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1.
Molecules ; 26(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199465

RESUMO

Hydrogen peroxide (H2O2) plays an important role in the human body and monitoring its level is meaningful due to the relationship between its level and diseases. A fluorescent sensor (CMB) based on coumarin was designed and its ability for detecting hydrogen peroxide by fluorescence signals was also studied. The CMB showed an approximate 25-fold fluorescence enhancement after adding H2O2 due to the interaction between the CMB and H2O2 and had the potential for detecting physiological H2O2. It also showed good biocompatibility and permeability, allowing it to penetrate cell membranes and zebrafish tissues, thus it can perform fluorescence imaging of H2O2 in living cells and zebrafish. This probe is a promising tool for monitoring the level of H2O2 in related physiological and pathological research.


Assuntos
Cumarínicos/química , Corantes Fluorescentes/química , Peróxido de Hidrogênio/análise , Animais , Membrana Celular , Humanos , Células MCF-7 , Imagem Óptica , Peixe-Zebra
2.
Int J Mol Sci ; 22(12)2021 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-34204524

RESUMO

The aim of this work is to develop a biomimetic interface between the natural tooth tissue and the restorative composite and to study it on the basis of synchrotron micro-FTIR mapping and multidimensional processing of the spectral data array. Using hierarchical cluster analysis of 3D FTIR data revealed marked improvements in the formation of the dentine/adhesive/dental hybrid interface using a biomimetic approach. The use of a biomimetic strategy (application of an amino acid-modified primer, alkaline calcium and a nano-c-HAp-modified adhesive) allowed the formation of a matrix that can be structurally integrated with natural dentine and dental composite. The biomimetic hybrid layer was characterised by homogeneous chemical composition and a higher degree of conversion of the adhesive during polymerisation, which should provide optimal integration of the dental composite with the dentine.


Assuntos
Biomimética , Odontologia , Espectroscopia de Infravermelho com Transformada de Fourier , Síncrotrons , Engenharia Tecidual , Dente , Biomimética/métodos , Odontologia/métodos , Humanos , Teste de Materiais , Nanotecnologia , Imagem Óptica/métodos
3.
Molecules ; 26(12)2021 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-34205289

RESUMO

The inadvertent severing of a ureter during surgery occurs in as many as 4.5% of colorectal surgeries. To help prevent this issue, several near-infrared (NIR) dyes have been developed to assist surgeons with identifying ureter location. However, the majority of these dyes exhibit at least some issue that precludes their widespread usage such as high levels of uptake in other tissues, overlapping emission wavelengths with other NIR dyes used for other fluorescence-guided surgeries, and/or rapid excretion times through the ureters. To overcome these limitations, we have synthesized and characterized the spectral properties and biodistribution of a new series of PEGylated UreterGlow derivatives. The most promising dye, UreterGlow-11 was shown to almost exclusively excrete through the kidneys/ureters with detectable fluorescence observed for at least 12 h. Additionally, while the excitation wavelength is similar to that of other NIR dyes used for cancer resections, the emission is shifted by ~30 nm allowing for discrimination between the different fluorescence-guided surgery probes. In conclusion, these new UreterGlow dyes show promising optical and biodistribution characteristics and are good candidates for translation into the clinic.


Assuntos
Abdome/cirurgia , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Ureter/cirurgia , Animais , Fluorescência , Corantes Fluorescentes/metabolismo , Humanos , Rim/cirurgia , Camundongos , Distribuição Tecidual/fisiologia , Ureter/metabolismo
7.
Nat Protoc ; 16(7): 3298-3321, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34075230

RESUMO

Molecule-specific techniques such as MALDI and desorption electrospray ionization mass spectrometry imaging enable direct and simultaneous mapping of biomolecules in tissue sections in a single experiment. However, neurotransmitter imaging in the complex environment of biological samples remains challenging. Our covalent charge-tagging approach using on-tissue chemical derivatization of primary and secondary amines and phenolic hydroxyls enables comprehensive mapping of neurotransmitter networks. Here, we present robust and easy-to-use chemical derivatization protocols that facilitate quantitative and simultaneous molecular imaging of complete neurotransmitter systems and drugs in diverse biological tissue sections with high lateral resolution. This is currently not possible with any other imaging technique. The protocol, using fluoromethylpyridinium and pyrylium reagents, describes all steps from tissue preparation (~1 h), chemical derivatization (1-2 h), data collection (timing depends on the number of samples and lateral resolution) and data analysis and interpretation. The specificity of the chemical reaction can also help users identify unknown chemical identities. Our protocol can reveal the cellular locations in which signaling molecules act and thus shed light on the complex responses that occur after the administration of drugs or during the course of a disease.


Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Neurotransmissores/metabolismo , Imagem Óptica , Espectrometria de Massas por Ionização por Electrospray/métodos , Animais , Limite de Detecção , Masculino , Ratos Sprague-Dawley , Padrões de Referência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
8.
Int J Mol Sci ; 22(9)2021 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-34062952

RESUMO

BACKGROUND: Dental implants are commonly used for missing teeth, for which success depends heavily on the quality of the alveolar bone. The creation of an ideal implant site is a key component in shortening the treatment time, which remains clinically challenging. Strontium ranelate (Protos) is an anti-osteoporotic agent which has previously been used to promote bone formation, however the systemic use of Protos has been linked to serious cardiovascular and venous thromboembolic events, thus local delivery strategies may be better suited for this purpose. In this study, a biodegradable, and biocompatible nanocarrier "polybutylcyanoacrylate" (PBCA) loaded with strontium was constructed and its ability to promote bone formation was assessed. METHODOLOGY: PBCA nanoparticles loaded with strontium (PBCA-Sr NPs) were synthesized using the emulsion polymerization method, and their physical properties (zeta potential, size and shape) and entrapment efficiency were characterized. Committed MSCs (osteoblasts) were derived from the differentiation of cultured rat mesenchymal stem cells (MSC), which were tested with the PBCA-Sr NPs for cytotoxicity, inflammatory response, bone formation and mineralization. Scanning electron microscopy was performed following a 7-day treatment of PBCA-Sr NPs on decellularized procaine mandibular bone blocks grafted with osteoblasts. RESULTS: Spherical PBCA-Sr NPs of 166.7 ± 2.3 nm, zeta potential of -1.15 ± 0.28 mV with a strontium loading efficiency of 90.04 ± 3.27% were constructed. The presence of strontium was confirmed by energy-dispersive X-ray spectroscopy. Rat committed MSCs incubated in PBCA-Sr NPs for 24 hrs showed viabilities in excess of 90% for concentrations of up to 250 ug/mL, the cellular expression of osteocalcin and alkaline phosphatase were 1.4 and 1.3 times higher than the untreated control, and significantly higher than those treated with strontium alone. Bone formation was evident following osteoblast engraftment on the decellularized procaine mandibular bone block with PBCA-Sr NPs, which appeared superior to those treated with strontium alone. CONCLUSION: Treatment of committed MSCs with PBCA-Sr NPs showed higher expression of markers of bone formation when compared with strontium alone and which corresponded to greater degree of bone formation observed on the 3-dimensinal decellularized procaine mandibular bone block. Further quantitative analysis on the extent of new bone formation is warranted.


Assuntos
Embucrilato/química , Mandíbula/crescimento & desenvolvimento , Nanopartículas/química , Osteogênese , Tiofenos/farmacologia , Fosfatase Alcalina/metabolismo , Animais , Calcificação Fisiológica/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Mandíbula/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanopartículas/ultraestrutura , Imagem Óptica , Osteocalcina/metabolismo , Tamanho da Partícula , Ratos Sprague-Dawley , Eletricidade Estática
9.
Anal Chim Acta ; 1171: 338655, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34112439

RESUMO

Cysteine (Cys), a kind of small molecule biological thiol, not only involves in the regulation of physiological processes, but also is considered a marker of tumor. However, it is challenging to develop suitable probe for detecting Cys in tumors. In this paper, a near-infrared (NIR) fluorescent probe named IX for Cys has been designed and synthesized. The probe shows a NIR emission peak at 770 nm with large Stokes shift (180 nm) upon adding Cys. It displays high sensitivity to Cys with 6-fold increase of fluorescence intensity. Meanwhile, IX has the high selectivity to Cys over other potential interference such as Hcy and GSH, which have similar structure with Cys. In addition, a possible mechanism of fluorescence enhancement is the reaction of IX with Cys to release IX-OH, which is verified by fluorescence spectra, MS and HPLC. Next, IX can selectively image Cys in HCT-116 cells thanks to the low cytotoxicity. Most important of all, the fluorescent probe IX has visualized Cys in HCT116-xenograft tumor mice due to the near-infrared properties with large Stokes shift.


Assuntos
Cisteína , Corantes Fluorescentes , Animais , Glutationa , Células HCT116 , Humanos , Camundongos , Imagem Óptica
10.
Nat Commun ; 12(1): 3798, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145235

RESUMO

Olfactory sensory neurons (OSNs) are functionally defined by their expression of a unique odorant receptor (OR). Mechanisms underlying singular OR expression are well studied, and involve a massive cross-chromosomal enhancer interaction network. Trace amine-associated receptors (TAARs) form a distinct family of olfactory receptors, and here we find that mechanisms regulating Taar gene choice display many unique features. The epigenetic signature of Taar genes in TAAR OSNs is different from that in OR OSNs. We further identify that two TAAR enhancers conserved across placental mammals are absolutely required for expression of the entire Taar gene repertoire. Deletion of either enhancer dramatically decreases the expression probabilities of different Taar genes, while deletion of both enhancers completely eliminates the TAAR OSN populations. In addition, both of the enhancers are sufficient to drive transgene expression in the partially overlapped TAAR OSNs. We also show that the TAAR enhancers operate in cis to regulate Taar gene expression. Our findings reveal a coordinated control of Taar gene choice in OSNs by two remote enhancers, and provide an excellent model to study molecular mechanisms underlying formation of an olfactory subsystem.


Assuntos
Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Neurônios Receptores Olfatórios/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Odorantes/metabolismo , Animais , Animais Geneticamente Modificados , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mucosa Olfatória/metabolismo , Imagem Óptica , Receptores Acoplados a Proteínas G/metabolismo , Olfato/genética , Peixe-Zebra/genética
11.
Methods Mol Biol ; 2276: 193-202, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060042

RESUMO

Brain is one of the most energy-demanding organs. Energy in the form of ATP is produced in brain cells predominantly in oxidative phosphorylation coupled to mitochondrial respiration. Any alteration of the mitochondrial metabolism or prolonged ischemic or anoxic conditions can lead to serious neurological conditions, including neurodegenerative disorders. Assessment of mitochondrial metabolism is important for understanding physiological and pathological processes in the brain. Bioenergetics in central nervous system is dependent on multiple parameters including neuron-glia interactions and considering this, in vivo or ex vivo, the measurements of mitochondrial metabolism should also be complimenting the experiments on isolated mitochondria or cell cultures. To assess the mitochondrial function, there are several key bioenergetic parameters which indicate mitochondrial health. One of the major characteristics of mitochondria is the mitochondrial membrane potential (ΔΨm) which is used as a proton motive force for ATP production and generated by activity of the electron transport chain. Major donor of electrons for the mitochondrial respiratory chain is NADH. Here we demonstrate how to measure mitochondrial NADH/NAD(P)H autofluorescence and ΔΨm in acute brain slices in a time-dependent manner and provide information for the identification of NADH redox index, mitochondrial NADH pool, and the rate of NADH production in the Krebs cycle. Additionally, non-mitochondrial NADH/NADPH autofluorescence can signify the level of activity of the pentose phosphate pathway.


Assuntos
Encéfalo/metabolismo , Potencial da Membrana Mitocondrial/fisiologia , Mitocôndrias/metabolismo , NADP/metabolismo , NAD/metabolismo , Imagem Óptica/métodos , Animais , Química Encefálica , Mitocôndrias/química , NAD/análise , NADP/análise , Oxirredução , Fosforilação Oxidativa
12.
Methods Mol Biol ; 2276: 259-270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060048

RESUMO

Mitochondrial dysfunction contributes to various injuries and diseases. A mechanistic understanding of how dysfunctional mitochondria modulates metabolism is of paramount importance. Three-dimensional (3D) optical cryo-imager is a custom-designed device that can quantify the volumetric bioenergetics of organs in small animal models. The instrument captures the autofluorescence of bioenergetics indices (NADH and FAD) from tissues at cryogenic temperature. The quantified redox ratio (NADH/FAD) is used as an optical indicator of mitochondrial redox state.


Assuntos
Flavina-Adenina Dinucleotídeo/análise , Imageamento Tridimensional/métodos , Rim/química , Mitocôndrias/química , NAD/análise , Imagem Óptica/métodos , Animais , Criopreservação , Metabolismo Energético , Flavina-Adenina Dinucleotídeo/metabolismo , Secções Congeladas , Rim/metabolismo , Rim/patologia , Mitocôndrias/metabolismo , Mitocôndrias/patologia , NAD/metabolismo , Oxirredução
13.
Methods Mol Biol ; 2276: 285-303, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060050

RESUMO

Changes to mitochondrial architecture are associated with various adaptive and pathogenic processes. However, quantification of changes to mitochondrial structures is limited by the yet unmet challenge of defining the borders of each individual mitochondrion within an image. Here, we describe a novel method for segmenting primary brown adipocyte (BA) mitochondria images. We describe a granular approach to quantifying subcellular structures, particularly mitochondria in close proximity to lipid droplets: peridroplet mitochondria. In addition, we lay out a novel machine-learning-based mitochondrial segmentation method that eliminates the bias of manual mitochondrial segmentation and improves object recognition compared to conventional thresholding analyses. By applying these methods, we discovered a significant difference between cytosolic and peridroplet BA mitochondrial H2O2 production and validated the machine-learning algorithm in BA via norepinephrine-induced mitochondrial fragmentation and comparing manual analyses to the automated analysis. This approach provides a high-throughput analysis protocol to quantify ratiometric probes in subpopulations of mitochondria in adipocytes.


Assuntos
Adipócitos Marrons/metabolismo , Ensaios de Triagem em Larga Escala/métodos , Processamento de Imagem Assistida por Computador/métodos , Gotículas Lipídicas/metabolismo , Aprendizado de Máquina , Mitocôndrias/metabolismo , Imagem Óptica/métodos , Adipócitos Marrons/citologia , Algoritmos , Humanos , Gotículas Lipídicas/química , Mitocôndrias/ultraestrutura
14.
Transl Vis Sci Technol ; 10(7): 11, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34110387

RESUMO

Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO) is a non-invasive imaging modality to investigate the human retina. This study compares FLIO lifetimes in different degenerative retinal diseases. Methods: Included were eyes with retinal pigment epithelium (RPE) and/or photoreceptor atrophy due to Stargardt disease (n = 66), pattern dystrophy (n = 18), macular telangiectasia type 2 (n = 49), retinitis pigmentosa (n = 28), choroideremia (n = 26), and geographic atrophy (n = 32) in age-related macular degeneration, as well as 37 eyes of 37 age-matched healthy controls. Subjects received Heidelberg Engineering FLIO, autofluorescence intensity, and optical coherence tomography imaging. Amplitude-weighted mean FLIO lifetimes (τm) were calculated and analyzed. Results: Retinal FLIO lifetimes show significant differences depending on the disease. Atrophic areas in geographic atrophy and choroideremia showed longest mean FLIO lifetimes. τm values within areas of RPE and outer nuclear layer atrophy were significantly longer than within areas with preserved outer nuclear layer (P < 0.001) or non-atrophic areas (P < 0.001). Conclusions: FLIO is able to contribute additional information regarding differences in chronic degenerative retinal diseases. Although it cannot replace conventional autofluorescence imaging, FLIO adds to the knowledge in these diseases and may help with the correct differentiation between them. This may lead to a more in-depth understanding of the pathomechanisms related to atrophy and types of progression. Translational Relevance: Differences between atrophic retinal diseases highlighted by FLIO may indicate separate pathomechanisms leading to atrophy and disease progression.


Assuntos
Telangiectasia Retiniana , Atrofia , Humanos , Oftalmoscopia , Imagem Óptica , Tomografia de Coerência Óptica
15.
Inorg Chem ; 60(12): 8826-8837, 2021 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-34060309

RESUMO

How to deliver nitric oxide (NO) to a physiological target and control its release quantitatively is a key issue for biomedical applications. Here, a water-soluble nitrosylruthenium complex, [(CH3)4N][RuCl3(5cqn)(NO)] (H5cqn = 5-chloro-8-quinoline), was synthesized, and its structure was confirmed with 1H NMR and X-ray crystal diffraction. Photoinduced NO release was investigated with time-resolved Fourier transform infrared and electron paramagnetic resonance (EPR) spectroscopies. The binding constant of the [RuCl3(5cqn)(NO)]- complex with human serum albumin (HSA) was determined by fluorescence spectroscopy, and the binding mode was identified by X-ray crystallography of the HSA and Ru-NO complex adduct. The crystal structure reveals that two molecules of the Ru-NO complex are located in the subdomain IB, which is one of the major drug binding regions of HSA. The chemical structures of the Ru complexes were [RuCl3(5cqn)(NO)]- and [RuCl3(Glycerin)NO]-, in which the electron densities for all ligands to Ru are unambiguously identified. EPR spin-trapping data showed that photoirradiation triggered NO radical generation from the HSA complex adduct. Moreover, the near-infrared image of exogenous NO from the nitrosylruthenium complex in living cells was observed using a NO-selective fluorescent probe. This study provides a strategy to design an appropriate delivery system to transport NO and metallodrugs in vivo for potential applications.


Assuntos
Complexos de Coordenação/metabolismo , Óxido Nítrico/metabolismo , Compostos de Rutênio/metabolismo , Albumina Sérica Humana/metabolismo , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Cristalografia por Raios X , Corantes Fluorescentes/química , Células HeLa , Humanos , Modelos Moleculares , Estrutura Molecular , Óxido Nítrico/química , Imagem Óptica , Processos Fotoquímicos , Compostos de Rutênio/química , Albumina Sérica Humana/química , Células Tumorais Cultivadas
16.
J Vis Exp ; (172)2021 06 05.
Artigo em Inglês | MEDLINE | ID: covidwho-1278530

RESUMO

As the COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to evolve, it has become evident that the presence of neutralizing antibodies against the virus may provide protection against future infection. Thus, as the creation and translation of effective COVID-19 vaccines continues at an unprecedented speed, the development of fast and effective methods to measure neutralizing antibodies against SARS-CoV-2 will become increasingly important to determine long-term protection against infection for both previously infected and immunized individuals. This paper describes a high-throughput protocol using vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein to measure the presence of neutralizing antibodies in convalescent serum from patients who have recently recovered from COVID-19. The use of a replicating pseudotyped virus eliminates the necessity for a containment level 3 facility required for SARS-CoV-2 handling, making this protocol accessible to virtually any containment level 2 lab. The use of a 96-well format allows for many samples to be run at the same time with a short turnaround time of 24 h.


Assuntos
Anticorpos Neutralizantes/análise , Anticorpos Antivirais/análise , Teste Sorológico para COVID-19/métodos , COVID-19/imunologia , Imagem Óptica/métodos , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Vacinas contra COVID-19/imunologia , Humanos , Testes de Neutralização , Vírus da Estomatite Vesicular Indiana/imunologia
17.
Sci Rep ; 11(1): 11778, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: covidwho-1258598

RESUMO

The COVID-19 pandemic has generated many concerns about cross-contamination risks, particularly in hospital settings and Intensive Care Units (ICU). Virus-laden aerosols produced by infected patients can propagate throughout ventilated rooms and put medical personnel entering them at risk. Experimental results found with a schlieren optical method have shown that the air flows generated by a cough and normal breathing were modified by the oxygenation technique used, especially when using High Flow Nasal Canulae, increasing the shedding of potentially infectious airborne particles. This study also uses a 3D Computational Fluid Dynamics model based on a Lattice Boltzmann Method to simulate the air flows as well as the movement of numerous airborne particles produced by a patient's cough within an ICU room under negative pressure. The effects of different mitigation scenarii on the amount of aerosols potentially containing SARS-CoV-2 that are extracted through the ventilation system are investigated. Numerical results indicate that adequate bed orientation and additional air treatment unit positioning can increase by 40% the number of particles extracted and decrease by 25% the amount of particles deposited on surfaces 45s after shedding. This approach could help lay the grounds for a more comprehensive way to tackle contamination risks in hospitals, as the model can be seen as a proof of concept and be adapted to any room configuration.


Assuntos
Microbiologia do Ar , COVID-19/transmissão , Tosse/virologia , Síndrome do Desconforto Respiratório/virologia , Aerossóis , Humanos , Unidades de Terapia Intensiva , Modelos Teóricos , Imagem Óptica , Ventilação/métodos
18.
Nat Struct Mol Biol ; 28(7): 573-582, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34158638

RESUMO

SARS-CoV-2 ORF3a is a putative viral ion channel implicated in autophagy inhibition, inflammasome activation and apoptosis. 3a protein and anti-3a antibodies are found in infected patient tissues and plasma. Deletion of 3a in SARS-CoV-1 reduces viral titer and morbidity in mice, suggesting it could be an effective target for vaccines or therapeutics. Here, we present structures of SARS-CoV-2 3a determined by cryo-EM to 2.1-Å resolution. 3a adopts a new fold with a polar cavity that opens to the cytosol and membrane through separate water- and lipid-filled openings. Hydrophilic grooves along outer helices could form ion-conduction paths. Using electrophysiology and fluorescent ion imaging of 3a-reconstituted liposomes, we observe Ca2+-permeable, nonselective cation channel activity, identify mutations that alter ion permeability and discover polycationic inhibitors of 3a activity. 3a-like proteins are found across coronavirus lineages that infect bats and humans, suggesting that 3a-targeted approaches could treat COVID-19 and other coronavirus diseases.


Assuntos
Microscopia Crioeletrônica , Nanoestruturas , SARS-CoV-2 , Proteínas Viroporinas/química , Proteínas Viroporinas/ultraestrutura , Animais , Cálcio/metabolismo , Quirópteros/virologia , Coronaviridae , Eletrofisiologia , Fluorescência , Humanos , Transporte de Íons , Lipossomos , Modelos Moleculares , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fases de Leitura Aberta , Imagem Óptica , Reprodutibilidade dos Testes , SARS-CoV-2/química , SARS-CoV-2/ultraestrutura , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/ultraestrutura , Proteínas Viroporinas/antagonistas & inibidores
19.
ACS Sens ; 6(6): 2270-2280, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34100604

RESUMO

Fluorescence guided surgery (FGS) has been highlighted in the clinical site for guiding surgical procedures and providing the surgeon with a real-time visualization of the operating field. FGS is a powerful technique for precise surgery, particularly tumor resection; however, clinically approved fluorescent dyes have often shown several limitations during FGS, such as non-tumor-targeting, low in vivo stability, insufficient emission intensity, and low blood-brain barrier penetration. In this study, we disclose a fluorescent dye complex, peptide, and protein for the targeted visualization of human glioblastoma (GBM) cells and tissues. Our noble triple receptor-targeting fluorescent complex (named BSA-OXN-SIWV) consists of (i) dipolar oxazepine dye (OXN), which has high stability, low cytotoxicity, bright fluorescence, and two-photon excitable, (ii) tetra-peptide (SIWV) for the targeting of the caveolin-1 receptor, and (iii) bovine serum-albumin (BSA) protein for the targeting of albondin (gp60) and secreted protein acidic and rich in cysteine receptor. The photophysical properties and binding mode of BSA-OXN-SIWV were analyzed, and the imaging of GBM cell lines and human clinical GBM tissues were successfully demonstrated in this study. Our findings hold great promise for the application of BSA-OXN-SIWV to GBM identification and the surgery at clinical sites, as a new FGS agent.


Assuntos
Glioblastoma , Animais , Bovinos , Glioblastoma/diagnóstico por imagem , Humanos , Imagem Óptica , Osteonectina , Peptídeos , Soroalbumina Bovina
20.
Anal Chim Acta ; 1169: 338605, 2021 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-34088368

RESUMO

Melanoma, the skin cancer with the highest mortality rate, can be diagnosed at the early stage by detecting unique biomarkers. Over-expressed tyrosinase has been confirmed by dozens of clinical studies as an independent factor to evaluate the malignancy of melanoma. Using Enteromorpha Prolifera as the raw material, herein we develop a novel fluorescent probe, ECDY, which can sensitively detect the tyrosinase activity in different types of cells. More importantly, melanoma cells can be specifically distinguished through cell lysate measurements as well as the whole-cell imaging technique. Mechanically, the tyrosine groups on the surface of ECDY can be specifically recognized by tyrosinase and further converted into dopaquinone, which consequently causes the intramolecular fluorescence quenching of the probe through photoinduced electron transfer (PET). Tyrosinase can be detected within 20 min in the solution, and the detection limit is as low as 0.067 U mL-1. For the in vitro demonstration, we evaluate the fluorescence decay of ECDY in response to the intracellular tyrosinase activity within the lysate of various cell lines, including non-cancerous, non-melanoma cancerous, and mouse melanoma ones. The experimental results verify that ECDY can accurately measure the apparent tyrosinase activity in different cell lines and detect melanoma cell lysate specifically. The confocal fluorescence imaging experiments further demonstrate that ECDY can distinguish melanoma cells from others significantly. We believe that ECDY provides a new strategy for the efficient detection of tyrosinase and melanoma cells, and is expected to apply as a clinical diagnosis platform.


Assuntos
Melanoma , Monofenol Mono-Oxigenase , Animais , Transporte de Elétrons , Corantes Fluorescentes , Camundongos , Monofenol Mono-Oxigenase/metabolismo , Imagem Óptica
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