Amphiphilic Tetraphenylethene-Based Pyridinium Salt for Selective

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Biological and Medical Applications of Materials and Interfaces

Amphiphilic Tetraphenylethene-Based Pyridinium Salt for Selective Cell-Membrane Imaging and Room Light Induced Special Reactive Oxygen Species Generation Weijie Zhang, Yuhua Huang, Yilong Chen, Engui Zhao, Yuning Hong, Sijie Chen, Jacky W. Y. Lam, Yuncong Chen, Jianquan Hou, and Ben Zhong Tang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00643 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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ACS Applied Materials & Interfaces

Amphiphilic Tetraphenylethene-Based Pyridinium Salt for Selective Cell-Membrane Imaging and Room Light Induced Special Reactive Oxygen Species Generation Weijie Zhang,‡,† Yuhua Huang, ‡,† Yilong Chen, ⊥, § Engui Zhao,# Yuning Hong,|| Sijie Chen, § Jacky W. Y. Lam, § Yuncong Chen, § Jianquan Hou,*, † Ben Zhong Tang*,⊥, §,&

†Department

of Urology, The First Affiliated Hospital of Soochow University, 188 Shizi

RD, Suzhou 215006, China. E-mail: [email protected]

⊥HKUST

Shenzhen Research Institute, No. 9 Yuexing 1st RD, South Area, Hi-tech Park

Nanshan, Shenzhen 518057, China. E-mail: [email protected]

§Department

of Chemistry, The Hong Kong Branch of Chinese National Engineering

Research Center for Tissue Restoration and Reconstruction and Institute for Advanced

ACS Paragon Plus Environment

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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

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Study, The Hong Kong University of Science & Technology, Clear Water Bay, Kowloon, Hong Kong, China

#School

of Chemical Engineering and Energy Technology, Dongguan University of

Technology, 1st University Road, Songshan Lake District, Dongguan, 523808, China

||Department

of Chemistry and Physics, La Trobe Institute of Molecular Science, La

Trobe University, Melbourne Victoria, Australia 2086

&Center

for Aggregation-Induced Emission, SCUT-HKUST Joint Research Laboratory,

State Key Laboratory of Luminescent Materials and Devices, South China University of Technology, Guangzhou 510640, China

†Electronic Supplementary Information (ESI) available: Synthetic procedures and characterizations, other experimental details. See DOI: 10.1039/x0xx00000x

‡ These authors contributed equally.

KEYWORDS: aggregation-induced emission (AIE); cell-membrane imaging; reactive oxygen species (ROS); photodynamic therapy (PDT); bioimaging


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ABSTRACT.

Cell membrane is the protecting frontier of cells, which is crucial for maintaining cell integrity, and has closely relationship with cell growth and death. There is growing need for cell membrane imaging and monitoring in both living and dying cells. Herein, we report a new amphiphilic tetraphenylethene-based pyridinium salt (TPE-MEM) with aggregation-induced emission (AIE) features for discriminatory cell membrane imaging. The fluorogenic probe with high yield was synthesized following asymmetric McMurry reaction, Williamson ether synthesis reaction, Suzuki coupling and aldol condensation between

a

double-charged

pyridinium

salt

and

hexyloxytetraphenylethene

benzaldehyde. TPE-MEM shows good water-solubility, biocompatibility, and cell membrane specificity. Interestingly, reactive oxygen species (ROS) is produced by the molecule (TPE-MEM) under room light irradiation, which could destroy the integrity of the plasma membrane and cause the cell necrosis. This enables a visible observation of cell necrosis and phototherapeutic effect under a mild condition. Preliminary animal


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investigation also demonstrated the photodynamic therapy (PDT) effectiveness of TPEMEM on tumor growth inhibition. We conclude that TPE-MEM is potentially a cell membrane selective photosensitizer for PDT and it is worthy for further exploration of the phototherapeutic effect on animals systematically.

INTRODUCTION A promising clinical modality, Photodynamic therapy (PDT), is widely using for the treatment of superficial tumors and non-malignant lesions, which involves non-toxic photosensitizer, powerful visible light and oxygen.1-4 Those therapeutic effects of PDT are acted on a precise region of the tissues that are exposed to visible light. Thus, the PDT treatments are non-invasive and their side effects are under control and could be confined to a specific region. Cell death could take place in three pathways: apoptosis, necrosis, and autophagy5-8, among which photosensitizers caused cell necrosis are superlethal PDT processes and could take effect in only a few minutes.9-10

The cell death pathways are closely associated with the subcelullar localization and doses of PDT sensitizers.5-10 Plasma membranes, lysosomes, endoplasmic reticulum


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(ER), and mitochondria are commonly targeted by PDT sensitizers.5-10 It is believed that the photodynamic process of the sensitizer coincides with the intracellular localization within few nanometers due to the short lifetime and limited diffusion radius of photogenerated reactive oxygen species (ROS).11 It has been found that photosesitizers targeting the mitochondria or the ER preferentially promote cell apoptosis within a certain PDT dose, while those photoactive molecules selectively bounding to the plasma membranes predispose the cells to necrosis primarily. 5-10 This is likely because of the oxidation of cholesterol and other unsaturated phospholipids by the locally generated ROS in the membrane, which results in the changes of membrane permeability, and the losses of membrane fluidity and integrity.12-13

During the cell necrosis process, the morphology of the cells is changing followed by cell swelling, blebbing, breakdown of the cell membrane and finally release of cellular contents into the extracellular content to induce inflammatory response. Therefore, the design of cell membrane targeting photosensitizers for PDT is a particularly important strategy of making effective PDT agents for practical tumor treatment. However,


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photosensitizers targeting plasma membranes are seldom available due to their delicate structural design.14-16 The cell death pathologies of PDT process are complicated and of great importance. They are mainly studied by using indirect optical techniques like flow cytometry and cell viability quantifica-tions.1-16 Direct observation of the cell death during the PDT process by using a photosensitizer’s own fluorescence, to avoid additive interference and to offer spatiotemporal imaging resolution, is barely reported.1-10,14-16 Ideally, if a photosensitizer could serve as both a ROS generator and a fluorescent plasma membrane probe, the cell death pathology could be monitored during the PDT process without the need of additional fluorescent probe. However, the cell membrane photosensitizers reported are normally less photostable. They can easily undergo either photooxidation or photobleaching during the light irradiation.14-16 To date, CellMaskTM Orange (CMO) and Deep Red (CMDR) are two of the best cell membrane dyes available commercially.17 However, their chemical structures and ingredients become commercial secret for their difficulty in structure deign and synthesis.


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It is found that most of the amphiphilic fluorophores encounters a quenching problem when they form micelles or aggregates in aqueous medium resulting in partially or completely quenching of their light emission. This phenomenon of aggregation-caused quenching (ACQ) has restricted their high-tech appliance.18 Recently, we and other researchers discovered an uncommon phenomenon of aggregation-induced emission (AIE) on a group of molecules with propeller-like structures, for instance, tetraphenylethene (TPE) and hexaphenylsilole19-20. Instead of fluorescence quenching by ACQ effect, the molecules emit effectually in the solid or aggregated state in spite of their faint emissions in the solution state. Systematic studies indicate that the main cause of the abnormal AIE phenomenon is restriction of intramolecular motion.21 During the past period, many AIE-active materials have been developed for electronics,22-24 optics25 and biological science.25-27 In biological science, AIE-active materials were widely investigated and utilized for bio-detection,28-31 cell25,27,32-37 and bacteria38 imaging.


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Particularly, several AIE bioprobes were found to be capable of long-term cell tracking,32-34 highly selective lysosome,21 mitochondria25,27 and lipid droplet37 imaging, taking advantages of their high fluorescent quantum yields, excellent photostabilities and less cytotoxicity. These findings also trigger us to develop new cell membranespecific dyes with AIE characteristics.39

In order to selectively targeting the cell membrane, the plasma membrane stain bearing both positive charges and the balance of the hydrophilicity and hydrophobicity (Scheme 1) would be crucial to approach and stay on the membrane. The molecules cannot target the cell membrane selectively if the molecule is either too hydrophilic or too hydrophobic. Our group has successfully synthesized TTAPE31,40 and TPE-Py25 (Chart 1) having positive charges. TTAPE, bearing four positive charges, can not target and sustain on the cell membrane. Compared with TTAPE, TPE-Py with only one positive charge, which is relatively hydrophobic, can easily pass through cell membrane and selectively target to the cell mitochondria. Fine tuning the molecular structure is needed for the design of a good cell membrane stain. Recently, a new organic dye


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derived from 1,8-naphthalimide was reported to show AIE property and good cell membrane targeting capability.39 The authors demonstrated the good cell membrane selectivity, photostability and long-term cell membrane traceability of the cell membrane probe. However, it emits in the blue region, which may suffer from the interference from cell autofluorescence.

Scheme 1. Illustration of cell membrane staining and cell necrosis induced by compound TPE-MEM.

N+ Br

N+ Br

-

O

O

O

O

N+ Br-

PF6-

N+ Br

-

TTAPE

TPE-Py

N+


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Chart 1. Chemical structures of TTAPE and TPE-Py.

In this paper, we report a yellow-emissive cell membrane specific bioprobe (TPEMEM, Scheme 2) derived from TPE based on the design strategy discussed above. The water soluble TPE-MEM with medium amphiphilicity lighted up the cell membrane selectively. The high image quality is comparable to CMDR. Interestingly, ROS can be generated by mild and white room light illumination, which oxidizes the substances in the plasma membrane and destroy the integrity of the membrane, enabling the real-time induction and observation of cell necrosis in situ.

All the results hint that TPE-MEM is not only an excellent cell membrane–specific probe, but also an effective visible phototherapeutic agent activated by mild, easily obtainable and harmless light source.


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O HO

O

CH3(CH2)5Br

Br

K2CO3, DMF, 70 oC

Zn/TiCl4, THF, Reflux

HO

1 (HO)2B

H3C(H2C)5O

Br

H3C(H2C)5O CHO

Br

Pd(PPh3)4, K2CO3, THF/H2O

2

N+ Br-

3

CHO

O

N+ Br-

EtOH, reflux

TPE-MEM

N+ Br-

N+ Br-

Scheme 2. Synthesis of TPE-MEM.

Dye Synthesis and Photophysical Properties.

TPE-MEM was synthesized by the synthetic way indicated in Scheme 2. The asymmetric TPE core (1) was simply synthesized by cross McMurry coupling reaction.41 In this reaction, 1 and two byproducts were obtained at the same time. By adding excess 4-bromobenzophenone, compound 1 was produced preferentially. Thanks to the large difference in polarity, routine silica gel chromatography was used to separate 1 by


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hexane and ethyl acetate (v/v, 10/1) as elution solvent. Following simple Williamson ether synthesis reaction and Suzuki coupling reaction, 1 was transformed to 3 to introduce a flexible alkyl chain and a benzaldehyde group. Finally, com-pound 3 and 1(3-trimethylammoniopropyl)-4-methylpyridinium dibromide were refluxed in ethanol to give TPE-MEM in reasonable yield.25 Unexpectedly, TPE-MEM can be purified with silica column by using high polarity solvent mixture (DCM: MeOH = 2:1).

Routine HRMS, 1H and

13C

NMR were used to characterize the compounds 1, 2, 3

and TPE-MEM. all giving satisfactory analysis results respecting to their chemical structures (Figures S1−S8). Due to the double charges, TPE-MEM has poor solubility in nonpolar solvent, such as THF and DCM, but it is soluble in polar solvent like water, DMF, DMSO and methanol. Figure S10 shows the UV and photoluminescence (PL) spectra of TPE-MEM in aqueous solution (40 µM). The absorption maximum of TPEMEM is located at 395 nm. To facile its bioapplications, 405 nm excitation light was utilized PL measurement. Photoexcitation of the aqueous solution induces a yellow emission peaked at 590 nm, giving a large stock shift of 195 nm by reason of the


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extended conjugation as well as intramolecular charge transfer (ICT) effect from the electron-donating TPE moiety to the electron-accepting pyridinium unit.25

Figure 1. (A) PL spectra of TPE-MEM (25 M) in THF/DMSO mixtures with different amount of THF (fTHF); λex: 405 nm. (B) Plot of emission intensity of TPE-MEM at 625 nm versus fTHF in the THF/DMSO mixtures (25 M). Inset: photographs of TPE-MEM in DMSO and in THF/DMSO mixture (fTHF = 99 %) taken under 365 nm UV irradiation.

The DMSO solution of TPE-MEM (25 µM) was weakly emissive (Figure 1), which becomes strongly emissive at 625 nm at 99 % THF fraction, demonstrating typical reversed AIE features, due to its highly polar nature. Considering the molecular structure of TPE-MEM, we surmised that the micelles was formed at high


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concentrations, owing to amphiphilic feature of TPE-MEM. Taking benefit of the AIE property, the critical micelle concentration (CMC) of TPE-MEM can be measured using its own fluorescence intensity (Figure 2A). TPE-MEM is molecularly dissolved at concentrations below CMC, and thus non-fluorescent. The PL intensity increases sharply when its concentrations were above 0.01 mM. Two lines was produced by plotting the PL intensity versus the dye concentration, the intersection of which determines the CMC to be 0.02 mM. The formation of nanoaggregates at high dye concentration and CMC value are also verified by means of transmission electron micrographs and Zeta potential particle size analyzer (Figure 2B and Figure S11). Particles are not detected when TPE-MEM is molecular dissolved below CMC, while particles are observed above CMC (0.02 mM).


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90

A

B

1

10

0.1

0.2

M

M

mM

mM

60

30

300 nm

CMC = 0.02 mM 1E-3

0.01

0.1

Concentration (mM)

Particle size (nm)

PL intensity (au)

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


0 1E-3

0.01

0.1

1

Concentration (mM)

Figure 2. (A) Plot of PL intensity at 600 nm versus the concentrations of TPE-MEM. Inset: Photographs of TPE-MEM with different concentrations taken under 365 nm UV irradiation. (B) Particle sizes at various concentration of TPE-MEM measured by Zeta potential particle size analyzer. Inset: Particle size of TPE-MEM (100 M) in aqueous solution measured by TEM.

The effective diameter of the hydrated micelle is 77.4 nm, which shrinks to ~40 nm upon dehydration. The inflexion point is identical with the CMC value obtained by PL measurement. Additionally, the size of the nanoaggregates is appropriate for both in vitro cell uptake and in vivo circulation and accumulation in tumor.


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Specific Cell Membrane Imaging

Prior to cell imaging, the cell viability or cytotoxicity of TPE-MEM was evaluated on HeLa cells using MTT assay. The cells were treated by diverse concentration (0, 2.5, 5, 10, 20 M) of TPE-MEM for 6 h in dark incubator at 37 ℃and then incubated in fresh culture medium for another 18 h for cell proliferation (Figure S12). The result shows that TPE-MEM is generally noncytotoxic at the concentrations up to 20 M in dark. Besides, the quantum yield of TPE-MEM (5 μM) in DMSO-water solution (99% water fraction) was determined to be 0.5%, which would be favorable for wash-free imaging due to the very low background signal. Then TPE-MEM was applied to cell imaging. Cervical cancer HeLa cells were incubated with 5 M TPE-MEM for 10 min at room temperature, and excess dyes were washed away by PBS solution. As shown in Figure 3A-C, TPEMEM stains specifically the membrane region surrounding the HeLa cell. The thin layer structures of the cell membrane are lighted up TPE-MEM and give bright yellow emission. The boundary of the vicinal cells could be clearly observed and defined.


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A

B

C

10 m

D

E

F

20 m

Figure 3. (A) Laser scanning confocal microscope, (B) brightfield and (C) merged images of HeLa cells stained with TPE-MEM (5 M) for 10 min at room temperature (λex = 405 nm and λem = 610±65 nm). Laser scanning confocal microscope images of HeLa cells co-stained with (D) TPE-MEM (5 M, λex = 405 nm and λem = 550±70 nm) and (E) Cell MaskTM Deep Red plasma membrane stain (C10046, 5 g/mL, λex = 633 nm and λem = 685±55 nm), (F) merged image of A/B. Overlap coefficient of A and B is calculated to be 72%.

To evaluate the specificity of TPE-MEM, CMDR, a commercially available cell membrane imaging agent, was utilized for costaining. As shown in Figure 3D-F, the


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fluorescence from TPE-MEM overlap very well with that from CMDR, indicating that the emission of TPE-MEM is really from the cell membrane of the living HeLa cells (Figure 3D-F). The overlap coefficient between Figure 3(D) and 3(E) evaluated with the software of the confocal microscope (CLSM LSM7; Carl Zeiss, Germany) was 72%. The overlap coefficient is reasonably high due to the thin layer cell membrane structure and the binding competition between the two dyes. As compared with CMDR, TPE-MEM shows comparably less internalization and gives better imaging resolution of the cell membrane. What’s more, the cell microvilli are also clearly visualized by TPE-MEM. In addition to the costaining experiment, the Z-stack CLSM scanning was carried out.

The images of HeLa cell were reconstructed to an ortho image (Figure 4) and threedimentional (3D) video (SI, Video 1), which also confirm the specific targeting of TPEMEM on cell membrane.


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10 m

Figure 4. Ortho laser scanning confocal image of HeLa cells stained with TPE-MEM (5 M, λex = 405 nm and λem = 610±65 nm) for 10 min at room temperature.

The cell membranes consist primarily of a thin bilayer of amphiphilic phospholipids and show a large negative potential across the membrane. Thus, cell membrane targeting dyes are generally amphiphilic and cationic in nature.42-43 Tuning the balance of the hydrophobicity and hydrophilicity of the molecules to meet the cell membrane amphiphilicity is critical for the design of the new cell membrane staining dyes.8 TPEMEM is both suitably amphiphilic and positively charged and thus an excellent probe for specific targeting the cell membrane in living cell. The amphiphilic TPE-MEM spontaneously arrange in the phospholipid bilayer so that the long alkyl could interact


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with the “non-polar tail” of the membrane while the pyridinium part are electrostatically attracted to the negative charged “polar head” of the cell membrane (Scheme 1). Forces such as electrostatic and van der Waals interactions both contribute to the specific targeting.18,39, 42-43

Photostability is one of the most important criteria for a cell imaging stain. Several AIE dyes for specific cell staining are highly photostable.25,27,39 The propeller molecular structure and its aggregation formation could prevent the oxygen diffusion into the AIE particles which may oxidize the fluorophore and bleach its emission.27 Similar photostability results were obtained in living cell as shown in Figure 5 and video 2 in the SI. The fluorescent intensities were normalized against the initial fluorescent intensity. As shown in Figure 5, the signal loss of TPE-MEM is less than 40 % with total irradiation time of ~5 min (30 scans). Slightly decline of the signal loss and the image brightness from time 0 to 325.7 s may be due to the diffusion of the dye and movement of the cells. Figure S13 shows the PL signal loss of the dye in aqueous solution scanning for around 4 h. No obvious signal loss was observed.


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120 100

Signal loss (%)

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


80 60

325.7 s

0s

40 20 20 m

0

0

70

140

210

280

350

Laser scanning time (s)

Figure 5. Signal loss (%) in TPE-MEM (solid circle) emission with increasing time of scans. Inset: ﬂuorescent images of living HeLa cells stained with TPE-MEM (5 M) before and after continuous scanning for 325.7 s. λex = 405 nm and λem = 550±50 nm

Room-Light Induced ROS Generation.

Interestingly, bubble generation was observed during the CLSM time series scanning as shown in Figure 5 and video 2 in the SI. Obviously, upon laser scanning, the cell microvilli shrunk and disappeared, followed by cells swelling and blebbing. Clearly, the cell plasma membrane becomes discontinuous and leaky, suggesting the cell death


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was induced by the laser scanning. This phenomenon inspired us to explore the reasons behind. Phenothiazinium based molecules having a positive charge in the πconjugation system are widely used for ROS generation and phototherapy.44-45 Similar to phenothiazinium, TPE-MEM also has a positive charge in the ð-conjugation system, which may also lead to light induced ROS generation and cause the cell death. In order to prove the hypothesis, H2DCFDA, a commercial ROS fluorescence probe, was utilized for ROS detection.46 H2DCFDA itself does not emit light. Reaction with ROS could turn on its emission at 535 nm (λex = 488 nm). Surprisingly, normal white room light (LED light bulb, 4 mW/cm2) was sufficient to induce ROS generation when irradiating the solution of TPE-MEM. The three PBS solutions containing H2DCFDA, TPE-MEM and both of them were irradiated under the same conditions. The PL spectra of the samples at different irradiation time were recorded (Figure 6A, λex = 488 nm) and the peak intensities at 535 nm were plot against irradiation time (Figure 6B). In Figure 6A, in the presence of both H2DCFDA and TPE-MEM, the characteristic peak at 535 nm of oxidized H2DCFDA appeared and increased with light irradiating. The PL intensity keeps increasing even up to 120 min irradiation with room light. The PL


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intensity of the individual H2DCFDA and TPE-MEM solutions changed little under the same conditions. Similar results were obtained by using singlet probe (Figure S14), Singlet Oxygen Sensor Green (SOSG), indicating that the ROS, including singlet oxygen, indeed are generated when light irradiating on TPE-MEM, which causes the cell damage and death. 500

500 B

400

400

TPE-MEM + H2DCFDA H2DCFDA TPE-MEM

120 min

300

300

200

200 0 min

100

100

0 510

PL intensity (au)

A

PL intensity (au)

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


560

610

Wavelength (nm)

660 0

20

40

60

80

100

0 120

Irradiation time (min)

Figure 6. (A) PL spectra of H2DCFDA (1 M) in the presence of TPE-MEM (10 M) upon irradiation by room light. Excitation wavelength: 488 nm. (B) Effect of the irradiation time on the PL intensity at 535 nm of the solution containing TPE-MEM, H2DCFDA, or both.

In order to study the light wavelength dependence of ROS generations, especially the singlet oxygen generations, lasers with different wavelength (405, 488, 560 and 633


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nm) were applied to the samples containing SOSG in the absence and in the presence of TPE-MEM (Figure S15-18). Compared with other lasers, the green emission of oxidized SOSG is turned on only by the irradiation of 405 nm laser in presence of TPEMEM. The figures also show that the cell morphology has obviously changed with irradiation by 405 and 488 nm lasers containing TPE-MEM. All the results strongly indicate that even though the ROS, including singlet oxygen, can be generated by white room-light irradiation, only the blue region of the light can be absorbed by TPE-MEM and facilitate ROS generation.

However, the pathology of the cell death is still a mystery. Change in plasma membrane integrity is one of the most characteristic which could differentiate apoptosis from necrosis.47-48 During the cell necrosis, the cell swells, its membrane becomes leaky, disrupted and finally the cell exchanges substances with the surrounding environment.42-43,48 Propidium iodide (PI) is cell membrane-impermeant and as a marker for membrane integrity and necrosis. PI is commonly used to stain the nuclei of dead cell, of which the cell membrane is leaky. In our case, TPE-MEM was added to the


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culture medium with the live cell (Figure 7) for labeling. Then PI was introduced to the observation medium. Before irradiation (Figure 7A-D), only yellow light from TPE-MEM (channel I) on the cell membrane was detected. When the cells were irradiated for about 5 min (30 scans) and settled for another 5 min for PI uptaking, the cell morphology changed and red emission from PI (Channel II) was observed (Figure 7EH) in the cell cytoplasma and the nuclei inside the cell. The red emission from PI in the cell nuclei is due to the PI intercalating to the DNA which turns on the PI red emission, while the red signal in the cytoplasma may due to the nucleus lysis into the cytoplasma. In the control experiment without TPE-MEM, no red signal (channel II) was detected both before (data not shown) and after irradiation (Figure 7I-L). Merged confocal images of channel I, channel II and bright-field images of HeLa cells costained with TPE-MEM and PI at varying irradiation time were also recorded in Figure 8 and video 3 in the SI.


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Figure 7. Confocal images of HeLa cells (A-H) costained with TPE-MEM and PI (A-D) before irradiation and (E-H) after irradiation, and (I-L) stained with PI only after irradiation. (C, G, K) Bright-field and (D, H, L) merged images of A/B/C, E/F/G and I/J/K. [TPE-MEM] = 5 μM; [PI] = 3 μM. Channel I: λex = 405 nm; λem = 550±50 nm. Channel II: λex = 560 nm; λem = 620±65 nm


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Figure 8. Merged confocal images of channel I, channel II and bright-field images of HeLa cells costained with TPE-MEM and PI at varying irradiation time (A:0s, B:204s, C:432s, D:659s, E:886s, F:1113s). [TPE-MEM] = 5 μM; [PI] = 3 μM. Channel I: λex = 405 nm; λem = 550±50 nm. Channel II: λex = 560 nm; λem = 620±65 nm. The arrows indicate where clear blebbing regions observed

The red signal of PI was almost evenly turned on in the whole cell, and no clear pathway was observed. All the observations indicate that the cell membrane becomes leaky and suggest that cell necrosis was induced by light irradiation in the presence of TPE-MEM.


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All the results indicate that TPE-MEM facilitates the ROS generation under room light irradiation. In addition to its cell membrane selectivity and excellent photostability, TPEMEM could potentially serve as a phototherapeutic drug for cancer. To evaluate the phototherapeutic effect of TPE-MEM with normal room light irradiation on HeLa cancer cell proliferation, the cell viability with and without 2 hour room light irradiation in presence of different concentration of TPE-MEM was evaluated by MTT assay (Figure 9). The cell viabilities were calculated by normalizing the MTT values against the MTT value obtained from the sample without irradiation in the absence of TPE-MEM. The results in Figure 9 show that neither TPE-MEM (up to 10 M) nor room light irradiation alone exerts obvious influence on HeLa cell. However, in the presence of both TPEMEM (10 M) and room light irradiation, the cell viability drops to 47%. Large differences in cell viability were obtained between the samples with and without room light irradiation in the presence of TPE-MEM.


28

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120

Without Irradiation With Irradiation

100

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


80 60 40 20 0 0

2.5

5

7.5

10

Concentration (M)

Figure 9. Effect of TPE-MEM with and without room light irradiation on cell proliferation of HeLa cells evaluated by MTT assay.

Figure 10. Magnified images of mice with subcutaneous tumors treated with and without TPE-MEM (intratumoral injection twice a week, inject 0.1 mL of 1 mg/mL TPE-MEM in


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PBS solution in each time) and laser irradiation (473 nm laser source, 30 mW, daily exposure of laser shining for 30 min). Black circles highlight the tumor region

The white room light irradiation is mild, easily obtainable and cheap, which make the photo-therapy harmless in combination with high yield of ROS generation, photostability and low dark toxicity. All the advantages make TPE-MEM a good photosensitizer.

In Vivo PDT Effect. Finally, the in vivo PDT effect of the TPE-MEM under laser irradiation was evaluated on nude mice bearing subcutaneous melanoma tumor. Under laser irradiation (473 nm laser source, 30 mW), mice treated with TPE-MEM showed obvious tumor growth inhibition and suppression compared with those control groups with no mouse death during the treatment (Figure 10 and Figure S19). The tumor sizes of control groups augment gradually as time passed, while the tumor sizes of experiment groups were inhibited or even shrunk a little bit. Obvious scabs formed in the tumors of the mice treated with both TPE-MEM and laser irradiation, while escharosis was not observed in the control groups. The laser irradiation on the TPE-MEM induces ROS generation on


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the plasma membrane causing the cell necrosis, and thus necrotizing tumors. Therefore, tumor growth is inhibited.

CONCLUSION

In summary, an asymmetrical and amphiphilic TPE-based pyridinium salt (TPE-MEM) with AIE characteristic has been synthesized and utilized for plasma membrane staining. Thanks to its cationic and amphiphilic nature, TPE-MEM possesses high specificity to cell membranes and excellent photostability in living cell. Surprisingly, TPE-MEM induced ROS generation effectively under irradiation by normal room light, which caused the cell necrosis. These unique properties allow real-time observation of cell necrosis and phototherapeutic process in situ. The in vivo animal verified the PDT effectiveness of the TPE-MEM under laser irradiation on inhibition of tumor growth. It is anticipated that the findings on ROS generation and phototherapy will trigger new research enthusiasm and effort for creation of new AIE phototherapeutic drug for cancers. Further studies on the tumor imaging and phototherapeutic treatment of cancer in animal are ongoing in our laboratories.


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EXPERIMENTAL

Materials and Instruments

4-Bromobenzophenone,

4-hydroxybenzophenone,

1-bromohexane,

4-

formylphenylboronic acid, tetrakis(triphenylphosphine)palladium, propidium iodide (PI), 2’,7’-dichlorofluorescin diacetate (H2DCFDA) and other chemicals were purchased from Sigma-Aldrich, and used without further purification unless specified otherwise. CellMaskTM Deep Red plasma (C10046) and Singlet Oxygen Sensor Green (S36002) were obtained from Invitrogen. THF were distilled from sodium benzophenone ketyl under

a nitrogen atmosphere prior

to

use.

1-(3-Trimethylammoniopropyl)-4-

methylpyridinium dibromide was prepared following the literature method.8 NMR spectra were recorded on Bruker ARX 400 NMR spectrometers using CDCl3 and methanold4 as the deuterated solvent. High-resolution mass spectra (HRMS) were measured on a Finnigan MAT TSQ 7000 Mass Spectrometer System operating in a MALDI-TOF mode. UV-vis absorption spectra were recorded on a Biochrom UV visible spectrometer. Photoluminescence (PL) spectra were performed on a Perkin-Elmer spectrofluorometer


32

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LS 55. Particle sizes were measured on a Zeta potential analyzer (Brookhaven, ZETAPLUS). The aggregation morphology of TPE-MEM was investigated using Transmission Electron Microscopy (Japan, JEOL JEM 100CXII) at an accelerating voltage of 100 kV. HeLa cells were imaged under LSM7 DUO Confocal Laser Scanning Microscope (CLSM; Carl Zeiss, Germany). All the lasers (405, 488, 560 and 633 nm) used for irradiation during the wavelength dependence testing are powered 37 µW. For cytotoxicity experiment, the cells were treated with MTT and the absorbance was recorded on a 1420 Victor Multi-Label Counter (Perkin Elmer Life Science). Room light irradiation is carried out under a desktop lamb with an LED light bulb (Philips, 4 mW/cm2). 4-(2-(4-Bromophenyl)-1,2-diphenylvinyl)phenol (1). It was synthesized by modified procedure from the literature.26 Into a 500 mL two-necked flask equipped with a condenser

were

added

4-hydroxybenzophenone

(1.98

g,

10

mmol),

4-

bromobenzophenone (5.20 g, 20 mmol) and Zinc powder (5.88 g, 90 mmol) in 200 mL of THF in nitrogen. The reaction mixture was cooled to -78 ℃ and TiCl4 was then charged drop-wisely. The reaction was refluxed overnight with nitrogen protection.


33


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Following by cooling down to room temp, this reaction mixture was acidified by hydroxy chloride (1 M) to pH < 2. The organic mixture was moved from DCM. Following by dring with anhydrous sodium sulfate, The crude product was purified by running silica column eluting by solvent mixture (Hexane: Ethyl Acetate = 10 : 1) to give 1 as a white solid (3.1 g, 72 %). 1H NMR (400 MHz, CDCl3, δ): 7.25-7.19 (m, 2H), 7.14-6.98 (m, 10H), 6.926.86 (m, 4H), 6.60-6.55 (m, 2H), 4.885 (d, 1H, J = 22 Hz, OH);

13C

NMR (100 MHz,

CDCl3, δ): 153.662, 153.551, 142.928, 142.877, 142.852, 142.353, 142.255, 140.468, 138.241, 138.204, 135.380, 135.266, 132.362, 132.061, 132.040, 130.643, 130.269, 130.173, 127.215, 127.113, 127.018, 126.042, 125.939, 125.868, 119.625, 114.177, 113.993; HRMS (MALDI-TOF) m/z: calcd, 426.0619 [M]; found, 428.0613 [M+2H]+. (1-(4-Bromophenyl)-2-(4-(hexyloxy)phenyl)ethene-1,2-diyl)dibenzene (2). Into a 250 mL two-necked flask equipped with a condenser were placed 1 (2.4 g, 5.6 mmol) and K2CO3 (1.9381 g, 14.0 mmol). DMF (50 mL) and 1-bromohexane (1.8557 g, 11.3 mmol) were added and the reaction was stirred overnight under nitrogen at 70 ℃. After cooling down to room temperature, the mixture was extracted with DCM. The organic phase was washed with distilled water 5 times and then dehydrated with anhydrous


34

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MgSO4. The crude product was purified by a silica gel column using hexane and DCM mixture (10:1, v/v) as eluent to give 2 as a pale yellow viscous oil (2.5 g, 87 %). 1H NMR (400 MHz, CDCl3, δ): 7.257-7.200 (m, 2H), 7.140-6.988 (m, 10H), 6.935-6.876 (m, 4H), 6.678-6.620 (m, 2H), 3.923-3.862 (m, 2H), 1.784-1.725 (m, 2H), 1.473-1.422 (m, 2H), 1.363-1.314 (m, 4H), 0.922 (t, 3H, J = 5.1 Hz);

13C

NMR (100 MHz, CDCl3, δ): 157.297,

157.208, 143.082, 143.014, 142.971, 142.885, 142.455, 142.399, 140.653, 138.044, 137.998, 134.914, 134.810, 132.393, 131.838, 131.815, 130.711, 130.681, 130.267, 130.159, 127.215, 127.184, 127.098, 126.987, 125.990, 125.887, 125.814, 119.570, 113.151, 112.978, 67.243, 67.196, 30.980, 28.642, 25.112, 21.972, 13.418; HRMS (MALDI-TOF) m/z: calcd, 510.1558 [M]; found, 510.1567 [M]. 4'-(2-(4-(Hexyloxy)phenyl)-1,2-diphenylvinyl)-[1,1'-biphenyl]-4-carbaldehyde (3). Into a 250 mL two-necked flask equipped with a condenser were placed 2 (2.20 g, 4.3 mmol), 4-formylphenylboronic acid (0.77 g, 5.1 mmol), K2CO3 (2.90 g, 21 mmol) and Pd(PPh3)4 (0.30 g, 0.26 mmol) in distilled THF (80 mL) and water (20 mL) under nitrogen. The reaction was refluxed overnight under nitrogen protection. Following by cooling down to the room temperature, the mixture was removed from with DCM. The mixture was


35


Page 36 of 54

washed with distilled water several times and dried with anhydrous magnesium sulfate. The crude product was purified by running silica column eluting by solvent mixture (Hexane : DCM = 5:1) to give 3 as yellow viscous oil (1.9 g, 83 %). 1H NMR (400 MHz, CDCl3, δ): 10.032 (d, 1H, J = 4 Hz), 7.931-7.895 (m, 2H), 7.715 (t, 2H, J = 9.0 Hz), 7.694-7.380 (m, 2H), 7.172-7.047 (m, 12H), 6.991-6.930 (m, 2H), 6.656 (t, 2H, J = 8.6 Hz), 3.885 (t, 2H, J = 6.4 Hz), 1.763-1.710 (m, 2H), 1.452-1.315 (m, 6H), 0.921 (d, 3H, J = 6.4 Hz);13C NMR (100 MHz, CDCl3, δ): 191.298, 157.277, 157.191, 146.079, 143.994, 143.933, 143.251, 143.170, 140.734, 138.551, 138.516, 136.395, 135.060, 134.367, 131.929, 131.904, 131.443, 130.807, 130.777, 129.605, 129.585, 127.224, 127.142, 127.104, 126.992, 126.698, 125.953, 125.875, 125.841, 125.789, 113.100, 112.967, 67.221, 67.195, 30.984, 28.637, 25.115, 21.973, 13.426; HRMS (MALDI-TOF) m/z: calcd, 536.2715 [M]; found, 536.2722 [M]. 4-(2-(4'-(2-(4-(Hexyloxy)phenyl)-1,2-diphenylvinyl)-[1,1'-biphenyl]-4-yl)vinyl)-1(3-(trimethylammonio)propyl)pyridinium

dibromide

(TPE-MEM).

An

ethanol

solution of 1-(3-trimethylammoniopropyl)-4-methylpyridinium di-bromide (0.50 g, 1.4 mmol) and 3 (1.50 g, 2.8 mmol) was refluxed under nitrogen catalyzed by piperidine. After

cooling

to

ambient

temperature,

the

solvent

was

evaporated

using

a


36

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rotary evaporator. The crude product was purified by a silica gel column using DCM and methanol mixture (2:1 v/v) as eluent to afford a yellow product TPE-MEM (0.72 g, 59 %). 1H NMR (400 MHz, methanol-d4, δ): 8.948 (d, 2H, J = 6.8 Hz), 8.213-8.190 (m, 2H), 7.946 (6, 1H, J = 16 Hz), 7.737-7.717 (m, 2H), 7.576 (d, 2H, J = 8 Hz), 7.430-7.331 (m, 3H), 7.061-6.843 (m, 14H), 6.605-6.570 (m, 2H), 4.698 (t, 2H, J = 15.2 Hz), 3.797 (t, 2H, J = 14.8 Hz), 3.644 (t, 2H, J = 16.4 Hz), 3.240 (s, 9H), 2.663-2.581 (m, 2H), 1.693-1.619 (m, 2H), 1.421-1.262 (m, 6H), 0.871 (t, 3H, J = 14.4 Hz);

13C

NMR (100 MHz, CDCl3,

δ): 157.362, 157.260, 153.875, 143.411, 143.306, 143.250, 143.227, 143.198, 141.993, 140.990, 140.597, 138.760, 136.680, 135.055, 133.295, 131.619, 131.584, 131.140, 130.482, 128.135, 126.902, 126.855, 126.798, 126.683, 126.281, 125.676, 125.507, 125.457, 125.216, 125.115, 123.440, 121.585, 112.874, 112.700, 66.856, 61.787, 55.965, 52.066, 30.728, 30.716, 28.311, 24.791, 24.216, 21.625, 12.399; HRMS (MALDI-TOF) m/z: calcd, 791.3571 [M-Br-]+; found, 791.3570 [M-Br-]+. Cell Culture and Cell Imaging. The minimum essential medium (MEM), including 10% FBS, 100 U/mL penicillin G and 100 μg/mL streptomycin, was used to culture Hela cells in a 5% CO2, 90% relative humidity incubator at 37°C. Before cell imaging, preparation of stock solution was made by dissolving TPE-MEM in DMSO at the concentration of 10 mM. HeLa cells were cultured overnight on a cover slip install in a 35 mm Petri dish and then dyed with TPE-MEM (5 μM) for 10 min at room temperature. The cells were rinsed three times by phosphate buffer solution prior to imaging with the confocal laser


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scanning microscope. For costaining, the cells were stained with TPE-MEM (5 μM) for 5 min, and CellMaskTM Deep Red plasma (C10046, 5 g/mL) was added to the cell for another 5 min at room temperature. The cells were washed 3 times with 1× PBS before imaging under the confocal laser scanning microscopes. For cell necrosis imaging, the cells stained with TPE-MEM (5 μM) were cultured for 10 min, and then the cells were rinsed 3 times with 1× PBS. PI (3 M) was added to the cell prior to imaging under the confocal laser scanning microscopes. The cell images in channel I (λex = 405 nm; λem = 550 ± 50 nm), channel II (λex= 560 nm; λem= 620 ± 65 nm) and bright-field were taken before 405 nm laser irradiation. For The cells were irradiated with 405 nm laser for 325.7 s (30 scans). After that, the cells were settled for 5 min and imaged in different channels. A control experiment was carried out with PI (3  only. For photostability study, the cells were stained with TPE-MEM (5 μM) for 10 min, and then irradiated under 405 nm laser for 30 scans (325.7 s). For wavelength dependent studies, the cells were stained with TPE-MEM (5 μM) for 10 min, and then the cells were rinsed three times with 1× PBS. SOSG (2.5 was added to the cell prior to imaging under the confocal laser scanning microscopes. The cell images in Channel I (λex= 405 nm; λem=


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560 ± 70 nm), Channel II (λex= 488 nm; λem = 545 ± 55 nm) and bright-field were taken before 405 nm laser irradiation. The cells were irradiated with lasers (405, 488, 560 and 633 nm, 37 μW) for ~12 min (50 scans). After that, the cells were imaged in different channels for comparison. ROS and Singlet Oxygen Detection. TPE-MEM (10 μM) and H2DCFDA (1 μM) or SOSG (2.5 μM) were mixed in PBS and irradiated under a desktop lamb with an LED light bulb (Philips, 4 mW/cm2). Their PL spectra were taken at different time intervals. The PBS solutions of individual TPE-MEM, H2DCFDA and SOSG were also treated at the same way as controls. Cell Proliferation Assay. HeLa cells were seeded with the density of the 8 × 103 per well into a 96-well plate over-night before the treatment of compound TPE-MEM at different concentrations. After 6 hours of culture in a light-free incubator, the cells were rinsed 3 times with buffer solution, and then was treated by fresh culture medium for another 18 hours. MTT assay was used to evaluate cell proliferation. Simply, 10 μL of 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) solution was added into the each well. The concentration of MTT is 5 mg/mL in PSA. After incubation at 37 °C for 4


39


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hours, 100 μL of solubilization solution, which contains 10% SDS in 0.01 M HCl, was supplied to melt the purple crystals. After incubation for 12 hours, a plate reader was used to read the optical density at 595 nm. Each experiment was carried out three times. For the cell proliferation experiment under irradiation, different concentrations of TPE-MEM were added to the medium for cell culture. Then the cells were kept at room temperature with or without room light irradiation (desktop lamb with an LED light bulb, Philips, 4 mW/cm2) for 2 h in fresh culture medium and the cell were incubated for 22 h without light shining. The cell proliferation was evaluated by MTT assay following the manufacturer’s instructions as before. In vivo PDT Effect. Melanoma cells (MDA-MB435) were injected subcutaneously into 4week-old nude mice. Briefly, the cells (1 × 106) in 100 μL of PBS solution were injected subcutaneously. All procedures were agreed by the animal care advisory agency of animal and plant care facility. After cell inoculation, we randomly separated the mice into four groups. Solutions of TPE-MEM (100l) solutions were injected intratumorally twice a week. Tumor regions were treated by a 473 nm laser (Changchun New


40

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Industries, LDWL206, China) using energy at 30 mW for 30 min per day. Photographs of the mice were captured by Canon digital camera. ASSOCIATED CONTENT

Supporting Information is available free of charge. NMR and mass spectra of TPE-MEM and intermediates are supplied.

AUTHOR INFORMATION

Corresponding Author *Corresponding author: J.Q. Hou (email: [email protected]) and B. Z. Tang (email: [email protected])

Author Contributions W. Zhang and Y. H. Huang performed the biological experiments, analyzed the data and wrote the paper. Y. L. Long and E. G. Zhao designed and synthesized TPE-MEM and the intermediates. Y. N. Hong, S. J. Chen, J. W. Y. Lam and Y. C. Chen helped


41


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prepare the manuscript. H. J. Quan and B. Z. Tang helped supervise and support the research.

‡These authors contributed equally.

Notes No financial conflict of interest.

ACKNOWLEDGMENT This work was supported by the National Science Foundation of China (21788102, 81472401, 81772708), the Research Grants Council of Hong Kong (16308016, C201415G, C6009-17G, A-HKUST605/16 and N-HKUST604/14), the Innovation and Technology Commission (ITC-CNERC14SC01 and ITS/254/17), the Jiangsu Provincial Key Medical Discipline (ZDXKA2016012) and the Science and Technology Plan of Shenzhen

(JCYJ20160229205601482,

JCYJ20170818113602462

and

JCYJ20160428150429072).

ABBREVIATIONS


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AIE, aggregation-induced emission; AIEgens, AIE luminogens; CAMs, cell adhesion molecules; DCM, dichloromethane; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum,

ft, toluene fraction; PBS, phosphate-buffered saline; PL, photoluminescence; Py, pyridium/pyridinium salt; QDs, quantum dots; TICT, twisted intramolecular charge transfer; TMS, tetramethylsilane.

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Table of Contents (TOC) Graphic

A plasma membrane-specific bioprobe (TPE-MEM) with AIE feature is developed. Under white light irradiation, the TPE-MEM can generate ROS to cause plasma membrane disruption and lead to cell death. This AIE bioprobe could be a promising photosensitizer for image-guided PDT.


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Amphiphilic Tetraphenylethene-Based Pyridinium Salt for Selective

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