidrug resistance-associated protein (MRP) activity by rifampicin in human multidrug-resistant lung tumor cells. Cancer Letters, 1999. 139(1): p. 97-104.
[3] Floss, H.G. and T.-W. Yu, Rifamycin: Mode of Action, Resistance, and Biosynthesis. Chemical Reviews, 2005. 105(2): p. 621-632.
[4] Mendes, L.P., et al., Surface-engineered polyethyleneimine-modified liposomes as novel carrier of siRNA and chemotherapeutics for combination treatment of drug-resistant cancers. Drug delivery, 2019. 26(1): p. 443-458.

Acknowledgements: Authors do not have any conflict of interest to disclaimer.
Authors acknowledge the financial support from Sao Paulo Research Foundation (FAPESP process number 2018/06475-4, 2021/12452-0, 2022/06228-2).


Figure 1. Graphical representation of methods employed in this study.


Figure 2. Cell viability study of unloaded liposomes, free rifamycin solution and rifamycin-loaded liposomes on 2D model of U87, SKOV-3 and A549 at 24h after treatments. Results expressed as average ± standard deviation, n=4, ***p < 0.001.


Figure 3. Cell viability study of unloaded liposomes, free rifamycin solution and rifamycin-loaded liposomes on 3D model of U87, SKOV-3 and A549 at 24h and 48h after treatments. Results expressed as average ± standard deviation, n=4, *p < 0.05, **p < 0.01, ***p < 0.001.