Targeted Bioimaging and Photodynamic Therapy of Cancer Cells with

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Targeted Bioimaging and Photodynamic Therapy of Cancer Cells with an Activatable Red Fluorescent Bioprobe Fang Hu,†,§ Yanyan Huang,†,§ Guanxin Zhang,† Rui Zhao,*,† Hua Yang,*,‡ and Deqing Zhang*,† †

Beijing National Laboratory for Molecular Sciences, CAS Key Laboratories of Organic Solids and Analytical Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of Sciences, Zhongguancun North First Street 2, Beijing 100190, China ‡ Department of Cell Biology, School of Basic Medical Sciences, Peking University, Beijing 100191, China S Supporting Information *

ABSTRACT: A new red-emissive bioprobe TPE-red-2AP2H was developed by taking advantage of the unique emission feature of tetraphenylethylene and a cancer cell-specific peptide. By responding to the target protein and the acidic microenvironment of tumor cells, activated fluorescence bioimaging was achieved with high signal-to-noise ratio and without involving mutiple washing steps. Apart from targeting the membrane-anchored LAPTM4B proteins, TPE-red-2AP2H was successfully utilized to trace the intracellular movement of LAPTM4B protein. The generation of 1O2 under visible light irradiation makes this bioprobe also promising for targetedphotodynamic therapy. By discriminating the expression level of the target protein, TPE-red-2AP2H can respond to the progression status of tumors with different photodynamic therapy effect.

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cytotoxic reactive oxygen species, especially singlet oxygen (1O2). Various agents, including porphyrins, phthalocyanines, metal complexes, and nanoparticles, have been examined as photosensitizers.39−46 However, highly selective photosensitizers are still desirable for accurate localization to minimize side effect and more efficient theraputic outcome.41 Since most photosensitizers are potential fluorophores, fluorescence imageguided photodynamic therapy is attractive for improving the theraputic accuracy.31,47 Furthermore, most photosensitizers are hydrophobic and strongly aggregate in aqueous media. This aggregation significantly reduces their photosensitizing efficacy because only monomeric species are appreciably photoactive.48−50 In this context, AIE luminogens are also attractive as efficient photosensitizers. Herein we report a dual-functional red-emissive AIE luminogen (TPE-red, Scheme 1) with which targetedfluorescent imaging of tumor cells and simultaneous photodynamic therapy are achieved. The molecular design is based on the following considerations: (1) TPE is AIE active, and the AIE characteristic is usually retained after chemical modification.19−23,51,52 (2) Incorporation of electron donating moieties (e.g., alkoxyl) and electron accepting moieties (e.g., PhC C(CN)2) in TPE-red is expected to shift the emission to the long wavelength region; thus, red-emissive AIE-luminogen can

remendous efforts have been exerted toward the development of smart molecules for the imaging detection and therapy of cancers. The major goal falls into two aspects: specificity and sensitivity. To this end, novel fluorescent agents capable of tracing cancer-related cell processes at molecular level are attracting growing interest.1−5 However, conventional fluorophores tend to aggregate in aqueous medium, thus causing the ubiquitous aggregation-caused quenching (ACQ) effect.6−11 Moreover, these “always on” fluorophores are also emissive in nontarget sites as well as the physiological buffer, leading to poor target-to-background ratios. As a result, multiple washing steps or natural clearance are required for in vitro and in vivo imaging to minimize the background interference. To address these issues, several fluorophores such as silole and tetraphenylethylene (TPE) have been found to exhibit abnormal aggregation-induced emission (AIE) behaviors: they are nonemissive or weakly fluorescent in solutions, but their fluorescence can be switched on after aggregation.7−18 More recently, such activatable fluorophores were also found to respond to binding events, and successfully utilized for biosensors19−23 and bioimaging.24−30 However, both parent silole and TPE show blue-green emission after aggregation, and it is well-known that red and near-IR emissive fluorophores are more advantageous for bioimaging.31−35 Photodynamic therapy is an emerging therapeutic modality using photosensitizers and light irradiation for the noninvasive treatment of cancer.36−38 Its therapeutic effect is activated by photoexcitation of the localized photosensitizer to generate © 2014 American Chemical Society

Received: June 6, 2014 Accepted: July 7, 2014 Published: July 7, 2014 7987

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Scheme 1. Chemical Structures and Synthetic Routes to TPE-red and TPE-red-2AP2H

and 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) were purchased from Siam (Chicago, IL). FMOC-amino acid-Wang Resin was from Advanced ChemTech (Louisville, KY). The extracellular fragment (EL2) of LAPTM4B protein was synthesized using an FMOC strategy on a PS3 automated solid-phase peptide synthesizer (Protein Technologies Inc.) and purified by HPLC. Propargylglycine conjugated AP2H peptide was provided by China Peptides Co., Ltd. Bovine serum albumin (BSA, 66.4 kDa, pI = 4.7), insulin (5808 Da, pI = 5.3), glutathione (GSH, 307.3 Da, pI = 5.9), ribonuclease A (Rnase A, ∼13.7 kDa, pI = 9.6), immunoglobulin G (IgG, 150 kDa), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC) were purchased from Sigma-Aldrich and used as received. Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (California). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was obtained from Sigma-Aldrich. Hoechst 33342 was obtained from Solarbio (Beijing, China). LysoTracker Green DND-26 was from life technologies (Thermo Fisher Scientific). Phosphate buffered saline (PBS) consisting of NaCl (137 mmol L−1), KCl (2.7 mmol L−1), Na2HPO4 (10 mmol L−1), and KH2PO4 (2.0 mmol L−1) was prepared according to the standard protocol. Ultrapure water from a Milli Q water purification system (Millipore, Bedford, MA) was used throughout. Other materials used for synthesis are commercial available and used as received. All solvents were purified and dried following standard procedures unless otherwise stated. Cells. HepG2 cells, HeLa cells, U2OS cells, and HEK293 cells were from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Cells were cultured at 37 °C and 5% CO2 in high-glucose DMEM

be expected after proper substitution of electron donating and accepting moieties.52 (3) The presence of [PhCC(CN)2] moiety may enable TPE-red to function as a photosensitizer to induce the formation of 1O2 after light irradiation. (4) The incorporation of the specific peptide AP2H (IHGHHIISVG) into TPE-red will allow TPE-red-2AP2H (Scheme 1) to be a highly selective probe for cancer cells, because AP2H can selectively bind the hydrophilic extracellular loop (EL2, PYRDDVMSVN, MW 1194.5) of a tumor-related protein, lysosomal protein transmembrane 4 beta (LAPTM4B), which overexpresses in majority of solid tumors.53−55 With the expected AIE behavior and distinct red emission, the designed TPE-red-2AP2H was successfully utilized for high-contrast fluorescence imaging of live cancer cells, including tracing the dynamic translocation of the target LAPTM4B protein and responding to the tumor acidosis. The generation of singlet oxygen by TPE-red-2AP2H was also realized in the cytoplasm of cancer cells facilitated by its good cell penetrability via LAPTM4B transportation. The targetable photodynamic therapy of TPE-red-2AP2H was demonstrated by phototoxicity to cancer cells and nontoxicity to normal cells. This work provides a new insight for the development of dual-functional AIE luminogen for the targeted bioimaging and photodynamic therapy. To the best of our knowledge, TPE-red-2AP2H is the first demonstration of dual-function AIE luminogen for both targeted-bioimaging of cancer cells and targeted-photodynamic therapy.

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EXPERIMENTAL SECTION Materials. Compound 1,56 ADPA,57 and 1-azido-3-tosyloxypropane58 were synthesized according to reported procedures. 9-Fluorenylmethoxycarbonyl (FMOC)-protected amino acids 7988

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UPLSAPO 100× oil-immersion objective (Olympus). Image processing and analysis was performed on Olympus software (FV10-ASW). For costaining assay with Hoechst, the TPE-red-2AP2H loaded HeLa cells were subjected for the incubation with Hoechst 33342 solution for 20 min. After washed with PBS, the cell samples were observed with CLSM. Hoechst 33342 was excited by a 50 mW, 405 nm Laser Head FV5-LD405-2 and collected with a band-pass filter within the range 425−475 nm. For colocalization experiments with GFP-tagged LAPTM4B protein, the cell samples were excited with a FV5-LAMAR 488 nm laser. The green-emission from GFP-tagged LAPTM4B was collected with a band-pass filter within the range 500−550 nm, and the red-emission from TPE-red-2AP2H was collected with a band-pass filter within the range 580−680 nm. For costaining assay with lysosomal tracker, the TPE-red-2AP2H loaded HeLa cells were incubated with LysoTracker Green solution (60 nM in PBS) at 37 °C for 30 min. After replacing the dye solution with PBS, the cell samples were observed with CLMS. A FV5LAMAR 488 nm laser was used as the excitation source. The green fluorescence from lysosomal tracker was collected with a band-pass filter within the range 500−550 nm, and the redemission from TPE-red-2AP2H was collected with a band-pass filter within the range 580−680 nm. The colocalization efficiency was analyzed with Olympus FV10-ASW software. The calculated overlap coefficient was in the range 0−1.0, with 0 representing no overlap between two images, whereas 1.0 indicates the perfect overlap between two images (perfect image registration). Photocytotoxicity Assays. The cells were seeded in 96well plates at a density of about 5000−6000 cells/mL and cultured for 24 h. Serial dilutions of TPE-red-2AP2H were prepared in cultured medium and added to the wells. The final concentrations of TPE-red-2AP2H ranged from 0.01 to 20 μM. After incubation in the dark for 1.5 h (37 °C, 5% CO2), the TPE-red-2AP2H-treated cells were photoirradiated for 15 min using a xenon lamp (500 W) with a filter passing light of 450 nm (power density 12 mW cm−2). As a control, a dark plate of TPEred-2AP2H-treated cells was also prepared and left in the incubator. Both the irradiated and the dark plates were subjected for a further incubation of 24 h at 37 °C (5% CO2). Cell viability was determined by MTT assay. For comparison, compound 5 was also used as the substitute for TPE-red-2AP2H to treat cells in the photocytotoxicity assay. The EC50 value for phototoxicity was calculated from sigmoidal fits of the dose response curve of TPE-red-2AP2H. MTT Assay. The viability of different cells was evaluated by the standard MTT assay. The culture medium was carefully removed, and 100 μL of freshly prepared MTT solution (0.5 mg mL−1 in culture medium) was added into each well. After incubation at 37 °C for 4.0 h, the MTT solution was removed, and 100 μL of DMSO was added to dissolve the formazan crystals. The plate was shaken for 10 min to fully dissolve formazan and homogenize. Absorbance values of the wells were read with a microplate reader at 490 nm (BIO RAD, iMark). The cell viability rate (VR) was calculated from the following equation: VR = A/A0 × 100%, where A is the absorbance of the experimental group and A0 is the absorbance from the cells cultured in serum-supplemented medium without any treatment. All data were obtained from three repeatedly parallel experiments.

supplemented with 10% fetal bovine serum (Hyclone). HEK293 cell line was used in the control experiments. Synthesis of TPE-red-2AP2H. The synthesis and characterization of 2, 3, 4, 5, and TPE-red are described in Supporting Information. Propargylglycine-conjugated AP2H peptide (9.2 mg, 7.90 μmol) and compound 5 (1.8 mg, 3.64 μmol) were dissolved in 200 μL of DMSO. Sodium ascorbate (3.1 mg, 15.65 μmol) in 5 μL of deionized water and CuBr (1.1 mg, 7.67 μmol) in 5 μL of triethylamine were added into the above mixture subsequently. After being shaken at room temperature for 5.0 h, the reaction mixture was purified with HPLC on a Kromasil C8 column (250 mm × 10 mm i.d.). TPE-red-2AP2H (2.7 mg, 0.89 μmol) was obtained as an orange-red solid in 24.4% yield. The chemical structure of TPE-red-2AP2H was characterized with highresolution mass spectroscopic techniques. HRMS (ESI, positive) m/z [(M + 2H)/2]+ calcd 1506.2697; found 1506.2916 (see Supporting Information Figure S-2). The purity was verified by HPLC on a Dikma Diamosil C18 column (4.6 mm × 250 mm) at a flow rate of 1.0 mL/min (see Supporting Information Figure S-3). Fluorescence Measurement. A stock solution of TPEred-2AP2H was prepared by dissolving the solid with DMSO to a concentration of 2.0 mM. For free TPE-red-2AP2H, the fluorescence spectra were measured directly by diluting 1.5 μL stock solution with PBS to a final volume of 300 μL. To measure the binding between TPE-red-2AP2H and different analytes, the stock solutions of each binding candidate were also prepared with PBS buffer, respectively. Then, 1.5 μL of TPE-red-2AP2H stock solution was mixed with each analyte, respectively, followed by the dilution with PBS to a final volume of 300 μL. The final concentration of the added analytes was 21 μM. After the incubation at room temperature for 60 min, the corresponding fluorescence spectrum was recorded; the excitation wavelength was 445 nm, and the emission was collected from 480 to 800 nm. Detection of 1O2 with ADPA. The generation of 1O2 was detected chemically according to literature using the disodium salt of ADPA as a 1O2 sensor.59 ADPA was bleached by 1O2 to its corresponding endoperoxide. The reaction was monitored spectrophotometrically by recording the decrease of absorbance at 378 nm. The ADPA (final concentration 100 μM) was mixed with 1.0 μL stock solution of TPE-red-2AP2H (final concentration 10 μM) in D2O to a final volume of 200 μL. The solution was irradiated with xenon lamp (500 W) with a filter passing light of 450 nm (power density 12 mW cm−2), and its absorbance at 378 nm was recorded every 2.0 min in a UV− vis spectrophotometer. Cell Lysis. Cells were lysed with NETN-100 buffer [10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1 mM EDTA, 0.05% NP40, 1 mM PMSF and protease inhibitors cocktail (P8340, Sigma)] for 30 min on ice and centrifuged at 12 000 rpm at 4 °C for 10 min. Cell lysates were collected for further binding and fluorescence assay with the bioprobe. Fluorescence Imaging. In 35 mm glass-bottomed dishes, the cells (approximately 1.0 × 105 mL−1) were seeded and cultured overnight for adhesion. After carefully washed with PBS three times, the cells were treated with TPE-red-2AP2H solution (200 μL, final concentration 10 μM). After a certain period of incubation, the cells were subjected to imaging analysis directly without further washing processes. Fluorescence imaging experiments were performed on a FV 1000-IX81 CLSM (Olympus, Japan). The objective used for imaging was a 7989

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Figure 1. (a) UV−vis absorption spectrum of TPE-red (10 μM) in THF. (b) Fluorescence spectra (λex = 445 nm) of TPE-red (10 μM) in THF/H2O mixtures with different volume fraction of H2O; inset shows photographs of TPE-red in THF and THF/water (v/v, 1:99) under light (>420 nm) illumination.

Figure 2. (a) Fluorescence spectra (λex = 445 nm) of TPE-red-2AP2H in the PBS buffer (10 μM, pH 7.4, containing 0.5% DMSO) before and after the addition of EL2. Inset shows the photographs of these solutions taken under light (>420 nm) illumination. (b) Dynamic light scattering data for TPE-red-2AP2H (10 μM) in the absence and presence of EL2 (21 μM) in PBS containing 0.5% DMSO.

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RESULTS AND DISCUSSION Synthesis. The synthesis of TPE-red and TPE-red-2AP2H is shown in Scheme 1, and details were provided in Supporting Information. Compound 1 was prepared according to the reported procedures.56 Compound 1 was first treated with nbutyl lithium at −78 °C, followed by the addition of trimethyl borate and hydrochloric acid leading to 2, which was transformed into 3 via Suzuki-coupling of 2 with benzoyl chloride. TPE-red was obtained in 92% yield by the reaction of 3 with malononitrile in the presence of titanium tetrachloride and pyridine. Removal of two methyl groups in TPE-red yielded 4, which was allowed to react with 3-azidopropyl pmethylbenzenesulfonate in the presence of K2CO3, affording 5 in 82% yield. The connection of AP2H to TPE-red was completed by the copper-catalyzed click reaction between 5 and the propargylglycine-derived AP2H, and TPE-red-2AP2H was obtained in 24.4% yield after HPLC purification. The chemical structures of TPE-red and TPE-red-2AP2H were characterized with NMR and HRMS data, and their purities were confirmed by either elemental analysis or analytical HPLC (Figures S1−3 of Supporting Information). AIE Behavior of TPE-red. Figure 1 shows the absorption and fluorescence spectra of TPE-red which absorbs in the range 360−540 nm with absorption maximum at 420 nm (εmax = 1.76 × 104 L mol−1 cm−1). Clearly, the absorption of TPE-red is hypsochromically shifted to visible region compared to the unsubstituted TPE. This is owing to the intramolecular electron donor−acceptor interaction between OMe and [PhC C(CN)2] moieties within TPE-red.52 As depicted in Figure 1, TPE-red is almost nonemissive in solution, but it becomes red-

emissive with maximum at 630 nm after introducing water to the THF solution of TPE-red; the fluorescence intensity at 630 nm increases by 170 times when the volume percentage of water is higher than 80%. Such fluorescence enhancement can be nakedeye detected as displayed in the inset of Figure 1b, where photos of TPE-red in THF and THF/water (1:99, v/v) are displayed under light (>420 nm) irradiation. Thus, TPE-red exhibits typical AIE behavior. This is probably owing to the restriction of internal rotations within TPE-red after aggregation according to previous studies.7−11 Generation of Singlet Oxygen. The incorporation of [PhCC(CN)2] moiety was expected to functionalize TPEred as a photosensitizer to induce the formation of 1O2 after light irradiation. The fact TPE-red-2AP2H also can generate 1 O2 is corroborated by measuring the absorption spectra of the mixture of TPE-red-2AP2H and disodium salt of 9,10anthracenedipropionic acid (ADPA) in D2O upon light irradiation. As depicted in Supporting Information Figure S-4, the absorptions at 358, 378, and 398 nm due to the anthracene moiety in ADPA decreased gradually after exposure the solution to 450 nm light. For instance, the absorbance at 378 nm decreased continuously by prolonging the irradiation time (Supporting Information Figure S-4b). It is known that ADPA can efficiently trap 1O2 by fast reaction with the anthracene moiety. The gradual decrease of absorptions at 358, 378, and 398 nm indicates the formation of 1O2 according to previous studies.59 The formation of 1O2 from TPE-red-2AP2H upon light irradiation can be simply understood as for normal photosensitizers: intersystem crossing from singlet excited state to the triplet state, followed by the energy transfer to the triplet of oxygen, leading to the formation of 1O2.60−62 7990

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Figure 3. (a) Variation of the relative fluorescence enhancement of TPE-red-2AP2H (10 μM) upon addition of different proteins, peptides, lipids, or their mixture: the concentration of each protein, peptide, and lipid was 21 μM; the concentration of BSA, insulin, GSH, Rnase, IgG, and PC in the mix was 21 μM; the concentration of EL2 was 21 μM. (b) Variation of the relative fluorescence enhancement of TPE-red-2AP2H (10 μM) responding to different cell lysates. I and I0 are the fluorescence intensities of TPE-red-2AP2H in the presence and absence of the corresponding proteins, peptides, lipid, mixtures, or cell lysates at 625 nm.

Fluorescence Turn-on of TPE-red-2AP2H upon TargetBinding. The presence of two AP2H residues endows TPEred-2AP2H with improved hydrophilicity. As a result TPE-red2AP2H can be dissolved in the PBS buffer [pH 7.4, containing 0.5% (volume percentage) DMSO], and the buffer solution is almost nonemissive. In order to examine the targeted-binding of TPE-red-2AP2H with LAPTM4B protein, the fluorescence of TPE-red-2AP2H was investigated after the addition of EL2, the hydrophilic extracellular loop of LAPTM4B protein. After incubation of TPE-red-2AP2H and EL2 in the buffer solution, the emission of TPE-red-2AP2H was switched on and a significantly red-emissive solution was resulted (see inset of Figure 2a); the fluorescence intensity at 625 nm was 150-fold enhanced. DLS (dynamic light scattering) data indicates that particles with the average diameter of 662 nm were formed in the buffer solution after incubation. Formation of such particles can be interpreted as the specific binding of AP2H with EL2 which initiates the aggregation of TPE-red-2AP2H.28 To explore the selectivity of TPE-red-2AP2H for targeting LAPTM4B protein, fluorescent spectra of TPE-red-2AP2H were measured after incubation with different biomolecules including bovine serum albumin (BSA), insulin, glutathione (GSH), ribonuclease A (Rnase A), immunoglobulin G (IgG), phosphatidylcholine (PC), and their mixture. As depicted in Figure 3a, either an individual biomolecule or their mixture could not turn on the fluorescence of TPE-red-2AP2H under the same condition. In particular, TPE-red-2AP2H still kept weakly emissive after incubation with phosphatidylcholine (PC) being a major component of cell membrane (Figure 3a). This result manifests that the interference from lipid can be avoided during imaging the membrane-located LAPTM4B protein. However, the fluorescence was significantly enhanced when the mixture of biomolecules contained EL2. These results verify that TPE-red-2AP2H can specifically discriminate EL2 from other biomolecules. In order to further demonstrate the high selectivity of TPEred-2AP2H toward LAPTM4B protein in a complex biosystem, the fluorescence variation for TPE-red-2AP2H was examined in the presence of different cell lysates. As depicted in Figure 3b the treatment of HEK293 cell lysate with TPE-red-2AP2H led to negligible fluorescence due to the lack of LAPTM4B protein in HEK293 cells. In comparison, large fluorescence enhancement was detected after incubation with the EL2-spiked HEK293 cell lysate. Moreover, the fluorescence of TPE-red-2AP2H was also switched on after treatment with the HeLa cell lysate. Since the

target LAPTM4B protein is highly expressed in HeLa cells, TPE-red-2AP2H specifically recognizes the EL2 site on LAPTM4B, resulting in the inhibition of internal rotations within this bioprobe and thus turning on the fluorescence. Targeted-Bioimaging of Live Cancer Cells. Since LAPTM4B protein is a unique broad-spectrum biomarker overexpressed in majority of solid tumors, three cell lines from different origins (hepatoblastoma cell line, HepG2; cervical cancer cell line, HeLa; osteosarcoma cell line, U2OS) were incubated separately with TPE-red-2AP2H on ice for 60 min to demonstrate its ability to specifically image cancer cells. As observed by CLSM (confocal laser scanning microscopy), bright red-fluorescence images were observed on the surfaces of three cancer cells, but no images from their interiors were detected (Figure 4). Z-stacking scan was further employed to analyze the

Figure 4. CLSM images of HepG2 cells, HeLa cells, U2OS cells, and HEK293 cells after incubation with TPE-red-2AP2H (10 μM) in neutral (pH 7.4) and acidic (pH 5.5) environments, respectively; the scale bar is 20 μm. TPE-red-2AP2H was excited with a 488 nm laser, and the emission was collected with the 580−680 nm filter.

spatial distribution of TPE-red-2AP2H. When scanned at the cross sections of the cells along the Z-axis, the shape of circular fluorescence changed continuously and was identical with the borders of these cells (see Supporting Information Figure S-5). These fluorescent images confirm that molecules of TPE-red2AP2H were bound to the respective whole cell membrane. For comparison, HEK293 cells from normal human kidney were also incubated with TPE-red-2AP2H under the same condition, and barely any fluorescent images were detected as shown in Figure 4. Notably, owing to the AIE characteristics of this redemissive luminogen, the imaging of cancer cells with TPE-red2AP2H can be carried out without any washing step. Fluorescence from background was negligible, and the average 7991

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Figure 5. (a, b) Fluorescence images for costaining of HeLa cells with TPE-red-2AP2H and a nucleus tracker Hoechst 33342, and fluorescence intensity profiles along the yellow lines after incubation at 0 °C (a) and 37 °C (b). (c) Colocalization of TPE-red-2AP2H with GFP-tagged LAPTM4B protein in HeLa cells treated with TPE-red-2AP2H at 37 °C. (d) Costaining of TPE-red-2AP2H (10 μM) with lysosomal tracker Green in HeLa cells. The scale bar is 10 μm. Hoechst 33342 was excited with a 405 nm laser, and the emission was collected with the 425−475 nm filter. GFP-tagged LAPTM4B protein, LysoTracker Green, and TPE-red-2AP2H were excited with a 488 nm laser; the 500−550 nm filter was used for GFP-tagged LAPTM4B and LysoTracker, whereas the 580−680 nm filter was utilized for TPE-red-2AP2H.

running across the cell (Figure 5a). In the intensity profile, red-emissions were only detected at the two joint points between the yellow line and the cell membrane. However, when the incubation temperature increased to 37 °C, red-emissive image appeared around the blue-stained nucleus inside the cell as depicted in Figure 5b. In the intensity profile, redfluorescence was detected as multiple peaks scattered close to the blue-emission signals (due to Hoechst). These results clearly confirm the intracellular delivery of TPE-red-2AP2H into cells via the specific binding of AP2H and LAPTM4B protein. It is noticeable that no red emission was detected from the nucleus. This agrees well with the fact that LAPTM4B protein is not located at the nucleus.53 For a clearer demonstrate the intracellular tracking of LAPTM4B protein by TPE-red-2AP2H, fluorescence colocalization assay was carried out. GFP (green fluorescent protein) was used to tag LAPTM4B protein in HeLa cells by standard molecular cloning technique and verified by western blot (Suporting Information, Figure S-11).66,67 Thus, the green fluorescence from GFP can be used to indicate the location of LAPTM4B protein. After incubation of these GFP-taggedLAPTM4B HeLa cells with TPE-red-2AP2H at 37 °C, both red- and green-emission images were observed (Figure 5c); the red image should be due to TPE-red-2AP2H, whereas the green image should come from the GFP-tagged LAPTM4B protein. Importantly, the yellow image was formed when the red- and green-emission images were merged as shown in Figure 5c. The overlap coefficient between the red and green images was measured to be 0.95 (with a value of 1.0 for perfect overlap),

signal-to-noise ratio is as high as 930. Competitive binding assay was also carried out to confirm that such fluorescence turn-on is attributed to the specific binding of AP2H residues of TPE-red2AP2H with LAPTM4B proteins on the membranes of cancer cells (see Supporting Information, Figure S-6). It is known that solid tumors are usually associated with low extracellular pH.63−65 For this reason the respective cancer cells were also incubated with TPE-red-2AP2H in the buffer solutions with pH 5.5. To our delight, brighter red-fluorescence images were detected for three cancer cells after incubation with TPE-red-2AP2H at pH 5.5, in comparison with those at pH 7.4 as shown in Figure 4. The average signal-to-noise ratio is 1530. This may be attributed to the fact that AP2H contains multiple basic histidine residues which may allow AP2H to bind LAPTM4B protein more strongly in acidic conditions. A similar situation was described for TPE with AP2H residue for fluorescent imaging of cancer cells.28 Thus, TPE-red-2AP2H can simultaneously target LAPTM4B proteins and sense the characteristic low-pH microenvironment of tumor cells. This advantage will allow TPE-red-2AP2H to be useful in more specific and accurate tracing of live cancer cells. Apart from targeting the membrane-anchored LAPTM4B proteins, TPE-red-2AP2H was further used to trace the intracellular movement of LAPTM4B protein. Figure 5 shows the costaining assay images with TPE-red-2AP2H and blueemissive Hoechst 33342 (nucleus indicator) at 0 and 37 °C. Red-emission was detected from the cell membrane, whereas blue-emission appeared from the nucleus region at 0 °C. The emission intensities were measured along the yellow line 7992

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Figure 6. (a) Comparison of cell viability for HeLa cells, U2OS cells, and HEK293 cells under different conditions; [TPE-red-2AP2H] = [compound 5] = 10 μM. (b) Variation of cell viability for HeLa cells vs the concentration of TPE-red-2AP2H in the incubation solution. (c) Comparison of cell viability for transfected HEK293 cells with low (L), moderate (M), and high (H) expression levels of LAPTM4B protein.

subsequently exposed to light irradiation (450 nm, 12 mW cm−2) for 15 min. Dramatic changes in cell morphology were detected; the cells became smaller, round up and detached as illustrated by light microscopy images shown in Supporting Information Figure S-10a. Such morphological shrinking and deformation indicate cell apoptosis. For quantitative evaluation, cell viability was calculated by standard MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) assay. The absorption intensities of formazan solutions were measured and normalized to those of untreated cells (Supporting Information). The cell viability was measured to be 20% for HeLa cells after incubation with TPE-red-2AP2H and further light irradiation as depicted in Figure 6a. In comparison, no damages occurred to HeLa cells that were treated with TPEred-2AP2H without light irradiation (Figure 6a and Supporting Information Figure S-10), suggesting no dark cytotoxicity of this bioprobe. Cell viability was kept high, and no cell morphological damage occurred under the same light irradiation for HeLa cells that were not incubated with TPE-red-2AP2H (Figure 6 and Supporting Information Figure S-10). Also, cellular death was not detected under light irradiation for HeLa cells that were treated with compound 5 (Scheme 1). This result manifests that the presence of AP2H residue in TPE-red-2AP2H is beneficial for the photodynamic therapy. Considering the broad expression of LAPTM4B protein in different solid tumors, U2OS cells were treated with TPE-red2AP2H and light irradiation under the same conditions as for HeLa cells. As depicted in Figure 6a, obvious cellular death was also detected, and the cell viability for U2OS cells was 37%. Thus, TPE-red-2AP2H also displays effective photodynamic therapy toward U2OS cells. In comparison, no damage occurred to normal cells (HEK293) that entail no LAPTM4B protein after the same treatment with TPE-red-2AP2H and light irradiation (see Figure 6a and Supporting Information Figure S-

indicating the good colocalization of the green-emission image with the red one. This result reveals that TPE-red-2AP2H is localized together with LAPTM4B protein after internalization, and thus, this bioprobe can be employed for intracelular tracking of LAPTM4B protein. The possible interaction between GFP protein and TPE-red-2AP2H can be ruled out because no redemission image was detected after incubation of TPE-red2AP2H with GFP-tagged cells without LAPTM4B protein (Supporting Information Figure S-7). In addition, costaining assay with lysosomal tracker (LysoTracker Green) was performed by incubation with TPEred-2AP2H loaded HeLa cells. As displayed in Figure 5d, the red-emissive image from TPE-red-2AP2H was well overlapped with the green-emissive image from lysosomal tracker. This clearly indicates that TPE-red-2AP2H accumulated in lysosome after binding with LAPTM4B protein and further trafficking into cancer cells. This observation is indeed consistent with previous studies that LAPTM4B protein is intracellularly sorted to lysosome and endosome.53,66,67 To examine the photostability of the bioprobe, continuous scanning was performed on cancer cells stained with TPE-red2AP2H. After 60 scans, the signal loss of TPE-red-2AP2H stained cells was only 12.8% (Supporting Information, Figure S8), while FITC labeled AP2H resulted in 58.3% signal loss only after 20 scans. Such good photostability provides TPE-red2AP2H with additional advantage for real-time monitoring LAPTM4B proteins in live cells and target-fluorescence imaging of live cancer cells. Targeted-Photodynamic Therapy with TPE-red2AP2H. Apart from targeted-bioimaging of cancer cells, TPEred-2AP2H can also be utilized for photodynamic therapy. After incubation of HeLa cells with TPE-red-2AP2H at 37 °C for 1.5 h, bright red-emission images were detected in cytoplasma (see Supporting Information Figure S-9), indicating the efficient uptake of TPE-red-2AP2H. The stained HeLa cells were 7993

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of cancer cells and photodynamic therapy. The bright red fluorescence of TPE-red-2AP2H was switched on by targeting the LAPTM4B protein, and this bioprobe was more sensitive to the characteristic low-pH microenvironment of tumor cells. Moreover, subcellular localization and intracellular translocation of LAPTM4B protein were also observed with TPE-red2AP2H. Note that the targeted-bioimaging of cancer cells with TPE-red-2AP2H can be carried out without involving multiple washing steps. As a photosensitizer, TPE-red-2AP2H was able to induce the formation of 1O2 within cancer cells under visible light irradiation, leading to effective cell damage. Notably, by recognizing LAPTM4B protein, such photodynamic therapy effect of TPE-red-2AP2H toward cancer cells can be tuned according to the expression level of LAPTM4B protein and thus the progression status of tumors, whereas it exhibits no phototoxicity toward normal cells. To conclude, TPE-red2AP2H is dual-functional for both target-bioimaging of cancer cells and target-photodynamic therapy. These results further demonstrate that TPE-red is a robust red-emissive luminogen which promises for wide applications in biological studies after conjugation with appropriate biomolecules.

10). Thus, TPE-red-2AP2H exhibits no phototoxicity toward normal cells. On the basis of the above observations, the photodynamic therapy effect of TPE-red-2AP2H toward cancer cells can be understood as follows: the specific binding of TPE-red-2AP2H with LAPTM4B protein can inhibit the internal rotations within the chromophore, and thus, the deactivation pathways for the excited state are reduced; as a result TPE-red-2AP2H can function as an effective photosensitizer to generate cytotoxic species such as 1O2 under light irradiation, and thus, cancer cells can be killed as reported previously.31 As discussed above, TPEred-2AP2H can be transported by LAPTM4B protein into cancer cells and may accumulate in certain membrane organelles, such as endosomes and lysosomes;53,68 thus, the generated 1O2 can damage these organelles, and accordingly, cancer cells can be more efficiently killed. In comparison, TPEred-2AP2H cannot bind the normal cells, and accordingly, the excited state is largely quenched owing to internal rotations. Thus, 1O2 cannot be generated efficiently. This agrees with the observation that TPE-red-2AP2H shows no phototoxicity toward normal cells. The photodynamic therapy effect is also dependent on the amount of TPE-red-2AP2H used for incubation with cancer cells. As an example, HeLa cells were incubated with different concentrations of TEP-red-2AP2H ranging from 10 nM to 20 μM, and each sample was then exposed to light irradiation under the same condition. As shown in Figure 6b, cell viability decreases by increasing the concentration of TPE-red-2AP2H in incubation solution. Cell viability was about 82% when the cells were treated with TPE-red-2AP2H with a concentration of 100 nM, whereas it decreased to 17% when the concentration of TPE-red-2AP2H in the incubation solution reached 20 μM. On the basis of the variation of cell viability versus the concentration of TPE-red-2AP2H in the incubation solution, the corresponding EC50 for the phototoxicity was estimated to be 3.1 μM. It is known that the expression level of LAPTM4B protein is closely correlated with invasiveness and aggressiveness of tumors.69 Therefore, it is interesting to investigate the dependence of the photodynamic therapy effect of TPE-red2AP2H on the expression level of LAPTM4B protein. For this purpose, modified HEK293 cells expressing low, moderate, and high levels of LAPTM4B protein were constructed by transfection assay (Supporting Information) and characterized by western blot (Supporting Information Figure S-11).70,71 The photodynamic therapy effect was investigated by incubating these modified cells with TPE-red-2A2H (10 μM) separately and further exposing them to light irradiation as discussed above. On the basis of the MTT assay, the cell viability was measured to be 50%, 33%, and 11% for the modified HEK293 cells with low, moderate, and high levels of LAPTM4B protein, respectively (Figure 6c). In comparison, HEK293 cells without LAPTM4B protein remained unaffected after the same incubation and light irradiation. These results reveal that the photodynamic therapy effect of TPE-red-2A2H is related with the expression level of LAPTM4B protein which is correlated with the progression status of tumors.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization of new compounds, establishment of transfected cells, NMR and MS spectra and HPLC analysis, singlet oxygen generation, consecutive cross-section scanning data, competitive binding assay, photostability of this bioprobe, optical microscopic characterization of HeLa cells under different conditions, and western blot assay. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Author Contributions §

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by grants from National Natural Science Foundation of China (21105105, 21321003, 21135006, 21190032), Ministry of Science and Technology of China, and Chinese Academy of Sciences.

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REFERENCES

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CONCLUSIONS A new red-emissive luminogen TPE-red with typical aggregation-induced emission feature was designed and synthesized. After conjugation with LAPTM4B-targeting AP2H peptide, TPE-red was transformed into TPE-red2AP2H, which was successfully utilized for targeted-bioimaging 7994

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