Doxorubicin (DOX) is an anthracyline drug first extracted from Streptomyces peucetius var. caesius in the 1970’s and used in clinical as a broad spectrum anti-cancer drug to treat several cancers. It intercalates between base pairs in the DNA helix and inhibit topoisomerase II, preventing DNA replication. However, cardio toxicity and renal damage limited its application. Thus, how to accumulate the doxorubicin at the tumor site becomes a critical step of better therapeutic outcome. We have previously developed a strategy to render saturated and PEGylated liposomes pH-sensitive: a protonation-induced conformational flip of lipid tails, using trans-2-aminocyclohexanol moiety (TACH) as a molecular trigger (Fig. 1). Such pH-sensitive liposomes are called fliposomes. The traditional two-dimensional (2D) monolayer cell cultures lack certain structure and intercellular interaction, and therefore do not sufficiently represent the biological phenotype. To overcome this challenge, three-dimensional (3D) cell cultures have been developed, including multicellular spheroids (MCS). MCS are 20-1000 μm diameter clusters of cells that self-aggregate when cultured in low attached surface of flask or plates. 3D MCS have been reported to generate heterogeneity and to preserve the physiological microenvironment of tumor, such as lowered pH.
In this study, we aim to 1) establish 3D MCS of cancer cells as an in vitro model of solid tumors and 2) to evaluate the anticancer activity of fliposomes on 3D MCS.
Select fliposomes and control liposomes were prepared by thin film hydration, freeze-annealing -thawing and polycarbonate membrane extrusion. Then doxorubicin (DOX) was remotely loaded into the liposomes by transmembrane manganese sulfate gradient followed by removing unloaded DOX with ion exchange resin. Encapsulation efficiency, mean size diameter and ζ-potential were characterized.
3D multicellular spheroids (MCS) were established to mimic solid tumors containing acidic interstitium. MDA-MB-231 cells were seeded with/without 1% collagen into ultra-low attachment microplate for 15 days before treatment. To evaluate anticancer activity, free DOX solution, DOX-loaded fliposomes, and DOX-loaded control liposomes were tested in both monolayer cell cultures and 3D MCS. The concentration required to inhibit the cell viability by 50% (IC50) were obtained for monolayer model. Inhibition of 3D MCS growth was observed under microscope and measured every other day.
By controlling the time of thawing above transition temperature (Tm), various fliposomes and control liposome containing DOX were successfully constructed with appropriate encapsulation efficiency (>90%), mean particle size (<120 nm), and neutral ζ-potential. Compact 3D MCS of consistent size (~300 µm) were obtained with collagen I bovine, while loose, invasive 3D MCS (600 µm with spread edges) were generated in the absence of collagen. In monolayer cells, IC50 of free DOX solution, fliposomes and control liposomes were similar (1.11µM, 1.35 µM and 1.68 µM, respectively) after 72 hours. In contrast, DOX-loaded fliposome showed better anticancer activity on 3D MCS by reducing the size (Figure 1), disrupting the invasive edge (Figure 2), and loosening the dense core (Figure 2) after 6 days of treatment.
DOX-loaded fliposomes exhibited enhanced anticancer activity on 3D MCS in vitro, which is more representative of solid tumors. The anticancer activity of fliposome in vivo is currently under investigation.
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Yifan Lu, Shen Zhao and Xin Guo. "Conformational Switch-based pH-sensitive Liposomes Enhance the Anticancer Activity of Doxorubicin on 3D Multicellular Spheroids" (2017)
Available at: http://works.bepress.com/xin-guo/6/