br Bioactive VES modified cationic peptides enhance their
3.2. Bioactive VES-modified cationic peptides enhance their retention in breast cancer cells
To investigate mitochondrial targeting of the conjugated peptides, EMT6/AR-1 ZVADFMK were incubated with fluorescein-labeled Str-H8R8 and VES-H8R8. Within 3 h of incubation in full medium, membrane asso-ciation and cell penetration were evident. The fluorescence from both Str-H8R8 and VES-H8R8 indicate colocalization with the Mitotracker dye, which stains the mitochondria (Fig. S4A, B) . Similarly to lipid-modified octaarginines, the cell uptake of both Str-H8R8 and VES-H8R8 are expected through the endolysosomal pathway . The re-sultant bright and punctate signals, suggestive of endolysosomes, make it difficult to visualize peptide uptake into the mitochondria. Therefore, we quantified the peptide uptake in isolated mitochondria from peptide treated EMT6/AR-1 cells. Intact cells were incubated with either fluorescein-labeled Str-H8R8,VES-H8R8, H8R8, or free fluorescein for 3 h, and mitochondria were isolated through differential centrifugation . The concentration of fluorescein in the isolated mitochondria was quantified and both Str-H8R8 and VES-H8R8 treated cells exhibited significantly higher uptake relative to H8R8 and free fluorescein (p < .001, Fig. 3A). Relative to Str-H8R8, VES-H8R8 treated cells ex-hibited significantly higher peptide uptake in the mitochondrial, pos-sibly due to efflux inhibition on the plasma membrane and enhanced mitochondrial targeting (p < .05) . Importantly, both Str-H8R8 and VES-H8R8 treated cells exhibited significantly higher peptide up-take in the mitochondria relative to H8R8, suggesting that the lipid modification of H8R8 is crucial for mitochondrial targeting (p < .001). Lipid-modified cationic peptides exhibit increased cell uptake due to enhanced membrane association and translocation, and hence in-creased mitochondrial uptake . Therefore, we investigated the uptake of fluorescein-labeled Str-H8R8,VES-H8R8, H8R8, and free fluor-escein in isolated mitochondria from untreated EMT6/AR-1 cells for 1 h. Isolated mitochondria treated with either Str-H8R8 or VES-H8R8 exhibited significantly higher peptide uptake relative to H8R8, Journal of Controlled Release 305 (2019) 210–219
confirming that lipid modification is required for mitochondrial uptake (p < .001, Fig. 3B). Interestingly, isolated mitochondria treated with VES-H8R8 exhibited significantly higher peptide uptake relative to Str-H8R8 (p < .001). VES is more hydrophobic than stearyl, which may be beneficial in penetrating the hydrophobic inner mitochondrial mem-brane . Doxorubicin-resistant breast cancer cells have been shown to express efflux pumps on the mitochondrial membranes, such as breast cancer resistance protein (BCRP) and multi-drug resistance protein (MRP1) . Relative to Str-H8R8, VES-H8R8 may also inhibit efflux pumps found on the mitochondrial membranes.
To investigate the role of VES-H8R8 on efflux pump inhibition , we compared the uptake and retention of fluorescein-labeled VES-H8R8 to that of Str-H8R8 with MDR breast cancer EMT6/AR-1 cells by flow cytometry. Interestingly, cell uptake was similar after 2 h of incubation; however, cells treated with VES-H8R8 showed significantly more fluorescein uptake at 5 and 24 h compared to those treated with Str-H8R8 (p < .001, Fig. 3C). To evaluate long-term retention of the pep-tides in the EMT6/AR-1 cells, the peptide-containing medium was re-moved after 24 h, and cells were left incubating for an additional 24 h in fresh medium. In these conditions, VES-H8R8 led to a ~8-fold higher retention in cells relative to that of Str-H8R8. Given that both amphi-philes should exhibit similar proteolytic stability, the increased uptake and retention observed for VES-H8R8 likely results from specific Pgp efflux inhibition. This observation is consistent with other studies where both vitamin E and VES have been shown to inhibit Pgp efflux in cancer cells . Furthermore, PEGylated-VES is an established Pgp efflux inhibitor that has higher anti-cancer activity and greater efflux inhibition than VES alone . Co-incubating VES and fluorescein-Str-H8R8 with EMT6/AR-1 cells led to a 12-fold increase in retention at 48 h vs. fluorescein-Str-H8R8 alone, further confirming the role of VES in Pgp efflux inhibition (Fig. S5). Thus, VES-H8R8 is an attractive amphiphile towards MDR treatment and would be more efficacious with the fluorescein replaced with a chemotherapeutic, such as doxorubicin, which would otherwise be effluxed by Pgp .
3.3. Lipid-modified cationic peptides affect mitochondria polarization and bioenergetics
We compared the effect of H8R8-based amphiphiles on mitochondria depolarization, using the JC-1 probe, to a series of controls, including the positive control of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), which showed the lowest mitochondrial membrane potential (Fig. 4A). EMT6/AR-1 cells treated with H8R8-based amphiphiles showed significantly reduced mitochondrial membrane potential re-lative to all the controls (i.e., VES, stearic acid, unmodified peptide (H8R8), PEG-H8R8 and DMSO). Similar results were obtained with the parental cell line (Fig. S6A). These results suggest that mitochondria depolarization is involved in the cytotoxic mechanism of action of lipid-modified cationic peptides.