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1.
BMC Bioinformatics ; 20(1): 522, 2019 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-31655541

RESUMO

BACKGROUND: Protein subcellular localization plays a crucial role in understanding cell function. Proteins need to be in the right place at the right time, and combine with the corresponding molecules to fulfill their functions. Furthermore, prediction of protein subcellular location not only should be a guiding role in drug design and development due to potential molecular targets but also be an essential role in genome annotation. Taking the current status of image-based protein subcellular localization as an example, there are three common drawbacks, i.e., obsolete datasets without updating label information, stereotypical feature descriptor on spatial domain or grey level, and single-function prediction algorithm's limited capacity of handling single-label database. RESULTS: In this paper, a novel human protein subcellular localization prediction model MIC_Locator is proposed. Firstly, the latest datasets are collected and collated as our benchmark dataset instead of obsolete data while training prediction model. Secondly, Fourier transformation, Riesz transformation, Log-Gabor filter and intensity coding strategy are employed to obtain frequency feature based on three components of monogenic signal with different frequency scales. Thirdly, a chained prediction model is proposed to handle multi-label instead of single-label datasets. The experiment results showed that the MIC_Locator can achieve 60.56% subset accuracy and outperform the existing majority of prediction models, and the frequency feature and intensity coding strategy can be conducive to improving the classification accuracy. CONCLUSIONS: Our results demonstrate that the frequency feature is more beneficial for improving the performance of model compared to features extracted from spatial domain, and the MIC_Locator proposed in this paper can speed up validation of protein annotation, knowledge of protein function and proteomics research.


Assuntos
Espaço Intracelular/metabolismo , Proteínas/análise , Algoritmos , Bases de Dados de Proteínas , Humanos , Espaço Intracelular/química , Transporte Proteico , Proteínas/metabolismo
2.
Nat Commun ; 10(1): 4764, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31628307

RESUMO

Water is arguably the most common and yet least understood material on Earth. Indeed, the biophysical behavior of water in crowded intracellular milieu is a long-debated issue. Understanding of the spatial and compositional heterogeneity of water inside cells remains elusive, largely due to a lack of proper water-sensing tools with high sensitivity and spatial resolution. Recently, stimulated Raman excited fluorescence (SREF) microscopy was reported as the most sensitive vibrational imaging in the optical far field. Herein we develop SREF into a water-sensing tool by coupling it with vibrational solvatochromism. This technique allows us to directly visualize spatially-resolved distribution of water states inside single mammalian cells. Qualitatively, our result supports the concept of biological water and reveals intracellular water heterogeneity between nucleus and cytoplasm. Quantitatively, we unveil a compositional map of the water pool inside living cells. Hence we hope SREF will be a promising tool to study intracellular water and its relationship with cellular activities.


Assuntos
Microscopia de Fluorescência/métodos , Microscopia Óptica não Linear/métodos , Análise de Célula Única/métodos , Água/metabolismo , Núcleo Celular/química , Núcleo Celular/metabolismo , Fenômenos Fisiológicos Celulares , Cor , Citoplasma/química , Citoplasma/metabolismo , Células HeLa , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Reprodutibilidade dos Testes , Solventes/química , Vibração , Água/química
3.
J Ind Microbiol Biotechnol ; 46(12): 1669-1683, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31531745

RESUMO

Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10-7 M, thus providing a detection range for Hg2+ quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Mercúrio/análise , Transporte Biológico , Sobrevivência Celular , Escherichia coli/química , Células HEK293 , Humanos , Espaço Intracelular/química , Saccharomyces cerevisiae/química
4.
Int J Nanomedicine ; 14: 5713-5728, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413571

RESUMO

Purpose: The levels of reactive oxygen species (ROS) in tumor cells are much higher than that in normal cells, and rise rapidly under the influence of exogenous or endogenous inducing factors, eventually leading to the apoptosis of tumor cells. Therefore, this study prepared a dual pH/reducing-responsive poly (N-isopropylacrylamide-co-Cinnamaldehyde-co-D-α-tocopheryl polyethylene glycol 1000 succinate, PssNCT) nanogels, which employed two exogenous ROS inducers, cinnamaldehyde (CA) and D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), to selectively induce apoptosis by regulating ROS levels in tumor cells. Methods: The PssNCT nanogels were prepared by the free radical precipitation polymerization under the crosslink between pH-sensitive hydrazone and reducing-sensitive disulfide bonds, followed by the physicochemical and morphological characteristics investigations. Plasma stability, dual pH/reducing responsive degradation and in vitro release were also assessed. In cell experiments, cytotoxicity in different cells were first detected. The intracellular ROS levels and mitochondrial functions of tumor cells were then evaluated. Moreover, the apoptosis and western-blot assays were employed to verify the association between ROS levels elevation and apoptosis in tumor cells. Results: The nanogels exhibited a round-like hollow structure with the diameter smaller than 200nm. The nanogels were stable in plasma, while showed rapid degradation in acidic and reducing environments, thus achieving significant release of CA and TPGS in these media. Furthermore, the sufficient amplification of ROS signals was induced by the synergistically function of CA and TPGS on mitochondria, which resulted in the opening of the mitochondrial apoptotic pathway and enhanced cytotoxicity on MCF-7 cells. However, nanogels barely affected L929 cells owing to their lower intracellular ROS basal levels. Conclusion: The specific ROS regulation method achieved by these nanogels could be explored to selectively kill tumor cells according to the difference of ROS signals in different kinds of cells.


Assuntos
Apoptose , Espaço Intracelular/química , Polietilenoglicóis/farmacologia , Polietilenoimina/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Acroleína/análogos & derivados , Acroleína/farmacologia , Animais , Apoptose/efeitos dos fármacos , Doxorrubicina/farmacologia , Liberação Controlada de Fármacos , Humanos , Concentração de Íons de Hidrogênio , Células MCF-7 , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Vitamina E/síntese química , Vitamina E/química
5.
Methods Appl Fluoresc ; 7(4): 044002, 2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31412329

RESUMO

Induced pluripotent stem cells (iPSC) are a promising tool for personalized cell therapy, in particular, in the field of dermatology. Metabolic plasticity of iPSC are not completely understood due to the fact that iPSC have a mixed mitochondrial phenotype, which still resembles that of somatic cells. In this study we investigated the metabolic changes in iPSC undergoing differentiation in two directions, dermal and epidermal, using two-photon fluorescence microscopy combined with FLIM. Directed differentiation of iPSC into dermal fibroblasts and keratinocyte progenitor cells was induced. Cellular metabolism was examined on the basis of the fluorescence of the metabolic cofactors NAD(P)H and FAD. The optical redox ratio (FAD/NAD(P)H) and the fluorescence lifetimes of NAD(P)H and FAD were traced using two-photon fluorescence microscopy combined with FLIM. Evaluation of the intracellular pH was carried out with the fluorescent pH sensor SypHer-2 and fluorescence microscopy. In this study, evaluation of the metabolic status of iPSC during dermal and epidermal differentiation was accomplished for the first time with the use of optical metabolic imaging. Based on the data on the FAD/NAD(P)H redox ratio and on the fluorescence lifetimes of protein-bound form of NAD(P)H and closed form of FAD, we registered a metabolic shift toward a more oxidative status in the process of iPSC differentiation into dermal fibroblasts and keratinocyte progenitor cells. Biosynthetic processes occurring in dermal fibroblasts associated with the synthesis of fibronectin and versican, that stimulate increased energy metabolism and lower the intracellular pH. No intracellular pH shift is observed in the culture of keratinocyte progenitor cells, which reflects the incomplete process of differentiation in this type of cells. Presented results provide the basis for further understanding the metabolic features of iPSC during differentiation process, which is essential for developing new treatment strategies in cell therapy and tissue engineering.


Assuntos
Diferenciação Celular , Derme/citologia , Epiderme/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Espaço Intracelular/química , Calibragem , Humanos , Concentração de Íons de Hidrogênio
6.
Mater Sci Eng C Mater Biol Appl ; 102: 863-875, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31147058

RESUMO

The frequent occurrence of multidrug resistance (MDR) in solid tumors is the major obstacle for nano-drug delivery systems (nDDS) to realize the successful cancer chemotherapy. Herein, we had prepared pH-responsive nanogels via cross-linking TPGS-grafted soy protein with an acid-labile ortho ester cross-linker (OEAM) to realize the efficient intracellular drugs release and accumulation, and subsequently enhance therapeutic effect in MDR tumor cells. These nanogels displayed a uniform size (~200 nm) and morphology, and the introduction of ortho ester bonds endowed nanogels stability in neutral environment and acid-degradability in acidic conditions. Cisplatin (CDDP) was successfully loaded into nanogels, which exhibited an accelerated drug release at low pH. The modification of TPGS efficiently improved cellular internalization and drug accumulation in A549/DDP cells by inhibiting the function of drug efflux pumps (MRP2 and ATP7A/7B), leading to higher cytotoxicity and apoptosis. Moreover, TPGS-grafted nanogels also showed better drug accumulation and penetration in tumor-like spheroids, and then remarkably inhibited tumor growth owing to the rapid drug release in acidic organelles. As a result, the TPGS-grafted and pH-sensitive soy protein nanogels have a great potential as a drugs carrier for the efficient cancer treatment.


Assuntos
Liberação Controlada de Fármacos , Resistencia a Medicamentos Antineoplásicos , Espaço Intracelular/química , Metacrilatos/química , Polietilenoglicóis/química , Polietilenoimina/química , Proteínas de Soja/química , Vitamina E/química , Células A549 , Trifosfato de Adenosina/metabolismo , Apoptose , Endocitose , Humanos , Hidrodinâmica , Concentração Inibidora 50 , Potencial da Membrana Mitocondrial , Tamanho da Partícula , Espectroscopia de Prótons por Ressonância Magnética , Esferoides Celulares/patologia
7.
Int J Nanomedicine ; 14: 1119-1130, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30863049

RESUMO

Background: Protein or peptide drugs are emerging therapeutics for treating human diseases. However, current protein drugs are typically limited to acting on extracellular/cell membrane components associated with the diseases, while intracellular delivery of recombinant proteins replaces or replenishes faulty/missing proteins and remains inadequate. In this study, we developed a convenient and efficient intracellular protein delivery vehicle. Materials and methods: A cationic liposomal polyethylenimine and polyethylene glycol complex (LPPC) was developed to noncovalently capture proteins for protein transfer into cells via endocytosis. ß-glucuronidase (ßG) was used in vitro and in vivo as a model enzyme to demonstrate the enzymatic activity of the intracellular transport of a protein. Results: The endocytosed protein/LPPC complexes escaped from lysosomes, and the bound protein dissociated from LPPC in the cytosol. The enzymatic activity of ßG was well preserved after intracellular delivery in vitro and in vivo. Conclusion: Using LPPC as an intracellular protein transporter for protein therapeutics, we illustrated that LPPC may be an effective and convenient tool for studying diseases and developing therapeutics.


Assuntos
Espaço Intracelular/química , Polietilenoglicóis/química , Polietilenoimina/análogos & derivados , Proteínas/uso terapêutico , Células 3T3 , Adsorção , Animais , Bovinos , Morte Celular , Fluoresceína-5-Isotiocianato/química , Fluorescência , Glucuronidase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células Hep G2 , Humanos , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Tamanho da Partícula , Polietilenoimina/química , Estabilidade Proteica , Soroalbumina Bovina/química , Eletricidade Estática
8.
Anal Chim Acta ; 1056: 79-87, 2019 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-30797464

RESUMO

Combination antiretroviral therapy (cART) regimens are recommended for HIV patients to better achieve and maintain plasma viral suppression. Despite adequate plasma viral suppression, HIV persists inside the brain, which is, in part thought to result from poor brain penetration of antiretroviral drugs. In this study, a simple and ultra-sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell lysates of an immortalized human brain microvascular endothelial cell line (hCMEC/D3) was developed and validated. Analytes were separated on a reverse phase C18 column using water and 0.1% formic acid in acetonitrile as mobile phases. The analytes were detected using positive electrospray ionization mode with multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.1-100 ng mL-1 for all analytes. Intra- and inter-assay precision and accuracy were within ±13.33% and ±10.53%, respectively. This approach described herein was used to determine the intracellular accumulation of tenofovir, emtricitabine, dolutegravir simultaneously in hCMEC/D3 cells samples.


Assuntos
Fármacos Anti-HIV/análise , Encéfalo/citologia , Cromatografia Líquida/métodos , Células Endoteliais/citologia , Espaço Intracelular/química , Espectrometria de Massas em Tandem/métodos , Métodos Analíticos de Preparação de Amostras , Fármacos Anti-HIV/isolamento & purificação , Linhagem Celular , Emtricitabina/análise , Emtricitabina/isolamento & purificação , Compostos Heterocíclicos com 3 Anéis/análise , Compostos Heterocíclicos com 3 Anéis/isolamento & purificação , Humanos , Modelos Lineares , Reprodutibilidade dos Testes , Tenofovir/análise , Tenofovir/isolamento & purificação
9.
J Mol Histol ; 50(2): 141-154, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30659401

RESUMO

The sperm produced in the seminiferous tubules pass through the rete testis, efferent ducts, and epididymis. The epididymis has three distinct regions known as caput, corpus, and cauda. The transit through the epididymis is an essential process in sperm maturation. The lumen of each epididymal region has a unique fluid composition regulated by many ion channels and transporters in the epithelial cells. The objective of this study was to map the sites of localization of ion channels ENaC and CFTR along the length of the mouse and rat epididymis using confocal microscopic imaging. The integrity of the fine structure of the tissues was verified by fluorescent phalloidin staining of actin filaments visualized by high-resolution confocal microscopy. The 2D and 3D images showed preservation of the stereocilia. Based on these images we determined morphometric parameters of the epithelial cells and ducts. ENaC and CFTR immunofluorescence appeared almost continuously on the apical membrane of caput and in smooth muscle myoid cells. In cauda, CFTR expression was observed continuously in long stretches of epithelium interrupted by clusters of cells that showed no CFTR expression. Similar patterns of localization were observed in both mouse and rat samples. Mutations in the CFTR gene are known to result in male infertility. Based on the widespread presence of ENaC along the epididymis we suggest that mutations in ENaC subunits may also be associated with male infertility. The diverse phenotypes associated with CFTR mutations may be due to malfunction of CFTR at specific subcellular locations in the male reproductive system.


Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/análise , Epididimo/química , Canais Epiteliais de Sódio/análise , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Células Epiteliais/química , Canais Epiteliais de Sódio/genética , Imunofluorescência , Infertilidade Masculina/genética , Espaço Intracelular/química , Masculino , Camundongos , Microscopia Confocal , Mutação , Ratos , Distribuição Tecidual
10.
J Environ Sci (China) ; 76: 1-11, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30528000

RESUMO

Coagulation is the best available method for removing intracellular organic matter (IOM), which is released from algae cells and is an important precursor to disinfection by-products in drinking water treatment. To gain insight into the best strategy to optimize IOM removal, the coagulation performance of two Al salts, i.e., aluminum chloride (AlCl3) and polyaluminum chloride (PACl, containing 81.2% Al13), was investigated to illuminate the effect of Al species distribution on IOM removal. PACl showed better removal efficiency than AlCl3 with regard to the removal of turbidity and dissolved organic carbon (DOC), owing to the higher charge neutralization effect and greater stability of pre-formed Al13 species. High pressure size exclusion chromatography analysis indicated that the superiority of PACl in DOC removal could be ascribed to the higher binding affinity between Al13 polymer and the low and medium molecular weight (MW) fractions of IOM. The results of differential log-transformed absorbance at 254 and 350 nm indicated more significant formation of complexes between AlCl3 and IOM, which benefits the removal of tryptophan-like proteins thereafter. Additionally, PACl showed more significant superiority compared to AlCl3 in the removal of <5 kDa and hydrophilic fractions, which are widely viewed as the most difficult to remove by coagulation. This study provides insight into the interactions between Al species and IOM, and advances the optimization of coagulation for the removal of IOM in eutrophic water.


Assuntos
Alumínio/química , Espaço Intracelular/química , Compostos Orgânicos/química , Compostos Orgânicos/isolamento & purificação , Polímeros/química , Eutrofização , Microcystis/citologia , Microcystis/crescimento & desenvolvimento , Peso Molecular
11.
Nat Commun ; 9(1): 4817, 2018 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-30446665

RESUMO

Molecular dyes, plasmonic nanoparticles and colloidal quantum dots are widely used in biomedical optics. Their operation is usually governed by spontaneous processes, which results in broad spectral features and limited signal-to-noise ratio, thus restricting opportunities for spectral multiplexing and sensing. Lasers provide the ultimate spectral definition and background suppression, and their integration with cells has recently been demonstrated. However, laser size and threshold remain problematic. Here, we report on the design, high-throughput fabrication and intracellular integration of semiconductor nanodisk lasers. By exploiting the large optical gain and high refractive index of GaInP/AlGaInP quantum wells, we obtain lasers with volumes 1000-fold smaller than the eukaryotic nucleus (Vlaser < 0.1 µm3), lasing thresholds 500-fold below the pulse energies typically used in two-photon microscopy (Eth ≈ 0.13 pJ), and excellent spectral stability (<50 pm wavelength shift). Multiplexed labeling with these lasers allows cell-tracking through micro-pores, thus providing a powerful tool to study cell migration and cancer invasion.


Assuntos
Espaço Intracelular/química , Lasers , Nanoestruturas/química , Nanotecnologia/métodos , Animais , Movimento Celular , Macrófagos/ultraestrutura , Camundongos , Células NIH 3T3 , Neurônios/ultraestrutura , Permeabilidade , Cultura Primária de Células , Semicondutores , Linfócitos T/ultraestrutura
12.
J Biomed Opt ; 23(10): 1-14, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30298706

RESUMO

Oxygen (O2) is one of the most important biometabolites. In abundance, it serves as the limiting terminus of aerobic respiratory chains in the mitochondria of higher organisms; in deficit, it is a potent determinant of development and regulation of other physiological and therapeutic processes. Most knowledge on intracellular and interstitial concentration ([O2]) is derived from mitochondria isolated from cells or tissue biopsies, providing detailed but nonnative insight into respiratory chain function. The possible loss of essential metabolites during isolation and disruption of the normal interactions of the organelle with the cytoskeleton may cause these data to misrepresent intact cells. Several optical methodologies were also developed, but they are often unable to detect heterogeneity of metabolic characteristics among different individual cells in the same culture, and most cannot detect heterogeneous consumption within different areas of a single cell. Here, we propose a noninvasive and highly sensitive fluorescence lifetime microscopy probe, myoglobin-mCherry, appropriate to intracellular targeting. Using our probe, we monitor mitochondrial contributions to O2 consumption in A549 nonsmall cell lung cancer cells and we reveal heterogeneous [O2] within the intracellular environments. The mitochondrial [O2] at a single-cell level is also mapped by adding a peptide to target the probe to the mitochondria.


Assuntos
Corantes Fluorescentes/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Mioglobina/metabolismo , Oxigênio/análise , Células A549 , Corantes Fluorescentes/análise , Humanos , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Mitocôndrias/química , Mitocôndrias/metabolismo , Mioglobina/genética , Oxigênio/química , Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção
13.
Biosens Bioelectron ; 121: 236-242, 2018 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-30219723

RESUMO

In this work, we report a novel sensor for colorimetric and fluorometric H2O2 sensing which is based on silver nanoparticles decorated and tetraphenylethene probe doped silica nanoparticles (Ag@TPE-SiO2 NPs). A positively charged tetraphenylethene (TPE) probe is doped into silica nanoparticles, and the nanoparticles exhibit strong fluorescence emission due to aggregation-induced emission (AIE) of the TPE probe. Ag nanoparticles (AgNPs) are prepared in situ on the surface of the silica nanoparticles. AgNPs serve as a nanoquencher which can quench the AIE emission of the TPE-SiO2 NPs efficiently. However, AgNPs can be oxidized to Ag+ by H2O2, which leads to fluorescence recovery and color fading of the Ag@TPE-SiO2 NPs. The dual-readout strategy allows sensitive analysis of H2O2. The detection limit of the fluorometric and colorimetric assay is 0.28 and 2.1 µM, respectively. And the nanosensor also shows good selectivity. In addition, analysis of H2O2 in human serum and intracellular imaging of H2O2 are both demonstrated. With the good analytical properties of merit, the proposed nanoprobe has a promising potential for H2O2 related bioanalysis and biomedical applications.


Assuntos
Técnicas Biossensoriais/métodos , Análise Química do Sangue/métodos , Peróxido de Hidrogênio/análise , Nanopartículas/química , Dióxido de Silício/química , Prata/química , Estilbenos/química , Técnicas Biossensoriais/instrumentação , Colorimetria , Fluorometria , Humanos , Peróxido de Hidrogênio/sangue , Espaço Intracelular/química , Limite de Detecção , Nanopartículas Metálicas/química , Imagem Molecular
14.
ACS Appl Mater Interfaces ; 10(38): 31998-32005, 2018 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-30178654

RESUMO

Nowadays, efficient endo/lysosomal escape and the subsequent release of drugs into the cytosol are the major obstacles for nanoplatform-based cancer therapy. Herein, we first report a metal-organic framework-based nanoplatform (doxorubicin@ZIF-8@AS1411) for intracellular environment-responsive endo/lysosomal escape and enhanced cancer therapy. In our system, the nanoplatform was first targeted toward the cancer cells. Then, it was entrapped in endo/lysosomes, where pH-responsive decomposition occurred and abundant Zn ions were released. The released Zn ions could induce an influx of counterions, promote reactive singlet oxygen (ROS) generation to rupture the endo/lysosomal membrane, and accelerate the release of anticancer drugs in the cytosol. Finally, the released drugs and the generation of ROS could synergistically enhance cancer therapy. With excellent biocompatibility, effective endo/lysosomal escape, and enhanced therapeutic effect, the novel drug delivery systems are supposed to become a promising anticancer agent for cancer therapy and bring more opportunities for biomedical application.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Endossomos/metabolismo , Espaço Intracelular/química , Lisossomos/metabolismo , Estruturas Metalorgânicas/química , Antineoplásicos/administração & dosagem , Doxorrubicina/administração & dosagem , Humanos , Neoplasias/tratamento farmacológico
15.
Methods Mol Biol ; 1831: 95-109, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30051427

RESUMO

MicroRNAs are small noncoding RNAs that function as powerful endogenous regulators of gene expression. Dysregulation of MicroRNA biogenesis has been correlated with the onset and progression of many human diseases. MicroRNA therapy involves the re-equilibration of aberrant intracellular MicroRNA expression profiles for long-term disease management. Despite the significant potential of MicroRNA therapy, the utilization of MicroRNA-based therapeutics has been drastically hindered in practice by the lack of a targeted and translatable delivery vehicle. CD44 is a cell surface glycoprotein that facilitates cellular communication and motility through cell-cell and cell-extracellular matrix interactions. CD44 has been shown to be elevated in multiple disease states including cancer making it a potential diagnostic biomarker and an ideal receptor for targeted drug delivery systems. We describe a method for targeting CD44 using a lipid nanocarrier for the cytoplasmic delivery of active MicroRNA.


Assuntos
Receptores de Hialuronatos/metabolismo , Lipídeos/química , MicroRNAs/uso terapêutico , Nanopartículas/química , Linhagem Celular , Portadores de Fármacos/química , Técnicas de Silenciamento de Genes , Humanos , Ácido Hialurônico/química , Espaço Intracelular/química , Nanopartículas/ultraestrutura , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Reprodutibilidade dos Testes
16.
Langmuir ; 34(28): 8170-8177, 2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-29924935

RESUMO

The advances in boron incorporated organics have captured overwhelming interest on account of their outstanding properties and promising applications in various fields. Mostly, triarylborane compounds (TAB) are exploited as sensors of F- and CN- anions at the expense of the intrinsic Lewis acidic nature of boron. New molecular probes 1 and 2 for detection of toxic thiophenol were designed by conjugating highly fluorescent borylanilines with the luminescent quencher 2,4-dinitrobenzene based sulfonamides (DNBS), wherein the electrophilicity of the DNBS moiety has been modulated by fine-tuning the intrinsic Lewis acidity of boron. The interplay between PET (photoinduced electron transfer) and ICT have been employed for developing the TAB tethered turn-on fluorescent sensor for thiophenol with high selectivity for the first time. The newly developed probes showed very fast response toward thiophenol (within ∼5 min) with limits of detection (LOD) lying in the micromolar range, clearly pointing to their potential. Further, compounds 1 and 2 were explored for detecting thiophenol in the intracellular environment by discriminating biothiols. DFT and TD-DFT calculations were performed to support the sensing mechanism.


Assuntos
Técnicas de Química Analítica/métodos , Corantes Fluorescentes , Espaço Intracelular/química , Fenóis , Compostos de Sulfidrila , Luminescência , Fenóis/análise , Fenóis/química , Compostos de Sulfidrila/análise , Compostos de Sulfidrila/química
17.
Anal Chem ; 90(13): 8058-8064, 2018 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-29847925

RESUMO

Acidified extracellular pH (pHe) is directly related to various disorders such as tumor invasion and the resistance to drugs. In this study, we developed two-photon-excitable emission ratiometric probes (XBH1-3) for the in situ measurement of pHe. These probes, based on benzimidazole and polar solubilizing groups, exhibited a strong two-photon-induced fluorescence and sensitive blue-to-green emission color changes with p Ka values of 5.1-5.7. XBH1, containing a carboxylic acid, stained the extracellular region in neutral media; it entered the cell under acidic media, thereby allowing a precise measurement of the extra- and intra-cellular pH values in the acidified tissue. XBH2, containing the sulfonate peripheral unit, facilitated the monitoring of the pHe value only. Ratiometric two-photon microscopy imaging revealed that XBH1 can directly monitor the pH values both inside and outside the cells in colon cancer tissue; there is also the morphological aspect. This could be useful for cancer analyses and drug development.


Assuntos
Ácidos Carboxílicos/química , Neoplasias do Colo/patologia , Espaço Extracelular/química , Corantes Fluorescentes/química , Espaço Intracelular/química , Fótons , Linhagem Celular Tumoral , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Solubilidade , Espectrometria de Fluorescência , Ácidos Sulfônicos/química , Fatores de Tempo
18.
Biomaterials ; 178: 546-558, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29657093

RESUMO

These years, transformable drug delivery systems (DDSs), which hold the capability of changing formulation morphology and subsequent functionality at the desired disease site, have shown great promise in control of spatio-temporal drug delivery/release manner and enhanced treatment efficacy. Equipped with controllability and design flexibility, the transformable DDSs are being increasingly pursued for the development of precision drug delivery platforms for biomedical applications. In this review, we describe the recently developed intracelluarly and extracellularly transformable DDSs, especially associated with assembly or disassociation of the original formulation units, for achieving various functionalities, including prolonged retention time, inhibited endocytosis and enhanced cytotoxicity. Furthermore, the different stimuli, such as pH, enzyme, light, temperature, redox and mechanical force that trigger the transformation process are also introduced. The future outlook and challenges are discussed in the end.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Animais , Espaço Extracelular/química , Humanos , Espaço Intracelular/química , Microbolhas , Nanopartículas/química , Nanopartículas/ultraestrutura , Neoplasias/diagnóstico , Neoplasias/patologia
19.
Biotechnol Lett ; 40(6): 981-987, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29619743

RESUMO

OBJECTIVE: Through heterologous expression of the tetrahydrocannabinolic acid synthase (THCAS) coding sequence from Cannabis sativa L. in Nicotiana benthamiana, we evaluated a transient plant-based expression system for the production of enzymes involved in cannabinoid biosynthesis. RESULTS: Thcas was modularized according to the GoldenBraid grammar and its expression tested upon alternative subcellular localization of the encoded catalyst with and without fusion to a fluorescent protein. THCAS was detected only when ER targeting was used; cytosolic and plastidal localization resulted in no detectable protein. Moreover, THCAS seems to be glycosylated in N. benthamiana, suggesting that this modification might have an influence on the stability of the protein. Activity assays with cannabigerolic acid as a substrate showed that the recombinant enzyme produced not only THCA (123 ± 12 fkat g FW-1 activity towards THCA production) but also cannabichromenic acid (CBCA; 31 ± 2.6 fkat g FW-1 activity towards CBCA production). CONCLUSION: Nicotiana benthamiana is a suitable host for the generation of cannabinoid producing enzymes. To attain whole pathway integration, careful analysis of subcellular localization is necessary.


Assuntos
Canabinoides/metabolismo , Espaço Intracelular/enzimologia , Oxirredutases Intramoleculares , Engenharia Metabólica/métodos , Proteínas de Plantas , Tabaco/enzimologia , Cannabis/enzimologia , Cannabis/genética , Espaço Intracelular/química , Espaço Intracelular/metabolismo , Oxirredutases Intramoleculares/química , Oxirredutases Intramoleculares/genética , Oxirredutases Intramoleculares/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tabaco/citologia , Tabaco/genética , Tabaco/metabolismo
20.
Int J Food Microbiol ; 266: 289-294, 2018 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-29274485

RESUMO

This study was designed to assess the efficiency of eight extraction methods regarding their ability to release superficial (exogenous) and intracellular (endogenous) DNA from B. cereus spores for subsequent analysis by quantitative PCR (qPCR). B. cereus spore suspensions were subjected to both commercial DNA extraction kits and mechanical DNA extraction methods. The spores were observed by transmission electron microscopy to evaluate any damage caused during extraction. The efficiency of both extraction and purification were assessed using a qPCR assay targeting the bclA gene. Most of the extraction methods assessed, except the passage through the French press or the use of the QIAamp DNA Blood Mini kit without 95°C treatment, allowed the amplification of significant amounts of DNA. By using propidium monoazide, which is a photoreactive DNA-binding dye, the presence of non-negligible amounts of amplifiable DNA at the spore surface was highlighted. A further set of extraction assays was then performed on spores previously treated with PMA. The results of this study show that both superficial and intracellular spore DNA can be released by extraction methods to a greater or lesser extent and then further amplified by qPCR. The Precellys extraction allowed the detection of both intracellular and superficial DNA, the DNeasy Blood & Tissue kit the specific detection of intracellular DNA, while the Instagene kit detected only superficial DNA. Of the methods tested in this study, the Precellys extraction was the most efficient in terms of further DNA detection. SIGNIFICANCE AND IMPACT OF THE STUDY: In order to verify the presence or absence of B. cereus spores in food or on surfaces in the food environment, the use of an efficient extraction method is required, followed by a qPCR analysis on the DNA released. Conversely, in order to quantify the population of Bacillus spores, any superficial DNA must be blocked, e.g. with PMA, prior to intracellular DNA extraction and further amplification.


Assuntos
Bacillus/genética , DNA Bacteriano/isolamento & purificação , Técnicas Genéticas/normas , Esporos Bacterianos/genética , Azidas/química , Bacillus/química , DNA Bacteriano/genética , Espaço Intracelular/química , Propídio/análogos & derivados , Propídio/química , Reação em Cadeia da Polimerase em Tempo Real , Esporos Bacterianos/química
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