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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.
J Am Chem Soc ; 142(35): 14843-14847, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32790294

RESUMO

In this study, we report lubrication properties of physisorbed zwitterionic bottlebrush polymers in the presence of multivalent ions using the surface force apparatus. Unlike polyelectrolyte brushes, the lubrication properties of which diminish drastically in the presence of multivalent ions at concentrations as low as 0.1 mM, zwitterionic bottlebrush polymers exhibit friction coefficients as low as ∼10-3 at such concentrations of multivalent ions up to intermediate normal loads. This lubrication ability persists until surface wear occurs at high normal loads. The surface wear is demonstrated to be triggered by the multivalent ions bridging the polymer chains and dehydrating the zwitterionic moieties. Finally, the analysis of the polymer film stability suggests that the partial desorption of polymers in the presence of the ions does not affect the lubrication performance. Therefore, even in the physisorbed state, zwitterionic brushes perform significantly better than covalently grafted polyelectrolyte brushes in the presence of multivalent ions.

3.
Mol Immunol ; 121: 144-158, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32222585

RESUMO

Most of therapeutic monoclonal antibodies belong to the immunoglobulin G1 (IgG1) family; they interact with the Fcγ receptors (FcγRs) at the surface of immune cells to trigger effector functions. The IgG1-Fc N-glycans impact the interaction with FcγRs and are considered a critical quality attribute. Pioneer studies on FcγR N-glycans have unveiled an additional complexity in that the N-glycan linked on the Asn-162 of FcγRIIIa was shown to be directly involved in the strong affinity for afucosylated IgG1. The last few years have thus seen the emergence of many studies investigating the complex influence of FcγRIIIa N-glycans on the interaction with IgG1 through their glycosylation sites or their glycoprofiles. In this context, we performed site-directed mutagenesis along with glycoengineering on FcγRs (FcγRI, FcγRIIaH131/b and FcγRIIIaV158/F158) in an effort to elucidate the impact of FcγRs N-glycans on the interaction with IgG1. Furthermore, we assessed their binding to various trastuzumab glycoforms with an enhanced surface plasmon resonance assay. The FcγRIIIa N-glycans had the highest impact on the interaction with IgG1. More specifically, the N162 glycan positively influenced the affinity (15-fold) for afucosylated IgG1 while the N45 glycan presented a negative impact (2-fold) regardless of the IgG1 glycoforms. Interestingly, only the FcγRIIIa glycoprofile had an impact on the interaction with IgG1 with a 1.5-fold affinity increase when FcγRIIIa displays high-mannose glycans. These results provide invaluable insights into the complex and strong influence of N-glycosylation upon FcγRs/IgG1 binding and are instrumental to further understand the impact of FcγRs N-glycosylation in their natural forms.


Assuntos
Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Animais , Células CHO , Cricetulus , Glicosilação , Células HEK293 , Humanos , Imunoglobulina G/imunologia , Manose/metabolismo , Mutagênese Sítio-Dirigida , Polissacarídeos/metabolismo , Engenharia de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Receptores de IgG/genética , Receptores de IgG/imunologia
4.
Methods Mol Biol ; 2095: 285-293, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31858474

RESUMO

To streamline cell culture process development, surface plasmon resonance (SPR) biosensors offer a versatile platform for the rapid quantification and quality analysis of recombinant proteins. As a representative case study, the present chapter details a procedure employing a SPR biosensor for determining the differential sialylation levels of recombinant interferon α2b contained in cell culture samples, using immobilized Sambucus nigra lectin. Of interest, this semiquantitative approach can be adapted to work with other lectins with unique carbohydrate-binding specificities, enabling a wide range of product characterization analysis.

5.
Langmuir ; 35(48): 15535-15542, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31478669

RESUMO

Challenges associated with nonspecific adsorption of proteins on sensor surfaces have steered the development of novel antifouling materials and strategies. Inspired by human synovial fluid composition and structure, we designed synergistic antifouling coatings with mixtures of hyaluronic acid (HA) and a zwitterionic bottlebrush polymer (BB). Using a fast and convenient online surface modification method, the polymers were immobilized on the Au surface, significantly increasing its hydrophilicity. Using surface plasmon resonance (SPR), a 10:1 ratio of HA to BB was found optimal to provide the best antifouling performance. Bovine serum albumin (BSA) adsorption on HA-BB coated surfaces was 0.2 ng/cm2, which was 60 times lower than BB or HA alone and 25 times lower than the commonly accepted ultralow adsorption limit (<5 ng/cm2), demonstrating the synergistic effect of HA and BB against nonspecific protein adsorption. This was found to be independent of BSA concentration up to physiological concentrations. Furthermore, the antifouling performance of HA-BB coated surfaces was tested against milk and serum, showing almost 92% lower protein adsorption than that on bare surfaces, suggesting the potential efficacy of this antifouling coating in real life settings.

6.
Langmuir ; 35(48): 15585-15591, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31333025

RESUMO

Using the surface force apparatus (SFA), the interaction forces between mica surfaces across ionic liquid (IL) solutions are studied. The IL solution, 1-hexyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide in propylene carbonate solvent, is used at different concentrations to elucidate the ions' conformation at the interface from the analysis of short-range structural forces. A direct correlation between the ion layer thickness at the interface and the IL molar fraction in the solution is observed, suggesting conformational changes relative to the ion packing density. In addition, effects of large microscopic and macroscopic water domains at the interface are investigated. The microscopic water domains induced significant adhesion at contact because of the long-range capillary forces, which are found to depend on solvent concentration. The macroscopic water domains entirely cover the interaction area, ensuring that the long-range interfacial interactions occur entirely across the aqueous electrolyte solution with dissolved IL ions as the electrolyte. These results help elucidate the interfacial interactions in IL-charged solid interfaces with practical importance in green energy storage, catalysis, and lubrication.

7.
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.

8.
MAbs ; 11(3): 435-452, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30822189

RESUMO

The N-glycosylation profile of immunoglobulin G (IgG) is considered a critical quality attribute due to its impact on IgG-Fc gamma receptor (FcγR) interactions, which subsequently affect antibody-dependent cell-based immune responses. In this study, we investigated the impact of the FcγR capture method, as well as FcγR N-glycosylation, on the kinetics of interaction with various glycoforms of trastuzumab (TZM) in a surface plasmon resonance (SPR) biosensor assay. More specifically, we developed a novel strategy based on coiled-coil interactions for the stable and oriented capture of coil-tagged FcγRs at the biosensor surface. Coil-tagged FcγR capture outperformed all other capture strategies applied to the SPR study of IgG-FcγR interactions, as the robustness and reproducibility of the assay and the shelf life of the biosensor chip were excellent (> 1,000 IgG injections with the same biosensor surface). Coil-tagged FcγRs displaying different N-glycosylation profiles were generated either by different expression systems, in vitro glycoengineering or by size-exclusion chromatography, and roughly characterized by lectin blotting. Of salient interest, the overlay of their kinetics of interaction with several TZM glycoforms revealed key differences on both association and dissociation kinetics, confirming a complex influence of the FcγR N-glycosylation and its inherent heterogeneity upon receptor interaction with mAbs. This work is thus an important step towards better understanding of the impact of glycosylation upon binding of IgGs, either natural or engineered, to their receptors.


Assuntos
Imunoglobulina G/química , Receptores de IgG/química , Ressonância de Plasmônio de Superfície , Trastuzumab/química , Animais , Células CHO , Cricetulus , Glicosilação , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , Receptores de IgG/metabolismo , Trastuzumab/metabolismo
9.
Angew Chem Int Ed Engl ; 58(5): 1308-1314, 2019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30426644

RESUMO

Demand for long-lasting antifouling surfaces has steered the development of accessible, novel, biocompatible and environmentally friendly materials. Inspired by lubricin (LUB), a component of mammalian synovial fluid with excellent antifouling properties, three block polymers offering stability, efficacy, and ease of use were designed. The bottlebrush-structured polymers adsorbed strongly on silica surfaces in less than 10 minutes by a simple drop casting or online exposure method and were extremely stable in high-salinity solutions and across a wide pH range. Antifouling properties against proteins and bacteria were evaluated with different techniques and ultralow fouling properties demonstrated. With serum albumin and lysozyme adsorption <0.2 ng cm-2 , the polymers were 50 and 25 times more effective than LUB and known ultralow fouling coatings. The antifouling properties were also tested under MPa compression pressures by direct force measurements using surface forces apparatus. The findings suggest that these polymers are among the most robust and efficient antifouling agents currently known.

10.
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
11.
Sci Rep ; 8(1): 12223, 2018 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-30111772

RESUMO

Photo-initiated chemical vapor deposition (PICVD) has been adapted for use in a jet-assisted fluidized bed configuration, allowing for the encapsulation of magnetic iron oxide nanoparticles on a larger scale than ever reported (5 g). This new methodology leads to a functional coating with a thickness of 1.4-10 nm, confirmed by HRTEM and TGA. XPS and TOF-SIMS characterization confirm that the coating is composed of both aliphatic and polymerized carbon chains, with incorporated organometallic bonds and oxygen-containing moieties. UV-Vis absorbance spectra show that the coating improved dispersion in non-polar solvents, such as n-dodecane. This process represents a first step towards the large-scale, solvent-free post-synthesis processing of nanoparticles to impart a functional coating.

12.
Biotechnol Prog ; 34(2): 494-504, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29314777

RESUMO

Lactate and ammonia accumulation is a major factor limiting the performance of fed-batch strategies for mammalian cell culture processes. In addition to the detrimental effects of these by-products on production yield, ammonia also contributes to recombinant glycoprotein quality deterioration. In this study, we tackled the accumulation of these two inhibiting metabolic wastes by culturing in glutamine-free fed-batch cultures an engineered HEK293 cell line displaying an improved central carbon metabolism. Batch cultures highlighted the ability of PYC2-overexpressing HEK293 cells to grow and sustain a relatively high viability in absence of glutamine without prior adaptation to the culture medium. In fed-batch cultures designed to maintain glucose at high concentration by daily feeding a glutamine-free concentrated nutrient feed, the maximum lactate and ammonia concentrations did not exceed 5 and 1 mM, respectively. In flask, this resulted in more than a 2.5-fold increase in IFNα2b titer in comparison to the control glutamine-supplied fed-batch. In bioreactor, this strategy led to similar reductions in lactate and ammonia accumulation and an increase in IFNα2b production. Of utmost importance, this strategy did not affect IFNα2b quality with respect to sialylation and glycoform distribution as confirmed by surface plasmon resonance biosensing and LC-MS, respectively. Our strategy thus offers an attractive and simple approach for the development of efficient cell culture processes for the mass production of high-quality therapeutic glycoproteins. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:494-504, 2018.


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Reatores Biológicos , Glicoproteínas/biossíntese , Proteínas Recombinantes/genética , Amônia/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Meios de Cultura/química , Glucose/metabolismo , Glutamina/metabolismo , Células HEK293 , Humanos , Ácido Láctico/metabolismo , Proteínas Recombinantes/química
13.
Appl Microbiol Biotechnol ; 101(21): 7837-7851, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28924963

RESUMO

Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFNα2b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFNα2b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions.


Assuntos
Interferon-alfa/isolamento & purificação , Interferon-alfa/metabolismo , Engenharia Metabólica/métodos , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Amônia/metabolismo , Sobrevivência Celular , Meios de Cultura/química , Expressão Gênica , Glucose/metabolismo , Células HEK293 , Humanos , Interferon alfa-2 , Interferon-alfa/química , Interferon-alfa/genética , Lactatos/metabolismo , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ressonância de Plasmônio de Superfície
14.
J Biomed Mater Res A ; 105(8): 2171-2181, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28380658

RESUMO

P15-CSP is a biomimetic cationic fusion peptide that stimulates osteogenesis and inhibits bacterial biofilm formation when coated on 2-D surfaces. This study tested the hypothesis that P15-CSP coatings enhance 3-D osteogenesis in a porous but otherwise hydrophobic poly-(ɛ-caprolactone) (PCL) scaffold. Scaffolds of 84 µm and 141 µm average pore size were coated or not with Layer-by-Layer polyelectrolytes followed by P15-CSP, seeded with adult primary human mesenchymal stem cells (MSCs), and cultured 10 days in proliferation medium, then 21 days in osteogenic medium. Atomic analyses showed that P15-CSP was successfully captured by LbL. After 2 days of culture, MSCs adhered and spread more on P15-CSP coated pores than PCL-only. At day 10, all constructs contained nonmineralized tissue. At day 31, all constructs became enveloped in a "skin" of tissue that, like 2-D cultures, underwent sporadic mineralization in areas of high cell density that extended into some 141 µm edge pores. By quantitative histomorphometry, 2.5-fold more tissue and biomineral accumulated in edge pores versus inner pores. P15-CSP specifically promoted tissue-scaffold integration, fourfold higher overall biomineralization, and more mineral deposits in the outer 84 µm and inner 141 µm pores than PCL-only (p < 0.05). 3-D Micro-CT revealed asymmetric mineral deposition consistent with histological calcium staining. This study provides proof-of-concept that P15-CSP coatings are osteoconductive in PCL pore surfaces with 3-D topography. Biomineralization deeper than 150 µm from the scaffold edge was optimally attained with the larger 141 µm peptide-coated pores. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2171-2181, 2017.


Assuntos
Materiais Revestidos Biocompatíveis/química , Colágeno/química , Células-Tronco Mesenquimais/citologia , Osteogênese , Fragmentos de Peptídeos/química , Poliésteres/química , Tecidos Suporte/química , Adulto , Diferenciação Celular , Células Cultivadas , Humanos , Porosidade , Engenharia Tecidual
15.
Biointerphases ; 12(1): 010501, 2017 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-28325051

RESUMO

In an effort to rationalize and optimize an antiapoptotic coating combining chondroitin sulfate (CS) and epidermal growth factor (EGF) for vascular applications, the authors here report the comparison of two grafting strategies aiming to display EGF in an oriented fashion on CS. For that purpose, the authors produced, purified, and characterized a chimeric protein corresponding to EGF that was N-terminally fused to a cysteine and a coil peptide. The chimera was covalently immobilized via its free thiol group or captured via coiled-coil interactions at the surface of a biosensor or on a chondroitin sulfate coating in multiwell plates, mimicking the coating that was previously developed by them for stent-graft surfaces. The interactions of grafted EGF with the soluble domain of its receptor or the impact of grafted EGF upon vascular smooth muscle survival in proapoptotic conditions indicated that the coiled-coil based tethering was the best approach to display EGF. These results, combined to direct enzyme-linked immunosorbent assay measurements, indicated that the coiled-coil tethering approach allowed increasing the amount of bioavailable EGF when compared to covalent coupling, rather than the total amount of grafted EGF, while using much lower concentrations of tagged EGF during incubation.


Assuntos
Sulfatos de Condroitina/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacocinética , Proteínas Imobilizadas/metabolismo , Proteínas Imobilizadas/farmacocinética , Animais , Disponibilidade Biológica , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/fisiologia , Ligação Proteica , Ratos
16.
Langmuir ; 33(8): 1780-1791, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28182436

RESUMO

Syngas is the product of gasification processes and is used for the production of petrochemicals. Little attention has been paid to its use in the production of oligomeric thin films under ambient conditions. Herein, the nature of the photoinitiated chemical vapor deposition of films made from syngas using high-wavelength ultraviolet light is discussed, including an exploration of the oligomeric films' structure, synthesis mechanism, and growth kinetics. Specifically, X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry analyses provide insight into the chemical structure, illustrating the effect of photogenerated radicals in the formation of aliphatic, anhydride, and cyclic structures. The films are covalently bonded to the substrate and chemically uniform. Electron and atomic force microscopy identify an islandlike morphology for the deposit. These insights into the mechanism and structure are linked to processing parameters through a study on the effect of residence time and treatment duration on the deposition rate, as determined through profilometry.

17.
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
18.
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
19.
Biomacromolecules ; 18(3): 965-975, 2017 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-28122454

RESUMO

We have developed a heterodimeric coiled-coil system based on two complementary peptides, namely (EVSALEK)5 and (KVSALKE)5, or E and K, for the attachment of E-tagged biomolecules onto K-decorated biomaterials. We here explore two approaches to control the strength and the stability of the E/K coiled-coil complex, and thus its potential for the controlled release of biomolecules. Those are Leucine-to-Alanine mutations in the K peptide (4 peptides with 0 to 3 mutations) and multivalent presentation of the E peptide (6 bio-objects from monomeric to dimeric and n-meric). Using E-tagged growth factors and nanoparticles as models, SPR-based assays performed under continuous flow indicated that the release rate was strongly affected by both approaches independently, and that the strength of the capture could be finely tuned over a wide range (apparent dissociation constant from 0.12 pM to 270 nM). Further release assays carried out in well-plates showed that the multivalent presentation only had a significant influence in this setup since the wells were not rinsed under continuous flow.


Assuntos
Materiais Biocompatíveis/química , Preparações de Ação Retardada/química , Peptídeos/química , Sequência de Aminoácidos , Dicroísmo Circular , Dimerização , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Modelos Moleculares , Mutação , Nanopartículas/química
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