Liposome-Mediated Delivery of Isolated Mitochondria to Mitochondria-Depleted (ρ⁰) Cancer Cells: A Nanotechnology-Based Strategy for Mitochondrial Restoration

Abstract

Mitochondrion is a vital organelle, which regulates cellular metabolism and apoptosis and has a primary role in energy production (Wallace, 2012). In cancer cell models, particularly pharmacologically induced Rho0 (rho-zero) cells, mitochondrial DNA (mtDNA) undergoes depletion, which causes vulnerability on glycolysis and disruption of oxidative phosphorylation (OXPHOS) (King & Attardi, 1996). Such cell models are used to study mitochondrial dysfunction, drug sensitivity and restoration of mitochondrial activity. Recent advances in mitochondrial transplantation such as direct mitochondrial injection, tunneling nanotube-mediated transfer, and cell-penetrating peptides represent potential of mitochondrial dysfunction restoration in depleted cells (Cruz-Gregorio et al., 2023). However, these methods remain highly invasive. Liposome-based or nanocarrier-mediated delivery, in contrast, can be considered as a non-invasive, scalable, and targeted alternative for transplantation of mitochondria into recipient cells (Sanchez-Aranguren et al., 2025; Kim et al., 2023). This project aims to design, optimize a liposome-based nanocarrier system to encapsulate isolated, functional mitochondria into liposomes into Rho0 cancer cells to restore mitochondrial function and OXPHOS. Another aim is to compare the efficiency of MitoCeption - transfer mitochondria between cells as a form of intercellular communication with liposome-based delivery (Caicedo et al., 2015). The first phase focused on MitoCeption, where A549 cells undergo depletion through CCCP and EtBr treatment, which triggers mitophagy, selective degradation of mitochondria. Isolated mitochondria from the adipose-derived mesenchymal stem cells (ADMSCs) and then transferred into recipient Rho0 cancer cell lines. Successful mitochondrial transfer and functional incorporation was confirmed by qPCR mitochondrial DNA copy number (mtDNA_CN) 4 quantification, flow cytometry and confocal imaging. Using donor stem cells indicated how their mitochondrial identity affects cancer metabolism, either proliferating it or restraining malignancy. The second phase focused on liposome-based delivery where liposomes were prepared using POPC, DOTAP and Lissamine Rhodamine B. Encapsulation of isolated mitochondria and liposomes were characterized by DLS, zeta potential, and electron microscopy. A549 cells underwent the same depletion through CCCP and EtBr treatment and then were incubated with liposome-mitochondria complexes. The efficiency of delivery was analysed with MitoTracker dyes, confocal microscopy, and flow cytometry as listed previously. This research with outcomes of successful delivery and functional integration of mitochondria into depleted cells establishes foundational applications for mitochondrial replacement therapy for cancer models using stem cells.