Energetics of ß-lactoglobulin-flavor compounds interactions.

Interaction of bovine ß-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).

Chitosan polyplexes: Energetics of formation and conformational changes in DNA upon binding and release.

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

Effect of Chitosan on the Activity of Water-Soluble and Hydrophobic Porphyrin Photosensitizers Solubilized by Amphiphilic Polymers.

In this work, we studied the photocatalytic activity of photosensitizers (PSs) of various natures solubilized with polyvinylpyrrolidone (PVP) and ternary block copolymer ethylene and propylene oxide Pluronic F127 (F127) in a model reaction of tryptophan photo-oxidation in water in the presence of chitosan (CT). Water-soluble compounds (dimegin and trisodium salt of chlorin e6 (Ce6)) and hydrophobic porphyrins (tetraphenylporphyrin (TPP) and its fluorine derivative (TPPF20)) were used as PSs. It was shown that the use of chitosan (Mw ~100 kDa) makes it possible to obtain a system whose activity is comparable to that of the photosensitizer-amphiphilic polymer systems. Thus, the previously observed drop in the photosensitizing activity of PS in the presence of a polysaccharide and amphiphilic polymers (AP) was absent in this case. At the same time, chitosan had practically no inhibitory effect on hydrophobic porphyrins solubilized by Pluronic F127.

Biodegradable thermoresponsive oligochitosan nanoparticles: Mechanisms of phase transition and drug binding-release.

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 ß-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.

Salt-Induced Thermoresponsivity of Cross-Linked Polymethoxyethylaminophosphazene Hydrogels: Energetics of the Volume Phase Transition.

Biodegradable hydrogels of cross-linked polymethoxyethylaminophosphazenes (PMOEAPs) of various cross-linking density and apparent subchain hydrophobicity were investigated by high-sensitivity differential scanning calorimetry and equilibrium swelling measurements. The volume phase transition of the hydrogels was found to be induced by salts of weak polybasic acids. The transition parameters were determined depending on the pH, phosphate concentration, cross-linking density, and apparent hydrophobicity of the gels. The transition enthalpy increased three times and reached 60 J g-1 at the phosphate concentrations 5-100 mM. The transition temperature decreased by 60 °C when the pH changed from 6 to 8. A decrease in the transition temperature (by ∼20 °C) was achieved due to incorporation of 9.4 mol % of some alkyl groups into the gel subchains. The classic theory of the collapse of polymer gels coupled with the data of protein science on hydration energetics for various molecular surfaces reproduces correctly thermodynamics of the collapse of PMOEAP hydrogels.

Cryostructuring of Polymeric Systems. 49. Unexpected "Kosmotropic-Like" Impact of Organic Chaotropes on Freeze⁻Thaw-Induced Gelation of PVA in DMSO.

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.

Interactions of Non-Phosphorous Glycerolipids with DNA: Energetics, Molecular Docking and Topoisomerase I Attenuation.

The phosphorus-containing glycerolipid based antitumor drugs (edelfosine as a prototype) are currently in clinical trials. To avoid the use of potentially harmful phosphoric reagents in the preparation of biologically active glycerolipids, and to obtain the compounds without the phosphoester bond cleavable inside the cells, we developed the synthesis of non-phosphorous glycerolipids (NPGLs) with neutral or cationic polar 'heads'. In this study, we analyzed the ability of novel NPGLs L1-L5 to interact with duplex DNA and interfere with the DNA modifying enzyme topoisomerase I (topo I). In cell-free systems, NPGLs formed highly affine complexes with DNA. Molecular docking revealed that NPGLs fitted very well into the DNA minor groove. Compounds L2 (with two long hydrophobic 'tails') and L4 (with ethylimidazolium cationic group), the most affine DNA binders, showed the best calculated energies of complex formation with DNA and topo I. The models demonstrated the binding of NPGLs to the topo I site known for interaction with conventional inhibitors. Each NPGL attenuated the topo I mediated unwinding of supercoiled DNA. Again, L2 and, to a lesser extent, L4 were the most potent topo I inhibitors. Thus, NPGLs with polar 'heads' emerge as a new class of DNA ligands and interfacial topo I antagonists.

Binding affinity of thermoresponsive polyelectrolyte hydrogels for charged amphiphilic ligands. A DSC approach.

Controlled drug binding and release stand among top requirements postulated for targeted drug delivery systems of the new generations. "Smart" polymers and gels are highly suitable for the controlled delivery due to their structural sensitivity to minor environmental variations. The aim of this work was to study thermoresponsive polyanionic and polycationic hydrogels of N-isopropylacrylamide copolymers with acrylic acid and N-aminopropylmethacrylamide in terms of their interaction with two widely used drugs, propranolol and ibuprofen. Binding energetics of these drugs by the gels in swollen and collapsed state was estimated by means of high-sensitivity differential scanning calorimetry. Thermodynamic parameters of the gel collapse (transition temperature, enthalpy, heat capacity increment, and width) were determined as a dependence of the drug concentrations. From these data the excess free energy of collapse was calculated as a function of drug concentration. Deconvolution of this function resulted in the evaluation of binding parameters and contributions from interactions of various types to the free energy of binding. The binding mechanism of both drugs to the swollen and collapsed gels was elucidated. Its main features are the cooperative character of the drug binding by the collapsed gel and the predominant role of the hydrophobicity of drugs in their affinity for the swollen gel.

Ternary interpolyelectrolyte complexes insulin-poly(methylaminophosphazene)-dextran sulfate for oral delivery of insulin.

Ternary interpolyelectrolyte complexes of insulin with biodegradable synthetic cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP), and dextran sulfate (DS) were investigated by means of turbidimetry, dynamic light scattering, phase analysis, and high-sensitivity differential scanning calorimetry. Formation of ternary insoluble stoichiometric Insulin-PMAP-DS complexes was detected under conditions imitating the human gastric environment (pH 2, 0.15 M NaCl). A complete immobilization of insulin in the complexes was observed in a wide range of the reaction mixture compositions. The ternary complexes were shown to dissolve and dissociate under conditions imitating the human intestinal environment (pH 8.3, 0.15 M NaCl). The products of the complex dissociation were free insulin and soluble binary Insulin-PMAP complexes. The conformational stability of insulin in the soluble complexes of various compositions was investigated by high-sensitivity differential scanning calorimetry. The dependence of the excess denaturation free energy of insulin in these complexes on the PMAP content was obtained. The binding constants of the folded and unfolded forms of insulin to the PMAP polycation were estimated. Proteolysis of insulin involved in the insoluble ternary complexes by pepsin was investigated under physiological conditions. It was found that the complexes ensure an almost 100% protection of insulin against proteolytic degradation. The obtained results provide a perspective basis for development of oral insulin preparations.

Polyplexes of poly(methylaminophosphazene): energetics of DNA melting.

The interaction of DNA with a synthetic biocompatible and biodegradable cationic polymer, poly(methylaminophosphazene) hydrochloride (PMAP·HCl), was investigated by high-sensitivity differential scanning calorimetry under conditions of strong and weak electrostatic interactions of the macroions. Thermodynamic parameters of the DNA double-helix melting were determined as a function of pH and the PMAP·HCl/DNA weight ratio. PMAP·HCL was shown to reveal two functions with respect to DNA: the polyelectrolyte function and the donor-acceptor one. The first function stabilizes the helical conformation of DNA, and the second one destabilizes it. The stabilizing effect of PMAP·HCl is of entropic origin, related to a displacement of mobile counterions from the DNA's nearest surroundings by the poly(methylaminophosphazene) charged groups. The donor-acceptor function of poly(methylaminophosphazene) dominates when its electrostatic interaction with DNA is either saturated (in the complex coacervate phase at high poly(methylaminophosphazene) concentrations) or completely suppressed (in a salt medium when the polycation carries a small charge). Under these conditions, poly(methylaminophosphazene) destabilizes DNA. It preferentially binds to the DNA coil form likely via the formation of multiple labile hydrogen bonds with the donor-acceptor groups of DNA.

Conformational energetics of interpolyelectrolyte complexation between ι-carrageenan and poly(methylaminophosphazene) measured by high-sensitivity differential scanning calorimetry.

The interaction of poly(methylaminophosphazene) hydrochloride (PMAP·HCl) of varying degrees of ionization (f) with the potassium salt of ι-carrageenan was studied by high-sensitivity differential scanning calorimetry at a KCl concentration of 0.15 M, which is included for the purpose of stabilizing the helix conformation of the polysaccharide up to 55 °C. The conditions of strong (pH 3.8, I = 0.15), moderate (pH 7.4, I = 0.15), and weak (pH 7.4, I = 0.25) electrostatic interactions of the polyelectrolytes were considered. The thermodynamic parameters of the helix-coil transition of ι-carrageenan were determined as a function of the polycation/polyanion ratio. We show that the interpolyelectrolyte reaction between PMAP·HCl and ι-carrageenan results in a complete unfolding of the polysaccharide helix under conditions of strong electrostatic interaction and increases its stability under conditions of medium and weak electrostatic interactions. The formation of stoichiometric PMAP-carrageenan interpolyelectrolyte complexes proceeded via a cooperative mechanism at pH 3.8 (f = 0.5) and pH 7.4 (f = 0.2) at an ionic strength of 0.15. In contrast, the complexation at pH 7.4 and an ionic strength of 0.25 could be considered to be a consecutive competitive binding of charged units of poly(methylaminophosphazene) to the oppositely charged polysaccharide matrix in the helix or coil conformation. Binding constants of the polycation to the helix and coil forms of ι-carrageenan were estimated. They revealed a preferential binding of the polycation to the helix form of the polysaccharide.

Temperature-sensitive chitosan-poly(N-isopropylacrylamide) interpenetrated networks with enhanced loading capacity and controlled release properties.

Interpenetrated polymer networks (IPN) of poly(N-isopropylacrylamide) (PNIPA) and chitosan (two grades) were prepared by free radical polymerisation and cross-linking of PNIPA (700 mM) with bis(acrylamide) (20 mM) in chitosan solutions (1.5 wt.% in acetic acid), and subsequent immersion in glutaraldehyde solutions (0 to 0.7 vol.%) to post-cross-link the chitosan. The amount of chitosan that remained in the IPNs, after washing, was proportional to the glutaraldehyde concentration used in the post-cross-linking step; being only 50% of the theoretical when the post-cross-linking was omitted (semi-IPN). The temperature-induced phase transitions of the IPNs were followed by the changes in the swelling degree and in the thermodynamic parameters (temperature, enthalpy, heat capacity, and width of the transition), which were evaluated using high-sensitivity differential scanning calorimetry (HS-DSC). An increase in the post-cross-linking degree of chitosan caused a decrease in the enthalpy of the transition, and in the absolute value of the transition heat capacity increment (delta(t)C(p)), as well as a broadening of the heat capacity peak. This behaviour is a consequence of the subdivision, in the IPNs, of the PNIPA network in microdomains, some regions of which (surface or outer) cannot be involved in the transitions. On the other hand, changes in pH from 8 to 3 only increased the transition temperature from about 32 to 34 degrees C, despite the considerable modification that this caused in the ionisation degree of chitosan. The PNIPA/chitosan IPNs had a notably greater affinity for diclofenac than the pure PNIPA hydrogel and were able to sustain the drug release for more than 8 h in 0.9% NaCl solutions or pH 8 phosphate buffer. The IPNs with lower chitosan post-cross-linking degree showed the higher temperature-sensitive release patterns. In contrast, the temperature did not significantly affect the release rate from the most cross-linked IPNs, in which the PNIPA microdomains are smaller and the volume phase transitions are less sharper. Therefore, PNIPA microdomains play an important role in controlling the release process. In summary, the interpenetration of networks with complementary properties, such as those made with PNIPA and chitosan, make it possible to develop drug delivery systems with improved drug loading capacity (owing to chitosan) and sustained release behaviour (owing to PNIPA).