RESUMO
The purpose of this study was to develop novel hybrid organic-inorganic materials based on poly(vinyl alcohol) (PVA) polymer chemically crosslinked network to be tested as solid support on bovine herpesvirus immunoassay. Hybrids were synthesized by reacting PVA with three different alkoxysilanes modifying chemical groups: tetraethoxysilane (TEOS), 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-glycidoxypropyltrimethoxysilane (GPTMS). PVA-derived hybrids were also modified by chemically crosslinking with glutaraldehyde (GA) during the synthesis reaction. In order to investigate the structure in the nanometer-scale, PVA-derived hybrids were characterized by using small-angle x-ray scattering synchrotron radiation (SAXS) and x-ray diffraction (XRD). PVA hybrids' chemical functionalities and their interaction with herpesviruses were also characterized by Fourier transform infrared spectroscopy (FTIR). The bioactivity assays were tested through enzyme linked immunosorbent assay (ELISA). SAXS results have indicated nano-ordered disperse domains for PVA hybrids with different x-ray scattering patterns for PVA polymer and PVA-derived hybrids. FTIR spectra have shown major vibration bands associated with organic-inorganic chemical groups present in the PVA, PVA-derived by silane modifier and PVA chemically crosslinked by GA. The immunoassay results have shown that PVA hybrids with chemically functionalized structures regulated to some extent the specific bioimmobilization of herpesvirus onto solid phase. We think that it is due to the overall balance of forces associated with van der Waals interaction, hydrophilic and hydrophobic forces and steric hindrance acting at the surface. PVA and PVA-derived hybrid materials were successfully produced with GA crosslinking in a nanometer-scale network. Also, such a PVA-based material could be advantageously used in immunoassays with enhanced specificity for diagnosis.
Assuntos
Materiais Biocompatíveis/química , Ensaio de Imunoadsorção Enzimática/instrumentação , Ensaio de Imunoadsorção Enzimática/métodos , Compostos Inorgânicos/química , Nanoestruturas/química , Compostos Orgânicos/química , Álcool de Polivinil/química , Desenho de Equipamento , Análise de Falha de Equipamento , Teste de Materiais , Nanoestruturas/ultraestrutura , Tamanho da PartículaRESUMO
The last 3 decades have been a revolution in the area of sol-gel-derived materials. They can be used to encapsulate biomolecules such as enzymes, antibodies, hormones, and proteins retaining their functional state. Proteins can be immobilized in many ways but it is crucial that they retain their native conformational structure and, therefore, bioactivity. Porous silica gel matrixes with modified surfaces offer unlimited possibilities to control the protein-solid interaction behavior. The bioimmobilization process on sol-gel biomaterials with chemically engineered surface has driven applications on solid-phase materials, affinity chromatography, biosensors and many others. In the present work, we have aimed to produce surface-modified silica glass materials obtained via sol-gel route to be used as solid support on drug delivery systems and as solid-phase in immunodiagnostic. The functionalization process was carried out by reacting alkoxysilanes with 5 different silane surface modifying chemical groups: tetraethoxysilane (TEOS), 3-mercaptopropyltrimethoxysilane (MPTMS) and 3-aminopropyltriethoxysilane (APTES), 3-glycidoxypropyltrimethoxysilane (GPTMS) and 3-isocyanatopropyltriethoxysilane (ICPES). The bioactivity assays were based on two main tests: (a) An in vivo bioresponse of rats with sol-gel disk implants with insulin protein incorporated. In vivo tests with adult male rats were used to verify the immobilized insulin bioactivity after implantation of different biomaterial with functionalized surfaces. All surface modified materials have presented hypoglycemic peak response associated with the insulin bioactivity. (b) The produced solid-phase sol-gel disks with protein substrates were tested through Enzyme Linked Immuno Sorbent Assay (ELISA). The immunoassay results have showed that glasses with chemically functionalized surfaces regulated the extent of bioimmobilization of protein. The amine, thiol and hydroxyl terminated porous gels have showed significant interaction with the antibody-antigen, during the coupling process. We believe that it is due to balance of forces associated with Van der Waals interaction, hydrophilic and hydrophobic forces and steric hindrance acting at the surface. Therefore, such novel biomaterial could be advantageously used in drug delivery systems and in immunoassays of diagnostic kits.
Assuntos
Materiais Biocompatíveis/química , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas/química , Animais , Bovinos , Géis/química , Implantes Experimentais , Insulina/administração & dosagem , Masculino , Conformação Proteica , Ratos , Venenos de Escorpião/química , Sílica Gel , Dióxido de Silício/química , Propriedades de SuperfícieRESUMO
This study describes an approach to obtaining 3-D scaffolds for tissue engineering that allows the incorporation and release of biologically active proteins to stimulate cell function. Laminin was adsorbed on the textured surfaces of binary 70S30C (70 mol % SiO(2), 30 mol % CaO) and ternary 58S (60 mol % SiO(2), 36 mol % CaO, 4 mol % P(2)O(5)) foams. The covalent bonds between the binding sites of the proteins and the ligands on the scaffolds' surfaces did not denaturate the proteins. In vitro studies show that the foams modified with chemical groups and coated with laminin were bioactive, as demonstrated by the formation of a crystalline hydroxy carbonate apatite (HCA) layer formed on the surfaces of the foams upon exposure to simulated body fluid (SBF). The release of proteins from the foams also was investigated. Sustained and controlled release from the scaffolds over a 30-day period was achieved. Laminin release from the bioactive foams followed the dissolution rate of the material network. These results suggest that bioactive foams have the potential to act as scaffolds for soft-tissue engineering with a controlled release of proteins that can induce tissue formation or regeneration.
Assuntos
Materiais Biocompatíveis/metabolismo , Proteínas/metabolismo , Apatitas/síntese química , Apatitas/química , Materiais Biocompatíveis/química , Cinética , Laminina/metabolismo , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The aim of this work was to use sol-gel processing to develop bioactive materials to serve as scaffolds for tissue engineering that will allow the incorporation and release of proteins to stimulate cell function and tissue growth. We obtained organofunctionalized silica with large content of amine and mercaptan groups (up to 25%). The developed method can allow the incorporation and delivery of proteins at a controlled rate. We also produced bioactive foams with binary SiO(2)-CaO and ternary SiO(2)-CaO-P(2)O(5) compositions. In order to enhance peptide-material surface properties, the bioactive foams were modified with amine and mercaptan groups. These materials exhibit a highly interconnected macroporous network and high surface area. These textural features together with the incorporation of organic functionally groups may enable them to be used as scaffolds for the engineering of soft tissue.
RESUMO
Biomolecules can be immobolized in many different ways. They can also be entrapped or tightly adsorbed within porous gels, clays, membranes, resins, and several other materials, but it is crucial that they retain their active conformation after the incorporation procedure. Porous gel matrixes with functionalized surfaces offer unlimited possibilities to control the protein-substrate interaction behavior. In the present work, we have studied the adsorption and the relative stability of bovine serum albumin (BSA) and porcine insulin(PI) incorporated in gels of SiO2 glass matrixes. The porous gel matrixes were obtained using tetramethoxysilane (TMOS)/metanol and functionalized with (3-mercaptopropyl) trimethoxysilane and (3-aminopropyl) triethoxysilane. The relative adsorption kinetics and stability of BSA and PI incorporated in glass networks were evaluated by immersion in phosphate buffer saline (PBS) and alkaline elution media for different periods of time. The kinetics of protein release from the gel matrix was monitored by UV-visible spectroscopy. A significantly larger PI release was observed compared to BSA in PBS solutions. We believe this is mainly associated with the difference on protein interactions with the modified surface, according to the characterization results of porosity, surface area, and contact angle of different functionalized gel matrixes. We could not observe any evidence of denaturation with either proteins after their desorption from gel matrixes using the ultraviolet spectroscopy technique. These results have also been confirmed with the strong bioactivity response from "in vivo" test conducted in rats, where porous gels with PI incorporated were implanted, showing that released proteins retained their native conformation.