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. Mostly full vectors demonstrated a rapid decrease in molecular weight corresponding to the release of capsid DNA, followed by slow aggregation. In contrast, empty vectors demonstrated immediate, rapid colloid-type aggregation. Mixtures of full and empty capsids showed a pronounced decrease in initial aggregation that cannot be explained by a linear superposition of empty and full degradation scattering signatures, indicating interactions between capsids and ejected DNA that influenced aggregation mechanisms. This demonstrates key interactions between AAV capsids and their cargo that influence capsid degradation, aggregation, and DNA release mechanisms in a physiological solution.

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. coli (ETEC) clinical vaccines. We observed biochemical changes to dmLT protein with formulation or thermal stress, including aggregation or subunit dissociation or alternatively resistance against these changes with specific buffer compositions. However, upon injection or mucosal vaccination with ETEC fimbriae adhesin proteins or inactivated polio virus, experimental findings indicated immunization route and co-administered antigen impacted vaccine immunogenicity more so than dmLT formulation stability (or instability). These results indicate the importance of both biochemical and vaccine-derived immunity assessment in formulation optimization. In addition, these studies have implications for use of dmLT in clinical settings and for delivery in resource poor settings.

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

It is known that oxygen (O2) stops radical polymerization (RP). Here, it was found that the reaction turn-off occurs abruptly at a threshold concentration of O2, [O2]t, for both free RP and reversible addition-fragmentation chain-transfer polymerization (RAFT). In some reactions, there was a spontaneous re-start of conversion. Three cases were investigated: RP of (i) acrylamide (Am) and (ii) sodium styrene sulfonate (SS) and (iii) Am RAFT polymerization. A controlled flow of O2 into the reactor was employed. An abrupt turn-off was observed in all cases, where polymerization stops sharply at [O2]t and remains stopped when [O2] > [O2]t. In (i), Am acts as a catalytic radical-transfer agent during conversion plateau, eliminating excess [O2], and polymerization spontaneously resumes at [O2]t. In no reaction, the initiator alone was capable of eliminating O2. N2 purge was needed to re-start reactions (ii) and (iii). For (i) and (ii), while [O2] < [O2]t, O2 acts a chain termination agent, reducing the molecular weight (Mw) and reduced viscosity (RV). O2 acts as an inhibitor for [O2] > [O2]t in all cases. The radical-transfer rates from Am* and SS* to O2 are >10,000× higher than the initial chain propagation step rates for Am and SS, which causes [O2]t at very low [O2].

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. These sites are all located at protuberances of preorganized secondary structure, and although stronger at low pH, are still apparent when ubiquitin possesses no net charge. These results demonstrate the traceability of these Hofmeister phenomena and suggest new strategies for understanding the supramolecular properties of proteins.

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. Measurable depolarization was found for a variety of organic liquids and suspensions of both polyfluoroethylene and latex spheres. No detectable depolarization was found for various polymers.

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. "Global stressors" affect the entire protein population, for example, temperature. "Local stressors" affect only a partial population at a given instant, for example, stirring. The instrumental error distribution (IED) allows stochasticity to be identified for AR distributions (ARDs) broader than IED. For ARD at the limit of the IED, the behavior is "minimally stochastic" or "operationally deterministic." A stochastic index is defined in terms of the ratio of the standard deviation (SD) of log(AR) data and the SD of IED. Thermal aggregation was operationally deterministic for BSA and mAbX, although significant lot-to-lot variations for BSA were found. ARD from contact-stir-stress was stochastic for BSA and mAb. Despite this, log(AR) decreases logarithmically with rpm. These trends may hold for other global and local stressors.

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 m2/g specific surface area, and mesoporous structure (7.19 nm pore width) were prepared and shown to be chemically modifiable using chlorosulfonic acid (CSA) and 3-CHloro-2 hydroxypropyl) trimethylammonium chloride solution (CHPACl) to obtain sulfonic acid and quaternary amine group containing modified p(DA) particles, m-p(DA)-CSA and m-p(DA)-CHPACl particles, respectively. The hydrolytic degradation of p(DA) particles at different pHs, including 1, 7.4 and 11, was carried out at 37.5 °C. These degradation studies revealed that p(DA) is slightly degradable at pH 1 and pH 7.4 with weight losses of 13.01 ± 0.08% and 7.26 ± 0.23% in 11 days, respectively. At pH 11, a sustained degradation that is almost linear degradation with time was observed for up to 30 days, with a total weight loss of 21.42 ± 0.88%. Furthermore, p(DA) particles were tested for cell toxicity against COS-1 cells and found non-toxic up to 50 µg/mL with 95.6 ± 4.5% cell viability as compared to 37.5 ± 0.03% for DA molecules. The p(DA) particles and DA were also compared for their ability to inhibit α-glucosidase; both inhibited α-glucosidase inhibition activity a concentration-dependent fashion: at concentrations of 500-4000 µg/mL, p(DA) provided 8.52-27.67% inhibition while DA inhibited 42.8-67.7% over the same concentration range. Furthermore, p(DA) particles were found to be blood compatible e.g., non-hemolytic with 1.87 ± 0.97% hemolysis ratio up to 50 µg/mL concentration and with 86.7% blood clotting index. Interestingly, p(DA) particle can be considered as an effective antioxidant with 33.5 ± 3.9 µg/ mL total phenol content in terms of gallic acid equivalency and 0.89 ± 0. 30 µmol/g trolox equivalent antioxidant capacity (TEAC). Finally, p(DA) particles and their modified forms, m-p(DA)-CSA, and m-p(DA)-CHPACl, were shown to be useful as active agent/drug delivery devices by using acyclovir as a model drug that can be readily loaded into particles and released at longer times at higher amounts for the modified p(DA) particles at physiological conditions.

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. The rate constant (k) was dependent on reaction pH and TEMPO concentration. The amount of -COOH per g of polysaccharide, at pH 9, in the presence and absence of TEMPO was different, 0.215 and 0.395mmolg-1, respectively. This suggested a secondary and non-selective polysaccharide oxidation occurring at a lower rate in the absence of catalyst. TEMPO protects the polysaccharide from degradation caused by secondary oxidant species, acting as a catalyst and "sacrificial molecule" at higher concentrations.

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. The present report compares these measures of aggregation rates across a range of solution conditions that result in different aggregation mechanisms for anti-streptavidin (AS) immunoglobulin gamma-1 (IgG1). The results illustrate how the two methods provide complementary information when deducing aggregation mechanisms, as well as cases where they provide new mechanistic details that were not possible to deduce in previous work. Criteria are presented for when the two techniques are expected to give equivalent results for quantitative rates, the potential limitations when solution non-idealities are large, as well as a comparison of the temperature dependence of AS-IgG1 aggregation rates with published data for other antibodies.

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. Well above the cmc dopant is solubilized by micelles. The SA appear above the cmc, dependent on dopant level and solution ionic strength, as seen by an abrupt increase in LS. Hence, the micelles do not simply release their hydrophobic payload at the cmc; rather, the dopant causes a morphological change from normal micelles above the LS peak concentration regime into SA as the LS regime is entered from above the cmc. Below the cmc the LS peak has a cutoff concentration corresponding to the solubility limit of dodecanol in water, which can be termed a "critical supramicellar assembly concentration" (csac). A three-component model is proposed that self-consistently yields maximum micellar dopant loading, SA mass, and dopant solubility in solution. The constancy of SA molar mass under widely different ionic strength and dopant levels conditions is surprising and not currently understood.

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. The Arrhenius regimes comprise two thermal regimes whose breakpoint occurs near the first thermal unfolding temperature of the mAb domain structure. The Stochastic regime occurs for T⩽40°C. Rates yielded activation energies and temperature and concentration crossovers among rate-limiting regimes. Virial coefficients were closely related to aggregation kinetics. Hydrodynamic diameter relationship to virial coefficients provided further insight into stability. SMSLS detected as few as three dimerization events among 1000 monomeric proteins. Although early phase aggregation is linear in time and reproducible, aggregation becomes chaotic in later phases. SMSLS dramatically increases protein monitoring throughput, providing continuous monitoring for hours, weeks, and longer. New samples can be changed in and out without affecting other sample measurements in progress.

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. From this, a model was used to connect the latter slope behavior to fractional ionization of counterions. With this modeled fractional ionization versus the instantaneous fractional amount of the charged comonomer incorporated into copolyelectrolyte, which is directly and continuously monitored during synthesis, it was possible to compute linear charge density xi and the corresponding average xi distribution.

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. This approach, hence, brings together the previously disparate fields of synthetic chemistry of copolymers and physical chemical properties of polyelectrolytes. Monitoring was achieved with a new implementation of the ACOMP (automatic continuous online monitoring of polymerization reactions) platform. Reactivity ratios determined by ACOMP were rQ9 = 0.47 and rAm = 1.10. Opposite trends in composition drift and final molar mass were found; low starting percentage of Q9 led to low composition drift and high molar mass, whereas the opposite was found at high starting percentage of Q9. Complementary end-product analysis by multidetector gel permeation chromatography supported the ACOMP results. End-product polyelectrolyte properties were characterized by automatic continuous mixing, revealing that combined electrostatic persistence length and excluded volume effects led to the expected large changes in polyelectrolyte conformation and interactions. These results set the groundwork for semibatch control of molar mass, composition, and xi, and eventually for monitoring and control for inverse emulsion-based reactions of this type.

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. Adsorbed amounts were nearly independent of the substrate. However, subtleties in the adsorption kinetics suggested different layer structures for the different systems. On hydrophilic silica, aggregates adsorbed at the transport limited rate until surface saturation, and associated interfacial structures were likely retained. On the hydrophobic surface, a subset of the copolymers exhibited retarded late stage adsorption kinetics suggestive of brush formation. This work demonstrates how subtle differences in adsorption kinetics provide insight into potential interfacial layer structures.

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. Composition drift was related to the evolution of conductivity. In some cases bimodal populations of copolyelectrolyte and homopolymeric poly(acrylamide) resulted, i.e., blends of copolyelectrolyte and neutral homopolymer. The end-product scattering behavior depended on whether the end-product was bimodal or not, as demonstrated using automatic continuous mixing (ACM) in conjunction with light scattering and viscosity. Negative light-scattering third virial coefficients were found for bimodal end-products. This combined approach may allow connecting the synthesis kinetics to the resulting "trivariate" distribution of composition, molar mass, and linear charge density, which in turn controls the properties of end-product solutions, such as chain conformations, interparticle interactions, viscosity, interactions with colloids and other polymers, phase separation, etc. Unified results may allow testing and improvement of existing polyelectrolyte theories, development of new quantitative physicochemical models, provide advanced characterization methods, set the stage for studying more complex copolyelectrolytes, such as hydrophobically modified ones, and provide tools for ultimately controlling and tailoring the synthesis and properties of copolyelectrolytes.

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. This inversion into a stable portion of the polymer/surfactant phase diagram is accomplished in tens of seconds, yielding dilute solutions containing acrylamide (Aam), polyacrylamide (PA), oil droplets, and small quantities of surfactant, initiator and other debris, and low molecular weight compounds. After establishing the means of making ACOMP measurements, a first application of the method is made to resolving some of the kinetic issues involved in emulsion polymerization, including the evolution of molecular mass, and the simultaneous action of an "intrinsic" initiator and an added chemical initiator.

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. The signature also provides estimates of the reaction rate and weight average molecular mass M(w), and its shape can help identify mechanistic aspects, for example, controlled versus free radical polymerization, presence of impurities, etc. The method is inherently adapted to small sample volumes and requires no special sample preparation or postpolymerization characterization. The demonstration here involved the free radical polymerization of acrylamide under varying conditions and should be readily applicable to a wide variety of other reactions. Results were cross-checked with multi-detector gel permeation chromatography.

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. This led to an investigation of whether the mechanical properties of the scaffolds were affected by solvent choice. Porous 75/25 PLGA scaffolds were created with the use of the solvent casting/particulate leaching technique with three different solvents: acetone, chloroform, and methylene chloride. Compression testing resulted in stiffness values of 21.7 +/- 4.8 N/mm for acetone, 18.9 +/- 4.2 N/mm for chloroform, and 30.2 +/- 9.6 N/mm for methylene chloride. Permeability testing found values of 3.9 +/- 1.9 x 10(-12) m2 for acetone, 3.6 +/- 1.3 x 10(-12) m2 for chloroform, and 2.4 +/- 1.0 x 10(-12) m2 for methylene chloride. Additional work was conducted to uncouple polymer/solvent interactions from evaporation dynamics, both of which may affect the scaffold properties. The results suggest that solvent choice creates small but significant differences in scaffold properties, and that the rate of evaporation is more important in affecting scaffold microstructure than polymer/solvent interactions.