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.

Design and Synthesis of a Mitochondrial-Targeted JNK Inhibitor and Its Protective Effect on Parkinson's Disease Phenotypes.

C-Jun N-terminal kinase (JNK) is a key mediator involved in a variety of physiological processes. JNK activation is regulated in a complex manner by upstream kinases and phosphatases, and plays an important role in physiological processes such as the immune response and neuronal function. Therefore, JNK has become a therapeutic target for neurodegenerative diseases, ankylosing spondylitis, psoriasis, arthritis and other diseases. Inhibition of JNK activation in mitochondria holds great potential for Parkinson's disease (PD) therapy. However, no specific mitochondrial-targeted JNK inhibitor has been reported. We have developed a mitochondrial-targeted JNK inhibitor, P2, by linking a mitochondrial-specific cell-penetrating peptide to SP600125 (SP), a commercialized specific inhibitor of JNK. We found that P2 specifically inhibited mitochondrial JNK phosphorylation instead of nuclear JNK signaling. Further studies showed that P2 effectively rescued PD phenotypes both in vitro and in vivo, thus indicating that it is a potential therapeutic for PD.

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.

Pyrimidine-Based Fluorescent Probe for Monitoring Mitophagy via Detection of Mitochondrial pH Variation.

Impaired mitophagy hinders the clearance of damaged mitochondria, inducing pathological states. Knowledge of this phenomenon is key to diagnosing certain diseases and understanding their pathogenesis. Mitophagy involves an acidization process that could serve as an ideal detection target. In this work, we designed and synthesized a mitochondrial-targeting fluorescence probe, Z2, for evaluating pH variation. This probe exhibited remarkable "turn-on" fluorescence under acidic conditions. In biological applications, Z2 showed a strong, specific pH detection capacity in Parkin-overexpressing HeLa cells during the mitophagy process. The "turn-on" fluorescence property of Z2 was also used to detect pH variations in Caenorhabditis elegans. This probe, as a novel pH assessment tool, may facilitate further research of mitophagy-associated pathological patterns.

Two-photon fluorescent turn-on probes for highly efficient detection and profiling of thiols in live cells and tissues.

Thiols are important units in amino acids such as cysteine and peptides like glutathione. Development of chemical sensors capable of precise detection of thiols is important in cancer diagnosis and therapy. We have developed novel two-photon fluorescent turn-on probes for selective detection of thiols. The probes displayed excellent sensitivity and low detection limits. The dual-purpose probes have been demonstrated to be suitable for simultaneous imaging and proteome profiling in live cells and tumor tissues. The unique turn-on design endows the probes with excellent selectivity toward thiols in vitro and in situ, and can be further developed to support a thiol-quantification assay.

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.

A light-activated magnetic bead strategy utilized in spatio-temporal controllable exosomes isolation.

Tumor-derived exosomes are considered as a key biomarker in the field of liquid biopsy. However, conventional separation techniques such as ultracentrifugation, co-precipitation and column chromatography cannot isolate samples with high throughput, while traditional immunomagnetic separation techniques, due to steric effect of magnetic beads, reducing sensitivity of exosomes optical detection. Herein, we provide a novel and simple nanoplatform for spatiotemporally controlling extraction and elution of exosomes via magnetic separation and light-activated cargo release. In this system, magnetic beads are co-modified by photoresponsive groups -nitrobenzyl group and aptamers that are compatible with CD63-a highly expressed exosomal surface-specific protein. Through exosomes extracted from cell model and nude mice xenograft tumor model morphological characterization and proteomic analysis, results showed that our novel magnetic bead system outperformed current ultracentrifugation in serum exosome extraction in terms of extraction time, yield, and proportion of populations with high CD63 expression. This strategy may be a powerful tool for exosome isolation in clinical liquid biopsies of cancer disease.

Simultaneous Enhancement of the Long-Wavelength NIR-II Brightness and Photothermal Performance of Semiconducting Polymer Nanoparticles.

Theranostic agents with fluorescence in the second near-infrared (NIR-II) window, especially in its long-wavelength region, and NIR-II-excitable photothermal effect is promising but challenging in tumor diagnosis and therapy. Here, we report a simple but effective strategy to develop semiconducting polymer nanoparticles-based theranostic agents (PBQx NPs) and demonstrate their applications for long-wavelength NIR-II fluorescence imaging beyond 1400 nm and photothermal therapy (PTT) of tumors upon excitation at 1064 nm. Both experimental results and theory calculations show that the brightness and photothermal performance of PBQx NPs can be simultaneously improved by simply increasing the repeating unit number of semiconducting polymers. For example, PBQ45 NPs have 5-fold higher brightness than PBQ5 NPs and 6.7-fold higher photothermal effect (based on PCE × Îµ) than PBQ3 NPs, and exhibit promising applications in long-wavelength NIR-II fluorescence abdomen imaging, image-guided tumor resection, and image-guided PTT. This study demonstrates the effectiveness and importance of repeating unit numbers in regulating the theranostic performance, which has not received enough attention before.

Lignin-Incorporated Nanogel Serving As an Antioxidant Biomaterial for Wound Healing.

Oxidative phosphorylation is an important biological process in the body to produce energy, during which oxygen free radicals are generated as byproduct. Excessive oxygen free radicals cause cell death and reduce the rate of tissue regeneration and healing in a wound. Lignin is a natural antioxidant derived from plants, but its biomedical application is restricted because of the uncertain biocompatibility. In this work, we developed a lignin-incorporated nanogel and explored its application for wound healing. Lignin was extracted from coconut husks and determined to have strong antioxidant activity (IC50 = 25.7 ppm). Various amounts of lignin were incorporated into thermoresponsive nanogels, which were produced from polyurethane copolymers of polyethylene glycol (PEG), polypropylene glycol (PPG), and polydimethylsiloxane (PDMS). It was shown that the addition of lignin had minimal effects on the gelation and rheological properties of the nanogel but slightly increased the critical micelle concentration (CMC) of poly(PEG/PPG/PDMS urethane) copolymer from 3.38 × 10-4 g mL-1 to 4.61 × 10-4 g mL-1. The lignin-incorporated nanogels did not display detectable cytotoxicity. The lignin-incorporated nanogel possessed antioxidant activity, as it reduced the active oxygen level, protecting the LO2 cells from apoptosis caused by oxidative stress. More importantly, in vivo studies demonstrated that the lignin-incorporated nanogels accelerated the healing of burn wounds in mice as proved by the increased expression of Ki67, one marker of cell proliferation. The present work demonstrates that lignin-incorporated nanogel could serve as an antioxidant wound-dressing material and facilitate the wound healing.

A novel naphthofluorescein-based probe for ultrasensitive point-of-care testing of zinc(II) ions and its bioimaging in living cells and zebrafishes.

The combination of fluorescence method with paper-based diagnostic device is quite suitable in point-of-care testing (POCT). Herein, we designed a novel hybrid fluorescein-based probe ZN-2 and investigated its fluorescent properties thoroughly in the detection of Zn2+. In comparison with the fluorescein-based probe ZN-1, ZN-2 displayed better sensitivity, long-wavelength and faster response to Zn2+ within 20 min. Interestingly, we could achieve ultrasensitive, high-throughput and visual detection in the POCT analysis of Zn2+ by anchoring this probe ZN-2 on the paper-based device. This device with satisfied performance for Zn2+ detection was achieved in real samples including cytochylema, serum and lake water. Finally, the probe ZN-2 was further applied to visualize and monitor the level changes of Zn2+ in the living cells and zebrafishes.

A mitochondrion-targeting Mn(ii)-terpyridine complex for two-photon photodynamic therapy.

We developed a mitochondrion-targeting MnII-terpyridine complex (MTP) for two-photon photodynamic therapy. MTP was subjected to two-photon excitation in the NIR region to generate 1O2 and hence produce a PDT effect. This use of MTP overcame the drawbacks of traditional PDT agents. The MnII center of MTP apparently catalyzed the H2O2-mediated production of ROS and oxygen, which subsequently promoted the 1O2 generation, furthering the PDT effect. Additionally, the mitochondrion targeting of MTP provided a good spatial condition for the generation of 1O2, which further promoted the PDT effect.

Rational design of NIR fluorescence probes for sensitive detection of viscosity in living cells.

Developing near-infrared (NIR) fluorescence probes for detection of intracellular viscosity is still sufficiently challenging. In this work, three kinds of D-A-D type naphthyl and 2,1,3­benzoxadiazol hybrid NIR dyes functionalized with amino (NY1), N­methylamino (NY2) and N,N­dimethylamino (NY3) groups for intracellular micro-viscosity detection were designed and synthesized. All the probes exhibited very weak NIR emission in low viscosity environment and obvious fluorescence enhancement with the increased viscosity. Different substituent groups had a high impact on the photophysical properties and response sensitive of the probes to viscosity. The structure-property relationships were systematic investigated. The results showed that stronger electron-donating ability and larger steric effect of N,N­dimethylamino led to a narrower energy gap and more sensitive to viscosity environment. Therefore, NY3 exhibited higher signal noise ratio for viscosity detection and was successfully applied for imaging the changes of intracellular micro-viscosity. This work provides an efficient way to design powerful NIR fluorescence probes for viscosity detection.

A novel pyrimidine based deep-red fluorogenic probe for detecting hydrogen peroxide in Parkinson's disease models.

Parkinson's disease (PD) severely affects life quality of patients and has brought huge economic burden to health system worldwide. Previous studies have shown that the abnormal expression of hydrogen peroxide (H2O2) in the brain is closely related to the development of neurodegenerative diseases such as PD. Herein, we designed a novel deep-red H2O2 fluorogenic probe PB1 to detect the level of H2O2in vivo. PB1 showed a highly selectivity response to H2O2 over other reactants such as reactive oxygen/nitrogen species, biothiols and various ions in aqueous solution at physiological pH. We have demonstrated that PB1 possesses an excellent response to H2O2 in the cells and in the brain tissue of drosophila from confocal fluorescence imaging. These results suggested that PB1 holds great potential in the study of the relationship between H2O2 overexpression and PD.