Nitroreductase Research Articles

Design, Synthesis, and Evaluation of Doxifluridine Derivatives as Nitroreductase-Responsive Anticancer Prodrugs

Antimetabolite antitumor drugs interfere with nucleic acid and DNA synthesis, causing cancer cell death. However, they also affect rapidly dividing normal cells and cause serious side effects. Doxifluridine (5′-deoxy-5-fluorouridine [5′-DFUR]), a 5-fluorouracil (5-FU) prodrug converted to 5-FU by thymidine phosphorylase (TP), exerts antitumor effects. Since TP is distributed in tumor and normal tissues, 5′-DFUR features side effects. Here we designed a series of novel 5′-DFUR derivatives based on high nitroreductase (NTR) levels in the hypoxic microenvironment of tumor tissues by introducing nitro-containing moieties into the 5′-DFUR structure. These derivatives exert their antitumor effects by producing 5-FU under the dual action of TP and NTR in the tumor microenvironment. The derivatives were synthesized and their stability, release, and cytotoxicity evaluated in vitro and antitumor activity evaluated in vivo. Compound 2c, featuring nitrofuran fragments, was stable in phosphate-buffered saline and plasma at different pH values and reduced rapidly in the presence of NTR. The in vitro cytotoxicity evaluation indicated that compound 2c showed excellent selectivity in the MCF-7 and HT29 cell lines. Moreover, it exhibited antitumor effects comparable to those of 5′-DFUR in vivo without significant toxic side effects. These results suggest that compound 2c is a promising antitumor prodrug.

A Nitroreductase‐Activatable Metabolic Reporter for Covalent Labeling of Pathological Hypoxic Cells in Tumorigenesis

AbstractAberrant hypoxic stress will initiate a cascade of pathological consequence observed prominently in tumorigenesis. Understanding of hypoxia's role in tumorigenesis is highly essential for developing effective therapeutics, which necessitates reliable tools to specifically distinguish hypoxic tumor cells (or tissues) and correlate their dynamics with the status of disease in complex living settings for precise theranostics. So far, disparate hypoxia‐responsive probe molecules and prodrugs were designed via chemical or enzymatic reactions, yet their capability in real‐time reporting pathogenesis development is often compromised due to unrestricted diffusion and less selectivity towards the environmental responsiveness. Herein we present an oxygen‐insensitive nitroreductase (NTR)‐activatable glycan metabolic reporter (pNB‐ManNAz) capable of covalently labeling hypoxic tumor cells and tissues. Under pathophysiological hypoxic environments, the caged non‐metabolizable precursor pNB‐ManNAz exhibited unique responsiveness to cellular NTR, culminating in structural self‐immolation and the resultant ManNAz could incorporate onto cell surface glycoproteins, thereby facilitating fluorescence labeling via bioorthogonal chemistry. This NTR‐responsive metabolic reporter demonstrated broad applicability for multicellular hypoxia labeling, particularly in the dynamic monitoring of orthotopic tumorigenesis and targeted tumor phototherapy in vivo. We anticipate that this approach holds promise for investigating hypoxia‐related pathological progression, offering valuable insights for accurate diagnosis and treatment.

In Vitro Evaluation of New 5-Nitroindazolin-3-one Derivatives as Promising Agents against Trypanosoma cruzi

Chagas disease is a prevalent health problem in Latin America which has received insufficient attention worldwide. Current treatments for this disease, benznidazole and nifurtimox, have limited efficacy and may cause side effects. A recent study proposed investigating a wide range of nitroindazole and indazolone derivatives as feasible treatments. Therefore, it is proposed that adding a nitro group at the 5-position of the indazole and indazolone structure could enhance trypanocidal activity by inducing oxidative stress through activation of the nitro group by NTRs (nitroreductases). The study results indicate that the nitro group advances free radical production, as confirmed by several analyses. Compound 5a (5-nitro-2-picolyl-indazolin-3-one) shows the most favorable trypanocidal activity (1.1 ± 0.3 µM in epimastigotes and 5.4 ± 1.0 µM in trypomastigotes), with a selectivity index superior to nifurtimox. Analysis of the mechanism of action indicated that the nitro group at the 5-position of the indazole ring induces the generation of reactive oxygen species (ROS), which causes apoptosis in the parasites. Computational docking studies reveal how the compounds interact with critical residues of the NTR and FMNH2 (flavin mononucleotide reduced) in the binding site, which is also present in active ligands. The lipophilicity of the studied series was shown to influence their activity, and the nitro group was found to play a crucial role in generating free radicals. Further investigations are needed of derivatives with comparable lipophilic characteristics and the location of the nitro group in different positions of the base structure.

Assembly‐Glued Ligands and Warheads for Hypoxia‐Activatable Supramolecular Covalent Inhibitors

AbstractTargeted covalent inhibitors are promising for drug discovery and challenged by the strict structural features for target proteins to sustain proximity‐induced conjugations. Herein, an assembly‐glued strategy is reported to create surface‐displayed supramolecular covalent inhibitors that leverage the display‐induced proximity between noncovalent ligands and warheads. The ligand and warhead are obtained via attaching an epidermal growth factor receptor (EGFR)‐binding segment or nitroreductase (NTR)‐activated moiety to bola‐amphiphilic peptides, respectively. Co‐assembling the ligand and warhead with a filler peptide glues them and forms the surface‐displayed covalent inhibitor. While the ligand associates with EGFR, the warhead undergoes NTR‐induced cysteine conjugation facilitated by the ligand‐EGFR association. In vitro studies show the reliable hypoxia‐activated inhibition of EGFR phosphorylation and enhanced cytotoxicity of the covalent inhibitor under hypoxic condition. In addition, this strategy also allows for creating covalent inhibitors targeting proteins without small molecular covalent drugs, due to the display‐induced proximity between ligands and warheads. In vivo results illustrate the prolonged retention of the covalent inhibitor and its efficacy in inhibiting tumor growth. These findings demonstrate that simultaneous surface display of ligands and warheads via assembly adhesives allows for implementation of covalent inhibition functions, thus providing a new strategy for developing covalent inhibitors in the future.

PK-PD relationship of poorly absorbable active ingredients from traditional Chinese medicines explaining by metabolic enzyme of gut microbiota: A case study of Dehydrocorydaline

Many active ingredients in traditional Chinese medicines generally have the characteristic of poor oral absorption but definite efficacy. It is necessary to establish a comprehensive technical system to explain the “PK-PD relationship” of them. Dehydrocorydaline (DHC), the quality control component in the Chinese patent drug “Kedaling Tablets”, has poor oral absorption but clear efficacy for coronary heart disease. Using DHC as a model drug, the changes in absorption and pharmacological effects of DHC in rats before and after inhibiting nitroreductase (NR) from gut microbiota were studied. The results showed that after inhibiting of NR activity, the plasma concentration of DHC in rats was decreased, the serum level of total cholesterol, triglyceride and low-density lipoprotein cholesterol were significantly increased. The levels of tumor necrosis factor-α, interleukin-1β, hypersensitive C-reactive protein, intercellular cell adhesion molecule-1 and Monocyte chemoattractant protein-1 were significantly increased, and pathological sections also showed that the efficacy of DHC decreased after inhibiting the activity of NR. We further investigated the drug metabolism of DHC under NR and found that DHC was metabolized into a hydrogenated metabolite, which may have stronger membrane permeability. In summary, NR may mediate the absorption degree and efficacy of DHC in vivo by metabolizing DHC into absorbable metabolite.

A dual-responsive fluorescent probe for cellular hypoxia and tumor imaging based on nitroreductase and viscosity synergy

Nitroreductase (NTR), an enzyme that operates within the cytoplasm, relies on flavin mononucleotide or flavin adenine dinucleotide for its activity. In tumor cells, hypoxic conditions often lead to an increase in NTR levels. Consequently, the development of fluorescent probes capable of detecting NTR activity is crucial for the monitoring of hypoxia in biological systems. This study presents the one-step synthesis of a near-infrared fluorescent probe, DSM-NO2. The probe was highly sensitive to NTR and caused a fluorescence enhancement by 50-fold, with a detection limit as low as 1 ng/mL. Under hypoxic conditions in tumor cells, not only does NTR increase, but the cellular viscosity also rises. In high-viscosity environments, the fluorescence enhancement could reach up to 220-fold, and the detection limit could be further reduced to 55 pg/mL. The reaction mechanism was thoroughly investigated using mass spectrometry, molecular docking, and theoretical calculations. Moreover, the probe was successfully applied in the real-time monitoring and visualization analysis of NTR during the growth of Escherichia coli and in the hypoxic state of cancer cells. Additionally, through nebulized inhalation, the probe was successfully employed for in situ fluorescent imaging studies in a murine model of lung cancer.

Sensitive Cancer Hypoxia Detection via a Dual-Locking Fluorescence Response System Using Two Hypoxia Indicators.

Hypoxia is intricately associated with various diseases, including ischemia, vascular disorders, and cancer. Particularly in cancer cells, hypoxia promotes tumor growth, cell proliferation, migration, and invasion and enhances treatment resistance, making its detection crucial for cancer diagnosis and therapy. However, methods for detecting hypoxia are limited, often relying on single-detection systems. In this study, we developed a dual-lock-based fluorescent probe that selectively exhibits strong green fluorescence under hypoxic conditions due to simultaneous activity of nitroreductases (NTRs) and hydrogen sulfide (H2S), with a high signal-to-background ratio. The biocompatibility and photophysical properties of the probes were thoroughly investigated through both extracellular and intracellular experimental analyses. Among the synthesized naphthalimide-based probes, the dual-detection probe DNNC demonstrated excellent selectivity and sensitivity to the simultaneous activity of NTR/H2S compared to other single-detection probes. The performance of DNNC was applied to various organ-derived cancer cells and tumor tissue models such as HeLa cell sparoids, enabling spatiotemporal confocal fluorescence imaging and quantitative analysis of hypoxic levels in cancer. Our development of DNNC is expected to significantly advance cancer diagnosis and treatment by molecularly detecting hypoxia associated with cancer aggressiveness, therapy resistance, and unfavorable prognosis.

One-Pot Multienzyme Cascades for Stereodivergent Synthesis of Tetrahydroquinolines.

The tetrahydroquinoline (THQ) framework is commonly found in natural products and pharmaceutically relevant molecules. Apart from the use of transition metal catalysts and chiral phosphoric acids, the chiral 2-substituted 1,2,3,4-THQs are synthesized using amine oxidase biocatalysts. However, the use of imine reductases (IREDs) in their asymmetric synthesis remained unexplored. In the current work, IREDs are employed in telescopic multienzyme cascades to catalyze the intramolecular reductive amination leading to chiral 2-alkyl and 2-aryl substituted-1,2,3,4-tetrahydroquinolines starting from inexpensive nitroalkenones. The cascades containing NtDBR (an ene reductase), NfsB (a nitro reductase) with either Na2S2O4 or V2O5, various IREDs, and glucose dehydrogenase (for NADPH regeneration) are used to synthesize a broad range of (R)/(S)-2-alkyl-substituted (THQs) (26 examples) with high yield (up to 93%) and excellent ee (up to 99%) in one-pot. The method further facilitates the one-pot biocatalytic synthesis of chiral 2-aryl substituted THQs (26 examples) from amino chalcones. Lastly, the asymmetric synthesis of several (R)- and (S)-THQ based intermediates of Hancock alkaloids showed the practical application of the newly developed biocatalytic cascades.

Surface Au-H Species as Self-Generated Prosthetic Groups of a Formate Dehydrogenase-like Au Nanozyme to Engineer Multienzymatic Activities.

Although the past decade has witnessed a rapid development of oxidoreductase-mimicking nanozymes, the mimicry of cofactors that play key roles in mediating electron and proton transfer remains limited. This study explores how surface Au-H species conjugated to Au nanoparticles (NPs) that imitate formate dehydrogenase (FDH) can serve as cofactors, analogous to NADH in natural enzymes, offering diverse possibilities for FDH-mimicking Au nanozymes to mimic various enzymes. Once O2 is present, Au-H species assist Au NPs to complete the on-demand H2O2 generation for cascade reactions. Alternatively, when oxidizing organic molecules are introduced as substrates, Au-H species confer nitro reductase- and aldehyde reductase-like activities on Au NPs under anaerobic conditions. Furthermore, similar to the dehydrogenase-NADH complex, Au NPs possessing Au-H species are gifted with esterase-like activity for ester hydrolysis. By revealing that Au-H species are prosthetic groups for FDH-mimicking Au nanozymes, this work may inspire explorations into future self-generated cofactor mimics for nanozymes, thereby circumventing the need for exogenous cofactors.

A Nitroreductase-Activatable Metabolic Reporter for Covalent Labeling of Pathological Hypoxic Cells in Tumorigenesis.

Aberrant hypoxic stress will initiate a cascade of pathological consequence observed prominently in tumorigenesis. Understanding of hypoxia's role in tumorigenesis is highly essential for developing effective therapeutics, which necessitates reliable tools to specifically distinguish hypoxic tumor cells (or tissues) and correlate their dynamics with the status of disease in complex living settings for precise theranostics. So far, disparate hypoxia-responsive probe molecules and prodrugs were designed via chemical or enzymatic reactions, yet their capability in real-time reporting pathogenesis development is often compromised due to unrestricted diffusion and less selectivity towards the environmental responsiveness. Herein we present an oxygen-insensitive nitroreductase (NTR)-activatable glycan metabolic reporter (pNB-ManNAz) capable of covalently labeling hypoxic tumor cells and tissues. Under pathophysiological hypoxic environments, the caged non-metabolizable precursor pNB-ManNAz exhibited unique responsiveness to cellular NTR, culminating in structural self-immolation and the resultant ManNAz could incorporate onto cell surface glycoproteins, thereby facilitating fluorescence labeling via bioorthogonal chemistry. This NTR-responsive metabolic reporter demonstrated broad applicability for multicellular hypoxia labeling, particularly in the dynamic monitoring of orthotopic tumorigenesis and targeted tumor phototherapy in vivo. We anticipate that this approach holds promise for investigating hypoxia-related pathological progression, offering valuable insights for accurate diagnosis and treatment.

Dual-functional fluorescent probe for imaging ROS and hypoxia dynamic in cancer cells

Hypoxia is able to induce the overexpression of nitroreductase (NTR) in cancer cells. Moreover, there is still the controversy about reactive oxygen species (ROS) change under the hypoxic condition. Herein, we designed and synthesized a fluorescent probe WNO that could simultaneously visualize the fluctuation changes of NTR and ROS in hypoxic HeLa cells. WNO consists of 4-nitro-1,8-naphthalimide group and 2,3,3-trimethyl indolium salt. In buffer 4-nitro-1,8-naphthalimide group could selectively response to NTR in the presence of nicotinamide adenine dinucleotide phosphate (NADH), resulting in a strong green fluorescence. However, upon LED light irradiation 2,3,3-trimethyl indolium salt moiety could efficiently convert into Cy3 emitting a red fluorescence. Furthermore, the monitoring for NTR and photoconversion of WNO in hypoxic HeLa cells were investigated in detail by confocal fluorescence imaging through green and red channels, respectively. It was found that WNO could image the intracellular NTR or hypoxia level. Meanwhile, the WNO photoconversion was accelerated largely in hypoxic cells, which resulted from the production of excessive ROS. WNO and/or its photoconversion product mainly accumulated into the mitochondria. Moreover, MTT assay indicated that LED irradiation or hypoxia treatment made the cytotoxicity of WNO increase by 50 %. Additionally, based on ROS-stimulated the photoconversion of WNO, the intracellular ROS and NTR changes in mitochondrial dysfunction and oxidative stress were also investigated. It indicated that WNO was capable of sensitive and rapid imaging the intracellular ROS and NTR levels under both conditions. These findings would provide an excellent foundation for cancer diagnosis and therapy.

Hemicyanine-Based Highly Water-Soluble Probe for Extracellular Nitroreductase.

Nitroreductase (NTR) has long been a target of interest for its important role involved in the nitro compounds metabolism. Various probes have been reported for NTR analysis, but rarely able to distinguish the extracellular NTR from intracellular ones. Herein we reported a new NTR sensor, HCyS-NO2, which was a hemicyanine molecule with one nitro and two sulfo groups attached. The nitro group acted as the reporting group to respond NTR reduction. Direct linkage of nitro group into the hemicyanine π conjugate system facilitated the intramolecular electron transfer (IET) process and thus quenched the fluorescence of hemicyanine core. Upon reduction with NTR, the nitro group was rapidly converted into the hydroxylamino and then the amino group, eliminating IET process and thus restoring the fluorescence. The sulfo groups installed significantly increased the hydrophilicity of the molecule, and introduced negative charges at physiological pH, preventing the diffusion into bacteria. Both gram-negative and gram-positive bacteria were able to turn on the fluorescence of HCyS-NO2, without detectable diffusion into cells, providing a useful tool to probe the extracellular reduction process.

A Host–Guest Platform for Highly Efficient, Quantitative, and Rapid Detection of Nitroreductase

Nitroreductase (NTR) is an enzyme expressed at an abnormally high level in solid tumors, which is associated with the hypoxia level in tumors. The establishment of a high-performance and convenient fluorescent platform for the fast monitoring of NTR is of pivotal importance. Herein, a novel host–guest complex was created by encapsulating a fluorescent substrate GP-NTR within a metal–organic capsule Zn-MPB that included a NADH mimic for the detection of hypoxia via responding to nitroreductase (NTR) with fast responsiveness and good fluorescence imaging. Notably, the double-substrate process was streamlined to a single–substrate process by the host–guest supramolecular method in the catalytic process of NTR, which enabled the reaction to be independent of the cofactor NADH supply and shortened the distance between the substrate and the active site of NTR. The increasing fluorescence intensity of Zn-MPB⊃GP-NTR exhibits a linear relationship with NTR concentration and shows a fast response toward NTR in solution in tens of seconds. Zn-MPB⊃GP-NTR also displays high sensitivity to NTR with a low detection limit of 6.4 ng/mL. Cells and in vivo studies have confirmed that Zn-MPB⊃GP-NTR could be successfully applied for the fast imaging of NTR in NTR-overexpressed tumor cells and tumor-bearing animals. The host–guest platform not only provides a new avenue for the design and optimization of a fluorescence detection platform for the rapid and quantitative detection of NTR activity, but also offers an imaging tool for the early diagnosis of hypoxia-related tumors.

A nitroreductase responsive probe for early diagnosis of pulmonary fibrosis disease

Idiopathic pulmonary fibrosis (IPF) is a serious interstitial lung disease. However, the definitive diagnosis of IPF is impeded by the limited capabilities of current diagnostic methods, which may fail to capture the optimal timing for treatment. The main goal of this study is to determine the feasibility of a nitroreductase (NTR) responsive probe, 18F-NCRP, for early detection and deterioration monitoring of IPF. 18F-NCRP was obtained with high radiochemical purity (>95 %). BLM-injured mice were established by intratracheal instillation with bleomycin (BLM) and characterized through histological analysis. Longitudinal PET/CT imaging, biodistribution study and in vitro autoradiography were performed. The correlations between the uptake of 18F-NCRP and mean lung density (tested by CT), as well as histopathological characteristics were analyzed. In PET imaging study, 18F-NCRP exhibited promising efficacy in monitoring the progression of IPF, which was earlier than CT. The ratio of uptake in BLM-injured lung to control lung increased from 1.4-fold on D15 to 2.2-fold on D22. Biodistribution data showed a significant lung uptake of 18F-NCRP in BLM-injured mice. There was a strong positive correlation between the 18F-NCRP uptake in the BLM-injured lungs and the histopathological characteristics. Given that, 18F-NCRP PET imaging of NTR, a promising biomarker for investigating the underlying pathogenic mechanism of IPF, is attainable as well as desirable, which might lay the foundation for establishing an NTR-targeted imaging evaluation system of IPF.

A Novel Fluorescent Probe for Nitroreductase Detection and Imaging of cancer Cells under Hypoxia Conditions.

Nitroreductase (NTR) is to be pivotal in the biodegradation of nitroaromatics. NTR is produced in tumor tissues under hypoxic conditions, which is one of the markers for early tumor diagnosis. In this study, a novel probe FD-NTR was developed for NTR detection. Probe FD-NTR can exhibit a specific reaction with NTR in the presence of NADH. The probe displayed satisfactory selectivity and sensitivity towards NTR with a calculated detection limit of 12 ng/mL. Under the conditions of low cytotoxic hypoxia, the FD-NTR probe has shown successful application in imaging both MCF-7 cells and tumor tissues, which indicated that the FD-NTR probe holds promising application prospects for detecting NTR in tumors.

Biochemical, kinetic, and structural characterization of a Bacillus tequilensis nitroreductase.

Nitroreductases (NRs) are NAD(P)H-dependent flavoenzymes that reduce nitro aromatic compounds to their corresponding arylamines via the nitroso and hydroxylamine intermediates. Because of their broad substrate scope and versatility, NRs have found application in multiple fields such as biocatalysis, bioremediation, cell-imaging and prodrug activation. However, only a limited number of members of the broad NR superfamily (> 24 000 sequences) have been experimentally characterized. Within this group of enzymes, only few are capable of amine synthesis, which is a fundamental chemical transformation for the pharmaceutical, agricultural, and textile industries. Herein, we provide a comprehensive description of a recently discovered NR from Bacillus tequilensis, named BtNR. This enzyme has previously been demonstrated to have the capability to fully convert nitro aromatic and heterocyclic compounds to their respective primary amines. In this study, we determined its biochemical, kinetic and structural properties, including its apparent melting temperature (Tm) of 59 °C, broad pH activity range (from pH 3 to 10) and a notably low redox potential (-236 ± 1 mV) in comparison to other well-known NRs. We also determined its steady-state and pre-steady-state kinetic parameters, which are consistent with other NRs. Additionally, we elucidated the crystal structure of BtNR, which resembles the well-characterized Escherichia coli oxygen-insensitive NAD(P)H nitroreductase (NfsB), and investigated the substrate binding in its active site through docking and molecular dynamics studies with four nitro aromatic substrates. Guided by these structural analyses, we probed the functional roles of active site residues by site-directed mutagenesis. Our findings provide valuable insights into the biochemical and structural properties of BtNR, as well as its potential applications in biotechnology.

Site-Specific Cascade-Activatable Fluorogenic Nanomicelles Enable Precision and Accuracy Imaging of Pulmonary Metastatic Tumor.

The precise localization of metastatic tumors with subtle growth is crucial for timely intervention and improvement of tumor prognosis but remains a paramount challenging. To date, site-specific activation of fluorogenic probes for single-stimulus-based diagnosis typically targets an occult molecular event in a complex biosystem with limited specificity. Herein, we propose a highly specific site-specific cascade-activated strategy to enhance detection accuracy, aiming to achieve the accurate detection of breast cancer (BC) lung metastasis in a cascade manner. Specifically, cascade-activatable NIR fluorogenic nanomicelles HPNs were constructed using ultra-pH-sensitive (UPS) block copolymers as carriers and nitroreductase (NTR)-activated fluorogenic reporters. HPNs exhibit programmable cascade response characteristics by first instantaneous dissociating under in situ tumor acidity, facilitating deep tumor penetration followed by selective fluorescence activation through NTR-mediated enzymatic reaction resulting in high fluorescence ON/OFF contrast. Notably, this unique feature of HPNs enables high-precision diagnosis of orthotopic BC as well as its lung metastases with a remarkable signal-to-background ratio (SBR). This proposed site-specific cascade activation strategy will offer opportunities for a specific diagnosis with high signal fidelity of various insidious metastatic lesions in situ across different diseases.

A novel low-background nitroreductase fluorescent probe for real-time fluorescence imaging and surgical guidance of thyroid cancer resection

Thyroid cancer always appears insidiously with few noticeable clinical symptoms. Due to its limitations, conventional ultrasound imaging can lead to missed or misdiagnosed cases. Surgery is still the primary treatment method of thyroid cancer, but removal of surrounding healthy tissues to minimize recurrence leads to overtreatment and added patient suffering. To address this challenge, herein, a nitroreductase (NTR) fluorescent probe, Ox-NTR, has been developed for detecting thyroid cancer and tracking the surgical removal of thyroid tumors by fluorescence imaging. The conjugated structure of oxazine 1 was disrupted, significantly reducing the issue of high background signals, thus effectively achieving low background fluorescence. Under hypoxic conditions, the nitro group of Ox-NTR can be reduced to an amine and subsequently decomposed into oxazine 1, emitting intense red fluorescence. Ox-NTR has a low detection limit of 0.09 μg/mL for NTR with excellent photostability and selectivity. Cellular studies show that Ox-NTR can effectively detect NTR levels in hypoxic thyroid cancer cells. Moreover, the ability of Ox-NTR of rapid response to thyroid cancer in vivo is confirmed by fluorescence imaging in mice, distinguishing tumors from normal tissues due to its superior low background fluorescence. Utilizing this fluorescence imaging method during surgical resection can guide the removal of tumors, preventing both missed tumor tissues and accidental removal of healthy tissue. In summary, the novel Ox-NTR offers precise detection capabilities that provide significant advantages over traditional imaging methods for thyroid cancer diagnosis and treatment, making it a valuable tool to guide tumor removal in surgical procedures.

Totally Caged Type I Pro-Photosensitizer for Oxygen-Independent Synergistic Phototherapy of Hypoxic Tumors.

Activatable type I photosensitizers are an effective way to overcome the insufficiency and imprecision of photodynamic therapy in the treatment of hypoxic tumors, however, the incompletely inhibited photoactivity of pro-photosensitizer and the limited oxidative phototoxicity of post-photosensitizer are major limitations. It is still a great challenge to address these issues using a single and facile design. Herein, a series of totally caged type I pro-photosensitizers (Pro-I-PSs) are rationally developed that are only activated in tumor hypoxic environment and combine two oxygen-independent therapeutic mechanisms under single-pulse laser irradiation to enhance the phototherapeutic efficacy. Specifically, five benzophenothiazine-based dyes modified with different nitroaromatic groups, BPN 1-5, are designed and explored as latent hypoxia-activatable Pro-I-PSs. By comparing their optical responses to nitroreductase (NTR), it is identified that the 2-methoxy-4-nitrophenyl decorated dye (BPN 2) is the optimal Pro-I-PSs, which can achieve NTR-activated background-free fluorescence/photoacoustic dual-modality tumor imaging. Furthermore, upon activation, BPN 2 can simultaneously produce an oxygen-independent photoacoustic cavitation effect and a photodynamic type I process at single-pulse laser irradiation. Detailed studies in vitro and in vivo indicated that BPN 2 can effectively induce cancer cell apoptosis through synergistic effects. This study provides promising potential for overcoming the pitfalls of hypoxic-tumor photodynamic therapy.

A Chemically Inducible Muscle Ablation System for the Zebrafish.

An effective method for tissue-specific ablation in zebrafish is the nitroreductase (NTR)/metronidazole (MTZ) system. Expressing bacterial NTR in the presence of nitroimidazole compounds causes apoptotic cell death, which can be useful for understanding many biological processes. However, this requires tissue-specific expression of the NTR enzyme, and many tissues have yet to be targeted with transgenic lines that express NTR. We generated a transgenic zebrafish line expressing NTR in differentiated skeletal muscle. Treatment of embryos with MTZ caused muscle specific cell ablation. We demonstrate this line can be used to monitor muscle regeneration in whole embryos and in transplanted transgenic cells.