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1.
PLoS One ; 14(6): e0218975, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31247048

RESUMO

Bovine serum albumin (BSA) is often employed as a proteinaceous component for synthesis of luminescent protein-stabilized gold nanoclusters (AuNC): intriguing systems with many potential applications. Typically, the formation of BSA-AuNC conjugate occurs under strongly alkaline conditions. Due to the sheer complexity of intertwined chemical and structural transitions taking place upon BSA-AuNC formation, the state of albumin enveloping AuNCs remains poorly characterized. Here, we study the conformational properties of BSA bound to AuNCs using an array of biophysical tools including vibrational spectroscopy, circular dichroism, fluorescence spectroscopy and trypsin digestion. The alkaline conditions of BSA-AuNC self-assembly appear to be primary responsible for the profound irreversible disruption of tertiary contacts, partial unfolding of native α-helices, hydrolysis of disulfide bonds and the protein becoming vulnerable to trypsin digestion. Further unfolding of BSA-AuNC by guanidinium hydrochloride (GdnHCl) is fully reversible equally in terms of albumin's secondary structure and conjugate's luminescent properties. This suggests that binding to AuNCs traps the albumin molecule in a state that is both partly disordered and refractory to irreversible misfolding. Indeed, when BSA-AuNC is subjected to conditions favoring self-association of BSA into amyloid-like fibrils, the buildup of non-native ß-sheet conformation is less pronounced than in a control experiment with unmodified BSA. Unexpectedly, BSA-AuNC reveals a tendency to self-assemble into giant twisted superstructures of micrometer lengths detectable with transmission electron microscopy (TEM), a property absent in unmodified BSA. The process is accompanied by ordering of bound AuNCs into elongated streaks and simultaneous decrease in fluorescence intensity. The newly discovered self-association pathway appears to be specifically accessible to protein molecules with a certain restriction on structural dynamics which in the case of BSA-AuNC arises from binding to metal nanoclusters. Our results have been discussed in the context of mechanisms of protein misfolding and applications of BSA-AuNC.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Soroalbumina Bovina/química , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , Nanopartículas Metálicas/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Agregados Proteicos , Conformação Proteica , Desnaturação Proteica , Estabilidade Proteica , Soroalbumina Bovina/genética , Soroalbumina Bovina/ultraestrutura , Espectrometria de Fluorescência , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman
2.
Int J Biol Macromol ; 135: 1114-1122, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31173836

RESUMO

In a physiological fluid (e.g., blood), nanomaterials will strongly interact with proteins to form "protein corona". The structure and aggregation of protein corona around the nanoparticles are of vital importance in the safe application of nanomaterials in living organisms. Here, we combined systematic methods, including transmission electron microscopy, scanning electron microscopy equipped with energy dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, hydrogen/deuterium exchange techniques and fluorescence quenching to explore the conformational change, stability and aggregation of protein corona bound on magnetic nanoparticles. For the first time we observed that the conformational change of protein corona could induce proteins to aggregate. We believe that these findings will deepen our understanding of the protein corona.


Assuntos
Agregados Proteicos , Conformação Proteica , Coroa de Proteína/química , Ligações de Hidrogênio , Nanopartículas/química , Nanopartículas/uso terapêutico , Estabilidade Proteica , Soroalbumina Bovina/química , Soroalbumina Bovina/ultraestrutura , Espectroscopia de Infravermelho com Transformada de Fourier , Relação Estrutura-Atividade
3.
Molecules ; 23(4)2018 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-29561799

RESUMO

α-Dicarbonyl compounds, which are widely generated during sugar fragmentation and oil oxidation, are important precursors of advanced glycation end products (AGEs). In this study, the effect of glycation derived from glyoxal (GO), methylglyoxal (MGO) and diacetyl (DA) on the in vitro digestibility of bovine serum albumin (BSA) was investigated. Glycation from α-dicarbonyl compounds reduced digestibility of BSA in both gastric and intestinal stage of digestion according to measurement of degree of hydrolysis. Changes in peptide composition of digests induced by glycation were displayed, showing absence of peptides, occurrence of new peptides and formation of peptide-AGEs, based on the results obtained using liquid chromatography electron-spray-ionization tandem mass spectrometry (LC-ESI-MS/MS). Crosslinked glycation structures derived from DA largely reduced the sensitivity of glycated BSA towards digestive proteases based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results. Network structures were found to remain in the digests of glycated samples by transmission electron microscope (TEM), thus the impact of AGEs in unabsorbed digests on the gut flora should be an interest for further studies.


Assuntos
Produtos Finais de Glicação Avançada/metabolismo , Peptídeos/metabolismo , Proteômica/métodos , Soroalbumina Bovina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Glicosilação , Hidrólise , Agregados Proteicos , Conformação Proteica , Soroalbumina Bovina/química , Soroalbumina Bovina/ultraestrutura
4.
Med Sci Monit ; 24: 473-478, 2018 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-29364864

RESUMO

BACKGROUND Bovine serum albumin nanoparticles loaded with isoniazid and rifampicin (INH-RFP-BSA-NPs) were prepared and their release characteristics were studied in vitro. MATERIAL AND METHODS The INH-RFP-BSA-NPs were prepared by a modified self-emulsion solvent diffusion method, with albumin and polylactic acid used as carriers and to form the nanoparticles structure. Transmission electron microscopy was used to observe the morphology of the INH-RFP-BSA-NPs. The size distribution of the INH-RFP-BSA-NPs were assessed using a submicron particle-size analyzer for drug loadings, and the coating rate of the INH-RFP-BSA-NPs was measured by high-performance liquid chromatography. A dynamic membrane dialysis method was used to study the in vitro release characteristics of the INH-RFP-BSA-NPs. RESULTS The INH-RFP-BSA-NPs were smooth, sphere-like, relatively uniform in size, and well-dispersed, and the average diameter was 60.5±4.6 nm. Drug loading and entrapment efficiencies were high, at 19.8% and 87.8% for isoniazid, respectively, and 20.1% and 98.0% for rifampicin, respectively. Drug release was slow and sustained with 97.02% INH cumulative release at 6 days, and full release of RFP requiring 5 days. CONCLUSIONS INH-RFP-BSA-NPs exhibit uniform NP diameter, good dispersion, high drug loading and encapsulation rates, and have sustained release properties.


Assuntos
Isoniazida/farmacologia , Nanopartículas/química , Rifampina/farmacologia , Soroalbumina Bovina/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Nanopartículas/ultraestrutura , Padrões de Referência , Soroalbumina Bovina/ultraestrutura
5.
Spectrochim Acta A Mol Biomol Spectrosc ; 191: 116-124, 2018 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-29028503

RESUMO

Amyloid fibrils are playing key role in the pathogenesis of various neurodegenerative diseases. Generally anionic molecules are known to induce amyloid fibril in several proteins. In this work, we have studied the effect of anionic food additive dye i.e., tartrazine (TZ) on the amyloid fibril formation of human serum albumins (HSA) and bovine serum albumin (BSA) at pHs7.4 and 3.5. We have employed various biophysical methods like, turbidity measurements, Rayleigh Light Scattering (RLS), Dynamic Light Scattering (DLS), intrinsic fluorescence, Congo red assay, far-UV CD, transmission electron microscopy (TEM) and atomic force microscopy (AFM) to decipher the mechanism of TZ-induce amyloid fibril formation in both the serum albumins at pHs7.4 and 3.5. The obtained results suggest that both the albumins forms amyloid-like aggregates in the presence of 1.0 to 15.0mM of TZ at pH3.5, but no amyloid fibril were seen at pH7.4. The possible cause of TZ-induced amyloid fibril formation is electrostatic and hydrophobic interaction because sulfate group of TZ may have interacted electrostatically with positively charged amino acids of the albumins at pH3.5 and increased protein-protein and protein-TZ interactions leading to amyloid fibril formation. The TEM, RLS and DLS results are suggesting that BSA forms bigger size amyloids compared to HSA, may be due to high surface hydrophobicity of BSA.


Assuntos
Soroalbumina Bovina/química , Albumina Sérica Humana/química , Tartrazina/farmacologia , Dicroísmo Circular , Vermelho Congo/química , Difusão Dinâmica da Luz , Humanos , Hidrodinâmica , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Microscopia de Força Atômica , Modelos Moleculares , Nefelometria e Turbidimetria , Agregados Proteicos , Estrutura Secundária de Proteína , Soroalbumina Bovina/ultraestrutura , Albumina Sérica Humana/ultraestrutura , Tartrazina/química
6.
Proc Natl Acad Sci U S A ; 114(7): 1474-1479, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28087691

RESUMO

Imaging single proteins has been a long-standing ambition for advancing various fields in natural science, as for instance structural biology, biophysics, and molecular nanotechnology. In particular, revealing the distinct conformations of an individual protein is of utmost importance. Here, we show the imaging of individual proteins and protein complexes by low-energy electron holography. Samples of individual proteins and protein complexes on ultraclean freestanding graphene were prepared by soft-landing electrospray ion beam deposition, which allows chemical- and conformational-specific selection and gentle deposition. Low-energy electrons do not induce radiation damage, which enables acquiring subnanometer resolution images of individual proteins (cytochrome C and BSA) as well as of protein complexes (hemoglobin), which are not the result of an averaging process.


Assuntos
Holografia/métodos , Proteínas/ultraestrutura , Imagem Individual de Molécula/métodos , Animais , Bovinos , Citocromos c/ultraestrutura , Elétrons , Grafite , Hemoglobinas/ultraestrutura , Holografia/instrumentação , Soroalbumina Bovina/ultraestrutura , Imagem Individual de Molécula/instrumentação , Espectrometria de Massas por Ionização por Electrospray/métodos , Eletricidade Estática , Vácuo
7.
Spectrochim Acta A Mol Biomol Spectrosc ; 170: 214-25, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-27450119

RESUMO

The thermodynamic study of the binding of sulphanilic acid with model transport protein bovine serum albumin is a promising approach in the area of synthesizing new sulfa drugs with improved therapeutic effect. Thus, such binding studies play an important role in the rational drug design process. The binding between sulphanilic acid and bovine serum albumin has been studied using calorimetry, light scattering in combination with spectroscopic and microscopic techniques. The calorimetric data reveals the presence of two sequential nature of binding sites where the first binding site has stronger affinity (~10(4)M(-1)) and second binding site has weaker affinity (~10(3)M(-1)). However, the spectroscopic (absorption and fluorescence) results suggest the presence of single low affinity binding site (~10(3)M(-1)) on protein. The contribution of polar and non-polar interactions to the binding process has been explored in the presence of various additives. It is found that sulphanilic acid binds with high affinity at Sudlow site II and with low affinity at Sudlow site I of protein. Light scattering and circular dichroism measurements have been used to study the effect on the molecular topology and conformation of protein, respectively. Thus these studies provide important insights into the binding of sulphanilic acid with bovine serum albumin both quantitatively and qualitatively.


Assuntos
Fenômenos Químicos , Soroalbumina Bovina/química , Soroalbumina Bovina/metabolismo , Ácidos Sulfanílicos/química , Ácidos Sulfanílicos/metabolismo , Animais , Sítios de Ligação , Calorimetria , Bovinos , Dicroísmo Circular , Entropia , Cinética , Ligação Proteica , Estrutura Secundária de Proteína , Espectroscopia de Prótons por Ressonância Magnética , Soroalbumina Bovina/ultraestrutura , Solventes , Espectrometria de Fluorescência , Tensoativos , Termodinâmica
8.
Int J Biol Macromol ; 91: 703-9, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-27177461

RESUMO

The current study tried to establish a simple and fast method for the preparation of BSA and HSA nanoparticles, based on an improved desolvation procedure under the aspect of a controllable particle size around 100nm for drug delivery applications. The Procedure used for the nanoparticles preparation was simplified by using a designed apparatus for controlling the addition of ethanol and it was used instead of conventional tubing pump which enabled the preparation of nanoparticles under defined conditions. By using EDC as cross-linker instead of glutharaldehyde, the time of nanoparticles preparation procedure was reduced to 3h. Several factors of the preparation process, such as the volume of the albumin solution, desolvating agent volume, the amount of cross-linker, the presence of salts and protein concentration were evaluated. Nanoparticles with smaller size were obtained under experimental conditions without the presence of salts or the use of buffers, 250mg of protein/4ml water, 5mg cross-linker, the addition of 4 and 8ml ethanol by using the designed apparatus to the HSA and BSA solution, respectively. By using this improved method, BSA and HSA nanoparticles of the size around 100nm and polydispersity below 0.2 were obtained.


Assuntos
Nanopartículas/química , Nanotecnologia/métodos , Albumina Sérica/química , Animais , Bovinos , Reagentes para Ligações Cruzadas/química , Etanol/farmacologia , Humanos , Nanopartículas/ultraestrutura , Tamanho da Partícula , Sais/química , Albumina Sérica/ultraestrutura , Soroalbumina Bovina/química , Soroalbumina Bovina/ultraestrutura , Soluções
9.
Artigo em Inglês | MEDLINE | ID: mdl-27037764

RESUMO

Glycation induced amyloid fibrillation is fundamental to the development of many neurodegenerative and cardiovascular complications. Excessive non-enzymatic glycation in conditions such as hyperglycaemia results in the increased accumulation of advanced glycation end products (AGEs). AGEs are highly reactive pro-oxidants, which can lead to the activation of inflammatory pathways and development of oxidative stress. Recently, the effect of non-enzymatic glycation on protein structure has been the major research area, but the role of specific AGEs in such structural alteration and induction of fibrillation remains undefined. In this study, we determined the specific AGEs mediated structural modifications in albumin mainly considering carboxymethyllysine (CML), carboxyethyllysine (CEL), and argpyrimidine (Arg-P) which are the major AGEs formed in the body. We studied the secondary structural changes based on circular dichroism (CD) and spectroscopic analysis. The AGEs induced fibrillation was determined by Congo red binding and examination of scanning and transmission electron micrographs. The amyloidogenic regions in the sequence of BSA were determined using FoldAmyloid. It was observed that CEL modification of BSA leads to the development of fibrillar structures, which was evident from both secondary structure changes and TEM analysis.


Assuntos
Produtos Finais de Glicação Avançada/farmacologia , Soroalbumina Bovina/química , Soroalbumina Bovina/efeitos dos fármacos , Amiloide/química , Animais , Arginina/metabolismo , Bovinos , Dicroísmo Circular , Lisina/metabolismo , Estrutura Secundária de Proteína , Soroalbumina Bovina/ultraestrutura , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
10.
Sci Rep ; 5: 13379, 2015 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-26307515

RESUMO

Dense arrays of gold-supported silver nanowires of about 100 nm in diameter grown directly in the channels of nanoporous aluminium oxide membrane were fabricated and tested as a novel platform for the immobilization and retention of BSA proteins in the microbial-protective environments. Additional treatment of the silver nanowires using low-temperature plasmas in the inductively-coupled plasma reactor and an atmospheric-pressure plasma jet have demonstrated that the morphology of the nanowire array can be controlled and the amount of the retained protein may be increased due to the plasma effect. A combination of the neutral gold sublayer with the antimicrobial properties of silver nanowires could significantly enhance the efficiency of the platforms used in various biotechnological processes.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Gases em Plasma/química , Soroalbumina Bovina/química , Prata/química , Adsorção , Teste de Materiais , Tamanho da Partícula , Ligação Proteica , Soroalbumina Bovina/ultraestrutura , Propriedades de Superfície
11.
Phys Chem Chem Phys ; 16(38): 20471-82, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25140357

RESUMO

Various properties of gold nanoparticles (GNPs) are found to play crucial roles in their biological activity. Among them, the morphology and surface chemistry are extremely important. This is because of differences in surface energies of various crystal facets arising from a large fraction of edges, corners and vertices. In the present work, we provide a comparative study on the adsorption and binding affinities of bovine serum albumin (BSA) onto triangular gold nanoplates (TGNP) and gold nanorods (GNR). The results were compared with similar size of both CTAB and citrate stabilized spherical GNPs. Our data suggested stronger binding of BSA on citrate stabilized spherical GNPs whereas TGNP shows the weakest binding among all the GNPs. A blue shift of approximately 20 nm in tryptophan fluorescence was observed for all CTAB stabilized GNPs, indicating the local dielectric changes surrounding the tryptophan residue. Loss of the secondary structure was also observed for all CTAB stabilized GNPs. No spectral shift was observed for citrate stabilized spherical GNPs though maximum quenching of fluorescence and minimum structural loss was observed. With the help of molecular simulation recently developed by our group, a binding model is proposed to explain all the above experimental results.


Assuntos
Ouro/química , Nanopartículas Metálicas/química , Nanopartículas Metálicas/ultraestrutura , Modelos Químicos , Modelos Moleculares , Soroalbumina Bovina/química , Soroalbumina Bovina/ultraestrutura , Adsorção , Sítios de Ligação , Simulação por Computador , Teste de Materiais , Tamanho da Partícula , Propriedades de Superfície
12.
Int J Nanomedicine ; 9: 3193-201, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25061296

RESUMO

Controlled-release carriers for local drug delivery have attracted increasing attention for inner-ear treatment recently. In this paper, flower-shaped bovine serum albumin (FBSA) particles were prepared by a modified desolvation method followed by glutaraldehyde or heat denaturation. The size of the FBSA particles varied from 10 µm to 100 µm, and most were 50-80 µm. Heat-denatured FBSA particles have good cytocompatibility with a prolonged survival time for L929 cells. The FBSA particles were utilized as carriers to investigate the release behaviors of the model drug - rhodamine B. Rhodamine B showed a sustained-release effect and penetrated the round-window membrane of guinea pigs. We also confirmed the attachment of FBSA particles onto the round-window membrane by microscopy. The FBSA particles, with good biocompatibility, drug-loading capacity, adhesive capability, and biodegradability, may have potential applications in the field of local drug delivery for inner-ear disease treatment.


Assuntos
Preparações de Ação Retardada/química , Portadores de Fármacos/química , Janela da Cóclea/metabolismo , Soroalbumina Bovina/química , Soroalbumina Bovina/ultraestrutura , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/farmacocinética , Preparações de Ação Retardada/toxicidade , Portadores de Fármacos/farmacocinética , Portadores de Fármacos/toxicidade , Cobaias , Teste de Materiais , Camundongos , Tamanho da Partícula , Rodaminas/química , Rodaminas/farmacocinética , Janela da Cóclea/química , Soroalbumina Bovina/farmacocinética , Soroalbumina Bovina/toxicidade , Distribuição Tecidual
13.
J Nanosci Nanotechnol ; 14(7): 4899-905, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24757960

RESUMO

Binding interaction of biologically synthesized silver nanoparticles with bovine serum albumin (BSA) has been investigated by UV-Vis and fluorescence spectroscopic techniques. UV-Vis analysis implies the formation of the ground state complex between BSA and silver nanoparticles. The analysis of fluorescence spectrum and fluorescence intensity indicates that silver nanoparticles (SNP) have a strong ability to quench the intrinsic fluorescence of BSA by dynamic quenching mechanisms. The number of binding sites 'n' and binding constants 'K' were determined at different temperatures based on fluorescence quenching. The thermodynamic parameters namely deltaH, deltaG, and deltaS were calculated at different temperatures (20, 30, and 40 degrees C) and the results indicate that both hydrophobic and electrostatic interactions were predominantly present in the SNP-BSA complex. Negative deltaG values imply that the binding process is spontaneous.


Assuntos
Nanopartículas Metálicas/química , Mapeamento de Interação de Proteínas/métodos , Soroalbumina Bovina/química , Prata/química , Análise Espectral/métodos , Animais , Sítios de Ligação , Bovinos , Teste de Materiais , Nanopartículas Metálicas/ultraestrutura , Tamanho da Partícula , Ligação Proteica , Soroalbumina Bovina/ultraestrutura
14.
Artigo em Inglês | MEDLINE | ID: mdl-24730839

RESUMO

The differences in phase behavior of anionic silica nanoparticles (88 Å) in the presence of two globular proteins [cationic lysozyme (molecular weight (MW) 14.7 kD) and anionic bovine serum albumin (BSA) (MW 66.4 kD)] have been studied by small-angle neutron scattering. The measurements were carried out on a fixed concentration (1 wt %) of Ludox silica nanoparticles with varying concentrations of proteins (0-5 wt %) at pH = 7. It is found that, despite having different natures (opposite charges), both proteins can render to the same kind of aggregation of silica nanoparticles. However, the concentration regions over which the aggregation is observed are widely different for the two proteins. Lysozyme with very small amounts (e.g., 0.01 wt %) leads to the aggregation of silica nanoparticles. On the other hand, silica nanoparticles coexist with BSA as independent entities at low protein concentrations and turn to aggregates at high protein concentrations (>1 wt %). In the case of lysozyme, the charge neutralization by the protein on the nanoparticles gives rise to the protein-mediated aggregation of the nanoparticles. The nanoparticle aggregates coexist with unaggregated nanoparticles at low protein concentrations, whereas, they coexist with a free protein at higher protein concentrations. For BSA, the nonadsorbing nature of the protein produces the depletion force that causes the aggregation of the nanoparticles at higher protein concentrations. The evolution of the interaction is modeled by the two Yukawa potential, taking account of both attractive and repulsive terms of the interaction in these systems. The nanoparticle aggregation is found to be governed by the short-range attraction for lysozyme and the long-range attraction for BSA. The aggregates are characterized by the diffusion limited aggregate type of mass fractal morphology.


Assuntos
Modelos Químicos , Muramidase/química , Nanopartículas/química , Soroalbumina Bovina/química , Dióxido de Silício/química , Animais , Bovinos , Simulação por Computador , Modelos Moleculares , Muramidase/ultraestrutura , Nanopartículas/ultraestrutura , Transição de Fase , Ligação Proteica , Soroalbumina Bovina/ultraestrutura
15.
Colloids Surf B Biointerfaces ; 117: 512-9, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24698146

RESUMO

The main challenge in the oral delivery of protein drugs is to enhance their oral bioavailability. Herein, we report the uniform-sized liposphere prepared by premix membrane emulsification combined with W1/O/W2 double-emulsion method as a potential oral carrier for proteins. The protein-loaded liposphere was composed of a hydrophobic poly (D, L-lactide-co-glycolide) (PLGA) core and the lipid molecules self-assembled at the interface of W1/O and O/W2. During the preparation, the protein structure was effectively maintained. Compared with PLGA microsphere, the liposphere achieved a higher loading capacity (LC, 20.18%), entrapment efficiency (EE, 90.82%) and a lower initial burst (24.73%). Importantly, the lipospheres also showed high transcytotic efficiency with human microfold cell (M cell) model, leading to a potential enhancement of intestinal absorption. This result, together with the above studies supported that the PLGA-lipid liposphere could be a promising platform for enhancing the proteins oral bioavailability.


Assuntos
Sistemas de Liberação de Medicamentos , Ácido Láctico/química , Lipídeos/química , Microesferas , Ácido Poliglicólico/química , Soroalbumina Bovina/administração & dosagem , Administração Oral , Animais , Bovinos , Linhagem Celular Tumoral , Humanos , Microscopia Eletrônica de Varredura , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Soroalbumina Bovina/metabolismo , Soroalbumina Bovina/ultraestrutura , Transcitose
16.
Opt Express ; 22(3): 2807-20, 2014 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-24663572

RESUMO

Optical microresonator biosensors have proven to be a valid tool to perform affinity analysis of a biological binding event. However, when these microresonators are excited with a single optical mode they can not distinguish between a thin dense layer of biomolecules or a thick sparse layer. This means the sensor is "blind" to changes in shape of bound biomolecules. We succeeded in exciting a Silicon-on-Insulator (SOI) microring with TE and TM polarisations simultaneously by using an asymmetrical directional coupler and as such were able to separately determine the thickness and the density (or refractive index) of a bound biolayer. A proof-of-concept is given by determining both parameters of deposited dielectric layers and by analysing the conformational changes of Bovine Serum Albumin (BSA) proteins due to a change in pH of the buffer.


Assuntos
Técnicas Biossensoriais/instrumentação , Lentes , Refratometria/instrumentação , Soroalbumina Bovina/química , Soroalbumina Bovina/ultraestrutura , Silício/química , Desenho de Equipamento , Análise de Falha de Equipamento , Miniaturização , Conformação Proteica , Soroalbumina Bovina/análise
17.
J Pharm Sci ; 103(3): 810-20, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24415208

RESUMO

The dehydration of biologics is commonly employed to achieve solid-dose formulation and enhanced stability during long-term preservation. We have developed a novel process, Microglassification™, which can rapidly and controllably dehydrate protein solutions into solid amorphous microspheres at room temperature. Single bovine serum albumin (BSA) microdroplets were suspended in pentanol or decanol using a micropipette, and the dynamic changes in droplet dissolution were observed in real-time and correlated to protein's water of hydration, medium's water activity, and microsphere protein concentration. Microglassification™ was also carried out at bulk scale, and changes in BSA secondary structure were analyzed by Fourier transform infrared spectroscopy and fluorescence spectroscopy; multimer formation was detected by native gel electrophoresis. BSA concentration in the microsphere increased with solvent exposure time and decreasing water activity. Image analysis at single particle and bulk scale showed the formation of solid BSA microspheres with a maximum protein concentration of 1147 ± 32 mg/mL. The native BSA samples were dehydrated to approximately 450 waters per BSA, which is well below monolayer coverage of 1282 waters per BSA. The secondary structure of Microglassified™ BSA reverted to native-like conformation upon rehydration with only minor irreversible aggregation (2.7%). Results of the study establish the efficacy of the Microglassification™ for the successful dehydration of biologics.


Assuntos
Produtos Biológicos/química , Dessecação/métodos , Soroalbumina Bovina/química , Animais , Produtos Biológicos/metabolismo , Bovinos , Fenômenos Químicos , Álcoois Graxos/química , Estudos de Viabilidade , Microquímica/métodos , Microesferas , Tamanho da Partícula , Pentanóis/química , Desnaturação Proteica , Renaturação Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Soroalbumina Bovina/metabolismo , Soroalbumina Bovina/ultraestrutura , Solubilidade , Solventes/química , Vitrificação , Água/análise
18.
Food Chem ; 141(2): 695-701, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23790836

RESUMO

In polymeric nanoparticle preparation, despite similar conditions, large fluctuations in particle size distributions are usually observed. Herein, we demonstrate that the intermittent addition of a desolvating agent can improve reproducibility in the preparation of polymeric bovine serum albumin (BSA) nanoparticles. Using this modification, BSA nanoparticles of controlled size can be manufactured with narrow particle size distributions. In our study, ethanol as a desolvating agent was added intermittently to 1% BSA solutions at different pHs with stirring at 700rpm. The effect of the preparation parameters on size and optical density of the fabricated nanoparticles were studied. The average particles sizes of BSA nanoparticles prepared at pH values of 6, 7 and 9 were approximately 100, 200 and 300nm, respectively. As ethanol addition increased, desolvation of BSA molecules resulted in formation of loose-structured particles with pH-dependent size. Beyond that, only particle density increased, but size remained unchanged with further addition of ethanol. Consistently uniform particle size distribution was achieved by adding ethanol intermittently.


Assuntos
Etanol/química , Nanopartículas/química , Soroalbumina Bovina/química , Concentração de Íons de Hidrogênio , Microscopia Eletrônica de Transmissão , Tamanho da Partícula , Polimerização , Soroalbumina Bovina/ultraestrutura
19.
Int J Biol Macromol ; 53: 42-53, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23148944

RESUMO

The manuscript describes the study of the oligomerization process of bovine serum albumin (BSA) in two different structural monomeric forms: the extended-form (E) at pH 2.0 and the basic-form (B) at pH 9.0. The study was conducted at low protein concentration (1mg/ml) and relatively short incubation time (maximum 56 days) in order to investigate early oligomerization events rather than the formation of mature fibrils. The comparison between the two isoforms show that oligomers form much faster (∼6 days) in the E-form than in the B-form where formation of oligomers requires ∼4 weeks. The oligomers appear to be limited to a maximum of tetramers with size <30 nm. Hydrophobic interactions from exposed neutral amino acid residues in the elongated E-form are the likely cause for the quick formation of aggregates at acidic pH. We used an array of biophysical techniques for the study and determined that oligomerization occurs without further large changes in the secondary structure of the monomers. Under the conditions adopted in this study, aggregation does not seem to exceed the formation of tetramers, even though a very small amount of much larger aggregates seem to form.


Assuntos
Soroalbumina Bovina/química , Algoritmos , Amiloide/química , Animais , Bovinos , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Luz , Microscopia de Força Atômica , Modelos Moleculares , Tamanho da Partícula , Multimerização Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Espalhamento de Radiação , Soroalbumina Bovina/ultraestrutura , Soluções , Espectrometria de Fluorescência
20.
Acta Biomater ; 8(10): 3652-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22705046

RESUMO

Photo-crosslinked networks were prepared from fumaric acid monoethyl ester-functionalized poly(D,L-lactic acid) oligomers and N-vinyl-2-pyrrolidone. Two model proteins, lysozyme and albumin, were incorporated into the network films as solid particles and their release behavior was studied. By varying the NVP content and macromer molecular weight the degradation behavior and protein release profiles of the prepared networks could be tuned. The more hydrophilic and less densely crosslinked networks released albumin and lysozyme at a faster rate. Although active lysozyme was released from the networks over the complete release period, lysozyme release was often incomplete. This was most likely caused by electrostatic and/or hydrophobic interactions between the protein and the degrading polymer network.


Assuntos
Reagentes para Ligações Cruzadas/química , Fumaratos/síntese química , Ácido Láctico/síntese química , Luz , Muramidase/metabolismo , Polímeros/síntese química , Pirrolidinonas/síntese química , Soroalbumina Bovina/metabolismo , Animais , Bovinos , Preparações de Ação Retardada , Fumaratos/química , Géis , Vidro/química , Hidrólise , Ácido Láctico/química , Peso Molecular , Muramidase/ultraestrutura , Poliésteres , Polímeros/química , Pirrolidinonas/química , Soroalbumina Bovina/ultraestrutura , Temperatura de Transição , Água/química
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