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1.
Int J Nanomedicine ; 15: 6137-6152, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32884268

RESUMO

Background: Beyond clinical atherosclerosis imaging of vessel stenosis and plaque morphology, early detection of inflamed atherosclerotic lesions by molecular imaging could improve risk assessment and clinical management in high-risk patients. To identify inflamed atherosclerotic lesions by molecular imaging in vivo, we studied the specificity of our radiotracer based on maleylated (Mal) human serum albumin (HSA), which targets key features of unstable atherosclerotic lesions. Materials and Methods: Mal-HSA was radiolabeled with a positron-emitting metal ion, zirconium-89 (89Zr4+). The targeting potential of this probe was compared with unspecific 89Zr-HSA and 18F-FDG in an experimental model of atherosclerosis (Apoe-/- mice, n=22), and compared with wild-type (WT) mice (C57BL/6J, n=21) as controls. Results: PET/MRI, gamma counter measurements, and autoradiography showed the accumulation of 89Zr-Mal-HSA in the atherosclerotic lesions of Apoe-/- mice. The maximum standardized uptake values (SUVmax) for 89Zr-Mal-HSA at 16 and 20 weeks were 26% and 20% higher (P<0.05) in Apoe-/- mice than in control WT mice, whereas no difference in SUVmax was observed for 18F-FDG in the same animals. 89Zr-Mal-HSA uptake in the aorta, as evaluated by a gamma counter 48 h postinjection, was 32% higher (P<0.01) for Apoe-/- mice than in WT mice, and the aorta-to-blood ratio was 8-fold higher (P<0.001) for 89Zr-Mal-HSA compared with unspecific 89Zr-HSA. HSA-based probes were mainly distributed to the liver, spleen, kidneys, bone, and lymph nodes. The phosphor imaging autoradiography (PI-ARG) results corroborated the PET and gamma counter measurements, showing higher accumulation of 89Zr-Mal-HSA in the aortas of Apoe-/- mice than in WT mice (9.4±1.4 vs 0.8±0.3%; P<0.001). Conclusion: 89Zr radiolabeling of Mal-HSA probes resulted in detectable activity in atherosclerotic lesions in aortas of Apoe-/- mice, as demonstrated by quantitative in vivo PET/MRI. 89Zr-Mal-HSA appears to be a promising diagnostic tool for the early identification of macrophage-rich areas of inflammation in atherosclerosis.


Assuntos
Aterosclerose/diagnóstico por imagem , Maleatos/química , Imagem Molecular/métodos , Radioisótopos , Albumina Sérica Humana/química , Zircônio , Animais , Aorta/diagnóstico por imagem , Aorta/patologia , Aterosclerose/patologia , Autorradiografia , Modelos Animais de Doenças , Feminino , Fluordesoxiglucose F18 , Humanos , Marcação por Isótopo , Macrófagos/patologia , Imagem por Ressonância Magnética , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Sondas Moleculares/química , Sondas Moleculares/farmacocinética , Placa Aterosclerótica/diagnóstico por imagem , Placa Aterosclerótica/patologia , Tomografia por Emissão de Pósitrons , Radioisótopos/química , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/química , Distribuição Tecidual , Zircônio/química , Zircônio/farmacocinética
2.
Int J Nanomedicine ; 15: 4607-4623, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636621

RESUMO

Aim: The interaction of NPs with biological systems may reveal useful details about their pharmacodynamic, anticancer and antibacterial effects. Methods: Herein, the interaction of as-synthesized Co3O4 NPs with HSA was explored by different kinds of fluorescence and CD spectroscopic methods, as well as molecular docking studies. Also, the anticancer effect of Co3O4 NPs against leukemia K562 cells was investigated by MTT, LDH, caspase, real-time PCR, ROS, cell cycle, and apoptosis assays. Afterwards, the antibacterial effects of Co3O4 NPs against three pathogenic bacteria were disclosed by antibacterial assays. Results: Different characterization methods such as TEM, DLS, zeta potential and XRD studies proved that fabricated Co3O4 NPs by sol-gel method have a diameter of around 50 nm, hydrodynamic radius of 177 nm with a charge distribution of -33.04 mV and a well-defined crystalline phase. Intrinsic, extrinsic, and synchronous fluorescence as well as CD studies, respectively, showed that the HSA undergoes some fluorescence quenching, minor conformational changes, microenvironmental changes as well as no structural changes in the secondary structure, after interaction with Co3O4 NPs. Molecular docking results also verified that the spherical clusters with a dimension of 1.5 nm exhibit the most binding energy with HSA molecules. Anticancer assays demonstrated that Co3O4 NPs can selectively lead to the reduction of K562 cell viability through the cell membrane damage, activation of caspase-9, -8 and -3, elevation of Bax/Bcl-2 mRNA ratio, ROS production, cell cycle arrest, and apoptosis. Finally, antibacterial assays disclosed that Co3O4 NPs can stimulate a promising antibacterial effect against pathogenic bacteria. Conclusion: In general, these observations can provide useful information for the early stages of nanomaterial applications in therapeutic platforms.


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Cobalto/química , Cobalto/farmacologia , Nanopartículas Metálicas/química , Óxidos/química , Óxidos/farmacologia , Albumina Sérica Humana/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cobalto/metabolismo , Escherichia coli/efeitos dos fármacos , Humanos , Células K562 , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Óxidos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Albumina Sérica Humana/química , Staphylococcus aureus/efeitos dos fármacos , Difração de Raios X
3.
Food Chem ; 330: 127241, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32540526

RESUMO

Curcumin (CUR) is a promising edible phytochemical compound with ideal ulcerative colitis (UC) treatment activity; however, it is characteristically instable in the digestive tract and has a short retention time in colon. Therefore, we designed and fabricated an oral food-grade nanocarrier composed of tannic acid (TA)-coated, Genipin (Gnp)-crosslinked human serum albumin (HSA) to encapsulate CUR (TA/CUR-NPs). The resulting CUR nanoparticles (NPs) were about 220 nm and -28.8 mV. With the assistance of TA layer and Gnp-crosslinking, the entire nano-scaled system effectively delayed CUR release in simulated gastric fluid, prolonged its colon adhesion and increased its uptake in Caco-2 cells. As expected, TA/CUR-NPs oral administration significantly alleviated colitis symptoms in DSS-treated mice when compared with controls by inhibiting the TLR4-linked NF-κB signaling pathway. Collectively, this study indicates that we have developed a convenient, eco-friendly, nano-scaled vehicle for oral delivery of CUR with anti-UC benefit.


Assuntos
Colite Ulcerativa/tratamento farmacológico , Curcumina/química , Iridoides/química , Albumina Sérica Humana/química , Taninos/química , Administração Oral , Animais , Células CACO-2 , Curcumina/administração & dosagem , Sistemas de Liberação de Medicamentos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química
4.
Phys Chem Chem Phys ; 22(19): 10934-10940, 2020 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-32373844

RESUMO

The structure of interfacial water determines atmospheric chemistry, wetting properties of materials, and protein folding. The challenge of investigating the properties of specific interfacial water molecules has frequently been confronted using surface-specific sum-frequency generation (SFG) vibrational spectroscopy using the O-H stretch mode. While perfectly suited for the water-air interface, for complex interfaces, a potential complication arises from the contribution of hydroxyl or amine groups of non-water species present at the surface, such as surface hydroxyls on minerals, or O-H and N-H groups contained in proteins. Here, we present a protocol to extract the hydrogen bond strength selectively of interfacial water, through the water bending mode. The bending mode vibrational frequency distribution provides a new avenue for unveiling the hydrogen bonding structure of interfacial water at complex aqueous interfaces. We demonstrate this method for the water-CaF2 and water-protein interfaces. For the former, we show that this method can indeed single out water O-H groups from surface hydroxyls, and that with increasing pH, the hydrogen-bonded network of interfacial water strengthens. Furthermore, we unveil enhanced hydrogen bonding of water, compared to bulk water, at the interface with human serum albumin proteins, a prototypical bio-interface.


Assuntos
Água/química , Fluoreto de Cálcio/química , Deutério/química , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Albumina Sérica Humana/química , Análise Espectral/métodos , Propriedades de Superfície , Vibração
5.
Toxicology ; 438: 152446, 2020 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-32278049

RESUMO

Chiral pollutants are widely distributed in the environment; however, the enantioselective toxic effects of these chemicals have still not fully been clarified. Using wet experiments and computational toxicology, this story was to explore the static and dynamic toxic reactions between chiral diclofop-methyl and target protein at the enantiomeric level, and further unveil the microscopic mechanism of enantioselective toxicity of chiral pesticide. Steady-state and time-resolved results indicated that both (R)-/(S)-enantiomers can form the stable toxic conjugates with target protein and the bioaffinities were 1.156 × 104 M-1/1.734 × 104 M-1, respectively, and significant enantioselectivity was occurred in the reaction. Results of the modes of toxic action revealed that diclofop-methyl enantiomers located in the subdomain IIA, and the strength of important noncovalent interactions between (S)-diclofop-methyl and the residues was greater than that of (R)-diclofop-methyl. The Gibbs free energies of the chiral reactions were -26.89/-29.40 kJ mol-1 and -25.79/-30.08 kJ mol-1, respectively, which was consistent with the outcomes of photochemistry and site-specific competitive assay. Dynamic enantioselective processes explained that the impact of intrinsic protein conformational flexibility on the toxic reaction of (R)-diclofop-methyl was lower than that of (S)-diclofop-methyl, which originates from the conformational changes and spatial displacement of the four loop regions (i.e. h6↔h7, h1↔h2, h5↔h6, and h8↔h9). The quantitative data of circular dichroism spectra confirmed such results. Energy decomposition displayed that the electrostatic energy of the target protein-(S)-diclofop-methyl system (-25.86 kJ mol-1) was higher than that of the target protein-(R)-diclofop-methyl complex (-18.21 kJ mol-1). Some crucial residues such as Lys-195, Lys-199, Ser-202, and Trp-214 have been shown to be of different importance for the enantioselective toxicity of chiral diclofop-methyl. Obviously this scenario will contribute mechanistic clues to assessing the potential hazards of chiral environmental pollutants to the body.


Assuntos
Poluentes Ambientais/toxicidade , Éteres Difenil Halogenados/toxicidade , Praguicidas/toxicidade , Albumina Sérica Humana/metabolismo , Animais , Sítios de Ligação , Poluentes Ambientais/química , Poluentes Ambientais/metabolismo , Éteres Difenil Halogenados/química , Éteres Difenil Halogenados/metabolismo , Humanos , Simulação de Acoplamento Molecular , Praguicidas/química , Praguicidas/metabolismo , Ligação Proteica , Domínios Proteicos , Medição de Risco , Albumina Sérica Humana/química , Estereoisomerismo , Relação Estrutura-Atividade
6.
Chemistry ; 26(27): 5965-5969, 2020 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-32237164

RESUMO

Nanoparticles are widely studied as carrier vehicles in biological systems because their size readily allows access through cellular membranes. Moreover, they have the potential to carry cargo molecules and as such, these factors make them especially attractive for intravenous drug delivery purposes. Interest in protein-based nanoparticles has recently gained attraction due to particle biocompatibility and lack of toxicity. However, the production of homogeneous protein nanoparticles with high encapsulation efficiencies, without the need for additional cross-linking or further engineering of the molecule, remains challenging. Herein, we present a microfluidic 3D co-flow device to generate human serum albumin/celastrol nanoparticles by co-flowing an aqueous protein solution with celastrol in ethanol. This microscale co-flow method resulted in the formation of nanoparticles with a homogeneous size distribution and an average size, which could be tuned from ≈100 nm to 1 µm by modulating the flow rates used. We show that the high stability of the particles stems from the covalent cross-linking of the naturally present cysteine residues within the particles formed during the assembly step. By choosing optimal flow rates during synthesis an encapsulation efficiency of 75±24 % was achieved. Finally, we show that this approach achieves significantly enhanced solubility of celastrol in the aqueous phase and, crucially, reduced cellular toxicity.


Assuntos
Microfluídica/métodos , Nanopartículas/química , Albumina Sérica Humana/química , Sistemas de Liberação de Medicamentos , Humanos , Dispositivos Lab-On-A-Chip , Albumina Sérica Humana/metabolismo , Solubilidade
7.
Inorg Chem ; 59(8): 5243-5246, 2020 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-32255347

RESUMO

The Anderson-type hexamolybdoaluminate functionalized with lauric acid (LA), (TBA)3[Al(OH)3Mo6O18{(OCH2)3CNHCOC11H23}]·9H2O (TBA-AlMo6-LA, where TBA = tetrabutylammonium), was prepared via two synthetic routes and characterized by thermogravimetric and elemental analyses, mass spectrometry, IR and 1H NMR spectroscopy, and powder and single-crystal X-ray diffraction. The interaction of TBA-AlMo6-LA with human serum albumin (HSA) was investigated via fluorescence and circular dichroism spectroscopy. The results revealed that TBA-AlMo6-LA binds strongly to HSA (63% quenching at an HSA/TBA-AlMo6-LA ratio of 1:1), exhibiting static quenching. In contrast to TBA-AlMo6-LA, the nonfunctionalized polyoxometalate, Na3(H2O)6[Al(OH)6Mo6O18]·2H2O (AlMo6), showed weak binding toward HSA (22% quenching at a HSA/AlMo6 ratio of 1:25). HSA binding was confirmed by X-ray structure analysis of the HSA-Myr-AlMo6-LA complex (Myr = myristate). These results provide a promising lead for the design of novel polyoxometalate-based hybrids that are able to exploit HSA as a delivery vehicle to improve their pharmacokinetics and bioactivity.


Assuntos
Compostos de Alumínio/metabolismo , Ácidos Láuricos/metabolismo , Albumina Sérica Humana/metabolismo , Compostos de Alumínio/síntese química , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Humanos , Ácidos Láuricos/síntese química , Molibdênio/química , Ligação Proteica , Albumina Sérica Humana/química , Espectrometria de Fluorescência , Triptofano/química
8.
Chemosphere ; 253: 126698, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32302901

RESUMO

Organophosphates insecticides (OPs) are one of the major environmental pollutants and their interaction with human serum albumin (HSA) has been shown to have significant effects on their bioavailability which is related to toxicokinetics and toxicodynamics in human body. In this research, solid-phase microextraction methods were developed to analyse the free concentrations of three OPs (chlorpyrifos, parathion-methyl and malathion) in buffered HSA solution and that provide a useful method for the determination of binding affinity constants (Ka), binding forces and binding location. Polydimethylsiloxane fibers were selected for analysing the free concentrations of OPs, with an external calibration approach. Good linearities conducted in PBS solution were observed in the range of 0.0025-1.7 µmol L-1 (R2 = 0.9975) for chlorpyrifos, 1.0-27 µmol L-1 (R2 = 0.9974) for parathion-methyl, and 0.5-70 µmol L-1 (R2 = 0.9973)for malathion, respectively. The LODs for instrument response were 1 ng, 5 ng and 10 ng for chlorpyrifos, parathion-methyl and malathion, respectively. The Ka values for chlorpyrifos, parathion-methyl and malathion showed that they were positively correlated with hydrophobicity and negatively correlated with temperature. The OP binding sites on HSA were confirmed by site marker competition test and further proven by computational approaches. The recognition region of parathion-methyl was situated within residues 199-292 in subdomain IIA. Malathion bonded to residues 404-558 in subdomain IIIA. The mode of action between HSA-parathion-methyl and HSA-malathion is found to involve mainly by H-bonds, π-π stacking and hydrophobic effects. These results clearly demonstrate the noncovalent binding of OPs with HSA and provide new insight into solid-phase microextraction, thermodynamics and computational approaches.


Assuntos
Inseticidas/toxicidade , Compostos Organofosforados/toxicidade , Clorpirifos , Dimetilpolisiloxanos , Humanos , Inseticidas/química , Inseticidas/metabolismo , Malation/análise , Metil Paration , Compostos Organofosforados/química , Compostos Organofosforados/metabolismo , Albumina Sérica Humana/química , Albumina Sérica Humana/metabolismo , Microextração em Fase Sólida , Temperatura , Termodinâmica
9.
J Phys Chem Lett ; 11(9): 3345-3349, 2020 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-32294382

RESUMO

Serum albumin-gold complexes exhibit UV-excitable red luminescence (λem = 640 nm) with unusual Stokes shifts compared with the innate UV/blue fluorescence arising from the aromatic residues. In order to understand the mechanism of this luminescence, we employed limited proteolysis and molecular cloning techniques and assessed the domain containing the red luminophore in bovine serum albumin (BSA) and human serum albumin (HSA). We identified that the luminophore is localized in a domain of serum albumin, residing within the N-terminus half.


Assuntos
Ouro/química , Soroalbumina Bovina/química , Albumina Sérica Humana/química , Luminescência , Domínios Proteicos
10.
J Chromatogr A ; 1620: 460940, 2020 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-32183982

RESUMO

Protein adsorption plays a role in many fields, where in some it is desirable to maximize the amount adsorbed, in others it is important to avoid protein adsorption altogether. Therefore, theoretical methods are needed for a better understanding of the underlying processes and for the prediction of adsorption quantities. In this study, we present a proof-of-concept that the calculation of protein adsorption isotherms by molecular dynamics (MD) simulations is possible using the steric mass action (SMA) theory. Here we are investigating the adsorption of bovine/human serum albumin (BSA/HSA) and hemoglobin (bHb) on Q Sepharose FF. Protein adsorption isotherms were experimentally determined and modeled. Free energy profiles of protein adsorption were calculated by MD simulations to determine the Henry isotherms as a first step. Although each simulation contained only one protein, notably the calculated isotherms are in reasonably good agreement with the experimental isotherms. Hence, we could show that MD data can lead to protein adsorption data in good agreement with experimental data. The results were critically discussed and requirements for future applications are identified.


Assuntos
Proteínas/química , Adsorção , Animais , Bovinos , Hemoglobinas/química , Humanos , Simulação de Dinâmica Molecular , Sefarose , Soroalbumina Bovina/química , Albumina Sérica Humana/química
11.
Sci Rep ; 10(1): 4166, 2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32139799

RESUMO

A nitrobenzoxadiazole-based fluoroprobe (NBD-Bu) is designed to probe cellular metabolic activity in cancer and normal cells. NBD-Bu shows a significant fluorescence enhancement upon selective binding to the transport protein serum albumin in PBS buffer at ambient conditions. Encouraged by this finding, the site- specificity of NBD-Bu has been explored through a competitive displacement assay in the presence of site-specific markers such as warfarin and ibuprofen. Notably, even at micromolar concentrations, the probe possesses the ability to displace the site marker drug ibuprofen, efficiently. Subsequently, high-resolution fluorescence imaging results consolidated the potential of NBD-Bu for detection of abnormal cellular metabolic activity.


Assuntos
4-Cloro-7-nitrobenzofurazano/química , Albumina Sérica/química , Animais , Células CHO , Linhagem Celular , Cricetulus , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Confocal , Soroalbumina Bovina/química , Albumina Sérica Humana/química
12.
J Photochem Photobiol B ; 205: 111825, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32142995

RESUMO

In recent years research based on kaempferol (KMP) has shown its potential therapeutic applications in medicinal chemistry and clinical biology. Therefore, to understand its molecular recognition mechanism, we studied its interactions with the carrier proteins, namely, human serum albumin (HSA), bovine hemoglobin (BHb) and hen egg white lysozyme (HEWL). The ligand, KMP was able to quench the intrinsic fluorescence of these three proteins efficiently through static quenching mode. The binding constant (Kb) for the interactions of KMP with these three proteins were found in the following order: HSA-KMP > BHb-KMP > HEWL-KMP. Different non-covalent forces such as hydrogen bonding and hydrophobic forces played a major role in the binding of KMP with HSA and HEWL, whereas hydrogen bonding and van der Waals forces contribute to the complexation of BHb with KMP. KMP was able to alter the micro-environment near the Trp fluorophore of the proteins. KMP altered the secondary structural component of all three proteins. The putative binding sites and the residues surrounding the KMP molecule within the respective protein matrix were determined through molecular docking and molecular dynamics (MD) simulation studies. The conformational flexibility of the ligand KMP and the three individual proteins were also evident from the MD simulation studies.


Assuntos
Hemoglobinas/química , Quempferóis/química , Muramidase/química , Albumina Sérica Humana/química , Dicroísmo Circular , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Espectrometria de Fluorescência , Termodinâmica
13.
Biochemistry ; 59(14): 1410-1419, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32208682

RESUMO

Somapacitan, a human growth hormone derivative that binds reversibly to albumin, was investigated for human serum albumin (HSA) and HSA domain binding. Isothermal titration calorimetry (ITC) binding profiles showed high-affinity binding (∼100-1000 nM) of one somapacitan molecule and low-affinity binding (∼1000-10000 nM) of one to two somapacitan molecules to HSA. The high-affinity site was identified in HSA domain III using size exclusion chromatography (SEC) and ITC. SEC studies showed that the neonatal Fc receptor shields one binding site for somapacitan, indicating its position in domain III. A crystal structure of somapacitan in complex with HSA optimized for neonatal Fc receptor binding, having four amino acid residue replacements, identified a low-affinity site in fatty acid-binding site 6 (domain II). Surface plasmon resonance (SPR) showed these replacements affect the kinetics of the high-affinity binding site. Furthermore, small-angle X-ray scattering and SPR brace two somapacitan-binding sites on HSA.


Assuntos
Hormônio do Crescimento/química , Albumina Sérica Humana/química , Sítios de Ligação , Hormônio do Crescimento/análogos & derivados , Hormônio do Crescimento/metabolismo , Humanos , Cinética , Ligação Proteica , Domínios Proteicos , Albumina Sérica Humana/metabolismo , Ressonância de Plasmônio de Superfície
14.
Mikrochim Acta ; 187(4): 230, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170435

RESUMO

An interferometric reflectance spectroscopy-based biosensor for the determination of cathepsin B (Cat B) as a cancer-related enzyme has been fabricated. For this purpose, the nanoporous anodic alumina (NAA) was fabricated electrochemically. The NAA was then modified with the amino-silane coupling agent. After that, human serum albumin (HSA) was immobilized into the NAA pores by using glutaraldehyde as a cross-linking agent. Subsequently, the carboxylic group of HSA was activated with N-ethyl-N'-(3-(dimethylamino)propyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to attach to thionine (TH) as a photoprobe to fabricate the labeled HSA (HSA-TH). HSA-TH plays a significant role in this sensor to determine cathepsin B as a model analyte for the development of the interferometric reflectance spectroscopy-based biosensor for the measurement of protease. The attached TH adsorbed the illuminated white light on NAA modified with HSA-TH. Therefore, the intensity of the reflected light to the charge-coupled device (CCD) detector decreased in the wavelength range 450-1050 nm. In the presence of Cat B, HAS-TH cleaved into short peptide fragments and washed away by flow cell system. Since TH was removed from NAA, the intensity of the reflected light increased. The peak area has a logarithmic relationship with the concentration of Cat B in the range 0.5 to 64.0 nM. The limit of detection of the biosensor sensor was 0.08 nM. The optical sensor was used for the determination of Cat B in a human serum sample. Graphical abstract Schematic presentation of biosensor for the determination of the cathepsin B which is based on nanoporous anodic alumina modified with HSA-thionine. The principle response of the optical biosensor is based on detecting changes in the intensity of the reflected light after cleaving the immobilized HSA-thionine by cathepsin B into short peptide fragments.


Assuntos
Óxido de Alumínio/química , Técnicas Biossensoriais , Catepsina B/análise , Técnicas Eletroquímicas , Fenotiazinas/química , Albumina Sérica Humana/química , Catepsina B/metabolismo , Eletrodos , Humanos , Fenômenos Ópticos , Tamanho da Partícula , Porosidade , Propriedades de Superfície
15.
Phys Chem Chem Phys ; 22(8): 4490-4500, 2020 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-32067002

RESUMO

Once introduced into the human body, nanoparticles often interact with blood proteins, which in turn undergo structural changes upon adsorption. Although protein corona formation is a widely studied phenomenon, the structure of proteins adsorbed on nanoparticles is far less understood. We propose a model to describe the interaction between human serum albumin (HSA) and nanoparticles (NPs) with arbitrary coatings. Our model takes into account the competition between protonated and unprotonated polymer ends and the curvature of the NPs. To this end, we explored the effects of surface ligands (citrate, PEG-OMe, PEG-NH2, PEG-COOH, and glycan) on gold nanoparticles (AuNPs) and the pH of the medium on structural changes in the most abundant protein in blood plasma (HSA), as well as the impact of such changes on cytotoxicity and cellular uptake. We observed a counterintuitive effect on the ζ-potential upon binding of negatively charged HSA, while circular dichroism spectroscopy at various pH values showed an unexpected pattern in the reduction of α-helix content, as a function of surface chemistry and curvature. Our model qualitatively reproduces the decrease in α-helix content, thereby offering a rationale based on particle curvature. The simulations quantitatively reproduce the charge inversion measured experimentally through the ζ-potential of the AuNPs in the presence of HSA. Finally, we found that AuNPs with adsorbed HSA display lower toxicity and slower cell uptake rates, compared to functionalized systems in the absence of protein. Our study allows examining and explaining the conformational dynamics of blood proteins triggered by NPs and corona formation, thereby opening new avenues toward designing safer NPs for drug delivery and nanomedical applications.


Assuntos
Ouro/química , Ouro/metabolismo , Nanopartículas Metálicas/química , Albumina Sérica Humana/química , Humanos , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Ligação Proteica , Estrutura Terciária de Proteína , Albumina Sérica Humana/metabolismo , Eletricidade Estática , Propriedades de Superfície
16.
Int J Nanomedicine ; 15: 151-167, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021171

RESUMO

Purpose: Combination therapy for tumors is an important and promising strategy to improve therapeutic efficiency. This study aims at combining tumor targeting, chemo-, and photodynamic therapies to improve the anti-tumor performance. Patients and Methods: Human serum albumin (HSA), as a nontoxic and biodegradable drug carrier, was used to load hydrophobic photosensitizers (mono-substituted ß-4-pyridyloxy phthalocyanine zinc, mPPZ) by a dilution-incubation-purification (DIP) strategy to form molecular complex HSA:mPPZ. This complex was cross-linked as nanoparticles, and then chemotherapy drug doxorubicin (DOX) was adsorbed into the nanoparticles to achieve combined photodynamic therapy and chemotherapy. Next, the surface of the obtained composite was modified by a tumor surface receptor (urokinase receptor) targeting agent (ATF-HSA) using a noncovalent method to obtain the final product (ATF-HSA@HSA:mPPZ:DOX nanoparticles, AHmDN). Results: AHmDN exhibited strong stability, remarkable cytotoxicity and higher uptake to tumor cells. Cell imaging analysis indicated that DOX was separated from AHmDN and uniformly distributed in cell nucleus while mPPZ localized in cytoplasm. The PDT activity of all the samples had been confirmed by the detection of intracellular ROS. In animal experiments, AHmDN was demonstrated to have a prominent tumor-targeting effect using a 3D imaging system. In addition, the enhanced antitumor effect of AHmDN in tumor-bearing mice was also been observed. Importantly, the tumor-targeting effect of such nanoparticles lasted for about 14 days after one injection. Conclusion: These albumin nanoparticles with combined functions of tumor targeting, chemotherapy and photodynamic therapy can highly enhance the anti-tumor effect. This drug delivery system can be applied to package other hydrophobic photosensitizers and chemotherapy drugs for improving therapeutic efficacy to tumors.


Assuntos
Doxorrubicina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Citoplasma/efeitos dos fármacos , Doxorrubicina/farmacocinética , Portadores de Fármacos/química , Feminino , Humanos , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Camundongos , Nanopartículas/química , Neoplasias/tratamento farmacológico , Paclitaxel/administração & dosagem , Paclitaxel/farmacocinética , Fármacos Fotossensibilizantes/química , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Albumina Sérica Humana/química
17.
Int J Nanomedicine ; 15: 263-273, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32021179

RESUMO

Purpose: Combined superoxide dismutase (SOD)/catalase mimetics have attracted much attention because of their efficacy against reactive oxygen species-associated diseases; however, their application is often limited owing to their poor stability and the absence of favorable grafting sites. To address this, we developed a new class of SOD/catalase mimetics based on hybrid nanoflowers, which exhibit superior stability and possess the desired grafting sites for drugs and endogenous molecules. Methods: In this work, for the first time, we used polynitroxylated human serum albumin (PNA) to mediate the formation of hybrid copper-based nanoflowers. H2O2 depletion and O2 evolution assays were first performed to determine the catalase-like activity of the hybrid nanoflowers. Next, the xanthine oxidase/cytochrome c method was used to assay the SOD-like activity of the nanoflowers. Further characteristics of the nanoflowers were evaluated using scanning electron microscopy (SEM), electron paramagnetic resonance (EPR), and Fourier-transform infrared spectroscopy (FTIR). Operational stability was assessed via the reusability assay. Results: The H2O2 depletion and O2 evolution assays indicated that PNA-incorporated nanoflowers have genuine catalase-like activity. Kinetic analysis revealed that the reactions of the incorporated nanoflowers with H2O2 not only obey Michaelis-Menton kinetics, but that the nanoflowers also possess a higher affinity for H2O2 than that of native catalase. The FTIR spectra corroborated the presence of PNA in the hybrid nanoflowers, while the EPR spectra confirmed the intermolecular interaction of nitroxides bound to the human serum albumin incorporated into the nanoflowers. The remarkable operational reproducibility of the hybrid nanoflowers in catalase-like and SOD-like reactions was verified across successive batches. Conclusion: Herein, a comparison of Michaelis constants showed that the hybrid nanoflower, a catalase mimetics, outperforms the native catalase. Acting as a "better-than-nature" enzyme mimetics, the hybrid nanoflower with superior stability and desired ligand grafting sites will find widespread utilization in the medical sciences.


Assuntos
Catalase/metabolismo , Nanoestruturas/química , Superóxido Dismutase/metabolismo , Catalase/química , Cobre/química , Citocromos c/química , Citocromos c/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Cinética , Microscopia Eletrônica de Varredura , Mimetismo Molecular , Óxidos de Nitrogênio/química , Oxigênio/química , Oxigênio/metabolismo , Reprodutibilidade dos Testes , Albumina Sérica Humana/química , Espectroscopia de Infravermelho com Transformada de Fourier , Superóxido Dismutase/química , Xantina Oxidase/química , Xantina Oxidase/metabolismo
18.
Nanoscale ; 12(7): 4573-4585, 2020 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-32043104

RESUMO

Nanoparticles (NPs) will inevitably interact with proteins and form protein coronas once they are exposed to biological fluids. This conventional model for nano-bio interactions has been used for over twenty years. Growing numbers of new nanomaterials are emerging every year. Among them, noble metal nanoclusters (NMNCs) are new types of fluorescent nanomaterials with considerable advantages in biomedical applications. Compared with NPs (typically >10 nm) like Au NPs, carbon nanotubes, etc., NMNCs have ultrasmall sizes (∼2 nm), so when NMNCs are exposed to biological milieu, will they form protein coronas like NPs? Due to a lack of characterization techniques for ultrasmall nanoparticles (USNPs), to date, studies on the binding stoichiometries of USNPs to proteins have been heavily hampered. To address this challenge, we combined the characteristics of various methods and selected human serum albumin (HSA) and transferrin (Trf) as model proteins to study their interactions with dihydrolipoic acid (DHLA) protected gold nanoclusters (DHLA-AuNCs). Steady-state fluorescence, transient fluorescence spectroscopy and isothermal titration calorimetry (ITC) were used to study the thermodynamic parameters (K, ΔH, ΔS, ΔG) and interaction mechanisms. The results showed that the intrinsic fluorescence of both proteins was quenched by DHLA-AuNCs, and the quenching process of HSA was an endothermic dynamic process. In contrast, the quenching process of Trf was an exothermic static process. The combination of ITC, agarose gel electrophoresis (AGE) and zeta potential showed that one HSA could bind 8 ± 1 DHLA-AuNCs and one Trf could bind 7 ± 2 DHLA-AuNCs, which was quite different from the conventional model of protein coronas. Based on these findings, the "protein complex" was termed for proteins upon binding with USNPs. Dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) showed that DHLA-AuNCs could induce the agglomeration of proteins. Circular dichroism (CD) and synchronous fluorescence spectroscopy showed that DHLA-AuNCs had a very minor effect on the secondary structures of HSA and Trf, which demonstrated the good biocompatibility of DHLA-AuNCs at the molecular scale. This work has shed light on a new interaction model beyond the protein corona, indicating a possible biological identity of USNPs.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Modelos Químicos , Coroa de Proteína/química , Albumina Sérica Humana/química , Transferrina/química , Humanos , Termodinâmica
19.
Biomed Res Int ; 2020: 1693602, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104676

RESUMO

Salbutamol (SBAL), a kind of short-acting beta 2-adrenergic agonist, has been mainly used to treat bronchial asthma and other allergic airway diseases clinically. In this study, the interaction mechanism between salbutamol and human serum albumin was researched by the multispectral method and molecular docking. The fluorescence intensity of HSA could be regularly enhanced with the increase of SBAL concentration. Both the results of the multispectral method and molecular docking showed that SBAL could bind HSA with van der Waals force and hydrogen bonds. The binding mechanism was further analysed by UV-Vis and synchronous fluorescence spectra. The contents of the secondary structure of free HSA and SBAL-HSA complex were evaluated using CD spectra.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/química , Albuterol/química , Simulação de Acoplamento Molecular , Albumina Sérica Humana/química , Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Albuterol/uso terapêutico , Asma/tratamento farmacológico , Humanos , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/efeitos dos fármacos
20.
Molecules ; 25(3)2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-32012739

RESUMO

The aim of this study was to analyze the binding interactions between a common antihypertensive drug (ramipril, R) and the widely distributed plant flavonoid quercetin (Q), in the presence of human serum albumin (HSA). From the observed fluorescence spectra of the (HSA + R) system we can assume that ramipril is also one of the Site 3 ligands-similar to fusidic acid-the binding of which has been proven by RTG crystallography. Our claim is supported by near-UV CD spectroscopy, microscale themophoresis and molecular modeling. The presence of R slightly inhibited the subsequent binding of Q to HSA and, on the contrary, the pre-incubation of HSA with Q caused a stronger binding of R, most likely due to allosteric interactions. At high concentrations, R is also able to displace Q from its binding site. The dissociation constant KD for the binding of R is more than hundredfold larger than for Q, which means that R is a very weak binder to HSA. The knowledge of qualitative and quantitative parameters of R, as well as the methods used in this study, are important for future research into HSA binding. This study shows the importance of implementing other methods for KD determination. Microscale thermophoresis has proved to be a novel, practical and accurate method for KD determination on HSA, especially in cases when fluorescence spectroscopy is unable to produce usable results.


Assuntos
Quercetina/metabolismo , Ramipril/metabolismo , Albumina Sérica Humana/metabolismo , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Quercetina/química , Ramipril/química , Albumina Sérica Humana/química
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