pH-Modulated Nanoarchitectonics for Enhancement of Multivalency-Induced Vesicle Shape Deformation at Receptor-Presenting Lipid Membrane Interfaces.

Multivalent ligand-receptor interactions between receptor-presenting lipid membranes and ligand-modified biological and biomimetic nanoparticles influence cellular entry and fusion processes. Environmental pH changes can drive these membrane-related interactions by affecting membrane nanomechanical properties. Quantitatively, however, the corresponding effects on high-curvature, sub-100 nm lipid vesicles are scarcely understood, especially in the multivalent binding context. Herein, we employed the label-free localized surface plasmon resonance (LSPR) sensing technique to track the multivalent attachment kinetics, shape deformation, and surface coverage of biotin ligand-functionalized, zwitterionic lipid vesicles with different ligand densities on a streptavidin receptor-coated supported lipid bilayer under varying pH conditions (4.5, 6, 7.5). Our results demonstrate that more extensive multivalent interactions caused greater vesicle shape deformation across the tested pH conditions, which affected vesicle surface packing as well. Notably, there were also pH-specific differences, i.e., a higher degree of vesicle shape deformation was triggered at a lower multivalent binding energy in pH 4.5 than in pH 6 and 7.5 conditions. These findings support that the nanomechanical properties of high-curvature lipid membranes, especially the membrane bending energy and the corresponding responsiveness to multivalent binding interactions, are sensitive to solution pH, and indicate that multivalency-induced vesicle shape deformation occurs slightly more readily in acidic pH conditions relevant to biological environments.

Ionizable lipids in bio-inspired nanocarriers.

In applications of bio-inspired nanoparticles (NPs), their composition is often optimised by including ionizable lipids. I use a generic statistical model to describe the charge and potential distributions in lipid nanoparticles (LNPs) containing such lipids. The LNP structure is considered to contain the biophase regions separated by narrow interphase boundaries with water. Ionizable lipids are uniformly distributed at the biophase-water boundaries. The potential is there described at the mean-filed level combining the Langmuir-Stern equation for ionizable lipids and the Poisson-Boltzmann equation for other charges in water. The latter equation is used outside a LNP as well. With physiologically reasonable parameters, the model predicts the scale of the potential in a LNP to be rather low, smaller or about [Formula: see text], and to change primarily near the LNP-solution interface or, more precisely, inside an NP near this interface because the charge of ionizable lipids becomes rapidly neutralized along the coordinate towards the center of a LNP. The extent of dissociation-mediated neutralization of ionizable lipids along this coordinate increases but only slightly. Thus, the neutralization is primarily due to the negative and positive ions related to the ionic strength in solution and located inside a LNP.

Release of molecules from nanocarriers.

Release of drugs or vaccine molecules from macro-, micro-, and nano-sized carriers is usually considered to be limited by diffusion and/or carrier dissolution and/or erosion. The corresponding experimentally observed kinetics are customarily fitted by using the empirical Weibull and Korsemeyer-Peppas expressions. With decreasing size of carriers down to about 100 nm, the timescale of diffusion decreases, and accordingly the release can be kinetically limited, i.e., controlled by jumps of molecules located near the carrier-solution interface. In addition, nanocarriers (e.g., lipid nanoparticles) are often structurally heterogeneous so that the absorption of molecules there can be interpreted in terms of energetic heterogeneity, i.e., distribution of energies corresponding to binding sites and activation barriers for release. Herein, I present a general kinetic model aimed at such situations. For illustration, the deviation of the molecule binding energy from the maximum value was considered to be about 4-8 kcal mol-1. With this physically reasonable (for non-covalent interaction) scale of energetic heterogeneity, the predicted kinetics (i) are linear in the very beginning and then, with increasing time, become logarithmic and (ii) can be nearly perfectly fitted by employing the Weibull or Korsmeyer-Peppas expressions with the exponent in the range from 0.6 to 0.75. Such values of the exponent are often obtained in experiments and customarily associated with non-Fickian diffusion. My analysis shows that the energetic heterogeneity can be operative here as well.

Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein.

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).

Dissimilar Deformation of Fluid- and Gel-Phase Liposomes upon Multivalent Interaction with Cell Membrane Mimics Revealed Using Dual-Wavelength Surface Plasmon Resonance.

The mechanical properties of biological nanoparticles play a crucial role in their interaction with the cellular membrane, in particular for cellular uptake. This has significant implications for the design of pharmaceutical carrier particles. In this context, liposomes have become increasingly popular, among other reasons due to their customizability and easily varied physicochemical properties. With currently available methods, it is, however, not trivial to characterize the mechanical properties of nanoscopic liposomes especially with respect to the level of deformation induced upon their ligand-receptor-mediated interaction with laterally fluid cellular membranes. Here, we utilize the sensitivity of dual-wavelength surface plasmon resonance to probe the size and shape of bound liposomes (∼100 nm in diameter) as a means to quantify receptor-induced deformation during their interaction with a supported cell membrane mimic. By comparing biotinylated liposomes in gel and fluid phases, we demonstrate that fluid-phase liposomes are more prone to deformation than their gel-phase counterparts upon binding to the cell membrane mimic and that, as expected, the degree of deformation depends on the number of ligand-receptor pairs that are engaged in the multivalent binding.

Unraveling How Cholesterol Affects Multivalency-Induced Membrane Deformation of Sub-100 nm Lipid Vesicles.

Cholesterol plays a critical role in modulating the lipid membrane properties of biological and biomimetic systems and recent attention has focused on its role in the functions of sub-100 nm lipid vesicles and lipid nanoparticles. These functions often rely on multivalent ligand-receptor interactions involving membrane attachment and dynamic shape transformations while the extent to which cholesterol can influence such interaction processes is largely unknown. To address this question, herein, we investigated the attachment of sub-100 nm lipid vesicles containing varying cholesterol fractions (0-45 mol %) to membrane-mimicking supported lipid bilayer (SLB) platforms. Biotinylated lipids and streptavidin proteins were used as model ligands and receptors, respectively, while the localized surface plasmon resonance sensing technique was employed to track vesicle attachment kinetics in combination with analytical modeling of vesicle shape changes. Across various conditions mimicking low and high multivalency, our findings revealed that cholesterol-containing vesicles could bind to receptor-functionalized membranes but underwent appreciably less multivalency-induced shape deformation than vesicles without cholesterol, which can be explained by a cholesterol-mediated increase in membrane bending rigidity. Interestingly, the extent of vesicle deformation that occurred in response to increasingly strong multivalent interactions was less pronounced for vesicles with greater cholesterol fraction. The latter trend was rationalized by taking into account the strong dependence of the membrane bending energy on the area of the vesicle-SLB contact region and such insights can aid the engineering of membrane-enveloped nanoparticles with tailored biophysical properties.

Time-Resolved and Label-Free Evanescent Light-Scattering Microscopy for Mass Quantification of Protein Binding to Single Lipid Vesicles.

In-depth understanding of the intricate interactions between biomolecules and nanoparticles is hampered by a lack of analytical methods providing quantitative information about binding kinetics. Herein, we demonstrate how label-free evanescent light-scattering microscopy can be used to temporally resolve specific protein binding to individual surface-bound (∼100 nm) lipid vesicles. A theoretical model is proposed that translates protein-induced changes in light-scattering intensity into bound mass. Since the analysis is centered on individual lipid vesicles, the signal from nonspecific protein binding to the surrounding surface is completely avoided, offering a key advantage over conventional surface-based techniques. Further, by averaging the intensities from less than 2000 lipid vesicles, the sensitivity is shown to increase by orders of magnitude. Taken together, these features provide a new avenue in studies of protein-nanoparticle interaction, in general, and specifically in the context of nanoparticles in medical diagnostics and drug delivery.

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.

Interplay of Cellular mRNA, miRNA and Viral miRNA during Infection of a Cell.

The understanding of the kinetics of gene expression in cells infected by viruses is currently limited. As a rule, the corresponding models do not take viral microRNAs (miRNAs) into account. Such RNAs are, however, operative during the replication of some viruses, including, e.g., herpesvirus. To clarify the kinetics of this category (with emphasis on the information available for herpesvirus), I introduce a generic model describing the transient interplay of cellular mRNA, protein, miRNA and viral miRNA. In the absence of viral miRNA, the cellular miRNA is considered to suppress the populations of mRNA and protein due to association with mRNA and subsequent degradation. During infection, the viral miRNA suppresses the population of cellular miRNA and via this pathway makes the mRNA and protein populations larger. This effect becomes appreciable with the progress of intracellular viral replication. Using biologically reasonable parameters, I investigate the corresponding mean-field kinetics and show the scale of the effect of viral miRNAs on cellular miRNA and mRNA. The scale of fluctuations of the populations of these species is illustrated as well by employing Monte Carlo simulations.

Virology from the perspective of theoretical colloid and interface science.

Viral infections occur at very different length and time scales and include various processes, which can often be described using the models developed and/or employed in colloid and interface science. Bearing in mind the currently active COVID-19, I discuss herein the models aimed at viral transmission via respiratory droplets and the contact of virions with the epithelium. In a more general context, I outline the models focused on penetration of virions via the cellular membrane, initial stage of viral genome replication, and formation of viral capsids in cells. In addition, the models related to a new generation of drug delivery vehicles, for example, lipid nanoparticles with size about 100-200 nm, are discussed as well. Despite the high current interest in all these processes, their understanding is still limited, and this area is open for new theoretical studies.

Mechanistic Aspects of the Evolution of 3D Cholesterol Crystallites in a Supported Lipid Membrane via a Quartz Crystal Microbalance with Dissipation Monitoring.

The irreversible formation of cholesterol monohydrate crystals within biological membranes is the leading cause of various diseases, including atherosclerosis. Understanding the process of cholesterol crystallization is fundamentally important and could also lead to the development of improved therapeutic strategies. This has driven several studies investigating the effect of the environmental parameters on the induction of cholesterol crystallite growth and the structure of the cholesterol crystallites, while the kinetics and mechanistic aspects of the crystallite formation process within lipid membranes remain poorly understood. Herein, we fabricated cholesterol crystallites within a supported lipid bilayer (SLB) by adsorbing a cholesterol-rich bicellar mixture onto a glass and silica surface and investigated the real-time kinetics of cholesterol crystallite nucleation and growth using epifluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D) monitoring. Microscopic imaging showed the evolution of the morphology of cholesterol crystallites from nanorod- and plate-shaped habits during the initial stage to mostly large, micron-sized three-dimensional (3D) plate-shaped crystallites in the end, which was likened to Ostwald ripening. QCM-D kinetics revealed unique signal responses during the later stage of the growth process, characterized by simultaneous positive frequency shifts, nonmonotonous energy dissipation shifts, and significant overtone dependence. Based on the optically observed changes in crystallite morphology, we discussed the physical background of these unique QCM-D signal responses and the mechanistic aspects of Ostwald ripening in this system. Together, our findings revealed mechanistic details of the cholesterol crystallite growth kinetics, which may be useful in biointerfacial sensing and bioanalytical applications.

Kinetic aspects of virus targeting by nanoparticles in vivo.

One of the suggested ways of the use of nanoparticles in virology implies their association with and subsequent deactivation of virions. The conditions determining the efficiency of this approach in vivo are now not clear. Herein, I propose the first kinetic model describing the corresponding processes and clarifying these conditions. My analysis indicates that nanoparticles can decrease concentration of infected cells by a factor of one order of magnitude, but this decrease itself (without feedback of the immune system) is insufficient for full eradication of infection. It can, however, induce delay in the progress of infection, and this delay can help to form sufficient feedback of the immune system.

Metal-polymer hybrid nanomaterials for plasmonic ultrafast hydrogen detection.

Hydrogen-air mixtures are highly flammable. Hydrogen sensors are therefore of paramount importance for timely leak detection during handling. However, existing solutions do not meet the stringent performance targets set by stakeholders, while deactivation due to poisoning, for example by carbon monoxide, is a widely unsolved problem. Here we present a plasmonic metal-polymer hybrid nanomaterial concept, where the polymer coating reduces the apparent activation energy for hydrogen transport into and out of the plasmonic nanoparticles, while deactivation resistance is provided via a tailored tandem polymer membrane. In concert with an optimized volume-to-surface ratio of the signal transducer uniquely offered by nanoparticles, this enables subsecond sensor response times. Simultaneously, hydrogen sorption hysteresis is suppressed, sensor limit of detection is enhanced, and sensor operation in demanding chemical environments is enabled, without signs of long-term deactivation. In a wider perspective, our work suggests strategies for next-generation optical gas sensors with functionalities optimized by hybrid material engineering.

Ligand-receptor-mediated attachment of lipid vesicles to a supported lipid bilayer.

The interaction of exosomes (cell-secreted [Formula: see text]100 nm-sized extracellular vesicles) or membrane-enveloped virions with cellular lipid membranes is often mediated by relatively weak ligand-receptor bonds. Interactions of this type can be studied using vesicles and observing their attachment to receptors located in a lipid bilayer formed at a solid surface. The contact region between a vesicle and the supported lipid bilayer and accordingly the number of ligand-receptor pairs there can be increased by deforming a vesicle. Herein, I (i) estimate theoretically the corresponding deformation energy assuming a disk-like or elongated shape of vesicles, (ii) present the equations allowing one to track such deformations by employing total internal reflection fluorescence microscopy and surface plasmon resonance, and (iii) briefly discuss some related experimental studies.

Single-Molecule Detection with Lightguiding Nanowires: Determination of Protein Concentration and Diffusivity in Supported Lipid Bilayers.

Determining the surface concentration and diffusivity of cell-membrane-bound molecules is central to the understanding of numerous important biochemical processes taking place at cell membranes. Here we use the high aspect ratio and lightguiding properties of semiconductor nanowires (NWs) to detect the presence of single freely diffusing proteins bound to a lipid bilayer covering the NW surface. Simultaneous observation of light-emission dynamics of hundreds of individual NWs occurring on the time scale of only a few seconds is interpreted using analytical models and employed to determine both surface concentration and diffusivity of cholera toxin subunit B (CTxB) bound to GM1 gangliosides in supported lipid bilayer (SLB) at surface concentrations down to below one CTxB per µm2. In particular, a decrease in diffusivity was observed with increasing GM1 content in the SLB, suggesting increasing multivalent binding of CTxB to GM1. The lightguiding capability of the NWs makes the method compatible with conventional epifluorescence microscopy, and it is shown to work well for both photostable and photosensitive dyes. These features make the concept an interesting complement to existing techniques for studying the diffusivity of low-abundance cell-membrane-bound molecules, expanding the rapidly growing use of semiconductor NWs in various bioanalytical sensor applications and live cell studies.

How the partial-slip boundary condition can influence the interpretation of the DLS and NTA data.

Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) are widely used to determine the size of biological nanoparticles in liquid. In both cases, one first measures the nanoparticle diffusion coefficient and then converts it to the nanoparticle radius via the Stokes-Einstein relation. This relation is based on the no-slip boundary condition. Now, there is evidence that this condition can be violated in biologically relevant cases (e.g., for vesicles) and that in such situations the partial-slip boundary condition is more suitable. I show (i) how the latter condition can be employed in the context of DLS and NTA and (ii) that the use of the former condition may result in underestimation of the nanoparticle radius by about 10 nm compared with the nominal one.

Competition for Membrane Receptors: Norovirus Detachment via Lectin Attachment.

Virus internalization into the host cells occurs via multivalent interactions, in which a single virus binds to multiple receptors in parallel. Because of analytical and experimental limitations this complex type of interaction is still poorly understood and quantified. Herein, the multivalent interaction of norovirus-like particles (noroVLPs) with H or B type 1 glycosphingolipids (GSLs), embedded in a supported phospholipid bilayer, is investigated by following the competition between noroVLPs and a lectin (from Ralstonia solanacearum) upon binding to these GSLs. Changes in noroVLP and lectin coverage, caused by competition, were monitored for both GSLs and at different GSL concentrations using quartz crystal microbalance with dissipation monitoring. The study yields information about the minimum GSL concentration needed for (i) noroVLPs to achieve firm attachment to the bilayer prior to competition and to (ii) remain firmly attached to the bilayer during competition. We show that these two concentrations are almost identical for the H type 1-noroVLP interaction but differ for B type 1, indicating an accumulation of B type 1 GSLs in the noroVLP-bilayer interaction area. Furthermore, the GSL concentration required for firm attachment is significantly larger for H type 1 than for B type 1, indicating a higher affinity of noroVLP toward B type 1. This finding is supported by extracting the energy of single noroVLP-H type 1 and noroVLP-B type 1 bonds from the competition kinetics, which were estimated to be 5 and 6 kcal/mol, respectively. This demonstrates the potential of utilizing competitive binding kinetics to analyze multivalent interactions, which has remained difficult to quantify using conventional approaches.

Influence of NaCl Concentration on Bicelle-Mediated SLB Formation.

The deposition of two-dimensional bicellar disks on hydrophilic surfaces is an emerging approach to fabricate supported lipid bilayers (SLBs) that requires minimal sample preparation, works at low lipid concentrations, and yields high-quality SLBs. While basic operating steps in the fabrication protocol mimic aspects of the conventional vesicle fusion method, lipid bicelles and vesicles have distinct architectural properties, and understanding how experimental parameters affect the efficiency of bicelle-mediated SLB formation remains to be investigated. Herein, using the quartz crystal microbalance-dissipation and localized surface plasmon resonance techniques, we investigated the effect of bulk NaCl concentration on bicelle-mediated SLB formation on silicon dioxide surfaces. For comparison, similar experiments were conducted with vesicles as well. In both cases, SLB formation was observed to occur rapidly provided that the NaCl concentration was sufficiently high (>50 mM). Under such conditions, the effect of NaCl concentration on SLB formation was minor in the case of bicelles and significant in the case of vesicles where it is expected to be related primarily to osmotic pressure. At lower NaCl concentrations, bicelles also formed SLBs but slowly, whereas adsorbed vesicles remained intact. These findings were complemented by time-lapsed fluorescence microscopy imaging and fluorescence recovery after photobleaching measurements that corroborated bicelle-mediated SLB formation across the range of tested NaCl concentrations. The results are discussed by comparing the architectural properties of bicelles and vesicles along with theoretical analysis of the corresponding adsorption kinetics.

Slow relaxation during and after perturbation of bistable kinetics of gene expression.

After a short perturbation of a bistable genetic network, it returns to its initial steady state or transits to another steady state. The time scale characterizing such transient regimes can be appreciably longer compared to those of the degradation of the perturbed mRNAs and proteins. The author shows in detail the specifics of this slowdown of the transient kinetics using mean-field kinetic equations and Monte Carlo simulations. Attention is focused on nanocarrier-mediated delivery and release of short non-coding RNA (e.g., miRNA or siRNA) into cells with subsequent suppression of the populations of the targeted mRNA and corresponding protein.

Spatio-temporal aspects of the interplay of cancer and the immune system.

The conventional mean-field kinetic models describing the interplay of cancer and the immune system are temporal and predict exponential growth or elimination of the population of tumour cells provided their number is small and their effect on the immune system is negligible. More complex kinetics are associated with non-linear features of the response of the immune system. The generic model presented in this communication takes into account that the rates of the birth and death of tumour cells inside a tumour spheroid can significantly depend on the radial coordinate due to diffusion limitations in the supply of nutrients and/or transport of the species (cells and proteins) belonging to the immune system. In this case, non-trivial kinetic regimes are shown to be possible even without appreciable perturbation of the immune system.