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2.
Int J Nanomedicine ; 14: 6971-6988, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31507319

RESUMO

Background and purpose: Ginsenoside Rg5 (Rg5), a triterpene saponin, extracted from the natural herbal plant ginseng, is one of the most potent anticancer drugs against various carcinoma cells. However, the therapeutic potential of Rg5 is limited by its low solubility in water, poor bioavailability, and nontargeted delivery. Therefore, we prepared folic acid (FA)-modified bovine serum albumin (BSA) nanoparticles (FA-Rg5-BSA NPs) to improve the therapeutic efficacy and tumor targetability of Rg5. Methods: Various aspects of the FA-Rg5-BSA NPs were characterized, including size, polydispersity, zeta potential, morphology, entrapment efficiency (EE), drug loading (DL), in vitro drug release, thermal stability, in vitro cytotoxicity, cell apoptosis, cellular uptake, in vivo antitumor effects and in vivo biodistribution imaging. Results: The FA-Rg5-BSA NPs showed a particle size of 201.4 nm with a polydispersity index of 0.081, uniform spherical shape, and drug loading of 12.64±4.02%. The aqueous solution of FA-Rg5-BSA NPs had favorable stability for 8 weeks at 4°C. The FA-Rg5-BSA NPs dissolved under acidic conditions. Moreover, the Rg5-BSA NPs and FA-Rg5-BSA NPs had advanced anticancer activity compared with Rg5 in MCF-7 cells, while poor cytotoxicity was observed in L929 cells. The FA-Rg5-BSA NPs facilitated cellular uptake and induced apoptosis in MCF-7 cells. In addition, in an MCF-7 xenograft mouse model, the in vivo antitumor evaluation revealed that FA-Rg5-BSA NPs were more effective in inhibiting tumor growth than Rg5 and Rg5-BSA NPs. The in vivo real-time bioimaging study showed that the FA-Rg5-BSA NPs exhibited superior tumor accumulation ability. Conclusion: The results suggested that FA-Rg5-BSA NPs could serve as a promising system to improve the antitumor effect of Rg5.


Assuntos
Ácido Fólico/química , Ginsenosídeos/uso terapêutico , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Soroalbumina Bovina/química , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Portadores de Fármacos , Liberação Controlada de Fármacos , Humanos , Hidrodinâmica , Concentração Inibidora 50 , Células MCF-7 , Camundongos , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Distribuição Tecidual/efeitos dos fármacos
3.
Chem Commun (Camb) ; 55(80): 12052-12055, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31535680

RESUMO

In this paper we report the kinetics based detection of single nucleotide variation (SNV) at room temperature by allele specific extension with different concentrations and types of crowding agents. In general, the crowding conditions enhanced the specificity in the detection of SNV.


Assuntos
DNA/genética , Nucleotídeos/genética , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Pareamento Incorreto de Bases , Técnicas Biossensoriais/métodos , Bovinos , Dextranos/química , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Cinética , Polietilenoglicóis/química , Soroalbumina Bovina/química , Temperatura Ambiente
4.
J Agric Food Chem ; 67(33): 9371-9381, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31379162

RESUMO

A major obstacle to the clinical use of curcumin (CUR) is its reduced bioavailability because of the drug's hydrophobic nature, low intestinal absorption, and rapid metabolism. In this study, a novel oral drug delivery system was constructed for improving the stability and enhancing mucoadhesion of CUR in the gastrointestinal (GI) tract. First, CUR was encapsulated in the bovine serum albumin nanoparticles (CUR-BSA-NPs). Then, N-acetyl cysteine (NAC)-modified CUR-BSA-NPs (CUR-NBSA-NPs) were obtained. The average particle size and zeta potential of CUR-NBSA-NPs were 251.6 nm and -30.66 mV, respectively; encapsulation efficiency and drug loading were 85.79 and 10.9%, respectively. CUR-NBSA-NPs exhibited a sustained release property and prominently enhanced stability in simulated GI conditions. Additionally, enhanced mucoadhesion of CUR-NBSA-NPs was also observed. An MTT study showed that the CUR-NBSA-NPs were safe for oral administration. Overall, NAC-modified BSA-NPs may potentially serve as an oral vehicle for improving CUR stability in the GI tract and enhancing mucoadhesion.


Assuntos
Acetilcisteína/química , Curcumina/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/instrumentação , Trato Gastrointestinal/metabolismo , Nanopartículas/química , Soroalbumina Bovina/química , Animais , Células CACO-2 , Bovinos , Curcumina/metabolismo , Estabilidade de Medicamentos , Humanos , Tamanho da Partícula
5.
Chem Commun (Camb) ; 55(67): 9959-9962, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31364996

RESUMO

On the basis of BSA stabilized tetraphenylethylene nanocrystals (BSA-TPE NCs) as aggregation-induced enhanced electrochemiluminescence (ECL) emitters with high ECL efficiency and good biocompatibility, as well as molecular recognition between ß-CD and ferrocene, an ultrasensitive and versatile ECL biosensing platform was constructed to achieve microRNA detection in cancer cells.


Assuntos
Materiais Biocompatíveis/química , Corantes Fluorescentes/química , MicroRNAs/análise , Nanopartículas/química , Soroalbumina Bovina/química , Estilbenos/química , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Bovinos , Linhagem Celular Tumoral , Técnicas Eletroquímicas , Compostos Ferrosos/química , Humanos , Limite de Detecção , Medições Luminescentes , Metalocenos/química , Sensibilidade e Especificidade , beta-Ciclodextrinas/química
6.
Food Chem ; 301: 125254, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31398672

RESUMO

Researches based on metal complexes of plant-derived phenolic acids have attracted much attention due to their beneficial applications in the development of functional food products, dietary supplements and pharmacology. Binding of phenolic acids with serum proteins greatly influences their pharmacological properties. In this context, interactions of a naturally occurring phenolic acid, sinapic acid (SA) and its Cu2+ complex with a model transport protein, bovine serum albumin (BSA), have been explored by means of different spectroscopic and theoretical tools. Spectroscopic studies revealed that the interaction of SA and its Cu2+ complex with BSA occurred through quenching of intrinsic fluorescence of BSA. Site-specific experimental and docking studies were performed to predict the binding site. The geometies of bound Cu2+ and interacting residues of protein were predicted from a solution dynamics study. Interestingly, the complexation of SA with Cu2+ enhanced the antioxidant activity of SA.


Assuntos
Cobre/química , Ácidos Cumáricos/química , Ácidos Cumáricos/metabolismo , Simulação de Acoplamento Molecular , Compostos Organometálicos/química , Compostos Organometálicos/metabolismo , Soroalbumina Bovina/metabolismo , Animais , Sítios de Ligação , Bovinos , Ligação Proteica , Conformação Proteica , Soroalbumina Bovina/química , Espectrometria de Fluorescência , Termodinâmica
7.
Chemistry ; 25(56): 13017-13024, 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31393027

RESUMO

As some stimuli utilized in conventional drug delivery systems can also be found in normal cells, it is inevitable that encapsulated drugs escape from carriers into normal cells. Based on mutual interactions among proteins, polyphenol compounds, and metal ions, we developed a serial-stimuli-responsive drug delivery system. With multi-crosslinking structure, nanocapsules can maintain the integrity of the framework, even with a certain amount of stimuli present, and eventually reach tumor cells to initiate apoptosis, and protect normal cells from being damaged. Meanwhile, the fluorescence of DOX will be quenched when encapsulated in nanocapsules. This property means that the DOX that is released from nanocapsules can be monitored in real-time based on the recovery of fluorescence. These versatile nanocapsules exhibit great potentials to treat cancer.


Assuntos
Doxorrubicina/química , Nanocápsulas/química , Animais , Bovinos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Células Hep G2 , Humanos , Metais/química , Polifenóis/química , Soroalbumina Bovina/química
8.
Chemistry ; 25(56): 12916-12919, 2019 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-31397017

RESUMO

Inorganic cells bearing calcium silicate membranes were prepared and resembled closed chemical gardens. It was demonstrated that these inorganic cells can successfully be loaded with natural products, proteins and plasmid DNA, and their cargo can be released in a controlled manner. These cells demonstrated the ability of chemical gardens to act as platforms for the sustained delivery of biomolecules and are expected to introduce chemical gardens in the field of biosciences.


Assuntos
Portadores de Fármacos/química , Animais , Compostos de Cálcio/química , Bovinos , Plasmídeos/química , Plasmídeos/metabolismo , Rutina/química , Rutina/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Silicatos/química
9.
Chemistry ; 25(55): 12820-12829, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411775

RESUMO

An inorganic sandwich molecule, Na[Co(C2 B9 H11 )2 ], able to produce vesicles through self-assembly and known to produce strong dihydrogen-bond interactions with amine groups is capable of interacting with proteins. This dual non-bonding ability of Na[Co(C2 B9 H11 )2 ] is what makes this molecule unique: it can be firmly anchored to a protein surface and is capable of extending over it. To prove this, the widely available bovine serum albumin (BSA), which has many pendant amino groups in its structure, has been taken as the model protein. It has been found that around 100 molecules of Na[Co(C2 B9 H11 )2 ] preserve the native structure of BSA, while endorsing it with a significantly increased stability with respect to chemical- and thermal-induced denaturation due to efficient encapsulation. The advantages of this encapsulation technique are two-fold; the first is its simplicity as it relies on the anchoring capacity of Na[Co(C2 B9 H11 )2 ] to the surface of the protein through the amine-containing residues and the second is its self-assembling capacity allowing it to spread across the surface. The dense shield of protection offered by Na[Co(C2 B9 H11 )2 ] has been demonstrated by the inhibition of BSA pseudo-esterase activity, which indicates that the inorganic corset around BSA protects its reactive surface residues, thereby preventing their acetylation.


Assuntos
Boranos/química , Compostos Organometálicos/química , Soroalbumina Bovina/metabolismo , Animais , Desnaturação Proteica , Soroalbumina Bovina/química
10.
Life Sci ; 233: 116710, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31369762

RESUMO

AIMS: The naturally occurring compound curcumin has been proposed for a number of pharmacological applications. In spite of the promising chemotherapeutic properties of the molecule, the use of curcumin has been largely limited by its chemical instability in water. In this work, we propose the use of water soluble proteins to overcome this issue in perspective applications to photodynamic therapy of tumors. MATERIALS AND METHODS: Curcumin was bound to bovine serum albumin and its photophysical properties was studied as well as its effect on cell viability after light exposure through MTT assay and confocal imaging. KEY FINDINGS: Bovine serum albumin binds curcumin with moderate affinity and solubilizes the hydrophobic compound preserving its photophysical properties for several hours. Cell viability assays demonstrate that when bound to serum albumin, curcumin is an effective photosensitizer for HeLa cells, with better performance than curcumin alone. Confocal fluorescence imaging reveals that when curcumin is delivered alone, it preferentially associates with mitochondria, whereas curcumin bound to bovine serum albumin is found in additional locations within the cell, a fact that may be related to the higher phototoxicity observed in this case. SIGNIFICANCE: The higher bioavailability of the photosensitizing compound curcumin when bound to serum albumin may be exploited to increase the efficiency of the drug in photodynamic therapy of tumors.


Assuntos
Apoproteínas/metabolismo , Apoptose/efeitos dos fármacos , Curcumina/farmacologia , Sistemas de Liberação de Medicamentos , Mioglobina/metabolismo , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Soroalbumina Bovina/metabolismo , Animais , Apoproteínas/química , Apoptose/efeitos da radiação , Bovinos , Sobrevivência Celular , Curcumina/química , Células HeLa , Cavalos , Humanos , Mioglobina/química , Fármacos Fotossensibilizantes/química , Soroalbumina Bovina/química
11.
Chemistry ; 25(53): 12275-12280, 2019 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-31389071

RESUMO

A range of oxobis(phenyl-1,3-butanedione) vanadium(IV) complexes have been successfully synthesized from cheap starting materials and a simple and solvent-free one-pot dry-melt reaction. This direct, straightforward, fast and alternative approach to inorganic synthesis has the potential for a wide range of applications. Analytical studies confirm their successful synthesis, purity and solid-state coordination, and we report the use of such complexes as potential drug candidates for the treatment of cancer. After a 24 hour incubation of A549 lung carcinoma cells with the compounds, they reveal cytotoxicity values elevenfold greater than cisplatin and remain non-toxic towards normal cell types. Additionally, the complexes are stable over a range of physiological pH values and show the potential for interactions with bovine serum albumin.


Assuntos
Antineoplásicos/farmacologia , Cisplatino/toxicidade , Complexos de Coordenação/síntese química , Vanádio/química , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Bovinos , Cisplatino/química , Complexos de Coordenação/química , Complexos de Coordenação/farmacologia , Humanos , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Solventes
12.
Soft Matter ; 15(38): 7583-7589, 2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31465079

RESUMO

Injectable hydrogels are adapted to irregularities in the desired location by injection as a liquid and gelation in situ. However, traditional slow-gelling injectable hydrogels may result in loss of cargo (cells/drugs) as well as diffusion at the target site, and extremely rapid gelation may lead to undesired premature coagulation. These practical problems can be solved by using self-healing hydrogels. Herein, through the reduction of disulfide bonds in BSA protein by using a reducing agent, the disulfide bonds between the individual BSA protein molecules are re-matched to form a network structure, thereby forming a protein hydrogel. This hydrogel shows an efficient and rapid self-healing property, and the broken protein hydrogel can be fast repaired within 1-2 minutes in response to H2O2 stimulation, and the repair efficiency reached up to 100%. The hydrogel can be extruded using only a pinhole syringe, and cytotoxicity experiments have demonstrated excellent biocompatibility of the protein hydrogel. This non-toxic, injectable, fast self-healing protein hydrogel is expected to be widely used in biomedical, tissue engineering, injectable gel, 3D bioprinting, and other applications.


Assuntos
Materiais Biocompatíveis/química , Hidrogéis/química , Soroalbumina Bovina/química , Animais , Bovinos , Sobrevivência Celular , Humanos , Peróxido de Hidrogênio/metabolismo , Injeções , Células MCF-7 , Oxirredução , Reologia , Resistência à Tração
13.
Int J Nanomedicine ; 14: 4833-4847, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31308661

RESUMO

Background: The use of functionalized graphene oxide (fGO) has led to a new trend in the sensor field, owing to its high sensitivity with regards to sensing characteristics and easy synthesis procedures. Methods: In this study, we developed an ultra-sensitive carboxyl-graphene oxide (carboxyl-GO)-based surface plasmon resonance (SPR) aptasensor using peptides to detect human chorionic gonadotropin (hCG) in clinical serum samples. The carboxyl-GO based SPR aptasensor provided high affinity and stronger binding of peptides, which are great importance to allow for a non-immunological label-free mechanism. Also, it allows the detection of low concentrations of hCG, which are in turn considered to be important clinical parameters to diagnose ectopic pregnancies and paraneoplastic syndromes. Results: The high selectivity of the carboxyl-GO-based SPR aptasensor for hCG recombinant protein was verified by the addition of the interfering proteins bovine serum albumin (BSA) and human serum albumin (HSA), which did not affect the sensitivity of the sensor. The carboxyl-GO-based chip can enhance the assay efficacy of interactions between peptides and had a high affinity binding for a ka of 17×106 M-1S-1. The limit of detection for hCG in clinical serum samples was 1.15 pg/mL. Conclusion: The results of this study demonstrated that the carboxyl-GO-based SPR aptasensor had excellent sensitivity, affinity and selectivity, and thus the potential to be used as disease-related biomarker assay to allow for an early diagnosis, and possibly a new area in the field of biochemical sensing technology.


Assuntos
Técnicas Biossensoriais/instrumentação , Gonadotropina Coriônica/sangue , Grafite/química , Ressonância de Plasmônio de Superfície/instrumentação , Animais , Bovinos , Eletroquímica , Humanos , Peptídeos/química , Espectroscopia Fotoeletrônica , Soroalbumina Bovina/química
14.
Zhongguo Zhong Yao Za Zhi ; 44(12): 2559-2565, 2019 Jun.
Artigo em Chinês | MEDLINE | ID: mdl-31359724

RESUMO

Small molecules with physiological or pharmacological activities need to interact with biological macromolecules in order to function in the body. As the protein with the highest proportion of plasma protein,serum albumin is the main protein binding to various endogenous or exogenous small molecules. Serum albumin interacts with small molecules in a reversible non-covalent manner and transports small molecules to target sites. Bovine serum albumin( BSA) is an ideal target protein for drug research because of its low cost and high homology with human serum albumin. The research on the interaction between drugs and BSA has become a hotspot in the fields of pharmacy,medicine,biology and chemistry. In this research,molecular docking method was used to study the interaction between three small ginsenosides with high pharmacological value( Rg_1,Rb_1,Ro) and bovine serum albumin( BSA),and the binding mode information of three ginsenosides interacting with BSA was obtained. The results of molecular docking showed that ginsenosides and amino acid residues in the active pocket of proteins could be combined by hydrophobic action,hydrogen bonding and electrostatic action. The interaction between small ginsenosides and bovine serum albumin is not the only form,and their interaction has many forms of force. The interaction between these molecules and various weak forces is the key factor for the stability of the complex. The results of this study can provide the structural information of computer simulation for the determination of the interaction patterns between active components and proteins of ginseng.


Assuntos
Ginsenosídeos/química , Simulação de Acoplamento Molecular , Soroalbumina Bovina/química , Animais , Sítios de Ligação , Bovinos , Simulação por Computador , Ligação Proteica , Espectrometria de Fluorescência , Termodinâmica
15.
J Agric Food Chem ; 67(28): 7821-7831, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31260293

RESUMO

The mechanism of inhibition of advanced glycation end product (AGE) formation by protocatechuic acid and 3,4-dihydroxyphenylacetic acid (DHPA) has been studied using a widespread applied in vitro model system composed of bovine serum albumin (BSA) and supraphysiological glucose concentrations. Protocatechuic acid and DHPA inhibited the formation of Amadori compounds, fluorescent AGEs (IC50 = 62.1 ± 1.4 and 155.4 ± 1.1 µmol/L, respectively), and Nε-(carboxymethyl)lysine (IC50 = 535.3 ± 1.1 and 751.2 ± 1.0 µmol/L, respectively). BSA was pretreated with the two phenolic acids, and the formation of BSA-phenolic acid adducts was estimated by nanoflow liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry. Results showed that the tested phenolic acids bound key sites of glycation in BSA through a metal-catalyzed oxidative mechanism. The antiglycative activity mechanism involved the formation of BSA-phenolic acid adducts, and it is unlikely that this occurs in vivo. These results raise the problem to design in vitro models closer to physiological conditions to reach biologically sound conclusions.


Assuntos
Ácido 3,4-Di-Hidroxifenilacético/química , Hidroxibenzoatos/química , Lisina/química , Metais/química , Soroalbumina Bovina/química , Animais , Catálise , Bovinos , Cromatografia Líquida , Glicosilação , Oxirredução , Espectrometria de Massas por Ionização por Electrospray
16.
Food Chem ; 300: 125204, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31336275

RESUMO

Analytical chromatographic techniques for mycotoxins control are well established, but they often depend on costly immunoaffinity sample clean-up. Serum albumins, particularly that from bovine origin (BSA), have stable binding affinity towards some mycotoxins, and can be cheaper alternative receptors for sample clean-up due to their wide availability. Thus, this work used BSA immobilized in agarose beads as a novel solid-phase extraction method for quantification of ochratoxin A (OTA) in wine. Constructed BSA-agarose columns could extract OTA efficiently from red wine after its dilution (4-fold) in 0.1 M Tris pH 8.0. The method was linear (R2 = 0.9999) in the OTA concentration range studied (0.05 to 3.0 µg L-1), with recovery rates above 98%. It also showed low detection (0.017 µg L-1) and quantification (0.051 µg L-1) limits. The efficacy of the BSA-based method was further validated by direct comparison with commercial immunoaffinity columns. Portuguese wines analyzed by both methods had agreeing results.


Assuntos
Contaminação de Alimentos/análise , Ocratoxinas/análise , Soroalbumina Bovina/química , Extração em Fase Sólida/métodos , Vinho/análise , Cromatografia Líquida de Alta Pressão/métodos , Concentração de Íons de Hidrogênio , Limite de Detecção , Micotoxinas/análise , Reprodutibilidade dos Testes , Extração em Fase Sólida/instrumentação
17.
Pharm Res ; 36(9): 133, 2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31289919

RESUMO

PURPOSE: KRAS is the most frequently mutated gene in human cancers. Despite its direct involvement in malignancy and intensive effort, direct inhibition of KRAS via pharmacological inhibitors has been challenging. RNAi induced knockdown using siRNAs against mutant KRAS alleles offers a promising tool for selective therapeutic silencing in KRAS-mutant lung cancers. However, the major bottleneck for clinical translation is the lack of efficient biocompatible siRNA carrier systems. METHODS: Bovine serum albumin (BSA) nanoparticles were prepared by desolvation method to deliver siRNA targeting the KRAS G12S mutation. The BSA nanoparticles were characterized with respect to their size, zeta potential, encapsulation efficiency and nucleic acid release. Nanoparticle uptake, cellular distribution of nucleic acids, cytotoxicity and gene knock down to interfere with cancer hallmarks, uncontrolled proliferation and migration, were evaluated in KRAS G12S mutant A459 cells, a lung adenocarcinoma cell line. RESULTS: BSA nanoparticles loaded with siRNA resulted in nanoparticles smaller than 200 nm in diameter and negative zeta potentials, displaying optimal characteristics for in vivo application. Encapsulating and protecting the siRNA payload well, the nanoparticles enabled transport to A549 cells in vitro, could evade endosomal entrapment and mediated significant sequence-specific KRAS knockdown, resulting in reduced cell growth of siRNA transfected lung cancer cells. CONCLUSIONS: BSA nanoparticles loaded with mutant specific siRNA are a promising therapeutic approach for KRAS-mutant cancers.


Assuntos
Neoplasias Pulmonares/tratamento farmacológico , Nanopartículas/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RNA Interferente Pequeno/farmacologia , Soroalbumina Bovina/química , Células A549 , Animais , Apoptose/efeitos dos fármacos , Bovinos , Sobrevivência Celular , Técnicas de Silenciamento de Genes , Terapia Genética , Humanos , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Transfecção
18.
J Chromatogr A ; 1603: 190-198, 2019 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-31277950

RESUMO

The heat-induced (80 ±â€¯1 ℃, 1 h) aggregates of bovine serum albumin and ovalbumin at neutral pH and low ionic strength (10 mM sodium phosphate buffer, pH 6.9) were characterized using size-exclusion chromatography combined with small-angle X-ray scattering measurement. The values calculated for the radius of gyration and molecular weight of the eluted aggregates of the bovine serum albumin were 9-11 nm and 540,000-820,000, respectively. Those of ovalbumin were 11-16 nm and 500,000-1,820,000, respectively. The overall linear conformation of the bovine serum albumin aggregates slightly differed from that of the ovalbumin aggregates since the mass fractal dimensions were found from calculation to be 1.63-1.7 for the bovine serum albumin and 1.36-1.51 for the ovalbumin. The surface property of the aggregates of both proteins was suggested to be similar to that of their native monomer since all the surface fractal dimensions were almost equivalent. The dimensionless Kratky plots of the eluted aggregates indicated that the non-globular conformation of the bovine serum albumin aggregates differs from that of the ovalbumin aggregates. These analyses using size-exclusion chromatography combined with the solution X-ray scattering measurement will be helpful for characterization of the components of the denatured protein aggregation in solution.


Assuntos
Cromatografia em Gel/métodos , Temperatura Alta , Concentração Osmolar , Agregados Proteicos , Proteínas/química , Espalhamento de Radiação , Água/química , Animais , Bovinos , Galinhas , Fractais , Peso Molecular , Ovalbumina/química , Conformação Proteica , Desnaturação Proteica , Soroalbumina Bovina/química , Soluções , Raios X
19.
Sci Total Environ ; 686: 1039-1048, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31200302

RESUMO

2,7-Dibromocarbazole (2,7-DBCB) and 3,6-dibromocarbazole (3,6-DBCB) are emerging environmental pollutants, being potentially high risks to human health. In this study, interactions of the two compounds with human serum albumin (HSA) and bovine serum albumin (BSA) were investigated by molecular modeling, density functional theory calculations (DFT) and multispectral techniques. The static quenching interaction deduced in the fluorescence quenching experiment is confirmed by the time-resolved analyses. The interactions of the two compounds with HSA/BSA induce molecular microenvironment and conformation changes, as assessed by synchronous and 3D fluorescence spectra, together with a destruction of polypeptide carbonyl hydrogen bond network by circular dichroism and Fourier transform infrared analyses. The thermodynamic analysis indicated that the spontaneous interaction was hydrogen bonding and hydrophobic forces. The binding constant Ka at 298 K was 3.54 × 105 M-1 in HSA-2,7-DBCB, 6.63 × 105 M-1 in HSA-3,6-DBCB, 1.32 × 105 M-1 in BSA-2,7-DBCB and 2.17 × 105 M-1 in BSA-3,6-DBCB. These results indicates that 3,6-DBCB binds HSA/BSA more strongly than 2,7-DBCB, which was estimated with DFT calculations. Site marker competition experiments coupled with molecular modeling studies confirmed that both compounds bind HSA/BSA at site I (subdomain IIA). The results suggest that interactions between 2,7-DBCB and 3,6-DBCB with HSA and BSA may affect the normal physiological activities in human and animals.


Assuntos
Carbazóis/química , Poluentes Ambientais/química , Modelos Moleculares , Soroalbumina Bovina/química , Animais , Dicroísmo Circular , Teoria da Densidade Funcional , Fluorescência , Humanos , Ligações de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Albumina Sérica , Termodinâmica
20.
Pharm Res ; 36(9): 129, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31254106

RESUMO

PURPOSE: Immunogenicity against biotherapeutics can lead to the formation of drug/anti-drug-antibody (ADA) immune complexes (ICs) with potential impact on safety and drug pharmacokinetics (PK). This work aimed to generate defined drug/ADA ICs, characterized by quantitative (bio) analytical methods for dedicated determination of IC sizes and IC profile changes in serum to facilitate future in vivo studies. METHODS: Defined ICs were generated and extensively characterized with chromatographic, biophysical and imaging methods. Quantification of drug fully complexed with ADAs (drug in ICs) was performed with an acid dissociation ELISA. Sequential coupling of SEC and ELISA enabled the reconstruction of IC patterns and thus analysis of IC species in serum. RESULTS: Characterization of generated ICs identified cyclic dimers, tetramers, hexamers, and larger ICs of drug and ADA as main IC species. The developed acid dissociation ELISA enabled a total quantification of drug fully complexed with ADAs. Multiplexing of SEC and ELISA allowed unbiased reconstruction of IC oligomeric states in serum. CONCLUSIONS: The developed in vitro IC model system has been properly characterized by biophysical and bioanalytical methods. The specificity of the developed methods enable discrimination between different oligomeric states of ICs and can be bench marking for future in vivo studies with ICs.


Assuntos
Anticorpos Monoclonais/química , Complexo Antígeno-Anticorpo/análise , Animais , Anticorpos Monoclonais/sangue , Complexo Antígeno-Anticorpo/sangue , Complexo Antígeno-Anticorpo/química , Cromatografia Líquida , Dimerização , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Imunoglobulina G/química , Conformação Proteica , Ratos Wistar , Soroalbumina Bovina/química
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