From Fluorescent Probe to Anticancer Agent - ACS Publications

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Light-Induced Translocation of a Conjugated Polyelectrolyte in Cells: From Fluorescent Probe to Anticancer Agent Pan Wu, Naihan Xu, Chunyan Tan, Lei Liu, Ying Tan, Zhifang Chen, and Yuyang Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b00540 • Publication Date (Web): 13 Mar 2017 Downloaded from http://pubs.acs.org on March 15, 2017

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ACS Applied Materials & Interfaces

Light-Induced Translocation of a Conjugated Polyelectrolyte in Cells: From Fluorescent Probe to Anticancer Agent

Pan Wu,†,‡ Naihan Xu,*,§,ǁ Chunyan Tan,*,†,‡,§ Lei Liu,ǁ Ying Tan,†,‡,§ Zhifang Chen,†,‡ and Yuyang Jiang‡,┴ †

‡

Department of Chemistry, Tsinghua University, Beijing 100084, P. R. China.

The State Key Laboratory Breeding Base-Shenzhen Key Laboratory of Chemical

Biology, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P. R. China §

ǁ

Open FIESTA Center, Tsinghua University, Shenzhen 518055, P. R. China

The Key Lab in Health Science and Technology, Division of Life Science, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, P.R. China

┴

School of Pharmaceutical Sciences, Tsinghua University, Beijing 100084, P. R. China

*Corresponding authors: email: [email protected] TEL: 86-755-26036533; email: [email protected] TEL: 86-755-26036357

KEYWORDS Conjugated polyelectrolyte, autophagy, autolysosome, singlet oxygen, translocation

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ABSTRACT Dual-functional probes, which not only enable visualization of diseased cells but also induce therapeutic cellular responses, are essential to biological studies. In the current work, a conjugated polyelectrolyte, PPET3-N2, was designed and synthesized as a dual-functional probe.

The polyphenylene ethynylene terthiophene polymer backbone contributes to the

polymer’s light-harvesting property to ensure the strong fluorescence as well as photosensitization, whereas quantanary ammonium side chains interact with target organelle for localization. As a fluorescent probe, PPET3-N2 was endocytosed to lysosomes through clathrin-mediated endocytosis (CME) and macropinocytosis (MPC) pathways. Colocalization of the probe with commercial fluorescent lysosome labels confirmed that this probe localized on lysosomes with high specificity and photostability. Real-time monitoring of autolysosome formation in autophagic cells was also demonstrated, providing a viable platform for cell-based screening of autophagy inhibitors. Finally, as a photosensitizer, PPET3-N2 can efficiently generate singlet oxygen in living cells upon irradiation of white light, leading to the destruction of lysosome membrane and release of ROS and lysosomal enzymes in cytoplasma, causing cell death.


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INTRODUCTION Developing molecules that possess the function to “see”, “grab” and “poke” biological targets or processes has always been a challenge for chemists tasked with improving the study of biological systems.1 A multi-functional probe requires biomolecular identification or recognition, selective target perturbation, and signal transduction. Water-soluble conjugated polyelectrolytes (CPEs) are in a category of potential candidates to develop such multifunctional probes by their excellent optical and photosensitising properties2-3 due to their special structure features. Specifically speaking, the conjugated polymer backbone contributes to their light-harvesting property, which enables the probe to “see” by strong fluorescence, as well as to “poke” by singlet oxygen through photosensitization, whereas to “grab” for recognition and localization by ionic side chains interacting with target biomolecules or organelles. Therefore, CPEs consisting of electron-delocalized polymer backbones with ionic solubilizing side groups have attracted a significant amount of attention for over two decades.4 They have been widely studied as potential fluorescent chemosensors and biosensors for small molecules, metal ions, proteins, and DNAs on the basis of their excellent photophysical properties, as well as amplified fluorescent quenching properties.5-7 In recent years, CPEs have become a new material platform in cell imaging because of their strong fluorescence, good cell compatibility, low cytotoxicity, and excellent photostability.8-11 By nonspecific electrostatic and hydrophobic interaction with major membrane lipid components, some cationic CPEs were applied in cell-surface imaging, while other CPEs and conjugated polymer nanoparticles 3 ACS Paragon Plus Environment


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(CPNs)12-13 modified with recognition elements, such as folic acid,14-16 lipid,17 antibodies,18-19 and peptides,20-24 have been used in recognizing and differentiating tumor cells by specific cell imaging. Apart from these cell-surface imaging studies, CPEs have also been used in intracellular imaging through response to small molecules, including ATP and fluoride ions,25-26 as well as pH value fluctuations.27 Photodynamic therapy (PDT) based on reactive oxygen species (ROS) generation has been considered as an emerging strategy for treatment of cancers.28 ROS mainly include hydroxyl radicals (OH.), hydrogen peroxides (H2O2), peroxynitrites (ONOO−), superoxides (O2−) and singlet oxygen (1O2), which are constantly produced as regulators of redox homeostasis by both enzymatic and non-enzymatic reactions in living system.29 Many molecular structures have been investigated as photosensitizers from synthesized or natural small molecules, such as porphyrins, phenothiazinium, and chlorins, to nanomaterials.30 Since Whitten and co-workers first published the antibacterial activity of a poly(phenylene ethynylene) (PPE) polymer in 2005,2 cationic CPEs with backbones of PPEs, poly(phenylene vinylene)s (PPVs),3, 31-32 and polyfluorenes (PFs),33 have attracted considerable interest and have been applied as antimicrobial agents based on their generation of singlet oxygen, one type of ROS. However, because of the relatively low photosensitizing efficiency of CPEs,34 other photosensitizers, such as porphyrin derivatives and platinum complexes, have been used, either by complexation with, or direct covalent linkage to, CPEs, in order to improve ROS generation.35-36 In those systems, CPEs acted as light-harvesting materials, which can transfer the energy to photosensitizers. Thus, it remains


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challenging to use CPEs as both light-harvesting molecules and photosensitizers in PDT. Herein, we have developed a CPE-based dual-functional probe, PPET3-N2, which not only enables the visualization of autophagosomal–lysosomal fusion during late autophagy, but also acts as a potential PDT agent to induce cytotoxicity to cancer cells upon irradiation with light (Scheme 1).

Scheme 1. Illustration of PPET3-N2 as a cell imaging probe and an anticancer agent. EXPERIMENTAL SECTION Materials. Dulbecco’s Modified Eagle’s Medium was purchased from Corning. Methyl-β-cyclodextrin, or LY294002, Earle’s Balanced Salt Solution (EBSS), 4% paraformaldehyde,

chlorpromazine,

genistein,

and

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), fluorometric intracellular ROS kit (deep red fluorescence) were purchased from Sigma-Aldrich. 1×PBS buffer, LysoTracker Red and dimethyl sulfoxide (DMSO) were purchased 5 ACS Paragon Plus Environment


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from Beyotime. Primary antibodies against LAMP1 were purchased from Cell Signaling Technology. Alexa Fluor-conjugated secondary antibody was purchased from Invitrogen. PCherry-LC3 was purchased from Addgene. Lipofectamine 2000 was purchased from Life Technologies. Water used in all of the experiments was prepared on a Milli-Q water purification system and displayed a resistivity of ≥ 18.2 MΩ cm-1. The stock solution concentrations of PPET3-N2 were 550 µM, and concentrations were provided as polymer repeat unit concentration

(PRU). The stock solution was

diluted as needed to prepare solutions used for spectroscopic experiments.

Cell Culture. Human cervical carcinoma cells (HeLa cells) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 °C with 5% CO2 in a humidified incubator for 24 h. For all cell-based experiments, HeLa cells were harvested from subconﬂuent (< 80%) cultures using a trypsin-EDTA solution and then re-suspended in fresh medium. A subculture was performed every 2 days.

Real-Time Tracking of PPET3-N2 in HeLa Cells. Time-dependent fluorescence imaging of polymer in living cells was performed as follows. HeLa cells were incubated with PPET3-N2 (5 µM) in medium at 37 °C for 0.5 h, washed six times with 1×PBS buffer, and further incubated with medium for various lengths of time (0 h, 2 h, and 4 h) at 37 °C in a humidified 5% CO2 incubator. Then the cells were washed three times with 1 × PBS buffer and observed using an Olympus FV1000 confocal microscope.


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Endocytosis Inhibition Studies of PPET3-N2. For endocytosis inhibition studies, HeLa cells were seeded into a 6-well plate (~200,000/well) overnight. Then cells were treated without (control) or with the pharmaceutical inhibitors chlorpromazine (1.00 mM), genistein (1.00 mM), methyl-β-cyclodextrin (1.00 mM), or LY294002 (1.00 mM) for 30 min before PPET3-N2 treatment. HeLa cells were incubated with PPET3-N2 (5 µM) in medium at 37 °C for 0.5 h, washed six times with 1×PBS buffer, and then incubated with medium for 2 h at 37 °C in a humidified 5% CO2 incubator. After washing with 1×PBS buffer three times, cells were digested by trypsin, and then the sample was immediately analyzed on the Becton Dickinson FACS Canto II flow cytometer.

Intracellular Localization of PPET3-N2. To ensure the intracellular localization of PPET3-N2, HeLa cells were incubated with PPET3-N2 (5 µM) in medium at 37 °C for 0.5 h, washed six times with 1×PBS buffer, and then incubated with medium for 4 h at 37 °C in a humidified 5% CO2 incubator. The cells were washed three times with 1×PBS buffer. Next, the cells were stained with LysoTracker Red and anti-LAMP1, respectively, and then observed using an Olympus FV1000 confocal microscope. Confocal microscope conditions: blue channel for DAPI (excitation at 405 nm and emission 425-475 nm); green channel for PPET3-N2 (excitation at 405/488 nm and emission 500-600 nm); and red channel for LysoTracker Red and anti-LAMP1 (excitation at 559 nm and emission 575-675 nm).

Photostability Test. HeLa cells were treated with PPET3-N2 and LysoTracker Red. The dye-labelled HeLa cells were imaged by confocal microscope. Confocal



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microscope conditions: blue channel for DAPI: excitation at 405 nm and emission 425-475 nm; green channel for PPET3-N2: excitation at 405/488 nm and emission 500-600 nm; and red channel for LysoTracker Red: excitation at 559 nm and emission 575-675 nm.

Fluorescence

Imaging

of

Autolysosome.

Fluorescence

imaging

of

autophagosomal–lysosomal fusion on starvation-induced autophagy was performed as follows. HeLa cells were transfected with pCherry-LC3 vector using Lipofectamine 2000. After 4 h, cells were treated with PPET3-N2 (5 µM) in medium at 37 °C. After incubation for 0.5 h, the cells were washed six times with 1×PBS buffer and then incubated in culture medium for another 4 h. For nutrient starvation, HeLa cells were washed 3 times with 1×PBS buffer, cultured in Earle’s Balanced Salt Solution for various lengths of time (0 min, 30 min, 60min, 90 min, and 120 min) at 37 °C in a humidified 5% CO2 incubator, and then visualized by confocal microscopy. Confocal microscope conditions: blue channel for DAPI (excitation at 405 nm and emission 425-475 nm); green channel for PPET3-N2 (excitation at 405/488 nm and emission 500-600 nm); and red channel for pCherry-LC3 (excitation at 559 nm and emission 575-675 nm). PCC was calculated by Image-Pro Plus 6.0 software.

Visualization of Inhibition of Autophagosomal–Lysosomal Fusion by PPET3-N2.

HeLa

cells

were

transfected

with

pCherry-LC3

vector

using

Lipofectamine 2000. After 4 h, cells were treated with PPET3-N2 (5 µM) in medium at 37 °C. After incubation for 0.5 h, the cells were washed six times with 1×PBS buffer and then incubated in culture medium for another 4 h. For control group, HeLa


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cells were cultured with DMEM medium for 6 h. HeLa cells were also cultured with EBSS. For the experimental group, HeLa cells were treated with HCQ (30 µM, 6 h) and OC (30 µM, 6 h), respectively. DAPI was used to stain nuclei which were then visualized by confocal microscopy.

Light-Induced ROS Production of the Polymer. ROS production arrays were measured on 96-well plates (300 µL Corning) using the Tecan M1000 Pro plate reader. PPET3-N2 (10 µM, 200 µL) was added in each well of the 96-well plates, followed by addition of 1µL 1× ROS detection agent into the wells. After that, the solutions were irradiated under white light (100 mW cm-2) for 5 min, and emission intensity of ROS kit solution at 675nm was recorded every minute with the excitation wavelength of 640 nm.

Cell Viability. Cell viability was evaluated using MTT assay. HeLa cells were seeded into a 96-well plate, maintained overnight in medium containing 10% FBS, and then treated with different concentration of polymers (0 µM, 10 µM, 20 µM, 30 µM, 40 µM, and 50 µM) at 37 °C for 4 h. After that, cells were treated with white light irradiation (100 mW cm-2) for various length of time (0 min, 10 min, and 30 min). After

24

h,

20

µL

of

freshly

prepared

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (5 mg/mL in 1× phosphate-buffered saline (PBS)) were added to each well, and the wells were incubated for 4 h. The supernatant was removed, and the cells were lysed by adding 100 µL of DMSO per well. Then the optical density (OD) at 570 nm was measured by



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a microplate reader (Tecan M1000 Pro). Data was correlated to relative cell viability by the formula: Cell viability = 1 −

!

" × 100%

Imaging of Light-Induced Translocation of the Polymer. Fluorescence imaging of polymer translocation in living cells was performed as follows. HeLa cells were stained with PPET3-N2 (10 µM) in medium at 37 °C. After incubation for 0.5 h, the cells were washed six times with 1×PBS buffer and then incubated in culture medium for another 4 h. After that, cells were treated with white light irradiation (100 mW cm-2) for 10 min, and then cells were incubated with anti-LAMP1 and MitoTracker Red, respectively. DAPI was used to stain nuclei. All steps were performed at room temperature. Images were captured using an Olympus FV1000 confocal microscope.

RESULTS AND DISCUSSION

Design Principle of PPET3-N2. In the past decade, CPEs demonstrated their capability of generating ROS upon irradiation with light, through spin-forbidden intersystem crossing (ISC) to sensitize the formation of singlet oxygen 1O2. However, greater ISC efficiency is needed to improve the photosensitizing properties of CPEs. It was reported that the terthiophene unit helped to improve ISC efficiency, probably due to the heavy atom effect of sulfur atoms which enhanced spin-orbit coupling.34, 37 By incoporating terthiophene into the PPE backbone, we expect to obtain a new polymer with improved ISC efficiency. The polymer PPET3-N2 with alternating polyphenylene ethynylene and terthiophene units with quantanary ammonium side


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chains (structure shown in Scheme 1) was synthesized by a straightforward Pd-catalyzed Sonogashira coupling reaction.25

Figure 1. a) Real-time tracking of PPET3-N2 (5 µM) in HeLa cells for different time intervals, as indicated in each image. Cells were washed before imaging. Top, fluorescence images; bottom, bright-field images. b) Endocytosis inhibition study under pharmacological inhibitor treatments. Mean fluorescence intensity of HeLa cells (Control), PPET3-N2 only (5 µM), chlorpromazine (1 mM, CME), genistein (1 mM, CvME), methyl-β-cyclodextrin (1 mM, CvME/MPC), and LY294002 (1 mM, MPC) was measured using flow cytometry. Error bars represent standard deviation. Student -t test, *p ＜ 0.05, ***p ＜ 0.001.

Intracellular Localization of PPET3-N2. Our previous work showed that PPET3-N2 had a certain ability to permeate cell membrane based on its positive charge.25 In the current work, membrane permeability of the polymer was further studied at different incubation times. HeLa cells were incubated with PPET3-N2 (5 µM) for 0.5 h and then washed and incubated in culture medium for different length of time (0 h, 2 h, and 4 h). As shown in Figure 1a, a clear fluorescence of PPET3-N2 at the green channel was observed on the cell membrane at 0 h. When culture time was extended to 2 h, some PPET3-N2 was still detectable on the cell membrane, while most of it had entered the cell and localized around the perinuclear region. After 4 h of the culture, PPET3-N2 had totally accumulated around the perinuclear region. Further study on endocytosis pathways was then carried out. Hela cells pretreated with 11 ACS Paragon Plus Environment


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different pharmacological inhibitors of various endocytosis pathways were incubated with PPET3-N2 for 2 h, and the mean fluorescence intensity of live cells was monitored by flow cytometry.38-39 As shown in Figure 1b, after treatment with inhibitors, the uptake of PPET3-N2 by HeLa cells treated by clathrin-mediated endocytosis (CME) and macropinocytosis (MPC) was noticeably reduced, suggesting that the internalization of PPET3-N2 related to CME and MPC endocytosis pathways had caused the endocytosed material to eventually fuse with lysosome in living cells.40 To identify the polymer’s intracellular location, colocalization experiments of PPET3-N2 with two different lysosome stainings were further studied, among which one was a commercially available small molecular fluorescent dye, LysoTracker Red (emission peak at 590 nm), and the other was a fluorescent anti-LAMP1 (lysosome-associated marker for lysosomal membranes). It can be observed that PPET3-N2 displayed a bright green fluorescence image at the perinuclear region of the cell, which was well colocalized with LysoTracker Red (in red pseudo-color) in the merged picture (Figure 2a), with the Pearson correlation coefficient (PCC) at 0.92. However, some separation of PPET3-N2 and LysoTracker Red was observed as a result of the dye's instability. Therefore, we used anti-LAMP1 antibody, a marker for endosomal and lysosomal membranes. When HeLa cells were co-stained with PPET3-N2 and anti-LAMP1, PPET3-N2 showed even better colocalization with anti-LAMP1 than LysoTracker Red with PCC value at 0.94. These results strongly suggested that PPET3-N2 could localize on lysosomes and thus be used as a lysosomal staining fluorophore.


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Figure 2. a) Fluorescence imaging of HeLa cells costained with PPET3-N2 (5 µM), LysoTracker Red (50 nM), and anti-LAMP1 for localization analysis. Cells were washed before imaging. Probes are indicated on each image. Blue channel: DAPI. b) Loss of emission intensity of PPET3-N2 and LysoTracker, respectively, vs. the number of scans. c) Fluorescence imaging of HeLa cells stained with PPET3-N2 and LysoTracker Red before and after 15 continuous scans. The photostability of PPET3-N2 was evaluated by a photobleaching experiment. As shown in Figure 2b and 2c, fluorescence of PPET3-N2 and LysoTracker Red after 15 continuous scans remained at 73% and 63% of the initial intensity, respectively. This result suggested that the photostability of PPET3-N2 was comparable to LysoTracker Red.

Imaging of Autolysosomes for Autophagy Studies. Autophagy is a natural process of degradation of cellular components. With its significant impact on cell growth, proliferation and death, autophagy is a potential therapeutic target for applications in the treatment of cancer, neurodegeneration and other diseases.41-42 During the dynamic and multistep autophagy process, several structures of the autophagic compartment are observed, including phagophores, or isolation membranes, autophagosomes, and autolysosomes.43 Among these structures, autolysosomes have attracted significant attention because the formation of autolysosomes by the fusion of



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Figure 3. Visualization of autophagosomal-lysosomal fusion. HeLa cells expressing pCherry-LC3 were treated with PPET3-N2 (5 µM) and incubated in EBSS medium for different times, as indicated in the figure. Cells were washed before imaging. Scale bar: 5 µm. autophagosomes and lysosomes is the key step of late-stage autophagy in order to degrade the contents by lysosomal hydrolases.44-45 Thus, real-time monitoring of autolysosomes in cells is essential for studying autophagy signaling, as well as developing drugs that interfere with autophagy pathways. With the advantages of high brightness, lysosome specificity, as well as good photostability, we used PPET3-N2 to monitor the formation of autolysosomes. In our study, HeLa cells were transfected with

pCherry-LC3

to

assay

the

autophagy

biomarker

LC3

expressed

on

autophagosomes. To real-time monitor autophagosome-lysosome fusion, we examined the colocalization of pCherry-LC3 and PPET3-N2 after nutrient starvation. As shown 14 ACS Paragon Plus Environment

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in Figure 3, starvation induced a remarkable increase of pCherry-LC3 red puncta, indicating the formation of autophagosomes. As a control group (0 h, without nutrient starvation), no colocalization of PPET3-N2 (green dots) and pCherry-LC3 (red dots) was detected in HeLa cells. When autophagy was induced by nutrient starvation for 1 h, the red dots gradually increased and started to merge with PPET3-N2 green dots. The formation of yellow dots indicates autophagosome-lysosome fusion. When starvation time was extended to 2 h, nearly all the pCherry-LC3 puncta were colocalized with PPET3-N2, suggesting that a large number of autophagosomes were fused with lysosomes. We used the PCC value to quantify the extent of colocalization of these two signals. Consistent with confocal microscopy observations, the PCC value of strarvation-induced cells (0-2h) was gradually increased from 0.11 to 0.73, indicating

that

PPET3-N2

could

provide

real-time

visualization

of

the

autophagosomal–lysosomal fusion in autophagy studies. Besides the possibility of monitoring autophagosomal–lysosomal fusion in autophagy studies, PPET3-N2 also provided a viable platform for cell-based screening of autophagy inhibitors toward drug development. Blocking autophagosome-lysosome fusion is an important way to suppress autophagy activity. In our experiments, two known autophagy inhibitors, hydroxychloroquine (HCQ) and oblongifolin C (OC),46 were examined. HeLa cells transfected with pCherry-LC3 were treated with PPET3N2, followed by treatment with individual inhibitors. As a positive control, HeLa cells were cultured in EBSS medium to induce nutrient starvation. As shown in Figure 4,



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Figure 4. Visualization of inhibition of autophagosomal-lysosomal fusion by known inhibitors HCQ and OC. PPET3-N2 (5 µM) treated HeLa cells expressing pCherry-LC3 were incubated in DMEM medium (control), EBSS medium (positive control), HCQ (30 µM), and OC (30 µM) for 2 h. Cells were washed before imaging. Scale bar: 5 µm. starvation induced a remarkable increase of pCherry-LC3 puncta, which were well colocalized with PPET3-N2, suggesting normal autolysosome formation during starvation upon autophagy activation. Notably, we found that HCQ- and OC-treated cells exhibited a separation of pCherry-LC3 and PPET3-N2 such that the PCC value of HCQ- or OC-treated cells was significantly lower than that under starvation treatment. This result was in good agreement with previous reports that HCQ and OC could block autophagosome-lysosome fusion.46 These results suggested that PPET3-N2 enabled living cell-based screening of autophagy inhibitors.

Light-Induced ROS Production. ROS production of PPET3-N2 in vitro was quantitatively compared with PPE (structure shown in the Supporting Information) using a commercially available ROS tracker assay, whose fluorescence at 675 nm was 16 ACS Paragon Plus Environment

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proportional to the quantity of ROS in the testing system. As shown in Figure 5a, the fluorescence intensity at 675 nm for PPET3-N2 increased much more significantly than that for PPE given 0-5 min of white light irradiation. This result confirmed that the introduction of terthiophene units into PPE polymer main chain increased singlet oxygen sensitization efficiency. ROS can oxidize many different biomolecules, potentially induce damage to various cellular structures,47 and induce oxidative stress and cell death through different mechanisms, including apoptosis, necrosis or autophagy.48 On the basis of these facts, we studied the viability of the PPET3-N2-treated HeLa cells upon light irradiation. As anticipated, cytotoxicity against HeLa cells was greater under light irradiation than in dark. As shown in Figure 5b, PPET3-N2 displayed prominent phototoxicity in HeLa cells with increasing light irradiation time and PPET3-N2 concentration.

Figure 5. a) Fluorescence intensity of ROS tracker (675 nm) in the presence of PPET3-N2 and PPE upon white light irradiation (0–5 min) with an excitation of 640 nm. b) Cell viability of HeLa cells treated with PPET3-N2 after 24 hours upon 10 min and 30 min of white light irradiation. Cells without white light irradiation were used as control. Exploration of subcellular damages upon light irradiation was carried out. HeLa cells were stained with PPET3-N2 and anti-LAMP1 in order to monitor the change of 17 ACS Paragon Plus Environment


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the lysosomal membrane.49 We observed an enhanced separation of the green fluorescence (PPET3-N2) from the red (anti-LAMP1) after irradiation of white light for 10 min, with the PCC value decreasing from 0.94 to 0.18 (Figure 6a and 6b). Immunoblot analysis also showed that PPET3-N2 treatment upon light irradiation resulted in approximately 28% decrease of LAMP1 (Figure 6c, integrated option density of the western blot intensities were calculated by Image-Pro Plus software). These results strongly suggested that the lysosome membrane had been destroyed and PPET3-N2 had escaped from endo/lysosomes under light irradiation in living cells.

Figure 6. Fluorescence imaging of HeLa cells costained with PPET3-N2 (10 µM), anti-LAMP1 upon light irradiation (10 min). Cells were washed before imaging. a) control b) upon 10 min white light irradiation. Scale bar: 20 µm. c) HeLa cells costained with or without PPET3-N2 (10 µM) upon different light irradiation times (0 h, 1 h). Samples were analyzed by Western blotting for cleaved LAMP1. GAPDH was used as a loading control. Further intracellular localization of PPET3-N2 after escaping from the lysosomes


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was studied. Noting that mitochondrial membranes have a strongly negative membrane potential,50 colocalization of PPET3-N2 with mitochondria were observed using MitoTracker Red. As shown in Figure 7, colocalization between PPET3-N2 and MitoTracker Red after irradiation was higher than the control in dark, with the PCC value decreased from 0.76 to 0.40. This result indicated that most PPET3-N2 had translocated from lysosome to mitochondria after the destruction of the lysosome membrane. The destruction of lysosome in living cells would cause the release of ROS and lysosomal enzymes in the cytoplasma, leading to, in turn, cell death.

Figure 7. Translocation of PPET3-N2 in HeLa cells. HeLa cells costained with PPET3-N2 (10 µM) and MitoTracker Red upon light irradiation (10 min). Cells were washed before imaging. a) control; b) upon 10 min white light irradiation. Scale bar: 20 µm. CONCLUSION In summary, we have developed a CPE-based dual-functional probe, PPET2-N2, for visualization of autolysosome/autophagy in living cells, as well as killing cancer cells by singlet oxygen through photosensitization. This CPE was readily synthesized by conventional Sonogashira coupling, and it offered good cellular internalization and 19 ACS Paragon Plus Environment


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compatibility, excellent selectivity towards lysosomes, as well as low cellular fluorescence background interference. Therefore, PPET3-N2 could be applied as a useful research tool for tracking autolysosomes during autophagy and for developing autophagy therapies. The probe can also function as an efficient photosensitizer to generate singlet oxygen in living cells. Photocytotoxicity was induced by irradiation of PPET3-N2 by the destruction of lysosomes and release of ROS and lysosomal enzymes into cytoplasma.

ASSOCIATED CONTENT Supporting Information Available. Experimental details and additional figures, including fluorescence spectra at various pH values, confocal images, quantification of PCC values. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS This work is supported by grants from the National Natural Science Foundation of China (No. 21572115)

and

Shenzhen

Municipal

Government

(JCYJ20160301153959476

and

JCYJ20160324163734374).

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Table of Contents Graphic



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Scheme 1. Illustration of PPET3-N2 as a cell imaging probe and an anticancer agent. 99x80mm (300 x 300 DPI)

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Figure 1. a) Real-time tracking of PPET3-N2 (5 µM) in HeLa cells for different time intervals, as indicated in each image. Cells were washed before imaging. Top, fluorescence images; bottom, bright-field images. b) Endocytosis inhibition study under pharmacological inhibitor treatments. Mean fluorescence intensity of HeLa cells (Control), PPET3-N2 only (5 µM), chlorpromazine (1 mM, CME), genistein (1 mM, CvME), methyl-bcyclodextrin (1 mM, CvME/MPC), and LY294002 (1 mM, MPC) was measured using flow cytometry. Error bars represent standard deviation. Student -t test, *p ＜ 0.05, ***p ＜ 0.001. 170x54mm (300 x 300 DPI)



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Figure 2. a) Fluorescence imaging of HeLa cells costained with PPET3-N2 (5 µM), LysoTracker Red (50 nM), and anti-LAMP1 for localization analysis. Cells were washed before imaging. Probes are indicated on each image. Blue channel: DAPI. b) Loss of emission intensity of PPET3-N2 and LysoTracker, respectively, vs. the number of scans. c) Fluorescence imaging of HeLa cells stained with PPET3-N2 and LysoTracker Red before and after 15 continuous scans. 150x42mm (300 x 300 DPI)


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Figure 3. Visualization of autophagosomal-lysosomal fusion. HeLa cells expressing pCherry-LC3 were treated with PPET3-N2 (5 µM) and incubated in EBSS medium for different times, as indicated in the figure. Cells were washed before imaging. Scale bar: 5 µm. 80x100mm (300 x 300 DPI)



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Figure 4. Visualization of inhibition of autophagosomal-lysosomal fusion by known inhibitors HCQ and OC. PPET3-N2 (5 µM) treated HeLa cells expressing pCherry-LC3 were incubated in DMEM medium (control), EBSS medium (positive control), HCQ (30 µM), and OC (30 µM) for 2 h. Cells were washed before imaging. Scale bar: 5 µm. 80x78mm (300 x 300 DPI)


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Figure 5. a) Fluorescence intensity of ROS tracker (675 nm) in the presence of PPET3-N2 and PPE upon white light irradiation (0–5 min) with an excitation of 640 nm. b) Cell viability of HeLa cells treated with PPET3-N2 after 24 hours upon 10 min and 30 min of white light irradiation. Cells without white light irradiation were used as control. 170x60mm (300 x 300 DPI)



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Figure 6. Fluorescence imaging of HeLa cells costained with PPET3-N2 (10 µM), anti-LAMP1 upon light irradiation (10 min). Cells were washed before imaging. a) control b) upon 10 min white light irradiation. Scale bar: 20 µm. c) HeLa cells costained with or without PPET3-N2 (10 µM) upon different light irradiation times (0 h, 1 h). Samples were analyzed by Western blotting for cleaved LAMP1. GAPDH was used as a loading control. 98x85mm (300 x 300 DPI)


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Figure 7. Translocation of PPET3-N2 in HeLa cells. HeLa cells costained with PPET3-N2 (10 µM) and MitoTracker Red upon light irradiation (10 min). Cells were washed before imaging. a) control; b) upon 10 min white light irradiation. Scale bar: 20 µm. 80x41mm (300 x 300 DPI)


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