To My Daughter

<p>Equal numbers of macaque CD34⁺ cells were transduced in 3-d transduction cultures with either the HOXB4GFP or YFP vector and then cultured for an additional 9 d (T02266) or 6 d (K03290 and J02152) in the presence of SCF, TPO, Flt-3L, and G-CSF. All the transduced and expanded cells were infused into myeloablated animals. The percentage of HOXB4GFP⁺ and YFP⁺ granulocytes was assessed by flow cytometry. Shown is the engraftment of HOXB4GFP⁺ and YFP⁺ granulocytes after transplantation. (A) T02266, (B) K03290, and (C) J02152. </p

Rational Design of NIR-II G‑Quadruplex Fluorescent Probes for Accurate In Vivo Tumor Metastasis Imaging

Accurate in vivo imaging of G-quadruplexes (G4) is critical for understanding the emergence and progression of G4-associated diseases like cancer. However, existing in vivo G4 fluorescent probes primarily operate within the near-infrared region (NIR-I), which limits their application accuracy due to the short emission wavelength. The transition to second near-infrared (NIR-II) fluorescent imaging has been of significant interest, as it offers reduced autofluorescence and deeper tissue penetration, thereby facilitating more accurate in vivo imaging. Nonetheless, the advancement of NIR-II G4 probes has been impeded by the absence of effective probe design strategies. Herein, through a “step-by-step” rational design approach, we have successfully developed NIRG-2, the first small-molecule fluorescent probe with NIR-II emission tailored for in vivo G4 detection. Molecular docking calculations reveal that NIRG-2 forms stable hydrogen bonds and strong π–π interactions with G4 structures, which effectively inhibit twisted intramolecular charge transfer (TICT) and, thereby, selectively illuminate G4 structures. Due to its NIR-II emission (940 nm), large Stokes shift (90 nm), and high selectivity, NIRG-2 offers up to 47-fold fluorescence enhancement and a tissue imaging depth of 5 mm for in vivo G4 detection, significantly outperforming existing G4 probes. Utilizing NIRG-2, we have, for the first time, achieved high-contrast visualization of tumor metastasis through lymph nodes and precise tumor resection. Furthermore, NIRG-2 proves to be highly effective and reliable in evaluating surgical and drug treatment efficacy in cancer lymphatic metastasis models. We are optimistic that this study not only provides a crucial molecular tool for an in-depth understanding of G4-related diseases in vivo but also marks a promising strategy for the development of clinical NIR-II G4-activated probes

Evolving an Ultra-Sensitive Near-Infrared β‑Galactosidase Fluorescent Probe for Breast Cancer Imaging and Surgical Resection Navigation

Early diagnosis and therapy are clinically crucial in decreasing mortality from breast carcinoma. However, the existing probes have difficulty in accurately identifying the margins and contours of breast carcinoma due to poor sensitivity and specificity. There is an urgent need to develop high-sensitive fluorescent probes for the diagnosis of breast carcinoma and for differentiating tumors from normal tissues during surgery. β-Galactosidase is a significant biomarker, whose overexpression is closely associated with the progression of breast tumors. Herein, we have constructed a β-galactosidase-activated fluorescent probe NIR-βgal-2 through rational design and molecular docking engineering simulations. The probe displayed superior sensitivity (detection limit = 2.0 × 10–3 U/mL), great affinity (Km = 1.84 μM), and catalytic efficiency (kcat/Km = 0.24 μM–1 s–1) for β-galactosidase. Leveraging this probe, we demonstrated the differentiation of cancer cells overexpressing β-galactosidase from normal cells and then applied the probe for intraoperative guided excision of breast tumors. Moreover, we exhibited the application of NIR-βgal-2 for the successful resection of orthotopic breast tumors by “in situ spraying” and monitored a good prognostic recovery. This work may promote the application of enzyme-activated near-infrared fluorescent probes for the development of carcinoma diagnosis and image-guided surgery

Efficient Two-Photon Fluorescent Probe for Nitroreductase Detection and Hypoxia Imaging in Tumor Cells and Tissues

Hypoxia plays an important role in tumor progression, and the development of efficient methods for monitoring hypoxic degree in living systems is of great biomedical importance. In the solid tumors, the nitroreductase level is directly corresponded with the hypoxic status. Many one-photon excited fluorescent probes have been developed for hypoxia imaging in tumor cells via the detection of nitroreductase level. However, two-photon excited probes are more suitable for bioimaging. In this work, a two-photon probe 1 for nitroreductase detection and hypoxic status monitoring in living tumor cells and tissues was reported for the first time. The detection is based on the fact that the nitro-group of probe 1 could be selectively reduced to an amino-group by nitroreductase in the presence of reduced NADH, following by a 1,6-rearrangement-elimination to release the fluorophore, resulting in the enhancement of fluorescence. The probe exhibited both one-photon and two-photon excited remarkable fluorescence enhancement (∼70-fold) for nitroreductase, which afforded a high sensitivity for nitroreductase, with a detection limit of 20 ng/mL observed. Moreover, the applications of the probe for fluorescent bioimaging of hypoxia in living cells and two-photon bioimaging in tissues were carried out, with tissue-imaging depths of 70–160 μm observed, which demonstrates its practical application in complex biosystems

A Bioluminescent Probe for Imaging Endogenous Peroxynitrite in Living Cells and Mice

Peroxynitrite (ONOO^–), an extremely reactive nitrogen species (RNS), is implicated in diverse pathophysiological conditions, including cancer, neurodegenerative diseases, and inflammation. Sensing and imaging of ONOO^– in living systems remains challenging due to the high autofluorescence and the limited light penetration depth. In this work, we developed a bioluminescent probe <b>BP-PN</b>, based on luciferase–luciferin pairs and the ONOO^–-responded group α-ketoamide, for highly sensitive detection and imaging of endogenous ONOO^– in living cells and mice for the first time. Attributed to the BL without external excitation, the probe <b>BP-PN</b> exhibits a high signal-to-noise ratio with relatively low autofluorescence. Furthermore, we examine the application of the probe <b>BP-PN</b> using the mice model of inflammation, and <b>BP-PN</b> shows high sensitivity for imaging endogenous ONOO^– in inflamed mice. This newly developed bioluminescent probe would be a potentially useful tool for in vivo imaging of ONOO^– in wider physiological and pathological processes

Bifunctional Fluoroionphore-Ionic Liquid Hybrid for Toxic Heavy Metal Ions: Improving Its Performance via the Synergistic Extraction Strategy

Several heavy metal ions (HMIs), such as Cd²⁺, Pb²⁺, and Hg²⁺, are highly toxic even at very low concentrations. Although a large number of fluoroionphores have been synthesized for HMIs, only a few of them show detection limits that are below the maximum contamination levels in drinking water (usually in the nM range), and few of them can simultaneously detect and remove HMIs. In this work, we report a new fluoroionphore-ionic liquid hybrid-based strategy to improve the performance of classic fluoroionphores via a synergistic extraction effect and realize simultaneous instrument-free detection and removal of HMIs. As a proof-of-concept, Hg²⁺ was chosen as a model HMI, and a rhodamine thiospirolactam was chosen as a model fluoroionphore to construct bifunctional fluoroionphore-ionic liquid hybrid <b>1</b>. The new sensing system could provide obviously improved sensitivity by simply increasing the aqueous-to-ionic liquid phase volume ratio to 10:1, resulting in a detection limit of 800 pM for Hg²⁺, and afford extraction efficiencies larger than 99% for Hg²⁺. The novel strategy provides a general platform for highly sensitive detection and removal of various HMIs in aqueous samples and holds promise for environmental and biomedical applications

A General Method To Increase Stokes Shift by Introducing Alternating Vibronic Structures

Fluorescent dyes have enabled much progress in the broad range of biomedical fields. However, many commercially available dyes suffer from small Stokes shifts, resulting in poor signal-to-noise ratio and self-quenching on current microscope configurations. In this work, we have developed a general method to significantly increase the Stokes shifts of common fluorophores. By simply appending a 1,4-diethyl-decahydro-quinoxaline (DQ) moiety onto the conjugated structure, we introduced a vibronic backbone that could facilely expand the Stokes shifts, emission wavelength, and photostability of 11 different fluorophores by more than 3-fold. This generalizable method could significantly improve the imaging efficiency of commercial fluorophores. As a demonstration, we showed that the DQ derivative of hemicyanine generated 5-fold signal in mouse models over indocyanine green. Furthermore, the DQ-modified fluorophores could pair with their parent molecules to conduct one-excitation, multiple emission imaging, allowing us to study the cell behavior more robustly. This approach shows promise in generating dyes suitable for super-resolution microscopy and second window near-infrared imaging

Near Infrared Graphene Quantum Dots-Based Two-Photon Nanoprobe for Direct Bioimaging of Endogenous Ascorbic Acid in Living Cells

Ascorbic acid (AA), as one of the most important vitamins, participates in various physiological reactions in the human body and is implicated with many diseases. Therefore, the development of effective methods for monitoring the AA level in living systems is of great significance. Up to date, various technologies have been developed for the detection of AA. However, few methods can realize the direct detection of endogenous AA in living cells. In this work, we for the first time reported that near-infrared (NIR) graphene quantum dots (GQD) possessed good two-photon fluorescence properties with a NIR emission at 660 nm upon exciting with 810 nm femtosecond pulses and a two-photon (TP) excitation action cross-section (δΦ) of 25.12 GM. They were then employed to construct a TP nanoprobe for detection and bioimaging of endogenous AA in living cells. In this nanosystem, NIR GQDs (NGs), which exhibited lower fluorescence background in living system to afford improved fluorescence imaging resolution, were acted as fluorescence reporters. Also CoOOH nanoflakes were chosen as fluorescence quenchers by forming on the surface of NGs. Once AA was introduced, CoOOH was reduced to Co²⁺, which resulted in a “turn-on” fluorescence signal of NGs. The proposed nanoprobe demonstrated high sensitivity toward AA, with the observed LOD of 270 nM. It also showed high selectivity to AA with excellent photostability. Moreover, the nanoprobe was successfully used for TP imaging of endogenous AA in living cells as well as deep tissue imaging

Development of Dual-Responsive Fluorescent Probe for Drug Screening of Diabetes Cardiomyopathy

For specific drug research and development, a drug-screening strategy (DSS) plays an indispensable role in the biomedical field. Unfortunately, traditional strategies are complicated and insufficiently accurate due to the widely used single-target screening method. Herein, a simple dual-target-based drug-screening strategy (dt-DSS) is proposed to screen highly effective drugs by fluorescence imaging. As a proof of concept, we utilized a dual-responsive fluorescence probe to screen drugs for diabetic cardiomyopathy (DCM). We first developed and took advantage of a dual-response probe HDB to detect reactive oxygen species (ROS) and mitophagy levels in cellular starvation and high glucose models. Based on this, HDB was utilized to study the effects of different drugs in the mitophagy process caused by the high-glucose cell model for DCM. Combined with Western blotting assays, we found that Drp-1 inhibitors could fundamentally reduce mitophagy caused by the high-glucose cells model. Compared with commercial single-target antioxidant drugs, the drugs with simultaneous antioxidant capacity and Drp-1 inhibition screened by dt-DSS, such as resveratrol and icariin, could treat DCM better. Therefore, HDB as an effective tool could accurately and quickly screen high-potency drugs for DCM. We believe that this work provides an attractive strategy to explore the pathogenesis of diabetic cardiomyopathy and precisely screen for highly effective drugs

Two-Photon DNAzyme–Gold Nanoparticle Probe for Imaging Intracellular Metal Ions

RNA-cleaving DNAzymes have been demonstrated as a promising platform for sensing metal ions. However, the poor biological imaging performance of RNA-cleaving DNAzyme-based fluorescent probes has limited their intracellular applications. Compared with traditional one-photon fluorescence imaging, two-photon (TP) fluorescent probes have shown advantages such as increased penetration depth, lower tissue autofluorescence, and reduced photodamage. Herein, for the first time, we developed an RNA-cleaving DNAzyme-based TP imaging probe (TP-8–17ES–AuNP) for Zn²⁺ detection in living cells by modifying a Zn²⁺-specific DNAzyme (8–17) with a TP fluorophore (TP-8–17ES) and using gold nanoparticles (AuNPs) for intracellular delivery. The modified TP-8–17ES exhibits good two-photon properties and excellent photostability. For the TP-8–17ES–AuNP, in the absence of Zn²⁺, the TP fluorophore is quenched by both AuNPs and the molecular quencher. Only in the presence of Zn²⁺ does the DNAzyme cleave the TP fluorophore-labeled substrate strand, resulting in fluorescence enhancement and TP imaging. Such probe shows remarkable selectivity of Zn²⁺ over other metal ions existing in the biological environment. Benefiting from the labeled TP fluorophore, the near-infrared (NIR) excited probe has the capability of TP imaging of Zn²⁺ in living cells and tissue with a deep tissue penetration up to 160 μm. This method can be generally applied to detect other metal ions in biological systems under TP imaging with higher tissue penetration ability and lower phototoxicity