Synthesis of lysozyme imprinted column with macroporous structure and enhanced selectivity: Utilization of cryogelation technique and electrostatic functional monomers

Applicability of molecularly imprinted polymers (MIP) in conventional protein separation processes demands monolithic construction of columns with macroporous structure in addition to the high specificity and adsorption capacity. In this study, therefore, lysozyme (Lyz) imprinted monolithic cryogel columns were synthesized using electrostatic functional monomers (EFMs) to provide strong interactions between template and polymer, leading to specific recognition and capture of Lyz. SEM images and FTIR spectroscopy analysis confirmed the macroporous structure and presence of EFMs in the samples. Adsorption isotherms, heterogeneity, and breakthrough curves as well as selectivity of the molecularly imprinted cryogels (EFMs‐MIC) and non‐imprinted cryogels (EFMs‐NIC) were investigated. Results showed effective imprinting with a maximum adsorption capacity of 211 mg/g and a high imprinting factor (IF) of 4.2 at low Lyz concentrations. A high relative selectivity coefficient of 7.24 was obtained for Lyz over cytochrome c, a competing protein, indicating that the imprinted sites could well distinguish Lyz. Reusability of MICs was also examined, where insignificant changes were observed in the cryogel adsorption/desorption characteristics after four cycles. Therefore, it is suggested to use EFMs and cryogelation in the synthesis of imprinted monolithic cryogels column for application in conventional protein separation processes. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 42880.     

Preparation and characterization of poly(vinyl alcohol) cryogel‐silver nanocomposites and evaluation of blood compatibility,cytotoxicity, and antimicrobial behaviors

In this investigation, cryogels composed of poly(vinyl alcohol) (PVA) were prepared by repeated freeze‐thaw method. The prepared cryogels served as templates for producing highly stable and uniformly distributed silver nanoparticles via in situ reduction of silver nitrate (AgNO₃) using alkaline formaldehyde solution as reducing agent. The structure of the PVA/Ag cryogel nanocomposites was characterized by a Fourier transform infrared and Raman spectroscopy. The morphologies of pure PVA cryogels and PVA/Ag nanocomposites were observed by a scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques. The SEM analysis suggested that cryogels show a well defined porous morphology whereas TEM micrographs revealed the presence of nearly spherical and well separated Ag nanoparticles with diameter about 100 nm. XRD results showed all relevant Bragg's reflections for crystal structure of silver nanoparticles. The amount of silver in cryogel nanocomposites and thermal stability were determined by inductively coupled plasma atomic emission spectrometry (ICP‐AES) and thermogravimetric analysis measurements. Mechanical properties of nanocomposites were observed in terms of tensile strength. The antibacterial studies of the synthesized nanosilver containing cryogels showed good antibacterial activity against both gram‐negative and gram‐positive bacteria. The prepared PVA/Ag nanocomposites were also investigated for swelling and deswelling behaviors. The results reveal that both the swelling and deswelling process depends on the chemical composition of the cryogel silver nanocomposites, number of freeze‐Thaw cycles and pH and temperature of the swelling medium. The biocompatibility of the prepared nanocomposites was judged by in vitro methods of percent hemolysis and protein (BSA) adsorption. POLYM. COMPOS., 36:1983–1997, 2015. © 2014 Society of Plastics Engineer     

Preparation and characterization of biocompatible spongy cryogels of poly(vinyl alcohol)–gelatin and study of water sorption behaviour

Porous biocompatible spongy hydrogels of poly(vinyl alcohol) (PVA)–gelatin were prepared by the freezing–thawing method and characterized by infrared and differential scanning calorimetry. The prepared so‐called ‘cryogels’ were evaluated for their water‐uptake potential and the influence of various factors, such as the chemical architecture of the spongy hydrogels, pH and the temperature of the swelling bath, on the degree of water sorption by the cryogels was investigated. It was found that the water sorption capacity constantly decreased with increasing concentration of PVA while initially an increase and thereafter a decrease in swelling was obtained with increasing amounts of gelatin in the cryogel. The water sorption capacity decreased with an increase in the number of freeze–thaw cycles. The hydrogels were also swollen in salt solutions and various simulated biological fluids and a fall in swelling ratio was noticed. The effect of the drying temperature of the cryogel on its water sorption capacity was also investigated, and a decrease in swelling was obtained with increasing temperature of drying. The biocompatibility of the prepared materials was assessed by in vitro methods of blood‐clot formation, platelet adhesion, and per cent haemolysis. It was noticed that with increasing concentration of PVA and gelatin the biocompatibility increased, while a reduced biocompatibility was noted with an increasing number of freeze–thaw cycles. Copyright © 2005 Society of Chemical Industry     

Poly(ethoxytriethyleneglycol acrylate) cryogels as novel sustained drug release systems for oral application

Novel temperature sensitive cryogels of poly(ethoxytriethyleneglycol acrylate) (PETEGA), with in situ entrapped active substance, are synthesized employing the UV irradiation technique and tested as matrix for controlled release of the hydrophilic drug verapamil hydrochloride. PETEGA cryogels are non-toxic materials and, due to the macroporous structure, exhibit a reversible, ultra-rapid volume phase transition at temperature ca. 31 °C. Carriers based on PETEGA cryogels possess sustained release of verapamil hydrochloride over a period of more than 8 h, which is attributed to the hydrophobic state of the polymer network at physiological temperature and the method of drug immobilization. Drug release characteristics of PETEGA cryogels are compared with another cryogel systems, based on polyacrylamide (PAAm), poly(N-isopropylacrylamide) (PNIPAAm) and poly(2-hydroxyethyl methacrylate) (PHEMA), obtained via the same method.     

Pullulan/poly(N-vinylimidazole) cryogel: An efficient adsorbent for methyl orange

Pullulan/poly(N-vinylimidazole) (PNVI) hybrid cryogels were synthesized under free radical polymerization and chemical crosslinking conditions in an alkaline, aqueous solution of pullulan (PUL), N-vinylimidazole (NVI), ammonium persulfate and epicholorohydrin (ECH) at −18°C. PUL and PNVI cryogels alone were also synthesized under similar conditions. Optimum cryogel formation conditions were determined by considering product yields and gel fractions of the samples. The products were characterized by elemental analysis, FTIR-ATR spectrometry, Thermal gravimetric analysis, and scanning electron microscopy (SEM) analyses. It has been found that PUL/PNVI hybrid samples bear improved physicochemical properties compared to ECH crosslinked PUL and PNVI samples alone. They act as hydrogels in aqueous medium reaching equilibrium swelling capacity values of the order of 600%. Dried PUL/PNVI cryogels show higher thermal stability than the dried cryogels of the parent polymers and maintain their physical integrity over a prolonged time period. Macroporous morphology was revealed by SEM analysis. Having 54.2 mg/g maximum equilibrium adsorption capacity in 200 ppm methyl orange solution and maintaining 95% of its adsorption capacity at the end of seven consecutive adsorption/desorption cycles, PUL/PNVI cryogel proved to be an efficient and durable dye adsorbent using methyl orange as the model compound in aqueous solution.     

Swelling–deswelling kinetics of ionic poly(acrylamide) hydrogels and cryogels

A series of ionic poly(acrylamide) (PAAm) gels was prepared by free‐radical crosslinking copolymerization of acrylamide and N,N′‐methylenebisacrylamide in aqueous solutions. The gels were prepared both below and above the bulk freezing temperature of the polymerization solvent water, which are called as the cryogels and the hydrogels, respectively. The deswelling behavior of swollen gels in acetone as well as the reswelling behavior of the collapsed gels in water were investigated. It was shown that the cryogels respond against the external stimuli much faster than the hydrogels. The interior morphology of the cryogel networks exhibits a discontinuity and a two‐phase structure, compared to the continuous morphology of the hydrogel networks. Introduction of the ionic units in the network chains further increased the response rate of the cryogels. In contrast to these advantages of cryogels, they exhibit lower swelling capacities than the conventional hydrogels. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 99: 319–325, 2006     

Preparation and characterization of polyacrylamide cryogels produced from a high‐molecular‐weight precursor. I. Influence of the reaction temperature and concentration of the crosslinking agent

A new type of cryogel was prepared through a reaction of high‐molecular‐weight polyacrylamide (viscosity‐average molecular weight ≈ 3 × 10⁶ Da) with glutaraldehyde in a moderately frozen aqueous medium. The influence of the crosslinking agent concentration and temperature of the reaction on the gel fraction yield, swelling characteristics, and morphology of the cryogels was investigated. The dependence of the gel fraction yield on the reaction temperature was bell‐shaped. The recognized regularities of the formation of this new type of polyacrylamide cryogel based on a high‐molecular‐weight precursor were very similar to those observed earlier for polyacrylamide cryogels synthesized through the cryopolymerization of monomeric precursors. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 2007     

Silver hydroxyapatite reinforced poly(vinyl alcohol)—starch cryogel nanocomposites and study of biodegradation,compressive strength and antibacterial activity

In the present work polyvinyl alcohol‐starch/silver hydroxyapatite (PVA‐starch/AgHap) cryogel nanocomposites were prepared by successive freezing‐thawing of a blend of PVA and starch solutions to fabricate a cryogel followed by its reinforcement with silver hydroxyapatite (AgHap). The prepared macroporous cryogel nanocomposites were characterized by Infra‐red spectroscopy (FTIR), environmental scanning electron microscopy (ESEM), and particle size and charge analysis. The amylase induced enzymatic degradation of nanocomposites was studied gravimetrically in phosphate buffer saline (PBS) and effect of various parameters like chemical composition of the nanocomposite, number of freeze‐thaw cycles, and enzyme activity were assessed on the extent of degradation of the nanocomposite. The influence of chemical composition and experimental conditions like the number of freeze thaw cycles was studied on the elastic modulii of the cryogels. The in vitro cytotoxicity and antibacterial activity of nanocomposites was also evaluated against L‐529 fibroblast cells and gram positive and gram negative bacteria, respectively. POLYM. ENG. SCI., 59:254–263, 2019. © 2018 Society of Plastics Engineers     

Study of cryostructuration of polymer systems. XII. Poly(vinyl alcohol) cryogels: Influence of low-molecular electrolytes

The influence of the presence of low-molecular electrolytes in initial solutions of poly(vinyl alcohol) (PVA) on the results of cryotropic gelation of the polymer (gelation caused by the freezing thawing) and on the swelling characteristics of PVA cryogels prepared in a salt-free medium were studied. The reinforcing ability of the electrolytes with respect to the gel strength for both alkaline element cations and simple low-molecular anions has been shown to be in agreement with the positions of these ions in corresponding lyotropic (Hofmeister) series. Namely, the ions (chaotropic ones), which are capable to interfere the H-bonding, disturbed somewhat the cryotropic gel-formation of PVA and facilitated the marked additional swelling of cryogels preliminary prepared in pure water medium, whereas the ions (antichaotropic ones), which are capable to promote the H-bonding, caused the formation of reinforced cryogels and resulted in the shrinking of cryogels prepared without salt additives. More pronounced effects were observed for anions as compared with cations. Some anomalous swelling behavior of PVA cryogel in Cs⁺-containing solutions was supposed to be associated with the formation of weak chelates. In addition to antichaotropic inorganic salts like NaF, rather high salting-out effects in respect to PVA were exhibited by wellsoluble amino acid salts: glycine zwitter-ions, lysine monochlorohydrate, and monosodium aspartate. © 1996 John Wiley & Sons, Inc.     

Swelling characteristics of thermo‐ sensitive poly[(2‐diethylaminoethyl methacrylate)‐co‐(N,N‐dimethylacrylamide)] porous hydrogels

Temperature‐sensitive poly[(2‐diethylaminoethyl methacrylate)‐co‐(N,N‐dimethylacrylamide)] [P(DEAEMA‐co‐DMAAm)] hydrogels with five different DMAAm contents were synthesized with and without the addition of sodium carbonate as porosity generator. The synthesized hydrogels were characterized with dry gel density measurements, scanning electron microscopy observation and the determination of swelling ratio. The influence of the pore‐forming agent and content of DMAAm on swelling ratio and network parameters such as polymer–solvent interaction parameter (χ), average molecular mass between crosslinks (M?_c) and mesh size (ζ) of the cryogels are reported and discussed. The swelling and deswelling rates of the porous hydrogels are much faster than for the same type of hydrogels prepared via conventional methods. At a temperature below the volume phase transition temperature, the macroporous hydrogels also absorbed larger amounts water compared to that of conventional hydrogels and showed obviously higher equilibrated swelling ratios in aqueous medium. In particular, the unique macroporous structure provided numerous water channels for water diffusion in or out of the matrix and, therefore, an improved response rate to the external temperature changes during the deswelling and swelling processes. These properties are attributed to the macroporous and regularly arranged network of the porous hydrogels. Scanning electron micrographs reveal that the macroporous network structure of the hydrogels can be adjusted by applying porosity generation methods during the polymerization reaction. Copyright © 2007 Society of Chemical Industry     

Removal of organic water pollutant using magnetite nanomaterials embedded with ionic copolymers of 2‐acrylamido‐2‐methylpropane sodium sulfonate cryogels

Magnetite cryogel composites as macroporous crosslinked matrices have received wide attention and attract much interest in the water purification and desalination industry. They can be used to produce effective adsorbents with high adsorption rate, capacity and desorption for water pollutants. In this work, the incorporation of magnetite nanoparticles into cryogels by the in situ method is proposed to increase the dispersion of nanoparticles in the gel composites and to produce effective magnetic materials with high adsorption capacities. Ionic sodium‐2‐acrylamido‐2‐methylpropane sulfonate (Na‐AMPS) monomer was selected to prepare cryogels as the homopolymer or copolymers with 2‐hydroxyethyl methacrylate (HEMA) or N‐vinyl pyrrolidone (VP) by the crosslinking polymerization technique in the frozen state. Magnetite nanoparticles were introduced into the cryogel by the in situ co‐precipitation method after introducing iron cations into the cryogel networks. The surface morphologies, crystal structure, magnetite content, thermal stability and magnetic properties were determined for the cryogels and their magnetite composites. The magnetite cryogel composites show significantly enhanced methylene blue dye removal in short times with higher adsorption efficiencies and good regeneration to form an effective adsorbent for water treatment. © 2017 Society of Chemical Industry     

Synthesis and characterization of injectable chitosan cryogel microsphere scaffolds

Treatment of tissue defects involves invasive processes such as implanting the tissue engineered scaffold to the defected area. Injectable scaffolds are increasingly being developed to achieve tissue regeneration in a less invasive manner. In this study, injectable chitosan cryogels in the form of microspheres were synthesized combining the water in oil emulsification method with the crosslinking of microspheres during cryogelation. The effects of polymer ratio, crosslinker concentration, cryogelation temperature, and stirring speed on the resulting cryogels’ chemistry, pore morphology, microsphere size, swelling ratio, degree of crosslinking, and degradation rate were examined for a possible noninvasive tissue engineering application. Microspheres with optimized properties were developed with an average pore and particle size of 5.50?±?0.63 and 220.11?±?25.58?µm at a chitosan ratio of 1%, glutaraldehyde concentration of 3%, reaction temperature of ?16°C, and stirring rate of 1,000?rpm.     

Influence of grafting conditions on the properties of polyacrylamide-based cation-exchange cryogels grafted with 2-acrylamido-2-methyl-1-propanesulfonic acid

The effects of grafting conditions on the properties of a novel cation-exchange cryogel by grafting of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA) onto polyacrylamide-based cryogel (pAAm cryogel) using potassium diperiodatocuprate (Cu(III) solution) as initiator were investigated experimentally. The basic cryogels were saturated with the initiator solution followed by the solution of monomers to get the expected sulfo groups under various grafting conditions, such as concentrations of monomers and initiator, graft reaction time, graft temperature and the volume ratio of Cu(III) to NaOH. The results showed that these factors influenced the liquid dispersion behaviors and the flow resistance as well as the protein binding capacity of the grafted cryogels. Protein binding capacity increased with the increase of concentration of AMPSA and Cu(III), the graft temperature and the graft reaction time in a wide range under the present conditions. Compared with these factors, the volume ratio of Cu(III) to NaOH in the initiator solution in the considered situations had a relatively weaker influence on the cryogel microstructures and protein binding capacity. The maximum binding capacity of lysozyme reached 2.5 mg/mL cryogel in these grafted cryogels.     

HSA immobilized novel polymeric matrix as an alternative sorbent in hemoperfusion columns for bilirubin removal

HSA immobilized poly(2-hydroxyethylmethacrylate), HSA-P[HEMA], cryogel monolith was examined as an alternative sorbent to be used in hemoperfusion columns for bilirubin removal from serum. The cryogel monolith synthesis was performed by free radical polymerization using MBAA as crosslinker, APS and TEMED as redox pair. Cyanogen bromide (CNBr) was used as a matrix-activating agent for the preparation of immobilized cryogels. Control cryogel monolith, P[HEMA], and HSA-P[HEMA] were characterized by swelling test, SEM images, porosity and surface area measurements, and blood compatibility tests. Activation and immobilization processes were optimized. The removal of bilirubin from plasma samples overloaded with bilirubin was performed using P[HEMA] cryogel monoliths containing different amounts of immobilized HSA in continuous mode. Several factors affecting adsorption capacity of the matrix such as incubation time, HSA concentration, bilirubin concentration in plasma and temperature were analysed. The maximum bilirubin removal from plasma was 25.4 mg/g at 37.5 °C. The desorption agent was 0.1 M NaOH and 1.0 M NaCl containing solution. The reusability was tested for 10 consecutive adsorption–desorption cycles. The adsorption isotherm models and kinetics of process were also studied.     

Preparation of dextran cryogels for separation processes of binary dye and pesticide mixtures from aqueous solutions

We present mechanically strong macroporous, squeezable dextran cryogels as a column filling material for the removal and separation of binary organic dye and pesticide mixtures from aquatic medium. Dextran cryogels were prepared from aqueous solutions of dextran of various molecular weights (MWs) in the presence of 20 to 50 mol% divinyl sulfone (DVS) as a cross-linker at −18°C. The cryogels have interconnected irregular pores of 100 μm in sizes, and exhibit 69-84% reversible squeezability without damaging the 3D dextran network. Their total open pore volumes (6.3-10 mL g⁻¹), weight swelling ratios in water (1380%-2200%), and mechanical parameters could easily be adjusted by both DVS mol% and MW of dextran. Dextran cryogel with the highest modulus (3.8 ± 0.5 MPa), compressive stress (8 ± 2 MPa) and plateau stress (0.46 ± 0.04 MPa) was obtained at 50 mol% DVS using dextran with a MW of 15 to 25 kg·mol⁻¹. Dextran cryogels are hydrolysable at pH = 1 and 9 but stable at 7.4 independent on both the degree of cross-linking and MW of dextran. At below 50 mol% DVS, they are blood compatible and possess slight thrombogenic effect with blood clotting index value of 98% ± 5%. They are also capable to separate binary dye and pesticide mixtures from aqueous solutions via ionic interactions.     

Preparation of polyacrylamide-based supermacroporous monolithic cryogel beds under freezing-temperature variation conditions

Continuous supermacroporous monolithic cryogel is a novel sponge-like chromatographic adsorbent for bioseparation in downstream process and it is always prepared under a constant freezing-temperature condition. In this work, polyacrylamide-based supermacroporous monolithic cryogel beds in different inner diameter glass columns (I.D. 10, 16 and 26 mm) have been prepared by radical cryogenic copolymerization process under the condition of freezing-temperature variation. The matrix microstructure morphologies of these cryogels were analyzed by scanning electron microscopy (SEM) and the axial liquid dispersion characteristics in these cryogel beds were also analyzed by measuring the residence time distributions (RTDs). The formation of cryogel bed under the present situation has been considered as a coinstantaneous process of solvent crystal growth and polymeric monomer copolymerization. Effects of freezing-temperature variation route, cooling rate and redox initiator concentration on the cryogel microstructure and the liquid dispersion characteristics were investigated experimentally to reveal the cooperative mechanisms of these two processes on the formation of cryogel bed. The results showed that the cryogel pore-structures and the axial liquid dispersion coefficients in the cryogel beds depended strongly on the cooling condition, which controls the solvent crystallization process, and the redox initiator concentration, which influences the monomer polymerization.     

Adsorption behaviours of lysozyme onto poly-hydroxyethyl methacrylate cryogels containing methacryloyl antipyrine-Ce(III)

In this study, Ce³⁺-based cryogel with methacryloyl antipyrine (MAAP) and 2-hydroxyethyl methacrylic acid (HEMA) [p(HEMA-MAAP-Ce³⁺] was prepared. MAAP-Ce³⁺ complex was characterized by UV–near infrared and energy-dispersive X-ray spectroscopy. Cryogel was characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and swelling test. Pore size of the cryogel was found to be about 30–50?µm. The effects of flow rate, pH, temperature, and initial enzyme concentration have been investigated. Maximum adsorption capacity was found to be 57.84?mg?g^?1 cryogel at pH 6.0. After seven times of adsorption–desorption cycles of same cryogel, it was observed that there is negligible decrease in the adsorption capacity.     

Preparation and characterization of spongy cryogels of poly(vinyl alcohol)–casein system: water sorption and blood compatibility study

Polyvinyl alcohol (PVA) and casein are biocompatible and biodegradable macromolecules, which have been widely applied in biomedical fields. In this paper, novel physically crosslinked hydrogels composed of PVA and casein were prepared by repeated freezing–thawing treatment of aqueous solutions of PVA and casein and characterized by IR, differential scanning calorimetry and scanning electron microscopy techniques. The prepared so‐called ‘cryogels’ were evaluated for their water uptake, and the influences of various factors, such as their chemical architecture and the pH and temperature of the swelling bath, were investigated in terms of water sorption. The effect of salt and various simulated biological fluids on the swelling of cryogels was investigated. The in vitro biocompatibility of the prepared cryogels was also judged by methods such as protein (bovine serum albumin) adsorption, blood clot formation and percentage haemolysis measurements. Copyright © 2005 Society of Chemical Industry     

Tough and hierarchical porous cryogel scaffolds preparation using n-butanol as a non-solvent

Hierarchical porous chitosan cryogels were prepared by combination of cryogelation and phase separation techniques. n-Butanol was included as a non-solvent in the initial polymer solutions to enable phase separation. The internal morphology of the resulting cryogels revealed hierarchical porosity where larger pores of between 25–50?µm were formed via cryogelation, whereas smaller pores of between 4–10?µm were produced by phase separation. The control sample prepared without n-butanol exhibited monotype porosity. Any crack development was not observed when samples were compressed up to 80% strain. The results demonstrated hierarchical porous and tough scaffolds with potential use in tissue engineering.     

活性自由基聚合法制备温度敏感聚(N-异丙基丙烯酰胺-co-丙烯酰胺)水凝胶及其性能研究

以水与二甲基甲酰胺作混合溶剂,选用五甲基二乙烯基三胺作ATRP的催化剂配体,实现了ATPR法制备P(NIPAM-co-AM)温敏性智能型水凝胶。通过提高组分中AM的摩尔分率,水凝胶溶胀率增加,其LCST也随之提高,溶胀速率加快。相对于传统自由基聚合法,利用ATRP法制备的P(NIPAM-co-AM)水凝胶具有最低临界温度(LCST)低,溶胀速率快,保水量高等特点。