In vitro two-dimensional (2D) and three-dimensional (3D) evaluation of the effect of doxorubicin-loaded microbubbles on breast cancer

Aim: Chemotherapeutics used in breast cancer have systemic side effects. In recent years, targeted treatment strategies have been investigated in order to eliminate or reduce these side effects. Mainly it is aimed to target ultrasound contrast agent micro-bubbles to breast cancer cells by loading doxorubicin (DOX) which is used in the treatment of breast cancer. In our previous studies, the ultrasound contrast agent was bound to the microbubbles were produced; DOX, a chemotherapeutic drug, was encapsulated in liposomes in the liposomes of DOX, and this structure was attached to microbubbles. In this study, two - dimensional (2D) and three - dimensional (3D) effects of DOX-loaded microbubbles and liposomal doxorubicin were investigated in in vitro breast cancer cell lines. Method: In this study, the effects of DOX, LipoDOX and DOX-loaded microbubbles on breast cancer were investigated in in vitro two and three dimensional levels. For this purpose, breast cancer cell lines of different characteristics (4T1, MDA-MB231, MCF-7) were produced in two and three dimensions (matrijel-based). The cytotoxic effects of different doses and duration were evaluated by WST-1, trypan blue, colony forming assay, Annexin V-PI, and immunohistochemistry (Ki-67, caspase 3,8,9). The results obtained were statistically compared with nonparametric tests at p <0.05 level. Results: After the DOX administration, both in the 2D and 3D models proliferation index were decreased and both intrinsic and extrinsic apoptotic pathways were activated in all three breast cancer cell lines. However, these effects were lower in the 3D model due to the insufficient diffusion of DOX into spheroids. However, 3D modeling was found suitable for breast cancer cell culture model as supported by formation of ductal structures and spheroids. Microbubble experiments could not be continued due to the fact that the floading of microbubbles in 2D and remaining above the matrigel as a result of their big sizes in 3D. Conclusion: DOX shows anti-proliferative and apoptosis inducing effects in breast cancer cell culture. But this effect vary among different characterized cell lines and also according to 2D and 3D modeling type. Therefore, 3D spheroid modeling is found to be important especially for drug efficacy studies. Keywords: 3D cell culture, breast cancer, microbubbles, drug delivery