Deposition and water repelling of temperature-responsive nanopesticides on leaves.

Pesticides are widely used to increase agricultural productivity, however, weak adhesion and deposition lead to low efficient utilization. Herein, we prepare a nanopesticide formulation (tebuconazole nanopesticides) which is leaf-adhesive, and water-dispersed via a rapid nanoparticle precipitation method, flash nanoprecipitation, using temperature-responsive copolymers poly-(2-(dimethylamino)ethylmethylacrylate)-b-poly(ε-caprolactone) as the carrier. Compared with commercial suspensions, the encapsulation by the polymer improves the deposition of TEB, and the contact angle on foliage is lowered by 40.0°. Due to the small size and strong van der Waals interactions, the anti-washing efficiency of TEB NPs is increased by 37% in contrast to commercial ones. Finally, the acute toxicity of TEB NPs to zebrafish shows a more than 25-fold reduction as compared to commercial formulation indicating good biocompatibility of the nanopesticides. This work is expected to enhance pesticide droplet deposition and adhesion, maximize the use of pesticides, tackling one of the application challenges of pesticides.

Rheology and Gelation of Hyaluronic Acid/Chitosan Coacervates.

Hyaluronic acid (HA) and chitosan (CHI) are biopolyelectrolytes which are interesting for both the medical and polymer physics communities due to their biocompatibility and semi-flexibility, respectively. In this work, we demonstrate by rheology experiments that the linear viscoelasticity of HA/CHI coacervates depends strongly on the molecular weight of the polymers. Moduli for coacervates were found significantly higher than those of individual HA and CHI physical gels. A remarkable 1.5-fold increase in moduli was noted when catechol-conjugated HA and CHI were used instead. This was attributed to the conversion of coacervates to chemical gels by oxidation of 3,4-dihydroxyphenylalanine (DOPA) groups in HA and CHI to di-DOPA crosslinks. These rheological results put HA/CHI coacervates in the category of strong candidates as injectable tissue scaffolds or medical adhesives.

Chondro-inductive hyaluronic acid/chitosan coacervate-based scaffolds for cartilage tissue engineering.

Injuries related to articular cartilage are among the most challenging musculoskeletal problems because of poor repair capacity of this tissue. The lack of efficient treatments for chondral defects has stimulated research on cartilage tissue engineering applications combining porous biocompatible scaffolds with stem cells in the presence of external stimuli. This work presents the role of rat bone marrow mesenchymal stem cell (BMSC) encapsulated-novel three-dimensional (3D) coacervate scaffolds prepared through complex coacervation between different chitosan salts (CHI) and sodium hyaluronate (HA). The 3D architecture of BMSC encapsulated scaffolds (HA/CHI) was shown by scanning electron microscopy (SEM) to have an interconnected structure to allow cell-cell and cell-matrix interactions. Chondrogenic induction of encapsulated BMSCs within HA/CHI coacervates demonstrated remarkable cellular viability in addition to the elevated expression levels of chondrogenic markers such as sex determining region Y-box 9 protein (SOX9), aggrecan (ACAN), cartilage oligomeric matrix protein (COMP) and collagen type II (COL2A1) by immunofluorescence staining, qPCR and ELISA test. Collectively, HA/CHI coacervates are promising candidates for future use of these scaffolds in cartilage tissue engineering applications.

Effect of Protein Surface Charge Distribution on Protein-Polyelectrolyte Complexation.

Charge anisotropy or the presence of charge patches at protein surfaces has long been thought to shift the coacervation curves of proteins and has been used to explain the ability of some proteins to coacervate on the "wrong side" of their isoelectric point. This work makes use of a panel of engineered superfolder green fluorescent protein mutants with varying surface charge distributions but equivalent net charge and a suite of strong and weak polyelectrolytes to explore this concept. A patchiness parameter, which assessed the charge correlation between points on the surface of the protein, was used to quantify the patchiness of the designed mutants. Complexation between the polyelectrolytes and proteins showed that the mutant with the largest patchiness parameter was the most likely to form complexes, while the smallest was the least likely to do so. The patchiness parameter was found to correlate well with the phase behavior of the protein-polymer mixtures, where both macrophase separation and the formation of soluble aggregates were promoted by increasing the patchiness depending on the polyelectrolyte with which the protein was mixed. Increasing total charge and increasing strength of the polyelectrolyte promote interactions for oppositely charged polyelectrolytes, while charge regulation is also key to interactions for similarly charged polyelectrolytes, which must interact selectively with oppositely charged patches.

Hyaluronic Acid/Chitosan Coacervate-Based Scaffolds.

Chitosan-chloride (CHI) and sodium hyaluronate (HA), two semiflexible biopolymers, self-assemble to form nonstoichiometric coacervates. The effect of counterions was briefly investigated by preparing HA/CHI coacervates in either CaCl2 or NaCl solutions to find only a small difference in their tendency to coacervate. Higher water content in coacervates within CaCl2 was attributed to the chaotropic nature of Ca2+ ions. This effect was also evidenced with smaller pore sizes for coacervates in NaCl. Besides, for coacervation of chitosan-glutamate (CHI-G) with HA, dynamic light scattering at different charge ratios indicated a wider coacervation region for the HA/CHI-G pair than the HA/CHI. This was attributed to the chaotropic and "soft" ion nature of glutamate compared to chloride as a counterion of chitosan. Positive zeta potential values for both coacervate suspensions were explained by the contribution of charge mismatch, chain semiflexibility, and intra- and intercomplex disproportionation. Finally, HA/CHI coacervates were used to encapsulate bone marrow stem cells. While cell viabilities in HA/CHI coacervates were remarkable up to 21 days, their well-spread morphology has proved that HA/CHI coacervates are promising scaffolds for cartilage tissue engineering.

Thermodynamics of complex coacervation.

Isothermal titration calorimetry has routinely been used to understand the thermodynamic characteristics of complexation and coacervation. Most commonly, built-in models that assume independent binding sites have been employed in these studies. However, the non-covalent nature of interactions and steric effects accompanying macromolecules require (i) usage of new models such as overlapping binding sites and Satake-Yang's two-state binding models and (ii) reformed interpretations of the data as two-stage structuring. Fitting data with these models, forces driving the interaction of polyelectrolytes with oppositely charged polyelectrolytes, surfactants, and proteins have been identified as electrostatics and/or counterion release with possible contributions from hydrogen bonding and hydrophobic interactions. Additionally, for surfactant-polyelectrolyte coacervation, ITC signals indicated separate regions for formation of polymer-induced micelles and free micelles. Regardless of the type of the coacervation system, thermodynamics of coacervation is affected by the following parameters: pH and ionic strength of the medium, charge density, molecular weight of the polyelectrolyte, concentration, and mixing order of macroions. Lastly, we present a brief comparison between ITC on one hand and surface plasmon resonance or capillary electrophoresis on the other regarding their application in coacervation.

Construction of Compact Polyelectrolyte Multilayers Inspired by Marine Mussel: Effects of Salt Concentration and pH As Observed by QCM-D and AFM.

Biomimetic multilayers based on layer-by-layer (LbL) assembly were prepared as functional films with compact structure by incorporating the mussel-inspired catechol cross-linking. Dopamine-modified poly(acrylic acid) (PAADopa) was synthesized as a polyanion to offer electrostatic interaction with the prelayer polyethylenimine (PEI) and consecutively cross-linked by zinc to generate compact multilayers with tunable physicochemical properties. In situ layer-by-layer growth and cross-linking were monitored by a quartz crystal microbalance with dissipation (QCM-D) to reveal the kinetics of the process and the influence of Dopa chemistry. Addition of Dopa enhanced the mass adsorption and led to the formation of a more compact structure. An increase of ionic strength induced an increase in mass adsorption in the Dopa-cross-linked multilayers. This is a universal approach for coating of various surfaces such as Au, SiO2, Ti, and Al2O3. Roughness observed by AFM in both wet and dry conditions was compared to confirm the compact morphology of Dopa-cross-linked multilayers. Because of the pH sensitivity of Dopa moiety, metal-chelated Dopa groups can be turned into softer structure at higher pH as revealed by reduction of Young's modulus determined by MFP-3D AFM. A deeper insight into the growth and mechanical properties of Dopa-cross-linked polyelectrolyte multilayers was addressed in the present study. This allows a better control of these systems for bioapplications.

"Bio-glues" to enhance slipperiness of mucins: improved lubricity and wear resistance of porcine gastric mucin (PGM) layers assisted by mucoadhesion with chitosan.

A synergetic lubricating effect between porcine gastric mucin (PGM) and chitosan based on their mucoadhesive interaction is reported at a hydrophobic interface comprised of self-mated polydimethylsiloxane (PDMS) surfaces. In acidic solution (pH 3.2) and low concentrations (0.1 mg mL(-1)), the interaction of PGM with chitosan led to surface recharge and size shrinkage of their aggregates. This resulted in higher mass adsorption on the PDMS surface with an increasing weight ratio of [chitosan]/[PGM + chitosan] up to 0.50. While neither PGM nor chitosan exhibited slippery characteristics, the coefficient of friction being close to 1, their mixture improved considerably the lubricating efficiency (the coefficient of friction is 0.011 at an optimum mixing ratio) and wear resistance of the adsorbed layers. These findings are explained by the role of chitosan as a physical crosslinker within the adsorbed PGM layers, resulting in higher cohesion and lower interlayer chain interpenetration and bridging.

Tunable adsorption of bovine serum albumin by annealed cationic spherical polyelectrolyte brushes.

By combining turbidimetric titration, dynamic light scattering (DLS), and zeta potential methods, we demonstrated that the adsorption of bovine serum albumin (BSA) in annealed cationic spherical polyelectrolyte brushes (SPB) can be controlled by modulating the pH, ionic strength, BSA concentration of the mixed solution, and SPB thickness. The SPB consist of a polystyrene core with a diameter around 80 nm and a dense shell of poly (2-aminoethylmethacrylate hydrochloride) (PAEMH) with a thickness from 10 to 60 nm covalently attached on the core surface. Results revealed the existence of three pH regions, corresponding to (1) adsorption of BSA in SPB, (2) aggregation of SPB induced by BSA adsorption, and (3) desorption of BSA from SPB. The extent of the pH regions can be modulated by ionic strength, BSA concentration, or SPB thickness. Adsorption measurements demonstrated that the adsorbed amount of BSA in SPB was affected by pH, ionic strength, and SPB thickness. These findings lay the foundation for protein separation by SPB.

Complexation and coacervation of polyelectrolytes with oppositely charged colloids.

Polyelectrolyte-colloid coacervation could be viewed as a sub-category of complex coacervation, but is unique in (1) retaining the structure and properties of the colloid, and (2) reducing the heterogeneity and configurational complexity of polyelectrolyte-polyelectrolyte (PE-PE) systems. Interest in protein-polyelectrolyte coacervates arises from preservation of biofunctionality; in addition, the geometric and charge isotropy of micelles allows for better comparison with theory, taking into account the central role of colloid charge density. In the context of these two systems, we describe critical conditions for complex formation and for coacervation with regard to colloid and polyelectrolyte charge densities, ionic strength, PE molecular weight (MW), and stoichiometry; and effects of temperature and shear, which are unique to the PE-micelle systems. The coacervation process is discussed in terms of theoretical treatments and models, as supported by experimental findings. We point out how soluble aggregates, subject to various equilibria and disproportionation effects, can self-assemble leading to heterogeneity in macroscopically homogeneous coacervates, on multiple length scales.

Protein specificity of charged sequences in polyanions and heparins.

Long-range electrostatic interactions are generally assigned a subordinate role in the high-affinity binding of proteins by glycosaminoglycans, the most highly charged biopolyelectrolytes. The discovery of high and low sulfation domains in heparan sulfates, however, suggests selectivity via complementarity of their linear sulfation patterns with protein charge patterns. We examined how charge sequences in anionic/nonionic copolymers affect their binding to a protein with prominent charge anisotropy. Experiments and united-atom Monte Carlo simulations, together with Delphi electrostatic modeling for the protein, confirm strongest binding when polyanion sequences allow for optimization of repulsive and attractive electrostatics. Simulations also importantly identified retention of considerable polyion conformational freedom, even for strong binding. The selective affinity for heparins of high and low charge density found for this protein is consistent with nonspecific binding to distinctly different protein charge domains. These findings suggest a more nuanced view of specificity than previously proposed for heparinoid-binding proteins.

Heterogeneity of polyelectrolyte diffusion in polyelectrolyte-protein coacervates: a 1H pulsed field gradient NMR study.

Proton pulsed field gradient (PFG) NMR was used to study the diffusion of poly(diallyldimethylammonium chloride) (PDADMAC) in coacervates formed from this polycation and the protein bovine serum albumin (BSA). Application of high (up to 30 T/m) magnetic field gradients in PFG NMR measurements allowed probing the diffusion of PDADMAC on a length scale of displacements as small as 100 nm in coacervates formed at different pH's and ionic strengths, i.e., conditions of varying protein-polycation interaction energy. Studies were carried out for a broad range of diffusion times and corresponding values of the mean square displacements. Several ensembles of PDADMAC polycations with different diffusivities were observed in the measured range of diffusion times. The existence of these ensembles and the pattern of their changes with increasing diffusion time support the hypothesis about the microscopic heterogeneity of PDADMAC-BSA coacervates and also provide evidence for the dynamic disintegration and reformation of dense domains.

Effect of polyelectrolyte structure on protein-polyelectrolyte coacervates: coacervates of bovine serum albumin with poly(diallyldimethylammonium chloride) versus chitosan.

Electrostatic interactions between synthetic polyelectrolytes and proteins can lead to the formation of dense, macroion-rich liquid phases, with equilibrium microheterogeneities on length scales up to hundreds of nanometers. The effects of pH and ionic strength on the rheological and optical properties of these coacervates indicate microstructures sensitive to protein-polyelectrolyte interactions. We report here on the properties of coacervates obtained for bovine serum albumin (BSA) with the biopolyelectrolyte chitosan and find remarkable differences relative to coacervates obtained for BSA with poly(diallyldimethylammonium chloride) (PDADMAC). Coacervation with chitosan occurs more readily than with PDADMAC. Viscosities of coacervates obtained with chitosan are more than an order of magnitude larger and, unlike those with PDADMAC, show temperature and shear rate dependence. For the coacervates with chitosan, a fast relaxation time in dynamic light scattering, attributable to relatively unrestricted protein diffusion in both systems, is diminished in intensity by a factor of 3-4, and the consequent dominance by slow modes is accompanied by a more heterogeneous array of slow apparent diffusivities. In place of a small-angle neutron scattering Guinier region in the vicinity of 0.004 A-1, a 10-fold increase in scattering intensity is observed at lower q. Taken together, these results confirm the presence of dense domains on length scales of hundreds of nanometers to micrometers, which in coacervates prepared with chitosan are less solidlike, more interconnected, and occupy a larger volume fraction. The differences in properties are thus correlated with differences in mesophase structure.

Effect of pore size on adsorption of a polyelectrolyte to porous glass.

The adsorption of quaternized poly(vinylpyridine) (QPVP) on controlled pore glass (CPG) size, over the ionic strength range 0.001-0.5 M was found to display nonmonotonic behavior as a function of pore size. Both adsorption kinetics and ionic strength effects deviated dramatically from behavior typical of adsorption on flat surfaces when the ratio of the pore radius Rp to the polymer hydrodynamic radius Rh became smaller than ca. 2. Ionic strength enhancement of adsorption for small pore sizes was observed at much higher salt concentrations than is typical for polycation adsorption on flat surfaces. The amount of polymer adsorbed per unit surface area of glass GammaA, in 0.5 M NaCl, exhibited a shallow maximum at Rp/Rh approximately 5. Since the value of GammaA for small pore size CPG is strongly depressed by the large surface area, an alternative and more interesting observation is that the amount of polymer adsorbed per gram of CPG, Gammaw, displays a strong maximum when Rp is equal to or slightly smaller than Rh. The efficiency with which QPVP binds anionic micelles to (negatively charged) CPG (grams of surfactant/grams of QPVP) increases strongly with diminishing pore size, indicating that the configuration of polycation bound to small pores favors micelle binding. Since the micelles are larger than small pores, the results indicate that when Rp < Rh, adsorbed polycation molecules reside only partially within the pore. The results of this study are supported by simulations of polyelectrolytes within cylindrical cavities.

Mesophase separation and probe dynamics in protein-polyelectrolyte coacervates.

Protein-polyelectrolyte coacervates are self-assembling macroscopically monophasic biomacromolecular fluids whose unique properties arise from transient heterogeneities. The structures of coacervates formed at different conditions of pH and ionic strength from poly(dimethyldiallylammonium chloride) and bovine serum albumin (BSA), were probed using fluorescence recovery after photobleaching. Measurements of self-diffusion in coacervates were carried out using fluorescein-tagged BSA, and similarly tagged Ficoll, a non-interacting branched polysaccharide with the same size as BSA. The results are best explained by temporal and spatial heterogeneities, also inferred from static light scattering and cryo-TEM, which indicate heterogeneous scattering centers of several hundred nm. Taken together with previous dynamic light scattering and rheology studies, the results are consistent with the presence of extensive dilute domains in which are embedded partially interconnected 50-700 nm dense domains. At short length scales, protein mobility is unobstructed by these clusters. At intermediate length scales, proteins are slowed down due to tortuosity effects within the blind alleys of the dense domains, and to adsorption at dense/dilute domain interfaces. Finally, at long length scales, obstructed diffusion is alleviated by the break-up of dense domains. These findings are discussed in terms of previously suggested models for protein-polyelectrolyte coacervates. Possible explanations for the origin of mesophase separation are offered.

Effects of polyelectrolyte chain stiffness, charge mobility, and charge sequences on binding to proteins and micelles.

The binding affinities of polyanions for bovine serum albumin in NaCl solutions from I = 0.01-0.6 M, were evaluated on the basis of the pH at the point of incipient binding, converting each such pH(c) value into a critical protein charge Zc. Analogous values of critical charge for mixed micelles were obtained as the cationic surfactant mole fraction Yc. The data were well fitted as Yc or Zc = KI a, and values of K and a were considered as a function of normalized polymer charge densities (tau), charge mobility, and chain stiffness. Binding increased with chain flexibility and charge mobility, as expected from simulations and theory. Complex effects of tau were related to intrapolyanion repulsions within micelle-bound loops (seen in the simulations) or negative protein domain-polyanion repulsions. The linearity of Zc with radicalI at I < 0.3 M was explained by using protein electrostatic images, showing that Zc at I < 0.3 M depends on a single positive "patch"; the appearance of multiple positive domains I > 0.3 M (lower pH(c)) disrupts this simple behavior.