The use of hydrophilic poly(N,N-dimethylacrylamide) for promoting engulfment of magnetic gamma-Fe2O3 nanoparticles by mammalian cells.

gamma-Fe2O3 nanoparticles obtained by coprecipitation of Fe(II) and Fe(III) chlorides with a base and subsequent oxidation were coated with a shell of hydrophilic biocompatible poly(N,N-dimethylacrylamide) (PDMAAm). Various initiators were attached to the iron oxide surface to enable the use of the "grafting-from" approach for immobilization of PDMAAm. They included 2,2'-azobis(2-methylpropanimidamide) dihydrochloride (AMPA), 2,2'-azobis(N-hydroxy-2-methylpropanimidamide) dihydrochloride (ABHA) and 4-cyano-4-{[1-cyano-3-(N-hydroxycarbamoyl)-1-methylpropyl]azo}pentanoic acid (CCHPA). Engulfment of PDMAAm-coated y-Fe2O3 nanoparticles by murine J774.2 macrophages was investigated. Only some nanoparticles were engulfed by the macrophages. PDMAAm-AMPA-gamma-Fe2O3 and PDMAAm-ABHA-y-Fe2O3 nanoparticles were rapidly engulfed by the cells. In contrast, neat y-Fe2O3 and PDMAAm-CCHPA-gamma-Fe2O3 particles induced formation of transparent vacuoles indicating toxicity of the particles. Thus, PDMAAm-coated AMPA- and ABHA-gamma-Fe2O3 nanoparticles can be recommended as non-toxic labels for mammalian cells.

Pentapeptide-modified poly(N,N-diethylacrylamide) hydrogel scaffolds for tissue engineering.

Poly(N,N-diethylacrylamide) (PDEAAm) hydrogel scaffolds were prepared by radical copolymerization of N,N-diethylacrylamide (DEAAm), N,N'-methylenebisacrylamide and methacrylic acid in the presence of (NH4)2SO4 or NaCl. The hydrogels were characterized by low-vacuum scanning electron microscopy in the water-swollen state, water and cyclohexane regain, and by mercury porosimetry. The pentapeptide, YIGSR-NH2, was immobilized on the hydrogel. Human embryonic stem cells (hESCs) were cultured with the hydrogels to test their biocompatibility. The results suggest that the PDEAAm hydrogel scaffolds are nontoxic and support hESC attachment and proliferation, and that interconnected pores of the scaffolds are important for hESC cultivation. Immobilization of YIGSR-NH2 pentapeptide on the PDEAAm surface improved both adhesion and growth of hESCs compared with the unmodified hydrogel. The YIGSR-NH2-modified PDEAAm hydrogels may be a useful tool for tissue-engineering purposes.

Synthesis of zirconia-immobilized copper chelates for catalytic decomposition of hydrogen peroxide and the oxidation of polycyclic aromatic hydrocarbons.

Chelating sorbents with diethylenetriaminepenta(methylene-phosphonic acid) (DTPMPA) and ethylenediaminetetraacetic acid ligands immobilized on zirconia matrix were prepared and subsequently saturated with Cu(II). All the Cu chelates catalyzed decomposition of H(2)O(2) yielding highly reactive hydroxyl radicals. All of them were also able to catalyze degradation of polycyclic aromatic hydrocarbons (anthracene, benzo[a]pyrene and benzo[b]fluoranthene). The most effective DTPMPA-based catalysts G-32 and G-35 (10 mg ml(-1) with 100 mmol H(2)O(2)) caused almost complete decomposition of 15 ppm anthracene and benzo[a]pyrene during a five day catalytic cycle at 30 degrees C. Anthracene-1,4-dione was the main product of anthracene oxidation by all catalysts. The catalysts were active in several cycles without regeneration.

Study of pepsin phosphorylation using immobilized metal affinity chromatography.

The interactions of pepsin with immobilized trivalent metal ions and the participation of the enzyme phosphate group in this process were investigated using high performance immobilized metal affinity chromatography. Two different sorbents were used: the newly prepared one, consisting of Ga(3+ )chelate of (6-amino-1-hydroxyhexane-1,1-diyl) bis(phosphonic acid) covalently bound to a methacrylate support (BP-Ga(3+)), and the commercial one, containing immobilized Fe(3+ )ions (POROS MC20-Fe(3+)). The comparison of the behavior of porcine pepsin A and its partially dephosphorylated form on both sorbents showed that both forms of pepsin were adsorbed under the same conditions. To eliminate the participation of free carboxyl groups in pepsin adsorption, both enzyme forms were modified by amidation or esterification. Native enzyme and its partially dephosphorylated form both with modified carboxyl groups differed in their interaction with immobilized Ga(3+ )and Fe(3+). Phosphorylated pepsin molecules with esterified carboxyl groups were adsorbed on both sorbents while nonphosphorylated ones with esterified carboxyl groups were not adsorbed.

Preparation and properties of magnetic nano- and microsized particles for biological and environmental separations.

The paper presents a critical overview on magnetic nanoparticles and microspheres used as separation media in different fields of chemistry, biochemistry, biology, and environment protection. The preparation of most widely used magnetic iron oxides in appropriate form, their coating or encapsulation in polymer microspheres, and functionalization is discussed in the first part. In the second part, new developments in the main application areas of magnetic composite particles for separation and catalytical purposes are briefly described. They cover separations and isolations of toxic inorganic and organic ions, proteins, and other biopolymers, cells, and microorganisms. Only selected number of relevant papers could be included due to the restricted extent of the review.

Methacrylate-based chromatographic media.

This review summarizes the preparation and application of chromatographic separation media based on methacrylate monomers with a major focus on highly crosslinked macroporous beads prepared from 2-hydroxyethyl methacrylate and glycidyl methacrylate, respectively. The effects of process variables such as composition of the polymerization mixture that includes monomers, porogenic solvents, and free radical initiator, suspension stabilizer, reaction temperature, and stirring are detailed for both classical and templated suspension polymerization. In addition, specific features of the preparation of monodisperse beads are also discussed. The performance of methacrylate-based separation media is demonstrated on numerous separations in a variety of chromatographic modes.

Affinity chromatography of porcine pepsin A using quinolin-8-ol as ligand.

Stationary phase containing quinolin-8-ol immobilized on macroporous methacrylate support for the affinity chromatography of porcine pepsin A is described. Optimized chromatographic conditions for separation of porcine pepsin A on this stationary phase were found investigating the influence of pH, concentration, ionic strength and chemical composition of the used mobile phases. The stationary phase shows a good reproducibility of chromatographic analyses (relative standard deviation, +/-2%), a high recovery (ca. 93%) and a satisfactory capacity (13 mg pepsin A/1 mL stationary phase) for porcine pepsin A. The obtained findings confirm the applicability of affinity chromatography on the stationary phase with immobilized quinolin-8-ol to the isolation and determination of porcine pepsin A.

Ferrite supports for isolation of DNA from complex samples and polymerase chain reaction amplification.

The influence of cobalt ferrite particles, with non-modified or modified surface, on the course of polymerase chain reaction (PCR) was investigated. DNA isolated from bacterial cells of Bifidobacterium bifidum was used in PCR evaluation of magnetic microspheres. The presence of cobalt ferrite particles inhibits PCR amplification. The effect is not dependent on the functional groups of the modifying reagents used (none, amino, carboxyl). Amplification was improved after the magnetic separation of magnetic particles. Proposed indirect method enabled verification of the suitability of designed particles for their application in PCR assays. Magnetic particles coated with alginic acid under high PEG and sodium chloride concentration were used for the isolation of PCR-ready bacterial DNA from various dairy products. DNA was isolated from crude bacterial cell lysates without phenol extraction of samples. Bifidobacterium and Lactobacillus DNAs were identified in dairy products using PCR.

Isolation of genomic DNA using magnetic cobalt ferrite and silica particles.

Adsorption separation techniques as an alternative to laborious traditional methods (e.g., based on phenol extraction procedure) have been applied for DNA purification. In this work we used two types of particles: silica and cobalt ferrite (unmodified or modified with a reagent containing weakly basic aminoethyl groups, aminophenyl groups, or alginic acid). DNA from chicken erythrocytes and DNA isolated from bacteria Lactococcus lactis were used for testing of adsorption/desorption properties of particles. The cobalt ferrite particles modified with different reagents were used for isolation of PCR-ready bacterial DNA from different dairy products.

Cleavage of double stranded plasmid DNA by lanthanide complexes.

The use of free lanthanide ions and their complexes for plasmid DNA pBR322 and chromosomal DNA cleavage was studied. Plasmid pBR322 DNA was treated by lanthanide chlorides (Eu(3+), La(3+), Nd(3+), Pr(3+), Gd(3+)) in HEPES buffer (pH 7.0, 7.5 and 8.0) at 24, 37, 50, 63, and 76 degrees C. The formation of linear and nicked plasmid forms was investigated depending on the reaction conditions. Heterogeneous lanthanide complexes of ethylenediamine tetraacetic acid (EDTA) immobilized on insoluble methacrylate support and iminodiacetic acid (IDA) immobilized on styrene support were used as catalysts plasmid for DNA pBR322 cleavage, too. The temperature of reaction mixture had substantial influence on cleavage rate. The precipitation of DNA occurred during the measurement of interactions between chromosomal DNA and La(3+) ions.

Chromatographic behaviour and purification of linear lambda phage and plasmid DNA molecules on 2-hydroxyethyl methacrylate-ethylene dimethacrylate-based supports.

The HEMA-BIO 1000 support, which is based on a copolymer of 2-hydroxyethyl methacrylate and ethylene dimethacrylate, was used for separation of lambda DNA and its fragments and plasmid pBR322 DNA. The separation of fragments greater than 6.6 kbp was demonstrated according to the slalom chromatography mechanism on column for size-exclusion chromatography in the case of linear lambda DNA fragments. The influence of particle size of column packing, mobile phase rate, and KCl concentration in mobile phase is discussed. The purification of plasmid DNA pBR322 using size-exclusion chromatography was more rapid compared to gel electrophoresis. The presence of salts in the eluate is not disadvantageous. DNA can be recovered from the eluate by ethanol precipitation. Plasmid DNA pBR322 isolated in this way was suitable for different biological applications (cleavage with restrictases, electrotransformation into bacterial cells).

Characterization of deoxyribonuclease I immobilized on magnetic hydrophilic polymer particles.

Magnetic bead cellulose particles and magnetic poly(HEMA-co-EDMA) microspheres with immobilized DNase I were used for degradation of chromosomal and plasmid DNAs. Magnetic bead particles were prepared from viscose and magnetite powder. Magnetic poly(HEMA-co-EDMA) microspheres were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite. Divalent cations (Mg(2+), Ca(2+), Mn(2+) and Co(2+)) were used for the activation of DNase I. A comparison of free and immobilized enzyme (magnetic bead particles) activities was carried out in dependence on pH and activating cation. The maximum of the activity of immobilized DNase I was shifted to lower pH compared with free DNase I. DNase I immobilized on magnetic bead cellulose was used 20 times in the degradation of chromosomal DNA. Its residual activity was influenced by the nature of activating divalent cation. The immobilized enzyme with decreased activity was reactivated by Co(2+) ions.