C8-glycosphingolipids preferentially insert into tumor cell membranes and promote chemotherapeutic drug uptake

AbstractInsufficient drug delivery into tumor cells limits the therapeutic efficacy of chemotherapy. Co-delivery of liposome-encapsulated drug and synthetic short-chain glycosphingolipids (SC-GSLs) significantly improved drug bioavailability by enhancing intracellular drug uptake. Investigating the mechanisms underlying this SC-GSL-mediated drug uptake enhancement is the aim of this study. Fluorescence microscopy was used to visualize the cell membrane lipid transfer intracellular fate of fluorescently labeled C6-NBD-GalCer incorporated in liposomes in tumor and non-tumor cells. Additionally click chemistry was applied to image and quantify native SC-GSLs in tumor and non-tumor cell membranes. SC-GSL-mediated flip-flop was investigated in model membranes to confirm membrane-incorporation of SC-GSL and its effect on membrane remodeling. SC-GSL enriched liposomes containing doxorubicin (Dox) were incubated at 4°C and 37°C and intracellular drug uptake was studied in comparison to standard liposomes and free Dox.SC-GSL transfer to the cell membrane was independent of liposomal uptake and the majority of the transferred lipid remained in the plasma membrane. The transfer of SC-GSL was tumor cell-specific and induced membrane rearrangement as evidenced by a transbilayer flip-flop of pyrene-SM. However, pore formation was measured, as leakage of hydrophilic fluorescent probes was not observed. Moreover, drug uptake appeared to be mediated by SC-GSLs. SC-GSLs enhanced the interaction of doxorubicin (Dox) with the outer leaflet of the plasma membrane of tumor cells at 4°C. Our results demonstrate that SC-GSLs preferentially insert into tumor cell plasma membranes enhancing cell intrinsic capacity to translocate amphiphilic drugs such as Dox across the membrane via a biophysical process

Liposomal Nanomedicine with Short Chain Sphingolipids Modulate Tumor Cell Membrane Permeability Modulate Tumor Cell Membrane Permeability and Improve Chemotherapy

markdownabstract__Abstract__ Chapter 6 discusses the significance of the results described in this thesis and future perspectives. The main goal of the thesis was the application of SCS enriched liposomes to improve chemotherapy outcome, by enhancing drug bioavailability in target tumor cells. Development of SCS (C8-GluCer or C8-GalCer) enriched liposomes containing Dox and MTO was successfully achieved with optimal characteristics from a pharmaceutical point of view, for use in vitro and vivo studies and to advance pharmaceutical development of such formulations toward clinical application, which is currently ongoing for C8-GluCer-Dox-liposomes. In vitro studies revealed that the SCS drug uptake enhancing and cytotoxic properties were displayed preferentially in tumor cells. Mechanistic studies demonstrated that this was related to preferential accumulation of SCS in tumor cell membranes. Intravital microscopic imaging proved that intratumoral bioavailable drug levels for SCS-liposomal treatment exceeded standard liposomal treatment in a MDA MB-231 tumor model. Therapeutic studies in an orthotopic breast cancer model demonstrated improved therapeutic efficacy of C8-GluCer enriched MTO-liposomes compared to standard MTO-liposomes. SCS-enriched liposomal chemotherapy represents a novel attractive drug delivery approach, which combines the benefits of reduced toxicity and improved tumor accumulation of drugs through nanoliposomal encapsulation with enhanced intracellular drug delivery by SCS-mediated tumor cell membrane permeabilization. In these studies we’ve broadened the application using different glycosphingolipids to formulate two chemotherapeutic drugs. Given their proven therapeutic potential, the performed pharmaceutical optimization of formulations will contribute to ultimate near future clinical application. Next to a translational direction, future studies will aim at further broadening of this drug delivery platform to include novel chemotherapeutic drugs, new SCS lipids and the search for optimal SCS – drug combinations for improved drug delivery. A further understanding of the working mechanism, especially in relation to the tumor cell membrane lipid composition will help to find efficient tumor cell-specific drug delivery routes for various chemotherapeutic drugs