Rectifying artificial nanochannels with multiple interconvertible permeability states.

Transmembrane channels play a vital role in regulating the permeation process, and have inspired recent development of biomimetic channels. Herein, we report a class of artificial biomimetic nanochannels based on DNAzyme-functionalized glass nanopipettes to realize delicate control of channel permeability, whereby the surface wettability and charge can be tuned by metal ions and DNAzyme-substrates, allowing reversible conversion between different permeability states. We demonstrate that the nanochannels can be reversibly switched between four different permeability states showing distinct permeability to various functional molecules. By embedding the artificial nanochannels into the plasma membrane of single living cells, we achieve selective transport of dye molecules across the cell membrane. Finally, we report on the advanced functions including gene silencing of miR-21 in single cancer cells and selective transport of Ca2+ into single PC-12 cells. In this work, we provide a versatile tool for the design of rectifying artificial nanochannels with on-demand functions.

Holistic Prediction of AuNP Aggregation in Diverse Aqueous Suspensions Based on Machine Vision and Dark-Field Scattering Imaging.

The localized surface-plasmon resonance of the AuNP in aqueous media is extremely sensitive to environmental changes. By measuring the signal of plasmon scattering light, the dark-field microscopic (DFM) imaging technique has been used to monitor the aggregation of AuNPs, which has attracted great attention because of its simplicity, low cost, high sensitivity, and universal applicability. However, it is still challenging to interpret DFM images of AuNP aggregation due to the heterogeneous characteristics of the isolated and discontinuous color distribution. Herein, we introduce machine vision algorithms for the training of DFM images of AuNPs in different saline aqueous media. A visual deep learning framework based on AlexNet is constructed for studying the aggregation patterns of AuNPs in aqueous suspensions, which allows for rapid and accurate identification of the aggregation extent of AuNPs, with a prediction accuracy higher than 0.96. With the aid of machine learning analysis, we further demonstrate the prediction ability of various aggregation phenomena induced by both cation species and the concentration of the external saline solution. Our results suggest the great potential of machine vision frameworks in the accurate recognition of subtle pattern changes in DFM images, which can help researchers build predictive analytics based on DFM imaging data.

Schiff Base Reaction in a Living Cell: In Situ Synthesis of a Hollow Covalent Organic Polymer To Regulate Biological Functions.

Artificially performing chemical reactions in living biosystems to attain various physiological aims remains an intriguing but very challenging task. In this study, the Schiff base reaction was conducted in cells using Sc(OTf)3 as a catalyst, enabling the in situ synthesis of a hollow covalent organic polymer (HCOP) without external stimuli. The reversible Schiff base reaction mediated intracellular Oswald ripening endows the HCOP with a spherical, hollow porous structure and a large specific surface area. The intracellularly generated HCOP reduced cellular motility by restraining actin polymerization, which consequently induced mitochondrial deactivation, apoptosis, and necroptosis. The presented intracellular synthesis system inspired by the Schiff base reaction has strong potential to regulate cell fate and biological functions, opening up a new strategic possibility for intervening in cellular behavior.

Engineered Extracellular Vesicle-Encapsuled Nanoreactors for Effective Targeting and Cascade Killing of Cancer Cells.

Nanomaterials have presented great potential for cancer therapy. However, their therapeutic efficacy is not always satisfied because of inefficient biocompatibility and targeting efficacy. Here, we report engineered extracellular vesicle (EV)-encapsuled nanoreactors for the targeting and killing of cancer cells. EVs are extracted from engineered cancer cells with surface N-glycans cut and intracellular microRNA-21 (miR-21) silenced to generate cancer-targeting membranes for the following coating of gold-polydopamine (PDA) core-shell nanoparticles. The encapsuled nanoparticles are decorated with doxorubicin (Dox), glucose oxidase (GOx), and miR-21-indicative DNA tags. Once endocytosed, the acidic pH, together with the photothermal effect of the PDA shell, can promote the release of Dox and GOx-catalyzed H2O2 generation/glucose consumption, while the DNA tags allow enhanced fluorescence imaging of miR-21 to indicate the targeting effect. The coadministration of EV-assisted delivery and cascade treatment represents a promising strategy for combination therapy.

Dual-Modal Apoptosis Assay Enabling Dynamic Visualization of ATP and Reactive Oxygen Species in Living Cells.

ATP and reactive oxygen species (ROS) are considered significant indicators of cell apoptosis. However, visualizing the interplay between apoptosis-related ATP and ROS is challenging. Herein, we developed a metal-organic framework (MOF)-based nanoprobe for an apoptosis assay using duplex imaging of cellular ATP and ROS. The nanoprobe was fabricated through controlled encapsulation of gold nanorods with a thin zirconium-based MOF layer, followed by modification of the ROS-responsive molecules 2-mercaptohydroquinone and 6-carboxyfluorescein-labeled ATP aptamer. The nanoprobe enables ATP and ROS visualization via fluorescence and surface-enhanced Raman spectroscopy, respectively, avoiding the mutual interference that often occurs in single-mode methods. Moreover, the dual-modal assay effectively showed dynamic imaging of ATP and ROS in cancer cells treated with various drugs, revealing their apoptosis-related pathways and interactions that differ from those under normal conditions. This study provides a method for studying the relationship between energy metabolism and redox homeostasis in cell apoptosis processes.

DNAzyme-Powered DNA Walker for Cooperative Expression Imaging of Mutant p53 and Telomerase in Cancer Cells.

Cooperative expression of multiple cancer biomarkers is of great significance in influencing cell pathways and drug treatment. However, the simultaneous analysis of low-abundance biomarkers in living cells remains a challenge. Here, we report a DNAzyme-powered DNA walker to visualize the cooperative expression of mutant p53 and telomerase in living cells. The activation of the DNA walker is orthogonally powered by mutated p53 and telomerase, which enables the unlocking of the walking strand and the subsequently repeated substrate cleavage, producing fluorescence recovery for the imaging of the two target molecules in living cells. The DNA walker allows for real-time monitoring of the expression profile of mutant p53 and active telomerase in cancer cells under various antitumor drug treatments, and the results demonstrate the cooperative expression of mutant p53 and telomerase via the Akt pathway, which may bring new insights into the study of cancer pathway-relevant biomarkers.

Combination Cancer Treatment: Using Engineered DNAzyme Molecular Machines for Dynamic Inter- and Intracellular Regulation.

Despite the promise of combination cancer therapy, it remains challenging to develop targeted strategies that are nontoxic to normal cells. Here we report a combination therapeutic strategy based on engineered DNAzyme molecular machines that can promote cancer apoptosis via dynamic inter- and intracellular regulation. To achieve external regulation of T-cell/cancer cell interactions, we designed a DNAzyme-based molecular machine with an aptamer and an i-motif, as the MUC-1-selective aptamer allows the specific recognition of cancer cells. The i-motif is folded under the tumor acidic microenvironment, shortening the intercellular distance. As a result, T-cells are released by metal ion activated DNAzyme cleavage. To achieve internal regulation of mitochondria, we delivered another DNAzyme-based molecular machine with mitochondria-targeted peptides into cancer cells to induce mitochondria aggregation. Our strategy achieved an enhanced killing effect in zinc deficient cancer cells.

A plasmonic nanoparticle-embedded polydopamine substrate for fluorescence detection of extracellular vesicle biomarkers in serum and urine from patients with systemic lupus erythematosus.

There is an unmet clinical need to develop noninvasive liquid biopsy tools for systemic lupus erythematosus (SLE) diagnosis and therapeutic effect evaluation. Extracellular vesicles (EVs), which are abundant in body fluids, have emerged as a valuable resource for liquid biopsy. Herein, we describe a simple and robust EV detection platform that is based on a plasmonic nanoparticle-embedded polydopamine substrate that is modified with EV-capture molecules and detection probes. We investigated three EV biomarkers, namely, programmed cell death protein-1 (PD-1), microRNA-146a (miRNA-146a) and sialic acid (SA), in serum and urine from SLE patients and healthy controls. This platform prevents complex pretreatment while enabling highly efficient EV capture to the substrate surface, and the multiple functionalization of the detection interface with specific biomarker probes enables simultaneous detection of PD-1, miRNA-146a and SA that are carried by EVs via fluorescence (FL) imaging at the single-vesicle level. Via comparison of EV biomarker profiles, SLE patients can be distinguished from normal controls and classified into treated and untreated groups. Due to its ease of preparation, simplicity and stability, our approach shows good potential in the design of EV-based biosensors for clinical use.

Dynamic Visualization of Endoplasmic Reticulum Stress in Living Cells via a Two-Stage Cascade Recognition Process.

The endoplasmic reticulum (ER) is crucial for the regulation of multiple cellular processes, such as cellular responses to stress and protein synthesis, folding, and posttranslational modification. Nevertheless, monitoring ER physiological activity remains challenging due to the lack of powerful detection methods. Herein, we built a two-stage cascade recognition process to achieve dynamic visualization of ER stress in living cells based on a fluorescent carbon dot (CD) probe, which is synthesized by a facile one-pot hydrothermal method without additional modification. The fluorescent CD probe enables two-stage cascade ER recognition by first accumulating in the ER as the positively charged and lipophilic surface of the CD probe allows its fast crossing of multiple membrane barriers. Next, the CD probe can specifically anchor on the ER membrane via recognition between boronic acids and o-dihydroxy groups of mannose in the ER lumen. The two-stage cascade recognition process significantly increases the ER affinity of the CD probe, thus allowing the following evaluation of ER stress by tracking autophagy-induced mannose transfer from the ER to the cytoplasm. Thus, the boronic acid-functionalized cationic CD probe represents an attractive tool for targeted ER imaging and dynamic tracking of ER stress in living cells.

A Squalene-Based Nanoemulsion for Therapeutic Delivery of Resiquimod.

Agonists for toll-like receptors (TLRs) have shown promising activities against cancer. In the present study, a squalene-based nanoemulsion (NE) was loaded with resiquimod, a TLR7/8 agonist for therapeutic delivery. R848 NE was developed and characterized for long-term stability. In vitro and in vivo antitumor immunity of R848 NE were also evaluated in combination with SD-101, a CpG-containing TLR9 agonist. In vitro studies demonstrated strong long-term stability and immune responses to R848 NE. When combined with SD-101, strong antitumor activity was observed in MC38 murine colon carcinoma model with over 80% tumor growth inhibition. The combination treatment showed a 4-fold increase in systemic TNFa production and a 2.6-fold increase in Cd8a expression in tumor tissues, suggesting strong cell-mediated immune responses against the tumor. The treatment not only demonstrated a strong antitumor immunity by TLR7/8 and TLR9 activations but also induced PD-L1 upregulation in tumors, suggesting a potential therapeutic synergy with immune checkpoint inhibitors.

Nanopipette-Based Nanosensor for Label-Free Electrochemical Monitoring of Cell Membrane Rupture under H2O2 Treatment.

H2O2 is an essential signaling molecule in living cells that can cause direct damage to lipids, proteins, and DNA, resulting in cell membrane rupture. However, current studies mostly focus on probe-based sensing of intracellular H2O2, and these methods usually require sophisticated probe synthesis and instruments. In particular, local H2O2 treatment induces cell membrane rupture, but the level of cell membrane destruction is unknown because the mechanical properties of the cell membrane are difficult to accurately determine. Therefore, highly sensitive and label-free methods are required to measure and reflect mechanical changes in the cell membrane. Here, using an ultrasmall quartz nanopipette with a tip diameter less than 90 nm as a nanosensor, label-free and noninvasive electrochemical single-cell measurement is achieved for real-time monitoring of cell membrane rupture under H2O2 treatment. By spatially controlling the nanopipette tip to precisely approach a specific location on the membrane of a single living cell, stable cyclic membrane oscillations are observed under a constant direct current voltage. Specifically, upon nanopipette advancement, the mechanical status of the cell membrane can be sensibly displayed by continuous current versus time traces. The electrical signals are collected and processed, ultimately revealing the mechanical properties of the cell membrane and the degree of cell apoptosis. This nanopipette-based nanosensor paves the way for developing a facile, label-free, and noninvasive strategy to assay the mechanical properties of the cell membrane during external stimulation at the single-cell level.

In Situ Monitoring of Hydrogen Peroxide Released from Living Cells Using a ZIF-8-Based Surface-Enhanced Raman Scattering Sensor.

Hydrogen peroxide (H2O2) widely involves in intracellular and intercellular redox signaling pathways, playing a vital role in regulating various physiological events. Nevertheless, current analytical methods for the H2O2 assay are often hindered by relatively long response time, low sensitivity, or self-interference. Herein, a zeolitic imidazolate framework-8 (ZIF-8)-based surface-enhanced Raman scattering (SERS) sensor has been developed to detect H2O2 released from living cells by depositing ZIF-8 over SERS active gold nanoparticles (AuNPs) grafted with H2O2-responsive probe molecules, 2-mercaptohydroquinone. Combining the superior fingerprint identification of SERS and the highly efficient enrichment and selective response of H2O2 by ZIF, the ZIF-8-based SERS sensor exhibits a high anti-interference ability for H2O2 detection, with a limit of detection as low as 0.357 nM. Satisfyingly, owing to the enhanced catalytic activity derived from the successful integration of AuNPs and ZIF, the response time as short as 1 min can be obtained, demonstrating the effectiveness of the SERS sensor for rapid H2O2 detection. Furthermore, the developed SERS sensor enables real-time detection of H2O2 secreted from living cells under phorbol myristate acetate stimulation, as cells can be cultured on-chip. This study will pave the way toward the development of a metal-organic framework-based SERS platform for application in the fields of biosensing and early disease diagnosis associated with H2O2 secretion, thus exhibiting promising potential for future therapies.

Recent Progress and Perspectives on Cell Surface Modification.

The cell membrane is a biological interface consisting of phospholipid bilayer, saccharides and proteins that maintains a stable metabolic intracellular environment as well as regulating and controlling the exchange of substances inside and outside the cell. Cell membranes provide a highly complex biological surface carrying a variety of essential surfaces ligands and receptors for cells to receive various stimuli of external signals, thereby inducing corresponding cell responses regulating the life activities of the cell. These surface receptors can be manipulated via cell surface modification to regulate cellular functions and behaviors Thus, cell surface modification has attracted considerable attention due to its significance in cell fate control, cell engineering and cell therapy. In this minireview, we describe the recent developments and advances of cell surface modification, and summarize the main modification methods with corresponding functions and applications. Finally, the prospect for the future development of the modification of the living cell membrane is discussed.

Cell Surface Engineering Using DNAzymes: Metal Ion Mediated Control of Cell-Cell Interactions.

Regulating cell-cell interactions and cell behaviors via cell surface engineering is of significance for biological research such as cell fate control and cell therapy. While extensive efforts have been made to induce cell-cell assembly via various cell surface modifications triggered by macromolecules or organic metabolites, controllable cell-cell interactions that include both assembly and disassembly triggered by metal ions remain a challenge. Herein, we report a strategy based on DNAzymes to realize controllable cell-cell interactions, triggered by metal ions. The metal-dependent DNAzyme-based cleavage can effectively manipulate cell behaviors, including cell-cell conjunctions and disaggregation. Using a Zn2+-specific DNAzyme, a Mg2+-specific DNAzyme, and their respective substrate strands as the building blocks, the corresponding DNA double-chain switches enabling two-factor disassembly are demonstrated. Moreover, the method has been applied to control the assembly and disassembly between two cell spheroids. Since a wide variety of metal-specific DNAzymes are available, this method can be readily applied to construct cell dynamic systems controlled by other metal ions, providing a smart and versatile platform to regulate dynamic cell behavior.

Exploiting deep learning for predictable carbon dot design.

In this study, we developed a deep convolution neural network (DCNN) model for predicting the optical properties of carbon dots (CDs), including spectral properties and fluorescence color under ultraviolet irradiation. These results demonstrate the powerful potential of DCNN for guiding the synthesis of CDs.

Sialidase-Conjugated "NanoNiche" for Efficient Immune Checkpoint Blockade Therapy.

Reactivation of T-cell immunity by blocking the PD-1/PD-L1 immune checkpoint has been considered a promising strategy for cancer treatment. However, the recognition of PD-L1 by antibodies is usually suppressed due to the N-linked glycosylation of PD-L1. In this study, we present an effective PD-L1-blocking strategy based on a sialidase-conjugated "NanoNiche" to improve the antitumor effect via T-cell reactivation. Molecularly imprinted by PD-L1 N-glycans, NanoNiche can specifically recognize glycosylated PD-L1 on the tumor cell surface, thereby resulting in more efficient PD-L1 blockade. Moreover, sialidase modified on the surface of NanoNiche can selectively strip sialoglycans from tumor cells, enhancing immune cell infiltration. In vitro studies confirmed that NanoNiche can specifically bind with PD-L1 while also desialylate the tumor cell surface. The proliferation of PD-L1-positive MDA-MB-231 human breast cancer cells under T-cell killing was significantly inhibited after NanoNiche treatment. In vivo experiments in solid tumors show enhanced therapeutic efficacy. Thus, the NanoNiche-sialidase conjugate represents a promising approach for immune checkpoint blockade therapy.

A polydopamine-based biomimetic multifunctional nanoplatform for multilayer imaging of cancer biomarkers carried by extracellular vesicles.

Cancer-derived extracellular vesicles (EVs) have attracted considerable attention for clinical diagnosis. However, a limiting factor in current EV assays is the ability to detect various EV cancer biomarkers expressed at different locations. Here, we report a biomimetic multifunctional nanoplatform for multilayer imaging of cancer biomarkers from the EV surface to the interior without complex pretreatment. Constructed from polydopamine-wrapped gold nanoparticles modified with multiple functional molecules, this nanoplatform can capture EVs from complex samples and target different EV cancer biomarkers for imaging analysis at the single-vesicle level. Combined with 96-well plates, this assay can distinguish cancer cell-derived EVs from normal ones in a high-throughput manner. Using serum samples, EVs from hepatocellular carcinoma (HCC) patients can be distinguished from healthy controls. This convenient workflow represents a promising tool for EV-based cancer diagnosis.

A DNA encoding loop program: the snowball effect enhanced microRNA visualization in living cells.

Here we report a convenient, universal "DNA encoding loop program (DELP)" strategy that significantly enhanced the sensitivity of subcellular imaging of microRNA. The assay relies on the dynamic, ultrafast clustering of multiple plasmonic gold nanoparticles actuated by a DNA-programmed recycling process.

Nanopipette-assisted single cell metabolic glycan labeling.

Here, we report a single cell glycan labeling strategy by combining nanoscale intracellular glass electrodes with bioorthogonal reaction. With the tip diameter less than 100 nm, the nanopipette electrode can be spatially controlled to inject artificial monosaccharides into single living cells with minimal invasion. The injection process can be precisely regulated by electroosmotic flow inside the nanopipette, and fluorescence labeling of sialic acid at single cell level is achieved.