Fitness Factors for Bioorthogonal Chemical Probes.

Bioorthogonal chemistry has offered an invaluable reactivity-based tool to chemical biology owing to its exquisite specificity in tagging a diverse set of biomolecules in their native environment. Despite tremendous progress in the field over the past decade, designing a suitable bioorthogonal chemical probe to investigate a given biological system remains a challenge. In this Perspective, we put forward a series of fitness factors that can be used to assess the performance of bioorthogonal chemical probes. The consideration of these criteria should encourage continuous innovation in bioorthogonal probe development as well as enhance the quality of biological data.

In Vivo Near-Infrared Fluorescence and Photoacoustic Dual-Modal Imaging of Endogenous Alkaline Phosphatase.

Alkaline phosphatase (ALP) is distributed widely in living organisms and is an important biomarker closely related to many physiological and pathological processes. However, in vivo real-time detection of ALP remains a significant challenge. Herein, we developed a turn-on molecular probe (denoted as LET-3) to visualize ALP activity in tumor tissues through near-infrared fluorescence (NIRF) and photoacoustic (PA) dual-modal imaging. LET-3, composed of NIR hemicyanine dye (LET-CyOH) and a phosphate moiety, showed a 23-fold NIRF enhancement at 730 nm and 27-fold PA enhancement at 710 nm upon activation by ALP. More importantly, both in vitro and in vivo diagnostic experiments indicated that LET-3 has a high sensitivity and good selectivity for ALP. These findings provide a promising strategy for in vivo ALP detection using NIRF and PA dual-channel turn-on probes.

Au@SiO2@CuInS2-ZnS/Anti-AFP fluorescent probe improves HCC cell labeling.

BACKGROUND: Clear tumor imaging is essential to the resection of hepatocellular carcinoma (HCC). This study aimed to create a novel biological probe to improve the HCC imaging. METHODS: Au nano-flower particles and CuInS2-ZnS core-shell quantum dots were synthesized by hydrothermal method. Au was coated with porous SiO2 and combined with anti-AFP antibody. HCC cell line HepG2 was used to evaluate the targeting efficacy of the probe, while flow cytometry and MTT assay were used to detect the cytotoxicity and bio-compatibility of the probe. Probes were subcutaneously injected to nude mice to explore light intensity and tissue penetration. RESULTS: The fluorescence stability of the probe was maintained 100% for 24 h, and the brightness value was 4 times stronger than that of the corresponding CuInS2-ZnS quantum dot. In the targeting experiment, the labeled HepG2 emitted yellow fluorescence. In the cytotoxicity experiments, MTT and flow cytometry results showed that the bio-compatibility of the probe was fine, the inhibition rate of HepG2 cell with 60% Cu-QDs/Anti-AFP probe and Au-QDs/Anti-AFP probe solution for 48 h were significantly different (86.3%±7.0% vs. 4.9%±1.3%, t = 19.745, P<0.05), and the apoptosis rates were 83.3%±5.1% vs. 4.4%±0.8% (P<0.001). In the animal experiment, the luminescence of the novel probe can penetrate the abdominal tissues of a mouse, stronger than that of CuInS2-ZnS quantum dot. CONCLUSIONS: The Au@SiO2@CuInS2-ZnS/Anti-AFP probe can targetedly recognize and label HepG2 cells with good bio-compatibility and no toxicity, and the strong tissue penetrability of luminescence may be helpful to surgeons.

Cell penetrable, clickable and tagless activity-based probe of human cathepsin L.

Human cathepsin L is a ubiquitously expressed endopeptidase and is known to play critical roles in a wide variety of cellular signaling events. Its overexpression has been implicated in numerous human diseases, including highly invasive forms of cancer. Inhibition of cathepsin L is therefore considered a viable therapeutic strategy. Unfortunately, several redundant and even opposing roles of cathepsin L have recently emerged. Selective cathepsin L probes are therefore needed to dissect its function in context-specific manner before significant resources are directed into drug discovery efforts. Herein, the development of a clickable and tagless activity-based probe of cathepsin L is reported. The probe is highly efficient, active-site directed and activity-dependent, selective, cell penetrable, and non-toxic to human cells. Using zebrafish model, we demonstrate that the probe can inhibit cathepsin L function in vivo during the hatching process. It is anticipated that the probe will be a highly effective tool in dissecting cathepsin L biology at the proteome levels in both normal physiology and human diseases, thereby facilitating drug-discovery efforts targeting cathepsin L.

Alkyne-Tagged Analogue of Jaspine†B: New Tool for Identifying Jaspine†B Mode of Action.

The first biologically relevant clickable probe related to the antitumor marine lipid jaspine B is reported. The concise synthetic route to both enantiomers relied on the supercritical fluid chromatography (SFC) enantiomeric resolution of racemic materials. The eutomeric dextrogyre derivative represents the first jaspine B analogue with enhanced cytotoxicity with IC50 down to 30 nm. These enantiomeric probes revealed a chiralitydependent cytoplasmic imaging of U2OS cancer cells by in situ click labeling.

Two-step activity-based protein profiling of diacylglycerol lipase.

Diacylglycerol lipases (DAGL) produce the endocannabinoid 2-arachidonoylglycerol, a key modulator of neurotransmitter release. Chemical tools that visualize endogenous DAGL activity are desired. Here, we report the design, synthesis and application of a triazole urea probe for DAGL equipped with a norbornene as a biorthogonal handle. The activity and selectivity of the probe was assessed with activity-based protein profiling. This probe was potent against endogenous DAGLα (IC50 = 5 nM) and it was successfully applied as a two-step activity-based probe for labeling of DAGLα using an inverse electron-demand Diels-Alder ligation in living cells.

A multifunctional targeting probe with dual-mode imaging and photothermal therapy used in vivo.

BACKGROUND: Ag2S has the characteristics of conventional quantum dot such as broad excitation spectrum, narrow emission spectrum, long fluorescence lifetime, strong anti-bleaching ability, and other optical properties. Moreover, since its fluorescence emission is located in the NIR-II region, has stronger penetrating ability for tissue. Ag2S quantum dot has strong absorption during the visible and NIR regions, it has good photothermal and photoacoustic response under certain wavelength excitation. RESULTS: 200 nm aqueous probe Ag2S@DSPE-PEG2000-FA (Ag2S@DP-FA) with good dispersibility and stability was prepared by coating hydrophobic Ag2S with the mixture of folic acid (FA) modified DSPE-PEG2000 (DP) and other polymers, it was found the probe had good fluorescent, photoacoustic and photothermal responses, and a low cell cytotoxicity at 50 µg/mL Ag concentration. Blood biochemical analysis, liver enzyme and tissue histopathological test showed that no significant influence was observed on blood and organs within 15 days after injection of the probe. In vivo and in vitro fluorescence and photoacoustic imaging of the probe further demonstrated that the Ag2S@DP-FA probe had good active targeting ability for tumor. In vivo and in vitro photothermal therapy experiments confirmed that the probe also had good ability of killing tumor by photothermal. CONCLUSIONS: Ag2S@DP-FA was a safe, integrated diagnosis and treatment probe with multi-mode imaging, photothermal therapy and active targeting ability, which had a great application prospect in the early diagnosis and treatment of tumor.

Photoluminescence Lifetime Imaging of Synthesized Proteins in Living Cells Using an Iridium-Alkyne Probe.

Designing probes for real-time imaging of dynamic processes in living cells is a continuous challenge. Herein, a novel near-infrared (NIR) photoluminescence probe having a long lifetime was exploited for photoluminescence lifetime imaging (PLIM) using an iridium-alkyne complex. This probe offers the benefits of deep-red to NIR emission, a long Stokes shift, excellent cell penetration, low cytotoxicity, and good resistance to photobleaching. This example is the first PLIM probe applicable to the click reaction of copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with remarkable lifetime shifts of 414 ns, before and after click reaction. The approach fully eliminates the background interference and distinguishes the reacted probes from the unreacted probes, thus enabling the wash-free imaging of the newly synthesized proteins within single living cells. Based on the unique properties of the iridium complexes, it is anticipated to have applications for imaging other processes within living cells.

A new "on-off-on" fluorescent probe containing triarylimidazole chromophore to sequentially detect copper and sulfide ions.

A novel compound TPI-H containing triphenylimidazole chromophore is synthesized and employed as fluorescent probe for sequential detection of Cu2+ and S2-. With three binding sites in its molecular structure, TPI-H exhibits highly selective binding towards Cu2+ and results in an apparent fluorescence "on-off" behavior. Fluorescence intensity is linear with the Cu2+ concentration, and the detection limit can be down to 8.7nM. Furthermore, the in-situ generated ensemble between TPI-H and Cu2+ (TPI-H-Cu(II)) can be used to detect S2- with a low detection limit of 15.6nM through Cu2+ displacement method. In addition, the potential utility of the probe for the detection of Cu2+ and further S2- in biological system is investigated by cell imaging.

Aggregation induced emission enhancement (AIEE) characteristics of quinoline based compound - A versatile fluorescent probe for pH, Fe(III) ion, BSA binding and optical cell imaging.

Novel benzimidazoquinoline derivative (AVT) was synthesized through a substitution reaction and characterized by various spectral techniques. Analyzing the optical properties of AVT under absorption and emission spectral studies in different environments exclusively with respect to solvents and pH, intriguing characteristics viz. aggregation induced emission enhancement (AIEE) in the THF solvent and 'On-Off' pH sensing were found at neutral pH. Sensing nature of AVT with diverse metal ions and bovine serum albumin (BSA) was also studied. Among the metal ions, Fe3+ ion alone tunes the fluorescence intensity of AVT probe in aqueous medium from "turn-on" to "turn-off" through ligand (probe) to metal charge transfer (LMCT) mechanism. The probe AVT in aqueous medium interacts strongly with BSA due to Fluorescence Resonance Energy Transfer (FRET) and the conformational change in BSA was further analyzed using synchronous fluorescence techniques. Docking study of AVT with BSA reveals that the active site of binding is tryptophan residue which is also supported by the experimental results. Interestingly, fluorescent AVT probe in cells was examined through cellular imaging studies using BT-549 and MDA-MB-231 cells. Thus, the single molecule probe based detection of multiple species and stimuli were described.

`Folate-Conjugated Phosphorescent Silica Nanoparticles for Bioimaging and Cancer Cell Targeting.

Besides their superior photophysical properties, phosphorescent transition-metal complexes have some drawbacks for biological applications because of their poor solubility in aqueous solutions and toxicity. In order to avoid the biological environmental impacts on their optical function and solve the problems of water-solubility and toxicity in sensing and bioimaging, two biocompatible organic/inorganic hybrid phosphorescent nanoprobes (Ir-MSN) by using mesoporous silica nanoparticles (MSN) as carriers and an iridium(III) complex as signaling unit were prepared in the present study. In addition, folic acid (FA) was covalently attached to them to endow the particles with characteristic of tumor targeting. The photophysical properties, cell staining and tumor cell targeting functions of FA-ligated Ir-MSN were investigated. These results demonstrated that such a design strategy of phosphorescent nanoprobes is an effective way to develop excellent phosphorescent cellular probes for living cell applications.

Development of ethynyl-2'-deoxyuridine chemical probes for cell proliferation.

A common method of evaluating cellular proliferation is to label DNA with chemical probes. 5-Ethynyl-2'-deoxyuridine (EdU) is a widely utilized chemical probe for labeling DNA, and upon incorporation, EdU treatment of cells is followed by a reaction with a small molecule fluorescent azide to allow detection. The limitations when using EdU include cytotoxicity and a reliance on nucleoside active transport mechanisms for entry into cells. Here we have developed six novel EdU pro-labels that consist of EdU modified with variable lipophilic acyl ester moieties. This pro-label:chemical probe relationship parallels the prodrug:drug relationship that is employed widely in medicinal chemistry. EdU and EdU pro-labels were evaluated for their labeling efficacy and cytotoxicity. Several EdU pro-label analogues incorporate into DNA at a similar level to EdU, suggesting that nucleoside transporters can be bypassed by the pro-labels. These EdU pro-labels also had reduced toxicity compared to EdU.

When molecular probes meet self-assembly: an enhanced quenching effect.

We demonstrate that the incorporation of one or two amino acids of phenylalanine (F) or 4-fluoro phenylalanine ((f)F) will greatly lower the background fluorescence intensities of conventional quenched probes with quenchers. This enhanced quenching effect was due to the synergetic effect of the aggregation caused quenching and the presence of a quencher. Such strategy will not greatly affect the enzyme recognition properties to the probes. We also demonstrated that our self-assembled nanoprobe with the enhanced quenching effect showed a better performance in cells for the detection of cell apoptosis than the unassembled probes. Our study demonstrates that using molecular self-assembly can optimize and improve the performance of molecular probes and it provides a simple but very useful strategy to boost the signal-to-noise ratios of fluorescence probes.

Cryptophane-folate biosensor for (129)xe NMR.

Folate-conjugated cryptophane was developed for targeting cryptophane to membrane-bound folate receptors that are overexpressed in many human cancers. The cryptophane biosensor was synthesized in 20 nonlinear steps, which included functionalization with folate recognition moiety, solubilizing peptide, and Cy3 fluorophore. Hyperpolarized (129)Xe NMR studies confirmed xenon binding to the folate-conjugated cryptophane. Cellular internalization of biosensor was monitored by confocal laser scanning microscopy and quantified by flow cytometry. Competitive blocking studies confirmed cryptophane endocytosis through a folate receptor-mediated pathway. Flow cytometry revealed 10-fold higher cellular internalization in KB cancer cells overexpressing folate receptors compared to HT-1080 cells with normal folate receptor expression. The biosensor was determined to be nontoxic in HT-1080 and KB cells by MTT assay at low micromolar concentrations typically used for hyperpolarized (129)Xe NMR experiments.

Transferring biomarker into molecular probe: melanin nanoparticle as a naturally active platform for multimodality imaging.

Developing multifunctional and easily prepared nanoplatforms with integrated different modalities is highly challenging for molecular imaging. Here, we report the successful transfer of an important molecular target, melanin, into a novel multimodality imaging nanoplatform. Melanin is abundantly expressed in melanotic melanomas and thus has been actively studied as a target for melanoma imaging. In our work, the multifunctional biopolymer nanoplatform based on ultrasmall (<10 nm) water-soluble melanin nanoparticle (MNP) was developed and showed unique photoacoustic property and natural binding ability with metal ions (for example, (64)Cu(2+), Fe(3+)). Therefore, MNP can serve not only as a photoacoustic contrast agent, but also as a nanoplatform for positron emission tomography (PET) and magnetic resonance imaging (MRI). Traditional passive nanoplatforms require complicated and time-consuming processes for prebuilding reporting moieties or chemical modifications using active groups to integrate different contrast properties into one entity. In comparison, utilizing functional biomarker melanin can greatly simplify the building process. We further conjugated αvß3 integrins, cyclic c(RGDfC) peptide, to MNPs to allow for U87MG tumor accumulation due to its targeting property combined with the enhanced permeability and retention (EPR) effect. The multimodal properties of MNPs demonstrate the high potential of endogenous materials with multifunctions as nanoplatforms for molecular theranostics and clinical translation.

Peptide-bridged dinuclear Ru(II) complex for mitochondrial targeted monitoring of dynamic changes to oxygen concentration and ROS generation in live mammalian cells.

A novel mitochondrial localizing ruthenium(II) peptide conjugate capable of monitoring dynamic changes in local O2 concentrations within living cells is presented. The complex is comprised of luminescent dinuclear ruthenium(II) polypyridyl complex bridged across a single mitochondrial penetrating peptide, FrFKFrFK-CONH2 (r = D-arginine). The membrane permeability and selective uptake of the peptide conjugate at the mitochondria of mammalian cells was demonstrated using confocal microscopy. Dye co-localization studies confirmed very precise localization and preconcentration of the probe at the mitochondria. This precision permitted collection of luminescent lifetime images of the probe, without the need for co-localizing dye and permitted semiquantitative determination of oxygen concentration at the mitochondria using calibration curves collected at 37 °C for the peptide conjugate in PBS buffer. Using Antimycin A the ability of the probe to respond dynamically to changing O2 concentrations within live HeLa cells was demonstrated. Furthermore, based on lifetime data it was evident that the probe also responds to elevated reactive oxygen species (ROS) levels within the mitochondria, where the greater quenching capacity of these species led to luminescent lifetimes of the probe at longer Antimycin A incubation times which lay outside of the O2 concentration range. Although both the dinuclear complex and a mononuclear analogue conjugated to an octaarginine peptide sequence exhibited some cytotoxicity over 24 h, cells were tolerant of the probes over periods of 4 to 6 h which facilitated imaging. These metal-peptide conjugated probes offer a valuable opportunity for following dynamic changes to mitochondrial function which should be of use across domains in which the metabolic activity of live cells are of interest from molecular biology and drug discovery.

Smart vesicle kit for in situ monitoring of intracellular telomerase activity using a telomerase-responsive probe.

A smart vesicle kit was designed for in situ imaging and detection of cytoplasmic telomerase activity. The vesicle kit contained a telomerase primer (TSP) and a Cy5-tagged molecular beacon (MB) functionalized gold nanoparticle probe, which were encapsulated in liposome for intracellular delivery. After the vesicle kit was transfected into cytoplasm, the released TSP could be extended in the presence of telomerase to produce a telomeric repeated sequence at the 3' end, which was just complementary with the loop of MB assembled on probe surface. Thus, the MB was opened upon hybridization to switch the fluorescent state from "off" to "on". The fluorescence signal depended on telomerase activity, leading to a novel strategy for in situ imaging and quantitative detection of the cytoplasmic telomerase activity. The cytoplasmic telomerase activity was estimated to be 3.2 × 10(-11), 2.4 × 10(-11), and 8.6 × 10(-13) IU in each HeLa, BEL tumor and QSG normal cell, respectively, demonstrating the capability of this approach to distinguish tumor from normal cells. The proposed method could be employed for dynamic monitoring of the cytoplasmic telomerase activity in response to a telomerase-based drug, suggesting the potential application in discovery and screening of telomerase-targeted anticancer drugs.

Etchable plasmonic nanoparticle probes to image and quantify cellular internalization.

There is considerable interest in using nanoparticles as labels or to deliver drugs and other bioactive compounds to cells in vitro and in vivo. Fluorescent imaging, commonly used to study internalization and subcellular localization of nanoparticles, does not allow unequivocal distinction between cell surface-bound and internalized particles, as there is no methodology to turn particles 'off'. We have developed a simple technique to rapidly remove silver nanoparticles outside living cells, leaving only the internalized pool for imaging or quantification. The silver nanoparticle (AgNP) etching is based on the sensitivity of Ag to a hexacyanoferrate-thiosulphate redox-based destain solution. In demonstration of the technique we present a class of multicoloured plasmonic nanoprobes comprising dye-labelled AgNPs that are exceptionally bright and photostable, carry peptides as model targeting ligands, can be etched rapidly and with minimal toxicity in mice, and that show tumour uptake in vivo.

A robust probe for lighting up intracellular telomerase via primer extension to open a nicked molecular beacon.

A nicked molecular beacon (MB)-functionalized probe has been designed for in situ imaging and detection of intracellular telomerase activity. The nick separates the MB into two segments: a shorter telomerase primer (TSP) sequence as a part of the 5'-end stem and a longer sequence to form a loop with one thiol-labeled 3'-end stem. The MB can be opened by substitutional hybridization of the telomerase-triggered stem elongation product, which leads to separation of the Cy5 at the 5'-end nick from the gold nanoparticle (AuNP) as the nanocarrier and thus inhibits the energy transfer from Cy5 to AuNP. Upon endocytosis of the probe, the TSP can be extended by intracellular telomerase at its 3' end to produce the telomeric repeated sequence, which leads to the inner chain substitution and thus turns on the fluorescence of Cy5. The probe provides a one-step incubation technique for quantification and monitoring of the telomerase activity in living cells. The practicality of the proposed approach for distinguishing tumor cells from normal cells and monitoring the decrease of telomerase activity during treatment with antitumor drugs demonstrates its potential in clinical diagnostic and therapeutic monitoring.