Zwitterionic Lipid Research Articles

Zwitterionic Polymer-Functionalized Lipid Nanoparticles for the Nebulized Delivery of mRNA.

Lipid nanoparticles (LNPs) have great potential to enable inhaled delivery of mRNA to treat pulmonary diseases. However, this potential has been limited by the challenge of nebulizing the LNPs. Nebulization of LNPs can cause LNPs to aggregate and release encapsulated mRNA, limiting their delivery efficacy. To overcome this challenge, LNPs are developed whereby the PEG-lipid is fully replaced with a zwitterionic polymer (ZIP)-lipid conjugate to greatly enhance the nebulizer stability. LNPs formulated with ZIP-lipids (ZIP-LNPs) were stable to nebulization across a wide range of formulation parameters. The optimized ZIP-LNP formulation, containing reduced cholesterol content relative to traditional PEG-lipid LNPs, demonstrated improved inhaled mRNA delivery in both healthy and mucoobstructed mouse lungs. Repeat administration of the optimized ZIP-LNP formulation was well tolerated and did not result in pulmonary inflammation. This study demonstrates the potential of zwitterionic polymer-lipid conjugates for improving the performance of inhaled mRNA-LNP formulations.

Interaction of Arginine-Rich Surfactant-like Peptide Nanotubes with Liposomes.

The interaction of the surfactant-like peptide (SLP) R3L12 bearing three cationic arginine residues with model liposomes is investigated in aqueous solution at various pH values, under conditions for which the SLP self-assembles into nanotubes. The structure of liposomes of model anionic lipid DPPG [1,2-dipalmitoyl-sn-glycero-3-phospho-rac-(1-glycerol)], or zwitterionic lipid DPPE [1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine] is probed using small-angle X-ray scattering and cryogenic-transmission electron microscopy. The unilamellar vesicles of DPPG are significantly restructured in the presence of R3L12, especially at low pH, and multilamellar vesicles of DPPE are also restructured under these conditions. The SLP promotes the release of cargo encapsulated in the vesicles as probed by calcein fluorescence, with notably higher release for anionic DPPG vesicles. Laurdan fluorescence experiments to probe membrane fluidity (lipid chain ordering) show that R3L12 destabilizes the lipid gel phase, especially for anionic DPPG. This model nanotube-forming SLP has promise as a pH-sensitive release system for vesicle-encapsulated cargo.

Dual Role of Anionic Lipids in Amyloid Aggregation.

Neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's, affect millions worldwide and share a common feature: the aggregation of intrinsically disordered proteins into toxic oligomers that interact with cell membranes. In Alzheimer's disease (AD), amyloid-beta (Aβ) peptides accumulate and bind to plasma membranes, potentially disrupting cellular function. The complex interplay between amyloidogenic peptides and lipid membranes, particularly the role of anionic lipids, is crucial in disease pathogenesis but challenging to characterize experimentally. The literature presents conflicting results on the influence of anionic lipids on peptide aggregation kinetics, highlighting a knowledge gap. To address this, we used coarse-grained molecular dynamics (CG-MD) simulations to study interactions between a model amyloidogenic peptide, amyloid-β's K16LVFFAE22 fragment (Aβ16-22), and mixed lipid bilayers. We used phosphatidylserine (PS) and phosphatidylcholine (PC) as representative anionic and zwitterionic lipids, respectively, examining the mixed bilayer compositions of 0% PS-100% PC, 10% PS-90% PC, and 30% PS-70% PC. Our simulations revealed that membranes enriched in anionic lipids enhance peptide adsorption and interaction kinetics. The aggregation dynamics was modulated by two competing factors: increased local peptide concentration near negatively charged membranes, which promoted aggregation, and peptide-lipid interactions, which slowed it down. Higher percentages of anionic lipids led to smaller and more ordered aggregates and enhanced lipid demixing, leading to the formation of PS clusters. These findings contribute to understanding membrane-mediated peptide aggregation in neurodegenerative disorders, potentially guiding new therapeutic strategies targeting the early stages of protein aggregation in various neurodegenerative diseases.

Role of zwitterionic lipid headgroups on monolayer formation and interfacial dilatational rheology in binary mixtures of phospholipids and cholesterol: A pendant drop tensiometer study

The complex composition of biological membranes, comprising a diverse array of lipids with unique moieties, has garnered increased attention due to the recognized roles of lipids in membrane stability and biological processes. Even subtle changes in phospholipid headgroups and fatty acyl tails profoundly affect the formation and interfacial dynamics of lipid monolayers at the air-water interface. However, the molecular-level understanding of their intermolecular forces and interactions during these processes, directly relating to the lipid chemical structures, is not well-explored. To better understand these complex physicochemical phenomena, simplified model monolayers with precise control over lipid types and compositions are utilized. In this study, we employ the pendant drop tensiometer technique to investigate the formation and interfacial rheology of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) monolayers, with varying amounts of cholesterol (CHOL) for the first time. These two phospholipids, with identical C16:0 acyl tails but different headgroups, exhibit marked differences in their interfacial interactions with CHOL and water molecules, consequently affecting monolayer formation and rheology. In the absence of CHOL, DPPE monolayers typically display a lower dilatational modulus than DPPC, attributed to increased headgroup hydration. However, introducing CHOL reverses this trend, resulting in stiffer DPPE-CHOL monolayers compared to DPPC-CHOL. With CHOL, we observe its well-known condensation effect on DPPC monolayers, yet for DPPE monolayers, both condensation and expansion effects are noted, contingent on CHOL amount. We anticipate this work will not only deepen our fundamental understanding of the structure-composition-property relationships in lipid molecules but also provide a robust foundation for comprehending more intricate biological systems.

Mapping of the Lipid-Binding Regions of the Antifungal Protein NFAP2 by Exploiting Model Membranes.

Fungal infections with high mortality rates represent an increasing health risk. The Neosartorya (Aspergillus) fischeri antifungal protein 2 (NFAP2) is a small, cysteine-rich, cationic protein exhibiting potent anti-Candida activity. As the underlying mechanism, pore formation has been demonstrated; however, molecular level details on its membrane disruption action are lacking. Herein, we addressed the lipid binding of NFAP2 using a combined computational and experimental approach to simple lipid compositions with various surface charge properties. Simulation results revealed binding preferences for negatively charged model membranes, where selectivity is mediated by anionic lipid components enriched at the protein binding site but also assisted by zwitterionic lipid species. Several potential binding routes initiated by various anchoring contacts were observed, which resulted in one main binding mode and a few variants, with NFAP2 residing on the membrane surface. Region 10NCPNNCKHKKG20 of the flexible N-terminal part of the protein showed potency to insert into the lipid bilayer, where the disulfide bond-stabilized short motif 11CPNNC15 could play a key role. In addition, several areas, including the beginning of the N-terminal (residues 1-8), played roles in facilitating initial membrane contacts. Besides, individual roles of residues such as Lys24, Lys32, Lys34, and Trp42 were also revealed by the simulations. Combined data demonstrated that the solution conformation was not perturbed markedly upon membrane interaction, and the folded part of the protein also contributed to stabilizing the bound state. Data also highlighted that the binding of NFAP2 to lipid vesicles is sensitively affected by environmental factors such as ionic strength. Electrostatic interactions driven by anionic lipids were found pivotal, explaining the reduced membrane activity observed under high salt conditions. Experimental data supported the lipid-selective binding mechanisms and pointed to salt-dependent effects, particularly to protein-assisted vesicle aggregation at low ionic strength. Our findings can contribute to the development of NFAP2-based anti-Candida agents and studies aiming at future medical use of peptide-based natural antifungal compounds.

Antisense Oligonucleotide Embedded Context Responsive Nanoparticles Derived from Synthetic Ionizable Lipids for lncRNA Targeted Therapy of Breast Cancer.

The long noncoding RNAs (lncRNA) are primarily associated with several essential gene regulations but are also connected to cancer metabolism and progression. HOTAIR and MALAT1 are two such lncRNAs that are detected in malignancies of various origins and are responsible for the poor prognosis of cancer patients. Due to these factors, the lncRNAs have emerged as prime targets for the development of anticancer therapeutics. However, nonviral delivery of lncRNA-targeted antisense oligonucleotides (ASOs) still remains a critical challenge while maintaining their structural and functional integrity. Herein, we have designed and synthesized a new series of ionizable lipids with variations in their head groups to prepare lipid nanoparticle (LNP) formulation along with cholesterol-based twin cationic lipid and amphiphilic zwitterionic lipid. The context responsiveness of these formulations in delivering the ASOs has been thoroughly investigated by various bioanalytical techniques, and an optimum formulation has been identified. The LNPs are utilized to deliver the ASOs targeting HOTAIR lncRNA in human cancer cell lines and MALAT1 lncRNA in mouse models. This study thus standardizes an advanced nanomaterial system for nonviral gene delivery that has been validated by a considerable reduction in the target lncRNA level under in vitro and a significant reduction in tumor volume under in vivo settings.

Salting-out and Competitive Adsorption of Ethanol into Lipid Bilayer Membranes: Conflicting Effects of Salts on Ethanol-Membrane Interactions Studied by Molecular Dynamics Simulations.

Small amphiphilic molecules, such as ethanol, disturb the structure of lipid bilayer membranes to increase the membrane permeability, which is important for applications such as drug delivery, disinfection, and fermentation. To investigate how and the extent to which coexisting salts affect membrane disturbance, we performed molecular dynamics (MD) simulations on lipid bilayer membranes composed of zwitterionic lipids in aqueous ethanol solutions containing 0-631 mM NaCl, KCl, and KI salts. The addition of salts at low concentrations induced cationic adsorption on the lipid membrane, which competes with ethanol adsorption, thereby reducing the hydrogen bonds between ethanol and lipid molecules. This competitive adsorption mitigated the membrane disturbance and decreased the permeation of ethanol molecules into the membrane. In contrast, higher salt concentrations enhanced the membrane disturbance and permeability, which was caused by the salting-out of ethanol from the aqueous phase to the lipid bilayer. These conflicting effects appearing at different concentrations were stronger with the chloride salts than with the iodide salt. Among the two chloride salts, NaCl and KCl, the latter showed a greater enhancement in ethanol permeation at high concentrations. This seeming anti-Hofmeister salting-out behavior resulted from greater Na+ adsorption, preventing the ethanol-lipid interactions.

Elucidating the role of lipid interactions in stabilizing the membrane protein KcsA

The significant effects of lipid binding on the functionality of potassium channel KcsA have been validated by brilliant studies. However, the specific interactions between lipids and KcsA, such as binding parameters for each binding event, have not been fully elucidated. In this study, we employed native mass spectrometry to investigate the binding of lipids to KcsA and their effects on the channel. The tetrameric structure of KcsA remains intact even in the absence of lipid binding. However, the subunit architecture of the E71A mutant, which is constantly open at low pH, relies on tightly associated copurified lipids. Furthermore, we observed that lipids exhibit weak binding to KcsA at high pH when the channel is at a closed/inactivation state in the absence of permeant cation K+. This feeble interaction potentially facilitates the association of K+ ions, leading to the transition of the channel to a resting closed/open state. Interestingly, both anionic and zwitterionic lipids strongly bind to KcsA at low pH when the channel is in an open/inactivation state. We also investigated the binding patterns of KcsA with natural lipids derived from E. coli and Streptomyces lividans. Interestingly, lipids from E. coli exhibited much stronger binding affinity compared to the lipids from S. lividans. Among the natural lipids from S. lividans, free fatty acids and triacylglycerols demonstrated the tightest binding to KcsA, whereas no detectable binding events were observed with natural phosphatidic acid lipids. These findings suggest that the lipid association pattern in S. lividans, the natural host for KcsA, warrants further investigation. In conclusion, our study sheds light on the role of lipids in stabilizing KcsA and highlights the importance of specific lipid-protein interactions in modulating its conformational states.

Time course of fluorescent-labelled oligonucleotide accumulation in cells with the use of folate receptor-targeted cationic liposomes

INTRODUCTION. The use of cationic liposomes is a promising approach to the delivery of therapeutic nucleic acids to target cells because liposomes can protect nucleic acids from degradation by extracellular nucleases. However, to ensure selective delivery to the site of action, this approach needs modification, including liposome surface functionalisation with targeting ligands.AIM. This study aimed to compare the time courses of the accumulation of a fluorescent-labelled oligonucleotide (FITC-ODN), which simulated a nucleic acid-based medicinal product, in cells with the use of folate receptor-targeted (F) and conventional (L) cationic liposomes.MATERIALS AND METHODS. F- and L-liposomes were prepared using the polycationic amphiphile 2X3, the zwitterionic helper lipid DOPE, and the folate lipoconjugate F12. Physicochemical characterisation of the liposomes was performed using dynamic light scattering and transmission electron microscopy. Liposome–FITC-ODN complexes were formed at various nitrogen to phosphate (N/P) charge ratios. Flow cytometry, fluorescence microscopy, and confocal microscopy methods were used to study the accumulation of liposome–FITC-ODN complexes in human cervical carcinoma (KB-3-1) and human embryonic kidney (HEK 293) cells.RESULTS. The prepared F- and L-liposomes were spherical particles with a diameter of 75–100 nm. The authors selected the optimal N/P ratio of 2/1 to obtain complexes of F- and L-liposomes with the FITC-ODN. This N/P ratio yielded homogeneous liposome–FITC-ODN complexes having a polydispersity index below 0.200 and a size of 112.4–125.1 nm. F-liposomes were 25% more efficient than L-liposomes in FITC-ODN delivery to KB-3-1 cells at 90, 120, and 240 minutes after transfection. In the first few minutes of cell transfection, fluorescence and confocal microscopy data on the distribution of liposome–FITC-ODN complexes showed that cationic liposome fluorescence signals colocalised with FITC-ODN signals. Later, FITC-ODN accumulation in the cytoplasm was observed.CONCLUSIONS. Cationic liposomes demonstrated efficient FITC-ODN delivery into the cytoplasm of cancer cells. F-liposomes enhanced the percentage of transfected cells and improved FITC-ODN delivery compared with L-liposomes. The results obtained can be used in the further development of targeted medicinal products based on therapeutic nucleic acids and liposomes.

The effect of nanobubbles on Langmuir-Blodgett films

Nanobubbles (NBs) are classified in two distinct categories: surface and bulk. Surface NBs are readily observed using atomic force microscopy (AFM), while the existence of bulk NBs has been a subject of debate, conflicting with the diffusion theory's predictions. Current methodologies for identifying bulk NBs yield inconclusive results. In this study, Langmuir Blodgett (LB) technique and AFM, are utilized to visualize NB imprints on anionic, cationic and zwitterionic lipid films deposited on glass-slide substrates. Our analysis of Langmuir monolayers compression isotherms reveals the impact of bulk NBs on lipid monolayer development. AFM scans of the deposited lipid films consistently show NB imprints. Notably, cationic and zwitterionic film depositions exhibit NB formations from the 1st layer, whereas in anionic films, these formations are observed only after the 3rd layer. These results suggest that the origin of these imprinted formations may be attributed to bulk NBs.

Effect of cationic lipid concentration on monomer transfer kinetics between zwitterionic lipid vesicles

The kinetics of spontaneous monomer transfer between vesicles consisting of zwitterionic phospholipids is dictated by the difference in desorption rate of lipid monomers from their donor vesicles and the concentration imbalance in the dispersion. In a system with two lipid species with the same headgroup, transfer is asymmetric, and takes place from the population of donor vesicles consisting of shorter chain lipids to acceptor ones of longer chain. Transfer typically proceeds until equilibrium is reached, resulting in populations of vesicles consisting of a binary mixture of both lipid species, whose concentration depends on the number of lipids in the precursor donor and acceptor vesicles before transfer.Upon the introduction of a second lipid species in the donor vesicle population, the desorption rate of monomers should change with time, since the composition of donor vesicles changes when monomers of a given lipid type desorb. To tackle this problem, we added a cationic lipid, 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP), into donor zwitterionic lipid vesicles and assessed how the concentration of DMTAP affects the lipid transfer process. Lipid transfer is the result of the interplay between the initial concentration of DMTAP in the donor vesicles (and related probability of desorption at short transfer times) and their concomitant time-dependent concentration (and thus desorption rate) change due to the depletion of monomer species as the transfer process proceeds.

Impact of Lipid Composition on Vesicle Protein Adsorption: A BSA Case Study.

Investigating the interaction between liposomes and proteins is of paramount importance in the development of liposomal formulations with real potential for bench-to-bedside transfer. Upon entering the body, proteins are immediately adsorbed on the liposomal surface, changing the nanovehicles' biological identity, which has a significant impact on their biodistribution and pharmacokinetics and ultimately on their therapeutic effect. Albumin is the most abundant plasma protein and thus usually adsorbs immediately on the liposomal surface. We herein report a comprehensive investigation on the adsorption of model protein bovine serum albumin (BSA) onto liposomal vesicles containing the zwitterionic lipid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), in combination with either cholesterol (CHOL) or the cationic lipid 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP). While many studies regarding protein adsorption on the surface of liposomes with different compositions have been performed, to the best of our knowledge, the differential responses of CHOL and DOTAP upon albumin adsorption on vesicles have not yet been investigated. UV-vis spectroscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a strong influence of the phospholipid membrane composition on protein adsorption. Hence, it was found that DOTAP-containing vesicles adsorb proteins more robustly but also aggregate in the presence of BSA, as confirmed by DLS and TEM. Separation of liposome-protein complexes from unadsorbed proteins performed by means of centrifugation and size exclusion chromatography (SEC) was also investigated. Our results show that neither method can be regarded as a golden experimental setup to study the protein corona of liposomes. Yet, SEC proved to be more successful in the separation of unbound proteins, although the amount of lipid loss upon liposome elution was higher than expected. In addition, coarse-grained molecular dynamics simulations were employed to ascertain key membrane parameters, such as the membrane thickness and area per lipid. Overall, this study highlights the importance of surface charge and membrane fluidity in influencing the extent of protein adsorption. We hope that our investigation will be a valuable contribution to better understanding protein-vesicle interactions for improved nanocarrier design.

Configurational Sampling of All-Atom Solvated Membranes Using Hybrid Nonequilibrium Molecular Dynamics Monte Carlo Simulations.

All-atom simulations are a powerful approach to study the structure and dynamics of biological membranes. However, sampling the atomic configurations of inhomogeneous membranes can be challenging due to the slow lateral diffusion of the various constituents. To address this issue, a hybrid nonequilibrium molecular dynamics Monte Carlo (neMD/MC) simulation method is proposed in which randomly chosen lipid molecules are swapped to generate configurations that are subsequently accepted or rejected according to a Metropolis criterion based on the alchemical work for the attempted swap calculated via a short trajectory. A dual-topology framework constraining the common atoms of the exchanging molecules yields a good acceptance probability using switching trajectories as short as 10 ps. The performance of the hybrid neMD/MC algorithm and its ability to sample the distribution of lipids near a transmembrane helix carrying a net charge are illustrated for a binary mixture of charged and zwitterionic lipids.

Design, Synthesis, and Anticancer Activity of N‐1 Terminal Segment of α‐(2, 5,7‐Tri‐tert‐butylindol‐3‐yl)alanine‐Incorporated Short Peptides

AbstractUsing a minimallised approach,we designed and synthesized N‐terminal modified peptides W‐KRGNKWLA‐CONH2 (P1) and W‐ARGDGGNGRGA‐CONH2 (P2) using Fmoc‐Chemistry solid phase peptide synthesis protocol. High‐performance liquid chromatograms and mass spectrometry (LCMS and analytical HPLC) were employed to determine the purity and molecular weight of the peptide. Surface tension measurements are used to evaluate peptide surface activity. The primary structure of the peptideis anlaysed by NMR spectroscopy. Circular dichroism studies demonstrate that the secondary structure of the peptide changes minimally when it attached to anionic lipid bilayers (POPC : POPG). According to a steady‐state fluorescence experiment, peptides P1 and P2 preferentially interact with anionic (POPC : POPG) lipid bilayers rather than zwitterionic (POPC) lipid bilayers. The peptide P2 interacts preferentially with anionic lipid membranes and has an anticancer effect due to RGD and NGR binding motifs. The cytotoxicity of the MTT experiment demonstrated that both normal and cancerous cells could lower peptide concentrations below their IC50 levels. Molecular docking also supports in‐vitro anticancer research. Both peptides P1 and P2 were non‐toxic to vero cells and appeared to be more promising as it demonstrated wide spectrum anticancer activity.

Membrane-Mediated Allosteric Action of Serotonin on a Noncognate G-Protein-Coupled Receptor.

The structure and dynamics of the lipid membrane can affect the activity of membrane proteins. Therefore, small lipophilic molecules that alter membrane properties (such as the neurotransmitter serotonin) can potentially modulate receptor activity without binding to the receptor. Here, we investigated how the activity of neuropeptide Y type 4 receptor (Y4R, reconstituted in lipid bicelles) is modulated by serotonin, which has no known interaction with Y4R. We found a serotonin-concentration-dependent decrease (down to 0.1 mM of serotonin) in the ligand affinity of Y4R. This effect correlates with a serotonin-induced reduction of the resistance of the bilayer to indentation (measured by atomic force microscopy) and bilayer thickness (measured by solid state NMR) in two different types of zwitterionic lipid bicelles. Our findings indicate a "membrane-mediated allosteric effect" of serotonin on the activation of Y4R and suggest the potential for developing pharmacophores, which can modulate cellular signaling without directly interacting with any receptor.

Antivesiculation and Complete Unbinding of Tail-Tethered Lipids.

We report the effect of tail-tethering on vesiculation and complete unbinding of bilayered membranes. Amphiphilic molecules of a bolalipid, resembling the tail-tethered molecular structure of archaeal lipids, with two identical zwitterionic phosphatidylcholine headgroups self-assemble into a large flat lamellar membrane, in contrast to the multilamellar vesicles (MLVs) observed in its counterpart, monopolar nontethered zwitterionic lipids. The antivesiculation is confirmed by small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cyro-TEM). With the net charge of zero and higher bending rigidity of the membrane (confirmed by neutron spin echo (NSE) spectroscopy), the current membrane theory would predict that membranes should stack with each other (aka "bind") due to dominant van der Waals attraction, while the outcome of the nonstacking ("unbinding") membrane suggests that the theory needs to include entropic contribution for the nonvesicular structures. This report pioneers an understanding of how the tail-tethering of amphiphiles affects the structure, enabling better control over the final nanoscale morphology.

Insight into the hydration friction of lipid bilayers.

Hydration layers formed on charged sites play crucial roles in many hydration lubrication systems in aqueous media. However, the underlying molecular mechanism is not well understood. Herein, we explored the hydration friction of lipid bilayers with different charged headgroups at the nanoscale through a combination of frequency-modulation atomic force microscopy and friction force microscopy. The nanoscale friction experiments showed that the hydration friction coefficient and frictional energy dissipation of a cationic lipid (DPTAP) were much lower than those of zwitterionic (DPPE) and anionic (DPPG) lipids. The hydration layer probing at the surfaces of different lipid bilayers clearly revealed the relationship between the charged lipid headgroups and hydration layer structures. Our detailed analysis demonstrated that the cationic lipid had the largest hydration force in comparison with zwitterionic and anionic lipids. These friction and hydration force results indicated that the difference of the lipid headgroup charge resulted in different hydration strengths which led to the difference of hydration friction behaviors. The findings in this study provide molecular insights into the hydration friction of lipid bilayers, which has potential implications for the development of efficient hydration lubrication systems with boundary lipid bilayers in aqueous media.

Peripheral adsorption of polylysine on one leaflet of a lipid bilayer reduces the lipid diffusion of both leaflets.

Understanding polycation-lipid interaction is essential not only in molecular biology but also in the biomedical industry and pharmacology. However, the effect of the polycation-lipid interaction on the molecular properties of lipids in biomembranes remains elusive. Here, two fluorescence correlation spectroscopies (FCSs), pulse-interleaved excitation (PIE) FCS and lifetime-based FCS, were performed to elucidate the change in the lipid diffusion of a model biomembrane induced by polylysine (PLL) adsorption. The results of PIE-FCS showed that the diffusions of both anionic and zwitterionic lipids become slower in the presence of PLL but the mobility of the anionic lipids is much reduced, suggesting the preferential interaction between the PLL and the anionic lipids due to the electrostatic attraction. Furthermore, leaflet-specific lipid diffusion analysis by lifetime-based FCS clearly showed that PLL adsorption on one leaflet of the membrane reduces the lipid diffusion of both leaflets in the same manner. This clearly indicates that the interleaflet coupling is strong in the presence of PLL.

Magnetic micromotors crossing lipid membranes.

Nano/micromotors are self-propelled particles that show enhanced motion upon being triggered by a stimulus. Their use in nanomedicine has been widely explored, with special focus on imaging or drug delivery. However, a thorough understanding of the requirements for more efficient locomotion is still lacking. In this paper, we assembled magnetically propelled motors of different sizes (i.e., 0.5, 1 and 4 μm) and surface chemistries (positive charge or PEGylated) and assessed their motion in the presence of giant unilamellar lipid vesicles (GUVs) of varying compositions (zwitterionic, negatively charged and saturated lipids). Unexpectedly, the size does not seem to be the dominating characteristics that governs the ability of the motors to cross lipid membranes. Specifically, the 0.5 μm PEGylated motors have very limited ability to cross the lipid membrane of GUVs due to their non-interacting nature compared to their equally sized positively charged counterparts. Furthermore, membranes made of saturated lipids and, in particular, in combination with a weak magnetic field facilitate motors' crossing, regardless of their size. The results were validated by in-house data-driven statistical analysis that employs experimental data to allow for the identification of individual motor motion in the ensemble when meeting the lipid membranes. Altogether, we provide insight into motor locomotion when they interact with a biological barrier considering both the entire ensemble and the individual motors, which has the potential to support considerations of future motor designs.

The molecular basis for the increased stability of the FUS-LC fibril at the anionic membrane- and air-water interfaces.

Self-organization of biomolecules can lead to the formation of liquid droplets, hydrogels, and irreversible aggregates that bear immense significance in biology and diseases. Despite the considerable number of studies conducted on biomolecular condensation in bulk solution, there is still a lack of understanding of how different surfaces regulate the condensation process. In this context, recent studies showed that, in contrast to zwitterionic lipid membranes, anionic membranes promoted the production of liquid droplets of FUsed in Sarcoma Low Complexity domain (FUS-LC) despite exhibiting no specific protein-lipid interactions. Moreover, the air-water interface led to a solid fibril-like aggregate of FUS-LC. The molecular mechanism of condensation/aggregation of proteins in response to surfaces of various charged states or levels of hydrophobicity remains to be better elucidated. Here, we provide initial insights into this question by investigating the stability of a small β fibril state of FUS-LC in bulk solution vs. membrane- and air-water interfaces. We perform multiple independent molecular dynamics simulations with distinct starting conformations for each system to demonstrate the statistical significance of our findings. Our study demonstrates the stability of the FUS-LC fibril in the presence of anionic membranes on the μs timescale while the fibril falls apart in bulk solution. We observe that a zwitterionic membrane does not enhance the stability of the fibril and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) has a higher propensity to stabilize the fibril than dioleoylphosphatidylglycerol (DOPG), in qualitative agreement with experiments. We further show that the fibril becomes more stable at the air-water interface. We pinpoint interfacial solvation at the membrane- and air-water interfaces as a key factor that contributes to the stabilization of the peptide assembly.