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.     

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.     

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.     

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.     

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.     

Self-Immolative Activation of β-Galactosidase-Responsive Probes for In Vivo MR Imaging in Mouse Models

Our lab has developed a new series of self-immolative MR agents for the rapid detection of enzyme activity in mouse models expressing β-galactosidase (β-gal). We investigated two molecular architectures to create agents that detect β-gal activity by modulating the coordination of water to Gd^III. The first is an intermolecular approach, wherein we designed several structural isomers to maximize coordination of endogenous carbonate ions. The second involves an intramolecular mechanism for q modulation. We incorporated a pendant coordinating carboxylate ligand with a 2, 4, 6, or 8 carbon linker to saturate ligand coordination to the Gd^III ion. This renders the agent ineffective. We show that one agent in particular (6-C pendant carboxylate) is an extremely effective MR reporter for the detection of enzyme activity in a mouse model expressing β-gal.     

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.     

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.     

Low-Fouling Fluoropolymers for Bioconjugation and In Vivo Tracking

The conjugation of hydrophilic low-fouling polymers to therapeutic molecules and particles is an effective approach to improving their aqueous stability, solubility, and pharmacokinetics. Recent concerns over the immunogenicity of poly(ethylene glycol) has highlighted the importance of identifying alternative low fouling polymers. Now, a new class of synthetic water-soluble homo-fluoropolymers are reported with a sulfoxide side-chain structure. The incorporation of fluorine enables direct imaging of the homopolymer by ¹⁹F MRI, negating the need for additional synthetic steps to attach an imaging moiety. These self-reporting fluoropolymers show outstanding imaging sensitivity and remarkable hydrophilicity, and as such are a new class of low-fouling polymer for bioconjugation and in vivo tracking.     

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.     

A Novel Fluorescent Probe for Lead Ions

A new fluorescent probe for lead ions, p-nitrophenyl 3H-phenoxazin-3-one-7-yl phosphoric acid (NPPA), has been synthesized by linking resorufLn (serving as a fluorophore and electron acceptor) to p-nitrophenol (serving as a fluorescence quencher and electron donor) through phosphodiester bonds. When NPPA was irradiated with light, intramolecular fluorescence self-quenching took place due to the PET (photoinduced electron transfer) from the donor to the acceptor. However, upon addition of Pb^Ⅱ, the phosphate ester bonds in the probe were cleaved and the fluorophore was released, accompanying the retrievement of fluorescence.     

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 “In Vivo Self-assembly” Strategy for Constructing Superstructures for Biomedical Applications

The interfacing study of biopolymer and supramolecular chemistry enables a better understanding of fundamental biochemical processes and the creating of new high-performance biomaterials. In this review, we introduced an "in vivo self-assembly" strategy which means in situ construction of functional self-assembled superstructures in specific physiological or pathological conditions in cell, tissue or animal levels that exhibit diverse biomedical effects. By using this strategy, unexpected phenomena and insights, e.g,assembly/aggregation induced retention(AIR) effect have been demonstrated where the self-assembled nanostructures showed extraordinary enhanced accumulation and retention of therapeutics in targeted sites.     

A Generalizable and Noncovalent Strategy for Interfacing Aptamers with a Microelectrode for the Selective Sensing of Neurotransmitters In Vivo

The selective sensing of neurochemicals is essential for understanding the chemical basis of brain function and pathology. Interfacing the excellent recognition features of aptamers with in vivo compatible carbon fiber microelectrode (CFE)-based electroanalytical systems offers a plausible means to achieve this end. However, this is challenging in terms of coupling chemistry, stability, and versatility. Here, we present a new interfacial functionalization strategy based on the assembly of aptamer cholesterol amphiphiles (aptCAs) on the alkyl chain-functionalized CFE. The noncovalent cholesterol-alkyl chain interactions effectively immobilize aptamers onto the CFE surface, allowing the generation of a highly selective system for probing neurochemical dynamics in living systems and opening up a vast array of new opportunities for designing in vivo sensors for exploring brain chemistry.     

A Novel Fluorescent Reagent for Analysis of Hydrogen Peroxide

IntroductionTheoxidationofmanyclinicalsubstancesinbodyfluidsproducesaquantityofhydrogenperoxide ,sothedetermina tionoftracehydrogenperoxideisofconsiderableimportanceinclinicalchemistry .1Further,themonitoringofhydrogenperoxideisalsonecessarytoenvironmentalsciencesinceitisakeyspeciesinthereactionsofthetroposphere,beingin volvedinimportantreactionssuchasthecatalyzedoruncat alyzedaqueousphaseoxidationofSO2 andtheultraviolet en hancedaqueousphaseoxidationoforganicspecies.2 Uptonow ,variousmethods…     

Conjugated Polymer Fluorescent Chemosensor： Design Principle and the Concept for Molecular Assembly

共轭高分子作为“分子导线”所特有的信号放大效应,使其在构建高灵敏度的荧光化学传感体系中具有特别的优势。本文首先从分子识别、传感机理和信号输出等方面概述了传感分子设计中需要考虑的一些基本问题;然后以聚对芳撑乙炔类共轭高分子为例,提出基于模板排列组合的分子组装概念,并将之用于合成一系列结构可调的荧光化学传感分子,以满足不同的检测目的;最后通过文献中的具体实例,对多种聚对芳撑乙炔类共轭高分子进行了分子结构和传感应用方面的解析。     

Real-Time In Vivo Detection of Cellular Senescence through the Controlled Release of the NIR Fluorescent Dye Nile Blue

In vivo detection of cellular senescence is accomplished by using mesoporous silica nanoparticles loaded with the NIR-FDA approved Nile blue (NB) dye and capped with a galactohexasaccharide ( S3 ). NB emission at 672 nm is highly quenched inside S3 , yet a remarkable emission enhancement is observed upon cap hydrolysis in the presence of β-galactosidase and dye release. The efficacy of the probe to detect cellular senescence is tested in vitro in melanoma SK-Mel-103 and breast cancer 4T1 cells and in vivo in palbociclib-treated BALB/cByJ mice bearing breast cancer tumor.     

Hyperbranched Polyglycerol-Induced Porous Silica Nanoparticles as Drug Carriers for Cancer Therapy In Vitro and In Vivo

Mesoporous silica-based nanoparticles are generally accepted as a potential platform for drug loading with a lot of advantages, except for their complex purification procedures and structures that are difficult to decompose. In this work, biocompatible hyperbranched polyglycerol is introduced to synthesize mesoporous silica nanoparticles (MSNs). The materials possess good biocompatibility, controlled release, and biodegradability. They also show passive targeting capability through the enhanced permeability and retention effect and can be excreted from the biological system. The method avoids the needs to employ traditional surfactants and complicated purified procedures, which make these MSNs an efficient delivery system for cancer therapy.     

Imidazole-Based Synthetic Lipidoids for In Vivo mRNA Delivery into Primary T Lymphocytes

Engineering T lymphocytes is an emerging approach in a variety of biomedical applications. However, delivering large biologics to primary T lymphocytes directly in vivo is technically challenging due to the low transfection efficacy. Herein, we investigated a library of synthetic lipid-like molecules (lipidoids) for their capability of delivering mRNA into primary T lymphocytes both ex vivo and in vivo. We initially screened a library with a large structural variety of lipidoids ex vivo and identified imidazole-containing lipidoids that are particularly potent in T lymphocytes transfection. We further optimized lipidoid structures by constructing and screening a detailed lipidoid library containing imidazole or imidazole analogues to perform a structure–activity correlation analysis. Using the lead lipidoid as a delivery vehicle for Cre mRNA in vivo through intravenous injection, we achieved 8.2 % gene recombination in mouse T lymphocytes.     

Development of a Versatile Protein Labeling Tool for Live-Cell Imaging Using Fluorescent β-Lactamase Inhibitors

To understand the function of protein in live cells, real-time monitoring of protein dynamics and sensing of their surrounding environment are important methods. Fluorescent labeling tools are thus needed that possess fast labeling kinetics, high efficiency, and long-term stability. We developed a versatile chemical protein-labeling tool based on fluorophore-conjugated diazabicyclooctane β-lactamase inhibitors (BLIs) and wild-type TEM-1 β-lactamase protein tag. The fluorescent probes efficiently formed a stable carbamoylated complex with β-lactamase, and the labeled proteins were visualized over a long period of time in live cells. Moreover, use of an α-fluorinated carboxylate ester-based BLI prodrug enabled the probe to permeate cell membranes and stably label intracellular proteins after unexpected spontaneous ester hydrolysis. Lastly, combining the labeling tool with a pH-activatable fluorescent probe allowed visual monitoring of lysosomal protein translocation during autophagy.