Dual Imaging Guided Oxidative-Photothermal Combination Anticancer

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Biological and Medical Applications of Materials and Interfaces

Dual Imaging Guided Oxidative-Photothermal Combination Anticancer Therapeutics Joungyoun Noh, Eunkyeong Jung, Donghyuck Yoo, Changsun Kang, Chun-Ho Kim, Sangjun Park, Gilson Khang, and Dongwon Lee ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b14968 • Publication Date (Web): 01 Nov 2018 Downloaded from http://pubs.acs.org on November 1, 2018

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ACS Applied Materials & Interfaces

Dual Imaging Guided Oxidative-Photothermal Combination Anticancer Therapeutics Joungyoun Noh, Eunkyeong Jung,§ Donghyuck Yoo,§ Changsun Kang,§ Chunho Kim,‡ Sangjun Park,‡ Gilson Khang,,§ and Dongwon Lee,§,* Department

of PolymerNano Science and Technology, Chonbuk National University, Jeonju,

Chonbuk, 54896, Republic of Korea §Department

of BIN Convergence Technology, Chonbuk National University, Jeonju, Chonbuk,

54896, Republic of Korea ‡Korea

Institute of Radiological & Medical Sciences, Nowonro 75, Nowon-gu, Seoul, 01812,

Republic of Korea Corresponding Author Dongwon Lee, Email: [email protected].

ACS Paragon Plus Environment

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Abstract

Heme oxygenase-1 (HO-1) is a stress-response protein with potent cytoprotective and antioxidant activity and its expression in cancer cells is enhanced in response to chemotherapy and radiotherapy. HO-1 is known to serve as a shield to protect cancer cells from anticancer therapy and attenuate apoptotic signals. It can be therefore reasoned that inhibition of HO-1 reduces the antioxidant level, making cancer cells more sensitive to photothermal heating. In this work, we developed dual imaging-guided oxidative-photothermal combination nanotherapeutics (OPCN) consisting of amphiphilic polymers conjugated with ZnPP (zinc protoporphyrin) as a HO-1 inhibitor and fluorescent IR820 as a photothermal agent, respectively. Combination of OPCN and near infrared (NIR) laser irradiation markedly increased temperature and exerted significant toxicity through induction of apoptosis. In a mouse model of xenografts, tumors were identified by the strong fluorescence and photoacoustic signals. OPCN


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combined with NIR laser irradiation resulted in effective and complete thermal ablation of tumors without discernable side effects and tumor recurrence. We believe that OPCN hold tremendous translational potential for dual imaging-guided oxidative-photothermal combination anticancer therapy.

Keywords: heme oxygenase-1, photothermal therapy, cancer, imaging, combination therapy

1. Introduction

Chemotherapy is one of cancer treatment regimens that employs one or more anticancer drugs and is the most widely used in the management of cancer patients.1 However,

chemotherapeutic

drugs

have

several

drawbacks

such

as

poor


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pharmacokinetics, low solubility, lack of ability to target cancer cells and the possibility to trigger multidrug resistance.2 In order to address these challenges, tremendous efforts have been made to develop other cancer treatment modalities such as photothermal therapy (PTT), photodynamic therapy (PDT) and radiotherapy.1,3-4 PTT is a recently emerging therapeutic strategy that employs a photothermal agent (photothermal sensitizer) and laser.5-6 Under the laser irradiation with an appropriate wavelength, photothermal agents are excited by photon energy and quickly release vibrational energy (heat), which could destroy tumors. PTT exhibits unique advantages over conventional therapeutic modalities including high specificity and precise spatial-temporal selectivity.7-8 Various organic dyes and gold nanostructures with an emission wavelength of NIR have been extensively utilized as a photoabsorber.5,9 In particular, indocyanine green (ICG), a NIR imaging agent has been extensively studied as a photoabsorber for PTT due to its biocompatibility and sufficient photothermal conversion efficiency.10 However, applications of ICG have been hampered by its non-specific binding to plasma proteins, concentration-dependent aggregation, poor aqueous stability and lack of specificity.11 There is therefore increasing interest in the development of photothermal agents with high stability in the physiological environment and high photothermal conversion efficiency.


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Recently, oxidation therapy has attracted increasing attentions and has been emerging as a promising anticancer therapeutic strategy.12-17 Oxidation therapy exploits the biochemical and biological differences between normal and cancer cells.18 Compared to normal cells, cancer cells are under oxidative stress because of overproduction of reactive oxygen species (ROS) which results from unrestrained growth and metabolic and signaling aberrations.15 Paradoxically, cancer cells are also equipped with a large amount of antioxidants to counterbalance the detrimental effects of ROS.19 Therefore, cancer cells are more vulnerable to exogenous agents that elevate oxidative stress or deplete antioxidant defense systems.20-21 One of antioxidants which can be discussed as being involved in oxidation therapy is heme oxygenase-1 (HO-1). As one of inducible heat shock proteins, HO-1 protects cells from oxidative injury and cellular stress by attenuating apoptotic signals.22 HO-1 is induced at low levels in most mammalian cells under normal conditions, but is highly induced under stress conditions such as chemotherapeutic agents, UV irradiation, heat shock, hypoxia and hydrogen peroxide.23-24 HO-1 is overexpressed in various cancer cells to protect them from oxidative stress by providing antioxidant bilirubin.25 Cancer cells are known to utilize the cytoprotective and anti-apoptotic HO-1 as a shield from chemotherapy and PDT. Additionally, cancer cells overexpressing HO-1 are resistant to chemotherapy, PDT and radiotherapy.26-27 In this context, a number of studies have targeted HO-1 inhibition as a promising strategy to sensitize cancer cells to additional therapy.22 Accumulative evidence has demonstrated that zinc protoporphyrin (ZnPP) inhibits significantly the induction of HO-1 in various cancer cells and suppresses tumor growth.24,28 However, the clinical applications of ZnPP as an anticancer agent are limited by its poor water solubility. In order to overcome this hurdle, water soluble ZnPP derivatives have been developed by conjugating poly(ethylene glycol), PEG.24-25 Despite selective tumor accumulation


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and targeted inhibition of HO-1 in tumor, the PEGylated ZnPP had critical drawbacks such as its low ZnPP loading content (~1.5 wt%) and undesirable high viscosity.29 Therefore, it is critical to

develop a strategy to achieve higher loading of ZnPP, while maintaining beneficial properties such as tumor targeting and aqueous solubility. Scheme 1. A schematic diagram of OPCN for dual imaging-guided oxidativephotothermal combination anticancer therapy.

The hypothesis of this work is that ZnPP-mediated inhibition of HO-1 provides a unique and promising strategy to make cancer cells more susceptible to photothermal heating and significantly enhances therapeutic efficacy of PTT (Scheme 1). In combination therapy, simultaneous and sufficient delivery of multiple therapeutic agents to the same site is critical because therapeutic success is hampered by insufficient tumor delivery when delivered independently. In order to realize full potential of combination of oxidation therapy and PTT, we developed oxidative-photothermal combination nanotherapeutics (OPCN) composed of ZnPP-


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conjugated polymer and IR820-conjugated polymer. Acid-sensitive amphiphilic poly(-amino ester) was developed as a framework of nanotherapeutics, to which ZnPP or IR820 was conjugated. As a derivative of ICG, IR820 has proven its potential for fluorescence imaging and photoacoustic imaging due to enhanced stability and long imaging time.30-32 In this study, OPCN were also exploited as a fluorescent and photoacoustic dual imaging agent for tumor detection. Here, we report the potential of OPCN as a dual imaging-guided combination anticancer therapeutic agent in the studies of cell culture models and xenograft mouse models.

2. Materials and Methods 2.1. Materials. Hexanediol diacrylate, protoporphyrin IX zinc (II), IR820 and poly(ethylene glycol) methyl ether acrylate of average Mn ~2,000 were purchased from Sigma-Aldrich (St. Louis, MO). Dichloromethane (DVM) and tetrahydrofuran (THF) were obtained from Samchun (Korea) and was dried with calcium hydride before the use. A549 (lung cancer cell line) and SW620 (colon cancer cell line) were obtained from Korean Cell Line Bank (Korea). 2.2. Synthesis of OPCN. 4.4'-Trimethylene-dipiperidine (1.2 mmol), ethanol amine (0.11 mmol), poly(ethylene glycol) methyl ether acrylate (0.12 mmol) and hexanediol diacrylate (1.1 mmol) were dissolved in 5 mL of dry DCM. The polymerization reaction was allowed with gentle stirring at 38C. After 72 h of polymerization reaction, the reaction mixture was poured into the cold hexane to terminate the reaction. Solid white polymer was obtained from vacuum drying. The chemical structure of the obtained poly(β-amine ester) was verified by nuclear magnetic resonance (NMR) and its molecular weight was determined using gel permeation chromatography (Waters, Milford, MA) equipped with a refractive index detector. To prepare IR820-conjugated poly(β-amine ester), poly(β-amine ester) and IR820 were dissolved in 5 mL of


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dimethyl formamide, to which triethylamine was added. For conjugation of ZnPP to poly(βamine ester), poly(β-amine ester) and ZnPP were dissolved in 5 mL of dimethyl sulfoxide (DMSO) containing EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) and DMAP (4dimethylaminopyridine). IR820-conjugated polymer and ZnPP-conjugated polymer were obtained from extraction using water/DCM and dried under vacuum at room temperature. 2.3. Preparation and characterization of OPCN. OPCN were prepared by self-assembly in aqueous solutions. ZnPP-conjugated polymer and IR820-conjugated polymers were dissolved in 100 µL of THF to yield a concentration of 10 mg/mL at a different weight ratio. The polymer solution was added into 10 mL of pH 7.4 PBS (phosphate buffer solution) and the mixture was gently stirred. OPCN were prepared by evaporating THF using a rotary evaporator. The hydrodynamic size of OPCN was measured by dynamic light scattering using a particle size analyzer (90Plus, Brookhaven Instrument Corp., Holtsville, NY) in presence or absence of FBS (fetal bovine serum). The morphology of OPCN was observed using a transmission electron microscope (Bio-TEM, HITACHI Corp., Japan) after staining with a tungstic acid solution. A UV-Vis spectrometer (S-3100, Scinco, Korea) was employed to observe the UV-Vis absorption spectra of the OPCN in a quartz cuvette. The fluorescence emission of OPCN was investigated using a fluorescence spectrometer (FP-6500, Jasco, Japan). A continuous wave 808 nm laser diode (Changchun New Industries Optoelectronics, China) was used to investigate the photothermal effects of OPCN. 2.4. Cellular uptake of OPCN. Cells (A549 cell line) were cultured in a glass bottom dish (MatTek Corp., Ashland, MA) containing 10% FBS in an incubator with 5% CO2 at 37C. Cells were treated with OPCN for 3 h or 6 h. Cells were gently washed with fresh medium twice and


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then their fluorescence images were obtained using a confocal laser scanning microscope (LSM510 Meta, Carl Zeiss, Germany). 2.5. Cytotoxicity of OPCN. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to evaluate

the cell viability after the treatment of OPCN

combined with NIR laser irradiation. After the treatment with various formulations, cells were given 100 µL MTT solution and then incubated for another 2 h. Each well was given 900 µL of DMSO to dissolve the resulting formazan crystals. The cell viability was determined by measuring the absorbance of cells at 570 nm with a microplate reader (Biotek Instruments, Winooski, VT). 2.6. Flow cytometry. After the treatment with various formulations, cells were gently washed with fresh medium and then resuspended in 1 binding buffer at a concentration of 1105 cells/mL. The cell suspensions were transferred to a round-bottom tube. The cells were stained with Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide for 15 min and then were given 400 µL of 1 binding buffer. The cells were analyzed using a FACS caliber (Becton Dickinson, San Jose, CA) with a minimum event of 1  104. 2.7. Immunoblot analysis. The level of apoptosis-related proteins in cells was determined by western blotting. A549 cells (2106 per well) were treated with IR820 micelles, ZnPP micelles or OPCN for 6 h and then washed with fresh PBS twice. Cells were lysed in a lysis buffer and proteins were extracted. Proteins were separated by electrophoresis on a 10% polyacrylamide gel and then transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were incubated with HO-1 antibody (Cell Signaling Technology, Beverly, MA), Bcl-2 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX), Bax antibody (Santa Cruz Biotechnology,


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Dallas, TX), caspase-3 antibody (Cell Signaling Technology, Beverly, MA), PARP antibody (Cell Signaling Technology, Beverly, MA), β-actin (Santa Cruz Biotechnology, Dallas, TX) and HRP-linked anti-rabbit IgG (Cell Signaling Technology, Beverly, MA), which is used as a secondary antibody. The immunoblots were developed with ultrachemiluminescent reagent (Pierce, Rockford, IL) and the images were cropped for presentation. 2.8. Fluorescence and photoacoustic imaging of tumor bearing mice. Nude mice (4 weeks old, Orient Bio, Korea) were injected with A549 cells (1106 cells) in a flank to establish subcutaneous tumors. When tumors reached a volume of ~200 mm3, mice were injected with OPCN (5 or 10 mg/kg) intravenously through a tail vein. The fluorescence imaging of tumors was made using a fluorescence imaging system (FOBI, Neoscience, Korea). Photoacoustic imaging was obtained using a laboratory prototype of a photoacoustic system developed by coupling a SonixTouch Ultrasound machine (Ultrasonix, Canada) with a laser system (SL III-10, Continuum, San Jose, CA) operating at 808 nm wavelength with 3 Hz pulse repetition and 5-7 ns pulse duration. The photoacoustic signals in tumors were detected using an unfocused ultrasonic transducer (central frequency: 7.5 MHz and aperture: 1.4 cm). 2.9. Evaluation of anticancer activity of the combination of OPCN with NIR laser irradiation. A549 cells (3106) were directly injected into a flank of mice (4 weeks old, Orient Bio, Korea) to develop subcutaneous tumors. Tumor-bearing mice were randomly divided into 8 groups. OPCN were intravenously injected into tumor-bearing mice through a tail vein at a dose of 5 mg/kg on days 0, 2 and 4. The tumors were irradiated with 808 nm laser (1 W/cm2) for 10 min at days 1, 3 and 5. The tumor volume and body weight of mice were recorded every 2 days. Tumor volume calculation was obtained using the formula: (width2  length)/2. All the experiments using animals were approved by the Institutional Animal Care and Use Committee


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of Chonbuk National University (CBNU2016-73) and carried out in accordance with the guidelines of the institution animal ethical committee. 2.10. Toxicity of OPCN. Healthy mice (Orient Bio, Korea) were intravenously injected with OPCN (5 mg/kg) through a tail vein on days 0, 2 and 4. Liver and heart were excised on day 10, and the tissues were fixed with formalin and embedded in paraffin. The tissue blocks were sectioned, ~5 µm thick. The tissue sections were stained with H&E (hematoxylin and eosin) and TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling). The stained tissue sections were observed under a microscope (Eclipse, Nikon, Japan) and a confocal laser scanning microscope (LSM510 Meta, Carl Zeiss, Inc. Germany). 2.11. Statistical analyses. Values were expressed as mean ± SD (standard deviation). Comparison between groups was conducted with one-way ANOVA using GraphPad Prism 5.0 (San Diego, CA, USA). Probability (p) value of

Dual Imaging Guided Oxidative-Photothermal Combination Anticancer

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