Structurally Modulated Formation of Cyanine J-Aggregates with Sharp and Tunable Spectra for Multiplexed Optoacoustic and Fluorescence Bioimaging.

J-aggregation brings intriguing optical and electronic properties to molecular dyes and significantly expands their applicability across diverse domains, yet the challenge for rationally designing J-aggregating dyes persists. Herein, we developed a large number of J-aggregating dyes from scratch by progressively refining structure of a common heptamethine cyanine. J-aggregates with sharp spectral bands (full-width at half-maximum≤38 nm) are attained by introducing a branched structure featuring a benzyl and a trifluoroacetyl group at meso-position of dyes. Fine-tuning the benzyl group enables spectral regulation of J-aggregates. Analysis of single crystal data of nine dyes reveals a correlation between J-aggregation propensity and molecular arrangement within crystals. Some J-aggregates are successfully implemented in multiplexed optoacoustic and fluorescence imaging in animals. Notably, three-color multispectral optoacoustic tomography imaging with high spatiotemporal resolution is achieved, owing to the sharp and distinct absorption bands of the J-aggregates.

Visualization detection of mycotoxin patulin in fruit juices by a small-molecule fluorescent probe.

The mycotoxin patulin is a common contaminant in rotten fruits, posing severe food safety risks and threats to human health. Developing a convenient, sensitive and reliable method for patulin detection is of utmost importance but remains challenging. In this study, we have successfully designed and synthesized a small-molecule fluorescent probe, FITC-Lys, which demonstrates good sensitivity in detecting patulin. Upon contact with patulin, the terminal Lys group of the FITC-Lys probe reacts with patulin, resulting in the formation of the fluorescein dimer that subsequently quenches fluorescence. This variation of fluorescence enables the visualization and sensitive detection of patulin. The probe exhibits good sensitivity with a low LOD of 8 ng mL-1 for the fluorescence spectrum method and a LOD of 12 ng mL-1 for the fluorescence imaging method. Moreover, we have validated the probe's capability for patulin detection in apple and pear juices, achieving good recoveries ranging from 98.60% to 103.80%. Notably, the probe FITC-Lys is the first small-molecule fluorescent probe that has proven successful in visualizing patulin in juices derived from decayed apples and pears. Consequently, this probe holds great potential as a practical tool for monitoring patulin in foodstuffs, thereby contributing to enhanced food safety standards.

Aminopeptidase N Activatable Nanoprobe for Tracking Lymphatic Metastasis and Guiding Tumor Resection Surgery via Optoacoustic/NIR-II Fluorescence Dual-Mode Imaging.

Lymphatic metastasis is a crucial mechanism by which the cancer cells break away from the primary (original) tumor and travel to the closest regional lymph node(s) and ultimately to other organs or parts of the body, which is closely associated with tumor recurrence and reduced survival. Thus, tracking tumor lymphatic metastasis and realizing imaging-guided lymphoma resection surgery is of great significance. In this study, an activatable nanoprobe is developed for precisely tracking lymphatic metastasis of tumors and imaging-guided resection of the primary tumor and metastatic lymphoma. The molecular probe contains tricyanofuran as the electron-accepting unit (electron acceptor), xanthene as the electron-donating unit (electron donor), and alanine as the responsive unit (recognition moiety) for aminopeptidase N, and the probe molecules form the nanoprobe with bovine serum albumin as the matrix. The nanoprobe can respond specifically to aminopeptidase N overproduced in the tumor, thereby transmuting the alanine into an amino group, and correspondingly the nanoprobe is activated. Strong optoacoustic and NIR-II fluorescence signals emitted by the activated nanoprobe can be utilized for visualizing the lymphatic metastasis of tumors. Moreover, the nanoprobe with the aid of three-dimensional multispectral optoacoustic tomography (3D MSOT) imaging can accurately locate the tumor site of lymphatic metastasis, and ultimately, both the primary tumor and the metastatic lymphoma can be excised with resection surgery under the guidance of NIR-II fluorescence imaging.

Water-Dispersible Activatable Nanoprobe for Detecting Cadmium-Ion-Induced Oxidative Stress in Edible Crops via Near-Infrared Second-Window Fluorescence Imaging.

Edible crops are important in terms of food security and sustainable agriculture. Heavy-metal-ion contamination of water/soil has deleterious impacts on the growth of edible crops. Among the heavy metals, cadmium (Cd) is toxic to plants, people, and animals, as it is widely used in industry; it has become the most important metal ion in the soil/water pollution. Once the toxic Cd ion enters edible crops via the water/soil in which the crops grow, it will induce oxidative stress (overproduction of reactive oxygen species with H2O2 being the most abundant) in the crops, and strong oxidative stress leads to the crops' growth depression or inhibition. Hence, it is of great significance to accurately monitor the oxidative stress induced by Cd ions in edible crops, as the monitoring results could be employed for the early warning of Cd-ion pollution in water/soil. Herein, we design an activatable nanoprobe that can detect Cd-ion-induced oxidative stress in edible crops via near-infrared second-window (NIR-II) fluorescence imaging. The molecular probe IXD-B contains the diphenylamine-modified xanthene group acting as the electron-donating unit, bis(methylenemalononitrile)indan as the electron-accepting unit, and the methenephenylboronic acid group as the recognition moiety for H2O2 and the fluorescence quencher. The probe molecules being encapsulated by the amphiphilic DSPE-PEG2000 render the water-dispersible nanoprobe (IXD-B@DSPE-PEG2000). When the nanoprobe enters the edible crops, it can be activated by the overexpressed H2O2 therein and consequently emit strong NIR-II fluorescence signals for visualizing and tracking the oxidative stress in edible crops induced by Cd ions.

Optical molecular imaging and theranostics in neurological diseases based on aggregation-induced emission luminogens.

Optical molecular imaging and image-guided theranostics benefit from special and specific imaging agents, for which aggregation-induced emission luminogens (AIEgens) have been regarded as good candidates in many biomedical applications. They display a large Stokes shift, high quantum yield, good biocompatibility, and resistance to photobleaching. Neurological diseases are becoming a substantial burden on individuals and society that affect over 50 million people worldwide. It is urgently needed to explore in more detail the brain structure and function, learn more about pathological processes of neurological diseases, and develop more efficient approaches for theranostics. Many AIEgens have been successfully designed, synthesized, and further applied for molecular imaging and image-guided theranostics in neurological diseases such as cerebrovascular disease, neurodegenerative disease, and brain tumor, which help us understand more about the pathophysiological state of brain through noninvasive optical imaging approaches. Herein, we focus on representative AIEgens investigated on brain vasculature imaging and theranostics in neurological diseases including cerebrovascular disease, neurodegenerative disease, and brain tumor. Considering different imaging modalities and various therapeutic functions, AIEgens have great potential to broaden neurological research and meet urgent needs in clinical practice. It will be inspiring to develop more practical and versatile AIEgens as molecular imaging agents for preclinical and clinical use on neurological diseases.

Dual-Responsive Curcumin-Loaded Nanoparticles for the Treatment of Cisplatin-Induced Acute Kidney Injury.

Acute kidney injury (AKI) has been a global public health concern leading to high patient morbidity and mortality in the world. Nanotechnology-mediated antioxidative therapy has facilitated the treatment of AKI. Herein, a hierarchical curcumin-loaded nanodrug delivery system (NPS@Cur) was fabricated for antioxidant therapy to ameliorate AKI. The nanoplatform could respond to subacidic and reactive oxygen species (ROS) microenvironments. The subacidic microenvironment led to a smaller size (from 140.9 to 99.36 nm) and positive charge (from -4.9 to 12.6 mV), contributing to the high accumulation of nanoparticles. An excessive ROS microenvironment led to nanoparticle degradation and drug release. In vitro assays showed that NPS@Cur could scavenge excessive ROS and relieve oxidative stress in H2O2-induced HK-2 cells through reduced apoptosis, activated autophagy, and decreased endoplasmic reticulum stress. Results from cisplatin-induced AKI models revealed that NPS@Cur could effectively alleviate mitochondria injury and protect kidneys via antioxidative protection, activated autophagy, decreased endoplasmic reticulum stress, and reduced apoptosis. NPS@Cur showed excellent biocompatibility and low toxicity to primary tissues in mice. These results revealed that NPS@Cur may be a potential therapeutic strategy for efficiently treating cisplatin or other cause-induced AKI.

Rational design of stable heptamethine cyanines and development of a biomarker-activatable probe for detecting acute lung/kidney injuries via NIR-II fluorescence imaging.

Developing high-quality dyes to construct activatable probes for analyte sensing via NIR-II fluorescence is critical for attaining enhanced imaging depths and resolution. Heptamethine cyanines can serve this purpose; however, they usually have poor stability and a tendency to self-aggregate. Herein, we present a design strategy involving the installation of pyridinium and tert-butyl groups onto the central cyclohexenyl core to increase steric crowding, enhance water solubility, and provide a site for the incorporation of analyte-responsive elements. The resulting NP-N dyes emit NIR-II light and can outperform benchmark heptamethine cyanines such as ICG. Using HP-N1, we developed HP-H2O2 and showed that NIR-II fluorescence signals could be enhanced when treating with H2O2. HP-H2O2 was subsequently evaluated in murine models of acute lung injury and acute kidney injury. This strategy unlocks the potential of heptamethine cyanines and is applicable to examples with extended conjugation.

Emerging contrast agents for multispectral optoacoustic imaging and their biomedical applications.

Optoacoustic imaging is a hybrid biomedical imaging modality which collects ultrasound waves generated via photoexciting contrast agents in tissues and produces images of high resolution and penetration depth. As a functional optoacoustic imaging technique, multispectral optoacoustic imaging, which can discriminate optoacoustic signals from different contrast agents by illuminating samples with multi-wavelength lasers and then processing the collected data with specific algorithms, assists in the identification of a specific contrast agent in target tissues and enables simultaneous molecular and physiological imaging. Moreover, multispectral optoacoustic imaging can also generate three-dimensional images for biological tissues/samples with high resolution and thus holds great potential in biomedical applications. Contrast agents play essential roles in optoacoustic imaging, and they have been widely explored and applied as probes and sensors in recent years, leading to the emergence of a variety of new contrast agents. In this review, we aim to summarize the latest advances in emerging contrast agents, especially the activatable ones which can respond to specific biological stimuli, as well as their preclinical and clinical applications. We highlight their design strategies, discuss the challenges and prospects in multispectral optoacoustic imaging, and outline the possibility of applying it in clinical translation and public health services using synthetic contrast agents.

Fluorophore-Dapagliflozin Dyad for Detecting Diabetic Liver/Kidney Damages via Fluorescent Imaging and Treating Diabetes via Inhibiting SGLT2.

Type II diabetes is a prevalent disease; if left untreated, it could cause serious complications including liver and kidney damages. Hence, early diagnosis for these damages and effective treatment of diabetes are of high importance. Herein, a fluorophore-dapagliflozin dyad (DX-B-DA) has been developed as a theranostic system that can be triggered by intrahepatic/intrarenal reactive oxygen species (ROS) to concomitantly release a near-infrared (NIR) fluorescent dye (DX) and a SGLT2 inhibitor dapagliflozin (DA). In this dyad (DX-B-DA), the NIR fluorophore (DX) and the drug DA were covalently linked through a boronate ester bond which serves as the fluorescence quencher as well as the ROS-responsive moiety that can be cleaved by pathological levels of ROS in diabetics. The in vitro experiments indicate that, in the absence of hydrogen peroxide, the dyad is weakly emissive and keeps its drug moiety in an inactive state, while upon responding to hydrogen peroxide, the dyad simultaneously releases the NIR dye and the drug DA, suggesting that it can serve as an activatable probe for detecting and imaging diabetic liver/kidney damages as well as a prodrug for diabetes treatment upon being triggered by ROS. The dyad was then injected in mouse model of type II diabetes, and it is found that the dyad can not only offer visualized diagnosis for diabetes-induced liver/kidney damages but also exhibit high efficacy in treating type II diabetes and consequently ameliorating diabetic liver/kidney damages.

A Targeted Nanosystem for Detection of Inflammatory Diseases via Fluorescent/Optoacoustic Imaging and Therapy via Modulating Nrf2/NF-κB Pathways.

Inflammatory diseases are sometimes devastating and notoriously difficult to treat. Precisely modulating inflammatory signaling pathways is a promising approach for treating inflammatory diseases. Herein, a multifunctional nanosystem is developed for active targeting, activatable imaging and on-demand therapy against inflammatory diseases through modulating inflammatory pathways. A chromophore-drug dyad (QBS-FIS) is synthesized by linking a chromophore and a Nrf2 (nuclear factor E2-related factor) activator fisetin through boronate bond which serves as fluorescence quencher and ROS (reactive oxygen species)-responsive linker. QBS-FIS molecules form nanoparticles in water and are coated with macrophage cell membrane to ensure active targeting toward inflammation site. To further improve therapeutic efficacy, a NF-kB (nuclear-factor kappa-light-chain-enhancer of activated B cells) inhibitor thalidomide is co-encapsulated to afford the nanosystem (QBS-FIS&Thd@MM). Upon administration into mice, the nanosystem migrates to inflammatory site and pathological ROS therein cleaves the boronate bonds, thereby activating the chromophore for imaging liver/kidney inflammatory diseases for disease diagnosis and recovery evaluation via fluorescence and optoacoustic imaging as well as releasing the active drugs for treating acute liver inflammation through activating Nrf2 pathway and inhibiting NF-kB pathway. The 3D multispectral optoacoustic tomography imaging is applied to precisely locate the inflammatory foci in a spatiotemporal manner.

Activatable Nanocomposite Probe for Preoperative Location and Intraoperative Navigation for Orthotopic Hepatic Tumor Resection via MSOT and Aggregation-Induced Near-IR-I/II Fluorescence Imaging.

The precise location of tumor and completeness of surgical resection are critical to successful tumor surgery; thus, the method capable of preoperatively locating a tumor site and intraoperatively determining tumor margins would be highly ideal. Herein, an activatable nanocomposite probe was developed for preoperatively locating orthotopic hepatic tumor via multispectral optoacoustic tomography imaging and for intraoperative navigation via near-IR-1 (NIR-I) and NIR-II fluorescence imaging. The molecular probe comprises an electronic donor, an acceptor, and a recognition moiety and forms the nanocomposite probe with bovine serum albumin. The probe specifically responds to nitroreductase overexpressed in tumor cells, which transforms the aromatic nitro group into an electron-donating amino group and thus activates the probe. The activated probe with the aggregation-induced emission feature generates strong NIR-I/NIR-II fluorescence and optoacoustic signals for dual-mode imaging. Owing to the in situ response toward nitroreductase in tumor cells in the hepatic region, the probe is found capable of detecting early stage orthotopic liver tumors. Furthermore, with the nanocomposite probe, we can obtain the 3D MSOT images to accurately locate orthotopic liver tumors preoperatively and the NIR-I/NIR-II fluorescence images to provide intraoperative guidance for tumor resection surgery.

An Activatable Nano-Prodrug for Treating Tyrosine-Kinase-Inhibitor-Resistant Non-Small Cell Lung Cancer and for Optoacoustic and Fluorescent Imaging.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the cause of high rate of mortality. The epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors are used to treat NSCLC, yet their curative effects are usually compromised by drug resistance. This study demonstrates a nanodrug for treating tyrosine-kinase-inhibitor-resistant NSCLC through inhibiting upstream and downstream EGFR signaling pathways. The main molecule of the nanodrug is synthesized by linking a tyrosine kinase inhibitor gefitinib and a near-infrared dye (NIR) on each side of a disulfide via carbonate bonds, and the nanodrug is then obtained through nanoparticle formation of the main molecule in aqueous medium and concomitant encapsulation of a serine threonine protein kinase (Akt) inhibitor celastrol. Upon administration, the nanodrug accumulates at the tumor region of NSCLC-bearing mice and releases the drugs for tumor inhibition, and the dye for fluorescence and optoacoustic imaging. Through suppressing the phosphorylation of upstream EGFR and downstream Akt in the EGFR pathway by gefitinib and celastrol, respectively, the nanodrug exhibits high inhibition efficacy against orthotopic NSCLC in mouse models.

Tetranitrile-anthracene as a probe for fluorescence detection of viscosity in fluid drinks via aggregation-induced emission.

Viscosity is an important quality parameter for fluid drinks, which can serve as an indicator for the extent of food spoilage, since the viscosity of fluid drinks varies during the spoilage process. In this study, a near-infrared fluorophore based on tetranitrile-anthracene (TPAEQ) was designed for viscosity determination via aggregation-induced emission (AIE). With increased viscosity, TPAEQ showed enhanced emission at around 759 nm with a large Stokes shift of 195 nm in water. The probe TPAEQ was successfully used to monitor the viscosity changes during the food spoilage process for fluid drinks. Moreover, the probe TPAEQ has effectively been utilized to determine the mass concentrations of food thickeners added in the real fruit drinks. The approach for the viscosity determination could enable the on-site direct detection with convenient operation in food safety inspection applications.

Nanoaggregate Probe for Breast Cancer Metastasis through Multispectral Optoacoustic Tomography and Aggregation-Induced NIR-I/II Fluorescence Imaging.

An activatable nanoprobe for imaging breast cancer metastases through near infrared-I (NIR-I)/NIR-II fluorescence imaging and multispectral optoacoustic tomography (MSOT) imaging was designed. With a dihydroxanthene moiety serving as the electron donor, quinolinium as the electron acceptor and nitrobenzyloxydiphenylamino as the recognition element, the probe can specifically respond to nitroreductase and transform into an activated D-π-A structure with a NIR emission band extending beyond 900 nm. The activated nanoprobe exhibits NIR emission enhanced by aggregation-induced emission (AIE) and produces strong optoacoustic signal. The nanoprobe was used to detect and image metastases from the orthotopic breast tumors to lymph nodes and then to lung in two breast cancer mouse models. Moreover, the nanoprobe can monitor the treatment efficacy during chemotherapeutic course through fluorescence and MSOT imaging.

Diagnosing Drug-Induced Liver Injury by Multispectral Optoacoustic Tomography and Fluorescence Imaging Using a Leucine-Aminopeptidase-Activated Probe.

Drug-induced liver injury (DILI) is a frequent cause of hepatic dysfunction as well as the single most frequent reason for removing approved medications from the market, and multispectral optoacoustic tomography (MSOT) is an emerging and noninvasive imaging modality for diagnosing and monitoring diseases. Herein, we report an activatable optoacoustic probe for imaging DILI through detecting the activity of leucine aminopeptidase (LAP). In this probe, an N-terminal leucyl moiety serving as the LAP recognition element is linked with a chromene-benzoindolium chromophore via 4-aminobenzylalcohol group. The elevated expression of hepatic LAP as a result of DILI cleaves the leucyl moiety and causes the red-shift of the probe's absorption band, thereby generating prominent optoacoustic signals for MSOT imaging. During this process, the probe also exhibits prominent NIR fluorescence, which can be utilized for fluorescent imaging. More importantly, by rendering stacks of cross-sectional images as maximal intensity projection (MIP) images, we could precisely locate the focus of drug-induced liver injury in mice. This probe is expected to serve a powerful tool for studying physiological and pathological processes related to LAP.

A Gold Nanocage/Cluster Hybrid Structure for Whole-Body Multispectral Optoacoustic Tomography Imaging, EGFR Inhibitor Delivery, and Photothermal Therapy.

Gold nanocages (AuNCs) and gold nanoclusters (AuClusters) are two classes of advantageous nanostructures with special optical properties, and many other attractive properties. Integrating them into one nanosystem may achieve greater and smarter performance. Herein, a hybrid gold nanostructure for fluorescent and optoacoustic tomography imaging, controlled release of drugs, and photothermal therapy (PTT) is demonstrated. For this nanodrug (EA-AB), an epidermal growth factor receptor (EGFR) inhibitor erlotinib (EB) is loaded into AuNCs, which are then capped and functionalized by biocompatible AuCluster@BSA (BSA = bovine serum albumin) conjugates via electrostatic interaction. Upon cell internalization, the lysosomal proteases and low pH cause the release of EB from EA-AB, and also induce fluorescence restoration of the AuCluster for imaging. Irradiation with near-infrared light further promotes the drug release and affords a PTT effect as well. The AuNC-based nanodrug is optoacoustically active, and its biodistribution and metabolic process have been successfully monitored by whole-body and 3D multispectral optoacoustic tomography imaging. Owing to the combined actions of PTT and EGFR pathway blockage, EA-AB exhibits marked tumor inhibition efficacy in vivo.

An Unsymmetrical Squaraine-Based Activatable Probe for Imaging Lymphatic Metastasis by Responding to Tumor Hypoxia with MSOT and Aggregation-Enhanced Fluorescent Imaging.

Optoacoustic imaging has great potential for preclinical research and clinical practice, and designing robust activatable optoacoustic probes for specific diseases is beneficial for its further development. Herein, an activatable probe has been developed for tumor hypoxia imaging. For this probe, indole and quinoline were linked on each side of an oxocyclobutenolate core to form an unsymmetrical squaraine. A triarylamine group was incorporated to endow the molecule with the aggregation enhanced emission (AEE) properties. In aqueous media, the squaraine chromophore aggregates into the nanoprobe, which specifically responds to nitroreductase and produces strong optoacoustic signals due to its high extinction coefficient, as well as prominent fluorescence emission as a result of its AEE feature. The nanoprobe was used to image tumor metastasis via the lymphatic system both optoacoustically and fluorescently. Moreover, both the fluorescence signals and three-dimensional multispectral optoacoustic tomography signals from the activated nanoprobe allow us to locate the tumor site and to map the metastatic route.

A fluorescent probe based on aggregation-induced emission for hydrogen sulfide-specific assaying in food and biological systems.

A fluorescent probe based on a triphenylamine benzopyridine platform for hydrogen sulfide (H2S) assaying has been designed and synthesized. As a result of the H2S-triggered cleavage reaction, the disappearance of the quenching effect of dinitrophenyl and the increased hydrophobicity in a poor solvent lead to the aggregation-induced emission (AIE) effect; consequently an obvious 'turn-on' fluorescence signal can be observed in this process. The probe TPANF features high selectivity towards H2S, low detection limit (0.17 µM), and good photostability and biocompatibility. Moreover, it has been successfully utilized to monitor H2S in food samples to distinguish the extent of food deterioration and to identify the H2S concentration variation in living cells. In addition, endogenous H2S in HCT-116 xenograft tumor tissues was imaged by using this probe. The approach could provide useful insight for the development of other activatable AIE-based probes that are potentially helpful for specific assaying in food chemistry and biological systems.

Real-Time Monitoring of Endogenous Cysteine Levels In Vivo by near-Infrared Turn-on Fluorescent Probe with Large Stokes Shift.

Cysteine (Cys), as an important biothiol, plays a major role in many physiological processes like protein synthesis, detoxification and metabolism, and also is closely associated with a variety of diseases; thus the design of novel highly selective and sensitive near-infrared (NIR) fluorescent probes for Cys detection in vivo is of great significance. Herein, we report a selective and sensitive NIR turn-on fluorescent probe (CP-NIR) with large Stokes shift for detecting Cys in vivo. Upon addition of Cys to the solution of the probe, it is absorption wavelength shifts from 550 to 600 nm, accompanying with an obvious enhancement of NIR fluorescence emission centering around 760 nm. This Michael-addition reaction-based probe shows a large Stokes shift (160 nm), low detection limit (48 nM), fast response time, and low toxicity. Moreover, this novel NIR probe with good cell permeability was successfully applied to monitoring endogenous Cys in living cells and in a mouse model.

Oligo(ethylene glycol)-Functionalized Squaraine Fluorophore as a Near-Infrared-Fluorescent Probe for the In Vivo Detection of Diagnostic Enzymes.

Squaraine dyes have excellent photostability with intensive absorption and strong fluorescence in the near-infrared (NIR) region. However, they display a strong tendency to aggregate in aqueous media because of their poor water solubility, often causing fluorescence quenching that severely limits their in vivo applications, especially for detecting or imaging diagnostic enzymes. In this work, an oligo(ethylene glycol)-functionalized squaraine fluorophore has been developed as an NIR-fluorescent probe that can detect and image the activities of a diagnostic enzyme (leucine aminopeptidase) both in vitro and in vivo. The probe shows near-infrared absorption and emission, a low detection limit (0.61 ng/mL), relatively good aqueous solubility, high selectivity, and little toxicity. In addition, the probe herein was successfully used to track endogenous leucine aminopeptidase both in vitro and in vivo with a nude-mouse model.