The conformational stability of pea vicilin was studied by high-sensitivity differential scanning calorimetry and sedimentation velocity at different pHs. It was found that vicilin completely conserved its trimeric 7S structure over the pH range from 3 to 10, and only partly dissociated into 4S subunits at pH 2 and 11. The temperature dependences of the partial heat capacity of vicilin were measured at different pHs. The pre-denaturation linear segments of these dependences were in quantitative agreement with a universal Freire function thus suggesting a native folding of the vicilin preparation. The denaturation curves of vicilin were deconvoluted into two independent cooperative transitions. This result implied the presence of two energetically different structural domains in vicilin subunits. The standard Gibbs free energies of denaturation for each domain were determined as functions of pH. These dependences were well approximated by the Harold Scheraga model, which considered the contributions of hydrogen bonds between definite ionogenic groups of a protein to its stability. The number and types of such bonds were determined. These data were generally confirmed by the analysis of the three-dimensional structure of the vicilin molecule.

Copolymers of N-vinylcaprolactam (NVCL) and N-vinylimidazole (NVI) of different compositions were synthesized by the solvent-free free radical copolymerization. The order-disorder conformational transitions of the copolymers in diluted aqueous solutions at pH 7.4 (20 mm imidazole buffer) were studied by high-sensitivity differential scanning calorimetry. Dependences of the transition temperature, enthalpy, and heat capacity increment of the copolymers on the NVI content were obtained. The transition temperature did not depend on the composition of the copolymers (Tt = 34.8 +/- 2.0 degrees C). Alternatively, the transition enthalpy decreased, and the heat capacity increment approached zero upon the increase in the NVI content in the copolymers. These trends displayed a decrease in the content of the long enough NVCL sequences in the copolymer structure capable of the cooperative order-disorder transitions. The Schr & ouml;der-Van Laar equation fitted well to the integral transition curves of the copolymers at a quite appropriate polymerization degree of the Kuhn segment for NVCL sequences, nK = 10.7 +/- 1.3. A copolymer with an NVI content of 45 mol. % was found to demonstrate a protein-like behavior in salt-free solutions. It underwent a cooperative order-disorder transition without precipitation from solution.

Polylactides (PLA) are insoluble in acetone under normal conditions. Upon heating, the PLA solubility in acetone increases sharply up to complete dissolution. The PLA transition from the solid semi-crystalline state to the soluble one is first studied by the high-sensitivity differential scanning calorimetry. The PLA dissolution thermograms are obtained. Thermodynamic characteristics of the dissolution, the transition temperature, and enthalpy (Tt = 51.0 +/- 0.3 degrees C and Delta th=${{\Delta }_t}\bar{h} = $22 +/- 2 J g-1), are shown to be independent of the polymer content in the system (1-7 mg mL-1). The dissolution thermograms are transformed to the temperature dependences of the relative PLA solubility (0 <= alpha(T) <= 1). These dependences are well approximated by the model of phase equilibrium between the solid polymer and its ideal solution (correlation coefficient of 0.999). The parameters of the model, the apparent transition enthalpy and the apparent transition temperature, do not depend on the PLA concentration: Delta mh* = 420 +/- 50 kJ mol-1, and Tm & lowast;=$T_m<^>* = $324.9 +/- 0.2 K. As a result of the consistent thermodynamic analysis of the calorimetric data, the PLA crystallization degree (20%) and the average molecular weight of the crystalline domains of PLA macromolecules (4000 Da) are estimated.

Interaction of bovine β-lactoglobulin (BLG) with several flavor compounds (FC) (2-methylpyrazine, vanillin, 2-acetylpyridine, 2- and 3-acetylthiophene, methyl isoamyl ketone, heptanone, octanone, and nonanone) was studied by high-sensitivity differential scanning calorimetry. The denaturation temperature, enthalpy, and heat capacity increment were determined at different FC concentrations. It was found that the denaturation temperature and heat capacity increment do not depend on the FC concentration, while the denaturation enthalpy decreases linearly with the FC concentration. These thermodynamic effects disclose the preferential FC binding to the unfolded form of BLG. By the obtained calorimetric data, the free energies of FC binding vs. the FC concentrations were calculated. These dependences were shown to be linear. Their slope relates closely to the overall FC affinity for the unfolded BLG in terms of the Langmuir binding model. The overall BLG affinity for FC varies from 20 M-1 (2-methylpyrazine) up to 360 M-1(nonanone). The maximal stoichiometry of the BLG-FC complexes was roughly estimated as a ratio of the length of the unfolded BLG to the molecular length of FC. Using these estimates, the apparent BLG-FC binding constants were determined. They are in the range of 0.3-8.0 M-1 and correlated strictly with the FC lipophilicity descriptor (logP).

beta-lactoglobulin-oligochitosan (MW 9500 Da) system was studied under conditions of protein-polysaccharide incompatibility, the dipole-charge, and charge-charge protein-polysaccharide interactions at pH 3.0, 5.5, and 6.0, respectively, using ITC, DSC, and DLS. At pH 5.5 and 6.0, the ITC data revealed a complex formation between beta-lactoglobulin and oligochitosan. The binding curves of the protein to the polysaccharide were obtained. The binding parameters, namely, the number of sites (n) and the binding constant (K-b), were estimated in terms of the Langmuir equation. These are n = 2.8 +/- 0.1 and K-b = 10(6.4 +/- 0.1) M-1 at pH 5.5, while n = 1.5 +/- 0.1 and K-b = 10(5.6 +/- 0.1) M-1 at pH 6.0. At pH 3.0, when both the protein and polysaccharide are positively charged, the denaturation parameters of beta-lactoglobulin in the beta-lactoglobulin-oligochitosan system and in the absence of the polysaccharide coincide independently of the system composition. Under conditions of the complexation, the denaturation parameters of beta-lactoglobulin (the denaturation temperature, enthalpy, entropy, and width) displayed complicated dependences on the oligochitosan content. Their analysis supported by measurements of the diffusion coefficients implied that the conformational stability of beta-lactoglobulin in the beta-lactoglobulin-oligochitosan complexes was defined by the complex composition and supramolecular structure of oligochitosan.

Energetics of chitosan (CS) polyplexes and conformational stability of bound DNA were studied at pH 5.0 by ITC and HS-DSC, respectively. The CS-DNA binding isotherm was well approximated by the McGhee-von Hippel model suggesting the binding mechanism to be a cooperative attachment of interacting CS ligands to the DNA matrix. Melting thermograms of polyplexes revealed the transformation of different conformational forms of bound DNA in dependence on the CS/DNA weight ratio rw. At 0<rw<0.7 two conformational forms were observed and assigned, respectively, to the intact (free) and globular (bound) DNA. A contribution of the globular DNA to the melting enthalpy approached 100 % at the equivalent weight CS content. At higher CS contents a further stabilization of the globular DNA conformation was detected and assigned to the DNA globules in overcharged polyplexes. The polyplex dissociation was studied at pH 7.4 under conditions of zero and a physiological concentration of NaCl. Phase separation of the system was observed in both cases. DNA was almost completely immobilized in the lower phase enriched with CS. At physiological ionic strength, DNA converted from the globular to the intact form although remained to be immobilized in the CS phase.

The interaction between ovalbumin (OVA) and fucoidan (FVF), a branched sulfated polysaccharide of seaweeds, was studied by turbidimetry, velocity sedimentation, and high-sensitivity differential scanning calorimetry (HS-DSC). The boundary conditions of the existence of soluble OVA-FVF complexes were determined as a function of pH and/or OVA-FVF mixture composition. The yield of soluble OVA-FVF complexes was determined from the velocity sedimentation data at pH 4.0 and 5.0 in 5 mM NaCl. It achieved a maximal value of similar to 90% at pH 5.0 and the OVA/FVF weight ratio of about 6. According to HS-DSC data, the denaturation behavior of OVA bound to FVF changed significantly as compared with that of the free protein. Depending on pH and OVA-FVF mixture composition, one or two heat capacity peaks were observed on the thermograms assigned either to the free or to bound protein. The denaturation temperature of the bound OVA was about 20 degrees C lower than that of the free OVA. Based on the HS-DSC results, the OVA to FVF binding curves were derived at pH 5.0 and 5.5. Both were well approximated by the Langmuir equation with n = 66 +/- 3 and Kb = 10(5.2 +/- 0.1) M-1 at pH 5.0, and n = 1.1 +/- 0.2 and Kb = 10(4.6 +/- 0.2) M-1 at pH 5.5. Some emulsifying and foaming properties of the OVA-FCD complexes were characterized and compared with those of OVA alone. The complexes showed a significantly lower emulsifying threshold. The foaming ability of the complexes could exceed that of OVA by more than twice. These results imply a high potential of the OVA-FCD complexes as emulsion and foam stabilizers.

Interaction of beta-lactoglobulin (BLG) with a sulfated polysaccharide isolated from the brown alga Fucus vesiculosus (fucoidan, FVF), was studied by nephelometry, high-sensitivity differential scanning calorimetry, and sedimentation velocity as a function of pH and the polysaccharide/protein ratio. FVF-BLG complexes were found over the pH 2.5-6.0 range. The complexation was accompanied by the decrease in the conformational stability of BLG at pH < pI 5.2 of the protein where FVF and BLG were oppositely charged. Near pI of the protein and at the pH values larger a bit than pI, where BLG has a large dipole moment or carries a small negative charge, the complexation appeared as changes in the oligomeric state of the protein. Binding curves of BLG to FVF were obtained at pH 3.0 and 6.0 according to the DSC and sedimentation velocity data. Binding constants were evaluated: 3.6 x 10(3) M-1 at pH 3.0 and 2 x 104 M-1 at pH 6.0. Correlations between the denaturation enthalpy and temperature for the free and bound protein in terms of Kirchhoff's law indicated that the denaturation of both forms of the protein is associated with comparable changes in the accessible surface area of the BLG molecule. It means that the binding of BLG to FVF does not perturb the hydrophobic core of the protein.

Interactions of a major whey protein, beta-lactoglobulin (BLG), with an anionic polysaccharide, dextran sulfate (DS), were investigated by nephelometric titration and high-sensitivity differential scanning calorimetry at pH 2.5-7.0 and NaCl concentrations of 0-500 mM. The pH-dependences of the light scattering of the BLG-DS system revealed a maximum. Its position was dependent on the polysaccharide/protein ratio and salt concentration. Thermodynamic parameters of the denaturation of BLG in mixtures with DS were determined at different pH and salt concentrations. Conformational stability of BLG in the BLG-DS complexes was shown to either increase or decrease depending on the solution conditions and complex composition. The binding curves of BLG to DS were calculated from the light scattering and calorimetric data and analyzed using various binding models including the Langmuir and McGhee-von Hippel models as well as a stoichiometric interpolyelectrolyte reaction. Mechanisms responsible for changes in the BLG oligomer structure and conformational stability induced by the complex formation with the polysaccharide were proposed.

The poly(vinyl alcohol) (PVA) physical cryogels that contained the additives of the H2N-(CH2)(n)-COOH-type amino acids (n = 1-5) have been prepared, and their physico-chemical characteristics, macroporous morphology and the solute release dynamics have been evaluated. These low-molecular solutes with respect of the PVA cryotropic gelation exhibited the kosmotropic influence, i.e. commencing from their concentration of about 0.5 mol/L the additives facilitated the growth in the rigidity and the heat endurance of the resultant cryogels. The efficiency of such influence was reduced with an increase in the length of oligomethylene spacer between the amino and carboxyl groups in the amino acid molecules. The average size of macropores also depended on the additive type and its concentration in the initial PVA solution; the cross-section of the pores decreased with increasing the solutes concentration. Upon the cryogel-entrapped amino acids release from the macroporous gel matrix a gradual transition from the release mechanism by a free diffusion for the hydrophilic glycine to the release according to the space-limited Fickian diffusion for the more hydrophobic e-aminocaproic acid was found. This effect was stipulated by the hydrophobic interactions between the carbochain core of the PVA macromolecules and the aliphatic moieties of the respective amino acid.

The phase transitions of poly(N-isopropylacrylamide) (PNIPAM) in water-methanol mixed solutions were studied in detail by high-sensitivity differential scanning calorimetry. From this study, the dependences of the transition temperature, enthalpy, heat capacity increment, and width on the methanol molar fraction (x(MeOH)) were obtained. The transition temperature passed through a minimum at the methanol molar fraction x x(MeOH)* similar to 0.35. At x(MeOH) < x(MeOH)*) the transition enthalpy decreased quickly with the methanol content and became so small that it could not be measured, even with an increase in the polymer concentration by a hundred times (up to 150 mg mL(-)(1)). Furthermore, over this x meoH range, the transition heat capacity increment being negative remained practically constant, but the transition width sharply increased. The transition thermograms were quantitatively described by the Okada-Tanaka theory, which takes into account the role of the polymer-solvent interaction cooperativity in the polymer thermoresponsivity. In terms of this approach, it is assumed that over the defined range of methanol content, PNIPAM possesses the cooperative hydro-solvation structure in the form of water-methanol complexes. The energetics of this structure smoothly decreases with the increase in the methanol content up to a complete disappearance of the structure at x(MeOH) < x(MeOH)*. In this range of the methanol content, the phase behavior of PNIPAM seems to be dictated by regularities typical of polymer solutions in organic solvents, that is, how the Flory-Huggins parameter depends on temperature.

Responsiveness of drug delivery systems (DDS) against internal and external stimuli attracts wide interest as a mechanism that can provide both site-specific release at the target place and feedback regulated release rate. Biological environment is quite complex and the effects that the intricate medium may have on the effectiveness of the stimulus have received certain attention. Differently, the impact that the drug loaded may have itself on the responsiveness of the DDS has been underestimated. Most drugs are not merely trapped in the polymer network, but they effectively interact with some polymer moieties. Nearly all drugs, including therapeutic proteins, are ionizable amphiphilic molecules, and thus ionic, hydrogen bonding and hydrophobic interactions are commonly exploited to increase the loading yield. If the moiety involved in drug binding is also responsible for (or at least partially involved in) the stimuli responsiveness, a strong impact of the drug on the behavior of the DDS can be expected. This review gathers relevant examples of how the drug may modify the sensitiveness (stimulus threshold) and the responsiveness (actuation) of the DDS to therapeutically relevant stimulus, and aims to shed light on the different drug binding modes of the swollen and collapsed states, which in turn modify drug release patterns. The information evidences that drug loading and release may trigger phase transitions in hydrogels non-intended to be drug-responsive (i.e., a priori not analyte-responsive networks). A better knowledge about the effect of the drug on the responsiveness is a required step forward for the clinical application of smart hydrogels and may also unveil novel uses of the stimuli-responsive DDS.

Oligochitosan, a low molecular weight derivative of the cationic biopolymer, chitosan, currently shows a great potential of application as a biodegradable non-toxic stimuli-sensitive drug carrier. This paper aimed to elucidate the thermoresponsive potential of oligochitosan and the temperature-controlled drug binding and release to shed light on oligochitosan potential in stimuli-responsive drug delivery. Mechanisms of thermoresponsive behavior of oligochitosan induced by β-glycerophosphate (GP) were investigated using ITC, DSC, and DLS. Upon heating, the aqueous oligochitosan solution underwent a cooperative transition of the microphase separation type resulting in the formation of stable nano-sized particles. Energetics of the GP-oligochitosan interaction (evaluated by ITC) revealed a positive enthalpy of the GP binding to oligochitosan, which pointed to a notable contribution of dehydration and the related rearrangement of the polysaccharide hydration shell. Energetics of the thermal phase transition of oligochitosan was investigated by DSC upon variation of the solvent dielectric constant and GP concentration. The dependences of the transition parameters on these variables were determined and used for the analysis of the oligochitosan thermoresponsivity mechanism. The binding of ibuprofen to the thermotropic oligochitosan nanogel particles and its release from them were evaluated under near-physiological conditions. Relevantly, the oligochitosan nanoparticles surpassed some reference macromolecular adsorbers by the affinity for the drug and by the delayed release kinetics.

In the present study, we report the synthesis of a series of copper(II) complexes with a wide range of ligands and their testing in the copper catalyzed Chan-Evans-Lam (CEL) coupling of aniline and phenylboronic acid. The efficiency of the coupling was directly connected with the ease of the reduction of Cu(II) to Cu(I) of the complexes. The most efficient catalyst was derived from 4-t-butyl-2,5-bis[(quinolinylimino)methyl]phenolate and two Cu(II) ions. Depending on the counter-anion nature and the concentration of the reaction mixture, the reaction can be directed to predominant C-N-bond formation. Forty-three derivatives of diphenylamine were prepared under the optimized conditions. The proposed mechanism of the catalysis was based on the reduction potential of a series of complexes, molecular weight measurements of the catalytic complex in MeOH and the kinetic studies of aniline and phenylboronic acid coupling. In addition, an H-1 NMR experiment in a sealed NMR tube, without external oxygen supply available, proved that no complete Cu(II) to Cu(I) conversion was observed under the condition, ruling out the usually accepted mechanism of the C-N coupling, which included the oxygenation of the intermediately formed Cu(I) complexes after the key step of C-N conversion had already been completed. Instead, a mechanism was proposed, involving an oxygen molecule coordinated to two copper ions in the key C-N bond formation without any detectable conversion of the Cu(II) complexes to Cu(I).

Nano- and micro-sized stimuli-responsive polymer containers capable of an effective controlled binding and release of ionic amphiphilic drugs are highly demanded in drug delivery. Thermoresponsive cross-linked microgels based on N-isopropylacrylamide (NIPAM) were synthesized by precipitation polymerization in aqueous media. The microgels were functionalized by the introduction of an ionic component (acrylic acid, AA, or vinyl sulfonate, VSA) either as a comonomer (NIPAM-co-AA and NIPAM-co-VSA microgels) or interpenetrating polymer network (PNIPAM-PAA microgel). The thermoresponsive behavior of the microgels was investigated by high-sensitivity differential scanning calorimetry. Thermodynamic parameters of the phase transitions (the transition temperature, enthalpy, and width) for the copolymer and interpenetrating microgels were determined. The copolymer microgels involving weak and strong ionogenic groups differ drastically by the transition energetics. This implies different types of their primary structures provided by either comonomer affinity or segregation in the reaction mixture under the polymerization conditions. The microgel functionalization via interpenetrating networks does not affect notably the transition temperature typical of the reference parent PNIPAM microgel but reduces the transition cooperativity. An analysis of the heat capacity profile of the microgel phase transition reveals some features of the mechanism of the thermoresponsivity in the microgels.

Thermoresponsivity of chitosan induced by beta-glycerophosphate (GP) in diluted aqueous solutions has been first studied by high-sensitivity differential scanning calorimetry. It has been found that the GP solutions of chitosan undergo a first-order phase transition upon heating. The onset of this transition coincides with the cloud point of the system. This allows one to identify the thermoresponsivity of chitosan as a macroscopic demonstration of the phase separation transition. The transition temperature, enthalpy, heat capacity increment, and width were determined as functions of GP and chitosan concentrations, and the dielectric constant of the solvent. Based on this data, we suggested that GP binds cooperatively to the chitosan matrix at low temperatures. The standard free energy of GP binding (Delta(b) g(int) = -6 +/- 1 kJ mol(-1)) was estimated from the DSC data. It was shown that the Okada-Tanaka model of cooperative hydration of polymers adequately describes the thermogram of the GP induced phase transition of chitosan.

Changes in the affinity of the swollen and collapsed forms of a thermoresponsive polymer gel for targeted ligands can be directly estimated using a thermodynamic approach based on high-sensitivity differential scanning calorimetry (HS-DSC). For macromolecular ligands (proteins) bound to the gel, this method provides information on changes in their conformational stability, which is of crucial importance for the biological or pharmaceutical activity of the protein. We used HS-DSC for the study of interactions of two widely administrated drugs-gemfibrozil and ibuprofen-and two globular proteins-α-lactalbumin and BSA-with hydrogels of the cross-linked poly(methoxyethylaminophosphazene). The gel collapse resulted in a substantial increase in the gel affinity for the drugs. We obtained quantitative estimations of the affinity of the collapsed gels depending on the gel structure, pH, concentration of NaCl, and phosphate buffer (an inductor of the thermoresponsivity). The gels retained a high affinity for the drugs in the near-physiological conditions (ionic composition and pH). The binding curves of globular proteins to the gels in the swollen and collapsed states were obtained. The different proteins demonstrated the preferential binding to the swollen or collapsed state of the gels, presumably depending on the protein surface hydrophobicity. The proteins bound to the gel subchains retain their native tertiary structure and, therefore, maintain their functionality when immobilized in the polyphosphazene hydrogels.

The volume phase transition of a hydrogel of the polyampholyte network N-isopropylacrylamide (6000) - N-(3-aminopropylmethacrylamide) (120) - Acrylic acid (120) - N,N'-Methylenebisacrylamide (40) was investigated by high-sensitivity differential scanning calorimetry in relation to pH, concentrations of NaCl and two oppositely charged drug ligands (ibuprofen and propranolol). The transition temperature, enthalpy, heat capacity increment and width against these thermodynamic variables were determined. The experimental data were used for calculation of the excess free energy functions of the phase transition which are measures of the relative stability of the dense (collapsed) state of the polyampholyte network under different conditions. By analogy with globular proteins, the stability of the network dense state is maximal at the isoelectric point of the polyampholyte and is increased by the kosmotropic salt and specific ligands. We demonstrate that the polyampholyte hydrogel consists of independent cooperative domains. The sizes of these domains in the hydrogel (9-15 kDa) are comparable with those in globular proteins.

We investigated energetics of binding of multifunctional pyranine ligands to hydrogels of the cross-linked poly(methoxyethylaminophosphazene) (PMOEAP) from data on the thermotropic volume phase transition of the gels by means of high-sensitivity differential scanning calorimetry. Dependences of the transition temperature, enthalpy, and width on the concentration of pyranines were obtained, and the excess transition free energy as a function of the pyranine concentration was calculated. We found that the affinity of the gels for the pyranine ligands increased very significantly upon the gel collapse. The intrinsic binding constants and free energies of binding of the ligands to the gels in the collapsed state were estimated from the DSC data. They revealed a significant increase in the hydrogel affinity for pyranines proportional to the number of anionic groups in the ligand structure. The affinity of the PMOEAP hydrogels for the multifunctional ligands was not affected by an increase in the cross-linking density of the gels and only slightly reduced by physiological salt concentrations.

Urea (URE) and guanidine hydrochloride (GHC) possessing strong chaotropic properties in aqueous media were added to DMSO solutions of poly(vinyl alcohol) (PVA) to be gelled via freeze–thaw processing. Unexpectedly, it turned out that in the case of the PVA cryotropic gel formation in DMSO medium, the URE and GHC additives caused the opposite effects to those observed in water, i.e., the formation of the PVA cryogels (PVACGs) was strengthened rather than inhibited. Our studies of this phenomenon showed that such “kosmotropic-like” effects were more pronounced for the PVACGs that were formed in DMSO in the presence of URE additives, with the effects being concentration-dependent. The additives also caused significant changes in the macroporous morphology of the cryogels; the commonly observed trend was a decrease in the structural regularity of the additive-containing samples compared to the additive-free gel sample. The viscosity measurements revealed consistent changes in the intrinsic viscosity, Huggins constant, and the excess activation heat of the viscosity caused by the additives. The results obtained evidently point to the urea-induced decrease in the solvation ability of DMSO with respect to PVA. As a result, this effect can be the key factor that is responsible for strengthening the structure formation upon the freeze–thaw gelation of this polymer in DMSO additionally containing additives such as urea, which is capable of competing with PVA for the solvent.