title: Enhanced Cytotoxicity of Folic Acid-Targeted Liposomes Co-Loaded with C6 Ceramide and Doxorubicin: In Vitro Evaluation on HeLa, A2780-ADR, and H69-AR Cells.
abstract: Current research in cancer therapy is beginning to shift toward the use of combinational drug treatment regimens. However, the efficient delivery of drug combinations is governed by a number of complex factors in the clinical setting. Therefore, the ability to synchronize the pharmacokinetics of the individual therapeutic agents present in combination not only to allow for simultaneous tumor accumulation but also to allow for a synergistic relationship at the intracellular level could prove to be advantageous. In this work, we report the development of a novel folic acid-targeted liposomal formulation simultaneously co-loaded with C6 ceramide and doxorubicin [FA-(C6+Dox)-LP]. In vitro cytotoxicity assays showed that the FA-(C6+Dox)-LP was able to significantly reduce the IC50 of Dox when compared to that after the treatment with the doxorubicin-loaded liposomes (Dox-LP) as well as the untargeted drug co-loaded (C6+Dox)-LP on HeLa, A2780-ADR, and H69-AR cells. The analysis of the cell cycle distribution showed that while the C6 liposomes (C6-LP) did not cause cell cycle arrest, all the Dox-containing liposomes mediated cell cycle arrest in HeLa cells in the G2 phase at Dox concentrations of 0.3 and 1 μM and in the S phase at the higher concentrations. It was also found that this arrest in the S phase precedes the progression of the cells to apoptosis. The targeted FA-(C6+Dox)-LP were able to significantly enhance the induction of apoptotic events in HeLa cell monolayers as compared to the other treatment groups. Next, using time-lapse phase holographic imaging microscopy, it was found that upon treatment with the FA-(C6+Dox)-LP, the HeLa cells underwent rapid progression to apoptosis after 21 h as evidenced by a drastic drop in the average area of the cells after loss of cell membrane integrity. Finally, upon evaluation in a HeLa spheroid cell model, treatment with the FA-(C6+Dox)-LP showed significantly higher levels of cell death compared to those with C6-LP and Dox-LP. Overall, this study clearly shows that the co-delivery of C6 ceramide and Dox using a liposomal platform significantly correlates with an antiproliferative effect due to cell cycle regulation and subsequent induction of apoptosis and thus warrants its further evaluation in preclinical animal models.
authors.affiliation: Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University , Boston, Massachusetts 02115, United States.
authors.forename: Shravan Kumar
authors.initials: SK
authors.lastname: Sriraman
authors.affiliation: Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University , Boston, Massachusetts 02115, United States.
authors.forename: Jiayi
authors.initials: J
authors.lastname: Pan
authors.affiliation: Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University , Boston, Massachusetts 02115, United States.
authors.forename: Can
authors.initials: C
authors.lastname: Sarisozen
authors.affiliation: Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University , Boston, Massachusetts 02115, United States.
authors.forename: Ed
authors.initials: E
authors.lastname: Luther
authors.affiliation: Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University , Boston, Massachusetts 02115, United States.|Department of Biochemistry, Faculty of Science, King Abdulaziz University , Jeddah, Saudi Arabia.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Torchilin
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000737:chemistry / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D002453:Cell Cycle / Q000187:drug effects; D002518:Ceramides / Q000737:chemistry*; D004317:Doxorubicin / Q000737:chemistry / Q000494:pharmacology*; D004337:Drug Carriers; D016503:Drug Delivery Systems; D005260:Female; D005492:Folic Acid / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D008081:Liposomes / Q000008:administration & dosage* / Q000737:chemistry; D008175:Lung Neoplasms / Q000188:drug therapy / Q000473:pathology; D010051:Ovarian Neoplasms / Q000188:drug therapy / Q000473:pathology; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000188:drug therapy / Q000473:pathology
chemical_list: D000903:Antibiotics, Antineoplastic; D002518:Ceramides; D004337:Drug Carriers; D008081:Liposomes; D004317:Doxorubicin; D005492:Folic Acid
keywords: A2780-ADR; C6 ceramide; H69-AR; HeLa; apoptosis; cancer; cancer cell spheroids; cell cycle arrest; combination drug therapy; doxorubicin; drug resistance; folic acid targeting; imaging cytometry; liposomes; nanomedicine; nanoparticles; phase holographic imaging; receptor targeting