Light-driven proton pumps, such as proteorhodopsin, have been proposed as an energy source in the field of synthetic biology. Energy is required to power biochemical reactions within artificially created reaction compartments like proto-or nanocells, which are typically based on either lipid or polymer membranes. The insertion of membrane proteins into these membranes is delicate and quantitative studies comparing these two systems are needed. Here we present a detailed analysis of the formation of proteoliposomes and proteopolymersomes and the requirements for a successful reconstitution of the membrane protein proteorhodopsin. To this end, we apply design of experiments to provide a mathematical framework for the reconstitution process. Mathematical optimization identifies suitable reconstitution conditions for lipid and polymer membranes and the obtained data fits well to the predictions. Altogether, our approach provides experimental and modeling evidence for different reconstitution mechanisms depending on the membrane type which resulted in a surprisingly similar performance.

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There is an increasing interest in using the zebrafish (Danio rerio) larva as a vertebrate screening model to study drug disposition. Since the pronephric kidney of zebrafish larvae shares high similarity with the anatomy of nephrons in higher vertebrates including humans, we explored in the present study whether 3 to 4 days old zebrafish larvae have a fully functional pronephron. Intravenous injection of fluorescent polyethylene glycol and dextran derivatives of different molecular weight revealed a cut-off of 4.4 to 7.6 nm in hydrodynamic diameter for passive glomerular filtration, which is in agreement with corresponding values in rodents and humans. Distal tubular reabsorption of a FITC-folate conjugate, covalently modified with PEG2000, via the folate receptor 1 was shown. Transport experiments of fluorescent substrates were assessed in the presence and absence of specific inhibitors in the blood systems. Thereby, functional expression in the proximal tubule of oat/slc22, mrp1/abcc1, mrp2/abcc2, mrp4/abcc4 and the zebrafish larvaes' p-glycoprotein analog abcb4 was shown. In addition, non-renal clearance of fluorescent substrates and plasmaprotein binding characteristics were assessed in vivo. Results of transporter studies were confirmed by extrapolation to ex vivo experiments in killifish (Fundulus heteroclitus) proximal kidney tubules. We conclude that the zebrafish larva has a fully functional pronephron at 96 hours post fertilization and is therefore an attractive translational vertebrate screening model to bridge the gap between cell culture-based test systems and pharmacokinetic experiments in higher vertebrates.

Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. Here, we describe a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods. nPMVs are produced by homogenizing giant plasma membrane vesicles following cell membrane blebbing and apoptotic body secretion induced by chemical stressors. nPMVs showed no significant differences compared to native EVs from the same cell line in cryo-TEM analysis, in vitro cellular interactions, and in vivo biodistribution studies in zebrafish larvae. Proteomics and lipidomics, on the other hand, suggested substantial differences consistent with the divergent origin of these two EV types and indicated that nPMVs primarily derive from apoptotic extracellular vesicles. nPMVs may provide an attractive source for developing EV-based pharmaceutical therapeutics.

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