Phospholipid tail asymmetry allows cellular adaptation to anoxic environments.

Membrane biophysical properties are critical to cell fitness and depend on unsaturated phospholipid acyl tails. These can only be produced in aerobic environments since eukaryotic desaturases require molecular oxygen. This raises the question of how cells maintain bilayer properties in anoxic environments. Using advanced microscopy, molecular dynamics simulations, and lipidomics by mass spectrometry we demonstrated the existence of an alternative pathway to regulate membrane fluidity that exploits phospholipid acyl tail length asymmetry, replacing unsaturated species in the membrane lipidome. We show that the fission yeast, Schizosaccharomyces japonicus, which can grow in aerobic and anaerobic conditions, is capable of utilizing this strategy, whereas its sister species, the well-known model organism Schizosaccharomyces pombe, cannot. The incorporation of asymmetric-tailed phospholipids might be a general adaptation to hypoxic environmental niches.

Drug reformulation for a neglected disease. The NANOHAT project to develop a safer more effective sleeping sickness drug.

BACKGROUND: Human African trypanosomiasis (HAT or sleeping sickness) is caused by the parasite Trypanosoma brucei sspp. The disease has two stages, a haemolymphatic stage after the bite of an infected tsetse fly, followed by a central nervous system stage where the parasite penetrates the brain, causing death if untreated. Treatment is stage-specific, due to the blood-brain barrier, with less toxic drugs such as pentamidine used to treat stage 1. The objective of our research programme was to develop an intravenous formulation of pentamidine which increases CNS exposure by some 10-100 fold, leading to efficacy against a model of stage 2 HAT. This target candidate profile is in line with drugs for neglected diseases inititative recommendations. METHODOLOGY: To do this, we evaluated the physicochemical and structural characteristics of formulations of pentamidine with Pluronic micelles (triblock-copolymers of polyethylene-oxide and polypropylene oxide), selected candidates for efficacy and toxicity evaluation in vitro, quantified pentamidine CNS delivery of a sub-set of formulations in vitro and in vivo, and progressed one pentamidine-Pluronic formulation for further evaluation using an in vivo single dose brain penetration study. PRINCIPAL FINDINGS: Screening pentamidine against 40 CNS targets did not reveal any major neurotoxicity concerns, however, pentamidine had a high affinity for the imidazoline2 receptor. The reduction in insulin secretion in MIN6 ß-cells by pentamidine may be secondary to pentamidine-mediated activation of ß-cell imidazoline receptors and impairment of cell viability. Pluronic F68 (0.01%w/v)-pentamidine formulation had a similar inhibitory effect on insulin secretion as pentamidine alone and an additive trypanocidal effect in vitro. However, all Pluronics tested (P85, P105 and F68) did not significantly enhance brain exposure of pentamidine. SIGNIFICANCE: These results are relevant to further developing block-copolymers as nanocarriers, improving BBB drug penetration and understanding the side effects of pentamidine.

The delivered dose: Applying particokinetics to in vitro investigations of nanoparticle internalization by macrophages.

PURPOSE: The aim of this study was to investigate the impact of nanoparticle dosimetry on the interpretation of results from in vitro experiments involving particle-cell interactions. Three different dose metrics were evaluated: 1) The administered dose (particle mass, number or surface area administered per volume media at the onset of an experiment), 2) the delivered dose (particle mass, number or surface area to reach the cell monolayer via diffusion and sedimentation over the duration of an experiment) and 3) the cellular dose (particle mass, number or surface area internalized by the cells during the experiment). The In Vitro Sedimentation and Diffusion and Dosimetry model (ISDD) was used to calculate particle sedimentation and diffusion in cell culture media to predict delivered dose values. These were compared with administered doses and experimentally determined cellular dose values. METHODS: Dosing conditions and predicted delivered dose values were computed in silico using ISDD. In vitro cell association experiments were performed by exposing fluorescently labelled polystyrene beads of 50, 100, 200, 700 and 1000nm diameter to J774A.1 macrophage-like cells and determining the internalized particle content (cellular dose) via fluorescence spectroscopy. Experiments were repeated using lipopolysachharide (LPS) to activate and cytochalasin D to inhibit phagocytosis. RESULTS: Only a small fraction (0.03-0.33%) of the administered dose was able to interact with the cells for all particle sizes tested. Measured cellular doses in non-activated J774A.1 cells corresponded well with computed delivered dose values for all particle sizes tested under six different exposure conditions. When cellular doses were averaged and normalized to their corresponding delivered doses, the percentage values of cell-associated particles were: 36 ± 10%(50 nm), 15 ± 3%(100 nm), 22 ± 6%(200 nm), 18 ± 4%(700 nm), and 42 ± 19%(1000 nm). Activation of J774A.1 cells with LPS significantly increased the cellular dose (normalized to the delivered dose) in all particle sizes except 50 nm, while cytochalasin D treatment significantly reduced the cellular dose of 100, 200 and 1000 nm particles. CONCLUSIONS: This study demonstrates that dose correction using the ISDD model (i.e. normalization of cellular dose values to the delivered dose) is essential for accurate interpretation of results derived from in vitro particle-cell interaction studies (e.g. particle uptake, cytotoxicity, mechanisms of action, pharmacodynamic studies, etc.). It is of particular relevance to the field of particulate drug delivery systems, because the low density nature of most biomaterials used as drug carriers will result in very low fractions of the administered particle dose reaching the cell monolayer under most commonly used experimental conditions.