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1.
Bioelectrochemistry ; 131: 107397, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31706117

RESUMO

A new polyclonal antibody that recognizes the CP4 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS), which provides resistance to glyphosate in soybean (Roundup Ready®, RR soybean), was produced. New Zealand rabbits were injected with a synthetic peptide (Pc_312-324, (PEP)) present in the soybean CP4-EPSPS protein. The anti-PEP antibodies production was evaluated by electrophoresis (SDS-PAGE) and an enzyme-linked immunosorbent assay (ELISA) was developed in order to study their specificity. The ELISA showed that the polyclonal antibody was specific to PEP. In addition, the anti- PEP was immobilized onto a gold disk electrode and the antigen-antibody interaction was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Moreover, the EIS showed that the electron transfer resistance of the modified electrode increased after incubation with solutions containing CP4-EPSPS protein from RR transgenic soybean, while no changes were detected after incubation with no-RR soybean proteins. These results suggest that the CP4-EPSPS was immobilized onto the electrode, due to the specific interaction with the anti-PEP. These results show that this antigen-antibody interaction can be detected by electrochemical techniques, suggesting that the anti-PEP produced can be used in electrochemical immunosensors development to quantify transgenic soybean.


Assuntos
Anticorpos/metabolismo , Formação de Anticorpos , Técnicas Eletroquímicas/métodos , Peptídeos/metabolismo , Plantas Geneticamente Modificadas/imunologia , Soja/imunologia , Técnicas Biossensoriais , Ensaio de Imunoadsorção Enzimática/métodos
2.
Chem Soc Rev ; 48(22): 5488-5505, 2019 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-31552920

RESUMO

Glycans - simple or complex carbohydrates - play key roles as recognition determinants and modulators of numerous physiological and pathological processes. Thus, many biotechnological, diagnostic and therapeutic opportunities abound for molecular recognition entities that can bind glycans with high selectivity and affinity. This review begins with an overview of the current biologically and synthetically derived glycan-binding scaffolds that include antibodies, lectins, aptamers and boronic acid-based entities. It is followed by a more detailed discussion on various aspects of their generation, structure and recognition properties. It serves as the basis for highlighting recent key developments and technical challenges that must be overcome in order to fully deal with the specific recognition of a highly diverse and complex range of glycan structures.


Assuntos
Anticorpos/química , Aptâmeros de Nucleotídeos/química , Ácidos Borônicos/química , Lectinas/química , Polissacarídeos/química , Receptores Artificiais/química , Anticorpos/metabolismo , Aptâmeros de Nucleotídeos/metabolismo , Ácidos Borônicos/metabolismo , Humanos , Lectinas/metabolismo , Polissacarídeos/síntese química , Polissacarídeos/metabolismo , Receptores Artificiais/metabolismo
3.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 653-658, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537250

RESUMO

Objective To produce rabbit polyclonal antibodies against human retinol-binding protein (RBP). Methods RBP cDNA was amplified by reverse transcription polymerase chain reaction (RT-PCR) and then the amplified products were inserted into prokaryotic expression vector pET-28a(+) to construct recombinant plasmid pET-28a(+)-RBP. The established plasmid was then transformed into E. coli. Isopropylthio-ß-D-thiogalactoside (IPTG) was used to induce the expression of recombinant protein His-RBP in E. coli. The expression products were identified by SDS-PAGE from different clones of E. coli to screen positive bacteria, followed by amplifying culture. His-RBP protein was purified from the expression products of positive clones. The purified recombinant His-RBP was used to immunize New Zealand white rabbits. Antisera were acquired after four times of booster immunization. The prepared purified polyclonal antibodies were identified by SDS-PAGE, ELISA and Western blotting. Results We successfully constructed the recombinant plasmid pET-28a(+)-RBP, and acquired recombinant protein His-RBP of high purity. ELISA showed that the antibody titer reached 1:512 000. Conclusion The rabbit polyclonal antibodies against human RBP have been successfully prepared.


Assuntos
Anticorpos/metabolismo , Escherichia coli , Proteínas de Ligação ao Retinol/biossíntese , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Vetores Genéticos , Humanos , Plasmídeos , Coelhos
4.
Nat Biotechnol ; 37(9): 1080-1090, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31427819

RESUMO

Spatial mapping of proteins in tissues is hindered by limitations in multiplexing, sensitivity and throughput. Here we report immunostaining with signal amplification by exchange reaction (Immuno-SABER), which achieves highly multiplexed signal amplification via DNA-barcoded antibodies and orthogonal DNA concatemers generated by primer exchange reaction (PER). SABER offers independently programmable signal amplification without in situ enzymatic reactions, and intrinsic scalability to rapidly amplify and visualize a large number of targets when combined with fast exchange cycles of fluorescent imager strands. We demonstrate 5- to 180-fold signal amplification in diverse samples (cultured cells, cryosections, formalin-fixed paraffin-embedded sections and whole-mount tissues), as well as simultaneous signal amplification for ten different proteins using standard equipment and workflows. We also combined SABER with expansion microscopy to enable rapid, multiplexed super-resolution tissue imaging. Immuno-SABER presents an effective and accessible platform for multiplexed and amplified imaging of proteins with high sensitivity and throughput.


Assuntos
Anticorpos/imunologia , Anticorpos/metabolismo , Imuno-Histoquímica/métodos , Proteínas/metabolismo , Coloração e Rotulagem , Animais , Linhagem Celular , DNA/análise , Código de Barras de DNA Taxonômico , Corantes Fluorescentes , Humanos , Hibridização in Situ Fluorescente/métodos , Camundongos , Microscopia de Fluorescência/métodos , Retina/citologia
5.
Int J Nanomedicine ; 14: 5611-5622, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413566

RESUMO

Background: Multimodal imaging probes have become a powerful tool for improving detection sensitivity and accuracy, which are important in disease diagnosis and treatment. Methods: In this study, novel bifunctional magnetic resonance imaging (MRI)/fluorescence probes were prepared by loading gadodiamide into fluorescent silica nanoparticles (NPs) (Gd@Cy5.5@SiO2-PEG-Ab NPs) for targeting of prostate cancer (PCa). The physicochemical characteristics, biosafety and PCa cell targeting ability of the Gd@Cy5.5@SiO2-PEG-Ab NPs were studied in vitro and in vivo. Results: The Gd@Cy5.5@SiO2-PEG-Ab NPs had a spherical morphology with a relatively uniform size distribution and demonstrated high efficiency for Gd loading. In vitro and in vivo cell-targeting experiments demonstrated a high potential for the synthesized NPs to target prostate-specific membrane antigen (PSMA) receptor-positive PCa cells, enabling MRI and fluorescence imaging. In vitro cytotoxicity assays and in vivo hematological and pathological assays showed that the prepared NPs exhibited good biological safety. Conclusion: Our study demonstrates that the synthesized Gd@Cy5.5@SiO2-PEG-Ab NPs have great potential as MRI/fluorescence contrast agents for specific identification of PSMA receptor-positive PCa cells.


Assuntos
Gadolínio DTPA/química , Imagem por Ressonância Magnética , Nanopartículas/química , Polietilenoglicóis/química , Neoplasias da Próstata/diagnóstico por imagem , Dióxido de Silício/química , Animais , Anticorpos/metabolismo , Linhagem Celular Tumoral , Meios de Contraste , Endocitose , Fluorescência , Glutamato Carboxipeptidase II/metabolismo , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/toxicidade , Nanopartículas/ultraestrutura , Especificidade de Órgãos , Polietilenoglicóis/síntese química , Padrões de Referência
6.
Biotechnol Lett ; 41(8-9): 963-977, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31325004

RESUMO

OBJECTIVES: The relationships of manipulation of culture temperature and medium circulation rate on the metabolic parameters were regressed by multiple linear regression analysis in hollow fiber bioreactors (HFB). RESULTS: The high circulation rate could significantly enhance the oxygen consumption of the hybridoma cells and the medium's oxidation-reduction potential. A mildly hypothermic condition of 36 °C and a circulation rate of 182.5 mL/min could support the hybridoma had the maximal antibody titer of 60.75 µg/mL for 20 days. When the ammonium ion was 65 ppm or lactate close to 2.6 g/L, the medium was replaced to maintain the stable and healthy cells at the high cell concentration of 3.33 × 108/mL for continuous antibody production. Two serum-free media could be successfully applied to this perfusion system and maintain hybridoma growth and antibody production. CONCLUSION: The single-use HFBs could provide the advantages including high cell density, low shear stress, and continuous antibody production.


Assuntos
Anticorpos/metabolismo , Reatores Biológicos , Contagem de Células , Hibridomas/metabolismo , Anticorpos/genética , Meios de Cultura/química , Análise Multivariada , Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente
7.
J Biochem ; 166(3): 205-212, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31251348

RESUMO

Signal enhancing systems have been introduced to enable detection of cell surface antigens by flow cytometry. Cell surface antigens are important targets that describe the function and lineage of cells. Although flow cytometry is an effective tool for analysing cell surface antigens, this technique has poor sensitivity, which prohibits the detection of many important antigens on cell membranes. Thus, signal amplification is essential for developing practical tools for evaluating cell surface antigens by flow cytometry. Using a bright fluorophore or fluorescent polymer incorporated into antibodies is a straightforward strategy to improve flow cytometry sensitivity but may affect the functional characteristics of the labelled antibody. In contrast, enzymatic signal amplification is a more practical and efficient strategy to improve sensitivity that should not affect antibody activity. Although enzymatic signal amplification still has a number of drawbacks, this approach is a promising strategy to analyse cell surface antigens.


Assuntos
Antígenos de Superfície/análise , Citometria de Fluxo , Anticorpos/química , Anticorpos/imunologia , Anticorpos/metabolismo , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Humanos , Polímeros/química , Polímeros/metabolismo
8.
J Chromatogr A ; 1602: 284-299, 2019 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-31230875

RESUMO

A great number of protein-binding peptides are known and utilized as drugs, diagnostic reagents, and affinity ligands. Recently, however, peptide mimetics have been proposed as valuable alternative to peptides by virtue of their excellent biorecognition activity and higher biochemical stability. This poses the need to develop a strategy for translating known protein-binding peptides into peptoid analogues with comparable or better affinity. This work proposes a route for translation utilizing the IgG-binding peptide HWRGWV as reference sequence. An ensemble of peptoid analogues of HWRGWV were produced by adjusting the number and sequence arrangement of residues containing functional groups that resemble both natural and non-natural amino acids. The variants were initially screened via IgG binding tests in non-competitive mode to select candidate ligands. A set of selected peptoids were studied in silico by docking onto putative binding sites identified on the crystal structures of human IgG1, IgG2, IgG3, and IgG4 subclasses, returning values of predicted binding energy that aligned well with the binding data. Selected peptoids PL-16 and PL-22 were further characterized by binding isotherm analysis to determine maximum capacity (Qmax ˜ 48-57 mg of IgG per mL of adsorbent) and binding strength on solid phase (KD ˜ 5.4-7.8 10-7 M). Adsorbents PL-16-Workbeads and PL-22-Workbeads were used for purifying human IgG from a cell culture supernatant added with bovine serum, affording high values of IgG recovery (up to 85%) and purity (up to 98%) under optimized binding and elution conditions. Both peptoid ligands also proved to be stable against proteolytic enzymes and strong alkaline agents. Collectively, these studies form a method guiding the design of peptoid variants of cognate peptide ligands, and help addressing the challenges that, despite the structural similarity, the peptide-to-peptoid translation presents.


Assuntos
Anticorpos/metabolismo , Afinidade de Anticorpos , Peptídeos/química , Peptoides/química , Adsorção , Álcalis/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Bovinos , Cricetinae , Cricetulus , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/metabolismo , Ligantes , Simulação de Acoplamento Molecular , Ligação Proteica , Proteólise , Temperatura Ambiente
9.
Mater Sci Eng C Mater Biol Appl ; 102: 315-323, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31147004

RESUMO

The conjugation of nanoparticles with antibodies has been successfully applied in sandwich immunoassays for detecting cancer biomarkers. However, two antibodies are necessary to perform such experiment, being one of them functionalized with a signal label for optical or electrochemical assay. This approach is time and cost consuming compared to direct label-free immunoassays. In this study, we propose the synthesis of gold nanoparticles conjugated to anti-PSA antibody to produce a label-free impedimetric immunosensors based on nanostructured Layer-by-Layer (LbL) films. Prostate-specific antigen (PSA) detection was performed by electrochemical impedance spectroscopy demonstrating a detection mechanism governed by Langmuir-Freundlich adsorption model. This strategy provided a significant sensitivity using 10-fold less antibody than conventional immunosensors, i.e. decreasing costs using a simple approach, with a limit of detection of 0.17 ng mL-1 and an analytical range of 0.1-20 ng mL-1 indicating that our sensor is potentially useful for clinical applications.


Assuntos
Anticorpos/metabolismo , Ouro/química , Imunoensaio/métodos , Nanopartículas Metálicas/química , Coloração e Rotulagem , Técnicas Biossensoriais , Nanopartículas Metálicas/ultraestrutura , Antígeno Prostático Específico/metabolismo , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
10.
Mater Sci Eng C Mater Biol Appl ; 102: 492-501, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31147020

RESUMO

The technology of an immunoassay detection platform is critical to clinical disease diagnoses, especially for developing a medical diagnostic system. A polymer-based immunoassay platform was fabricated on nonwoven fabric polypropylene (PP) using a photografting reaction to graft 2-hydroxyethyl methacrylate (HEMA) and sulfobetaine (SBMA). The antifouling properties of PP-g-P(HEMA-co-SBMA) were investigated by fibrinogen adsorption and platelet adhesion. Carbonyldiimidazole was employed to activate the pendant hydroxyl groups in HEMA moieties and covalently coupled antibody molecules. The detection of the limit of the immunoassay platform was as low as 10 pg/mL. Antibody amount and bioactivity affected the availability of antibody and the sensitivity of immunoassay. The immune efficiency was dependent on the strategies of antibody immobilization. The immune efficiency of Au-g-P(SBMA-co-HEMA) and Au-SH surfaces measured by QCM-D was 165% and 35.7%, respectively. The covalently binding antibody via hydrophilic polymer chains as spacers could retain fragment antigen-binding up orientation, maintain the bioactivity of antibody, and mainly improve the accessibility of antibody molecules via adjusting the conformations of polymer chains when the antibodies recognized the antigens. Therefore, grafting hydrophilic polymers, such as zwitterionic PSBMA and reactive PHEMA onto nonwoven fabric PP, and binding antibody by covalent strategy had the potential to be developed as a commercial immunoassay platform.


Assuntos
Imunoensaio/métodos , Processos Fotoquímicos , Polipropilenos/química , Adsorção , Animais , Anticorpos/metabolismo , Antígenos/metabolismo , Fibrinogênio/metabolismo , Fluorescência , Proteínas Imobilizadas/metabolismo , Limite de Detecção , Teste de Materiais , Coelhos , Razão Sinal-Ruído , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície
11.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1122-1123: 83-89, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31173996

RESUMO

For magnetic control of cells for biomedical applications such as targeting of immune cells to tumors, cells must be magnetizable. For that, cells are incubated with superparamagnetic iron oxide nanoparticles (SPIONs) to take them up and thus become magnetizable. When using adherent cells, non-ingested SPIONs can be easily removed by rinsing of the particles regardless of their colloidal stability in cell culture medium. However, if suspension cells such as T cells are to be loaded with SPIONs, established methods to separate excess nanoparticles from cells are based on physicochemical parameters such as density, size or magnetizability. Thus, colloidal stability of the particles is of great importance, since only colloidally stable SPIONs can be completely separated from the cells due to their physicochemical differences. Aggregates of colloidally meta- or unstable particles cannot, however, be separated from cells due to their overlapping sizes and densities. Thus, development of an alternative method for the separation of nanoparticle aggregates from suspension cells is urgently needed. Here, we present an affinity chromatographic separation method based on immunohistochemical properties of the respective cells. A desthiobiotinylated antibody against a cellular surface antigen (here CD90.2 receptor on EL4 T cells) is immobilized on a streptavidin agarose column optimized for cell purification. Subsequently the column is loaded with the particle/cell suspension so that the cells bind to the column. After removing the particles by washing, the cells can be gently eluted with biotin solution under physiological conditions. This allows >95% of the excess iron concentration to be removed while maintaining cell viability.


Assuntos
Cromatografia de Afinidade/métodos , Separação Imunomagnética/métodos , Nanopartículas de Magnetita/química , Animais , Anticorpos/metabolismo , Biotina/química , Linhagem Celular , Sobrevivência Celular/fisiologia , Coloides/química , Camundongos , Estreptavidina/química
12.
Int J Mol Sci ; 20(9)2019 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-31083520

RESUMO

Alpha-synuclein is considered the major pathological protein associated with Parkinson's disease, but there is still no effective immunotherapy which targets alpha-synuclein. In order to create a safer and more effective therapy against PD, we are targeting an epitope of alpha-synuclein rather than full-length alpha-synuclein. We have selected several antigenic domains (B-cell epitope) through antigenicity prediction, and also made several recombinant protein fragments from alpha-synuclein upon antigenicity prediction in an E. coli system. We then tested the function of each of the peptides and recombinant fragments in aggregation, their toxicity and antigenicity. We have discovered that the full-length recombinant (aa1-140) can aggregate into oligomers or even fibrils, and fragment aa15-65 can promote the aggregation of aa1-140. It is worth noting that it not only promotes whole protein aggregation, but also self-aggregates as seen by western blotting and silver staining assays. We have tested all candidates on primary neurons for their toxicity and discovered that aa15-65 is the most toxic domain compared to all other fragments. The antibody targeting this domain also showed both anti-aggregation activity and some therapeutic effect. Therefore, we believe that we have identified the most potent therapeutic domain of alpha synuclein as a therapeutic target.


Assuntos
Doença de Parkinson/tratamento farmacológico , alfa-Sinucleína/química , alfa-Sinucleína/uso terapêutico , Animais , Anticorpos/metabolismo , Mapeamento de Epitopos , Feminino , Humanos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/toxicidade , Agregados Proteicos , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/toxicidade
13.
ACS Appl Mater Interfaces ; 11(21): 19495-19505, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31058488

RESUMO

Rapid and early diagnosis of respiratory viruses is key to preventing infections from spreading and guiding treatments. Here, we developed a sensitive and quantitative surface-enhanced Raman scattering-based lateral flow immunoassay (SERS-based LFIA) strip for simultaneous detection of influenza A H1N1 virus and human adenovirus (HAdV) by using Fe3O4@Ag nanoparticles as magnetic SERS nanotags. The new type of Fe3O4@Ag magnetic tags, which were conjugated with dual-layer Raman dye molecules and target virus-capture antibodies, performs the following functions: specific recognition and magnetic enrichment of target viruses in the solution and SERS detection of the viruses on the strip. Based on this strategy, the magnetic SERS strip can directly be used for real biological samples without any sample pretreatment steps. The limits of detection for H1N1 and HAdV were 50 and 10 pfu/mL, respectively, which were 2000 times more sensitive than those from the standard colloidal gold strip method. Moreover, the proposed strip is easy to operate, rapid, stable, and can achieve high throughput and is thus a potential tool for early detection of virus infection.


Assuntos
Adenovírus Humanos/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Magnetismo , Análise Espectral Raman , Anticorpos/metabolismo , Coloides/química , Compostos Férricos/química , Ouro/química , Humanos , Nanopartículas de Magnetita/química , Nanopartículas de Magnetita/ultraestrutura , Sensibilidade e Especificidade , Prata/química
14.
Int J Nanomedicine ; 14: 2451-2464, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31040668

RESUMO

Background: Acute myocardial infarction (AMI), usually caused by atherosclerosis of coronary artery, is the most severe manifestation of coronary artery disease which results in a large amount of death annually. A new diagnosis approach with high accuracy, reliability and low measuring-time-consuming is essential for AMI quick diagnosis. Purpose: The objective of this study was to develop a new point-of-care testing system with high accuracy and reliability for AMI quick diagnosis. Patients and methods: 50 plasma samples of acute myocardial infarction patients were analyzed by developed Smartphone-Assisted Pressure-Measuring-Based Diagnosis System (SPDS). The concentration of substrate was firstly optimized. The effect of antibody labeling and matrix solution on measuring result were then evaluated. And standard curves for cTnI, CK-MB and Myo were built for clinical sample analysis. The measuring results of 50 clinical samples were finally evaluated by comparing with the measuring result obtained by CLIA. Results: The concentration of substrate H2O2 was firstly optimized as 30% to increase measuring signal. A commercial serum matrix was chosen as the matrix solution to dilute biomarkers for standard curve building to minimize matrix effect on the accuracy of clinical plasma sample measuring. The standard curves for cTnI, CK-MB and Myo were built, with measuring dynamic range of 0-25 ng/mL, 0-33 ng/mL and 0-250 ng/mL, and limit of detection of 0.014 ng/mL, 0.16 ng/mL and 0.85 ng/mL respectively. The measuring results obtained by the developed system of 50 clinical plasma samples for three biomarkers matched well with the results obtained by chemiluminescent immunoassay. Conclusion: Due to its small device size, high sensitivity and accuracy, SPDS showed a bright potential for point-of-care testing (POCT) applications.


Assuntos
Pressão Sanguínea , Infarto do Miocárdio/diagnóstico , Infarto do Miocárdio/fisiopatologia , Smartphone , Anticorpos/metabolismo , Biomarcadores/sangue , Catálise , Feminino , Humanos , Peróxido de Hidrogênio/análise , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Nanopartículas/química , Platina/química , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Eletricidade Estática
15.
Biomed Res Int ; 2019: 7528609, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31139649

RESUMO

Beta-glucan (ß-glucan) is a macromolecule structure where glucose unit has bonded through ß-glycosidic bond at 1 and 3 positions. It is well known as a natural immunomodulator without exhibiting any side effects via enhancing immunity. Mushroom contains a large amount of ß-glucan and it has anticancerous and antioxidant efficacy. Structure and physical properties of ß-glucan are highly influenced by the types of mushroom. In particular, Grifola frondosa has ß-1, 3 and ß-1, 6 bonds in their structure. It has been noted that ß-glucan content also depends upon the size of mushroom particles. The exact content of ß-glucan and their immunological activity by a particle size of G. frondosa have yet to be fully elucidated. Herein, ß-glucan contents were analyzed according to the particle size of leaf mushroom followed by cell activation and immunoactivity analysis. The highest ß-glucan content was observed at a particle size of 20-30 µm (27.65 ± 0.30 w/w). All samples showed ~ 103% cell activation compared to the control and greater cell activity was observed at higher concentration. The significant increase in cytokines secretion was observed in the presence of 20-30 µm particle size of G. frondosa compared to the control. This study suggested that 20-30 µm size is the suitable size of G. frondosa that can be used as a health supplement and food additive to act as an immune booster, hypotensive agent, and hypoglycemic agent.


Assuntos
Grifola/química , Imunidade/efeitos dos fármacos , Polissacarídeos/farmacologia , Anticorpos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Tamanho da Partícula , Espectroscopia de Prótons por Ressonância Magnética , beta-Glucanas/química , beta-Glucanas/isolamento & purificação
16.
Yonsei Med J ; 60(6): 509-516, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31124333

RESUMO

PURPOSE: This study was conducted to verify the induction and mechanism of selective apoptosis in G361 melanoma cells using anti-HER2 antibody-conjugated gold nanoparticles (GNP-HER2). MATERIALS AND METHODS: Following GNP-HER2 treatment of G361 cells, cell cycle arrest and apoptosis were measured by WST-1 assay, Hemacolor staining, Hoechst staining, immunofluorescence staining, fluorescence-activated cell sorting analysis, and Western blotting. RESULTS: G361 cells treated with GNP-HER2 showed condensation of nuclei, which is an apoptotic phenomenon, and translocation of apoptosis-inducing factor and cytochrome c from mitochondria into the nucleus and cytoplasm, respectively. Increases in BAX in cells undergoing apoptosis, activation of caspase-3 and -9, and fragmentation of poly (ADP-ribose) polymerase and DNA fragmentation factor 45 (inhibitor of caspase-activated DNase) were observed upon GNP-HER2 treatment. Following GNP-HER2 treatment, an increase of cells in sub-G1 phase, which is a signal of cell apoptosis, was observed. This resulted in the down-regulation of cyclin A, cyclin D1, cyclin E, cdk2, cdk4, and cdc2 and the up-regulation of p21. Thus, GNP-HER2 treatment was confirmed to induce the cessation of cell cycle progression. Also, decreases in phospho-focal adhesion kinase and phospho-human epidermal growth factor receptor, which activate cellular focal adhesion, and decreases in phospho-paxillin, which stimulates the disassembly of filamentous actin, were observed. Reduced cell adhesion and disassembly of the intracellular structure indicated cell deactivation. CONCLUSION: GNP-HER2 can selectively kill G361 melanoma cells without affecting normal cells. The mechanism of G361 cell death upon treatment with GNP-HER2 was apoptosis accompanied by activation of caspases.


Assuntos
Anticorpos/metabolismo , Apoptose , Ouro/química , Melanoma/patologia , Nanopartículas Metálicas/química , Receptor ErbB-2/metabolismo , Actinas/metabolismo , Fator de Indução de Apoptose/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular , Citocromos c/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Fase G1 , Humanos , Nanopartículas Metálicas/ultraestrutura
17.
Int J Nanomedicine ; 14: 2829-2846, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31114197

RESUMO

Background: Vitamin D3 possesses anti-inflammatory and modulatory properties in addition to its role in calcium and phosphate homeostasis. Upon activation, macrophages (M) can initiate and sustain pro-inflammatory cytokine production in inflammatory disorders and play a pathogenic role in certain cancers. Purpose: The main purpose of this study was to encapsulate and specifically target calcitriol to macrophages and investigate the anti-inflammatory properties of calcitriol in vitro and in vivo. Methods: In this study we have designed and developed near-infrared calcitriol PEGylated nanoparticles (PEG-LNP(Cal)) using a microfluidic mixing technique and modified lipid nanoparticles (LNPs) to target the M specific endocytic receptor CD163. We have investigated LNP cellular uptake and anti-inflammatory effect in LPS-induced M in vitro by flow cytometry, confocal microscopy and gene expression analyses. LNP pharmacodynamics, bio-distribution and organ specific LNP accumulation was also investigated in mice in vivo. Results: In vitro, we observed the specific uptake of PEG-LNP(Cal)-hCD163 in human M, which was significantly higher than the non-specific uptake of control PEG-LNP(Cal)-IgG(h) in M. Pretreatment with encapsulated calcitriol was able to attenuate intracellular TNF-expression, and M surface marker HLA-DR expression more efficiently than free calcitriol in LPS-induced M in vitro. Encapsulated calcitriol diminished mRNA gene levels of TNF-, NF-B, MCP-1 and IL-6, while upregulating IL-10. TNF- and IL-6 protein secretion also decreased. In mice, an in vivo pharmacodynamic study of PEG-LNP(Cal) showed a rapid clearance of IgG and CD163 modified LNPs compared to PEG-LNP(Cal). Antibody modified PEG-LNP(Cal) accumulated in the liver, spleen and kidney, whereas unmodified PEG-LNP(Cal) accumulation was only observed in the liver. Conclusion: Our results show that calcitriol can be effectively targeted to M. Our data confirms the anti-inflammatory properties of calcitriol and this may be a potential way to deliver high dose bioactive calcitriol to M during inflammation in vivo.


Assuntos
Anti-Inflamatórios/farmacologia , Calcitriol/administração & dosagem , Calcitriol/farmacologia , Lipídeos/química , Macrófagos/metabolismo , Nanopartículas/química , Animais , Anticorpos/metabolismo , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Calcitriol/farmacocinética , Quimiocinas/metabolismo , Composição de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tamanho da Partícula , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Distribuição Tecidual/efeitos dos fármacos
18.
IET Nanobiotechnol ; 13(1): 90-99, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30964044

RESUMO

Epithelial cell adhesion molecule (EpCAM) is a transmembrane glycoprotein involved in cell proliferation and differentiation. Ribosomal inactivating proteins derived from plants specifically target ribosomes and irreversibly inhibit protein synthesis. EpCAM antibody and saporin were conjugated using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide chemistry. The mass of the conjugates were characterised using matrix-assisted laser desorption ionisation (MALDI). The saporin-EpCAM (SAP-EpAB) conjugates were tested in-vitro against MCF-7 (breast cancer cells), WERI-Rb1 (retinoblastoma) cells. The flow cytometry and fluorescence microscopy were performed to show the binding efficiency of SAP-EpAB conjugate. Whole transcriptome changes of sap-conjugate treated cells were studied using affymetrix microarrays. MALDI-TOF analysis and polyacrylamide gel electrophoresis confirmed the conjugation of SAP with EpCAM antibody. Flow cytometry and fluorescent microscopy analysis revealed the binding of SAP-EpAB conjugates to the MCF-7, WERI-Rb1 cells. Apoptosis assay by annexin-V has shown an increased apoptotic and necrotic population in conjugate treated cells. MTT assay confirmed the tumour cell death and had shown the IC50 value of 0.8 µg for conjugate in MCF-7 (breast cancer cells), and 1 µg for WERI-Rb1 (retinoblastoma) cells. The microarray analysis revealed downregulation of the tumourigenic genes and upregulation of pro-apoptotic genes leading to apoptosis of tumour cells.


Assuntos
Anticorpos/metabolismo , Antineoplásicos/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Molécula de Adesão da Célula Epitelial/metabolismo , Saporinas/química , Anticorpos/química , Anticorpos/imunologia , Anticorpos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Molécula de Adesão da Célula Epitelial/imunologia , Humanos , Células MCF-7 , Saporinas/metabolismo
19.
Mater Sci Eng C Mater Biol Appl ; 100: 23-29, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948057

RESUMO

Emulsions are crucial in the treatment of snake bites to bust the antibody response of the inmunogen. The widely used Freund's emulsion typically combines 50/50 water-oil (W/O) phase. However, its use is limited because it is associated with tissue damage. We formulated and characterized a Pickering Emulsion 70/30 (W/O) that uses a chemically modified hydrophobic hydroxyapatite as surfactant. This Pickering emulsion has similar rheologic behavior to Freund's emulsion 50/50, but with lower oil and surfactant concentration. Evaluation of cell recruitment, antibody response and adhering tissue in mice immunized with B. asper of Pacific venom and treated with Freund's and Pickering 70/30 emulsions resulted in similar adjuvant activity (only 18% lower in Pickering 70/30 emulsion). However, Pickering 70/30 emulsions minimized negative side effects in the host animals and showed better ease of flow that favors injection of the host. Our results open up room for optimization and improvement of Pickering emulsion based on modified nanoparticles for medical applications.


Assuntos
Adjuvantes Imunológicos/química , Anticorpos/metabolismo , Durapatita/química , Emulsões/química , Nanopartículas/química , Venenos de Serpentes/imunologia , Animais , Camundongos , Venenos de Serpentes/química , Serpentes/metabolismo , Tensoativos/química
20.
Mater Sci Eng C Mater Biol Appl ; 100: 424-432, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30948078

RESUMO

This work reports on the development of a label-free immunosensor technology, based on nanoplasmonic Au-TiO2 thin films. The Au-TiO2 thin films were prepared by cost-effective reactive DC magnetron sputtering, followed by a thermal annealing procedure. The latter promoted the growth of the Au nanoparticles throughout the TiO2 matrix and induced some morphological changes, which are the base for the immunosensor device functionality. A posterior plasma etching treatment was required to partially expose the nanoparticles to the biological environment. It gave rise to a 6-fold increase of the total area of gold exposed, allowing further possibilities for the sensor sensitivity enhancement. Experimental results demonstrated the successful functionalization of the films' surface with antibodies, with the immobilization occurring preferentially in the exposed nanoparticles and negligibly on the TiO2 matrix. Antibody adsorption surface coverage studies revealed antibody low affinity to the film's surface. Nevertheless, immunoassay development experiments showed a strong and active immobilized antibody monolayer at an optimized antibody concentration. This allowed a 236 signal-to-noise-ratio in a confocal microscope, using mouse IgG and 100 ng/ml of Fab-specific anti-mouse IgG-FITC conjugated. Label-free detection of the optimized antibody monolayer on Au-TiO2 thin films was also tested, revealing an expected redshift in the LSPR band, which demonstrates the suitability for the development of cost-effective, label-free LSPR based immunosensor devices.


Assuntos
Técnicas Biossensoriais/métodos , Ouro/química , Imunoensaio/métodos , Coloração e Rotulagem , Titânio/química , Adsorção , Animais , Anticorpos/metabolismo , Proteínas Imobilizadas/metabolismo , Camundongos , Nanopartículas/química , Nanopartículas/ultraestrutura , Fenômenos Ópticos , Propriedades de Superfície
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