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1.
Pharm Res ; 36(11): 154, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31482205

RESUMO

PURPOSE: Conjugation of nanocarriers with antibodies that bind to specific membrane receptors that are overexpressed in cancer cells enables targeted delivery. In the present study, we developed and synthesised two PAMAM dendrimer-trastuzumab conjugates that carried docetaxel or paclitaxel, specifically targeted to cells which overexpressed HER-2. METHODS: The 1H NMR, 13C NMR, FTIR and RP-HPLC were used to analyse the characteristics of the products and assess their purity. The toxicity of PAMAM-trastuzumab, PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates was determined using MTT assay and compared with free trastuzumab, docetaxel and paclitaxel toward HER-2-positive (SKBR-3) and negative (MCF-7) human breast cancer cell lines. The cellular uptake and internal localisation were studied using flow cytometry and confocal microscopy, respectively. RESULTS: The PAMAM-drug-trastuzumab conjugates in particular showed extremely high toxicity toward the HER-2-positive SKBR-3 cells and very low toxicity towards to HER-2-negative MCF-7 cells. As expected, the HER-2-positive SKBR-3 cell line accumulated trastuzumab from both conjugates rapidly; but surprisingly, although a large amount of PAMAM-ptx-trastuzumab conjugate was observed in the HER-2-negative MCF-7 cells. Confocal microscopy confirmed the intracellular localisation of analysed compounds. The key result of fluorescent imaging was the identification of strong selective binding of the PAMAM-doc-trastuzumab conjugate with HER-2-positive SKBR-3 cells only. CONCLUSIONS: Our results confirm the high selectivity of PAMAM-doc-trastuzumab and PAMAM-ptx-trastuzumab conjugates for HER-2-positive cells, and demonstrate the utility of trastuzumab as a targeting agent. Therefore, the analysed conjugates present an promising approach for the improvement of efficacy of targeted delivery of anticancer drugs such as docetaxel or paclitaxel.


Assuntos
Antineoplásicos/química , Neoplasias da Mama/tratamento farmacológico , Dendrímeros/química , Docetaxel/química , Paclitaxel/química , Receptor ErbB-2/metabolismo , Trastuzumab/química , Antineoplásicos/farmacologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Docetaxel/farmacologia , Interações de Medicamentos , Liberação Controlada de Fármacos , Corantes Fluorescentes/química , Humanos , Paclitaxel/farmacologia , Propriedades de Superfície , Trastuzumab/farmacologia , Resultado do Tratamento
2.
Chem Commun (Camb) ; 55(66): 9869-9872, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31364617

RESUMO

In this work, we designed and synthesized an aggregation-induced emission (AIE)-active tetraphenylethene-decorated pseudo-trisialic acid (TPE3S) and validated its high affinity for Siglecs using microscale thermophoresis techniques. TPE3S was a unique binding-on fluorescent trivalent sialocluster which was successfully utilized for the visualization of Siglecs expressed on the surface of mammalian cells.


Assuntos
Metabolismo dos Carboidratos , Lectinas/metabolismo , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico/metabolismo , Animais , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Células HeLa , Humanos , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Células PC12 , Ligação Proteica , Ratos
3.
Chem Commun (Camb) ; 55(67): 9955-9958, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31364619

RESUMO

A silver nanocluster-based ratiometric fluorescent nanosensor was developed for the determination of ATP in the cerebrospinal fluid of a mouse brain. Using this useful tool with good stability and high selectivity as well as a wide linear detection range, it was found that the ATP concentration in a mouse brain with Alzheimer's disease was 2300-fold higher than that in a normal one.


Assuntos
Trifosfato de Adenosina/líquido cefalorraquidiano , Química Encefálica , DNA/química , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Encéfalo/citologia , Encéfalo/patologia , Córtex Cerebral/química , Hipocampo/química , Camundongos , Conformação de Ácido Nucleico , Prata/química , Espectrometria de Fluorescência/métodos
5.
Chem Commun (Camb) ; 55(69): 10312-10315, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31397446

RESUMO

Herein, we report a novel magnet-mediated antibody-boronate sandwich-typed assay (ABSTA) strategy for the ultrasensitive, specific, rapid, and enzyme-free detection of glycoproteins in complex samples. The proposed ABSTA method exhibited ultrahigh sensitivity for HCG with a detection limit of 0.19 mIU mL-1, which is approximately 40-fold lower than that of conventional sandwich enzyme immunoassay.


Assuntos
Anticorpos Imobilizados/química , Ácidos Borônicos/química , Gonadotropina Coriônica/sangue , Corantes Fluorescentes/química , Imãs/química , Anticorpos Monoclonais/química , Gonadotropina Coriônica/análise , Fluorescência , Humanos , Imunoensaio/métodos , Limite de Detecção
6.
Tumour Biol ; 41(8): 1010428319866369, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31402761

RESUMO

Gaining a better understanding of the biological properties of cell-free DNA constitutes an important step in the development of clinically meaningful cell-free DNA-based tests. Since the in vivo characterization of cell-free DNA is complicated by the immense heterogeneity of blood samples, an increasing number of in vitro cell culture experiments, which offer a greater level of control, are being conducted. However, cell culture studies are currently faced with three notable caveats. First, the concentration of cell-free DNA in vitro is relatively low. Second, the median amount and size of cell-free DNA in culture medium varies greatly between cell types. Third, the amount and size of cell-free DNA in the culture medium of a single cell line fluctuates over time. Although these are interesting findings, it can also be a great source of experimental confusion and emphasizes the importance of method optimization and standardization. Therefore, in this study, we compared five commonly used cell-free DNA quantification methods, including quantitative polymerase chain reaction, Qubit Double-Stranded DNA High Sensitivity assay, Quant-iT PicoGreen Assay, Bioanalyzer High Sensitivity DNA assay, and NanoDrop Onec. Analysis of the resulting data, along with an interpretation of theoretical values (i.e. the theoretical detection and quantification limits of the respective methods), enables the calculation of optimal conditions for several important preanalytical steps pertaining to each quantification method and different cell types, including the (1) time-point at which culture medium should be collected for cell-free DNA extraction, (2) amount of cell culture supernatant from which to isolate cell-free DNA, (3) volume of elution buffer, and (4) volume of cell-free DNA sample to use for quantification.


Assuntos
Ácidos Nucleicos Livres/química , Meios de Cultura/química , Técnicas de Cultura de Células , Corantes Fluorescentes/química , Humanos , Compostos Orgânicos/química
8.
Chem Commun (Camb) ; 55(68): 10128-10131, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31386708

RESUMO

Fueled by the therapeutic potential of the epigenetic machinery, BET bromodomains have seen high interest as drug targets. Herein, we introduce different linkers to a BET bromodomain benzodiazepine ligand (I-BET762) to gauge its implications in the development of hybrid drugs, imaging probes and small molecule drug conjugates. Biophysical studies confirmed minimal disruption to binding of the BRD4 cavity by the synthesized entities, which includes imaging probes. Target engagement was confirmed in a cellular context, but poor membrane diffusion was found despite efficient localization in the nuclei after membrane disruption. Our study highlights challenges and opportunities for the successful design of benzodiazepine-derived drug-delivery systems.


Assuntos
Benzodiazepinas/farmacologia , Fluoresceínas/farmacologia , Corantes Fluorescentes/farmacologia , Proteínas Nucleares/antagonistas & inibidores , Benzodiazepinas/síntese química , Benzodiazepinas/química , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Desenho de Drogas , Fluoresceínas/síntese química , Fluoresceínas/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Ligantes , Estrutura Molecular , Proteínas Nucleares/química , Domínios Proteicos
9.
Life Sci ; 234: 116758, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31421083

RESUMO

In this work, fluorescent copper oxide nanoparticles (CuO NPs) were green synthesized using viable cells, cell lysate supernatant (CLS) and protein extracts of luminescent Vibrio sp. VLC. Biogenic CuO NPs were then characterized by XRD, FTIR, UV/Vis spectroscopy, TEM, DLS, and PL spectroscopy. Results showed that CLS method was more efficient for CuO NPs production, therefore CuO NPs synthesized by this method from copper sulfate (CuO NPs-1) and/or copper nitrate (CuO NPs-2) were used for further studies. The crystallite size of polydispersed CuO NPs-1 and CuO NPs-2 were about 8.83 and 8.77 nm, respectively indicating their suitability for biological applications. Antibacterial activity of CuO NPs was determined using broth microdilution, well diffusion agar, and time-kill curves methods. Both CuO NP-1 and CuO NP-2 inhibited bacterial growth at the minimum inhibitory concentration (MIC) of 625 mg/L except St. mutants (MIC = 1250 mg/L). Emission of fluorescent light from the surface of NPs was increased when exposed to Cd2+, As2+ and Hg2+ ions but decreased by Pb2+ ions. Results showed that CuO NP-1 had anticancer properties against KYSE30 esophageal cancer cell line (IC50 = 13.96 mg/L) while no higher cytotoxic effects were observed on Human Dermal Fibroblasts (HDF) (IC50 = 48.88 mg/L).


Assuntos
Antibacterianos/farmacologia , Antineoplásicos/farmacologia , Cobre/farmacologia , Neoplasias Esofágicas/tratamento farmacológico , Corantes Fluorescentes/química , Metais Pesados/análise , Nanopartículas/química , Antibacterianos/química , Antineoplásicos/química , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Linhagem Celular Tumoral , Cobre/química , Química Verde/métodos , Humanos , Espectrometria de Fluorescência/métodos , Vibrio/química
10.
J Agric Food Chem ; 67(38): 10726-10733, 2019 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-31469953

RESUMO

A turn-on fluorescent probe, HOCD-RB, for monitoring singlet oxygen (1O2) was developed by linking rhodamine B as fluorophore with dimethylhomoocoerdianthrone (HOCD) as 1O2 reaction site and fluorescence quencher due to the intramolecular energy transfer (ET) between rhodamine B and HOCD moieties. Upon exposure to 1O2 it rapidly forms endoperoxide with HOCD and turns on the fluorescence of rhodamine B by 18-fold. Taking advantage of the HOCD-RB probe that shows fast response, high sensitivity, and selectivity for 1O2, it is applied for imaging of endogenous 1O2 in living cells and the fluorometric assay for evaluating 1O2 quenching activity of selected common flavonoids found in our daily diets. The results show that the 1O2 scavenging activity of flavonoids depends on not only the structure of individual flavonoid but also the competitive interactions between mixed flavonoids. The best antioxidant capacity for individual and mixed flavonoids is epigallocatechin gallate and the mixture of catechin gallate with kaempferol, respectively. Overall, this work provided a new tool for detection and imaging of singlet oxygen activity in a biological system as well as an efficient fluorometric assay of 1O2 scavenging activity.


Assuntos
Flavonoides/química , Corantes Fluorescentes/química , Fluorometria/métodos , Depuradores de Radicais Livres/química , Rodaminas/química , Oxigênio Singlete/química , Fluorescência , Estrutura Molecular
11.
Soft Matter ; 15(33): 6660-6676, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31389467

RESUMO

The dynamic behavior of monoclonal antibodies (mAbs) at high concentration provides insight into protein microstructure and protein-protein interactions (PPI) that influence solution viscosity and protein stability. At high concentration, interpretation of the collective-diffusion coefficient Dc, as determined by dynamic light scattering (DLS), is highly challenging given the complex hydrodynamics and PPI at close spacings. In contrast, self-diffusion of a tracer particle by Brownian motion is simpler to understand. Herein, we develop fluorescence correlation spectroscopy (FCS) for the measurement of the long-time self-diffusion of mAb2 over a wide range of concentrations and viscosities in multiple co-solute formulations with varying PPI. The normalized self-diffusion coefficient D0/Ds (equal to the microscopic relative viscosity ηeff/η0) was found to be smaller than η/η0. Smaller ratios of the microscopic to macroscopic viscosity (ηeff/η) are attributed to a combination of weaker PPI and less self-association. The interaction parameters extracted from fits of D0/Ds with a length scale dependent viscosity model agree with previous measurements of PPI by SLS and SAXS. Trends in the degree of self-association, estimated from ηeff/η with a microviscosity model, are consistent with oligomer sizes measured by SLS. Finally, measurements of collective diffusion and osmotic compressibility were combined with FCS data to demonstrate that the changes in self-diffusion between formulations are due primarily to changes in the protein-protein friction in these systems, and not to protein-solvent friction. Thus, FCS is a robust and accessible technique for measuring mAb self-diffusion, and, by extension, microviscosity, PPI and self-association that govern mAb solution dynamics.


Assuntos
Anticorpos Monoclonais/química , Fenômenos Biofísicos , Difusão , Fluorescência , Corantes Fluorescentes/química , Microscopia de Fluorescência , Modelos Químicos , Multimerização Proteica , Estabilidade Proteica , Soluções , Viscosidade
12.
Chem Commun (Camb) ; 55(72): 10713-10716, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31429427

RESUMO

A red light-triggered reaction based on cyclic oligonucleotide substrates that is accelerated over 30-fold by specific nucleic acid templates and generates a bright fluorescent probe was developed. We confirmed that this reaction is compatible with fluorescence correlation spectroscopy (FCS) thereby allowing detection of nucleic acids down to 1 nM.


Assuntos
Luz , Ácidos Nucleicos/análise , Oligonucleotídeos/química , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Estrutura Molecular , Espectrometria de Fluorescência
13.
Chem Commun (Camb) ; 55(72): 10776-10779, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31432809

RESUMO

Herein, we present a dual-site ratiometric fluorescent probe based on the ICT-PET-FRET mechanism for the detection of pH in the ER. The probe showed a highly sensitive response to pH in the range of 5.0-7.2, and could be applied for the quantitative measurement of the pH values in the ER during ER stress and dexamethanose-induced stimulation.


Assuntos
Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Dexametasona/farmacologia , Transporte de Elétrons , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio
14.
Chem Commun (Camb) ; 55(72): 10740-10743, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31432813

RESUMO

We constructed a two-photon fluorescence ratio probe (CST) for in situ quantitative real-time detection of mitochondrial O2˙-. Fluorescence imaging showed that O2˙- was over-generated from mitochondria and conveyed to the cytoplasm via voltage-dependent anion channels in hepatic ischemia-reperfusion mice, damaging the important functional protein aconitase in the cytoplasm.


Assuntos
Fígado/metabolismo , Mitocôndrias/química , Imagem Óptica , Fótons , Traumatismo por Reperfusão/metabolismo , Superóxidos/química , Animais , Ânions/química , Ânions/metabolismo , Transporte Biológico , Corantes Fluorescentes/química , Camundongos , Mitocôndrias/metabolismo , Estrutura Molecular , Superóxidos/metabolismo
15.
Chem Commun (Camb) ; 55(71): 10615-10618, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31428753

RESUMO

A programmable molecular beacon (MB) with a good discrimination capability for mature and precursor microRNAs was loaded onto the surface of Mo2C quantum dots (QDs) for accurate detection of intracellular mature microRNAs.


Assuntos
Corantes Fluorescentes/química , MicroRNAs/análise , Molibdênio/química , Pontos Quânticos/química , Animais , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Humanos , Limite de Detecção , Camundongos , Imagem Óptica/métodos , Precursores de RNA/análise , Propriedades de Superfície
16.
Chem Commun (Camb) ; 55(70): 10424-10427, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31407744

RESUMO

The development of sensitive and reliable fluorescent probes for the early diagnosis of Alzheimer's disease (AD) is highly challenging and plays an important role in achieving effective treatments. Herein, we designed and synthesized an indole-based fluorophore for TTR in human plasma, an important hallmark of AD pathogenesis. This robust and simple fluorescent method allows quantification of TTR in the complex biological matrix.


Assuntos
Doença de Alzheimer/diagnóstico , Corantes Fluorescentes/química , Pré-Albumina/metabolismo , Doença de Alzheimer/sangue , Peptídeos beta-Amiloides/metabolismo , Humanos , Limite de Detecção , Espectrometria de Fluorescência
17.
Chem Commun (Camb) ; 55(70): 10456-10459, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31411211

RESUMO

A bio-orthogonal chemistry-based approach for fluorescent labelling of ribosomal RNA is described. It involves an adenosine analogue modified with trans-cyclooctene and masked 5'-phosphate group using aryl phosphoramidate. The incorporation into rRNA has been confirmed using agarose gel electrophoresis, as well as a highly sensitive UHPLC-MS/MS method. Fluorescent labelling of rRNA has been achieved in live HeLa cells via an inverse electron demand Diels-Alder reaction with a tetrazine conjugated to an Oregon Green fluorophore. This communication describes the stepwise approach that led to the development and characterization of the probe. The results demonstrate a new strategy towards development of future fluorescent probes to investigate the biochemistry of nucleic acids.


Assuntos
Corantes Fluorescentes/química , RNA Ribossômico/química , Cromatografia Líquida de Alta Pressão/métodos , Reação de Cicloadição , Células HeLa , Humanos , Espectrometria de Massas em Tandem/métodos
18.
Eur J Histochem ; 63(3)2019 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-31455073

RESUMO

RNA interference is a powerful approach to understand gene function both for therapeutic and experimental purposes. Since the lack of knowledge in the gene silencing of various hepatic cell lines, this work was aimed to compare two transfection agents, the liposome-based Lipofectamine™ RNAiMAX and the HepG2-specific, polymer-based GenMute™, in two cellular models of human hepatoma, HepG2 and Huh7.5. In the first part, we assessed transfection efficiency of a fluorescent Cy3-labeled negative control siRNA by cell imaging analysis; we found that cells treated with GenMute present a higher uptake of the fluorescent negative control siRNA when compared to Lipofectamine RNAiMAX-transfected cells, both in HepG2 and in Huh7.5 cells. In the second part, we evaluated GAPDH silencing with the two transfection reagents by RT-PCR similar GAPDH mRNA expression after each transfection treatment. Finally, we measured cell viability by the MTT assay, observing that cells transfected with GenMute have higher viability with respect to Lipofectamine RNAiMAX-administered cells. These results suggest that GenMute reagent might be considered the most suitable transfection agent for hepatic gene silencing.


Assuntos
Técnicas de Transferência de Genes , Lipídeos/química , Polímeros/química , RNA Interferente Pequeno/genética , Transfecção/métodos , Sequência de Bases , Carbocianinas/química , Carbocianinas/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Inativação Gênica , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Metabolismo dos Lipídeos , Lipídeos/toxicidade , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/toxicidade , Neoplasias Hepáticas/genética , Polímeros/metabolismo , Polímeros/toxicidade , Interferência de RNA
19.
Chem Commun (Camb) ; 55(67): 9959-9962, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31364996

RESUMO

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


Assuntos
Materiais Biocompatíveis/química , Corantes Fluorescentes/química , MicroRNAs/análise , Nanopartículas/química , Soroalbumina Bovina/química , Estilbenos/química , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Bovinos , Linhagem Celular Tumoral , Técnicas Eletroquímicas , Compostos Ferrosos/química , Humanos , Limite de Detecção , Medições Luminescentes , Metalocenos/química , Sensibilidade e Especificidade , beta-Ciclodextrinas/química
20.
Chem Commun (Camb) ; 55(67): 9971-9974, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31367709

RESUMO

Photodynamic therapy (PDT) is a clinically approved cancer treatment that uses light, oxygen and a photosensitizer to produce localized reactive oxygen species (ROS). Due to the short lifetime of ROS, the location of the photosensitizer in the cell is believed to be the key determinant governing the outcome of PDT. To explore the effect of direct association between a photosensitizer and DNA a well know DNA-binding dye, DAPI, was converted into a photosensitizer. Br-DAPI - unlike native DAPI - upon irradiation produces ROS. We demonstrate that the ROS are only effective in inducing dsDNA breaks when Br-DAPI is bound to DNA. In cancer cells (A549) Br-DAPI causes rapid light dependent cell death. This work supports the design of photosensitizers which bind with high affinity to the DNA of target cells for potentially more effective PDT.


Assuntos
Bromo/química , DNA/química , Indóis/química , Fármacos Fotossensibilizantes/química , Células A549 , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA , Corantes Fluorescentes/química , Humanos , Luz , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Estudo de Prova de Conceito , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo
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