Mechanism‐Guided Design and Synthesis of a Mitochondria‐Targeting Artemisinin Analogue with Enhanced Anticancer Activity

Understanding the mechanism of action (MOA) of bioactive natural products will guide endeavor to improve their cellular activities. Artemisinin and its derivatives inhibit cancer cell proliferation, yet with much lower efficiencies than their roles in killing malaria parasites. To improve their efficacies on cancer cells, we studied the MOA of artemisinin using chemical proteomics and found that free heme could directly activate artemisinin. We then designed and synthesized a derivative, ART‐TPP, which is capable of targeting the drug to mitochondria where free heme is synthesized. Remarkably, ART‐TPP exerted more potent inhibition than its parent compound to cancer cells. A clickable probe ART‐TPP‐Alk was also employed to confirm that the attachment of the TPP group could label more mitochondrial proteins than that for the ART derivative without TPP (AP1). This work shows the importance of MOA study, which enables us to optimize the design of natural drug analogues to improve their biological activities.     

Platinum(II)–Gadolinium(III) Complexes as Potential Single‐Molecular Theranostic Agents for Cancer Treatment

Theranostic agents are emerging multifunctional molecules capable of simultaneous therapy and diagnosis of diseases. We found that platinum(II)–gadolinium(III) complexes with the formula [{Pt(NH₃)₂Cl}₂GdL](NO₃)₂ possess such properties. The Gd center is stable in solution and the cytoplasm, whereas the Pt centers undergo ligand substitution in cancer cells. The Pt units interact with DNA and significantly promote the cellular uptake of Gd complexes. The cytotoxicity of the Pt–Gd complexes is comparable to that of cisplatin at high concentrations (≥0.1 mM ), and their proton relaxivity is higher than that of the commercial magnetic resonance imaging (MRI) contrast agent Gd–DTPA. T₁‐weighted MRI on B6 mice demonstrated that these complexes can reveal the accumulation of platinum drugs in vivo. Their cytotoxicity and imaging capabilities make the Pt–Gd complexes promising theranostic agents for cancer treatment.     

手术导航用荧光探针

荧光成像技术因具有操作简便、分辨率高、安全性好且可实时成像等优势,在术中导航中具有广阔的应用前景.虽然目前还没有靶向荧光探针在临床上得到批准,但已经有相当一部分荧光探针进入了临床试验阶段.最早进入临床试验的是一些偶联肿瘤靶向配体的荧光染料,例如近红外菁染料(IRDye800CW)标记的肿瘤特异抗体,叶酸标记的异硫氰酸荧...     

FRET‐Based Mitochondria‐Targetable Dual‐Excitation Ratiometric Fluorescent Probe for Monitoring Hydrogen Sulfide in Living Cells

Hydrogen sulfide (H₂S) is connected with various physiological and pathological functions. However, understanding the important functions of H₂S remains challenging, in part because of the lack of tools for detecting endogenous H₂S. Herein, compounds Ratio‐H₂S 1/2 are the first FRET‐based mitochondrial‐targetable dual‐excitation ratiometric fluorescent probes for H₂S on the basis of H₂S‐promoted thiolysis of dinitrophenyl ether. With the enhancement of H₂S concentration, the excitation peak at λ≈402 nm of the phenolate form of the hydroxycoumarin unit drastically increases, whereas the excitation band centered at λ≈570 nm from rhodamine stays constant and can serve as a reference signal. Thus, the ratios of fluorescence intensities at λ=402 and 570 nm (I₄₀₂/I₅₇₀) exhibit a drastic change from 0.048 in the absence of H₂S to 0.36 in the presence of 180 μM H₂S; this is a 7.5‐fold variation in the excitation ratios. The favorable properties of the probe include the donor and acceptor excitation bands, which exhibit large excitation separations (up to 168 nm separation) and comparable excitation intensities, high sensitivity and selectivity, and function well at physiological pH. In addition, it is demonstrated that the probe can localize in the mitochondria and determine H₂S in living cells. It is expected that this strategy will lead to the development of a wide range of mitochondria‐targetable dual‐excitation ratiometric probes for other analytes with outstanding spectral features, including large separations between the excitation wavelengths and comparable excitation intensities.     

Three‐Arm,Biotin‐Tagged Carbazole–Dicyanovinyl–Chlorambucil Conjugate: Simultaneous Tumor Targeting,Sensing, and Photoresponsive Anticancer Drug Delivery

The design, synthesis, and in vitro biological studies of a biotin–carbazole–dicyanovinyl–chlorambucil conjugate (Bio‐CBZ‐DCV‐CBL; 6 ) are reported. This conjugate ( 6 ) is a multifunctional single‐molecule appliance composed of a thiol‐sensor DCV functionality, a CBZ‐derived phototrigger as well as fluorescent reporter, and CBL as the anticancer drug, and Bio as the cancer‐targeting ligand. In conjugate 6 , the DCV bond undergoes a thiol–ene click reaction at pH<7 with intracellular thiols, thereby shutting down internal charge transfer between the donor CBZ and acceptor DCV units, resulting in a change of the fluorescence color from green to blue, and thereby, sensing the tumor microenvironment. Subsequent photoirradiation results in release of the anticancer drug CBL in a controlled manner.     

Construction of a Selective Fluorescent Probe for GSH Based on a Chloro‐Functionalized Coumarin‐enone Dye Platform

Glutathione (GSH), the most abundant intracellular biothiol, protects cellular components from damage caused by free radicals and reactive oxygen species (ROS), and plays a crucial role in human pathologies. A fluorescent probe that can selectively sense intracellular GSH would be very valuable for understanding of its biological functions and mechanisms of diseases. In this work, a 3,4‐dimethoxythiophenol‐substituted coumarin‐enone was exploited as a reaction‐type fluorescent probe for GSH based on a chloro‐functionalized coumarin‐enone platform. In the probe, the 3,4‐dimethoxythiophenol group functions not only as a fluorescence quencher through photoinduced electron transfer (PET) to ensure a low background fluorescence, but also as a reactive site for biothiols. The probe displays a dramatic fluorescence turn‐on response toward GSH with the long‐wavelength emission (600 nm) and significant Stokes shift (100 nm). The selectivity of the probe toward GSH over cysteine (Cys), homocysteine (Hcy), and other amino acids was demonstrated. Assisted by laser‐scanning confocal microscopy, we have demonstrated that the probe could specifically sense GSH over Cys/Hcy in human renal cell carcinoma SiHa cells.     

Integrin‐Targeting Knottin Peptide–Drug Conjugates Are Potent Inhibitors of Tumor Cell Proliferation

Antibody–drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide–drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a knottin peptide–drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor‐associated integrins. This KDC binds to tumor cells with low‐nanomolar affinity, is internalized by an integrin‐mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine‐resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug‐conjugate toolkit to include non‐antibody protein scaffolds.     

A Highly Selective Mitochondria‐Targeting Fluorescent K+ Sensor

Regulation of intracellular potassium (K⁺) concentration plays a key role in metabolic processes. So far, only a few intracellular K⁺ sensors have been developed. The highly selective fluorescent K⁺ sensor KS6 for monitoring K⁺ ion dynamics in mitochondria was produced by coupling triphenylphosphonium, borondipyrromethene (BODIPY), and triazacryptand (TAC). KS6 shows a good response to K⁺ in the range 30–500 mM , a large dynamic range (F_max/F₀≈130), high brightness (?_f=14.4 % at 150 mM of K⁺), and insensitivity to both pH in the range 5.5–9.0 and other metal ions under physiological conditions. Colocalization tests of KS6 with MitoTracker Green confirmed its predominant localization in the mitochondria of HeLa and U87MG cells. K⁺ efflux/influx in the mitochondria was observed upon stimulation with ionophores, nigericin, or ionomycin. KS6 is thus a highly selective semiquantitative K⁺ sensor suitable for the study of mitochondrial potassium flux in live cells.     

Ultrafluorogenic Coumarin–Tetrazine Probes for Real‐Time Biological Imaging

We have developed a series of new ultrafluorogenic probes in the blue‐green region of the visible‐light spectrum that display fluorescence enhancement exceeding 11 000‐fold. These fluorogenic dyes integrate a coumarin fluorochrome with the bioorthogonal trans‐cyclooctene(TCO)–tetrazine chemistry platform. By exploiting highly efficient through‐bond energy transfer (TBET), these probes exhibit the highest brightness enhancements reported for any bioorthogonal fluorogenic dyes. No‐wash, fluorogenic imaging of diverse targets including cell‐surface receptors in cancer cells, mitochondria, and the actin cytoskeleton is possible within seconds, with minimal background signal and no appreciable nonspecific binding, opening the possibility for in vivo sensing.     

Multifunctional Core‐Shell‐Corona‐Type Polymeric Micelles for Anticancer Drug‐Delivery and Imaging

We have developed core‐shell‐corona‐type polymeric micelles that can integrate multiple functions in one system, including the capability of accommodating hydrophobic dyes into core and hydrophilic drug into the shell, as well as pH‐triggered drug‐release. The neutral and hydrophilic corona sterically stabilizes the multifunctional polymeric micelles in aqueous solution. The mineralization of calcium phosphate (CaP) on the PAA domain not only enhances the diagnostic efficacy of organic dyes, but also works as a diffusion barrier for the controlled release.     

Photoelectrocyclization as an Activation Mechanism for Organelle‐Specific Live‐Cell Imaging Probes

Photoactivatable fluorophores are useful tools in live‐cell imaging owing to their potential for precise spatial and temporal control. In this report, a new photoactivatable organelle‐specific live‐cell imaging probe based on a 6π electrocyclization/oxidation mechanism is described. It is shown that this new probe is water‐soluble, non‐cytotoxic, cell‐permeable, and useful for mitochondrial imaging. The probe displays large Stokes shifts in both pre‐activated and activated forms, allowing simultaneous use with common dyes and fluorescent proteins. Sequential single‐cell activation experiments in dense cellular environments demonstrate high spatial precision and utility in single‐ or multi‐cell labeling experiments.     

Near‐Infrared Light and pH‐Responsive Polypyrrole@Polyacrylic acid/Fluorescent Mesoporous Silica Nanoparticles for Imaging and Chemo‐Photothermal Cancer Therapy

We have rationally designed a new theranostic agent by coating near‐infrared (NIR) light‐absorbing polypyrrole (PPY) with poly(acrylic acid) (PAA), in which PAA acts as a nanoreactor and template, followed by growing small fluorescent silica nanoparticles (fSiO₂ NPs) inside the PAA networks, resulting in the formation of polypyrrole@polyacrylic acid/fluorescent mesoporous silica (PPY@PAA/fmSiO₂) core–shell NPs. Meanwhile, DOX‐loaded PPY@PAA/fmSiO₂ NPs as pH and NIR dual‐sensitive drug delivery vehicles were employed for fluorescence imaging and chemo‐photothermal synergetic therapy in vitro and in vivo. The results demonstrate that the PPY@PAA/fmSiO₂ NPs show high in vivo tumor uptake by the enhanced permeability and retention (EPR) effect after intravenous injection as revealed by in vivo fluorescence imaging, which is very helpful for visualizing the location of the tumor. Moreover, the obtained NPs inhibit tumor growth (95.6 % of tumors were eliminated) because of the combination of chemo‐photothermal therapy, which offers a synergistically improved therapeutic outcome compared with the use of either therapy alone. Therefore, the present study provides new insights into developing NIR and pH‐stimuli responsive PPY‐based multifunctional platform for cancer theranostics.     

Active‐Site‐Matched Fluorescent Probes for Rapid and Direct Detection of Vicinal‐Sulfydryl‐Containing Peptides/Proteins in Living Cells

Vicinal‐sulfydryl‐containing peptides/proteins (VSPPs) play a crucial role in human pathologies. Fluorescent probes that are capable of detecting intracellular VSPPs in vivo would be useful tools to explore the mechanisms of some diseases. In this study, by regulating the spatial separation of two maleimide groups in a fluorescent dye to match that of two active cysteine residues contained in the conserved amino acid sequence (–CGPC–) of human thioredoxin, two active‐site‐matched fluorescent probes, o‐Dm‐Ac and m‐Dm‐Ac, were developed for real‐time imaging of VSPPs in living cells. As a result, the two probes can rapidly respond to small peptide models and reduced proteins, such as WCGPCK (W‐6), WCGGPCK (W‐7), and WCGGGPCK (W‐8), reduced bovine serum albumin (rBSA), and reduced thioredoxin (rTrx). Moreover, o‐Dm‐Ac displays a higher binding sensitivity with the above‐mentioned peptides and proteins, especially with W‐7 and rTrx. Furthermore, o‐Dm‐Ac was successfully used to rapidly and directly detect VSPPs both in vitro and in living cells. Thus, a novel probe‐design strategy was proposed and the synthesized probe applied successfully in imaging of target proteins in situ.     

Peptide‐Conjugated Long‐Lived Theranostic Imaging for Targeting GRPr in Cancer and Immune Cells

Gastrin‐releasing peptide receptor (GRPr) plays proliferative and inflammatory roles in living systems. Here, we report a highly selective GRPr antagonist (JMV594)‐tethered iridium(III) complex for probing GRPr in living cancer cells and immune cells. This probe exhibited desirable photophysical properties and also displayed negligible cytotoxicity, overcoming the inherent toxicity of the iridium(III) complex. Its long emission lifetime enabled its luminescence signal to be readily distinguished from the interfering fluorescence of organic dyes by using a time‐resolved technique. This probe selectively visualized living cancer cells via specific binding to GRPr, while it also modulated the function of GRPr on TNF‐α secretion in immune cells. To our knowledge, this is the first peptide‐conjugated iridium(III) complex developed as a GRPr bioimaging probe and modulator of GRPr activity. This theranostic agent shows great potential at unmasking the diverse roles of GRPr in living systems.     

Reversible Fluorescent Probes for Biological Redox States

The redox chemistry of the cell is key to its function and health, and the development of chemical tools to study redox biology is important. While fluxes in oxidative state are essential for healthy cell function, a chronically elevated oxidative capacity is linked to disease. It is therefore essential that probes of biological redox states distinguish between these two conditions by the reversible sensing of changes over time. In this review, we discuss the current progress towards such probes, and identify key directions for future research in this nascent field of vital biological interest.