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

Through-Bond Energy Transfer-Based Ratiometric Two-Photon Probe for Fluorescent Imaging of Pd²⁺ Ions in Living Cells and Tissues

Palladium can cause severe skin and eye irritation once it enters the human body. Ratiometric two-photon fluorescent probes can both eliminate interference from environmental factors and realize deep-tissue imaging with improved spatial localization. To quantitatively track Pd²⁺ in biosystems, we report here a colorimetric and two-photon ratiometric fluorescent probe, termed <b>Np</b>–<b>Rh</b>–<b>Pd</b>, which consists of a two-photon fluorophore (naphthalene derivative with a D-π-A structure) and a rhodamine B dye. The two fluorophores are directly linked to form a two-photon ratiometric fluorescent probe for Pd²⁺ based on a through-bond energy transfer (TBET) strategy. It exhibits highly efficient energy transfer (90%) with two well-resolved emission peaks (wavelength difference of 100 nm), which could efficiently diminish the cross talk between channels and is especially favorable for ratiometric bioimaging applications. A signal-to-background ratio of 31.2 was observed for the probe, which affords a high sensitivity for Pd²⁺ with a detection limit of 2.3 × 10^–7 M. It was also found that acidity does not affect the fluorescent response of the probe to Pd²⁺, which is favorable for its applications in practical samples. The probe was further used for fluorescence imaging of Pd²⁺ ions in live cells and tissue slices under two-photon excitation, which showed significant tissue-imaging depths (90–270 μm) and a high resolution for ratiometric imaging

“Four-In-One” Design of a Hemicyanine-Based Modular Scaffold for High-Contrast Activatable Molecular Afterglow Imaging

Afterglow luminescence (long persistent luminescence) holds great potential for nonbackground molecular imaging. However, current afterglow probes are mainly nanoparticles, and afterglow imaging systems based on organic small molecules are still lacking and have rarely been reported. Moreover, the lack of reactive sites and a universal molecular scaffold makes it difficult to design activatable afterglow probes. To address these issues, this study reports a novel kind of hemicyanine-based molecule scaffolds with stimuli-responsive afterglow luminescence, which is dependent on an intramolecular cascade photoreaction between 1O2 and the afterglow molecule to store the photoenergy for delayed luminescence after light cessation. As a proof of concept, three modular activatable molecular afterglow probes (MAPs) with a “four-in-one” molecular design by integrating a stimuli-responsive unit, 1O2-generating unit, 1O2-capturing unit, and luminescent unit into one probe are customized for quantification and imaging of targets including pH, superoxide anions, and aminopeptidase. Notably, MAPs show higher sensitivity in afterglow imaging than in fluorescence imaging because the responsive unit simultaneously controls the initiation of fluorescence (S1 to S0) and 1O2 generation (S1 to T1). Finally, MAPs are applied for high-contrast afterglow imaging of drug-induced hepatotoxicity, which is poorly evaluated in clinics and drug discovery. By reporting the sequential occurrence of oxidative stress and upregulation of aminopeptidase, such activatable afterglow probes allow noninvasive imaging of hepatotoxicity earlier than the serological and histology manifestation, indicating their promise for early diagnosis of hepatotoxicity

Highly Selective Fluorescent Probe Design for Visualizing Hepatic Hydrogen Sulfide in the Pathological Progression of Nonalcoholic Fatty Liver

Hydrogen sulfide (H2S), emerging as an important gaseous signal, has attracted more and more attention for its key role in chronic fatty liver diseases. However, lacking tools for H2S-specific in situ detection, the changes of endogenous hepatic H2S levels in the pathological progression of chronic liver diseases are still unclear. To this end, we adopted a strategy of combining molecular probe design and nanofunctionalization to develop a highly selective near-infrared (NIR) fluorescent probe, which allows in vivo real-time monitoring of hepatic H2S levels in the process of nonalcoholic fatty liver disease (NAFLD). As a proof of strategy demonstration, we first designed NIR molecular probes for H2S sensing through chemical design and probe screening and then loaded molecular probes into mesoporous silicon nanomaterials (MSNs) with surface encapsulation using poly­(ethylene glycol) to construct a highly selective probe MSN@CSN@PEG, with significantly improved selectivity and photostability. Moreover, MSN@CSN@PEG exhibited high selectivity and sensitivity for endogenous H2S in cells and tumors in vivo, eliminating the interference of a high concentration of biothiols and sulfhydryl proteins. Furthermore, the probe was applied to in situ intravital imaging and systematic assessment of hepatic H2S levels in different stages of NAFLD for the first time, which may offer a promising tool for the future study of fatty liver diseases and other chronic liver diseases

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

Efficient Two-Photon Fluorescent Probe with Red Emission for Imaging of Thiophenols in Living Cells and Tissues

Thiophenols, a class of highly toxic and pollutant compounds, are widely used in industrial production. Some aliphatic thiols play important roles in living organisms. Therefore, the development of efficient methods to discriminate thiophenols from aliphatic thiols is of great importance. Although several one-photon fluorescent probes have been reported for thiophenols, two-photon fluorescent probes are more favorable for biological imaging due to its low background fluorescence, deep penetration depth, and so on. In this work, a two-photon fluorescent probe for thiophenols, termed NpRb1, has been developed for the first time by employing 2,4-dinitrobenzene-sulfonate (DNBS) as a recognition unit (also a fluorescence quencher) and a naphthalene-BODIPY-based through-bond energy transfer (TBET) cassette as a fluorescent reporter. The TBET system consists of a D-π-A structured two-photon naphthalene fluorophore and a red-emitting BODIPY. It displayed highly energy transfer efficiency (93.5%), large pseudo-Stokes shifts upon one-photon excitation, and red fluorescence emission (λ_em = 586 nm), which is highly desirable for bioimaging applications. The probe exhibited a 163-fold thiophenol-triggered two-photon excited fluorescence enhancement at 586 nm. It showed a high selectivity and excellent sensitivity to thiophenols, with a detection limit of 4.9 nM. Moreover, it was successfully applied for practical detection of thiophenol in water samples with a good recovery, two-photon imaging of thiophenol in living cells, and tissues with tissue-imaging depths of 90–220 μm, demonstrating its practical application in environmental samples and biological systems

Catalytic and Molecular Beacons for Amplified Detection of Metal Ions and Organic Molecules with High Sensitivity

The catalytic beacon has emerged as a general platform for sensing metal ions and organic molecules. However, few reports have taken advantage of the true potential of catalytic beacons in signal amplification through multiple enzymatic turnovers, as existing designs require either equal concentrations of substrate and DNAzyme or an excess of DNAzyme in order to maintain efficient quenching, eliminating the excess of substrate necessary for multiple turnovers. On the basis of the large difference in the melting temperatures between the intramolecular molecular beacon stem and intermolecular products of identical sequences, we here report a general strategy of catalytic and molecular beacon (CAMB) that combines the advantages of the molecular beacon for highly efficient quenching with the catalytic beacon for amplified sensing through enzymatic turnovers. Such a CAMB design allows detection of metal ions such as Pb2+ with a high sensitivity (LOD = 600 pM). Furthermore, the aptamer sequence has been introduced into DNAzyme to use the modified CAMB for amplified sensing of adenosine with similar high sensitivity. These results together demonstrate that CAMB provides a general platform for amplified detection of a wide range of targets

Molecular Engineering of pH-Responsive NIR Oxazine Assemblies for Evoking Tumor Ferroptosis via Triggering Lysosomal Dysfunction

Ferroptosis, a newly discovered form of regulated cell death, is emerging as a promising approach to tumor therapy. However, the spatiotemporal control of cell-intrinsic Fenton chemistry to modulate tumor ferroptosis remains challenging. Here, we report an oxazine-based activatable molecular assembly (PTO-Biotin Nps), which is capable of triggering the lysosomal dysfunction-mediated Fenton pathway with excellent spatiotemporal resolution via near-infrared (NIR) light to evoke ferroptosis. In this system, a pH-responsive NIR photothermal oxazine molecule was designed and functionalized with a tumor-targeting hydrophilic biotin-poly(ethylene glycol) (PEG) chain to engineer well-defined nanostructured assemblies within a single-molecular framework. PTO-Biotin Nps possesses a selective tropism to lysosome accumulation inside tumor cells, accommodated by its enhanced photothermal activity in the acidic microenvironment. Upon NIR light activation, PTO-Biotin Nps promoted lysosomal dysfunction and induced cytosolic acidification and impaired autophagy. More importantly, photoactivation-mediated lysosomal dysfunction via PTO-Biotin Nps was found to markedly enhance cellular Fenton reactions and evoke ferroptosis, thereby improving antitumor efficacy and mitigating systemic side effects. Overall, our study demonstrates that the molecular engineering approach of pH-responsive photothermal oxazine assemblies enables the spatiotemporal modulation of the intrinsic ferroptosis mechanism, offering a novel strategy for the development of metal-free Fenton inducers in antitumor therapy

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