In situ cell condensation-based cartilage tissue engineering via immediately implantable high-density stem cell core and rapidly degradable shell microgels.

Formation of chondromimetic human mesenchymal stem cells (hMSCs) condensations typically required in vitro culture in defined environments. In addition, extended in vitro culture in differentiation media over several weeks is usually necessary prior to implantation, which is costly, time consuming and delays clinical treatment. Here, this study reports on immediately implantable core/shell microgels with a high-density hMSC-laden core and rapidly degradable hydrogel shell. The hMSCs in the core formed cell condensates within 12 hours and the oxidized and methacrylated alginate (OMA) hydrogel shells were completely degraded within 3 days, enabling spontaneous and precipitous fusion of adjacent condensed aggregates. By delivering transforming growth factor-ß1 (TGF-ß1) within the core, the fused condensates were chondrogenically differentiated and formed cartilage microtissues. Importantly, these hMSC-laden core/shell microgels, fabricated without any in vitro culture, were subcutaneously implanted into mice and shown to form cartilage tissue via cellular condensations in the core after 3 weeks. This innovative approach to form cell condensations in situ without in vitro culture that can fuse together with each other and with host tissue and be matured into new tissue with incorporated bioactive signals, allows for immediate implantation and may be a platform strategy for cartilage regeneration and other tissue engineering applications.

Response strategies and biological applications of organic fluorescent thermometry: cell- and mitochondrion-level detection.

Temperature homeostasis is critical for cells to perform their physiological functions. Among the diverse methods for temperature detection, fluorescent temperature probes stand out as a proven and effective tool, especially for monitoring temperature in cells and suborganelles, with a specific emphasis on mitochondria. The utilization of these probes provides a new opportunity to enhance our understanding of the mechanisms and interconnections underlying various physiological activities related to temperature homeostasis. However, the complexity and variability of cells and suborganelles necessitate fluorescent temperature probes with high resolution and sensitivity. To meet the demanding requirements for intracellular/subcellular temperature detection, several strategies have been developed, offering a range of options to address this challenge. This review examines four fundamental temperature-response strategies employed by small molecule and polymer probes, including intramolecular rotation, polarity sensitivity, Förster resonance energy transfer, and structural changes. The primary emphasis was placed on elucidating molecular design and biological applications specific to each type of probe. Furthermore, this review provides an insightful discussion on factors that may affect fluorescent thermometry, providing valuable perspectives for future development in the field. Finally, the review concludes by presenting cutting-edge response strategies and research insights for mitigating biases in temperature sensing.

The involvement of the mitochondrial membrane in drug delivery.

Treatment effectiveness and biosafety are critical for disease therapy. Bio-membrane modification facilitates the homologous targeting of drugs in vivo by exploiting unique antibodies or antigens, thereby enhancing therapeutic efficacy while ensuring biosafety. To further enhance the precision of disease treatment, future research should shift focus from targeted cellular delivery to targeted subcellular delivery. As the cellular powerhouses, mitochondria play an indispensable role in cell growth and regulation and are closely involved in many diseases (e.g., cancer, cardiovascular, and neurodegenerative diseases). The double-layer membrane wrapped on the surface of mitochondria not only maintains the stability of their internal environment but also plays a crucial role in fundamental biological processes, such as energy generation, metabolite transport, and information communication. A growing body of evidence suggests that various diseases are tightly related to mitochondrial imbalance. Moreover, mitochondria-targeted strategies hold great potential to decrease therapeutic threshold dosage, minimize side effects, and promote the development of precision medicine. Herein, we introduce the structure and function of mitochondrial membranes, summarize and discuss the important role of mitochondrial membrane-targeting materials in disease diagnosis/treatment, and expound the advantages of mitochondrial membrane-assisted drug delivery for disease diagnosis, treatment, and biosafety. This review helps readers understand mitochondria-targeted therapies and promotes the application of mitochondrial membranes in drug delivery. STATEMENT OF SIGNIFICANCE: Bio-membrane modification facilitates the homologous targeting of drugs in vivo by exploiting unique antibodies or antigens, thereby enhancing therapeutic efficacy while ensuring biosafety. Compared to cell-targeted treatment, targeting of mitochondria for drug delivery offers higher efficiency and improved biosafety and will promote the development of precision medicine. As a natural material, the mitochondrial membrane exhibits excellent biocompatibility and can serve as a carrier for mitochondria-targeted delivery. This review provides an overview of the structure and function of mitochondrial membranes and explores the potential benefits of utilizing mitochondrial membrane-assisted drug delivery for disease treatment and biosafety. The aim of this review is to enhance readers' comprehension of mitochondrial targeted therapy and to advance the utilization of mitochondrial membrane in drug delivery.

Gene and Photothermal Combination Therapy: Principle, Materials, and Amplified Anticancer Intervention.

Gene therapy (GT) and photothermal therapy (PTT) have emerged as promising alternatives to chemotherapy and radiotherapy for cancer treatment, offering noninvasiveness and reduced side effects. However, their efficacy as standalone treatments is limited. GT exhibits slow response rates, while PTT is confined to local tumor ablation. The convergence of GT and PTT, known as GT-PTT, facilitated by photothermal gene nanocarriers, has attracted considerable attention across various disciplines. In this integrated approach, GT reciprocates PTT by sensitizing cellular response to heat, while PTT benefits GT by improving gene translocation, unpacking, and expression. Consequently, this integration presents a unique opportunity for cancer therapy with rapid response and improved effectiveness. Extensive efforts over the past few years have been dedicated to the development of GT-PTT, resulting in notable achievements and rapid progress from the laboratory to potential clinical applications. This comprehensive review outlines recent advances in GT-PTT, including synergistic mechanisms, material systems, imaging-guided therapy, and anticancer applications. It also explores the challenges and future prospects in this nascent field. By presenting innovative ideas and insights into the implementation of GT-PTT for enhanced cancer therapy, this review aims to inspire further progress in this promising area of research.

Rational Design and Biological Application of Hybrid Fluorophores.

Fluorophores are considered powerful tools for not only enabling the visualization of cell structures, substructures, and biological processes, but also making for the quantitative and qualitative measurement of various analytes in living systems. However, most fluorophores do not meet the diverse requirements for biological applications in terms of their photophysical and biological properties. Hybridization is an important strategy in molecular engineering that provides fluorophores with complementarity and multifunctionality. This review summarizes the basic strategies of hybridization with four classes of fluorophores, including xanthene, cyanine, coumarin, and BODIPY with a focus on their structure-property relationship (SPR) and biological applications. This review aims to provide rational hybrid ideas for expanding the reservoir of knowledge regarding fluorophores and promoting the development of newly produced fluorophores for applications in the field of life sciences.

Small-molecule probes from bench to bedside: advancing molecular analysis of drug-target interactions toward precision medicine.

Over the past decade, remarkable advances have been witnessed in the development of small-molecule probes. These molecular tools have been widely applied for interrogating proteins, pathways and drug-target interactions in preclinical research. While novel structures and designs are commonly explored in probe development, the clinical translation of small-molecule probes remains limited, primarily due to safety and regulatory considerations. Recent synergistic developments - interfacing novel chemical probes with complementary analytical technologies - have introduced and expedited diverse biomedical opportunities to molecularly characterize targeted drug interactions directly in the human body or through accessible clinical specimens (e.g., blood and ascites fluid). These integrated developments thus offer unprecedented opportunities for drug development, disease diagnostics and treatment monitoring. In this review, we discuss recent advances in the structure and design of small-molecule probes with novel functionalities and the integrated development with imaging, proteomics and other emerging technologies. We further highlight recent applications of integrated small-molecule technologies for the molecular analysis of drug-target interactions, including translational applications and emerging opportunities for whole-body imaging, tissue-based measurement and blood-based analysis.

Ultrafast Detection of Monoamine Oxidase A in Live Cells and Clinical Glioma Tissues Using an Affinity Binding-Based Two-Photon Fluorogenic Probe.

Abnormal expression of monoamine oxidase A (MAO-A) has been implicated in the development of human glioma, making MAO-A a promising target for therapy. Therefore, a rapid determination of MAO-A is critical for diagnosis. Through in silico screening of two-photon fluorophores, we discovered that a derivative of N,N-dimethyl-naphthalenamine (pre-mito) can effectively fit into the entrance of the MAO-A cavity. Substitutions on the N-pyridine not only further explore the MAO-A cavity, but also enable mitochondrial targeting ability. The aminopropyl substituted molecule, CD1, showed the fastest MAO-A detection (within 20 s), high MAO-A affinity and selectivity. It was also used for in situ imaging of MAO-A in living cells, enabling a comparison of the MAO-A content in human glioma and paracancerous tissues. Our results demonstrate that optimizing the affinity binding-based fluorogenic probes significantly improves their detection rate, providing a general approach for rapid detection probe design and optimization.

Red-emissive Dual-state Fluorogenic Probe for Wash-free Imaging of Lipid Droplets in Living Cells and Fatty Liver Tissues.

Lipid droplet (LD) dysfunction can result in various diseases, such as nonalcoholic fatty liver disease. Imaging agents built on dual-state emission (DSE) molecules that fluoresce in both dilute solutions and the aggregated state are receiving attention as this type of probe could provide bright fluorescence signals at variable concentrations, avoiding false signal readout caused by the concentration fluctuation in living systems. Herein, we identified a red emissive molecule featuring DSE, from three newly synthesized molecules, for specific detection of LDs in live cells. The bioimaging abilities have been well confirmed by optical spectroscopies, theoretical calculations, cell experiments, as well as animal studies. The DSE probe is effective for LD detection at concentrations ranging from 1 µM to 100 µM while retaining high brightness and signal fidelity. This study provides a knowledge base for the future design of DSE-active fluorescent probes for understanding LD-related diseases.

Conjugating Coumarin with Tetraphenylethylene to Achieve Dual-State Emission for Reversible Mechanofluorochromism and Live Cell Imaging.

Dual-state emission luminogens (DSEgens) are receiving research interest in the construction of multifunctional materials due to their inherent advantage of high emission efficiency in both the molecularly dispersed solution state and the solid state. However, it remains challenging in synthesizing DSEgens via a delicate manipulation of the molecular structures. This work presents an example of bright DSEgen synthesis by tuning the molecular electronic structures and conformations. Three coumarin-tetraphenylethylene (TPE) molecules with a donor-acceptor electronic structure and highly twisting conformations have been synthesized. While compound resulting from direct conjugation of coumarin with a TPE unit shows aggregation-induced emission, compound with an N,N-diaminoethyl modification on the 7-position of coumarin and compound with a further phenyl linker between coumarin and TPE units feature strong dual-state emission. Benefiting from their strong solid emission and twisting conformations, these fluorophores display reversible mechanofluorochromism. Finally, applications for rewritable information storage in the solid state and live-cell imaging in the solution state were demonstrated.

4D Cell-Condensate Bioprinting.

4D bioprinting techniques that facilitate formation of shape-changing scaffold-free cell condensates with prescribed geometries have yet been demonstrated. Here, a simple 4D bioprinting approach is presented that enables formation of a shape-morphing cell condensate-laden bilayer system. The strategy produces scaffold-free cell condensates which morph over time into predefined complex shapes. Cell condensate-laden bilayers with specific geometries are readily fabricated by bioprinting technologies. The bilayers have tunable deformability and microgel (MG) degradation, enabling controllable morphological transformations and on-demand liberation of deformed cell condensates. With this system, large cell condensate-laden constructs with various complex shapes are obtained. As a proof-of-concept study, the formation of the letter "C"- and helix-shaped robust cartilage-like tissues differentiated from human mesenchymal stem cells (hMSCs) is demonstrated. This system brings about a versatile 4D bioprinting platform idea that is anticipated to broaden and facilitate the applications of cell condensation-based 4D bioprinting.

A NIR Aggregation-Induced Emission Fluoroamphiphile as Visually Trackable and Serum-Tolerant Nonviral Gene Carrier.

Functional fluorescence (FL) nonviral gene vectors with high serum tolerance bear broad application prospects in gene delivery. Fluorination has been widely utilized as an effective strategy to enhance serum tolerance. Herein, we show the combination of fluorination and aggregation-induced emission (AIE) for the construction of a nonviral gene vector with low cytotoxicity, visual tracking ability, and high serum tolerance. Large π-conjugation triphenylamine (TPA) derivative with a characteristic D-π-A structure was modified with two polar [12]aneN3 heads and a long fluorocarbon tail, giving the vector molecule FluoTPA. FluoTPA features near-infrared (NIR) emission, large Stokes shift, and strong binding affinity toward nucleic acids. Liposomes consisting of FluoTPA and dioleoylphosphatidylethanolamine (DOPE) (FluoTPA/DOPE) can effectively deliver both plasmid DNAs (pDNAs) and siRNAs into cells. Impressively, FluoTPA/DOPE showed comparable transfection efficiency (TE) in the presence of serum content up to 30% with that in the serum-free condition and achieved 7.4 times higher TE than the commercial transfection agent lipofectamine 2000 at the same condition. Finally, spatiotemporal tracking of the delivery process in cells was demonstrated. The results in this work suggest that FluoTPA could be a reliable theranostic platform for the nonviral delivery of nucleic acid therapeutics in serum condition.

Alkyl-Engineered Dual-State Luminogens with Pronounced Odd-Even Effects: Quantum Yields with up to 48% Difference and Crystallochromy with up to 22 nm Difference.

Alkyl chain-resulted odd-even effects in fluorescence quantum yield (FLQY) have also been reported in organic luminescent materials (OLMs). However, the odd-even effects in FLQY caused by the alkyl substitutes in OLMs are generally very weak, with only single-digit differences. Here, we report a series of alkyl-substituted dual-state luminogens (DSEgens) showing extremely high solid-state FLQY in even-numbered analogues (>90% FLQY) and a dramatically pronounced odd-even effect in FLQY. The odd-even effect in FLQY is over 26% alternation, and a maximum of 48% difference in FLQY was observed between the compounds C1 and C2 with a methyl and ethyl substitution, respectively. C1 and C2 also displayed a crystallochromy with a 22 nm difference in emission wavelength. In addition, odd-even effects in the melting point and decomposition temperature were also observed. With these bright DSEgens, applications such as specific recognition of picric acid and ultrasensitive trace water detection have been demonstrated.

Jammed Micro-Flake Hydrogel for Four-Dimensional Living Cell Bioprinting.

4D bioprinting is promising to build cell-laden constructs (bioconstructs) with complex geometries and functions for tissue/organ regeneration applications. The development of hydrogel-based 4D bioinks, especially those allowing living cell printing, with easy preparation, defined composition, and controlled physical properties is critically important for 4D bioprinting. Here, a single-component jammed micro-flake hydrogel (MFH) system with heterogeneous size distribution, which differs from the conventional granular microgel, has been developed as a new cell-laden bioink for 4D bioprinting. This jammed cytocompatible MFH features scalable production and straightforward composition with shear-thinning, shear-yielding, and rapid self-healing properties. As such, it can be smoothly printed into stable 3D bioconstructs, which can be further cross-linked to form a gradient in cross-linking density when a photoinitiator and a UV absorber are incorporated. After being subject to shape morphing, a variety of complex bioconstructs with well-defined configurations and high cell viability are obtained. Based on this system, 4D cartilage-like tissue formation is demonstrated as a proof-of-concept. The establishment of this versatile new 4D bioink system may open up a number of applications in tissue engineering.

4D biofabrication via instantly generated graded hydrogel scaffolds.

Formation of graded biomaterials to render shape-morphing scaffolds for 4D biofabrication holds great promise in fabrication of complex structures and the recapitulation of critical dynamics for tissue/organ regeneration. Here we describe a facile generation of an adjustable and robust gradient using a single- or multi-material one-step fabrication strategy for 4D biofabrication. By simply photocrosslinking a mixed solution of a photocrosslinkable polymer macromer, photoinitiator (PI), UV absorber and live cells, a cell-laden gradient hydrogel with pre-programmable deformation can be generated. Gradient formation was demonstrated in various polymers including poly(ethylene glycol) (PEG), alginate, and gelatin derivatives using various UV absorbers that present overlap in UV spectrum with that of the PI UV absorbance spectrum. Moreover, this simple and effective method was used as a universal platform to integrate with other hydrogel-engineering techniques such as photomask-aided microfabrication, photo-patterning, ion-transfer printing, and 3D bioprinting to fabricate more advanced cell-laden scaffold structures. Lastly, proof-of-concept 4D tissue engineering was demonstrated in a study of 4D bone-like tissue formation. The strategy's simplicity along with its versatility paves a new way in solving the hurdle of achieving temporal shape changes in cell-laden single-component hydrogel scaffolds and may expedite the development of 4D biofabricated constructs for biological applications.

Induction of 4D spatiotemporal geometric transformations in high cell density tissues via shape changing hydrogels.

Developing and healing tissues begin as a cellular condensation. Spatiotemporal changes in tissue geometry, transformations in the spatial distribution of the cells and extracellular matrix, are essential for its evolution into a functional tissue. 4D materials, 3D materials capable of geometric changes, may have the potential to recreate the aforementioned biological phenomenon. However, most reported 4D materials are non-degradable and/or not biocompatible, which limits their application in regenerative medicine, and to date there are no systems controlling the geometry of high density cellular condensations and differentiation. Here, we describe 4D high cell density tissues based on shape-changing hydrogels. By sequential photocrosslinking of oxidized and methacrylated alginate (OMA) and methacrylated gelatin (GelMA), bi-layered hydrogels presenting controllable geometric changes without any external stimuli were fabricated. Fibroblasts and human adipose-derived stem cells (ASCs) were incorporated at concentrations up to 1.0 × 108 cells/mL to the 4D constructs, and controllable shape changes were achieved in concert with ASCs differentiated down chondrogenic and osteogenic lineages. Bioprinting of the high density cell-laden OMA and GelMA permitted the formation of more complex constructs with defined 4D geometric changes, which may further expand the promise of this approach in regenerative medicine applications.

Cell-Laden Multiple-Step and Reversible 4D Hydrogel Actuators to Mimic Dynamic Tissue Morphogenesis.

Shape-morphing hydrogels bear promising prospects as soft actuators and for robotics. However, they are mostly restricted to applications in the abiotic domain due to the harsh physicochemical conditions typically necessary to induce shape morphing. Here, multilayer hydrogel actuator systems are developed using biocompatible and photocrosslinkable oxidized, methacrylated alginate and methacrylated gelatin that permit encapsulation and maintenance of living cells within the hydrogel actuators and implement programmed and controlled actuations with multiple shape changes. The hydrogel actuators encapsulating cells enable defined self-folding and/or user-regulated, on-demand-folding into specific 3D architectures under physiological conditions, with the capability to partially bioemulate complex developmental processes such as branching morphogenesis. The hydrogel actuator systems can be utilized as novel platforms for investigating the effect of programmed multiple-step and reversible shape morphing on cellular behaviors in 3D extracellular matrix and the role of recapitulating developmental and healing morphogenic processes on promoting new complex tissue formation.

Two-Photon Near-Infrared AIE Luminogens as Multifunctional Gene Carriers for Cancer Theranostics.

Construction of multifunctional nonviral gene vectors to execute defined tasks holds great potential for the precise and effective treatment of gene-associated diseases. Herein, we have developed four large π-conjugation triphenylamine derivatives bearing two polar [12]aneN3 heads and a lipophilic tail for applications in gene delivery, one/two-photon-triggered near-infrared (NIR) fluorescence bioimaging, and combined photodynamic therapy (PDT) and gene therapy of cancer. These compounds possess typical NIR aggregation-induced emission characteristics, mega Stokes shifts, strong two-photon excitation fluorescence, and excellent DNA condensation abilities. Among them, vector 4 with a tail of n-hexadecane realized a transfection efficiency as high as 6.7 times that of the commercial transfection agent Lipofectamine 2000 in HEK293T cell lines. Using vector 4 as an example, transfection process tracking and ex vivo/in vivo tumoral imaging and retention with high resolution, high brightness, deep tissue penetration, and good biosafety were demonstrated. In addition, efficient singlet oxygen (1O2) generation by the DNA complex formed by vector 4 (4/DNA) resulted in effective PDT. Combined with anticancer gene therapy, collaborative cancer treatment with a dramatically enhanced cancer cell-killing effect was achieved. The development of this "three birds, one stone" approach suggests a new and promising strategy for better cancer treatment and real-time tracking of gene delivery.

A 1,8-naphthalimide-[12]aneN3 derivative for efficient Cu2+ recognition, lysosome staining and siRNA delivery.

Development of multifunctional compounds as both fluorescence probes and non-viral vectors is still difficult till date. It is necessary to overcome many hurdles such as the balance of hydrophilic and hydrophobic moieties, binding affinity between multifunctional compound and targeting substrate, the cytotoxicity of multifunctional compound, and so on. In this work, the performances of compound 1 on Cu2+ recognition, lysosome staining and siRNA (small interfering RNA) delivery were investigated. It was found that compound 1 exhibited high selectivity and sensitivity toward Cu2+ in aqueous solutions. The fluorescence emission of 1 was quenched by a factor of 42-fold in the presence of Cu2+ ions. Even in the common pure organic solutions, still more than 8-fold fluorescence quenching was achieved. Due to its high sensitivity to the pH, the complex of 1-Cu was also successfully applied in selective staining of lysosome in HeLa cells. Furthermore, cellular uptake experiment revealed that compound 1 showed good RNA delivery ability in HeLa, HepG2, U2Os and MC3T3-E1 cells, and its performance was better than commercial agents lipofectamine 2000 and 25 kDa PEI (Polyethylenimine). The RNA interference effect mediated by compound 1 was further evaluated by real-time fluorescent quantitative PCR experiment. Compound 1 showed much higher transfection efficacy than lipofectamine 2000 in MC3T3-E1 cells. Our study demonstrated that 1,8-naphthalimide- [12]aneN3 compound 1 with low cytotoxicity, high specificity towards Cu2+ and lysosome, high transfection efficacy, and low cost is an efficient multifunctional material both in molecular recognition and gene delivery.

Enhanced Synergetic Catalytic Effect of Mo2C/NCNTs@Co Heterostructures in Dye-Sensitized Solar Cells: Fine-Tuned Energy Level Alignment and Efficient Charge Transfer Behavior.

A highly efficient and stable electrocatalyst with the novel heterostructure of Co-embedded and N-doped carbon nanotubes supported Mo2C nanoparticles (Mo2C/NCNTs@Co) is creatively constructed by adopting the one-step metal catalyzed carbonization-nitridation strategy. Systematic characterizations and density functional theory (DFT) calculations reveal the advanced structural and electronic properties of Mo2C/NCNTs@Co heterostructure, in which the Co-embedded and N-doped CNTs with tunable diameters present electron-donating effect and the work function is correspondingly regulated from 4.91 to 4.52 eV, and the size-controlled Mo2C nanoparticles exhibit Pt-like 4d electronic structure and the well matched work function (4.85 eV) with I-/I3- redox couples (4.90 eV). As a result, the conductive NCNTs@Co substrate with fine-tuned energy level alignment accelerates the electron transportation and the electron migration from NCNTs@Co to Mo2C, and the active Mo2C shows high affinity for I3- adsorption and high charge transfer ability for I3- reduction, which reach a decent synergetic catalytic effect in Mo2C/NCNTs@Co heterostructure. The DSSC with Mo2C/NCNTs@Co CE achieves a high photoelectric conversion efficiency of 8.82% and exceptional electrochemical stability with a residual efficiency of 7.95% after continuous illumination of 200 h, better than Pt-based cell. Moreover, the synergistic catalytic mechanism toward I3- reduction is comprehensively studied on the basis of structure-activity correlation and DFT calculations. The advanced heterostructure engineering and electronic modulation provide a new design principle to develop the efficient, stable, and economic hybrid catalysts in relevant electrocatalytic fields.