Techniques to characterize dynamics in biomaterials microenvironments: XPCS and microrheology of alginate/PEO-PPO-PEO hydrogels.

Many recent studies have highlighted the timescale for stress relaxation of biomaterials on the microscale as an important factor in regulating a number of cell-material interactions, including cell spreading, proliferation, and differentiation. Relevant timescales on the order of 0.1-100 s have been suggested by several studies. While such timescales are accessible through conventional mechanical rheology, several biomaterials have heterogeneous structures, and stress relaxation mechanisms of the bulk material may not correspond to that experienced in the cellular microenvironment. Here we employ X-ray photon correlation spectroscopy (XPCS) to explore the temperature-dependent dynamics, relaxation time, and microrheology of multicomponent hydrogels comprising of commercial poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymer F127 and alginate. Previous studies on this system have shown thermoreversible behavior in the bulk oscillatory shear rheology. At physiological temperatures, bulk rheology of these samples shows behavior characteristic of a soft solid, with G' > G'' and no crossover between G' and G'' over the measurable frequency range, indicating a relaxation time >125 s. By contrast, XPCS-based microrheology shows viscoelastic behavior at low frequencies, and XPCS-derived correlation functions show relaxation times ranging from 10-45 s on smaller length scales. Thus, we are able to use XPCS to effectively probe the viscoelasticity and relaxation behavior within the material microenvironments.

Emulsions Stabilized by Inorganic Nanoclays and Surfactants: Stability, Viscosity, and Implications for Applications.

Pickering emulsions, or emulsions with solid particles at the interface, have attracted significant interest in Enhanced Oil Recovery (EOR) processes, cosmetics, and drug delivery systems due to their ability to resist coalescence. Here, a synthetic clay nanoparticle, laponite®, is utilized to create oil-in-water (o/w) emulsions, and the addition of small-molecule surfactants induces a more stable emulsion. In this study, the stability of laponite® Pickering emulsions with and without the surfactants (dodecyltrimethylammonium bromide (DTAB), Pluronic F68 (F68), and sodium dodecyl sulfate (SDS) is investigated using dynamic light scattering (DLS), ζ-potential, optical microscopy, and rheology. With laponite® and no added surfactants, the DLS and ζ-potential results show formation of emulsion droplets with a diameter of 3 µm and a ζ-potential of -90 mV. With the addition of surfactants, both the droplet diameter and ζ-potential increase, suggesting adsorption of surfactants on the surface of laponite® particle. Optical microscopy suggests that the Pickering emulsion without surfactant undergoes flocculation, while the emulsion becomes stable to coalescence and creaming with addition of surfactants due to formation of a network structure. Regardless of the formation of network structure, the laponite®-F68 emulsion rheologically behaves as a Newtonian fluid, while the laponite®-SDS and laponite®-DTAB emulsions display shear thinning behavior. The difference in the rheological behavior can be attributed to the weak adsorption of F68 on laponite® and electrostatic interactions between laponite® and charged surfactants at oil-water interface.

Impact of stereochemistry on rheology and nanostructure of PLA-PEO-PLA triblocks: stiff gels at intermediate l/d-lactide ratios.

We report rheology and structural studies of poly(lactide)-poly(ethylene oxide)-poly(lactide) (PLA-PEO-PLA) triblock copolymer gels with various ratios of l-lactide and d-lactide in the PLA blocks. These materials form associative micellar gels in water, and previous work has shown that stereoregular triblocks with a l/d ratio of 100/0 form much stiffer gels than triblocks with a 50/50 l/d ratio. Our systems display an unexpected maximum in the storage modulus, G', of the hydrogels at intermediate l/d ratio. The impact of stereochemistry on the rheology is very striking; gels with an l/d ratio of 85/15 have storage moduli that are ∼1-2 orders of magnitude higher than hydrogels with l/d ratios of 100/0. No stereocomplexation is observed in the gels, although PLLA crystals are found for gels with l/d ratios of 95/5 and 90/10, and SANS results show a decrease in the intermicellar spacing for intermediate l/d ratios. We expect the dominant contribution to the elasticity of the gels to be intermicellar bridging chains and attribute the rheology to a competition between an increase in the time for PLA endblocks to pull out of micelles as the l/d ratio is increased and PLLA crystallization occurs, and a decrease in the number of bridging chains for micelles with crystalline PLA domains, as formation of bridges may be hindered by crowded crystalline PLA domains. These results provide a new strategy for controlling the rheology of PLA-based hydrogels for potential applications in biomaterials, as well as fundamental insights into how intermicellar interactions can be tuned via stereochemistry.

Inhibition of antithrombin and bovine serum albumin native state aggregation by heparin.

Protein native state aggregation, a major problem in pharmaceutical and biological processes, has been addressed pharmacologically by the addition of protein-binding excipients. Heparin (Hp), a highly sulfated polysaccharide, interacts with numerous proteins with moderate to high affinity, but reports about its effect on protein aggregation are contradictory. We studied the pH dependence of the aggregation of antithrombin (AT) and bovine serum albumin (BSA) in the presence and absence of heparin. High-precision turbidimetry showed strong aggregation for both AT and BSA in I = 10 mM NaCl, conditions at which electrostatically driven Hp binding and aggregation both occur, with more obvious aggregation of heparin-free AT appearing as larger aggregate size. Aggregation of AT was dramatically inhibited at Hp: protein 6:1 (mole ratio); however, the effect at 0.5:1 Hp:protein was greater for BSA. Frontal analysis capillary electrophoresis showed a much larger equilibrium association constant Kobs between Hp and AT, in accord with the onset of Hp binding at a higher pH; both effects are explained by the higher charge density of the positive domain for AT as revealed by modeling with DelPhi. The corresponding modeling images showed that these domains persist at high salt only for AT, consistent with the 160-fold drop in Kobs at 100 mM salt for BSA-Hp binding. The smaller inhibition effect for AT arises from the tendency of its uncomplexed monomer to form larger aggregates more rapidly, but the stronger binding of Hp to AT does not facilitate Hp-induced aggregate dissolution which occurs more readily for BSA. This can be attributed to the higher density of AT aggregates evidenced by higher fractal dimensions. Differences between inhibition and reversal by Hp arise because the former may depend on the stage at which Hp enters the aggregation process and the latter on aggregate size and morphology.

pH-Dependent aggregation and disaggregation of native ß-lactoglobulin in low salt.

The aggregation of ß-lactoglobulin (BLG) near its isoelectric point was studied as a function of ionic strength and pH. We compared the behavior of native BLG with those of its two isoforms, BLG-A and BLG-B, and with that of a protein with a very similar pI, bovine serum albumin (BSA). Rates of aggregation were obtained through a highly precise and convenient pH/turbidimetric titration that measures transmittance to ±0.05 %T. A comparison of BLG and BSA suggests that the difference between pHmax (the pH of the maximum aggregation rate) and pI is systematically related to the nature of protein charge asymmetry, as further supported by the effect of localized charge density on the dramatically different aggregation rates of the two BLG isoforms. Kinetic measurements including very short time periods show well-differentiated first and second steps. BLG was analyzed by light scattering under conditions corresponding to maxima in the first and second steps. Dynamic light scattering (DLS) was used to monitor the kinetics, and static light scattering (SLS) was used to evaluate the aggregate structure fractal dimensions at different quench points. The rate of the first step is relatively symmetrical around pHmax and is attributed to the local charges within the negative domain of the free protein. In contrast, the remarkably linear pH dependence of the second step is related to the uniform reduction in global protein charge with increasing pH below pI, accompanied by an attractive force due to surface charge fluctuations.

Controlling Rheology of Fluid Interfaces through Microblock Length of Sequence-Controlled Amphiphilic Copolymers.

Previous studies have demonstrated that films of sequence-controlled amphiphilic copolymers display contact angles that depend on microblock size. This suggests that microblock length may provide a means of tuning surface and interfacial properties. In this work, the interfacial rheology of a series of sequence-controlled copolymers, prepared through the addition of bicyclo[4.2.0]oct-1(8)-ene-8-carboxamide (monomer A) and cyclohexene (monomer B) to generate sequences up to 24 monomeric units composed of (A m B n ) i microblocks, where m, n, and i range from 1 to 6. Interfacial rheometry is used to measure the mechanical properties of an air-water interface with these copolymers. As the microblock size increases, the interfacial storage modulus, G', increases, which may be due to an increase in the size of interfacial hydrophobic domains. Small-angle X-ray scattering shows that the copolymers have a similar conformation in solution, suggesting that any variations in the mechanics of the interface are due to assembly at the interface, and not on solution association or bulk rheological properties. This is the first study demonstrating that microblock size can be used to control interfacial rheology of amphiphilic copolymers. Thus, the results provide a new strategy for controlling the dynamics of fluid interfaces through precision sequence-controlled polymers.

XPCS Microrheology and Rheology of Sterically Stabilized Nanoparticle Dispersions in Aprotic Solvents.

X-ray photon correlation spectroscopy (XPCS) microrheology and conventional bulk rheology were performed on silica nanoparticle dispersions associated with battery electrolyte applications to probe the properties of these specific complex materials and to explore the utility of XPCS microrheology in characterizing nanoparticle dispersions. Sterically stabilized shear-thickening electrolytes were synthesized by grafting poly(methyl methacrylate) chains onto silica nanoparticles. Coated silica dispersions containing 5-30 wt % nanoparticles dispersed in propylene carbonate were studied. In general, both XPCS microrheology and conventional rheology showed that coated silica dispersions were more viscous at higher concentrations, as expected. The complex viscosity of coated silica dispersions showed shear-thinning behavior over the frequency range probed by XPCS measurements. However, measurements using conventional mechanical rheometry yielded a shear viscosity with weak shear-thickening behavior for dispersions with the highest concentration of 30% particles. Our results indicate that there is a critical concentration needed for shear-thickening behavior, as well as appropriate particle size and surface polymer chain length, for this class of nanoparticle-based electrolytes. The results of this study can provide insights for comparing XPCS microrheology and bulk rheology for related complex fluids and whether XPCS microrheology can capture expected macroscopic rheological properties by probing small-scale particle dynamics.

Cationic Amphiphilic Alternating Copolymers with Tunable Morphology.

A series of ionic amphiphilic alternating copolymers were characterized via SAXS, TEM and DLS to help understand factors that could potentially affect self-assembly, including the degree of polymerization, the length of hydrophobic spacers between ionic units, the distance between charged groups and polymer backbone, solvent envrioment and counterions.

The triphenylmethane dye brilliant blue G is only moderately effective at inhibiting amyloid formation by human amylin or at disaggregating amylin amyloid fibrils, but interferes with amyloid assays; Implications for inhibitor design.

The development of inhibitors of islet amyloid formation is important as pancreatic amyloid deposition contributes to type-2 diabetes and islet transplant failure. The Alzheimer's Aß peptide and human amylin (h-amylin), the polypeptide responsible for amyloid formation in type-2 diabetes, share common physio-chemical features and some inhibitors of Aß also inhibit amyloid formation by h-amylin and vice versa. Thus, a popular and potentially useful strategy to find lead compounds for anti-amylin amyloid agents is to examine compounds that have effects on Aß amyloid formation. The triphenylmethane dye, brilliant blue G (BBG, Sodium;3-[[4-[(E)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-N-ethyl-3-methylanilino]methyl]benzenesulfonate) has been shown to modulate Aß amyloid formation and inhibit Aß induced toxicity. However, the effects of BBG on h-amylin have not been examined, although other triphenylmethane derivatives inhibit h-amylin amyloid formation. The compound has only a modest impact on h-amylin amyloid formation unless it is added in significant excess. BBG also remodels preformed h-amylin amyloid fibrils if added in excess, however BBG has no significant effect on h-amylin induced toxicity towards cultured ß-cells or cultured CHO-T cells except at high concentrations. BBG is shown to interfere with standard thioflavin-T assays of h-amylin amyloid formation and disaggregation, highlighting the difficulty of interpreting such experiments in the absence of other measurements. BBG also interferes with ANS based assays of h-amylin amyloid formation. The work highlights the differences between inhibition of Aß and h-amylin amyloid formation, illustrates the limitation of using Aß inhibitors as leads for h-amylin amyloid inhibitors, and reinforces the difficulties in interpreting dye binding assays of amyloid formation.

Hierarchical assembly in PLA-PEO-PLA hydrogels with crystalline domains and effect of block stereochemistry.

Understanding the development of microstructure (e.g., structures with length scales roughly 0.5-500 µm) in hydrogels is crucial for their use in several biomedical applications. We utilize ultra-small-angle neutron scattering (USANS) and confocal microscopy to explore microstructure of poly(lactide)-poly(ethylene oxide)-poly(lactide) (PLA-PEO-PLA) triblock copolymer hydrogels with varying l/d-lactide ratio. We have previously found that these polymers self-assemble on the nanoscale into micelles. Here, we observe large-scale structures with diverse morphologies, including highly porous self-similar networks with characteristic sizes spanning approximately 120 nm-200 µm. These structural features give rise to power-law scattering indicative of fractal structures in USANS. Mass fractal and surface fractal structures are found for gels with l/d ratios of 80/20 and 50/50, respectively. Confocal microscopy shows microscale water-filled channels and pores that are more clearly evident in gels with a higher fraction of l-lactide in the PLA block as compared to the 50/50 hydrogels. Tuning block stereochemistry may provide a means of controlling the self-assembly and structural evolution at both the nanoscale and microscale, impacting application of these materials in tissue engineering and drug delivery.