Ligand-Receptor Interaction Triggers Hopping and Sliding Motions on Living Cell Membranes.

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.

Burst of hopping trafficking correlated reversible dynamic interactions between lipid droplets and mitochondria under starvation.

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.

Nanozyme-Triggered Cascade Reactions from Cup-Shaped Nanomotors Promote Active Cellular Targeting.

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.

Shining at the Tips: Anisotropic Deposition of Pt Nanoparticles Boosting Hot Carrier Utilization for Plasmon-Driven Photocatalysis.

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.

Charged Tubular Supramolecule Boosting Multivalent Interactions for the Drastic Suppression of Aß Fibrillation.

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.

Supramolecular Bioimaging through Signal Amplification by Combining Indicator Displacement Assay with Förster Resonance Energy Transfer.

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.

Cell membrane coated smart two-dimensional supraparticle for in vivo homotypic cancer targeting and enhanced combinational theranostics.

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.

Gramicidin A-based unimolecular channel: cancer cell-targeting behavior and ion transport-induced apoptosis.

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.

Length-Dependent Distinct Cytotoxic Effect of Amyloid Fibrils beyond Optical Diffraction Limit Revealed by Nanoscopic Imaging.

Fibrillar species have been proposed to play an essential role in the cytotoxicity of amyloid peptide and the pathogenesis of neurodegenerative diseases. Discrimination of Aß aggregates in situ at high spatial resolution is therefore significant for the development of a therapeutic method. In this work, we adopt a rhodamine-like structure as luminescent centers to fabricate carbonized fluorescent nanoparticles (i.e., carbon dots, RhoCDs) with tunable emission wavelengths from green to red and burst-like photoblinking property for localization-based nanoscopic imaging. These RhoCDs contain lipophilic cationic and carboxyl groups which can specifically bind with Aß1-40 aggregates via electrostatic interaction and hydrogen bonding. According to the nanoscopic imaging in the Aß1-40 fibrillation and disaggregation process, different types of Aß1-40 aggregates beyond the optical diffraction limit have been disclosed. Additionally, length-dependent toxic effect of Aß1-40 aggregates beyond the optical diffraction limit is unveiled. Short amyloid assemblies with length of 187 ± 3.9 nm in the early stage are more toxic than the elongated amyloid fibrils. Second, disassembly of long fibrils into short species by Gramicidin S (GS-2) peptide might enhance the cytotoxicity. These results lay the foundation to develop functional fluorophore for nanoscopic imaging and also provide deep insight into morphology-dependent cytotoxicity from amyloid peptides.

Artificial Aquaporin That Restores Wound Healing of Impaired Cells.

Artificial aquaporins are synthetic molecules that mimic the structure and function of natural aquaporins (AQPs) in cell membranes. The development of artificial aquaporins would provide an alternative strategy for treatment of AQP-related diseases. In this report, an artificial aquaporin has been constructed from an amino-terminated tubular molecule, which operates in a unimolecular mechanism. The artificial channel can work in cell membranes with high water permeability and selectivity rivaling those of AQPs. Importantly, the channel can restore wound healing of the cells that contain function-lost AQPs.

Deep Red Blinking Fluorophore for Nanoscopic Imaging and Inhibition of ß-Amyloid Peptide Fibrillation.

Deposition and aggregation of ß-amyloid (Aß) peptides are demonstrated to be closely related to the pathogenesis of Alzheimer's disease (AD). Development of functional molecules capable of visualizing Aß1-40 aggregates with nanoscale resolution and even modulating Aß assembly has attracted great attention recently. In this work, we use monocyanine fluorophore as the lead structure to develop a set of deep red carbazole-based cyanine molecules, which can specifically bind with Aß1-40 fibril via electrostatic and van der Waals interactions. Spectroscopic and microscopic characterizations demonstrate that one of these fluorophores, (E)-1-(2-(2-methoxyethoxy)ethyl)-4-(2-(9-methyl-9H-carbazol-3-yl)vinyl) quinolinium iodide (me-slg) can bind to Aß1-40 aggregates with strong fluorescence enhancement. The photophysical properties of me-slg at the single-molecule level, including low "on/off" duty cycle, high photon output, and sufficient switching cycles, enable real-time nanoscopic imaging of Aß1-40 aggregates. Morphology-dependent toxic effect of Aß1-40 aggregates toward PC12 cells is unveiled from in situ nanoscopic fluorescence imaging. In addition, me-slg displays a strong inhibitory effect on Aß1-40 fibrillation in a low inhibitor-protein ratio (e.g., I:P = 0.2). A noticeably reduced cytotoxic effect of Aß1-40 after the addition of me-slg is also confirmed. These results afford promising applications in the design of a nanoscopic imaging probe for amyloid fibril as well as the development of inhibitors to modulate the fibrillation process.

Recent advances of plasmonic nanoparticle-based optical analysis in homogeneous solution and at the single-nanoparticle level.

Plasmonic nanoparticles with special localized surface plasmon resonance (LSPR) characters have been widely applied for optical sensing of various targets. With the combination of single nanoparticle imaging techniques, dynamic information of reactions and biological processes is obtained, facilitating the deep understanding of their principle and design of outstanding nanomaterials. In this review, we summarize the recently adopted optical analysis of diverse analytes based on plasmonic nanoparticles both in homogeneous solution and at the single-nanoparticle level. A brief introduction of LSPR is first discussed. Colorimetric and fluorimetric homogeneous detection examples by using different sensing mechanisms and strategies are provided. Single plasmonic nanoparticle-based analysis is concluded in two aspects: visualization of chemical reactions and understanding of biological processes. The basic sensing mechanisms and performances of these systems are introduced. Finally, this review highlights the challenges and future trend of plasmonic nanoparticle-based optical analysis systems.

Gold nanoparticles enumeration with dark-field optical microscope for the sensitive glycoprotein sandwich assay.

Protein glycosylation is an important post-translational modification and glycoproteins are associated with many crucial metabolic progresses of life. In order to detect glycoproteins sensitively, we propose a gold nanoparticles (GNPs) enumeration method based on boronate affinity sandwich system, which is constructed between the boronic acid polymer functionalized magnetic nanoparticles (Fe3O4@MPS@VPBA NPs) and 4-mercaptophenylboronic acid modified GNPs (GNPs-MPBA) by the targeted glycoproteins as the linker. Therefore, the sandwich complex is formed, resulting in the decrease of GNPs-MPBA counts in the solution. Based on the dark-field microscope (DFM) imaging technique, the sensitive GNPs enumeration assay is developed for glycoproteins quantitation. Immunoglobulin (IgG), as one of the important glycoproteins, is introduced to evaluate the proposed method. A low detection limit of 1.22 ng mL-1 for IgG analysis is obtained. The result indicates that the proposed GNPs enumeration method offers a simple, effective, label-free and highly sensitive strategy without signal amplification. It also possesses great potential for various target molecules determination at the single-particle level in the future.

Single-Particle Tracking with Scattering-Based Optical Microscopy.

Different from traditional ensemble measurement methods, single-particle tracking (SPT) is a powerful approach to study the distribution of dynamic processes in a complex environment, providing crucial information from individual objects. This Feature summarizes the optical microscopic techniques and data analysis methods for scattering-based SPT. Some essential SPT-based applications within the cell are also delineated.

Mitochondrion-Specific Blinking Fluorescent Bioprobe for Nanoscopic Monitoring of Mitophagy.

Dynamic changes of mitochondrial morphology play an important role in cellular metabolism. Real-time monitoring mitochondrial ultrastructural dynamics at nanometer-scale resolution is crucially desired for further understanding of the mitochondria-based cellular function. In this work, we introduce a fluorescent carbon dot, which can selectively target mitochondria in live cells (named as MitoCD). MitoCD can effectively accumulate in mitochondria regardless of the decrease or vanishing of mitochondrial membrane potential (MMP), enabling the exploration of MMP-independent mitochondrial process. Moreover, the MitoCD is a thiol-based reaction-free probe that target mitochondria without consuming the thiol groups from mitochondrial proteins. Additionally, the MitoCD possesses good photophysical properties under physiological conditions, such as burst-like blinking, high photon counts, and low "on"/"off" ratio, which are specifically suitable for localization-based nanoscopic imaging. According to the optical microscopic imaging results, dynamical fission and fusion processes from mitochondria have been observed in live cells. During mitophagy, it is found that reticular formation of the mitochondria gradually collapsed, and then a portion of mitochondria split and vanished. Owing to the attractive biological and special photophysical properties, this probe displays promising application in a variety of super-resolution based biological studies and will provide deep insight in mitochondrial metabolism.

Cell Membrane-Coated Porphyrin Metal-Organic Frameworks for Cancer Cell Targeting and O2-Evolving Photodynamic Therapy.

Photodynamic therapy (PDT) has attracted great attention as an alternative tumor treatment method. Unfortunately, it suffers from some limitations like poor targeting capability and insufficient therapeutic efficiency caused by tumor hypoxia. In this work, we introduce a novel O2-evolving PDT nanoparticle for homologous cancer cell targeting as well as dual-mode imaging [i.e., magnetic resonance imaging (MRI) and fluorescence imaging]. Specifically, the nanostructure consists of a MnO2 nanosheet-coated metal-organic framework core and cancer cell membrane shell (defined as CM-MMNPs). The MnO2 layer displays H+ and H2O2 responsiveness, which can produce O2 to enhance O2-mediated singlet oxygen (1O2) generation for PDT. Moreover, the resulted Mn2+ can also be used as an optimal MRI contrast agent. The introduction of cell membrane and membrane proteins endow the CM-MMNPs with good stability and integrity in the process of cellular endocytosis, as well as strong homologous cell-targeting ability. This multifunctional nanoparticle has the potential to overcome the hypoxia of cancer cells in PDT, and provides a new paradigm for tumor targeting, detection, and therapy, which is promising for biomedical applications in the future.

Single-Particle LRET Aptasensor for the Sensitive Detection of Aflatoxin B1 with Upconversion Nanoparticles.

Contamination of foods and feeds by aflatoxins is a universal yet serious problem all over the world. Particularly, aflatoxin B1 (AFB1) is the most primary form and readily leads to terrible damages to human health. In this work, we construct a sensitive aptasensor based on single-particle detection (SPD) to analyze AFB1 in peanut samples with luminescence resonance energy transfer (LRET) between the aptamer-modified upconversion nanoparticles (UCNPs-aptamer) and gold nanoparticles (GNPs). The UCNP-aptamer plays as the luminescence donor, while GNP acts as the energy acceptor. In the absence of AFB1, GNPs would adsorb onto the surface of UCNPs-aptamer because of the association between aptamers and GNPs, leading to luminescence quenching. However, the luminescence of UCNPs-aptamer is recovered gradually in the presence of AFB1, because the aptamers possess stronger affinity toward AFB1 than GNPs. Through statistically counting the number of luminescent particles on the glass slide surface, the concentration of AFB1 in solution is accurately determined. The linear dynamic range for AFB1 detection is from 3.13 to 125.00 ng/mL. The limit-of-detection (LOD) is 0.17 ng/mL, which is much lower than the allowable concentration in foods. As a result, this method would provide promising application for the sensitive detection of AFB1 in foods and feeds, which might make a meaningful contribution to food safety and public health in the future.

Laser illumination-induced dramatic catalytic activity change on Au nanospheres.

Understanding morphology dependent catalytic kinetics from a single nanoparticle plays a significant role in the development of robust nano-catalysts with high efficiency. Unfortunately, detailed knowledge of the morphology dependent catalytic properties of single nanoparticles after shape transitions is lacking. In this work, the distinct catalytic properties of a single gold nanoparticle (GNP) after symmetry breaking were disclosed at the single-particle level for the first time. The morphology of the spherical GNP was elongated into a rod shape (i.e., gold nanorod, GNR) with a tightly focused Gaussian laser beam based on the photothermal effect. By using the fluorogenic oxidation reaction (i.e., amplex red to resorufin) as a model reaction, noticeable variation in catalytic efficiency after the shape modulation process was found at the single-particle level. The GNP displays noticeably higher catalytic efficiency which might be ascribed to the heterogeneous lattice structure on the particle surface as confirmed by transmission electron microscopy (TEM) characterization. Rearrangement of surface atoms after shape modulation normally generates a more ordered crystal structure, resulting in a lower surface energy for catalytic reaction. However, both of these nanoparticles still exhibit dynamic activity fluctuation in a temporal dependent route, indicating a distinct spontaneous dynamic surface restructuring process. These kinetic evidences might facilitate the development nanoparticle-based heterogeneous catalysts, particularly based on the morphology effect.

Single-particle enumeration-based ultrasensitive enzyme activity quantification with fluorescent polymer nanoparticles.

Acetylcholinesterase (AChE) plays a vital role in nerve conduction through rapidly hydrolyzing the neurotransmitter acetylcholine (ACh) and is correlated with Alzheimer's disease. In this work, a label-free single-particle enumeration (SPE) method for the quantitative detection of acetylcholinesterase (AChE) activity is developed. The design is based on the fluorescence resonance energy transfer (FRET) between fluorescent conjugated polymer nanoparticles (FCPNPs) and MnO2 nanosheets. The fluorescence of FCPNPs can be effectively quenched by MnO2 nanosheets via hydrogen bonding interaction. In the presence of acetylcholinesterase (AChE), acetylthiocholine (ATCh) could be hydrolyzed to thiocholine (TCh), which can reduce MnO2 to Mn2+ and trigger the decomposition of MnO2 nanosheets. As a result, the fluorescence of FCPNPs is restored. Taking advantage of the superior brightness and stable fluorescence emission from individual FCPNPs, the accurate quantification of AChE is achieved by statistically counting the fluorescent particles on the glass slide surface. A linear range from 5 to 1600 µU mL-1 is obtained for AChE assay and the limit-of-detection (LOD) is 1.02 µU mL-1, which is far below the spectroscopic measurements in bulk solution. In the human serum sample, satisfactory recovery efficiencies are determined in a range of 91.0%-103.0%. Furthermore, pesticide carbaryl as an inhibitor of AChE activity was detected. The LOD is 1.12 pg mL-1 with linear responses ranging from 5 to 300 pg mL-1, which demonstrates the feasibility of this approach for AChE inhibitor screening. As a consequence, the label-free SPE-based method affords a promising platform for the sensitive detection of target molecules in the future.

Efficient Modulation of ß-Amyloid Peptide Fibrillation with Polymer Nanoparticles Revealed by Super-Resolution Optical Microscopy.

ß-Amyloid peptide (Aß) aggregation is the essential hallmark of neurodegenerative disorders such as Alzheimer's disease. Efficient inhibitors are highly desired for the prevention of Aß assembly that has been considered as the primary therapeutic strategy for neurodegenerative diseases. Apart from this, visualization of the aggregates and morphology at high spatial resolution is widely considered of crucial significance on biological treatment. In this work, we have developed small-sized (with diameter of ∼4.7 nm) and positively charged fluorescent conjugated polymer nanoparticles (CPNPs) with strong inhibition effect on Aß1-40 peptides fibrillation. Interestingly, the CPNPs also possess excellent photophysical properties, including high photon counts, robust blinking, and repetitive fluorescence switching, that are especially suitable for localization-based super-resolution imaging. Spatial resolution of ∼20 nm for these blinking CPNPs is readily achieved. According to the optical microscopic results, it was found that binding of CPNPs to the terminal of seed fibrils can effectively inhibit the fibrillation process. Owing to these attractive biological and unique photophysical properties, the small-sized CPNPs show high potential in a variety of super-resolution based biological applications.