Versatile Fluorescent Probes for Imaging the Superoxide Anion in Living Cells and In Vivo

The superoxide anion (O₂^.−) is widely engaged in the regulation of cell functions and is thereby intimately associated with the onset and progression of many diseases. To ascertain the pathological roles of O₂^.− in related diseases, developing effective methods for monitoring O₂^.− in biological systems is essential. Fluorescence imaging is a powerful tool for monitoring bioactive molecules in cells and in vivo owing to its high sensitivity and high temporal-spatial resolution. Therefore, increasing numbers of fluorescent imaging probes have been constructed to monitor O₂^.− inside live cells and small animals. In this minireview, we summarize the methods for design and application of O₂^.−-responsive fluorescent probes. Moreover, we present the challenges for detecting O₂^.− and suggestions for constructing new fluorescent probes that can indicate the production sites and concentration changes in O₂^.− as well as O₂^.−-associated active molecules in living cells and in vivo.     

NIR-II Chemiluminescence Molecular Sensor for In Vivo High-Contrast Inflammation Imaging

Chemiluminescence (CL) sensing without external excitation by light and autofluorescence interference has been applied to high-contrast in vitro immunoassays and in vivo inflammation and tumor microenvironment detection. However, conventional CL sensing usually operates in the range of 400–850 nm, which limits the performance of in vivo imaging due to serious light scattering effects and signal attenuation in tissue. To address this challenge, a new type of CL sensor is presented that functions in the second near-infrared window (NIR-II CLS) with a deep penetration depth (≈8 mm). Successive CL resonance energy transfer (CRET) and Förster resonance energy transfer (FRET) from the activated CL substrate to two rationally designed donor-acceptor-donor fluorophores BTD540 and BBTD700 occurs. NIR-II CLS can be selectively activated by hydrogen peroxide over other reactive oxygen species (ROSs). Moreover, NIR-II CLS is capable of detecting local inflammation in mice with a 4.5-fold higher signal-to-noise ratio (SNR) than that under the NIR-II fluorescence modality.     

An Activatable NIR-II Fluorescent Reporter for In Vivo Imaging of Amyloid-β Plaques

Fluorescence imaging in the second near-infrared (NIR-II) window holds great promise for in vivo visualization of amyloid-β (Aβ) pathology, which can facilitate characterization and deep understanding of Alzheimer's disease (AD); however, it has been rarely exploited. Herein, we report the development of NIR-II fluorescent reporters with a donor-π-acceptor (D-π-A) architecture for specific detection of Aβ plaques in AD-model mice. Among all the designed probes, DMP2 exhibits the highest affinity to Aβ fibrils and can specifically activate its NIR-II fluorescence after binding to Aβ fibrils via suppressed twisted intramolecular charge transfer (TICT) effect. With suitable lipophilicity for ideal blood–brain barrier (BBB) penetrability and deep-tissue penetration of NIR-II fluorescence, DMP2 possesses specific detection of Aβ plaques in in vivo AD-model mice. Thus, this study presents a potential agent for non-invasive imaging of Aβ plaques and deep deciphering of AD progression.     

Synthesis of Small-Molecule Fluorescent Probes for the In Vitro Imaging of Calcium-Activated Potassium Channel KCa3.1

Small-molecule probes for the in vitro imaging of K_Ca3.1 channel-expressing cells were developed. Senicapoc, showing high affinity and selectivity for the K_Ca3.1 channels, was chosen as the targeting component. BODIPY dyes 15 – 20 were synthesized and connected by a Cu^I-catalyzed azide–alkyne [3+2]cycloaddition with propargyl ether senicapoc derivative 8 , yielding fluorescently labeled ligands 21 – 26 . The dimethylpyrrole-based imaging probes 25 and 26 allow staining of K_Ca3.1 channels in NSCLC cells. The specificity was shown by removing the punctate staining pattern by pre-incubation with senicapoc. The density of K_Ca3.1 channels detected with 25 and by immunostaining was identical. The punctate structure of the labeled channels could also be observed in living cells. Molecular modeling showed binding of the senicapoc-targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.     

A Far-Red Emitting Fluorescent Chemogenetic Reporter for In Vivo Molecular Imaging

Far-red emitting fluorescent labels are highly desirable for spectral multiplexing and deep tissue imaging. Here, we describe the generation of frFAST (far-red Fluorescence Activating and absorption Shifting Tag), a 14-kDa monomeric protein that forms a bright far-red fluorescent assembly with (4-hydroxy-3-methoxy-phenyl)allylidene rhodanine (HPAR-3OM). As HPAR-3OM is essentially non-fluorescent in solution and in cells, frFAST can be imaged with high contrast in presence of free HPAR-3OM, which allowed the rapid and efficient imaging of frFAST fusions in live cells, zebrafish embryo/larvae, and chicken embryos. Beyond enabling the genetic encoding of far-red fluorescence, frFAST allowed the design of a far-red chemogenetic reporter of protein–protein interactions, demonstrating its great potential for the design of innovative far-red emitting biosensors.     

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click-to-Release” Bioorthogonal Tumour Imaging

The bioorthogonal inverse-electron-demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans-cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single-walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO-caged molecule was used to deliver active effector molecules. To optimize a turn-on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near-infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real-time, non-invasive tumour visualization with a high target-to-background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine-functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off-site activation of fluorophore/drug.     

In Vivo Solid-Phase Microextraction for Sampling of Oxylipins in Brain of Awake,Moving Rats

Oxylipins are key lipid mediators of important brain processes, including pain, sleep, oxidative stress, and inflammation. For the first time, an in-depth profile of up to 52 oxylipins can be obtained from the brains of awake moving animals using in vivo solid-phase microextraction (SPME) chemical biopsy tool in combination with liquid chromatography–high resolution mass spectrometry. Among these, 23 oxylipins are detectable in the majority of healthy wildtype samples. This new approach successfully eliminates the changes in oxylipin concentrations routinely observed during the analysis of post-mortem samples, allows time-course monitoring of their concentrations with high spatial resolution in specific brain regions of interest, and can be performed using the same experimental set-up as in vivo microdialysis (MD) thus providing a new and exciting tool in neuroscience and drug discovery.     

A Systematic Study of Coumarin–Tetrazine Light-Up Probes for Bioorthogonal Fluorescence Imaging

Fluorescent probes that light-up upon reaction with complementary bioorthogonal reagents are superior tools for no-wash fluorogenic bioimaging applications. In this work, a thorough study is presented on a set of seventeen structurally diverse coumarin–tetrazine probes that produce fluorescent dyes with exceptional turn-on ratios when reacted with trans-cyclooctene (TCO) and bicyclononyne (BCN) dienophiles. In general, formation of the fully aromatic pyridazine-containing dyes resulting from the reaction with BCN was found superior in terms of fluorogenicity. However, evaluation of the probes in cellular imaging experiments revealed that other factors, such as reaction kinetics and good cell permeability, prevail over the fluorescence turn-on properties. The best compound identified in this study showed excellent performance in live cell-labeling experiments and enabled no-wash fluorogenic imaging on a timescale of seconds.     

In Vivo Imaging of the Tumor-Associated Enzyme NCEH1 with a Covalent PET Probe

Herein, we report the development of an ¹⁸F-labeled, activity-based small-molecule probe targeting the cancer-associated serine hydrolase NCEH1. We undertook a focused medicinal chemistry campaign to simultaneously preserve potent and specific NCEH1 labeling in live cells and animals, while permitting facile ¹⁸F radionuclide incorporation required for PET imaging. The resulting molecule, [¹⁸F]JW199, labels active NCEH1 in live cells at nanomolar concentrations and greater than 1000-fold selectivity relative to other serine hydrolases. [¹⁸F]JW199 displays rapid, NCEH1-dependent accumulation in mouse tissues. Finally, we demonstrate that [¹⁸F]JW199 labels aggressive cancer tumor cells in vivo, which uncovered localized NCEH1 activity at the leading edge of triple-negative breast cancer tumors, suggesting roles for NCEH1 in tumor aggressiveness and metastasis.     

An Extended NIR-II Superior Imaging Window from 1500 to 1900 nm for High-Resolution In Vivo Multiplexed Imaging Based on Lanthanide Nanocrystals

High-resolution in vivo optical multiplexing in second near-infrared window (NIR-II, 1000–1700 nm) is vital to biomedical research. Presently, limited by bio-tissue scattering, only luminescent probes located at NIR-IIb (1500–1700 nm) window can provide high-resolution in vivo multiplexed imaging. However, the number of available luminescent probes in this narrow NIR-IIb region is limited, which hampers the available multiplexed channels of in vivo imaging. To overcome the above challenges, through theoretical simulation we expanded the conventional NIR-IIb window to NIR-II long-wavelength (NIR-II-L, 1500–1900 nm) window on the basis of photon-scattering and water-absorption. We developed a series of novel lanthanide luminescent nanoprobes with emission wavelengths from 1852 nm to 2842 nm. NIR-II-L nanoprobes enabled high-resolution in vivo dynamic multiplexed imaging on blood vessels and intestines, and provided multi-channels imaging on lymph tubes, tumors and intestines. The proposed NIR-II-L probes without mutual interference are powerful tools for high-contrast in vivo multiplexed detection, which holds promise for revealing physiological process in living body.     

Phenotypic Assays for β-Amyloid in Mouse Embryonic Stem Cell-Derived Neurons

1. Download : Download high-res image (138KB)
2. Download : Download full-size image

     

Theoretical Studies of β-Peptide Models

Recently, the β-peptides, which consist entirely of β-amino acids instead of α-amino acids, have received intensive attention because of their interesting secondary structures.^1-3 Depending upon side chain substitution pattern, β-sheets, 14-helices, and 12-helices all have been observed.¹ Due to great variety of substitution patterns,the easiness of formation of secondary structures with even 4-6 residues compared to about 15 for natural peptides,and ready formation of cyclic compounds that stack into tube structures,⁴ β-peptides have generated great excitement. In addition,β-amino acids also frequently occur in natural products, especially cyclic peptides. It has been found that β-amino acids have excellent stability toward proteases. Therefore, they have wide applications in drug development.⁶     

Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic Gene Silencing In Vivo

Special (lipid) delivery: The role of the ionizable lipid pK(a) in the in?vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pK(a) value and silencing of the mouse FVII gene (FVII ED(50) ) was found, with an optimal pK(a) range of 6.2-6.5. The most potent cationic lipid from this study has ED(50) levels around 0.005?mg?kg(-1) in mice and less than 0.03?mg?kg(-1) in non-human primates.     

A Fluorescent Probe for Rapid,High-Contrast Visualization of Folate-Receptor-Expressing Tumors In Vivo

Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs in vivo, the near-infrared (NIR) region (650–900 nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1 , utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30 min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.     

Light-Induced Chiral Iron Copper Selenide Nanoparticles Prevent β-Amyloidopathy In Vivo

The accumulation and deposition of β-amyloid (Aβ) plaques in the brain is considered a potential pathogenic mechanism underlying Alzheimer's disease (AD). Chiral l/d -Fe_xCu_ySe nanoparticles (NPs) were fabricated that interfer with the self-assembly of Aβ42 monomers and trigger the Aβ42 fibrils in dense structures to become looser monomers under 808 nm near-infrared (NIR) illumination. d -Fe_xCu_ySe NPs have a much higher affinity for Aβ42 fibrils than l -Fe_xCu_ySe NPs and chiral Cu_2−xSe NPs. The chiral Fe_xCu_ySe NPs also generate more reactive oxygen species (ROS) than chiral Cu_2−xSe NPs under NIR-light irradiation. In living MN9D cells, d -NPs attenuate the adhesion of Aβ42 to membranes and neuron loss after NIR treatment within 10 min without the photothermal effect. In-vivo experiments showed that d -Fe_xCu_ySe NPs provide an efficient protection against neuronal damage induced by the deposition of Aβ42 and alleviate symptoms in a mouse model of AD, leading to the recovery of cognitive competence.     

A Smart,Autocatalytic, DNAzyme Biocircuit for in Vivo,Amplified, MicroRNA Imaging

DNAzymes have been recognized as promising transducing agents for visualizing endogenous biomarkers, but their inefficient intracellular delivery and limited amplification capacity (including insufficient cofactor supply) preclude their extensive biological application. Herein, an autocatalytic DNAzyme (ACD) biocircuit is constructed for amplified microRNA imaging in vivo based on a hybridization chain reaction (HCR) and DNAzyme biocatalysis, sustained by a honeycomb MnO₂ nanosponge (hMNS). The hMNS not only delivers DNA probes, but also supplies Mn²⁺ as a DNAzyme cofactor and magnetic resonance imaging (MRI) agent. Through the subsequent cross-activation of HCR and DNAzyme amplicons, the ACD amplifies the limited signal resulting from miRNA recognition. The hMNS/ACD system was used to image microRNA in vivo, thus demonstrating its great promise in cancer diagnosis.     

An Activatable NIR-II Nanoprobe for In Vivo Early Real-Time Diagnosis of Traumatic Brain Injury

Traumatic brain injury (TBI) is one of the most dangerous acute diseases resulting in high morbidity and mortality. Current methods remain limited with respect to early diagnosis and real-time feedback on the pathological process. Herein, a targeted activatable fluorescent nanoprobe (V&A@Ag₂S) in the second near-infrared window (NIR-II) is presented for in vivo optical imaging of TBI. Initially, the fluorescence of V&A@Ag₂S is turned off owing to energy transfer from Ag₂S to the A1094 chromophore. Upon intravenous injection, V&A@Ag₂S quickly accumulates in the inflamed vascular endothelium of TBI based on VCAM1-mediated endocytosis, after which the nanoprobe achieves rapid recovery of the NIR-II fluorescence of Ag₂S quantum dots (QDs) owing to the bleaching of A1094 by the prodromal biomarker of TBI, peroxynitrite (ONOO⁻). The nanoprobe offers high specificity, rapid response, and high sensitivity toward ONOO⁻, providing a convenient approach for in vivo early real-time assessment of TBI.     

In vitro and in vivo evaluation of β-cyclodextrin-based nanosponges of telmisartan

Telmisartan (TEL) is a BCS Class II drug having dissolution rate limited bioavailability. The aim of work was to enhance the solubility of TEL so that bioavailability problems are solved. β-Cyclodextrin (β-CD) based nanosponges (NSs) were formed by cross-linking β-CD with carbonate bonds, which were porous as well as nanosized. Drug was incorporated by solvent evaporation method. The effect of ternary component alkalizer (NaHCO₃) on solubility of TEL was studied. In order to find out the solubilization efficiency of NS, phase solubility study was carried out. Saturation solubility and in vitro dissolution study of β-CD complex of TEL was compared with plain TEL and NS complexes of TEL. The NS and NS complexes of TEL were characterized by differential scanning calorimetry, powder X-ray diffraction, Fourier transform infrared spectroscopy, nuclear magnetic resonance and scanning electron microscope. It was found that solubility of TEL was increased by 8.53-fold in distilled water; 3.35-fold in 0.1 N HCl and 4.66-fold in phosphate buffer pH 6.8 by incorporating NaHCO₃ in drug–NS complex than TEL. It was found that the NaHCO₃ in NS based complex synergistically enhanced dissolution of TEL by modulating microenvironmental pH and by changing amorphization of the drug. The highest solubility and in vitro drug release was observed in inclusion complex prepared from NS and NaHCO₃. An increase of 54.4 % in AUC was seen in case the ternary NS complex whereas β-CD ternary complex exhibited an increase of 79.65 %.     

Blood Levels of Hexavalent Chromium in Rats. “In Vitro” and “In Vivo” Experiments

Abstract

For the Cr(VI) selective separation from biological materials we have developed a highly rapid extraction-separation method with liquid anion exchanger as Amberlite LA-1 or LA-2. The analytical determination of Cr(VI) in organic phase was carried out using electrothermal atomic absorption spectroscopy (ETA-AAS).

After i.v. administration of 0.5 and 2.5mg/kg b.w. of K₂Cr₂O₇ in male Wistar rats the biological samples, collected at different times, were immediately analyzed. Cr(VI) was not detected in whole blood one minute after administration of the lower dose. In blood of rats receiving higher dose an incomplete reduction of Cr(VI) was observed.

Such data demonstrate a highly rapid but limited metabolic capacity of hematic compartment to reduce Cr(VI) to trivalent status.

These results obtained with a new and specific analytical method, confirmed a trigger role of red cells in Cr(VI) metabolism.

“In vitro” incubation of K₂Cr₂O₇ (4 μM) with rat erythrocytes or plasma at 37°C showed a rapid reduction of Cr(VI) in red cells while plasma samples demonstrated a limited reductive power.