Induction of human ESC-derived and adult primary multipotent limbal stem cells into retinal pigment epithelial cells and corneal stromal stem cells.

Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.

Beyond single cells: microfluidics empowering multiomics analysis.

Single-cell multiomics technologies empower simultaneous measurement of multiple types of molecules within individual cells, providing a more profound comprehension compared with the analysis of discrete molecular layers from different cells. Microfluidic technology, on the other hand, has emerged as a pivotal facilitator for high-throughput single-cell analysis, offering precise control and manipulation of individual cells. The primary focus of this review encompasses an appraisal of cutting-edge microfluidic platforms employed in the realm of single-cell multiomics analysis. Furthermore, it discusses technological advancements in various single-cell omics such as genomics, transcriptomics, epigenomics, and proteomics, with their perspective applications. Finally, it provides future prospects of these integrated single-cell multiomics methodologies, shedding light on the possibilities for future biological research.

Well-TEMP-seq as a microwell-based strategy for massively parallel profiling of single-cell temporal RNA dynamics.

Single-cell RNA sequencing (scRNA-seq) reveals the transcriptional heterogeneity of cells, but the static snapshots fail to reveal the time-resolved dynamics of transcription. Herein, we develop Well-TEMP-seq, a high-throughput, cost-effective, accurate, and efficient method for massively parallel profiling the temporal dynamics of single-cell gene expression. Well-TEMP-seq combines metabolic RNA labeling with scRNA-seq method Well-paired-seq to distinguish newly transcribed RNAs marked by T-to-C substitutions from pre-existing RNAs in each of thousands of single cells. The Well-paired-seq chip ensures a high single cell/barcoded bead pairing rate (~80%) and the improved alkylation chemistry on beads greatly alleviates chemical conversion-induced cell loss (~67.5% recovery). We further apply Well-TEMP-seq to profile the transcriptional dynamics of colorectal cancer cells exposed to 5-AZA-CdR, a DNA-demethylating drug. Well-TEMP-seq unbiasedly captures the RNA dynamics and outperforms the splicing-based RNA velocity method. We anticipate that Well-TEMP-seq will be broadly applicable to unveil the dynamics of single-cell gene expression in diverse biological processes.

Degradable ZnS-Supported Bioorthogonal Nanozymes with Enhanced Catalytic Activity for Intracellular Activation of Therapeutics.

Bioorthogonal catalysis using transition-metal catalysts (TMCs) provides a toolkit for the in situ generation of imaging and therapeutic agents in biological environments. Integrating TMCs with nanomaterials mimics key properties of natural enzymes, providing bioorthogonal "nanozymes". ZnS nanoparticles provide a platform for bioorthogonal nanozymes using ruthenium catalysts embedded in self-assembled monolayers on the particle surface. These nanozymes uncage allylated profluorophores and prodrugs. The ZnS core combines the non-toxicity and degradability with the enhancement of Ru catalysis through the release of thiolate surface ligands that accelerate the rate-determining step in the Ru-mediated deallylation catalytic cycle. The maximum rate of reaction (Vmax) increases ∼2.5-fold as compared to the non-degradable gold nanoparticle analogue. The therapeutic potential of these bioorthogonal nanozymes is demonstrated by activating a chemotherapy drug from an inactive prodrug with efficient killing of cancer cells.

Decoding Expression Dynamics of Protein and Transcriptome at the Single-Cell Level in Paired Picoliter Chambers.

Simultaneous analysis of mRNAs and proteins at the single-cell level provides information about the dynamics and correlations of gene and protein expressions in individual cells, enabling a comprehensive study of cellular heterogeneity and expression patterns. Here, we present a platform for about 1000 cellular indexing of mRNAs and membrane proteins, named multi-Paired-seq, with high cell utilization, accurate molecular measurement, and low cost. Based on hydrodynamic differential flow resistance, multi-Paired-seq largely improves cell utilization in the percentage of cells measured in population (>95%). Combined with the pump/valve structure, cell-free antibodies and mRNAs can be removed completely for highly accurate detection (R = 0.96) of protein copies. The picoliter reaction chambers allow high detection sensitivity for both mRNA transcripts and protein copies and low sequencing cost. Using multi-Paired-seq, three clusters of known breast cancer cell types are identified according to multimodal measurements, and the expression correlations between mRNAs and proteins under altered conditions are quantified. Multi-Paired-seq provides multimodal measurements at the single-cell level, which offers a new tool for cell biology, developmental biology, drug discovery, and precision medicine.

Direct and Simultaneous Identification of Multiple Mitochondrial Reactive Oxygen Species in Living Cells Using a SERS Borrowing Strategy.

Identification of different mitochondrial reactive oxygen species (ROS) simultaneously in living cells is vital for understanding the critical roles of different ROS in biological processes. To date, it remains a great challenge to develop ROS probes for direct and simultaneous identification of multiple ROS with high specificity. Herein, we report a SERS-borrowing-strategy-based nanoprobe (Au@Pt core-shell nanoparticles) for simultaneous and direct identification of different ROS by their distinct Raman fingerprints. Isotope substitution experiments and DFT calculations confirmed the ability of Au@Pt nanoprobe to capture and identify different mitochondrial ROS (i.e. ⋅OOH, H2 O2 , and ⋅OH). When functionalized with triphenylphosphine (TPP), the Au@Pt-TPP nanoprobe located to mitochondria and detected multiple ROS simultaneously in living cells under oxidative stimulation. Our method offers a new tool for the study of the functions of various ROS in biological processes.

Microfluidic single-cell transcriptomics: moving towards multimodal and spatiotemporal omics.

Cells are the basic units of life with vast heterogeneity. Single-cell transcriptomics unveils cell-to-cell gene expression variabilities, discovers novel cell types, and uncovers the critical roles of cellular heterogeneity in biological processes. The recent advances in microfluidic technologies have greatly accelerated the development of single-cell transcriptomics with regard to throughput, sensitivity, cost, and automation. In this article, we review state-of-the-art microfluidic single-cell transcriptomics, with a focus on the methodologies. We first summarize six typical microfluidic platforms for isolation and transcriptomic analysis of single cells. Then the on-going trend of microfluidic transcriptomics towards multimodal omics, which integrates transcriptomics with other omics to provide more comprehensive pictures of gene expression networks, is discussed. We also highlight single-cell spatial transcriptomics and single-cell temporal transcriptomics that provide unprecedented spatiotemporal resolution to reveal transcriptomic dynamics in space and time, respectively. The emerging applications of microfluidic single-cell transcriptomics are also discussed. Finally, we discuss the current challenges to be tackled and provide perspectives on the future development of microfluidic single-cell transcriptomics.

Stimulus-Responsive Microfluidic Interface Enables Efficient Enrichment and Cytogenetic Profiling of Circulating Myeloma Cells.

Minimal residual disease (MRD) provides an independent prognostic factor for multiple myeloma (MM) patients. However, clinical MRD assays suffer from highly invasive sampling, insufficient detection sensitivity, and high cost. Herein, a stiMulus-Responsive ligand-Decorated microfluidic chip (MRD-Chip) was developed for efficient capture and controlled release of circulating myeloma cells (CMCs) in the peripheral blood for noninvasive myeloma evaluation. The CD138 antibody-decorated herringbone chip with a disulfide linker was designed to enhance the collision probability between blood cells and capture antibodies, leading to high capture efficiency of CMCs. More importantly, the captured CMCs can be nondestructively released via a thiol-exchange reaction, allowing them to be used for subsequent cellular and molecular analysis. By fluorescence in situ hybridization assay, we successfully identified the cytogenetic abnormalities (chromosome 1q21 amplification and p53 deletion) of CMCs in clinical samples. Overall, with the merits of noninvasive sampling, high capture efficiency (70.93%), high throughput (1.5 mL/h), and nondestructive release of target cells (over 90% viability) for downstream analysis, our strategy provides new opportunities for myeloma evaluation, such as prognosis assessment, efficacy monitoring, and mechanism research of disease relapse and drug resistance.

Fe3O4@GO magnetic nanocomposites protect mesenchymal stem cells and promote osteogenic differentiation of rat bone marrow mesenchymal stem cells.

Fe3O4 nanoparticles (Fe3O4 NPs) are typical magnetic materials for bone tissue regeneration. However, the accompanying oxidative stress during the reaction process of Fe3O4 NPs and H2O2 in bone remodeling and disease may hinder their application. In order to reduce this side effect, we selected graphene oxide (GO) to modify Fe3O4 NPs. We showed that Fe3O4@GO magnetic nanocomposites (Fe3O4@GO MNCs) eliminated 30% of H2O2 in 3 h, and reduced the amount of ˙OH, the intermediate product of the Fenton reaction. The cellular study demonstrated that Fe3O4@GO MNCs reduced the cell damage caused by reactive oxygen species (ROS) and improved the activity of mesenchymal stem cells (MSCs). Moreover, when the magnetic field and bone morphogenetic protein-2 (BMP2) delivered by Fe3O4@GO MNCs worked together, osteogenic differentiation of MSCs in vitro was well promoted.

Integrated cascade nanozyme catalyzes in vivo ROS scavenging for anti-inflammatory therapy.

Here, an integrated cascade nanozyme with a formulation of Pt@PCN222-Mn is developed to eliminate excessive reactive oxygen species (ROS). This nanozyme mimics superoxide dismutase by incorporation of a Mn-[5,10,15,20-tetrakis(4-carboxyphenyl)porphyrinato]-based metal-organic framework compound capable of transforming oxygen radicals to hydrogen peroxide. The second mimicked functionality is that of catalase by incorporation of Pt nanoparticles, which catalyze hydrogen peroxide disproportionation to water and oxygen. Both in vitro and in vivo experimental measurements reveal the synergistic ROS-scavenging capacity of such an integrated cascade nanozyme. Two forms of inflammatory bowel disease (IBD; i.e., ulcerative colitis and Crohn's disease) can be effectively relieved by treatment with the cascade nanozyme. This study not only provides a new method for constructing enzyme-like cascade systems but also illustrates their efficient therapeutic promise in the treatment of in vivo IBDs.

Multifunctional STING-Activating Mn3 O4 @Au-dsDNA/DOX Nanoparticle for Antitumor Immunotherapy.

The promise of immunotherapy for cancer therapy has not been fully fulfilled because portions of tumors are immunosuppressive. To tackle this challenge, the initiation of immune system by stimulator of interferon genes (STING) pathway is explored and multifunctional STING-activating nanoparticles are rationally designed for synergistic antitumor therapy. The STING-activating nanoparticles have a formulation of Mn3 O4 @Au-dsDNA/DOX, where dsDNA is used to activate STING for immunotherapy and doxorubicin (DOX) is chosen as a model drug for chemotherapy. The STING-mediated immunity is activated, inducing interferon-ß (IFN-ß) production, increasing T cell priming, and enhancing effector T cell infiltration. Combined with chemotherapy, STING-mediated immunotherapy shows good antitumor efficacy by inhibiting tumor growth and prolonging survival rate in vivo. The promise of cancer immunotherapy can be fulfilled by combining novel antitumor immunity with innovative nanotechnology, and chemotherapy and targeted therapies.

Copper Tannic Acid Coordination Nanosheet: A Potent Nanozyme for Scavenging ROS from Cigarette Smoke.

The global tobacco epidemic is still a devastating threat to public health. Toxic reactive oxygen species (ROS) in the cigarette smoke cannot be efficiently eliminated by currently available cigarette filters. The resultant oxidative stress causes severe lung injury and further diseases. To tackle this challenge, herein, a novel copper tannic acid coordination (CuTA) nanozyme is reported as a highly active and thermostable ROS scavenger. The CuTA nanozyme exhibits intrinsic superoxide dismutase-like activity, catalase-like activity, and hydroxyl radical elimination capacity. These synergistic antioxidant abilities make the CuTA nanozyme a promising candidate for the improvement of commercial cigarette filters. Mouse model results show that commercial cigarettes loaded with CuTA nanozyme efficiently scavenge ROS in the cigarette smoke, reduce oxidative stress-induced lung inflammation, and minimize the resultant acute lung injury. The developed CuTA nanozyme offers an efficient ROS scavenger with multiple antioxidant ability and opens up new opportunities for the modification of cigarette filters to reduce the toxic effects of cigarette smoke.

eg occupancy as an effective descriptor for the catalytic activity of perovskite oxide-based peroxidase mimics.

A peroxidase catalyzes the oxidation of a substrate with a peroxide. The search for peroxidase-like and other enzyme-like nanomaterials (called nanozymes) mainly relies on trial-and-error strategies, due to the lack of predictive descriptors. To fill this gap, here we investigate the occupancy of eg orbitals as a possible descriptor for the peroxidase-like activity of transition metal oxide (including perovskite oxide) nanozymes. Both experimental measurements and density functional theory calculations reveal a volcano relationship between the eg occupancy and nanozymes' activity, with the highest peroxidase-like activities corresponding to eg occupancies of ~1.2. LaNiO3-δ, optimized based on the eg occupancy, exhibits an activity one to two orders of magnitude higher than that of other representative peroxidase-like nanozymes. This study shows that the eg occupancy is a predictive descriptor to guide the design of peroxidase-like nanozymes; in addition, it provides detailed insight into the catalytic mechanism of peroxidase-like nanozymes.

Nucleobase-mediated synthesis of nitrogen-doped carbon nanozymes as efficient peroxidase mimics.

Carbon nanozymes are catalytic carbon nanomaterials with intrinsic enzyme-like activities. They are advantageous over their natural counterparts in terms of higher stability, lower preparation cost, and better robustness. However, the peroxidase-like activities of the most developed carbon nanozymes were moderate due to the imperfection of active centers and limited tuning strategies. Herein, we designed a novel class of efficient peroxidase-mimicking carbon nanozymes with nitrogen atom doping. The N-doped carbon nanozymes were facilely synthesized by direct pyrolysis of different nucleobases at controlled temperatures without other treatments. A high ratio of nitrogen atoms was doped into the carbon skeleton. For example, 8.77 wt% of N remained in the guanine-derived carbon nanozyme with a pyrolysis temperature of 900 °C. The dominant graphitic N species greatly boosted the peroxidase-like activities of nucleobase-derived carbon nanozymes. Moreover, nucleobases are cheap, abundant, and environmentally friendly. We have demonstrated that nitrogen-rich nucleobases are ideal starting materials for the large-scale and cost-effective synthesis of N-doped carbon nanozymes. The carefully designed N-doped carbon nanozymes with superior activities were further used to construct effective biosensors for bioactive molecules (i.e., H2O2 and glucose). Highly sensitive and selective detection of H2O2 and glucose was achieved using the N-doped carbon nanozymes as efficient peroxidase mimics. This study offers an economical and sustainable approach for the scalable preparation of N-doped carbon nanozymes and creates a new path for the rational design of efficient peroxidase-mimicking carbon nanozymes by heteroatom doping.

ROS scavenging Mn3O4 nanozymes for in vivo anti-inflammation.

Reactive oxygen species (ROS)-induced oxidative stress is linked to various diseases, including cardiovascular disease and cancer. Though highly efficient natural ROS scavenging enzymes have been evolved, they are sensitive to environmental conditions and hard to mass-produce. Therefore, enormous efforts have been devoted to developing artificial enzymes with ROS scavenging activities. Among them, ROS scavenging nanozymes have recently attracted great interest owing to their enhanced stability, multi-functionality, and tunable activity. It has been implicated that Mn-contained nanozymes would possess efficient ROS scavenging activities, however only a few such nanozymes have been reported. To fill this gap, herein we demonstrated that Mn3O4 nanoparticles (NPs) possessed multiple enzyme mimicking activities (i.e., superoxide dismutase and catalase mimicking activities as well as hydroxyl radical scavenging activity). The Mn3O4 nanozymes therefore significantly scavenged superoxide radical as well as hydrogen peroxide and hydroxyl radical. Moreover, they were not only more stable than the corresponding natural enzymes but also superior to CeO2 nanozymes in terms of ROS elimination. We showed that the Mn3O4 NPs not only exhibited excellent ROS removal efficacy in vitro but also effectively protected live mice from ROS-induced ear-inflammation in vivo. These results indicated that Mn3O4 nanozymes are promising therapeutic nanomedicine for treating ROS-related diseases.

Monitoring of Heparin Activity in Live Rats Using Metal-Organic Framework Nanosheets as Peroxidase Mimics.

Metal-organic framework (MOF) nanosheets are a class of two-dimensional (2D) porous and crystalline materials that hold promise for catalysis and biodetection. Although 2D MOF nanosheets have been utilized for in vitro assays, ways of engineering them into diagnostic tools for live animals are much less explored. In this work, a series of MOF nanosheets are successfully engineered into a highly sensitive and selective diagnostic platform for in vivo monitoring of heparin (Hep) activity. The iron-porphyrin derivative is selected as a ligand to synthesize a series of archetypical MOF nanosheets with intrinsic heme-like catalytic sites, mimicking peroxidase. Hep-specific AG73 peptides as recognition motifs are physically adsorbed onto MOF nanosheets, blocking active sites from nonspecific substrate-catalyst interaction. Because of the highly specific interaction between Hep and AG73, the activity of AG73-MOF nanosheets is restored upon the binding of Hep, but not Hep analogues and other endogenous biomolecules. Furthermore, by taking advantages of biocompatibility and diagnostic property enabled by AG73-MOF nanosheets, the elimination process of Hep in live rats is quantitatively monitored by coupling with microdialysis technology. This work expands the biomedical applications of 2D MOF nanomaterials and provides access to a promising in vivo diagnostic platform.

Boosting the Peroxidase-Like Activity of Nanostructured Nickel by Inducing Its 3+ Oxidation State in LaNiO3 Perovskite and Its Application for Biomedical Assays.

Catalytic nanomaterials with intrinsic enzyme-like activities, called nanozymes, have recently attracted significant research interest due to their unique advantages relative to natural enzymes and conventional artificial enzymes. Among the nanozymes developed, particular interests have been devoted to nanozymes with peroxidase mimicking activities because of their promising applications in biosensing, bioimaging, biomedicine, etc. Till now, lots of functional nanomaterials have been used to mimic peroxidase. However, few studies have focused on the Ni-based nanomaterials for peroxidase mimics. In this work, we obtained the porous LaNiO3 nanocubes with high peroxidase-like activity by inducing its 3+ oxidation state in LaNiO3 perovskite and optimizing the morphology of LaNiO3 perovskite. The peroxidase mimicking activity of the porous LaNiO3 nanocubes with Ni3+ was about 58~fold and 22~fold higher than that of NiO with Ni2+ and Ni nanoparticles with Ni0. More, the porous LaNiO3 nanocubes exhibited about 2-fold higher activity when compared with LaNiO3 nanoparticles. Based on the superior peroxidase-like activity of porous LaNiO3 nanocubes, facile colorimetric assays for H2O2, glucose, and sarcosine detection were developed. Our present work not only demonstrates a useful strategy for modulating nanozymes' activities but also provides promising bioassays for clinical diagnostics.

Surface-Enhanced Raman Scattering Active Gold Nanoparticles with Enzyme-Mimicking Activities for Measuring Glucose and Lactate in Living Tissues.

Gold nanoparticles (AuNPs) with simultaneous plasmonic and biocatalytic properties provide a promising approach to developing versatile bioassays. However, the combination of AuNPs' intrinsic enzyme-mimicking properties with their surface-enhanced Raman scattering (SERS) activities has yet to be explored. Here we designed a peroxidase-mimicking nanozyme by in situ growing AuNPs into a highly porous and thermally stable metal-organic framework called MIL-101. The obtained AuNPs@MIL-101 nanozymes acted as peroxidase mimics to oxidize Raman-inactive reporter leucomalachite green into the active malachite green (MG) with hydrogen peroxide and simultaneously as the SERS substrates to enhance the Raman signals of the as-produced MG. We then assembled glucose oxidase (GOx) and lactate oxidase (LOx) onto AuNPs@MIL-101 to form AuNPs@MIL-101@GOx and AuNPs@MIL-101@LOx integrative nanozymes for in vitro detection of glucose and lactate via SERS. Moreover, the integrative nanozymes were further explored for monitoring the change of glucose and lactate in living brains, which are associated with ischemic stroke. The integrative nanozymes were then used to evaluate the therapeutic efficacy of potential drugs (such as astaxanthin for alleviating cerebral ischemic injuries) in living rats. They were also employed to determine glucose and lactate metabolism in tumors. This study not only demonstrated the great promise of combining AuNPs' multiple functionalities for versatile bioassays but also provided an interesting approach to designing nanozymes for biomedical and catalytic applications.