Evaluation of Liposomal and Microbubbles Mediated Delivery of Doxorubicin in Two-Dimensional (2D) and Three-Dimensional (3D) Models for Breast Cancer

Abstract

Objective: Liposomal cancer treatment strategies are useful in removing the side effects that were the main concern in recent years. In this study, we prepared microbubble (MBs) conjugated with DOX-loaded liposomes (DOX-loaded MBs) and investigated their effectiveness in in vitro breast cancer cells in two dimensions (2D) and three dimensions (3D).Materials and Methods: With this aim, breast cancer cells with different features (4T1, MDA-MB231, MCF-7) were growth in 2D and 3D dimensions. The cytotoxic and cell death effects under different conditions, durations and doses were evaluated with WST-1, trypan-blue, colony counts. Apoptotic effects were investigated with flow cytometric Annexin-V-PI and immunohistochemical (Ki-67, caspase 3, 8, 9) methods.Results: After free DOX and LipoDOX were applied, the proliferation index of three cell lines reduced. Intrinsic and extrinsic apoptotic pathways were activated in both 2D and 3D models. However, this effect was observed at lower levels in the 3D model due to the difficulty of diffusion of DOX into the spheroids. Additionally, the suitability of the 3D model for breast cancer cells was supported by formation of ductus-like structures and spheroids. Cell deaths were not observed significantly with the DOX-loaded microbubbles due to rising of MBs to the surface and not reaching spheroids held in matrigel of 3D model. Conclusion:DOX and LipoDOX showed anti-proliferative and apoptosis-inducing effects in breast cancer cells. However, these effects indicated variability depending on the cell lines and 2D or 3D model types.

A Microfluidic Platform for Evaluating the Internalization of Liposome Drug Carriers in Tumor Spheroids

“…Based on the dose–response curves, MTS sensitivity to the drug was measured as the area above the curve (AAC), with the calculated AAC values of 53 and 34% for DOX and Doxil, respectively. A higher efficacy of DOX aligned with earlier reports. , …”

“…As a proof of concept, we screened the efficacy of FDA-approved Doxil formulation and compared it with the free drug (DOX). An inferior uptake of the Doxil liposomes by the MTSs aligned with the results of previous studies. ,, Capping of Doxil with poly­(ethylene glycol) hindered their intracellular transport, in agreement with earlier reports; ,, however, Doxil was effective in the delivery of DOX to the MTSs. Notably, the perfusion of Doxil and DOX led to the complete penetration of the MTSs, a phenomenon that improved Doxil efficacy and was not previously observed in static three-dimensional (3D) culture experiments …”

“…A higher efficacy of DOX aligned with earlier reports. 45,46 The relationship between the dose-equivalent of DOX and the number of proliferating cells was analyzed to show MTS response to DOX and its liposomal formulation Doxil (Figure 5d). The proliferation activity of MCF-7 cells in the MTSs cultured in drug-free culture media was taken as 100%.…”

Impact of Doxorubicin on Cell-Substrate Topology

“…Chinese Hamster Ovary (CHO) cells were transfected with a membrane-associated Green Fluorescent Protein (GFP), and the distance between the fluorescent cell membrane and a glass slide, upon which cells were growing as monolayers, was calculated for all pixels from individual images acquired by Variable-Angle Total Internal Reflection Fluorescence Microscopy (VA-TIRFM) [ 17 ]. This technique was used in order to measure early cellular responses caused by doxorubicin within 2–24 h, since conventional tests proved cytotoxicity only after an incubation time t ≥ 24 h for 2D cell cultures [ 18 , 19 , 20 ] and after t = 48–96 h for 3D cultures [ 16 ]. In addition, established cytotoxicity tests require considerably more time for evaluation (up to about 7 days for a colony formation assay [ 21 ]).…”