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1.
Artigo em Inglês | MEDLINE | ID: mdl-32036254

RESUMO

Antibody-drug conjugates (ADCs) have become major biopharmaceutical drugs in the field of oncology. Traditional ADCs possess a stochastic distribution of cytotoxic payloads linked to several different amino acid residues of the antibody. This heterogeneous nature of stochastic ADCs results in a complex conjugation-site characterization. To improve upon traditional ADC technology, we have developed a chemical conjugation platform, termed AJICAP™, for site-specific modification of native antibodies using a class of IgG Fc-affinity reagents (Yamada et al., 2019). Here, we report further investigation focusing on peptide mapping of the AJICAP™-ADC to confirm the exact conjugation position of the first generation AJICAP™-ADC. Neutral pH pretreatment for peptide mapping prevented undesired PTMs such as succinimide ring hydrolysis. Mirroring comparison using the purified ADC visibly indicated that Lys248 in the Fc region was conjugated to the drug-linker. MS/MS analysis also provided evidence to support Lys248 conjugation. Finally, extracted ion-chromatogram methodology suggested the site-specificity of AJICAP™ conjugation. Purified ADCs by preparative HIC-HPLC showed clear visual results and more than 93% sequence coverage by a single enzymatic digestion. The analytical strategy described herein demonstrated a robust analytical methodology for revealing the conjugation site of ADCs.


Assuntos
Antineoplásicos/química , Imunoconjugados/química , Antineoplásicos/farmacologia , Sítios de Ligação , Imunoconjugados/farmacologia , Fragmentos Fc das Imunoglobulinas/química , Lisina/química , Oligopeptídeos/química , Mapeamento de Peptídeos , Ligação Proteica , Succinimidas/química , Trastuzumab/química
2.
Comput Biol Chem ; 84: 107161, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31787580

RESUMO

The present study is focused on a series of newly synthesized 1-aryl-3-ethyl-3-methylsuccinimide derivatives, as potential anticonvulsants. The retention behavior of eleven succinimide derivatives was determined by using reversed phase high performance liquid chromatography (RP-HPLC) and reversed phase high performance thin layer chromatography (RP-HPTLC). The estimated retention behavior was correlated with partition (logP) and distribution coefficients (logD). These high correlations pointed out that the determined retention parameters (logk0 and RM0) can be considered chromatographic (anisotropic) lipophilicity of the studied succinimide derivatives. The structural properties, which dominantly affect the chromatographic lipophilicity, were determined as well. The significant correlations between the chromatographic lipophilicity and plasma protein binding (PPB), Madin-Darby Canine Kidney (MDCK) cells permeability, volume of distribution (Vd) and absorption constant (Ka) indicate the strong influence of lipophilicity on pharmacokinetics of 1-aryl-3-ethyl-3-methylsuccinimide derivatives. These derivatives have also been tested applying Comprehensive Medicinal Chemistry (CMC) drug-like rules which confirmed their drug-like properties. Besides, their blood-brain penetration (BBB) ability has been estimated applying the set of Clark's rules and by using Pre-ADMET software. Regarding toxicity, it was predicted that only one compound from the set might have toxic effects by blocking the hERG potassium channel. The present study reveals which molecular features in the structure of novel succinimide derivatives could be crucial for their lipophilicity, and consequently for their pharmacokinetic properties. The results indicate that the newly synthesized series of succinimide derivatives should be further considered in design of novel anticonvulsants.


Assuntos
Anticonvulsivantes/química , Succinimidas/química , Animais , Anisotropia , Anticonvulsivantes/farmacocinética , Células CACO-2 , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Delgada , Simulação por Computador , Cães , Humanos , Interações Hidrofóbicas e Hidrofílicas , Absorção Intestinal , Células Madin Darby de Rim Canino , Succinimidas/farmacocinética
3.
Molecules ; 24(19)2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591356

RESUMO

The work reports the use of polyamidoamine dendrimers (PAMAM) and a cross-linking agent, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) or 4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), for the thermal stabilization of dermal bovine collagen. The efficiency of EDC/NHS/PAMAM and DMTMM/PAMAM in the cross-linking of collagen is correlated to the increase of the collagen shrinkage temperature (Ts), measured by differential scanning calorimetry (DSC). An alternative enzymatic protocol was adopted to measure the degradability of EDC/NHS/PAMAM tanned hides; these data are correlated to the thermal stability values measured by DSC. In the presence of PAMAMs, EDC/NHS provides very high stabilization of bovine dermal collagen, giving Ts of up to 95 °C, while DMTMM achieves lower stabilization. Preliminary tanning tests carried out in best reaction conditions show that EDC/NHS/PAMAM could be an interesting, environmentally-sustainable tanning system which is completely free of metals, formaldehyde, and phenols. Two new unreported dendrimeric species were synthesized and employed.


Assuntos
Colágeno/química , Reagentes para Ligações Cruzadas/química , Dendrímeros/química , Animais , Varredura Diferencial de Calorimetria , Bovinos , Colagenases/metabolismo , Ensaios Enzimáticos , Succinimidas/química , Curtume , Temperatura
4.
Nanoscale ; 11(32): 15120-15130, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31369017

RESUMO

The piezoelectricity of collagen is purported to be linked to many biological processes including bone formation and wound healing. Although the piezoelectricity of tissue-derived collagen has been documented across the length scales, little work has been undertaken to characterise the local electromechanical properties of processed collagen, which is used as a base for tissue-engineering implants. In this work, three chemically distinct treatments used to form structurally and mechanically stable scaffolds-EDC-NHS, genipin and tissue transglutaminase-are investigated for their effect on collagen piezolectricity. Crosslinking with EDC-NHS is noted to produce a distinct self-assembly of the fibres into bundles roughly 300 nm in width regardless of the collagen origin. These fibre bundles also show a localised piezoelectric response, with enhanced vertical piezoelectricity of collagen. Such topographical features are not observed with the other two chemical treatments, although the shear piezoelectric response is significantly enhanced upon crosslinking. These observations are reconciled by a proposed effect of the crosslinking mechanisms on the molecular and nanostructure of collagen. These results highlight the ability to modify the electromechanical properties of collagen using chemical crosslinking methods.


Assuntos
Colágeno/química , Reagentes para Ligações Cruzadas/química , Módulo de Elasticidade , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Iridoides/química , Iridoides/metabolismo , Microscopia de Força Atômica , Nanoestruturas/química , Succinimidas/química , Engenharia Tecidual , Transglutaminases/química , Transglutaminases/metabolismo
5.
Eur J Med Chem ; 179: 805-827, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31295714

RESUMO

A novel series of benzylidene-succinimide derivatives were synthesized, characterized and evaluated for their cytotoxicities against HCT116, and SW480 cancer cells and NCM460 normal human cells. Their antiangiogenic capabilities were evaluated using a chick chorioallantoic membrane (CAM) assay. The compound, XCF-37b, was selected as the most potent antiangiogenic inhibitor with noncytotoxicity to evaluate the pharmacological effects on human umbilical vein endothelial cells (HUVECs) and cancer cells in vivo and in vitro. The results showed that XCF-37b inhibited HT29-cell colon tumor growth in vivo, without showing cytotoxicity against the five other cancer cell lines in vitro. Experiments confirmed that XCF-37b had obvious antiangiogenic activity by HUVEC migration and invasion and rat aortic ring angiogenesis ex vivo. Mechanism studies showed that XCF-37b inhibited the AKT/mTOR and VEGFR2 signaling pathways, as evidenced by decreased expressions of phosphor-AKT (p-AKT), p-mTOR, p-VEGFR2 (Tyr175), p-Src (Tyr416), p-FAK (Tyr925), and p-Erk1/2 (Thr202/Tyr204). Moreover, XCF-37b significantly decreased the protein expressions of matrix metalloproteinase-2 (MMP-2), MMP-9 and hypoxia-inducible factor-1α (HIF-1α). XCF-37b generally regulated angiogenic inhibition through several regulatory pathways, without significantly interfering with colorectal cancer cell growth.


Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Compostos de Benzilideno/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neovascularização Patológica/tratamento farmacológico , Succinimidas/farmacologia , Inibidores da Angiogênese/síntese química , Inibidores da Angiogênese/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Compostos de Benzilideno/química , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Galinhas , Neoplasias Colorretais/patologia , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Neovascularização Patológica/patologia , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Succinimidas/química , Cicatrização/efeitos dos fármacos
6.
Int J Mol Sci ; 20(13)2019 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-31261853

RESUMO

Conjugation of latent growth factors to superparamagnetic iron oxide nanoparticles (SPIONs) is potentially useful for magnetically triggered release of bioactive macromolecules. Thus, the goal of this work was to trigger the release of active Transforming Growth-Factor Beta (TGF-ß) via magnetic hyperthermia by binding SPIONs to the latent form of TGF-ß, since heat has been shown to induce release of TGF-ß from the latent complex. Commercially available SPIONS with high specific absorption rates (SAR) were hydrolyzed in 70% ethanol to create surface carboxylic acid conjugation sites for carbodiimide chemistry. Fourier-Transform Infra-Red (FTIR) analysis verified the conversion of maleic anhydride to maleic acid. 1-Ethyl-2-(3-dimethyulaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide (Sulfo-NHS) were used to bind to the open conjugation sites of the SPION in order to graft latent TGF-ß onto the particles. The resulting conjugated particles were imaged with transmission electron microscopy (TEM), and the complexed particles were characterized by dynamic light scattering (DLS) and superconducting quantum interference device (SQUID) magnetometry. Enzyme-linked immunosorbent assay (ELISA) was used to assess the thermally triggered release of active TGF-ß from the latent complex, demonstrating that conjugation did not interfere with release. Results showed that latent TGF-ß was successfully conjugated to the iron oxide nanoparticles, and magnetically triggered release of active TGF-ß was achieved.


Assuntos
Carbodi-Imidas/química , Nanopartículas Metálicas/química , Nanoconjugados/química , Fator de Crescimento Transformador beta/química , Liberação Controlada de Fármacos , Compostos Férricos/química , Campos Magnéticos , Succinimidas/química , Fator de Crescimento Transformador beta/administração & dosagem
7.
Molecules ; 24(14)2019 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-31340597

RESUMO

Quorum sensing (QS) signaling system is important for bacterial growth, adhesion, and biofilm formation resulting in numerous infectious diseases. Dihydropyrrol-2-ones (DHPs) represent a novel class of antimicrobial agents that inhibit QS, and are less prone to develop bacterial resistance due to their non-growth inhibition mechanism of action which does not cause survival pressure on bacteria. DHPs can prevent bacterial colonization and quorum sensing when covalently bound to substrates. In this study, the role of orientation of DHP compounds was investigated after covalent attachment by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling reaction to amine-functionalized glass surfaces via various positions of the DHP scaffold. The functionalized glass surfaces were characterized by X-ray photoelectron spectroscopy (XPS) and contact angle measurements and tested for their in vitro biological activity against S. aureus and P. aeruginosa. DHPs attached via the N-1 position resulted in the highest antibacterial activities against S. aureus, while no difference was observed for DHPs attached either via the N-1 position or the C-4 phenyl ring against P. aeruginosa.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pirrolidinonas/farmacologia , Percepção de Quorum/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/síntese química , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Carbodi-Imidas/química , Reagentes para Ligações Cruzadas/química , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pirrolidinonas/síntese química , Staphylococcus aureus/crescimento & desenvolvimento , Relação Estrutura-Atividade , Succinimidas/química
8.
Int J Biol Macromol ; 138: 1-12, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31302127

RESUMO

In this study, a CotA laccase from Bacillus subtilis cjp3 was successfully immobilized onto magnetic graphene oxide (MGO) nanomaterials via covalent bonding with hydrochloride/N-hydroxysuccinimide (EDC/NHS). The morphology, structure, and properties of the MGO-laccase were then characterized by scanning-electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray-photoelectron spectroscopy (XPS), and a magnetic-property-measurement system (MPMS). The magnetic composite exhibited an extremely high binding capacity of ~145.04mg/g and maintained maximal relative enzyme activities at 25°C, pH7, and a reaction time of 2h. The pH, thermal, operational, and storage stabilities of MGO-laccase were significantly improved over those of free laccase. Moreover, MGO-laccase exhibited a higher tolerance than that of free laccase in the presence of organic solvents, inhibitors, metal ions, and salts. Furthermore, MGO-laccase showed good decolorization performance of malachite green (MG), with decolorization rates reaching 99% after 5h of reaction at 30°C and pH6. In addition, the maximum saturation magnetization of MGO-laccase was 27.7emu/g, allowing for rapid magnetic separation. Accordingly, magnetic separation allowed MGO-laccase to maintain 75% of its activity after ten consecutive decolorization cycles.


Assuntos
Bacillus subtilis/enzimologia , Grafite/química , Lacase/química , Lacase/metabolismo , Imãs/química , Corantes de Rosanilina/metabolismo , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática , Enzimas Imobilizadas/antagonistas & inibidores , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Cinética , Lacase/antagonistas & inibidores , Metais/farmacologia , Corantes de Rosanilina/isolamento & purificação , Sais/farmacologia , Solventes/farmacologia , Succinimidas/química
9.
Protein Pept Lett ; 26(10): 758-767, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31215362

RESUMO

BACKGROUND: Herein we report the multigram-scale synthesis, characterization and application of a rhodamine B-based fluorophore (ROSA) suitable for fluorescent studies in biological applications. This fluorophore is devoid of rhodamine spirolactone formation and furthermore characterized by a high molar extinction coefficient (ϵ=87250 ± 1630 M-1cm-1) and quantum yield (φ) of 0.589 ± 0.070 in water. Reported here is also the application of ROSA towards synthesis of a ROSA-PEG-GRGDS-NH2 fluorescent probe suitable for live cell imaging of αvß3 integrins for in vitro assays. OBJECTIVES: The main objective of this study is to efficiently prepare rhodamine B derivative, devoid of spirolactone formation that would be suitable for bioconjugation and subsequent bioimaging. METHODS: Rhodamine B was transformed into rhodamine B succinimide ester (RhoB-OSu) using N-hydroxysuccinimide. RhoB-OSu was further coupled to sarcosine to obtain rhodamine Bsarcosine dye (ROSA) in good yield. The ROSA dye was then coupled to a αvß3 integrin binding sequence using standard solid-phase conditions. Resulting ROSA-PEG-GRGDS-NH2 probe was used to image integrins on cancer cells. RESULTS: The rhodamine B-sarcosine dye (ROSA) was obtained in multigram scale in good total yield of 47%. Unlike rhodamine B, the ROSA dye does not undergo pH-dependent spirolactone/spirolactam formation as compared with rhodamine B-glycine. It is also characterized by excellent quantum yield (φ) of 0.589 ± 0.070 in water and high molar extinction coefficient of 87250 ± 1630 M-1cm-1. ROSA coupling to the RGD-like peptide was proved to be efficient and straightforward. Imaging using standard filters on multimode plate reader and confocal microscope was performed. The αvß3 integrins present on the surface of live WM-266-4 (melanoma) and MCF- 7 (breast cancer) cells were successfully imaged. CONCLUSION: We successfully derivatized rhodamine B to create an inexpensive, stable and convenient to use fluorescent probe. The obtained derivative has excellent photochemical properties and it is suitable for bioconjugation and many imaging applications.


Assuntos
Corantes Fluorescentes/síntese química , Integrina alfaVbeta3/química , Imagem Óptica/métodos , Rodaminas/síntese química , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Oligopeptídeos/química , Succinimidas/química
10.
ACS Appl Mater Interfaces ; 11(26): 22973-22978, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31252497

RESUMO

Misfolding and abnormal assembly of proteins cause many intractable diseases. The modulation of the assembly process of these proteins could contribute to understanding and controlling amyloid protein aggregation. Previous works focused mainly on the inhibition of the assembly process. To broaden the interaction modality of modulators with proteins for developing new modulators, in this work, we designed and synthesized two reactive poly ( p-phenylene vinylene) polymers, respectively, functionalized with N-hydroxysuccinimide ester (PPV-NHS) and pentafluorophenol ester (PPV-PFP), which exhibited the prevention or co-assembly effect on the aggregation process of islet amyloid polypeptide (IAPP). Cell assays demonstrated that both of the two polymers could effectively eliminate the cytotoxicity of IAPP. Moreover, PPV-NHS also could irreversibly disrupt preformed IAPP fibrils. We envision that PPV-NHS and PPV-PFP might offer a new design method for the modulation of protein assembly.


Assuntos
Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Polímeros/química , Agregação Patológica de Proteínas/tratamento farmacológico , Sequência de Aminoácidos/genética , Ésteres/síntese química , Ésteres/química , Ésteres/farmacologia , Fluorbenzenos/síntese química , Fluorbenzenos/química , Fluorbenzenos/farmacologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/síntese química , Polipeptídeo Amiloide das Ilhotas Pancreáticas/farmacologia , Fenóis/síntese química , Fenóis/química , Fenóis/farmacologia , Polímeros/síntese química , Polímeros/farmacologia , Agregação Patológica de Proteínas/genética , Succinimidas/síntese química , Succinimidas/química , Succinimidas/farmacologia
11.
PLoS One ; 14(6): e0218686, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31242246

RESUMO

Immunochemical techniques are the workhorse for sample enrichment and detection of a large variety of analytes. In contrast to classical microtiter plate-based assays, microparticles are a next generation solid support, as they promote automation of immunoassays using flow-based techniques. Antibody immobilization is a crucial step, as these reagents are expensive, and inefficient coupling can result in low sensitivities. This paper proposes a general procedure for efficient immobilization of antibodies onto TentaGel particles, via N-hydroxysuccinimide chemistry. The goal was the preparation of solid supports with optimum immunorecognition, while increasing the sustainability of the process. The influence of buffer composition, activation and coupling time, as well as the amount of antibody on the immobilization efficiency was investigated, resorting to fluorophore-labeled proteins and fluorescence imaging. Buffer pH and activation time are the most important parameters for efficient coupling. It is demonstrated, that the hydrolysis of N-hydroxysuccinimide esters occurs at similar rates as in solution, limiting the utilizable time for coupling. Finally, applicability of the generated material for automated affinity extraction is demonstrated on the mesofluidic platform lab-on-valve.


Assuntos
Anticorpos Imobilizados/química , Imunoensaio/métodos , Imunoconjugados/química , Animais , Automação/métodos , Carbamazepina/análise , Carbamazepina/imunologia , Ensaio de Imunoadsorção Enzimática , Desenho de Equipamento , Humanos , Imunoensaio/instrumentação , Imunoglobulina G/química , Dispositivos Lab-On-A-Chip , Camundongos , Microesferas , Poliestirenos , Succinimidas/química
12.
Int J Mol Sci ; 20(10)2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31096657

RESUMO

Aspartic acid (Asp) residues are prone to non-enzymatic stereoinversion, and Asp-residue stereoinversion is believed to be mediated via a succinimide (SI) intermediate. The stereoinverted Asp residues are believed to cause several age-related diseases. However, in peptides and proteins, few studies have reported the stereoinversion of glutamic acid (Glu) residues whose structures are similar to that of Asp. We previously presumed that Glu-residue stereoinversion proceeds via a glutarimide (GI) intermediate and showed that the calculated activation barriers of SI- and GI-intermediate stereoinversion are almost equivalent in the gas phase. In this study, we investigated the stereoinversion pathways of the l-GI intermediate in the aqueous phase using B3LYP density functional methods. The calculated activation barrier of l-GI-intermediate stereoinversion in the aqueous phase was approximately 36 kcal·mol-1, which was much higher than that in the gas phase. Additionally, as this activation barrier exceeded that of Asp-residue stereoinversion, it is presumed that Glu-residue stereoinversion has a lower probability of proceeding under physiological conditions than Asp-residue stereoinversion.


Assuntos
Ácido Aspártico/química , Resistência a Medicamentos , Ácido Glutâmico/química , Piperidonas/química , Estereoisomerismo , Água/química , Catálise , Estrutura Molecular , Peptídeos/química , Proteínas/química , Succinimidas/química
13.
Luminescence ; 34(5): 512-519, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30972942

RESUMO

Acridinium salts, due to their chemiluminogenic properties, have found several applications in biomedical analysis as labels and indicators, where the assessment of emission intensity is used for the end-point detection. This work presents the use of chemiluminescent indicators in the form of selected acridinium esters in order to determine the antioxidant properties of exemplary formulations, namely quercetin, vitamin C and the dietary supplement, Apiextract. The principle of measurements is based on a change in the kinetics of emission decay derived from the acridinium cations in alkaline solutions of hydrogen peroxide in the presence of an antioxidant (the analyte). The proposed system makes a beneficial alternative to related methods, which mostly rely on the assessment of emission efficiency and use the luminometric standard luminol - due to superior parameters of acridinium chemiluminescence, among others - high temporary emission efficiency. The features of the proposed method are manifested by a shorter time period of analysis and lower background signals associated with the environmental influences, as compared to typical approaches. The chromatographic (RP-HPLC) analyses of the substrates and products generated during chemiluminogenic oxidation of acridinium cations under assay conditions are also presented.


Assuntos
Acridinas/química , Antioxidantes/química , Suplementos Nutricionais/análise , Medições Luminescentes/métodos , Succinimidas/química , Cinética , Luminescência , Luminol/química
14.
Int J Nanomedicine ; 14: 2127-2144, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30988613

RESUMO

Purpose: A promising vascular scaffold must possess satisfying mechanical properties, great hemocompatibility, and favorable tissue regeneration. Combining natural with synthetic materials is a popular method of creating/enhancing such scaffolds. However, the effect of additional modification on the materials requires further exploration. Materials and methods: We selected polycaprolactone (PCL), which has excellent mechanical properties and biocompatibility and can be combined with collagen. Electrospun fibers created using a PCL/collagen solution were used to fashion mixed nanofibers, while separate syringes of PCL and collagen were used to create separated nanofibers, resulting in different pore sizes. Mixed and separated nanofibers were cross-linked with glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and genipin; hence, we named them as mixed GA, mixed EDC (ME), mixed genipin (MG), separated GA, separated EDC (SE), and separated genipin (SG). Results: Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction showed that cross-linking did not affect the main functional groups of fibers in all groups. ME, MG, SE, and SG met the requisite mechanical properties, and they also resisted collagenase degradation. In hemocompatibility assays, only ME and MG demonstrated ideal safety. Furthermore, ME and MG presented the greatest cytocompatibility. For vascular scaffolds, rapid endothelialization helps to prevent thrombosis. According to human umbilical vein endothelial cell migration on different nanofibers, ME and MG are also successful in promoting cell migration. Conclusion: ME and MG may be promising candidates for vascular tissue engineering. The study suggests that collagen cross-linked by EDC/N-hydroxysuccinimide or genipin facilitates endothelial cell regeneration, which could be of great benefit in tissue engineering of vascular scaffolds.


Assuntos
Colágeno/química , Células Endoteliais/citologia , Iridoides/química , Nanofibras/administração & dosagem , Poliésteres/química , Succinimidas/química , Tecidos Suporte/química , Animais , Materiais Biocompatíveis/química , Carbodi-Imidas/química , Movimento Celular , Células Cultivadas , Reagentes para Ligações Cruzadas/química , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Teste de Materiais , Nanofibras/química , Adesividade Plaquetária , Coelhos , Ratos , Regeneração , Engenharia Tecidual/métodos
15.
Anal Sci ; 35(7): 811-813, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-30930352

RESUMO

An immunosensor based on surface plasmon resonance was applied to detect mast cells expressing c-Kit. Sufficient detection of the mast cells was achieved by covalent immobilization of gelatin firstly on the sensor surface and followed by covalent binding of the anti-c-Kit antibody to lysine residues in the gelatin molecules through bis(sulfosuccinimidyl)suberate (BS3) treatment. By using BS3, which is a homo-bifunctional reagent, the lysine residues of the anti-c-Kit antibody easily bound to the lysine residues of the gelatin in the physiological condition. The lower limit of detection was 104 cells/mL.


Assuntos
Anticorpos Imobilizados/química , Gelatina/química , Regulação da Expressão Gênica , Imunoensaio/métodos , Mastócitos/citologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos Imobilizados/imunologia , Ácidos Decanoicos/química , Humanos , Limite de Detecção , Mastócitos/metabolismo , Camundongos , Proteínas Proto-Oncogênicas c-kit/imunologia , Succinimidas/química
16.
Biotechnol Appl Biochem ; 66(4): 564-573, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31021013

RESUMO

The rheological behaviors of a polyanionic collagen, fabricated using poly(γ-glutamic acid)-N-hydroxysuccinimide (γ-PGA-NHS) as a novel modifier, were investigated in this study. It was found that both of the native and modified collagen solutions were pseudoplastic fluids, as shown from the steady-shear tests. While the storage modulus and loss modulus of collagen increased with increasing the amount of γ-PGA-NHS, or with increasing the degree of esterification of γ-PGA-NHS; meanwhile, the dynamic denaturation temperature determined by dynamic temperature sweep was also increased, indicating an improved thermal stability of collagen solution modified by γ-PGA-NHS. The creep-recovery measurements showed that the resistance to deformation was enhanced for modified collagen, probably due to the cross-linking occurred between the ε-amino groups of collagen molecules and α-COOH groups of γ-PGA-NHS, as well as the electrostatic interaction and hydrogen-bond interactions between the two molecules. Furthermore, the aggregation of collagen fibers was promoted due to these interactions between collagen and γ-PGA-NHS as observed by atomic force morphology. In addition, the modified collagen exhibited good cytocompatibility as demonstrated by cell growth culturing. The obtained information was expected to give valuable clues to the design and fabrication of controlled stable collagen-based products for applications in various biomedical fields.


Assuntos
Colágeno/química , Ésteres/química , Ácido Poliglutâmico/análogos & derivados , Polímeros/química , Succinimidas/química , Estrutura Molecular , Ácido Poliglutâmico/química , Reologia , Temperatura
17.
Eur J Pharm Biopharm ; 139: 161-167, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30898541

RESUMO

AIM: The aim of the study was to establish a novel type of covalently mucus-binding polymers by targeting selectively amino groups within mucus glycoproteins. METHODS: N-Hydroxysuccinimide (NHS) was attached to carboxylic groups of polyacrylic acid (PAA). The reaction was mediated by the coupling reagent N,N'-dicyclohexylcarbodiimide (DCC) achieving polymeric NHS esters being able to form amide bonds with free amino groups. The chemical structure of the obtained conjugates was characterized via FTIR- and UV spectroscopy. Reactivity towards mucosal amino groups was evaluated UV spectrometrically upon addition of L-glycine. Furthermore, tensile force evaluations on intestinal mucosa as well as rheological experiments with mucus were performed in order to prove mucoadhesive potential. RESULTS: Depending on the amount of NHS added to the synthesis, coupling rates of 876 to 1820 µmol NHS per gram polymer were obtained. Kinetic studies of amide bond formation showed a substrate dependent reaction velocity. Rheological synergism of PAA-NHS was proven by a 7.9-fold increased mucus viscosity compared to the control polymer. In further mucoadhesion studies PAA-NHS showed a 5.5-fold improved adhesion time compared to unmodified PAA. Tensile force evaluation confirmed these results with a 1.7-fold higher maximum detachment force (MDF) and 2.7-fold increased total work adhesion (TWA) for PAA-NHS compared to the unmodified control polymer. CONCLUSION: The results of the present study provide strong evidence that coupling NHS to polymers could be a promising tool for the development of novel mucoadhesive excipients.


Assuntos
Resinas Acrílicas/química , Sistemas de Liberação de Medicamentos/métodos , Excipientes/química , Succinimidas/química , Adesividade , Células CACO-2 , Química Farmacêutica , Humanos , Mucosa Intestinal/metabolismo , Reologia
18.
BMC Microbiol ; 19(1): 67, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30909866

RESUMO

BACKGROUND: Neisseria gonorrhoeae is an obligate human pathogen and its adherence to host cells is essential for its pathogenesis. Gonococcal adherence assays are based on the enumeration of bacteria attached to human cells on solid media. Because conventional adherence assays are based on bacterial counts, they are often time consuming to perform and prone to observer bias. A flow cytometry based method, using the cell-permeable fluorescent dye 5'-carboxyfluoroscein succidyl ester (CFSE), was developed to dramatically increase the number of adherent N. gonorrhoeae quantified per assay while improving repeatability and removing observer bias. Piliated N. gonorrhoeae F62 were stained with CFSE then the staining reaction was quenched with foetal bovine serum. Human cervical ME-180 cells were infected with CFSE-stained N. gonorrhoeae (multiplicity of the infection 100:1) for 2 h. Infected cells were washed to remove loosely adhered bacteria. Flow cytometry was used to quantify the percentage of ME-180 cells associated with CFSE-stained N. gonorrhoeae and a minimum of 30,000 events were recorded. Real time-PCR analysis targeting opa gene (encoding N. gonorrhoeae opacity associated gonococcal outer membrane protein) was performed on infected ME-180 cells to confirm the flow cytometric adherence assay results. A rabbit was immunized with heat-killed N. gonorrhoeaeF62 to generate hyperimmune serum. The functional compatibility of the assay was confirmed by studying the effect of N. gonorrhoeae F62 antiserum on blocking adherence/invasion of CFSE-stained bacteria to ME-180 cells. RESULTS: We observed that 20.3% (+/- 1.0) ME-180 cells were associated with CFSE-stained N. gonorrhoeae. Heat-inactivated hyperimmune serum, at 1:10 to 1:80 dilutions, significantly inhibited gonococcal adherence by 6 and 3 fold, respectively. Real time-PCR analysis targeting opa gene confirmed that hyperimmune serum blocked adherence/invasion of N. gonorrhoeae to the ME-180 cells in a dilution-dependent manner. CONCLUSIONS: Flow cytometric analysis was amenable to quick, easy and high-throughput quantification of the association of N. gonorrhoeae with ME-180 cells and was functionally confirmed using PCR analysis. These approaches may be adapted for in vitro and in vivo adherence studies related to gonococcal pathogenesis.


Assuntos
Aderência Bacteriana , Citometria de Fluxo/métodos , Fluoresceínas/química , Corantes Fluorescentes/química , Neisseria gonorrhoeae/fisiologia , Succinimidas/química , Proteínas da Membrana Bacteriana Externa/genética , Linhagem Celular , Colo do Útero/citologia , Células Epiteliais/microbiologia , Feminino , Humanos , Neisseria gonorrhoeae/genética
19.
Biologicals ; 59: 1-5, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30910309

RESUMO

Therapeutic recombinant monoclonal antibodies are subject to various modifications during cell culture, and to a lesser degree, during purification. These modifications are expected to remain relatively constant during storage with the protection of appropriate formulations. However, after administration to patients, the levels of modifications may vary over time in circulation, where the recombinant monoclonal antibodies are exposed to the physiological conditions. Scientific understanding of those in vivo modifications can help drug candidate selection to choose the most stable molecules and set appropriate specifications for product release, which ultimately ensures safety and efficacy.


Assuntos
Anticorpos Monoclonais/química , Preparações Farmacêuticas/química , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Asparagina/química , Asparagina/metabolismo , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Glutamina/química , Glutamina/metabolismo , Glicosilação , Humanos , Lisina/química , Lisina/metabolismo , Preparações Farmacêuticas/metabolismo , Proteínas Recombinantes/sangue , Proteínas Recombinantes/metabolismo , Succinimidas/química , Succinimidas/metabolismo , Fatores de Tempo
20.
Nucleic Acids Res ; 47(6): 3223-3232, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30759226

RESUMO

Natural products that target the eukaryotic ribosome are promising therapeutics to treat a variety of cancers. It is therefore essential to determine their molecular mechanism of action to fully understand their mode of interaction with the target and to inform the development of new synthetic compounds with improved potency and reduced cytotoxicity. Toward this goal, we have previously established a short synthesis pathway that grants access to multiple congeners of the lissoclimide family. Here we present the X-ray co-crystal structure at 3.1 Å resolution of C45, a potent congener with two A-ring chlorine-bearing stereogenic centers with 'unnatural' configurations, with the yeast 80S ribosome, intermolecular interaction energies of the C45/ribosome complex, and single-molecule FRET data quantifying the impact of C45 on both human and yeast ribosomes. Together, these data provide new insights into the role of unusual non-covalent halogen bonding interactions involved in the binding of this synthetic compound to the 80S ribosome.


Assuntos
Produtos Biológicos/química , Diterpenos/química , Modelos Moleculares , Ribossomos/química , Succinimidas/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Diterpenos/síntese química , Células Eucarióticas/química , Humanos , Ligação Proteica , RNA Ribossômico/química , RNA Ribossômico/genética , Ribossomos/genética , Saccharomyces cerevisiae/química , Succinimidas/síntese química
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