Lighting Up Nucleolus To Report Mitochondria Damage Using a Mitochondria-to-Nucleolus Migration Probe.

Visualization of the mitochondrial state is crucial for tracking cell life processes and diagnosing disease, while fluorescent probes that can accurately assess mitochondrial status are currently scarce. Herein, a fluorescent probe named "SYN" was designed and prepared, which can target mitochondria via the mitochondrial membrane potential. Upon pathology or external stimulation, SYN can be released from the mitochondria and accumulate in the nucleolus to monitor the status of mitochondria. During this process, the brightness of the nucleolus can then serve as an indicator of mitochondrial damage. SYN has demonstrated excellent photostability in live cells as well as an extremely inert fluorescence response to bioactive molecules and the physiological pH environment of live cells. Spectroscopic titration and molecular docking studies have revealed that SYN can be lit up in nucleoli due to the high viscosity of the nucleus and the strong electrostatic interaction with the phosphate backbone of RNA. This probe is expected to be an exceptional tool based on its excellent imaging properties for tracking mitochondrial state in live cells.

Long-Chain Fluorescent Probe for Straightforward and Nondestructive Staining Mitochondria in Fixed Cells and Tissues.

Normally, small-molecule fluorescent probes dependent on the mitochondrial membrane potential (MMP) are invalid for fixed cells and tissues, which limits their clinical applications when the fixation of pathological specimens is imperative. Given that mitochondrial morphology is closely associated with disease, we developed a long-chain mitochondrial probe for fixed cells and tissues, DMPQ-12, by installing a C12-alkyl chain into the quinoline moiety. In fixed cells stained with DMPQ-12, filament mitochondria and folded cristae were observed with confocal and structural illumination microscopy, respectively. In titration test with three major phospholipids, DMPQ-12 exhibited a stronger binding force to mitochondria-exclusive cardiolipin, revealing its targeting mechanism. Moreover, mitochondrial morphological changes in the three lesion models were clearly visualized in fixed cells. Finally, by DMPQ-12, three kinds of mitochondria with different morphologies were observed in situ in fixed muscle tissues. This work breaks the conventional concept that organic fluorescent probes only stain mitochondria with normal membrane potentials and opens new avenues for comprehensive mitochondrial investigations in research and clinical settings.

Influence of preoperative simulation on the reduction quality and clinical outcomes of open reduction and internal fixation for complex proximal humerus fractures.

PURPOSE: Proximal humerus fractures (PHFs) are common. With the development of locking plates, open reduction and internal fixation (ORIF) of the proximal humerus can provide excellent clinical outcomes. The quality of fracture reduction is crucial in the locking plate fixation of proximal humeral fractures. The purpose of this study was to determine the impact of 3-dimensional (3D) printing technology and computer virtual technology assisted preoperative simulation on the reduction quality and clinical outcomes of 3-part and 4-part proximal humeral fractures. METHOD: A retrospective comparative analysis of 3-part and 4-part PHFs undergoing open reduction internal fixation was performed. Patients were divided into 2 groups according to whether computer virtual technology and 3D printed technology were used for preoperative simulation: the simulation group and the conventional group. Operative time, intraoperative bleeding, hospital stay, quality of fracture reduction, Constant scores, American Society for Shoulder and Elbow Surgery (ASES) scores, shoulder range of motion, complications, and revision surgeries were assessed. RESULTS: This study included 67 patients (58.3%) in the conventional group and 48 patients (41.7%) in the simulation group. The patient demographics and fracture characteristics were comparable in these groups. Compared with the conventional group, the simulation group had shorter operation time and less intraoperative bleeding (P < 0.001, both). Immediate postoperative assessment of fracture reduction showed a higher incidence of greater tuberosity cranialization of < 5 mm, neck-shaft angle of 120° to 150°, and head shaft displacement of < 5 mm in the simulation group. The incidence of good reduction was 2.6 times higher in the simulation group than in the conventional group (95% CI, 1.2-5.8). At the final follow-up, the chance of forward flexion > 120° (OR 5.8, 95% CI 1.8-18.0) and mean constant score of > 65 (OR 3.4, 95% CI 1.5-7.4) was higher in the simulation group than the conventional group, as well as a lower incidence of complications in the simulation group was obtained (OR 0.2, 95% CI 0.1-0.6). CONCLUSIONS: This study identified that preoperative simulation assisted by computer virtual technology and 3D printed technology can improve reduction quality and clinical outcomes in treatment of 3-part and 4-part PHFs.

Monocomponent Nanodots with Dichromatic Output Regulated by Synergistic Dual-Stimuli for Cervical Cancer Tissue Imaging and Photodynamic Tumor Therapy.

Inflammation exists in the microenvironment of most, if not virtually all, tumors, which greatly exacerbates the difficulty of cancer treatment. Considering the superiority of activatable photosensitizers (PSs), a novel strategy of 'making friends with the enemy' for tumor treatment was proposed. In this strategy, the "enemy" refers to inflammatory cytokines and the tumor site is targeted by detecting the enemy. Upon detection, a dichromatic fluorescence signal is released and the PS is activated specifically by the inflammatory cytokines. In this study, a multifunctional PS (TPE-PTZ-Py) was rationally designed, which can be activated specifically under the synergistic action of hypochlorous acid (HClO) (one kind of inflammatory cytokines) and acid (one typical marker of tumor), and output a ratiometric fluorescence signal simultaneously. The sulfoxide analogue (TPE-PTZO-PyH) as the response product effectively produced 1O2 (1.8-fold higher than that obtained with Rose Bengal) and showed high phototoxicity (IC50 < 7.6 µM). More importantly, imaging analyses confirmed that TPE-PTZ-Py could be activated in human cervical cancer tissue. To date, several phenothiazine (PTZ)-based fluorescent probes have been developed for the selective sensing and imaging of HClO in subcellular organelles; however, this is the first phenothiazine-based nanodrug designed for the treatment of inflammation-associated tumors with a few side effects.

Two Ratiometric Fluorescent Probes Based on the Hydroxyl Coumarin Chalcone Unit with Large Fluorescent Peak Shift for the Detection of Hydrazine in Living Cells.

Hydrazine is widely used in industrial and agricultural production, but excessive hydrazine possesses a serious threat to human health and environment. Here two new ratiometric fluorescence probes, DDP and DDC, with the hydroxyl coumarin chalcone unit as the sensing site are developed, which can achieve colorimetric and ratiometric recognition for hydrazine with good sensitivity, excellent selectivity, and anti-interference. The calculated fluorescence limits of detections are 0.26 µM (DDC) and 0.14 µM (DDP). The ratiometric fluorescence response to hydrazine is realized through the adjustment of donor and receptor units in coumarin conjugate structure terminals, accompanied by fluorescence peak shift about 200 nm (DDC, 188 nm; DDP, 229 nm). Stronger electropositivity in the carbon-carbon double bond is helpful to the first phase addition reaction between the probe and hydrazine. Higher phenol activity in the hydroxyl coumarin moiety will facilitate the following dihydro-pyrazole cyclization reaction. In addition, both of these probes realized the convenient detection of hydrazine vapor. The probes were also successfully applied to detect hydrazine in actual water samples, different soils, and living cells.

A Nuclear-Targeted AIE Photosensitizer for Enzyme Inhibition and Photosensitization in Cancer Cell Ablation.

The nucleus is considered the ideal target for anti-tumor therapy because DNA and some enzymes in the nucleus are the main causes of cell canceration and malignant proliferation. However, nuclear target drugs with good biosafety and high efficiency in cancer treatment are rare. Herein, a nuclear-targeted material MeTPAE with aggregation-induced emission (AIE) characteristics was developed based on a triphenylamine structure skeleton. MeTPAE can not only interact with histone deacetylases (HDACs) to inhibit cell proliferation but also damage telomere and nucleic acids precisely through photodynamic treatment (PDT). The cocktail strategy of MeTPAE caused obvious cell cycle arrest and showed excellent PDT anti-tumor activity, which offered new opportunities for the effective treatment of malignant tumors.

Ratiometric Fluorescence Imaging for the Distribution of Nucleic Acid Content in Living Cells and Human Tissue Sections.

The misregulation of nucleic acids behavior leads to cell dysfunction and induces serious diseases. A ratiometric fluorescence probe is a powerful tool to study the dynamic behavior and function relationships of nucleic acids. However, currently, no such effective probe has been reported for in situ, real-time tracking of nucleic acids in living cells and tissue sections. Herein, the unique probe named QPP-AS was rationally designed for ratiometric fluorescence response to nucleic acids through skillful regulation of the intramolecular charge-transfer capabilities of the electron acceptor and donor. Encouraged by the advantages of the selective nucleic acid response, ideal biocompatibility, and high signal-to-noise ratio, QPP-AS has been applied for in situ, real-time ratiometric fluorescence imaging of nucleic acids in living cells for the first time. Furthermore, we have demonstrated that QPP-AS is capable of visualizing the dynamic behavior of nucleic acids during different cellular processes (e.g., cell division and apoptosis) by ratiometric fluorescence imaging. More significantly, QPP-AS has been successfully used for ratiometric fluorescence imaging of nucleic acids in human tissue sections, which provides not only the cell contour, nuclear morphology, and nuclear-plasma ratio but also the nucleic acid content information and may greatly improve accuracy in clinicopathological diagnosis.

Multifunctional AIE iridium (III) photosensitizer nanoparticles for two-photon-activated imaging and mitochondria targeting photodynamic therapy.

Developing novel photosensitizers for deep tissue imaging and efficient photodynamic therapy (PDT) remains a challenge because of the poor water solubility, low reactive oxygen species (ROS) generation efficiency, serve dark cytotoxicity, and weak absorption in the NIR region of conventional photosensitizers. Herein, cyclometalated iridium (III) complexes (Ir) with aggregation-induced emission (AIE) feature, high photoinduced ROS generation efficiency, two-photon excitation, and mitochondria-targeting capability were designed and further encapsulated into biocompatible nanoparticles (NPs). The Ir-NPs can be used to disturb redox homeostasis in vitro, result in mitochondrial dysfunction and cell apoptosis. Importantly, in vivo experiments demonstrated that the Ir-NPs presented obviously tumor-targeting ability, excellent antitumor effect, and low systematic dark-toxicity. Moreover, the Ir-NPs could serve as a two-photon imaging agent for deep tissue bioimaging with a penetration depth of up to 300 µm. This work presents a promising strategy for designing a clinical application of multifunctional Ir-NPs toward bioimaging and PDT.

Spatial Matching Selectivity and Solution Structure of Organic-Metal Hybrid to Quadruplex-Duplex Hybrid.

The sequence-dependent DNA secondary structures possess structure polymorphism. To date, studies on regulated ligands mainly focus on individual DNA secondary topologies, while lack focus on quadruplex-duplex hybrids (QDHs). Here, we design an organic-metal hybrid ligand L1 Pt(dien), which matches and selectively binds one type of QDHs with lateral duplex stem-loop (QLDH) with high affinity, while shows poor affinity for other QDHs and individual G4 or duplex DNA. The solution structure of QLDH MYT1L-L1 Pt(dien) complex was determined by NMR. The structure reveals that L1 Pt(dien) presents a chair-type conformation, whose large aromatic "chair surface" intercalates into the G-quadruplex-duplex interface via π-π stacking and "backrest" platinum unit interacts with duplex region through hydrogen bonding and electrostatic interactions, showing a highly matched lock-key binding mode. Our work provided guidance for spatial matching design of selectively targeting ligands to QDH structures.

A Polarity-Sensitive Ratiometric Fluorescence Probe for Monitoring Changes in Lipid Droplets and Nucleus during Ferroptosis.

Ferroptosis regulates cell death through reactive oxygen species (ROS)-associated lipid peroxide accumulation, which is expected to affect the structure and polarity of lipid droplets (LDs), but with no clear evidence. Herein, we report the first example of an LD/nucleus dual-targeted ratiometric fluorescent probe, CQPP, for monitoring polarity changes in the cellular microenvironment. Due to the donor-acceptor structure of CQPP, it offers ratiometric fluorescence emission and fluorescence lifetime signals that reflect polarity variations. Using nucleus imaging as a reference, CQPP was applied to report the increase in LD polarity and the homogenization of polarity between LDs and cytoplasm in the ferroptosis model. This LD/nucleus dual-targeted fluorescent probe shows the great potential of using fluorescence imaging to study ferroptosis and ferroptosis-related diseases.

Quantitative Detection of G-Quadruplex DNA in Live Cells Based on Photon Counts and Complex Structure Discrimination.

G-quadruplex DNA show structural polymorphism, leading to challenges in the use of selective recognition probes for the accurate detection of G-quadruplexes in vivo. Herein, we present a tripodal cationic fluorescent probe, NBTE, which showed distinguishable fluorescence lifetime responses between G-quadruplexes and other DNA topologies, and fluorescence quantum yield (Φf ) enhancement upon G-quadruplex binding. We determined two NBTE-G-quadruplex complex structures with high Φf values by NMR spectroscopy. The structures indicated NBTE interacted with G-quadruplexes using three arms through π-π stacking, differing from that with duplex DNA using two arms, which rationalized the higher Φf values and lifetime response of NBTE upon G-quadruplex binding. Based on photon counts of FLIM, we detected the percentage of G-quadruplex DNA in live cells with NBTE and found G-quadruplex DNA content in cancer cells is 4-fold that in normal cells, suggesting the potential applications of this probe in cancer cell detection.

Multiple-Color Platinum Complex with Super-Large Stokes Shift for Super-Resolution Imaging of Autolysosome Escape.

It is of great significance to track the platinum drugs in real time with super-resolution to elucidate their mechanism of action, such as their behavior and distribution in live cells. Such information is required for further drug development. However, it is always challenging to design platinum complexes suitable for such research. Herein, we design a luminescent building block (L) for metal complexes and a dinuclear platinum complex (Pt2 L) for super-resolution imaging. Because of its super-large Stokes shift and excellent photophysical properties, Pt2 L is capable of serving as an ideal candidate for super-resolution imaging with extremely low luminescence background and high photobleaching resistance. Moreover, upon light stimulation, a matter flux of Pt2 L escaping from autolysosomes to nucleus was observed, which represents a new transportation path. Utilizing the photoactivated escape properties, we can regulate the nuclear accessibility of Pt2 L form autolysosomes with photo-selectivity, which provides a new way to improve the targeting of platinum drugs.

Aggregation-induced emission-active iridium (III)-based mitochondria-targeting nanoparticle for two-photon imaging-guided photodynamic therapy.

The efficacy of imaging-guided photodynamic therapy (PDT) is compromised by the attenuation of fluorescence and decline in reactive oxygen species (ROS) generation efficiency in the physiological environment of conventional photosensitizers, limited near-infrared (NIR) absorption, and high systemic cytotoxicity. This paper presents the synthesis of two cyclometalated Ir (III) complexes (Ir-thpy and Ir-ppy) by using a triphenylamine derivative (DPTPA) as the primary ligand and their encapsulation into an amphiphilic phospholipid to form nanoparticles (NPs). These complexes exhibit aggregation-induced emission features and remarkably enhanced ROS generation compared to Chlorin e6 (Ce6). Moreover, Ir-thpy NPs possess the unique ability to selectively target mitochondria, leading to depolarization of the mitochondrial membrane potential and ultimately triggering apoptosis. Notably, Ir-thpy NPs exhibit exceptional photocytotoxicity even towards cisplatin-resistant A549/DDP tumor cells. In vivo two-photon imaging verified the robust tumor-targeting efficacy of Ir-thpy NPs. The in vivo results unequivocally demonstrate that Ir-thpy NPs exhibit excellent tumor ablation along with remarkable biocompatibility. This study presents a promising approach for the development of multifunctional Ir-NPs for two-photon imaging-guided PDT and provides novel insights for potential clinical applications in oncology.

Dual-targeted fluorescent probe for tracking polarity and phase transition processes during lipophagy.

Eukaryotic cells regulate various cellular processes through membrane-bound and membrane-less organelles, enabling active signal communication and material exchange. Lysosomes and lipid droplets are representative organelles, contributing to cell lipophagy when their interaction and metabolism are disrupted. Our limited understanding of the interacting behaviours and physicochemical properties of different organelles during lipophagy hinders accurate diagnosis and treatment of related diseases. In this contribution, we report a fluorescent probe, PTZ, engineered for dual-targeting of lipid droplets and lysosomes. PTZ can track liquid-liquid phase separation and respond to polarity shifts through ratiometric fluorescence emission, elucidating the lipophagy process from the perspective of organelle behavior and physicochemical properties. Leveraging on the multifunctionality of PTZ, we have successfully tracked the polarity and dynamic changes of lysosomes and lipid droplets during lipophagy. Furthermore, an unknown homogeneous transition of lipid droplets and lysosomes was discovered, which provided a new perspective for understanding lipophagy processes. And this work is expected to serve as a reference for diagnosis and treatment of lipophagy-related diseases.

"Two-Stage Rocket-Propelled" Strategy Boosting Theranostic Efficacy with Mitochondria-Specific Type I-II Photosensitizers.

Imaging-guided photodynamic therapy (PDT) holds great potential for tumor therapy. However, achieving the synergistic enhancement of the reactive oxygen species (ROS) generation efficiency and fluorescence emission of photosensitizers (PSs) remains a challenge, resulting in suboptimal image guidance and theranostic efficacy. The hypoxic tumor microenvironment also hinders the efficacy of PDT. Herein, we propose a "two-stage rocket-propelled" photosensitive system for tumor cell ablation. This system utilizes MitoS, a mitochondria-targeted PS, to ablate tumor cells. Importantly, MitoS can react with HClO to generate a more efficient PS, MitoSO, with a significantly improved fluorescence quantum yield. Both MitoS and MitoSO exhibit less O2-dependent type I ROS generation capability, inducing apoptosis and ferroptosis. In vivo PDT results confirm that this mitochondrial-specific type I-II cascade phototherapeutic strategy is a potent intervention for tumor downstaging. This study not only sheds light on the correlation between the PS structure and the ROS generation pathway but also proposes a novel and effective strategy for tumor downstaging intervention.

Blood-Brain Barrier Penetrating Nanovehicles for Interfering with Mitochondrial Electron Flow in Glioblastoma.

Glioblastoma multiforme (GBM) is the most aggressive and lethal form of human brain tumors. Dismantling the suppressed immune microenvironment is an effective therapeutic strategy against GBM; however, GBM does not respond to exogenous immunotherapeutic agents due to low immunogenicity. Manipulating the mitochondrial electron transport chain (ETC) elevates the immunogenicity of GBM, rendering previously immune-evasive tumors highly susceptible to immune surveillance, thereby enhancing tumor immune responsiveness and subsequently activating both innate and adaptive immunity. Here, we report a nanomedicine-based immunotherapeutic approach that targets the mitochondria in GBM cells by utilizing a Trojan-inspired nanovector (ABBPN) that can cross the blood-brain barrier. We propose that the synthetic photosensitizer IrPS can alter mitochondrial electron flow and concurrently interfere with mitochondrial antioxidative mechanisms by delivering si-OGG1 to GBM cells. Our synthesized ABBPN coloaded with IrPS and si-OGG1 (ISA) disrupts mitochondrial electron flow, which inhibits ATP production and induces mitochondrial DNA oxidation, thereby recruiting immune cells and endogenously activating intracranial antitumor immune responses. The results of our study indicate that strategies targeting the mitochondrial ETC have the potential to treat tumors with limited immunogenicity.

A two-photon iridium(III) complex probe for sensitive detection of SO2 derivatives in living cell mitochondria.

The derivatives of sulfur dioxide (HSO3-) formed in the biological environment play a vital role in the circulation system. Excessive SO2 derivatives will cause serious damage to the living system. Herein, a two-photon phosphorescent probe based on Ir(III) complex (named as Ir-CN) was designed and synthesized. Ir-CN is extremely selective and sensitive to SO2 derivatives with significant phosphorescent enhancement and increased phosphorescent lifetime. The detection limit of Ir-CN for SO2 derivatives reaches 0.17 µM. More importantly, Ir-CN preferentially accumulates in mitochondria, so bisulfite derivatives can be detected at subcellular level, which enriching the application of metal complex probe in biological detection. In addition, both single-photon and two-photon images can clearly show that Ir-CN is targeted to mitochondria. Benefits from its good biocompatibility, Ir-CN may be used as a reliable tool to detect SO2 derivatives in mitochondrion of living cells.

Mitochondrial nucleoid condensates drive peripheral fission through high membrane curvature.

Mitochondria are dynamic organelles that undergo fusion and fission events, in which the mitochondrial membrane and DNA (mtDNA) play critical roles. The spatiotemporal organization of mtDNA reflects and impacts mitochondrial dynamics. Herein, to study the detailed dynamics of mitochondrial membrane and mtDNA, we rationally develop a dual-color fluorescent probe, mtGLP, that could be used for simultaneously monitoring mitochondrial membrane and mtDNA dynamics via separate color outputs. By combining mtGLP with structured illumination microscopy to monitor mitochondrial dynamics, we discover the formation of nucleoid condensates in damaged mitochondria. We further reveal that nucleoid condensates promoted the peripheral fission of damaged mitochondria via asymmetric segregation. Through simulations, we find that the peripheral fission events occurred when the nucleoid condensates interacted with the highly curved membrane regions at the two ends of the mitochondria. Overall, we show that mitochondrial nucleoid condensates utilize peripheral fission to maintain mitochondrial homeostasis.

Dynamic Adjust of Non-Radiative and Radiative Attenuation of AIE Molecules Reinforces NIR-II Imaging Mediated Photothermal Therapy and Immunotherapy.

Due to the aggregation-caused quenching effect and near-infrared I poor penetration capabilities of common fluorescent molecules, their applications in visualized imaging and photoactivated treatment are limited. Therefore, new near-infrared II (NIR-II) molecule (named TST), which had the abilities of aggregation-induced emission (AIE) and photothermal therapy are synthesized. Moreover, in order to further improve its fluorescent yield and therapeutic effect, camptothecin prodrug (CPT-S-PEG) and novel immune checkpoint inhibitor AZD4635 are used to co-assemble with TST into nanoparticles for drug delivery. On account of the strong interaction of camptothecin and TST, the intramolecular rotation of TST is limited, thereby inhibiting non-radiation attenuation and promoting fluorescence generation when the nanoparticles are intact. As nanoparticles uptake by cancer cells, redox sensitive CPT-S-PEG is degraded and the nanoparticles disintegrate. The released TST enhances non-radiative attenuation and expedites photothermal conversion because of the removal of the constraint of camptothecin. Furthermore, photothermal therapy induces immunogenic cell death of cancer cells and releases abundant ATP into the tumor microenvironment to recruit immune cells. However, superfluous ATP is converted into immunosuppressive adenosine through the CD39-CD73-A2AR pathway. The AZD4635 released by photothermal disintegration of the nanoparticles just blocks this pathway timely, achieving favorable synergistic effect of photothermal therapy, chemotherapy, and immunotherapy.

A benzothiazole-based near-infrared fluorescent probe for sensing SO2 derivatives and viscosity in HeLa cells.

The unbalanced metabolism of sulfur dioxide can cause various diseases, such as neurological disorders and lung cancer. Until now, some researches revealed that the normal function of lysosomes would be disrupted by its abnormal viscosity. As a signal molecule, sulfur dioxide (SO2) plays an important role in lysosome metabolism. However, the connection of metabolism between the SO2 and viscosity in lysosomes is still unknown. Herein, we developed a benzothiazole-based near-infrared (NIR) fluorescent probe (Triph-SZ), which can monitor the SO2 derivatives and respond to the change of viscosity in lysosomes through two-photon imaging. Triph-SZ present high sensitivity and selectivity fluorescence response with the addition of SO2 derivatives based on the nucleophilic addition, and it also exhibits a sensitive fluorescence enhancement to environmental viscosity, which allows Triph-SZ to be employed to monitor the level of HSO3- and viscosity changes in lysosomes by the two-photon fluorescence lifetime imaging microscopy.