Polysaccharide functionalization reduces lipid vesicle stiffness.

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.

Encapsulated bacteria deform lipid vesicles into flagellated swimmers.

We study a synthetic system of motile Escherichia coli bacteria encapsulated inside giant lipid vesicles. Forces exerted by the bacteria on the inner side of the membrane are sufficient to extrude membrane tubes filled with one or several bacteria. We show that a physical coupling between the membrane tube and the flagella of the enclosed cells transforms the tube into an effective helical flagellum propelling the vesicle. We develop a simple theoretical model to estimate the propulsive force from the speed of the vesicles and demonstrate the good efficiency of this coupling mechanism. Together, these results point to design principles for conferring motility to synthetic cells.

Modulating Lipid Membrane Morphology by Dynamic DNA Origami Networks.

Membrane morphology and its dynamic adaptation regulate many cellular functions, which are often mediated by membrane proteins. Advances in DNA nanotechnology have enabled the realization of various protein-inspired structures and functions with precise control at the nanometer level, suggesting a viable tool to artificially engineer membrane morphology. In this work, we demonstrate a DNA origami cross (DOC) structure that can be anchored onto giant unilamellar vesicles (GUVs) and subsequently polymerized into micrometer-scale reconfigurable one-dimensional (1D) chains or two-dimensional (2D) lattices. Such DNA origami-based networks can be switched between left-handed (LH) and right-handed (RH) conformations by DNA fuels and exhibit potent efficacy in remodeling the membrane curvatures of GUVs. This work sheds light on designing hierarchically assembled dynamic DNA systems for the programmable modulation of synthetic cells for useful applications.

DNA Origami Vesicle Sensors with Triggered Single-Molecule Cargo Transfer.

Interacting with living systems typically involves the ability to address lipid membranes of cellular systems. The first step of interaction of a nanorobot with a cell will thus be the detection of binding to a lipid membrane. Utilizing DNA origami, we engineered a biosensor with single-molecule Fluorescence Resonance Energy Transfer (smFRET) as transduction mechanism for precise lipid vesicle detection and cargo delivery. The system hinges on a hydrophobic ATTO647N modified single-stranded DNA (ssDNA) leash, protruding from a DNA origami nanostructure. In a vesicle-free environment, the ssDNA coils, ensuring high FRET efficiency. Upon vesicle binding to cholesterol anchors on the DNA origami, hydrophobic ATTO647N induces the ssDNA to stretch towards the lipid bilayer, reducing FRET efficiency. As the next step, the sensing strand serves as molecular cargo that can be transferred to the vesicle through a triggered strand displacement reaction. Depending on the number of cholesterols on the displacer strands, we either induce a diffusive release of the fluorescent load towards neighboring vesicles or a stoichiometric release of a single cargo-unit to the vesicle on the nanosensor. Ultimately, our multi-functional liposome interaction and detection platform opens up pathways for innovative biosensing applications and controllable stoichiometric loading of vesicles with single-molecule control.

Anionic lipid vesicles have differential effects on the aggregation of early onset-associated α-synuclein missense mutants.

α-synuclein (αS) is the key component of synucleinopathies such as Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. αS was first linked to PD through the identification of point mutations in the SNCA gene, causing single amino acid substitutions within αS and familial autosomal dominant forms of PD that profoundly accelerated disease onset by up to several decades. At least eight single-point mutations linked to familial PD (A30G/P, E46K, H50Q, G51D, and A53T/E/V) are located in proximity of the region preceding the non-ß amyloid component (preNAC) region, strongly implicating its pathogenic role in αS-mediated cytotoxicity. Furthermore, lipids are known to be important for native αS function, where they play a key role in the regulation of synaptic vesicle docking to presynaptic membranes and dopamine transmission. However, the role of lipids in the function of mutant αS is unclear. Here, we studied αS aggregation properties of WT αS and five of the most predominant single-point missense mutants associated with early onset PD in the presence of anionic 1,2-dimyristoyl-sn-glycero-3-phospho-l-serine lipid vesicles. Our results highlight significant differences between aggregation rates, the number of aggregates produced, and overall fibril morphologies of WT αS and the A30P, E46K, H50Q, G51D, and A53T missense mutants in the presence of lipid vesicles. These findings have important implications regarding the interplay between the lipids required for αS function and the individual point mutations known to accelerate PD and related diseases.

A penetratin-derived peptide reduces the membrane permeabilization and cell toxicity of α-synuclein oligomers.

Parkinson's disease is a neurodegenerative movement disorder associated with the intracellular aggregation of α-synuclein (α-syn). Cytotoxicity is mainly associated with the oligomeric species (αSOs) formed at early stages in α-syn aggregation. Consequently, there is an intense focus on the discovery of novel inhibitors such as peptides to inhibit oligomer formation and toxicity. Here, using peptide arrays, we identified nine peptides with high specificity and affinity for αSOs. Of these, peptides p194, p235, and p249 diverted α-syn aggregation from fibrils to amorphous aggregates with reduced ß-structures and increased random coil content. However, they did not reduce αSO cytotoxicity and permeabilization of large anionic unilamellar vesicles. In parallel, we identified a non-self-aggregating peptide (p216), derived from the cell-penetrating peptide penetratin, which showed 12-fold higher binding affinity to αSOs than to α-syn monomers (Kdapp 2.7 and 31.2 µM, respectively). p216 reduced αSOs-induced large anionic unilamellar vesicle membrane permeability at 10-1 to 10-3 mg/ml by almost 100%, was not toxic to SH-SY5Y cells, and reduced αSOs cytotoxicity by about 20%. We conclude that p216 is a promising starting point from which to develop peptides targeting toxic αSOs in Parkinson's disease.

A DNA Segregation Module for Synthetic Cells.

The bottom-up construction of an artificial cell requires the realization of synthetic cell division. Significant progress has been made toward reliable compartment division, yet mechanisms to segregate the DNA-encoded informational content are still in their infancy. Herein, droplets of DNA Y-motifs are formed by liquid-liquid phase separation. DNA droplet segregation is obtained by cleaving the linking component between two populations of DNA Y-motifs. In addition to enzymatic cleavage, photolabile sites are introduced for spatio-temporally controlled DNA segregation in bulk as well as in cell-sized water-in-oil droplets and giant unilamellar lipid vesicles (GUVs). Notably, the segregation process is slower in confinement than in bulk. The ionic strength of the solution and the nucleobase sequences are employed to regulate the segregation dynamics. The experimental results are corroborated in a lattice-based theoretical model which mimics the interactions between the DNA Y-motif populations. Altogether, engineered DNA droplets, reconstituted in GUVs, can represent a strategy toward a DNA segregation module within bottom-up assembled synthetic cells.

Impact of ergosterol content on acetic and lactic acids toxicity to Saccharomyces cerevisiae.

Organic acid stress often represents a major hurdle in industrial bio-based microbial processes. Organic acids can be released from lignocellulosic feedstocks pretreatment and can also be desirable products obtained by microbial fermentation with applications in different industrial sectors. Yeasts are prominent cell factories. However, the presence of organic acids can compromise yeast metabolism, impairing fermentation performances and limiting the economic feasibility of the processes. Plasma membrane remodeling is deeply involved in yeast tolerance to organic acids, but the detailed mechanisms and potentials of this phenomenon remain largely to be studied and exploited. We investigated the impact of ergosterol on Saccharomyces cerevisiae tolerance against organic acid stress by coupling in vitro and in vivo assays. In the in vitro assay, synthetic lipid vesicles were prepared containing different concentrations of ergosterol. We observed changes in organic acids diffusion through the membrane as a function of ergosterol content. Then, we extended our approach in vivo, engineering S. cerevisiae with the aim of changing the ergosterol content of cells. We focused on ECM22, an important transcription factor, involved in the regulation of ergosterol biosynthesis. The overexpression of ECM22 was sufficient to increase ergosterol levels in S. cerevisiae, resulting in an enhanced tolerance toward lactic acid stress. In this work we propose an in vitro approach, using synthetic lipid vesicles, as a complementary method to be used when studying the impact of the plasma membrane lipid composition on the diffusion of organic acids.

Differential Effects of Lipid Bilayers on αPSM Peptide Functional Amyloid Formation.

Phenol-soluble modulins (PSMs) are key virulence factors of S. aureus, and they comprise the structural scaffold of biofilm as they self-assemble into functional amyloids. They have been shown to interact with cell membranes as they display toxicity towards human cells through cell lysis, with αPSM3 being the most cytotoxic. In addition to causing cell lysis in mammalian cells, PSMs have also been shown to interact with bacterial cell membranes through antimicrobial effects. Here, we present a study on the effects of lipid bilayers on the aggregation mechanism of αPSM using chemical kinetics to study the effects of lipid vesicles on the aggregation kinetics and using circular dichroism (CD) spectroscopy, Fourier-transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM) to investigate the corresponding secondary structure of the aggregates. We found that the effects of lipid bilayers on αPSM aggregation were not homogeneous between lipid type and αPSM peptides, although none of the lipids caused changes in the dominating aggregation mechanism. In the case of αPSM3, all types of lipids slowed down aggregation to a varying degree, with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) having the most pronounced effect. For αPSM1, lipids had opposite effects, where DOPC decelerated aggregation and lipopolysaccharide (LPS) accelerated the aggregation, while 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG) had no effect. For αPSM4, both DOPG and LPS accelerated the aggregation, but only at high concentration, while DOPC showed no effect. None of the lipids was capable of inducing aggregation of αPSM2. Our data reveal a complex interaction pattern between PSMs peptides and lipid bilayers that causes changes in the aggregation kinetics by affecting different kinetic parameters along with only subtle changes in morphology.

Function Investigations and Applications of Membrane Proteins on Artificial Lipid Membranes.

Membrane proteins play an important role in key cellular functions, such as signal transduction, apoptosis, and metabolism. Therefore, structural and functional studies of these proteins are essential in fields such as fundamental biology, medical science, pharmacology, biotechnology, and bioengineering. However, observing the precise elemental reactions and structures of membrane proteins is difficult, despite their functioning through interactions with various biomolecules in living cells. To investigate these properties, methodologies have been developed to study the functions of membrane proteins that have been purified from biological cells. In this paper, we introduce various methods for creating liposomes or lipid vesicles, from conventional to recent approaches, as well as techniques for reconstituting membrane proteins into artificial membranes. We also cover the different types of artificial membranes that can be used to observe the functions of reconstituted membrane proteins, including their structure, number of transmembrane domains, and functional type. Finally, we discuss the reconstitution of membrane proteins using a cell-free synthesis system and the reconstitution and function of multiple membrane proteins.

Synthetic Membraneless Droplets for Synaptic-Like Clustering of Lipid Vesicles.

In eukaryotic cells, the membraneless organelles (MLOs) formed via liquid-liquid phase separation (LLPS) are found to interact intimately with membranous organelles (MOs). One major mode is the clustering of MOs by MLOs, such as the formation of clusters of synaptic vesicles at nerve terminals mediated by the synapsin-rich MLOs. Aqueous droplets, including complex coacervates and aqueous two-phase systems, have been plausible MLO-mimics to emulate or elucidate biological processes. However, neither of them can cluster lipid vesicles (LVs) like MLOs. In this work, we develop a synthetic droplet assembled from a combination of two different interactions underlying the formation of these two droplets, namely, associative and segregative interactions, which we call segregative-associative (SA) droplets. The SA droplets cluster and disperse LVs recapitulating the key functional features of synapsin condensates, which can be attributed to the weak electrostatic interaction environment provided by SA droplets. This work suggests LLPS with combined segregative and associative interactions as a possible route for synaptic clustering of lipid vesicles and highlights SA droplets as plausible MLO-mimics and models for studying and mimicking related cellular dynamics.

Exploring fast proton transfer events associated with lateral proton diffusion on the surface of membranes.

Proton diffusion (PD) across biological membranes is a fundamental process in many biological systems, and much experimental and theoretical effort has been employed for deciphering it. Here, we report on a spectroscopic probe, which can be tightly tethered to the membrane, for following fast (nanosecond) proton transfer events on the surface of membranes. Our probe is composed of a photoacid that serves as our light-induced proton source for the initiation of the PD process. We use our probe to follow PD, and its pH dependence, on the surface of lipid vesicles composed of a zwitterionic headgroup, a negative headgroup, a headgroup that is composed only from the negative phosphate group, or a positive headgroup without the phosphate group. We reveal that the PD kinetic parameters are highly sensitive to the nature of the lipid headgroup, ranging from a fast lateral diffusion at some membranes to the escape of protons from surface to bulk (and vice versa) at others. By referring to existing theoretical models for membrane PD, we found that while some of our results confirm the quasi-equilibrium model, other results are in line with the nonequilibrium model.

Diffusion of Lipid Nanovesicles Bound to a Lipid Membrane Is Associated with the Partial-Slip Boundary Condition.

During diffusion of nanoparticles bound to a cellular membrane by ligand-receptor pairs, the distance to the laterally mobile interface is sufficiently short for their motion to depend not only on the membrane-mediated diffusivity of the tethers but also in a not yet fully understood manner on nanoparticle size and interfacial hydrodynamics. By quantifying diffusivity, velocity, and size of individual membrane-bound liposomes subjected to a hydrodynamic shear flow, we have successfully separated the diffusivity contributions from particle size and number of tethers. The obtained diffusion-size relations for synthetic and extracellular lipid vesicles are not well-described by the conventional no-slip boundary condition, suggesting partial slip as well as a significant diffusivity dependence on the distance to the lipid bilayer. These insights, extending the understanding of diffusion of biological nanoparticles at lipid bilayers, are of relevance for processes such as cellular uptake of viruses and lipid nanoparticles or labeling of cell-membrane-residing molecules.

The Use of Chitosan-Coated Nanovesicles in Repairing Alcohol-Induced Damage of Liver Cells in Mice.

Background and Objectives In the past few decades, the studies concerning the natural polysaccharide chitosan have been centered on a new direction: its hepatoprotective action. The aim of our study was to evaluate the influence of previously designed chitosan lipid vesicles on the liver damage induced by alcohol consumption in mice. Materials and Methods The study involved the oral administration of substances in one daily dose as follows: Group 1 (control): water; Group 2 (control alcohol): 5% alcohol in water; Group 3 (CHIT): 0.1 mL/10 g body weight chitosan solution in animals treated with alcohol; Group 4 (CHIT-ves): 0.1 mL/10 g body chitosan vesicles in animals treated with alcohol; Group 5 (AcA): 200 mg/kg body ascorbic acid in animals treated with alcohol. In order to evaluate liver damage after alcohol consumption, the following hematological parameters were tested: the activity of alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase; serum values of urea and creatinine; the phagocytic capacity of polymorphonuclear neutrophilsin peripheral blood;serum opsonic capacity;bactericidal capacity of peritoneal macrophages; and the activity of malondialdehyde, glutathione peroxidase, superoxide dismutase and lactate dehydrogenase. Results and Conclusions The treatment with chitosan vesicles decreased liver enzyme activity and reduced the oxidative stress disturbances in alcoholic mice, thus repairing the hepatic functional and structural damages. These beneficial activities of chitosan vesicles were comparable with ascorbic acid effects in alcoholic mice.

Scalable Fabrication of Quasi-One-Dimensional Gold Nanoribbons for Plasmonic Sensing.

Plasmonic nanostructures have a wide range of applications, including chemical and biological sensing. However, the development of techniques to fabricate submicrometer-sized plasmonic structures over large scales remains challenging. We demonstrate a high-throughput, cost-effective approach to fabricate Au nanoribbons via chemical lift-off lithography (CLL). Commercial HD-DVDs were used as large-area templates for CLL. Transparent glass slides were coated with Au/Ti films and functionalized with self-assembled alkanethiolate monolayers. Monolayers were patterned with lines via CLL. The lifted-off, exposed regions of underlying Au were selectively etched into large-area grating-like patterns (200 nm line width; 400 nm pitch; 60 nm height). After removal of the remaining monolayers, a thin In2O3 layer was deposited and the resulting gratings were used as plasmonic sensors. Distinct features in the extinction spectra varied in their responses to refractive index changes in the solution environment with a maximum bulk sensitivity of ∼510 nm/refractive index unit. Sensitivity to local refractive index changes in the near-field was also achieved, as evidenced by real-time tracking of lipid vesicle or protein adsorption. These findings show how CLL provides a simple and economical means to pattern large-area plasmonic nanostructures for applications in optoelectronics and sensing.

Investigation of Shape Transformations of Vesicles, Induced by Their Adhesion to Flat Substrates Characterized by Different Adhesion Strength.

The adhesion of lipid vesicles to a rigid flat surface is investigated. We examine the influence of the membrane spontaneous curvature, adhesion strength, and the reduced volume on the stability and shape transformations of adhered vesicles. The minimal strength of the adhesion necessary to stabilize the shapes of adhered vesicles belonging to different shape classes is determined. It is shown that the budding of an adhered vesicle may be induced by the change of the adhesion strength. The importance of the free vesicle shape for its susceptibility to adhesion is discussed.

Synergistic Action of Antimicrobial Lung Proteins against Klebsiella pneumoniae.

As key components of innate immunity, lung antimicrobial proteins play a critical role in warding off invading respiratory pathogens. Lung surfactant protein A (SP-A) exerts synergistic antimicrobial activity with the N-terminal segment of the SP-B proprotein (SP-BN) against Klebsiella pneumoniae K2 in vivo. However, the factors that govern SP-A/SP-BN antimicrobial activity are still unclear. The aim of this study was to identify the mechanisms by which SP-A and SP-BN act synergistically against K. pneumoniae, which is resistant to either protein alone. The effect of these proteins on K. pneumoniae was studied by membrane permeabilization and depolarization assays and transmission electron microscopy. Their effects on model membranes of the outer and inner bacterial membranes were analyzed by differential scanning calorimetry and membrane leakage assays. Our results indicate that the SP-A/SP-BN complex alters the ultrastructure of K. pneumoniae by binding to lipopolysaccharide molecules present in the outer membrane, forming packing defects in the membrane that may favor the translocation of both proteins to the periplasmic space. The SP-A/SP-BN complex depolarized and permeabilized the inner membrane, perhaps through the induction of toroidal pores. We conclude that the synergistic antimicrobial activity of SP-A/SP-BN is based on the capability of this complex, but not either protein alone, to alter the integrity of bacterial membranes.

Assessment of the In Vivo Release and Biocompatibility of Novel Vesicles Containing Zinc in Rats.

This paper is focused on the in vivo release and biocompatibility evaluation in rats of some novel systems entrapping zinc chloride in lipid vesicles. The particles were prepared by zinc chloride immobilization inside lipid vesicles made using phosphatidylcholine, stabilized with 0.5% chitosan solution, and dialyzed for 10 h to achieve a neutral pH. The submicrometric systems were physico-chemically characterized. White Wistar rats, assigned into four groups of six animals each, were treated orally with a single dose, as follows: Group I (control): deionized water 0.3 mL/100 g body weight; Group II (Zn): 2 mg/kg body weight (kbw) zinc chloride; Group III (LV-Zn): 2 mg/kbw zinc chloride in vesicles; Group IV (LVC-Zn): 2 mg/kbw zinc chloride in vesicles stabilized with chitosan. Haematological, biochemical, and immune parameters were assessed after 24 h and 7 days, and then liver fragments were collected for histopathological examination. The use of zinc submicrometric particles-especially those stabilized with chitosan-showed a delayed zinc release in rats. No substantial changes to blood parameters, plasma biochemical tests, serum complement activity, or peripheral neutrophils phagocytic capacity were noted; moreover, the tested substances did not induce liver architectural disturbances. The obtained systems provided a delayed release of zinc, and showed good biocompatibility in rats.

Normalizing polydiacetylene colorimetric assays of vesicle binding across lipid systems.

Mixed polydiacetylene (PDA) lipid vesicles mimic cell membranes and exhibit a colorimetric response induced by mechanical stress, which can be used to determine the affinity of proteins or molecules for lipid membranes. Due to a simple spectroscopic readout, PDA assays are amenable to high-throughput screens; however, these assays exhibit batch-to-batch variability. Sensitivity of the assay is also influenced by physicochemical properties associated with different lipids. Here, a method of normalizing PDA assays to reduce variability and enable direct comparison across lipid systems is described.

Highly oriented photosynthetic reaction centers generate a proton gradient in synthetic protocells.

Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP. Herein we show that synthetic protocells, based on giant lipid vesicles embedding an oriented population of reaction centers, are capable of generating a photoinduced proton gradient across the membrane. Under continuous illumination, the protocells generate a gradient of 0.061 pH units per min, equivalent to a proton motive force of 3.6 mV⋅min-1 Remarkably, the facile reconstitution of the photosynthetic reaction center in the artificial lipid membrane, obtained by the droplet transfer method, paves the way for the construction of novel and more functional protocells for synthetic biology.