Conformational control of antimicrobial peptide amphiphilicity: consequences for boosting membrane interactions and antimicrobial effects of photocatalytic TiO2 nanoparticles.

This study reports on the effects of conformationally controlled amphiphilicity of antimicrobial peptides (AMPs) on their ability to coat TiO2 nanoparticles (NPs) and boost the photocatalytic antimicrobial effects of such NPs. For this, TiO2 NPs were combined with AMP EFK17 (EFKRIVQRIKDFLRNLV), displaying a disordered conformation in aqueous solution but helix formation on interaction with bacterial membranes. The membrane-bound helix is amphiphilic, with all polar and charged amino acid residues located at one side and all non-polar and hydrophobic residues on the other. In contrast, the d-enantiomer variant EFK17-d (E(dF)KR(dI)VQR(dI)KD(dF)LRNLV) is unable to form the amphiphilic helix on bacterial membrane interaction, whereas the W-residues in EFK17-W (EWKRWVQRWKDFLRNLV) boost hydrophobic interactions of the amphiphilic helix. Circular dichroism results showed the effects displayed for the free peptide, to also be present for peptide-coated TiO2 NPs, causing peptide binding to decrease in the order EFK17-W > EFK17 > EFK17-d. Notably, the formation of reactive oxygen species (ROS) by the TiO2 NPs was essentially unaffected by the presence of peptide coating, for all the peptides investigated, and the coatings stabilized over hours of UV exposure. Photocatalytic membrane degradation from TiO2 NPs coated with EFK17-W and EFK17 was promoted for bacteria-like model bilayers containing anionic phosphatidylglycerol but suppressed in mammalian-like bilayers formed by zwitterionic phosphatidylcholine and cholesterol. Structural aspects of these effects were further investigated by neutron reflectometry with clear variations observed between the bacteria- and mammalian-like model bilayers for the three peptides. Mirroring these results in bacteria-like model membranes, combining TiO2 NPs with EFK17-W and EFK17, but not with non-adsorbing EFK17-d, resulted in boosted antimicrobial effects of the resulting cationic composite NPs already in darkness, effects enhanced further on UV illumination.

Cation and buffer specific effects on the DNA-lipid interaction.

Knowledge of DNA - lipid layer interactions is key for the development of biosensors, synthetic nanopores, scaffolds, and gene-delivery systems. These interactions are strongly affected by the ionic composition of the solvent. We have combined quartz crystal microbalance (QCM) and ellipsometry measurements to reveal how pH, buffers and alkali metal chloride salts affect the interaction of DNA with lipid bilayers (DOTAP/DOPC 30:70 in moles). We found that the thickness of the DNA layer adsorbed onto the lipid bilayer decreased in the order citrate > phosphate > Tris > HEPES. The effect of cations on the thickness of the DNA layer decreased in the order (K+ > Na+ > Cs+ ∼ Li+). Rationalization of the experimental results requires that adsorption, due to cation specific charge screening, is driven by the simultaneous action of two mechanisms namely, the law of matching water affinities for kosmotropes (Li+) and ion dispersion forces for chaotropes (Cs+). The outcome of these two opposing mechanisms is a "bell-shaped" specific cations sequence. Moreover, a superimposed buffer specificity, which goes beyond the simple effect of pH regulation, further modulated cation specificity. In summary, DNA-lipid bilayer interactions are maximized if citrate buffer (50 mM, pH 7.4) and KCl (100 mM) are used.

Nanoscale structural and mechanical characterization of thin bicontinuous cubic phase lipid films.

The mechanical response of lipid membranes to nanoscale deformations is of fundamental importance for understanding how these interfaces behave in multiple biological processes; in particular, the nanoscale mechanics of non-lamellar membranes represents a largely unexplored research field. Among these mesophases, inverse bicontinuous cubic phase QII membranes have been found to spontaneously occur in stressed or virally infected cells and to play a role in fundamental processes, such as cell fusion and food digestion. We herein report on the fabrication of thin ( Ì´150 nm) supported QII cubic phase lipid films (SQIIFs) and on their characterization via multiple techniques including Small Angle X-Ray Scattering (SAXS), Ellipsometry and Atomic Force Microscopy (AFM). Moreover, we present the first nanomechanical characterization of a cubic phase lipid membrane, through AFM-based Force Spectroscopy (AFM-FS). Our analysis reveals that the mechanical response of these architectures is strictly related to their topology and structure. The observed properties are strikingly similar to those of macroscopic 3D printed cubic structures when subjected to compression tests in material science; suggesting that this behaviour depends on the 3D organisation, rather than on the length-scale of the architecture. We also show for the first time that AFM-FS can be used for characterizing the structure of non-lamellar mesophases, obtaining lattice parameters in agreement with SAXS data. In contrast to classical rheological studies, which can only probe bulk cubic phase solutions, our AFM-FS analysis allows probing the response of cubic membranes to deformations occurring at length and force scales similar to those found in biological interactions.

Enrichment of Charged Monomers Explains Non-monotonic Polymer Volume Fraction Profiles of Multi-stimulus Responsive Copolymer Brushes.

Multi-stimulus responsive poly(2-(2-methoxyethoxy)ethyl methacrylate-co-2-(diethylamino)ethyl methacrylate) [P(MEO2MA-co-DEA)] 80:20 mol % copolymer brushes were synthesized on planar silica substrates via surface-initiated activators continuously regenerated via electron transfer atom transfer radical polymerization. Brush thickness was sensitive to changes in pH and temperature as monitored with ellipsometry. At low pH, the brush is charged and swollen, while at high pH, the brush is uncharged and more collapsed. Clear thermoresponsive behavior is also observed with the brush more swollen at low temperatures compared to high temperatures at both high and low pH. Neutron reflectometry was used to determine the polymer volume fraction profiles (VFPs) at various pH values and temperatures. A region of lower polymer content, or a depletion region, near the substrate is present in all of the experimental polymer VFPs, and it is more pronounced at low pH (high charge) and less so at high pH (low charge). Polymer VFPs calculated through numerical self-consistent field theory suggest that enrichment of DEA monomers near the substrate results in the experimentally observed non-monotonic VFPs. Adsorption of DEA monomers to the substrate prior to initiation of polymerization could give rise to DEA segment-enriched region proximal to the substrate.

Poly(N-isopropylacrylamide) Response to Salt Concentration and Anion Identity: A Brush-on-Brush Study.

The stability of poly(N-isopropylacrylamide) (PNIPAM) brush-modified colloidal silica particles was compared to asymmetric and symmetric PNIPAM brush direct force measurements in the presence of 1, 10, and 500 mM aqueous salt solution of KCl, KNO3, and KSCN between 10 and 45 °C. Dynamic light scattering measurements highlighted subtle variations in the salt-mediated thermoresponse, while atomic force microscopy (AFM) force curves between a bare silica or PNIPAM brush-modified colloid probe and a planar PNIPAM brush elucidated differences in brush interactions. The AFM force curves in the presence of KCl primarily revealed steric interactions between the surfaces, while KNO3 and KSCN solutions exhibited electrosteric interactions on approach as a function of the chaotropic nature of the ion and the solution concentration. The symmetric PNIPAM brush interaction highlighted significant variations between KCl and KSCN at 1 and 500 mM concentrations, while the approach and retraction force curves were relatively similar at 10 mM concentration. The combination of these techniques enabled the stability of PNIPAM brush-modified colloidal dispersions in the presence of electrolyte to be better understood with specific ion binding and the solution Debye length playing a significant role.

Temperature dependent specific ion effects in mixed salt environments on a thermoresponsive poly(oligoethylene glycol methacrylate) brush.

The temperature induced swelling/collapse transition of poly(oligoethylene glycol methacrylate) (POEGMA) brushes has been investigated in electrolyte solutions comprised of multiple anions. The behaviour of a POEGMA brush in mixed salt environments of potassium acetate (KCH3COO, causes collapse) and thiocyanate (KSCN, causes swelling), two ions at opposite ends of the Hofmeister series, has been monitored with neutron reflectometry (NR) and quartz crystal microbalance with dissipation (QCM-D). These techniques revealed that the balance of the swelling/collapse influence of the two ions on the structure of the brush is temperature dependent. At low temperatures in mixed salt environments, the influence of the acetate and thiocyanate ions appears additive, antagonistic and approximately equal in magnitude, with brush thickness and dissipation similar to the brush in the absence of electrolyte. At higher temperatures, the influence of the acetate ion diminishes, resulting in an increase in the relative influence of the thiocyanate ion on the brush conformation. These temperature dependent specific ion effects are attributed to increased steric crowding in the brush, along with an increased affinity of the thiocyanate ion for the polymer at higher temperatures.

Combined Experimental and Theoretical Study of Weak Polyelectrolyte Brushes in Salt Mixtures.

The swelling behavior of a hydrophobic poly(2diisopropylamino)ethyl methacrylate (PDPA) brush immersed in aqueous solutions of single and mixed salts has been investigated using ellipsometry and numerical self-consistent field (nSCF) theory. As a function of solution ionic strength, the osmotic and salted brush regimes of weak polyelectrolyte brushes as well as substantial specific anion effects in the presence of K+ salts of Cl-, NO3-, and SCN- are found. For solutions containing mixtures of NO3- and Cl-, the brush swelling is the same as one would expect on the basis of the concentration-weighted average of the brush behavior in the single salt solutions. However, in mixtures of SCN- and Cl-, the swelling response is more complicated and substantial divergence from ideal behavior is observed. Mean-field theory shows excellent qualitative agreement with the ellipsometry findings. nSCF reveals that for the SCN-/Cl- cases the swelling behavior of the PDPA brush most likely arises from the predominant localization of the weakly hydrated SCN- within the brush compared to the more strongly hydrated Cl-.

Influence of molecular weight on PNIPAM brush modified colloidal silica particles.

The effect of molecular weight and temperature on the phase transition and internal structure of poly(N-isopropylacrylamide) brush modified colloidal silica particles was investigated using dynamic light scattering (DLS) and small angle neutron scattering (SANS) between 15 and 45 °C. Dry particle analysis utilising transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) all confirmed the thickness of the polymer brush shell increased as a function of polymerisation time. Hydrodynamic diameter and electrophoretic mobility results revealed that the brush modified particles transitioned from swollen shells to a collapsed conformation between 15 and 35 °C. The dispersions were electrosterically stabilised over the entire temperature range investigated, with minimal thermal hysteresis recorded. Modelling of the hydrodynamic diameter enabled the calculation of a lower critical solution temperature (LCST) which increased as a function of brush thickness. The internal structure determined via SANS showed a swollen brush at low temperatures (18 and 25 °C) which decayed radially away from the substrate, while a collapsed block-like conformation with 60% polymer volume fraction was present at 40 °C. Radial phase separation was evident at intermediate temperatures (30 and 32.5 °C) with the lower molecular weight sample having a greater volume fraction of polymer in the dense inner region at these temperatures.

Specific ion effects on thermoresponsive polymer brushes: Comparison to other architectures.

Thermoresponsive polymers have received significant research attention as smart materials with particular interest in biomedical applications. The composition and architecture are known to strongly influence the thermoresponsive properties of the materials. For example, the strong overlap of end-grafted polymer chains in polymer brushes leads to a broader collapse transition relative to linear ungrafted chains as well as temperature dependent adhesion. The temperature response of free polymer has been widely reported to depend on the concentration and identity of ions in solution and is further modified by the composition of the solvent and presence of cosolutes. However, the influence of polymer architecture on these specific ion effects is relatively unknown. Herein, we compare the current understanding of specific ion effects on free polymer chains and gels with recent studies of polymer brushes. Further studies on mixed salt systems are found to be the next step to predicting the behaviour of these materials in biological systems.

Effect of ionic strength and salt identity on poly(N-isopropylacrylamide) brush modified colloidal silica particles.

HYPOTHESIS: The thermoresponse of poly(N-isopropylacrylamide) stabilised particles is influenced by the presence of salt and is dependent on the concentration, and ions present. The conformation and electrophoretic mobility of core/shell PNIPAM brush modified silica particles is expected to vary as a function of these specific ion effects. EXPERIMENTS: The thermoresponse of PNIPAM brush modified silica particles was investigated via dynamic light scattering and electrophoretic mobility measurements between 5 and 45 °C in the presence of 11 different salt solutions. FINDINGS: Specific ion effects were observed in the presence of salt solutions for concentrations between 50 and 1000 mM. The temperature response could be mapped to a master curve unlike PNIPAM brush behaviour on planar substrates. The magnitude of brush layer lower critical solution temperature reduction was found to follow the order F- > CH3CO2- > Cl- > NO3- ∼ Br- > I- > SCN- for the potassium series and Na+ > K+ > Cs+ > Li+ ∼ NH4+ for the chloride salts. The electrophoretic mobility of the modified particles in the presence of 100 mM potassium salts increased in magnitude as the brush layer collapsed and also with the chaotropic nature of the anion.

Enhanced specific ion effects in ethylene glycol-based thermoresponsive polymer brushes.

The thermoresponse of poly(di(ethyleneglycol) methyl ether methacrylate) (PMEO2MA) brushes has been investigated in the presence of monovalent anions at either end of the Hofmeister series using ellipsometry, neutron reflectometry (NR) and colloid probe atomic force microscopy (AFM). NR measurements in deuterium oxide showed no evidence of vertical phase separation perpendicular to the grafting substrate with a gradual transition between a block-like, dense structure at 45°C and an extended, dilute conformation at lower temperatures. All three techniques revealed a shift to a more collapsed state for a given temperature in kosmotropic potassium acetate solutions, while more swollen structures were observed in chaotropic potassium thiocyanate solutions. No difference was observed between 250mM and 500mM thiocyanate for a 540Å brush studied by ellipsometry, while the lower molecular weight ∼200Å brushes used for NR and AFM measurements continued to respond with increasing salt concentration. The effect of thiocyanate on the temperature response was greatly enhanced relative to PNIPAM with the shift in temperature response at 250mM being five times greater than a PNIPAM brush of similar thickness and grafting density.

Specific ion modulated thermoresponse of poly(N-isopropylacrylamide) brushes.

The influence of specific anions on the equilibrium thermoresponse of poly(N-isopropylacrylamide) (pNIPAM) brushes has been studied using in situ ellipsometry, quartz crystal microbalance with dissipation (QCM-D) and static contact angle measurements between 20 and 45 °C in the presence of up to 250 mM acetate and thiocyanate anions in water. The thickness and changes in dissipation exhibited a broad swelling transition spanning approximately 15 °C from collapsed (high temperatures) to swollen conformation (low temperatures) while the brush surface wettability changed over approximately 2 °C. In the presence of the kosmotropic acetate anions, the measured lower critical solution temperature (LCST) by the three techniques was very similar and decreased linearly as a function of ionic strength. Conversely, increasing the concentration of the chaotropic thiocyanate anions raised the LCST of the pNIPAM brushes with variation in the measured LCST between the three techniques increasing with ionic strength. The thickness of the pNIPAM brush was seen to progressively increase with increasing thiocyanate concentration at all temperatures. It is proposed that specific ion binding of the chaotropic thiocyanate anion with pNIPAM amide moieties increases the electrostatic intra- and intermolecular repulsion within and between pNIPAM chains. This allows the brush to begin to swell at higher temperatures and to an overall greater extent.

Bridging Translation by Improving Preclinical Study Design in AKI.

Despite extensive research, no therapeutic interventions have been shown to prevent AKI, accelerate recovery of AKI, or reduce progression of AKI to CKD in patients. This failure in translation has led investigators to speculate that the animal models being used do not predict therapeutic responses in humans. Although this issue continues to be debated, an important concern that has not been addressed is whether improvements in preclinical study design can be identified that might also increase the likelihood of translating basic AKI research into clinical practice using the current models. In this review, we have taken an evidence-based approach to identify common weaknesses in study design and reporting in preclinical AKI research that may contribute to the poor translatability of the findings. We focused on use of N-acetylcysteine or sodium bicarbonate for the prevention of contrast-induced AKI and use of erythropoietin for the prevention of AKI, two therapeutic approaches that have been extensively studied in clinical trials. On the basis of our findings, we identified five areas for improvement in preclinical study design and reporting. These suggested and preliminary guidelines may help improve the quality of preclinical research for AKI drug development.

Anion-specific effects on the behavior of pH-sensitive polybasic brushes.

The anion-specific solvation and conformational behavior of weakly basic poly(2-dimethylamino)ethyl methacrylate (poly(DMA)), poly(2-diethylamino)ethyl methacrylate (poly(DEA)), and poly(2-diisopropylamino)ethyl methacrylate (poly(DPA)) brushes, with correspondingly increasing inherent hydrophobicity, have been investigated using in situ ellipsometric and quartz crystal microbalance with dissipation (QCM-D) measurements. In the osmotic brush regime, as the initial low concentration of salt is increased, the brushes osmotically swell by the uptake of solvent as they become charged and the attractive hydrophobic inter- and intrachain interactions are overcome. With increased ionic strength, the brushes move into the salted brush regime where they desolvate and collapse as their electrostatic charge is screened. Here, as the brushes collapse, they transition to more uniform and rigid conformations, which dissipate less energy, than similarly solvated brushes at lower ionic strength. Significantly, in these distinct regimes brush behavior is not only ionic strength dependent but is also influenced by the nature of the added salt based on its position in the well-known Hofmeister or lyotropic series, with potassium acetate, nitrate, and thiocyanate investigated. The strongly kosmotropic acetate anions display low affinity for the hydrophobic polymers, and largely unscreened electrosteric repulsions allow the brushes to remain highly solvated at higher acetate concentrations. The mildly chaotropic nitrate and strongly chaotropic thiocyanate anions exhibit a polymer hydrophobicity-dependent affinity for the brushes. Increasing thiocyanate concentration causes the brushes to collapse at lower ionic strength than for the other two anions. This study of weak polybasic brushes demonstrates the importance of all ion, solvent, and polymer interactions.

Hydrophobic effects within the dynamic pH-response of polybasic tertiary amine methacrylate brushes.

The solvation and swelling behaviour of three dialkylaminoethyl methacrylate polymer brushes, of varying hydrophobicity, have been investigated using a combination of in situ ellipsometry and a quartz crystal microbalance with dissipation (QCM-D). At low pH the tertiary amine groups of the three polymers are protonated and all three brushes are significantly solvated and swell by adopting an extended conformation. As the pH is increased the weak polybasic brushes become increasingly deprotonated and collapse via solvent expulsion. By employing high temporal resolution measurements we have found that monomer hydrophobicity has a direct influence on the dynamics of this pH-response. The most hydrophobic poly(2-diisopropylamino)ethyl methacrylate (poly(DPA)) brush exhibits the fastest maximum swelling rate. This maximum swelling rate is reduced with decreasing monomer hydrophobicity for the 2-diethylamino, and even further for the 2-dimethylamino analogues. For all three brushes, the corresponding collapse transition is slower and compounded by an induction time that decreases with monomer hydrophobicity. Here also, the maximum collapse rate is greatest for the most hydrophobic polymer. This domination of the pH-response kinetics by monomer hydrophobicity is attributed to attractive hydrophobic forces between polymer segments overcoming the repulsive electrostatic forces between the tertiary amine residues.

Critical salt effects in the swelling behavior of a weak polybasic brush.

The swelling behavior of poly(2-(diethylamino)ethyl methacrylate) (PDEA) brushes in response to changes in solution pH and ionic strength has been investigated. The brushes were synthesized by ARGET ATRP methodology at the silica-aqueous solution interface via two different surface-bound initiator approaches: electrostatically adsorbed cationic macroinitiator and covalently anchored silane-based ATRP initiator moieties. The pH-response of these brushes is studied as a function of the solvated brush thickness in a constant flow regime that elucidates the intrinsic behavior of polymer brushes. In situ ellipsometry equilibrium measurements show the pH-induced brush swelling and collapse transitions are hysteretic in nature. Furthermore, high temporal resolution kinetic studies demonstrate that protonation and solvent ingress during swelling occur much faster than the brush charge neutralization and solvent expulsion during collapse. This hysteresis is attributed to the formation of a dense outer region or skin during collapse that retards solvent egress. Moreover, at a constant pH below its pKa, the PDEA brush exhibited a critical conformational change in the range 0.5-1 mM electrolyte, a range much narrower than predicted by the theory of the osmotic brush regime. This behavior is attributed to the hydrophobicity of the collapsed brush. The swelling and collapse kinetics for this salt-induced transition are nearly identical. This is in contrast to the asymmetry in the rate of the pH-induced response, suggesting an alternative mechanism for the two processes dependent on the nature of the environmental trigger.