A Dual Anticancer Efficacy Molecule: A Selective Dark Cytotoxicity

Oct 17, 2016 - Unlike traditional binary nanostructures that construct chemotherapy drugs and photodynamic therapy photosensitizers, we introduce a ...
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A Dual Anticancer Efficacy Molecule: A Selective Dark Cytotoxicity Photosensitizer Jyun-Wei Chen† and Cheng-Chung Chang*,‡ †

Department of Chemical Engineering, National Chung Hsing University, Taichung 402, Taiwan Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan



S Supporting Information *

ABSTRACT: Unlike traditional binary nanostructures that construct chemotherapy drugs and photodynamic therapy photosensitizers, we introduce a molecule with a chemo-photodynamic dual therapy function. A water-soluble aggregation-induced emission enhancement (AIEE) fluorogen, NV-12P, was designed and synthesized based on asymmetric 1,6disubstituted naphthalene and can generate particular reactive oxygen species to undergo type I photodynamic therapy under irradiation. Furthermore, this compound can specifically localize in mitochondria and, after biological evaluation, can cause mitochondrial dysfunction and potent cytotoxicity to cancer cells but not normal cells. We conclude that this compound is a potential dual-toxic efficacy molecule because it exhibits selective dark cytotoxicity and efficient photodamage in cancer cells. Additionally, we also supported the optimal combinational treatment course for the best chemo-phototherapy efficacy. KEYWORDS: photosensitizers, aggregation-induced emission enhancement, reactive oxygen species, mitochondrial dysfunction, chemo-phototherapy



INTRODUCTION Photodynamic therapy (PDT), consisting of a photosensitizer (PS), a light source, and oxygen, is a new emerging cancer therapy method that promises local and minimally invasive treatment of various cancers.1 In principle, an excited photosensitizer can undergo type I (electron transfer pathway, producing superoxide radicals and hydroxyl radicals, for example) and/or type II (energy transfer pathway, producing singlet oxygen) reactions to generate reactive oxygen species (ROS) upon light irradiation followed by necrosis and/or apoptosis of exposed cells.1,2 In general, most biological results in PDT studies infer that the singlet oxygen formation of type II ROS is the primary response3 and conclude that type I and II PDT are two competing mechanisms. However, some studies showed that type I and II pathways can take place simultaneously.4,5 Recently, we prepared and verified that phenothiazine derivatives, precursors of methylene blue, and borondipyrromethene (BODIPY) derivatives may simultaneously perform type I and II PDT.6,7 Moreover, we found that the carbazole derivative BMVC presents only type I PDT behavior rather than generating singlet oxygen.8 One strategy to promote PDT efficiency is to control the intracellular localization of the photosensitizer, which is known to influence the cell death mechanism strongly. The biological PDT efficacy to tumors of most sensitizers depends on their efficient cell membrane permeabilities and efficient targeting to specific intracellular organelles.7 Thus, target ability is crucial to consider the rational design to improve PDT efficiency. © XXXX American Chemical Society

Therefore, the strategy will achieve selective photodamage, which is a major criterion for evaluating biological efficacy of PDT reagents. The functions of mitochondria include apoptosis, cell redox, calcium signaling, molecular metabolism, and energy production of intracellular biological processes.9,10 Photodamage of mitochondria is expected to cause cell apoptosis in PDT treatment, and eventually, conjugate mitochondria-targeting functions to photosensitizers or nanocarriers loaded with therapeutic reagents became popular strategies to generate mitochondria-targeted PDT.11−13 A more advanced strategy to maximize and potentiate PDT efficiency is combinational therapy, which uses both PDT and chemotherapy, thus utilizing the synergistic effect of the two strategies.14,15 Numerous studies have demonstrated that PDT is associated with chemotherapeutic agents, antioxidants, immunomodulatory agents, receptor inhibitors, surgical techniques, or even radiotherapy for cancer therapy based on nanobiotechnologies.16,17 In principle, a multifunctional nanostructure serves as a carrier platform to allow the photosensitizer and chemotherapy drug to coload in the platform and then to assemble to become an effective drug delivery nanohybrid system, improving the uptake and reducing the therapeutic doses of the photosensitizer and anticancer drug and resulting in minimized side effects.18−20 In addition, the Received: June 25, 2016 Accepted: October 17, 2016 Published: October 17, 2016 A

DOI: 10.1021/acsami.6b07715 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces Scheme 1. Synthesis Procedure of NV-12Pa

a

(i) 1,12-Dibromododecane, KOH, KI, ethanol, reflux overnight. (ii) Piperidine, K2CO3, THF/H2O = 30/1, reflux overnight. (iii) 4-Vinylpyridine, Pd(OAc)2, (o-tol)3P, MeCN/Et3N = 2/1, reflux 3 days. (iv) CH3I, acetone, reflux overnight.

Figure 1. FON illustrations for 10 μM NV-12P compounds. (a) Absorption (left) and emission (right) spectra variations with aqueous solutions that contained 0, 25, 50, 75 and 100% THF (excitation (ex) = 400 nm). The inset shows the relatively visible emission photographs under UV light (365 nm). (b and c) TEM images prepared from 75 and 0% THF mixed aqueous solutions, respectively. (d) Fluorescent microscopy images prepared from the 75% FON solution in panel a (cube: ex, 390/10 nm bandpass (bp) filter; emission (em), 410 nm long pass (lp) filter).

apoptosis, in one molecule to implement selective dark cytotoxicity and phototoxicity.

combination of PDT and chemotherapy can induce antitumor immunity.21,22 As expected, successful multiple therapeutic approaches may provide new and encouraging methods in the development of versatile cancer therapy and may even be highly effective for their ability to impact multiple disease pathways. However, most of the performed studies consist of at least two organic/ inorganic molecules developed into binary or ternary nanosystems. Evaluating drug loading and releasing courses in an optimally controlled manner to afford a high efficiency is challenging.23 Thus, we sought to develop a dual anticancer efficacy molecule that can induce apoptosis in target cancer cells in the dark and photodamage in cancer cells when exposed to irradiation. In this respect, we designed a strategy that combines two therapy functions, AIEE (aggregation-induced emission enhancement) PDT and mitochondria-targeting



EXPERIMENTAL SECTION

General Information. The chemicals, instruments, methods for optical properties investigation (UV−vis absorption and fluorescence spectrometer), nanoparticle investigation (TEM), PDT measurements, cell culture conditions with and without compound, and the cellular fluorescence images used in this study are same as those described in our previous publications.6,24,25 Assay and Measurement. The phototoxicity,6,7 cell death morphology,6 and MTT assay24 were similar to those previously reported. Herein, the only changes are the concentration and treatment time periods. All of the assays were performed in three individual runs, and the average result is presented. The procedure of dual cell staining of the tracker and compound were similar to those in the previous report.24 Herein, the only change is the tracker. B

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ACS Applied Materials & Interfaces Colony Formation Assay. Cells were seeded into a 6 cm tissue culture plate with 1 × 103 cells in each plate for 24 h, and each desired compound at different concentrations was added for 5 d. After that, the cells were washed twice with PBS and fixed with 4% formaldehyde for 2 h at room temperature. After fixation, the cells were washed twice with PBS and stained with 0.01% crystal violet (Sigma, United States) in 25% methanol for 24 h. Cells were washed twice with PBS and counted. Measurement of ROS and PDT. The light source came from a 20 W xenon lamp that passes through a 400−700 nm mirror module to generate a light power of 20 mW/cm2 of white light (measured on the dish surface). The singlet oxygen quantum yield and the ROS evaluation from the compound were determined using 1,3diphenylisobenzofuran as the scavenger in DMSO and using TEMPO-9ac and H2DCF-DA as the detector in DD water in the photosteady state, respectively. Synthesis. Details of the synthesis of NV-12P are in the Supporting Information.



RESULTS AND DISCUSSION Molecular Design. As mentioned above, the functional group vinylpyridine exhibited the potential to generate

Figure 4. Concentration-dependent phototoxicities of HeLa cells, CL1-0 cancer cells, and MRC-5 normal cells treated with and without (/Co) NV-12P for 24 h followed by phototreatment. Cells were irradiated with (a) 400−700 nm and (b) UV-A+ 400−700 nm light sources (average 20 mW/cm2) for 50 min and incubated overnight three times. Cell death was assessed overnight. Each data point represents the average of three separate experiments, and the irradiation condition details are described in the Experimental Section.

Figure 2. (a) Fluorescent images of HeLa cells stained with NV-12P (5 μM) for 30 min (cube: ex, 390/10 nm; em, 410 nm lp filter). (b) Several selected fluorescent and bright-field photodamage images from a real-time video of HeLa cancer cells incubated with 10 μM NV-12P for 12 h and then irradiated with a 390/10 nm light source from fluorescent microscopy. The yellow arrows in panel b indicate growth of a bleb.

particular ROS when it became the cationic form vinylpyridinium, particularly when it was conjugated with an electron donor core scaffold.8 In addition, N-dodecyl-Nmethyl-piperidinium helped its conjugates accumulate in mitochondria and facilitate DNA binding molecules to interact with mitochondrial DNA (mtDNA). Consequently, cancer cell death occurs without damaging normal cells,26,27 which exhibits

Figure 3. Release of ROS monitored by H2DCF-DA. (a) Fluorescent spectral growth at 525 nm H2DCF-DA (10 μM) with mixed with 10 μM NV12P upon time-dependent irradiation (400−700 nm). Excitation wavelength: 495 nm. Inset shows the fluorescent intensity curves of (i) the mixing solution (at 525 nm), (ii) pure NV-12P (at 585 nm), and (iii) pure H2DCF-DA (at 525 nm). (b−d) Time-dependent irradiation (ex, 390/10 nm) fluorescent microscope images of the variations of HeLa cells incubated with 5 μM NV-12P for 10 h and then stained with H2DCF-DA (5 μM) for 30 min. (b) Bright-field and (c) fluorescence images from the UV cube (ex, 390/20 nm; em, 410 nm lp filter; collect emission of NV-12P). (d) Fluorescence images from the blue cube (ex, 470/20 nm; em, 515 lp filter; collect emission of DCF). C

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H2O/THF mixing solutions are generally greater than those in pure H2O and pure THF, this fluorophore may exhibit selfaggregation and accompanied emission enhancement due to the AIEE effect.29−32 Figure 1a reveals that the fluorescence intensities (570−590 nm) for NV-12P reached their maximum values in a 75% THF volume fraction of the aqueous solution. The TEM image, prepared from the same solution, clearly showed fine spherical-shaped nanoparticles (Figure 1b) with the mean diameter of FONs ranging from 5 to approximately 100 nm. Interestingly, in aqueous solution, we also observed similar patterns under TEM with larger FONs ranging from 100 to approximately 250 nm (Figure 1c). Results from previous studies suggested that NV-12P may serve as a surfactant-like molecule due to their amphiphilic characteristic, which may further self-assemble to form micelle-like nanoparticles.24,33 Under this aggregated condition, the thermal relaxation of the excited state was suppressed due to the restriction of intramolecular rotation, and then AIEE was achieved.34 Thus, we observe the water-soluble fluorescent property of NV-12P. Furthermore, Figure 1d shows that bright FONs can be easily prepared after immediate removal of the mixing solvent (75%, v/v, THF/H2O), and the bright spots were observed in the solid state under fluorescence microscopy. It was noted that the synthetic product powder showed no fluorescence when crystallizing from other organic solvents. Nevertheless, we can now prepare a fluorescent aqueous solution and a fluorescent solid powder from the FON condition. Cellular Permeability and New ROS. On the basis of the molecular design strategy, we initially assessed the cellular permeability of compound NV-12P. Figure 2a displays a characteristic mitochondrial localization pattern (merging well with the commercial mitochondria-specific tracker MitoTrackers Red CMXRos, Figure S2), revealing that the N-dodecyl-Nmethyl-piperidinium chain of NV-12P can effectively drive this compound to accumulate on mitochondria. After that, we examined whether NV-12P can be used as an intracellular photosensitizer. Figure 2b illustrates the several extracted photographs from real-time fluorescence microscope of HeLa cells incubated with NV-12P. The photodamage result is clear that plasma membrane bleb formation35 (as indicated by arrows) and acute cell death occurred under continuous irradiation with a light source from a 390 ± 20 nm cube. Here, we also observed that photobleaching of compound NV-12P occurs with increasing irradiation time, resulting in weaker emission signals. There are a number of spectral approaches to measure ROS; specifically, the fluorescence ROS probes are more suitable in microscopic imaging. Here, DPBF (1,3-diphenylisobenzofuran) is known as the singlet oxygen acceptor, which belonged to the type II mechanism of PDT. The disappearance of the 410 nm absorbance band is the detection criterion once the photosensitizer-generated singlet oxygen reaction was achieved.36 In addition, the hydroxyl radicals and superoxide probe TEMPO9-ac (4-((9-acridinecarbonyl) amino)-2,2,6,6-tetramethylpiperidin-1-oxyl) is known as an indicator for a type I-progressed photosensitizer, which can capture radicals and results in fluorescence enhancement (λ ex/em = 358/440 nm).37 We find that both absorptions of the indicators (DPBF and TEMPO-9ac) are very close to the NV-12P and could be simultaneously excited when compound NV-12P was irradiated, consequently disturbing the detecting signals from the compound. Figure S3a shows that direct irradiation of DPBF causes a considerable

Figure 5. (a) Cell viability assays of HeLa, CL1-0 cancer, and MRC-5 normal cell lines after incubation with and without (/Co) NV-12P for 72 h as measured using the MTT assay. (b) Colony-forming ability of HeLa cells after NV-12P treatment. Cells (1 × 103) were seeded into each 6 cm culture plate for 24 h, and then different concentrations of the compound were added to the corresponding well for an additional 5 days. Colonies were stained with crystal violet and quantified.

an alternative biological function compared with another mitochondrial functional group triphenylphosphonium.28 Combining these two molecular design factors, we synthesized molecule 1,6-bis((E)-2-(N-methylpyridinium-4-yl) vinyl)-2(12-(N-methylpiperidinium-1-yl) dodecyloxy) naphthalene triiodide (NV-12P, Scheme 1). Here, the 4-vinylpyridinium moiety also serves as the electron acceptor and hydrophilic group with respect to naphthalene, and the cation aliphatic side chain (N-alkyl-N-dodecylpiperidinum cations) was chosen as the lipophilicity and mitochondria-targeting regulator. More importantly, this molecule will be the first asymmetric structure to combine chemotherapy and PDT. Particular Optical Properties (AIEE-Based Fluorescent Organic Nanoparticle (FON)). On the basis of spectral properties in shown in Figure S1, broader absorption peaks with bathochromic shifts and very low quantum yields were obtained in low-polarity solvents (ET < 40), whereas higher quantum yields were obtained in high ET30 polar solvents (ET > 40). Here, we are interested in the quantum yield when NV12P is soluble in water because it is rare to obtain the watersoluble fluorogen. In previous studies, we applied AIEE properties to describe the fluorescence behavior of the solid state and water-soluble fluorogen and observed the formation of FONs.29−31 The AIEE phenomenon was easily checked by measuring the emission spectra of the compound in H2O/THF mixing solvents with various ratios. In principle, once the emission intensities of the fluorophore in a certain ratio of D

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Figure 6. (a) Time-dependent intracellular accumulation illustration of NV-12P in MRC-5 normal and HeLa cancer cells. The UV cube (ex 390/10 nm, 410 lp filter) was used to collect the compound-staining green color mitochondria images (pattern in b), and the blue cube (ex 470/20 nm, 515 lp filter) was used to collect the intracellular yellow-orange speckle image (pattern in c and d). Patterns b−d depict the development of mitochondrial swelling in HeLa cells cultured with 5 μM NV-12P for 8, 24, and 48 h, respectively. The zoomed-in images reveal (e) the bright nodes (spot) between tubular-like mitochondria during the short incubation time, (f) disconnected rods or reticulum, and (g) and (h) fragments, hollow or even granules, with a long incubation time.

H2DCF-DA, which can be oxidized to a highly fluorescent DCF (λ ex/em = 495/525 nm) in the presence of most general types of ROS (superoxide, hydroxyl radical, peroxyl radical, and singlet oxygen). More importantly, H2DCF-DA can be used to detect the ROS signal in cells.38,39 Given the above experience, the solution of NV-12P containing H2DCF-DA was irradiated with the light source from a 400−700 nm cube; the emission signal of H2DCF-DA was turned on, and the emission intensity was gradually intensified (Figure 3a). After short-duration light exposure, the intensity of H2DCF-DA at 525 nm had a greater than 100-fold increase compared with the emission intensity before irradiation. Controlled experiments showed that either NV-12P or H2DCF-DA along the solution remained non- or weakly emissive upon irradiation. Furthermore, to observe the ROS generation performance of NV-12P in living cells under light irradiation directly,39 HeLa cells were incubated with H2DCF-DA (5 μM) and NV-12P (5 μM). Before irradiation, the emission from H2DCF-DA was undetectable in the cell, while the green emission of H2DCF-DA was observed gradually after light irradiation for 1 min (Figures 3b−d). In comparison to the control cells incubated with H2DCF-DA alone, the emission signal remained almost invisible under the same irradiation condition (Figure S4). These results prove that NV12P promotes the generation of ROS upon light irradiation both in solution and in cells. Selective Photodamage and Dark Toxicity to Cancer Cells. The cellular photodamage of the compound was confirmed. However, given the selectivity criterion of the PDT system, the gradational irradiation treatment was adapted as the optimal experiment condition so that we can observe the NV-12P presented at different photoinduced cytotoxicity levels between cancer and normal cells. Here, every phototoxicity

Figure 7. Photoinduced fluorescence image variations of HeLa cells after culture with 5 μM NV-12P for 48 h and staining with H2DCFDA 30 min. (a) Photobleaching of bright yellow-orange speckles of the compound that were collected from the UV light cube (ex, 390/10 nm; em, 410 nm lp filter). (b) Emission enhancement due to the oxidation of H2DCF-DA by ROS collected from the blue light cube (ex, 470/20 nm; em, 515 lp filter).

decrease in its absorption. Then, on the basis of our deconvolution results, the differences between the decreasing absorption rates of DPBF with and without NV-12P are not apparent (Figure S3d), whereas an increase in the emission of TEMPO was noted once the NV-12P was added (Figures S3c and 3e). This result indicates that this compound may undergo the type I but not type II PDT effect under irradiation. Thus, the photogeneration ROS property of NV-12P was assessed by E

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Figure 8. (a) Evaluation of the cellular viability for several combinational therapy courses. Cancer cells were treated with 5 μM NV-12P for 1 (red), 2 (green), or 3 days (blue curve). Then, the cells were exposed to UV irradiation for 40 min followed by further incubation in the dark for 12 h (one cycle of irradiation treatment). The arrow represents the beginning of irradiation. Combinational therapies 1, 2, and 3 indicate 4, 3, and 2 cycles of irradiation treatment, respectively. (b) Following the “combinational therapy 3” experimental condition of panel a, the cellular viability organization on the 3rd, 4th, and 5th day after 1, 3, or 5 μM NV-12P combination treatment is shown. Here, similar treatment was applied to MRC-5 normal cells as a control.

source, we also observed a result similar to that above. Together, the compound dose and light illumination combination treatments resulted in quantitative cellular toxicity. Thus, NV-12P is a potential PDT candidate due to its selective photosensitization regardless of its application in vis light, UVA−vis light, or sunlight. However, we sought to determine why the gradational irradiation cycle is necessary for the compound to present selective photodamage to cancer cells. Figure 5a illustrates the dark toxicity effects of NV-12P as demonstrated by MTT assays of HeLa and CL1-0 cancer cells with MRC-5 normal fibroblasts. The cytotoxicity EC50 values of NV-12P on HeLa and CL1-0 cancer cells were approximately 5 μM with approximately negligible effects on normal MRC-5 cells. This result indicated that the mitochondrial activity was suppressed by the compound. Another long-term treatment assay showed that NV-12P presented a significant inhibitory effect on the colony formation of HeLa and CL1-0 cancer cells (Figure 5b). This finding is a clear indication that NV-12P can selectively suppress the proliferation of cancer cells but not MRC-5 normal fibroblasts. Interestingly, compound NV-12P presented dark toxicity with more pronounced suppression than the killing of cancer cells. Nevertheless, on the basis of the observation above, we propose that the selective dark toxicity of NV-12P to cancer cells may explain why the gradational irradiation cycle is necessary for this compound to present selective photodamage to cancer cells Intracellular Accumulation and Localization. We expect that the compound NV-12P can be a chemophototherapy reagent. Thus, the mechanism by which NV-

treatment was divided into several periods of irradiating stages; the cells were irradiated with a visible light source (400−700 nm) for 50 min followed by incubation for 12 h at each stage. Finally, cell survival numbers were counted after being incubated overnight. The real-time photodamage phase contrast images in Figure S4 clearly illustrate that human lung adenocarcinoma CL1-0 cells become condensed with gradational cell death within every irradiation cycle and acute cell death after three cycles of irradiation. By contrast, the MRC-5 normal cells were still healthy under controlled experimental conditions. Figure 4a reveals that gradational irradiation with a visible light dose presented significant lightinduced toxicities with EC50 values of ∼6.5 and ∼4.5 μM for HeLa and CL1-0 cells, respectively. Here, we concluded the very important message that compound NV-12P leads to apparent photodamage to HeLa and CL1-0 cells but not to normal cells MRC-5 under optimized irradiation conditions as we described above, either extending the incubation time of the compound or increasing the cycles of irradiation. The photodamage efficiency to cancer cells of NV-12P became more apparent when the system was subjected to an additional UV-A light source (340−700 nm). Under this condition, as shown in Figure 4b, we observed photodamage to HeLa and CL1-0 cells with EC50 values of ∼4.5 and ∼3.5 μM, respectively. However, more apparent photodamage was also observed for MRC-5 cells. This finding indicates that the UV region transition absorption of NV-12P (from approximately 340 to 400 nm) can also transfer light energy to ROS and contributes phototoxicity. Moreover, under the same phototoxicity protocol but a different solar generator as the light F

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after 12 h. The particular bright speckles were not clearly observed until 24 h of compound incubation (Figure S6). To summarize the uptake of NV-12P in cells, the compound first accumulates on mitochondria and then aggregates to form bright speckles, and the uptake behavior period is different between cancer and normal cells. Thus, we speculated that the cellular uptake and localization might be the main origins for compound NV-12P to cause selective dark toxicity to cancer cells. It is noteworthy to discuss that the fluorescence signal on mitochondria apparently decreased after incubation for 24 h (Figure 6a). On the basis of our observation, the cause might be mitochondrial swelling. Through fission, fusion, and mitophagic events, mitochondria dynamics are essential to maintain organelle stability and function.40 We carefully assessed the morphology pattern of the mitochondria of HeLa cells. Figure 6e reveals that the bright nodes (spot), which differ from the bright speckles observed in Figure 6d (red cycle), appear between tubular-like mitochondria during the short incubation time, implying that the compound may interfere with the mitochondrial dynamic balance between fusion and fission. Consequently, changes in mitochondrial morphology occur. The long tubal-like structures became disconnected reticulum shapes (Figure 6f) and eventually dispersed small fragments or even granules appeared when the incubation time was greater than 24 h (Figure 6g and h). This type of mitochondrial swelling implied the depolarization of the mitochondrial membrane potential.41 It is possible that oxidation stress occurred in the NV-12P targeting mitochondria and drove the apoptotic pathway, in which cytochrome c is released into the cytosol, which activates the caspase cascade and initiates cell apoptosis.42,43 Cell Photodamage Mechanism. The intracellular mitochondrial localization of NV-12P seems to follow our molecular design. However, we were interested in the intracellular accumulated location of bright speckles because they provide extra PDT efficiency from the long-term culture of NV-12P. Given that the intracellular localization trackers Lyso, ER, and Golgi Tracker Red all present less than 5% colocalization (merge) with these fluorescent spots (Figure S2), we also considered the possibility that compound NV-12P was encased in the endosome. Nevertheless, we proposed that these spots may result from the aggregation of the compound followed by spreading in the cytoplasm. Thus, AIEE-related biology should be considered in this manuscript. Recently, there is increasing interest in the development of AIEE molecules for biological sensing, imaging, and cancer therapy applications.44 Particularly, there are several AIEE conjugates that present effective ROS generation with application to AIEE-PDT.24,33,45,46 We concluded that compound NV-12P can form FONs via AIEE behavior in aqueous and polar solvent conditions (Figure 1). In addition, the ROS characteristics of Figure 3a were measured in the aqueous condition. This finding suggested that compound NV12P is an AIEE-PDT candidate. Thus, it is reasonable to propose that the bright speckles in cells, whose fluorescence was gradually enhanced coming from the long-term NV-12P incubation, were caused by the AIEE of the compound. On the other hand, the time-dependent cell photodamage images in Figures 2 and 3 showed that these bright yellow-orange speckles were quenched immediately following irradiation with respect to the green mitochondrial pattern. This result hints that the excited singlet state of NV-12P may possibly transfer

Figure 9. Following the experimental condition of Figure 8, HeLa cells were costained with Hoechst and PI after culture with the compound for (a) 12 or (b) 72 h followed by irradiation. The mutual intensities between the blue and red emissions are illustrated as (left) before irradiation, (middle) after irradiation, and after culture overnight (right).

12P causes selective photodamage and dark toxicity to cancer cells should be explored. In general, the uptake and/or intracellular localization of the compound should be the dominant cause(s) of the selectivity therapy. Thus, the quantitative determination of the intracellular accumulation and localization of the compound are critical. To recognize these signals, we prolonged the incubation period and carefully assessed the time-dependent fluorescent cellular image under fluorescence microscopy with color CCD, as illustrated in Figure 6a. At the initial NV-12P incubation period of 30 min to 2 h, green fluorescence signals in HeLa cells appeared on mitochondria and then gradually increased in brightness (Figure 6b). Next, yellow-orange colored bright speckles appeared around the nucleus at 2−4 h (Figures 6c and d, red cycle). The fluorescence intensities of these bright speckles became increasingly strong with the prolongation of the incubation time and consequently surpassed the signal intensity of mitochondria. However, it takes much longer to observe similar phenomena in normal MRC-5 cells. Before the initial incubation of the compound for 4 h, very weak fluorescence is distributed in the cytoplasm, and mitochondria accumulation appeared after 4 h of compound incubation and became clearer G

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protocol can be used to discriminate live cells, apoptotic cells, and dead cells by use of the two fluorescent color populations. The interaction between Hoechst and condensed (chromatin) DNA brings about blue emission in the nucleus, and red emission in the nucleus from PI is ascribed to nuclear membrane damage. Figure 9 shows that, after short-term combinational therapy (12 h of culture and then irradiation treatment with overnight incubation), red fluorescence in the nucleus was dominant. After long-term combinational therapy (72 h of culture and then irradiation treatment with overnight incubation), both the red and blue fluorescence in the nucleus were clear. It is proposed that short-term combinational therapy leads to cell death via necrosis, and long-term combinational therapy leads to cell death via apoptosis and necrosis, as discussed above. Eventually, to resolve the cytotoxicity mechanism of NV-12P in cancer cells, the PDT pathway may occur both after short-term (on mitochondria) and long-term culture (on mitochondria + on bright speckle), whereas dark chemotherapy occurs only with long-term compound incubation (on mitochondria). We conclude that NV-12P can be treated as a dual anticancer efficacy molecule.

energy to the triplet state and generate ROS. The intracellular ROS distribution supports this finding. Following the experimental condition as shown in Figure 3, Figure 7 indicated that the bright green DCF fluorescence (oxidation of H2DCFDA by ROS) was mainly merged with the speckles, which undergo breaching immediately upon irradiation. Thus, we concluded that the intracellular bright speckles of NV-12P support extra PDT achievement with respect to mitochondrial NV-12P. Combinational Treatment. Finally, we expected that the combination strategy, which includes PDT and chemotherapy in a single compound NV-12P, should increase the therapeutic effectiveness. A decisive impact is to stipulate the treatment course in which PDT begins to switch on. On the basis of our data, PDT efficiency is represented by bright intracellular speckles dependent on NV-12P accumulation. The preferred treatment process for the combinational therapy of NV-12P is as follows: preweakening or suppressing the activity of cancer cells such that they become more susceptible to later PDT treatment.47 The detailed compound treatment course and relative combinational therapy are described in Figure 8a. For example, HeLa cells were treated with 5 μM NV-12P for 1, 2, and 3 days and then irradiated by light for 40 min followed by a further 12 h incubation in the dark (this irradiation and incubation period together for one cycle of irradiation treatment). The optimized course of treatment is three days of incubation followed by two cycles of irradiation treatment (blue curve in Figure 8a). In fact, cancer cells also completely die on the fifth day when only one cycle of irradiation treatment is administered on the third day. We also observed complete cell death with three and four cycles of irradiation treatment for two days (green curve) and one day (red curve) of incubation, respectively. Although the combination strategy experimental results of CL1-0 are less efficient, in the short term incubation combination strategy, we still collected similar results, as shown in Figure 8a. On the other hand, whatever irradiated cells after treatment with the compound for 1, 2, or 3 days, the cells numbers in these cases all presented dramatic decreases in a time interval of 2.5−3.5 days, which suggests that this is prime time for PDT because the dark toxicity effect became prominent. Further, in the incubation with 3 μM NV-12P, HeLa and CL1-0 cells were also completely killed, but 3 cycles of irradiation treatment after 3 days of incubation were needed (Figure S7). Here, it is necessary to incubate cancer cells with the compound for three days because the other conditions (one and two days of incubation) cannot totally extirpate the cancer cells after several cycles of irradiation treatment. Thus, HeLa and CL1-0 cancer cells were treated with different concentrations of NV-12P for 3 days with irradiation. On the basis of the discussion above, considering the whole treatment course (chemotherapy + PDT), we focused on observing the cell numbers on the fourth and fifth days, and the results are illustrated in Figure 8b. The dose of 3 μM NV-12P can totally extirpate cancer cells on the 5th day after appropriate irradiation treatment (3 cycles and overnight incubation). This combination therapy strategy greatly enhanced PDT efficiency with suppression of the activity of cancer cells first and then PDT treatment because, upon review of the data of Figures 4 and 5, complete cell death was not observed in either short-term PDT or long-term MTT assays at the 3 or 5 μM compound dose. Additionally, cell death morphology was detected by Hoechst 33342 and propidium iodide (PI) double staining.48 This



CONCLUSION The water-soluble fluorogen NV-12P depolarizes the mitochondria membrane potential and selectively exerts potent chemocytotoxicity in cancer cells but not normal cells. We also proved that intracellular NV-12P, particularly the AIEE of NV-12P, can efficiently generate ROS with irradiation, which further enhanced the anticancer effect. Thus, NV-12P is a dual-therapy functional molecule that simultaneously performs chemophotodynamic therapy. Eventually, we conclude that the preferred combinational therapy treatment of NV-12P is preweakening or suppressing the activity of cancer cells followed by PDT treatment.



ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b07715. Figures S1−S7: synthesis and characteristic data (PDF)



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported financially by the Ministry of Science and Technology (MOST 104-2119-M-005-007-MY3) of ROC. REFERENCES

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DOI: 10.1021/acsami.6b07715 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.6b07715 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX