Bioconjugation and Reaction-Induced Tumor Therapy via Alkynamide-Based Thiol-Yne Click Reaction.

Ferroptosis is associated with the occurrence and development of many diseases, which is the result of an imbalance in cellular metabolism and oxidation-reduction balance. Therefore, it is an effective therapeutic strategy that simultaneously regulating the intracellular oxidation-reduction system. Herein, a click reaction of alkynylamide with thiol groups in the presence of amine or in PBS (pH = 7.4) is developed, which can react efficiently with thiol substances, such as cysteine (Cys), glutathione (GSH), and bovine serum albumin (BSA). Notably, MBTB-PA, an aggregation-induced emission (AIE) photosensitizer with an alkynylamide unit, is synthesized and its intracellular behavior is visualized in situ by fluorescence imaging, demonstrating its excellent ability to target the endoplasmic reticulum. Furthermore, MBTB-PA reacted with proteins in tumor cells, consumed reducing substances, and triggered intracellular oxidative stress, resulting in cell death. Based on this reaction therapy strategy, click reaction is combined with photodynamic therapy to achieve effective killing of tumor cells by simultaneously raising the intracellular oxidative state and reducing the reductive state. This work not only develops an application of click reaction of alkynamide with thiol in bioconjugation and anti-tumor therapy, but also provides feasible ideas for organic reactions in the exploration of organisms.

Size Optimization of Organic Nanoparticles with Aggregation-Induced Emission Characteristics for Improved ROS Generation and Photodynamic Cancer Cell Ablation.

Aggregation-induced emission (AIE) fluorogens provide new opportunities to promote efficient reactive oxygen species (ROS) production in aggregates, which represent the promising candidates to construct theranostic nanoparticles for photodynamic therapy (PDT), but the size effect has been rarely explored. Herein, a universal method to fabricate organic nanoparticles with controllable sizes is reported and it demonstrates that ≈45 nm is the optimal size of AIE nanoparticles for PDT. Different from conventional Ce6 nanoparticles which show largely reduced fluorescence and ROS generation with increasing nanoparticle size, AIE nanoparticles show gradually enhanced brightness and ROS generation upon increasing the sizes from 6 to ≈45 nm. Further increasing sizes could continue to intensify the nanoparticle's brightness at the expense of ROS production, with the optimal size for ROS generation being achieved at ≈45 nm. Both 2D monolayer cell and 3D multicellular spheroid experiments confirm that 45 nm AIE nanoparticles have the highest cellular uptake, the deepest penetration depth, and the best photodynamic killing effect. Such a study not only manifests the advantages of AIE photosensitizers, but also delivers the optimal size ranging for efficient PDT, which shall provide an attractive paradigm to guide the development of phototheranostic nanoparticles besides molecular design to further promote PDT applications.

Effective Therapy of Drug-Resistant Bacterial Infection by Killing Planktonic Bacteria and Destructing Biofilms with Cationic Photosensitizer Based on Phosphindole Oxide.

Developing effective therapies to fight against biofilm-associated infection is extremely urgent. The complex environment of biofilm forces the bacteria to evade the elimination of antibiotics, resulting in recalcitrant chronic infections. To address this issue, a cationic antibacterial agent based on phosphindole oxide (ß-PM-PIO) is designed and prepared. The unique molecular structure endows ß-PM-PIO with aggregation-induced emission feature and efficient singlet oxygen generation ability. ß-PM-PIO shows excellent visual diagnostic function to planktonic bacteria and biofilm. In addition, owing to the synergistic effect of phototoxicity and dark toxicity, ß-PM-PIO can achieve superb antibacterial and antibiofilm performance against Gram-positive bacteria with less potential of developing drug resistance. Notably, ß-PM-PIO also holds excellent anti-infection capacity against drug-resistant bacteria in vivo with negligible side effects. This work offers a promising platform to develop advanced antibacterial agents against multidrug-resistant bacterial infection.

Recent Progress in Type I Aggregation-Induced Emission Photosensitizers for Photodynamic Therapy.

In modern medicine, precision diagnosis and treatment using optical materials, such as fluorescence/photoacoustic imaging-guided photodynamic therapy (PDT), are becoming increasingly popular. Photosensitizers (PSs) are the most important component of PDT. Different from conventional PSs with planar molecular structures, which are susceptible to quenching effects caused by aggregation, the distinct advantages of AIE fluorogens open up new avenues for the development of image-guided PDT with improved treatment accuracy and efficacy in practical applications. It is critical that as much of the energy absorbed by optical materials is dissipated into the pathways required to maximize biomedical applications as possible. Intersystem crossing (ISC) represents a key step during the energy conversion process that determines many fundamental optical properties, such as increasing the efficiency of reactive oxygen species (ROS) production from PSs, thus enhancing PDT efficacy. Although some review articles have summarized the accomplishments of various optical materials in imaging and therapeutics, few of them have focused on how to improve the phototherapeutic applications, especially PDT, by adjusting the ISC process of organic optics materials. In this review, we emphasize the latest advances in the reasonable design of AIE-active PSs with type I photochemical mechanism for anticancer or antibacterial applications based on ISC modulation, as well as discuss the future prospects and challenges of them. In order to maximize the anticancer or antibacterial effects of type I AIE PSs, it is the aim of this review to offer advice for their design with the best energy conversion.

Activatable Persistent Luminescence from Porphyrin Derivatives and Supramolecular Probes with Imaging-Modality Transformable Characteristics for Improved Biological Applications.

Persistent luminescence without excitation light and tissue autofluorescence interference holds great promise for biological applications, but is limited by available materials with long-wavelength emission and excellent clinical potential. Here, we report that porphyrin derivatives can emit near-infrared persistent luminescence over 60 min after cessation of excitation light or on interaction with peroxynitrite. A plausible mechanism of the successive oxidation of vinylene bonds was demonstrated. A supramolecular probe with a ß-sheet structure was constructed to enhance the tumor targeting ability and the photoacoustic and persistent luminescence signals. Such probes featuring light-triggered function transformation from photoacoustic imaging to persistent luminescence imaging permit advanced image-guided cancer surgery. Furthermore, peroxynitrite-activated persistent luminescence of the supramolecular probe also enables rapid and precise screening of immunogenic cell death drugs.

An Activatable Near-Infrared Afterglow Theranostic Prodrug with Self-Sustainable Magnification Effect of Immunogenic Cell Death.

Herein, we report an activatable near-infrared (NIR) afterglow theranostic prodrug that circumvents high background noise interference caused by external light excitation. The prodrug can release hydroxycamptothecin (HCPT) in response to the high intratumoral peroxynitrite level associated with immunogenic cell death (ICD), and synchronously activate afterglow signal to monitor the drug release process and cold-to-hot tumor transformation. The prodrug itself is an ICD inducer achieved by photodynamic therapy (PDT). PDT initiates ICD and recruits first-arrived neutrophils to secrete peroxynitrite to trigger HCPT release. Intriguingly, we demonstrate that HCPT can significantly amplify PDT-mediated ICD process. The prodrug thus shows a self-sustainable ICD magnification effect by establishing an "ICD-HCPT release-amplified ICD" cycling loop. In vivo studies demonstrate that the prodrug can eradicate existing tumors and prevent further tumor recurrence through antitumor immune response.

Design of superior phototheranostic agents guided by Jablonski diagrams.

Phototheranostics represents a promising direction for modern precision medicine, which has recently attracted great research interest from multidisciplinary research areas. Organic optical agents including small molecular fluorophores, semiconducting/conjugated polymers, aggregation-induced emission luminogens, etc. with tuneable photophysical properties, high biosafety and biocompatibility, facile processability and ease of functionalization have delivered encouraging performance in disease phototheranostics. This review summarizes the recent progress of organic phototheranostic agents with an emphasis on the main strategies to manipulate the three excitation energy dissipation pathways, namely, radiative decay, thermal deactivation, and intersystem crossing, with the assistance of a Jablonski diagram, which particularly showcases how the Jablonski diagram has been guiding the design of organic agents from molecule to aggregate levels to promote the disease phototheranostic outcomes. Molecular design and nanoengineering strategies to modulate photophysical processes of organic optical agents to convert the absorbed photons into fluorescent/phosphorescent/photoacoustic signals and/or photodynamic/photothermal curing effects for improved disease phototheranostics are elaborated. Noteworthily, adaptive phototheranostics with activatable and transformable functions on demand, and regulation of excitation such as chemiexcitation to promote the phototheranostic efficacies are also included. A brief summary with the discussion of current challenges and future perspectives in this research field is further presented.

Fast and High-Throughput Evaluation of Photodynamic Effect by Monitoring Specific Protein Oxidation with MALDI-TOF Mass Spectrometry.

In antibacterial practices by photodynamic treatment, bacteria are incubated with photosensitizers and then oxidized to death by generating reactive oxygen species (ROS) under light irradiation. Generally, Luria-Bertani (LB) agar colony is a conventional method to evaluate the photodynamic effect. However, this method is time consuming, easily disturbed by pollutants, and limited to the analysis of a pure bacteria sample. Herein, we introduce a novel method of photodynamic effect evaluation through in situ detection of specific protein oxidation by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) with only 1 µL of sample in a fast (less than 1 min per sample) and high-throughput (up to 384 samples per run) way. The oxidation rates of specific proteins stayed highly consistent with bactericidal rates and thus MALDI-TOF MS might be able to replace the LB agar colony to evaluate the photodynamic effect. With the present method, several experimental conditions including different photosensitizer types, dosage controls, and different illumination times were easily screened to optimize photodynamic effect. Photodynamic effects of various bacteria species, cancer cells, and even mixture samples were also evaluated. The results demonstrate the promising application of MALDI-TOF MS in evaluating the photodynamic effect of each component in a mixture sample without any separation or purification, which could not be achieved by the traditional LB agar colony method.

Aggregation-Induced Emission (AIE) Dots: Emerging Theranostic Nanolights.

Theranostic nanolights refer to luminescent nanoparticles possessing both imaging and therapeutic functions. Their shape, size, surface functions, and optical properties can be precisely manipulated through integrated efforts of chemistry, materials, and nanotechnology for customized applications. When localized photons are used to activate both imaging and therapeutic functions such as photodynamic or photothermal therapy, these theranostic nanolights increase treatment efficacy with minimized damage to surrounding healthy tissues, which represents a promising noninvasive nanomedicine as compared to conventional theranostic approaches. As one of the most promising theranostic nanolights, organic dots with aggregation-induced emission (AIE dots) are biocompatible nanoparticles with a dense core of AIE fluorogens (AIEgens) and protective shells, whose sizes are in the range of a few to tens of nanometers. Different from conventional fluorophores that suffer from aggregation-caused quenching (ACQ) due to π-π stacking interaction in the aggregate state, AIEgens emit strongly as nanoaggregates due to the restriction of intramolecular motions. Through precise molecular engineering, AIEgens could also be designed to show efficient photosensitizing or photothermal abilities in the aggregate state. Different from ACQ dyes, AIEgens allow high loading in nanoparticles without compromised performance, which makes them the ideal cores for theranostic nanolights to offer high brightness for imaging and strong photoactivities for theranostic applications. In this Account, we summarize the recent advance of AIE dots and highlight their great potential as theranostic nanolights in biomedical applications. Starting from the design of AIEgens, the fabrication of AIE dots and their bioimaging applications are discussed. The exceptional advantages of superbrightness, high resistance to photobleaching, lack of emission intermittency, and excellent biocompatibility have made them reliable cross platform contrast agents for different imaging techniques such as confocal microscopy, multiphoton fluorescence microscopy, super-resolution nanoscopy, and light-sheet ultramicroscopy, which have been successfully applied for cell tracking, vascular disease diagnosis, and image-guided surgery. The integration of therapeutic functions with customized AIEgens has further empowered AIE dots as an excellent theranostic platform for image-guided phototherapy. Of particular interest is AIE photosensitizer dots, which simultaneously show bright fluorescence and high photosensitization, yielding superior performance to commercial photosensitizer nanoparticles in image-guided therapy. Further development in multiphoton excited photodynamic therapy has offered precise treatment with up to 5 µm resolution at 200 µm depth, while chemiexcited photodynamic therapy has completely eliminated the limitation of penetration depth to realize power-free imaging and therapy. With this Account, we hope to stimulate more collaborative research interests from different fields of chemistry, materials, biology, and medicine to promote translational research of AIE dots as the theranostic nanolights.

Aggregation-Induced Emission Probe for Specific Turn-On Quantification of Soluble Transferrin Receptor: An Important Disease Marker for Iron Deficiency Anemia and Kidney Diseases.

Transferrin receptor (TfR) is overexpressed on the surface of many cancer cells due to its vital roles in iron circulation and cellular respiration. Soluble transferrin receptor (sTfR), a truncated extracellular form of TfR in serum, is an important marker of iron deficiency anemia (IDA) and bone marrow failure in cancer patients. More recently, sTfR level in urine has been related to a specific kidney disease of Henoch-Schönlein purpura nephritis (HSPN). Despite the universal significance of sTfR, there is still a lack of a simple and sensitive method for the quantification of sTfR. Furthermore, it is desirable to have a probe that can detect both TfR and sTfR for further comparison study. In this work, we developed a water-soluble AIE-peptide conjugate with aggregation-induced emission (AIE) characteristics. Taking advantage of the negligible emission from molecularly dissolved tetraphenylethene (TPE), probe TPE-2T7 was used for the light-up detection of sTfR. The probe itself is nonemissive in aqueous solution, but it turns on its fluorescence upon interaction with sTfR to yield a detection limit of 0.27 µg/mL, which is much lower than the sTfR level in IDA patients. Furthermore, a proof-of-concept experiment validates the potential of the probe for diagnosis of HSPN by urine test.

Multifunctional Conjugated Polymer Nanoparticles for Image-Guided Photodynamic and Photothermal Therapy.

A multifunctional theranostic platform based on conjugated polymer nanoparticles (CPNs) with tumor targeting, fluorescence detection, photodynamic therapy (PDT), and photothermal therapy (PTT) is developed for effective cancer imaging and therapy. Two conjugated polymers, poly[9,9-bis(2-(2-(2-methoxyethoxy)ethoxy)-ethyl)fluorenyldivinylene]-alt-4,7-(2,1,3-benzothiadiazole) with bright red emission and photosensitizing ability and poly[(4,4,9,9-tetrakis(4-(octyloxy)phenyl)-4,9-dihydro-s-indacenol-dithiophene-2,7-diyl)-alt-co-4,9-bis(thiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-thiadiazolo-quinoxaline] with strong near-infrared absorption and excellent photothermal conversion ability are co-loaded into one single CPN via encapsulation approach using lipid-polyethylene glycol as the matrix. The obtained co-loaded CPNs show sizes of around 30 nm with a high singlet oxygen quantum yield of 60.4% and an effective photothermal conversion efficiency of 47.6%. The CPN surface is further decorated with anti-HER2 affibody, which bestows the resultant anti-HER2-CPNs superior selectivity toward tumor cells with HER2 overexpression both in vitro and in vivo. Under light irradiation, the PDT and PTT show synergistic therapeutic efficacy, which provides new opportunities for the development of multifunctional biocompatible organic materials in cancer therapy.

Real-time naked-eye multiplex detection of toxins and bacteria using AIEgens with the assistance of graphene oxide.

Toxins and bacteria in water or food pose a threat to human life and could potentially be exploited for bioterrorism. Real-time naked-eye detection of these contaminants is highly desirable to provide a direct and simple analytical method and address the challenges of the existing strategies. Using the detection of ricin and B. subtilis as an example, a naked-eye multiplex detection model is established. In this work, a green fluorogen with aggregation-induced emission (AIE) characteristics was encapsulated in silica nanoshells. The resulting green AIE nanoparticles (NPs) were further functionalized with ricin binding aptamers (RBA), which were used together with graphene oxide (GO) to provide a fluorescence turn-on approach recognizable by naked eye for the specific sensing of ricin. The platform is compatible with a red emissive fluorescent light-up probe (AIE-2Van) for B. subtilis detection. The success of the multiplex is validated by different colours, that is, green for ricin and red for B. subtilis, which are clearly recognizable by naked eye in the same solution.

Antibacterial Narrow-Band-Gap Conjugated Oligoelectrolytes with High Photothermal Conversion Efficiency.

Two conjugated oligoelectrolytes (COEs), WMG1 and WMG2, were designed with the goal of achieving near infrared absorption and high photothermal conversion efficiency. Specifically, electron-rich thiophene and electron-poor benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole subunits were introduced into the conjugated core to modulate the optical gap and to reduce the fluorescence emission efficiency. WMG1 and WMG2 show absorption maxima at around 800 nm, which favors tissue penetration. Although relatively small in size, WMG1 and WMG2 exhibit photothermal conversion efficiencies of circa 60 % and 54 %, respectively. WMG1 shows dark toxicity to the Gram positive bacterium B. subtilis and good photothermal killing efficiency toward both B. subtilis and Gram negative E. coli, features that demonstrate the promising potential of the COE molecular design for photothermal applications.

Real-Time Specific Light-Up Sensing of Transferrin Receptor: Image-Guided Photodynamic Ablation of Cancer Cells through Controlled Cytomembrane Disintegration.

Transferrin receptor (TfR) represents a unique target for specific imaging of cancer cells and targeted delivery of therapeutic reagents. Detection and qualification of TfR is thus of great importance for cancer diagnosis and therapy. In this contribution, a light-up probe TPETH-2T7 was developed by conjugating a red-emissive photosensitizer with aggregation-induced emission (AIE) characteristics to a TfR-targeting peptide T7. The probe is almost nonemissive by itself, but it gives turn-on fluorescence in the presence of TfR with a detection limit of 0.45 µg/mL. Cellular experiments show that the probe specifically binds to TfR-overexpressed cancer cells. Real-time imaging results reveal that the probe stains the MDA-MB-231 cell membrane in 30 min, which is followed by probe internalization. Experiments on image-guided photodynamic cancer ablation show that the therapeutic performance is better when TPETH-2T7 is localized on the cell membrane as compared to that being internalized into cells. Confocal laser scanning microscopy (CLSM) study reveals that cytomembrane disintegration allows quick ablation of MDA-MB-231 cells.

Specific Light-Up Probe with Aggregation-Induced Emission for Facile Detection of Chymase.

Human chymases are important proteases abundant in mast cell granules. The elevated level of chymases and other serine proteases is closely related to inflammatory and immunoregulatory functions. Monitoring of the chymase level is very important, however, the existing methods remain limited and insufficient. In this work, a light-up probe of TPETH-2(CFTERD3) (where CFTERD is Cys-Phe-Thr-Glu-Arg-Asp) was developed for chymase detection. The probe has low fluorescent signal in aqueous media, but its solubility can be changed after hydrolysis by chymase, giving significant fluorescence turn-on with a high signal-to-noise (S/N) ratio. The probe has excellent selectivity to chymase compared to other proteins and can effectively differentiate chymase from other enzymes (e.g., chymotrypsin and trypsin) in the same family (E.C. 3.4.21). The detection limit is calculated to be 0.1 ng/mL in PBS buffer with a linear range of 0-9.0 ng/mL. A comparison study using TPETH-2(CFTERD2) as the probe reveals the importance of molecular design in realizing the high S/N ratio. TPETH-2(CFTERD3) thus represents a simple turn-on probe for chymase detection, with real-time and direct readout and also excellent sensitivity and selectivity.

Multifunctional AIEgens for Future Theranostics.

The combination of diagnosis and therapeutics into one theranostics system has attracted great interest in life science and biomedical fields. The current theranostic platform largely relies on the integration of multiple materials with different functionalities. The all-in-one approach has the risk of high complicity with reduced reproducibility. Smart design of simple molecules born with multifunctions should represent one of the future directions in theranostics. Fluorogens with aggregation-induced emission (AIEgens) are one type of such smart materials, which have attracted increasing attentions in recent years. In this concept, the key frontier developments of simple AIEgens with multifunctions for imaging and therapy are presented, which include fluorescence-photoacoustic imaging, fluorescence-magnetic resonance imaging, fluorescence image-guided photodynamic therapy, fluorescence image-guided chemotherapy and photoacoustic image-guided photothermal therapy. The smart molecular design to endow each AIEgen with strong capability to simultaneously offer two or more theranostic functions should attract more scientists into this exciting research direction.

A Porphyrin-Based Conjugated Polymer for Highly Efficient In Vitro and In Vivo Photothermal Therapy.

Conjugated polymers have been increasingly studied for photothermal therapy (PTT) because of their merits including large absorption coefficient, facile tuning of exciton energy dissipation through nonradiative decay, and good therapeutic efficacy. The high photothermal conversion efficiency (PCE) is the key to realize efficient PTT. Herein, a donor-acceptor (D-A) structured porphyrin-containing conjugated polymer (PorCP) is reported for efficient PTT in vitro and in vivo. The D-A structure introduces intramolecular charge transfer along the backbone, resulting in redshifted Q band, broadened absorption, and increased extinction coefficient as compared to the state-of-art porphyrin-based photothermal reagent. Through nanoencapsulation, the dense packing of a large number of PorCP molecules in a single nanoparticle (NP) leads to favorable nonradiative decay, good photostability, and high extinction coefficient of 4.23 × 104 m-1 cm-1 at 800 nm based on porphyrin molar concentration and the highest PCE of 63.8% among conjugated polymer NPs. With the aid of coloaded fluorescent conjugated polymer, the cellular uptake and distribution of the PorCP in vitro can be clearly visualized, which also shows effective photothermal tumor ablation in vitro and in vivo. This research indicates a new design route of conjugated polymer-based photothermal therapeutic materials for potential personalized theranostic nanomedicine.

Silole-Based Red Fluorescent Organic Dots for Bright Two-Photon Fluorescence In vitro Cell and In vivo Blood Vessel Imaging.

Robust luminescent dyes with efficient two-photon fluorescence are highly desirable for biological imaging applications, but those suitable for organic dots fabrication are still rare because of aggregation-caused quenching. In this work, a red fluorescent silole, 2,5-bis[5-(dimesitylboranyl)thiophen-2-yl]-1-methyl-1,3,4-triphenylsilole ((MesB)2 DTTPS), is synthesized and characterized. (MesB)2 DTTPS exhibits enhanced fluorescence efficiency in nanoaggregates, indicative of aggregation-enhanced emission (AEE). The organic dots fabricated by encapsulating (MesB)2 DTTPS within lipid-PEG show red fluorescence peaking at 598 nm and a high fluorescence quantum yield of 32%. Upon excitation at 820 nm, the dots show a large two-photon absorption cross section of 3.43 × 10(5) GM, which yields a two-photon action cross section of 1.09 × 10(5) GM. These (MesB)2 DTTPS dots show good biocompatibility and are successfully applied to one-photon and two-photon fluorescence imaging of MCF-7 cells and two-photon in vivo visualization of the blood vascular of mouse muscle in a high-contrast and noninvasive manner. Moreover, the 3D blood vasculature located at the mouse ear skin with a depth of over 100 µm can also be visualized clearly, providing the spatiotemporal information about the whole blood vascular network.

Structure-Dependent cis/trans Isomerization of Tetraphenylethene Derivatives: Consequences for Aggregation-Induced Emission.

The isomerization and optical properties of the cis and trans isomers of tetraphenylethene (TPE) derivatives with aggregation-induced emission (AIEgens) have been sparsely explored. We have now observed the tautomerization-induced isomerization of a hydroxy-substituted derivative, TPETH-OH, under acidic but not under basic conditions. Replacing the proton of the hydroxy group in TPETH-OH with an alkyl group leads to the formation of TPETH-MAL, for which the pure cis and trans isomers were obtained and characterized by HPLC analysis and NMR spectroscopy. Importantly, cis-TPETH-MAL emits yellow fluorescence in DMSO at -20 °C whereas trans-TPETH-MAL shows red fluorescence under the same conditions. Moreover, the geometry of cis- and trans-TPETH-MAL remains unchanged when they undergo thiol-ene reactions to form cis- and trans-TPETH-cRGD, respectively. Collectively, our findings improve our fundamental understanding of the cis/trans isomerization and photophysical properties of TPE derivatives, which will guide further AIEgen design for various applications.

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.