H. B. Bohidar

Kinetics of sol–gel transition in thermoreversible gelation of gelatin

The sol–gel transition in dilute gels of gelatin are studied by differential scanning calorimetry (DSC) and static light scattering techniques. The sol–gel phase diagram clearly shows the existence of an upper critical solution temperature in this system. In addition, the DSC data conclusively exhibits two more phase curves in the sol state—one pertaining to the coexistence of monomers and aggregates and the second one separating the random coil and helix domains in the solution phase. The Ferry–Eldridge equation has been used to determine the enthalpy of the melting of the gel structure (ΔHg) which is equal to (30±2.0) kcal mol−1. The gelation temperature Tg is correlated to Flory’s statistical model of gelation, which gives the enthalpy of melting of pure gelatin crystallites (ΔHv) as ΔHv=(35±2.0) kcal cm−3 and crystallite melting temperature (Tm) as Tm=(588±20.0) K. The Flory–Huggins interaction parameter (χ) has been determined as χ=0.49±0.05. Interesting scaling behavior has been observed through lig...

Effect of ionic strength on surface-selective patch binding-induced phase separation and coacervation in similarly charged gelatin-agar molecular systems.

Coacervate is defined as a polymer-rich dense phase, which remains in thermodynamic equilibrium with its low concentrated phase called the supernatant. The effect of ionic strength (I = 0-0.1 M NaCl) on the mechanism of surface patch binding-induced protein-polysaccharide interaction leading to complex coacervation, between agar (a polyanionic polysaccharide) and gelatin B (a polyampholyte protein), both having similar net charge, at a particular mixing ratio, [gelatin]/[agar] = 1, was studied at various temperatures (20-40 °C). The coacervation transition was probed by turbidity and zeta-potential measurements. The intermolecular association had the signature of surface-selective binding, and a model calculation could explain the potential energy of interactions operative in such processes. The thermo-mechanical features of the coacervates were found to be strongly dependent on ionic strength, which has been interpreted as originating from formation of salt-bridges between the biopolymers. The microstructure of the coacervate materials was analyzed using rheology and small angle neutron scattering (SANS) techniques, which probed the heterogeneity prevailing in the system that had characteristic length in the range 1.3-2.0 nm, and the same data yielded the correlation length of concentration fluctuations, which was estimated to lay in the range 2.4-4 nm. It is concluded that the coacervation transition driven by surface-selective binding is not influenced by the ionic strength of the solution, but the mobile ions participate in the structural organization of the interacting polyions in the coacervate.

Study of sol‐state properties of aqueous gelatin solutions

Aqueous gelatin solutions have been studied in a systematic and exhaustive manner in a neutral buffer medium, in the temperature range of 30–60 °C, by static (SLS) and dynamic laser light scattering. It has been clearly established that upon cooling the sol from 60 °C, the gelatin chains form thermoreversible aggregates until the gelation is encountered below 30 °C. The radius of gyration of the chains (Rg) at any temperature was found to scale with corresponding molecular weight as Rg∼M0.57±0.03w. The ratio of Rg to hydrodynamic radius RH has been found to be Rg/RH=1.82±0.03 as expected for random coils. The translational diffusion coefficient of the chains Dz(c) exhibited linear concentration dependence; Dz(c)=D0(1+KDc) and D0∼M−0.57±0.03. The second virial coefficient (A2) of osmotic pressure obtained through SLS could be excellently related to KD through KD≂2A2Mw for all temperatures. The Flory–Huggins interaction parameter χ was determined to be χ≂0.48 and it showed negligible temperature dependence....

Kinetics of coacervation transition versus nanoparticle formation in chitosan-sodium tripolyphosphate solutions.

Chitosan (deacetylation=75-85%) and sodium tripolyphosphate (TPP) solutions were observed to undergo spontaneous coacervation transition or nanoparticle formation depending on the chitosan concentration and the volumetric mixing ratio [chitosan/TPP]. Three distinct conditions have been identified: (i) [chitosan]<or=0.5mg/ml, 0.5<or=[chitosan/TPP]<or=2 and pH<5.5, yields spontaneous coacervation, (ii) 0.5<or=[chitosan]<or=2.5mg/ml, [chitosan/TPP]>or=2 and 3.5<pH<5.5, produces chitosan nanoparticles and (iii) 0.5<or=[chitosan]<or=3mg/ml, 3<or=[chitosan/TPP]<or=6 and 5<pH<7.5, favors induced coacervation. Sizing done by TEM and dynamic light scattering studies revealed that chitosan nanoparticles had typical diameter in the range approximately 150-350 nm depending on polymer concentration and chitosan/TPP mixing ratio, which reduced by approximately 40% when polyethylene glycol (PEG) was added to these solutions. The long-time light scattering probing of the solutions revealed that residual interactions continuously produced soluble intermolecular complexes over extended period of time, a process that enabled the generation of coacervate droplets seamlessly. The coacervates (formed spontaneously or induced), and chitosan and chitosan-PEG nanoparticles, were used for encapsulating hydrophobic protein synthesis inhibitor model drug cycloheximide. The comparative in vitro release profiles in phosphate buffer and simulated intestinal fluid was monitored at 37 degrees C.

Interaction of Gelatin with Room Temperature Ionic Liquids: A Detailed Physicochemical Study

Interaction of gelatin (G) with room temperature ionic liquids (ILs), 3-methyl-1-octylimidazolium chloride [C(8)mim][Cl] and 1-butyl-3-methylimidazolium octylsulfate [C(4)mim][C(8)OSO(3)], have been investigated through tensiometry, conductivity, steady-state fluorescence, turbidity, and dynamic light scattering (DLS). We have observed that the nature of interactions in G-[C(8)mim][Cl] system are remarkably different as compared to G-[C(4)mim][C(8)OSO(3)] system. At low concentrations, much below the critical micelle concentration (cmc) of IL, the IL monomers are adsorbed at the native G at the interface forming G-IL (monomer) complex, whereas both the monomers and lower IL aggregates are interacted with G in bulk leading to G-IL (aggregate) complex. The increased hydrophobic character of the G-IL complexes is evidenced from pyrene fluorescence. Turbidity measurements showed interestingly distinguished coacervation characteristics in the investigated systems. In case of G-[C(4)mim][C(8)OSO(3)] system, the coacervates dissolve in the free micellar solution whereas G-[C(8)mim][Cl] coacervates remain stable up to very high concentration. DLS provided useful information about the changes in size of gelatin and the nature of interactions between gelatin and ILs. Thermodynamic parameters of micellization with and without gelatin have been derived and compared.

Universal charge quenching and stability of proteins in 1-methyl-3-alkyl (hexyl/octyl) imidazolium chloride ionic liquid solutions.

This study reports pH dependent stability of protein dispersions of five common proteins, bovine serum albumin (BSA), human serum albumin (HSA), immunoglobulin (IgG), β-lactoglobulin (β-Lg), and gelatin-B (Gel-B), all having isoelectric pH, pI ≈ 5, in room temperature ionic liquid solutions of 1-methyl-3-alkyl (hexyl/octyl) imidazolium chloride (concentration 0-0.2% w/v). Molecular hydrophobicity index, (H-index = hydrophobicity/hydrophilicity) of these molecules spanned the range 0.43-0.87. Electrophoretic characteristics, surface tension data and hydrodynamic size information revealed that IL solutions provide dispersion stability owing to specific protein-IL binding which did not alter their pI values though their surface charge was considerably screened. Change in maximum (ζ(max)) and minimum (ζ(min)) zeta potential values observed at pH ~3 (maximum protonated state) and pH ~8 (maximum deprotonated state) could be described universally as function of IL concentration, c as Δζ(x) = [1 - exp(-ac)] where Δζ(x) is either |(ζ(max) - ζ(w))|/ζ(w) or |(ζ(min) - ζ(w))|/ζ(w), and ζ(w) is the corresponding value in water. Tensiometry data showed two major stages of protein-IL interactions: (i) for c < cmc of IL, the IL molecules selectively bind with imidazolium cation through electrostatic forces forming protein-IL (complex) and (ii) for c> cmc free IL-aggregates begin to form. Similarly, we can define Δγ(x) as either |(γ(max) - γ(w))|/γ(w) at pH 3 or |(γ(min) - γ(w))|/γ(w) at pH 8. Both Δζ(x) and Δγ(x) showed linear dependence with c, Δγ(min, max) (or Δζ(min, max)) = (1 - K(γ) (or K(ζ)) H-index), where the slopes K(ζ) and K(γ) defined intermolecular interactions. Hydrodynamic radii data revealed protein stabilization, circular dichroism spectra implied retention of secondary structures, and Raman spectra confirmed a marginal increase in water structure. Results concluded that selective binding of IL molecules to protein surface in the form of bilayer screen protein surface charge, thereby, contributing to its dispersion stability.

Hydrodynamic properties of gelatin in dilute solutions

Aggregation properties of Gelatin chains in neutral aqueous solutions, are reported in the temperature range T = 35-60 degrees C, from the measured intrinsic viscosity [eta], diffusion coefficient, D(o), molecular weight Mw, and radius of gyration (Rg) data. Gelatin chains doubled their size as the solution was cooled to 35 degrees C from 60 degrees C. The intermolecular interaction was found to be repulsive which showed significant decrease as the temperature was reduced. The data provides excellent fitting to the scaling relations Mw[eta] = (1.96 +/- 0.06) x 10(-26)(Re,eta/Re,D)3(D(o) eta o/T)-3 and (D(o)n1/2)-1 approximately equal to (6 1/2 pie eta o chi beta/kB/T)[1 + 0.201(v/beta 3)n1/2] where n is the number of segments in the chain. The ratio of the hydrodynamic radius (Re,D) (deduced from D(o)) and Rg, (Re,D/Rg = zeta) was found to be 0.555. From the known solvent viscosity eta o, the segment length beta, was deduced to be (15 +/- 2) A. The deduced excluded volume was v approximately equal to (4.1 A)3. The Flory-Huggins interaction parameter (zeta) did not show observable temperature dependence.

DNA–Gelatin Complex Coacervation, UCST and First-Order Phase Transition of Coacervate to Anisotropic ion gel in 1-Methyl-3-octylimidazolium Chloride Ionic Liquid Solutions

Study of kinetics of complex coacervation occurring in aqueous 1-octyl-3-methylimidazolium chloride ionic liquid solution of low charge density polypeptide (gelatin A) and 200 base pair DNA, and thermally activated coacervate into anisotropic gel transition, is reported here. Associative interaction between DNA and gelatin A (GA) having charge ratio (DNA:GA = 16:1) and persistence length ratio (5:1) was studied at fixed DNA (0.005% (w/v)) and varying GA concentration (C(GA) = 0-0.25% (w/v)). The interaction profile was found to be strongly hierarchical and revealed three distinct binding regions: (i) Region I showed DNA-condensation (primary binding) for C(GA) < 0.10% (w/v), the DNA ζ potential decrease from -80 to -5 mV (95%) (partial charge neutralization), and a size decrease by ≈60%. (ii) Region II (0.10 < C(GA) < 0.15% (w/v)) indicated secondary binding, a 4-fold turbidity increase, a ζ potential decrease from -5 to 0 mV (complete charge neutralization), which resulted in the appearance of soluble complexes and initiation of coacervation. (iii) Region III (0.15 < C(GA) < 0.25% (w/v)) revealed growth of insoluble complexes followed by precipitation. The hydration of coacervate was found to be protein concentration specific in Raman studies. The binding profile of DNA-GA complex with IL concentration revealed optimum IL concentration (=0.05% (w/v)) was required to maximize the interactions. Small angle neutron scattering (SANS) data of coacervates gave static structure factor profiles, I(q) versus wave vector q, that were remarkably similar and invariant of protein concentration. This data could be split into two distinct regions: (i) for 0.0173 < q < 0.0353 Å(-1), I(q) ~ q(-α) with α = 1.35-1.67, and (ii) for 0.0353 < q < 0.35 Å(-1), I(q) = I(0)/(1 + q(2)ξ(2)). The correlation length found was ξ = 2 ± 0.1 nm independent of protein concentration. The viscoelastic length (≈8 nm) was found to have value close to the persistence length of the protein (≈10 nm). Rheology data indicated that the coacervate phase resided close to the gelation state of the protein. Thus, on a heating-cooling cycle (heating to 50 °C followed by cooling to 20 °C), the heterogeneous coacervate exhibited an irreversible first-order phase transition to an anisotropic ion gel. This established a coacervate-ion gel phase diagram having a well-defined UCST.

Sorbitan Ester Organogels for Transdermal Delivery of Sumatriptan

ABSTRACT The partial phase behavior, rheological, and drug release characteristics of an organogel (OG) composed of water, isooctane and sorbitan esters, sorbitan monopalmitate (Span-40) and poly(oxyethylene)sorbitan monostearate (Polysorbate-60) were studied. Phase diagrams showed decreasing areas of optically isotropic organogel region depending on the surfactant ratio, Kw and drug incorporation. The nonbirefringent, clear isotropic solution suggested the reverse micellar/microemulsion nature of the organogel without any molecular ordering. The increase in drug concentration in OGs leads to increase in the viscosity and sol-gel transition temperature (Tg). Fractal dimension (df) values calculated for different compositions suggested that the density of the tubular network increases with increasing drug concentration in OGs. The release rate of the drug from OGs was found to be non-Fickian through the dialysis membrane. The permeation rate of sumatriptan from pig skin was 0.231 mg/h/cm2 (781.9 nmol/h/cm2). The study indicates potential of OG as a reservoir system for transdermal drug delivery.

Universal Growth of Microdomains and Gelation Transition in Agar Hydrogels

Investigations were carried out on aqueous sols and gels of agar (extracted from red seaweed Gelidiella acerosa) to explore the growth of microdomains en route to gelation. Isothermal frequency sweep studies on gel samples revealed master plots showing power-law dependence of gel elastic modulus, |G*|, on oscillation frequency, omega as |G*| approximately omegan, independent of temperature, with 0.5<or=n<or=1.4. Dynamic structure factor data from sol samples comprised of a double-exponential relaxation function, S(q,t)=A exp(-DSq2t)+B exp(-DLq2t) where DS and DL are the two translational diffusion coefficients and q is the scattering wave vector. This yielded hydrodynamic radii (from DS), with RS varying from approximately 20 nm (for sol) to 250 nm (at gelation point). The second hydrodynamic radius (from DL) obtained was RL in the range of approximately 200-500 nm (for sol) to approximately 1000 nm (at 38 degrees C, gelation point). These data could be universally fitted to RS approximately epsilon(-3/5) and RL approximately epsilon-1/3 (epsilon=(T/Tg-1), T>Tg). The S(q,t) behavior close to the gel transition point (Tg approximately (38+/-3 degrees C determined from rheology) followed a stretched exponential function: S(t)=A exp(-t/ts)beta. The beta factor increased from 0.25 to 1 as the gel temperature approached 25 degrees C from Tg, and relaxation time, ts, showed a peak at T approximately 30 degrees C. The SLS data (in the sol state) suggested the scaling of scattered intensity, Is(q) approximately epsilon(-gamma) (epsilon=(T/Tg-1), T>Tg) with gamma=0.13+/-0.03, and the presence of two distinct domains characterized by a Guinier regime (low q) and a power-law regime (high q). Close to and above Tg (+2 degrees C), IS(q) scaled with q as Is(q) approximately q(-alpha) with alpha=2.2+/-0.2, which decreased to 1.4+/-1 just below Tg (-2 degrees C), implying a coil-helix transition for 0.2% (w/v) and 0.3% (w/v) samples. For a 0.01% sample, alpha=3.5+/-0.5 which indicated the presence of spherical microgels.