Graphical Abstract

Elesclomol (ELC) is an anticancer drug inducing mitochondria cytotoxicity through reactive oxygen species. Here, for the first time, we encapsulate the poorly water soluble ELC in monoolein-based cubosomes stabilized with Pluronic F127. Cellular uptake and nanocarrier accumulation close to the mitochondria with sub-micrometer distance is identified via three-dimensional (3D) confocal microscopy and edge-to-edge compartment analysis. To monitor the therapeutic effect of the ELC nanocarrier, we apply for the first time, label-free time-lapse multi-photon fluorescence lifetime imaging microscopy (MP-FLIM) to track NAD(P)H cofactors with sub-cellular resolution on live cells exposed to an anticancer nanocarrier. Improved in vitro cytotoxicity is verified when loading the pre-complexed ELC with copper (ELC-Cu). Importantly, for equivalent copper concentration, cubosomes loaded with ELC-Cu show higher cytotoxicity compared to the free drug. The novel nanocarrier shows promising features for systemic ELC-Cu administration, and furthermore we establish the MP-FLIM technique for the assessment of anticancer drug delivery systems.
Weinberg, S. E.; Chandel, N. S. Targeting mitochondria metabolism for cancer therapy. Nat. Chem. Biol. 2015, 11, 9-15.
Fulda, S.; Galluzzi, L.; Kroemer, G. Targeting mitochondria for cancer therapy. Nat. Rev. Drug Discov. 2010, 9, 447-464.
Raza, M. H.; Siraj, S.; Arshad, A.; Waheed, U.; Aldakheel, F.; Alduraywish, S.; Arshad, M. ROS-modulated therapeutic approaches in cancer treatment. J. Cancer Res. Clin. Oncol. 2017, 143, 1789-1809.
Modica-Napolitano, J. S.; Weissig, V. Treatment strategies that enhance the efficacy and selectivity of mitochondria-targeted anticancer agents. Int. J. Mol. Sci. 2015, 16, 17394-17421.
Trachootham, D.; Alexandre, J.; Huang, P. Targeting cancer cells by ros-mediated mechanisms: A radical therapeutic approach? Nat. Rev. Drug Discov. 2009, 8, 579-591.
Berkenblit, A.; Eder, J. P. Jr.; Ryan, D. P.; Seiden, M. V.; Tatsuta, N.; Sherman, M. L.; Dahl, T. A.; Dezube, B. J.; Supko, J. G. Phase I clinical trial of STA-4783 in combination with paclitaxel in patients with refractory solid tumors. Clin. Cancer Res. 2007, 13, 584-590.
O'Day, S.; Gonzalez, R.; Lawson, D.; Weber, R.; Hutchins, L.; Anderson, C.; Haddad, J.; Kong, S.; Williams, A.; Jacobson, E. Phase Ⅱ, randomized, controlled, double-blinded trial of weekly elesclomol plus paclitaxel versus paclitaxel alone for stage IV metastatic melanoma. J. Clin. Oncol. 2009, 27, 5452-5458.
Hedley, D.; Shamas-Din, A.; Chow, S.; Sanfelice, D.; Schuh, A. C.; Brandwein, J. M.; Seftel, M. D.; Gupta, V.; Yee, K. W. L.; Schimmer, A. D. A phase I study of elesclomol sodium in patients with acute myeloid leukemia. Leuk. Lymphoma 2016, 57, 2437-2440.
Nagai, M.; Vo, N. H.; Shin Ogawa, L.; Chimmanamada, D.; Inoue, T.; Chu, J.; Beaudette-Zlatanova, B. C.; Lu, R. Z.; Blackman, R. K.; Barsoum, J. et al. The oncology drug elesclomol selectively transports copper to the mitochondria to induce oxidative stress in cancer cells. Free Radic. Biol. Med. 2012, 52, 2142-2150.
Blackman, R. K.; Cheung-Ong, K.; Gebbia, M.; Proia, D. A.; He, S. Q.; Kepros, J.; Jonneaux, A.; Marchetti, P.; Kluza, J.; Rao, P. E. et al. Mitochondrial electron transport is the cellular target of the oncology drug elesclomol. PLoS One 2012, 7, e29798.
Kirshner, J. R.; He, S. Q.; Balasubramanyam, V.; Kepros, J.; Yang, C. Y.; Zhang, M.; Du, Z. J.; Barsoum, J.; Bertin, J. Elesclomol induces cancer cell apoptosis through oxidative stress. Mol. Cancer Ther. 2008, 7, 2319-2327.
Hasinoff, B. B.; Wu, X.; Yadav, A. A.; Patel, D.; Zhang, H.; Wang, D. S.; Chen, Z. S.; Yalowich, J. C. Cellular mechanisms of the cytotoxicity of the anticancer drug elesclomol and its complex with Cu(Ⅱ). Biochem. Pharmacol. 2015, 93, 266-276.
Hasinoff, B. B.; Yadav, A. A.; Patel, D.; Wu, X. The cytotoxicity of the anticancer drug elesclomol is due to oxidative stress indirectly mediated through its complex with Cu(Ⅱ). J. Inorg. Biochem. 2014, 137, 22-30.
Yadav, A. A.; Patel, D.; Wu, X.; Hasinoff, B. B. Molecular mechanisms of the biological activity of the anticancer drug elesclomol and its complexes with Cu(Ⅱ), Ni(Ⅱ) and Pt(Ⅱ). J. Inorg. Biochem. 2013, 126, 1-6.
Karami, Z.; Hamidi, M. Cubosomes: Remarkable drug delivery potential. Drug Discov. Today 2016, 21, 789-801.
Allen, T. M.; Cullis, P. R. Liposomal drug delivery systems: From concept to clinical applications. Adv. Drug Deliv. Rev. 2013, 65, 36-48.
Larsson, K. Aqueous dispersions of cubic lipid-water phases. Curr. Opin. Colloid Interface Sci. 2000, 5, 64-69.
Demurtas, D.; Guichard, P.; Martiel, I.; Mezzenga, R.; Hébert, C.; Sagalowicz, L. Direct visualization of dispersed lipid bicontinuous cubic phases by cryo-electron tomography. Nat. Commun. 2015, 6, 8915.
Seddon, J. M.; Templer, R. H. Cubic phases of self-assembled amphiphilic aggregates. Philos. Trans. Roy. Soc. A Math. Phys. Eng. Sci. 1993, 344, 377-401.
Oliveira, A. C. N.; Raemdonck, K.; Martens, T.; Rombouts, K.; Simón-Vázquez, R.; Botelho, C.; Lopes, I.; Lúcio, M.; González-Fernández, Á.; Real Oliveira, M. E. C. D. et al. Stealth monoolein-based nanocarriers for delivery of siRNA to cancer cells. Acta Biomater. 2015, 25, 216-229.
Spicer, P. T.; Hayden, K. L.; Lynch, M. L.; Ofori-Boateng, A.; Burns, J. L. Novel process for producing cubic liquid crystalline nanoparticles (cubosomes). Langmuir 2001, 17, 5748-5756.
Kulkarni, C. V.; Wachter, W.; Iglesias-Salto, G.; Engelskirchen, S.; Ahualli, S. Monoolein: A magic lipid? Phys. Chem. Chem. Phys. 2011, 13, 3004-3021.
Yaghmur, A.; Glatter, O. Characterization and potential applications of nanostructured aqueous dispersions. Adv. Colloid Interface Sci. 2009, 147-148, 333-342.
Zhai, J. L.; Tran, N.; Sarkar, S.; Fong, C.; Mulet, X.; Drummond, C. J. Self-assembled lyotropic liquid crystalline phase behavior of monoolein-capric acid-phospholipid nanoparticulate systems. Langmuir 2017, 33, 2571-2580.
Azmi, I. D.; Moghimi, S. M.; Yaghmur, A. Cubosomes and hexosomes as versatile platforms for drug delivery. Ther. Deliv. 2015, 6, 1347-1364.
Rizwan, S. B.; Assmus, D.; Boehnke, A.; Hanley, T.; Boyd, B. J.; Rades, T.; Hook, S. Preparation of phytantriol cubosomes by solvent precursor dilution for the delivery of protein vaccines. Eur. J. Pharm. Biopharm. 2011, 79, 15-22.
Nazaruk, E.; Majkowska-Pilip, A.; Bilewicz, R. Lipidic cubic-phase nanoparticles—Cubosomes for efficient drug delivery to cancer cells. Chempluschem 2017, 82, 570-575.
Mat Azmi, I. D.; Nilsson, C.; Stürup, S.; Østergaard, J.; Gammelgaard, B.; Moghimi, S. M.; Urtti, A. Characterization of cisplatin-loaded cubosomes and hexosomes: Effect of mixing with human plasma. J. Geriatr. Oncol. 2013, 4, S62.
Nasr, M.; Ghorab, M. K.; Abdelazem, A. In vitro and in vivo evaluation of cubosomes containing 5-fluorouracil for liver targeting. Acta Pharm. Sin. B 2015, 5, 79-88.
Maulucci, G.; Bačić, G.; Bridal, L.; Schmidt, H. H. H. W.; Tavitian, B.; Viel, T.; Utsumi, H.; Yalçın, A. S.; De Spirito, M. Imaging reactive oxygen species-induced modifications in living systems. Antioxid. Redox Signal. 2016, 24, 939-958.
Blacker, T. S.; Duchen, M. R. Investigating mitochondrial redox state using nadh and NADPH autofluorescence. Free Radic. Biol. Med. 2016, 100, 53-65.
Wang, Z. W.; Zheng, Y. P.; Zhao, D. Q.; Zhao, Z. W.; Liu, L. X.; Pliss, A.; Zhu, F. Q.; Liu, J.; Qu, J. L.; Luan, P. Applications of fluorescence lifetime imaging in clinical medicine. J. Innov. Opt. Health Sci. 2018, 11, 1830001.
Kolenc, O. I.; Quinn, K. P. Evaluating cell metabolism through autofluorescence imaging of NAD(P)H and FAD. Antioxid. Redox Signal. , in press, https://doi.org/10.1089/ars.2017.7451.
Blacker, T. S.; Mann, Z. F.; Gale, J. E.; Ziegler, M.; Bain, A. J.; Szabadkai, G.; Duchen, M. R. Separating NADH and NADPH fluorescence in live cells and tissues using FLIM. Nat. Commun. 2014, 5, 3936.
Lakowicz, J. R.; Szmacinski, H.; Nowaczyk, K.; Johnson, M. L. Fluorescence lifetime imaging of free and protein-bound NADH. Proc. Natl. Acad. Sci. USA 1992, 89, 1271-1275.
Alexiev, U.; Volz, P.; Boreham, A.; Brodwolf, R. Time-resolved fluorescence microscopy (FLIM) as an analytical tool in skin nanomedicine. Eur. J. Pharm. Biopharm. 2017, 116, 111-124.
Lin, L. L.; Grice, J. E.; Butler, M. K.; Zvyagin, A. V.; Becker, W.; Robertson, T. A.; Soyer, H. P.; Roberts, M. S.; Prow, T. W. Time-correlated single photon counting for simultaneous monitoring of zinc oxide nanoparticles and NAD(P)H in intact and barrier-disrupted volunteer skin. Pharm. Res. 2011, 28, 2920-2930.
Walsh, A. J.; Cook, R. S.; Sanders, M. E.; Aurisicchio, L.; Ciliberto, G.; Arteaga, C. L.; Skala, M. C. Quantitative optical imaging of primary tumor organoid metabolism predicts drug response in breast cancer. Cancer Res. 2014, 74, 5184-5194.
Shirmanova, M. V.; Druzhkova, I. N.; Lukina, M. M.; Dudenkova, V. V.; Ignatova, N. I.; Snopova, L. B.; Shcheslavskiy, V. I.; Belousov, V. V.; Zagaynova, E. V. Chemotherapy with cisplatin: Insights into intracellular pH and metabolic landscape of cancer cells in vitro and in vivo. Sci. Rep. 2017, 7, 8911.
Alam, S. R.; Wallrabe, H.; Svindrych, Z.; Chaudhary, A. K.; Christopher, K. G.; Chandra, D.; Periasamy, A. Investigation of mitochondrial metabolic response to doxorubicin in prostate cancer cells: An NADH, FAD and tryptophan FLIM assay. Sci. Rep. 2017, 7, 10451.
Shah, A. T.; Diggins, K. E.; Walsh, A. J.; Irish, J. M.; Skala, M. C. In vivo autofluorescence imaging of tumor heterogeneity in response to treatment. Neoplasia 2015, 17, 862-870.
Tilley, A. J.; Drummond, C. J.; Boyd, B. J. Disposition and association of the steric stabilizer Pluronic® F127 in lyotropic liquid crystalline nano-structured particle dispersions. J. Colloid Interface Sci. 2013, 392, 288-296.
Honary, S.; Zahir, F. Effect of zeta potential on the properties of nano-drug delivery systems—A review (part 2). Trop. J. Pharm. Res. 2013, 12, 265-273.
Yingchoncharoen, P.; Kalinowski, D. S.; Richardson, D. R. Lipid-based drug delivery systems in cancer therapy: What is available and what is yet to come. Pharmacol. Rev. 2016, 68, 701-787.
Choi, K. Y.; Silvestre, O. F.; Huang, X. L.; Min, K. H.; Howard, G. P.; Hida, N.; Jin, A. J.; Carvajal, N.; Lee, S. W.; Hong, J. I. et al. Versatile RNA interference nanoplatform for systemic delivery of RNAs. ACS Nano 2014, 8, 4559-4570.
Rampersad, S. N. Multiple applications of alamar blue as an indicator of metabolic function and cellular health in cell viability bioassays. Sensors 2012, 12, 12347-12360.
Hinton, T. M.; Grusche, F.; Acharya, D.; Shukla, R.; Bansal, V.; Waddington, L. J.; Monaghan, P.; Muir, B. W. Bicontinuous cubic phase nanoparticle lipid chemistry affects toxicity in cultured cells. Toxicol. Res. 2014, 3, 11-22.
Tran, N.; Mulet, X.; Hawley, A. M.; Hinton, T. M.; Mudie, S. T.; Muir, B. W.; Giakoumatos, E. C.; Waddington, L. J.; Kirby, N. M.; Drummond, C. J. Nanostructure and cytotoxicity of self-assembled monoolein-capric acid lyotropic liquid crystalline nanoparticles. RSC Adv. 2015, 5, 26785-26795.
Zhai, J. L.; Suryadinata, R.; Luan, B.; Tran, N.; Hinton, T. M.; Ratcliffe, J.; Hao, X. J.; Drummond, C. J. Amphiphilic brush polymers produced using the RAFT polymerisation method stabilise and reduce the cell cytotoxicity of lipid lyotropic liquid crystalline nanoparticles. Faraday Discuss. 2016, 191, 545-563.
Zhang, S. L.; Gao, H. J.; Bao, G. Physical principles of nanoparticle cellular endocytosis. ACS Nano 2015, 9, 8655-8671.
Liu, Y.; Workalemahu, B.; Jiang, X. Y. The effects of physicochemical properties of nanomaterials on their cellular uptake in vitro and in vivo. Small 2017, 13, 1701815.
Gilles, J. F.; Dos Santos, M.; Boudier, T.; Bolte, S.; Heck, N. DiAna, an ImageJ tool for object-based 3D co-localization and distance analysis. Methods 2017, 115, 55-64.
Rosa, A.; Murgia, S.; Putzu, D.; Meli, V.; Falchi, A. M. Monoolein-based cubosomes affect lipid profile in HeLa cells. Chem. Phys. Lipids 2015, 191, 96-105.
Rambold, A. S.; Cohen, S.; Lippincott-Schwartz, J. Fatty acid trafficking in starved cells: Regulation by lipid droplet lipolysis, autophagy, and mitochondrial fusion dynamics. Dev. Cell 2015, 32, 678-692.
Beloribi-Djefaflia, S.; Vasseur, S.; Guillaumond, F. Lipid metabolic reprogramming in cancer cells. Oncogenesis 2016, 5, e189.
Falchi, A. M.; Rosa, A.; Atzeri, A.; Incani, A.; Lampis, S.; Meli, V.; Caltagirone, C.; Murgia, S. Effects of monoolein-based cubosome formulations on lipid droplets and mitochondria of HeLa cells. Toxicol. Res. 2015, 4, 1025-1036.
Ying, W. H. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: Regulation and biological consequences. Antioxid. Redox Signal. 2008, 10, 179-206.
Vinogradov, A. D.; Grivennikova, V. G. Oxidation of NADH and ROS production by respiratory complex I. Biochim. Biophys. Acta Bioenerg. 2016, 1857, 863-871.
Chaderjian, W. B.; Chin, E. T.; Harris, R. J.; Etcheverry, T. M. Effect of copper sulfate on performance of a serum-free CHO cell culture process and the level of free thiol in the recombinant antibody expressed. Biotechnol. Prog. 2005, 21, 550-553.
Lin, M.; Wang, D. D.; Liu, S. W.; Huang, T. T.; Sun, B.; Cui, Y.; Zhang, D. Q.; Sun, H. C.; Zhang, H.; Sun, H. et al. Cupreous complex-loaded chitosan nanoparticles for photothermal therapy and chemotherapy of oral epithelial carcinoma. ACS Appl. Mater. Interfaces 2015, 7, 20801-20812.
Wellcome Sanger Institute. Genomics of Drug Sensitivity in Cancer[Online]. http://www.cancerrxgene.org/translation/Drug/1031 (accessed Dec 6, 2017)
Enderlein, J.; Erdmann, R. Fast fitting of multi-exponential decay curves. Opt. Commun. 1997, 134, 371-378.