Synthesis of sp2-Iminosugar Selenoglycolipids as Multitarget Drug Candidates with Antiproliferative, Leishmanicidal and Anti-Inflammatory Properties

sp2-Iminosugar glycolipids (sp2-IGLs) represent a consolidated family of glycoconjugate mimetics encompassing a monosaccharide-like glycone moiety with a pseudoamide-type nitrogen replacing the endocyclic oxygen atom of carbohydrates and an axially-oriented lipid chain anchored at the pseudoanomeric position. The combination of these structural features makes them promising candidates for the treatment of a variety of conditions, spanning from cancer and inflammatory disorders to parasite infections. The exacerbated anomeric effect associated to the putative sp2-hybridized N-atom imparts chemical and enzymatic stability to sp2-IGLs and warrants total α-anomeric stereoselectivity in the key glycoconjugation step. A variety of O-, N-, C- and S-pseudoglycosides, differing in glycone configurational patterns and lipid nature, have been previously prepared and evaluated. Here we expand the chemical space of sp2-IGLs by reporting the synthesis of α-d-gluco-configured analogs with a bicyclic (5N,6O-oxomethylidene)nojirimycin (ONJ) core incorporating selenium at the glycosidic position. Structure–activity relationship studies in three different scenarios, namely cancer, Leishmaniasis and inflammation, convey that the therapeutic potential of the sp2-IGLs is highly dependent, not only on the length of the lipid chain (linear aliphatic C12 vs. C8), but also on the nature of the glycosidic atom (nitrogen vs. sulfur vs. selenium). The ensemble of results highlights the α-dodecylseleno-ONJ-glycoside as a promising multitarget drug candidate.

The Optimal Lipid Chain Length of a Membrane-Permeabilizing Lipopeptide Results From the Balance of Membrane Partitioning and Local Damage

Pseudodesmin A (PSD) is a cyclic lipodepsipeptide produced by Pseudomonas that kills certain bacteria at MIC1/2 in the single micromolar range, probably by permeabilizing their cellular membranes. Synthetic PSD variants, where the native decanoic (C10) acyl chain is varied in length from C4 to C8 and C12 to C14 carbons, were described to be not or less active against a panel of gram-positive strains, as compared to native PSD-C10. Here, we test the membrane-permeabilizing activity of PSD-C4 through PSD-C14 in terms of calcein release from liposomes, which is characterized in detail by the fluorescence-lifetime based leakage assay. Antagonistic concentrations and their chain length dependence agree well for liposome leakage and antimicrobial activity. The optimal chain length is governed by a balance between membrane partitioning (favoring longer chains) and the local perturbation or “damage” inflicted by a membrane-bound molecule (weakening for longer chains). Local perturbation, in turn, may involve at least two modes of action. Asymmetry stress between outer and inner leaflet builds up as the lipopeptides enter the outer leaflet and when it reaches a system-specific stability threshold, it causes a transient membrane failure that allows for the flip of some molecules from the outer to the inner leaflet. This cracking-in may be accompanied by transient, incomplete leakage from the aqueous cores of the liposomes observed, typically, for some seconds or less. The mismatch of the lipopeptide with the lipid leaflet geometry, expressed for example in terms of a spontaneous curvature, has two effects. First, it affects the threshold for transient leakage as described. Second, it controls the rate of equilibrium leakage proceeding as the lipopeptide has reached sufficient local concentrations in both leaflets to form quasi-toroidal defects or pores. Both modes of action, transient and equilibrium leakage, synergize for intermediate chain lengths such as the native, i.e., for PSD-C10. These mechanisms may also account for the reported chain-length dependent specificities of antibiotic action against the target bacteria.

The influence of lipid digestion on the fate of orally administered drug delivery vehicles

This review will focus on orally administered lipid-based drug delivery vehicles and specifically the influence of lipid digestion on the structure of the carrier lipids and their entrained drug cargoes. Digestion of the formulation lipids, which are typically apolar triglycerides, generates amphiphilic monoglycerides and fatty acids that can self-assemble into a diverse array of liquid crystalline structures. Tracking the dynamic changes in self-assembly of the lipid digestion products during digestion has recently been made possible using synchrotron-based small angle X-ray scattering. The influence of lipid chain length and degree of unsaturation on the resulting lipid structuring will be described in the context of the critical packing parameter theory. The chemical and structural transformation of the formulation lipids can also have a dramatic impact on the physical state of drugs co-administered with the formulation. It is often assumed that the best strategy for drug development is to maximise drug solubility in the undigested formulation lipids and to incorporate additives to maintain drug solubility during digestion. However, it is possible to improve drug absorption using lipid digestion in cases where the solubility of the dosed drug or one of its polymorphic forms is greater in the digested lipids. Three different fates for drugs administered with digestible lipid-based formulations will be discussed: (1) where the drug is more soluble in the undigested formulation lipids; (2) where the drug undergoes a polymorphic transformation during lipid digestion; and (3) where the drug is more soluble in the digested formulation lipids.

Skeletal Muscle Cell Growth Alters the Lipid Composition of Extracellular Vesicles

We sought to characterize the lipid profile of skeletal muscle cell-derived Extracellular Vesicles (EVs) to determine if a hypertrophic stimulus would affect the lipid composition of C2C12 myotube-derived EVs. Analyses included C2C12 murine myoblasts differentiated into myotubes and treated with Insulin-Like Growth Factor 1 (IGF-1) for 24 h to induce hypertrophic growth. EVs were isolated from cell culture media, quantified using Nanoparticle Tracking Analysis (NTA) and analyzed using Transmission Electron Microscopy (TEM). EVs were homogenized and lipids extracted for quantification by Mass Spectrometry followed by downstream lipid class enrichment and lipid chain analysis. IGF-1 treatment elicited an increase in CD63 and CD81 levels (39% and 21%) compared to the controls (16%), respectively. Analysis revealed that skeletal muscle-derived EVs are enriched in bioactive lipids that are likely selectively incorporated into EVs during hypertrophic growth. IGF-1 treatment of myotubes had a significant impact on the levels of diacylglycerol (DG) and ceramide (Cer) in secreted EVs. Specifically, the proportion of unsaturated DG was two- to three-fold higher in EVs derived from IGF-treated cells, as compared to those from control cells. The levels of saturated DG were unaffected. Selective increases were similarly seen in C16- and C24-Cer but not in other species. Levels of free sphingoid bases tended to decrease, while those of sphingosine-1-phosphate was unaffected. Our results suggest that the lipid composition and biogenesis of skeletal muscle-derived EVs, are specific and highly selective during hypertrophic growth.

Effects of (R)- and (S)-α-Hydroxylation of Acyl Chains in Sphingosine, Dihydrosphingosine, and Phytosphingosine Ceramides on Phase Behavior and Permeability of Skin Lipid Models

Ceramides (Cers) with α-hydroxylated acyl chains comprise about a third of all extractable skin Cers and are required for permeability barrier homeostasis. We have probed here the effects of Cer hydroxylation on their behavior in lipid models comprising the major SC lipids, Cer/free fatty acids (C 16-C 24)/cholesterol, and a minor component, cholesteryl sulfate. Namely, Cers with (R)-α-hydroxy lignoceroyl chains attached to sphingosine (Cer AS), dihydrosphingosine (Cer AdS), and phytosphingosine (Cer AP) were compared to their unnatural (S)-diastereomers and to Cers with non-hydroxylated lignoceroyl chains attached to sphingosine (Cer NS), dihydrosphingosine (Cer NdS), and phytosphingosine (Cer NP). By comparing several biophysical parameters (lamellar organization by X-ray diffraction, chain order, lateral packing, phase transitions, and lipid mixing by infrared spectroscopy using deuterated lipids) and the permeabilities of these models (water loss and two permeability markers), we conclude that there is no general or common consequence of Cer α-hydroxylation. Instead, we found a rich mix of effects, highly dependent on the sphingoid base chain, configuration at the α-carbon, and permeability marker used. We found that the model membranes with unnatural Cer (S)-AS have fewer orthorhombically packed lipid chains than those based on the (R)-diastereomer. In addition, physiological (R)-configuration decreases the permeability of membranes, with Cer (R)-AdS to theophylline, and increases the lipid chain order in model systems with natural Cer (R)-AP. Thus, each Cer subclass makes a distinct contribution to the structural organization and function of the skin lipid barrier.

The Importance of Free Fatty Chain Length on the Lipid Organization in the Long Periodicity Phase

The skin’s barrier ability is an essential function for terrestrial survival, which is controlled by intercellular lipids within the stratum corneum (SC) layer. In this barrier, free fatty acids (FFAs) are an important lipid class. As seen in inflammatory skin diseases, when the lipid chain length is reduced, a reduction in the barrier’s performance is observed. In this study, we have investigated the contributing effects of various FFA chain lengths on the lamellar phase, lateral packing. The repeat distance of the lamellar phase increased with FFA chain length (C20–C28), while shorter FFAs (C16 to C18) had the opposite behaviour. While the lateral packing was affected, the orthorhombic to hexagonal to fluid phase transitions were not affected by the FFA chain length. Porcine SC lipid composition mimicking model was then used to investigate the proportional effect of shorter FFA C16, up to 50% content of the total FFA mixture. At this level, no difference in the overall lamellar phases and lateral packing was observed, while a significant increase in the water permeability was detected. Our results demonstrate a FFA C16 threshold that must be exceeded before the structure and barrier function of the long periodicity phase (LPP) is affected. These results are important to understand the lipid behaviour in this unique LPP structure as well as for the understanding, treatment, and development of inflammatory skin conditions.

Influence of a Single Ether Bond on Assembly, Orientation, and Miscibility of Phosphocholine Lipids at the Air-Water Interface

How does a small change in the structure of a phospholipid affect its supramolecular assembly? In aqueous suspensions, the substitution of one ester linkage in DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) by an ether linkage alters its phase behaviour completely. To unravel the effect of replacing a phospholipid’s ester linkage by an ether linkage in lipid monolayers, we characterized pure monolayers of the model lipid DPPC and its sn-2 ether analogue PHPC (1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine) as well as mixtures of both by measurements of surface pressure – molecular area (π–A_mol) isotherms. In addition, we used infrared reflection absorption spectroscopy (IRRAS) and epifluorescence microscopy to study lipid condensation, lipid chain orientation, headgroup hydration, and lipid miscibility in all samples.

Influence of a Single Ether Bond on Assembly, Orientation, and Miscibility of Phosphocholine Lipids at the Air-Water Interface

How does a small change in the structure of a phospholipid affect its supramolecular assembly? In aqueous suspensions, the substitution of one ester linkage in DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) by an ether linkage alters its phase behaviour completely. To unravel the effect of replacing a phospholipid’s ester linkage by an ether linkage in lipid monolayers, we characterized pure monolayers of the model lipid DPPC and its sn-2 ether analogue PHPC (1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine) as well as mixtures of both by measurements of surface pressure – molecular area (π–A_mol) isotherms. In addition, we used infrared reflection absorption spectroscopy (IRRAS) and epifluorescence microscopy to study lipid condensation, lipid chain orientation, headgroup hydration, and lipid miscibility in all samples.

Influence of a Single Ether Bond on Assembly, Orientation, and Miscibility of Phosphocholine Lipids at the Air-Water Interface

How does a small change in the structure of a phospholipid affect its supramolecular assembly? In aqueous suspensions, the substitution of one ester linkage in DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) by an ether linkage alters its phase behaviour completely. To unravel the effect of replacing a phospholipid’s ester linkage by an ether linkage in lipid monolayers, we characterized pure monolayers of the model lipid DPPC and its sn-2 ether analogue PHPC (1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine) as well as mixtures of both by measurements of surface pressure – molecular area (π–A_mol) isotherms. In addition, we used infrared reflection absorption spectroscopy (IRRAS) and epifluorescence microscopy to study lipid condensation, lipid chain orientation, headgroup hydration, and lipid miscibility in all samples.