Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 48
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
ACS Med Chem Lett ; 12(4): 563-571, 2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33859796

RESUMO

This study evaluated the potential use of senescence-inducing small molecules in the treatment of melanoma. We screened commercially available small-molecule libraries with high-throughput screening and high-content screening image-based technology. Our findings showed an initial hit with the embedded N-arylpiperidine-3-carboxamide scaffold-induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. A focused library containing diversely modified analogues were constructed and examined to evaluate the structure-activity relationship of N-arylpiperidine-3-carboxamide derivatives starting from hit 1. This work identified a novel compound with remarkable antiproliferative activity in vitro and demonstrated the key structural moieties within.

2.
Eur J Med Chem ; 209: 112550, 2021 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-33268144

RESUMO

Current research suggests therapy-induced senescence (TIS) of cancer cells characterized by distinct morphological and biochemical phenotypic changes represent a novel functional target that may enhance the effectiveness of cancer therapy. In order to identify novel small-molecule inducers of cellular senescence and determine the potential to be used for the treatment of melanoma, a new method of high-throughput screening (HTS) and high-contents screening (HCS) based on the detection of morphological changes was designed. This image-based and whole cell-based technology was applied to screen and select a novel class of antiproliferative agents on cancer cells, 4H-chromeno[2,3-d]pyrimidin-4-one derivatives, which induced senescence-like phenotypic changes in human melanoma A375 cells without serious cytotoxicity against normal cells. To evaluate structure-activity relationship (SAR) study of 4H-chromeno[2,3-d]pyrimidin-4-one scaffold starting from hit 3, a focused library containing diversely modified analogues was constructed and which led to the identification of 38, a novel compound to have remarkable anti-melanoma activity in vitro with good metabolic stability.


Assuntos
Antineoplásicos/farmacologia , Benzopiranos/farmacologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Melanoma/tratamento farmacológico , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Benzopiranos/química , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Masculino , Melanoma/patologia , Camundongos Endogâmicos BALB C , Pirimidinas/química
3.
Antiviral Res ; 129: 39-46, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26850830

RESUMO

Upon screening synthetic small molecule libraries with the infectious hepatitis C virus (HCV) cell culture system, we identified a benzothiazepinecarboxamide (BTC) scaffold that inhibits HCV. A structure-activity relationship (SAR) study with BTCs was performed, and modifications that led to nanomolar antiviral activity and improved the selective index (CC50/EC50) by more than 1000-fold were identified. In addition, a pharmacophore modeling study determined that the tricyclic core and positive charge on the piperidine moiety were essential for antiviral activity. Furthermore, we demonstrated that BTC interferes with HCV glycoprotein E1/E2-mediated viral entry and the generation of infectious virions by using HCV pseudoparticle and cell culture supernatant transfer assays, respectively. BTC showed potent antiviral activity against HCV genotype 2 (EC50 = 0.01 ± 0.01 µM), but was less potent against a genotype 1/2 chimeric virus (EC50 = 2.71 ± 0.05 µM), which expressed the structural proteins of HCV genotype 1. In summary, we identified, optimized, and characterized novel BTC inhibitors that interfere with early and late steps of the HCV viral life cycle.


Assuntos
Antivirais/farmacologia , Descoberta de Drogas , Hepacivirus/efeitos dos fármacos , Tiazepinas/farmacologia , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/síntese química , Antivirais/química , Linhagem Celular , Genótipo , Hepacivirus/genética , Hepacivirus/fisiologia , Humanos , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Tiazepinas/síntese química , Tiazepinas/química , Vírion/efeitos dos fármacos
4.
J Nanosci Nanotechnol ; 15(2): 1708-13, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26353719

RESUMO

Efficient oligonucleotide probe design and synthesis based on polymer-coated CdSe/ZnS quantum dot (QD) is demonstrated for detection of telomeres in human monocyte and Leishmania major, a protozoan pathogenic parasite. The highly photoluminescent polymer-coated QDs conjugated with various length of telomere probe sequences were prepared via carbodiimide chemistry and characterized. Specific detection of telomere was observed when DNA sequence was (CCCAAT)n (n = 5 or 3) probe sequence, rather than (GGGTTA)n (n = 3, 5, 8). The sensitivity and specificity were comparable with commercially available PNA probe for human telomere detection.


Assuntos
DNA/genética , Hibridização in Situ Fluorescente/métodos , Leishmania major/genética , Pontos Quânticos , Análise de Sequência de DNA/métodos , Telômero/genética , Sequência de Bases , DNA/química , Sondas de DNA/genética , Desenho de Equipamento , Análise de Falha de Equipamento , Teste de Materiais , Dados de Sequência Molecular , Nanoconjugados/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
5.
J Biomol Screen ; 20(1): 70-81, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25342146

RESUMO

Human African trypanosomiasis (HAT) is a vector-transmitted tropical disease caused by the protozoan parasite Trypanosoma brucei. High-throughput screening (HTS) of small-molecule libraries in whole-cell assays is one of the most frequently used approaches in drug discovery for infectious diseases. To aid in drug discovery efforts for HAT, the SYBR Green assay was developed for T. brucei in a 384-well format. This semi-automated assay is cost- and time-effective, robust, and reproducible. The SYBR Green assay was compared to the resazurin assay by screening a library of 4000 putative kinase inhibitors, revealing a superior performance in terms of assay time, sensitivity, simplicity, and reproducibility, and resulting in a higher hit confirmation rate. Although the resazurin assay allows for comparatively improved detection of slow-killing compounds, it also has higher false-positive rates that are likely to arise from the assay experimental conditions. The compounds with the most potent antitrypanosomal activity were selected in both screens and grouped into 13 structural clusters, with 11 new scaffolds as antitrypanosomal agents. Several of the identified compounds had IC50 <1 µM coupled with high selectivity toward the parasite. The core structures of the scaffolds are shown, providing promising new starting points for drug discovery for HAT.


Assuntos
Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala , Doenças Negligenciadas/tratamento farmacológico , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Linhagem Celular , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Fluorometria/métodos , Humanos , Oxazinas , Bibliotecas de Moléculas Pequenas , Tripanossomicidas/uso terapêutico , Xantenos
6.
Eur J Med Chem ; 84: 395-403, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25036797

RESUMO

A high-throughput (HTS) and high-content screening (HCS) campaign of a commercial library identified 2,3-dihydroimidazo[1,2-a]benzimidazole analogues as a novel class of anti-parasitic agents. A series of synthetic derivatives were evaluated for their in vitro anti-leishmanial and anti-trypanosomal activities against Leishmania donovani and Trypanosoma cruzi, which have been known as the causative parasites for visceral leishmaniasis and Chagas disease, respectively. In the case of Leishmania, the compounds were tested in both intracellular amastigote and extracellular promastigote assays. Compounds 4 and 24 showed promising anti-leishmanial activity against intracellular L. donovani (3.05 and 5.29 µM, respectively) and anti-trypanosomal activity against T. cruzi (1.10 and 2.10 µM, respectively) without serious cytotoxicity toward THP-1 and U2OS cell lines.


Assuntos
Antiparasitários/síntese química , Antiparasitários/farmacologia , Benzimidazóis/síntese química , Benzimidazóis/farmacologia , Compostos Heterocíclicos com 3 Anéis/síntese química , Compostos Heterocíclicos com 3 Anéis/farmacologia , Leishmania donovani/efeitos dos fármacos , Trypanosoma cruzi/efeitos dos fármacos , Antiparasitários/química , Benzimidazóis/química , Células Cultivadas , Relação Dose-Resposta a Droga , Compostos Heterocíclicos com 3 Anéis/química , Humanos , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
7.
Chem Commun (Camb) ; 49(55): 6182-4, 2013 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-23732395

RESUMO

A new binding strategy of linking quantum dots (QDs) to magnetic nanoparticles (MNPs) using DNA interaction with metal coordination bonding was developed. Platinum was selected for binding QDs to DNA. This novel self-assembled nanoconjugate would be a new probe for diagnosing a specific disease more accurately with its double modalities, fluorescence and magnetic property.


Assuntos
Complexos de Coordenação/química , DNA/química , Nanoconjugados/química , Platina/química , Pontos Quânticos , Dimetil Sulfóxido/química , Fenômenos Magnéticos
8.
J Nanosci Nanotechnol ; 13(1): 417-22, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23646748

RESUMO

Developing highly selective probes for subcellular regions such as nucleus and cytoplamic organelles is of great interest for cellular imaging and high content screening analysis for biology and medicine. Cytoplasmic delivery of QDs has been well-understood, while nuclear delivery of QDs has been a challenge due to the unique structural characteristics of cell nucleus. In this study, we systematically investigated nucleus penetrating properties of small-sized ligand-exchanged QDs with either positive or negative surface charges in the similar size range of hydrodynamic diameter (7-10 nm). We found that the positively-charged QDs efficiently stain the nucleus in fixed HeLa cells as well as label nucleolar compartments in live HeLa cells. In contrast, the negatively charged QDs with the similar size range stain only the cytoplam in either fixed or live cells. The charge-dependent labeling pattern allowed us to simultaneously perform multiplex imaging of nuclues and cytoplasm. This study offers an insight into efficient nuclear delivery of nanoparticles such as QDs of which surface charge and size are critical for intracelllar localization and delivery.


Assuntos
Núcleo Celular/química , Nanocápsulas/química , Pontos Quânticos , Ânions , Células HeLa , Humanos , Nanocápsulas/ultraestrutura , Eletricidade Estática
9.
Nanotechnology ; 24(14): 145601, 2013 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-23508062

RESUMO

Highly monodispersed CdSe quantum dots (QDs) were prepared without an injection procedure. A series of Cd salts of long chain fatty acids, including Cd-myristate (C14), Cd-palmitate (C16) and Cd-stearate (C18) was prepared, and all metallic precursors and surfactants were mixed together followed by increasing the temperature in a controlled manner. The reaction resulted in highly monodisperse and bright zinc blende QDs. In addition, the effects of specific ligands which have been known to lead anisotropic growth of the nanocrystals in the injection method were investigated. The use of alkyl phosphonic acid and alkyl amine was found to produce extremely monodisperse CdSe QDs with a high quantum yield. This procedure was proven to be able to yield a large quantity of zinc blende CdSe QDs (2 g) in a one-pot reaction. The use of a controlled amount of tetradecylphosphonic acid and octadecylamine resulted in tetrapod- and match-shaped QDs, the first reported by a non-injection method. These results clearly demonstrate that appropriate combination of precursors can provide high quality of CdSe nanocrystals in terms of quantum yield, monodispersity and shape control by a non-injection method.

10.
Nanotechnology ; 24(7): 075101, 2013 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-23358444

RESUMO

Elucidation of unknown target proteins of a drug is of great importance in understanding cell biology and drug discovery. There have been extensive studies to discover and identify target proteins in the cell. Visualization of targets using drug-conjugated probes has been an important approach to gathering mechanistic information of drug action at the cellular level. As quantum dot (QD) nanocrystals have attracted much attention as a fluorescent probe in the bioimaging area, we prepared drug-conjugated QD to explore the potential of target discovery. As a model drug, we selected a well-known anticancer drug, methotrexate (MTX), which has been known to target dihydrofolate reductase (DHFR) with high affinity binding (K(d) = 0.54 nM). MTX molecules were covalently attached to amino-PEG-polymer-coated QDs. Specific interactions of MTX-conjugated QDs with DHFR were identified using agarose gel electrophoresis and fluorescence microscopy. Cellular uptake of the MTX-conjugated QDs in living CHO cells was investigated with regard to their localization and distribution pattern. MTX-QD was found to be internalized into the cells via caveolae-medicated endocytosis without significant sequestration in endosomes. A colocalization experiment of the MTX-QD conjugate with antiDHFR-TAT-QD also confirmed that MTX-QD binds to the target DHFR. This study showed the potential of the drug-QD conjugate to identify or visualize drug-target interactions in the cell, which is currently of great importance in the area of drug discovery and chemical biology.


Assuntos
Metotrexato/farmacologia , Pontos Quânticos , Tetra-Hidrofolato Desidrogenase/metabolismo , Animais , Células CHO , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Ensaio de Desvio de Mobilidade Eletroforética , Endocitose/efeitos dos fármacos , Escherichia coli/enzimologia , Polímeros/química , Ligação Proteica/efeitos dos fármacos , Espectrofotometria Ultravioleta
11.
Korean J Radiol ; 14(1): 30-7, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23323028

RESUMO

OBJECTIVE: Authors aimed to determine the targeting ability of vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated quantum dots (QDs) in vitro, and apply it for a xenograft prostate cancer mouse model. MATERIALS AND METHODS: Conjugation reaction of QDs was performed by using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and sulfo-(N-hydroxysulfosuccinimide) (Sulfo-NHS). The human umbilical vein cord endothelial cells (HUVECs) were incubated with QDs, conjugated with antiVGFR2, to see a specific binding in vitro. Fluorescent cell images were taken by a confocal microscope. The human prostate cancer cells (PC3) were injected to five nude mice on hind limbs to make the xenograft tumor model. QD-antiVEGFR2 antibody complex was injected into the tumor model and fluorescence measurements were performed at 1, 4, 9, 12, 15, and 24 hours after the injection. RESULTS: The specific interaction between HUVECs and QD-antiVEGFR2 antibody was clearly shown in vitro. The in vivo fluorescence image disclosed that there was an increased signal of tumor, 12 hours after the injection of QDs. CONCLUSION: By showing endothelial cells binding with QDs-antiVEGFR2 antibodyand an experimental application of the antibody for VEGFR2 imaging in the prostate cancer xenograft mouse model, we suggests that the antibody-conjugated QDs can be a potential imaging tool for angiogenesis of the cancer.


Assuntos
Neovascularização Patológica/patologia , Neoplasias da Próstata/patologia , Pontos Quânticos , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Carbodi-Imidas/farmacologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Eletroforese em Gel de Ágar , Fluorescência , Técnicas In Vitro , Masculino , Camundongos , Camundongos Nus , Microscopia Confocal , Succinimidas/farmacologia , Transplante Heterólogo
12.
J Nucl Med ; 54(1): 96-103, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23232276

RESUMO

UNLABELLED: Multimodal nanoparticles have been extensively studied for target-specific imaging and therapy of various diseases, including cancer. In this study, radiolabeled arginine-glycine-aspartic acid (RGD)-functionalized Er(3+)/Yb(3+) co-doped NaGdF(4) upconversion nanophosphors (UCNPs) were synthesized and evaluated as a multimodal PET/MR/optical probe with tumor angiogenesis-specific targeting properties. METHODS: A dimeric cyclic RGDyk ((cRGDyk)(2)) peptide was conjugated to polyacrylic acid-coated NaGdF(4):Yb(3+)/Er(3+) UCNPs along with polyethylene glycol molecules and was consecutively radiolabeled with (124)I. In vitro cytotoxicity testing was performed for 3 d. Upconversion luminescence imaging of (cRGDyk)(2)-UCNP was performed on U87MG cells with a laboratory-made confocal microscope. In vivo small-animal PET and clinical 3-T T1-weighted MR imaging of (124)I-labeled RGD-functionalized UCNPs was acquired with or without blocking of cyclic RGD peptide in a U87MG tumor model. Inductively coupled plasma mass spectrometry and biologic transmission electron microscopy were done to evaluate gadolinium concentration and UCNP localization, respectively. RESULTS: Polymer-coated UCNPs and dimeric RGD-conjugated UCNPs were monodispersely synthesized, and those of hydrodynamic size were 30 ± 8 nm and 32 ± 9 nm, respectively. (cRGDyk)(2)-UCNPs have a low cytotoxic effect on cells. Upconversion luminescence signals of (cRGDyk)(2)-UCNP were specifically localized on the surface of U87MG cells. (124)I-c(RGDyk)(2)-UCNPs specifically accumulated in U87MG tumors (2.8 ± 0.8 vs. 1.3 ± 0.4 percentage injected dose per gram in the blocking experiment), and T1-weighted MR images showed significant positive contrast enhancement in U87MG tumors. Tumor localization of (124)I-c(RGDyk)(2)-UCNPs was confirmed by inductively coupled plasma mass spectrometry and biologic transmission electron microscopy analysis. CONCLUSION: These results suggest that (124)I-labeled RGD-functionalized UCNPs have high specificity for α(v)ß(3) integrin-expressing U87MG tumor cells and xenografted tumor models. Multimodal UCNPs can be used as a platform nanoparticle with multimodal imaging for cancer-specific diagnoses.


Assuntos
Érbio/química , Fluoretos/química , Gadolínio/química , Glioblastoma/irrigação sanguínea , Imagem Molecular/métodos , Nanoestruturas , Oligopeptídeos/química , Itérbio/química , Animais , Linhagem Celular Tumoral , Dimerização , Estudos de Viabilidade , Glioblastoma/diagnóstico , Glioblastoma/diagnóstico por imagem , Glioblastoma/patologia , Humanos , Integrina alfaVbeta3/metabolismo , Radioisótopos do Iodo , Substâncias Luminescentes/química , Medições Luminescentes , Imageamento por Ressonância Magnética , Camundongos , Nanoestruturas/toxicidade , Neovascularização Patológica , Oligopeptídeos/toxicidade , Polietilenoglicóis/química , Tomografia por Emissão de Pósitrons
13.
Anal Chem ; 84(20): 8595-601, 2012 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-22954333

RESUMO

We have developed a novel fluorogenic nanoprobe prepared from the assembly of CdSe/ZnS quantum dot (QD) and gold (Au) nanoparticles in which QD was conjugated with a specifically designed ß-secretase (BACE1) substrate peptide, which was allowed to bind to the Ni-nitrilotriacetate (Ni-NTA) modified Au nanoparticles. This coordination-mediated binding of the QD with Au nanoparticles via Ni-NTA-histidine (His) interaction resulted in highly efficient quenching of QD fluorescence through a distance-dependent fluorescence resonance energy transfer (FRET) phenomenon. The prequenched QD-Au assembly recovered the fluorescence in the presence of the BACE1 enzyme after incubation in vitro. The high quenching efficiency of AuNP and robust QD fluorescence signal recovery upon BACE1 enzymatic digestion enabled us to visualize BACE1 activity in living cells, which further allowed us to generate the half maximal inhibitory concentration (IC(50)) values for BACE1 inhibitors in the cell-based assay utilizing a high throughput system (HTS). These results suggest the potential application of QD-AuNP assembly toward the HTS drug screening system as a robust and efficient probe to identify active molecules in BACE1-related diseases such as Alzheimer's disease.


Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Transferência Ressonante de Energia de Fluorescência/métodos , Ouro/química , Nanopartículas/química , Pontos Quânticos , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Corantes Fluorescentes/química , Células HEK293 , Humanos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo
14.
Chembiochem ; 13(10): 1503-8, 2012 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-22692979

RESUMO

Cellular imaging has emerged as an important tool to unravel biological complexity and to accelerate the drug-discovery process, including cell-based screening, target identification, and mechanism of action studies. Recently, semiconductor nanoparticles known as quantum dots (QDs) have attracted great interest in cellular imaging applications due to their unique photophysical properties such as size, tunable optical property, multiplexing capability, and photostability. Herein, we show that QDs can also be applied to assay development and eventually to high-throughput/content screening (HTS/HCS) for drug discovery. We have synthesized QDs modified with PEG and primary antibodies to be used as fluorescent probes for a cell-based HTS system. The G protein-coupled receptor (GPCR) family is known to be involved in most major diseases. We therefore constructed human osteosarcoma (U2OS) cells that specifically overexpress two types of differently tagged GPCRs: influenza hemagglutinin (HA) peptide-tagged κ-opioid receptors (κ-ORs) and GFP-tagged A3 adenosine receptors (A3AR). In this study, we have demonstrated that 1) anti-HA antibody-conjugated QDs could specifically label HA-tagged κ-ORs, 2) subsequent treatment of QD-tagged GPCR agonists allowed agonist-induced translocation to be monitored in real time, 3) excellent emission spectral properties of QD permitted the simultaneous detection of two GPCRs in one cell, and 4) the robust imaging capabilities of the QD-antibody conjugates could lead to reproducible quantitative data from high-content cellular images. These results suggest that the present QD-based GPCR inhibitor screening system can be a promising platform for further drug screening applications.


Assuntos
Pontos Quânticos , Receptores Acoplados a Proteínas G/agonistas , Anticorpos/química , Anticorpos/imunologia , Linhagem Celular Tumoral , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemaglutininas/imunologia , Humanos , Microscopia Confocal , Polietilenoglicóis/química , Receptor A3 de Adenosina/genética , Receptor A3 de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
15.
Analyst ; 137(6): 1440-5, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22310726

RESUMO

We proposed an effective strategy for evaluating the targeting specificity of an antibody-conjugated quantum dot (QD) nanoprobe in a coculture system mimicking an in vivo-like tumor microenvironment in which cancer cells grow with normal cells. Analysis of the images was performed with automated confocal microscopy. We have employed a melanoma-melanocyte coculture model to assess the specific binding of QDs conjugated with melanoma antibodies. Conjugation of antibodies to the QD significantly improved the melanoma specificity, while unconjugated antibody alone suffered from non-specific binding to melanocytes. Concentration-dependent binding and competitive inhibition studies with QD-antibody conjugates reproducibly proved the specificity to melanoma cells against melanocytes. The specificity and targeting efficiency of nanoprobes evaluated in a simple coculture model may provide a reasonable assessment for the in vitro diagnosis of early stage melanoma development before in vivo studies. Further, a rapid and sensitive cancer cell detection system demonstrated herein may allow for the development of high-throughput screening platforms for early cancer diagnosis and anti-cancer therapeutics.


Assuntos
Linhagem Celular Tumoral , Técnicas de Cocultura , Ensaios de Triagem em Larga Escala/métodos , Melanoma/metabolismo , Pontos Quânticos , Anticorpos/metabolismo , Humanos , Melanócitos/citologia , Melanoma/diagnóstico , Melanoma/patologia , Modelos Biológicos , Estrutura Molecular
16.
Bioconjug Chem ; 22(8): 1576-86, 2011 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-21718016

RESUMO

Imaging of specific intracellular target proteins in living cells has been of great challenge and importance for understanding intracellular events and elucidating various biological phenomena. Highly photoluminescent and water-soluble semiconductor nanocrystal quantum dots (QDs) have been extensively applied to various cellular imaging applications due to the long-term photostability and the tunable narrow emission spectra with broad excitation. Despite the great success of various bioimaging and diagnostic applications, visualization of intracellular targets in live cells still has been of great challenge. Nonspecific binding, difficulty of intracellular delivery, or endosomal trapping of nanosized QDs are the main reasons to hamper specific target binding in live cells. In this context, we prepared the polymer-coated QDs (pcQD) of which the surface was optimized for specific intracellular targeting in live cells. Efficient intracellular delivery was achieved through PEGylation and subsequent cell penetrating peptide (i.e., TAT) conjugation to the pcQD in order to avoid significant endosomal sequestration and to facilitate internalization of the QDs, respectively. In this study, we employed HEK293 cell line overexpressing endothelin A receptor (ET(A)R), a family of G-protein coupled receptor (GPCR), of which the cytosolic c-terminal site is genetically engineered to possess green fluorescent protein (GFP) as our intracellular protein target. The fluorescence signal of the target protein and the well-defined intracellular behavior of the GPCR help to evaluate the targeting specificity of QDs in living cells. To test the hypothesis that the TAT-QDs conjugated with antibody against intracellular target of interest can find the target, we conjugated anti-GFP antibody to TAT-PEG-pcQD using heterobifunctional linkers. Compared to the TAT-PEG-pcQD, which was distributed throughout the cytoplasm, the antiGFP-functionalized TAT-PEG-pcQD could penetrate the cell membrane and colocalize with the GFP. An agonist (endothelin-1, ET-1) treatment induced GFP-ET(A)R translocation into pericentriolar region, where the GFP also significantly colocalized with antiGFP-TAT-PEG-pcQD. These results demonstrate that stepwise optimization of PEG-pcQD conjugation with both a cell penetrating peptide and an antibody against a target of interest allows specific binding to the intracellular target protein with minimized nonspecific binding.


Assuntos
Imagem Molecular/métodos , Proteínas/análise , Pontos Quânticos , Anticorpos , Peptídeos Penetradores de Células , Diagnóstico por Imagem , Proteínas de Fluorescência Verde , Células HEK293 , Humanos , Sondas Moleculares/síntese química , Polietilenoglicóis , Ligação Proteica , Proteínas/imunologia , Receptor de Endotelina A
17.
Malar J ; 10: 118, 2011 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-21554712

RESUMO

BACKGROUND: Malaria infects over 300 million people every year and one of the major obstacles for the eradication of the disease is parasite's resistance to current chemotherapy, thus new drugs are urgently needed. Quantum dot (QD) is a fluorescent nanocrystal that has been in the spotlight as a robust tool for visualization of live cell processes in real time. Here, a simple and efficient method using QD to directly label Plasmodium falciparum-infected erythrocytes (iRBCs) was searched in order to use the QD as a probe in an anti-malarial drug-screening assay. METHODS: A range of QDs with different chemical coatings were tested for their ability to specifically bind iRBCs by immunofluorescence assay (IFA). One QD was selected and used to detect parasite growth and drug sensitivity by flow cytometry. RESULTS: PEGylated-cationic QD (PCQD) was found to specifically label infected erythrocytes preferentially with late stage parasites. The detection of QD-labelled infected erythrocytes by flow cytometry was sensitive enough to monitor chloroquine anti-malarial toxicity with a drug incubation period as short as 24 h (EC50 = 113nM). A comparison of our assay with another widely used anti-malarial drug screening assay, the pLDH assay, showed that PCQD-based assay had 50% improved sensitivity in detecting drug efficacy within a parasite life cycle. An excellent Z-factor of 0.8 shows that the QD assay is suitable for high-throughput screening. CONCLUSIONS: This new assay can offer a rapid and robust platform to screen novel classes of anti-malarial drugs.


Assuntos
Antimaláricos/farmacologia , Eritrócitos/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/crescimento & desenvolvimento , Pontos Quânticos , Animais , Citometria de Fluxo/métodos , Técnica Direta de Fluorescência para Anticorpo/métodos , Humanos , Testes de Sensibilidade Parasitária/métodos , Fatores de Tempo
18.
Appl Radiat Isot ; 69(1): 56-62, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20888778

RESUMO

In this study, the radioiodination and biodistribution of quantum dots (QDs) using Bolton-Hunter reagent were investigated. Radioiodination yield was 33.4 ± 2.0%. Fluorescent intensity of radioiodinated QDs decreased to 75.4% of the maximum prior to radioiodination. In biodistribution and ex vivo fluorescence imaging, radioiodinated QDs were highly accumulated in reticuloendothelial system (liver and spleen) and had low level bone uptakes and slow clearance from body. These results suggest that the radioiodination method of nanoparticles using Bolton-Hunter reagent could be easily used in the biodistribution and quantification of nanoparticles in vivo.


Assuntos
Radioisótopos do Iodo/química , Pontos Quânticos , Compostos Radiofarmacêuticos/síntese química , Succinimidas/química , Animais , Feminino , Radioisótopos do Iodo/farmacocinética , Radioisótopos do Iodo/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Espectrometria de Fluorescência , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único
19.
Chem Commun (Camb) ; 46(48): 9146-8, 2010 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-21049123

RESUMO

A novel QD-peptide complex for detecting HIV-1 protease activity was prepared from simple one step electrostatic interaction. Fluorescence recovery of the pre-quenched QD through fluorescence resonance energy transfer allowed for in vitro assay and live cell imaging of the protease activity in HIV-1 transfected cells, proving the potential for cell-based protease inhibitor screening.


Assuntos
Diagnóstico por Imagem/métodos , Protease de HIV/metabolismo , Peptídeos , Pontos Quânticos , Corantes Fluorescentes/análise , Humanos
20.
Chem Commun (Camb) ; 46(47): 8959-61, 2010 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-20976317

RESUMO

An oligonucleotide modified with Ni-nitrilotriacetate (NTA) was successfully synthesized and used for the stoichiometric functionalization of QDs. This synthetic approach allowed for the facile preparation of DNA-QD conjugates with a defined DNA/QD ratio using well-known Ni-histidine coordination chemistry. A FRET based DNA-QD nanoprobe was prepared using this method highlighting the great potential of this synthetic strategy.


Assuntos
Níquel/química , Oligonucleotídeos/química , Pontos Quânticos , Transferência Ressonante de Energia de Fluorescência , Histidina/química , Ácido Nitrilotriacético/química
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...