The protective effect of biomineralized BSA-Mn3O4 nanoparticles on HUVECs investigated by atomic force microscopy.

Manganese-based nanozymes have been widely used in the field of cell protection due to their various enzyme-mimicking activities, but their effect on the mechanical properties of cells is not yet known. Here, bovine serum albumin-modified Mn3O4 nanoparticles (BSA-Mn3O4 NPs) with good biocompatibility were synthesized by a one-step biomineralization method using BSA as a template. BSA-Mn3O4 NPs possess scavenging activity against superoxide free radicals (O2˙-), hydroxyl radicals (˙OH) and hydrogen peroxide (H2O2). The excellent reactive oxygen species (ROS) scavenging activity of BSA-Mn3O4 NPs enables them to effectively reduce the intracellular ROS level, thus mitigating the damage of oxidative stress on human umbilical vein endothelial cells (HUVECs). Subsequently, the intracellular antioxidant mechanism of the BSA-Mn3O4 NPs was further investigated. The results show that the BSA-Mn3O4 NPs could inhibit the depolymerization of F-actin, help cells maintain their normal morphology, and reduce the decrease in Young's modulus of cells caused by oxidative stress.

Combination of Rapid Intrinsic Activity Measurements and Machine Learning as a Screening Approach for Multicomponent Electrocatalysts.

Machine learning (ML) coupled with quantum chemistry calculations predicts catalyst properties with high accuracy; however, ML approaches in the design of multicomponent catalysts primarily rely on simulation data because obtaining sufficient experimental data in a short time is difficult. Herein, we developed a rapid screening strategy involving nanodroplet-mediated electrodeposition using a carbon nanocorn electrode as the support substrate that enables complete data collection for training artificial intelligence networks in one week. The inert support substrate ensures intrinsic activity measurement and operando characterization of the irreversible reconstruction of multinary alloy particles during the oxygen evolution reaction. Our approach works as a closed loop: catalyst synthesis-in situ measurement and characterization-database construction-ML analysis-catalyst design. Using artificial neural networks, the ML analysis revealed that the entropy values of multicomponent catalysts are proportional to their catalytic activity. The catalytic activities of high-entropy systems with different components varied little, and the overall catalytic activity was greater than that of the medium-low-entropy system. These findings will serve as a guideline for the design of catalysts.

Aptamer conjugated polydopamine-coated gold nanoparticles as a dual-action nanoplatform targeting ß-amyloid peptide for Alzheimer's disease therapy.

The accumulation and deposits of amyloid beta (Aß) peptide are an important pathological hallmark of Alzheimer's disease (AD). The development of multifunctional agents that can effectively clear Aß aggregates is one of the potential strategies to treat AD. Herein, aptamer conjugated polydopamine-coated gold nanoparticles (Au@PDA-Apt NPs) for targeting Aß1-40 peptides were designed. Au@PDA-Apt NPs exhibited a strong capability to inhibit Aß1-40 monomer fibrillization and disaggregate mature Aß1-40 fibrils. In addition, Au@PDA-Apt NPs could effectively alleviate Aß1-40-triggered cytotoxicity. Importantly, AFM quantitative nanomechanical measurements indicated that Au@PDA-Apt NPs could prevent cell membrane damage and decrease of cell mechanical properties caused by Aß1-40 aggregation. Taken together, this study provided a new dual-action nanoplatform for Aß-targeted AD therapy.

Imaging and quantifying analysis the binding behavior of PD-L1 at molecular resolution by atomic force microscopy.

Immunotherapy has emerged as an effective treatment modality for cancer. The interaction of programmed cell death ligand-1 (PD-L1) and programmed cell death protein-1 (PD-1) plays a key role in tumor-related immune escape and has become one of the most extensive targets for immunotherapy. Herein, we investigated the interaction of PD-L1 with its antibody and PD-1 using atomic force microscopy-based single molecule force spectroscopy for the first time. It was found that the PD-L1/anti-PD-L1 antibody complex was easier to dissociate than PD-L1/PD-1. The unbinding forces of specific interaction of PD-L1 on T24 cells with its antibody and PD-1 were quantitatively measured and similar to those on substrate. In addition, the location of PD-L1 on T24 cells was mapped at the single-molecule level by force-volume mapping. The force maps revealed that PD-L1 randomly distributed on T24 cells surface. The recognition events on cells obviously increased after INF-γ treatment, which proved that INF-γ up-regulated the expression of PD-L1 on T24 cells. These findings enrich our understanding of the molecular mechanisms by which PD-L1 interacts with its antibody and PD-1. It provides useful information for the physical factors that is needed to be considered in the design of inhibitors for tumor immunology.

Size-effect on the intracellular antioxidative activity of Prussian blue nanoparticles investigated by atomic force microscopy.

Nanoparticles-based antioxidative therapy has been highlighted in a series of diseases triggered by excessive reactive oxygen species (ROS). Prussian blue nanoparticles (PBNPs), as a representative artificial nanozyme, have been proved as highly effective ROS scavengers. However, its detailed intracellular antioxidant mechanism is not clear yet. Herein, a series of PBNPs with different particle sizes were synthesized and their intracellular antioxidant activities were studied by atomic force microscopy (AFM) from a biomechanical perspective. We first validated the ROS scavenging ability of PBNPs in vitro. It indicated that PBNPs had great scavenging effect on multiple ROS, such as hydroxyl radicals (•OH), superoxide radicals (O2•-) and hydrogen peroxide (H2O2). By observing the changes in morphology and mechanical properties of human umbilical vascular endothelium cells (HUVECs), it was further found that PBNPs could apparently alleviate the decrease of Young's modulus caused by oxidative stress damage and kept cells in their normal morphology. In addition, the distribution of F-actin revealed that the enhancement of cytoskeleton stability by PBNPs might be a key way to protect HUVECs from oxidative damage. Importantly, the antioxidant activities of PBNPs were found to be size-dependent, which indicated the smaller particle size had better antioxidant activities compared with the larger particle size. This study serves as a novel medium to reveal the mechanism of nanoparticles on cells at the single-cell level and demonstrates the great potential of atomic force microscopy in studying the application of nanoparticles in cell biology.

Studying the effect of PDA@CeO2 nanoparticles with antioxidant activity on the mechanical properties of cells.

Studying the influence of nanomaterials on the microstructure and mechanical properties of cells is essential to guide the biological applications of nanomaterials. In this article, the effects of the first synthesized PDA@CeO2 nanoparticles (NPs) with multiple ROS scavenging activities on cell ultra-morphology and mechanical properties were investigated by atomic force microscopy (AFM). After the cells were exposed to PDA@CeO2 NPs, there was no obvious change in cell morphology, but the Young's modulus of the cells was increased. On the contrary, after the cells were damaged by H2O2, the secreted molecules appeared on the cell surface, and the Young's modulus was decreased significantly. However, PDA@CeO2 NPs could effectively inhibit the reduction of the Young's modulus caused by oxidative stress damage. PDA@CeO2 NPs could also protect F-actin from oxidative stress damage and maintain the stability of the cytoskeleton. This work investigates the intracellular antioxidant mechanism of nanomaterials from the changes in the microstructure and biomechanics of living cells, providing a new analytical approach to explore the biological effects of nanomaterials.

Investigation of the interaction between MeCP2 methyl-CpG binding domain and methylated DNA by single molecule force spectroscopy.

MeCP2 is an essential transcriptional repressor that mediates transcriptional inhibition by binding to methylated DNA. The binding specificity of MeCP2 protein to methylated DNA was considered to depend on its methyl-CpG binding domain (MBD). In this study, we used atomic force microscope based single-molecular force spectroscopy to investigate the interaction of MeCP2 MBD and methylated DNA. The specific interaction forces of the MeCP2 MBD-methylated DNA complexes were measured for the first time. The dynamics was also investigated by measuring the unbinding force of the complex at different loading rates. In addition, the distribution of unbinding forces and binding probabilities of MeCP2 MBD and different DNA were studied at the same loading rate. It was found that MeCP2 MBD had weak interaction with hemi-methylated and unmethylated DNA compared to methylated DNA. This work revealed the binding characteristics of MeCP2 MBD and methylated DNA at the single-molecule level. It provides a new idea for exploring the molecular mechanism of MeCP2 in regulating methylation signals.

Interaction of vascular endothelial growth factor and heparin quantified by single molecule force spectroscopy.

Heparin, as an effective anticoagulant, has been increasingly used in clinical practice, but the binding characteristics and influence of exogenous heparin on heparin-affinity proteins in the body are still unclear. Vascular endothelial growth factor A (VEGF-A) is a kind of protein with heparin affinity involved in the pathogenesis and progression of many angiogenesis-dependent diseases including cancer. As an important step in the angiogenesis-related cascade, it is necessary to clarify the interaction between VEGF165 (the major form of VEGF-A) and heparin. In this work, we investigated this interaction based on single molecule force spectroscopy (SMFS) and molecular dynamics (MD) simulation. From the SMFS study, binding forces between VEGF165 and heparin at different loading rates were quantified under near-physiological conditions. Meanwhile, the kinetic and thermodynamic parameters of the VEGF165/heparin complex dissociation process were also obtained. Results of MD simulation visually displayed the most likely binding conformation of VEGF165/heparin* complex, indicating that hydrogen bonding and hydrophobic interaction play a positive role in the binding between the two molecules. This work provides a new insight into the binding between VEGF165 and heparin and offers a research framework to study the interaction between heparin and multiple heparin affinity proteins, which is helpful for guiding the safe application of heparin in the clinic.

Microcantilever Array Biosensor for Simultaneous Detection of Carcinoembryonic Antigens and α-Fetoprotein Based on Real-Time Monitoring of the Profile of Cantilever.

A microcantilever array biosensor based on a sandwich structure has been developed for simultaneously measuring two biomarkers carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) via an optical readout technique-real-time monitoring of the profile of cantilever. First, the aptamers of CEA and AFP were self-assembled on their respective cantilevers. After the adsorption of the mixture of CEA and AFP, further specific interaction was performed via the addition of the antibodies specific to each target. The compressive stress on the cantilever was generated by the aptamer-antigen-antibody sandwich structure formed on the gold surface, resulting in cantilever bending. The profile of cantilever could be monitored in real time. The relationship between the deflection value at the 90% position of the cantilever and the target concentration served as a calibration curve, and the detection sensitivity was 1.3 ng/mL for CEA and 0.6 ng/mL for AFP, respectively. This work demonstrated the ability of simultaneously measuring two biomarkers via a microcantilever array biosensor, giving great potential for further application in detecting several targets simultaneously for early clinical diagnosis.

Evaluating the protective mechanism of panax notoginseng saponins against oxidative stress damage by quantifying the biomechanical properties of single cell.

Panax notoginseng saponins (PNS) have shown to be the biologically active constituents responsible for the therapeutic action of panax notoginseng. PNS could help to restrain the oxidative stress, however, whether biomechanical properties of the single cell involve in the protective effect exerted by PNS against oxidative stress injury remains unclear. In this work, we investigated the protective mechanism of PNS against oxidative stress based on the PeakForce Tapping technology firstly, focusing on the biomechanical properties of single human umbilical vascular endothelium cell (HUVEC). PNS display distinct inhibition on the reduction of the young's modulus of cells caused by oxidative stress damage. Combining with immunofluorescence assay, it indicates that improving the stability of cytoskeleton is a significant way for PNS to play a protective role in HUVEC cells during oxidative damage. This work provides a new idea for exploring the functional mechanism of traditional Chinese medicine at the single cell level, and reveals great potential of the atomic force microscope in studying the drug mechanism.

Directly observing alterations of morphology and mechanical properties of living cancer cells with atomic force microscopy.

Epithelial-mesenchymal transition (EMT) is a biological process during which cells lose their characteristic structure and biochemical properties then adopt typical features of a mesenchymal phenotype. Alterations in the morphology, structure, and mechanical properties of cells during EMT are associated with a series of pathological processes. In this work, atomic force microscopy (AFM) is used for investigating effects of TGF-ß1 on morphology and mechanical properties of living bladder cancer cells (T24) during EMT for the first time. High-resolution topography and Young's modulus images of T24 living cell are obtained simultaneously. The results show that TGF-ß1 is able to induce EMT, leading to the increased F-actin stress fibers and much higher Young's modulus values of T24 living cells. It reveals that the cytoskeletal-associated cell architecture is closely related to the mechanical dynamics of T24 cells during EMT. This work provides new insights into the changes of cell morphology and mechanical properties during EMT. It enables us to gain a deeper understanding of the growth, development and metastasis of the bladder cancer cell therefore it is of great significance for studying the pathological mechanism of cells at single-cell level.