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  • br Introduction br Mitochondria are the powerhouse of the


    1. Introduction
    Mitochondria are the powerhouse of the cell and serve as an at-tractive target for cancer treatment. In cellular processes, these orga-nelles provide 3X FLAG Peptide through oxidative phos-phorylation, constitute the main source of reactive oxygen species (ROS), and participate in oxidative signaling, thereby playing a critical role in the rapid proliferation of cancer cells [1–3]. Oncogenic activa-tion leads to increased mitochondrial metabolism and higher mi-tochondrial membrane potential compared to that of non-cancer cells [1,4]. Moreover, multi-drug resistant (MDR) cancer cells exhibit in-creased mitochondrial mass with more polarized mitochondria relative to non-MDR cells [5]. As MDR arises from the overexpression of drug efflux pumps, which requires ATP from mitochondria, mitochondrial targeting is a particularly sensible option for the treatment of drug-resistant cancer cells [1,6,7]. Hence, the more 3X FLAG Peptide polarized mitochondria
    membranes in cancer cells together with the ATP-dependent drug efflux introduces an important target in MDR cancer cells.
    Targeting mitochondria requires penetration through cellular and mitochondrial membranes [8]. Cationic amphiphilic structures are ex-cellent candidates for mitochondrial targeting as they can interact with negatively charged cellular and mitochondrial membranes [8,9,10]. Mitochondrial-penetrating peptides (MPPs) have been designed with alternating amino acids containing delocalized lipophilic cationic side groups, such as arginine, and hydrophobic amino acids, such as cy-clohexylalanine [8]. Primarily used at low concentrations for mi-tochondria imaging, these peptides have been shown to potentiate the activity of chemotherapeutics or reduce the side effects of anti-microbials [8,11]. At a threshold concentration, they inhibit oxidative phosphorylation and electron transport chain, collapse mitochondria and induce apoptosis [8]. Similarly, cationic amphiphilic polymers, such as poly(ethylenimine) (PEI), form pores in mitochondria
    Corresponding author at: Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, ON M5S 3E5, Canada. E-mail address: (M.S. Shoichet).
    Fig. 1. Schematic of the mechanism of cancer cell death following treatment with vitamin E succinate modified octaarginine-octahistidine (VES-H8R8). VES-H8R8 is taken up through electrostatic interac-tion with the plasma membrane and depolarizes the mitochondria through a mitochondrial permeability transition pore (mPTP)-dependent pathway (dotted red line). Consequently, bioenergetics of the mi-tochondria are inhibited, ROS are elevated, both apoptosis and necrosis are induced, and cells are arrested at the G1 phase. VES-H8R8 was shown to decrease the efflux capability of permeation glyco-protein (Pgp). VES represents vitamin E succinate moiety and Str represents a stearyl moiety. (For in-terpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    membranes, causing proton leakage and depolarization, and thereby induce apoptosis [12]. Interestingly, lipid-modified cationic peptides, composed of a cationic cell penetrating peptide and a fatty acid chain, closely resemble MPPs in structural properties; yet, their anti-mi-tochondrial activity remain understudied and poorly understood. This knowledge gap prompted us to investigate the activity of lipid-modified cationic peptides as mitochondria-targeting drugs for cancer treatment. We hypothesized that such lipid-modified cationic peptides could se-lectively target and depolarize the mitochondria of MDR breast cancer cells (Fig. 1).
    Using the octahistidine-octaarginine (H8R8) peptide as a common cationic cell penetrating peptide with endosomal escape capabilities, we investigated, for the first time, the intracellular fate and effects of lipid-modified cationic peptides in breast cancer cells [13,14]. First, we studied the anticancer activity of H8R8-based amphiphiles in relation to hydrophobicity and degradability. Comparing a biologically inert hy-drophobic stearyl chain-modified H8R8 to the bioactive MDR sensitizer, vitamin E succinate (VES)-modified peptide, we investigated cancer cell toxicity and cancer selectivity relative to healthy cells [15,16]. We explored the intracellular effects of H8R8-based cationic lipids and evaluated mitochondrial inhibition, induction of ROS accumulation, apoptosis, necrosis, and cell cycle arrest. VES-H8R8 was also in-vestigated for inhibition of permeation glycoprotein (Pgp) efflux in MDR breast cancer cells. Herein, we highlight H8R8-based amphiphiles, and specifically vitamin E-based prodrugs for MDR breast cancer cell targeting.