Near-infrared Fluorescent Probe Research Articles

Imaging gastrointestinal damage due to acute mercury poisoning using a mitochondria-targeted dual near-infrared fluorescent probe

Mercury (Hg) is one of the most widespread pollutants that pose serious threats to public health and the environment. People are inevitably exposed to Hg via different routes, such as respiration, dermal contact, drinking or diet. Hg poisoning could cause gingivitis, inflammation, vomiting and diarrhea, respiratory distress or even death. Especially during the developmental stage, there is considerable harm to the brain development of young children, causing serious symptoms such as intellectual disability and motor impairments, and delayed neural development. Therefore, it’s of great significance to develop a specific, quick, practical and labor-saving assay for monitoring Hg2+. Herein, a mitochondria-targeted dual (excitation 700 nm and emission 728 nm) near-infrared (NIR) fluorescent probe JZ-1 was synthesized to detect Hg2+, which is a turn-on fluorescent probe designed based on the rhodamine fluorophore thiolactone, with advantages of swift response, great selectivity, and robust anti-interference capability. Cell fluorescence imaging results showed that JZ-1 could selectively target mitochondria in HeLa cells and monitor exogenous Hg2+. More importantly, JZ-1 has been successfully used to monitor gastrointestinal damage of acute mercury poisoning in a drug-induced mouse model, which provided a great method for sensing Hg species in living subjects, as well as for prenatal diagnosis.

Ultrabright NIR AIEgen nanoparticles‐enhanced lateral flow immunoassay platform for accurate diagnostics of complex samples

AbstractAccurate and sensitive near‐infrared (NIR) luminescent lateral flow immunoassay (LFIA) has attracted considerable attention in the field of point‐of‐care testing (POCT). However, the detection accuracy and sensitivity are often compromised by the low fluorescence quantum efficiency (<10%) of the NIR fluorescent probe. Herein, ultrabright NIR AIEgen nanoparticles (PS@AIE830NPs) composed of polystyrene (PS) nanoparticles and NIR aggregation‐induced emission luminogen (AIEgen) with the maximum emission at 830 nm (AIE830) is reported, and its potential to promote an accurate and sensitive detection of complex samples by LFIA is described. The relative quantum yield (QY) of the PS@AIE830NPs was 14.76%, which was superior to that of the polymer embedding method and indocyanine green (ICG)‐based NIR nanoparticles. The PS@AIE830NPs immunolabeled‐LFIA combined with laboratory‐built NIR‐LFIA portable quantitative instruments (detected light range > 800 nm) completely eliminated background interference and allowed highly accurate and sensitive detection without any pre‐treatment steps. The limits of detection (LODs) for aflatoxin B1 (AFB1) in soy sauce, alpha hemolysin (Hla) of Staphylococcus aureus biomarker in joint fluid, and C‐reactive protein (CRP) in human haemolysed samples were 0.01 ng mL−1, 0.02 µg mL−1, and 0.156 mg L−1, respectively, commensurating with those of the corresponding gold standard assays and covering the detection range of interests. It is anticipated that the ultrabright NIR AIEgen nanoparticles will serve as a universally applicable signal probe for NIR‐LFIA diagnostics, promising to expand the range of applications for quantitative detection of complex samples.

Discovery of Natural Products Alleviating Renal Fibrosis with a Viscosity-Responsive Molecular Probe.

Renal fibrosis poses a significant threat to individuals suffering from chronic progressive kidney disease. Given the absence of effective medications for treating renal fibrosis, it becomes crucial to assess the extent of fibrosis in real time and explore the development of novel drugs with substantial therapeutic benefits. Due to the accumulation of renal tissue damage and the uncontrolled deposition of fibrotic matrix during the course of the disease, there is an increase in viscosity both intracellularly and extracellularly. Therefore, a viscosity-sensitive near-infrared fluorescence (NIRF) and photoacoustic (PA) imaging probe, BDP-KY, was developed to detect aberrant changes in viscosity during fibrosis. Furthermore, BDP-KY has been applied to screen the effective components of herbal medicine, rhubarb, resulting in the identification of potential antirenal fibrotic compounds such as emodin-8-glucoside and chrysophanol 8-O-glucoside. Ultrasound, PA, and NIRF imaging of a unilateral uretera obstruction mice model show that different concentrations of emodin-8-glucoside and chrysophanol 8-O-glucoside effectively reduce viscosity levels during the renal fibrosis process. The histological results showed a significant decrease in fibrosis factors α-smooth muscle actin and collagen deposition. Combining these findings with their pharmacokinetic characteristics, these compounds have the potential to fill the current market gap for effective antirenal fibrosis drugs. This study demonstrates the potential of BDP-KY in the evaluation of renal fibrosis, and the two identified active components from rhubarb hold great promise for the treatment of renal fibrosis.

Rational design of a near-infrared fluorescent probe for monitoring butyrylcholinesterase activity and its application in development of inhibitors

Butyrylcholinesterase (BChE) is widely expressed in multiple tissues and has a vital role in several key human disorders, such as Alzheimer’s disease and tumorigenesis. However, the role of BChE in human disorders has not been investigated. Thus, to quantitatively detect and visualize dynamical variations in BChE activity is essential for exploring the biological roles of BChE in the progression of a number of human disorders. Herein, based on the substrate characteristics of BChE, we customized and synthesized three near-infrared (NIR) fluorescent probe substrates with cyanine-skeleton, and finally selected a NIR fluorescence probe substrate named CYBA. The CYBA demonstrated a significant increase in fluorescence when interacting with BChE, but mainly avoided AChE. Upon the addition of BChE, CYBA could be specifically hydrolyzed to TBO, resulting in a significant NIR fluorescence signal enhancement at 710 nm. Systematic evaluation revealed that CYBA exhibited exceptional chemical stability in complex biosamples and possessed remarkable selectivity and sensitivity towards BChE. Moreover, CYBA was successfully applied for real-time imaging of endogenous BChE activity in two types of nerve-related living cells. Additionally, CYBA demonstrated exceptional stability in the detection of complex biological samples in plasma recovery studies (97.51%–104.01%). Furthermore, CYBA was used to construct a high-throughput screening (HTS) method for BChE inhibitors using human plasma as the enzyme source. We evaluated inhibitory effects of a series of natural products and four flavonoids were identified as potent inhibitors of BChE. Collectively, CYBA can serve as a practical tool to track the changes of BChE activity in complicated biological environments due to its excellent capabilities.

Ratiometric Near-Infrared Fluorescent Probe Monitors Ferroptosis in HCC Cells by Imaging HClO in Mitochondria.

Hypochlorous acid (HClO) is a typical endogenous ROS produced mainly in mitochondria, and it has strong oxidative properties. Abnormal HClO levels lead to mitochondrial dysfunction, strongly associated with various diseases. It has been shown that HClO shows traces of overexpression in cells of both ferroptosis and hepatocellular carcinoma (HCC). Therefore, visualization of HClO levels during ferroptosis of HCC is important to explore its physiological and pathological roles. So far, there has been no report on the visualization of HClO in ferroptosis of HCC. Thus, we present a ratiometric near-infrared (NIR) fluorescent probe Mito-Rh-S which visualized for the first time the fluctuation of HClO in mitochondria during ferroptosis of HCC. Mito-Rh-S has an ultrafast response rate (2 s) and large emission shift (115 nm). Mito-Rh-S was constructed based on the PET sensing mechanism and thus has a high signal-to-noise ratio. The cell experiments of Mito-Rh-S demonstrated that Fe2+- and erastin-induced ferroptosis in HepG2 cells resulted in elevated levels of mitochondrial HClO and that high concentration levels of Fe2+ and erastin cause severe mitochondrial damage and oxidative stress and had the potential to kill HepG2 cells. By regulating the erastin concentration, erastin induction time, and treatment of the ferroptosis model, Mito-Rh-S can accurately detect the fluctuation of mitochondrial HClO levels during ferroptosis in HCC.

Near-Infrared Fluorescence Probe with a New Recognition Moiety for the Specific Detection of Cysteine to Study the Corresponding Physiological Processes in Cells, Zebrafish, and Arabidopsis thaliana.

Cysteine (Cys), as one of the biological thiols, is related to many physiological and pathological processes in humans and plants. Therefore, it is necessary to develop a sensitive and selective method for the detection and imaging of Cys in biological organisms. In this work, a novel near-infrared (NIR) fluorescent probe, Probe-Cys, was designed by connecting furancarbonyl, as a new recognition moiety, with Fluorophore-OH via the decomposition of IR-806. The use of the furan moiety is anticipated to produce more effective fluorescence quenching because of the electron-donating ability of the O atom. Probe-Cys has outstanding properties, such as a new recognition group, an emission wavelength in the infrared region at 710 nm, a linear range (0-100 μM), a low detection limit of 0.035 μM, good water solubility, excellent sensitivity, and selectivity without the interference of Hcy, GSH, and HS-. More importantly, Probe-Cys could achieve the detection of endogenous Cys by reacting with the stimulant 1,4-dimercaptothreitol (DTT) and the inhibitor N-ethylmaleimide (NEM) in HepG2 cells and zebrafish. Ultimately, it was successfully applied to obtain images of Arabidopsis thaliana, revealing that the content of Cys in the meristematic zone was higher than that in the elongation zone, which was the first time that the NIR fluorescence probe was used to obtain images of Cys in A. thaliana. The superior properties of the probe exhibit its great potential for use in biosystems to explore the physiological and pathological processes associated with Cys.

Near-Infrared Fluorescent Probe for the Detection of Cysteine

Hemicyanine dyes are an ideal structure for building near-infrared fluorescent probes due to their excellent emission wavelength properties and biocompatibility in biological imaging field. Developing a near-infrared fluorescent probe capable of detecting cysteine (Cys) was the aim of this study. A novel developed fluorescent probe P showed high selectivity and sensitivity to Cys in the presence of various analytes. The detection limit of P was found to be 0.329 μM. The MTT assay showed that the probe was essentially non-cytotoxic. Furthermore, the probe was successfully used as cysteine imaging in living cells and mice.

Near-infrared fluorescent probe based on carboxylesterase 2 and viscosity cascade response for early diagnosis of thyroid cancer

Carboxylesterase 2 (CES2) plays a critical role in the metabolism of ester- and amide-containing drugs and thus has an impact on drug efficacy and toxicity. This study introduces QVJ-CES2, a near-infrared fluorescent probe designed to enhance thyroid cancer diagnosis through CES2 enzyme activity and distinct cellular viscosity of thyroid cells. The probe's dual-response mechanism significantly amplified fluorescence in high-viscosity environments, thus had high sensitivity. Our findings revealed that QVJ-CES2 could detect CES2 activity with a limit of detection as low as 1.9ng/mL. In vitro experiments confirmed the rapid and selective response of QVJ-CES2 to CES2 and the ability to distinguish cancer cells from their normal cells with high specificity. Most importantly, the fluorescence intensity difference between cancerous and normal cells after incubation with the probe was as high as 7-fold, attributable to the synergistic enzyme and viscosity cascade response. Further, in vivo studies demonstrated that the probe was effective in accurately identifying thyroid cancer tissues, showing that it is a promising tool for early cancer detection and precise surgical interventions.

Clarifying the role of carboxyl functionality in squaraines towards aggregation and self-assembly through photophysical characterization and BSA interactions

The demand for near-infrared (NIR) probes with robust and stable fluorescence signals has surged, driven by their applications in optical sensing and bioimaging. Squraine dyes as NIR fluorescent probes have gained significant traction. However, understanding their behavior in aqueous media is important for their successful application. Herein we report the synthesis and photophysical characterization of carboxy-functionalized unsymmetrical squaraine dyes capable of light absorption and emission in the near-infrared (NIR) wavelength region. Structural variations in alkyl chain length, and spacer chain length between the primary chromophore and –COOH group were systematically introduced to investigate their impact on self-assembly in aqueous media leading to dye aggregation and their differential interactions with Bovine Serum Albumin (BSA) as a model protein. In phosphate buffer (PB) the absorption intensity decreased drastically along with pronounced shoulders due to dye aggregation leading to complete fluorescence quenching. However, in the presence of BSA, the intensity of shoulder bands reduced coupled with a sharp increase in the absorption associated with monomeric dye with the increasing concentration of BSA, indicating the prevention of dye aggregation owing to dye-BSA interactions. The prevention of dye aggregation due to dye-BSA interaction also led to a tremendous increase in the fluorescence intensities as a function of the increasing BSA concentrations. Site-selective study off BSA showed that these dyes bind preferably at Site-II using hydrophilic interactions. Thus, these dyes show great potential as FRET or fluorescent on/off probes for biosensing, non-covalent labeling of proteins, and fluorescent probes for bioimaging.

ONOO–-activatable and LD-traced NIR fluorescent probe for mechanism study and early diagnosis of NAFLD and diabetes induced liver injury

Lipid metabolism disorder and oxidative stress play an essential role in the occurrence and development of metabolic diseases such as nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). Here, we constructed an ONOO-activatable and lipid drops (LDs) traced near-infrared (NIR) fluorescent probe BDP-NIR-Py+ through structural modification, and evaluated oxidative stress and lipid metabolism disorders in relevant disease by monitoring changes in ONOO and LDs levels in cells and mice. With the dual monitoring of ONOO and LDs, BDP-NIR-Py+ realized real-time visualization of the occurrence and development of NAFLD and T2DM, and early diagnosis of liver injury, which is faster and more sensitive than tissue analysis and blood detection. Collectively, the ability of BDP-NIR-Py+ to simultaneously monitor ONOO and LDs makes it has unlimited potential in the early diagnosis and mechanism research of diseases such as NAFLD and T2DM in which oxidative stress and lipid metabolism disorder interact.

Discovery of Near-Infrared Heptamethine Cyanine Probes for Imaging-Guided Surgery in Solid Tumors.

Near-infrared (NIR) fluorescence imaging has attracted much attention in image-guided interventions with unique advantages. However, the clinical translation rate of fluorescence probes is extremely low, primarily due to weak lesion signal contrast and poor specificity. To address this dilemma, a series of small-molecule near-infrared fluorescence probes have been designed for tumor imaging. Among them, YQ-04-03 showed notable optical stability and remarkable sensitivity toward tumor targeting. Moreover, within a specific concentration and time range against oxidizing reducing agents and laser, it demonstrated better stability than ICG. The retention time of YQ-04-03 in tumors was significantly longer compared to other nonspecific uptake sites in the subjects, and its tumor-to-normal tissue ratio (TNR) outperformed ICG. Successful resection of in situ hepatocarcinoma and peritoneal carcinoma was achieved using probe imaging guidance, with the smallest visual lesion resected measuring approximately 1 mm3. Ultimately, this probe holds great potential for advancing tumor tracer.

Dicyanoisophorone-based near-infrared fluorescent probe with large Stokes shift for the monitoring and bioimaging of hypochlorite

Hypochlorite (ClO−), as one of reactive oxygen species (ROS), is closely linked to various illnesses and is essential for the proper functioning of immune system. Hence, monitoring and assessing ClO− levels in organisms are extremely important for the clinical diagnosis of ClO−-related disorders. In this study, a novel ClO−-selective fluorescent probe, DCP-ClO, was synthesized with dicyanoisophorone-xanthene unit as parent fluorophore, which displayed excellent selectivity towards ClO−, near-infrared emission (755 nm), large Stokes shift (100 nm), real-time response to ClO−, high sensitivity (LOD = 3.95 × 10−8 M), and low cytotoxicity. The recognition mechanism of DCP-ClO towards ClO− was confirmed to be a typical ICT process by HPLC-MS, HR-MS, 1H NMR and theoretical calculations. Meanwhile, DCP-ClO demonstrated remarkable efficacy in monitoring ClO− levels in water samples and eye-catching ability in imaging endogenous/exogenous ClO− in living organisms, which verified its potential as a powerful tool for the recognition of ClO− in complex biological systems.

Theoretical design of novel fluorophores for near-infrared fluorescent probes of peroxynitrite

On the basis of the experimentally reported near-infrared (NIR) fluorescent probe for peroxynitrite anions (ONOO–) DDAO-PN (DDAO = 1,3-dichloro-7-oxygen-9,9-dimethyl-2(9H)-acridinone, PN = 4- methylene benzeneboronic acid pinacol ester, DDAO-PN connecting 7-oxygen of DDAO with 4- methylene of PN), six DDAO derivatives DDAO-Px (x = 1–6) were designed. The fluorescence response mechanism and characteristics of the DDAO-PN probe to ONOO– were explored and the molecular structures, molecular orbital, and electron transfer during excitation and emission in DDAO-PN and DDAO-Px (x = 1–6) were comparatively analyzed in detail through the density functional theory (DFT) and time-dependent DFT calculations. The significant intramolecular charge transfer (ICT) effect in DDAO-PN were found to be suppressed when PN (as the recognition domain of ONOO– in the recognition process) was removed, rendering DDAO-PN being an effective “off-on” NIR ONOO– fluorescent probe. The introduction of different electron-donating/accepting substituents in DDAO-Px significantly changed the electronic structures and thus the fluorescent properties, which finally modified the probe efficiency. This work clarifies the structure-property relationship in DDAO-Px type NIR probes and thus provides theoretical guidance for designing new NIR fluorescent probe.

Hypoxia-activated and improved cell uptake near-infrared fluorescent probe for precise guiding surgical resection

Tumor imaging and fluorescence-guided surgical resection offers a promising therapeutic approach for the treatment of cancer with simple operation. Hypoxia, as a crucial feature in solid tumor, is closely related to the design of hypoxia-activated fluorescent probes to distinguish tumor and normal tissue. In present, many hypoxia-activated fluorescent probes have received widespread attention in various imaging applications. However, as far as we know, hypoxia-activated near-infrared fluorescent probe for tumor fluorescence-guided surgical resection is not currently reported. Therefore, a hypoxia-activated near-infrared fluorescent probes Fu-Cy was synthesized. It was able to produce near-infrared fluorescence (780 nm) in specific response to overexpressed nitroreductase in hypoxic tumors with rapid response. However, the uptake of the probe in the cell will affect the imaging ability of the probe. Therefore, acetazolamide (AZ), a well-known inhibitor of carbonic anhydrase IX (CAIX), was coupled to Fu-Cy by clicking reaction to obtain the improved probe Az-Fu-Cy, which is more absorbed by cancer cells than Fu-Cy. Thus, Az-Fu-Cy can effectively distinguish between tumors and normal organs by spraying. Interestingly, it showed accurate guidance during surgical resection.

Monitoring Hg2+ ions in food and environmental matrices using a novel ratiometric NIR fluorescent sensor via carbonothioate-deprotection reaction

Mercury toxicity and its environmental impact are significant concerns for public health and environmental protection. Therefore, the development of effective, rapid, and reliable detection methods for trace levels of Hg2+ is crucial. Herein, a cyanine dye bearing a carbonothioate group is reported as a potential NIR fluorescent probe for Hg2+ detection. The spectral properties of the free probe have been characterized by the presence and absence of a series of analytes. The addition of Hg2+ leads to significant changes in the fluorescence signal with distinct red coloration compared to other competing analytes, indicating that the probe is highly selective for Hg2+. The fluorescence quantum yield increases from 0.073 to 0.315. The detection limit is 0.10 μM, indicating the high sensitivity of the probe to low Hg2+ levels. The most prominent sensing features of the probe include NIR fluorescence, low cytotoxicity, ratiometric fluorescence response, and fast response compared to most of the currently available fluorescent probes. In addition, the probe can detect Hg2+ in actual samples such as foodstuff, soil, water, and live cells. Bioimaging studies have demonstrated that the present probe is highly efficient in targeting mitochondria and possesses good imaging abilities for detecting Hg2+ in cells. Therefore, these results suggest that it can be proposed as a powerful NIR fluorescent probe for the highly sensitive detection of Hg2+.

Abstract 4167: Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes

Abstract Tumors consist of a complex mixture of resident and infiltrating cells that can support their survival and evasion of treatment. Selective detection of genetically stable cells in the tumor microenvironment (TME) offers insight into tumor biology beyond the malignant cells, which are the focus of most molecular imaging approaches to cancer detection. Motivated by the goal of non-invasively understanding the primary breast TME, we have developed fluorescent molecular probes that target cancer-associated fibroblasts (CAFs), a major component of the stromal compartment of many tumors, and emit light in the near-infrared (NIR) spectral window, providing access to deeper tissues than visible probes. We synthesized and characterized a series of CAF-targeted antibody- and small molecule-based fluorescent imaging agents utilizing a highly hydrophilic NIR dye and ligands that target Fibroblast Activation Protein (FAP). FAP is a key cell-surface marker of breast cancer CAFs with an S1 phenotype, which are modulators of immunosuppression, metastatic spread, and are associated with worse outcomes, especially in HER2+ and triple-negative breast cancer. Modifications to the chemical structure of the linker and targeting ligands allowed us to alter the specificity and brightness of the CAF-probes in cellulo. Imaging of a co-implanted tumor model of SKBR3 human breast cancer cells and FAP expressing fibroblasts after administering our lead small-molecule CAF imaging agent revealed selective tumor targeting of CAF-rich tumors compared to cancer cell predominant tumors with favorable pharmacokinetics and tumor-to-background ratio (TBR) greater than 2. Similar imaging studies with MMTV-PyMT transgenic mice, an established murine model of breast cancer, showed analogous enhanced uptake in mammary tumors (TBR > 2) and clearance over 24 hours. NIR fluorescence also correlated with CAFs isolated from PyMT tumor tissue via flow cytometry, consistent with our hypothesis that this agent can specifically detect endogenous CAFs. This study identified a novel, bright, and water-soluble FAP-specific NIR agent for in vivo imaging, setting the stage for simultaneous optical imaging of malignant cells and activated fibroblasts in the TME to elucidate cancer-host interactions. Citation Format: Megan S. Michie, Junwei Du, Jiayu (Jennifer) Ye, Kathleen Duncan, Mikhail Y. Berezin, Samuel Achilefu. Selective imaging of cancer-associated fibroblasts in the breast cancer microenvironment with near-infrared fluorescent probes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4167.

Near-infrared dual-response fluorescent probe for detection of N2H4 and intracellular viscosity changes in biological samples and various water samples

N2H4 is a common raw material used in the production of pesticides and has good water solubility, so it may contaminate water sources and eventually enter living organisms, causing serious health problems. Viscosity is an important indicator of the cellular microenvironment and an early warning signal for many diseases. The high reactivity of hydrazine depletes glutathione (GSH) in hepatocytes, causing oxidative stress ultimately leading to significant changes in intracellular viscosity and even death. Therefore, it is particularly important to develop an effective method to detect N2H4 and viscosity in environmental and biological systems. On this basis, we developed two fluorescent probes, BDD and BHD, based on xanthene and 2-benzothiazole acetonitrile. The experimental results show that BHD and BDD have good imaging capabilities for N2H4 in cells, zebrafish and Arabidopsis. BHD and BDD also showed sensitive detection and fluorescence enhancement in the near-infrared region when the intracellular viscosity was changed. Notably, the probe BDD has also successfully imaged N2H4 in a variety of real water samples.

A near-infrared fluorescence probe with large Stokes shift for selectively monitoring nitroreductase in living cells and mouse tumor models

Hypoxia is commonly regarded as a typical feature of solid tumors, which originates from the insufficient supply of oxygen. Herein, the development of an efficient method for assessing hypoxia levels in tumors is strongly desirable. Nitroreductase (NTR) is an overexpressed reductase in the solid tumors, has been served as a potential biomarker to evaluate the degrees of hypoxia. In this work, we elaborately synthesized a new near-infrared (NIR) fluorescence probe (MR) to monitor NTR activity for assessment of hypoxia levels in living cells and in tumors. Upon exposure of NTR, the nitro-unit of MR could be selectively reduced to amino-moiety with the help of nicotinamide adenine dinucleotide. Moreover, the obtained fluorophore emitted a prominent NIR fluorescence, because it possessed a classical “push-pull” structure. The MR displayed several distinguished characters toward NTR, including intense NIR fluorescent signals, large Stokes shift, high selectivity and low limit of detection (46 ng/mL). Furthermore, cellular confocal fluorescence imaging results validated that the MR had potential of detecting NTR levels in hypoxic cells. Significantly, using the MR, the elevated of NTR levels were successfully visualized in the tumor-bearing mouse models. Therefore, this detecting platform based on this probe may be tactfully constructed for monitoring the variations of NTR and estimating the degrees of hypoxia in tumors.

Acidity-Triggered "Sticky Spotlight": CCK2R-Targeted TME-Sensitive NIR Fluorescent Probes for Tumor Imaging In Vivo.

Cancer which causes high mortality globally threatens public health seriously. There is an urgent need to develop tumor-specific near-infrared (NIR) imaging agents to achieve precise diagnosis and guide effective treatment. In recent years, imaging probes that respond to acidic environments such as endosomes, lysosomes, or acidic tumor microenvironments (TMEs) are being developed. However, because of their nonspecific internalization by both normal and tumor cells, resulting in a poor signal-to-noise ratio in diagnosis, these pH-sensitive probes fail to be applied to in vivo tumor imaging. To address this issue, a cholecystokinin-2 receptor (CCK2R)-targeted TME-sensitive NIR fluorescent probe R2SM was synthesized by coupling pH-sensitive heptamethine cyanine with a CCK2R ligand, minigastrin analogue 11 (MG11) for in vivo imaging, in which MG11 would target overexpressed CCK2Rs in gastrointestinal stromal tumors (GISTs). Cell uptake assay demonstrated that R2SM exhibited a high affinity for CCK2R, leading to receptor-mediated internalization and making probes finally accumulated in the lysosomes of tumor cells, which suggested in the tumor tissues, the probes were distributed in the extracellular acidic TME and intracellular lysosomes. With a pKa of 6.83, R2SM can be activated at the acidic TME (pH = 6.5-6.8) and lysosomes (pH = 4.5-5.0), exhibiting an apparent pH-dependent behavior and generating more intense fluorescence in these acidic environments. In vivo imaging showed that coupling of MG11 with a pH-sensitive NIR probe facilitated the accumulation of probe and enhanced the fluorescence in CCK2R-overexpressed HT-29 tumor cells. A high signal was observed in the tumor region within 0.5 h postinjection, indicating its potential application in intraoperative imaging. Fluorescence imaging of R2SM exhibited higher tumor-to-liver and tumor-to-kidney ratios (2.1:1 and 2.3:1, respectively), compared separately with the probes that are lipophilic, pH-insensitive, or MG11-free. In vitro and in vivo studies demonstrated that the synergistic effect of tumor targeting with pH sensitivity plays a vital role in the high signal-to-noise ratio of the NIR imaging probe. Moreover, different kinds of tumor-targeting vectors could be conjugated simultaneously with the NIR dye, which would further improve the receptor affinity and targeting efficiency.

A novel pH-sensitive hemi-cyanine containing tetrahydropyridine ring near-infrared fluorescence probe with lysosome-targeting ability

In recent years, hemi-cyanine dyes have been widely used as biological probes due to their red-light emission characteristics and high fluorescence quantum yield. In this study, we synthesized a novel hemi-cyanine dye containing a tetrahydropyridine ring. A lysosomal target was introduced into its structure to create a new pH-sensitive near-infrared fluorescent probe that successfully targeted lysosomes. The results showed that when the probe solution was excited at the absorption wavelength of 650 nm, its fluorescence emission wavelength was about 700 nm, and the peak intensity changed with different pH values in a wide range. Therefore, this probe enabled non-invasive detection of changes in the acidic environment of lysosomes in living organisms and showed good imaging capabilities. Moreover, the probe displays high sensitivity and good stability. The theoretical calculation of a probe structure has also been completed to discuss the relationship between structure and property.