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Screening of natural products found in the marine environment is a highly competitive field where pharmaceutical companies have their interests. Systematic understanding of an isolated bio-interface such as plasma membrane vesicle when other complex biochemical processes within the cells are excluded, may be a prerequisite for many future innovations. Unique to photosynthetic marine microalga Dunaliella is the absence of a rigid cell wall. Dunaliella possesses the glycocalyx type of cell envelope and therefore, softness and deformability will be harnessed in the isolation of its plasma membrane by osmotic stress. Isolated plasma membrane vesicle could mimics a biological membrane since it possesses a natural complex assembly of proteins, sugars, and a mixture of lipids, which is a step up from the commonly used model system such as a phospholipid vesicle. Different imaging techniques have been employed in order to examine structural characterization of vesicle and to image the efficiency of intracellular release. We compared qualitatively molecular transport of fluorescent dye through the membrane systems of different level of complexities (plasma membrane vs. model lipid membrane). The observed differences in transport are ascribed to differences in membranes’ structural features and physicochemical properties.

From the biotechnological perspective, it is of interest to explore the hidden potentials of bio-available algal cell products and in such a way to link the ocean and human health. The purpose of our study is to compare qualitatively molecular transport of fluorescent dyes through membrane systems of different level of complexities (algal plasma membrane vs. model phospholipid membrane). Plasma membrane vesicles were reconstructed from the native membrane material released upon rupturing algal cells by osmotic stress. Reconstructed plasma membranes retain the natural heterogeneous composition of proteins, lipids and carbohydrates, which is a step up from simple, artificial phospholipid vesicles. Reconstructed vesicles are of sufficient size to be observable under a microscope, and their membrane curvature and tension are close to natural systems. Structural characterization of reconstructed vesicles shows a thick envelope with nearly empty vesicle interior. The envelope contains surface globular structures which may be derived from surface coat proteins. Confocal imaging shows that plasma membrane vesicles are auto-fluorescent due to the chlorophyll pigments within the membrane. Physico-chemical characterization of plasma membrane vesicles in terms of adhesion behavior at the interface shows that vesicles are soft and easily deformable, which is in line with the low stiffness of a few kPa measured for algal cells. Transport of fluorescent dyes into the plasma membrane vesicle or into giant unilamellar vesicles shows striking differences in process spontaneity, probably due to membrane structural features and physicochemical properties. Naturally reconstructed plasma membrane models appear highly relevant and convenient to probe membrane-related processes, since complex biochemical processes within the cells are eliminated. This study may contribute to the design of bio-inspired carriers for advanced biotechnological applications. Acknowledgment This work is supported by the Croatian Science Foundation Project No IP-2018-01-5840. PTV is supported by United States Air Force Office of Scientific Research (AFOSR) grant FA9550-14-1- 0023 (a collaborative effort with FA9550-14-1- 0018) and by AFOSR MURI grant FA9550-15-1-0517 on "Nanoelectropulse- Induced Electromechanical Signaling and Control of Biological Systems", administered through Old Dominion University.

In aqueous media, where a majority of biochemical processes occur, continuum electrostatic models describe externally applied electric fields that are uniform and well behaved far from the point of origin. However, perturbations at charged aqueous interfaces caused by the interaction of ions, organic molecules, vesicles, and cells significantly affect nearby electrochemical phenomena. Experiments have attempted to quantify interfacial interactions by measuring charge displacement current during nonspecific vesicle adhesion that could be mediated by specific interactions of positively charged phospholipid choline head groups with negatively charged electrodes. Experiments cannot directly validate these interactions at nanometer length scales ; therefore, we have run united-atom molecular dynamics simulations to observe how zwitterionic and anionic lipids, in addition to model hydrocarbons, change their conformations and binding kinetics in the vicinity of charged interfaces. This enables direct comparisons between the binding affinities measured in experiment to those obtained in simulations, while elucidating the interfacial interactions that facilitate lipid vesicle adhesion at charged interfaces. Moreover, the implementation of a charged interface in molecular dynamics simulations provides an advantageous alternative for the generation of large electric fields across phospholipid bilayers, especially in the presence of periodic boundary conditions, and may be useful for simulations of membrane electropermeabilization.