Dialysis Monitoring of Ionic Strength and Denaturant Effects, and Their Reversibility, for Various Classes of Macromolecules.

Monitoring membrane-mediated dialysis in real time with static and dynamic light scattering revealed distinctive differences, including reversibility/irreversibility, in the effects of ionic strength (NaCl) and the denaturant guanidine-HCl (Gd) on a synthetic polyelectrolyte and several types of biomacromolecules: protein, polysaccharide, and polyampholyte. Dialysis cycles against aqueous NaCl and Gd, and reverse back to the original aqueous solution, were monitored. The behavior of Na-polystyrenesulfonate was reversible and yielded a detailed polymer physics description.

DNA Released by Adeno-Associated Virus Strongly Alters Capsid Aggregation Kinetics in a Physiological Solution.

While adeno-associated virus is a leading vector for gene therapy, significant gaps remain in understanding AAV degradation and stability. In this work, we study the degradation of an engineered AAV serotype at physiological pH and ionic strength. Viral particles of varying fractions of encapsulated DNA were incubated between 30 and 60 °C, with changes in molecular weight measured by changes in total light scattering intensity at 90° over time.

Route and antigen shape immunity to dmLT-adjuvanted vaccines to a greater extent than biochemical stress or formulation excipients.

A key aspect to vaccine efficacy is formulation stability. Biochemical evaluations provide information on optimal compositions or thermal stability but are routinely validated by ex vivo analysis and not efficacy in animal models. Here we assessed formulations identified to improve or reduce stability of the mucosal adjuvant dmLT being investigated in polio and enterotoxigenic E.

Observation and Modeling of a Sharp Oxygen Threshold in Aqueous Free Radical and RAFT Polymerization.

It is known that oxygen (O) stops radical polymerization (RP). Here, it was found that the reaction turn-off occurs abruptly at a threshold concentration of O, [O], for both free RP and reversible addition-fragmentation chain-transfer polymerization (RAFT). In some reactions, there was a spontaneous re-start of conversion.

Anion binding to ubiquitin and its relevance to the Hofmeister effects.

Although the non-covalent interactions between proteins and salts contributing to the Hofmeister effects have been generally mapped, there are many questions regarding the specifics of these interactions. We report here studies involving the small protein ubiquitin and salts of polarizable anions. These studies reveal a complex interplay between the reverse Hofmeister effect at low pH, the salting-in Hofmeister effect at higher pH, and six anion binding sites in ubiquitin at the root of these phenomena.

A polarization sensitive light scattering unit for high throughput screening.

A simple, effective light scattering prototype sensitive to both polarized and depolarized scattering was constructed, and its performance was tested on a variety of pure liquids and optically isotropic and anisotropic polymer solutions and colloidal suspensions. The results, performance, and means to further improvement are reported here. Because of its simplicity and low cost, many identical units can be produced to construct a simultaneous multiple sample light scattering platform that can be used to monitor polymer and colloid solution stability, phase changes, aggregation, degradation, etc.

On the Reproducibility of Early-Stage Thermally Induced and Contact-Stir-Induced Protein Aggregation.

Is aggregation kinetics for a protein under given conditions reproducible? Is aggregation inherently deterministic, stochastic, or even chaotic? Because protein aggregation in ex vivo formulations is complex, with many origins and manifestations, the question of aggregation reproducibility for a given protein, formulation, and stressor is of both fundamental and practical significance. This work concerns temperature-induced and contact-stir-induced aggregation of bovine serum albumin (BSA) and a monoclonal antibody (mAbX). It assesses reproducibility via early-stage aggregation rates (ARs) from light scattering.

Polydopamine particles as nontoxic, blood compatible, antioxidant and drug delivery materials.

Herein, the potential biomedical application of poly(3,4-dihyroxyphenyl)ethylamine, (poly(dopamine)-p(DA)) particles is reported. P(DA) particles with the size about 100 nm, 18.05 m/g specific surface area, and mesoporous structure (7.

Polysaccharide depolymerization from TEMPO-catalysis: Effect of TEMPO concentration.

Polysaccharide TEMPO-oxidation was monitored using automatic continuous online monitoring of polymerization reactions (ACOMP). The products of oxidation, obtained at different pHs (9, 7 and 5) and different concentrations of catalyst TEMPO, were evaluated by Automatic Continuous Mixing (ACM) and Size Exclusion Chromatography (SEC). The degree of oxidation was higher at pH 9 and polysaccharide degradation was observed under different pH conditions, but was much higher without catalyst TEMPO.

Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering.

Parallel temperature initial rates (PTIR) from chromatographic separation of aggregating protein solutions are combined with continuous simultaneous multiple sample light scattering (SMSLS) to make quantitative deductions about protein aggregation kinetics and mechanisms. PTIR determines the rates at which initially monomeric proteins are converted to aggregates over a range of temperatures, under initial-rate conditions. Using SMSLS for the same set of conditions provides time courses of the absolute Rayleigh scattering ratio, IR(t), from which a potentially different measure of aggregation rates can be quantified.

Enhanced surfactant supramicellar assembly by hydrophobic dopants.

The light scattering peak (LS peak) near the surfactant critical micelle concentration (cmc) demonstrates that low hydrophobic dopant levels can cause supramolecular assembly of surfactants into metastable structures much more massive than dopant-free micelles. These supramicellar assemblies (SA) exist over the entire LS peak region, which extends from above the cmc down to well below the cmc. Dodecanol (D) was the dopant, and sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium bromide (DTABr) were the surfactants in this work.

Monitoring protein aggregation kinetics with simultaneous multiple sample light scattering.

A simultaneous multiple sample light scattering (SMSLS) prototype instrument was built to simultaneously measure light scattering from many independent monoclonal antibody (mAb) solutions in order to monitor their time-dependent aggregation behavior and to characterize, via absolute Rayleigh scattering ratios, their molecular masses and second, third, and fourth virial coefficients under non-aggregating conditions at concentrations up to 190mg/ml. One stable mAb and another prone to aggregation were studied. Early phase aggregation rates spanned six orders of magnitude over temperatures 30 to 83°C for both mAbs and divided into "Arrhenius" and "Stochastic" regimes.

Experimental observation of crossover from noncondensed to counterion condensed regimes during free radical polyelectrolyte copolymerization under high-composition drift conditions.

By combining detailed online kinetics of comonomer consumption with light scattering, viscosity, and conductivity data, experimental detection of changing degrees of counterion condensation was achieved by using comonomers of widely separated reactivity ratios that produced large composition drifts during synthesis. Endproducts of such syntheses contained mixtures of chains of widely varying linear charge density. Evidence of a smooth transition from noncondensed to counterion condensed regimes was found during individual synthesis reactions of copolymeric polyelectrolytes, or "copolyelectrolytes", from the changing slope of conductivity versus [ionic comonomer] incorporated in the high-composition drift syntheses.

Monitoring the synthesis and properties of copolymeric polycations.

The kinetics; evolution of molar mass; solution conductivity, sigma; intrinsic viscosity; and average composition drift; and distribution were determined by monitoring the synthesis of copolymeric polycations of acrylamide (Am) and [2-(acryloyloxy)ethyl]-trimethylammonium chloride (Q9). The quantitative relationship between diminishing sigma and charged co-monomers incorporation was monitored for the first time and provided novel data on counterion condensation, which occurs gradually over a broad composition regime. This new capability allows predictions concerning the relationship between copolymer composition and linear charge density, xi, to be tested and models of trivariate mass, composition, and xi distributions to be built.

Adsorption of copolymers aggregates: from kinetics to adsorbed layer structure.

We examined the adsorption, on hydrophobic and hydrophilic surfaces, of 4 rake-type poly(dimethyl siloxane) (PDMS) copolymers varying the amount of poly(ethylene glycol) (PEG) graft arms from 41 to 72%. The copolymers formed large aggregates in solution, complicating their adsorption kinetics and layer structures. We found the adsorption process always to be dominated by the adsorption of large aggregates, with strongly bound layers resistant to rinsing in adsorbing buffer.

Evolution of composition, molar mass, and conductivity during the free radical copolymerization of polyelectrolytes.

Despite their importance in biological and technological contexts, copolymeric polyelectrolytes (or "copolyelectrolytes") continue to present challenges to theorists and experimentalists. The first results of a unified approach to the kinetics and mechanisms of copolyelectrolyte synthesis and the physical characteristics of the resulting polymers are presented. The free radical copolymerization of 4-vinylbenzenesulfonic acid sodium salt and acrylamide was monitored using automatic continuous online monitoring of polymerization reactions (ACOMP), from which the average bivariate composition and mass distributions were determined.

Online monitoring of polymerization reactions in inverse emulsions.

Automatic continuous online monitoring of polymerization reactions (ACOMP) was adapted to the monitoring of acrylamide polymerization in inverse emulsions. This is the first application of ACOMP to heterogeneous phase polymerization. The conversion and reduced viscosity were monitored by continuously inverting and diluting the emulsion phase using a small reactor sample stream and a breaker surfactant solution, followed by UV absorption and viscometric detection.

Simultaneous in-situ monitoring of parallel polymerization reactions using light scattering; a new tool for high-throughput screening.

A recently introduced technique, simultaneous multiple sample light scattering (SMSLS), was used to monitor parallel polymerization reactions in situ. SMSLS is designed for real-time, high-throughput screening and provides a time-dependent light scattering signature for each reaction, which contains both qualitative and semiquantitative information. Qualitatively, the signature immediately indicates whether the reaction occurs or not, whether there is an initial lag period, and how long the reaction takes until it stops.

Solvent effects on the microstructure and properties of 75/25 poly(D,L-lactide-co-glycolide) tissue scaffolds.

Poly(lactide-co-glycolide) (PLGA) is used in many biomedical applications because it is biodegradable, biocompatible, and FDA approved. PLGA can also be processed into porous tissue scaffolds, often through the use of organic solvents. A static light scattering experiment showed that 75/25 PLGA is well solvated in acetone and methylene chloride, but forms aggregates in chloroform.