Artemisinin and AIEgen Conjugate for Mitochondria-Targeted and

Subscriber access provided by Oregon Health & Science University Library

Biological and Medical Applications of Materials and Interfaces

Artemisinin and AIEgen Conjugate for Mitochondria-Targeted and Image-Guided Chemo- and Photodynamic Cancer Cell Ablation Guangxue Feng, Jie Liu, Chong-Jing Zhang, and Bin Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b01960 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Artemisinin and AIEgen Conjugate for Mitochondria-Targeted and Image-Guided Chemoand Photodynamic Cancer Cell Ablation Guangxue Feng,† Jie Liu,† Chong-Jing Zhang†‡* and Bin Liu†* †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, Singapore 117585, Singapore ‡

State Key Laboratory of Bioactive Substances and Functions of Natural Medicines and Beijing

Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of Materia Medica, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China 100050, China KEYWORDS: Artemisinin, mitochondrial targeting, fluorescence imaging, photodynamic therapy, combination cancer therapy

ABSTRACT Cell organelle targeting is a promising approach for cancer therapy. We herein report a light-up probe (TPETH-Mito-1ART) to co-deliver artemisinin (ART) and an AIE photosensitizer (PS) to cancer cell mitochondria for image-guided combination cancer cell ablation. This probe contains an AIEgen core of TPETH, two mitochondria targeting arms with ART on one arm, which show

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 23

high specificity towards cancer cells over normal ones, predominant accumulation and fluorescence turn-on in mitochondria. The fresh heme produced in mitochondria quickly activates ART, and the direct generation of reactive oxygen species at mitochondria promotes photodynamic therapy (PDT) performance. The corporation of ART and PDT leads to largely improved cancer cell ablation efficacy with a synergistic effect, which could quickly depolarize mitochondrial membrane, and largely reduce cancer migration activity. This co-delivery strategy provides great potentials for subcellular organelle-targeted and image-guided combination cancer cell ablation.

Introduction As a natural product isolated from an annual wormwood (Artemisia annua), Artemisinin (ART) is well recognized as the antimalarial reagents.1-2 Recently, ART analogues were reported to possess anticancer properties, where they could inhibit cell proliferation and angiogenesis, increase cellular oxidative stress and induce apoptosis.3-6 However, they only showed low and limited lesion towards certain cancer cells.7 Very recently, through a mode of action study, we demonstrated that heme is able to activate ART to promiscuously target multiple proteins to kill parasite and cancer cells,8 and that delivering ART to cancer cell mitochondria could largely improve the therapeutic efficacy.9 However, ART itself is not emissive, it becomes vitally important to design fluorescent ART analogues to offer real-time monitoring of the subcellular localization without affecting their activities.10 Design of fluorescent ART analogues with mitochondria targeting ability therefore is highly desirable for theranostic cancer treatment. On the other hand, several fluorophores are reported to generate toxic reactive oxygen species (ROS) to precisely kill cancer cells under localized light irradiation, which is known as


2

Page 3 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


photodynamic therapy (PDT).11-14 Benefiting from the short lifetime and radius of action of ROS, PDT shows minimal damage to surrounding health tissues, which, however, leads to low therapeutic efficiency when they are randomly localized inside cancer cells.15-16 As the primary target of ROS, the dysfunction of mitochondria induces cell apoptosis during the early stage of PDT.17 Delivery of photosensitizers (PSs) to mitochondria thus represents an efficient strategy to improve PDT efficacy.18-19 However, the overall PDT performance is yet unsatisfactory. One reason is that conventional PSs tend to form aggregates, which usually leads to quenched fluorescence and reduced ROS production due to π-π stacking interaction.20 Moreover, the generation of anti-apoptotic factors such as NF-κB during PDT also plays a negative role.21 Since ART analogues have been reported to inhibit the generation of anti-apoptotic factors,22-23 it holds great promise to co-deliver effective PSs and ART to mitochondria for combination cancer cell ablation. Recently, a novel class of fluorogens with aggregation-induced emission characteristics (AIE) has demonstrated great performance and potentials in biological applications.24-26 These AIE fluorogens (AIEgens) are almost not emissive as isolated molecules but show largely intensified fluorescence in solid state or aggregates.27-28 The unique features of AIEgens facilitate light-up probe design via specific targeting and binding for biosensing and bioimaging with high signal-to-noise ratios.29-30 Very recently, some AIEgens are designed to possess ROS production, which demonstrate efficient photosensitizing ability in aggregate state.31-33 This, together with their unique light-up feature and facile chemical modification, makes AIEgens an ideal candidate for the design of subcellular-targeted delivery platforms with high signal-to-noise ratios and largely improved PDT performance.34-36


3


Page 4 of 23

Herein, we designed a light-up probe for simultaneous delivery of ART and AIE PS to mitochondria of cancer cells for image-guided chemo- and photodynamic combination cancer cell

ablation.

This

probe

(TPETH-Mito-1ART)

contains

an

AIE

core,

tetraphenylethenethiophene (TPETH), as the fluorescent reporter and PS, two armed quaternary ammonium salts as the cancer cell mitochondria targeting moiety,37-38 with ART on one arm (Figure 1a). Benefiting from the AIE feature, TPETH-Mito-1ART shows very weak emission in solution, but intensified fluorescence upon aggregate formation. TPETH-Mito-1ART shows predominant accumulation and fluorescence turn-on in mitochondria of cancer cells, while they show poor uptake towards normal cells due to the less cell plasm and mitochondria membrane potentials of normal cells.39-40 The activated ART by heme at mitochondrial site leads to chemotherapeutic effect to cancer cells. Upon light irradiation, TPETH-Mito-1ART could efficiently generate toxic ROS to quickly oxidize mitochondrial membrane and damage the mitochondria, which further promotes the therapeutic efficacy with a synergistic effect (Figure 1b). Results and Discussion


4

Page 5 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. a) Chemical structures of TPETH-Mito, TPETH-Mito-1ART, and TPETH-Mito2ART. b) Schematic illustration of TPETH-Mito-1ART for combination cancer cell ablation. c) PL spectra of TPETH analogues in water/DMSO (v/v = 99:1) mixture. d) Fluorescence intensity changes

of

TPETH-Mito,

TPETH-Mito-1ART,

and

TPETH-Mito-2ART

in

diethyl

ether/acetonitrile with increased diethyl ether fractions. The synthetic routes towards TPETH-Mito, TPETH-Mito-1ART, and TPETH-Mito2ART, and the confirmation of their structures via 1H NMR spectra are provided in the Supporting Information (Figures S1-S3). TPETH shows an absorption maximum at 350 nm with a broad shoulder ranging from 400 to 550 nm (Figure S4). The broad absorption band makes TPETH compatible with most confocal and fluorescence microscopes. The emission peak of TPETH is localized at around 635 nm in water/DMSO (v/v = 99/1) mixture, the large Stokes shift minimizes the interference from excitation sources. The incorporation of the mitochondrial targeting moieties and ART led to largely reduced emission intensities in water/DMSO (v/v = 99/1) mixture due to the increased water solubility (Figure 1c). Moreover, TPETH-Mito, TPETH-Mito-1ART, and TPETH-Mito-2ART inherit the AIE features of TPETH, as indicated


5


Page 6 of 23

by their intensified emission in diethyl ether/acetonitrile mixture with increased diethyl ether fractions to induce the aggregate formation (Figure 1d). The brightness enhancement factors for TPETH-Mito, TPETH-Mito-1ART, and TPETH-Mito-2ART in aggregate state as compared to these in molecular state were measured to be 124, 131, and 173 folds, respectively. This, together with their negligible emission in aqueous solution, makes them ideal for fluorescence imaging with high signal-to-noise ratios.

Figure 2. Confocal images of a-c) HeLa cancer cells, d) NIH-3T3 and e) HEK-293T normal cells after incubation with a) TPETH-Mito, b, d, e) TPETH-Mito-1ART or c) TPETH-Mito2ART. The cells were co-stained with MitoTracker Green FM. Red channel: Ex = 405, Em = > 650 nm; Green channel: Ex = 488 nm, Em = 505 - 520 nm. All images share the same scale bar of 10 µm.


6

Page 7 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Next, we tested their potentials for mitochondria imaging. HeLa cells were inoculated with these AIE probes (5 µM) for 2 h, which were further co-stained with MitoTracker Green FM dye. Figure 2a-c show the confocal images of HeLa cells. The red fluorescence originated from TPETH probes overlaps very well with the green signals from MitoTracker Green FM, indicating their ability to target and image cancer cell mitochondria. Pearson correlation coefficients were 0.97, 0.93, and 0.84 for TPETH-Mito, TPETH-Mito-1ART, and TPETH-Mito2ART, respectively. Similar cancer cell mitochondria imaging was also found for MDA-MB-231 cancer cells (Figure S5). It should be noted that although incorporation of ART to TPETH-Mito probe leads to the descended Pearson correlation coefficients, both TPETH-Mito-1ART and TPETH-Mito-2ART still possess excellent mitochondria targeting ability as their Pearson correlation coefficients are above 0.8. On the other hand, when these probes were used to label normal cells such as NIH-3T3 fibroblast cells and human embryonic kidney 293T cells (HEK293T), very weak fluorescent signals were found inside the cells (Figures 2d-e and S5). The mean fluorescence intensities inside HeLa cells and MDA-MB-231 cells are over 8 times higher than that inside NIH-3T3 and HEK-293T cells. Moreover, very poor co-localization between AIE probes and MitoTracker Green FM was found for NIH-3T3 and HEK-293T normal cells, where the Pearson correlation coefficients of all three AIE probes were smaller than 0.3. The mitochondria targeting ability for these AIE probes should be contributed by their two quaternary ammonium salt arms,37-38 which lead to predominately mitochondrial accumulation via enhanced electro-potentials due to high mitochondria membrane potentials of cancer cells.3940

These results clearly demonstrate that the AIE probes could not only selectively enter cancer

cells over normal cells, but also show predominate accumulation in cancer cell mitochondria, favoring mitochondria-targeted cancer cell ablation.


7


TPETH-Mito ART

TPETH-Mito-1ART TPETH-Mito-2ART

b) 150 TPETH-Mito ART

Cell Viability (%)

Cell Viability (%)

a) 150 100

50

HeLa

0 3.0

3.5

4.0

4.5

5.0

50

MDA-MB-231 3.0

3.5

4.0

4.5

5.0

Log(conc/nM)

Log(conc/nM) TPETH-Mito ART


100

0

c) 150 Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 23

d)


IC50 (µM)

MDA-MB231

HeLa

NIH3T3

ART

73.1

88.8

>100.0

100

50

NIH-3T3

0 3.0

3.5

4.0

4.5

TPETH-Mito

100.0

94.4

>100.0

TPETH-Mito1ART

13.9

11.1

>100.0

TPETH-Mito2ART

3.7

3.1

12.6

5.0

Log(conc/nM)

Figure 3. Viabilities of a) HeLa, b) MDA-MB-231, and c) NIH-3T3 cells after 24 h incubation with ART, TPETH-Mito, TPETH-Mito-1ART or TPETH-Mito-2ART in dark. d) IC50 values of these probes towards different cell lines. As heme could activate the anticancer activities of ART,8 and mitochondria are the main place for fresh heme generation, the delivery of ART to mitochondria should largely enhance its anticancer performance. To test this hypothesis, we investigated the cancer lesion effects by the accumulation of TPETH-Mito-1ART and TPETH-Mito-2ART to mitochondria via an MTT assay. Besides these two ART analogues, TPETH-Mito and ART alone were also used as controls. HeLa cancer cells, MDA-MB-231 cancer cells, and NIH-3T3 normal cells were treated with these probes and controls at different concentrations for 24 h in dark. Their toxicity effects towards different cell lines are shown in Figure 3. ART alone shows very low cell toxicities to all these tested cells, with half maximal inhibitory concentration (IC50) values of over 70 µM. Similarly, the accumulation of TPETH-Mito itself in mitochondria does not cause significant cytotoxicity. On the other hand, delivery of ART to mitochondria could largely improve its


8

Page 9 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


killing effects towards cancer cells, where TPETH-Mito-2ART shows IC50 values of 3.1 and 3.7 µM towards HeLa and MDA-MB-231 cells, and TPETH-Mito-1ART exhibited IC50 values of 11.1 and 13.9 µM towards HeLa and MDA-MB-231 cells, respectively. The significant toxicity towards cancer cells clearly reveals that directing ART to mitochondria could largely improve its anticancer activity. However, due to the high loading amount of ART, TPETH-Mito-2ART also demonstrates a severe cytotoxicity towards NIH-3T3 normal cells, with an IC50 value of 12.6 µM, which makes it less suitable to serve as a non-invasive anticancer drug. In contrast, TPETHMito-1ART is much less toxicity to NIH-3T3 cells (IC50 100 µM). The selective toxicity between cancer cells and normal cells makes TPETH-Mito-1ART an excellent non-invasive anticancer drug, which was selected for further PDT experiments.

Figure 4. a) Absorption spectra of ABDA (50 µM) in the presence of TPETH-Mito-1ART after white light irradiation with different irradiation time. b) Decomposition rates of ABDA by different TPETH analogues under white light irradiation, where A0 and A are ABDA absorbance at 399 nm. c) Intracellular ROS detection inside HeLa cells by DCF-DA after treatment with ART, TPETH-Mito, or TPETH-Mito-1ART and white light irradiation.


9


Page 10 of 23

TPETH itself is a PS, which could generate ROS efficiently in aggregate state.41 We next investigated whether the TPETH-Mito-1ART probe also inherits such photosensitizing ability. 9,10-Anthracenediylbis(methylene)dimalonic acid (ABDA) which could react with ROS to show decreased absorbance was selected to assess ROS production in aqueous solution. Upon incubation ABDA with TPETH-Mito-1ART, white light irradiation leads to quickly decreased ABDA absorbance (Figure 4a), indicating its efficient ROS production. The ABDA decomposition rates by TPETH, TPETH-Mito, TPETH-Mito-1ART were very similar (Figures 4b and S6), indicating that the incorporation of mitochondria targeting arms and ART could not significantly affect their photosensitizing ability. In addition, the singlet oxygen production efficiencies of TPETH-Mito and TPETH-Mito-1ART were measured to be 69.2% and 68.1%, respectively, using Rose Bengal (RB, 75%) as the reference (Figure S6), much higher than clinically used photosensitizers such as Photofrin (28%) or Laserphyrin (48%).42 To evaluate intracellular ROS production of the AIE probes, dichlorofluorescein diacetate (DCF-DA) that could react with ROS to show strong green fluorescence was chosen and added to HeLa cells treated with the probes and light irradiation. As shown in Figure 4c, strong green fluorescence is observed inside the HeLa cells treated with TPETH-Mito and TPETH-Mito-1ART, but not for cells treated with ART, demonstrating the efficient intracellular ROS generation by TPETH probes, which makes them promising for photodynamic cancer cell ablation.


10

Page 11 of 23

b) 120

90

90

60

30

Cell Viability (%)

a) 120 Cell Viability (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TPETH-Mito-1ART HeLa + Dark HeLa + Light NIH-3T3 + Dark NIH-3T3 + Light

0

60 TPETH-Mito HeLa + Dark HeLa + Light 30 NIH-3T3 + Dark NIH-3T3 + Light 0

3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4

3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8

Log(conc/nM)

Log(conc/nM)

Figure 5. Viabilities of HeLa cells after treatment with a) TPETH-Mito-1ART or b) TPETHMito for 2.5 h with or without white light treatment (60 mWcm-2, 10 min). We next evaluated the photodynamic cancer cell ablation by TPETH-Mito and TPETHMito-1ART. With white light irradiation, TPETH-Mito-1ART showed a much higher cancer cell killing efficiency as compared to that without light irradiation (Figure 5a). The IC50 value of TPETH-Mito-1ART towards HeLa cells is reduced from 14.5 µM to 8.1 µM upon white light irradiation, while its IC50 values remain over 100 µM for NIH-3T3 cells under dark or irradiation. Moreover, TPETH-Mito shows an IC50 value of ~78.1 µM (Figure 5b) towards HeLa cells under white light irradiation. Live and dead staining via fluorescein diacetate (FDA) and propidium iodide (PI) were further applied to visualize the therapeutic performance (Figure S7). Only green fluorescence from FDA was observed inside ART-treated HeLa cells, indicating its low toxicity towards cancer cells. The occurrence of cells with red emission from PI clearly indicated the chemotherapy effect of TPETH-Mito-1ART in dark. With white light irradiation, TPETH-Mito-1ART led to a large population of red emissive dead HeLa cells with only few green emissive live ones. To quantitatively evaluate the combination therapy effect of ART and PDT, we also measured the combination index (C.I.) from the dose-effect profiles,43 where the C.I. values of TPETH-Mito-1ART were all smaller than 1 at different cancer cell killing levels


11


Page 12 of 23

(Figure S8), which demonstrated the synergistic combination effect of TPETH-Mito-1ART by the co-delivery of ART and TPETH to cancer cell mitochondria.

Figure 6. Migration ratios of HeLa cells after treatment with different probes with or without white light irradiation. As the major energy supplier for cell activities, the dysfunction of mitochondria largely affects cancer cell proliferation and migration.44 The effect of ART and/or PDT treatment on mitochondria activity was then evaluated by a cell migration assay. As shown in Figures 6 and S9, ART itself shows a minimal effect on HeLa cell migration, while treatment of TPETH-Mito in dark shows a decreased migration ratio of 70%. This should be attributed to the accumulation of probes onto mitochondria,45

which displays low toxicity to cells but could influence

mitochondrial activity to a certain degree. HeLa cells treated with TPETH-Mito-1ART in dark shows a very low migration ratio of ~36%, indicating the largely affected cell migration by ART activation. White light irradiation further lowers the migration ratio to ~8%, revealing that the mitochondria dysfunction caused by ART and PDT could further reduce the possibility of cancer cell migration.


12

Page 13 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. a) Confocal images of JC-1 dye labeled HeLa cells. The HeLa cells were pretreated with ART or TPETH-Mito-1ART followed by dark or white light treatment and then JC-1 dye staining. b) JC-1 monomer or aggregate fluorescence intensity changes in HeLa cells with different treatments.

To provide more insight into the mitochondria damage, we further evaluated the mitochondrial membrane potential (MMP) changes of HeLa cells with JC-1 dye, which shows potential-dependent emission color changes in mitochondria. It forms red emissive aggregates on normal mitochondria, while it shifts to green emissive monomers on depolarized mitochondria. As indicated by the green and red intensity changes of JC-1 dye (Figure 7), ART itself did not show any damage to MMP as indicated by the absence of green signal, while TPETH-Mito-1ART in dark led to a moderate depolarization of the mitochondria membrane, and


13


Page 14 of 23

the white light irradiation further enhanced its damage to MMP and almost no red signal was detectable.

Conclusions In conclusion, we have designed a light-up probe based on an AIE PS to visualize the delivery of ART to cancer cell mitochondria for combination cancer cell ablation with a synergistic effect. This mitochondria light-up probe contains TPETH as the core, two mitochondria targeting arms, with ART on one arm. Benefiting from the AIE feature of TPETH, the obtained TPETH-Mito1ART probe is almost not emissive in solution but can turn on its fluorescence upon selective binding to mitochondria of cancer cells. The delivery of ART to mitochondria could quickly activate its anticancer activities, and direct ROS generation on mitochondria maximizes its PDT efficiency, which further promotes final therapeutic efficacy with a synergistic effect. Further analysis reveals that the co-delivery of AIE PSs and ART to mitochondria could quickly depolarize the mitochondria membrane, and reduce cell migration, which should further enforce its great potentials for cancer therapy via not only directly killing cancer cells but also reducing the chances of cancer migration. With the advantage of AIE PSs and organelle targeting moieties, we foresee the great potentials of AIEgen conjugates for organelle targeted theranostics. Experimental Section Synthesis of TPETH-Mito-1ART and TPETH-Mito-2ART. 80 µL of MeI (1.28 mmol) was added to the 2 mL acetonitrile solution of 4 (18 mg, 0.018 mmol). The reaction is conducted for 24 h at room temperature under continuous stirring. After solvent removal, 2 mL of


14

Page 15 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dichloromethane (DCM) was added to dissolve the residue, followed by addition of 0.2 mL of trifluoroacetic acid (TFA) (0.2 mL). After 4 h reaction, the obtained residue was dissolved in 2 mL of dimethyl sulfoxide (DMSO). Compound 6 (9.4 mg, 0.019 mmol) and DIEA (7.4 mg, 0.057 mmol) was then added, and the reaction was further conducted for 4 h at room temperature under continuous stirring to obtain the final products. The mixture was purified with HPLC using acetonitrile and water as elution buffer to give TPETH-Mito-1ART (3.2 mg, 12.8% yield) and TPETH-Mito-2ART (4.3 mg, 13.8% yield). TPETH-Mito-1ART: 1H NMR (400 MHz, DMSOd6) δ 8.27 (m, 1H), 8.23 (brs, 3H), 7.68 (m, 1H), 7.45 (m, 1H), 7.33-7.39 (m, 3H), 7.10-7.19 (m, 5H), 7.01-7.03 (m, 2H), 6.84-6.90 (m, 4H), 6.68-6.73 (m, 4H), 5.90 (m, 0.32 H), 5.29 (m, 0.6 H), 3.91-3.97 (m, 7H), 3,05-3.09 (m, 12H), 1.91-2.17 (m, 4H), 1.54-1.81 (m, 16H), 1.16-1.36 (m, 8H), 0.86-0.92 (m, 4H), 0.77-0.79 (m, 3H); MS (ESI) calcd for [M-2CF3COO]2+: 580.31, found: 580.34. TPETH-Mito-2ART: 1H NMR (400 MHz, DMSO-d6) δ 8.27 (dd, J1 = 1.2 Hz, J2 = 4.8 Hz, 1H), 7.67 (dd, J1 = 1.2 Hz, J2 = 4.0 Hz, 1H), 7.45 (t, J = 5.2 Hz, 2H), 7.33-7.38 (m, 3H), 7.09-7.19 (m, 5H), 7.03 (m, 2H), 6.91 (m, 2H), 6.85 (m, 2H), 6.68-6.73 (m, 4H), 5.29 (s, 1H), 5.28 (s, 1H), 3.91-4.03 (m, 10 H), 3.37-3.41 (m, 10 H), 3.04 (s, 12H), 2.43 (m, 2H), 2.07-2.16 (m, 2H), 1.99 (m, 3H), 1.69-1.81 (m, 14H), 1.50-1.54 (m, 8H), 1.23-1.35 (m, 17H), 1.13-1.17 (m, 2H), 0.83-0.90 (m, 6H), 0.76-0.79 (m, 6H); MS (ESI) calcd for [M-2CF3COO]2+: 756.41, found: 756.94. Solution ROS detection. ABDA was used to detect the solution ROS production of AIE probes upon irradiation. A DMSO solution of ABDA (10 mM, 5 µL) was added into AIE probe aqueous solution (5 µM, 1 mL). The mixture’s absorption spectra upon light irradiation (60 mWcm-2) were recorded, where the descending ABDA absorbance after designated irradiation time


15


Page 16 of 23

intervals indicates the solution ROS production of AIE probes. The singlet oxygen production efficiencies of AIE probes were determined by the following equation:

=

(1)

where η is the singlet oxygen production efficiency, K is the decomposition rate constants of ABDA, A is total light absorbed by the photosensitizer. K is determined by the plot ln(Abs0/Abs) vs. irradiation time, where Abs0 and Abs are the ABDA absorbance before and after designated irradiation time. Rose Bengal is selected as the referenced photosensitizer, which has a η of 75% in water. Cell imaging. HeLa cancer cells, MDA-MB-231 cancer cells, NIH-3T3 normal fibroblast cells, and human embryonic kidney 293T cells (HEK-293T) were selected for cell imaging, which were cultured in an 8-well chamber at 37 °C. Upon reaching 80% confluence, the cells were treated with TPETH-Mito, TPETH-Mito-1ART, or TPETH-Mito-2ART (5 µM) for 2 h. After washing with 1 × PBS twice, these cells were co-stained with MitoTracker Green FM (200 nM) for 20 min. Leica SP8 was used to acquire the living cell imaging. Red AIE channel: Ex = 405 nm, Em = >650 nm. Green MitoTracker Green FM channel: Ex = 488 nm, Em = 505-520 nm. Intracellular ROS detection. HeLa cancer cells were selected for intracellular ROS detection. After treatment with ART, TPETH-Mito, or TPETH-Mito-1ART (5 µM) for 2.5 h, HeLa cells were further incubated with dichlorofluorescein diacetate (DCF-DA) (10 µM) for 20 min. 10 min white light irradiation (60 mWcm-2) was then applied to these cells before imaging by Leica SP8 CLSM. Green fluorescence channel: Ex = 488nm, Em = 505-560 nm. Cell toxicity study. Methylthiazolyldiphenyltetrazolium bromide (MTT) assay was used to access the cytotoxicity of AIE probes. HeLa, MDA-MB-231 and NIH-3T3 cells were seeded in


16

Page 17 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


96-well plate at a density of 4000 cells per well. After overnight culturing, the cell culture media were replaced with fresh ones containing AIE or ART probes, followed by 24 h incubation in dark. The cells were further treated with MTT containing cell culture medium (0.5 mg/mL) for 3 h and DMSO (100 µL/well) for 10 min in sequence. The MTT absorbance at 570 nm was then recorded by a microplate reader. Cell viabilities were calculated based on the MTT absorbance changes where the viability of control cells without any probe treatment is arbitrarily determined to be 100%. For photodynamic therapy treatment, these cells were incubated with ART, TPETHMito, TPETH-Mito-1ART for 2.5 h in dark at varied concentrations. After changing the medium to fresh ones, selected wells were treated with white light irradiation (60 mWcm-2) for 10 min. All the wells were further cultured for 24 h. The standard MTT assay procedures were then repeated, and the cell viability was calculated accordingly. Cell migration study. The cells were incubated for 2.5 h with ART, TPETH-Mito, TPETHMito-1ART (5 µM, 400 µL) containing cell culture medium in an 8-well chamber. After changing the medium to fresh ones, selected wells were treated with white light irradiation (60 mWcm-2) for 10 min. A 10 µL tip was then used to scratch a wound gap on the monolayer HeLa cells. After rinsing with 1× PBS buffer to remove scratched cells, the wound gaps were imaged by Nikon-Ti-U microscope with a 10 × objection (Nikon-Ti-U), which were further imaged after 48 h culturing. The cell number of migrated to the wound gap reflects their cell migration ability under different treatments. Mitochondrial membrane potential (MMP) measurement. HeLa cells were with ART, TPETH-Mito, or TPETH-Mito-1ART (5 µM) for 2.5 h, followed by 10 min white light irradiation (60 mWcm-2). After these treatments, JC-1 dyes (5 µg/mL) were added to HeLa cell chambers and incubated for 20 min. The cells were immediately imaged by Leica SP8


17


Page 18 of 23

microscope after washing with 1× PBS buffer. Upon excitation at 488 nm, the green signal of JC-1 monomer was collected between 505 and 525 nm, and the red signal of JC-1 aggregate was collected above 590 nm.

ASSOCIATED CONTENT Supporting Information. The synthesis of AIE probes, 1H NMR spectra, the absorption and emission spectra of TPETH, confocal images of different cell lines treated with AIE probes, live/dead cell staining after different treatments; combination index plot of the TPETH-Mito1ART against HeLa cells, photographs of HeLa cell migrations. The supporting information is available free of charge via the Internet at http://pubs.acs.org. Corresponding Author *Email: [email protected] *Email: [email protected] ACKNOWLEDGMENT The authors are grateful to the Singapore National Research Foundation (R279-000-444-281), Singapore NRF Competitive Research Programme (R279-000-483-281), National University of Singapore (R279-000-482-133), CAMS Innovation Fund for Medical Sciences (2017-I2M-4005), CAMS Fund of Non-profit Central Research Institutes (2017RC31009) for financial support. REFERENCES


18

Page 19 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.

Tu, Y., The Discovery of Artemisinin (Qinghaosu) and Gifts from Chinese Medicine.

Nat. Med. 2011, 17, 1217-1220. 2.

Tu, Y., Artemisinin—A Gift from Traditional Chinese Medicine to the World (Nobel

Lecture). Angew. Chem. Int. Ed. 2016, 55, 10210-10226. 3.

Kong, L. Y.; Tan, R. X., Artemisinin, A Miracle of Traditional Chinese Medicine. Nat.

Prod. Rep. 2015, 32, 1617-1621. 4.

O’Neill, P. M.; Barton, V. E.; Ward, S. A., The Molecular Mechanism of Action of

Artemisinin—The Debate Continues. Molecules 2010, 15, 1705-1721. 5.

Krishna, S.; Bustamante, L.; Haynes, R. K.; Staines, H. M., Artemisinins: Their Growing

Importance in Medicine. Trends Pharmacol. Sci. 2008, 29, 520-527. 6.

Chen, H.-J.; Huang, X.-R.; Zhou, X.-B.; Zheng, B.-Y.; Huang, J.-D., Potential

Sonodynamic Anticancer Activities of Artemether and Liposome-Encapsulated Artemether. Chem. Commun. 2015, 51, 4681-4684. 7.

Ho, W. E.; Peh, H. Y.; Chan, T. K.; Wong, W. S. F., Artemisinins: Pharmacological

Actions Beyond Anti-malarial. Pharmacol. Ther. 2014, 142, 126-139. 8.

Wang, J.; Zhang, C.-J.; Chia, W. N.; Loh, C. C. Y.; Li, Z.; Lee, Y. M.; He, Y.; Yuan, L.-

X.; Lim, T. K.; Liu, M.; Liew, C. X.; Lee, Y. Q.; Zhang, J.; Lu, N.; Lim, C. T.; Hua, Z.-C.; Liu, B.; Shen, H.-M.; Tan, K. S. W.; Lin, Q., Haem-Activated Promiscuous Targeting of Artemisinin in Plasmodium Falciparum. Nat. Commun. 2015, 6, 10111. 9.

Zhang, C.-J.; Wang, J.; Zhang, J.; Lee, Y. M.; Feng, G.; Lim, T. K.; Shen, H.-M.; Lin,

Q.; Liu, B., Mechanism-Guided Design and Synthesis of a Mitochondria-Targeting Artemisinin Analogue with Enhanced Anticancer Activity. Angew. Chem. Int. Ed. 2016, 55, 13770-13774. 10.

Eckstein-Ludwig, U.; Webb, R. J.; van Goethem, I. D. A.; East, J. M.; Lee, A. G.;

Kimura, M.; O'Neill, P. M.; Bray, P. G.; Ward, S. A.; Krishna, S., Artemisinins Target the SERCA of Plasmodium Falciparum. Nature 2003, 424, 957-961. 11.

Castano, A. P.; Mroz, P.; Hamblin, M. R., Photodynamic Therapy and Anti-tumour

Immunity. Nat. Rev. Cancer 2006, 6, 535-545. 12.

Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K., Photodynamic Therapy for Cancer.

Nat. Rev. Cancer 2003, 3, 380-387. 13.

Li, X.; Yu, S.; Lee, D.; Kim, G.; Lee, B.; Cho, Y.; Zheng, B.-Y.; Ke, M.-R.; Huang, J.-

D.; Nam, K. T.; Chen, X.; Yoon, J., Facile Supramolecular Approach to Nucleic-Acid-Driven


19


Page 20 of 23

Activatable Nanotheranostics That Overcome Drawbacks of Photodynamic Therapy. ACS Nano 2018, 12, 681-688. 14.

Li, X.; Lee, S.; Yoon, J., Supramolecular Photosensitizers Rejuvenate Photodynamic

Therapy. Chem. Soc. Rev. 2018, 47, 1174-1188. 15.

Skovsen, E.; Snyder, J. W.; Lambert, J. D. C.; Ogilby, P. R., Lifetime and Diffusion of

Singlet Oxygen in a Cell. J. Phys. Chem. B 2005, 109, 8570-8573. 16.

Kim, S.; Tachikawa, T.; Fujitsuka, M.; Majima, T., Far-Red Fluorescence Probe for

Monitoring Singlet Oxygen during Photodynamic Therapy. J. Am. Chem. Soc. 2014, 136, 1170711715. 17.

Ngen, E. J.; Rajaputra, P.; You, Y., Evaluation of Delocalized Lipophilic Cationic Dyes

as Delivery Vehicles for Photosensitizers to Mitochondria. Bioorg. Med. Chem. 2009, 17, 66316640. 18.

Morgan, J.; Oseroff, A. R., Mitochondria-Based Photodynamic Anti-Cancer Therapy.

Adv. Drug Delivery Rev. 2001, 49, 71-86. 19.

Feng, G.; Qin, W.; Hu, Q.; Tang, B. Z.; Liu, B., Cellular and Mitochondrial Dual-

Targeted Organic Dots with Aggregation-Induced Emission Characteristics for Image-Guided Photodynamic Therapy. Adv. Healthcare Mater. 2015, 4, 2667-2676. 20.

Bennett, L. E.; Ghiggino, K. P.; Henderson, R. W., Singlet Oxygen Formation in

Monomeric and Aggregated Porphyrin C. J. Photochem. Photobiol. B 1989, 3, 81-89. 21.

Piette, J., Signalling Pathway Activation b Photodynamic Therapy: NF-κB at The

Crossroad Between Oncology and Immunology. Photochem. Photobiol. Sci. 2015, 14, 15101517. 22.

Mroz, P.; Yaroslavsky, A.; Kharkwal, G. B.; Hamblin, M. R., Cell Death Pathways in

Photodynamic Therapy of Cancer. Cancers (Basel) 2011, 3, 2516-2539. 23.

Wang, J.-X.; Hou, L.-F.; Yang, Y.; Tang, W.; Li, Y.; Zuo, J.-P., SM905, an Artemisinin

Derivative, Inhibited NO and Pro-Inflammatory Cytokine Production by Suppressing MAPK and NF-κb Pathways in RAW 264.7 Macrophages. Acta Pharmacol. Sin. 2009, 30, 1428-1435. 24.

Ding, D.; Li, K.; Liu, B.; Tang, B. Z., Bioprobes Based on AIE Fluorogens. Acc. Chem.

Res. 2013, 46, 2441-2453. 25.

Feng, G.; Liu, B., Multifunctional AIEgens for Future Theranostics. Small 2016, 12,

6528-6535.


20

Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26.

Qian, J.; Tang, B. Z., AIE Luminogens for Bioimaging and Theranostics: From

Organelles to Animals. Chem 2017, 3, 56-91. 27.

Feng, G.; Kwok, R. T. K.; Tang, B. Z.; Liu, B., Functionality and Versatility of

Aggregation-Induced Emission Luminogens. Appl. Phys. Rev. 2017, 4, 021307. 28.

Mei, J.; Leung, N. L. C.; Kwok, R. T. K.; Lam, J. W. Y.; Tang, B. Z., Aggregation-

Induced Emission: Together We Shine, United We Soar! Chem. Rev. 2015, 115, 11718-11940. 29.

Hu, F.; Liu, B., Organelle-Specific Bioprobes Based on Fluorogens with Aggregation-

Induced Emission (AIE) Characteristics. Org. Biomol. Chem. 2016, 14, 9931-9944. 30.

Liang, J.; Tang, B. Z.; Liu, B., Specific Light-up Bioprobes Based on AIEgen

Conjugates. Chem. Soc. Rev. 2015, 44, 2798-2811. 31.

Hu, F.; Huang, Y.; Zhang, G.; Zhao, R.; Yang, H.; Zhang, D., Targeted Bioimaging and

Photodynamic Therapy of Cancer Cells with an Activatable Red Fluorescent Bioprobe. Anal. Chem. 2014, 86, 7987-7995. 32.

Xu, S.; Yuan, Y.; Cai, X.; Zhang, C.-J.; Hu, F.; Liang, J.; Zhang, G.; Zhang, D.; Liu, B.,

Tuning the Singlet-Triplet Energy Gap: a Unique Approach to Efficient Photosensitizers with Aggregation-Induced Emission (AIE) Characteristics. Chem. Sci. 2015, 6, 5824-5830. 33.

Yuan, Y.; Feng, G.; Qin, W.; Tang, B. Z.; Liu, B., Targeted and Image-Guided

Photodynamic Cancer Therapy Based on Organic Nanoparticles with Aggregation-Induced Emission Characteristics. Chem. Commun. 2014, 50, 8757-8760. 34.

Yuan, Y.; Zhang, C.-J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B., Specific Light-Up

Bioprobe with Aggregation-Induced Emission and Activatable Photoactivity for the Targeted and Image-Guided Photodynamic Ablation of Cancer Cells. Angew. Chem. Int. Ed. 2015, 54, 1780-1786. 35.

Yuan, Y.; Zhang, C.-J.; Liu, B., A Photoactivatable AIE Polymer for Light-Controlled

Gene Delivery: Concurrent Endo/Lysosomal Escape and DNA Unpacking. Angew. Chem. Int. Ed. 2015, 54, 11419-11423. 36.

Zhang, C.-J.; Hu, Q.; Feng, G.; Zhang, R.; Yuan, Y.; Lu, X.; Liu, B., Image-Guided

Combination Chemotherapy and Photodynamic Therapy Using a Mitochondria-Targeted Molecular Probe with Aggregation-Induced Emission Characteristics. Chem. Sci. 2015, 6, 45804586.


21


37.

Page 22 of 23

Inácio, Â. S.; Costa, G. N.; Domingues, N. S.; Santos, M. S.; Moreno, A. J. M.; Vaz, W.

L. C.; Vieira, O. V., Mitochondrial Dysfunction Is the Focus of Quaternary Ammonium Surfactant Toxicity to Mammalian Epithelial Cells. Antimicrob. Agents Chemother. 2013, 57, 2631-2639. 38.

Chen, Z.; Zhang, L.; Song, Y.; He, J.; Wu, L.; Zhao, C.; Xiao, Y.; Li, W.; Cai, B.; Cheng,

H.; Li, W., Hierarchical Targeted Hepatocyte Mitochondrial Multifunctional Chitosan Nanoparticles for Anticancer Drug Delivery. Biomaterials 2015, 52, 240-250. 39.

Modica-Napolitano, J. S.; Aprille, J. R., Delocalized Lipophilic Cations Selectively

Target the Mitochondria of Carcinoma Cells. Adv. Drug Delivery Rev. 2001, 49, 63-70. 40.

Chen, L. B., Mitochondrial Membrane Potential in Living Cells. Annu. Rev. Cell Biol.

1988, 4, 155-181. 41.

Yuan, Y.; Zhang, C.-J.; Xu, S.; Liu, B., A Self-Reporting AIE Probe with a Built-in

Singlet Oxygen Sensor for Targeted Photodynamic Ablation of Cancer Cells. Chem. Sci. 2016, 7, 1862-1866. 42.

Ogilby, P. R., Singlet Oxygen: There Is Indeed Something New Under the Sun. Chem.

Soc. Rev. 2010, 39, 3181-3209. 43.

Chou, T.-C., Drug Combination Studies and Their Synergy Quantification Using the

Chou-Talalay Method. Cancer Res. 2010, 70, 440-446. 44.

McBride, H. M.; Neuspiel, M.; Wasiak, S., Mitochondria: More Than Just a Powerhouse.

Curr. Biol. 2006, 16, R551-R560. 45.

Hu, Q.; Gao, M.; Feng, G.; Liu, B., Mitochondria-Targeted Cancer Therapy Using a

Light-Up Probe with Aggregation-Induced-Emission Characteristics. Angew. Chem. Int. Ed. 2014, 53, 14225-14229.

TOC


22

Page 23 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



23

Artemisinin and AIEgen Conjugate for Mitochondria-Targeted and

Recommend Documents