Adsorption of Mono- and Divalent Ions onto Dendritic Polyglycerol Sulfate (dPGS) as Studied Using Isothermal Titration Calorimetry.

The effective charge of highly charged polyelectrolytes is significantly lowered by a condensation of counterions. This effect is more pronounced for divalent ions. Here we present a study of the counterion condensation to dendritic polyglycerol sulfate (dPGS) that consists of a hydrophilic dendritic scaffold onto which sulfate groups are appended. The interactions between the dPGS and divalent ions (Mg2+ and Ca2+) were analyzed using isothermal titration calorimetry (ITC) and showed no ion specificity upon binding, but clear competition between the monovalent and divalent ions. Our findings, in line with the latest theoretical studies, demonstrate that a large fraction of the monovalent ions is sequentially replaced with the divalent ions.

Respiratory viruses interacting with cells: the importance of electrostatics.

The COVID-19 pandemic has rekindled interest in the molecular mechanisms involved in the early steps of infection of cells by viruses. Compared to SARS-CoV-1 which only caused a relatively small albeit deadly outbreak, SARS-CoV-2 has led to fulminant spread and a full-scale pandemic characterized by efficient virus transmission worldwide within a very short time. Moreover, the mutations the virus acquired over the many months of virus transmission, particularly those seen in the Omicron variant, have turned out to result in an even more transmissible virus. Here, we focus on the early events of virus infection of cells. We review evidence that the first decisive step in this process is the electrostatic interaction of the spike protein with heparan sulfate chains present on the surface of target cells: Patches of cationic amino acids located on the surface of the spike protein can interact intimately with the negatively charged heparan sulfate chains, which results in the binding of the virion to the cell surface. In a second step, the specific interaction of the receptor binding domain (RBD) within the spike with the angiotensin-converting enzyme 2 (ACE2) receptor leads to the uptake of bound virions into the cell. We show that these events can be expressed as a semi-quantitative model by calculating the surface potential of different spike proteins using the Adaptive Poison-Boltzmann-Solver (APBS). This software allows visualization of the positive surface potential caused by the cationic patches, which increased markedly from the original Wuhan strain of SARS-CoV-2 to the Omicron variant. The surface potential thus enhanced leads to a much stronger binding of the Omicron variant as compared to the original wild-type virus. At the same time, data taken from the literature demonstrate that the interaction of the RBD of the spike protein with the ACE2 receptor remains constant within the limits of error. Finally, we briefly digress to other viruses and show the usefulness of these electrostatic processes and calculations for cell-virus interactions more generally.

Analysis of the Binding of Cytokines to Highly Charged Polymer Networks.

A model describing the binding of biological signaling proteins to highly charged polymer networks is presented. The networks are formed by polyelectrolyte chains for which the distance between two charges at the chain is smaller than the Bjerrum length. Counterion condensation on such highly charged chains immobilizes a part of the counterions. The Donnan-equilibrium between the polymer network and the aqueous solution with salt concentration c s b $c_s^b$ is used to calculate the salt concentration of the co- and counterions c s g $c_s^g$ entering the network. Two factors are decisive: i) The electrostatic interaction between the network and the protein is given by the Donnan-potential of the network and the net charge of the protein. In addition to this leading term, a second term describes the change in the Born-energy of the proteins when entering the network. ii) The interaction of the protein with the highly charged chains within the network is governed by counterion release: Patches of positive charge at the protein become multivalent counterions of the polyelectrolyte chains thus releasing a concomitant number of condensed counterions. The model compares favorably to experimental data obtained on a set of biohybrid polymer networks composed of crosslinked glycosaminoglycan chains that interact with a mixture of key signaling proteins.

Dendritic polyglycerolsulfate-SS-poly(ester amide) micelles for the systemic delivery of docetaxel: pushing the limits of stability through the insertion of π-π interactions.

Insufficient stability of micellar drug delivery systems is still the major limitation to their systematic application in chemotherapy. This work demonstrates novel π-electron stabilized polyelectrolyte block copolymer micelles based on dendritic polyglycerolsulfate-cystamine-block-poly(4-benzoyl-1,4-oxazepan-7-one)-pyrene (dPGS-SS-POxPPh-Py) presenting a very low critical micelle concentration (CMC) of 0.3 mg L-1 (18 nM), 55-fold lower than that of conventional amphiphilic block copolymer micelles. The drug loading capacities of up to 13 wt% allow the efficient encapsulation of the chemotherapeutic Docetaxel (DTX). The spherical morphology of the micelles was proven by cryogenic electron microscopy (cryo-EM). Gaussian Analysis revealed well-defined sizes of 57 nm and 80 nm in the unloaded/loaded state, respectively. Experiments by dynamic light scattering (DLS), ultraviolet-visible spectroscopy (UV-VIS), fluorescence spectroscopy, and cross-polarization solid-state 13C NMR studied the π-π interactions between the core-forming block segment of dPGS-SS-POxPPh-Py and DTX. The findings point to a substantial contribution of these noncovalent interactions to the system's high stability. By confocal laser scanning microscopy (CLSM), the cellular uptake of fluorescein-labelled FITC-dPGS-SS-POxPPh-Py micelles was monitored after one day displaying the successful cell insertion of the cargo-loaded systems. To ensure the drug release in cancerous cells, the disassembly of the micellar DTX-formulations was achieved by reductive and enzymatic degradation studied by light scattering and GPC experiments. Further, no size increase nor disassembly in the presence of human serum proteins after four days was detected. The precise in vitro drug release was also given by the high potency of inhibiting cancer cell growth, finding half-maximal inhibitory concentrations (IC50) efficiently reduced to 68 nM coming along with high viabilities of the empty polymer materials tested on tumor-derived HeLa, A549, and McF-7 cell lines after two days. This study highlights the substantial potential of micelles tailored through the combination of π-electron stabilization with dendritic polyglycerolsulfate for targeted drug delivery systems, enabling them to have a significant foothold in the clinical treatment of cancer.

Sulfated Hyperbranched and Linear Polyglycerols Modulate HMGB1 and Morphological Plasticity in Neural Cells.

The objective of this study was to establish if polyglycerols with sulfate or sialic acid functional groups interact with high mobility group box 1 (HMGB1), and if so, which polyglycerol could prevent loss of morphological plasticity in excitatory neurons in the hippocampus. Considering that HMGB1 binds to heparan sulfate and that heparan sulfate has structural similarities with dendritic polyglycerol sulfates (dPGS), we performed the experiments to show if polyglycerols can mimic heparin functions by addressing the following questions: (1) do dendritic and linear polyglycerols interact with the alarmin molecule HMGB1? (2) Does dPGS interaction with HMGB1 influence the redox status of HMGB1? (3) Can dPGS prevent the loss of dendritic spines in organotypic cultures challenged with lipopolysaccharide (LPS)? LPS plays a critical role in infections with Gram-negative bacteria and is commonly used to test candidate therapeutic agents for inflammation and endotoxemia. Pathologically high LPS concentrations and other stressful stimuli cause HMGB1 release and post-translational modifications. We hypothesized that (i) electrostatic interactions of hyperbranched and linear polysulfated polyglycerols with HMGB1 will likely involve sites similar to those of heparan sulfate. (ii) dPGS can normalize HMGB1 compartmentalization in microglia exposed to LPS and prevent dendritic spine loss in the excitatory hippocampal neurons. We performed immunocytochemistry and biochemical analyses combined with confocal microscopy to determine cellular and extracellular locations of HMGB1 and morphological plasticity. Our results suggest that dPGS interacts with HMGB1 similarly to heparan sulfate. Hyperbranched dPGS and linear sulfated polymers prevent dendritic spine loss in hippocampal excitatory neurons. MS/MS analyses reveal that dPGS-HMGB1 interactions result in fully oxidized HMGB1 at critical cysteine residues (Cys23, Cys45, and Cys106). Triply oxidized HMGB1 leads to the loss of its pro-inflammatory action and could participate in dPGS-mediated spine loss prevention. LPG-Sia exposure to HMGB1 results in the oxidation of Cys23 and Cys106 but does not normalize spine density.

Interaction of Heparin with Proteins: Hydration Effects.

We present a thermodynamic investigation of the interaction of heparin with lysozyme in the presence of potassium glutamate (KGlu). The binding constant Kb is measured by isothermal titration calorimetry (ITC) in a temperature range from 288 to 310 K for concentrations of KGlu between 25 and 175 mM. The free energy of binding ΔGb derived from Kb is strongly decreasing with increasing concentration of KGlu, whereas the dependence of ΔGb on temperature T is found to be small. The decrease of ΔGb can be explained in terms of counterion release: Binding of lysozyme to the strong polyelectrolyte heparin liberates approximately three of the condensed counterions of heparin, thus increasing the entropy of the system. The dependence of ΔGb on T, on the other hand, is traced back to a change of hydration of the protein and the polyelectrolyte upon complex formation. This dependence is quantitatively described by the parameter Δw that depends on T and vanishes at a characteristic temperature T0. A comparison of the complex formation in the presence of KGlu with the one in the presence of NaCl demonstrates that the parameters related to hydration are changed considerably. The characteristic temperature T0 in the presence of KGlu solutions is considerably smaller than that in the presence of NaCl solutions. The change of specific heat Δcp is found to become more negative with increasing salt concentration: This finding agrees with the model-free analysis by the generalized van't Hoff equation. The entire analysis reveals a small but important change of the free energy of binding by hydration. It shows that these ion-specific Hofmeister effects can be modeled quantitatively in terms of a characteristic temperature T0 and a parameter describing the dependence of Δcp on salt concentration.

Denaturation of proteins: electrostatic effects vs. hydration.

The unfolding transition of proteins in aqueous solution containing various salts or uncharged solutes is a classical subject of biophysics. In many cases, this transition is a well-defined two-stage equilibrium process which can be described by a free energy of transition ΔG u and a transition temperature T m. For a long time, it has been known that solutes can change T m profoundly. Here we present a phenomenological model that describes the change of T m with the solute concentration c s in terms of two effects: (i) the change of the number of correlated counterions Δn ci and (ii) the change of hydration expressed through the parameter Δw and its dependence on temperature expressed through the parameter dΔc p/dc s. Proteins always carry charges and Δn ci describes the uptake or release of counterions during the transition. Likewise, the parameter Δw measures the uptake or release of water during the transition. The transition takes place in a reservoir with a given salt concentration c s that defines also the activity of water. The parameter Δn ci is a measure for the gain or loss of free energy because of the release or uptake of ions and is related to purely entropic effects that scale with ln c s. Δw describes the effect on ΔG u through the loss or uptake of water molecules and contains enthalpic as well as entropic effects that scale with c s. It is related to the enthalpy of transition ΔH u through a Maxwell relation: the dependence of ΔH u on c s is proportional to the dependence of Δw on temperature. While ionic effects embodied in Δn ci are independent of the kind of salt, the hydration effects described through Δw are directly related to Hofmeister effects of the various salt ions. A comparison with literature data underscores the general validity of the model.

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.

Interaction of Linear Polyelectrolytes with Proteins: Role of Specific Charge-Charge Interaction and Ionic Strength.

We present a thermodynamic study of the interaction of synthetic, linear polyelectrolytes with bovine serum albumin (BSA). All polyelectrolytes are based on poly(allyl glycidyl ether) which has been modified by polymer-analogous reaction with anionic (-SO3Na), cationic (-NH3Cl or -NHMe2Cl) or zwitterionic groups (-NMe2(CH2)3SO3). While the anionic polymer shows a very weak interaction, the zwitterionic polymer exhibits no interaction with BSA (pI = 4.7) under the applied pH = 7.4, ionic strength (I = 23-80 mM) and temperature conditions (T = 20-37 °C). A strong binding, however, was observed for the polycations bearing primary amino or tertiary dimethyl amino groups, which could be analysed in detail by isothermal titration calorimetry (ITC). The analysis was done using an expression which describes the free energy of binding, ΔGb, as the function of the two decisive variables, temperature, T, and salt concentration, cs. The underlying model splits ΔGb into a term related to counterion release and a term related to water release. While the number of released counter ions is similar for both systems, the release of bound water is more important for the primary amine compared to the tertiary N,N-dimethyl amine presenting polymer. This finding is further traced back to a closer contact of the polymers' protonated primary amino groups in the complex with oppositely charged moieties of BSA as compared to the bulkier protonated tertiary amine groups. We thus present an investigation that quantifies both driving forces for electrostatic binding, namely counterion release and change of hydration, which contribute to a deeper understanding with direct impact on future advancements in the biomedical field.

Interaction of Polyelectrolytes with Proteins: Quantifying the Role of Water.

A theoretical model is presented for the free energy ΔGb of complex formation between a highly charged polyelectrolyte and a protein. The model introduced here comprises both the effect of released counterions and the uptake or release of water molecules during complex formation. The resulting expression for ΔGb is hence capable of describing the dependence of ΔGb on temperature as well as on the concentration of salt in the system: An increase of the salt concentration in the solution increases the activity of the ions and counterion release becomes less effective for binding. On the other hand, an increased salt concentration leads to the decrease of the activity of water in bulk. Hence, release of water molecules during complex formation will be more advantageous and lead to an increase of the magnitude of ΔGb and the binding constant. It is furthermore demonstrated that the release or uptake of water molecules is the origin of the marked enthalpy-entropy cancellation observed during complex formation of polyelectrolytes with proteins. The comparison with experimental data on complex formation between a synthetic (sulfated dendritic polyglycerol) and natural polyelectrolytes (DNA; heparin) with proteins shows full agreement with theory.

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.

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.

Solid Electrolyte Interphase Layer Formation during Lithiation of Single-Crystal Silicon Electrodes with a Protective Aluminum Oxide Coating.

The lithiation of crystalline silicon was studied over several cycles using operando neutron reflectometry over six cycles. A thin layer of aluminum oxide was employed as an artificial coating on the silicon to suppress the solid electrolyte interphase (SEI) layer-related aging effects. Initially, the artificial SEI prevented side effects but led to increased lithium trapping. This layer degraded after two cycles, followed by side reactions, which decrease the coulombic efficiency. No hint for electrode fracturization was found even though the lithiation depth exceeded 1 µm. Two distinct zones with high and low lithium concentrations were found, initially separated by a sharp interface, which broadens with cycling. The correlation of the reflectometry results with the electrochemical current showed the lithium fraction that is lithiated in the silicon and the lithium consumed in side reactions. Also, neutron reflectometry was used to quantify the amount of lithium that remained inside of the silicon. Additional electrochemical impedance spectroscopy was used to gain insights into the electrical properties of the sample via fitting to an equivalent circuit.

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.

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.

Cells Undergo Major Changes in the Quantity of Cytoplasmic Organelles after Uptake of Gold Nanoparticles with Biologically Relevant Surface Coatings.

Here, we use cryo soft X-ray tomography (cryo-SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insight about nanoparticle uptake for nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS-coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), with cryo-SXT, we detected ∼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed ∼5% of dPGS-AuNPs in a rarely identified subcellular site, namely, lipid droplets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1-6 h incubation period; the number of small vesicles and mitochondria significantly increased, and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethylenimine (PEI), used for nucleic acid delivery. PEI nanoparticles did not enter lipid droplets, but they induced similar, albeit less pronounced, changes in the quantity of cytoplasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.

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.

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.

Interaction of Proteins with a Planar Poly(acrylic acid) Brush: Analysis by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D).

We describe the preparation of a poly(acrylic acid) (PAA) brush, polymerized by atom transfer radical polymerization (ATRP) of tert-butyl acrylate (tBA) and subsequent acid hydrolysis, on the flat gold surfaces of quartz-crystal microbalance (QCM) crystals. The PAA brushes were characterized by Fourier transform infrared (FT-IR) spectroscopy, ellipsometry and water contact angle analysis. The interaction of the PAA brushes with human serum albumin (HSA) was studied for a range of ionic strengths and pH conditions by quartz-crystal microbalance with dissipation monitoring (QCM-D). The quantitative analysis showed a strong adsorption of protein molecules onto the PAA brush. By increasing the ionic strength, we were able to release a fraction of the initially bound HSA molecules. This finding highlights the importance of counterions in the polyelectrolyte-mediated protein adsorption/desorption. A comparison with recent calorimetric studies related to the binding of HSA to polyelectrolytes allowed us to fully analyze the QCM data based on the results of the thermodynamic analysis of the binding process.

Interaction of Lysozyme with a Dendritic Polyelectrolyte: Quantitative Analysis of the Free Energy of Binding and Comparison to Molecular Dynamics Simulations.

We present a comprehensive analysis of the energetics of the binding of lysozyme to dendritic polyglycerolsulfate (dPGS) in aqueous solution. This system is a perfect model for studying the interaction of proteins with polyelectrolytes. We discuss and model the free energy of binding ΔGb = -kBT ln Kb as the function of the two decisive variables, namely, the salt concentration cs and the temperature T. The system lysozyme/dPGS exhibits a strong enthalpy-entropy compensation throughout the entire range of temperature, similar to the one observed for the interaction of DNA with various proteins. Following a suggestion of Dragan et al. [Eur. Biophys. J. 2017, 46, 301], the free energy ΔGb can be split up into ΔGb = ΔGres + ΔGci, where ΔGci denotes the part due to counterion release, whereas ΔGres is the part obtained by extrapolation of ΔGb to 1 M salt concentration. Plots of dlog Kb/dlog cs lead to perfectly straight lines that can be extrapolated to cs = 1 M in order to obtain ΔGres. Both ΔGres and ΔGci can be independently obtained by implicit solvent molecular dynamics simulations made up to salt concentrations of 1 M. Good agreement of the experiment and simulation within prescribed limits of error is found. Moreover, ΔGres is shown to be caused by direct unscreened electrostatic contacts or salt bridges between dPGS and lysozyme. Because ΔGci = -TΔSci where ΔSci is the entropy due to counterion release, the entire binding entropy ΔSb can be split up as ΔSb = ΔSci + ΔSres. Plots of the binding enthalpy ΔHb versus ΔSres lead to a perfect master curve for the system dPGS/lysozyme. These findings suggest that the strong enthalpy-entropy cancellation found for this system is an entirely nonelectrostatic phenomenon solely due to solvation or desolvation by water. Thus, the results obtained here on the model system dPGS and lysozyme are in full agreement with the conclusion drawn by Dragan et al. for the binding of DNA to various proteins.