Dual-signal readout sensing of ATP content in single dental pulp stem cells during differentiation via functionalized glass nanopipettes.

Adenosine triphosphate (ATP) is regarded as the "energy currency" in living cells, so real-time quantification of content variation of intracellular ATP is highly desired for understanding some important physiological processes. Due to its single-molecule readout ability, nanopipette sensing has emerged as a powerful technique for molecular sensing. In this study, based on the effect of targeting-aptamer binding on ionic current, and fluorescence resonance energy transfer (FRET), we reported a dual-signal readout nanopipette sensing system for monitoring ATP content variation at the subcellular level. In the presence of ATP, the complementary DNA-modified gold nanoparticles (cDNAs-AuNPs) were released from the inner wall of the nanopipette, which leads to sensitive response variations in ionic current rectification and fluorescence intensity. The developed nanopipette sensor was capable of detecting ATP in single cells, and the fluctuation of ATP content in the differentiation of dental pulp stem cells (DPSCs) was further quantified with this method. The study provides a more reliable nanopipette sensing platform due to the introduction of fluorescence readout signals. Significantly, the study of energy fluctuation during cell differentiation from the perspective of energy metabolism is helpful for differentiation regulation and cell therapy.

A wearable electrostimulation-augmented ionic-gel photothermal patch doped with MXene for skin tumor treatment.

A wearable biological patch capable of producing multiple responses to light and electricity without interfering with daily activities is highly desired for skin cancer treatment, but remains a key challenge. Herein, the skin-mountable electrostimulation-augmented photothermal patch (eT-patch) comprising transparent ionic gel with MXene (Ti3C2Tx) doping is developed and applied for the treatment of melanoma under photostimulation at 0.5 W/cm2. The eT-patch designed has superior photothermal and electrical characteristics owing to ionic gels doped with MXene which provides high photothermal conversion efficiency and electrical conductivity as a medium. Simultaneously, the ionic gel-based eT-patch having excellent optical transparency actualizes real-time observation of skin response and melanoma treatment process under photothermal and electrical stimulation (PES) co-therapy. Systematical cellular study on anti-tumor mechanism of the eT-patch under PES treatment revealed that eT-patch under PES treatment can synergically trigger cancer cell apoptosis and pyroptosis, which together lead to the death of melanoma cells. Due to the obvious advantages of relatively safe and less side effects in healthy organs, the developed eT-patch provides a promising cost-effective therapeutic strategy for skin tumors and will open a new avenue for biomedical applications of ionic gels.

Molecular/Nanomechanical Insights into Electrostimulation-Inhibited Energy Metabolism Mechanisms and Cytoskeleton Damage of Cancer Cells.

Inhibiting energy metabolism of cancer cells is an effective way to treat cancer but remains a great challenge. Herein, electrostimulation (ES) is applied to effectively suppress energy metabolism of cancer cells to induce rapid cell death, and deeply reveal the underlying mechanisms at the molecular and nanomechanical levels by combined use of fluorescence imaging and atomic force microscopy. Cancer cells are found significantly less tolerant to ES than normal cells; and ES causes "domino effect" to induce mitochondrial dysfunction to impede electron transport chain (ETC) and tricarboxylic acid (TCA) cycle pathways, leading to fatal energy-supply crisis and death of cancer cells. From the perspective of cell mechanics, the Young's modulus decreases and cytoskeleton destruction of MCF-7 cell membranes caused by F-actin depolymerization occurs, along with down-regulation and sporadic distribution of glucose transporter 1 (GLUT1) after ES. Such a double whammy renders ES highly effective and promising for potential clinical cancer treatments.

Label-Free SERS Detection of Protein Damage in Organelles under Electrostimulation with 2D AuNPs-based Nanomembranes as Substrates.

Proteins as the material basis of life are the main undertakers of life activities. However, it is difficult to identify the related proteins in organelles during stimuli-induced stress responses in cells and remains a great challenge in early diagnosis and treatment of disease. Here, proteins in the cell nucleus and mitochondria of cells under the electrical stimulation (ES) process were collected and sensitively detected based on label-free surface-enhanced Raman spectroscopy (SERS) by using AuNP-based nanomembranes as high-performance SERS substrates. Due to the existence of rich "hot spots" on the 2D plasmonic sensing platform, high-quality SERS spectra of proteins were obtained with superior sensitivity and repeatability. From the SERS analyses in vitro, it was found that the conformation of some proteins in the two kinds of organelles from cancerous HCT-116 cells (compared with normal NCM-460 cells) changed significantly and the expression levels of tyrosine, phenylalanine, and tryptophan were significantly promoted during the stimulation process. Although currently the exact proteins are still unknown, the damage of proteins in the organelles of cells at the amino acid level under ES can be revealed by the method. The developed plasmonic SERS sensing platform would be promising for bioassay and cell studies.

Monitoring Stress Response Difference in Nucleolus Morphology and ATP Content Changes during Hyperthermia Cell Apoptosis with Plasmonic Fluorescent Nanoprobes.

The nucleolus, as a main "cellular stress receptor", is the hub of the stress response driving cancer development and has great research value in the field of organelle-targeting photothermal therapy. However, there are few studies focused on monitoring nucleolar stress response and revealing how the energy metabolism of cells regulates the nucleolar stress response during photothermal therapy. Herein, by designing a nucleolus-targeting and ATP- and photothermal-responsive plasmonic fluorescent nanoprobe (AuNRs-CDs) based on gold nanorods (AuNRs) and fluorescent carbon quantum dots (CDs), we achieved real-time fluorescence imaging of nucleus morphology while monitoring changes of ATP content at the subcellular level. We found that the green fluorescence diminished at 5 min of photothermal therapy, and the nucleolus morphology began to shrink and became smaller in cancerous HepG2 cells. In contrast, there is no significant change of green fluorescence in the nucleolar region of normal HL-7702 cells. ATP content monitoring also showed similar results. Apparently, in response to photothermal stimuli, cancerous cells produce more ATP (energy) along with obvious change in nucleolus morphology and state compared to normal cells under the hyperthermia-induced cell apoptosis. The developed AuNRs-CDs as a nucleolus imaging nanoprobe and effective photothermal agent present promising applications for nucleolar stress studies and targeted photothermal therapy.

Electrostimulus-triggered reactive oxygen species level in organelles revealed by organelle-targeting SERS nanoprobes.

Electrostimulation (ES) is an important therapeutic method for diseases caused by abnormal intracellular electrical activity. Also, it can induce apoptosis of cells, which is a potential tumor treatment method. At present, there are no relevant studies on changes in intracellular reactive oxygen species (ROS) levels produced in the process of ES, or on the effects of simultaneous implementation of conventional antioxidant inhibitor drugs and ES therapy. To reveal these, two organelle-targeting core-shell plasmonic probes were designed for measuring ROS produced during ES. The probes were delivered into target organelles (nucleus and mitochondrion) before the cells were electrically stimulated for different periods of time. Surface-enhanced Raman scattering (SERS) signals were detected in situ, and the sensing mechanism for the quantitative analysis of ROS is based on the signal reduction of SERS caused by the ROS-etching effect on the silver shell. The detection results revealed that ES could trigger ROS generation in cells, and the ROS levels localized around organelles were assessed by SERS. This study has great potential for exploring abnormal organelle microenvironments via organelle-targeting probes combined with SERS technology.

Electrostimulus-associated PD-L1 expression on cell membrane revealed by immune SERS nanoprobes.

Programmed cell death ligand 1 (PD-L1) is considered a major immune checkpoint protein that mediates antitumor immune suppression and response. Effectively regulating PD-L1 expression and dynamic monitoring has become a significant challenge in immunotherapy. Herein, we adopted smart surface-enhanced Raman scattering (SERS) nanoprobes to discriminate and monitor the dynamic expression of PD-L1 under external electrostimulation (ES). The PD-L1 expression levels in three cell lines (MCF-7 cells, HeLa cells, and H8 cells) were assessed before and after ES. The results reveal that ES could effectively and rapidly mediate a transformation in the PD-L1 content (or activity) on the cell membrane. Moreover, the molecular profiles of the cell membrane before and after ES were revealed by using the label-free SERS method with the help of immune plasmonic nanoparticles. The cell membrane protein information presented identifiable conformation changes after ES, showing a significant inhibitory effect on the bridge of PD-L1 and its antibody. This study indicates that ES is superior to chemical drugs due to lesser side effects because ES-based regulation does not depend on intracellular signalling pathways. This strategy is versatile and robust for discriminating and monitoring PD-L1 on cell membranes, thus providing potential clinical application value to PD-L1-mediated systems. This study also offers a practical way to assess the molecular profiles of cell membrane proteins in the presence of an external stimulus, which may be applicable to many membrane protein-related studies.

Fast Activation and Tracing of Caspase-3 Involved Cell Apoptosis by Combined Electrostimulation and Smart Signal-Amplified SERS Nanoprobes.

Caspase-3 is considered as one of the key proteases that can spontaneously regulate the life activities of cells, and its activation (usually is a slow process) will execute the apoptosis process of cells. Rapid activation of caspase-3 on demand in living-cells is therefore highly desired toward precise cancer therapy but it is still a key challenge. Herein, we applied electrostimulus (ES) to achieve fast activation of caspase-3 and trigger cell apoptosis, and developed a smart magnetic-plasmonic assembly nanoprobes (A-nanoprobes) to real-time trace cellular caspase-3 activation at the single cell level. The designer core-satellite A-nanoprobe, working specific to the activated caspase-3 via a disassembly tactic, provides strong "hot spots" to improve the sensitivity and therefore enables SERS sensing of cellular caspase-3 upon activated by ES. Single-cell analysis revealed that the ES can rapidly activate the apoptosis pathway of caspase-3 on demand to make the DNA fragmentation and ultimately induce the cell apoptosis. Such method and nanoplatform were further used to monitor ES-triggered caspase-3 activation in cell apoptosis process of different cell types, revealing that more caspase-3 will be activated for cancerous cells than normal cells during the ES to induce cells apoptosis. This strategy and platform are promising for detecting cellular caspase-3 and other enzymes in the process of cancer diagnosis and treatments.

Living-Cell Imaging of Mitochondrial Membrane Potential Oscillation and Phenylalanine Metabolism Modulation during Periodic Electrostimulus.

Special electrosensory cells are sensitive to electric fields and give responses upon stimulation, but little is known about normal regular cells and cancerous cells. Herein, by designing nucleus- and mitochondria-targeting SERS nanoprobes combined with fluorescent monitoring of the mitochondrial membrane potential (MMP) variations, we found an interesting electrosensory and self-healing response in MMP within cancerous and normal cells during periodic impulse electrostimulation (IES). More importantly, the key regulator role of phenylalanine (phe) was revealed by cell fluorescent imaging and SERS detection, whose expression level was increased in response to IES to induce cell apoptosis. During IES off-state, the self-repair function of cells was activated to reduce phe release. We also found that cancerous cells (MCF-7 and HeLa cells) demonstrated a response more remarkable than that of normal cells (L929 and H8 cells) to periodic IES. Our finding revealed a common electrosensory and self-repair biofunction of cells and its related phe metabolism response. Understanding the difference of biophysical/electrophysiological responses between cancerous and normal cells may broaden the view for cancer therapy in the future.

Smart Plasmonic Nanorobot for Real-Time Monitoring Cytochrome c Release and Cell Acidification in Apoptosis during Electrostimulation.

Cytochrome c (Cyt c) release and cellular pH change are two important mediators of apoptosis. Effective methods to regulate or monitor such two events are therefore highly desired for apoptosis research and cancer cell therapy. Herein, we exploited electrostimulation to regulate cellular Cyt c release and apoptosis process, and by designing and preparing a smart and efficient plasmonic nanorobot (with surface-modified Cyt c-specific aptamer and 4-mercaptobenzoic acid) that is capable of Cyt c capture and self-sensing, we achieved real-time SERS monitoring of dynamic Cyt c release and simultaneous cell acidification in apoptosis during electrostimulation. Distinctly different molecular stress responses in the two events for cancerous MCF-7 and HeLa cells and normal L929 cells were identified and revealed. The method and results are valuable and promising for apoptosis and Cyt c-mediated biology studies.

Nucleus and Mitochondria Targeting Theranostic Plasmonic Surface-Enhanced Raman Spectroscopy Nanoprobes as a Means for Revealing Molecular Stress Response Differences in Hyperthermia Cell Death between Cancerous and Normal Cells.

Metallic plasmonic nanoparticles have been intensively exploited as theranostic nanoprobes for plasmonic photothermal therapy (PPT) and surface-enhanced Raman spectroscopy (SERS) applications. But the underlying molecular mechanisms associated with PPT-induced apoptosis between cancerous and normal cells have remained largely unknown or disputed. In this study, we designed an organelle-targeting theranostic plasmonic SERS nanoprobe (CDs-Ag/Au NS) composed of porous Ag/Au nanoshell (p-Ag/Au NSs) and carbon dots (CDs) for nucleus and mitochondria targeted PPT of cells. The differences in molecular stress response in the PPT-induced hyperthermia cell death between cancerous HeLa and normal L929 and H8 cells have been revealed by site-specific single-cell SERS detection. The contents of tryptophan (Trp), phenylalanine (Phe), and tyrosine (Tyr) in HeLa cells were found more evidently increased than L929 and H8 cells during the PPT-induced cell-death process. And from the mitochondria point of view, we found that the PPT-induced cell apoptosis for HeLa cells mainly stems from (or is regulated through) cellular thermal stress-responsive proteins, while for L929 and H8 cells it seems more related to DNA. Understanding molecular stress response difference of the PPT-induced cell apoptosis between cancerous and normal cells is helpful for diagnosis and treatment of cancer, and the method will open an avenue for single-cell studies.