Facile Supramolecular Approach to Nucleic-Acid-Driven Activatable

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A Facile Supramolecular Approach to Nucleic Acid-Driven Activatable Nanotheranostics that Overcome Drawbacks of Photodynamic Therapy Xingshu Li, Sungsook Yu, Dayoung Lee, Gyoungmi Kim, Buhyun Lee, Yejin Cho, Bi-Yuan Zheng, Mei-Rong Ke, Jian-Dong Huang, Ki Taek Nam, Xiaoyuan Chen, and Juyoung Yoon ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b07809 • Publication Date (Web): 12 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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A Facile Supramolecular Approach to Nucleic AcidDriven Activatable Nanotheranostics that Overcome Drawbacks of Photodynamic Therapy Xingshu Li,1,2,# Sungsook Yu,3,# Dayoung Lee,2,# Gyoungmi Kim,2 Buhyun Lee,3 Yejin Cho,3 BiYuan Zheng,1 Mei-Rong Ke,1 Jian-Dong Huang,*,1 Ki Taek Nam,*,3 Xiaoyuan Chen, *,4 and Juyoung Yoon*,2 1

College of Chemistry, State Key Laboratory of Photocatalysis on Energy and Environment,

Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, Fuzhou University, Fuzhou 350108, China. 2

Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea.

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Severance Biomedical Science Institute, Brain Korea 21 PLUS Project for Medical Science,

Yonsei University College of Medicine, Seoul 120-752, South Korea. 4

Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of

Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland 20892, USA.

ACS Paragon Plus Environment

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ABSTRACT: Supramolecular chemistry provides a “bottom-up” method to fabricate nanostructures for biomedical applications. Herein, we report a facile strategy to directly assemble a phthalocyanine photosensitizer (PcS) with an anticancer drug mitoxantrone (MA) to form uniform nanostructures (PcS-MA), which not only display nanoscale optical properties but also have the capability of undergoing nucleic acid-responsive disassembly. These supramolecular assemblies possess activatable fluorescence emission and singlet oxygen generation associated with the formation of free PcS, mild photothermal heating, and a concomitant chemotherapeutic effect associated with the formation of free MA. In vivo evaluations indicate that PcS-MA have a high level of accumulation in tumor tissues, are capable of being used for cancer imaging, and have significantly improved anticancer effect than PcS. This study demonstrates an attractive strategy for overcoming the limitations of photodynamic cancer therapy.

KEYWORDS: nanotheranostics, supramolecular assembly, photodynamic therapy, activatable, nucleic acid-responsive disassembly

Photodynamic therapy (PDT) has become a clinically promising approach for cancer treatment owing to several features, including the spatiotemporal selectivity and noninvasive character.1-4 To date, several photosensitizers (PSs), such as Photofrin® and Foscan®, have been approved for clinical applications. Unfortunately, investigations have shown that PDT using these traditional PSs has “Achilles’ heels”.5,6 Specifically, patients need to avoid exposure to sunlight and even indoor light during and a relatively long period (usually 4-6 weeks) after PDT treatment. If not, the “always on” PSs induce harmful photosensitization effects on skin, eyes


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and other normal tissues.7-10 In addition, the hypoxic microenvironment of tumor tissues severely inhibits the PDT process, which operates through an oxygen-dependent mechanism.6,11,12 Moreover, PS-mediated oxygen consumption during PDT further enhances tumor hypoxia, which hampers therapeutic outcomes.13,14 Lastly, even at optimum wavelengths in the phototherapeutic window (650-850 nm), the tissue-penetration depth of light is limited to a few millimeters, making PDT ineffective for treatment of deep-seated tumors.5,15 Recently, many strategies have been developed to overcome the drawbacks of PDT. One strategy utilizes activatable PSs, substances that remain at an “off” state even under light irradiation but can be “turned on” at the target site by specific stimuli.16-23 Studies have shown that the activatable strategy minimizes unselective harmful effects on healthy tissues.24,25 To overcome the hypoxic and/or light penetration limitations of traditional PDT, appealing strategies, such as those utilizing oxygen self-enriching,26,27 self-illuminating28 and fractional PDTs,29 thermal-responsive endoperoxides,30 combination with other therapeutic modalities (e.g. radiotherapy, photothermal therapy (PTT)), and chemotherapy (CHT)),31-36 are being intensely investigated. Despite these efforts, strategies that lead to improved effects by overcoming all three problems of traditional PDT have not been reported thus far. Moreover, most of the existing approaches are based on complex nanocarrier systems, which suffer from tedious fabrication protocols and limited therapeutic agent loadings, and usually require extra materials for nanoconstruction and stabilization. The potential heterogeneity of formulations and the requirement for complex toxicity evaluations may pose challenges for future clinical translation of these methods.37 Inspired by the well-known prodrug concept and the successful use of concomitant drugs in clinical medicine, we embarked on a study aimed at developing an alternative approach to PDT.


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The effort focused on the design of a stimuli-responsive supramolecular nanostructure, comprised of a PS and an anticancer agent. This co-assembly was also designed to display nanoscale optical properties, as well as activatable singlet oxygen (1O2) generation and chemotherapeutic abilities. It was believed that a smart prodrug of this type, constructed using a supramolecular approach, would overcome the “Achilles’ heel” drawbacks of current protocols. In a screening study, the water-soluble PS, zinc(II) phthalocyanine tetra-substituted with 6,8disulphonate-2-naphthyloxy groups (PcS), and the common anticancer drug, mitoxantrone (MA) were found to be ideal host and guest molecules for supramolecular assembly (Figure 1). The ideal molecular recognition interactions occurring between PcS and MA enable spontaneous assembly to form uniform nanoparticles (PcS-MA) in water. Interestingly, PcS-MA can be dissociated in the presence of nucleic acids. Owing to these properties, PcS-MA has several advantageous features. Firstly, the PcS-MA consists of two properly designed components, both of which are therapeutic agents. Secondly, the super-quenched nature of PcS-MA prevents it from serving as a PS, and nucleic acid-activated 1O2 generation capability leads to minimal side effects in PDT. Thirdly, the partial disassembly of PcS-MA resulting from limited nucleic acid concentrations in a local area suggests that PcS-MA will also display a mild PTT effect, which in turn could enhance PDT by increasing intratumoral blood flow and relieving tumor hypoxia. Finally, the concomitantly produced chemotherapeutic agent, MA, gives the PcS-MA the potential to destroy deeply-located tumor cells that are out of the PDT range.


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Figure 1. (a) Structures of one of the possible C4h isomers of octasulfonated phthalocyanine (PcS) and mitoxantrone (MA). (b) Schematic illustration of the construction of a nanotheranostic agent based on supramolecular interaction between PcS and MA and its nucleic acid-driven activatable properties for fluorescent imaging and PDT synergized with PTT and CHT.

RESULTS AND DISCUSSION Screening of Host and Guest Molecules and Fabrication of Nano-Assemblies. Over the past few decades, supramolecular chemistry has undergone rapid development and drawn increasing interest from the scientific community.38,39 From the perspective of both fundamental and functional studies, more attention is now being given to applications of self-assembled (or co-assembled) nanoarchitectures rather than to their controlled fabrication.40 Recently, supramolecular assemblies have gained relevance to biomedicine.41-44 Several porphyrin-based nano-assemblies have been shown to be applicable to biophotonic imaging and therapy.45-52 For example, Liang et al. fabricated an interesting nanorod by co-assembly of 10hydroxycamptothecin and Chlorin e6 and showed that this supramolecular system can be


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employed in an effective strategy for combined chemo-photodynamic anticancer therapy.52 Our consideration of possible PSs to incorporate into the supramolecular assembly took into account the fact that phthalocyanines generally possess higher extinction coefficients and longer absorption wavelengths (usually λmax > 670 nm) than porphyrins. As a result, they are more suitable for in vivo phototheranostics.53 In addition, the results of several investigations show that supramolecular phthalocyanine assemblies can be constructed by controlling the chemical structures of the assembly units.54-56 Hence, to provide a higher possibility for host-gust interactions, the phthalocyanine, PcS, selected for this effort contains Zn2+ to promote photosensitizing ability,57 and anionic groups to reduce reorganization by the reticuloendothelial system and interactions with blood components.58 Other features that we believe would make PcS a good candidate include its non-cytotoxic activity in the dark, high photosensitization activity, along with the rapid excretion of PcS.59 Anthracenedione antineoplastic agents, such as MA and doxorubicin (DOX), were screened to identify ideal guests for the assembly. These substances contain a conjugated aromatic ring system and at least one amine group (Figure S1), which can participate in respective π-π stacking and metal-ligand interactions, and complementary hydrogen bonding with PcS. The screening effort led to identification of MA as the best guest candidate. Several methods were utilized to demonstrate that nanostructured assemblies form by mixing MA and PcS. Firstly, analysis of UV-vis titration curve provided information related to the binding affinity between MA and PcS. As shown in Figure S2, addition of one equivalent of MA to PcS induces the greatest change in the absorption spectrum. By fitting the data to an equation for 1:1 complex formation, the binding constant of MA with PcS was calculated to be K = 4.3 ± 0.8 × 105 M-1, revealing that strong non-covalent interactions take place between these


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substances.41,42 The binding stoichiometry of 1:1 was further confirmed by using a Job plot (Figure S3). Secondly, the formation of PcS-MA nanoparticles was verified by using dynamic light scattering (DLS) analysis. As shown in Figure S4, the hydrodynamic diameters of pure MA and pure PcS are both about 2 nm. However, mixing MA and PcS in water creates assemblies that have a mean hydrodynamic diameter of about 60 nm (Figure 2a), indicating that PcS-MA nano-assemblies have been formed. Thirdly, analysis of transmission electron microscope (TEM) images showed that PcS-MA exist as approximately oval shaped nanoparticles with unsmooth surfaces (Figure 2b). The storage stability of PcS-MA in RPMI 1640 culture medium over a month was assessed by monitoring changes in the electronic absorption spectrum (Figure S5). The results showed that PcS-MA remained unchanged (no disassembly or precipitation) over the investigated period, indicating that they possess an excellent stability for biomedical applications. Clearly, the results presented above demonstrate that MA is a desirable guest molecule that interacts with PcS to form a supramolecular nano-assembly. In contrast, DOX does not form a uniform assembly with PcS (Table S1 and Figure S6). As well, both an anthraquinone derivative containing two anionic groups (AQDS, Figure S1) and benzene derivative containing two amine groups (TMPD, Figure S1) do not strongly interact with PcS. Many other anticancer drugs, such as pemetrexed disodium, chlorambucil and 5-fluorouracil, are also not the desirable guest molecules. Therefore, strong π-π stacking and electrostatic interactions are required to form a host-guest nanostructured assembly with PcS. We also found that methylene blue (MB) interacts with PcS to form a supramolecular assembly with a mean size of about 180 nm. Because MB is not a chemotherapeutic anticancer agent, we have not included a discussion of the results of studies with this complex here.


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In the next stage of the development process, we explored the photophysical and photochemical properties of PcS-MA. The results of UV-vis spectroscopic studies show that, compared to that of PcS, the spectrum of PcS-MA has a decreased and broadened Q band (Figure 2c), which according to Kasha’s exciton theory60, H-aggregates likely exist in PcS-MA nanostructures. In addition, unlike PcS, PcS-MA do not fluoresce (Figure 2d). Consistent with its lack of fluorescence emission, PcS-MA does not promote 1O2 generation (96.7% quenching) (Figure 2e). Generally, the excited state of a photoactive agent can undergo one of the three relaxation pathways: emission of photons (fluorescence), intersystem crossing (e.g.

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generation), and vibrational relaxation (heating effect).61 The suppressed fluorescence emission and intersystem crossing suggest that PcS-MA might serve as a photothermal agent. As shown in Figure S7 that temperature elevation of an aqueous solution of PcS-MA is light intensity and PcS-MA concentration dependent. For instance, after 655 nm laser irradiation at 2.7 W/cm2 for 1 min, the temperature of a 12 µM solution of PcS-MA (the concentration of PcS used for coassembly) was elevated from 28 °C to 68.7 ± 0.9 °C. In contrast, the temperature of water not containing PcS-MA even in the presence of added PcS and MA was elevated to only about 34 °C under the same conditions (Figure 2f). The results demonstrate that PcS-MA effectively convert light into heat energy.


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Figure 2. Characterization of PcS-MA nano-assemblies and their photophysical and photochemical properties. (a) Size distribution of PcS-MA in water determined using DLS. (b) The morphology of PcS-MA determined using TEM. (c) Absorption and (d) fluorescence


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(excited at 610 nm) spectra of PcS, MA and PcS-MA (3 µM) in water. (e) Singlet oxygen generation of PcS, MA, and PcS-MA in water. (f) Temperature change curves of solutions of MA, PcS, and PcS-MA (3 µM) in water exposed to 655 nm laser (2.7 W/cm2). Data were expressed as the mean ± standard deviation (n = 3).

Activatable Photoactivity Based on Nucleic Acid-Driven Disassembly. A superior feature of supramolecular nanostructures is the fact that they result from non-covalent interactions. Consequently, disassembly of this type of supramolecular assemblies have dynamic-controllable capabilities and can be sensitive to external stimuli.40,41 It is known that the mechanism for anticancer activity of MA involves intercalation into DNA and the resulting inhibition of the action of topoisomerase II. Also, the binding constant of MA with DNA is about 105-106 M-1,62,63 which is comparable to that of MA with PcS. These observations suggest that DNA will likely compete with the interactions between PcS and MA. In studies designed to assess this possibility, we initially employed calf thymus DNA (ctDNA) as the target DNA because it has been commonly used for studying the interaction of MA with DNA in a test tube setting.62 As shown in Figure 3a, we observed that the absorption spectrum of PcS-MA gradually transformed to that of PcS when the concentration of DNA in the solution is increased. In addition, the intensity of fluorescence (Figure 3b) and the ability to generate 1O2 (Figure 3c and Figure S8) dramatically increased when DNA was added to a solution of PcS-MA. Importantly, significant changes in the emission intensity did not occur when PcS-MA was exposed to other stimuli, such as some proteins and metal ions (Figure S9). Collectively, the observations demonstrate that disassembly of PcS-MA is selectively promoted by nucleic acid.


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The photothermal behavior of PcS-MA in response to nucleic acid was also evaluated. As shown in Figure 3d, addition of ctDNA (300 µM, 100-fold higher than the molar concentration of MA) to a solution of PcS-MA causes a decrease in the magnitude of the irradiation induced temperature elevation. However, the temperature elevation effect caused by PcS-MA in the presence of ctDNA is still higher than that of pure water (control). Considering the limited concentrations of nucleic acid that are present in a local extracellular area or in an intracellular non-nucleus area, we expect that PcS-MA will likely promote a mild, irradiation induced heating effect in tumor tissues that could improve PDT.

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Figure 3. Nucleic acid-responsive photoactivities of PcS-MA. (a) Absorption and (b) fluorescence (excited at 610 nm) spectra of PcS-MA (3 µM) in the presence of different concentrations of ctDNA (µM) in water. (c) Singlet oxygen generation of PcS-MA (3 µM) in the presence of different concentrations of ctDNA (µM) in water. (d) Temperature change curves of pure water, PcS-MA and PcS-MA (3 µM) with ctDNA (300 µM) in water exposed to 655 nm laser (2.7 W/cm2). Data were expressed as the mean ± standard deviation (n = 3).

The DNA-responsive properties observed in these experiments encouraged an exploration of the activatable photoactivities of PcS-MA in cancer cells. As anticipated, the fluorescence signal of MCF7 cells in RPMI 1640 medium is much weaker immediately following addition of PcSMA (Figure 4a, 0 h and 1 h) as compared to the addition of PcS (Figure 4b, 0 h and 1 h). Moreover, the intensity of the fluorescence signal emanating from the PcS-MA treated cells increased with time (Figure 4a) while that arising from the PcS-incubated cells gradually decreased after washing and re-incubation in fresh culture medium (Figure 4b, 2 h, 2 h + 2 h and 2 h + 20 h). Furthermore, the intracellular production of reactive oxygen species (ROS) by the treated cells was also determined using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as the probe. Figure S10 showed that PcS treated cells had highly efficient production of ROS, which decreased after continuous incubation (2 h + 2 h and 2 h + 20 h). In contrast, PcS-MA incubated cells sustainably induced ROS over the entire incubation time period. The findings indicated that PcS-MA nanoparticles readily internalized into cancer cells and that they sustainably disassembled to produce PcS in concert with photosensitizing activity.


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Figure 4. In vitro activatable photoactivities of PcS-MA. Confocal images of MCF7 cells after incubation with (a) PcS-MA and (b) PcS (both at 4 µM) for 0, 1, 2, and 2 h followed by washing and further incubation in fresh medium for another 2 and 20 h (expressed as 2 h + 2 h and 2 h + 20 h). Scale bar, 100 µm. (c) and (d) The plots of changes in the intracellular fluorescence intensity. Data were expressed as the mean ± standard deviation (number of cells = 50). Solutions were excited at 635 nm and monitored at 645-750 nm.


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Tumor Accumulation of PcS-MA. To highlight the cancer phototherapeutic possibility of the supramolecular assembly, time-dependent biodistribution was determined using fluorescence imaging of several tumor models after systemic administration of PcS-MA, PcS and MA. As shown in Figures 5a and S11, after tail vein injection, PcS quickly spread throughout the whole body of MCF7 tumor-bearing mice. In addition, PcS quickly accumulated in the tumor and then quickly eliminated from the body over a very short period of time. This phenomenon was also observed in our previous study.59 In contrast, fluorescence from PcS, arising from disassembly of injected PcS-MA, reached a higher level and lasted for a longer period in the tumor tissue, likely due to an enhanced permeability and retention (EPR) effect64,65 and nucleic acid-driven disassembly of the supramolecular nanostructure. Similar results arose from studies using the SW620 tumor model (Figure 5b), indicating that this supramolecular approach is potentially suitable for the treatment of different kinds of cancers.


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Figure 5. In/ex vivo fluorescence images of tumor-bearing mice before and after intravenous injection of PcS-MA, PcS, and MA. (a) MCF7 tumor model. The dotted circles indicate tumor sites. (b) SW620 tumor model. H: Heart, Lu: Lung, Li: liver, K: Kidney, S: Spleen, T: Tumor. Fluorescence images were excited at 640 nm and monitored at 695-770 nm with IVIS Lumina II imaging system.


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Cancer Therapeutic Efficacy of PcS-MA. To evaluate the in vivo phototherapeutic efficacy of PcS-MA, MCF7 tumor mice were treated with PcS, MA or PcS-MA through intravenous injection and followed by laser irradiation. The tumor temperature irradiated with the laser at 1 W/cm2 for 5 min was determined by using a FLIR thermal camera. The temperature of tumor sites in PcS- and MA-treated mice (Figure S12) were found to be 36.8 ± 1.0 °C and 36.4 ± 1.2 °C, respectively, which were not significantly different from that of the control (34.7 ± 0.6 °C). However, the temperature of tumor sites in PcS-MA treated mice increased to 42.7 ± 0.7 °C under the same irradiation conditions. Even though the temperature elevation induced by PcSMA was not excessively high, the final temperature matched that required for hyperthermia (42 °C) for PTT.66 The results of earlier studies showed that pretreatment of tumor tissues with mild hyperthermia enables effective oxygenation of tumors, making them more susceptible to many therapeutic modalities including PDT.67-70 To confirm the ability of treatment of PcS-MA combined with laser irradiation to alleviate tumor hypoxia, immunofluorescence staining was performed in MCF7 tumor with different treatments using the anti HIF-1α antibody as the hypoxia marker and anti-CD31 antibody as blood vessels and angiogenesis marker. As shown in Figure S13a, MCF7 tumors in the control group showed severe hypoxia in both CD31 positive and CD31 negative regions. However, hypoxic levels were decreased across all areas of the tumor in a group that simultaneously received PcS-MA and laser irradiation. Quantitative analysis of HIF-1α-positive area in MCF7 tumor slices represented that the ratio of hypoxia area reduced from 32.6% to 15.6% after treatment of PcS-MA and laser irradiation (Figure S13b), suggesting that this mild PTT effect can improve the tumor oxygenation.


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As shown in Figure 6a, tumor growths in mice treated with PcS and MA in the presence of laser irradiation were moderately reduced as compared with that of the control group. In contrast, the tumor growth was significantly inhibited by administration of PcS-MA combined with laser irradiation. These findings clearly demonstrated the improved therapeutic potential gained by synergizing PDT with PTT and CHT. The excellent phototherapeutic outcome of PcS-MA was confirmed by histological analysis (Figure 6b).

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Figure 6. Phototherapeutic efficacy of PcS, MA and PcS-MA on mice bearing MCF7 tumors. (a) Tumor growth of mice after various treatments as indicated. Tumor volumes were normalized to their initial values. At 4 h after treatment (200 µM each, intravenous injection), tumor was laser irradiated at 690 nm (1 W/cm2 for 5 min). Data are expressed as mean ± SEM (n = 5). * indicates P < 0.05, ** indicates P < 0.01, compared to the control group. (b) Histological analysis of the MCF7 tumors acquired from mice at the 21st day after various treatment as indicated. Proliferation shown in brown signals was represented by Ki-67 antibody. Nuclei was counterstained by hematoxylin. Apotosis was tested by TUNEL assay. Green was positive signal. Nuclei were visualized by using DAPI (blue). Scale bars represent 50 µm for Ki-67 and 100 µm for TUNEL.

Additional studies showed that PcS did not have a cytotoxic effect in the absence of laser irradiation (Figure S14). However, PcS-MA without laser irradiation displayed a moderate tumor growth inhibition (44.7%), which was better than that of MA alone (22.8%). Thus, it appears that the supramolecular approach strengthens the CHT of MA because of its increased accumulation and prolonged action in tumors. Finally, to determine the biocompatibility of these treatments, several organs including mouse lung, liver, heart, kidneys and spleen were subjected to hematoxylin and eosin stain on the 21st day following treatment. The results showed that none of the mouse tissues treated PcS, MA or PcS-MA displayed pathological or other adverse changes (Figure 7). These results indicate that the supramolecular assembly approach to cancer treatment is both effective and biocompatible. We believe that future systematic investigations focusing on optimization of the


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drug dose, light interval after treatment, and light conditions (e.g. laser wavelength, intensity, and irradiation time) will further improve the treatment efficacy.

Figure 7. Histological analysis of the organs acquired from mice bearing MCF7 tumors at the 21st day after various treatments as indicated. Scale bars represent 200 µm.

CONCLUSIONS In summary, we have developed and successfully tested a facile supramolecular strategy for the design of nanostructured assembly based on photosensitizer PcS and chemotherapeutic drug MA. The oval shaped nanoassembly exhibits nucleic acid-dependent disassembly. Consequently, PcS-MA is an effective theranostic agent for cancer-targeted fluorescence imaging and for activatable PDT. In addition, because the assembly concomitantly releases a chemotherapeutic


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drug and has a mild heating effect in the tumor tissue, PcS-MA has significantly enhanced therapeutic effect over PcS PDT and MA chemotherapy. EXPERIMENTAL SECTION Materials

and

Instruments.

anthraquinone-1,5-disulfonic

acid

Dimethyl disodium

sulfoxide (DMSO), salt

(AQDS),

doxorubicin

(DOX),

N,N,N′,N′-Tetramethyl-p-

phenylenediamine dihydrochloride (TMPD), pemetrexed disodium, chlorambucil, 5-fluorouracil, methylene blue (MB), calf thymus DNA (ctDNA), 1,3-diphenylisobenzofuran (DPBF), and 2,7dichlorofluorescin diacetate (DCF) were purchased from Sigma-Aldrich Korea. Mitoxantrone (MA) was obtained from Dalian Mellon biological technology co., LTD (China). Octasulfonated phthalocyanine (PcS) was prepared using our previously described procedure.59 Dynamic light scattering (DLS) was measured using a Nanotrac Wave. TEM was observed using a JEM-2100F (JEOL). Electronic absorption spectra were detected on a SHIMADZU UV2450 spectrophotometer. Fluorescence spectra were carried out on an Edinburgh FL900/FS900 spectrofluorometer. Confocal imaging of cells was performed on a Leica TCS SPE laser fluorescent confocal microscope. 1

O2 Generation Tests. The studies of 1O2 generation in water solution were performed as

our previously described procedure.25 In Vitro Fluorescence Imaging Tests. About 1 × 104 MCF7 cells in RPMI 1640 medium were seeded in confocal dishes and incubated overnight at 37 oC under a humidified 5% CO2 atmosphere. After removing the medium, the cells were incubated with solutions of the drugs (PcS-MA, PcS, or MA) in the medium (4 µM, 400 µL) for 0 h, 1 h, 2 h, 2 h + 2 h, and 2 h + 20 h


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(cells after incubation with drugs for 2 h followed by washing and further incubation in fresh medium for another 20 h). After that, the cells with incubation time of 2 h, 2 h + 2 h, and 2 h + 20 h were rinsed with phosphate buffered saline (PBS) twice and imaged using a Leica laser fluorescent confocal microscope, while the cells with incubation time of 0 h and 1 h were directly imaged without changing the medium containing drugs. Excitation was at 635 nm and monitoring at 645-750 nm. The images were then digitized and analyzed by using the SPE ROI Fluorescence Statistics software. The average intracellular fluorescence intensities (a total of 50 cells for each sample) were also determined. In Vitro ROS Generation Tests. Intracellular reactive oxygen species (ROS) production was studied by measuring the fluorescence intensity of dichlorofluorescein (DCF). DCFH-DA, a non-fluorescent cell-permeable compound, is cleaved by endogenous esterases within the cell and the de-esterified product can be converted into the fluorescent compound DCF. About 1 × 104 MCF7 cells were cultured in 96-well plates and incubated overnight at 37 oC under a humidified 5% CO2 atmosphere. After removing the medium, the cells were incubated with the solution of drugs (PcS-MA, PcS, or MA) in the medium (4 µM, 100 µL) for 0 h, 1 h, 2 h, 2 h + 2 h, and 2 h + 20 h. Simultaneously, DCFH-DA (10 µM) was loaded into the cells. After that, all the cells were washed twice with PBS and then exposed to light irradiation (λ > 610 nm) for 20 min at the power density of 15 mW⋅cm-2. After irradiation, the fluorescence intensity of the treated cells was acquired using a microplate reader (Tecan Infinite M200Pro). For DCF detection, the excitation was 488 nm, and the emission was 526 nm. In/Ex Vivo Fluorescence Imaging. All animal procedures were approved by the Institutional Animal Care Committee at Yonsei University. The SW620 cancer cells (approximately 2×107


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cells per mouse) were subcutaneously injected into male BALB/c nude mice. For MCF7 transplantation, BALB/c nude mice were supplemented with 17-β-estradiol (0.72 mg/pellet, 60day release, Innovative Research of America, USA) into the dorsal flank subcutaneously. One week later, MCF7 cells (2×107 cells per mouse) were injected into fourth mammary fad pad. SW620 and MCF7 cancer cells were used in a 1:1 ratio with Matrigel (Corning, USA). When the tumor volumes reached approximately 100 mm3, the samples (PcS, MA, or PcS-MA) were intravenously injected into the tail of mice. In/ex vivo fluorescence images were captured at different time points after injecting the samples by using an animal optical imaging system (IVIS, Caliper Life Sciences). The samples were excited at 680 nm and monitored at 690-730 nm. In Vivo Therapeutic Efficacy Tests. MCF7 cancer cells (approximately 2×107 cells per mouse) were injected into fourth mammary fad of male BALB/c nude mice after transplantation of 17-β-estradiol as described above. When the tumor volumes reached approximately 100 mm3, the mice (5 mice each group) were intravenously injected with PcS (200 µM, 200 µL), MA (200 µM, 200 µL), PcS-MA (200 µM, 200 µL) or saline (200 µL). After 4 h, mice treated with PcS, MA, or PcS-MA were irradiated with a 690 nm laser (1 W/cm2, 5 min). The temperature change of tumors was detected using a FLIR thermal camera. The tumor sizes were determined using a caliper and measured for a duration of 21 d. The sizes were calculated using the following formula: volume = π × (width × height × depth)/6. Evaluation of tumor oxygenation. For ex vivo immunofluorescence staining to test the tumor hypoxia status, tumor slides from control group and PcS-MA combined with laser irradition-treated group were deparaffinized and rehydrated through series of graded ethanol.


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Antigen retrieval was performed using a pressure cooker and then incubated with protein blocking solution (Dako, Glostrup, Denmark) for 1h at room temperature. Primary antibody was incubated in a humid chamber at 4 °C overnight, then slides were incubated with Alexa488 rabbit IgG (Jackson Immno Research Laboratories, Inc., West Grove, USA) for anti-CD31 antibody and with Alexa568 mouse IgG (Jackson Immuno Research Laboratories, Inc., West Grove, USA) for anti-HIF1-α antibody for 1 h at room temperature. Nuclei were stained with DAPI (Vector Laboratories, INC, Burlingame, CA, USA), and mounted with ProLong Gold (Invitrogen). Apoptosis and Proliferation in Tumor Tissues. Twenty-one days after treatment, the mice were sacrificed, and the harvested tumors were fixed in in 4% paraformaldehyde and then embedded in paraffin. Sections (4 µm) were subjected to either a TUNEL assay (click-iT® Plus TUNEL Assay, invitrogen) or Ki-67 (abcam) antibody following the manufacturer's instructions. The numbers of TUNEL positive cells (green signal) and ki-67 positive cells (brown signals) were counted in four selected microscopic fields (× 100 or × 20). Experiments were repeated four times. Evaluation of Biocompatibility. Twenty-one days after treatment, the mice were sacrificed. To evaluate side effects in the lung, liver, heart, kidney and spleen, morphological changes were determined for all mice by a pathologist. Sections (4 µm) were stained with hematoxyline and eosin, and observed under the microscope (Olympus BX-43).

ASSOCIATED CONTENT The authors declare no competing financial interest.


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The Supporting Information is available free of charge on the ACS Publications website. Supplementary methods, and additional experimental data (Figures S1-S13). AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected] * E-mail: [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. # These authors contributed equally. ACKNOWLEDGMENT Jian-Dong Huang thanks to the support from National Natural Science Foundation of China (Grant Nos. 21473033, 21301031, 21172037). Juyoung Yoon thanks to the support from the National Research Foundation of Korea (NRF), which was funded by the Korea government (MSIP) (No. 2012R1A3A2048814). Ki Taek Nam thanks to the support from the Korea Mouse Phenotyping Project (NRF-2016M3A9D5A01952416) of the National Research Foundation, and the Brain Korea 21 PLUS Project for Medical Science, Yonsei University. Xiaoyuan Chen


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thanks to the support from the Intramural Research Program (IRP), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (NIH).

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MnSe@Bi2Se3 Fabricated

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a

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Exchange

Method

as

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Facile Supramolecular Approach to Nucleic-Acid-Driven Activatable

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