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

Abstract

Insufficient 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.

Synthetic Strategies for Modified Glycosphingolipids and Their Design as Probes

“…734 This method has been applied to the synthesis of alkyne-containing glycolipids. 735 Oleate can be converted to a saturated ω-alkyne fatty acid through bromination/debromination, followed by an acetylene zipper reaction. 736,737 Alkyne-terminated fatty acids have been generated using a convergent method from 1-bromo alkyl chains.…”

“…Saturated ω-alkyne fatty acids were generated through the oxidation of ω-hydroxy fatty acid esters, followed by conversion of the aldehyde to an alkyne with the Bestmann–Ohira reagent . This method has been applied to the synthesis of alkyne-containing glycolipids . Oleate can be converted to a saturated ω-alkyne fatty acid through bromination/debromination, followed by an acetylene zipper reaction. , Alkyne-terminated fatty acids have been generated using a convergent method from 1-bromo alkyl chains …”

Synthetic Glycosphingolipids for Live-Cell Labeling

“…The organic phase was dried with MgSO 4 and evaporated under reduced pressure. 40 was obtained as a colorless solid, which was used without further purification (3.46 (41). Lactoside 40 (2.8 g, 3.93 mmol) was dissolved in 30 mL dry DMF and treated with NaN 3 (2.54 g, 39 mmol).…”

“…This strategy has, for example, been adopted for the study of phospholipids, − sphingolipids, and lipids with a choline headgroup . However, synthetically demanding glycosphingolipids with chemical reporter groups have been reported only rarely . Here, we present the synthesis of azide- and alkyne-tagged glycosphingolipids and their visualization in cell membranes by subsequent click reaction with various fluorescent dyes.…”

Hallmarks of glycogene expression and glycosylation pathways in squamous and adenocarcinoma cervical cancer

“…Glycogenes, or genes associated with glycan synthesis, include those genes implicated in the glycosylation pathways, such as glycosyltransferases and glycosidases, and genes necessary for glycosynthesis, such as sugar-nucleotide synthases, sugar-nucleotide transporters, chaperones, genes necessary for glycolysis (Sachiko & Kiyohiko, 2019;Schjoldager et al, 2020). Several studies support that cancer displays an aberrant expression of those glycogenes implicated in glycosylation (Pinho & Reis, 2015;Schjoldager et al, 2020); and certainly, changes in the expression of glycogenes or cell surface glycans have been related to cancer progression and prognosis (Kannagi et al, 2008;Pinho et al, 2012;Pinho & Reis, 2015;Munkley & Elliott, 2016;Zhuo, Li & Guan, 2018;Schjoldager et al, 2020;Yip, Smollich & Götte, 2006;Liu et al, 2010;Cordeiro Pedrosa et al, 2015;Li et al, 2017;Lin et al, 2018;Salustiano et al, 2020). In this context, in silico models predicting cellular glycosylation are important for addressing the cancer glycome (Schjoldager et al, 2020).…”