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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 de...

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.

Poly(ethylaminophosphazene) (PEAP) in salt-free aqueous solution does not reveal thermoresponsive properties but acquires thermoresponsivity upon addition of various poly(protic acid)s. Energetics of the salt-induced phase separation transition of the PEAP solutions upon heating was investigated by high-sensitivity differential scanning calorimetry at pH 3.5. At low concentrations of poly(carboxylic acid)s (10–100 mM), the transition temperature and enthalpy vary from 90 to 25 °C and from 1.5 to 35 J g–1, respectively, depending on the pKa1 and the apparent hydrophobicity of the acids. Dependences of the transition parameters (Tt, Δth, and Δtcp) on the citrate buffer concentration were obtained. The binding curve of citrate anions to PEAP was derived. High concentrations of sulfate anions (0.1–0.8 M) induced a marginal phase transition at high temperatures. A mechanism of the PEAP thermoresponsivity is proposed. The Okado–Tanaka model for cooperative hydration of macromolecules is used to quantitatively d...

Bisstyrylpyridine dyes 1 and 2, in which two styrylpyridine moieties are connected to each other through the oxo- or azacrown ether unit, and their monomer analogues 3 and 4 have been synthesized and spectroscopically characterized. The prepared bisstyryl dyes 1 and 2 are the rare example of bisstyryls constructed in tail-to-tail mode. The binding of dyes 1–4 with salmon DNA has been evaluated using the UV–vis, CD, fluorescence, high-sensitivity differential scanning calorimetry, viscometric data and molecular modeling. The dyes were found to have negligible fluorescence in a buffer solution, but exhibited a significant emission increase upon binding to DNA. The results obtained are consistent with a groove binding mode between mono- and bisstyryls and DNA.

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.

Micellization of a poloxamer P105 in diluted aqueous solutions was first investigated by means of high-sensitivity differential scanning calorimetry at different pressures up to ∼160 MPa. The temperature dependence of partial heat capacity of the poloxamer clearly indicates that the unimers have the random coil conformation while the micelles are built of a “dry” PO core and hydrated EO shell. Independently of the polymer concentration, the micellization temperature increases upon increasing pressure while the enthalpy decreases. Based on these dependences, the micellization increments of the partial volume Δ t v as well as the coefficients of thermal volume expansion and compressibility, Δ t α and Δ t χ , of the poloxamer were calculated. They prove that the dehydration of PO hydrophobic blocks makes the main contribution to the micellization energetics. We demonstrate that poloxamer micelles are formed via a nucleation stage of unimer dimerization followed by consecutive energetically favored attachment of unimers to the dimer and further to the growing micelle.

Interactions of a major whey protein, β-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.

Spongy cryostructurates based on bovine serum albumin (BSA) have been preparedviafreezing the aqueous solutions of the protein followed by freeze-drying and subsequent cross-linking BSA macromolecules each together within the macropore walls using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) dissolved in ethanol. The gel-fraction yield values testifies high efficiency (>93%) of the protein building-up into the 3D polymeric network. Poor swelling of the pore walls of BSA-based sponges in water (1–2 g H2O per 1 g of dry polymer) and even in the powerful protein-solubilizing media (8murea, 5mguanidine hydrochloride, 1% SDS) indicates the multipoint character of albumin cross-linkingviathe pendant peptide bonds. As a result, strong cross-linking is able (as revealed by HS-DSC) to inhibit BSA thermal denaturation. The size of wide pores in the obtained cryostructures ranges from 40 to 250 μm and mainly depends on the freezing temperature.

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.

Interaction of a biodegradable anionic polyphosphazene, poly[di(carboxylatophenoxy)phosphazene] (PCPP), with a model globular protein, hen egg white lysozyme, was investigated under near-physiological conditions by means of turbidimetry, high-sensitivity differential scanning and isothermal titration calorimetry (ITC). The formation of soluble (non-stoichiometric) and insoluble (stoichiometric) interpolyelectrolyte complexes was demonstrated upon changes in the reaction mixture composition. The denaturation parameters of the bound lysozyme in the stoichiometric and non-stoichiometric complexes were determined. They point to a substantial decrease in conformational stability of the bound protein in the stoichiometric complex. In the non-stoichiometric complexes corresponding to a loose protein loading on the PCPP matrix an additional decrease in conformational stability of lysozyme was observed. It indicates unfolding of the protein at room temperature. ITC experiments were carried out at different temperatures in two modes: direct and reversible titrations. The direct titration curves were well approximated by the McGhee-von Hippel model that resulted in determination of the binding constant (∼1 × 10 6 M −1 ), apparent ligand size (∼10), cooperativity parameter (∼11) as well as the enthalpies of binding and ligand-ligand interaction. The heat capacity increments associated with the binding of the protein ligands and contact interaction between the bound ligands were comparable by value but opposite in sign. We discuss this effect in terms of changes in polar and apolar accessible surface areas of the bound protein ligands caused by the ligand-matrix and ligand-ligand interactions.

The thermoresponsive copolymers of N-isopropylacrylamide (NIPAM) with sodium styrene sulfonate (SSS) exhibit a protein-like transition behavior in aqueous medium. They undergo a coil-to-globule conformational transition without phase separation. In the present work we investigated a conformation-dependent binding of polyfunctional ligands to the protein-like NIPAM- b -SSS copolymers modelling their functional behavior by analogy with the enzyme-substrate interaction. Natural polyamines of various charge (putrescine, spermidine and spermine) were used as the polyfunctional ligands. The polyamine-copolymer binding was evaluated by high-sensitivity differential scanning calorimetry. The dependences of the transition parameters (transition temperature, enthalpy, heat capacity increment and thermodynamic width) for two NIPAM- b -SSS copolymers of low and high charge density on the polyamine concentrations were obtained. In general, they pointed to a preferential binding of polyamines to the copolymers in globular conformation, that is particularly pronounced for the copolymer of low charge density. The excess transition enthalpy was introduced as an apparent measure of the ligand binding to the copolymer. The apparent binding constants increased in the series putrescine

Chemically cross-linked wide-porous protein cryogels have been obtained by freezing aqueous bovine serum albumin (BSA) solutions (30–50 g/L) at–15,–20, or–25°C in the presence of water-soluble carbodiimide (CDI) as a coupling agent. It has been shown that the gel-fraction yield and the swelling extent of the polymer phase of the formed spongy matrices depend primarily on the initial concentration of albumin and the amount of CDI added to a system, while the morphometric characteristics of the porous microstructure of the BSA cryogels are mainly determined by the temperature of the cryogenic treatment. The sizes of macropores in the obtained cryogels range from ≈50 to ≈200 μm. A high-sensitivity differential scanning calorimetric study of the conformational state of protein macromolecules incorporated into the spatial network of the polymer phase (gel walls of macropores) of the cryogels has shown that, during the cryotropic gelation, the native structure of albumin globules is subjected to “cold denaturation,” and the partly unfolded conformation of the protein is, simultaneously, fixed by intermolecular covalent cross links.

Phase separation in aqueous solutions of poly(vinyl methyl ether) was first investigated by high-sensitivity differential scanning calorimetry at normal and high pressures. Thermograms of the phase separation had two singular points: a binodal point (Tt,1) and a point of the three phases coexistence (Tt,2). At normal pressure the temperature Tt,1 decreased slightly and the temperature Tt,2 was constant (36.8 ± 0.1°С) upon increasing the polymer concentration from 0.01 to 0.25%. The transition enthalpy and heat capacity increment did not depend on the polymer concentration (Δth = 88.0 ± 1.2 J g−1; Δtcp = −1.0 ± 0.1 J g−1 K−1). The calorimetric experiments at high pressures were carried out up to pressure 170 MPa in the range of polymer concentrations 0.1–1.1%. When pressure increased the temperature Tt,1 dropped by 10 °C, the temperature Tt,2 increased by 15 °C, the total transition enthalpy reduced 1.5 fold and the transition heat capacity increment remained virtually unchanged. These data reveal that the sequential phase transitions, which the diluted aqueous solutions of poly(vinyl methyl ether) undergo upon heating, have different nature.

Interaction of a synthetic biodegradable polymer, poly(methylaminophosphazene), (PMAP) with human recombinant zinc insulin at pH 7.4 and different ionic strengths was studied by means of turbidimetry, phase analysis and high-sensitivity differential scanning calorimetry. The formation of stoichiometric (insoluble) and non-stoichiometric (soluble) interpolyelectrolyte Insulin-PMAP complexes was detected. It was shown that binding of zinc insulin to PMAP is accompanied by conversion of the protein to its zinc-free form as a result of the Zn+2 release from the insulin native structure. The conformational stability of the bound zinc-free insulin is defined by composition of the complexes. It increases in case of the non-stoichiometric complexes and decreases in the stoichiometric ones. We found that soluble Insulin-PMAP complexes revealed thermoresponsive properties within physiological range of temperature and ionic strength.

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.

Most drugs besides their intended activity, express undesired side effects, including those with the engagement of cell membrane. Previously, such undesired nonspecific effects on the membrane have been shown for a number of widely used nonsteroidal anti-inflammatory drugs. In this paper, we study the mechanism of interaction between moxifloxacin (Mox), antibacterial drug of broad specificity, with lipid bilayer of the liposomes of various compositions as a model of cell membrane using a combination of spectroscopy methods, including ATR-FTIR spectroscopy, circular dichroism, UV and fluorescence spectroscopy. The fine structure of the moxifloxacin-liposome complex, localization of the drug in bilayer and the main sites of Mox interaction with lipid membrane were determined. Lipid composition of the liposome plays a key role in the interaction with moxifloxacin, drastically affecting the loading efficiency, strength and character of drug binding, lipid phase segregation and phase transition parameters. In case of anionic liposomes composed of dipalmitoylphosphatidylcholine (DPPC) and cardiolipin (CL2-) the electrostatic interaction of negatively charged nitrogen in heterocycle moiety of moxifloxacin with cardiolipin phosphate groups is a crucial factor for stable complex formation. The study of moxifloxacin-liposome complex behavior at phase transition in bilayer by DSC method revealed that in DPPC/CL2- liposomes system two microphases with different content of CL2- coexist and Mox interacts with both of these microphases resulting in the formation of two types of complexes with different structure and phase transition temperature. This binding stabilized the gel-state of the lipid bilayer with increasing the phase transition temperature Tm up to 3-5 °C. A different situation is observed for neutral DPPC liposomes: drug interaction with bilayer results in defects formation and a fluidization effect in lipid bilayer, resulted to decrease the Tm value by 2-4 °C. Moxifloxacin is not firmly binding in the membrane of DPPC and drug releases rapidly.

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.

Thermoresponsivity of chitosan induced by β-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 ( Δ 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.

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.

N-Acryloyl derivatives of histamine, monoethanolamine, and β-alanine, which contain imidazole, hydroxyl, and carboxyl functional groups, respectively, are synthesized and characterized. Copolymers, which are further divided into thermally precipitable and thermally nonprecipitable (soluble) fractions, are obtained via the free-radical copolymerization of N-vinylcaprolactam and the specified comonomers that is initiated by a persulfate–tertiary amine redox system in a medium of 10% aqueous dimethyl sulfoxide (DMSO) at 65°C. It is shown that the soluble fractions of the synthesized copolymers exhibit catalytic activity in the reaction of hydrolysis of 4-nitrophenyl propionate (NPP) at a temperature above the coil-to-globule transition.

Thermoresponsive diblock copolymers of N-vinylcaprolactam (VCL) with N-vinylimidazole (NVI) have been synthesized for the first time through two-stage controlled reversible addition–fragmentation chain transfer (RAFT) polymerization. The resulting diblock copolymers exhibit the same catalytic activity toward p-nitrophenyl propionate (NPP) hydrolysis as “protein-like” copolymers of VCL and NVI. The catalytic activity of the diblock copolymers is likely caused by their structure favorable for the manifestation of catalytic properties, namely, by micelle-like chain conformations with a relatively dense core formed by the more hydrophobic PVCL block and a periphery formed by the hydrophilic poly-N-vinylimidazole (PNVI).

The interaction of the synthetic biodegradable anionic polymer poly[di(carboxylatophenoxy)phosphazene]) with bovine serum albumin was studied at pH 4.5–7.4 in the ionic strength range from 0.05 to 0.2 by means of velocity sedimentation, dynamic light scattering, and high-sensitivity differential scanning calorimetry. It was found that a relatively low molecular weight poly[di(carboxylatophenoxy)phosphazene] (M sD ~ 2 × 104) can form insoluble and soluble complexes with bovine serum albumin. The conformational stability of bovine serum albumin was investigated in detail as a function of mixture composition. It was shown that the denaturation temperature and enthalpy of the protein steadily decrease with the content of polyphosphazene in the system, regardless of the ionic strength of the medium. On the basis of these data, dependences of the excess free energy of denaturation of bovine serum albumin on the polyphosphazene-to-protein ratio were calculated. An inspection of these dependences primarily suggests the preferential binding of poly[di(carboxylatophenoxy)phosphazene] by the unfolded form of the protein. At the same time, there is a certain affinity of the polymer ligand to the native form of the protein, which is related to some cooperative effects. Calorimetric studies showed that the formation of weak complexes of bovine serum albumin with the polyphosphazene considerably reduces the conformational stability of the protein, which determines its physiological functionality.

Poly(ethylene oxide), PEO, is known to undergo a phase separation transition in aqueous solutions upon heating. This LCST transition is first investigated by high-sensitivity differential scanning calorimetry. Dependences of the transition temperature (Tt), enthalpy (Δth) and heat capacity increment (Δtcp) on the polymer concentration are determined. The transition temperature and enthalpy of PEO decreased with increasing the polymer concentration and followed the Kirchhoff law. A substantial positive value of the transition heat capacity increment of PEO is detected suggesting that the transition is accompanied by an exposure of the polymer hydrophobic groups to water. Comparison of Δtcp with the corresponding change in the accessible surface area of PEO reveals that the LCST transition of PEO may involve cooperative conformational changes of the type of helix–coil transition in PEO macromolecules.

The phase separation of aqueous solutions of poly(N-isopropylacrylamide) at different pressures (up to 212 MPa) has been first investigated by high-sensitivity differential scanning calorimetry (HS-DSC). The dependences of the transition temperature, enthalpy and width on pressure have been determined. Upon increasing pressure, the transition temperature passes through a maximum, the enthalpy decreases and the thermodynamic width of the transition notably increases. The transition increment of partial volume of the polymer (Δtv) is found to be positive at low pressures and negative at high ones. The positivity of the increment Δtv points to a significant role of hydrophobic hydration in the transition mechanism. The negativity of Δtv can be related to domination of the interchain H-bond formation upon the system phase separation. The transition width increase reflects changes in the shape of the system binodal.

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.

Freeze-thaw processing of bovine serum albumin (BSA) aqueous solutions, which contain also the additives of denaturants (urea in this case) and thiol-bearing reductants [cysteine (Cys) in this case] leads to the formation of wide-pore cryogels. The properties and porous morphology of these spongy gel matrices were demonstrated to depend on the initial concentration of all precursors and on the freezing/frozen storage temperature. The optimum conditions for preparing such BSA-based cryogels were found to be as follows: [BSA] = 3-5 g dL(-1), [urea] = 0.5-2.0 mol L(-1), [Cys] = 0.01 mol L(-1), and freezing temperatures in the range of -15 to -20 °C. The size of gross pores in thus prepared cryogels is ∼50-150 μm. The spatial network of BSA-cryogels was shown to be cross-linked chemically via interchain disulfide bridges. The significant role of hydrophobic interactions in the stabilization of 3D networks of these cryogels is inferred, as well as the supposition about the relay-race sequence mechanism of the intermolecular disulfide cross-link formation is made.