Many responsive polymeric systems, important for pharmaceutical formulations, exhibit complex phase behavior which require both large and precise experimental data sets for identifying phase boundaries between various self-assemblies, morphologies, and solution properties. Datasets of these polymeric systems are often limited by the quantity of materials and the time required to prepare samples and collect data. Here we develop automated methods for sample preparation and analysis while using initial sample volumes on the order of 100 µL. This volume was small enough to enable a dataset of a model monoclonal antibody protein solution. Two sample-delivery approaches (syringe- or pneumatic-based) are used to control the flow and mixing within the same measurement platform. The sample is appropriately delivered to a commercial low-volume temperature-controlled spectroscopic flow cell for characterization using in-line ultraviolet-visible spectroscopy, turbidity, dynamic light scattering, and a separate microcapillary rheometer for viscosity measurements. This approach can be further optimized to expedite structure-property datasets for the development of materials with targeted properties.

We observed individual macromolecular chains with distinct ring topology when studying cryogenic electron microscopy (Cryo-EM) images of a high molar mass polyorganophosphazene in its vitrified aqueous solution. The presence of monocyclic chains was confirmed by visualizing samples of the same macromolecule in its absorbed form by using atomic force microscopy (AFM). The polymer, poly[di(carboxylatophenoxy)phosphazene], PCPP, is synthesized via a two-stage process, which includes a ring-opening polymerization (ROP) and a subsequent macromolecular substitution transformation. The visualization of macrocycles, which are 20-60 nm in diameter, provides direct proof of a ring extension polymerization (REP) mechanism occurring in the chain-growth process commonly employed for the synthesis of high molar mass polyphosphazenes. Asymmetric flow field flow fractionation (AF4) analysis shows the presence of a faster moving fraction, which can be potentially attributed to macrocycles.

We report that polar cosolvent-water mixtures offer a unique approach to controlling the liquid-liquid phase separation (LLPS) of polyelectrolyte complex solutions formed from degree of polymerization-matched mixtures of strong and weak polyelectrolytes─respectively, quaternary poly(N,N-dimethylaminoethyl methacrylate chloride) (qPDMAEMA) and sodium poly(acrylate) (PA). As observed in prior work, associative LLPS in water exhibits an upper-critical salt concentration with stoichiometric complexes and lower-critical solution temperature (LCST) behavior, where electrostatic correlations are believed to drive phase behavior. However, upon addition of a miscible cosolvent prior to mixing the individual polyelectrolytes at room temperature, we observe a shift in the LCST and the appearance of an upper-critical solution temperature (UCST). This new UCST feature corresponds to a segregative LLPS, whereby the polycation partitions out of the polyanion-rich dense phase and into the supernatant. This behavior arises with cosolvents that decrease (e.g., ethylene glycol) or increase (e.g., N-methyl formamide) the average solvent dielectric constant, suggesting that electrostatic correlations may not primarily control the phase behavior for cosolvated coacervate systems. A conceptual 3D phase surface summarizing these observations for the cosolvated system suggests that two distinct surfaces with critical lines appear on the polymer-salt-temperature phase diagram.

Advances in nucleic acid delivery have inspired efforts to mimic the function of natural viruses through the development of self-assemblies capable of gene delivery. RNA assemblies based on amphiphilic polymers are emerging as alternatives to lipid nanoparticles, but the factors that govern the self-assembly of RNA with polymeric amphiphiles are poorly understood. Here, we describe the structure of coacervate nanoparticle assemblies derived from RNA and synthetic cationic polymer amphiphiles based on Charge Altering Releasable Transporters (CARTs). CARTs are effective gene delivery agents derived from block copolymer amphiphiles. Cryogenic electron microscopy and tomography (CryoEM, CryoET), small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) reveal that the self-assembly of RNA with low molar mass (≤10,000 g/mol) CART amphiphiles generates nanoparticles with disordered bicontinuous internal morphologies composed of interpenetrating lipid and aqueous coacervate domains. Systematic variation of the cationic and lipophilic blocks in low molar mass CART amphiphiles demonstrates that both the internal domain spacings (6 to 8 nm) and the order of the resulting bicontinuous CART-RNA assemblies depend on the CART chemical structure and the oligonucleotide cargo (mRNA vs siRNA). Notably, the presence of RNA drives the formation of bicontinuous morphologies. In contrast, CART/RNA assemblies with higher molar mass (≥28,000 g/mol) CART amphiphiles fail to generate bicontinuous assemblies, instead yielding aggregates composed of particles approximately 10 to 20 nm in diameter. This work illuminates the internal morphologies of RNA assemblies with synthetic block copolymer amphiphiles, with implications for the rational design of polymer-based RNA delivery systems.

Polyorganophosphazenes are water-soluble macromolecules with immunoadjuvant activity that self-assemble with proteins to enable biological functionality. Direct imaging by cryogenic electron microscopy uncovers the coil structure of those highly charged macromolecules. Here, we successfully visualize individual polymer chains within the vitrified state in the absence of additives for contrast enhancement which is attributed to the high mass contrast of the inorganic backbone. Upon assembly with proteins, multiple protein copies bind at the single polymer chain level resulting in structures reminiscent of compact spherical complexes or stiffened coils. The outcome depends on protein characteristics and cannot be deduced by commonly used characterization techniques, such as light scattering, thus revealing direct morphological insights crucial for understanding biological activity. Atomic force microscopy supports the morphology outcomes while advanced analytical techniques confirm protein-polymer binding. The chain visualization methodology provides tools for gaining insights into the processes of supramolecular assembly and mechanistic aspects of polymer-enabled vaccine delivery. Polyorganophosphazenes are macromolecules that selfassemble with antigenic proteins to enable biological functionality. Here, direct imaging by cryogenic electron microscopy reveals the coil structure of their individual chains and compact stiffened coils of their complexes with proteins.

Evidence is shown that cosolvent mixtures control the coacervation of mixtures of oppositely charged polyelectrolytes. Binary and ternary solvent mixtures lead to non-monotonic solubility as a function of the average dielectric constants of the solvent mixtures. These data are rationalized by considering both electrostatic-driven phase separation and solvophobic-driven phase separation using group contribution effects on solubility parameters. These estimates are introduced into an effective Flory-Huggins interaction parameter within the framework of Voorn-Overbeek theory with variable dielectric constants and temperature dependences. Despite its simplicity, the model recovers salient experimental observations not only on their coacervate stabilities, but also on their lower critical solution temperature behaviors. These observations highlight the importance of weak van der Waals interactions in determining the phase behaviors of polyelectrolyte complexes relative to electrostatic correlations. Non-monotonic dependence of polyelectrolyte complex solubility on the average dielectric constants is observed in solvent mixtures, as rationalized in the framework of Voorn-Overbeek theory including an effective Flory-Huggins term.

A model zwitterionic polysulfobetaine, poly(3-(acrylamidopropyl-dimethyl-ammonium) propyl-1-sulfonate) (pAPAPS), phase separates upon cooling and exhibits an upper critical solution temperature (UCST) behavior with no added salt in deuterium oxide solutions. Dynamic light scattering measurements indicate the presence of distinct fast and slow diffusive modes, where the fast mode is interpreted as a collective diffusion coefficient and the slow mode is attributed to the diffusion of multi-chain dynamic clusters. The relative population of fast and slow modes varies systematically with temperature and concentration. A clustering temperature (T*) was assigned when the slow mode first appeared upon cooling. The slow mode then increases in relative scattering amplitude as the phase boundary is approached. The fast mode exhibits a concentration dependence above T* consistent with the virial expansion in the collective diffusion. The sign of the virial coefficient (kd) is negative, even in the good solvent region above the expected Flory temperature (Θ ≈ 39 °C), a behavior distinct from synthetic neutral polymers in organic solvents. The onset of multi-chain clustering at T < T* coincides with the poor solvent regime (T < Θ). Attractive dipolar interactions due to the zwitterionic sulfobetaine groups in pAPAPS are suggested as the origin of the multi-chain clusters with no salt. Upon the addition of 100 mM NaCl, the slow mode is suppressed, and the hydrodynamic radius is consistent with polyzwitterion chain dimensions in a dilute solution. We find that concentration dependent diffusion is highly linked to the theta temperature and the emergence of dynamic clusters as the polymer goes from good to poor solvent on approach to the UCST. The slow mode in the semidilute regime is reported along with preliminary small-angle neutron scattering data that show salt reduces clustering and leads to predominantly chain scattering.

Polyphosphazenes represent a class of intrinsically flexible polyelectrolytes with potent immunoadjuvant activity, which is enabled through non-covalent self-assembly with antigenic proteins by charge complexation. The formation of supramolecular complexes between polyphosphazene adjuvant, poly[di(carboxylatophenoxy)phosphazene] (PCPP), and a model vaccine antigen, hen egg lysozyme, was studied under physiological conditions using automated dynamic light scattering titration, asymmetric flow field flow fractionation (AF4), enzyme-linked immunosorbent assay (ELISA), and fluorescent quenching methods. Three regimes of self-assembly were observed covering complexation of PCPP with lysozyme in the nano-scale range, multi-chain complexes, and larger aggregates with complexes characterized by a maximum loading of over six hundred protein molecules per PCPP chain and dissociation constant in the micromolar range (Kd = 7 × 10-6 mol/L). The antigenicity of PCPP bound lysozyme, when compared to equivalent lysozyme solutions, was largely retained for all complexes, but observed a dramatic reduction for heavily aggregated systems. Routes to control the complexation regimes with elevated NaCl or KCl salt concentrations indicate ion-specific effects, such that more smaller-size complexes are present at higher NaCl, counterintuitive with respect to PCPP solubility arguments. While the order of mixing shows a prominent effect at lower stoichiometries of mixing, higher NaCl salt reduces the effect all together.

We investigate the hydration of poly(3-[2-(acrylamido) ethyldimethylammonio] propanesulfonate) over a range of temperatures in pure water and with the inclusion of 0.1 mol/L NaCl using atomistic molecular dynamics simulation. Drawing on concepts drawn from the field of glass-forming liquids, we use the Debye-Waller parameter () for describing the water mobility gradient around the polybetaine backbone extending to an overall distance ≈18 Å. The water mobility in this layer is defined through the mean-square water molecule displacement at a time on the order of water's β-relaxation time. The brushlike topology of polybetaines leads to two regions in the dynamic hydration layer. The inner region of ≈10.5 Å is explored by pendant group conformational motions, and the outer region of ≈7.5 Å represents an extended layer of reduced water mobility relative to bulk water. The dynamic hydration layer extends far beyond the static hydration layer, adjacent to the polymer.

We examine whether the mode-coupling theory of Kawasaki and Ferrell (KF) [Kawasaki, K. Kinetic Equations and Time Correlation Functions of Critical Fluctuations. Ann. Phys. 1970, 61 (1), 1-56; Ferrell, R. A. Decoupled-Mode Dynamical Scaling Theory of the Binary-Liquid Phase Transition. Phys. Rev. Lett. 1970, 24 (21), 1169-1172] can describe dynamic light scattering (DLS) measurements of the dynamic structure factor of near-critical polyelectrolyte complex (PC) solutions that have been previously shown to exhibit a theoretically unanticipated lower critical solution temperature type phase behavior, i.e., phase separation upon heating, and a conventional pattern of static critical properties (low angle scattering intensity and static correlation, ξs) as a function of reduced temperature. Good qualitative accord is observed between our DLS measurements and the KF theory. In particular, we observe that the collective diffusion coefficient Dc of the PC solutions obeys the generalized Stokes-Einstein equation (GSE), Dc = kBT/6πηξs, where ξs is specified from our previous measurements and where η is measured by capillary rheometry under the same thermodynamic conditions as in our previous study of these solutions, allowing for a no-free-parameter test of the GSE. We also find that even the wavevector (q)-dependent collective diffusion coefficient Dc(q), measured by varying the scattering angle in the DLS measurements over a large range, is also well-described by the mean-field version of the KF theory. We find it remarkable that the KF theory provides such a robust description of collective diffusion in these complex charged polyelectrolyte blends under near-critical conditions given that charge fluctuations and association of the polymers might be expected to lead to physical complications that would invalidate the standard model of uncharged fluid mixtures.

Polyorganophosphazenes are biodegradable macromolecules with potent immunoadjuvant activity that self-assemble with protein antigens to provide biological activity. Direct imaging by cryogenic electron microscopy reveals the coil structure of the highly-charged high molecular mass synthetic polyorganophosphazenes within the vitrified state without any additives for contrast enhancement for the first time. Upon mixing with protein antigens under a controlled stoichiometric ratio, multiple proteins bind at the single chain level revealing a structural change reminiscent of compact spherical complexes or stiffened coils depending on the bound protein antigen. The structural outcome depends on the protein charge density that cannot be deduced by methods, such as dynamic light scattering, thus revealing direct morphological insight necessary to understand in vivo biological activity. Complementary atomic force microscopy supports the binding morphology outcomes as well as additional analytical techniques that indicate binding. These observations open opportunities to understand supramolecular assembly of proteins and other biomacromolecules at the single chain level with highly charged polyelectrolytes for vaccines as well as important to developing fields such as polyelectrolyte complex coacervation.

Nanostructures similar to those found in the vividly blue wings of Morpho butterflies and colorful photonic crystals enable structural color through constructive interference of light waves. Different from commonly studied structure-colored materials using periodic structures to manipulate optical properties, we report a previously unrecognized approach to precisely control the structural color and light transmission via a novel photonic colloidal gel without long-range order. Nanoparticles in this gel form micrometer-sized bicontinuous domains driven by the microphase separation of binary solvents. This approach enables dynamic coloration with a precise wavelength selectivity over a broad range of wavelengths extended well beyond the visible light that is not achievable with traditional methods. The dynamic wavelength selectivity is thermally tunable, reversible, and the material fabrication is easily scalable.

AbstractPolyelectrolytes contain many units of charged moieties, often as either anionic or cationic. However, when a polymer comprises both anionic and cationic monomers, the result is a polyampholyte. Polyzwitterions are a type of polyampholyte that contain pairs of opposite charges within each monomer unit, ultimately leading to charge neutrality across the entire polymer structure. These special classes of ion‐containing polymers have many applications enabled by advances in synthesis. This article discusses the unique solution‐responsive behaviors of these ion‐containing polymers and how their chemistry is used to alter structure–property relationships.

Lipid vesicles are widely used as models for cell membranes and extracellular vesicles, hosts for membrane protein studies, and containers for hydrophilic biotherapeutic molecules. Vesicle solutions are usually prepared at a specific lipid concentration; however, because vesicles are solvent-filled structures, the corresponding volume fraction of vesicles is at least a factor or 3 times higher than the corresponding lipid volume fraction and critically depends on the vesicle radii. Seemingly low lipid concentrations correspond to significantly higher vesicle volume fractions and closer face-to-face distances between their surfaces than may be expected.

Responsive polyzwitterionic materials have become important for a range of applications such as environmental remediation and targeted drug delivery. Much is known about the macroscopic phase-behaviors of such materials, but how the smaller scale single-chain structures of polyzwitterions respond to external stimuli is not well understood, especially at temperatures close to their phase boundaries. Such chain conformation responses are important in directing larger-scale associative properties. Here, we study the temperature dependent single-chain structure of a model polysulfobetaine, poly[3-(acrylamidopropyl-dimethyl-ammonium) propyl-1-sulfonate], using small angle neutron scattering. In the absence of salt, we find that temperature has a large effect on solvent quality with a decreasing trend from good solvent conditions at 50 °C to poor solvent at 10 °C (a temperature just above the cloud point of 7.6 °C) and an estimated theta temperature of 39 °C. When 100 mM NaCl is present, the solvent quality is good with weak temperature dependence. Without salt present, the polymer chain appears to have a nearly Gaussian coil conformation and the backbone becomes slightly more rigid as the temperature is lowered to the cloud point as determined by the Debye-local rod model on a Kratky plot. The addition of salt has a notable effect on the intra-chain correlations where an increase in chain dimensions to a swollen coil conformation and an increase in chain rigidity is observed at 100 mM NaCl in D2O, however, with a negligible temperature dependence.

Polyampholytes are polymers with both cationic and anionic monomers that have many features in common with proteins. These molecules have many applications associated with their natural biocompatibility and their propensity to form viscoelastic fluids and gels due to their common tendency to form self-assembled structures in solution. Recently, advances in the synthesis capabilities have enabled the fine-tuning of the monomer structure and composition, such as the sequence and ratio of the charged and hydrophobic groups. We correspondingly investigate these molecular characteristics on the solution properties of a series of poly(carbonate)-based polyampholytes using both molecular dynamics simulations and small-angle neutron scattering measurements. Our simulations suggest the importance of hydrophobic groups for understanding the observed properties of our polyampholyte solutions, a well-known fact for protein solutions. In particular, our simulations indicate that the hydrophobic groups cause the polyampholyte chains to form compact micelle-like rather than diffuse clusters at low concentrations and greatly influence the concentration dependence of the correlation peak in the structure factor describing the correlation distance between the polyampholyte chains in solution. We also construct a polyampholyte solution association map where the polymer concentration and charge ratios lead to different propensities of association (or clustering) and determine a self-assembly boundary governing the transition between self-assembled clusters that resemble micelles and a transient polymer network that percolates the system.

Lipid vesicles are widely used as models for cell membranes, hosts for membrane protein studies, and containers for hydrophilic molecules. The vesicle solutions in these applications are usually prepared at a specific lipid concentration; however, because vesicles are solvent-filled structures, the corresponding volume fraction of vesicles is at least a factor of three times higher than the corresponding lipid volume fraction and critically depends on the vesicle radii. Here we show that these higher than may be expected vesicle volume fractions result in measurable interactions between the vesicles as well as affect the vesicle diffusion. We show that vesicle solutions prepared with lipid mass fractions, m L , as low as ≈ 0.004, which correspond to a lipid concentration of ≈ 4 mg/mL or 5 mmol/L (mM), not only have a measurable apparent structure factor (S′(q)) in small angle neutron scattering (SANS) experiments, but that this repulsive structure factor also affects the measured diffusion coefficient at small scattering vectors (q) such as those probed with dynamic light scattering (DLS). The measured diffusion coefficients are further affected by indirect solvent mediated interactions described by a hydrodynamic factor (H(q)). Accounting for the concentration-dependence of the vesicle diffusion shows that the lipid concentration dependence measured in neutron spin echo (NSE) spectroscopy is due to differences in the effective vesicle diffusion coefficients and not the membrane fluctuation dynamics. The results have practical implications for static and dynamic scattering experiments as well as provide interesting insights into the interactions between soft lipid vesicles.

An ultra-small angle light scattering setup with the ability of simultaneous registration of scattered light by a charge-coupled device camera and the transmitted direct beam by a pin photodiode was developed. A pinhole mirror was used to reflect the scattered light; the transmitted direct beam was focused and passed through the central pinhole with a diameter of 500 μm. Time-resolved static light scattering measurement was carried out over the angular range 0.2° ≤θ≤ 8.9° with a time resolution of ∼33 ms. The measured scattering pattern in the q-range between 5 × 10-5 and 1.5 × 10-3 nm-1 enables investigating structures of few micrometers to submillimeter, where q is the scattering vector. A LabVIEW-based graphical user interface was developed, which integrates the data acquisition of the scattering pattern and the transmitted intensity. The Peltier temperature-controlled sample cells of varying thicknesses allow for a rapid temperature equilibration and minimization of multiple scattering. The spinodal decomposition for coacervation (phase separation) kinetics of an aqueous mixture of oppositely charged polyelectrolytes was demonstrated.

The interfacial tension of coacervates, the liquidlike phase composed of oppositely charged polymers that coexists at equilibrium with a supernatant, forms the basis for multiple technologies. Here we present a comprehensive set of experiments and molecular dynamics simulations to probe the effect of molecular mass on interfacial tension γ, far from the critical point, and derive γ=γ_{∞}(1-h/N), where N is the degree of polymerization, γ_{∞} is the infinite molecular mass limit, and h is a constant that physically corresponds to the number of monomers of one chain within the coacervate correlation volume.