Photomanipulation of Minimal Synthetic Cells: Area Increase, Softening, and Interleaflet Coupling of Membrane Models Doped with Azobenzene-Lipid Photoswitches.

Light can effectively interrogate biological systems in a reversible and physiologically compatible manner with high spatiotemporal precision. Understanding the biophysics of photo-induced processes in bio-systems is crucial for achieving relevant clinical applications. Employing membranes doped with the photolipid azobenzene-phosphatidylcholine (azo-PC), a holistic picture of light-triggered changes in membrane kinetics, morphology, and material properties obtained from correlative studies on cell-sized vesicles, Langmuir monolayers, supported lipid bilayers, and molecular dynamics simulations is provided. Light-induced membrane area increases as high as ≈25% and a ten-fold decrease in the membrane bending rigidity is observed upon trans-to-cis azo-PC isomerization associated with membrane leaflet coupling and molecular curvature changes. Vesicle electrodeformation measurements and atomic force microscopy reveal that trans azo-PC bilayers are thicker than palmitoyl-oleoyl phosphatidylcholine (POPC) bilayers but have higher specific membrane capacitance and dielectric constant suggesting an increased ability to store electric charges across the membrane. Lastly, incubating POPC vesicles with azo-PC solutions results in the insertion of azo-PC in the membrane enabling them to become photoresponsive. All these results demonstrate that light can be used to finely manipulate the shape, mechanical and electric properties of photolipid-doped minimal cell models, and liposomal drug carriers, thus, presenting a promising therapeutic alternative for the repair of cellular disorders.

Leaflet Tensions Control the Spatio-Temporal Remodeling of Lipid Bilayers and Nanovesicles.

Biological and biomimetic membranes are based on lipid bilayers, which consist of two monolayers or leaflets. To avoid bilayer edges, which form when the hydrophobic core of such a bilayer is exposed to the surrounding aqueous solution, a single bilayer closes up into a unilamellar vesicle, thereby separating an interior from an exterior aqueous compartment. Synthetic nanovesicles with a size below 100 nanometers, traditionally called small unilamellar vesicles, have emerged as potent platforms for the delivery of drugs and vaccines. Cellular nanovesicles of a similar size are released from almost every type of living cell. The nanovesicle morphology has been studied by electron microscopy methods but these methods are limited to a single snapshot of each vesicle. Here, we review recent results of molecular dynamics simulations, by which one can monitor and elucidate the spatio-temporal remodeling of individual bilayers and nanovesicles. We emphasize the new concept of leaflet tensions, which control the bilayers' stability and instability, the transition rates of lipid flip-flops between the two leaflets, the shape transformations of nanovesicles, the engulfment and endocytosis of condensate droplets and rigid nanoparticles, as well as nanovesicle adhesion and fusion. To actually compute the leaflet tensions, one has to determine the bilayer's midsurface, which represents the average position of the interface between the two leaflets. Two particularly useful methods to determine this midsurface are based on the density profile of the hydrophobic lipid chains and on the molecular volumes.

Deoxyfluorination tunes the aggregation of cellulose and chitin oligosaccharides and highlights the role of specific hydroxyl groups in the crystallization process.

Cellulose and chitin are abundant structural polysaccharides exploited by nature in a large number of applications thanks to their crystallinity. Chemical modifications are commonly employed to tune polysaccharide physical and mechanical properties, but generate heterogeneous mixtures. Thus, the effect of such modifications is not well understood at the molecular level. In this work, we examined how deoxyfluorination (site and pattern) impact the solubility and aggregation of well-defined cellulose and chitin oligomers. While deoxyfluorination increased solubility in water and lowered the crystallinity of cellulose oligomers, chitin was much less affected by the modification. The OH/F substitution also highlighted the role of specific hydroxyl groups in the crystallization process. This work provides guidelines for the design of cellulose- and chitin-based materials. A similar approach can be imagined to prepare cellulose and chitin analogues capable of withstanding enzymatic degradation.

Budding and Fission of Nanovesicles Induced by Membrane Adsorption of Small Solutes.

Membrane budding and fission are essential cellular processes that produce new membrane compartments during cell and organelle division, for intracellular vesicle trafficking as well as during endo- and exocytosis. Such morphological transformations have also been observed for giant lipid vesicles with a size of many micrometers. Here, we report budding and fission processes of lipid nanovesicles with a size below 50 nm. We use coarse-grained molecular dynamics simulations, by which we can visualize the morphological transformations of individual vesicles. The budding and fission processes are induced by low concentrations of small solutes that absorb onto the outer leaflets of the vesicle membranes. In addition to the solute concentration, we identify the solvent conditions as a second key parameter for these processes. For good solvent conditions, the budding of a nanovesicle can be controlled by reducing the vesicle volume for constant solute concentration or by increasing the solute concentration for constant vesicle volume. After the budding process is completed, the budded vesicle consists of two membrane subcompartments which are connected by a closed membrane neck. The budding process is reversible as we demonstrate explicitly by reopening the closed neck. For poor solvent conditions, on the other hand, we observe two unexpected morphological transformations of nanovesicles. Close to the binodal line, at which the aqueous solution undergoes phase separation, the vesicle exhibits recurrent shape changes with closed and open membrane necks, reminiscent of flickering fusion pores (kiss-and-run) as observed for synaptic vesicles. As we approach the binodal line even closer, the recurrent shape changes are truncated by the fission of the membrane neck which leads to the division of the nanovesicle into two daughter vesicles. In this way, our simulations reveal a nanoscale mechanism for the budding and fission of nanovesicles, a mechanism that arises from the interplay between membrane elasticity and solute-mediated membrane adhesion.

Systematic Structural Characterization of Chitooligosaccharides Enabled by Automated Glycan Assembly.

Chitin, a polymer composed of ß(1-4)-linked N-acetyl-glucosamine monomers, and its partially deacetylated analogue chitosan, are abundant biopolymers with outstanding mechanical as well as elastic properties. Their degradation products, chitooligosaccharides (COS), can trigger the innate immune response in humans and plants. Both material and biological properties are dependent on polymer length, acetylation, as well as the pH. Without well-defined samples, a complete molecular description of these factors is still missing. Automated glycan assembly (AGA) enabled rapid access to synthetic well-defined COS. Chitin-cellulose hybrid oligomers were prepared as important tools for a systematic structural analysis. Intramolecular interactions, identified by molecular dynamics simulations and NMR analysis, underscore the importance of the chitosan amino group for the stabilization of specific geometries.

A multiscale coarse-grained model to predict the molecular architecture and drug transport properties of modified chitosan hydrogels.

Hydrogels constructed with functionalized polysaccharides are of interest in a multitude of applications, chiefly the design of therapeutic and regenerative formulations. Tailoring the chemical modification of polysaccharide-based hydrogels to achieve specific drug release properties involves the optimization of many tunable parameters, including (i) the type, degree (χ), and pattern of the functional groups, (ii) the water-polymer ratio, and (iii) the drug payload. To guide the design of modified polysaccharide hydrogels for drug release, we have developed a computational toolbox that predicts the structure and physicochemical properties of acylated chitosan chains, and their impact on the transport of drug molecules. Herein, we present a multiscale coarse-grained model to investigate the structure of networks of chitosan chains modified with acetyl, butanoyl, or heptanoyl moieties, as well as the diffusion of drugs doxorubicin (Dox) and gemcitabine (Gem) through the resulting networks. The model predicts the formation of different network structures, in particular the hydrophobically-driven transition from a uniform to a cluster/channel morphology and the formation of fibers of chitin chains. The model also describes the impact of structural and physicochemical properties on drug transport, which was confirmed experimentally by measuring Dox and Gem diffusion through an ensemble of modified chitosan hydrogels.

Weak carbohydrate-carbohydrate interactions in membrane adhesion are fuzzy and generic.

Carbohydrates such as the trisaccharide motif LeX are key constituents of cell surfaces. Despite intense research, the interactions between carbohydrates of apposing cells or membranes are not well understood. In this article, we investigate carbohydrate-carbohydrate interactions in membrane adhesion as well as in solution with extensive atomistic molecular dynamics simulations that exceed the simulation times of previous studies by orders of magnitude. For LeX, we obtain association constants of soluble carbohydrates, adhesion energies of lipid-anchored carbohydrates, and maximally sustained forces of carbohydrate complexes in membrane adhesion that are in good agreement with experimental results in the literature. Our simulations thus appear to provide a realistic, detailed picture of LeX-LeX interactions in solution and during membrane adhesion. In this picture, the LeX-LeX interactions are fuzzy, i.e. LeX pairs interact in a large variety of short-lived, bound conformations. For the synthetic tetrasaccharide Lac 2, which is composed of two lactose units, we observe similarly fuzzy interactions and obtain association constants of both soluble and lipid-anchored variants that are comparable to the corresponding association constants of LeX. The fuzzy, weak carbohydrate-carbohydrate interactions quantified in our simulations thus appear to be a generic feature of small, neutral carbohydrates such as LeX and Lac 2.

Sequential Linkage of Carbohydrate Antigens to Mimic Capsular Polysaccharides: Toward Semisynthetic Glycoconjugate Vaccine Candidates against Streptococcus pneumoniae Serotype 14.

Vaccines based on isolated polysaccharides successfully protect humans from bacterial pathogens such as Streptococcus pneumoniae. Because polysaccharide production and isolation can be technically challenging, glycoconjugates containing synthetic antigens are an attractive alternative. Typically, the shortest possible oligosaccharide antigen is preferable as syntheses of longer structures are more difficult and time-consuming. Combining several protective epitopes or polysaccharide repeating units as blocks by bonds other than glycosidic linkages would greatly reduce the synthetic effort if the immunological response to the polysaccharide could be retained. To explore this concept, we bridged the well-understood and immunologically potent RU of S. pneumoniae serotype 14 (ST14) with an aliphatic spacer and conjugated it to the carrier protein CRM197. Mice immunized with the spacer-bridged glycan conjugates produced high levels of specific antibodies after just one or two vaccine doses, while the tetrasaccharide repeating unit alone required three doses. The antibodies recognized specifically ST14 CPS, while no significant antibody levels were raised against the spacer or unrelated CPS. Synthetic vaccines generated antibodies with opsonic activity. Mimicking polysaccharides by coupling repeating unit antigens via an aliphatic spacer may prove useful also for the development of other glycoconjugate vaccine candidates, thereby reducing the synthetic complexity while enhancing a faster immune response.

Automated access to well-defined ionic oligosaccharides.

Ionic polysaccharides are part of many biological events, but lack structural characterisation due to challenging purifications and complex synthesis. Four monosaccharides bearing modifications not found in nature are used for the automated synthesis of a collection of ionic oligosaccharides. Structural analysis reveals how the charge pattern affects glycan conformation.

Asymmetrically Branched Precision Glycooligomers Targeting Langerin.

Asymmetrically branched precision glycooligomers are synthesized by solid-phase polymer synthesis for studying multivalent carbohydrate-protein interactions. Through the stepwise assembly of Fmoc-protected oligo(amidoamine) building blocks and Fmoc/Dde-protected lysine, straightforward variation of structural parameters such as the number and length of arms, as well as the number and position of carbohydrate ligands, is achieved. Binding of 1-arm and 3-arm glycooligomers toward lectin receptors langerin and concanavalin A (ConA) was evaluated where the smallest 3-arm glycooligomer shows the highest binding toward langerin, and stepwise elongation of one, two, or all three arms leads to decreased binding. When directly comparing binding toward langerin and ConA, we find that structural variation of the scaffold affects glycomimetic ligand binding differently for the different targets, indicating the potential to tune such ligands not only for their avidity but also for their selectivity toward different lectins.

Spherical Nanovesicles Transform into a Multitude of Nonspherical Shapes.

Nanovesicles are closed, bubblelike surfaces with a diameter between 20 and 200 nm, formed by lipid bilayers and biomembranes. Electron microscopy (EM) studies have shown that these vesicles can attain both spherical and nonspherical shapes. One disadvantage of EM methods is that they provide only a single snapshot of each vesicle. Here, we use molecular dynamics simulations to monitor the morphological transformations of individual nanovesicles. We start with the assembly of spherical vesicles that enclose a certain volume of water and contain a certain total number of lipids. When we reduce their volume, the spherical vesicles are observed to transform into a multitude of nonspherical shapes such as oblates and stomatocytes as well as prolates and dumbbells. This surprising polymorphism can be controlled by redistributing a small fraction of lipids between the inner and outer leaflets of the bilayer membranes. As a consequence, the inner and the outer leaflets experience different mechanical tensions. Small changes in the vesicle volume reduce the overall bilayer tension by 2 orders of magnitude, thereby producing tensionless bilayers. In addition, we show how to determine, for a certain total number of lipids, the unique spherical vesicle for which both leaflet tensions vanish individually. We also compute the local spontaneous curvature of the spherical membranes by identifying the first moment of the spherically symmetric stress profiles across the lipid bilayers with the nanoscopic torque as derived from curvature elasticity. Our study can be extended to other types of lipid membranes and sheds new light on cellular nanovesicles such as exosomes, which are increasingly used as biomarkers and drug delivery systems.

Tailoring the Chemical Modification of Chitosan Hydrogels to Fine-Tune the Release of a Synergistic Combination of Chemotherapeutics.

Combination chemotherapy with a defined ratio and sequence of drug release is a clinically established and effective route to treat advanced solid tumors. In this context, a growing body of literature demonstrates the potential of hydrogels constructed with chemically modified polysaccharides as depots for controlled release of chemotherapeutics. Identifying the appropriate modification in terms of physicochemical properties of the functional group and its degree of substitution (χ) to achieve the desired release profile for multiple drugs is, however, a complex multivariate problem. To address this issue, we have developed a computational toolbox that models the migration of a drug pair through a hydrated network of polysaccharide chains modified with hydrophobic moieties. In this study, we chose doxorubicin (DOX) and Gemcitabine (GEM) as model drugs, as their synergistic effect against breast cancer has been thoroughly investigated, and chitosan as the model polymer. Our model describes how the modification of chitosan chains with acetyl, butanoyl, and heptanoyl moieties at different values χ governs both the structure of the hydrogel network and drug migration through it. Our experimental data confirm the in silico predictions for both single- and dual-drug release and, most notably, the counterintuitive inversion of release vs χ that occurs when switching from a single- to a dual-drug system. Consensus between predicted and experimental data indicates that acetyl modifications (χ = 32-42%) and butanoyl modifications (χ = 19-24%) provide synergistic GEM/DOX release molar ratios (i.e., 5-10). Collectively, these results demonstrate the potential of this model in guiding the design of chemotherapeutic hydrogels to combat cancer.

Systematic Hydrogen-Bond Manipulations To Establish Polysaccharide Structure-Property Correlations.

A dense hydrogen-bond network is responsible for the mechanical and structural properties of polysaccharides. Random derivatization alters the properties of the bulk material by disrupting the hydrogen bonds, but obstructs detailed structure-function correlations. We have prepared well-defined unnatural oligosaccharides including methylated, deoxygenated, deoxyfluorinated, as well as carboxymethylated cellulose and chitin analogues with full control over the degree and pattern of substitution. Molecular dynamics simulations and crystallographic analysis show how distinct hydrogen-bond modifications drastically affect the solubility, aggregation behavior, and crystallinity of carbohydrate materials. This systematic approach to establishing detailed structure-property correlations will guide the synthesis of novel, tailor-made carbohydrate materials.

Interaction of SNARE Mimetic Peptides with Lipid bilayers: Effects of Secondary Structure, Bilayer Composition and Lipid Anchoring.

The coiled-coil forming peptides 'K' enriched in lysine and 'E' enriched in glutamic acid have been used as a minimal SNARE mimetic system for membrane fusion. Here we describe atomistic molecular dynamics simulations to characterize the interactions of these peptides with lipid bilayers for two different compositions. For neutral phosphatidylcholine (PC)/phosphatidylethanolamine (PE) bilayers the peptides experience a strong repulsive barrier against adsorption, also observed in potential of mean force (PMF) profiles calculated with umbrella sampling. For peptide K, a minimum of -12 kBT in the PMF provides an upper bound for the binding free energy whereas no stable membrane bound state could be observed for peptide E. In contrast, the electrostatic interactions with negatively charged phosphatidylglycerol (PG) lipids lead to fast adsorption of both peptides at the head-water interface. Experimental data using fluorescently labeled peptides confirm the stronger binding to PG containing bilayers. Lipid anchors have little effect on the peptide-bilayer interactions or peptide structure, when the peptide also binds to the bilayer in the absence of a lipid anchor. For peptide E, which does not bind to the PC bilayer without a lipid anchor, the presence of such an anchor strengthens the electrostatic interactions between the charged side chains and the zwitterionic head-groups and leads to a stabilization of the peptide's helical fold by the membrane.

Multivalent Interactions of Polyamide Based Sequence-Controlled Glycomacromolecules with Concanavalin A.

Binding of mannose presenting macromolecules to the protein receptor concanavalin A (ConA) is investigated by means of single-molecule atomic force spectroscopy (SMFS) in combination with dynamic light scattering and molecular modeling. Oligomeric (Mw ≈ 1.5-2.5 kDa) and polymeric (Mw ≈ 22-30 kDa) glycomacromolecules with controlled number and positioning of mannose units along the scaffolds accessible by combining solid phase synthesis and thiol-ene coupling are used as model systems to assess the molecular mechanisms that contribute to multivalent ConA-mannose complexes. SMFS measurements show increasing dissociation force from monovalent (≈57 pN) to pentavalent oligomers (≈75 pN) suggesting subsite binding to ConA. Polymeric glycomacromolecules with larger hydrodynamic diameters compared to the binding site spacing of ConA exhibit larger dissociation forces (≈80 pN), indicating simultaneous dissociation from multiple ConA binding sites. Nevertheless, although simultaneous dissociation of multiple ligands could be expected for such multivalent systems, predominantly single dissociation events are observed. This is rationalized by strong coiling of the macromolecules' polyamide backbone due to intramolecular hydrogen bonding hindering unfolding of the coil. Therefore, this study shows that the design of glycopolymers for multivalent receptor binding and clustering must consider 3D structure and intramolecular interactions of the scaffold.

Tailoring carbon nitride properties and photoactivity by interfacial engineering of hydrogen-bonded frameworks.

The rational synthesis of carbon nitride materials, ranging from polymeric carbon nitride to nitrogen-doped carbon, by supramolecular preorganization of their monomers is a powerful tool for the design of their morphology and photophysical and catalytic activities. Here we show a new facile and scalable approach for the synthesis of ordered CN materials with excellent photoactivity, which consists of supramolecular interfacial preorganization of CN monomers at the interface of two non-miscible solvents. Molecular dynamic simulations supported by experimental results reveal that an appropriate choice of monomers and solvents leads to the formation of a supramolecular assembly solely at the interface of the solvents. As a proof of concept, we show that the properties of the CN materials after thermal condensation can be tuned by adding an additional monomer to one solvent only. The advantages of the new method are demonstrated here through the tunable morphologies and surface area, the formation of new electronic junctions and high activity as a photocatalyst for hydrogen evolution and pollutant degradation of the CN materials.

Divalent Sialylated Precision Glycooligomers Binding to Polyomaviruses and the Effect of Different Linkers.

Divalent precision glycooligomers terminating in N-acetylneuraminic acid (Neu5Ac) or 3'-sialyllactose (3'-SL) with varying linkers between scaffold and the glycan portions are synthesized via solid phase synthesis for co-crystallization studies with the sialic acid-binding major capsid protein VP1 of human Trichodysplasia spinulosa-associated Polyomavirus. High-resolution crystal structures of complexes demonstrate that the compounds bind to VP1 depending on the favorable combination of carbohydrate ligand and linker. It is found that artificial linkers can replace portions of natural carbohydrate linkers as long as they meet certain requirements such as size or flexibility to optimize contact area between ligand and receptor binding sites. The obtained results will influence the design of future high affinity ligands based on the structures presented here, and they can serve as a blueprint to develop multivalent glycooligomers as inhibitors of viral adhesion.

Nanodroplets at Membranes Create Tight-Lipped Membrane Necks via Negative Line Tension.

The response of biomembranes to aqueous-phase separation and to the resulting water-in-water droplets has been recently studied on the micrometer scale using optical microscopy and elasticity theory. When such a droplet adheres to the membrane, it forms a contact area that is bounded by a contact line. For a micrometer-sized droplet, the line tension associated with this contact line can usually be ignored compared with the surface tensions. However, for a small nanoscopic droplet, this line tension is expected to affect the membrane-droplet morphology. Here, we use molecular simulations to study nanodroplets at membranes and to gain insight into these line tension effects. The latter effects are shown to depend strongly on another key parameter, the mechanical tension experienced by the membrane. For a large membrane tension, a droplet adhering to the membrane is only partially engulfed by the membrane, and the membrane-droplet system exhibits an axisymmetric morphology. A reduction of the membrane tension leads to an increase in the contact area and a decrease in the interfacial area of the droplet, initially retaining its axisymmetric shape, which implies a circular contact line and a circular membrane neck. However, when the tension falls below a certain threshold value, the system undergoes a morphological transition toward a non-axisymmetric morphology with a non-circular membrane neck. This morphology persists until the nanodroplet is completely engulfed by the membrane and the membrane neck has closed into a tight-lipped shape. The latter morphology is caused by a negative line tension, which is shown to be a robust feature of membrane-droplet systems. A closed membrane neck with a tight-lipped shape suppresses both thermally activated and protein-induced scission of the neck, implying a reduction in the cellular uptake of nanodroplets by pinocytosis and fluid-phase endocytosis. Furthermore, based on our results, we can also draw important conclusions about the time-dependent processes corresponding to the surface nucleation and growth of nanodroplets at membranes.

Fucose-Functionalized Precision Glycomacromolecules Targeting Human Norovirus Capsid Protein.

Norovirus infection is the major cause of nonbacterial gastroenteritis in humans and has been the subject of numerous studies investigating the virus's biophysical properties and biochemical function with the aim of deriving novel and highly potent entry inhibitors to prevent infection. Recently, it has been shown that the protruding P domain dimer (P-dimer) of a GII.10 Norovirus strain exhibits two new binding sites for l-fucose in addition to the canonical binding sites. Thus, these sites provide a novel target for the design of multivalent fucose ligands as entry inhibitors of norovirus infections. In this current study, a first generation of multivalent fucose-functionalized glycomacromolecules was synthesized and applied as model structures to investigate the potential targeting of fucose binding sites in human norovirus P-dimer. Following previously established solid phase polymer synthesis, eight precision glycomacromolecules varying in number and position of fucose ligands along an oligo(amidoamine) backbone were obtained and then used in a series of binding studies applying native MS, NMR, and X-ray crystallography. We observed only one fucose per glycomacromolecule binding to one P-dimer resulting in similar binding affinities for all fucose-functionalized glycomacromolecules, which based on our current findings we attribute to the overall size of macromolecular ligands and possibly to steric hindrance.

Area Increase and Budding in Giant Vesicles Triggered by Light: Behind the Scene.

Biomembranes are constantly remodeled and in cells, these processes are controlled and modulated by an assortment of membrane proteins. Here, it is shown that such remodeling can also be induced by photoresponsive molecules. The morphological control of giant vesicles in the presence of a water-soluble ortho-tetrafluoroazobenzene photoswitch (F-azo) is demonstrated and it is shown that the shape transformations are based on an increase in membrane area and generation of spontaneous curvature. The vesicles exhibit budding and the buds can be retracted by using light of a different wavelength. In the presence of F-azo, the membrane area can increase by more than 5% as assessed from vesicle electrodeformation. To elucidate the underlying molecular mechanism and the partitioning of F-azo in the membrane, molecular dynamics simulations are employed. Comparison with theoretically calculated shapes reveals that the budded shapes are governed by curvature elasticity, that the spontaneous curvature can be decomposed into a local and a nonlocal contribution, and that the local spontaneous curvature is about 1/(2.5 µm). The results show that exo- and endocytotic events can be controlled by light and that these photoinduced processes provide an attractive method to change membrane area and morphology.