Rafail Khalfin

Self-Assembly of Bovine β-Casein below the Isoelectric pH

Beta-casein is an intrinsically unstructured amphiphilic protein that self-assembles into micelles at neutral pH. This paper reports that beta-casein self-organizes into micelles also under acidic conditions. The protein association behavior and micelle characteristics at pH 2.6, well below the p I, are presented. The pH was found to strongly affect the micelle shape and dimensions. Cryogenic transmission electron microscopy (cryo-TEM) experiments revealed disk-like micelles of 20-25 nm in length and approximately 3.5 nm in height in acidic conditions. An aggregation number of 6 was determined by sedimentation equilibrium under these conditions. Isothermal titration calorimetry experiments verified the association below the p I and allowed determination of the micellization enthalpy, the critical micellar concentration, and the micellization relative cooperativity (MR). Small-angle X-ray scattering results at concentrations below the critical micellization concentration (CMC) suggest that the monomeric protein is likely in a premolten globule state at low pH. Calculations of the protein charge at acidic and neutral pH reveal a similar high net charge but considerable differences in the charge distribution along the protein backbone. Overall the results show that beta-casein is amphiphilic at low pH, but the distribution of charge along the protein chain creates packing constraints that affect the micelle organization, leading at concentrations above the CMC to the formation of disk micelles.

True molecular solutions of natural cellulose in the binary ionic liquid-containing solvent mixtures

Evidence is presented for the first time of true molecular dissolution of cellulose in binary mixtures of common polar organic solvents with ionic liquid. Cryogenic transmission electron microscopy, small-angle neutron-, X-ray- and static light scattering were used to investigate the structure of cellulose solutions in mixture of dimethyl formamide and 1-ethyl-3-methylimidazolium acetate. Structural information on the dissolved chains (average molecular weight ∼ 5 × 10(4)g/mol; gyration radius ∼ 36 nm, persistence length ∼ 4.5 nm), indicate the absence of significant aggregation of the dissolved chains and the calculated value of the second virial coefficient ∼ 2.45 × 10(-2)mol ml/g(2) indicates that this solvent system is a good solvent for cellulose. More facile dissolution of cellulose could be achieved in solvent mixtures that exhibit the highest electrical conductivity. Highly concentrated cellulose solution in pure ionic liquid (27 wt.%) prepared according to novel method, utilizing the rapid evaporation of a volatile co-solvent in binary solvent mixtures at superheated conditions, shows insignificant cellulose molecular aggregation.

About the albumin structure in solution and related electro-spinnability issues.

In this work, we study the relationship between the shape and form of bovine serum albumin (BSA) protein in different solutions and their ability to form electrospun nanofibers. Small-angle X-ray scattering (SAXS) of the BSA in a water environment, in a 2,2,2-trifluoroethanol (TFE) environment, and in a TFE/beta-mercaptoethanol (beta-ME) environment demonstrated an unfolding pathway; folded, partially unfolded and unfolded states of the protein, respectively. The scattering plot of BSA in water is characterized by a strong peak, thereby describing a solution of densely packaged globules. The scattering of BSA in TFE is attributed to a chain with compact folded-domains along its length, where the scattering of BSA in a mixture of TFE and beta-ME suggests that the protein molecule adopts a freely coiled conformation in this solution. The zeta potential for both solutions of BSA in TFE was found to have an almost zero net charge, while the BSA solution in the water was highly negatively charged. This unfolding between three conformational states was correlated with the changes in electro-spinnability. Results show that the unfolded BSA is the only spinnable solution, producing long and continuous fibers with good mechanical stability.

Celecoxib Encapsulation in β-Casein Micelles: Structure, Interactions, and Conformation.

β-Casein is a 24 kDa natural protein that has an open conformation and almost no folded or secondary structure, and thus is classified as an intrinsically unstructured protein. At neutral pH, β-casein has an amphiphilic character. Therefore, in contrast to most unstructured proteins that remain monomeric in solution, β-casein self-assembles into well-defined core-shell micelles. We recently developed these micelles as potential carriers for oral administration of poorly water-soluble pharmaceuticals, using celecoxib as a model drug. Herein we present deep and precise insight into the physicochemical characteristics of the protein-drug formulation, both in bulk solution and in dry form, emphasizing drug conformation, packing properties and aggregation state. In addition, the formulation is extensively studied in terms of structure and morphology, protein/drug interactions and physical stability. Particularly, NMR measurements indicated strong drug-protein interactions and noncrystalline drug conformation, which is expected to improve drug solubility and bioavailability. Small-angle X-ray scattering (SAXS) and cryogenic transmission electron microscopy (cryo-TEM) were combined for nanostructural characterization, proving that drug-protein interactions lead to well-defined spheroidal micelles that become puffier and denser upon drug loading. Dynamice light scattering (DLS), turbidity measurements, and visual observations complemented the analysis for determining formulation structure, interactions, and stability. Additionally, it was shown that the loaded micelles retain their properties through freeze-drying and rehydration, providing long-term physical and chemical stability. Altogether, the formulation seems greatly promising for oral drug delivery.

Characterization and quantification of chromate adsorption by layered porous iron oxyhydroxide: An experimental and theoretical study

The inner structure of iron oxyhydroxide agglomerates (IOAs) prepared from hydrolysis of ferric chloride was characterized and correlated to surface complexation of hexavalent chromium, Cr(VI), in a broad range of pH (3-12) and ionic strengths (0.0-5.0M). Evolution of particle size, morphology, and surface activity, combined with density functional theory (DFT) calculations, support the condensation reaction initiated formation of IOAs in three levels: iron nanoparticles to nanolayers to agglomerates. This agglomeration process led to a layered porous structure for aqueous-phase IOAs resulting in a rapid and high removal of Cr(VI) in batch tests. By integrating adsorption results, thermodynamic modeling, and quantum chemical calculations for the adsorption reactions, a quantitative distribution profile for each surface coordination of Cr(VI) ions (i.e., monodentate, bidentate, and hydrogen-bonding) was established. Results of this study are important to understand the fundamental mechanism of IOAs formation in aqueous phase and the intrinsic nature of surface complexations at the mineral-water interface for optimal Cr(VI) removal in hypersaline waste streams.

Conductive PVDF-HFP nanofibers with embedded TTF-TCNQ charge transfer complex.

Tetrathiafulvalene-tetracyanoquinodimethane charge-transfer complex (TTF-TCNQ CTC) represents a promising organic conductive system. However, application of this donor-acceptor pair is highly limited, because of its ultrafast crystallization kinetics and very low solubility. In this work, conductive organic nanofibers were generated via a coelectrospinning process of poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) with embedded TTF and TCNQ in the shell and core solutions, respectively. Upon supply of the polymer solutions, a core-shell droplet was formed at the exit of the spinneret. The electron donor TTF and the electron acceptor TCNQ migrated toward each other, within the compound droplet, to produce conductive CTC crystals. In the presence of a sufficiently strong electric field, jetting set in at the droplet tip, which yielded solidified PVDF-HFP nanofibers embedded with aligned CTC. Fiber diameters ranged between 100 and 500 nm. X-ray analysis showed strong equatorial reflections (110,200) of oriented copolymer PVDF-HFP crystals (β-phase) with copolymer chains oriented along the fiber axis, and of CTC (001), indicating that the CTC molecular planes were aligned parallel to the nanofiber axis. In addition, reflections of unreacted TCNQ (120,220) and TTF (110) crystals were observed. The electrospun nanofibers were collected to form a fiber mat, which was evaluated as a working electrode in a three-electrode cell system, exhibiting differential conductance of 5.23 μmho.

On the elongated domains in electrospun nanofibers of polyamide-6 blends

We report analysis of electrospun polymer nanofibers fabricated from blends of semicrystalline polyamide-6 (PA-6) and fully amorphous polyamide-6 (PA-6I) in the narrow diameter range of 200–250 nm. The combined differential scanning calorimetry and wide angle x-ray scattering results show the presence of α- and γ-phase crystals in PA-6 nanofibers. Simultaneous small- and wide-angle x-ray scattering investigations confirmed the existence of small elongated domains which were formed during the electrospinning process due to stretching of polymer chains. The Debye–Bueche analysis determined a cross-sectional width of elongated domains in the range of 10.8–16 A. Additionally, analysis by Rulands method revealed a decrease in the length of elongated domains with increasing crystalline phase content in the blends. Evaluation of Hermans orientation function suggested that the domains possessed higher molecular anisotropy along the c-axis in PA-6I compared to PA-6 nanofibers. These investigations and the suggested role of the amorphous phase in molecular stretching of polymer chains in electrospun nanofibers, may have broad implications for the size-dependent mechanical properties.