Igor Yu. Galaev

The potential of polymeric cryogels in bioseparation

This is a review discussing the production and properties of cryogels (from the Greek κριoσ (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.

Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns

Continuous supermacroporous chromatographic columns with anion-exchange ligands [2-(dimethylamino)ethyl group] and immobilized metal affinity (IMA) ligands (Cu2+-loaded iminodiacetic acid) have been developed allowing binding of Escherichia coli cells and the elution of bound cells with high recoveries. These poly(acrylamide)-based continuous supermacroporous columns have been produced by radical co-polymerization of monomers in aqueous solution frozen inside a column (cryo-polymerization). After thawing, the column contains a continuous matrix (so-called cryogel) with interconnected pores of 10-100 microm in size. The large pore size of the matrix makes it possible for E. coli cells to pass unhindered through a plain column containing no ligands. E. coli cells bound to an ion-exchange column at low ionic strength were eluted with 70-80% recovery at NaCl concentrations of 0.35-0.40 M, while cells bound to an IMA-column were eluted with around 80% recovery using either 10 mM imidazole or 20 mM EDTA solutions, respectively. The cells maintain their viability after the binding/elution procedure. These preliminary results indicate that microbial cells can be handled in a chromatographic mode using supermacroporous continuous columns. These columns are easy to manufacture from cheap and readily available starting materials, which make the columns suitable for single-time use.

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.

Cryogel applications in microbiology

There is a great demand for improved technologies with regard to rapid processing of nano- and microparticles. The handling of viruses in addition to microbial and mammalian cells requires the availability of appropriate adsorbents. Recent developments in macroporous gels produced at subzero temperatures (known as cryogels) have demonstrated an efficiency for processing cell and virus suspensions, cell separation and cell culture applications. Their unique combination of properties such as macroporosity, tissue-like elasticity and biocompatibility, physical and chemical stability and ease of preparation, renders these materials interesting candidates for a broad range of potential applications within microbiological research. This review describes current applications of macroporous cryogels in microbiology with a brief discussion of future perspectives.

Cell Chromatography: Separation of Different Microbial Cells Using IMAC Supermacroporous Monolithic Columns

Supermacroporous monolithic columns with Cu2+‐IDA ligands have been successfully used for chromatographic separation of different types of microbial cells. The bed of monolithic matrix is formed by a cryogel of poly(acrylamide) cross‐linked with methylenebis(acrylamide) and has a network of large (10–100 μm) interconnected pores allowing unhindered passage of whole cells through the plain cryogel column containing no ligands. Two model systems have been studied: the mixtures of wild‐type Escherichia coli(w.t. E. coli) and recombinant E. coli cells displaying poly‐His peptides (His‐tagged E. coli) and of w.t. E. coli and Bacillus haloduranscells. Wild‐type E. coli and His‐tagged E. coli were quantitatively captured from the feedstock containing equal amounts of both cell types and recovered by selective elution with imidazole and EDTA, with yields of 80% and 77%, respectively. The peak obtained after EDTA elution was 8‐fold enriched with His‐tagged E. colicells as compared with the peak from imidazole elution, which contained mainly weakly bound w.t. E. colicells. Haloalkalophilic B. haloduranscells had low affinity to the Cu2+‐IDA cryogel column and could be efficiently separated from a mixture with w.t. E. colicells, which were retained and recovered in high yields from the column with imidazole gradient. All the cells maintained their viability after the chromatographic procedure. The results show that chromatography on affinity supermacroporous monolithic columns is a promising approach to efficient separations of individual cell types.

Methods in Cell Separations

Research in the field of cell biology and biomedicine relies on technologies that fractionate cell populations and isolate rare cell types to high purity. A brief overview of methods and commercially available products currently used in cell separations is presented. Cell fractionation by size and density and highly selective affinity-based technologies such as affinity chromatography, fluorescence-activated cell sorting (FACS) and magnetic cell sorting are discussed in terms of throughput, yield, and purity.

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.

Smart polymers : applications in biotechnology and biomedicine

Phase Transition in Smart Polymer Solutions and Light Scattering in Biotechnology and Bioprocessing, S.V. Kazakov Responsive Polymer Brushes: A Theoretical Outlook, O.V. Borisov and E.B. Zhulina Conformational Transitions in Cross-Linked Ionic Gels: Theoretical Background, Recent Developments, and Applications, S. Starodubtsev, V. Vasilevskaya, and A. Khokhlov Thermally Responsive Polymers with Amphiphilic Grafts: Intelligent Polymers by Macromonomer Technique, A. Laukkanen and H. Tenhu Microgels from Smart Polymers, N. Kausar, B.Z. Chowdhry, and M.J. Snowden Protein-Based Smart Polymers, J.C. Rodriguez-Cabello, J. Reguera, S. Prieto, and M. Alonso Imprinting Using Smart Polymers, C. Alvarez-Lorenzo, A. Concheiro, J. Chuang, and A.Yu. Grosberg Smart Hydrogels, K. Jilie and M. Li Macroporous Hydrogels from Smart Polymers, O. Okay Smart Boronate-Containing Copolymers and Gels at Solid-Liquid Interfaces, Cell Membranes, and Tissues, A.E. Ivanov, I. Yu. Galaev, and B. Mattiasson Drug Delivery Using Smart Polymers: Recent Advances, N.A. Peppas Polymeric Carriers for Regional Drug Therapy, A. Mittal, D. Chitkara, N. Kumar, R. Pawar, A. Domb, and B. Corn Smart Polymers in Affinity Precipitation of Proteins, A. Kumar, I. Yu. Galaev, and B. Mattiasson Hydrogels in Microfluidics, J. Moorthy

Conjugation of Penicillin Acylase with the Reactive Copolymer of N‐Isopropylacrylamide: A Step Toward a Thermosensitive Industrial Biocatalyst

Conjugation of penicillin acylase (PA) to poly‐N‐isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10‐fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4‐fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 106 and, presumably, cross‐linked protein‐polymer aggregates of MW > 107. Michaelis constant for 5‐nitro‐3‐phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 μM) was found to be slightly higher than that of the free enzyme (12 μM), and evaluation of Vmax testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM‐cross‐linked PA retained its capacity to synthesize cephalexin from d‐phenylglycin amide and 7‐aminodeacetoxycephalosporanic acid. The synthesis‐hydrolysis ratios of free and polyNIPAM‐cross‐linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of β‐lactam antibiotics, can be successfully eliminated by cross‐linking penicillin acylase to a smart polymer (i.e., polyNIPAM).

Smart polymers for bioseparation and bioprocessing

Temperature and pH Sensitive Graft Copolymers. Synthesis of Novel Smart Polymers for Bioseparation and Bioprocessing. Affinity Precipitation of Proteins using Smart Polymers. Aqueous Two Phase Systems with Smart Polymers. Polycomplexes for Bioseparation and Bioprocessing. Controlled Permeation through Membranes Modified with Smart Polymers. Applications of Smart Hydrogels in Separation. Surfaces Coated with Smart Polymers: Chromatography and Cell Detachment. Smart Latexes for Bioseparation and Bioprocessing. Application of Water-Soluble Polymers and their Complexes for Immunoanalytical Purposes. Enzymes Immobilized in Smart Hydrogels. Stimuli Responsive Polymers in Bioprocessing.