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Brown adipose tissue (BAT), characterized by the presence of numerous mitochondria, plays a key role in metabolism and energy expenditure. Accurately reporting the presence and activation of BAT is beneficial to study obesity, diabetes, and other metabolic disorders. Near-infrared (NIR) fluorescence imaging has the advantages of high sensitivity, non-radioactivity, and simple operation. However, most NIR probes for BAT imaging exhibit small Stokes shifts, which may lead to self-quenching, reducing the signal-to-noise ratio, and introducing cross-talk. Herein, we rationally designed and synthesized a series of hemicyanine-based NIR fluorescent probes HCYBAT-1-3. Among them, HCYBAT-1 demonstrated favorable properties such as near-infrared emission (776 nm), large Stokes shift (139 nm), good biocompatibility and specific mitochondrial targeting (Pearson's colocalization coefficient of 0.87). Meanwhile, HCYBAT-1 was successfully employed to differentiate BAT from white adipose tissue (WAT). Quantitative analysis of NIR fluorescent images showed that HCYBAT-1 could be used for real-time monitoring of BAT activation in mice stimulated by norepinephrine (NE) and cold exposure. Overall, probe HCYBAT-1 showcased its efficacy in non-invasive evaluation of BAT metabolism in vivo with high selectivity and sensitivity.
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Tecido Adiposo Marrom , Corantes Fluorescentes , Imagem Óptica , Tecido Adiposo Marrom/diagnóstico por imagem , Tecido Adiposo Marrom/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Animais , Camundongos , Imagem Óptica/métodos , Carbocianinas/química , Carbocianinas/síntese química , Camundongos Endogâmicos C57BL , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Mitocôndrias/metabolismo , MasculinoRESUMO
In this work, we design a new approach for digital counting-based protein kinase activity assay by using plasmonic nanoparticle-assisted single-molecule dynamic binding. This method breaks the concentration-dependent limitation in single-molecule detection and displays good selectivity and sensitivity with a detection limit as low as 0.0001 U mL-1 for the protein kinase A (PKA) assay, which would find broad applications in drug screening and medical investigations.
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Nanopartículas Metálicas , Nanopartículas Metálicas/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/química , Ouro/química , Limite de DetecçãoRESUMO
Dynamic membrane contacts between lipid droplets (LDs) and mitochondria play key roles in lipid metabolism and energy homeostasis. Understanding the dynamics of LDs under energy stimulation is thereby crucial to disclosing the metabolic mechanism. Here, the reversible interactions between LDs and mitochondria are tracked in real-time using a robust LDs-specific fluorescent probe (LDs-Tags). Through tracking the dynamics of LDs at the single-particle level, spatiotemporal heterogeneity is revealed. LDs in starved cells communicate and integrate their activities (i.e., lipid exchange) through a membrane contact site-mediated mechanism. Thus the diffusion is intermittently alternated between active and confined states. Statistical analysis shows that the translocation of LDs in response to starvation stress is non-Gaussian, and obeys nonergodic-like behavior. These results provide deep understanding of the anomalous diffusion of LDs in living cells, and also afford guidance for rationally designing efficient transporter.
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Exploring the surface-capturing and releasing processes of nanocargo on the living cell membrane is critical for understanding the membrane translocation process. In this work, we achieve total internal reflection scattering (TIRS) illumination on a commercial dark-field optical microscope without the introduction of any additional optical components. By gradually reducing the diaphragm size in the excitation light path, the angle of the incident beam can be well manipulated. Under optimal conditions, the excitation light can be totally reflected at the glass/water interface, resulting in a thin layer of evanescent field for TIRS illumination. Due to the exponential decay feature of the evanescent field, the displacement of the nanocargo along the vertical direction can be directly resolved in the intensity track. With this method, we selectively monitor the dynamics of the transferrin-modified nanocargo on the living cell membrane. Transition between confined diffusion and long-range searching is involved in the binding site recognition process, which exhibits non-Gaussian and nonergodic-like behavior. More interestingly, 2D fast sliding and 3D hopping motions are also distinguished on the fluidic cell membrane, which is essentially modulated by the strength of ligand-receptor interactions, as revealed by the free-energy profiles. These heterogeneous and dynamic interactions together control the diffusion mode of the nanocargo on the lipid membrane and, thus, determine the cellular translocation efficiency.
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Microscopia , Ligantes , Membrana Celular/metabolismoRESUMO
The sensitive and accurate detection of biomarkers plays an important role in clinical diagnosis and drug discovery. Currently, amplification-based methods for biomarker detection are widely explored. However, the key challenges of these methods are limited reproducibility and high background noise. To overcome these limitations, we develop a robust plasmonic nanoparticle-coupled single-molecule kinetic fingerprinting (PNP-SMKF) method to achieve ultrasensitive detection of protein kinase A (PKA). Transient binding of a short fluorescent probe with the genuine target produces a distinct kinetic signature that is completely different from that of the background signal, allowing us to recognize PKA sensitively. Importantly, integrating a plasmonic nanoparticle efficiently breaks the concentration limit of the imager strand for single-molecule imaging, thus achieving a much faster imaging speed. A limit of detection (LOD) of as low as 0.0005 U/mL is readily realized. This method holds great potential as a versatile platform for enzyme detection and inhibitor screening in the future.
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Técnicas Biossensoriais , Nanopartículas , Reprodutibilidade dos Testes , Nanotecnologia , Biomarcadores , Corantes Fluorescentes/química , Limite de Detecção , Técnicas Biossensoriais/métodosRESUMO
The photooxygenation of amyloid-ß (Aß) protein is considered a promising strategy against Alzheimer's disease (AD). The inhibition of Aß aggregation or depolymerization of Aß aggregates can effectively alleviate and improve the condition of AD. Herein, we report a series of "off-on" near-infrared quinolinium photosensitizers (QM20-QM22) based on D-π-A structures using a target-sensing catalyst activation (TaSCAc) strategy. They exhibit turn-on fluorescence when bonded to Aß aggregates and generate singlet oxygen to achieve the specific imaging and photooxygenation of Aß aggregates, leading to attenuated Aß aggregates, enhancing their clearance through the microglial lysosomal pathway, decreasing their neurotoxicity. This study will shed light on the development of the photooxygenation of misfolded proteins for the treatment of neurodegenerative diseases.
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Doença de Alzheimer , Medicina de Precisão , Humanos , Peptídeos beta-Amiloides/química , Doença de Alzheimer/diagnóstico por imagem , Doença de Alzheimer/metabolismo , Oxigênio Singlete/química , Fármacos Fotossensibilizantes/farmacologiaRESUMO
Lipid droplets (LDs), as crucial organelles, play a significant role in some physiological processes. Monitoring the concentration of LDs and dynamic behaviors between LDs and other organelles during some physiological processes is important for studying their biological function and medical diagnosis. Herein, we report a series of aggregation-induced emission (AIE) probes AIE-Cbz-LD-Cn (n = 1, 3, 5, 7, OMe) based on the conjugation of quinoline-malononitrile (QM) and carbazole for tracking the dynamic changes of LDs and studying the association between LDs and lysosome/endoplasmic reticulum (ER). To our great delight, AIE-Cbz-LD-C3, AIE-Cbz-LD-C5, and AIE-Cbz-LD-C7 could aggregate in LDs accurately and light up the LDs with good photostability. Among them, AIE-Cbz-LD-C7 was used to visualize the interplay between LDs and lysosomes during lipophagy due to the excellent LD-specificity. We also succeeded in tracking the number of newborn LDs generated near the endoplasmic reticulum regions revealing that the number increased considerably during ferroptosis by using AIE-Cbz-LD-C7, which supplies useful evidence for the hypothesis that LDs generate from the ER. We expect the probe AIE-Cbz-LD-C7 would be a practical tool for tracking the physiological and pathological processes contacted with LDs.
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Ferroptose , Quinolinas , Autofagia , Carbazóis , Humanos , Recém-Nascido , Gotículas LipídicasRESUMO
The advent of macrocycle-based supramolecular chemistry can offer powerful strategies for regulating vital bioactivities in living systems and bring about emerging technology in biomedical science. Herein, we construct a supra-biomacromolecular nanosystem involving microtubules, cell-permeable porphyrins, and antimitotic peptide-decorated permethyl-ß-cyclodextrins for promoting cell apoptosis in a cooperative manner. Through specific polypeptide-tubulin recognition, cyclodextrin moieties are capable of anchoring to the tubulin surface and providing abundant hydrophobic microenvironments to accommodate the photosensitive porphyrins. Consequently, spherical tubulin aggregates are formed, and reactive oxygen species can be efficiently generated via the host-guest complexation. The combined usage of complexation-promoted photodynamic efficacy and tubulin aggregation gives more serious cell apoptosis under light irradiation in vitro and in vivo. To be envisioned, this supramolecularly enhanced photodynamic performance together with controlled aggregation of natural biomacromolecules may be developed as an innovative approach to improve the therapeutic potency against many diseases.
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Antimitóticos , Ciclodextrinas , Neoplasias , Porfirinas , beta-Ciclodextrinas , Ciclodextrinas/química , Humanos , Neoplasias/tratamento farmacológico , Peptídeos/farmacologia , Porfirinas/química , Porfirinas/farmacologia , Medicina de Precisão , Espécies Reativas de Oxigênio , Tubulina (Proteína) , Microambiente Tumoral , beta-Ciclodextrinas/químicaRESUMO
Self-propelled nanomotors have shown enormous potential in biomedical applications. Herein, we report on a nanozyme-powered cup-shaped nanomotor for active cellular targeting and synergistic photodynamic/thermal therapy under near-infrared (NIR) laser irradiation. The nanomotor is constructed by the asymmetric decoration of platinum nanoparticles (PtNPs) at the bottom of gold nanocups (GNCs). PtNPs with robust peroxidase- (POD-) like activity are employed not only as propelling elements for nanomotors but also as continuous O2 generators to promote photodynamic therapy via catalyzing endogenous H2O2 decomposition. Owing to the Janus structure, asymmetric propulsion force is generated to trigger the short-ranged directional diffusion, facilitating broader diffusion areas and more efficient cellular searching and uptake. This cascade strategy combines key capabilities, i.e., endogenous substrate-based self-propulsion, active cellular targeting, and enhanced dual-modal therapy, in one multifunctional nanomotor, which is crucial in advancing self-propelled nanomotors towards eventual therapeutic agents.
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Bimetallic nanostructures are a promising candidate for plasmon-driven photocatalysis. However, knowledge on the generation and utilization of hot carriers in bimetallic nanostructures is still limited. In this work, we explored Pt position-dependent photocatalytic properties of bimetallic Au nanobipyramids (Au NBPs) with single-molecule fluorescence imaging. Compared with all-deposited core-shell nanostructures (aPt-Au NBPs), single-molecule imaging and simulation results show that the end-deposited bimetallic nanostructures (ePt-Au NBPs) can maintain a strong electromagnetic (EM) field and further promote the generation and transfer of energetic hot electrons for photocatalysis. Even though the Pt lattice is more stable than Au, the strong EM field at the sharp tips can boost lattice vibration, where enhanced spontaneous surface restructuring for active reaction site generation takes place. Significantly enhanced catalytic efficiency from ePt-Au NBPs is observed in contrast to that of Au NBPs and aPt-Au NBPs. These microscopic evidences offer valuable guidelines to design plasmon-based photocatalysts, particularly for bimetallic nanostructures.
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[This corrects the article DOI: 10.1039/C8SC01828F.].
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Anti-Aß therapy has dominated clinical trials for the prevention and treatment of Alzheimer's disease (AD). However, suppressing Aß aggregation and disintegrating mature fibrils simultaneously remains a great challenge. In this work, we developed a new strategy using a charged tubular supramolecule (CTS) with pillar[5]arene as the backbone and modifying amino and carboxyl groups at the tubular terminals (noted as CTS-A, CTS-A/C, and CTS-C, respectively) to suppress Aß fibrillation for the first time. According to the spectroscopic and microscopic characterizations, Aß40 fibrillation can be efficiently suppressed by CTS-A in a very low inhibitor:peptide (I:P) molar ratio (1:10). A greatly alleviated cytotoxic effect of Aß peptides after the inhibition or disaggregation process is further disclosed. The well-organized supramolecular structure drives multivalent interaction and gains enhanced efficiency on amyloid fibrillar modulation. These results open a new path for the design of supramolecules in the application of AD treatment.
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Doença de Alzheimer , Peptídeos beta-Amiloides , Amiloide/química , Peptídeos beta-Amiloides/química , Humanos , Fragmentos de PeptídeosRESUMO
Small extracellular vesicles (sEVs) play a key role in intercellular communication. Cargo molecules carried by sEVs may affect the phenotype and function of recipient cells. Epithelial cancer cell-derived sEVs, particularly those enriched in CD151 or tetraspanin8 (TSPAN8) and associated integrins, promote tumour progression. The mechanism of binding and modulation of sEVs to recipient cells remains elusive. Here, we used genetically engineered breast cancer cells to derive TSPAN8-enriched sEVs and evaluated the impact of TSPAN8 on target cell membrane's diffusion and transport properties. The single-particle tracking technique showed that TSPAN8 significantly promoted sEV binding via confined diffusion. Functional assays indicated that the transgenic TSPAN8-sEV cargo increased cancer cell motility and epithelial-mesenchymal transition (EMT). In vivo, transgenic TSPAN8-sEV promoted uptake of sEVs in the liver, lung, and spleen. We concluded that TSPAN8 encourages the sEV-target cell interaction via forced confined diffusion and significantly increases cell motility. Therefore, TSPAN8-sEV may serve as an important direct or indirect therapeutic target.
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Neoplasias da Mama/metabolismo , Comunicação Celular/genética , Vesículas Extracelulares/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Transdução de Sinais/genética , Baço/metabolismo , Tetraspaninas/metabolismo , Animais , Neoplasias da Mama/patologia , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Técnicas de Silenciamento de Genes/métodos , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Tetraspanina 24/metabolismo , Tetraspaninas/genética , TransfecçãoRESUMO
Fluorescent chemosensors are powerful imaging tools in the fields of life sciences and engineering. Based on the principle of supramolecular chemistry, indicator displacement assay (IDA) provides an alternative approach for constructing and optimizing chemosensors, which has the advantages of simplicity, tunability, and modularity. However, the application of IDA in bioimaging continues to face a series of challenges, including interfering signals, background noise, and inconsistent spatial location. Accordingly, we herein report a supramolecular bioimaging strategy of Förster resonance energy transfer (FRET)-assisted IDA by employing macrocyclic amphiphiles as the operating platform. By merging FRET with IDA, the limitations of IDA in bioimaging were addressed. As a proof of concept, the study achieved mitochondria-targeted imaging of adenosine triphosphate in live cells with signal amplification. This study opens a non-covalent avenue for bioimaging with advancements in tunability, generality, and simplicity, apart from the covalent approach.
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Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Indicadores e Reagentes/química , Células Hep G2 , Humanos , Substâncias Macromoleculares/análise , Espectrometria de FluorescênciaRESUMO
A challenge for sensors targeting specific enzymes of interest in their native environment for direct imaging is that they rationally exploit a highly selective fluorescent probe with a high binding affinity to provide real-time detection. Immunohistochemical staining, proteomic analysis, or recent enzymatic fluorescent probes are not optimal for tracking specific enzymes directly in living cells. Herein, we introduce the concept of designing a highly effective fluorescent probe (BVQ1814) targeting phosphodiesterase 10A with a highly potent affinity and a >1000-fold subfamily selectivity by gaining insights into the three-dimensional structural information of the active site of the catalytic pocket. BVQ1814 showed an outstanding binding affinity for PDE10A in vitro and specifically detected PDE10A in living cells, indicating that most PDE10A was probably distributed in the lysosomes. We validated the PDE10A distribution in stable mCherry-PDE10A-overexpressing HepG2 cells. This probe delineated the profile of PDE10A in tissue sections and exhibited a remarkable therapeutic effect as a PDE10A inhibitor for treating pulmonary arterial hypertension. This concept will open up a new avenue for designing a highly effective fluorescent probe for tracking receptor proteins by taking full advantage of the structural information in the ligand-binding pocket of the target of interest.
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Corantes Fluorescentes/química , Lisossomos/química , Inibidores de Fosfodiesterase/química , Diester Fosfórico Hidrolases/química , Catálise , Domínio Catalítico , Células HeLa , Células Hep G2 , Humanos , Lisossomos/ultraestrutura , Imagem Óptica , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/ultraestrutura , Ligação Proteica , Conformação Proteica , ProteômicaRESUMO
Plasmonic hotspots can enhance hot charge carrier generation, offering new opportunities for improving the photocatalytic activity. In this work, eight types of heteronanostructures are synthesized by selectively depositing catalytic metals at the different sites of highly asymmetric Au nanocups for the photocatalytic oxidation of o-phenylenediamine. The oxidation of this molecule has so far mainly relied on the use of H2O2 as an oxidizing agent in the presence of an appropriate catalyst. The photocatalytic oxidation under visible light has not been reported before. The Au nanocups with AgPt nanoparticles grown at the opening edge and bottom exhibit the highest photocatalytic activity. The generated hot electrons and holes both participate in the reaction. The hot carriers from the interband and intraband transitions are both utilized. The optimal catalyst shows a favorable activity even under room light. Simulations reveal that the profound electric field enhancement at the hotspots boosts the hot-carrier density in the catalytic nanoparticles, explaining the overwhelming photocatalytic activity of the optimal catalyst.
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Development of intelligent and multifunctional nanoparticle for the diagnosis and treatment of cancer has drawn great attention recently. In this work, we design a smart two-dimensional (2D) supraparticle for tumor targeted magnetic resonance imaging (MRI)/photothermal imaging (PTI) and chemo/photothermal therapy (PTT). Methods: The nanoparticle consists of a manganese dioxide (MnO2) nanosheet coated gold nanorod (GNR) core (loading with chemotherapeutics doxorubicin (DOX)), and cancer cell membrane shell (denoted as CM-DOX-GMNPs). Decoration of cell membrane endows the nanoparticle with greatly improved colloidal stability and homotypic cancer cell targeting ability. Once the nanoparticles enter tumor cells, MnO2 nanosheets can be etched to Mn2+ by glutathione (GSH) and acidic hydrogen peroxide (H2O2) in the cytosol, leading to the release of DOX. Meanwhile, stimuli dependent releasing of Mn2+ can act as MRI contrast agent for tumor diagnosis. Illumination with near-infrared (NIR) light, photothermal conversion effect of GNRs can be activated for synergistic cancer therapy. Results:In vivo results illustrate that the CM-DOX-GMNPs display tumor specific MRI/PTI ability and excellent inhibition effect on tumor growth. Conclusion: This bioinspired nanoparticle presents an effective and intelligent approach for tumor imaging and therapy, affording valuable guidance for the rational design of robust theranostics nanoplatform.
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Nanopartículas/química , Neoplasias/terapia , Medicina de Precisão , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Membrana Celular , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Humanos , Imageamento por Ressonância Magnética , Neoplasias/diagnóstico por imagem , Terapia FototérmicaRESUMO
Alkaline phosphatase (ALP) as a necessary hydrolase in phosphate metabolism is closely related to various diseases. Ultrasensitive detection of ALP with a convenient and sensitive method is of fundamental importance. In this work, a fluorescence resonance energy transfer (FRET)-based single-particle enumeration (SPE) method is proposed for the quantitative analysis of ALP. This strategy is based on the effective fluorescence suppression by a polydopamine (PDA) shell on the surface of semiconducting polymer nanoparticles (SPNs). PDA with broadband absorption in the UV-vis region can serve as an excellent quencher for SPNs. However, ascorbic acid (AA), the product of the hydrolysis of 2-phosphate-l-ascorbic acid trisodium salt (AAP) in the presence of ALP, can effectively inhibit the self-polymerization of dopamine (DA) to form a PDA layer. Therefore, ALP can be accurately quantified by counting the concentration-related fluorescent particles in the fluorescence image. A linear range from 0.031 to 12.4 µU mL-1 and a limit-of-detection (LOD) of 0.01 µU mL-1 for ALP determination are achieved. The spiked recoveries for ALP determination in a human serum sample are between 90% and 108% with RSD less than 3.1%. In summary, this convenient and sensitive approach proposed here provides promising prospects for ALP detection in a complex biological matrix.
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Fosfatase Alcalina , Nanopartículas , Fosfatase Alcalina/metabolismo , Transferência Ressonante de Energia de Fluorescência , Humanos , Limite de Detecção , PolímerosRESUMO
Fibrinogen participates in many physiological processes and is a biomarker for a variety of diseases. On this account, the development of a sensitive method for fibrinogen assay is particularly important. Herein, we demonstrate a new color-coded single-particle detection (SPD) method for fibrinogen detection by using platelet membrane-coated fluorescent polystyrene nanoparticles (PNPs) as the probes. Due to the specific interactions between fibrinogen and integrin receptors on platelet membranes, PNPs can form aggregated structures in the presence of fibrinogen. Therefore, colocalization events between green and red PNPs and the corresponding Pearson's correlation coefficient vary with the concentrations of fibrinogen. The sensing ability shows a linear range of 30-300 µg mL-1 and a limit of detection (LOD) of 3.9 µg mL-1 (11.3 nM) for fibrinogen detection. Moreover, it has been validated that the proposed biosensor can selectively detect fibrinogen and shows a good performance in real sample applications.
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Fibrinogênio , Nanopartículas , Plaquetas , Limite de Detecção , PoliestirenosRESUMO
A series of glycoside-peptide conjugates were prepared by engineering at the N-terminus of the natural peptide gramicidin A. The conjugate containing galactose moiety formed a unimolecular transmembrane channel and mediated ion transport to induce apoptosis of cancer cells. More importantly, it exhibited liver cancer cell-targeting behavior due to the galactose-asialoglycoprotein receptor recognition.