Single-particle rotational microrheology enables pathological staging of macrophage foaming and antiatherosclerotic studies.

The formation of macrophage-derived foam cells has been recognized as the pathological hallmark of atherosclerotic diseases. However, the pathological evolution dynamics and underlying regulatory mechanisms remain largely unknown. Herein, we introduce a single-particle rotational microrheology method for pathological staging of macrophage foaming and antiatherosclerotic explorations by probing the dynamic changes of lysosomal viscous feature over the pathological evolution progression. The principle of this method involves continuous monitoring of out-of-plane rotation-caused scattering brightness fluctuations of the gold nanorod (AuNR) probe-based microrheometer and subsequent determination of rotational relaxation time to analyze the viscous feature in macrophage lysosomes. With this method, we demonstrated the lysosomal viscous feature as a robust pathological reporter and uncovered three distinct pathological stages underlying the evolution dynamics, which are highly correlated with a pathological stage-dependent activation of the NLRP3 inflammasome-involved positive feedback loop. We also validated the potential of this positive feedback loop as a promising therapeutic target and revealed the time window-dependent efficacy of NLRP3 inflammasome-targeted drugs against atherosclerotic diseases. To our knowledge, the pathological staging of macrophage foaming and the pathological stage-dependent activation of the NLRP3 inflammasome-involved positive feedback mechanism have not yet been reported. These findings provide insights into in-depth understanding of evolutionary features and regulatory mechanisms of macrophage foaming, which can benefit the analysis of effective therapeutical drugs as well as the time window of drug treatment against atherosclerotic diseases in preclinical studies.

Rational Design of a Double-Locked Photoacoustic Probe for Precise In Vivo Imaging of Cathepsin B in Atherosclerotic Plaques.

Atherosclerotic plaque rupture is a significant cause of acute cardiovascular events such as heart attack and stroke, triggered by the decomposition of fiber caps induced by cysteine cathepsin. However, the accurate measurement of cathepsin B (CTB) activity in plaques is challenging due to the low specificity and insufficient penetration depth of available atherosclerosis-associated cathepsin fluorescent probes, hampering reliable assessment of plaque vulnerability. To address these limitations, we added both lipophilic alkyl chain and hydrophilic CTB substrate to the hemicyanine scaffold to develop a lipid-unlocked CTB responsive probe (L-CRP) that uses lipids and CTB as two keys to unlock photoacoustic (PA) signals for measuring CTB activity in lipophilic environments. Such properties allow L-CRP for the reliable imaging of specific CTB activities in foam cells and atherosclerotic plaques while keeping in silence toward CTB in lipid-deficient environments, such as M1-type macrophages and LPS-induced inflammatory lesions. Moreover, the activatable PA signals of L-CRP exhibit a deeper tissue penetration ability (>1.0 cm) than current CTB probes based on near-infrared fluorescent imaging (∼0.3 cm), suitable for atherosclerosis imaging in living mice. In atherosclerotic mice, L-CRP dynamically reports intraplaque CTB levels, which is well-correlated with the plaque vulnerability characteristics such as fiber cap thickness, macrophage recruitment, and necrotic core size, thus enabling risk stratification of atherosclerotic mice complicated with pneumonia. Moreover, L-CRP successfully identifies atherosclerotic plaques in excised human artery tissues, promising for auxiliary diagnosis of plaque vulnerability in clinical application.

Virus-like Plasmonic Nanoprobes for Quick Analysis of Antiviral Efficacy and Mutation-Induced Drug Resistance.

As the pathogenic viruses and the variants of concern greatly threaten human health and global public safety, the development of convenient and robust strategies enabling rapid analysis of antiviral drug efficacy and mutation-induced resistance is quite important to prevent the spread of human epidemics. Herein, we introduce a simple single-particle detection strategy for quick analysis of anti-infective drugs against SARS-CoV-2 and mutation-induced drug resistance, by using the wild-type and mutant spike protein-functionalized AuNPs as virus-like plasmonic nanoprobes. Both the wild-type and mutant virus-like plasmonic nanoprobes can form core-satellite nanoassemblies with the ACE2@AuNPs, providing the opportunity to detect the drug efficacy and mutation-induced resistance by detecting the changes of nanoassemblies upon drug treatment with dark-field microscopy. As a demonstration, we applied the single-particle detection strategy for quantitative determination of antiviral efficacy and mutation-induced resistance of ceftazidime and rhein. The mutations in the receptor-binding domain of Omicron variant could lead to an increase of EC50 values of ceftazidime and rhein, formerly from 49 and 57 µM against wild-type SARS-CoV-2, to 121 and 340 µM, respectively. The mutation-induced remarkable decline in the inhibitory efficacy of drugs was validated with molecule docking analysis and virus-like plasmonic nanoprobe-based cell-incubation assay. Due to the generality and feasibility of the strategy for the preparation of virus-like plasmonic nanoprobes and single-particle detection, we anticipated that this simple and robust method is promising for the discovery and efficacy evaluation of anti-infective drugs against different pathogenic viruses.

Ratiometric Semiconducting Polymer Nanoparticle for Reliable Photoacoustic Imaging of Pneumonia-Induced Vulnerable Atherosclerotic Plaque in Vivo.

Acute pneumonia can greatly increase the vulnerable risk of atherosclerotic plaque and contribute to the mortality of cardiovascular disease. To accurately assess the rupture risk caused by acute pneumonia, we developed a novel kind of ratiometric semiconducting polymer nanoparticle (RSPN) for photoacoustic imaging of vulnerable plaque in apolipoprotein E-deficient mice complicated with pneumonia. Specifically, RSPN can react with O2•- and exhibit the enhanced photoacoustic signals at about 690 nm, while 800 nm is regarded as an internal photoacoustic reference. As a result, RSPN can provide reliable determination of O2•- within aortic atherosclerosis by analyzing the ratios of photoacoustic signals, which can successfully reflect the oxidative stress level in vulnerable plaque. Therefore, RSPN enable to specifically distinguish plaque-bearing mice and plaque-bearing mice complicated with pneumonia from healthy mice, which provides a promising tool to predict the vulnerability of plaque for reducing the mortality of atherosclerotic-induced cardiovascular disease.

Plasmonic Imaging of Dynamic Interactions between Membrane Receptor Clusters beyond the Diffraction Limit in Live Cells.

As a general mechanism, ligand-induced receptor clustering on cell membrane plays determinative roles in pattern recognition and transmembrane signaling. Nevertheless, probing the dynamic characteristics for the complicated interactions between receptor clusters remains difficult because of the lack of strategy for receptor cluster labeling and long-term monitoring in live cells. Herein, we proposed a data-mining-integrated plasmon coupling microscopy to study the dynamic cluster-cluster interactions on cell surface. The receptor clusters were activated and labeled with multivalent plasmonic nanoprobes, which enables the real-time monitoring of individual receptor clusters and the measurement of cluster-cluster interactions from the analysis of plasmonic coupling for the nanoprobe pairs beyond the diffraction limit. Using this method, we found that the protease-activated receptor 1 (PAR1) clusters would experience an initial contact and then form a weakly bound cluster-cluster complex, followed by cluster fusion to generate large-sized signaling complexes. The underlying state transitions for the cluster-cluster fusion process were uncovered using a data-mining technique named the K-means-based hidden Markov model with the scattering intensity of coupled nanoprobe pairs as observations. All of the findings from single-particle analysis and bulk measurements suggested that the allosteric inhibitors could suppress the dynamic transitions from the weakly bound cluster-cluster complexes to fused signaling complexes, leading to the subsequent downregulation of intracellular calcium signaling pathways. We believe that this strategy is promising for imaging and monitoring receptor clustering as well as protein phase separation on the cell surface in various biological and physiological processes.

Single-Particle Mobility Analysis Enables Ratiometric Detection of Cancer Markers under Darkfield Tracking Microscopy.

Here, we introduced a single-particle mobility analysis-based ratiometric strategy for quantitative detection of disease-related biomarkers using antibody-conjugated gold nanoparticles (AuNPs) as probes under darkfield tracking microscopy (DFTM). On the basis of the capability of discriminating nanoparticles with different hydrodynamic sizes and detecting the changes in hydrodynamic effect, single-particle mobility analysis enables us to determine the amount of aggregated and monodispersed nanoprobes for the sandwich-like immunoassay strategy, making it possible to quantify the biotargets by analyzing the relative changes in the aggregate-to-monomer ratio of nanoprobes. By using capture antibody and detection antibody conjugated AuNPs as nanoprobes, we demonstrated ratiometric detection of carcinoembryonic antigen (CEA) over a linear dynamic range from 50 to 750 pM, which is acceptable for clinical diagnostic analysis of CEA in tumor patients. This ratiometric detection technique exhibited excellent anti-interference ability in the presence of nonspecific proteins or complicated protein mixtures. It can be anticipated that this robust technique is promising for the accurate detection of disease biomarkers and other biomolecules for biochemical and diagnostic applications.

Single-particle rotational sensing for analyzing the neutralization activity of antiviral antibodies.

Due to the pathogen-specific targeting, neutralization capabilities, and enduring efficacy, neutralizing antibodies (NAs) have received widespread attentions as a critical immunotherapeutic strategy against infectious viruses. However, because of the high variability and complexity of pathogens, rapid determination of neutralization activity of antiviral antibodies remains a challenge. Here, we report a new method, named as out-of-plane polarization imaging based single-particle rotational sensing, for rapid analysis of neutralization activity of antiviral antibody against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using the spike protein functionalized gold nanorods (AuNRs) and angiotensin-converting enzyme 2 (ACE2) coated gold nanoparticles (AuNPs) as the rotational sensors and chaperone probes, we demonstrated the single-particle rotational sensing strategy for the measurement of rotational diffusion coefficient of the chaperone-bound rotational sensors caused by the specific spike protein-ACE2 interactions. This enables us to measure the neutralizing activity of neutralizing antibody from the analysis of dose-dependent changes in rotational diffusion coefficient (Dr) of the rotational sensors upon the treatment of SARS-CoV-2 antibody. With this technique, we achieved the quantitative determination of neutralization activity of a commercially available SARS-CoV-2 antibody (IC50, 294.1 ng/mL) with satisfying accuracy and anti-interference ability. This simple and robust method holds the potential for rapid and accurate evaluation of neutralization activity against different pathogenic viruses.

Activatable Photoacoustic/Near-Infrared Probes for the Detection of Copper Ions of Cardiovascular Disease In Vivo and in Urine.

Copper ions, implicated in processes such as oxidative stress and inflammation, are believed to play a crucial role in cardiovascular disease, a prevalent and deadly disease. Despite this, current diagnostic methods fail to detect early stage cardiovascular disease or track copper ion accumulation, limiting our understanding of the disease's progression. Therefore, the development of noninvasive techniques to image copper ions in cardiovascular disease is urgently needed to enhance diagnostic precision and therapeutic strategies. In this study, we report the successful synthesis and application of a copper ion-activated photoacoustic probe, CS-Cu, which exhibits high sensitivity and selectivity toward copper ions both in vitro and in vivo. CS-Cu was able to noninvasively monitor the changes in copper ion levels and differentiate between different mice based on copper ions in urine. Furthermore, the probe demonstrated good photoacoustic stability and exhibited no significant toxicity in the mice. These findings suggest that CS-Cu could be a promising tool for early detection and monitoring of Cu2+ levels in vivo and urine, providing a new perspective on the role of copper ions in cardiovascular disease.

Oxidative stress biomarker triggered multiplexed tool for auxiliary diagnosis of atherosclerosis.

Oxidative stress is integral in the development of atherosclerosis, but knowledge of how oxidative stress affects atherosclerosis remains insufficient. Here, we design a multiplexed diagnostic tool that includes two functions (photoacoustic imaging and urinalysis), for assessing intraplaque and urinary malondialdehyde (MDA), a well-recognized end-product of oxidative stress. Molecular design is conducted to develop the first near-infrared MDA-responsive molecule (MRM). Acid-unlocked ratiometric photoacoustic nanoprobe is designed to report intraplaque MDA, enabling it to reflect plaque burden. Furthermore, MRM is tailored for urinary MDA detection with excellent specificity in a blind study. Moreover, we found a significant difference in urinary MDA between healthy adults and atherosclerotic patients (more than 600 participants). Combining these two functions, such a multiplexed diagnostic tool can dynamically report intraplaque and systemic oxidative stress levels during atherosclerosis progression, pneumonia infection, and drug treatment in atherosclerotic mice, which is promising for the auxiliary diagnosis of atherosclerosis.

Probing Dynamic Features of Phagosome Maturation in Macrophage using Au@MnOx @SiO2 Nanoparticles as pH-Sensitive Plasmonic Nanoprobes.

Phagosome maturation in macrophage is essential to the clearance of pathogenic materials in host defence but the dynamic features remain difficult to be measured in real time. Herein, we reported the multilayered Au@MnOx @SiO2 nanoparticle as a robust pH-sensitive plasmonic nanosensor for monitoring the dynamic acidification features over the phagosome maturation process in macrophage under darkfield microscopy. For this multilayered nanosensor, the gold nanoparticle core plays a role of signal reporter, the MnOx shell and the outmost SiO2 act as the sensing layer and the protecting layer, respectively. After subject to the acidic buffer solution, the MnOx layer in the multilayered nanoprobe could be decomposed rapidly, resulting in a remarkable spectral shift and color change under darkfield microscopy. We demonstrated this nanosensor for the investigation of single phagosome acidification dynamics by monitoring the color changes of nanoprobes after phagocytosis over time. The nanoprobes after phagocytosized in macrophage displayed a slight color change within the first hour and then cost several minutes to change from red to green in the next stage, indicating the phagosome undergoes a slow first and then fast acidification feature as well as a slow-to-fast acidification translation over the phagosome maturation process. Moreover, we validated that the slow-to-fast acidification translation was dependent on the activation of V-ATPase from the ATP depletion assay. We believed that this nanosensor is promising for studying the dynamic acidification features as well as disorders in phagosome maturation in phagocytic cells, which might provide valuable information for understanding the disease pathogenesis related to phagosome dysfunctions.

A surface-engineered NIR light-responsive actuator for controllable modulation of collective cell migration.

Mechanical signal transduction is fundamental for maintaining and regulating cellular processes and functions. Here, we proposed a novel near-infrared (NIR) light-responsive optomechanical actuator for the directional regulation of collective cell adhesion and migration. This optomechanical actuator that is made up of a thermal-responsive copolymer hydrogel and gold nanorods (AuNRs), enables non-invasive activation by NIR light stimulation. The activation of the optomechanical actuator leads to hydrogel contraction and an increase in Young's modulus, which could be used for applying contraction force to cells cultured on the surface of the hydrogel actuator. By grafting cell adhesive peptide ligands, the cells could attach onto the surface of the actuator and displayed a NIR light illumination intensity dependent migration rate along a random orientation. To achieve the controllable modulation of cell behaviors, we employed a microcontact printing strategy for patterned presentation of adhesive ligands on this actuator and achieved directional cell alignment and cell migration through optomechanical actuation. These demonstrations suggest that this robust optomechanical actuator is promising for the optical modulation of cellular events and cell functions in various bioapplications.