Morphology Of Cryogels Research Articles

SUPERSOFT SPONGE-LIKE CRYOGEL AS AN IMPLANT TO TREAT GLIOBLASTOMA

Abstract AIMS Create cylindrical cryogels with well-defined size. Investigate clemastine drug loading capicaty and drug release patterns. Investigate in vitro and in vivo safety and antitumor efficacy. METHOD A 3D printed template was designed and printed to make cylindrical shape cryogels. The morphology of cryogels was characterized by bright field photos, SEM, FTIR and confocal microscopy photos. The mechanical property was evaluated by uniaxial compression tests. The orthotropic glioblastoma tumor model was established by engrafting BT12 human glioblastoma stem cells intracerebrally into immunocompromised nude mice. The tumor was surgically removed after two weeks and the cryogel was implanted into the resection cavity. Animals were euthanized after two weeks, and their brains were collected for the immunohistochemistry. RESULTS Cylindrical cryogels were prepared to exactly fit the biopsy resection cavity in the mouse glioblastoma tumor resected model. The macropore structure in the cryogel was observed under confocal microscopy. Young’s modulus of the cryogel was only 1.6 kPa. The loading capacity of clemastine in one cryogel was more than 100 μg. Clemastine could be sustained released for around a week. In vitro cell viability studies showed that empty cryogels were not toxic to the tumor cells and drug loaded cryogels killed the tumor cells in a time and concentration dependent manner. In vivo studies further confirmed that cryogels could be safely implanted into the brain. Although the implantation of clemastine loaded cryogels did not prevent the growth of the primary tumor, it reduced the amount of invasion tumor cells compared with the mice only resected the tumor. CONCLUSION Cryogels were easily created by the 3D printed templates. The soft material avoided mechanical mismatch with the brain tissue. The cryogel did not show any toxicity in in vitro and in vivo studies, which indicated it would be a promising material to be implanted into the brain.

Radiation-induced synthesis of hydrophobic cryogels with rapid and high absorption of organic solvents and oils

Herein, for the first time, macro-porous hydrophobic cryogels of poly( iso- decyl acrylate- co -ethylene glycol dimethacrylate) P(IDA- co -EGDMA) have been successfully fabricated through γ -ray radiation-induced cryo-polymerization. This synthesis route is highly advantageous over the reported redox and UV-radiation routes for the synthesis of hydrophobic cryogels considering the high energy and the penetrating depth of γ -ray, which favours the fabrication of larger cryogels within shorter duration. The effects of radiation time, dose rate, amount of cross-linker, solvent type and solvent amount on the cryo-polymerization were studied in detail, which were based on the measurements of gel fraction and contact angle as well as the characterizations of Fourier transform infrared spectroscopy, thermal gravimetric analysis and scanning electron microscopic examination. The fabricated macro-porous cryogels were used as a sorbent for different organic solvents and oils. Using benzene as a common organic solvent, the absorption equilibrium was reached within only 60 s and the absorption capacity varied in the range of 16–20 g/g depending on the cryogel morphology and crosslinking density. As for the halogenated solvents, P(IDA- co -EGDMA) cryogels absorbed 25–29 g/g of chlorobenzene, bromobenzene, iodobenzene and tetrachloromethane. Those cryogels withstood high temperature (150 °C) deprived of any alteration in absorption capability and pore morphology. The cryogels reusability was also confirmed with keeping absorption capacity and cryogel mass up to 10 cycles. Rapid synthesis of macroporous cryogels with rapid and good absorption for organic solvents have been attained through γ -ray radiation-induced cryo-polymerization. • Hydrophobic cryogels were obtained through γ -ray radiation. • Cryogel morphology was modulated with cryo-polymerization parameters. • Cryogels exhibited rapid and high absorption capacity. • Cryogels were recyclable, reusable and stable.

Hybrid cryogels composed of P(NIPAM-co-AMPS) and metal nanoparticles for rapid reduction of p-nitrophenol

Temperature-responsive macro-porous cryogels of poly(N-iospropylacryamide-co-acrylamido-2-methylpropane sulfonic acid) [P(NIPAM-co-AMPS)] were prepared through redox-initiated cryo-polymerization at −12, −18 and −24 °C. Silver or gold nanoparticles (AgNPs or AuNPs) were formed inside P(NIPAM-co-AMPS) cryogels through the thermal reduction of absorbed AgNO3 or HAuCl4, resulting in hybrid cryogels immobilized with AgNPs or AuNPs. Fourier transform infrared spectroscopy, thermal gravimetric analysis and X-ray diffraction confirmed the chemical composition. Cryogel morphology and nanoparticle distribution were investigated through scanning and transmission electron microscopies. By checking swelling ratio in neutral water, the temperature response of cryogels was identified. The hybrid cryogels were used as the catalysts to reduce p-nitrophenol with NaBH4 at different temperatures and pHs. The hybrid cryogel with AuNPs had the higher catalytic activity than the counterpart hybrid cryogel with AgNPs. The catalytic activity of hybrid cryogels increased with temperature and pH. The temperature response of cryogels facilitated their isolation from the reaction mixture. Since AuNPs were fixed tightly onto the cryogel pore-wall and hardly lost, hybrid cryogels with AuNPs demonstrated no decrease in catalytic activity for 5 cycles of p-nitrophenol reduction.

Dimethylacrylamide gels linked by dimethacrylate cross-linkers

Poly(N,N-dimethylacrylamide) (PDMA) is a water-soluble and biocompatible polymer useful in biomedical, biotechnological and pharmaceutical application areas. In this study, dimethylacrylamide (DMA) units were connected with dimethacrylate chemical cross-linkers (DMA-xl) with various chain lengths to get homogeneous, biocompatible and mechanically strong DMA hydrogels and cryogels. In the first step, the effect of the distance between the vinyl groups of the cross-linker molecules on swelling and mechanical properties was investigated. While cross-linkers bearing longer ethylene glycol units from DMA-1 to DMA-2 bond more water molecules, DMA-9’s equilibrium swelling values are the lowest, which may be due to the better movement capability of the chains with longer cross-linker units that will increase the physical interactions between the molecules and lead to the more compact structures. In the second step, cross-linker ratio was changed with a fixed dimethacrylate group. When the mol ratio of DMA-2 is decreasing step by step from the highest to lowest concentration for the gels after synthesis, the elastic modulus decreases gradually from 30 to 6 kPa, and the gels show similar mechanical behavior in the swollen state. In the last step, the amount of cross-link units on the morphology of cryogels was investigated with DMA-2 gels. The results explicitly indicated that while the mesh size of PDMA cryogels decreased with an increase in DMA-2 concentration, the mechanical stability of the gels was increased.

New cryogels based on polymers and zeolite L for controlled Enalapril maleate release

New composite cryogels for controlled release of Enalapril Maleate (EM) were developed based on two biodegradable polymers, poly(vinyl alcohol) (PVA) and pullulan (PU) as polymer matrix, and zeolite L (ZL) as inorganic filler. The cryogels were prepared by freeze-thaw technique using different concentrations of the components, the EM being introduced directly during the cryogel preparation. The samples were characterized by FTIR spectroscopy, scanning electron microscopy and water vapor sorption-desorption isotherms. The presence of PU and ZL in the cryogels led to the modification of FTIR characteristic absorption bands of EM and contributed to the modification of the cryogel morphology. The sample moisture absorption was in the range 5.28–18.20%, determined at a relative humidity RH = 82%. Cryogels based on PVA, PU and ZL components showed a longer EM release period compared to cryogels containing only PVA. The analysis of in vitro EM release kinetics was performed using three predictable models: zero and first order kinetics, and Korsmeyer-Peppas model. Korsmeyer-Peppas model best fitted the release data.

Nicotinamide-Modified poly(HEMA-GMA)-Nic Cryogels for Removal of Pesticides

Chlordane is only one of the persistent pesticides used in some countries despite the ban. Removal of chlordane, a severe threat to all living things, was performed using nicotinamide-modified poly (2-hydroxyethyl methacrylate-glycidyl methacrylate), poly(HEMA-GMA)-Nic, polymeric cryogels in this study. Pesticides are practically insoluble in water. For that reason, ethanol is used as a solvent which is not chemically dangerous and easily accessible in every laboratory. As an adsorbent, poly (HEMA-GMA) polymeric cryogels previously synthesized in the literature have been modified using nicotinamide. The modification of poly(HEMA-GMA) with nicotinamide is the first in the literature. Removal of chlordane in alcohol medium has been accomplished exploiting the alcoho-phobic interaction, which was the first indication in our previous study. Structural analysis of poly(HEMA-GMA)-Nic was performed using Fourier transform infrared spectroscopy (FT-IR) and elemental analysis methods. Scanning electron microscopy (SEM) was used to understand the surface morphology of cryogels. Surface area and cavity volume calculations were determined by applying N2 adsorption method and swelling test. The interaction time and maximum adsorption capacity were identified as 5 minutes and 64.61 mg chlordane/g cryogel for 300 mg/L chlordane concentration and 108.818 mg chlordane/g cryogel for 800 mg/L chlordane concentration during the adsorption experiments. Cyclohexane, toluene, chloroform, dichloromethane, acetone, and acetonitrile were used as solvent to observe the solvent effect on adsorption of chlordane onto the polymeric material. As expected, the removal of chlordane was performed with the highest adsorption performance in cyclohexane with the lowest dielectric constant.

A novel approach for fabricating nanocomposite materials by embedding stabilized core-shell micelles into polysaccharide cryogel matrix

We report a novel approach for fabricating nanocomposite polysaccharide-based carriers for sustained delivery of poorly-water-soluble drugs by embedding stabilized core-shell micelles (SPM) possessing hydrophobic cores into super-macroporous hydroxypropyl cellulose (HPC) cryogels. Firstly, nano-sized SPM were synthesized by loading and photochemical crosslinking of pentaerythritoltetraacrylate (PETA) in poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO19PPO29PEO19) core-shell micelles. Next, HPC cryogels containing different amount of SPM were fabricated by combination of cryogenic treatment and photo-crosslinking. A crosslinking agent, N,N′-methylenebisacrylamide, was used to enhance the density of polymer network. The effect of SPM concentration on gel fraction yield, swelling degree, cryogel morphology and mechanical properties were studied. Nanocomposite cryogels were loaded with curcumin and their encapsulation efficiency and drug release profile as a function of SPM content were investigated. The cytotoxic effect of blank and curcumin loaded nanocomposite cryogels was assessed as well.

Insight into the cryopolymerization to form a poly(N-isopropylacrylamide)/clay macroporous gel: structure and phase evolution

The cryopolymerization and formation of a macroporous poly(N-isopropylacrylamide) (PNIPA)/clay cryogel were investigated. The mechanism of the cryopolymerization and cryogel formation was elucidated. Two processes, cryostructuration and cryopolymerization, proceed simultaneously and their relative rates determine the structure evolution and the cryogel morphology - porosity. The cryostructuration in the PNIPA/clay system during freezing, controlled by the freezing temperature and the rate of cooling, includes both water and NIPA crystallization, formation of a highly concentrated non-frozen liquid phase (NFLP) and clay aggregation. The rate of cryopolymerization and gelation is governed by the following effects: by a low polymerization temperature and after freezing, by the high cryoconcentration and a steric confinement, manifested by a reduced reagent mobility. Moreover, it depends on the cooling rate and the evolution of cryostructuration. The progress of cryostructuration and cryopolymerization during freezing was described and experimentally proved step by step. Both the phase development during freezing and the progress of cryopolymerization including gelation were monitored in situ by NMR, DSC, chemorheology and SAXS. The morphology and porosity of the cryogels were characterized by SEM and TEM.

A study of cryostructuring of polymer systems. 43. Characteristics of microstructure of chitosan-containing complex and composite poly(vinyl alcohol) cryogels

The microstructure of complex and composite poly(vinyl alcohol) (PVA) cryogels containing water-soluble chitosan hydrochloride (ChHC) of dispersed particles of water-insoluble chitosan base (Ch), respectively, has been studied by optical microscopy and attenuated total reflection FTIR spectroscopy. The macroporous morphology of cryogels has been studied using preparations in the form of thin (~10 μm) sections and discs 1 mm thick. The introduction of non-gelling additives (NaCl and ChHC) into an initial PVA solution causes significant changes in the size and shapes of macropores in the complex cryogels formed by freezing–defrosting, as compared with the pores in the samples obtained under the same conditions without additives. The reasons for the changes are the process of phase segregation and the influence of low- and high-molecular-weight electrolytes on crystallization of ice, which plays the role of a porogen upon cryotropic gelation of aqueous PVA solutions. As a result of an alkaline treatment of the complex cryogels, which transforms ChHC into Ch, microcoagulation of chitosan yields discrete, almost spherical, particles with sizes of about 1–5 μm. IR spectral studies have shown that concentration gradients of the gelling and nongelling polymers arise along the thickness of the gel discs, with PVA concentration prevailing near the lower surface and ChHC or Ch concentration dominating near the upper surface of the disc.

Physically and chemically cross-linked cellulose cryogels: Structure, properties and application for controlled release

Porous cellulose matrices were prepared via cellulose dissolution in 8wt% NaOH-water, physical gelation and chemical cross-linking with epichlorohydrin (ECH), coagulation in water and lyophilisation. Cellulose and cross-linker concentration were varied. The behaviour of gels upon coagulation and the swelling of cryogels in water were analysed. An anomalous high swelling at cross-linker concentration around stoichiometric molar ratio with cellulose was observed. Cellulose cryogel morphology, crystallinity and density were studied. The influence of chemical cross-linking on cellulose swelling was explained by suggesting that ECH acts as a spacer preventing cellulose chains tight packing during coagulation. Cellulose was loaded with a model drug, procaine hydrochloride, and the kinetics of its release was investigated.

Complexation of macroporous amphoteric cryogels based on N,N‐dimethylaminoethyl methacrylate and methacrylic acid with dyes, surfactant, and protein

ABSTRACTMacroporous amphoteric cryogels based on N,N‐dimethylaminoethylmethacrylate and methacrylic acid p(DMAEM‐co‐MAA) crosslinked by N,N′‐methylenebisacrylamide (MBAA) were synthesized by radical copolymerization of monomer mixtures in cryoconditions. The structure and morphology of cryogels were evaluated by FTIR and SEM. Cryogels exhibited interconnected porous structure with pore size ranging from 40 to 80 µm, which depended on their crosslinking degree. The value of the isoelectric point (IEP) of equimolar amphoteric cryogel determined from the water flux was equal to 4.4, while the IEP of cryogel with the excess of DMAEM units was equal to 7.1. The mechanical strength of equimolar amphoteric cryogels increases with increasing amount of crosslinking agent. The complexation ability of amphoteric cryogels with respect to surfactant, dyes, and protein was demonstrated. The adsorption isotherms with respect to anionic surfactant—sodium dodecylbenzene sulfonate (SDBS) and protein—lysozyme correspond to Langmuir equation, while adsorption isotherms of anionic and cationic dyes—methylene blue (MB) and methyl orange (MO) are well described by Freundlich equation. It was found that the binding ability of p(DMAEM‐co‐MAA) with respect to various low‐ and high‐molecular weight compounds changes in the following order: SDBS > lysozyme ≫ MO > MB. The preferential adsorption of MB from the mixture of protein and MB was shown. The quantitative release of protein, surfactant and dye molecules from the matrix of cryogels takes place at the IEP of cryogel. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016, 133, 43784.

Preparation and characterization of modified nanosilica/PNIPAm hybrid cryogels

A series of macroporous hybrid cryogels based on poly(N-isopropylacrylamide) (PNIPAm) were synthesized by free radical polymerization in the presence of organically modified nanosilica. To improve the interaction of silica nanoparticles with polymer structure, the silica surface was first treated with 3-methacryloxypropyltrimethoxysilane (MPTMS). Then a series of hybrid cryogels each containing 1 wt.% nanosilica and various amounts of MPTMS (1–4 wt.% to monomer NIPAm) were prepared. The yield of methacrylate modification of the nanosilica surface determined by TGA analysis was 11.2 wt.%. The particle size distribution of modified nanosilica particles was investigated by dynamic light scattering technique (DLS) and the mean particle diameter with respect to number was found as 68 nm. The morphology of cryogels was evaluated by SEM. The improvement in the thermal and mechanical properties using the addition of modified silica nanoparticles was achieved.

Metal Complexes of Amphoteric Cryogels Based on Allylamine and Methacrylic Acid

AbstractA series of macroporous amphoteric cryogels based on allylamine, methacrylic acid and acrylamide were synthesized by radical copolymerization of monomers in cryoconditions. The average molar composition of amphoteric cryogels was found from the potentiometric and conductimetric titration curves. The morphology of cryogels was evaluated by SEM. Cryogels are highly elastic and have continuous macroporous structure with 50–200 µm pores. The values of the isoelectric pH determined from the swelling experiments arranged between 3.5 and 4.3. Complexation of amphoteric with transition metal ions was studied. Cryogels with adsorbed copper, nickel, or cobalt ions have an intense colour due to formation of coordination and ionic bonds between metal ions and amine and/or carboxylic groups of cryogels. Metal ions entrapped within the pores of cryogels were reduced by treating aqueous solution of NaBH4. Formation of micron sized metals on the inner surface of cryogels was observed by SEM.

Novel macroporous amphoteric gels: Preparation and characterization

Macroporous amphoteric gels based on allylamine, methacrylic acid and acrylamide crosslinked by N,N!-meth- ylenebisacrylamide were synthesized by radical copolymerization of monomers in cryoconditions. The composition of cryogels was determined by combination of potentiometric and conductimetric titrations. The morphology of cryogels was evaluated by scanning electron microscope (SEM). Cryogels exhibited sponge-like porous structure with pore size ranging from 50 to 200 µm. The values of the isoelectric points (IEPs) determined from the swelling experiments arranged between 3.5 and 4.3. The high adsorption-desorption capacity of amphoteric cryogels with respect to mM and trace concentrations of copper, nickel, and cobalt ions was demonstrated. It was shown that the macroporous amphoteric cryogels are able to adsorb up to 99.9% of copper, nickel, and cobalt ions from 10 -3 mol·L -1 aqueous solution.

Engineered (hep/pARG)2polyelectrolyte capsules for sustained release of bioactive TGF-β1

Spatial and temporal controlled delivery of growth factors which contribute to efficient repair upon tissue injury or failure remains a major challenge in tissue engineering. In this paper, we evaluated polyelectrolyte multilayer microcapsules to control spatiotemporal release of transforming growth factor-β1 (TGF-β1). Polyelectrolyte microcapsules were bio-specifically engineered to enhance their ability to bind growth factors. Myofibroblast differentiation and contraction demonstrated that the bioactivity of TGF-β1 was not altered upon encapsulation into polyelectrolyte multilayer microcapsules and subsequent incorporation into gelatin based hydrogels. Finally, polyelectrolyte microcapsules were incorporated within a synthetic tissue engineering cryogel scaffold composed of gelatin without affecting the cryogel morphology, cell population capacity or mechanical properties of the scaffold.

Cryostructuring of polymer systems. XXVI. Heterophase organic–inorganic cryogels prepared via freezing–thawing of aqueous solutions of poly(vinyl alcohol) with added tetramethoxysilane

AbstractComposite heterophase organic–inorganic hybrid cryogels of poly(vinyl alcohol) (PVA) containing silica constituents were prepared and studied. Such constituents were formed in the course of hydrolytic polycondensation (sol‐gel process) of tetramethoxysilane (TMOS) introduced in to the aqueous polymer solution prior to its freeze–thaw treatment. It was shown that moderate (over the range of −15 to −30°C) freezing, then frozen storage, and subsequent thawing of the water/PVA/TMOS systems resulted in the formation of macroporous composite cryogels filled with dispersed silica particles (discrete phase). The continuous phase of such gel materials represents the supramolecular PVA network, which is supposed to be additionally cured with the silicon‐containing oligomeric cross agents formed from TMOS in the course of hydrolytic polycondensation. The incorporated silica components influenced the morphology of cryogels. The effects of significant increase in gel strength and heat resistance with increasing TMOS concentration in the initial feed and with thawing rate decreasing have also been observed. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007

The potential of polymeric cryogels in bioseparation.

This is a review discussing the production and properties of cryogels (from the Greek kappa rho iota sigma (kryos) meaning frost or ice), immobilization of ligands in cryogels and the application of affinity cryogels in bioseparation. Cryotropic gel formation proceeds in a non-frozen liquid microphase existing in the macroscopically frozen sample. Due to the cryoconcentration of gel precursors in the non-frozen liquid microphase, cryogelation is characterised by a decrease in the critical concentration of gelation and an increase in gelation rates compared with traditional gelation at temperatures above freezing point. Cryogels can be obtained through the formation of both physically and covalently cross-linked heterogeneous polymer networks. Interconnected systems of macropores and sponge-like morphology are typical for cryogels, allowing unhindered diffusion of solutes of practically any size. Most of the water present in spongy cryogels is capillary bound and can be removed mechanically by squeezing. The properties of cryogels can be regulated by the temperature of cryogelation, the time the sample is kept in a frozen state and freezing/thawing rates, by the nature of the solvent and by the use of soluble and insoluble additives. The unique macroporous morphology of cryogels, in combination with osmotic, chemical and mechanical stability, makes them attractive matrices for chromatography of large entities such as protein aggregates, membrane fragments, viruses, cell organells and even whole cells. Special attention is given to immunosorption of viruses on cryogel-based sorbents. As chromatographic materials, cryogels can be used both in bead form and as spongy cylindrical blocks (monoliths) synthesized inside the chromatographic column. The macroporous nature of cryogels is also advantageous for their application as matrices in the immobilization of biocatalysts operating in both aqueous and organic solvents. New potential applications of cryogels are discussed.

Compartmentation in the poly(vinyl alcohol) cryogels. 1H NMR self-diffusion study

The self-diffusion of the poly(vinyl alcohol) (PVA) cryogels prepared by freezing–thawing the aqueous and water/DMSO solutions of PVA was studied with the NMR FT-PGSE method. The temperature dependencies of the self-diffusion coefficients, D s, for the PVA chains have a maximum at 45°C due to the syneresis of cryogels. They are quite different from the monotonous increase of D s for the aqueous solutions of PVA. Evaluated apparent activation energies, E a, of the self-diffusion for the PVA chains in the PVA solutions and cryogels in D 2O are practically the same and equal 22–24 kJ mol −1 below the crucial point. The proton spin-lattice relaxation times, T 1, of the PVA chain also coincide with one another for solutions and cryogels. It means that molecular packing in cryogels depends mainly on the dimensions of the ice and polymer microcrystallites formed by freezing the solution. Above the crucial point polymer compartments become firmer, and the chain mobility somewhat reduces. The strength of cryogels also increases along with growing the DMSO contents and decreases by the BSA addition. For an estimation of the cryogel morphology, effects of the restricted diffusion of both the water and PVA in a q-space have been taken into account. By introducing DMSO to cryogels, the solvent filled pores become smaller, and channels become much shorter. The diameter of the PVA filaments is similar to those for all the cryogels, but the length of filaments with D 2O is twice those for cryogels with a mixed solvent. Entrapment of BSA in the cryogel matrix by preparation leads to the increase of an average diameter of the water filled pores, and destroys molecular packing the cryogel.

1H NMR Self-Diffusion Study of Morphology and Structure of Polyvinyl Alcohol Cryogels

The multicomponent self-diffusion of the polyvinyl alcohol (PVA) cryogels prepared by a freezing–thawing treatment of aqueous and water–DMSO solutions of PVA has been studied with the NMR FT-PGSE method. The temperature dependencies of the self-diffusion coefficients,Ds, for the PVA chains have a maximum at 45°C due to the syneresis of cryogels. They are quite different from the monotonous increase ofDsfor the aqueous solutions of PVA. Evaluated apparent activation energies,Ea, of the self-diffusion for the PVA chains in the PVA solutions and cryogels in D2O are practically the same and equal 22–24 kJ/mol below the crucial point. The proton spin–lattice relaxation times,T1, of the PVA chain also coincide with one another for solutions and cryogels. This means that molecular packing in cryogels depends mainly on the dimensions of the ice and polymer microcrystallites formed by freezing the solution. Above the crucial point polymer compartments become firmer, and the chain mobility somewhat reduces. The strength of cryogels also increases along with growing the DMSO contents and decreases by the BSA addition. For estimation of the cryogel morphology, effects of the restricted diffusion of both the water and PVA in aq-space have been taken into account. By the introduction of DMSO to cryogels the solvent filled pores become smaller, and channels become much shorter. The diameter of the PVA filaments is similar to those for all the cryogels, but the length of filaments with D2O is twice that for cryogels with a mixed solvent. Entrapment of BSA in the cryogel matrix by preparation leads to the increase of an average diameter of the water filled pores and destroys molecular packing the cryogel.