Imaging the dynamics of messenger RNA with a bright and stable green fluorescent RNA.

Fluorescent RNAs (FRs) provide an attractive approach to visualizing RNAs in live cells. Although the color palette of FRs has been greatly expanded recently, a green FR with high cellular brightness and photostability is still highly desired. Here we develop a fluorogenic RNA aptamer, termed Okra, that can bind and activate the fluorophore ligand ACE to emit bright green fluorescence. Okra has an order of magnitude enhanced cellular brightness than currently available green FRs, allowing the robust imaging of messenger RNA in both live bacterial and mammalian cells. We further demonstrate the usefulness of Okra for time-resolved measurements of ACTB mRNA trafficking to stress granules, as well as live-cell dual-color superresolution imaging of RNA in combination with Pepper620, revealing nonuniform and distinct distributions of different RNAs throughout the granules. The favorable properties of Okra make it a versatile tool for the study of RNA dynamics and subcellular localization.

A novel adhesive dual-sensitive hydrogel for sustained release of exosomes derived from M2 macrophages promotes repair of bone defects.

The repair of bone defects remains a huge clinical challenge. M2 macrophage-derived exosomes (M2-Exos) can act as immunomodulators to promote fracture healing; however, how to retain the sustained release of exosomes to the target area remains a challenge. Here, we report a composite hydrogel loaded with M2-Exos aiming to accelerate bone defect healing. It was verified that the F127/HA-NB hydrogel had a dense network structure, tissue adhesiveness, and dual sensitivity to temperature and light. F127/HA-NB loaded with M2-Exos (M2-Exos@F127/HA-NB) exhibited good biocompatibility and achieved sustained release of exosomes for up to two weeks. The study showed that both M0-Exos and M2-Exos@F127/HA-NB significantly promoted osteogenic differentiation of rat bone marrow mesenchymal stem cells. The mechanism study implied that M2-Exos activates the Wnt/ß-catenin signaling pathway to promote osteogenic differentiation of BMSCs. Finally, we evaluated the osteogenetic effects of M2-Exos@F127/HA-NB in a rat cranial defect model, and the results showed that M2-Exos@F127/HA-NB had superior bone regeneration-promoting effects. This study provides a new strategy for cell-free treatment of bone defects.

Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells.

Fluorescent RNAs, aptamers that bind and activate small fluorogenic dyes, have provided a particularly attractive approach to visualizing RNAs in live cells. However, the simultaneous imaging of multiple RNAs remains challenging due to a lack of bright and stable fluorescent RNAs with bio-orthogonality and suitable spectral properties. Here, we develop the Clivias, a series of small, monomeric and stable orange-to-red fluorescent RNAs with large Stokes shifts of up to 108 nm, enabling the simple and robust imaging of RNA with minimal perturbation of the target RNA's localization and functionality. In combination with Pepper fluorescent RNAs, the Clivias enable the single-excitation two-emission dual-color imaging of cellular RNAs and genomic loci. Clivias can also be used to detect RNA-protein interactions by bioluminescent imaging both in live cells and in vivo. We believe that these large Stokes shift fluorescent RNAs will be useful tools for the tracking and quantification of multiple RNAs in diverse biological processes.

3D Embedded Printing of Complex Biological Structures with Supporting Bath of Pluronic F-127.

Biofabrication is crucial in contemporary tissue engineering. The primary challenge in biofabrication lies in achieving simultaneous replication of both external organ geometries and internal structures. Particularly for organs with high oxygen demand, the incorporation of a vascular network, which is usually intricate, is crucial to enhance tissue viability, which is still a difficulty in current biofabrication technology. In this study, we address this problem by introducing an innovative three-dimensional (3D) printing strategy using a thermo-reversible supporting bath which can be easily removed by decreasing the temperature. This technology is capable of printing hydrated materials with diverse crosslinked mechanisms, encompassing gelatin, hyaluronate, Pluronic F-127, and alginate. Furthermore, the technology can replicate the external geometry of native tissues and organs from computed tomography data. The work also demonstrates the capability to print lines around 10 µm with a nozzle with a diameter of 60 µm due to the extra force exerted by the supporting bath, by which the line size was largely reduced, and this technique can be used to fabricate intricate capillary networks.

Rapid fabrication of physically robust hydrogels.

Hydrogel materials show promise for diverse applications, particular as biocompatible materials due to their high water content. Despite advances in hydrogel technology in recent years, their application is often severely limited by inadequate mechanical properties and time-consuming fabrication processes. Here we report a rapid hydrogel preparation strategy that achieves the simultaneous photo-crosslinking and establishment of biomimetic soft-hard material interface microstructures. These biomimetic interfacial-bonding nanocomposite hydrogels are prepared within seconds and feature clearly separated phases but have a strongly bonded interface. Due to effective interphase load transfer, biomimetic interfacial-bonding nanocomposite gels achieve an ultrahigh toughness (138 MJ m-3) and exceptional tensile strength (15.31 MPa) while maintaining a structural stability that rivals or surpasses that of commonly used elastomer (non-hydrated) materials. Biomimetic interfacial-bonding nanocomposite gels can be fabricated into arbitrarily complex structures via three-dimensional printing with micrometre-level precision. Overall, this work presents a generalizable preparation strategy for hydrogel materials and acrylic elastomers that will foster potential advances in soft materials.

One-Step Manufacturing of Supramolecular Liquid-Crystal Elastomers by Stress-Induced Alignment and Hydrogen Bond Exchange.

Liquid-crystal elastomers (LCEs) capable of performing large and reversible deformation in response to an external stimulus are an important class of soft actuators. However, their manufacturing process typically involves a multistep approach that requires harsh conditions. For the very first time, LCEs with customized geometries that can be manufactured by a rapid one-step approach at room temperature are developed. The LCEs are hydrogen bond (H-bond) crosslinked main chain polymers comprising flexible short side chains. Applying a stretching/shear force to the LCE can simultaneously induce mesogen alignment and H-bond exchange, allowing for the formation of well-aligned LCE networks stabilized by H-bonds. Based on this working principle, soft actuators in fibers and 2D/3D objects can be manufactured by mechanical stretching or melt extrusion within a short time (e.g. <1 min). These actuators can perform reversible macroscopic motions with large, controlled deformations up to 38 %. The dynamic nature of H-bonds also provides the actuators with reprocessability and reprogrammability. Thus, this work opens the way for the one-step and custom manufacturing of soft actuators.

Design of a palette of SNAP-tag mimics of fluorescent proteins and their use as cell reporters.

Naturally occurring fluorescent proteins (FPs) are the most widely used tools for tracking cellular proteins and sensing cellular events. Here, we chemically evolved the self-labeling SNAP-tag into a palette of SNAP-tag mimics of fluorescent proteins (SmFPs) that possess bright, rapidly inducible fluorescence ranging from cyan to infrared. SmFPs are integral chemical-genetic entities based on the same fluorogenic principle as FPs, i.e., induction of fluorescence of non-emitting molecular rotors by conformational locking. We demonstrate the usefulness of these SmFPs in real-time tracking of protein expression, degradation, binding interactions, trafficking, and assembly, and show that these optimally designed SmFPs outperform FPs like GFP in many important ways. We further show that the fluorescence of circularly permuted SmFPs is sensitive to the conformational changes of their fusion partners, and that these fusion partners can be used for the development of single SmFP-based genetically encoded calcium sensors for live cell imaging.

Correction: Reinforced hydrogel network building by a rapid dual-photo-coupling reaction for 3D printing.

Correction for 'Reinforced hydrogel network building by a rapid dual-photo-coupling reaction for 3D printing' by Renjie Zhou et al., Chem. Commun., 2023, 59, 1963-1966, https://doi.org/10.1039/D2CC05677A.

Imaging intracellular metabolite and protein changes in live mammalian cells with bright fluorescent RNA-based genetically encoded sensors.

Fluorescent RNA (FR)-based genetically encoded sensors have been engineered to detect various essential metabolites in living systems. However, the unfavorable characteristics of FR impede sensor applications. Here, we describe a strategy for converting Pepper fluorescent RNA into a series of fluorescent sensors to detect their cognate targets both in vitro and in live cells. Compared to previously developed FR-based sensors, Pepper-based sensors exhibited expanded emission of up to 620 nm and markedly improved cellular brightness, allowing robust and real-time monitoring of the pharmacologic-triggered dynamics changes in the intracellular level of S-adenosylmethionine (SAM) and the optogenetic manipulated protein translocation in live mammalian cells. Furthermore, signal amplification in fluorescence imaging of the target was achieved using the CRISPR-display strategy by incorporating a Pepper-based sensor into the sgRNA scaffold. Together, these results demonstrate that Pepper can be readily developed into high-performance FR-based sensors to detect various cellular targets.

Visible-light activatable coumarin-based phototriggers for fluorescence imaging with ultra-high photolysis efficiency.

Photoactivated fluorophores (PAFs) are powerful imaging tools for observing subcellular structures and tracking dynamic biological processes. However, photoremovable protecting groups (PPGs) widely used to construct PAFs suffer from the drawbacks of short-wavelength excitation and/or low photolysis efficiency. Herein, a class of coumarin-based PPGs with electron-rich thiophene derived substitutions at the C3-position of a coumarin scaffold were prepared. The modification not only leads to the redshift of the absorption band to the blue light region (400-500 nm), but also the increases of uncaging quantum yield (Φu) as well as molar extinction coefficient (εmax), thus enhancing the photolysis efficiency (Φu × Îµmax) up to 34.2 × 103 M-1 cm-1. The exceptionally high photolysis efficiency enables efficient photolysis in blue light as weak as 2 mW cm-2 or in blue light from a Luminol chemiluminescence system. Based on the excellent photolysis properties, the PAF constructed by the new PPG exhibits fast photoactivation and a low background signal, and the resulting fluorescence images display a signal-to-noise ratio greater than 780. It is anticipated that the superior photolysis performance makes the PPGs a novel platform for the construction of photo responsive systems in a variety of applications.

A rigid-axle-based molecular rotaxane channel facilitates K+/Cl- co-transport across a lipid membrane.

Inspired by the design criteria of heteroditopic receptors for ion-pair binding, we herein describe a new strategy to construct a rotaxane transporter (RR[2]) for K+/Cl- co-transport. The use of a rigid axle improves the transport activity with an EC50 value of 0.58 µM, presenting a significant step toward developing rotaxane artificial channels.

A Facile and Versatile Approach to Construct Photoactivated Peptide Hydrogels by Regulating Electrostatic Repulsion.

Short peptides that can respond to external stimuli have been considered as the preferred building blocks to construct hydrogels for biomedical applications. In particular, photoresponsive peptides that are capable of triggering the formation of hydrogels upon light irradiation allow the properties of hydrogels to be changed remotely by precise and localized actuation. Here, we used the photochemical reaction of the 2-nitrobenzyl ester group (NB) to develop a facile and versatile strategy for constructing photoactivated peptide hydrogels. The peptides with high aggregation propensity were designed as hydrogelators, which were photocaged by a positively charged dipeptide (KK) to provide strong charge repulsion and prevent self-assembly in water. Light irradiation led to the removal of KK and triggered the self-assembly of peptides and the formation of hydrogel. Light stimulation endows spatial and temporal control, which enables the formation of hydrogel with precisely tunable structure and mechanical properties. Cell culture and behavior study indicated that the optimized photoactivated hydrogel was suitable for 2D and 3D cell culture, and its photocontrollable mechanical strength could regulate the spreading of stem cells on its surface. Therefore, our strategy provides an alternative way to construct photoactivated peptide hydrogels with wide applications in biomedical areas.

A light-activated polymer with excellent serum tolerance for intracellular protein delivery.

The design of efficient materials for intracellular protein delivery has attracted great interest in recent years; however, most current materials for this purpose are limited by poor serum stability due to the early release of cargoes triggered by abundant serum proteins. Here, we propose a light-activated crosslinking (LAC) strategy to prepare efficient polymers with excellent serum tolerance for intracellular protein delivery. A cationic dendrimer engineered with photoactivatable O-nitrobenzene moieties co-assembles with cargo proteins via ionic interactions, followed by light activation to yield aldehyde groups on the dendrimer and the formation of imine bonds with cargo proteins. The light-activated complexes show high stability in buffer and serum solutions, but dis-assemble under low pH conditions. As a result, the polymer successfully delivers cargo proteins green fluorescent protein and ß-galactosidase into cells with maintained bioactivity even in the presence of 50% serum. The LAC strategy proposed in this study provides a new insight to improve the serum stability of polymers for intracellular protein delivery.

Reinforced hydrogel network building by a rapid dual-photo-coupling reaction for 3D printing.

A facile hydrogel fabrication strategy based on a simultaneous dual-photo-coupling reaction (i.e., photoinduced S-nitrosylation and Schiff base reaction) was reported. This strategy allowed a strengthened three-arm crosslinking network to form in one step and the hydrogels obtained displayed rapid gelation, excellent mechanical strength and biocompatibility for cell encapsulated-3D printing in real time. Our hydrogel fabrication strategy will likely foster advances in biomaterials and the extreme speed and reinforced mechanical strength should significantly benefit 3D printing and related applications.

Continuous Glucose Monitoring Enabled by Fluorescent Nanodiamond Boronic Hydrogel.

Continuous monitoring of glucose allows diabetic patients to better maintain blood glucose level by altering insulin dosage or diet according to prevailing glucose values and thus to prevent potential hyperglycemia and hypoglycemia. However, current continuous glucose monitoring (CGM) relies mostly on enzyme electrodes or micro-dialysis probes, which suffer from insufficient stability, susceptibility to corrosion of electrodes, weak or inconsistent correlation, and inevitable interference. A fluorescence-based glucose sensor in the skin will likely be more stable, have improved sensitivity, and can resolve the issues of electrochemical interference from the tissue. This study develops a fluorescent nanodiamond boronic hydrogel system in porous microneedles for CGM. Fluorescent nanodiamond is one of the most photostable fluorophores with superior biocompatibility. When surface functionalized, the fluorescent nanodiamond can integrate with boronic polymer and form a hydrogel, which can produce fluorescent signals in response to environmental glucose concentration. In this proof-of-concept study, the strategy for building a miniatured device with fluorescent nanodiamond hydrogel is developed. The device demonstrates remarkable long-term photo and signal stability in vivo with both small and large animal models. This study presents a new strategy of fluorescence based CGM toward treatment and control of diabetes.

Microfluidic-templated cell-laden microgels fabricated using phototriggered imine-crosslinking as injectable and adaptable granular gels for bone regeneration.

Injectable granular gels consisting of densely packed microgels serving as scaffolding biomaterial have recently shown great potential for applications in tissue regeneration, which allow administration via minimally invasive surgery, on-target cargo delivery, and high efficiency in nutrient/waste exchange. However, limitations such as insufficient mechanical strength, structural integrity, and uncontrollable differentiation of the encapsulated cells in the scaffolds hamper their further applications in the biomedical field. Herein, we developed a new class of granular gels via bottom-up assembly of cell-laden microgels via photo-triggered imine-crosslinking (PIC) chemistry based on the microfluidic technique. The particulate nature of the granular gels rendered them with shear-thinning and self-healing behavior, thereby functioning as an injectable and adaptable cellularized scaffold for bone tissue regeneration. Specifically, single cell-laden, monodisperse microgels composed of methacrylate- and o-nitrobenzene-functionalized hyaluronic acid and gelatin were prepared using a high-throughput microfluidic technique with a production rate up to 3.7 × 108 microgels/hr, wherein the PIC chemistry alleviated the oxygen inhibition on free-radical polymerization and facilitated enhanced fabrication accuracy, accelerated gelation rate, and improved network strength. Further in vitro and in vivo studies demonstrated that the microgels can serve as carriers to support the activity of the encapsulated mesenchymal stem cells; these cell-laden microgels can also be used as cellularized bone fillers to induce the regeneration of bone tissues as evidenced by the in vivo experiment using the rat femoral condyle defect model. In general, these results represent a significant step toward the precise fabrication of engineered tissue mimics with single-cell resolution and high cell-density and can potentially offer a powerful tool for the design and applications of a next generation of tissue engineering strategy. STATEMENT OF SIGNIFICANCE: Using microfluidic droplet-based technology, we hereby developed a new class of injectable and moldable granular gels via bottom-up assembly of cell-laden microgels as a versatile platform for tissue regeneration. Phototriggered imine-crosslinking chemistry was introduced for microgel cross-linkage, which allowed for the fabrication of microgels with improved matrix homogeneity, accelerated gelation process, and enhanced mechanical strength. We demonstrated that the microgel building blocks within the granular gels facilitated the proliferation and differentiation of the encapsulated mesenchymal stem cells, which can further serve as a cellularized scaffold for the treatment of bone defects.

Ultrasensitive sensors reveal the spatiotemporal landscape of lactate metabolism in physiology and disease.

Despite its central importance in cellular metabolism, many details remain to be determined regarding subcellular lactate metabolism and its regulation in physiology and disease, as there is sensitive spatiotemporal resolution of lactate distribution, and dynamics remains a technical challenge. Here, we develop and characterize an ultrasensitive, highly responsive, ratiometric lactate sensor, named FiLa, enabling the monitoring of subtle lactate fluctuations in living cells and animals. Utilizing FiLa, we demonstrate that lactate is highly enriched in mammalian mitochondria and compile an atlas of subcellular lactate metabolism that reveals lactate as a key hub sensing various metabolic activities. In addition, FiLa sensors also enable direct imaging of elevated lactate levels in diabetic mice and facilitate the establishment of a simple, rapid, and sensitive lactate assay for point-of-care clinical screening. Thus, FiLa sensors provide powerful, broadly applicable tools for defining the spatiotemporal landscape of lactate metabolism in health and disease.

In vitro Cartilage Regeneration Regulated by a Hydrostatic Pressure Bioreactor Based on Hybrid Photocrosslinkable Hydrogels.

Because of the superior characteristics of photocrosslinkable hydrogels suitable for 3D cell-laden bioprinting, tissue regeneration based on photocrosslinkable hydrogels has become an important research topic. However, due to nutrient permeation obstacles caused by the dense networks and static culture conditions, there have been no successful reports on in vitro cartilage regeneration with certain thicknesses based on photocrosslinkable hydrogels. To solve this problem, hydrostatic pressure (HP) provided by the bioreactor was used to regulate the in vitro cartilage regeneration based on hybrid photocrosslinkable (HPC) hydrogel. Chondrocyte laden HPC hydrogels (CHPC) were cultured under 5 MPa HP for 8 weeks and evaluated by various staining and quantitative methods. Results demonstrated that CHPC can maintain the characteristics of HPC hydrogels and is suitable for 3D cell-laden bioprinting. However, HPC hydrogels with concentrations over 3% wt% significantly influenced cell viability and in vitro cartilage regeneration due to nutrient permeation obstacles. Fortunately, HP completely reversed the negative influences of HPC hydrogels at 3% wt%, significantly enhanced cell viability, proliferation, and extracellular matrix (ECM) deposition by improving nutrient transportation and up-regulating the expression of cartilage-specific genes, and successfully regenerated homogeneous cartilage with a thickness over 3 mm. The transcriptome sequencing results demonstrated that HP regulated in vitro cartilage regeneration primarily by inhibiting cell senescence and apoptosis, promoting ECM synthesis, suppressing ECM catabolism, and ECM structure remodeling. Evaluation of in vivo fate indicated that in vitro regenerated cartilage in the HP group further developed after implantation and formed homogeneous and mature cartilage close to the native one, suggesting significant clinical potential. The current study outlines an efficient strategy for in vitro cartilage regeneration based on photocrosslinkable hydrogel scaffolds and its in vivo application.

A Light-Driven Molecular Machine Controls K+ Channel Transport and Induces Cancer Cell Apoptosis.

The design of artificial ion channels with high activity, selectivity and gating function is challenging. Herein, we designed the light-driven motor molecule MC2, which provides new design criteria to overcome these challenges. MC2 forms a selective K+ channel through a single molecular transmembrane mechanism, and the light-driven rotary motion significantly accelerates ion transport, which endows the irradiated motor molecule with excellent cytotoxicity and cancer cell selectivity. Mechanistic studies reveal that the rotary motion of MC2 promotes K+ efflux, generates reactive oxygen species and eventually activates caspase-3-dependent apoptosis in cancer cells. Combined with the spatiotemporally controllable advantages of light, we believe this strategy can be exploited in the structural design and application of next-generation synthetic cation transporters for the treatment of cancer and other diseases.

A system for artificial light signal transduction via molecular translocation in a lipid membrane.

Light signal transduction pathways are the central components of mechanisms that regulate plant development, in which photoreceptors receive light and participate in light signal transduction. Chemical systems can be designed to mimic these biological processes that have potential applications in smart sensing, drug delivery and synthetic biology. Here, we synthesized a series of simple photoresponsive molecules for use as photoreceptors in artificial light signal transduction. The hydrophobic structures of these molecules facilitate their insertion into vesicular lipid bilayers, and reversible photoisomerization initiates the reciprocating translocation of molecules in the membrane, thus activating or deactivating the hydrolysis reaction of a precatalyst in the transducer for an encapsulated substrate, resulting in a light controllable output signal. This study represents the first example of using simplified synthetic molecules to simulate light signal transduction performed by complex biomolecules.