Photoresponsive micelle-incorporated doxorubicin for chemo

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Photoresponsive micelle-incorporated doxorubicin for chemophotodynamic therapy to achieve synergistic antitumor effects Da hye Kim, Hee Sook Hwang, and Kun Na Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00607 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Photoresponsive micelle-incorporated doxorubicin for chemophotodynamic therapy to achieve synergistic antitumor effects

Da Hye Kim‡, Hee Sook Hwang‡, Kun Na*

Center for Photomedicine, Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi do, 420-743, Korea

‡These authors contributed equally to this work. *Corresponding author: Kun Na, Ph.D. Center for Photomedicine, Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi do, 420-743, Korea. Tel: +82-2-2164-4832 Fax: +82-2-2164-4865; E-mail: [email protected]

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Abstract A combination of chemo-photodynamic therapy has been manifested as a promising strategy for efficient cancer treatment due to the enhanced therapeutic efficacy. Here, we designed doxorubicin (DOX)-loaded photoresponsive micelle (DPRMs) based on a combination of chlorin e6 (Ce6) and lipoic acid (LA) conjugated methoxy-poly(ethylene) glycol (mPEG-Ce6, mPEGLA) to achieve effective drug delivery using a single system. DPRMs were optimized with different molar ratios of mPEG-Ce6 and mPEG-LA which showed uniformly spherical morphology of size ~130 nm and approximately 9% of DOX loading contents. Photoresponsive lipoyl ring of mPEG-LA was incorporated in DPRMs in order to induce photo-mediated reduction resulting in 2~3-fold accelerated DOX release according to higher molar ratio of mPEG-LA and enhancement of light dose. The photoresponsive DOX release and ROS generation by Ce6 mediated cytotoxic effect of DPRMs were demonstrated in vitro using CT-26 (mouse colon cancer) and HCT-116 (human colon cancer) cells. We observed both the photosensitizer and the anticancer drug are colocalized in the tumor cells to achieve effective enhancement. Additionally, the DPRMs with laser irradiation successfully inhibited tumor growth in CT-26 tumor bearing mouse model and immunohistochemical staining verified apoptosis-mediated tumor growth inhibition. These observations demonstrated that the DPRMs showed a higher therapeutic effect than the other systems and PDT maximized the antitumor effect. Thus, DPRMs confirmed the advantages as a chemo-photodynamic dual-therapy with a synergistic therapeutic effect and great potential for cancer treatment.

Keywords Chemo-photodynamic therapy; photoresponsive micelle; doxorubicin; lipoic acid; drug release


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Introduction As a means of delivering the chemo-drug, nanotechnology has received enormous attention due to their unique size, dynamics and addible functional moiety according to the needs.1-4 Among the various anticancer drugs, doxorubicin (DOX) is commonly used to treat a diversity of cancers, which binds negatively charged nucleic acids and intercalates the base pairs of DNA strands through noncovalent interactions.5,

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Doxil® is a pegylated liposomal formulation and

FDA-approved drug that is formulated with smaller than 100 nm diameter and supplemented many huddles of free DOX having long circulation times by pegylation, lower cardiotoxicity, and active targeting of diseased sites.5,

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However, unexpected effects due to Doxil

administration, such as hand-foot syndrome because of the long circulation of the drug, have been reported.7, 9 To overcome the undesired side effects to normal tissues and acquire a synergistic antitumoral effects in vitro and in vivo, photodynamic therapy (PDT) has been introduced to chemotherapy.10-13 PDT, a light-responsive therapeutic method, has number of advantages including safe, minimally invasive, and tissue selective treatment for cancer therapy.14,

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Photosensitizer (PS), one of the essential factors for PDT, is used not only to trigger reactive oxygen species (ROS) generation but also to induce cell apoptosis or necrosis via PDT.16-18 Thus, the combination of PDT and DOX has a synergistic potential to boost the therapeutic effect regardless of the concentration of each agent since the cytotoxic action mode of PDT and DOX is different. 13 In PDT, generated singlet oxygen reacts with nearby cellular membranes causing cell death upon laser irradiation, whereas DOX interacts with DNA in cell nucleus and inhibits macromolecular biosynthesis.13, 19, 20 In addition, light-responsive PS is able to provide


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controlled drug release to acquire enhanced therapeutic effect and minimized side effect by introducing light-responsive moiety in the drug formulations.21 Based on these additive advantages,

we established a DOX-loaded photoresponsive

micelles (DPRMs) by self-assembly based on the conjugation of chlorin e6 (Ce6) as a PS, and lipoic acid (LA) as a photoresponsive material to polyethylene glycol (PEG) for ROS-triggered drug release and PDT for synergistic cancer therapy.22 Naturally synthesized LA has many biological functions in human body, such as quenching of ROS, antioxidants regeneration, and metal ions chelation.23-25 This unique delivery system involves the ROS sensitive moiety of LA in DPRMs, which triggers DOX release upon laser irradiation at target site (Scheme 1a). When DPRMs reach tumor site via enhanced permeability and retention (EPR) effect followed by laser

Scheme 1. Schematic illustration of (a) DOX loaded self-assembly micelles consisted of mPEG-LA and mPEG-Ce6 and (b) its delivery due to the enhanced permeability and retention (EPR) effect for effective tumor inhibition.


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irradiation, ROS is produced by Ce6 activation, disulfide bonds of LA are reduced by ROS, and thereby DOX is simultaneously released. Furthermore, a photodynamic effect will be induced because of remaining ROS at the same area and directly kill tumor cells (Scheme 1b). To confirm this hypothesis, we evaluated singlet oxygen generation ability of DPRMs, assessed the scavenger ability of mPEG-LA in DPRMs, and monitored cleavage of LA upon laser power. We also investigated 3 different molar ratios of mPEG-Ce6 to mPEG-LA and estimated particle size, morphologies using scanning electron microscope (SEM) image, critical micelle concentration (CMC) values before or after laser irradiation, and DOX release profile with laser irradiation. For in vitro experiments, we selected the 1 : 3 ratio of DPRMs which exhibited effective DOX release compared to others, characterized its cellular uptake using flow cytometry and CLSM images, and assessed the cytotoxicity with or without laser irradiation. In vivo tumor growth inhibition test, body weight changes, H&E staining, and TUNEL assay on tumors were also assessed in CT-26 xenograft mice model.

Materials and Methods Materials. Methoxy polyethylene glycol amine (mPEG-amine) with a molecular weight of 2,000 Da was purchased from Sunbio (Orinda, CA, USA). Chlorin e6 (Ce6) was purchased from Frontier Scientific, Inc. (Salt Lake City, UT, USA). Doxorubicin hydrochloride (DOX·HCl), N,N’-Dicyclohexylcarbodiimide (DCC), N-hydroxysuccinimide (NHS), (±)-α-Lipoic acid (LA, ≥ 99%), triethylamine (TEA), Hoechst 33342, 9,10-Dimethylanthracene (DMA), and 3-(4,5dimethyl-2-thiaxolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were obtained from SigmaAldrich Co. (St.Louis, MO, USA). N,N-Dimethyl formamide (DMF) and Dimethyl sulfoxide


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(DMSO) were purchased from Junsei Chemicals (Tokyo, Japan). The dialysis membrane (1 kD) was obtained from Spectrum Laboratories, Inc. (Rancho Dominguez, CA, USA). Phosphate buffered saline, 1X (PBS) was obtained from Welgene (Daegu, Korea). DMEM medium, RPMI 1640 medium, fetal bovine serum (FBS), antibiotics (penicillin/streptomycin), and Dulbecco's Phosphate-Buffered Saline (DPBS) were purchased from Gibco BRL (Invitrogen Corp., CA, USA). Finally, 4’,6-diamidino-2-phenylindole (DAPI) and Singlet Oxygen Sensor Green (SOSG) were purchased from Molecular Probes, Inc. (Eugene, USA). All other chemicals and solvents were analytical grade. Synthesis and Characterization of mPEG-LA and mPEG-Ce6. For mPEG-LA and mPEG-Ce6 conjugates, two of mPEG powder (0.5 g) were suspended in 5 mL of DMF and dissolved by vigorous stirring for 6 h at room temperature. Simultaneously, LA and Ce6 was dissolved respectively in DMF (3 mL) with DCC, NHS. The molar mass ratio of mPEG : LA : Ce6 was 1 : 1.2 : 1.2 and the molar mass ratio of LA : DCC : NHS was 1 : 1.2 : 1.2. After 6 h, the stirred solution of LA and Ce6 were added dropwise a solution of mPEG in DMF. Subsequently, the precipitate generated during the reaction was removed by filtration and the final mixtures were further stirred for 24 h at room temperature. The conjugates were isolated by precipitation and sedimentation in cold ether, washed several times with ether, and vacuum dried. To remove unconjugated Ce6 or LA, obtained solids were dissolved in DMSO (5 mL) and dialyzed against DI water using 1 kDa MWCO dialysis tube for 3 days. After dialysis, the final solution was freeze-dried and lyophilized. The formation of mPEG-LA and mPEG-Ce6 was verified by 1H-NMR (Bruker, Germany), and FT-IR spectroscopy (Nicolet, Magna IR 550). mPEG-LA ; 1H-NMR (500 MHz, DMSO-d6) : δ 3.64 (m, 1H), 3.51 (s, 4H), 3.31 (s, 3H), 3.18 (t, 2H), 2.40 (m, 2H), 1.65-1.50 (m, 2H), 1.34 (m, 2H). mPEG-Ce6 ; 1H-NMR (500 MHz, DMSO-


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d6) : δ 9.76 (s, 1H), 9.75 (s, 1H), 9.14 (s, 1H), 8.34 (dd, 1H), 6.46 (dd, 2H), 3.49 (s, 4H), 3.32 (s, 3H). The singlet oxygen generation (SOG) by mPEG-Ce6 was chemically detected using 9,10dimethylanthracene (DMA) and singlet oxygen sensor green (SOSG) as a probe in DMSO and DI water, respectively. To clarify the photoactivatable capability of Ce6 in DMSO, SOG was determined by DMA (20 × 10-3 mM). Free Ce6 and mPEG-Ce6 were dissolved in DMSO, and then irradiated with 20 mW/cm2 for 500 sec. DMA fluorescence was determined using an excitation wavelength of 375 nm and an emission wavelength of 436 nm after irradiation to determine the extent of SOG. The decrease in the fluorescence intensity of DMA as a result of the photochemical reaction was monitored. SOG was evaluated by observing the DMA fluorescence decrease using a spectrofluorophotometer (RF-5301; Shimadzu, Japan). The SOSG probe works via an intramolecular electron transfer, which quenches the fluorescence from the light-emitting chromophore prior to reaction with singlet oxygen. The reaction with singlet oxygen results in the formation of the endoperoxide, prohibiting electron transfer and thus leading to the recovery of fluorescence. Free Ce6 and mPEG-Ce6 were dissolved in deionized water and mixed with a SOSG solution (2 mM). The solution was irradiated with 671 nm light source (fiber coupled laser system, LaserLab®, Korea) at 10 J/cm2 (light density ; 20 mW/cm2, for 500 sec). The fluorescence intensity of SOSG (λex 494 nm, λem 534 nm) was recorded using fluorescence spectroscopy (RF-5301; Shimadzu, Japan). The characterization of lipoyl rings was observed with ultraviolet-visible (UV-Vis) spectrometry (UV-2450; Shimadzu, Japan). The sample solutions were adjusted to various irradiations for 0, 500 or 1000 sec (20 mW/cm2) using a 671 nm light source (fiber coupled laser system, LaserLab, Republic of Korea). The UV spectra of mPEG-LA in DMSO showed absorbance at 336 nm.


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Preparation and Characterization of DPRMs. Doxorubicin hydrochloride (DOX·HCl, 10 mg) and triethylamine (1.5 × DOX·HCl in moles) were allowed to react in DMSO (2 mL) to form the DOX basic adduct.26 mPEG-LA and mPEG-Ce6 were added in DOX solution (1 mg), and the mixture was stirred overnight at 4 ℃ in the dark; un-loaded micelles (PRMs) were prepared with mPEG-LA and mPEG-Ce6 only. The products were obtained at a molar ratio of mPEG-Ce6 : mPEG-LA = 1 : 0.3, 1 : 1, 1 : 3. The final reaction solution was then transferred to a wet dialysis tube (MWCO, 1 kDa) and dialyzed against DI water for 24 h at room temperature in a dark room. The solution was filtered with a syringe filter (0.45 µm, Millipore) to remove the precipitated material. To investigate the DOX loading efficiency (LE) and loading contents (LC) in DPRMs, the amount of DOX was quantified from a standard curve using DMSO/DI water = 8 : 2 (V/V %). The excitation and emission wavelengths of DOX was set to 490 and 590 nm, respectively. The LE and LC of DPRMs were calculated using the following equations. (1) LE (%) = [DOXin/DOXtotal] x 100 (2) LC (%) = DOXin/TM x 100 DOXin is the amount of DOX in DPRMs, DOXtotal is the feeding amount of DOX for the preparation of DPRMs, and TM is the total mass of DPRMs containing all polymers and drug used for micelles preparation. The size distribution of DPRMs with and without laser irradiation was analyzed using a dynamic laser scattering (DLS; Zetasizer Nano ZS, Malvern Instruments Ltd., UK) at 25 ℃. Morphological measurements were performed under field emission scanning electron microscopy (FE-SEM) (S-4700, Hitachi, Japan). The specimens were prepared as follows: a particle solution (10 µL) was dropped onto an 18 mm φ cover glass and then dried at room


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temperature overnight. The cover glass was mounted, sputter coated with platinum using an ion coater (10 mA, 45 sec), and then observed at an accelerating voltage of 3 kV. The critical micelle concentration (CMC) of DPRMs by different molar ratio in deionized water was measured at room temperature using a microplate reader with Hoechst 33342 as a fluorescent probe as previously reported (Bio-Tek, VT, USA). The final concentration of Hoechst 33342 was 7.0 × 10-4 M in each sample solution. The fluorescence was measured at an excitation of 355 nm and an emission of 457 nm, and the slit widths were ex = 3 nm and em = 3 nm, respectively. In vitro drug release test. In vitro drug release profiles of DPRMs were investigated in 10 mM phosphate buffered saline solution containing 0.1% Tween 20 (PBST, pH 7.4) using a dialysis membrane (MWCO = 1 kDa). DPRMs were transferred to a dialysis tube and then stirred at 100 rpm at 37 ℃. At a pre-determined sampling time, all media were removed and replaced with fresh PBST in the dark with two laser irradiations at 4 h and 8 h with 671 nm light for different duration (20 mW/cm2 for 0, 500, or 1000 sec). The concentration of DOX in the buffer was determined from a standard curve using DMSO/DI water = 8 : 2 (V/V %) (Correlation coefficient R2 = 0.9975). The excitation and emission wavelengths of DOX are 490 and 590 nm, respectively. Cell culture and incubation conditions. CT-26 (mouse colon cancer) and HCT-116 (human colon cancer) cells were obtained from the Korean Cell Line Bank and cultured in DMEM or RPMI 1640 medium that was supplemented with 10% heat inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin, which is referred to as complement medium (CM) in this study. The cells were cultured at 37 ℃ with 100% humidity and 5% CO2, and sub-cultured in new media every 2-3 days. The micelles were suspended in serum-free (SF) medium. Water-


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insoluble free DOX was dissolved in DMSO and then diluted in SF medium until the DMSO concentration reached < 0.1%. All of the reported concentrations refer to the free DOX equivalents. Untreated cells were maintained in the dark and used as a reference standard. In vitro cellular uptake test. To verify the cellular uptake of DPRMs at different incubation times, CT-26 cells and HCT-116 cells were seeded onto 6-well cell culture plates at a density of 1 × 105 cells per well and incubated for 24 h at 37 ℃ in 5% CO2. The medium was subsequently exchanged for serum-free (SF) medium containing Free DOX, Ce6 or DPRMs and the cells were incubated for determined times before rinsed, harvested and resuspended with DPBS. The cellular uptake was quantitatively analyzed using flow cytometry with a BectonDickinson FACS Canto℃ (San Jose, CA, USA). For each sample, 10,000 cells (gated events) were counted, and DOX and Ce6 fluorescence were detected with logarithmic settings (PE, Em = 578 nm/APC, Em = 665 nm). Each experiment was analyzed statistically using the FACS Diva software (BD). To observe the cellular localization of DPRMs at different incubation times, CT-26 cells and HCT-116 cells (1 × 105 cells per well in a 6-well plate) were treated with Free DOX or DPRMs for determined times. The cells were then washed twice with DPBS, fixed with 4% paraformaldehyde and stained with DAPI. The cells were visualized using a confocal laserscanning microscope (LSM 710 Meta, Carl Zeiss, Germany). Fluorescence images were analyzed using the LSM image browser software (Zen series, Carl Zeiss, Germany). IC50 of DOX. CT-26 cells and HCT-116 cells were seeded onto 6-well plates at a density of 3 × 105 cells/well in CM medium and incubated in a humidified 5% CO2 atmosphere for 24 h. The original medium was replaced with SF media containing free DOX (Dose of DOX = 0.001100 µg/mL). After 4 h incubation, the cells were washed twice with DPBS and fresh culture


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medium was added. The cells were then incubated at 37 ℃ for 48 h and cell viability was evaluated by MTT assay. The resulting formazan crystals were dissolved in DMSO (100 µL), and absorbance intensity was measured at 570 nm using a microplate reader (Bio-Tek, VT, USA).

Intracellular reactive oxygen species (ROS) generation efficacy and DOX release behavior. ROS generation inside cells upon laser irradiation was observed using Image-iT LIVE Green Reactive Oxygen Species Detection Kit (Invitrogen Corp., CA, USA). Briefly, HCT-116 cells were cultured in 35-mm culture dishes (3 × 105 cells/well) and incubated for 24 h at 37 ℃ in 5% CO2. Following treatment with DPRMs (1 µg/mL DOX, 1.5 µg/mL Ce6) were incubated for 4 h at 37 ℃. Cells were then gently washed three times with DPBS and incubated with carboxy-H2 dichlorofluorescein diacetate (DCFDA; 25 × 10-6 M, 30 min at 37 ℃) to detect ROS and Hoechst 33342 (1 × 10-6 M, for the last 5 min at 37 ℃) was used to stain nuclei. DCF-DA is non-fluorescent compound; however, it is oxidized to DCF by ROS and emits green fluorescence once internalized. The cells were washed three times with DPBS and then exposed to light irradiation using the 671 nm laser (0.2 J/cm2). After irradiation, the cells were washed three time with DPBS and fixed at 4 % paraformaldehyde. The cells were mounted in mounting medium (Dako, Glostrup, Denmark) and visualized using a CLSM (LSM 710 Meta, Carl Zeiss, Germany). In vitro cell cytotoxicity study. The CT-26 cells and HCT-116 cells were seeded onto 35 mm cell culture plates at a density of 3 × 105 cells per well and incubated for 24 h. Free DOX, PRMs or DPRMs (5 µg/mL of DOX; 7.5 µg/mL of Ce6) were added to each well in SF medium (1 mL), and the dishes were returned to the incubator for 4 h. After incubation, the wells were rinsed twice with DPBS to remove samples that had not been internalized into the cells, 2 mL of


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complement medium was added to the wells, and each well was irradiated using the predetermined 671 nm light source (0.6 J/cm2; 6.0 mW/cm2, 0 or 100 sec, fiber coupled laser system, LaserLab®, Korea). The cells were then incubated for another 48 h. The cell viability was assessed using the MTT colorimetric assay. The resulting formazan crystals were dissolved in DMSO and transferred to a new plate. The absorbance intensity was measured at 570 nm using a microplate reader (Bio-Tek, VT, USA). For the live and dead cell assay, the LIVE/DEAD Viability/Cytotoxicity Assay Kit (Molecular Probes, USA) containing calcein AM and ethidium homodimer-1 (EthD-1) recognizes live and dead cells as green and red emissions, respectively: calcein AM is converted to green fluorescent calcein after acetoxymethyl ester hydrolysis by intracellular esterase in live cells; and EthD-1 marks the nuclei in dead cells once the integrity of the cell membrane is compromised. HCT-116 cells and CT-26 cells (3 × 105 cells/well) were seeded onto 35 mm cell culture dishes and incubated for 24 h at 37 ℃ in 5% CO2. After incubation, the medium was removed, and the cells were incubated in medium containing free DOX, PRMs or DPRMs (5 µg/mL of DOX; 7.5 µg/mL of Ce6) for 4 h. The medium was removed, and the cells were rinsed twice with DPBS. Subsequently, irradiation (0.6 J/cm2; 6.0 mW/cm2, 100 sec) was performed using a 671 nm light source (fiber coupled laser system, LaserLab®, Korea). The cells were then incubated in complement medium for 48 h, and the cell viability was observed through CLSM (Zen series, Carl Zeiss, Germany). Animal model. The CT-26 cells were implanted into 5-week-old BALB/c nude mice. Briefly, the cells (1 × 105) in 100 µL of SF DMEM were subcutaneously injected. Tumor volume was calculated using the following equation: Volume = 0.5 × L × W2, where “W” and “L” are the width and length of the tumor, respectively. All procedures were approved by the


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Institutional Animal Care and Use Committee (IACUC) of College of Pharmacy, the Catholic University of Korea (Republic of Korea) and Use Committee in accordance with the “Principles of Laboratory Animal care”, NIH publication no.85-23, revised in 1985. In vivo tumor growth inhibition effect. The CT-26 bearing nude mice were randomly divided into six groups (n = 5). The treatments were conducted when tumor size reached approximately 50-100 mm3. Solutions of PBS, free DOX, PRMs with or without laser irradiation and DPRMs with or without laser irradiation were injected into the mice via the tail vein (dose : 2 mg/kg DOX and 3 mg/kg Ce6) at 15, 17, and 20 days after tumor bearing. Tumor regions were irradiated with a laser at power densities of 100 J/cm2 at 24 h post-injection. Tumor sizes and body weights were measured at each time point. Immunohistochemical analysis. To investigate histological analysis (H&E staining), the mice were sacrificed and the tumors were collected and fixed for 24 h in 4% paraformaldehyde. After deparaffinization, the tissue sections (5 µm) were stained with hematoxylin/eosin (H&E). Also the terminal deoxy-nucleotidyl transferase-mediated nick-end labelling (TUNEL) assay was performed using a commercial apoptosis detection kit (Promega Corp., WI, USA) with the following modifications. The mice were sacrificed and tumors were collected and fixed for 24 h in 4% paraformaldehyde. After 5-µm sections were taken by frozen section, the sections were washed twice with PBS and treated with 0.2% Triton X-100 for 10 min at room temperature. The samples were washed twice and incubated with equilibration buffer for 10 min at room temperature. The equilibration buffer was drained, and a reaction buffer containing equilibration buffer, nucleotide mix, and TdT enzyme was added to the tissue sections. Samples were incubated in a dark, humidified atmosphere at 37 ℃ for 1 h. The samples were then washed for 5


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min each to remove unincorporated fluorescein-TdT. The samples were stained with DAPI and mounted. The stained slides were observed by using a slide scanner (APERIO CS2, Leica, Germany). Statistical Analysis. Data are expressed as the mean ± standard deviation. Student’s t-test was used for statistical analysis.

Results and Discussion Synthesis and Characterization of mPEG-LA and mPEG-Ce6. mPEG-LA and mPEG-Ce6 were synthesized via DCC and NHS-mediated amide formation which was previously reported by our group (Figure S1).27 The synthesized mPEG-LA and mPEG-Ce6 were assessed via 1H-NMR analysis (Figure S2a,b). The complete disappearance of the peak at 12 ppm (-COOH in LA) confirmed successful synthesis of mPEG-LA. Similarly, the appearance of Ce6 peak at 9-10 ppm reported great polymerization. The FT-IR spectrum of mPEG-LA and mPEG-Ce6 confirmed the carboxyl groups of mPEG, as indicated by C=O stretching of – CONH– (amide bond) at 1674 cm-1 (Figure S3).


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To confirm ROS generated from Ce6 after laser irradiation at 20 mW/cm2 for 500 sec, we used DMA as a detector (Figure 1a). DMA irreversibly reacts with 1O2 in many organic solvents, which causes a decrease in the intensity of the DMA absorption at 360 nm. As expected, the

Figure 1. (a) Singlet oxygen generation efficacy of mPEG-Ce6 using 9,10-Dimethylanthracene (DMA) in DMSO comparison to free Ce6 without or with laser irradiation at 10 J/cm2. (b) Determination of reductive strength of lipoic acid using fluorescence intensity of singlet oxygen sensor green (SOSG) in DW comparison to other samples with laser irradiation at 10 J/cm2. (c) Changes of UV absorbance at 336 nm due to the reduction of lipoyl ring with or without laser irradiation. All samples were irradiated by 671 nm light source.

DMA concentration of free Ce6 and the mPEG-Ce6 group without irradiation was not decreased, however, a decreased concentration of DMA was observed in those groups after laser irradiation. Based on these results, we confirmed that laser irradiation promotes the activation of Ce6, leading to ROS generation. To observe the ROS scavenge capacity of lipoyl ring in LA, each solution of free Ce6, mPEG-Ce6, and a mixture of mPEG-Ce6 and mPEG-LA was irradiated with a laser at 20 mW/cm2 with light for 500 sec, where singlet oxygen sensor green (SOSG) was used as a probe in DI water (Figure 1b). SOSG does not have a fluorescence under normal conditions, however, it can be rapidly oxidized to a fluorescence molecule by ROS and detect singlet oxygen, which is a type of ROS.28 The singlet oxygen generation (SOG) yield of free Ce6 under laser irradiation


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was much lower than that of mPEG-Ce6. Generally, free Ce6 easily aggregates in SOSG solution and dose not show any differences in SOSG fluorescence intensity, indicating that it does not produce singlet oxygen under aqueous conditions. Unlike free Ce6, mPEG-Ce6 completely dissolved in DI water resulted in a 14-fold increase in SOSG fluorescence intensity, indicating the high generation of singlet oxygen upon laser irradiation. In case of mixture of mPEG-Ce6 and mPEG-LA, the SOSG fluorescence intensity of the mixture was increased approximately 4fold compared to free Ce6, but a significantly lower level of SOG production of was observed than that of mPEG-Ce6. This result denotes that LA was used to scavenge ROS generated from Ce6 under irradiation. When Figure 1b were converted to slope values depending on initiatory laser irradiation time as shown in Figure S4, the slope of mPEG-Ce6 and mPEG-LA mixture was much lower than that of mPEG-Ce6 approximately 2-fold due to the scavenger role of LA. To investigate the characteristics of lipoyl rings, we analyzed the UV spectra of the mixture with mPEG-Ce6 and mPEG-LA. As depicted in Figure 1c, the change of absorbance value at 336 nm was inversely proportional to the irradiation time. This result suggests that ROS generated from activated Ce6 reduces the lipoyl ring in LA. Preparation and physicochemical characterization of DPRMs. DPRMs were prepared via self-assembly of mPEG-LA and mPEG-Ce6, and purified using the dialysis method (MWCO 1000 Da). DOX was loaded into the micellar core via hydrophobic interactions. To determine the optimal ratio required to construct DPRMs, 3 different molar ratios of mPEG- Ce6


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: mPEG-LA were tested, where DOX feeding amount : polymer ratio was fixed to 1 : 8. At each molar ratio, DOX loading efficiency and loading contents were varied approximately 76~80% and 8~9%, respectively (Table 1). The hydrodynamic size and morphology of the DPRMs with various molar ratios depending on laser irradiation time were determined by dynamic laser scattering (DLS) and field emission-scanning electron microscopy (FE-SEM) to optimize photoregulated micelles. As described in Figure 2a, the average size of mPEG-Ce6 : mPEG-LA = 1 : 0.3, 1 : 1, 1 : 3 was 139, 182, and 123 nm, respectively. Upon laser irradiation at 20 mW/cm2 for 1000 sec, we observed that the particle size was increased which indicates that the lipoyl rings of LA were reduced by ROS, and the disulfide bonds become degraded and increase in size. In addition, FE-SEM images showed that the morphology of DPRMs was uniform, showing a spherical shape prior to laser irradiation (Figure 2b). However, after laser irradiation, the morphology of the DPRMs became irregular and the size of each particle was increased. Taken together, these results clearly demonstrated that DPRMs were disrupted by ROS-triggered LA reduction upon laser irradiation. To confirm the critical micelle concentration (CMC) of mPEG-LA and mPEG-Ce6 at each molar ratio, CMC was investigated using Hoechst 33342 as a fluorescence probe in DI water. When the concentration of total polymers (x-axis) was increased, the Hoechst 33342 fluorescence intensity was inversely proportional to those polymer concentration due to the strong quenching effect of the hydrophobic interaction between Ce6 and Hoechst 33342, known as the fluorescence resonance energy transfer effect.29

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In addition, the

change of CMC values of 1 : 3 DPRMs before (0.048 g/L) and after (0.157 g/L) laser irradiation was much higher than those of other molar ratio micelles. This demonstrates that the change of CMC is due to different molar ratio of mPEG-LA in micelles which shows different ROS responsiveness. Higher molar ratio of mPEG-LA sensitively responses to ROS generation and


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increases the hydrophilicity of lipoyl groups in mPEG-LA than other molar ratios; thus, more LA content in micelles performed as an important drug release factor that contributes to DPRMs

Figure 2. Physico-chemical characterization of micelles consisted of mPEG-Ce6 : mPEG-LA. (a) Size distribution of micelles created by each molar ratio before and after laser irradiation. (b) SEM images of micelles created by each molar ratio before and after laser irradiation. (Scale bar = 250 nm). (c) Critical micelle concentration of micelles using Hoechst 33342 before and after laser irradiation. All samples were irradiated at 20 J/cm2.


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destabilization. In vitro photoregulated drug release of DPRMs. The ROS-responsive controlled release of DPRMs was investigated depending on the molar ratio of DPRMs. As shown in Figure 3a-c, only 20% of DPRMs without laser irradiation were released after 24 h of incubation, because of hydrophobic interaction between DOX and Ce6 prohibits the efficient release of DOX. After 500 or 1000 sec laser irradiation, the final amount of DOX released from DPRMs was increased compared to non-irradiated group. As a result, the accumulative DOX release of DPRMs was increased to 32%, 42% and 50% within 24 h, respectively. A 1 : 3 molar ratio of DPRMs has the most LA content and showed fastest release after laser irradiation, leading to rapid destabilization via a reduction of lipoyl groups in LA which facilitates DOX release. These results describe that DPRMs with laser irradiation may accelerate DOX release due to ROS responsiveness of LA. In addition, the accumulation of DOX in 1 : 3 micelles was better than that in others, because more LA reduces faster by ROS and

Figure 3. The cumulative DRPMs release profile of various molar ratio between mPEG-Ce6 and mPEG-LA in phosphate buffer saline (pH 7.4, 0.1% Tween 20). a) 1 : 0.3, b) 1 : 1, c) 1 : 3. The red arrows indicate laser irradiation (671 nm light, 20 mw/cm2).


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disassembly. Based on the DOX release kinetics, we selected the molar ratio of 1 : 3 micelles for further experiment. In vitro cellular uptake and cytotoxicity of DPRMs. We next investigated the cellular internalization behavior of free DOX and DPRMs (DOX dose = 5.0 µg/mL, Ce6 = 7.5 µg/mL) by flow cytometry and confocal microscopy with murine colorectal cancer cell (CT-26) and human colon cancer (HCT-116) cells. When CT-26 and HCT-116 cells were treated with DPRMs, DOX fluorescence intensity was proportional to incubation time. As shown in Figure 4a and Figure S6a, flow cytometry revealed that the quantitative fluorescence intensity was shifted to the right according to increased incubation duration in free DOX and DPRMs. Free DOX (left) showed a slightly higher cellular uptake efficacy than DPRMs (right), because free DOX

Figure 4. Representative uptake efficiency of free DOX and DPRMs in CT-26 cells. Flow cytometry analysis of (a) Paragonby Plustime-dependent Environment conditions. (c) Confocal laser scanning free DOX or DPRMs and (b) free Ce6ACS or DPRMs microscopy images of each samples by time-dependent conditions. The scale bar is 20 µm.

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internalizes cells through rapid and nonspecific diffusion, while DPRMs enter cells by slower endocytosis.31 As mentioned above, Free Ce6 (left) showed more enhanced fluorescence than DPRMs (right) because of its hydrophobic characteristics (Figure 4b). Similar to the results of flow cytometry, the observed fluorescence signals in confocal images were increased in both groups as proportional to time (Figure 4b and Figure S6b). This result indicates that the red fluorescence from DOX was successfully accumulated in the cytoplasm delivered by DPRMs. Furthermore, we assessed intracellular ROS generation efficacy of DPRMs and in vitro DOX release behavior upon laser irradiation in HCT-116 cells using DCF-DA as a probe. As shown in Figure S7, green (DCF-DA) and red (released DOX) fluorescence of DPRMs with laser group was much higher than DPRMs without laser and control (non-treated) group. It means DPRMs could generate ROS upon laser irradiation due to Ce6 activation and accelerate DOX release successfully via reduction of lipoyl ring in LA. However, in case of DPRMs and free LA, the negligible fluorescence was observed regardless of laser irradiation. These results demonstrate that addition of free LA also plays a role in scavenging the generated ROS and it is not sufficient to release DOX in DPRMs because of competitive scavenging between free LA and remaining in DPRMs. To demonstrate the in vitro cytotoxicity, both cells were incubated with free DOX, PRMs, and DPRMs in the absence or presence of laser irradiation and subjected to the standard methyl thiaolyl tetrazolium (MTT) assay. According to our preliminary data (Figure S5), the IC50 value of DOX against CT-26 and HCT-116 is ~8.41 and ~10.46 µg/mL. The experiment was performed with laser irradiation (0.6 J/cm2 ; 6.0 mW/cm2, 100 sec) at a DOX concentration equivalent to 5 and 7 µg/mL of Ce6 concentration. Under laser irradiation, DPRMs showed significantly enhanced cytotoxicity compared to DPRMs without laser. Both DOX release and


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ROS generated from remaining Ce6 destroy cells and induce strong phototoxicity that results direct cell death (Figure 5a and Figure S8a). Free DOX showed cell cytotoxicity due to their small size and hydrophobicity regardless of irradiation. PRMs also showed ROS induced

Figure 5. In vitro cytotoxicity assay of CT-26 cells. (a) MTT assay of free DOX, PRMs and DPRMs (DOX conc.; 5 µg/mL, Ce6 conc.; 7.5 µg/mL) under no irradiation or laser irradiation of 0.6 J/cm2 (n = 3, ***P < 0.001). (b) Live & Dead assay after treatment free DOX, PRMs and DPRMs (DOX conc.; 5 µg/mL, Ce6 conc.; 7.5 µg/mL) with or without irradiation (Laser power = 0.6 J/cm2 ). The scale bar is 100 µm.

phototoxicity but not as much as DPRMs owing to the deficiency of DOX. As expected, PRMs without irradiation showed no significant cytotoxicity. To confirm these results, we performed a live and dead assay with Calcein-AM (green) and Ethidium homodimer (red). As shown in Figure 5b and Figure S8b, both green (live cells) and red fluorescence (dead cells) were observed in the free DOX group because of the good permeability of hydrophobic free DOX, regardless of irradiation. Without laser irradiation of the PRMs or DPRMs groups, there was almost green fluorescence due to the inactivation of Ce6. However, under laser irradiation, almost half of the PRMs group images were indicated by red fluorescence. In addition to the DPRMs group with laser irradiation displayed significantly enhanced red fluorescence due to combinatory chemo-photodynamic therapy. These results


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demonstrated that the DPRMs are able to efficiently release the encapsulated DOX upon ROS generation, leading to phototoxicity for synergistic effect under external laser irradiation. In vivo chemophotodynamic therapeutic effect of DPRMs in CT-26 tumor bearing mice. To evaluate the feasibility of DPRMs on tumor growth inhibition, we performed an in vivo tumor suppression experiment in nude mice bearing CT-26 tumors (Figure 6a). Here, we eventually chose colon cancer model which is hard to cure only by chemotherapy and also limited by dose-limiting toxicity.32 To overcome DOX monotherapy, we used DPRMs platform via cytotoxic ROS generation effect of Ce6 and reduced systemic toxicity by EPR effects. All samples were intravenously injected and some samples were irradiated under a laser at a power

Figure 6. In vivo tumor therapeutic efficacy of CT-26 subcutaneous mice model by intravenous injection with various formulations. (a) Tumor growth inhibition test after tail vein injection of PBS, free DOX, PRMs or DPRMs with or without laser irradiation (dose: 2 mg/kg DOX and 3 mg/kg Ce6, n = 4, **P < 0.005 , ***P < 0.001). The red arrows indicate sample injection and blue arrows mean laser irradiation. (b) Body weight measurement of CT-26 tumor bearing mice throughout the treatments (n=5). (c) Histological observation of the tumor tissues stained with H&E after intravenous injection of PBS, Free DOX, PRMs or DPRMs with or without laser irradiation of 100 J/cm2 (n=5). Nuclei were stained blue, and the extracellular matrix and cytoplasm were stained red in H&E staining. Paragon PlusScale Environment Brown color indicates apoptotic cells afterACS a TUNEL assay. bars are 100 µm.

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density of 100 J/cm2 (100 mW/cm2, 1000 sec). Mice injected with PBS, free DOX, and PRMs (with and without laser) demonstrated a rapid increase in the tumor volume over time. Interestingly, DPRMs with no laser irradiated group exhibited aggressive tumor growth, which explains why DPRMs without laser do not possess significant tumor suppression ability. However, the tumors of mice treated with DPRMs under laser irradiation exhibited remarkably more efficiency in tumor growth suppression compared to other groups. From these results, we concluded that efficient tumor growth inhibition could be achieved by efficiently released DOX from DPRMs with ROS production from remaining Ce6 resulting in phototoxicity. As shown in Figure 6b, no significant body weight loss was observed throughout the treatments in all groups, except for a slight decrease in free DOX. This result revealed that DPRMs are a safe drug delivery system for tumor therapy. At the end of the treatments, the tumors were isolated from the mice and used for further evaluation. Microscopic observation of tumor cells apoptosis and necrosis in vivo was conducted by staining with hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay. As shown in Figure 6c, only PRMs and DPRMs with laser irradiated groups demonstrated the destruction of tumor cells by photosensitizer-mediated phototoxicity. However, the DPRMs group generated more extensive apoptotic and necrotic cells and showed a superior treatment effect than the PRMs group. The tumor from DPRMs treatment significantly reduced the number of cancerous cells and increased the number of TUNELpositive tumor cells indicating the enhanced anti-tumor efficiency for inhibiting cell proliferation and inducing cell death. Thus, these results indicate that combination therapy with DPRMs leads to significant tumor growth inhibition.


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Conclusion In summary, we demonstrated that photoresponsive micelles-encapsulated doxorubicin combines both chemotherapy and photodynamic therapy for the spatiotemporally controlled release of anticancer drugs by external stimuli. In the designed DPRMs, DOX was encapsulated in a hydrophobic core of micelles, while ROS scavenger (lipoic acid) and ROS generator (Ce6) were covalently conjugated with mPEG to construct a single combinatory system. Under laser irradiation, the ROS generation of DPRMs induced the reduction of lipoyl groups in mPEG-LA, thereby promoting the release of DOX from micelles. In vitro and in vivo studies showed that treatment with DPRMs and laser irradiation resulted in higher therapeutic efficacy than chemotherapy alone. Importantly, this strategy can remarkably be utilized for efficient combination therapy in cancer treatment as a promising platform with great potential for clinical applications.

Associated Content Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Synthetic method by structures and its characterization ; H-NMR, FT-IR spectrum; Slope of singlet oxygen generation efficacy; IC50 of Doxorubicin in CT-26 and HCT-116 cells; Cellular uptake of free DOX and DRPMs in HCT-116 cells; In vitro reactive oxygen species generation efficacy and DOX release behavior in HCT-116 cells; Cell viability of


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free DOX, PRMs, and DPRMs with or without laser irradiation in HCT-116 cells via MTT and Live & Dead assay.

Author Information Corresponding Author * E-mail: [email protected] Contributions ‡ D.H.K. and H.S.H. contributed equally to this work. Conflict of interest statement The authors declare that they have no conflict of interest.

Acknowledgements This work was financially supported through a grant the Basic Research Laboratory (BRL) program (NRF-2015R1A4A1042350), the National Research Foundation of Korea (NRF) grant funded by the Korean government (Ministry of Science, ICT and future Planning).

Abbreviations PDT, photodynamic therapy; DOX, doxorubicin; PS, photosensitizer; ROS, reactive oxygen species; DPRMs, DOX-loaded photoresponsive micelles; Ce6, chlorin e6; LA, lipoic acid; PRMs, DOX-unloaded photoresponsive micelles


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