Charge Matters: Mutations in Omicron Variant Favor Binding to Cells.

Evidence is strengthening to suggest that the novel SARS-CoV-2 mutant Omicron, with its more than 60 mutations, will spread and dominate worldwide. Although the mutations in the spike protein are known, the molecular basis for why the additional mutations in the spike protein that have not previously occurred account for Omicron's higher infection potential, is not understood. We propose, based on chemical rational and molecular dynamics simulations, that the elevated occurrence of positively charged amino acids in certain domains of the spike protein (Delta: +4; Omicron: +5 vs. wild type) increases binding to cellular polyanionic receptors, such as heparan sulfate due to multivalent charge-charge interactions. This observation is a starting point for targeted drug development.

Toolbox of Biodegradable Dendritic (Poly glycerol sulfate)-SS-poly(ester) Micelles for Cancer Treatment: Stability, Drug Release, and Tumor Targeting.

In this paper, we present well-defined dPGS-SS-PCL/PLGA/PLA micellar systems demonstrating excellent capabilities as a drug delivery platform in light of high stability and precise in vitro and in vivo drug release combined with active targetability to tumors. These six amphiphilic block copolymers were each targeted in two different molecular weights (8 or 16 kDa) and characterized using 1H NMR, gel permeation chromatography (GPC), and elemental analysis. The block copolymer micelles showed monodispersed size distributions of 81-187 nm, strong negative charges between -52 and -41 mV, and low critical micelle concentrations (CMCs) of up to 1.13-3.58 mg/L (134-527 nM). The serum stability was determined as 94% after 24 h. The drug-loading efficiency for Sunitinib ranges from 38 to 83% (8-17 wt %). The release was selectively triggered by glutathione (GSH) and lipase, reaching 85% after 5 days, while only 20% leaching was observed under physiological conditions. Both the in vitro and in vivo studies showed sustained release of Sunitinib over 1 week. CCK-8 assays on HeLa lines demonstrated the high cell compatibility (1 mg/mL, 94% cell viability, 48 h) and the high cancer cell toxicity of Sunitinib-loaded micelles (IC50 2.5 µg/mL). By in vivo fluorescence imaging studies on HT-29 tumor-bearing mice, the targetability of dPGS7.8-SS-PCL7.8 enabled substantial accumulation in tumor tissue compared to nonsulfated dPG3.9-SS-PCL7.8. As a proof of concept, Sunitinib-loaded dPGS-SS-poly(ester) micelles improved the antitumor efficacy of the chemotherapeutic. A tenfold lower dosage of loaded Sunitinib led to an even higher tumor growth inhibition compared to the free drug, as demonstrated in a HeLa human cervical tumor-bearing mice model. No toxicity for the organism was observed, confirming the good biocompatibility of the system.

Understanding the Interaction of Polyelectrolyte Architectures with Proteins and Biosystems.

The counterions neutralizing the charges on polyelectrolytes such as DNA or heparin may dissociate in water and greatly influence the interaction of such polyelectrolytes with biomolecules, particularly proteins. In this Review we give an overview of studies on the interaction of proteins with polyelectrolytes and how this knowledge can be used for medical applications. Counterion release was identified as the main driving force for the binding of proteins to polyelectrolytes: Patches of positive charge become multivalent counterions of the polyelectrolyte and lead to the release of counterions from the polyelectrolyte and a concomitant increase in entropy. This is shown from investigations on the interaction of proteins with natural and synthetic polyelectrolytes. Special emphasis is paid to sulfated dendritic polyglycerols (dPGS). The Review demonstrates that we are moving to a better understanding of charge-charge interactions in systems of biological relevance. Research along these lines will aid and promote the design of synthetic polyelectrolytes for medical applications.

Polysulfates Block SARS-CoV-2 Uptake through Electrostatic Interactions*.

Here we report that negatively charged polysulfates can bind to the spike protein of SARS-CoV-2 via electrostatic interactions. Using a plaque reduction assay, we compare inhibition of SARS-CoV-2 by heparin, pentosan sulfate, linear polyglycerol sulfate (LPGS) and hyperbranched polyglycerol sulfate (HPGS). Highly sulfated LPGS is the optimal inhibitor, with an IC50 of 67 µg mL-1 (approx. 1.6 µm). This synthetic polysulfate exhibits more than 60-fold higher virus inhibitory activity than heparin (IC50 : 4084 µg mL-1 ), along with much lower anticoagulant activity. Furthermore, in molecular dynamics simulations, we verified that LPGS can bind more strongly to the spike protein than heparin, and that LPGS can interact even more with the spike protein of the new N501Y and E484K variants. Our study demonstrates that the entry of SARS-CoV-2 into host cells can be blocked via electrostatic interactions, therefore LPGS can serve as a blueprint for the design of novel viral inhibitors of SARS-CoV-2.

Mechanism of the Oxidation of 3,3',5,5'-Tetramethylbenzidine Catalyzed by Peroxidase-Like Pt Nanoparticles Immobilized in Spherical Polyelectrolyte Brushes: A Kinetic Study.

Experimental and kinetic modelling studies are presented to investigate the mechanism of 3,3',5,5'-tetramethylbenzidine (TMB) oxidation by hydrogen peroxide (H2 O2 ) catalyzed by peroxidase-like Pt nanoparticles immobilized in spherical polyelectrolyte brushes (SPB-Pt). Due to the high stability of SPB-Pt colloidal, this reaction can be monitored precisely in situ by UV/VIS spectroscopy. The time-dependent concentration of the blue-colored oxidation product of TMB expressed by different kinetic models was used to simulate the experimental data by a genetic fitting algorithm. After falsifying the models with abundant experimental data, it is found that both H2 O2 and TMB adsorb on the surface of Pt nanoparticles to react, indicating that the reaction follows the Langmuir-Hinshelwood mechanism. A true rate constant k, characterizing the rate-determining step of the reaction and which is independent on the amount of catalysts used, is obtained for the first time. Furthermore, it is found that the product adsorbes strongly on the surface of nanoparticles, thus inhibiting the reaction. The entire analysis provides a new perspective to study the catalytic mechanism and evaluate the catalytic activity of the peroxidase-like nanoparticles.

Thermodynamic Analysis of the Interaction of Heparin with Lysozyme.

Glycosaminoglycan (GAG)-protein binding governs critically important signaling events in living matter. Aiming at a quantitative analysis of the involved processes, we herein present a thermodynamic study of the interaction of the model GAG heparin and lysozyme in aqueous solution. Heparin is a highly charged linear polyelectrolyte with a charge parameter of 2.9 (37 °C). The binding constant Kb was determined by ITC as a function of the temperature and ionic strength adjusted through the concentration cs of added salt. The dependence on salt concentration cs was used to determine the net number of released counterions. Moreover, the binding constant at a reference salt concentration of 1 M Kb(1 M) was determined by extrapolation. The dependence on temperature of Kb was used to dissect the binding free energy ΔGb into the respective enthalpies ΔHb and entropies ΔSb together with the specific heat Δcp. A strong enthalpy-entropy cancelation was found similar to the results for many other systems. The binding free energy ΔGb could furthermore be split up into a part ΔGci due to counterion release and a residual part ΔGres. The latter quantity reflects specific contributions as, e.g., salt bridges, van der Waals interactions, or hydrogen bonds. The entire analysis shows that heparin-lysozyme interactions are mainly caused by counterion release; that is, ca. three counterions are being released upon binding one lysozyme molecule. Our reported approach of quantifying interactions between glycosaminoglycans and proteins is generally applicable and suitable to provide new insights in the physical modulation of biomolecular signals.

Thermodynamic Analysis of the Uptake of a Protein in a Spherical Polyelectrolyte Brush.

A thermodynamic study of the adsorption of Human Serum Albumin (HSA) onto spherical polyelectrolyte brushes (SPBs) by isothermal titration calorimetry (ITC) is presented. The SPBs are composed of a solid polystyrene core bearing long chains of poly(acrylic acid). ITC measurements done at different temperatures and ionic strengths lead to a full set of thermodynamicbinding constants together with the enthalpies and entropies of binding. The adsorption of HSA onto SPBs is described with a two-step model. The free energy of binding ΔGb depends only weakly on temperature because of a marked compensation of enthalpy by entropy. Studies of the adsorbed HSA by Fourier transform infrared spectroscopy (FT-IR) demonstrate no significant disturbance in the secondary structure of the protein. The quantitative analysis demonstrates that counterion release is the major driving force for adsorption in a process where proteins become multivalent counterions of the polyelectrolyte chains upon adsorption. A comparison with the analysis of other sets of data related to the binding of HSA to polyelectrolytes demonstrates that the cancellation of enthalpy and entropy is a general phenomenon that always accompanies the binding of proteins to polyelectrolytes dominated by counterion release.

Highly Dispersible Hexagonal Carbon-MoS2 -Carbon Nanoplates with Hollow Sandwich Structures for Supercapacitors.

MoS2 , a typical layered transition-metal dichalcogenide, is promising as an electrode material in supercapacitors. However, its low electrical conductivity could lead to limited capacitance if applied in electrochemical devices. Herein, a new nanostructure composed of hollow carbon-MoS2 -carbon was successfully synthesized through an l-cysteine-assisted hydrothermal method by using gibbsite as a template and polydopamine as a carbon precursor. After calcination and etching of the gibbsite template, uniform hollow platelets, which were made of a sandwich-like assembly of partial graphitic carbon and two-dimensional layered MoS2 flakes, were obtained. The platelets showed excellent dispersibility and stability in water, and good electrical conductivity due to carbon provided by the calcination of polydopamine coatings. The hollow nanoplate morphology of the material provided a high specific surface area of 543 m2 g-1 , a total pore volume of 0.677 cm3 g-1 , and fairly small mesopores (≈5.3 nm). The material was applied in a symmetric supercapacitor and exhibited a specific capacitance of 248 F g-1 (0.12 F cm-2 ) at a constant current density of 0.1 A g-1 ; thus suggesting that hollow carbon-MoS2 -carbon nanoplates are promising candidate materials for supercapacitors.

Interaction of Proteins with Polyelectrolytes: Comparison of Theory to Experiment.

We discuss recent investigations of the interaction of polyelectrolytes with proteins. In particular, we review our recent studies on the interaction of simple proteins such as human serum albumin (HSA) and lysozyme with linear polyelectrolytes, charged dendrimers, charged networks, and polyelectrolyte brushes. In all cases discussed here, we combined experimental work with molecular dynamics (MD) simulations and mean-field theories. In particular, isothermal titration calorimetry (ITC) has been employed to obtain the respective binding constants Kb and the Gibbs free energy of binding. MD simulations with explicit counterions but implicit water demonstrate that counterion release is the main driving force for the binding of proteins to strongly charged polyelectrolytes: patches of positive charges located on the surface of the protein become multivalent counterions of the polyelectrolyte, thereby releasing a number of counterions condensed on the polyelectrolyte. The binding Gibbs free energy due to counterion release is predicted to scale with the logarithm of the salt concentration in the system, which is verified by both simulations and experiment. In several cases, namely, for the interaction of proteins with linear polyelectrolytes and highly charged hydrophilic dendrimers, the binding constant could be calculated from simulations to very good approximation. This finding demonstrated that in these cases explicit hydration effects do not contribute to the Gibbs free energy of binding. The Gibbs free energy can also be used to predict the kinetics of protein uptake by microgels for a given system by applying dynamic density functional theory. The entire discussion demonstrates that the direct comparison of theory with experiments can lead to a full understanding of the interaction of proteins with charged polymers. Possible implications for applications, such as drug design, are discussed.

Counterion-Release Entropy Governs the Inhibition of Serum Proteins by Polyelectrolyte Drugs.

Dendritic polyelectrolytes constitute high potential drugs and carrier systems for biomedical purposes. Still, their biomolecular interaction modes, in particular those determining the binding affinity to proteins, have not been rationalized. We study the interaction of the drug candidate dendritic polyglycerol sulfate (dPGS) with serum proteins using isothermal titration calorimetry (ITC) interpreted and complemented with molecular computer simulations. Lysozyme is first studied as a well-defined model protein to verify theoretical concepts, which are then applied to the important cell adhesion protein family of selectins. We demonstrate that the driving force of the strong complexation, leading to a distinct protein corona, originates mainly from the release of only a few condensed counterions from the dPGS upon binding. The binding constant shows a surprisingly weak dependence on dPGS size (and bare charge) which can be understood by colloidal charge-renormalization effects and by the fact that the magnitude of the dominating counterion-release mechanism almost exclusively depends on the interfacial charge structure of the protein-specific binding patch. Our findings explain the high selectivity of P- and L-selectins over E-selectin for dPGS to act as a highly anti-inflammatory drug. The entire analysis demonstrates that the interaction of proteins with charged polymeric drugs can be predicted by simulations with unprecedented accuracy. Thus, our results open new perspectives for the rational design of charged polymeric drugs and carrier systems.

Charge and hydration structure of dendritic polyelectrolytes: molecular simulations of polyglycerol sulphate.

Macromolecules based on dendritic or hyperbranched polyelectrolytes have been emerging as high potential candidates for biomedical applications. Here we study the charge and solvation structure of dendritic polyglycerol sulphate (dPGS) of generations 0 to 3 in aqueous sodium chloride solution by explicit-solvent molecular dynamics computer simulations. We characterize dPGS by calculating several important properties such as relevant dPGS radii, molecular distributions, the solvent accessible surface area, and the partial molecular volume. In particular, as the dPGS exhibits high charge renormalization effects, we address the challenges of how to obtain a well-defined effective charge and surface potential of dPGS for practical applications. We compare implicit- and explicit-solvent approaches in our all-atom simulations with the coarse-grained simulations from our previous work. We find consistent values for the effective electrostatic size (i.e., the location of the effective charge of a Debye-Hückel sphere) within all the approaches, deviating at most by the size of a water molecule. Finally, the excess chemical potential of water insertion into dPGS and its thermodynamic signature are presented and rationalized.

Interaction of human serum albumin with dendritic polyglycerol sulfate: Rationalizing the thermodynamics of binding.

We study the thermodynamics of the interaction between human serum albumin (HSA) and dendritic polyglycerol sulfate (dPGS) of different sizes (generations) by isothermal titration calorimetry (ITC) and computer simulations. The analysis by ITC revealed the formation of a 1:1 complex for the dPGS-G2 of second generation. The secondary structure of HSA remained unchanged in the presence of dPGS-G2, as shown by circular dichroism. For higher generations, several HSA are bound to one polymer (dPGS-G4: 2; dPGS-G5.5: 4). The Gibbs free energy ΔG b was determined at different temperatures and salt concentrations. The binding constant K b exhibited a logarithmic dependence on the salt concentration thus indicating a marked contribution of counterion-release entropy to ΔG b. The number of released counterions (∼4) was found to be independent of temperature. In addition, the temperature dependence of ΔG b was small, whereas the enthalpy ΔH ITC was found to vary strongly with temperature. The corresponding heat capacity change ΔC p,ITC for different generations was of similar values [8 kJ/(mol K)]. The nonlinear van't Hoff analysis of ΔG b revealed a significant heat capacity change ΔC p,vH of similar magnitude [6 kJ/(mol K)] accompanied by a strong enthalpy-entropy compensation. ΔG b obtained by molecular dynamics simulation with implicit water and explicit ions coincided with experimental results. The agreement indicates that the enthalpy-entropy compensation assigned to hydration effects is practically total and the binding affinity is fully governed by electrostatic interactions.

Interaction of Charged Patchy Protein Models with Like-Charged Polyelectrolyte Brushes.

We study the adsorption of charged patchy particle models (CPPMs) on a thin film of a like-charged and dense polyelectrolyte (PE) brush (of 50 monomers per chain) by means of implicit-solvent, explicit-salt Langevin dynamics computer simulations. Our previously introduced set of CPPMs embraces well-defined one- and two-patched spherical globules, each of the same net charge and (nanometer) size, with mono- and multipole moments comparable to those of small globular proteins. We focus on electrostatic effects on the adsorption far away from the isoelectric point of typical proteins, i.e., where charge regulation plays no role. Despite the same net charge of the brush and globule, we observe large binding affinities up to tens of the thermal energy, kBT, which are enhanced by decreasing salt concentration and increasing charge of the patch(es). Our analysis of the distance-resolved potentials of mean force together with a phenomenological description of all leading interaction contributions shows that the attraction is strongest at the brush surface, driven by multipolar, Born (self-energy), and counterion-release contributions, dominating locally over the monopolar and steric repulsions.

Protein Immobilization onto Cationic Spherical Polyelectrolyte Brushes Studied by Small Angle X-ray Scattering.

The immobilization of bovine serum albumins (BSA) onto cationic spherical polyelectrolyte brushes (SPB) consisting of a solid polystyrene (PS) core and a densely grafted poly(2-aminoethyl methacrylate hydrochloride) (PAEMH) shell was studied by small-angle X-ray scattering (SAXS). The observed dynamics of adsorption of BSA onto SPB by time-resolved SAXS can be divided into two stages. In the first stage (tens of milliseconds), the added proteins as in-between bridge instantaneously caused the aggregation of SPB. Then BSA penetrated into the brush layer driven by electrostatic attractions, and reached equilibrium in the second stage (tens of seconds). The amount of BSA immobilized onto brush layer reached the maximum when pH was increased to about 6.1 and BSA concentration to 10 g/L. The cationic SPB were confirmed to provide stronger adsorption capacity for BSA compared to anionic ones.

Precise and Reversible Protein-Microtubule-Like Structure with Helicity Driven by Dual Supramolecular Interactions.

Protein microtubule is a significant self-assembled architecture found in nature with crucial biological functions. However, mimicking protein microtubules with precise structure and controllable self-assembly behavior remains highly challenging. In this work, we demonstrate that by using dual supramolecular interactions from a series of well-designed ligands, i.e., protein-sugar interaction and π-π stacking, highly homogeneous protein microtubes were achieved from tetrameric soybean agglutinin without any chemical or biological modification. Using combined cryo-EM single-particle reconstruction and computational modeling, the accurate structure of protein microtube was determined. The helical protein microtube is consisted of three protofilaments, each of which features an array of soybean agglutinin tetramer linked by the designed ligands. Notably, the microtubes resemble the natural microtubules in their structural and dynamic features such as the shape and diameter and the controllable and reversible assembly behavior, among others. Furthermore, the protein microtubes showed an ability to enhance immune response, demonstrating its great potential for biological applications.

Distribution of Sulfur in Carbon/Sulfur Nanocomposites Analyzed by Small-Angle X-ray Scattering.

The analysis of sulfur distribution in porous carbon/sulfur nanocomposites using small-angle X-ray scattering (SAXS) is presented. Ordered porous CMK-8 carbon was used as the host matrix and gradually filled with sulfur (20-50 wt %) via melt impregnation. Owing to the almost complete match between the electron densities of carbon and sulfur, the porous nanocomposites present in essence a two-phase system and the filling of the host material can be precisely followed by this method. The absolute scattering intensities normalized per unit of mass were corrected accounting for the scattering contribution of the turbostratic microstructure of carbon and amorphous sulfur. The analysis using the Porod parameter and the chord-length distribution (CLD) approach determined the specific surface areas and filling mechanism of the nanocomposite materials, respectively. Thus, SAXS provides comprehensive characterization of the sulfur distribution in porous carbon and valuable information for a deeper understanding of cathode materials of lithium-sulfur batteries.

Alzheimer's peptide amyloid-ß, fragment 22-40, perturbs lipid dynamics.

The peptide amyloid-ß (Aß) interacts with membranes of cells in the human brain and is associated with Alzheimer's disease (AD). The intercalation of Aß in membranes alters membrane properties, including the structure and lipid dynamics. Any change in the membrane lipid dynamics will affect essential membrane processes, such as energy conversion, signal transduction and amyloid precursor protein (APP) processing, and may result in the observed neurotoxicity associated with the disease. The influence of this peptide on membrane dynamics was studied with quasi-elastic neutron scattering, a technique which allows a wide range of observation times from picoseconds to nanoseconds, over nanometer length scales. The effect of the membrane integral neurotoxic peptide amyloid-ß, residues 22-40, on the in- and out-of-plane lipid dynamics was observed in an oriented DMPC/DMPS bilayer at 15 °C, in its gel phase, and at 30 °C, near the phase transition temperature of the lipids. Near the phase-transition temperature, a 1.5 mol% of peptide causes up to a twofold decrease in the lipid diffusion coefficients. In the gel-phase, this effect is reversed, with amyloid-ß(22-40) increasing the lipid diffusion coefficients. The observed changes in lipid diffusion are relevant to protein-protein interactions, which are strongly influenced by the diffusion of membrane components. The effect of the amyloid-ß peptide fragment on the diffusion of membrane lipids will provide insight into the membrane's role in AD.

Divergence of the third harmonic stress response to oscillatory strain approaching the glass transition.

The leading nonlinear stress response in a periodically strained concentrated colloidal dispersion is studied experimentally and by theory. A thermosensitive microgel dispersion serves as well-characterized glass-forming model, where the stress response at the first higher harmonic frequency (3ω for strain at frequency ω) is investigated in the limit of small amplitude. The intrinsic nonlinearity at the third harmonic exhibits a scaling behavior which has a maximum in an intermediate frequency window and diverges when approaching the glass transition. It captures the (in-) stability of the transient elastic structure. Elastic stresses in-phase with the third power of the strain dominate the scaling. Our results qualitatively differ from previously derived scaling behavior in dielectric spectroscopy of supercooled molecular liquids. This might indicate a dependence of the nonlinear response on the symmetry of the external driving under time reversal.

Reaction rate of a composite core-shell nanoreactor with multiple nanocatalysts.

We present a detailed theory for the total reaction rate constant of a composite core-shell nanoreactor, consisting of a central solid core surrounded by a hydrogel layer of variable thickness, where a given number of small catalytic nanoparticles are embedded at prescribed positions and are endowed with a prescribed surface reaction rate constant. Besides the precise geometry of the assembly, our theory accounts explicitly for the diffusion coefficients of the reactants in the hydrogel and in the bulk as well as for their transfer free energy jump upon entering the hydrogel shell. Moreover, we work out an approximate analytical formula for the overall rate constant, which is valid in the physically relevant range of geometrical and chemical parameters. We discuss in depth how the diffusion-controlled part of the rate depends on the essential variables, including the size of the central core. In particular, we derive some simple rules for estimating the number of nanocatalysts per nanoreactor for an efficient catalytic performance in the case of small to intermediate core sizes. Our theoretical treatment promises to provide a very useful and flexible tool for the design of superior performing nanoreactor geometries with optimized nanoparticle load.

Surface-Active Lipid Linings under Shear Load--A Combined in-Situ Neutron Reflectivity and ATR-FTIR Study.

We study shear effects in solid-supported lipid membrane stacks by simultaneous combined in-situ neutron reflectivity (NR) and attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR). The stacks mimic the terminal surface-active phospholipid (SAPL) coatings on cartilage in mammalian joints. Piles of 11 bilayer membranes of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) are immobilized at the interface of the solid silicon support and the liquid D2O backing phase. We replace the natural hyaluronic acid (HA) component of synovial fluid by a synthetic substitute, namely, poly(allylamine hydrochloride) (PAH), at identical concentration. We find the oligolamellar DMPC bilayer films strongly interacting with PAH resulting in a drastic increase of the membranes d spacing (by a factor of ∼5). Onset of shear causes a buckling-like deformation of the DMPC bilayers perpendicular to the applied shear field. With increasing shear rate we observe substantially enhanced water fractions in the membrane slabs which we attribute to increasing fragmentation caused by Kelvin-Helmholtz-like instabilities parallel to the applied shear field. Both effects are in line with recent theoretical predictions on shear-induced instabilities of lipid bilayer membranes in water (Hanasaki, I.; Walther, J. H.; Kawano, S.; Koumoutsakos, P. Phys. Rev. E 2010, 82, 051602). With the applied shear the interfacial lipid linings transform from their gel state Pß' to their fluid state Lα. Although in chain-molten state with reduced bending rigidity the lipid layers do not detach from their solid support. We hold steric bridging of the fragmented lipid bilayer membranes by PAH molecules responsible for the unexpected mechanical stability of the DMPC linings.