Dual-Targeting into the Mitochondria of Cancer Cells for Ratiometric Investigation of the Dynamic Fluctuation of Sulfur Dioxide and Formaldehyde with Two-Photon Integrated Semiconducting Polymer Dots.

Mitochondrial sulfur dioxide (SO2) and formaldehyde (FA) in cancer cells serve as important signal molecules in mediating multiple physiological and pathological activities. Accurate monitoring of the dynamic fluctuation of SO2 and FA in the mitochondria of cancer cells is important for insight into their relationships and functions in cancer, understanding cancer mechanism, and the role of mitochondrial homeostasis in cancer invasion and metastasis. Herein, a novel integrated two-photon semiconducting polymer dot (BF@Pdots) with dual-targeting (cancer cells and mitochondrial) and dual-emission in green and red regions, which is rationally designed through a four-step engineering strategy by using two newly synthesized functionalized polymers PFNA and FD-PSMA as precursors, has been developed for accurate tracking of the dynamic variation of SO2 and FA in the mitochondria of cancer cells. The sensing mechanism is on the basis of the fluorescence resonance energy transfer (FRET) process in BF@Pdots tuned by the reversible Michael addition reaction between the sensing-groups and SO2 (or FA). The integrated BF@Pdots nanoprobes display excellent performances in the accurate detection of the dynamic fluctuation of SO2 and FA such as precise positioning in the mitochondria of cancer cells, self-calibrating ratiometric, two-photon emission with long wavelength excitation, and fast reversible response. The BF@Pdots nanoprobes are also applied to the ratiometric detection of the dynamic fluctuation of exogenous and endogenous SO2 and FA in the mitochondria of cancer cells for the first time with satisfactory results. Taken together, this work will provide an attractive way to develop versatile integrated Pdots-based fluorescent probes through flexible molecular engineering for applications in accurate imaging of biomolecules in living systems.

Sulfone-containing Conjugated Polyimide 2D Nanosheets for Efficient Water Oxidation.

Water oxidation is a bottleneck in artificial photosynthesis that impedes its practicality for solar energy conversion and utilization. It is highly desired to significantly improve the efficacy of the existing catalysts or to rationally design new catalysts with improved performance. We report a novel conjugated and sulfone containing polyimide as a metal-free photocatalyst synthesized via a two-step method: (i) synthesis of precursor poly(amic acid) (PAA) (ii) solvothermal synthesis of polyimide through thermal imidization. The synthesis of the polyimide photocatalyst was demonstrated by the amide linkage in the FTIR spectrum. The obtained photocatalyst was semicrystalline in nature and possessed sheet-like morphology as illustrated by the diffraction pattern and the electron micrographic images, respectively. The thermogravimetric analysis of the polyimide nanosheets validated a thermally stable structure. The DFT calculations were performed which showed a suitable HOMO band position, favorable for water oxidation. The photoelectrocatalytic (PEC) performance of the polyimide nanosheets evaluated by studying water oxidation reaction without any sacrificial agent under 1-SUN showed enhanced PEC performance and good stability towards water oxidation at 0 V versus SCE.

Retained Functionality of Atherosclerotic Human Arteries Following Photoactivated Linking of the Extracellular Matrix by Natural Vascular Scaffolding Treatment.

In this study, we investigated natural vascular scaffolding (NVS) treatment on vascular functionality using freshly isolated human popliteal arteries in vitro. Arteries were exposed to intraluminal NVS treatment consisting of a compound (4 amino-1,8-naphthalimide) photoactivated by a 450-nm light-emitting light fiber placed inside the artery. This procedure results in covalent linking between the extracellular matrix proteins to achieve a larger vessel diameter post-angioplasty and minimizing elastic recoil. Immediately following NVS treatment, rings were cut from the treated arteries and mounted in organ baths for contractility testing in response to U46619 and sodium nitroprusside. We also investigated the effect of NVS treatment on IL-6 cytokine release from vascular rings following a 4-h organoculture post-NVS treatment. Based on our results, we conclude that exposure of the vessels to NVS treatment does not adversely affect the contractile responsiveness of the vascular smooth muscle and exerts no pro-inflammatory effect. Graphical abstract.

Directing charge transfer in perylene based light-harvesting antenna molecules.

Directing energy and charge transfer processes in light-harvesting antenna systems is quintessential for optimizing the efficiency of molecular devices for artificial photosynthesis. In this work, we report a novel synthetic method to construct two regioisomeric antenna molecules (1-D2A2 and 7-D2A2), in which the 4-(n-butylamino)naphthalene monoimide energy and electron donor is attached to the perylene monoimide diester (PMIDE) acceptor at the 1- and 7-bay positions, respectively. The non-symmetric structure of PMIDE renders a polarized distribution of the frontier molecular orbitals along the long axis of this acceptor moiety, which differentiates the electron coupling between the donor, attached at either the 1- or the 7-position, and the acceptor. We demonstrate that directional control of the photo-driven charge transfer process has been obtained by engineering the molecular structure of the light-harvesting antenna molecules.

A naphthalimide derivative can release COS and form H2S in a light-controlled manner and protect cells against ROS with real-time monitoring ability.

As an important gasotransmitter, hydrogen sulfide having multiple biological roles cannot be easily probed in cells. In this study, a light controllable H2S donor, Nap-Sul-ONB, derived from naphthalimide was developed. Under the irradiation of 365 nm light, a readily controlled stimulus, the donor could release COS to form H2S and exhibit turn on fluorescence to indicate the release of payload and its cellular location. Besides, the ROS scavenging ability and cell protective effect of Nap-Sul-ONB against endogenous and exogenous ROS were studied. The results showed that upon 365 nm light irradiation, Nap-Sul-ONB could reduce the cellular ROS level and increase the survival rate of PMA-treated cells.

A lysosome specific theranostic NO donor inhibits cancer cells by stimuli responsive molecular self-decomposition with an on-demand fluorescence pattern.

The anticancer mechanism of NO is difficult to study owing to its short lifetime and high reactivity. Thus, a theranostic anticancer NO donor assembled with NO on-demand release abilities, accurate lysosome location capabilities and signal feedback behavior was developed. Profiting from the theranostic properties, the specific mechanism was comprehensively studied. Spectral and cell imaging studies revealed that the as prepared NO donors could release NO in solution or within cancer cells. Fluorescence co-dyeing experiments demonstrated that Mo-Nap-NO entered lysosomes specifically and disrupted them after being triggered by light. Upon irradiation with 460 nm visible light, both the donors demonstrated considerable in vitro anticancer effects. A further mechanistic study showed that after entering the lysosome and being triggered by 460 nm irradiation, NO ruptured the lysosome, resulting in the release of cathepsin D into the cytosol, which activated the caspase3 mediated apoptosis pathway.

Tracking lysosomal polarity variation in inflamed, obese, and cancer mice guided by a fluorescence sensing strategy.

Elucidating lysosome polarity effect in complicated biosystems was impeded with the deficiency of lacking multi-disease models for researching the relation between lysosomal polarity and diseases. So far, dissecting the abnormal lysosome polarity in the inflamed and obese living mice has not been realized. To overcome this challenge, a robust probe MND-Lys was proposed for monitoring lysosomal polarity with two-photon emission. Using the probe, monitoring the intrinsic polarity variance in embryos and adult zebrafish has been achieved for the first time. Moreover, besides obviously discriminating tumors from normal ones, the probe also enabled tracing polarity changes in inflammatory and obese mice for the first time. The unique tracking and distinguishing polarity in lysosome make the probe a promising agent for fluorescence visualization studies of LD-lysosome related bioprocess and metabolism diseases.

A colorimetric and fluorescent lighting-up sensor based on ICT coupled with PET for rapid, specific and sensitive detection of nitrite in food.

An anthracene carboxyimide derivative was synthesized as a colorimetric and fluorogenic sensor to determine NO2- with a rapid response (<4 min), excellent selectivity and a low detection limit (84 nM). Paper strips containing the sensor were applied to visually determine the NO2- content in food.

A highly selective and ultrasensitive ratiometric fluorescent probe for peroxynitrite and its two-photon bioimaging applications.

In this work, taking full advantage of the intramolecular charge transfer (ICT) mechanism, a hydroxynaphthalimide-based ratiometric two-photon fluorescent probe RTP-PN was synthesized to detect ONOO-. Probe RTP-PN could accurately detect ONOO- in the range of 1.4 nM-1.4 µM with the detection limit of 1.4 nM by a ratiometric fluorescence spectroscopy method. Additionally, probe RTP-PN exhibited an ultrafast response for ONOO- than other various species including H2O2 and ClO-. Finally, probe RTP-PN was successfully adopted to detect intracellular ONOO- by the two-photon excitation microscopy.

Organic Fluorophore Coated Polycrystalline Ceramic LSO:Ce Scintillators for X-ray Bioimaging.

The current effort demonstrates that lutetium oxyorthosilicate doped with 1-10% cerium (Lu2SiO5:Ce, LSO:Ce) radioluminescent particles can be coated with a single dye or multiple dyes and generate an effective energy transfer between the core and dye(s) when excited via X-rays. LSO:Ce particles were surface modified with an alkyne modified naphthalimide (6-piperidin-1-yl-2-prop-2-yn-1-yl-1 H-benzo[ de]isoquinoline-1,3-(2 H)-dione, AlNap) and alkyne modified rhodamine B ( N-(6-diethylamino)-9-{2-[(prop-2-yn-1-yloxy)carbonyl]phenyl}-3 H-xanthen-3-ylidene)- N-ethylethanaminium, AlRhod) derivatives to tune the X-ray excited optical luminescence from blue to green to red using Förster Resonance Energy Transfer (FRET). As X-rays penetrate tissue much more effectively than UV/visible light, the fluorophore modified phosphors may have applications as bioimaging agents. To that end, the phosphors were incubated with rat cortical neurons and imaged after 24 h. The LSO:Ce surface modified with AlNap was able to be successfully imaged in vitro with a low-output X-ray tube. To use the LSO:Ce fluorophore modified particles as imaging agents, they must not induce cytotoxicity. Neither LSO:Ce nor LSO:Ce modified with AlNap showed any cytotoxicity toward normal human dermal fibroblast cells or mouse cortical neurons, respectively.

AIE active piperazine appended naphthalimide-BODIPYs: photophysical properties and applications in live cell lysosomal tracking.

Piperazine appended naphthalimide-BODIPYs (NPB1-NPB4) exhibiting solvatochromism and aggregation-induced emission with a large Stokes shift (up to 146 nm) have been described. Separation of naphthalimide and BODIPY fluorophores by piperazine in these conjugates creates a donor-acceptor system and induces twisted intramolecular charge transfer, in addition to photoinduced electron transfer. The crucial role of naphthalimide, the alkyl chain length, the piperazine ring, and the solid-state packing on AIE has been extensively investigated by various studies. Superior cell permeability coupled with bio-compatibility of these conjugates offers a unique opportunity for their potential applications in live cell lysosomal tracking.

High-depth fluorescence imaging using a two-photon FRET system for mitochondrial pH in live cells and tissues.

We developed a fluorescent pH probe (1) capable of two-photon excitation and far-visible-emission based on FRET, composed of naphthalimide-piperazine-rhodamine. It exhibited a pH-dependent reversible and fast ratiometric fluorescence change in the rhodamine emission. Probe 1 was applied to image the pH perturbations of mitochondria in living cells and tissues.

Two-photon imaging of the endoplasmic reticulum thiol flux in the brains of mice with depression phenotypes.

Depression is a common mental illness with high morbidity and mortality. Mounting evidence suggests that an imbalance of the oxidant-antioxidant defence system is strongly correlated with depression and the dysfunction of the endoplasmic reticulum (ER) is strongly related to the oxidative stress. Therefore, as vital and abundant antioxidants in the ER, biothiols may contribute to the etiology of depression. However, ideal two-photon (TP) fluorescent probes for in vivo imaging of ER-associated thiols in the brains of mice with depression phenotypes are still lacking. Hence, we describe a fluorescent probe (ER-SH) to visualize thiols in living systems. ER-SH displays high sensitivity, excellent ER-targeting ability, outstanding TP properties and low cytotoxicity. Using this ER-SH probe, we succeeded in revealing an increase in the endogenous thiol levels under ER stress induced by DTT. Significantly, TP in vivo imaging showed for the first time that the thiol levels are reduced in brains of mice with depression phenotypes. Collectively, this work can assist in further understanding the molecular mechanism of depression and offers a crucial dimension for diagnosis and anti-depression treatments.

Efficient Two-Photon Fluorescent Probe for Imaging of Nitric Oxide during Endoplasmic Reticulum Stress.

Nitric oxide (NO) is a vital gaseous signal molecule and plays an important role in diverse physiological and pathological processes including regulation of vascular functions. Endoplasmic reticulum (ER) stress is caused by the accumulation of misfolded or unfolded protein in the ER. Besides, ER stress induced by NO can be involved in the pathogenesis of various vascular diseases. Unfortunately, to the best of our knowledge, no ER-targeting probe for NO is reported to study the relationship between ER stress and the level of NO in a biological system. Herein, an ER-targeted fluorescent probe named ER-Nap-NO for imaging of NO is designed and synthesized. ER-Nap-NO consists of three main parts: naphthalimide (two-photon fluorophore), o-phenylenediamino (NO recognition group), and methyl sulfonamide (ER-targetable group). The probe itself is nonfluorescent because a photoinduced electron transfer (PET) process exists. After the addition of NO, the PET process is inhibited and thus strong fluorescence is released. Moreover, the response mechanism is confirmed by 1H NMR and mass spectra and DFT calculation in detail. In addition, from the experimental results, we can conclude that the probe displays several obvious advantages including high sensitivity, selectivity, and ER-targetable ability. Based on these excellent properties, the probe is used for the two-photon imaging of exogenous and endogenous NO in ER of living cells. Most importantly, the ER-targetable probe has potential capability as a tool for investigating the level of NO during tunicamycin-induced ER stress in cells and tissues, which is beneficial for revealing the role of NO in ER-associated vascular diseases.

Identification and Isolation of Glucosytransferases (GT) Expressed Fungi Using a Two-Photon Ratiometric Fluorescent Probe Activated by GT.

As is well-known, fungi are an important biocatalysis model of glucosylation and have been widely applied for bioactive compounds glucosylation mediated by the intracellular glucosytransferases (GTs). However, there is no efficient method for the real-time detection of GTs and the rapid isolation of the target fungi strains with the high expression of GTs. In the present work, we first developed a two-photon ratiometric fluorescent probe N-( n-butyl)-4-hydroxy-1,8-naphthalimide (NHN) for detecting the glucosyltransferases activity and intracellular imaging of GTs. Under UV light (365 nm), the transformed product of NHN mediated by intracellular glucosyltransferase displayed blue emission to guide the rapid isolation of fungal strains possessing overexpression of GTs from complex soil samples. Finally, by using the fluorescent probe, two target fungi were isolated and identified to be Rhizopus oryzae and Mucor circinelloides by molecular analysis, and they exhibited a robust capability for regio- and stereospecific O-glycosylation. Our results fully demonstrated that NHN may be a promising tool for guiding real-time GTs activity in fungal strains and even for developing natural fungal strains with GTs overexpression.

A photostable fluorescent probe for long-time imagining of lysosome in cell and nematode.

Lysosome fluorescent imaging has been widely used in the field of biological staining and diagnostics, which plays a key role in understanding intracellular metabolism and various physiological processes. However, for most currently used small-molecule lysotrackers, the photostability is often unsatisfactory when used for long-term and real-time visualization of lysosomal dynamics. Herein, we reported a new lysosome-targetable photostable fluorescent probe (i.e. MPL-NPA), and results showed that MPL-NAP possesses superior photostability, appreciable tolerance to pH change, low cytotoxicity and high lysosome targeting ability. These findings confirm that MPL-NAP is a well-suited imaging agent for targeting lysosome and enables long-term and real-time monitor of lysosome morphological changes under physiological processes.

Super-Resolution Monitoring of Mitochondrial Dynamics upon Time-Gated Photo-Triggered Release of Nitric Oxide.

Nitric oxide (NO) potentially plays a regulatory role in mitochondrial fusion and fission, which are vital to cell survival and implicated in health, disease, and aging. Molecular tools facilitating the study of the relationship between NO and mitochondrial dynamics are in need. We have recently developed a novel NO donor (NOD550). Upon photoactivation, NOD550 decomposes to release two NO molecules and a fluorophore. The NO release could be spatially mapped with subdiffraction resolution and with a temporal resolution of 10 s. Due to the preferential localization of NOD550 at mitochondria, morphology and dynamics of mitochondria could be monitored upon NO release from NOD550.

Lysosome-Targeted Turn-On Fluorescent Probe for Endogenous Formaldehyde in Living Cells.

As one of the simplest reactive carbonyl species, formaldehyde is implicated in nervous system diseases and cancer. Organelles play crucial roles in various physiological processes in living cells. Accordingly, the detection of endogenous formaldehyde at the subcellular level is of high interest. We herein describe the development of the first organelle-targeted fluorescent formaldehyde probe (Na-FA-Lyso). The new probe exhibits favorable features including a large fluorescence enhancement (about 350-fold) and a fast response to formaldehyde. Significantly, the novel probe Na-FA-Lyso was employed to visualize the endogenous formaldehyde in the lysosomes in living cells for the first time.