Synthesis and characterization of multifunctional poly(glycidyl methacrylate) microspheres and their use in cell separation

Multifunctional poly(glycidyl methacrylate) (PGMA) microspheres containing magnetic, fluorescent, and cancer cell-specific moieties were prepared in four steps: (i) preparation of parent PGMA microspheres by dispersion polymerization and their reaction with ethylenediamine to obtain amino groups, (ii) precipitation of iron ions (Fe²⁺ and Fe³⁺) to form Fe₃O₄ nanoparticles within the microspheres, (iii) consecutive reactions of folic acid with the amino groups on PGMA, and (iv) incorporation of fluorescein isothiocyanate into the microspheres. The microspheres were superparamagnetic, highly monodispersive, intensively fluorescent, and capable of recognizing and binding cancer cells that overexpress folic acid receptors. It was demonstrated that with these microspheres, HeLa cells could be captured from their suspension and easily moved in the direction of the externally applied magnetic field.     

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.     

Fabrication and characterization of tosyl‐activated magnetic and nonmagnetic monodisperse microspheres for use in microfluic‐based ferritin immunoassay

This article describes the preparation of tosyl‐activated nonmagnetic poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) [P(HEMA‐GMA)] microspheres by dispersion polymerization and tosyl‐activated magnetic poly(2‐hydroxyethyl methacrylate‐co‐ethylene dimethacrylate) [P(HEMA‐EDMA)] microspheres by multistep swelling polymerization method and precipitation of iron oxide inside the pores. These new approaches show that monodisperse microspheres, 2.3 µm, respectively 4.1 µm, in diameter can be produced in high yields avoiding aggregation and with the advantage of being free of aromatic moieties. To demonstrate their potential for diagnostic applications, both types of microparticles have been coated with capture and detection antibodies (DAs), respectively. Immunoassay protocols have then been developed for the dosage of ferritin using an automated affinity platform combining microchannel chips and electrochemical detection. The assay performance using the above magnetic microspheres has been compared with that obtained with commercial tosyl‐activated beads. Finally, the possibility to combine functionalized magnetic and nonmagnetic microspheres has been evaluated in view of amplifying the number of enzymatic labels in the immuno‐complex. At a ferritin concentration of 119.6 ng/mL, a signal‐to‐noise ratio of 150.5 is obtained using 0.2 mg/mL of anti‐ferritin‐coated P(HEMA‐GMA)‐DA microspheres against a value of 158.8 using free DA in solution. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 532–542, 2013     

Novel magnetic microspheres of P (GMA-b-HEMA): preparation, lipase immobilization and enzymatic activity in two phases

Magnetic oleic-acid-coated Fe?O? nanoparticles were first introduced into 1, 1-diphenylethylene (DPE)-controlled radical polymerization system to prepare superparamagnetic microspheres for enzyme immobilization by two steps of polymerization. In the presence of DPE, glycidyl methacrylate, 2-hydroxyethyl methacrylate and methacryloxyethyl trimethyl ammonium chloride with charge were selected as copolymering monomers based on their reactive functional group and excellent biocompatibility which were suitable for immobilization of Candida rugosa lipase (CRL). The resulting magnetic microspheres were characterized by means of scanning electron microscope, Fourier transform infrared spectrum, thermogravimetric analysis and vibrating sample magnetometry. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE analysis was also conducted to demonstrate whether CRL is covalently immobilized or only physically adsorbed. The results indicated that the polymerization was successfully carried out, and lipase was immobilized on the magnetic microspheres through ionic adsorption and covalent binding under mild conditions. The immobilized lipase exhibited high activity recovery (69.7%), better resistance to pH and temperature inactivation in aqueous phase, as well as superior reusability in nonaqueous phase. The data showed that the resulting carrier could hold an amphiphilic property.     

Recyclable antibacterial magnetic nanoparticles grafted with quaternized poly(2-(dimethylamino)ethyl methacrylate) brushes

Highly efficient recyclable antibacterial magnetite nanoparticles consisting of a magnetic Fe(3)O(4) core with an antibacterial poly(quaternary ammonium) (PQA) coating were prepared in an efficient four-step process. The synthetic pathway included: (1) preparation of Fe(3)O(4) nanoparticles via coprecipitation of Fe(2+)/Fe(3+) in the presence of an alkaline solution; (2) attachment of an ATRP initiating functionality to the surface of the nanoparticles; (3) surface-initiated atom transfer radical polymerization (ATRP) of 2-(dimethylamino)ethyl methacrylate (DMAEMA); and (4) transformation of PDMAEMA brushes to PQA via quaternization with ethyl bromide. The success of the surface functionalization was confirmed by FT-IR, thermal gravimetric analysis (TGA), elemental analysis, and transmission electron microscopy (TEM). The PQA-modified magnetite nanoparticles were dispersed in water and exhibited a response to an external magnetic field, making the nanoparticles easy to remove from water after antibacterial tests. The PQA-modified magnetite nanoparticles retained 100% biocidal efficiency against E. coli (10(5) to 10(6)E. coli/mg nanoparticles) during eight exposure/collect/recycle procedures without washing with any solvents or water.     

Process and formulation variables in the preparation of injectable and biodegradable magnetic microspheres

The aim of this study was to prepare biodegradable sustained release magnetite microspheres sized between 1 to 2 μm. The microspheres with or without magnetic materials were prepared by a W/O/W double emulsion solvent evaporation technique using poly(lactide-co-glycolide) (PLGA) as the biodegradable matrix forming polymer. Effects of manufacturing and formulation variables on particle size were investigated with non-magnetic microspheres. Microsphere size could be controlled by modification of homogenization speed, PLGA concentration in the oil phase, oil phase volume, solvent composition, and polyvinyl alcohol (PVA) concentration in the outer water phase. Most influential were the agitation velocity and all parameters that influence the kinematic viscosity of oil and outer water phase, specifically the type and concentration of the oil phase. The magnetic component yielding homogeneous magnetic microspheres consisted of magnetite nanoparticles of 8 nm diameter stabilized with a polyethylene glycole/polyacrylic acid (PEG/PAA) coating and a saturation magnetization of 47.8 emu/g. Non-magnetic and magnetic microspheres had very similar size, morphology, and size distribution, as shown by scanning electron microscopy. The optimized conditions yielded microspheres with 13.7 weight% of magnetite and an average diameter of 1.37 μm. Such biodegradable magnetic microspheres seem appropriate for vascular administration followed by magnetic drug targeting.     

Preparation of magnetic microspheres from water-in-oil emulsion stabilized by block copolymer dispersant

A new method for the preparation of magnetic microspheres is reported. The preparation involved first the dispersion of an aqueous phase, containing magnetite nanoparticles and a water-soluble homopolymer, into droplets in an organic medium using an amphiphilic block copolymer as the dispersant. This was followed by water distillation at a raised temperature from the aqueous droplets to yield polymer/magnetite particles. The structure of the particles was then locked in by a reagent being added to cross-link the water-soluble copolymer block and homopolymer. Since the hydrophobic block of the copolymer consisted of a protected polyester, the removal of the protective moieties from the coronal chains yielded poly(acrylic acid) or other functional polymers to render water dispersibility to the spheres and to enable biomolecule immobilization.     

Synthesis of novel porous magnetic silica microspheres as adsorbents for isolation of genomic DNA

An improved procedure is described for preparation of novel mesoporous microspheres consisting of magnetic nanoparticles homogeneously dispersed in a silica matrix. The method is based on a three-step process, involving (i) formation of hematite/silica composite microspheres by urea-formaldehyde polymerization, (ii) calcination of the composite particles to remove the organic constituents, and (iii) in situ transformation of the iron oxide in the composites by hydrogen reductive reaction. The as-synthesized magnetite/silica composite microspheres were nearly monodisperse, mesoporous, and magnetizable, with as typical values an average diameter of 3.5 microm, a surface area of 250 m(2)/g, a pore size of 6.03 nm, and a saturation magnetization of 9.82 emu/g. These magnetic particles were tested as adsorbents for isolation of genomic DNA from Saccharomyces cerevisiae cells and maize kernels. The results are quite encouraging as the magnetic particle based protocols lead to the extraction of genomic DNA with satisfactory integrity, yield, and purity. Being hydrophilic in nature, the porous magnetic silica microspheres are considered a good alternative to polystyrene-based magnetic particles for use in biomedical applications where nonspecific adsorption of biomolecules is to be minimized.     

Properties of RNase A immobilized on magnetic Poly(2-hydroxyethyl methacrylate) microspheres

Magnetic hydrogel microspheres 1.5 microm in size were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite, which formed the core of the particles. RNase A was coupled to the particles by the cyanuric chloride method. Gel electrophoresis of plasmid DNA pUC 19 (contaminated by bacterial RNA) confirmed RNA degradation with the immobilized enzyme. The effect of temperature and pH on the relative activity of immobilized RNase A was estimated after incubation of the samples at different temperatures (30-80 degrees C) and pH (4.0-8.0). Maximum relative activity was observed at 70 degrees C and pH 6.5. The matrices based on magnetic poly(HEMA) had a low tendency to adsorb RNA.     

One-pot synthesis of pegylated ultrasmall iron-oxide nanoparticles and their in vivo evaluation as magnetic resonance imaging contrast agents

A well-defined copolymer poly(oligo(ethylene glycol) methacrylate-co-methacrylic acid) P(OEGMA-co-MAA) was studied as a novel water-soluble biocompatible coating for superparamagnetic iron oxide nanoparticles. This copolymer was prepared via a two-step procedure: a well-defined precursor poly(oligo(ethylene glycol) methacrylate-co-tert-butyl methacrylate), P(OEGMA-co-tBMA) (M(n) = 17300 g mol(-1); M(w)/M(n) = 1.22), was first synthesized by atom-transfer radical polymerization in the presence of the catalyst system copper(I) chloride/2,2'-bipyridyl and subsequently selectively hydrolyzed in acidic conditions. The resulting P(OEGMA-co-MAA) was directly utilized as a polymeric stabilizer in the nanoparticle synthesis. Four batches of ultrasmall PEGylated magnetite nanoparticles (i.e., with an average diameter below 30 nm) were prepared via aqueous coprecipitation of iron salts in the presence of variable amounts of P(OEGMA-co-MAA). The diameter of the nanoparticles could be easily tuned in the range 10-25 nm by varying the initial copolymer concentration. Moreover, the formed PEGylated ferrofluids exhibited a long-term colloidal stability in physiological buffer and could therefore be studied in vivo by magnetic resonance (MR) imaging. Intravenous injection into rats showed no detectable signal in the liver within the first 2 h. Maximum liver accumulation was found after 6 h, suggesting a prolongated circulation of the nanoparticles in the bloodstream as compared to conventional MR imaging contrast agents.     

Development and application of thermo-sensitive magnetic immunomicrospheres for antibody purification

Ultrafine magnetite particles were prepared by a co-precipitation method. The poly-(styrene/N-isopropylacrylamide/methacrylic acid) latex particles containing ultrafine magnetite [magnetic P(St/NIPAM/MAA)] were prepared by two-step emulsifier-free emulsion polymerization. The minimum NaCl concentration for flocculation of these magnetic latex particles (critical flocculation concentration, CFC) decreased with increasing temperature. These temperature dependence of CFC, namely its thermo-sensitivity, originated from NIPAM. At a certain NaCl concentration, some of the magnetic latex particles showed reversible transition between flocculation and dispersion by controlling the temperature, and the thermo-flocculated magnetic latex particles were separated quickly in a magnetic field. Bovine serum albumin (BSA) was covalently immobilized onto the magnetic P(St/NIPAM/MAA) latex particles with high efficiency by the carbodiimide method. These thermo-sensitive magnetic immunomicrospheres were effective for the immunoaffinity purification of anti-BSA antibodies from antiserum.Correspondence to: A. Kondo     

Novel, fluorescent, magnetic, polysaccharide-based microsphere for orientation, tracing, and anticoagulation: preparation and characterization

A fluorescent, magnetic composite poly(styrene-maleic anhydride) microsphere, suitable for conjugation with polysaccharide, was synthesized using magnetite/europium phthalate particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite/europium phthalate particles were wrapped up by poly(ethylene glycol), which improved the affinity between the seed particles and the monomers. The composite microspheres obtained, with a diameter of 0.15-0.7 microm, contain 586-1013 microg of magnetite/g of microsphere and 0.5-16 mmol surface anhydride groups/g of microsphere. Heparin was conjugated with the reactive surface anhydride groups on the surface of the microspheres by covalent binding to obtain a fluorescent, magnetic, polysaccharide-based microsphere. The microspheres not only retain their bioactivities but also provide magnetic susceptibility and fluorescence. They can be used as a carrier with magnetic orientation and fluorescence tracer for potent drug targeting. The orientation, tracer, and anticoagulation of the fluorescence, magnetic, polysaccharide-based microspheres were studied. The anticoagulant activity of the microspheres and heparin binding capacity reached 54,212.8 U and 607.1 mg/g of dry microspheres. The activity recovery was 50.2%. The anticoagulant activity of the microspheres increases with the increase of the conjugated heparin on the surface of the microspheres and the decrease of the microsphere size. Furthermore, The fluorescent, magnetic, polysaccharide-based microspheres can be easily transported to a given position in a magnetic field and traced via their fluorescence.     

Facile synthesis of C8-functionalized magnetic silica microspheres for enrichment of low-concentration peptides for direct MALDI-TOF MS analysis

In this study, novel C8-functionalized magnetic polymer microspheres were prepared by coating single submicron-sized magnetite particle with silica and subsequent modification with chloro (dimethyl) octylsilane. The resulting C8-functionalized magnetic silica (C8-f-M-S) microspheres exhibit well-defined magnetite-core-silica-shell structure and possess high content of magnetite, which endow them with high dispersibility and strong magnetic response. With their magnetic property, the synthesized C8-f-M-S microspheres provide a convenient and efficient way for enrichment of low-abundance peptides from tryptic protein digest and human serum. The enriched peptides/proteins were subjected for MALDI-TOF MS analysis and the enrichment efficiency was documented. In a word, the facile synthesis and efficient enrichment process of the novel C8-f-M-S microspheres make them promising candidates for isolation of peptides even in complex biological samples such as serum, plasma, and urine.     

Cellular response to magnetic nanoparticles "PEGylated" via surface-initiated atom transfer radical polymerization

A new method to PEGylate magnetic nanoparticles with a dense layer of poly(poly(ethylene glycol) monomethacrylate) (P(PEGMA)) by surface-initiated atom transfer radical polymerization (ATRP) is reported. In this approach, an initiator for ATRP was first immobilized onto the magnetic nanoparticle surface, and then P(PEGMA) was grafted onto the surface of magnetic nanoparticle via copper-mediated ATRP. The modified nanoparticles were subjected to detailed characterization using FTIR, XPS, and TGA. The P(PEGMA)-immobilized nanoparticles dispersed well in aqueous media. The saturation magnetization values of the P(PEGMA)-immobilized nanoparticles were 19 emu/g and 11 emu/g after 2 and 4 h polymerization respectively, compared to 52 emu/g for the pristine magnetic nanoparticles. The response of macrophage cells to pristine and P(PEGMA)-immobilized nanoparticles was compared. The results showed that the macrophage cells are very effective in cleaning up the pristine magnetic nanoparticles. With the P(PEGMA)-immobilized nanoparticles, the amount of nanoparticles internalized into the cells is greatly reduced to <2 pg/cell over a 5 day period. With this amount of nanoparticles uptake, no significant cytotoxicity effects were observed.     

Latex particles with thermo-flocculation and magnetic properties for immobilization of alpha-chymotrypsin

Core-shell-type latex particles composed of styrene, N-isopropylacrylamide (NIPAAm), and N-acryloxysuccinimide (NAS) were synthesized by surfactant-free emulsion polymerization. The latex particles show thermo-flocculation behavior due to the presence of temperature-sensitive monomer NIPAAm and could be used for immobilization of alpha-chymotrypsin through covalent bonding with the reactive ester groups of NAS. Enzyme recycle could be accomplished in this immobilized enzyme system by sedimentation of the thermo-flocculated latex particles in 20 min at 30 degrees C by raising the salt (NaCl) concentration to 0.5 M. To further enhance the sedimentation rate, ultrafine magnetite particles were prepared and included during polymerization to produce magnetic temperature-sensitive latex particles (MTLP), which could be recovered 6 times faster after thermo-flocculation by applying a low magnetic field. However, a higher salt concentration was necessary to flocculate the MTLP under the same condition as a result of its increased surface hydrophilicity, which originates from different polymerization conditions and the incorporation of magnetite. The immobilized enzyme shows high activity even against macromolecular substrates (hemoglobin and casein) owing to limited diffusion resistance, with full activity retention for nonmagnetic latex but one-half reduction in activity if the magnetic property was introduced. Optimal enzyme immobilization pH and enzyme loading were determined, and properties of the immobilized enzyme were characterized. The immobilized enzyme was used in 10 repeated batch hydrolyses of casein with successive flocculation/dispersion cycles and showed less than 15% activity decrease at the end. Overall, introducing the magnetic property to the latex could effectively enhance the solid-liquid separation rate after thermo-flocculation and maintain enzyme activity after repeated use but adversely influence the activity of the immobilized enzyme.     

Preparation of porous hollow Fe3O4/P(GMA–DVB–St) microspheres and application for lipase immobilization

Bioprocess and Biosystems Engineering - Functional porous hollow microspheres with superparamagnetism, Fe3O4/P(GMA–DVB–St) microspheres, were prepared via a dispersion polymerization...     

Reversible immobilization of glucoamylase onto magnetic polystyrene beads with multifunctional groups

A novel and simple process for the surface functionalization of micron-sized monodisperse magnetic polystyrene (PS) microbeads was reported. The polystyrene seed particles were prepared prior to the dispersion polymerization method. Afterwards, series of surface chemical modifications on polystyrene microspheres were conducted, and three end-functional microspheres with carboxyl, imidazolyl and sulphydryl groups were obtained. The functional magnetic polystyrene microspheres were prepared by impregnation and subsequent precipitation of ferric and ferrous ions into the polystyrene particles. Finally, the functional magnetic polystyrene was used for the reversible immobilization of glucoamylase via metal-affinity adsorption. The results indicated that the obtained immobilized glucoamylase presented excellent reusability, applicability, magnetic response and regeneration of supports. The magnetic PS microspheres retained >65% of its initial activity at 65 °C over 6 h; and the lowest residual activity of immobilized glucoamylase prepared by regenerated supports still remained about 50% of the initial activity after the 10th cycles.     

Fabrication of novel magnetic nanoparticles-coated P(styrene-itaconic acid-divinylbenzene) microspheres

In this paper, P(styrene-itaconic acid-divinylbenzene) microspheres (P(St-IA-DVB) microspheres) based on styrene (St), itaconic acid (IA) and divinylbenzene (DVB) were prepared via water-in-oil emulsions method (W/O) in the presence of emulsifiers with the size of 5-10 μm. The magnetic nanoparticles (i.e. Fe₃O₄) coated tightly on the surface of P(St-IA-DVB) microspheres were prepared in water with a continuous stirring. The morphology of blank microspheres and magnetic nanoparticles-coated microspheres was investigated in this work. In vitro drug release behavior was studied using doxorubicin as a model drug, and these magnetic nanoparticles-coated P(St-IA-DVB) (MNPSID) microspheres might have great potential application in magnetically targeted and thermal therapy.     

Affinity selection of target cells from cell surface displayed libraries: a novel procedure using thermo-responsive magnetic nanoparticles

Biotinylated thermo-responsive magnetic nanoparticles for use in affinity selection from yeast cell surface display libraries were prepared by coating magnetite nanoparticles with a thermo-responsive polymer consisting of N-isopropyl acrylamide and a biotin derivative. These particles showed a reversible transition between flocculation and dispersion at around the lower critical solution temperature of 30 degrees C, above which the flocculated particles--which absorbed a large amount of avidin due to their large surface area--were quickly separable by magnet. The model library was constructed by mixing control yeast cells with target yeast cells co-displaying IgG binding protein (ZZ) and enhanced green fluorescence protein. Biotinylated IgG and avidin were subsequently added to the model library, and target cells were efficiently enriched with the biotinylated magnetic nanoparticles by avidin-biotin sandwich and ZZ-IgG interaction. The few target cells (0.001%) in the model library were enriched by up to 100% in only 5 days by an affinity selection procedure repeated four times. This novel method based on magnetic nanoparticles and a yeast cell surface display system could fulfill a wide range of applications in the analysis of protein-protein interactions and rapid isolation of novel biomolecules.     

Microbial production and characterization of superparamagnetic magnetite nanoparticles by Shewanella sp. HN-41

A facultative dissimilatory metal-reducing bacterium, Shewanella sp. strain HN-41, was used to produce magnetite nanoparticles from a precursor, poorly crystalline ironoxyhydroxide akaganeite (beta-FeOOH), by reducing Fe(III). The diameter of the biogenic magnetite nanoparticles ranged from 26 nm to 38 nm, characterized by dynamic light scattering spectrophotometry. The magnetite nanoparticles consisted of mostly uniformly shaped spheres, which were identified by electron microscopy. The magnetometry revealed the superparamagnetic property of the magnetic nanoparticles. The atomic structure of the biogenic magnetite, which was determined by extended X-ray absorption fine structure spectroscopic analysis, showed similar atomic structural parameters, such as atomic distances and coordinations, to typical magnetite mineral.