Affinity based and molecularly imprinted cryogels: Applications in biomacromolecule purification.

The publications in macro-molecularly imprinted polymers have increased drastically in recent years with the development of water-based polymer systems. The macroporous structure of cryogels has allowed the use of these materials within different applications, particularly in affinity purification and molecular imprinting based methods. Due to their high selectivity, specificity, efficient mass transfer and good reproducibility, molecularly imprinted cryogels (MICs) have become attractive for researchers in the separation and purification of proteins. In this review, the recent developments in affinity based cryogels and molecularly imprinted cryogels in protein purification are reviewed comprehensively.

Molecularly imprinted cryogels for human serum albumin depletion.

Molecularly imprinted polymers can be used for the selective capture of a target molecule from complex medium. Cryogels novel matrices, which characterized by their supermacropores that makes their use advantageous when studying with biological samples. By combining high selectivity of the molecular imprinting approach with using cryogel as a base polymer, in this protocol, preparation of the albumin-imprinted cryogels is described. This material is a useful candidate for the selective albumin depletion from the human serum sample prior to the detailed proteomic analysis.

Molecularly imprinted poly(hydroxyethyl methacrylate) based cryogel for albumin depletion from human serum.

Macroporous cryogels imprinted with human serum albumin (HSA) have been prepared by copolymerization of 2-hydroxyethyl methacrylate with a functional co-monomer of N-methacryloyl-L-phenylalanine. The cryogels were used for the depletion of HSA from human serum. HSA-imprinted cryogels were prepared with gel fraction yields up to 90%, and their chemical structure, morphology and porosity were characterized by FTIR-spectroscopy, scanning electron microscopy, swelling studies and flow dynamics. Selective binding experiments were performed in the presence of competitive proteins like human transferrin and myoglobin. Albumin-imprinted cryogel column was optimized for fast protein liquid chromatography. Sodium-dodecyl sulfate polyacrylamide gel electrophoresis was used to show the efficiency of albumin depletion.

Gelatin cryogels crosslinked with oxidized dextran and containing freshly formed hydroxyapatite as potential bone tissue-engineering scaffolds.

Gelatin-based cryogels were prepared by using a novel crosslinker, oxidized dextran, which was synthesized and used in the study. The cryogels were also loaded with freshly formed hydroxyapatite (HA) particles. These cryogels are opaque, spongy and highly elastic and have a pore structure with large interconnected pores. They swell about 500% in aqueous media and within a few minutes reach their final swollen forms. The elastic moduli of HA-containing cryogels were 18.5 ± 3.0 kPa, which is suitable for non-load-bearing bone tissue-engineering (TE) applications, especially for the craniofacial area.

Enzyme-catalyzed crosslinking in a partly frozen state: a new way to produce supermacroporous protein structures.

In this study a new way to produce supermacroporous protein structures was investigated. Enzyme-mediated crosslinking of gelatin or casein was performed in a partly frozen state, which yielded stable, protein-based cryogels. The reaction kinetics for the formation of cryogels were found to be fairly slow, most likely due to the low temperature (-12 °C) used or due to an increased viscosity owing to the cryo-concentration taking place. The produced cryogels were characterized with regards to their physical properties and in vitro degradation. Furthermore, cryogels produced from gelatin and casein were evaluated as potential scaffolds by fibroblast cultivation to confirm their in vitro biocompatibility. Gelatin- and casein-based scaffolds both supported cell proliferation and migration through the scaffold.

Formation of macroporous self-assembled hydrogels through cryogelation of Fmoc-Phe-Phe.

In this study, it was found that macroporous hydrogels were formed when self-assembly of fluorenyl-9-methoxycarbonyl (Fmoc)-diphenylalanine (Phe-Phe) peptides was induced using glucono-δ-lactone (GdL) in apparently frozen samples. Formed cryogels exhibited a heterogeneous structure with pore walls of densely packed fibres of assembled dipeptides and pores in the range 10-100 µm. Hydrogels formed from the same composition above the freezing point exhibited a homogenous structure without any apparent porosity. The formed gels were characterised using microscopy techniques, CD-spectroscopy and stress sweeps. The cryogels exhibited less mechanical strength than the hydrogels that might be due to the heterogeneous structure of the former. It appeared that the self-assembled peptide both in the cryo- and hydrogel maintained the ß-sheet structure commonly attributed to these.

An improved capillary model for describing the microstructure characteristics, fluid hydrodynamics and breakthrough performance of proteins in cryogel beds.

A capillary-based model modified for characterization of monolithic cryogels is presented with key parameters like the pore size distribution, the tortuosity and the skeleton thickness employed for describing the porous structure characteristics of a cryogel matrix. Laminar flow, liquid dispersion and mass transfer in each capillary are considered and the model is solved numerically by the finite difference method. As examples, two poly(hydroxyethyl methacrylate) (pHEMA) based cryogel beds have been prepared by radical cryo-copolymerization of monomers and used to test the model. The axial dispersion behaviors, the pressure drop vs. flow rate performance as well as the non-adsorption breakthrough curves of different proteins, i.e., lysozyme, bovine serum albumin (BSA) and concanavalin A (Con A), at various flow velocities in the cryogel beds are measured experimentally. The lumped parameters in the model are determined by matching the model prediction with the experimental data. The results showed that for a given cryogel column, by using the model based on the physical properties of the cryogel (i.e., diameter, length, porosity, and permeability) together with the protein breakthrough curves one can obtain a reasonable estimate and detailed characterization of the porous structure properties of cryogel matrix, particularly regarding the number of capillaries, the capillary tortuousness, the pore size distribution and the skeleton thickness. The model is also effective with regards to predicting the flow performance and the non-adsorption breakthrough profiles of proteins at different flow velocities. It is thus expected to be applicable for characterizing the properties of cryogels and predicting the chromatographic performance under a given set of operating conditions.

Apparent heterogeneity in the pIII-peptide fusion protein in single-phage clones isolated from peptide libraries.

Ligand homogeneity is an important issue in affinity chromatography. Using phages expressing peptides on the pIII protein, a heterogeneity in the binding of monoclonal phages was observed during affinity chromatography on supermacroporous cryogels. Fractions with different apparent binding affinities could be separated by stepwise elution. When these different fractions were re-applied, the respective differences in affinity were retained. However, when phage fractions with different apparent affinities were first amplified, an offspring was generated with again variable affinities. As the sequence of the peptide insert was the same, the heterogeneity must be ascribed to differences in avidity and although no direct evidence could be generated, we hypothesize that this is possibly due to phages displaying different numbers of the same peptide as a consequence of either proteolytic or packaging events during the amplification step in Escherichia coli.

Modulating the porosity of cryogels by influencing the nonfrozen liquid phase through the addition of inert solutes.

The freezing of monomeric mixtures is known to concentrate solutes in a nonfrozen phase in the area surrounding the ice crystals. The concentration of such solutes is determined by the freezing temperature. Although salts or solvents do not directly react in the polymerization reaction, they do change the composition and properties of the nonfrozen phase. In this study, we investigated the influence of the addition of various salts and solvents on the structure of macroporous hydrogels formed in a semifrozen state through aqueous free-radical polymerization. The change in composition of the nonfrozen phase was studied using NMR to monitor the freezing of water, and the structural changes of the gels were observed using scanning electron microscopy. It was found that the addition of methanol or acetone caused the formation of reaction-induced phase separation polymerization due to cryoconcentration, which caused a significant increase of methanol or acetone in the nonfrozen phase. This resulted in a material with bimodal pore size distribution with pores of 10-80 µm in diameter caused by cryogelation, and with pores in the polymeric matrix with a diameter of less than 1 µm due to the reaction-induced phase separation. Addition of salts to the monomeric mixture resulted in a structure with only pores of 10-80 µm in diameter due to cryogelation. Increasing the amount of salts added resulted in the formation of thicker pore walls and thus a slight reduction in pore size compared to a sample with no added solute. The possibility of changing the structure and properties of the gels by adding different solutes could open up new applications for these materials, for example, chromatography applications.

Grafting of poly[1-(N,N-bis-carboxymethyl)amino-3-allylglycerol-co-dimethylacrylamide] copolymer onto siliceous support for preconcentration and determination of lead (II) in human plasma and environmental samples.

A method is reported for surface grafting of polymer containing a functional monomer for metal chelating, poly[1-(N,N-bis-carboxymethyl)amino-3-allylglycerol-co-dimethylacrylamide] (poly(AGE/IDA-co-DMAA) onto silica modified by silylation with 3-mercaptopropyltrimethoxysilane. Monomer 1-(N,N-bis-carboxymethyl)amino-3-allylglycerol (AGE/IDA) was synthesized by reaction of allyl glycidyl ether with iminodiacetic acid. The resulting sorbent has been characterized using FT-IR, elemental analysis, thermogravimetric analysis (TGA), FT-Raman and scanning electron microscopy (SEM) and evaluated for the preconcentration and determination of trace Pb(II) in human biological fluid and environmental water samples. The optimum pH value for sorption of the metal ion was 5.5. The sorption capacity of functionalized resin was 15.06 mg g(-1). The chelating sorbent can be reused for 15 cycles of sorption-desorption without any significant change in sorption capacity. A recovery of 96.2% was obtained for the metal ion with 0.5 M nitric acid as eluting agent. The profile of lead uptake by the sorbent reflects good accessibility of the chelating sites in the poly(AGE/IDA-co-DMAA)-grafted silica gel. Scatchard analysis revealed that the homogeneous binding sites were formed in the polymers. The equilibrium adsorption data of Pb(II) by modified resin were analyzed by Langmuir, Freundlich, Temkin and Redlich-Peterson models. On the basis of equilibrium adsorption data the Langmuir, Freundlich and Temkin constants were determined as 0.70, 1.35 and 2.7, respectively at pH 5.5 and 20 degrees C. Isotherms have also been used to obtain the thermodynamic parameters such as free energy, enthalpy and entropy of adsorption.

Gelatin-fibrinogen cryogel dermal matrices for wound repair: preparation, optimisation and in vitro study.

Macroporous sponge-like gelatin-fibrinogen (Gl-Fg) scaffolds cross-linked with different concentrations (0.05-0.5%) of glutaraldehyde (GA) were produced using cryogelation technology, which allows for the preparation of highly porous scaffolds without compromising their mechanical properties, and is a more cost-efficient process than freeze-drying. The produced Gl-Fg-GA(X) scaffolds had a uniform interconnected open porous structure with a porosity of up to 90-92% and a pore size distribution of 10-120 microm. All of the obtained cryogels were elastic and mechanically stable, except for the Gl-Fg-GA(0.05) scaffolds. Swelling kinetics and degradation rate, but not the porous structure of the cryogels, were strongly dependent on the degree of cross-linking. A ten-fold increase in the degree of cross-linking resulted in an almost 80-fold decrease in the rate of degradation in a solution of protease. Cryogels were seeded with primary dermal fibroblasts and the densities observed on the surface, plus the expression levels of collagen types I and III observed 5 days post-seeding, were similar to those observed on a control dermal substitute material, Integra. Fibroblast proliferation and migration within the scaffolds were relative to the GA content. Glucose consumption rate was 3-fold higher on Gl-Fg-GA(0.1) than on Gl-Fg-GA(0.5) cryogels 10 days post-seeding. An enhanced cell motility on cryogels with reducing GA crosslinking was obtained after long time culture. Particularly marked cell infiltration was seen in gels using 0.1% GA as a crosslinker. The scaffold started to disintegrate after 42 days of in vitro culturing. The described in vitro studies demonstrated good potential of Gl-Fg-GA(0.1) scaffolds as matrices for wound healing.

Evaluation of boronate-containing polymer brushes and gels as substrates for carbohydrate-mediated adhesion and cultivation of animal cells.

Boronate-containing thin polyacrylamide gels (B-Gel), polymer brushes (B-Brush) and chemisorbed organosilane layers (B-COSL) were prepared on the surface of glass slides and studied as substrates for carbohydrate-mediated cell adhesion. B-COSL- and B-Brush-modified glass samples exhibited multiple submicron structures densely and irregularly distributed on the glass surface, as found by scanning electron microscopy and atomic force microscopy. B-Gel was ca. 0.1 mm thick and contained pores with effective size of 1-2 microm in the middle and of 5-20 microm on the edges of the gel sample as found by confocal laser scanning microscopy. Evidence for the presence of phenylboronic acid in the samples was given by time-of-flight secondary ion mass-spectrometry (ToF SIMS), contact angle measurements performed in the presence of fructose, and staining with Alizarin Red S dye capable of formation specific, fluorescent complexes with boronic acids. A comparative study of adhesion and cultivation of animal cells on the above substrates was carried out using murine hybridoma M2139 cell line as a model. M2139 cells adhered to the substrates in the culture medium without glucose or sodium pyruvate at pH 8.0, and then were cultivated in the same medium at pH 7.2 for 4 days. It was found that the substrates of B-Brush type were superior both regarding cell adhesion and viability of the adhered cells, among the substrates studied. MTT assay confirmed proliferation of M2139 cells on B-Brush substrates. Some cell adhesion was also registered in the macropores of B-Gel substrate. The effects of surface microstructure of the boronate-containing polymers on cell adhesion are discussed. Transparent glass substrates grafted with boronate-containing copolymers offer good prospects for cell adhesion studies and development of cell-based assays.

Ultrafast Responsive Poly- (N-isopropylacrylamide) Gel Produced by Cryostructuring of Self-crosslinkable Polymer Microgels.

A macroporous material composed of closely aggregated particles was prepared by cryo-structuration of N-isopropylacrylamide-co-N-hydroxymethylacrylamide (NIPA-co-HMAm) particle suspensions. The formed structure was maintained by the formation of covalent bonds through self-crosslinking between the particles while the system was in a semi-frozen state thus avoiding the need to freeze-dry the sample. This resulted in macroporous structure composed of closely aggregated thermoresponsive particles which exhibit an ultrafast temperature response. The response rate can be attributed both to the macroporous structure as well as the fast responsive properties of the individual particles.

Biomimetic macroporous hydrogels: protein ligand distribution and cell response to the ligand architecture in the scaffold.

Macroporous hydrogels (MHs), cryogels, are a new type of biomaterials for tissue engineering that can be produced from any natural or synthetic polymer that forms a gel. Synthetic MHs are rendered bioactive by surface or bulk modifications with extracellular matrix components. In this study, cell response to the architecture of protein ligands, bovine type-I collagen (CG) and human fibrinogen (Fg), immobilised using different methods on poly(2-hydroxyethyl methacrylate) (pHEMA) macroporous hydrogels (MHs) was analysed. Bulk modification was performed by cross-linking cryo-co-polymerisation of HEMA and poly(ethylene glycol)diacrylate (PEGA) in the presence of proteins (CG/pHEMA and Fg/pHEMA MHs). The polymer surface was modified by covalent immobilisation of the proteins to the active epoxy (ep) groups present on pHEMA after hydrogel fabrication (CG-epHEMA and Fg-epHEMA MHs). The concentration of proteins in protein/pHEMA and protein-epHEMA MHs was 80-85 and 130-140 mug/ml hydrogel, respectively. It was demonstrated by immunostaining and confocal laser scanning microscopy that bulk modification resulted in spreading of CG in the polymer matrix and spot-like distribution of Fg. On the contrary, surface modification resulted in spot-like distribution of CG and uniform spreading of Fg, which evenly coated the surface. Proliferation rate of fibroblasts was higher on MHs with even distribution of the ligands, i.e., on Fg-epHEMA and CG/pHEMA. After 30 days of growth, fibroblasts formed several monolayers and deposited extracellular matrix filling the pores of these MHs. The best result in terms of cell proliferation was obtained on Fg-epHEMA. The ligands displayed on surface of these scaffolds were in native conformation, while in bulk-modified CG/pHEMA MHs most of the proteins were buried inside the polymer matrix and were less accessible for interactions with specific antibodies and cells. The method used for MH modification with bioligands strongly affects spatial distribution, density and conformation of the ligand on the scaffold surface, which, in turn, influence cell-surface interactions. The optimal type of modification varies depending on intrinsic properties of proteins and MHs.

Modeling of protein breakthrough performance in cryogel columns by taking into account the overall axial dispersion.

A model considering the overall axial dispersion for describing protein adsorption and breakthrough in monolithic cryogel beds has been developed. The microstructure of cryogels was characterized by tortuous capillaries with a normal diameter distribution but a constant pore wall thickness. The axial dispersion within cryogel columns was described by using the overall axial dispersion coefficient, which can be easily obtained by matching the experimental breakthrough curves without adsorption or measuring residence time distributions (RTDs). Experimental breakthrough curves of lysozyme within a metal-chelated affinity cryogel by Persson et al. (Biotechnol. Bioeng. 2004, 88, 224-236) and a cation-exchange cryogel by Yao et al. (J. Chromatogr. A 2007, 1157, 246-251) were employed as examples to test the model. The results showed that by using the axial dispersion coefficient and assuming uniform radial concentration profile at a given cross-section of the cryogel along the bed height, the model can describe the detailed behaviors of the in-bed overall axial dispersion, the in-pore mass transfer, as well as the protein adsorption and breakthrough. For a known overall axial dispersion coefficient, the lumped parameter of the mass transfer coefficient between the bulk liquid and the capillary wall can be determined by fitting the protein breakthrough curve at a known chromatographic condition. Once this parameter is determined, the model can be used to predict the protein breakthrough profiles under different conditions based on the basic physical parameters of the cryogel bed and the properties of the fluid and protein. The effective capillary diameters employed in the model are close to the actual pore sizes observed from the images by SEM. The model predictions of lysozyme breakthrough profiles at various flow rates are also in good agreement with the experimental data in both the metal-chelated affinity and cation-exchange cryogel columns.

Dried-reswollen immobilized biocatalysts for detoxification of organophosphorous compounds in the flow systems.

New immobilized biocatalysts based on polypeptides containing N- or C-terminal polyhistidine sequences and possessing organophosphorus hydrolase activity were investigated for detoxification of organophosphorous neurotoxic compounds in the flow systems. The biocatalysts were revealed to have a high catalytic activity within wide pH and temperature ranges 7.5-12.5 degrees C and 15-65 degrees C, respectively. The immobilized biocatalysts can be dried and reswollen before use with 92-93% catalytic activity remaining after drying and rehydration procedures. The half-lives of the biocatalysts under wet and dry storage conditions were 420 and 540 days, respectively.

Building macroporous materials from microgels and microbes via one-step cryogelation.

Macroporous materials are prepared from microgels or microbes by one-step chemical cross-linking under semifrozen conditions. This avoids the use of freeze drying of the sample because a chemically stable structure is prepared under semifrozen conditions. Cryostructuration results in a material with pore walls composed of closely packed particles.

Chromato-panning: an efficient new mode of identifying suitable ligands from phage display libraries.

BACKGROUND: Phage Display technology is a well established technique for high throughput screening of affinity ligands. Here we describe a new compact chromato-panning procedure for selection of suitable binders from a phage peptide display library. RESULTS: Both phages and E. coli cells pass non-hindered through the interconnected pores of macroporous gel, so called cryogel. After coupling a ligand to a monolithic cryogel column, the phage library was applied on the column and non-bound phages were washed out. The selection of strong phage-binders was achieved already after the first panning cycle due to the efficient separation of phage-binders from phage-non-binders in chromatographic mode rather than in batch mode as in traditional biopanning procedures. E. coli cells were applied on the column for infection with the specifically bound phages. CONCLUSION: Chromato-panning allows combining several steps of the panning procedure resulting in 4-8 fold decrease of total time needed for phage selection.

Bilirubin recognition via molecularly imprinted supermacroporous cryogels.

Recent years molecular imprinting has received considerable attention as an excellent and simple approach to recognize small molecules and bioactive substances. The aim of this study is to prepare the bilirubin-imprinted supermacroporous cryogels which can be used for the adsorption of bilirubin from human plasma. N-methacryloyl-(L)-tyrosinemethylester (MAT) was chosen as the pre-organization monomer. In the first step, bilirubin was complexed with MAT and the bilirubin-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-tyrosine methylester) [BR-MIP] cryogel was produced by free radical polymerization initiated by N,N,N',N'-tetramethylene diamine (TEMED) and ammonium persulfate (APS) pair in an ice bath. After that, the template molecules (i.e., bilirubin) were removed from the polymeric structure using sodium carbonate and sodium hydroxide. The maximum bilirubin adsorption amount was 3.6 mg/g polymer. The relative selectivity coefficients of the BR-MIP cryogel for bilirubin/cholesterol and bilirubin/testosterone mixtures were 7.3 and 3.2 times greater than non-imprinted poly(HEMA-MAT) [NIP] cryogel, respectively. The BR-MIP cryogel could be used many times without decreasing bilirubin adsorption amount significantly. Therefore, as a reusable carrier possessing high selectivity, BR-MIP cryogel has a potential candidate as a clinical hemoperfusion material.

Boronate-containing polymer brushes: characterization, interaction with saccharides and mammalian cancer cells.

Boronate-containing polymer brushes were synthesized by free radical copolymerization of N,N-dimethylacrylamide (DMAA) and N-acryloyl-m-phenylboronic acid (NAAPBA) (9:1) on the surface of 3-mercaptopropyl-silylated glass plates and capillaries. The brushes were characterized with time-of-flight secondary ion mass-spectrometry (ToF SIMS), atomic force microscopy and contact angle measurements. Fructose caused a well-expressed drop spreading on the surface of copolymer-grafted glass, due to the strong interaction with the boronate groups. Sedimentation of murine hybridoma cells M2139 or human myeloid leukemia cells KG1 onto the DMAA-NAAPBA copolymer-grafted glass plates from 10 mM phosphate buffer solution (pH 8.0) resulted in the cell adhesion. The adhered M2139 and KG1 cells could be quantitatively detached from the grafted plates with 0.1 M fructose, which competed with cell surface carbohydrates for binding to the boronates. Evaluation of the binding strength between M2139 cells and the copolymer brush was performed by exposure of the adhered cells to a shear stress. Detachment of a fraction of 18% of the adhered M2139 cells was obtained at a shear force of 1400-2800 pN/cell generated by the running phosphate buffer (pH 8.0), whereas the remaining adhered cells (70%) could be detached with 0.1 M fructose dissolved in the same buffer. Possible applications of the boronate-containing polymer brushes to affinity cell separation can be based upon the facile recovery of the attached cells.