Activatable Fluorescence Research Articles

An activatable fluorescence probe for visualization of DAGL activity in hippocampal tissue of brain-injured mice

Diacylglycerol lipase (DAGL) is an important biosynthetic enzyme of endocannabinoid 2-arachidonoyl-glycerol (2-AG) that activates the endocannabinoid receptors to mediate basic biological processes, such as inflammation, pain sensation, epileptogenesis, synaptic plasticity, neuroprotection and adult neurogenesis. However, the changes of DAGL activity are still unclear in neurophysiological and pathological processes due to a lack of effective method for in situ detection of DAGL activity. Herein, based on the specific cleavage of the sn-1 acyl group of diacylglycerol (DAG) hydrolyzed by DAGL, we rational design an activatable fluorescence probe by simulating the substrate of DAGL for detecting the DAGL activity. Coumarin and rhodamine fluorophores are introduced into 1- and 2-site of glycerol through ester bonds to construct a fluorescence resonance energy transfer probe CGR. The probe CGR shows high selectivity and can be used to detect the activity of endogenous DAGL in tissue and brain. Notably, we used the probe CGR to reveal the changes of DAGL activity after brain injury.

Polymeric agents for activatable fluorescence, self-luminescence and photoacoustic imaging

Numerous polymeric agents have been widely applied in biology and medicine by virtue of the facile chemical modification, feasible nano-engineering approaches and fine-tuned pharmacokinetics. To endow polymeric imaging agents with ability to monitor and measure subtle molecular or cellular alterations at diseased sites, activatable polymeric probes that can elicit signal changes in response to biomolecular interactions or the analytes of interest have to be developed. Herein, this review aims to provide a systemic interpretation and summarization of the design methodology and imaging utility of recently emerged activatable polymeric probes. An introduction of activatable probes allowing for precise imaging and classification of polymeric imaging agents is reported first. Then, we give a detailed discussion of the contemporary design approaches toward activatable polymeric probes in diverse imaging modes for the detection of various stimuli and their imaging applications. Finally, current challenges and future advances are discussed and highlighted.

Redox-Responsive Breakup of a Nucleic Acids@CoOOH Nanocomplex Triggering Cascade Recycling Amplification for Sensitive Sensing of Alkaline Phosphatase.

Alkaline phosphatase (ALP), an essential hydrolase with crucial roles in living organisms, has widely been regarded as a biomarker for various human diseases in clinical diagnoses. Herein, taking advantage of cobalt oxyhydroxide (CoOOH) nanoflakes and nonenzymatic cascade recycling amplification (CRA), a highly sensitive and label-free fluorescence biosensing strategy for the determination of ALP activity is introduced. In our design, ALP can promote the dephosphorylation of l-ascorbic acid 2-phosphate (AAP) to reduce ascorbic acid (AA), which is then able to decompose CoOOH in a nucleic acids@CoOOH nanocomplex into Co2+ cofactors. Further, enzyme-free CRA was rapidly initiated by integrating DNAzyme recycling amplification and catalytic hairpin assembly, resulting in the generation of an abundance of G-quadruplex structure-contained DNA duplexes. In the presence of thioflavin T (ThT), analytical target ALP was converted in an amplified and activatable fluorescence signal. The experimental results show that this method can be applied for the quantitative analysis of ALP activity with a low detection limit of 0.027 mU/mL. Moreover, this developed biosensing approach exhibits excellent specificity, and the evaluation of ALP activity in the complex human serum samples was successfully realized, indicating that it can afford a reliable, robust, and cost-effective nanoplatform for an ALP-based clinical diagnosis and for biomedical research.

Specific diagnosis of lymph node micrometastasis in breast cancer by targeting activatable near-infrared fluorescence imaging

Axillary lymph node metastasis has always been defined as the most important prognostic factor in the treatment of early breast cancer. Ultrasound and MRI can detect only 10% of lymph node micrometastases in early breast cancer. Therefore, it is crucial to detect early breast cancer with lymph node metastasis, however, there is no current examination method for accurate diagnosis. When breast cancer presents a malignant tendency, colony stimulating factor-1 and chemokine CCL-2 absorb mononuclear cells from the surrounding environment and differentiate into M2 Tumor associated macrophages (TAM), which increase the invasion of tumor cells and further promote the development of tumors. Mannose, as a simple natural ligand, can selectively bind to TAM surface CD206 (macrophage mannose receptor, MMR). In this study, mannose was connected with near infrared dye (NIR) IR780 via disulfide bond to obtain Mannose-IR780 conjugate (MR780), which was further self-assembled into near infrared nanoprobe (MR780 NPs) with quenched fluorescence. When selectively targeting CD206 highly expressed on the surface of TAM, disulfide bond was cleaved by the glutathione enriched in the microenvironment, resulting in fluorescence recovery, thus achieving NIR fluorescence molecular imaging of TAM and diagnosis of tumor lymph node metastasis in mouse models. Our findings suggest that targeted imaging of TAM enable noninvasive and sensitive detection of metastatic lymph nodes in vivo, which is instructive for tumor therapy.

Activatable fluorescence molecular imaging and anti-tumor effects investigation of GSH-sensitive BRD4 ligands

Overexpression of bromodomain 4 (BRD4) is closely correlated with a variety of human cancers by regulating the histone post-translational modifications, which renders BRD4 a promising target for pharmacological discoveries of novel therapeutic agents for cancer therapy. We herein present the design, chemical synthesis, cellular imaging and biological assessment of a novel tumor-sensitive BRD4 ligand (compound 4) by introducing anticancer BRD4 inhibitor into naphthalimide moiety (fluorescent reporter) via a sulfonamide unit as glutathione (GSH)-specific cleavable linker. Upon reaction with abundant intramolecular GSH in cancer cells or free GSH in aqueous solution (pH = 7.4), sulfonamide cleavage of 4 occurs, leading to the release of BRD4 inhibitor and concomitant fluorescence-on. This activatable fluorescence molecular imaging was demonstrated to preferentially occur in tumor cells. Moreover, towards cancer cell lines MGC-803 cells and THP-1, compound 4 was identified to show better antitumor efficacy than net BRD4 inhibitor. Collectively, this study presents a drug delivery strategy, wherein the drug release can be directly monitored in the cellular content by fluorescence imaging, and provides a valuable compound 4 as a potential antitumor agent. Compound 4 may represent a useful tool for explorative studies of BRD4 inhibition, such as an improved understanding of BRD4 inhibitor release-related information.

Activatable NIR-II organic fluorescent probes for bioimaging.

NIR-II imaging is developed rapidly for noninvasive deep tissue inspection with high spatio-temporal resolution, taking advantage of diminished autofluorescence and light attenuation. Activatable NIR‐II fluorescence probes are widely developed to report pathological changes with accurate targeting, among which organic fluorescent probes achieve significant progress. Furthermore, the activatable NIR‐II fluorescent probes exhibited appealing characteristics like tunable physicochemical and optical properties, easy processability, and excellent biocompatibility. In the present review, we highlight the advances of activatable NIR-II fluorescence probes in design, synthesis and applications for imaging pathological changes like reactive oxygen species (ROS), reactive nitrogen species (RNS), reactive sulfur species (RSS), pH, hypoxia, viscosity as well as abnormally expressed enzymes. This non-invasive optical imaging modality shows a promising prospect in targeting the pathological site and is envisioned for potential clinical translation.

A carboxylesterase-activatable near-infrared phototheranostic probe for tumor fluorescence imaging and photodynamic therapy.

Phototheranostic probes have been proven to be a promising option for cancer diagnosis and treatment. However, near-infrared phototheranostic probes with specific tumor microenvironment responsiveness are still in demand. In this paper, a carboxylesterase (CES)-responsive near-infrared phototheranostic probe was developed by incorporating 6-acetamidohexanoic acid into a hemicyanine dye through an ester bond. The probe exhibits highly sensitive and selective fluorescence enhancement towards CES because CES-catalyzed cleavage of the ester bond leads to the release of the fluorophore. By virtue of its near-infrared analytical wavelengths and high sensitivity, the probe has been employed for endogenous CES activatable fluorescence imaging of tumor cells. Moreover, under 660 nm laser irradiation, the probe can generate toxic reactive oxygen species and efficiently kill tumor cells, with low cytotoxicity in dark. As far as we know, the probe was the first CES-responsive phototheranostic probe with both near-infrared analytical wavelengths and photosensitive capacity, which may be useful in the real-time and in situ imaging of CES as well as imaging-guided photodynamic therapy of tumors. Therefore, the proposed probe may have wide application prospect in cancer theranostics.

A gold nanobipyramid-based photothermal reagent with functions of targeting and activatable fluorescence labeling for visual photothermal therapy

A gold nanobipyramid-based photothermal reagent with functions of targeting and activatable fluorescence labeling was constructed for visual photothermal therapy.

Intracellular Enzyme-Responsive Profluorophore and Prodrug Nanoparticles for Tumor-Specific Imaging and Precise Chemotherapy.

Responsive drug delivery systems possess great potential in disease diagnosis and treatment. Herein, we develop an activatable prodrug and fluorescence imaging material by engineering the endogenous NAD(P)H:quinone oxidoreductase-1 (NQO1) responsive linker. The as-prepared nanomaterials possess the NQO1-switched drug release and fluorescence enablement, which realizes the tumor-specific chemotherapy and imaging in living mice. The enzyme-sensitive prodrug nanoparticles exhibit selectively potent anticancer performance to NQO1-positive cancer and ignorable off-target toxicity. This work provides an alternative strategy for constructing smart prodrug nanoplatforms with precision, selectivity, and practicability for advanced cancer imaging and therapy.

Target-Activating and Toehold Displacement Ag NCs/GO Biosensor-Mediating Signal Shift and Enhancement for Simultaneous Multiple Detection.

Herein, we demonstrate that a new multicolor silver nanoclusters/graphene oxide (Ag NCs/GO) hybrid material, upon target response, undergoes a configuration transformation, based on entropy-driven enzyme-free toehold-mediated strand displacement reaction, achieving emission shift and enhancement. To realize the aim above, two different synthesis routes (route I and II) of synthesizing fluorescent Ag NCs for constructing toehold displacement Ag NCs/GO biosensor is designed and performed. Influenza A virus subtype genes (H1N1 and H5N1) as a model can efficiently initiate the operation of entropy-driven displacement reaction, resulting in activatable fluorescence. Red-emitting and green-emitting Ag NCs tethering the complementary sequence of H1N1 (pDNA1) and H5N1 (pDNA2) are indirectly immobilized on GO surface through binding with capture DNA (cDNA1 and cDNA2), respectively, forming multicolor pDNA-Ag NCs/GO nanohybrid materials. However, they do not exhibit nearly fluorescence signals attributed to energy transfer from donor Ag NCs to acceptor GO. Upon adding targets H1N1 and H5N1 (tDNA1 and tDNA2), pDNA1-Ag NCs and pDNA2-Ag NCs detach from GO, based on toehold-mediated strand displacement reaction, which interferes the energy transfer and leads to significant fluorescence enhancement. More interestingly, the activatable process is accompanied by remarkable hypsochromic shift (19 nm) or bathochromic shift (21 nm) emission with quite high fluorescence recovery rates (823.35% and 693.62%). Therefore, based on these phenomena, a novel multiple approach has been developed with the assistance of toehold displacement and Ag NCs/GO nanohybrid materials. As for the remarkable emission recovery and multichannel signal, the proposed approach displays the promising application prospect in accurate diagnosis and treatment.

An Activatable Near-Infrared Fluorescence Probe for in Vivo Imaging of Acute Kidney Injury by Targeting Phosphatidylserine and Caspase-3.

Renal-clearable and target-responsive near-infrared (NIR) fluorescent imaging probes have been promising for in vivo diagnosis of acute kidney injury (AKI). However, designing an imaging probe that is renal-clearable and concurrently responsive toward multiple molecular targets to facilitate early detection of AKI with improved sensitivity and specificity is challenging. Herein, by leveraging the receptor-mediated binding and retention effect along with enzyme-triggered fluorescence activation, we design and synthesize an activatable small-molecule NIR fluorescent probe (1-DPA2) using a "one-pot sequential click reaction" approach. 1-DPA2 can target both the externalized phosphatidylserine (PS) and active caspase-3 (Casp-3), two essential biomarkers of apoptosis, producing enhanced 808 nm NIR fluorescence and a high signal-to-background ratio (SBR) amenable to detecting the onset of cisplatin-induced AKI in mice as early as 24 h post-treatment with cisplatin. We not only monitor the gradual activation of Casp-3 in the kidney of mice upon AKI progression but also can report on the progressive recovery of kidney functions in AKI mice following N-acetyl-l-cysteine (NAC) therapy via real-time fluorescence imaging by 1-DPA2. This study demonstrates the ability of 1-DPA2 for longitudinal monitoring of renal cell apoptosis by concurrently targeting PS externalization and Casp-3 activation, which is efficient for early diagnosis of AKI and useful for prediction of potential drug nephrotoxicity as well as in vivo screening of anti-AKI drugs' efficacy.

Multifunctional Thio-Stabilized Gold Nanoparticles for Near-Infrared Fluorescence Detection and Imaging of Activated Caspase-3.

Gold nanoparticles (AuNPs) are commonly used in nanomedicine because of their unique spectral properties, chemical and biological stability, and ability to quench the fluorescence of organic dyes attached to their surfaces. However, the utility of spherical AuNPs for activatable fluorescence sensing of molecular processes have been confined to resonance-matched fluorophores in the 500 nm to 600 nm spectral range to maximize dye fluorescence quenching efficiency. Expanding the repertoire of fluorophore systems into the NIR fluorescence regimen with emission >800 nm will facilitate the analysis of multiple biological events with high detection sensitivity. The primary goal of this study is to determine if spherical AuNP-induced radiative rate suppression of non-resonant near-infrared (NIR) fluorescent probes can serve as a versatile nanoconstruct for highly sensitive detection and imaging of activated caspase-3 in aqueous media and cancer cells. This required the development of activatable NIR fluorescence sensors of caspase-3 designed to overcome the nonspecific degradation and release of the surface coatings in aqueous media. We harnessed the fluorescence-quenching properties and multivalency of spherical AuNPs to develop AuNP-templated activatable NIR fluorescent probes to detect activated caspase-3, an intracellular reporter of early cell death. Freshly AuNPs were coated with a multifunctional NIR fluorescent dye-labeled peptide (LS422) consisting of an RGD peptide sequence that targets αvβ3-integrin protein (αvβ3) on the surface of cancer cells to mediate the uptake and internalization of the sensors in tumor cells; a DEVD peptide sequence for reporting the induction of cell death through caspase-3 mediated NIR fluorescence enhancement; and a multidentate hexacysteine sequence for enhancing self-assembly and stabilizing the multifunctional construct on AuNPs. The integrin binding affinity of LS422 and caspase-3 kinetics were determined by a radioligand competitive binding and fluorogenic peptide assays, respectively. Detection of intracellular caspase-3, cell viability, and the internalization of LS422 in cancer cells were determined by confocal NIR fluorescence spectroscopy and microscopy. Narrow size AuNPs (13 nm) were prepared and characterized by transmission electron microscopy and dynamic light scattering. When assembled on the AuNPs, the binding constant of LS422 for αvβ3 improved 11-fold from 13.2 nM to 1.2 nM. Whereas the catalytic turnover of caspase-3 by LS422-AuNPs was similar to the reference fluorogenic peptide, the binding affinity for the enzyme increased by a factor of 2. Unlike the αvβ3 positive, but caspase-3 negative breast cancer MCF-7 cells, treatment of the αvβ3 and caspase-3 positive lung cancer A549 cells with Paclitaxel showed significant fluorescence enhancement within 30 minutes, which correlated with caspase-3 specific activation of LS422-AuNPs fluorescence. Incorporation of a 3.5 mW NIR laser source into our spectrofluorometer increased the detection sensitivity by an order of magnitude (limit of detection ~0.1 nM of cypate) and significantly decreased the signal noise relative to a xenon lamp. This gain in sensitivity enabled the detection of substrate hydrolysis at a broad range of inhibitor concentrations without photobleaching the cypate dye. The multifunctional AuNPs demonstrate the use of a non-resonant quenching strategy to design activatable NIR fluorescence molecular probes. The nanoconstruct offers a selective reporting method for detecting activated caspase-3, imaging of cell viability, identifying dying cells, and visualizing the functional status of intracellular enzymes. Performing these tasks with NIR fluorescent probes creates an opportunity to translate the in vitro and cellular analysis of enzymes into in vivo interrogation of their functional status using deep tissue penetrating NIR fluorescence analytical methods.

Rapid fluorescence imaging of human hepatocellular carcinoma using the \u03b2-galactosidase-activatable fluorescence probe SPiDER-\u03b2Gal

Fluorescence imaging of tumours facilitates rapid intraoperative diagnosis. Thus far, a promising activatable fluorescence probe for hepatocellular carcinoma (HCC) has not been developed. Herein, the utility of the fluorescence imaging of HCC using a β-galactosidase (β-Gal)-activatable fluorescence probe SPiDER-βGal was examined. β-Gal activity was measured in cryopreserved tissues from 68 patients. Live cell imaging of HCC cell lines and imaging of tumour-bearing model mice were performed using SPiDER-βGal. Furthermore, fluorescence imaging was performed in 27 freshly resected human HCC specimens. In cryopreserved samples, β-Gal activity was significantly higher in tumour tissues than in non-tumour tissues. Fluorescence was observed in HCC cell lines. In mouse models, tumours displayed stronger fluorescence than normal liver tissue. In freshly resected specimens, fluorescence intensity in the tumour was significantly higher than that in non-tumour liver specimens as early as 2 min after spraying. Receiver operating characteristic curves were generated to determine the diagnostic value of SPiDER-βGal 10 min after its spraying; an area under the curve of 0.864, sensitivity of 85.2%, and specificity of 74.1% were observed for SPiDER-βGal. SPiDER-βGal is useful for the rapid fluorescence imaging of HCC. Fluorescence imaging guided by SPiDER-βGal would help surgeons detect tumours rapidly and achieve complete liver resection.

Fluorescence Imaging Using Enzyme-Activatable Probes for Real-Time Identification of Pancreatic Cancer.

IntroductionRadical resection is the only curative treatment for pancreatic cancer, which is a life-threatening disease. However, it is often not easy to accurately identify the extent of the tumor before and during surgery. Here we describe the development of a novel method to detect pancreatic tumors using a tumor-specific enzyme-activatable fluorescence probe.MethodsTumor and non-tumor lysate or small specimen collected from the resected specimen were selected to serve as the most appropriate fluorescence probe to distinguish cancer tissues from noncancerous tissues. The selected probe was sprayed onto the cut surface of the resected specimen of cancer tissue to acquire a fluorescence image. Next, we evaluated the ability of the probe to detect the tumor and calculated the tumor-to-background ratio (TBR) by comparing the fluorescence image with the pathological extent of the tumor. Finally, we searched for a tumor-specific enzyme that optimally activates the selected probe.ResultsUsing a library comprising 309 unique fluorescence probes, we selected GP-HMRG as the most appropriate activatable fluorescence probe. We obtained eight fluorescence images of resected specimens, among which four approximated the pathological findings of the tumor, which achieved the highest TBR. Finally, dipeptidyl-peptidase IV (DPP-IV) or a DPP-IV-like enzyme was identified as the target enzyme.ConclusionThis novel method may enable rapid and real-time visualization of pancreatic cancer through the enzymatic activities of cancer tissues.

Ratiometric Imaging of MMP-2 Activity Facilitates Tumor Detection Using Activatable Near-Infrared Fluorescent Semiconducting Polymer Nanoparticles.

Enzyme-activatable ratiometric near-infrared (NIR) fluorescent probes enabling noninvasive imaging of enzyme activity in vivo are promising for biomedical research; however, such probes with ratiometric fluorescence emissions both in NIR window under a single NIR light excitation are largely unexplored. Here, a quenched NIR fluorophore of Cy5.5 is integrated with NIR fluorescent poly[2,6-(4,4-bis-(2-ethylhexyl)-4H-cyclopenta[2,1-b;3,4-b']dithiophene)-alt-4,7(2,1,3-benzothiadiazole)] (PCPDTBT)-based semiconducting polymer nanoparticles (SPNs), and an αv β3 integrin-targeting and matrix metalloproteinase-2 (MMP-2)-activatable ratiometric fluorescent probe (SPN-MMP-RGD) is developed. Under excitation at 660nm, SPN-MMP-RGD shows "always-on" fluorescence of PCPDTBT (830nm) and activatable fluorescence of Cy5.5 (690nm) toward MMP-2, affording a remarkable ≈176-fold enhancement in fluorescence intensity ratio between 690 and 830nm (I690 /I830 ) for sensitive detection of MMP-2 activity in vitro and in tumor cells. By virtue of ratiometric fluorescence imaging independently of probe's concentration, SPN-MMP-RGD can not only accurately report on MMP-2 levels regarding different tumor sizes, but also noninvasively delineate MMP-2-positive tiny gastric tumors metastasis in vivo. The authors' study reveals the potential of SPN-MMP-RGD for ratiometric fluorescence imaging of MMP-2 activity via combining two independent NIR fluorophores, which can be amenable for the design of other enzyme-activatable ratiometric NIR fluorescent probes for reliable in vivo imaging.

An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

An Integration Strategy to Develop Dual-State Luminophores with Tunable Spectra, Large Stokes Shift, and Activatable Fluorescence for High-Contrast Imaging

\u03b2-Galactosidase is a target enzyme for detecting peritoneal metastasis of gastric cancer

Diagnosis of peritoneal metastasis in gastric cancer (GC) is essential for determining appropriate therapeutic strategies and avoiding non-essential laparotomy or gastrectomy. Recently, a variety of activatable fluorescence probes that can detect enzyme activities have been developed for cancer imaging. The aim of this study was to identify the key enzyme involved in peritoneal metastasis in GC. The enzymatic activity of gamma-glutamyl transpeptidase, dipeptidyl peptidase IV, and β-galactosidase (β-Gal) was assessed in lysates prepared from preserved human GC (n = 89) and normal peritoneal (NP; n = 20) samples. β-Gal activity was significantly higher in the human GC samples than in NP samples, whereas no differences were observed in the activities of the other enzymes. Therefore, we used SPiDER-βGal, a fluorescent probe that can be activated by β-Gal, for imaging GC cell lines, peritoneal metastasis in a mouse model, and fresh human resected GC samples (n = 13). All cell lines showed fluorescence after applying SPiDER-βGal, and metastatic nodules in the mice gradually developed high fluorescence that could be visualized with SPiDER-βGal. The human GC samples showed significantly higher fluorescence than NP samples. β-Gal is a useful target enzyme for fluorescence imaging of peritoneal metastasis in GC.

Plasmonic-Fluorescent Janus Ag/Ag2S Nanoparticles for In Situ H2O2-Activated NIR-II Fluorescence Imaging.

Silver sulfide (Ag2S) has gained widespread attention in second near-infrared (950-1700 nm, NIR-II) window imaging because of its high fluorescence quantum yield and low toxicity. However, its "always on" fluorescence shows inapplicability for targeted molecule-activated biomedical applications. Herein, we first developed a novel silver/silver sulfide Janus nanoparticle (Ag/Ag2S JNP) for specific activatable fluorescence imaging in the NIR-II window. Inner-particle electron compensation from Ag to Ag2S upon laser irradiation endowed JNPs an "off" state of fluorescence, whereas the oxidization of Ag incubated with H2O2, decreasing the electron-transfer effect and illuminating the NIR-II fluorescence of the Ag2S part. In contrast, the absorption of Ag/Ag2S JNPs slightly decreased in an H2O2-dependent manner, showing an activated photoacoustic imaging mechanism. The Ag/Ag2S JNPs were used for noninvasive location and diagnosis of diseases in vivo, such as for liver injury and cancer, with high sensitivity and accuracy.

A Smart "Sense-and-Treat" Nanoplatform Based on Semiconducting Polymer Nanoparticles for Precise Photothermal-Photodynamic Combined Therapy.

Integrated theranostic nanoplatforms with biomarker recognition and photothermal- and photodynamic (PTT/PDT) therapy is in high demand but remains challenging. Herein, a "sense-and-treat" nanoplatform based on semiconducting polymer nanoparticles (SPNs) for ratiometric bioimaging of phospholipase D (PLD) activity and PTT/PDT combined therapy was proposed. Semiconducting polymer nanoparticles (PSBTBT NPs) serve not only as photothermal agents but also as fluorescent quenchers of Rhodamine B (Rhod B) through a PLD-cleavable linker. Chlorin e6 (Ce6) was used as a photodynamic agent and fluorescence reference. The obtained nanoplatform (PSBTBT-Ce6@Rhod NPs) showed high PDT efficiency and photothermal performance upon single laser irradiation. The PTT/PDT combined therapy achieved more efficient tumor inhibition results as compared with single treatments. In addition, the overexpressed biomarker PLD in tumor tissue will cleave Rhod, leading to the fluorescence recovery of Rhod B and thus allowing the activatable fluorescence imaging of tumor and targeted phototherapy.

Fluorescence Probes for Imaging Basic Carboxypeptidase Activity in Living Cells with High Intracellular Retention.

Basic carboxypeptidases (basic CPs) cleave the C-terminal basic amino acid of peptides, and their activity is upregulated in some types of cancers. Therefore, detecting the activity of basic CPs in living cells would be important not only for studying the physiological functions of these enzymes but also for visualization of cancerous tissues. Here, we report two fluorescein diacetate (FDA)-based activatable fluorescence probes, named 5ArgAF-FDA and 5LysAF-FDA, in which the substrate amino acid arginine or lysine is conjugated to the benzene moiety via an azoformyl linker. In live-cell fluorescence imaging of CPM, one of the seven basic CPs, 5ArgAF-FDA showed a larger intracellular fluorescence increase than did 5LysAF-FDA within a few minutes. This increase was inhibited by coincubation with 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid (MGTA), an inhibitor of basic CPs. When 5ArgAF-FDA was applied to a coculture of two breast cancer cell lines with different CPM activities, the fluorescence increase in individual cells was correlated with the expression level of CPM, suggesting that 5ArgAF-FDA has the ability to distinguish cell lines having different levels of CPM activity, owing to its high intracellular retention. We believe these probes will be useful for imaging cancers with upregulated basic CP activity.