Development of an aptamer capable of multidrug resistance reversal for tumor combination chemotherapy.

Multidrug resistance (MDR) is a major factor in the failure of many forms of tumor chemotherapy. Development of a specific ligand for MDR-reversal would enhance the intracellular accumulation of therapeutic agents and effectively improve the tumor treatments. Here, an aptamer was screened against a doxorubicin (DOX)-resistant human hepatocellular carcinoma cell line (HepG2/DOX) via cell-based systematic evolution of ligands by exponential enrichment. A 50 nt truncated sequence termed d3 was obtained with high affinity and specificity for HepG2/DOX cells. Multidrug resistance protein 1 (MDR1) is determined to be a possible recognition target of the selected aptamer. Aptamer d3 binding was revealed to block the MDR of the tumor cells and increase the accumulation of intracellular anticancer drugs, including DOX, vincristine, and paclitaxel, which led to a boost to the cell killing of the anticancer drugs and lowering their survival of the tumor cells. The aptamer d3-mediated MDR-reversal for effective chemotherapy was further verified in an in vivo animal model, and combination of aptamer d3 with DOX significantly improved the suppression of tumor growth by treating a xenograft HepG2/DOX tumor in vivo. This work demonstrates the feasibility of a therapeutic DNA aptamer as a tumor MDR-reversal agent, and combination of the selected aptamer with chemotherapeutic drugs shows great potential for liver cancer treatments.

Tiny pH-Resolved DNA Nanospheres for Cellular Pyroptosis or Apoptosis Regulation.

The design and synthesis of nanomedicines capable of regulating programmed cell death patterns to enhance antitumor efficacy remain significant challenges in cancer therapy. In this study, we developed intelligent DNA nanospheres (NS) capable of distinguishing tiny pH changes between different endosomal compartments to regulate pyroptosis or apoptosis. These NS are self-assembled from two multifunctional DNA modules, enabling tumor targeting, acid-responsive disassembly, and photodynamic therapy (PDT) activation. By modifying the embedded i-motif sequence, the NS can be activated in early endosomes (EE) or lysosomes (Ly), producing singlet oxygen (1O2) at specific locations under laser irradiation. Our results demonstrate that EE-activated PDT induces gasdermin-E-mediated pyroptosis in tumor cells, enhancing antitumor efficacy and reducing systemic toxicity compared to Ly-activated apoptosis. This study offers new insights into the design of endosome-activated nanomedicines, advancing the biomedical applications of targeted cancer therapy.

Modular Assembled Localized Hybridization Chain Reaction for In Situ mRNA Amplified Imaging.

As a nonenzymatic DNA signal amplification technique, localized hybridization chain reaction (LHCR) was designed to improve the limitations in response speed and low sensitivity of conventional free diffusional HCR (hybridization chain reaction). However, it is still confronted with the challenges of complicated DNA scaffolds with low loading capacity and a time-consuming process of diffusion. Herein, we introduced modular assembly of a DNA minimal scaffold for coassembly of DNA hairpins for amplified fluorescence imaging of mRNA in situ. DNA hairpins were spatially bound to two Y-shaped modules to form H-shaped DNA modules, and then multiple H-shaped DNA modules can further assemble into an H-module-based hairpin scaffold (HHS). Benefiting from highly spatial localization and high loading capacity, the HHS system showed higher sensitivity and faster speed. It has also been proven to work perfectly in vitro and in vivo, which could provide a promising bioanalysis system for low abundance biomolecule detection.

Atomic Force Microscopy Lifetime Analysis: An Intuitive Method for Evaluating Receptor Tyrosine Kinase Dimer-Targeting Inhibitors.

Overexpression of receptor tyrosine kinases (RTKs) or binding to ligands can lead to the formation of specific unliganded and liganded RTK dimers, and these two RTK dimers are potential targets for preventing tumor metastasis. Traditional RTK dimer inhibitor analysis was mostly based on end point assays, which required cumbersome cell handling and behavior monitoring. There are still challenges in developing intuitive process-based analytical methods to study RTK dimer inhibitors, especially those used to visually distinguish between unliganded and liganded RTK dimer inhibitors. Herein, taking the mesenchymal-epithelial transition factor (MET) receptor, an intuitive method for evaluating MET inhibitors has been developed based on atomic force microscopy (AFM) lifetime analysis. The time interval between the start of the force and the bond break point was regarded as the bond lifetime, which could reflect the stability of the MET dimer. The results showed that there was a significant difference in the lifetime (τ) of unliganded MET dimers (τ1 = 207.87 ± 4.69 ms) and liganded MET dimers (τ2 = 330.58 ± 15.60 ms) induced by the hepatocyte growth factor, and aptamer SL1 could decrease τ1 and τ2, suggesting that SL1 could inhibit both unliganded and liganded MET dimers. However, heparin only decreased τ2, suggesting that it could inhibit only the liganded MET dimer. AFM-based lifetime analysis methods could monitor RTK dimer status rather than provide overall average results, allowing for intuitive process-based analysis and evaluation of RTK dimers and related inhibitors at the single-molecule level. This study provides a novel complementary strategy for simple and intuitive RTK inhibitor research.

Cell Membrane-Anchored AND Logic Gate Aptasensor for Tumor Cell-Specific Imaging with Improved Accuracy.

Accurate and rapid imaging of tumor cells is of vital importance for early cancer diagnosis and intervention. Aptamer-based fluorescence sensors have become a potent instrument for bioimaging, while false positives and on-target off-tumors linked to single-biomarker aptasensors compromise the specificity and sensitivity of cancer imaging. In this paper, we describe a sequential response aptasensor for precise cancer cell identification that is based on a DNA "AND" logic gate. Specifically, the sensor consists of three single-stranded DNA, including the P-strand that can sensitively respond to an acid environment, the L-strand containing the ATP aptamer sequence, and the R-strand for target cell anchoring. These DNA strands hybridize with one another to create a Y-shaped structure (named Y-ALGN). The aptamer in the R-strand is utilized to anchor the sensor to the target cell membrane primarily. Responding to the extracellular acidic environment of the tumor (input 1), the I-motif sequence forms a tetramer structure so that the P-strand is released from the Y-shaped structure and exposes the ATP binding sites in the L-strand. Extracellular ATP, as input 2, continuously operates the DNA aptasensor to complete the logic computation. Upon the sequential response of both protons and ATP molecules, the aptasensor is activated with restored fluorescence on a particular cancer cell membrane. Benefiting from the precise computation capacity of the "AND" logic gate, the Y-ALGN aptasensor can distinguish between MCF-7 cells and normal cells with high accuracy. As a simple and dual-stimuli-responsive strategy, this nanodevice would offer a fresh approach for accurately diagnosing tumor cells.

Synthetic Cells from Droplet-Based Microfluidics for Biosensing and Biomedical Applications.

Synthetic cells function as biological mimics of natural cells by mimicking salient features of cells such as metabolism, response to stimuli, gene expression, direct metabolism, and high stability. Droplet-based microfluidic technology presents the opportunity for encapsulating biological functional components in uni-lamellar liposome or polymer droplets. Verified by its success in the fabrication of synthetic cells, microfluidic technology is widely replacing conventional labor-intensive, expensive, and sophisticated techniques justified by its ability to miniaturize and perform batch production operations. In this review, an overview of recent research on the preparation of synthetic cells through droplet-based microfluidics is provided. Different synthetic cells including lipid vesicles (liposome), polymer vesicles (polymersome), coacervate microdroplets, and colloidosomes, are systematically discussed. Efforts are then made to discuss the design of a variety of microfluidic chips for synthetic cell preparation since the combination of microfluidics with bottom-up synthetic biology allows for reproductive and tunable construction of batches of synthetic cell models from simple structures to higher hierarchical structures. The recent advances aimed at exploiting them in biosensors and other biomedical applications are then discussed. Finally, some perspectives on the challenges and future developments of synthetic cell research with microfluidics for biomimetic science and biomedical applications are provided.

A glaucoma micro-stent with diverging channel and stepped shaft structure based on microfluidic template processing technology.

BACKGROUND: Minimally invasive glaucoma surgery (MIGS) has experienced a surge in popularity in recent years. Glaucoma micro-stents serve as the foundation for these minimally invasive procedures. Nevertheless, the utilization of these stents still presents certain short-term and long-term complications. This study aims to elucidate the creation of a novel drainage stent implant featuring a diverging channel, produced through microfluidic template processing technology. Additionally, an analysis of the mechanical properties, biocompatibility, and feasibility of implantation is conducted. RESULTS: The stress concentration value of the proposed stent is significantly lower, approximately two to three times smaller, compared to the currently available commercial XEN gel stent. This indicates a stronger resistance to bending in theory. Theoretical calculations further reveal that the initial drainage efficiency of the gradient diverging drainage stent is approximately 5.76 times higher than that of XEN stents. Notably, in vivo experiments conducted at the third month demonstrate a favorable biocompatibility profile without any observed cytotoxicity. Additionally, the drainage stent exhibits excellent material stability in an in vitro simulation environment. CONCLUSIONS: In summary, the diverging drainage stent presents a novel approach to the cost-effective and efficient preparation process of minimally invasive glaucoma surgery (MIGS) devices, offering additional filtering treatment options for glaucoma.

Photocaged amplified FRET nanoflares: spatiotemporal controllable of mRNA-powered nanomachines for precise and sensitive microRNA imaging in live cells.

There is considerable interest in creating a precise and sensitive strategy for in situ visualizing and profiling intracellular miRNA. Present here is a novel photocaged amplified FRET nanoflare (PAFN), which spatiotemporal controls of mRNA-powered nanomachine for precise and sensitive miRNA imaging in live cells. The PAFN could be activated remotely by light, be triggered by specific low-abundance miRNA and fueled by high-abundance mRNA. It offers high spatiotemporal control over the initial activity of nanomachine at desirable time and site, and a 'one-to-more' ratiometric signal amplification model. The PAFN, an unprecedented design, is quiescent during the delivery process. However, upon reaching the interest tumor site, it can be selectively activated by light, and then be triggered by specific miRNA, avoiding undesirable early activation and reducing nonspecific signals, allowing precise and sensitive detection of specific miRNA in live cells. This strategy may open new avenues for creating spatiotemporally controllable and endogenous molecule-powered nanomachine, facilitating application at biological and medical imaging.

Logic Nanodevice-Mediated Receptor Assembly for Nongenetic Regulation of Cell Behavior in Tumor-like Microenvironment.

The reprogramming of cell signaling and behavior through the artificial control of cell surface receptor oligomerization shows great promise in biomedical research and cell-based therapy. However, it remains challenging to achieve combinatorial recognition in a complicated environment and logical regulation of receptors for desirable cellular behavior. Herein, we develop a logic-gated DNA nanodevice with responsiveness to multiple environmental inputs for logically controlled assembly of heterogeneous receptors to modulate signaling. The "AND" gate nanodevice uses an i-motif and an ATP-binding aptamer as environmental cue-responsive units, which can successfully implement a logic operation to manipulate receptors on the cell surface. In the presence of both protons and ATP, the DNA nanodevice is activated to selectively assemble MET and CD71, which modulate the HGF/MET signaling, resulting in cytoskeletal reorganization to inhibit cancer cell motility in a tumor-like microenvironment. Our strategy would be highly promising for precision therapeutics, including controlled drug release and cancer treatment.

Spatially Controlled DNA Frameworks for Sensitive Detection and Specific Isolation of Tumor Cells.

High-affinity, specific, and sensitive probes are crucial for the specific recognition and identification of tumor cells from complex matrices. Multivalent binding is a powerful strategy, but the irrational spatial distribution of the functional moieties may reduce the probe performance. Here, we constructed a Janus DNA triangular prism nanostructure (3Zy1-JTP-3) for sensitive detection and specific isolation of tumor cells. Benefiting from spatial features of the triangular prism, the fluorescence intensity induced by 3Zy1-JTP-3 was almost 4 times that of the monovalent structure. Moreover, the DNA triangular prisms were connected to form hand-in-hand multivalent DNA triangular prism structures (Zy1-MTP), in which the fluorescence intensity and affinity were increased to 9-fold and 10-fold of 3Zy1-JTP-3, respectively. Furthermore, 3Zy1-JTP-3 and Zy1-MTP were combined with magnetic beads, and the latter showed higher capture efficiency (> 90%) in whole blood. This work provides a new strategy for the efficient capture of rare cells in complex biological samples.

Osmotic-Induced Reconfiguration and Activation in Membranized Coacervate-Based Protocells.

The design and construction of synthetic protocells capable of stimuli response and homeostatic regulation is an important challenge for synthetic protobiology. Here, we develop a step toward the construction of model protocells capable of a hypotonic stress-induced volume response that facilitates an increase in membrane permeability and the triggering of endogenous enzyme reactions. We describe a facile self-transformation process for constructing single- or multichambered molecularly crowded protocells based on the osmotic reconfiguration of lipid-coated coacervate droplets into multicompartmentalized coacervate vesicles. Hypotonic swelling broadens membrane permeability and increases transmembrane transport such that protease-based hydrolysis and enzyme cascades can be triggered and enhanced within the protocells by osmotically induced expansion. Specifically, we demonstrate how the enhanced production of nitric oxide (NO) within the swollen coacervate vesicles can be used to induce in vitro blood vessel vasodilation in thoracic artery rings. Our approach provides opportunities for designing reconfigurable model protocells capable of homeostatic volume regulation, dynamic structural reorganization, and adaptive functionality in response to changes in environment osmolarity, and could find applications in biomedicine, cellular diagnostics, and bioengineering.

DNA-Encoded Bidirectional Regulation of the Peroxidase Activity of Pt Nanozymes for Bioanalysis.

Rational regulation of nanozyme activity can promote biochemical sensing by expanding sensing strategies and improving sensing performance, but the design of effective regulatory strategies remains a challenge. Herein, a rapid DNA-encoded strategy was developed for the efficient regulation of Pt nanozyme activity. Interestingly, we found that the catalytic activity of Pt nanozymes was sequence-dependent, and its peroxidase activity was significantly enhanced only in the presence of T-rich sequences. Thus, different DNA sequences realized bidirectional regulation of Pt nanozyme peroxidase activity. Furthermore, the DNA-encoded strategy can effectively enhance the stability of Pt nanozymes at high temperatures, freezing, and long-term storage. Meanwhile, a series of studies demonstrated that the presence of DNA influenced the reduction degree of H2PtCl6 precursors, which in turn affected the peroxidase activity of Pt nanozymes. As a proof of application, the sensor array based on the Pt nanozyme system showed superior performance in the accurate discrimination of antioxidants. This study obtained the regulation rules of DNA on Pt nanozymes, which provided theoretical guidance for the development of new sensing platforms and new ideas for the regulation of other nanozyme activities.

Self-Priming DNA Polymerization-Propelled Stochastic Walkers on Magnetic Microbeads for Amplified Detection of miRNA.

Sensitive detection of miRNA targets in complex biological samples possesses great value in biopsy analysis and disease diagnosis but is still challenging because of low abundance and nonspecific interferences. In this work, self-primer DNA polymerization-propelled stochastic walkers (SWs) were proposed to detect miRNA-24 by combining magnetic microbeads (MMBs) and flow cytometry. The MMBs not only provide a three-dimensional interface for DNA walkers but also facilitate the enrichment and isolation of RNA targets from complex biological samples such as serum. The SWs can be initiated to walk through the entire surface of MMBs and transduce RNA walking into amplified fluorescence signals, with the detection limit of miRNA-24 at 0.95 pM. Moreover, this strategy integrating with flow cytometry was demonstrated to have good specificity with other homologous miRNAs. This platform offers promising applications in RNA biosensing and biomedical diagnostics.

A Versatile Integrated Microfluidic Chip Based on Sonic Toothbrush-Assisted Mixing for Analyses of Diverse Biomolecules.

Usually, different assays and instrumentation are required for different types of targets, e.g., nucleic acids, proteins, small molecules, etc., because of significant differences in their structures and sizes. To increase efficiency and reduce costs, a desirable solution is to develop a versatile platform suitable for diverse objectives. Here, we established a versatile detection technique: first, target separation and enrichment were carried out using magnetic beads (MBs); then, different targets were converted to same barcoded DNA strands (BDs) released from gold nanoparticles; finally, sensitive detection of three different targets (miRNA-21, digoxigenin antibody, and aflatoxin B1) was achieved through exonuclease III (Exo III) cyclic cleavage-assisted signal amplification. To simplify the operation, we integrated this technique into a microfluidic chip with multiple chambers in which the requisite reagents were prestored. Just by moving the MBs through different chambers with a magnet, multiple steps can be completed. Due to the limited space in microfluidic chips, the full mixing of MBs and solution is a key point to improve reaction efficiency. The mixing can be achieved by acoustic vibration generated by a small, portable sonic toothbrush. Based on the microfluidic chip, the detection limits of the above three targets were 0.76 pM, 0.16 ng/mL, and 0.56 nM, respectively. Furthermore, miRNA-21 and Digoxigenin antibody (Dig-Ab) in serum and AFB1 in corn powder were also used to demonstrate the performance of this chip. Our versatile platform is easy to operate and is expected to develop into an automatic "sample-to-answer" device.

Catalyst-Accelerated Circular Cascaded DNA Circuits: Simpler Design, Faster Speed, Higher Gain.

DNA cascaded circuits have great potential in detecting low abundance molecules in complex biological environment due to their powerful signal amplification capability and nonenzymatic feature. However, the problem of the cascaded circuits is that the design is relatively complex and the kinetics is slow. Herein, a new design paradigm called catalyst-accelerated circular cascaded circuits is proposed, where the catalyst inlet is implanted and the reaction speed can be adjusted by the catalyst concentration. This new design is very simple and only requires three hairpin probes. Meanwhile, the results of a series of studies demonstrate that the reaction speed can be accelerated and the sensitivity can be also improved. Moreover, endogenous mRNA can also be used as a catalyst to drive the circuits to amplify the detection of target miRNA in live cells and in mice. These catalyst-accelerated circular cascaded circuits can substantially expand the toolbox for intracellular low abundance molecular detection.

Construction of a DNA Nanoassembly Based on Spatially Ordered Recognition Elements for Inhibiting ß-Amyloid Aggregation.

A ß-amyloid (Aß) aggregation process is a spontaneous process where the original random coil or helical structure changes into a regularly arranged ß-sheet structure. The development of inhibitors with the features of low cost, high efficiency, and biosafety by targeting Aß self-aggregation is significant for Alzheimer's disease treatment. However, the issues of low inhibition efficiency under low concentrations of inhibitors and biological toxicity are currently to be addressed. To resolve the above problems, a DNA nanoassembly (HCR-Apt) based on spatially ordered recognition elements was constructed by targeted disruption of Aß ordered arrangement. It was discovered that HCR-Apt could inhibit effectively the fibrillation of Aß40 monomers and oligomers at substoichiometric ratios. This may be due to orderly arrangement of aptamers in rigid nanoskeletons for enhancing the recognition interaction between aptamers and Aß40. The strong interaction between HCR-Apt and Aß40 limited the flexible conformational conversion of Aß40 molecules, thereby inhibiting their self-assembly. Computational simulations and experimental analysis revealed the interactions of Apt42 with Aß40, which explained different inhibition effects on the fibrillation of Aß40 monomers and oligomers. Furthermore, the analysis of tyrosine intrinsic fluorescence spectra and surface plasmon resonance imaging showed that the interaction of HCR-Apt and Aß40 was stronger than that of Apt42 and Aß40. These findings contributed to establishing a promising method of boosting the recognition interaction by orderly arrangement of recognition elements. Taken together, this work is expected to provide a simple and efficient strategy for inhibiting Aß aggregation, expanding aptamer's application potential in neurodegenerative diseases.

"One suction and one extrusion" mode-based wash-free platform for determination of breast cancer cell-derived exosomes.

A simple and wash-free POCT platform based on microcapillary was developed, using breast cancer cell-derived exosomes as a model. This method adopted the "one suction and one extrusion" mode. The hybridized complex of epithelial cell adhesion molecule (EpCAM) aptamer and complementary DNA-horseradish peroxidase conjugate (CDNA-HRP) was pre-modified on the microcapillary's inner surface. "One suction" meant inhaling the sample into the functionalized microcapillary. The exosomes could specifically bind with the EpCAM aptamer on the microcapillary's inner wall, and then the CDNA-HRP complex was released. "One extrusion" referred to squeezing the shedding CDNA-HRP into the 3,3',5,5'-tetramethylbenzidine (TMB)/H2O2 solution, and then the enzyme-catalyzed reaction would occur to make the solution yellow using sulfuric acid as the terminator. Therefore, exosome detection could be realized. The limit of detection was 2.69 × 104 particles mL-1 and the signal value had excellent linearity in the concentration range from 2.75 × 104 to 2.75 × 108 particles⋅mL-1 exosomes. In addition, the wash-free POCT platform also displayed a favorable reproducibility (RSD = 2.9%) in exosome detection. This method could effectively differentiate breast cancer patients from healthy donors. This work provided an easy-to-operate method for detecting cancer-derived exosomes without complex cleaning steps, which is expected to be applied to breast cancer screening.

Size-Controllable and Self-Assembled DNA Nanosphere for Amplified MicroRNA Imaging through ATP-Fueled Cyclic Dissociation.

Visualizing intracellular microRNA (miRNA) is of great importance for revealing its roles in the development of disease. However, cell membrane barrier, complex intracellular environment and low abundance of target miRNA are three main challenges for efficient imaging of intracellular miRNA. Here, we report a size-controllable and self-assembled DNA nanosphere with ATP-fueled dissociation property for amplified miRNA imaging in live cells and mice. The DNA nanosphere was self-assembled from Y-shaped DNA (Y-DNA) monomers through predesigned base pair hybridization, and the size could be easily controlled by varying the concentration of Y-DNA. Once the nanosphere was internalized into cells, the intracellular specific target miRNA would trigger the cyclic dissociation of the DNA nanosphere driven by ATP, resulting in amplified FRET signal. The programmable DNA nanosphere has been proven to work well for detecting the expression of miRNA in cancer cells and in mice, which demonstrates its fairish cell penetration, stability and sensitivity.

DNA-Based Artificial Receptors as Transmembrane Signal Transduction Systems for Protocellular Communication.

The ability to reproduce signal transduction and cellular communication in artificial cell systems is significant in synthetic protobiology. Here, we describe an artificial transmembrane signal transduction through low pH-mediated formation of the i-motif and dimerization of DNA-based artificial membrane receptors, which is coupled to the occurrence of fluorescence resonance energy transfer and the activation of G-quadruplex/hemin-mediated fluorescence amplification inside giant unilamellar vesicles. Moreover, an intercellular signal communication model is established when the extravesicular H+ input is replaced by coacervate microdroplets, which activate the dimerization of the artificial receptors, and subsequent fluorescence production or polymerization in giant unilamellar vesicles. This study represents a crucial step towards designing artificial signalling systems with environmental response, and provides an opportunity to establish signalling networks in protocell colonies.

Membrane Protein and Extracellular Acid Heterogeneity-Driven Amplified DNA Logic Gate Enables Accurate and Sensitive Identification of Cancer Cells.

DNA logic gates, as a class of smart molecular devices with excellent biocompatibility and convenient information processing mode, have been widely used for identification of cancer cells based on logic analysis of cancer biomarkers. However, most of the developed DNA logic gates for identification of cancer cells are mainly driven by homogeneous biomarkers such as membrane proteins or RNAs, which may suffer from insufficient accuracy. Herein, we reported a membrane protein and extracellular acid heterogeneity-driven amplified DNA logic gate (HDLG) for accurate and sensitive identification of cancer cells by combining the superior signal amplification characteristics of the hybridization chain reaction (HCR) and the precise computation ability of the logic operation. In this strategy, a DNA aptamer was employed for membrane protein recognition, and a split i-motif was used for the response of the extracellular acid. Only when the two heterogeneous biomarkers existed simultaneously, the DNA logic gate could be driven to perform the "AND" logic operation and induce the formation of an intact trigger to initiate a HCR process on the cell surface, generating an amplified "ON" fluorescence signal. Benefiting from the design of heterogeneity-driven and signal amplification, this DNA logic gate could not only autonomously perform high-resolution fluorescence imaging on the surface of target cancer cells, but also perform sensitive analysis of target cancer cells with a cell number of 70 detected in 200 µL of buffer and desirable accuracy in differentiating target cancer cells from complicated cell mixtures. We anticipate that this novel HDLG is expected to be applied in precise disease diagnosis.