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1.
J Colloid Interface Sci ; 581(Pt A): 218-225, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32771733

RESUMO

We used the Surface Forces Apparatus to elucidate the interaction mechanism between grafted 5 heptad-long peptides engineered to spontaneously form a heterodimeric coiled-coil complex. The results demonstrated that when intimate contact between peptides is reached, binding occurs first via weakly interacting but more mobile distal heptads, suggesting an induced-fit association process. Precise control of the distance between peptide-coated surfaces allowed to quantitatively monitor the evolution of their biding energy. The binding energy of the coiled-coil complex increased in a stepwise fashion rather than monotonically with the overlapping distance, each step corresponding to the interaction between a quantized number of heptads. Surface forces data were corroborated to surface plasmon resonance measurements and molecular dynamics simulations and allowed the calculation of the energetic contribution of each heptad within the coiled-coil complex.

2.
Biomacromolecules ; 20(5): 1926-1936, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-30951296

RESUMO

Affinity-based systems represent a promising solution to control the delivery of therapeutics using hydrogels. Here, we report a hybrid system that is based on the peptidic E/K coiled coil affinity pair to mediate the release of gold nanoparticles (NPs) from alginate scaffolds. On one hand, the gold NPs were functionalized with the Ecoil-tagged epidermal growth factor (EGF). The bioactivity of the grafted EGF and the bioavailability of the Ecoil moiety were confirmed by EGF receptor phosphorylation assays and by capturing the functionalized NPs on a Kcoil-derivatized surface. On the other hand, alginate chains were modified with azido-homoalanine Kcoil (Aha-Kcoil) by azide-alkyne click chemistry. The hybrid system was formed by dispersing NPs functionalized with the Ecoil-tagged EGF in alginate hydrogels containing either 0, 10, or 20% of Kcoil-modified alginate (Alg-Kcoil). With 20% of Alg-Kcoil, the release of Ecoil-functionalized NPs was reduced by half when compared to the release of NPs without Ecoil, whereas little to no differences were noticed with either 0 or 10% of Alg-Kcoil. Differential dynamic microscopy was used to determine the diffusion coefficient of the NPs, and the results showed a decrease in the diffusion coefficient of Ecoil-functionalized NPs, when compared to bare PEGylated NPs. Altogether, our data demonstrated that the E/K coiled coil system can control the release of NPs in a high Kcoil content alginate gel, opening diverse applications in drug delivery.

3.
Bioconjug Chem ; 29(11): 3866-3876, 2018 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-30350572

RESUMO

Thiol(-click) chemistry has been extensively investigated to conjugate (bio)molecules to polymers. Handling of cysteine-containing molecules may however be cumbersome, especially in the case of fast-oxidizing coiled-coil-forming peptides. In the present study, we investigated the practicality of a one-pot process to concomitantly reduce and conjugate an oxidized peptide to a polymer. Three thiol-based conjugation chemistries (vinyl sulfone (VS), maleimide, and pyridyldithiol) were assayed along with three reducing agents (tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol, and ß-mercaptoethanol). Seven out of the nine possible combinations significantly enhanced the conjugation yield, provided that an adequate concentration of reductant was used. Among them, the coincubation of an oxidized peptide with TCEP and a VS-modified polymer displayed the highest level of conjugation. Our results also provide insights into two topics that currently lack consensus: TCEP is stable in 10 mM phosphate buffered saline and it reacts with thiol-alkylating agents at submillimolar concentrations, and thus should be carefully used in order to avoid interference with thiol-based conjugation reactions.


Assuntos
Química Click/métodos , Peptídeos/química , Polímeros/química , Substâncias Redutoras/química , Compostos de Sulfidrila/química , Alquilação , Maleimidas/síntese química , Maleimidas/química , Oxirredução , Peptídeos/síntese química , Polímeros/síntese química , Substâncias Redutoras/síntese química , Compostos de Sulfidrila/síntese química , Sulfonas/síntese química , Sulfonas/química
4.
Carbohydr Polym ; 161: 219-227, 2017 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-28189232

RESUMO

Dextran is one of the hydrophilic polymers that is used for hydrogel preparation. As any polysaccharide, it presents a high density of hydroxyl groups, which make possible several types of derivatization and crosslinking reactions. Furthermore, dextran is an excellent candidate for hydrogel fabrication with controlled cell/scaffold interactions as it is resistant to protein adsorption and cell adhesion. RGD peptide can be grafted to the dextran in order to promote selected cell adhesion and proliferation. Altogether, we have developed a novel strategy to graft the RGD peptide sequence to dextran-based hydrogel using divinyl sulfone as a linker. The resulting RGD functionalized dextran-based hydrogels were transparent, presented a smooth surface and were easy to handle. The impact of varying RGD peptide amounts, hydrogel porosity and topology upon human umbilical vein endothelial cell (HUVEC) adhesion, proliferation and infiltration was investigated. Our results demonstrated that 0.1% of RGD-modified dextran within the gel was sufficient to support HUVEC cells adhesion to the hydrogel surface. Sodium chloride was added (i) to the original hydrogel mix in order to form a macroporous structure presenting interconnected pores and (ii) to the hydrogel surface to create small orifices essential for cells migration inside the matrix.


Assuntos
Dextranos/química , Hidrogéis/química , Oligopeptídeos/química , Adesão Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Oligopeptídeos/farmacologia
5.
Acta Biomater ; 50: 198-206, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28069507

RESUMO

In the field of tissue engineering, the tethering of growth factors to tissue scaffolds in an oriented manner can enhance their activity and increase their half-life. We chose to investigate the capture of the basic Fibroblast Growth Factor (bFGF) and the Epidermal Growth Factor (EGF) on a gelatin layer, as a model for the functionalization of collagen-based biomaterials. Our strategy relies on the use of two high affinity interactions, that is, the one between two distinct coil peptides as well as the one occurring between a collagen-binding domain (CBD) and gelatin. We expressed a chimeric protein to be used as an adaptor that comprises one of the coil peptides and a CBD derived from the human fibronectin. We proved that it has the ability to bind simultaneously to a gelatin substrate and to form a heterodimeric coiled-coil domain with recombinant growth factors being tagged with the complementary coil peptide. The tethering of the growth factors was characterized by ELISA and surface plasmon resonance-based biosensing. The bioactivity of the immobilized bFGF and EGF was evaluated by a human umbilical vein endothelial cell proliferation assay and a vascular smooth muscle cell survival assay. We found that the tethering of EGF preserved its mitogenic and anti-apoptotic activity. In the case of bFGF, when captured via our adaptor protein, changes in its natural mode of interaction with gelatin were observed. STATEMENT OF SIGNIFICANCE: In an effort to functionalize collagen/gelatin-based biomaterials with growth factors, we have designed an adaptor protein corresponding to a collagen-binding domain fused to a coil peptide. In our strategy, this adaptor protein captures growth factors being tagged with the partner coil peptide in a specific, stable and oriented manner. We have found that the tethering of the Epidermal Growth Factor preserved its mitogenic and anti-apoptotic activity. In the case of the basic Fibroblast Growth Factor, the captured growth factor remained bioactive although its tethering via this adaptor protein modified its natural mode of interaction with gelatin. Altogether this strategy is easily adaptable to the simultaneous tethering of various growth factors.


Assuntos
Materiais Biocompatíveis , Fator de Crescimento Epidérmico , Fator 2 de Crescimento de Fibroblastos , Fibronectinas , Gelatina , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Imobilizadas , Proteínas Recombinantes de Fusão , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/química , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fibronectinas/química , Fibronectinas/farmacologia , Gelatina/química , Gelatina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/farmacologia , Teste de Materiais/métodos , Domínios Proteicos , Proteínas Recombinantes de Fusão/farmacologia , Engenharia Tecidual/métodos
7.
Biomacromolecules ; 18(1): 303-310, 2017 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-27997154

RESUMO

The patency of small-diameter (<6 mm) synthetic vascular grafts (VGs) is still limited by the absence of a confluent, blood flow-resistant monolayer of endothelial cells (ECs) on the lumen and of vascular smooth muscle cell (VSMC) growth into the media layer. In this research, electrospinning has been combined with bioactive coatings based on chondroitin sulfate (CS) to create scaffolds that possess optimal morphological and bioactive properties for subsequent cell seeding. We fabricated random and aligned electrospun poly(ethylene terephthalate), ePET, mats with small pores (3.2 ± 0.5 or 3.9 ± 0.3 µm) and then investigated the effects of topography and bioactive coatings on EC adhesion, growth, and resistance to shear stress. Bioactive coatings were found to dominate the cell behavior, which enabled creation of a near-confluent EC monolayer that resisted physiological shear-flow conditions. CS is particularly interesting since it prevents platelet adhesion, a key issue to avoid blood clot formation in case of an incomplete EC monolayer or partial cell detachment. Regarding the media layer, circumferentially oriented nanofibers with larger pores (6.3 ± 0.5 µm) allowed growth, survival, and inward penetration of VSMCs, especially when the CS was further coated with tethered, oriented epithelial growth factor (EGF). In summary, the techniques developed here can lead to adequate scaffolds for the luminal and media layers of small-diameter synthetic VGs.


Assuntos
Prótese Vascular , Sulfatos de Condroitina/química , Eletroquímica , Células Endoteliais da Veia Umbilical Humana/citologia , Músculo Liso Vascular/citologia , Nanofibras/química , Engenharia Tecidual/métodos , Animais , Aorta Torácica/citologia , Adesão Celular , Células Cultivadas , Humanos , Polietilenotereftalatos/química , Ratos , Estresse Mecânico , Tecidos Suporte
8.
Biomatter ; 6(1): e1231276, 2016 01.
Artigo em Inglês | MEDLINE | ID: mdl-27740881

RESUMO

In an effort to design biomaterials that may promote repair of the central nervous system, 3-dimensional scaffolds made of electrospun poly lactic acid nanofibers with interconnected pores were fabricated. These scaffolds were functionalized with polyallylamine to introduce amine groups by wet chemistry. Experimental conditions of the amination protocol were thoroughly studied and selected to introduce a high amount of amine group while preserving the mechanical and structural properties of the scaffold. Subsequent covalent grafting of epidermal growth factor was then performed to further tailor these aminated structures. The scaffolds were then tested for their ability to support Neural Stem-Like Cells (NSLCs) culture. Of interest, NSLCs were able to proliferate on these EGF-grafted substrates and remained viable up to 14 d even in the absence of soluble growth factors in the medium.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Neurônios/citologia , Poliésteres/química , Engenharia Tecidual/métodos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fator de Crescimento Epidérmico/química , Nanofibras/química , Neurônios/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacos , Propriedades de Superfície , Tecidos Suporte/química
9.
Acta Biomater ; 29: 239-247, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26485166

RESUMO

UNLABELLED: Growth factors (GFs) are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Co-immobilizing GFs on materials while preserving their bioactivity still represents a major challenge in the field of tissue regeneration and bioactive implants. In this study, we explore the potential of an oriented immobilization technique based on two high affinity peptides, namely the Ecoil and Kcoil, to allow for the simultaneous capture of the epidermal growth factor (EGF) and the vascular endothelial growth factor (VEGF) on a chondroitin sulfate coating. This glycosaminoglycan layer was selected as it promotes cell adhesion but reduces non-specific adsorption of plasma proteins. We demonstrate here that both Ecoil-tagged GFs can be successfully immobilized on chondroitin sulfate surfaces that had been pre-decorated with the Kcoil peptide. As shown by direct ELISA, changing the incubation concentration of the various GFs enabled to control their grafted amount. Moreover, cell survival studies with endothelial and smooth muscle cells confirmed that our oriented tethering strategy preserved GF bioactivity. Of salient interest, co-immobilizing EGF and VEGF led to better cell survival compared to each GF captured alone, suggesting a synergistic effect of these GFs. Altogether, these results demonstrate the potential of coiled-coil oriented GF tethering for the co-immobilization of macromolecules; it thus open the way to the generation of biomaterials surfaces with fine-tuned biological properties. STATEMENT OF SIGNIFICANCE: Growth factors are potent signaling molecules that act in a coordinated manner in physiological processes such as tissue healing or angiogenesis. Controlled coimmobilization of growth factors on biomaterials while preserving their bioactivity represents a major challenge in the field of tissue regeneration and bioactive implants. This study demonstrates the potential of an oriented immobilization technique based on two high affinity peptides to allow for the simultaneous capture of epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF). Our system allowed an efficient control on growth factor immobilization by adjusting the incubation concentrations of EGF and VEGF. Of salient interest, co-immobilizing of specific ratios of EGF and VEGF demonstrated a synergistic effect on cell survival compared to each GF captured alone.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Proteínas Imobilizadas/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacologia , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/agonistas , Fator de Crescimento Epidérmico/química , Células Endoteliais da Veia Umbilical Humana/citologia , Humanos , Proteínas Imobilizadas/agonistas , Proteínas Imobilizadas/química , Fator A de Crescimento do Endotélio Vascular/agonistas , Fator A de Crescimento do Endotélio Vascular/química
10.
Biomacromolecules ; 15(7): 2512-20, 2014 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-24927450

RESUMO

This study highlights the advantages of chondroitin sulfate (CS) as a sublayer combining selective low-fouling properties, low-platelet adhesion and pro-adhesive properties on endothelial cells, making CS promising for vascular graft applications. These properties were evaluated by comparing CS with well-known low-fouling coatings such as poly(ethylene glycol) (PEG) and carboxymethylated dextran (CMD), which were covalently grafted on primary amine-rich plasma polymerized (LP) films. Protein adsorption studies by quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence measurements showed that CS is as effective as PEG in reducing fibrinogen adsorption (~90% reduction). CS also largely reduced adsorption of bovine serum albumin (BSA) as well as fetal bovine serum (FBS) but to a lower extent than PEG and CMD surfaces (72% vs 85% for BSA and 66% vs 89% for FBS). Whole blood perfusion assays indicated that, while LP surfaces were highly reactive with platelets, PEG, CMD, and CS grafted surfaces drastically decreased platelet adhesion and activation to levels significantly lower than polyethylene terephthalate (PET) surfaces. Finally, while human umbilical vein endothelial cell (HUVEC) adhesion and growth were found to be very limited on PEG and CMD, they were significantly increased on CS compared to that on bare PET and reached similar values as those for tissue culture polystyrene positive controls. Interestingly, HUVEC retention during perfusion with blood was found to be excellent on CS but poor on PET. Overall, our results suggest that the CS surface has the advantage of promoting HUVEC growth and resistance to flow-induced shear stress while preventing fibrinogen and platelet attachment. Such a nonthrombogenic but endothelial-cell adhesive surface is thus promising to limit vascular graft occlusion.


Assuntos
Plaquetas/efeitos dos fármacos , Prótese Vascular , Adesão Celular/efeitos dos fármacos , Sulfatos de Condroitina/química , Materiais Revestidos Biocompatíveis/química , Sulfatos de Condroitina/farmacologia , Materiais Revestidos Biocompatíveis/farmacologia , Dextranos/química , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fibrinogênio/química , Células Endoteliais da Veia Umbilical Humana , Humanos , Adesividade Plaquetária/efeitos dos fármacos , Polietilenoglicóis/química , Polietilenotereftalatos/química , Poliestirenos/química , Proteínas/química , Técnicas de Microbalança de Cristal de Quartzo , Soroalbumina Bovina/química
11.
Methods Mol Biol ; 1172: 39-47, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24908293

RESUMO

In a "sandwich" enzyme-linked immunosorbent assay (ELISA) designed to detect an antigen in a complex protein mixture, the antigen is usually captured via an antibody adsorbed to the wells of a microplate. Plate preparation for standard assay involves a passive adsorption of capture antibodies followed by the incubation of blocking agents. Here, we describe a new strategy that replaces these two time-consuming adsorption steps (up to 15 h) by a unique step corresponding to the covalent grafting of the capture antibody on a carboxymethylated dextran (CMD) layer, a single step completed in 15 min. Taking advantage of the CMD low-fouling properties, blocking agent-free buffer solutions can be used as diluent in the improved approach.


Assuntos
Antígenos/análise , Dextranos/síntese química , Ensaio de Imunoadsorção Enzimática/métodos , Fator de Crescimento Epidérmico/análise , Adsorção , Anticorpos/química , Proteínas de Bactérias/química , Tampões (Química) , Carbodi-Imidas/química , Materiais Revestidos Biocompatíveis/síntese química , Ensaio de Imunoadsorção Enzimática/normas , Peroxidase do Rábano Silvestre/química , Humanos , Proteínas Recombinantes/análise , Soluções , Succinimidas/química
12.
Macromol Biosci ; 14(5): 720-30, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24469944

RESUMO

An anti-apoptotic coating combining chondroitin sulfate (CS) and coiled-coil-based tethering of epidermal growth factor (EGF) is designed for vascular applications. The oriented tethering strategy enables to reach higher EGF surface densities compared to the commonly used random covalent grafting, while using much lower concentrations of EGF during incubation. It also significantly improves vascular smooth muscle cell (VSMC) survival and resistance to apoptosis in serum-free conditions. The comparison of CS and low-fouling carboxymethylated dextran as a sublayer for growth factors highlights the tremendous benefit of CS thanks to its selective protein resistance and good cell adhesion properties. This approach can be tuned by capturing other growth factors on CS through coiled-coil interactions.


Assuntos
Sulfatos de Condroitina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Análise de Variância , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ensaio de Imunoadsorção Enzimática , Músculo Liso Vascular/citologia , Ratos
13.
J Biomed Nanotechnol ; 9(7): 1195-209, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23909134

RESUMO

This study examines the effect of electrospun polyethylene terephthalate mats fiber diameter, orientation, and surface properties on the Human Aortic Endothelial Cell behavior. Mats with two different average fiber diameters (740 +/- 200 nm and 1.8 +/- 0.2 microm); orientations (low and high); NaOH-treated and untreated were prepared. NaOH treatment altered mats physical properties. AlamarBlue assay revealed that all four test mats supported cell adhesion and growth. Cell growth was observed to be faster for mat with large fiber diameter than for the small fiber diameter mat. Fluorescent staining and scanning electron microscopy showed that fiber diameter and orientation influenced cell morphology. Cells were randomly spread on the 740-nm diameter fibers whereas most of them were oriented along the fibers with 1.8 microm diameter. Mat with higher fiber alignment showed higher cell orientation. Cells penetrated into the mats having 1.8 +/- 0.2 microm fiber diameter but remained on the surface of the mat with 740 +/- 200 nm, as determined from histological analysis. These findings highly suggest that the two mats may be potential materials to construct a two layer vascular graft scaffold in which the mat with small diameter fibers forms the luminal surface and the mat with larger fiber diameter the abluminal surface.


Assuntos
Aorta/citologia , Células Endoteliais/fisiologia , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Engenharia Tecidual/instrumentação , Tecidos Suporte , Aorta/fisiologia , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Eletroquímica/métodos , Células Endoteliais/citologia , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Teste de Materiais , Rotação
14.
Acta Biomater ; 9(6): 6806-13, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23485856

RESUMO

Chimeric growth factors may represent a powerful alternative to their natural counterparts for the functionalization of tissue-engineered scaffolds and applications in regenerative medicine. Their rational design should provide a simple, readily scalable production strategy while improving retention at the site of action. In that endeavor, we here report the synthesis of a chimeric protein corresponding to human vascular endothelial growth factor 165 being N-terminally fused to an E5 peptide tag (E5-VEGF). E5-VEGF was successfully expressed as a homodimer in mammalian cells. Following affinity purification, in vitro surface plasmon resonance biosensing and cell survival assays confirmed diffusible E5-VEGF ability to bind to its receptor ectodomains, while observed morphological phenotypes confirmed its anti-apoptotic features. Additional surface plasmon resonance assays highlighted that E5-VEGF could be specifically captured with high stability when interacting with covalently immobilized K5 peptide (a synthetic peptide designed to bind to the E5 moiety of chimeric hVEGF). This immobilization strategy was applied to glass substrates and chimeric hVEGF was shown to be maintained in a functionally active state following capture. Altogether, our data demonstrated that stable hVEGF capture can be performed via coiled-coil interactions without impacting hVEGF bioactivity, thus opening up the way to future applications in the field of tissue engineering and regenerative medicine.


Assuntos
Proteínas de Fluorescência Verde/química , Engenharia de Proteínas/métodos , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento/ultraestrutura , Proteínas Recombinantes de Fusão/síntese química , Fator A de Crescimento do Endotélio Vascular/química , Fator A de Crescimento do Endotélio Vascular/ultraestrutura , Sítios de Ligação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/ultraestrutura , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/ultraestrutura , Fator A de Crescimento do Endotélio Vascular/genética
15.
J Immunol Methods ; 389(1-2): 38-44, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23279947

RESUMO

We here report the development of a new generation of enzyme-linked immunosorbent assay (ELISA) that takes advantage of a low-fouling carboxymethylated dextran (CMD) layer chemically grafted on ELISA wells. In our approach, the overnight capture antibody adsorption step found in classical ELISA was replaced by a covalent attachment step to the CMD layer completed in 15 min. As a model, the potential of our approach was highlighted using commercially available anti-human epidermal growth factor (EGF) antibodies to quantify EGF present in various samples. Of interest, the grafted CMD layer was found to be as efficient as the commonly used bovine serum albumine (BSA) to reduce non-specific adsorption, thus eliminating the need of a time-consuming BSA blocking step normally required in classical ELISA. Our results demonstrated similar specificity, affinity, and intra- and inter-assay variations regardless of the diluent used in the assay (BSA-based diluent or protein-free buffer solution) when compared to standard ELISA. Finally, accuracy and precision of the CMD-based ELISA were verified by a spike and recovery test. Dilutions of recombinant human EGF in serum from healthy human volunteers showed almost-perfect linearity and mean recovery rates ranging between 90 and 110%.


Assuntos
Dextranos/química , Ensaio de Imunoadsorção Enzimática/métodos , Fator de Crescimento Epidérmico/sangue , Adulto , Feminino , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
Bioconjug Chem ; 22(8): 1690-9, 2011 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-21736371

RESUMO

Biocompatible polymers are commonly functionalized with specific moieties such as amino groups to modify their surface properties and/or to attach bioactive compounds. A reliable method is usually required to characterize amino group surface densities. In this study, aminated polyethylene terephthalate (PET) films were generated via an aminolysis reaction involving either ethylenediamine molecules (EtDA), in order to vary easily the amino group density on PET surfaces, or 25 kDa polyvinylamine (PVAm) as an alternative reagent preventing bulk damages resulting from the aminolysis reaction. Among commonly used dyes for amino group quantification, Orange II and Coomassie Brillant Blue (CBB) were selected to quantify the extent of amine grafting resulting from these derivatization procedures. Rapid and convenient colorimetric assays were compared to surface atomic compositions obtained from X-ray photoelectron spectroscopy (XPS) measurements. Orange II was found to be the most appropriate dye for quantifying primary amine groups in a reliable and specific way. Due to its unique negative charge and low steric hindrance compared to CBB, the Orange II dye was very sensitive and provided reliable quantification over a wide range of amino group surface densities (ca. 5 to at least 200 pmol/mm(2)). In order to further validate the use of the Orange II dye for amino group quantification, a heterobifunctional linker reacting with amino groups was then grafted on modified PET surfaces. Interestingly, the good correlation between the densities of adsorbed Orange II and covalently grafted linkers suggests that the Orange II method is a relevant, reliable, easy, and inexpensive method to predict the amount of amino groups available for subsequent functionalization of polymer surfaces.


Assuntos
Aminas/análise , Polímeros/química , Aminas/química , Compostos Azo , Benzenossulfonatos , Materiais Biocompatíveis/química , Colorimetria , Métodos , Polietilenotereftalatos/química , Propriedades de Superfície
17.
Biomaterials ; 32(6): 1591-600, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21106234

RESUMO

Deficient healing after endovascular aneurysm repair is thought to be related to the pro-apoptotic environment in abdominal aortic aneurysms and inertness of the graft materials. A bioactive coating containing both chondroitin sulfate (CS) and epidermal growth factor (EGF) was developed in order to increase the growth and resistance to apoptosis of vascular smooth muscle cells (VSMC) on biomaterials surfaces. CS and EGF were covalently grafted using carbodiimide chemistry and the coating was characterized and optimized using ellipsometry, static contact angle and ToF-SIMS. Its potential to improve cell adhesion, growth and resistance to apoptosis was assessed in vitro with rat aortic VSMC. Results showed that CS and EGF immobilization allowed for the creation of a uniform coating that increased cell adhesion, growth and resistance to apoptosis in serum-free medium. Overall, CS and EGF possess great potential as bioactive anti-apoptotic mediators for vascular repair.


Assuntos
Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Sulfatos de Condroitina/química , Sulfatos de Condroitina/farmacologia , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/farmacologia , Miócitos de Músculo Liso/citologia , Animais , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Ratos
18.
Bioconjug Chem ; 21(12): 2257-66, 2010 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-21058714

RESUMO

In an effort to evaluate the impact of various epidermal growth factor (EGF) grafting strategies upon cell surface receptor activation and cell adhesion, we generated low-fouling surfaces by homogeneously grafting carboxymethylated dextran (CMD) on amino-coated glass substrate. By preventing nonspecific cell adhesion while providing reactive groups facilitating subsequent protein grafting, CMD allowed achieving specific cell/tethered EGF interactions and therefore deriving unambiguous conclusions about various EGF grafting strategies. We demonstrate here that A-431 cell response to immobilized EGF is highly dependent on the bioactivity of the tagged protein being tethered, its proper orientation, and its surface density. Among all the approaches we tested, the oriented tethering of fully bioactive EGF via a de novo-designed coiled-coil capture system was shown to be the most efficient. That is, it led to the most intense and sustained phosphorylation of EGF receptors as well as to strong A-431 cell adhesion, the latter being comparable to that observed with amino-coated surfaces in the absence of CMD.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Proteínas Imobilizadas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Aminas/química , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dextranos/química , Fator de Crescimento Epidérmico/química , Fator de Crescimento Epidérmico/genética , Fator de Crescimento Epidérmico/farmacologia , Expressão Gênica , Vidro/química , Células HEK293 , Humanos , Proteínas Imobilizadas/química , Proteínas Imobilizadas/genética , Proteínas Imobilizadas/farmacologia , Fosforilação/efeitos dos fármacos , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Propriedades de Superfície/efeitos dos fármacos , Engenharia Tecidual/métodos
19.
J Immunol Methods ; 362(1-2): 161-7, 2010 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-20869967

RESUMO

The de novo designed heterodimeric E/K coiled-coil system has been previously demonstrated to be an excellent capture/dimerization system applicable to various needs in both biotechnology and pharmaceutical fields. Those include controlled protein dimerization, capture, purification and Western-blot detection. We here report the development of a new generation of ELISA test based on coiled-coil interactions for the direct quantitation of coil-tagged epidermal growth factor (EGF). The new approach was evaluated for its specificity, plate storability and reusability as well as for convenience when compared to commercially available systems. Our results show a similar affinity/sensitivity to standard capturing antibody-based ELISA systems and an improved affinity/sensitivity when compared to the commercially available Ni-NTA capture system. The E/K coiled-coil ELISA system was validated with respect to recovery, intra- and inter-assay variations. The practical working range was estimated to be between 5.2 and 34,000 pM. Furthermore, the storability and reusability of the plates was greater than the two aforementioned systems, suggesting that the E/K coiled-coil system is a good alternative to traditional tags such as poly-histidine for the development of ELISA tests aiming at quantitating coil-tagged proteins.


Assuntos
Fator de Crescimento Epidérmico/análise , Ensaio de Imunoadsorção Enzimática/métodos , Fator de Crescimento Epidérmico/imunologia , Humanos , Estrutura Secundária de Proteína , Sensibilidade e Especificidade
20.
Bioconjug Chem ; 20(8): 1569-77, 2009 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-19588921

RESUMO

We have elaborated and validated a novel approach for the oriented tethering of proteins such as the epidermal growth factor (EGF) on aminated surfaces. The grafting reactions were optimized to generate a dense and homogeneous EGF layer. Impact of EGF orientation on A-431 cellular response was investigated. Our results demonstrate that, in sharp contrast to responses obtained with soluble EGF supply or with randomly grafted EGF, oriented immobilization of EGF via a de novo designed coiled-coil capture system leads to a sustained phosphorylation of A-431 cell surface EGF receptors. Our results thus indicate that oriented protein immobilization via coiled-coil interactions is an efficient and versatile method to control tethering of bioactive molecules for future applications in the field of regenerative medicine and tissue engineering.


Assuntos
Fator de Crescimento Epidérmico/metabolismo , Receptores de Superfície Celular/metabolismo , Células Cultivadas , Humanos , Estrutura Molecular , Fosforilação , Propilaminas , Silanos/química , Silício/química , Propriedades de Superfície
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