title: Simultaneous active intracellular delivery of doxorubicin and C6-ceramide shifts the additive/antagonistic drug interaction of non-encapsulated combination.
abstract: Drug resistance remains the Achilles tendon undermining the success of chemotherapy. It has been recognized that success requires the identification of compounds that, when combined, lead to synergistic tumor inhibition while simultaneously minimizing systemic toxicity. However, in vivo application of such protocols is dependent on the ability to deliver the appropriate drug ratio at the tumor level. In this respect, nanotechnology-based delivery platforms, like liposomes, offer an elegant solution for the in vivo translation of such strategy. In this work, we propose the active intracellular delivery of combinations of doxorubicin and the pro-apoptotic sphingolipid, C6-ceramide, using our previously described cytosolic triggered release-enabling liposomes, targeting nucleolin with the F3 peptide. Combination of doxorubicin (DXR):C6-ceramide (C6-Cer) at 1:2 molar ratio interacted synergistically against drug resistant/triple negative MDA-MB-231 breast cancer cells, as well as drug sensitive MDA-MB-435S melanoma cells. Cell viability studies indicated that F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) performed similarly as targeted liposomal DXR (p[F3]SL), encapsulating twice the amount of DXR, at the IC50, for an incubation time of 24 h. Importantly, F3-targeted liposomes encapsulating DXR:C6-Cer 1:2 molar ratio (p[F3]DC12) enabled a cell death above 90% at 24 h of treatment against both DXR-resistant and sensitive cells, unattainable by the F3-targeted liposomal doxorubicin. Furthermore, a F3-targeted formulation encapsulating a mildly additive/antagonistic DXR:C6-Cer 1:1 molar ratio (p[F3]DC11) enabled an effect above 90% for an incubation period as short as 4 h, suggesting that the delivery route at the cell level may shift the nature of drug interaction. Such activity, including the one for p[F3]DC12, induced a marked cell and nucleus swelling at similar extent, consistent with necrotic cell death. Overall, these results demonstrated that F3-targeted intracellular delivery of different DXR/C6-Cer ratios, with diversed drug interactions, enabled a highly relevant increased efficacy against chemotherapy resistant cells.
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal; FFUC-Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. Electronic address: nuno.a.c.fonseca@gmail.com.
authors.forename: Nuno A
authors.initials: NA
authors.lastname: Fonseca
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal. Electronic address: ligia.cgs@gmail.com.
authors.forename: Lígia C
authors.initials: LC
authors.lastname: Gomes-da-Silva
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal; TREAT U, SA, Parque Industrial de Taveiro, Lote 44, 3045-508 Coimbra, Portugal. Electronic address: vera.moura@treatu.pt.
authors.forename: Vera
authors.initials: V
authors.lastname: Moura
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal; FFUC-Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. Electronic address: ssimoes@ci.uc.pt.
authors.forename: Sérgio
authors.initials: S
authors.lastname: Simões
authors.affiliation: CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Largo Marquês de Pombal, 3004-517 Coimbra, Portugal; FFUC-Faculty of Pharmacy, University of Coimbra, Pólo das Ciências da Saúde, Azinhaga de Santa Comba, 3000-548 Coimbra, Portugal. Electronic address: jmoreira@ff.uc.pt.
authors.forename: João Nuno
authors.initials: JN
authors.lastname: Moreira
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage* / Q000737:chemistry; D045744:Cell Line, Tumor; D002518:Ceramides / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical; D004317:Doxorubicin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000737:chemistry; D016503:Drug Delivery Systems; D004347:Drug Interactions; D019008:Drug Resistance, Neoplasm; D004357:Drug Synergism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry; D011092:Polyethylene Glycols / Q000008:administration & dosage / Q000737:chemistry
chemical_list: D000903:Antibiotics, Antineoplastic; D002518:Ceramides; D008081:Liposomes; C506643:liposomal doxorubicin; C101954:N-caproylsphingosine; D011092:Polyethylene Glycols; D004317:Doxorubicin
keywords: C6-ceramide; Combinatorial ratiometric drug design; F3-targeted delivery; Nanoparticles; Nucleolin