RESUMO
Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-Fe2O3) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 µm in diameter, contain 0.9 mM · g-1 of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.
Assuntos
Autoanticorpos/química , Autoanticorpos/isolamento & purificação , Doenças Autoimunes/imunologia , Microesferas , Esclerose Múltipla/imunologia , Miosina Tipo I/imunologia , Ácidos Polimetacrílicos/química , Autoanticorpos/sangue , Autoanticorpos/imunologia , Humanos , Imãs/química , Peso Molecular , Miosina Tipo I/químicaRESUMO
Three-dimensional and porous polypyrrole (PPy) aerogels were prepared using a facile two-step procedure in which cryogels were synthesized via the cryopolymerization of pyrrole with iron (III) chloride in the presence of supporting water-soluble polymers (poly(N-vinylpyrrolidone), poly(vinyl alcohol), gelatin, methylcellulose or hydroxypropylcellulose), followed by freeze-drying to obtain aerogels. The choice of supporting polymers was found to affect the morphology, porosity, electrical conductivity, and mechanical properties of PPy aerogels. PPy aerogels were successfully used as adsorbents to remove toxic Cr(VI) ions from aqueous solutions.
RESUMO
Magnetic maghemite (γ-Fe2O3) nanoparticles obtained by a coprecipitation of iron chlorides were dispersed in superporous poly(2-hydroxyethyl methacrylate) scaffolds containing continuous pores prepared by the polymerization of 2-hydroxyethyl methacrylate (HEMA) and ethylene dimethacrylate (EDMA) in the presence of ammonium oxalate porogen. The scaffolds were thoroughly characterized by scanning electron microscopy (SEM), vibrating sample magnetometry, FTIR spectroscopy, and mechanical testing in terms of chemical composition, magnetization, and mechanical properties. While the SEM microscopy confirmed that the hydrogels contained communicating pores with a length of ≤2 mm and thickness of ≤400 µm, the SEM/EDX microanalysis documented the presence of γ-Fe2O3 nanoparticles in the polymer matrix. The saturation magnetization of the magnetic hydrogel reached 2.04 Am2/kg, which corresponded to 3.7 wt.% of maghemite in the scaffold; the shape of the hysteresis loop and coercivity parameters suggested the superparamagnetic nature of the hydrogel. The highest toughness and compressive modulus were observed with γ-Fe2O3-loaded PHEMA hydrogels. Finally, the cell seeding experiments with the human SAOS-2 cell line showed a rather mediocre cell colonization on the PHEMA-based hydrogel scaffolds; however, the incorporation of γ-Fe2O3 nanoparticles into the hydrogel improved the cell adhesion significantly. This could make this composite a promising material for bone tissue engineering.
RESUMO
Research of degradable hydrogel polymeric materials exhibiting high water content and mechanical properties resembling tissues is crucial not only in drug delivery systems but also in tissue engineering, medical devices, and biomedical-healthcare sensors. Therefore, we newly offer development of hydrogels based on poly(2-hydroxyethyl methacrylate-co-2-(acetylthio) ethyl methacrylate-co-2-methacryloyloxyethyl phosphorylcholine) [P(HEMA-ATEMA-MPC)] and optimization of their mechanical and in vitro and in vivo degradability. P(HEMA-ATEMA-MPC) hydrogels differed in chemical composition, degree of crosslinking, and starting molar mass of polymers (15, 19, and 30 kDa). Polymer precursors were synthesized by a reversible addition fragmentation chain transfer (RAFT) polymerization using 2-(acetylthio)ethyl methacrylate containing protected thiol groups, which enabled crosslinking and gel formation. Elastic modulus of hydrogels increased with the degree of crosslinking (Slaughter et al., 2009) [1]. In vitro and in vivo controlled degradation was confirmed using glutathione and subcutaneous implantation of hydrogels in rats, respectively. We proved that the hydrogels with higher degree of crosslinking retarded the degradation. Also, albumin, γ-globulin, and fibrinogen adsorption on P(HEMA-ATEMA-MPC) hydrogel surface was tested, to simulate adsorption in living organism. Rat mesenchymal stromal cell adhesion on hydrogels was improved by the presence of RGDS peptide and laminin on the hydrogels. We found that rat mesenchymal stromal cells proliferated better on laminin-coated hydrogels than on RGDS-modified ones.
Assuntos
Hidrogéis , Células-Tronco Mesenquimais , Animais , Materiais Biocompatíveis/farmacologia , Metacrilatos , Poli-Hidroxietil Metacrilato , Ratos , Engenharia TecidualRESUMO
Heat-treated polyacrylonitrile (HT-PAN), also referred to as black orlon (BO), is a promising carbon-based material used for applications in tissue engineering and regenerative medicine. To the best of our knowledge, no such complex bone morphology-mimicking three-dimensional (3D) BO structure has been reported to date. We report that BO can be easily made into 3D cryogel scaffolds with porous structures, using succinonitrile as a porogen. The cryogels possess a porous morphology, similar to bone tissue. The prepared scaffolds showed strong osteoconductive activity, providing excellent support for the adhesion, proliferation, and mitochondrial activity of human bone-derived cells. This effect was more apparent in scaffolds prepared from a matrix with a higher content of PAN (i.e., 10% rather than 5%). The scaffolds with 10% of PAN also showed enhanced mechanical properties, as revealed by higher compressive modulus and higher compressive strength. Therefore, these scaffolds have a robust potential for use in bone tissue engineering.
Assuntos
Resinas Acrílicas/química , Osso e Ossos , Força Compressiva , Temperatura Alta , Humanos , Porosidade , Engenharia Tecidual , Alicerces TeciduaisRESUMO
The aim of the present study is to develop new magnetic polymer microspheres with functional groups available for easy protein and antibody binding. Monodisperse macroporous poly(2-hydroxyethyl methacrylate) (PHEMA-COOH) microspheres ~4 µm in diameter and containing â¼1 mmol COOH/g were synthesized by multistep swelling polymerization of 2-hydroxyethyl methacrylate (HEMA), ethylene dimethacrylate (EDMA), and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA), which was followed by MCMEMA hydrolysis. The microspheres were rendered magnetic by precipitation of iron oxide inside the pores, which made them easily separable in a magnetic field. Properties of the resulting magnetic poly(2-hydroxyethyl methacrylate) (mgt.PHEMA) particles with COOH functionality were examined by scanning and transmission electron microscopy (SEM and TEM), static volumetric adsorption of helium and nitrogen, mercury porosimetry, Fourier transform infrared (FTIR) and atomic absorption spectroscopy (AAS), and elemental analysis. Mgt.PHEMA microspheres were coupled with p46/Myo1C protein purified from blood serum of multiple sclerosis (MS) patients, which enabled easy isolation of monospecific anti-p46/Myo1C immunoglobulin G (IgG) antibodies from crude antibody preparations of mouse blood serum. High efficiency of this approach was confirmed by SDS/PAGE, Western blot, and dot blot analyses. The newly developed mgt.PHEMA microspheres conjugated with a potential disease biomarker, p46/Myo1C protein, are thus a promising tool for affinity purification of antibodies, which can improve diagnosis and treatment of MS patients.
Assuntos
Proteínas Imobilizadas/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Esclerose Múltipla/diagnóstico , Miosina Tipo I/imunologia , Poli-Hidroxietil Metacrilato/química , Animais , Humanos , Proteínas Imobilizadas/química , Magnetismo/métodos , Imãs/química , Camundongos , Microesferas , Esclerose Múltipla/imunologia , Miosina Tipo I/química , Isoformas de Proteínas/química , Isoformas de Proteínas/imunologiaRESUMO
Degradable poly(2-hydroxyethyl methacrylate) hydrogels were prepared from a linear copolymer (Mw = 49 kDa) of 2-hydroxyethyl methacrylate (HEMA), 2-(acethylthio)ethyl methacrylate (ATEMA), and zwitterionic 2-methacryloyloxyethyl phosphorylcholine (MPC). The deprotection of ATEMA thiol groups by triethylamine followed by their gentle oxidation with 2,2'-dithiodipyridine resulted in the formation of reductively degradable polymers with disulfide bridges. Finally, a hydrogel 3D structure with an oriented porosity was obtained by gelation of the polymer in the presence of needle-like sodium acetate crystals. The pore diameter and porosity of resulting poly(2-hydroxyethyl methacrylate-co-2-(acethylthio)ethyl methacrylate-co-2-methacryloyloxyethyl phosphorylcholine) [P(HEMA-ATEMA-MPC)] hydrogels varied between 59 and 65 µm and between 70 and 79.6 vol % according to Hg porosimetry, and complete degradation of these materials was reached in 86 days in 0.33 mmol solution of l-cysteine/L in phosphate buffer. The cross-linked P(HEMA-ATEMA-MPC) hydrogels were evaluated as a possible support for human mesenchymal stem cells (MSCs). No cytotoxicity was found for the un-cross-linked thiol-containing and protected P(HEMA-ATEMA-MPC) chains up to a concentration of 5 and 1 wt % in α-minimum essential medium, respectively.
Assuntos
Poli-Hidroxietil Metacrilato/química , Humanos , Hidrogéis , Porosidade , Engenharia TecidualRESUMO
Three-dimensional hydrogel supports for mesenchymal and neural stem cells (NSCs) are promising materials for tissue engineering applications such as spinal cord repair. This study involves the preparation and characterization of superporous scaffolds based on a copolymer of 2-hydroxyethyl and 2-aminoethyl methacrylate (HEMA and AEMA) crosslinked with ethylene dimethacrylate. Ammonium oxalate is chosen as a suitable porogen because it consists of needle-like crystals, allowing their parallel arrangement in the polymerization mold. The amino group of AEMA is used to immobilize RGDS and SIKVAVS peptide sequences with an N-γ-maleimidobutyryloxy succinimide ester linker. The amount of the peptide on the scaffold is determined using 125 I radiolabeled SIKVAVS. Both RGDS- and SIKVAVS-modified poly(2-hydroxyethyl methacrylate) scaffolds serve as supports for culturing human mesenchymal stem cells (MSCs) and human fetal NSCs. The RGDS sequence is found to be better for MSC and NSC proliferation and growth than SIKVAVS.
Assuntos
Células-Tronco Neurais/metabolismo , Oligopeptídeos , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Linhagem Celular , Humanos , Células-Tronco Mesenquimais/citologia , Metilmetacrilatos/química , Metilmetacrilatos/farmacologia , Células-Tronco Neurais/citologia , Oligopeptídeos/química , Oligopeptídeos/farmacologiaRESUMO
The efficient isolation and concentration of protein antigens from complex biological samples is a critical step in several analytical methods, such as mass spectrometry, flow cytometry and immunochemistry. These techniques take advantage of magnetic microspheres as immunosorbents. The focus of this study was on the development of new superparamagnetic polymer microspheres for the specific isolation of the tumor suppressor protein p53. Monodisperse macroporous poly(glycidyl methacrylate) (PGMA) microspheres measuring approximately 5 µm and containing carboxyl groups were prepared by multistep swelling polymerization of glycidyl methacrylate (GMA), 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA) and ethylene dimethylacrylate (EDMA) as a crosslinker in the presence of cyclohexyl acetate as a porogen. To render the microspheres magnetic, iron oxide was precipitated within their pores; the Fe content in the particles received â¼18 wt%. Nonspecific interactions between the magnetic particles and biological media were minimized by coating the microspheres with poly(ethylene glycol) (PEG) terminated by carboxyl groups. The carboxyl groups of the magnetic PGMA microspheres were conjugated with primary amino groups of mouse monoclonal DO-1 antibody using conventional carbodiimide chemistry. The efficiency of protein p53 capture and the degree of nonspecific adsorption on neat and PEG-coated magnetic microspheres were determined by western blot analysis.
Assuntos
Anticorpos Monoclonais Murinos/química , Microesferas , Ácidos Polimetacrílicos/química , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/isolamento & purificação , Animais , Linhagem Celular Tumoral , Camundongos , Polietilenoglicóis/químicaRESUMO
Identification and evaluation of small changes in ß-amyloid peptide (Aß) levels in cerebrospinal fluid is of crucial importance for early detection of Alzheimer's disease. Microfluidic detection methods enable effective preconcentration of Aß using magnetic microparticles coated with Aß antibodies. Poly(glycidyl methacrylate) microspheres are coated with α-amino-ω-methoxy-PEG5000 /α-amino-ω-Boc-NH-PEG5000 Boc groups deprotected and NH2 succinylated to introduce carboxyl groups. Capillary electrophoresis with laser-induced fluorescence detection confirms the efficient capture of Aß 1-40 peptides on the microspheres with immobilized monoclonal anti-Aß 6E10. The capture specificity is confirmed by comparing Aß 1-40 levels on the anti-IgG-immobilized particles used as a control.
Assuntos
Peptídeos beta-Amiloides/isolamento & purificação , Ácidos Carboxílicos/química , Imunoglobulina G/metabolismo , Microesferas , Fragmentos de Peptídeos/isolamento & purificação , Polietilenoglicóis/química , Ácidos Polimetacrílicos/química , Adsorção , Animais , Bovinos , Cromatografia de Afinidade , Eletroforese Capilar , Fenômenos Magnéticos , Microscopia de Força Atômica , Tamanho da Partícula , TermogravimetriaRESUMO
To minimize non-specific protein adsorption on macroporous poly(glycidyl methacrylate) and poly(2-hydroxyethyl methacrylate) microspheres containing amino and/or carboxyl groups, the microspheres are coated with α,ω-bis-carboxy poly(ethylene glycol) and amino-terminated poly(ethylene glycol-co-propylene glycol) or α-methoxy-ω-amino poly(ethylene glycol). Adsorption of bovine serum albumin (BSA), γ-globulin, (125) I-BSA, pepsin, and chymotrypsin on neat and PEGylated microspheres is determined by UV-VIS spectroscopy of supernatants and eluates or by measurement of radioactivity in an ionization chamber. Neat and PEGylated microspheres adsorb 0.8-70% and 0.02-44% of protein, respectively.
Assuntos
Biotecnologia/métodos , Microesferas , Polietilenoglicóis/química , Poli-Hidroxietil Metacrilato/química , Ácidos Polimetacrílicos/química , Adsorção , Quimotripsina/química , Radioisótopos do Iodo/química , Estrutura Molecular , Soroalbumina Bovina/química , gama-Globulinas/químicaRESUMO
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.