A Multifunctional Nanomicelle for Real‐Time Targeted Imaging and Precise Near‐Infrared Cancer Therapy

Simultaneous targeted cancer imaging, therapy and real‐time therapeutic monitoring can prevent over‐ or undertreatment. This work describes the design of a multifunctional nanomicelle for recognition and precise near‐infrared (NIR) cancer therapy. The nanomicelle encapsulates a new pH‐activatable fluorescent probe and a robust NIR photosensitizer, R16FP, and is functionalized with a newly screened cancer‐specific aptamer for targeting viable cancer cells. The fluorescent probe can light up the lysosomes for real‐time imaging. Upon NIR irradiation, R16FP‐mediated generation of reactive oxygen species causes lysosomal destruction and subsequently trigger lysosomal cell death. Meanwhile the fluorescent probe can reflect the cellular status and in situ visualize the treatment process. This protocol can provide molecular information for precise therapy and therapeutic monitoring.     

A FRET Probe for Cell‐Based Imaging of Ganglioside‐Processing Enzyme Activity and High‐Throughput Screening

Gangliosides are important signaling molecules in the cell membrane and are processed by several enzymes. Deficiencies in these enzymes can cause human lysosomal storage diseases. Building an understanding of the pathways of glycosphingolipid catabolism requires methods for the analysis of these enzymatic activities A GM3‐derived FRET probe was synthesized chemoenzymatically for the detection and quantitation of a range of ganglioside‐degrading enzymes, both in cell lysates and in living cells. This is the first substrate that enables the ratiometric fluorogenic assay of sphingolipid ceramide N‐deacylase and endoglycoceramidase and can detect and localize neuraminidase activity in living cells. It is therefore a valuable tool for building a better understanding of membrane‐confined enzymology. It also enables the robust and reliable assay of ganglioside‐degrading enzymes in a microtiter plate, thus opening the door to screening for novel or engineered biocatalysts or for new inhibitors.     

A Lysosome‐Compatible Near‐Infrared Fluorescent Probe for Targeted Monitoring of Nitric Oxide

The requirement for nitric oxide (NO) of lysosomes has motivated the development of a sophisticated fluorescent probe to monitor the distribution of this important biomolecule at the subcellular level in living cells. A near‐infrared (NIR) fluorescent Si‐rhodamine (SiRB)‐NO probe was designed based on the NO‐induced ring‐opening process of Si‐rhodamine. The probe exhibits fast chromogenic and fluorogenic responses, and high sensitivity and selectivity toward trace amounts of NO. Significantly, the spirolactam in Si‐rhodamine exhibits very good tolerance to H⁺, which in turn brings extremely low background fluorescence not only in the physiological environment but also under acidic conditions. The stability of the highly fluorescent product in acidic solution provides persistent fluorescence emission for long‐term imaging experiments. To achieve targeted imaging with improved spatial resolution and sensitivity, an efficient lysosome‐targeting moiety was conjugated to a SiRB‐NO probe, affording a tailored lysosome‐targeting NIR fluorescent Lyso‐SiRB‐NO probe. Inheriting the key advantages of its parent SiRB‐NO probe, Lyso‐SiRB‐NO is a functional probe that is suited for monitoring lysosomal NO with excellent lysosome compatibility. Imaging experiments demonstrated the monitoring of both exogenous and endogenous NO in real time by using the Lyso‐SiRB‐NO probe.     

Environment‐sensitive Fluorescent Turn‐on Chemical Probe for the Specific Detection of O‐Methylguanine‐DNA Methyltransferase (MGMT) in Living Cells

MGMT protein, which has been associated with resistance to antitumor alkylation drugs for many patients, is a very useful prognostic marker to provide a guide for therapeutic decisions. Considering the large number of cellular samples that have to be handled daily at the hospitals, it is thus important to develop a rapid and simple analytical method to distinguish MGMT activity in different types of cells. In this paper, we describe a MGMT‐activated fluorescence turn‐on probe for the rapid no‐wash imaging of MGMT in living cells. The probe consists of a specific MGMT suicide pseudosubstrate, O⁶‐benzyl‐guanine and an environment‐sensitive fluorophore, SBD. In the presence of MGMT, the enzyme transfers SBD to the protein active site where the hydrophobic surrounding causes the fluorophore to exhibit more than 50‐fold fluorescence enhancement. With this probe, bright fluorescence was observed for MGMT‐positive, Hela S3 and MCF‐7 cells, while MGMT‐deficient CHO cells displayed no fluorescence. We believe that this fluorescence activation probe design can also be extended to detect other transferases, for which there are still no effective methods to image them in living cells.     

Cysteine‐Mediated Intracellular Building of Luciferin to Enhance Probe Retention and Fluorescence Turn‐On

The development of sensitive and selective small molecular probes that enable real‐time detection of endogenous cysteine (Cys) has become an attractive topic because of the essential roles played by Cys in controlling the cellular nitrogen balance and in maintaining biological redox homeostasis. Herein, we report a Cys‐specific probe, 2‐cyanobenzothiazol‐6‐yl acrylate (CBTOA), that shows not only fluorescence turn‐on for sensitive detection of endogenous Cys but also enhanced probe retention inside cells for real‐time monitoring of Cys levels upon external stimulation. Cys‐mediated intracellular formation of luciferin from CBTOA was the key strategy leading to this new type of fluorogenic probe. CBTOA showed fast response to Cys in living cells and liver tissue slices with high sensitivity and selectivity. By using CBTOA as a real‐time probe, we were able to monitor the change in Cys levels in living HeLa cells under ROS‐induced oxidative stress as well as in human mesenchymal stem cells during adipogenic differentiation.     

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.     

Detection of LacZ‐Positive Cells in Living Tissue with Single‐Cell Resolution

The LacZ gene, which encodes Escherichia coli β‐galactosidase, is widely used as a marker for cells with targeted gene expression or disruption. However, it has been difficult to detect lacZ‐positive cells in living organisms or tissues at single‐cell resolution, limiting the utility of existing lacZ reporters. Herein we present a newly developed fluorogenic β‐galactosidase substrate suitable for labeling live cells in culture, as well as in living tissues. This precisely functionalized fluorescent probe exhibited dramatic activation of fluorescence upon reaction with the enzyme, remained inside cells by anchoring itself to intracellular proteins, and provided single‐cell resolution. Neurons labeled with this probe preserved spontaneous firing, which was enhanced by application of ligands of receptors expressed in the cells, suggesting that this probe would be applicable to investigate functions of targeted cells in living tissues and organisms.     

Bioorthogonal Conjugation Directed by a Sugar‐Sorting Pathway for Continual Tracking of Stressed Organelles

Selective and continuous tracking of dynamic organelles is crucial for modern biology. We herein report a ship‐in‐a‐bottle strategy for tagging lysosomes by a strain‐promoted azide–alkyne cycloaddition to couple a pH sensor (RC) with mannose‐6‐carboxylate (M6C) actively transported into lysosomes through cell sorting. In contrast to classical acidotropic sensors, which are prone to dissipate from lysosomes, M6C‐RC formed in situ is stably trapped in lysosomes without resort to lysosomal acidity and exhibits “always‐on” blue fluorescence to pinpoint lysosomes and red‐to‐blue fluorescence ratios indicative of the lysosomal pH value. These advantages enable tracking of stressed lysosomes, and necrosis to be differentiated from apoptosis on the basis of lysosomal pH changes. The cell‐sorting‐mediated bioorthogonal tagging strategy offers a new route to track stressed organelles with disrupted physiological organelle–probe affinity.     

A Ratiometric Two‐Photon Fluorescent Probe for Tracking Lysosomal ATP: Direct In Cellulo Observation of Lysosomal Membrane Fusion Processes

Vesicles exchange their contents through membrane fusion processes, kiss‐and‐run and full‐collapse fusion. Indirect observation of these fusion processes using artificial vesicles enhanced our understanding on the molecular mechanisms involved. Direct observation of the fusion processes in a real biological system, however, remains a challenge owing to many technical obstacles. We report a ratiometric two‐photon probe offering real‐time tracking of lysosomal ATP with quantitative information for the first time. By applying the probe to two‐photon live‐cell imaging, the lysosomal membrane fusion process in cells has been directly observed and the concentration of its content, lysosomal ATP, has been measured. Results show that the kiss‐and‐run process between lysosomes proceeds through repeated transient interactions with gradual content mixing, whereas the full‐fusion process occurs at once. Furthermore, it is confirmed that both the fusion processes proceed with conservation of the content. Such a small‐molecule probe exerts minimal disturbance and hence has potential for studying various biological processes associated with lysosomal ATP.     

Organelle‐Specific Activity‐Based Protein Profiling in Living Cells

A multimodal activity‐based probe for targeting acidic organelles was developed to measure subcellular native enzymatic activity in cells by fluorescence microscopy and mass spectrometry. A cathepsin‐reactive warhead conjugated to a weakly basic amine and a clickable alkyne, for subsequent appendage of a fluorophore or biotin reporter tag, accumulated in lysosomes as observed by structured illumination microscopy (SIM) in J774 mouse macrophage cells. Analysis of in vivo labeled J774 cells by mass spectrometry showed that the probe was very selective for cathepsins B and Z, two lysosomal cysteine proteases. Analysis of starvation‐induced autophagy, a catabolic pathway involving lysosomes, showed a large increase in the number of tagged proteins and an increase in cathepsin activity. The organelle‐targeting of activity‐based probes holds great promise for the characterization of enzyme activities in the myriad diseases linked to specific subcellular locations, particularly the lysosome.     

Fluorescent Detection of Hypochlorous Acid from Turn‐On to FRET‐Based Ratiometry by a HOCl‐Mediated Cyclization Reaction

Hypochlorous acid (HOCl), a reactive oxygen species (ROS), plays a significant biological role in living systems. However, abnormal levels of HOCl are implicated in many inflammation‐associated diseases. Therefore, the detection of HOCl is of great importance. In this work, we describe the HOCl‐promoted cyclization of rhodamine‐thiosemicarbazides to rhodamine‐oxadiazoles, which is then exploited as a novel design strategy for the development of a new fluorescence turn‐on HOCl probe 2 . On the basis of the fluorescence resonance energy transfer (FRET) signaling mechanism, 2 was further converted into 1 a and 1 b , which represent the first paradigm of FRET‐based ratiometric fluorescent HOCl probes. The outstanding features of 1 a and 1 b include well‐resolved emission peaks, high sensitivity, high selectivity, good functionality at physiological pH, rapid response, low cytotoxicity, and good cell‐membrane permeability. Furthermore, these excellent attributes enable us to demonstrate, for the first time, the ratiometric imaging of endogenously produced HOCl in living cells by using these novel ratiometric probes. We expect that 1 a and 1 b will be useful molecular tools for studies of HOCl biology. In addition, the HOCl‐promoted cyclization reaction of rhodamine‐thiosemicarbazides to rhodamine‐oxadiazoles should be widely applicable for the development of different types of fluorescent HOCl probes.     

Visible Light‐Triggered Photoswitchable Diarylethene‐Based Iridium(III) Complexes for Imaging Living Cells

A novel diarylethene‐based iridium(III) complex was synthesized as a phosphorescence probe for monitoring living cells. The switchable phosphorescence complex in solution and within living cells was controlled by two distinguishable visible‐light irradiations, which suggests that this complex can be developed as a promising probe with weak photodamage for biological samples.     

Photo‐induced and Rapid Labeling of Tetrazine‐Bearing Proteins via Cyclopropenone‐Caged Bicyclononynes

Inverse electron‐demand Diels–Alder cycloadditions (iEDDAC) between tetrazines and strained alkenes/alkynes have emerged as essential tools for studying and manipulating biomolecules. A light‐triggered version of iEDDAC (photo‐iEDDAC) is presented that confers spatio‐temporal control to bioorthogonal labeling in vitro and in cellulo. A cyclopropenone‐caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo‐DMBO) was designed that is unreactive towards tetrazines before light‐activation, but engages in iEDDAC after irradiation at 365 nm. Aminoacyl tRNA synthetase/tRNA pairs were discovered for efficient site‐specific incorporation of tetrazine‐containing amino acids into proteins in living cells. In situ light activation of photo‐DMBO conjugates allows labeling of tetrazine‐modified proteins in living E. coli. This allows proteins in living cells to be modified in a spatio‐temporally controlled manner and may be extended to photo‐induced and site‐specific protein labeling in animals.     

Reinventing Hsp90 Inhibitors: Blocking C‐Terminal Binding Events to Hsp90 by Using Dimerized Inhibitors

Heat shock protein 90 (Hsp90) is a molecular chaperone (90 kDa) that functions as a dimer. This protein facilitates the folding, assembly, and stabilization of more than 400 proteins that are responsible for cancer development and progression. Inhibiting Hsp90’s function will shut down multiple cancer‐driven pathways simultaneously because oncogenic clients rely heavily on Hsp90, which makes this chaperone a promising anticancer target. Classical inhibitors that block the binding of adenine triphosphate (ATP) to the N‐terminus of Hsp90 are highly toxic to cells and trigger a resistance mechanism within cells. This resistance mechanism comprises a large increase in prosurvival proteins, namely, heat shock protein 70 (Hsp70), heat shock protein 27 (Hsp27), and heat shock factor 1 (HSF‐1). Molecules that modulate the C‐terminus of Hsp90 are effective at inducing cancer‐cell death without activating the resistance mechanism. Herein, we describe the design, synthesis, and biological binding affinity for a series of dimerized C‐terminal Hsp90 modulators. We show that dimers of these C‐terminal modulators synergistically inhibit Hsp90 relative to monomers.     

A Native‐Chemical‐Ligation‐Mechanism‐Based Ratiometric Fluorescent Probe for Aminothiols

Thiol‐containing amino acids (aminothiols) such as cysteine (Cys) and homocysteine (Hcy) play a key role in various biological processes including maintaining the homeostasis of biological thiols. However, abnormal levels of aminothiols are associated with a variety of diseases. The native chemical ligation (NCL) reaction has attracted great attention in the fields of chemistry and biology. NCL of peptide segments involves cascade reactions between a peptide‐α‐thioester and an N‐terminal cysteine peptide. In this work, we employed the NCL reaction mechanism to formulate a Förster resonance energy transfer (FRET) strategy for the design of ratiometric fluorescent probes that were selective toward aminothiols. On the basis of this new strategy, the ratiometric fluorescent probe 1 for aminothiols was judiciously designed. The new probe is highly selective toward aminothiols over other thiols and exhibits a very large variation (up to 160‐fold) in its fluorescence ratio (I₄₅₈/I₆₀₃). The new fluorescent probe is capable of ratiometric detection of aminothiols in newborn calf and human serum samples and is also suitable for ratiometric fluorescent imaging of aminothiols in living cells.     

Hyaluronic Acid/Poly(β‐Amino Ester) Polymer Nanogels for Cancer‐Cell‐Specific NIR Fluorescence Switch

Cancer‐cell‐specific pH‐activatable polymer nanogels consisting of CD44‐receptor‐targeting hyaluronic acid (HA), pH‐sensitive poly(β‐amino ester) (PBAE), and near‐infrared (NIR) fluorescent indocyanine green (ICG) were synthesized and used to detect cancer cells. The HA/PBAE/ICG‐polymer‐nanogel‐based NIR probe was nonfluorescent outside of tumor cells. After internalization by CD44‐receptor‐mediated endocytosis, the probe accumulated in the late endosomes or lysosomes where the acidic pH solubilized the PBAE and caused instant disassembly of the polymer nanogel. During endosomal maturation, the encapsulated ICG was released from its quenched state, inducing strong NIR fluorescence recovery. The nanogels generate a highly tumor‐specific NIR signal with a reduced background signal.     

Catalytic Molecular Imaging of MicroRNA in Living Cells by DNA‐Programmed Nanoparticle Disassembly

Molecular imaging is an essential tool for disease diagnostics and treatment. Direct imaging of low‐abundance nucleic acids in living cells remains challenging because of the relatively low sensitivity and insufficient signal‐to‐background ratio of conventional molecular imaging probes. Herein, we report a class of DNA‐templated gold nanoparticle (GNP)–quantum dot (QD) assembly‐based probes for catalytic imaging of cancer‐related microRNAs (miRNA) in living cells with signal amplification capacity. We show that a single miRNA molecule could catalyze the disassembly of multiple QDs with the GNP through a DNA‐programmed thermodynamically driven entropy gain process, yielding significantly amplified QD photoluminescence (PL) for miRNA imaging. By combining the robust PL of QDs with the catalytic amplification strategy, three orders of magnitude improvement in detection sensitivity is achieved in comparison with non‐catalytic imaging probe, which enables facile and accurate differentiation between cancer cells and normal cells by miRNA imaging in living cells.     

Green Synthesis of Red‐Emitting Carbon Nanodots as a Novel “Turn‐on” Nanothermometer in Living Cells

Temperature measurements in biology and medical diagnostics, along with sensitive temperature probing of living cells, is of great importance; however, it still faces significant challenges. Herein, a novel “turn‐on” carbon‐dot‐based fluorescent nanothermometry device for spatially resolved temperature measurements in living cells is presented. The carbon nanodots (CNDs) are prepared by a green microwave‐assisted method and exhibit red fluorescence (λ_em=615 nm) with high quantum yields (15 %). Then, an on–off fluorescent probe is prepared for detecting glutathione (GSH) based on aggregation‐induced fluorescence quenching. Interestingly, the quenched fluorescence could be recovered by increasing temperature and the CNDs–GSH mixture could behave as an off–on fluorescent probe for temperature. Thus, red‐emitting CNDs can be utilized for “turn‐on” fluorescent nanothermometry through the fluorescence quenching and recovery processes, respectively. We employ MC3T3‐E1 cells as an example model to demonstrate the red‐emitting CNDs can function as “non‐contact” tools for the accurate measurement of temperature and its gradient inside a living cell.     

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.     

GRP78‐Targeted HPMA Copolymer‐Photosensitizer Conjugate for Hyperthermia‐Induced Enhanced Uptake and Cytotoxicity in MCF‐7 Breast Cancer Cells

Photodynamic therapy (PDT) is a promising cancer treatment approach. However, the photosensitizers (PS) used for PDT are often limited by their poor solubility and selectivity for tumors. The goal of this study is to improve water solubility and delivery of the photosensitizer 2‐[1‐hexyloxyethyl]‐2‐divinyl pyropheophorbide‐a (HPPH) to breast cancer cells. An N‐(2‐hydroxypropyl)methacrylamide (HPMA) copolymer–HPPH photosensitizer conjugate is synthesized with heat shock receptor glucose‐regulated protein 78 (GRP78), targeting to GRP78 receptors of MCF‐7 cells, which are upregulated under mild hyperthermia. It is found that the uptake of the GRP78 targeted pep‐HPMA‐HPPH copolymer conjugate in MCF‐7 cells is improved through heat induction. Under mild hyperthermia the targeted copolymers are more effective compared to free HPPH. These results show potential for the utility of mild hyperthermia and copolymer delivery vehicles to enhance the efficacy of photodynamic therapy.