The real-time monitoring of reactive oxygen species (ROS, particularly singlet oxygen) generation during photodynamic therapy is a great challenge due to the extremely short half-life and small radius of action. To tackle this issue, we herein report a bioprobe composed of a red emissive photosensitizer (PS) with aggregation-induced emission (AIE) characteristics and a fluorogenic green emissive rhodol dye conjugated via a singlet oxygen cleavable aminoacrylate (AA) linker. The probe emits red fluorescence in water, and the red emissive PS can be used for probe self-tracking. Upon image-guided light irradiation, the generated singlet oxygen cleaves the AA linker to yield green fluorescence turn-on of rhodol, which offers real-time and in situ monitoring of singlet oxygen generation during photodynamic ablation of cancer cells, providing a strategy for the early evaluation of the therapeutic effect.

The emergence of drug-resistant bacterial pathogens highlights an urgent need for new therapeutic options. Photodynamic therapy (PDT) has emerged as a potential alternative to antibiotics to kill bacteria, which has been used in clinical settings. PDT employs photosensitizers (PSs), light, and oxygen to kill bacteria by generating highly reactive oxygen species (ROS). PDT can target both external and internal structures of bacteria, which does not really require the PSs to enter bacteria. Therefore, bacteria can hardly develop resistance to PDT. However, most of the PSs reported so far are hydrophobic and tend to form aggregates when they interact with bacteria. The aggregation could cause fluorescence quenching and reduce ROS generation, which generally compromises the effects of both imaging and therapy. In this contribution, we report on a Zn(II)-tetradentate-coordinated red-emissive probe with aggregation-induced emission characterization. The probe could selectively image bacteria over mammalian cells. Moreover, the probe shows potent phototoxicity to both Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Bacillus subtilis).

Subcellular targeted cancer therapy and in situ monitoring of therapeutic effect are highly desirable for clinical applications. Herein, we report a series of probes by conjugating zero (TPECM-2Br), one (TPECM-1TPP) and two (TPECM-2TPP) triphenylphosphine (TPP) ligands to a fluorogen with aggregation-induced emission (AIE) characteristics. The probes are almost non-emissive as molecularly dissolved species, but they can light up in cell cytoplasm or mitochondria. TPECM-2TPP is found to be able to target mitochondria, depolarize mitochondria membrane potential and selectively exert potent chemo-cytotoxicity on cancer cells. Furthermore, it can efficiently generate singlet oxygen with strong photo-toxicity upon light illumination, which further enhances its anti-cancer effect. On the other hand, TPECM-1TPP can also target mitochondria and generate singlet oxygen to trigger cancer cell apoptosis, but it shows low cytotoxicity in dark. Meanwhile, TPECM-1TPP can report the cellular oxidative stress by visualizing the morphological changes of mitochondria. However, TPECM-2Br does not target mitochondria and shows no obvious anticancer effect either in dark or under light illumination. This study thus highlights the importance of molecular probe design, which yields a new generation of subcellular targeted molecular theranostic agents with multi-function, such as cancer cell imaging, chemotherapy, photodynamic therapy, and in situ monitoring of the therapeutic effect in one go.

Direct monitoring of multiple enzyme activities in a given biological process is extremely important for disease diagnosis. Herein, we report a single fluorescent probe that targets two caspase activities in living cells. The probe consists of three parts that includes two AIE fluorogens with distinctive green and red emission colors excitable at a single wavelength, and a hydrophilic peptide as the substrate of the apoptosis initiator caspase-8 and the effector caspase-3. The probe is non-fluorescent in aqueous media. The green and red fluorescence can be sequentially turned on when the peptide substrate is cleaved by the cascade activation of caspase-8 and caspase-3 in early apoptotic HeLa cells induced by hydrogen peroxide. This sequential fluorescence turn-on allows real-time monitoring of the caspase cascade activation during the apoptotic process, which was further explored for evaluating the therapeutic efficiency of anticancer drugs. The probe design strategy developed in this study also proved to be general, which opens a new avenue for real-time, multiplexed imaging of cellular enzyme activity in a biological process.

The efficiency of the intersystem crossing process can be improved by reducing the energy gap between the singlet and triplet excited states (ΔE_ST), which offers the opportunity to improve the yield of the triplet excited state. Herein, we demonstrate that modulation of the excited states is also an effective strategy to regulate the singlet oxygen generation of photosensitizers. Based on our previous studies that photosensitizers with aggregation-induced emission characteristics (AIE) showed enhanced fluorescence and efficient singlet oxygen production in the aggregated state, a series of AIE fluorogens such as TPDC, TPPDC and PPDC were synthesized, which showed ΔE_ST values of 0.48, 0.35 and 0.27 eV, respectively. A detailed study revealed that PPDC exhibited the highest singlet oxygen efficiency (0.89) as nanoaggregates, while TPDC exhibited the lowest efficiency (0.28), inversely correlated with their ΔE_ST values. Due to their similar optical properties, TPDC and PPDC were further encapsulated into nanoparticles (NPs). Subsequent surface modification with cell penetrating peptide (TAT) yielded TAT-TPDC NPs and TAT-PPDC NPs. As a result of the stronger singlet oxygen generation, TAT-PPDC NPs showed enhanced cancer cell ablation as compared to TAT-TPDC NPs. Fine-tuning of the singlet-triplet energy gap is thus proven to be an effective new strategy to generate efficient photosensitizers for photodynamic therapy.

To date, the exact targets and mechanism of action of curcumin, a natural product with anti-inflammatory and anti-cancer properties, remain elusive. Here we synthesized a cell permeable curcumin probe (Cur-P) with an alkyne moiety, which can be tagged with biotin for affinity enrichment, or with a fluorescent dye for visualization of the direct-binding protein targets of curcumin in situ. iTRAQ(TM) quantitative proteomics approach was applied to distinguish the specific binding targets from the non-specific ones. In total, 197 proteins were confidently identified as curcumin binding targets from HCT116 colon cancer cell line. Gene Ontology analysis showed that the targets are broadly distributed and enriched in the nucleus, mitochondria and plasma membrane, and they are involved in various biological functions including metabolic process, regulation, response to stimulus and cellular process. Ingenuity Pathway Analysis(TM) (IPA) suggested that curcumin may exert its anticancer effects over multiple critical biological pathways including the EIF2, eIF4/p70S6K, mTOR signaling and mitochondrial dysfunction pathways. Functional validations confirmed that curcumin downregulates cellular protein synthesis, and induces autophagy, lysosomal activation and increased ROS production, thus leading to cell death.