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Nov 22, 2016 - Madhappan Santha Moorthy 1, Hansu Seo 2, Nhat Quang Bui 3, and ...... Serum albumin has many biologically friendly effects and has vast.
Accepted Manuscript Title: Astaxanthin conjugated polypyrrole nanoparticles as a multimodal agent for photo-based therapy and imaging Author: Subramaniyan Bharathiraja Panchanathan Manivasagan Yun-Ok Oh Madhappan Santha Moorthy Hansu Seo Nhat Quang Bui Junghwan Oh PII: DOI: Reference:

S0378-5173(16)31151-6 http://dx.doi.org/doi:10.1016/j.ijpharm.2016.12.020 IJP 16291

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

13-10-2016 22-11-2016 9-12-2016

Please cite this article as: Bharathiraja, Subramaniyan, Manivasagan, Panchanathan, Oh, Yun-Ok, Moorthy, Madhappan Santha, Seo, Hansu, Bui, Nhat Quang, Oh, Junghwan, Astaxanthin conjugated polypyrrole nanoparticles as a multimodal agent for photo-based therapy and imaging.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2016.12.020 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ORIGINAL RESEARCH

Astaxanthin conjugated polypyrrole nanoparticles multimodal agent for photo-based therapy and imaging

Subramaniyan Bharathiraja

1,

Panchanathan Manivasagan

1,

as

Yun-Ok Oh

a

1,

Madhappan Santha Moorthy 1, Hansu Seo 2, Nhat Quang Bui 3, and Junghwan Oh 1, 2, 3*

1

Marine-Integrated Bionics Research Center, Pukyong National University, Busan 48513, Republic of Korea. 2

Department of Biomedical Engineering, Pukyong National University, Busan 48513, Republic of Korea. 3

Department of Biomedical Engineering and Center for Marine-Integrated Biotechnology (BK21 Plus), Pukyong National University, Busan 48513, Republic of Korea. ∗

Corresponding author at: Marine-Integrated Bionics Research Center, Pukyong National University, Busan 48513, Republic of Korea. Fax: +82 51 629 5779. E-mail address: [email protected] (J. O).

Graphical abstract

Abstract Polymeric nanoparticles are emerging as promising candidates for photo-based therapy and imaging due to their versatile chemical properties and easy fabrication and functionalization. 1

In the present study we synthesized polypyrrole nanoparticles by stabilization with astaxanthin conjugated bovine serum albumin polymer (PPy@BSA-Astx). The synthesized nanoparticles were biocompatible with MBA-MD-231 and HEK-293 cells. Interestingly, the fabricated nanoparticles produced reactive oxygen species under 808-nm laser exposure and exerted a hyperthermic effect when the power density of the laser was increased. The photodynamic efficiency of PPy@BSA-Astx was measured by DPBF assay, and it was found to generate sufficient amount of reactive radicals to kill the cells at a power density of 0.3 W/cm2. In photothermal aspect, the temperature level was reached to 57 ºC within 5 min at 1 W/cm2 power density, at the concentration of 50 µg/ml. The in vitro cell toxicity studies showed concentration dependent photothermal and photodynamic toxicity. Fluorescence microscopic investigation explored the cell death and intra-cellular organ destruction by photodynamic treatment. In addition, we observed a strong photoacoustic signal from a tissue mimicking phantom study of nanoparticle treated MBA-MD-231 cells. In conclusion, the fabricated PPy@BSA-Astx nanoparticles can be used as photoacoustic imaging based prognostic agents for photothermal or photodynamic treatment.

Keywords: albumin-polypyrrole, astaxanthin, photodynamic, photothermal, photoacoustic.

1. Introduction Phototherapy is emerging as a promising treatment strategy to kill infected cells due to its selective and localized therapeutic effect upon laser irradiation. Two major modalities of photo treatment are available. One of these is photothermal therapy (PTT), where a laser based heating effect is used to denature the cell’s biomolecules and its membrane. In another

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approach, photodynamic therapy (PDT), photosensitizers are used to propagate reactive oxygen species (ROS) to irreversibly damage the cell using a laser source. Ideal materials for phototherapy should have the following properties: biocompatibility, high absorption intensity, photo stability, and easy degradability (Nishiyama et al., 2003). The use of a near infrared (NIR) laser is another essential factor because blood and tissues absorb in the UVVis spectral region which leads to non-specific damage. In addition, water significantly absorbs spectral wavelengths above 900 nm which leads to a non-specific temperature rises in surrounding tissue during treatment. Metallic nanoparticles such as gold (Singh et al., 2016), copper (Wang et al., 2015), platinum (Manikandan et al., 2013), and silver (Huang and El-Sayed, 2011), and carbon nanomaterials (Hong et al., 2015) are widely used in photobased therapies. However, metallic nanoparticles are non-biodegradable and there are concerns regarding long-term toxicity due to their accumulation in tissues for a prolonged period of time. Currently polymeric nanoparticles have attracted much interest due to their biocompatibility, stability, drug loading capacity, and easy fabrication (Cheng et al., 2012). Of the various polymeric materials, polypyrrole, an organic conductive polymer, is an attractive candidate for photo-based therapies as it has a strong absorption in the NIR region. Traditionally, polypyrrole nanoparticles (PPy NPs) are synthesized using poly(vinyl alcohol) as a stabilizing agent (Yang et al., 2012). In the present work, we synthesized PPy NPs using astaxanthin (astx) conjugated bovine serum albumin (BSA) as a coating agent (PPy@BSAAstx). Here we used astx as a photosensitizer for possible induction of ROS in PDT modality and astx was already reported for free radical formation (Polyakov et al., 2010). Astx is a natural keto-carotenoid pigment which has anticancer activity and can trigger a cellular immune response to fight against diseases (Song et al., 2011). Moreover it has been reported for antibacterial and anti-inflammatory activities (Ambati et al., 2014). PPy NPs have been 3

demonstrated to have high photothermal conversion with a good photostability (Zha et al., 2013). Synthesis of nanoparticles for PTT combined with PDT has advantages over singular treatment modality. In addition, we tried to utilize PPy@BSA-Astx for photoacoustic imaging (PAI), a non-invasive imaging technology. The imaging of tissue using PAI was gradually investigated in order to enable image guided therapy (Chen et al., 2010). Imaging using same therapeutic nanocomposite can be supportive for diagnosis combined treatment and it can provide information about tumor localization and therapeutic efficiency (Song et al., 2014). In the present study, we synthesized PPy@BSA-Astx nanoparticles for PAI imaging combined PDT and PTT. 2. Materials and methods 2.1. Materials Astaxanthin (astx), bovine serum albumin (BSA), pyrrole monomer, dimethyl sulfoxide (DMSO), N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-hydrox -ysuccinimide (NHS), ferric chloride hexahydrate (FeCl3.6H2O), potassium bromide (KBr), 1,3-diphenyisobenzofuran (DPBF), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma Aldrich (St. Louis, MO, USA). Cellular staining reagents, such as acridine orange (AO), Hoechst 33342, rhodamine-123 (rho-123), and dichlorodihydrofluorescin diacetate (DCFH-DA), were also purchased from Sigma Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, 1X trypsin, and phosphate buffered saline (PBS) were purchased from HyClone (South Logan, UT, USA).

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2.2. Formulation of PPy@BSA-Astx To synthesize BSA-Astx conjugation, 0.596 mg of astx was dissolved in 40 µL of DMSO, an equal molar ratio of EDC and NHS (astx: EDC: NHS = 1:1:1) was added, and the mixture was vibrated gently for 30 min. An 8-mL PBS solution consisting 20 mg of BSA was added to the above reaction solution and allowed to stir overnight. Then, the solution was filtered using 10 kDa MWCO filters (Millipore, Billerica, MA, USA) to remove the excess astx, EDC, and NHS. Finally 20 mg of purified product was obtained. Based on Uv-Vis spectra analysis it was predicted that 2.5 molecules of astx were conjugated to each molecule of BSA. The concentration of BSA was determined using bicinchoninic acid (BCA). The purified BSA-Astx complex was dissolved in 6 mL of water. To this 20 µL of pyrrole monomer was added to synthesize PPy@BSA-Astx. The reaction mixture was stirred for 1 h and then 20 mg of FeCl3.6H2O was added to initiate the oxidation process. The mixture was then gently stirred for 24 h. Finally, the reaction mixture was purified using 100 kDa MWCO filters, and the final product was dissolved in water for further characterization and then freeze dried for subsequent experiments. BSA stabilized PPy NPs (PPy@BSA) were synthesized in the same way without astx for comparative studies. 2.3. Characterization The morphology of the synthesized particles was investigated using transmission electron microscopy (TEM) (JEM 1010 JEOL, Tokyo, Japan). An aqueous sample was deposited on a carbon-coated copper grid and dried and then mounted for TEM. A part of the liquid sample was taken to find its size distribution using dynamic light scattering (DLS). To analyze the functional groups of the final particles, an aliquot of freeze dried product was pelletized with KBr for FTIR analysis at a resolution of 4 cm−1 over a wavelength range of 500 to 4000 cm−1.

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2.4. Cellular biocompatibility MDA-MB-231 human breast cancer cells were used as a model cancer cell line. Normal human embryonic kidney cells (HEK 293) also were used to evaluate the toxicity of PPy@BSA-Astx. Both cells were cultured in DMEM medium containing 10% FBS and supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin. Cell cultures were maintained at 37 ºC with 5% CO2 in a humidified atmosphere. To evaluate the toxic effect of the formulated particles, MDA-MB-231 and HEK-293 cells were cultured separately in 96-well plates at 104 cells/well. Then, the cells were treated with different concentrations of PPy@BSA-Astx and incubated for 24 h. A standard MTT assay (Mosmann, 1983) was adopted to determine the cell viability for the different treatments used in this work. MTT is reduced to form an insoluble purple formazan product by dehydrogenase enzymes which are present in metabolically active cells. The insoluble purple formazan was dissolved with DMSO and its absorbance was measured at 570 nm to calculate the percentage of cell viability. 2.5. DPBF assay DPBF assay was used to measure the singlet oxygen production efficacy of synthesized particles under 808-nm illumination. Upon exposure to singlet oxygen, DPBF is converted to 0-dibenzoylbenzene (DBB) which can be monitored by measuring absorbance at 418 nm. Since DPBF can rapidly react with 1O2, it can be easily converted to DBB which has a lesser absorption than DPBF. The DPBF assay was performed based on previous protocol (Ai et al., 2015), In a typical experiment, 2 mL of 10 µg/mL PPy@BSA-Astx and an equal concentration of astx (0.5 µM calculated by mass extinction coefficient) and the synthesized astx unconjugated PPy@BSA (10 µg/ml) were placed in a separate cuvette along with 10 µM of DPBF dissolved in DMSO. After measuring the initial absorption at 418 nm, the samples were irradiated with an 808-nm laser at different (0.1 and 0.3 W/cm2) power densities in dark conditions for 20 min. The absorbance at 418

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nm was recorded for each 5 min for all the samples. A sample of PPy@BSA-Astx which had not been exposed to laser irradiation was retained for comparative analysis.

2.6. In vitro PDT effect To analyze the PDT mediated cell toxicity, MDA-MB-231 cells were seeded in a 96well plate at a concentration of 1 x 104 cells/well. The seeded cells were treated with different concentrations of PPy@BSA-Astx for 6 h and then the unbound particles were washed with PBS. After that, the cells were illuminated with an 808-nm continuous laser at power density 0.3 W/cm2 for 15 min with a 1 min gap for every 5 min. The laser treated cells were further incubated for 24 h, and cell viability was measured using the MTT assay. Cells exposed to laser irradiation without PPy@BSA-Astx were used as controls.

2.7. Morphology of PDT treated cells MDA-MB-231 cells were prepared in 12-well plate at density of 1 x 105 cells/well. The cells were treated with PPy@BSA-Astx at different concentrations for 6 h and unbound particles were rinsed with PBS buffer. Then, the cells were exposed to 808-nm irradiation at a power density of 0.3 W/cm2 for 15 min with a 1 min interval for every 5 min. To observe the morphology of the cells, the culture plate was mounted under a microscope (Leica Microsystems GmbH, Wetzlar, Germany) and bright field images of treated and untreated cells were captured. To examine cellular organs like nuclei and mitochondria, the treated cells were stained with Hoechst and rho-123 fluorescent stains, respectively, and observed under a fluorescent microscope. 2.8. Intracellular ROS measurement DCFH-DA assay was adapted to study the intracellular ROS level. DCFH-DA is a cell permeable probe that is hydrolyzed to form dichlorodihydrofluorescein (DCFH) in the intracellular environment. Upon reaction with intracellular ROS, DCFH form a fluorescent 7

product dichlorofluorescein (DCF) which can be visualized under fluorescence microscope. For DCFH-DA assay, the cells were seeded in 12-well plate, treated with different concentration of formulated PPy@BSA-Astx nanoparticles, and incubated for 6 h. After washing the unbound particles with PBS, cells were illuminated with an 808-nm laser with a power density of 0.3 W/cm2 for 15 min. In addition, cells were incubated for 1 h, and then 10 µM of DCFH-DA was added and the cells incubated for further 30 min. After this, excess stain was washed away with PBS and green fluorescent intensity was measured using a microplate reader and the cells were micrographed using a fluorescent microscope. 2.9. Heating experiment We next evaluated the photothermal effect of PPy@BSA-Astx using an 808-nm continues wave laser at a power density of 1 W/cm2. The nanoparticles were dissolved in water at different concentrations (10, 20, 30, 40, and 50 µg/mL) and placed in 12-well plate. Each well which contained a nanoparticle solution was then irradiated using an 808-nm laser with a power density of 1 W/cm2 for 5 min. The temperature was monitored using a thermal fiber which was connected to a MASTECH-MS6514 thermometer (MASTECH, CA, USA). Simultaneously, thermograph images were captured using an IR camera. 2.10. In vitro PTT toxicity To study the heating effect of PPy@BSA-Astx on breast cancer cells, MBA-MD-231 cells (1 x 104) were seeded in 96-well plate and treated with different concentrations of nanoparticles. After 6 h incubation the unbound particles were rinsed with PBS and the cells were irradiated with an 808-nm laser at 1 W/cm2 power density. Again, the incubation of cells in the CO2 incubator continued for 24 h, and then cell viability was determined using a standard MTT assay.

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2.11. Staining of PTT treated cells MDA-MB-231 cells were grown at high-density in 12-well plate and treated with 50 µg/ml PPy@BSA-Astx nanoparticles for 6 h, after which the free nanoparticles were removed with PBS. The treated cells were illuminated under an 808-nm laser at power density of 1 W/cm2 for 5 min. Then, the cells were incubated for 3 h. After incubation, the cells were stained with AO stain and observed under a fluorescence microscope. 2.12. Photoacoustic imaging PPy@BSA-Astx was screened for the production of ultrasound signals under 808-nm laser irradiation to image the treated cells. In this experiment, pre-seeded MBA-MD-231 cells were treated with different concentrations of PPy@BSA-Astx for 6 h and then the cells were harvested and loaded with 2% gelatin on a tissue mimicking phantom which was prepared with 8% poly vinyl alcohol and 0.2% silica. Then, the cells were covered with the same 2% gelatin mixture and allowed to solidify. The non-invasive PAI system was previously developed and described by Bui et al. (2015) and we followed the same to capture photoacoustic images. A non-ionizing laser system integrated with a pulsed Nd-YAD Qswitched laser (Surelite III, San Jose, CA, USA), in which the laser light can be tuned from 650 to 1064 at 10 Hz with 5 ns pulse operation, was used. The wavelength was fixed at 808nm to obtain the photoacoustic images. The input optical fiber connected plano-convex lens had a focal length of 50 mm (Thorlabs, Newton, NJ, USA). The output end of the fiber was connected to a focused transducer (Olympus NDT, Waltham, MA, USA) and aligned to the center of the illuminated area. Then, the signals were digitized and stored using a data acquisition (DAQ) system in coordination with a laser system to capture photoacoustic signals. To control the DAQ system and the actuators, a custom LabView program (Version: 2010, National Instruments, Austin, TX, USA) was developed. Finally, the envelope signals

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were used to construct a 3D photoacoustic image using a home-made Matlab program (Version: 2013, MathWorks, Natick, MA, USA). 2.13. Statistical analysis The data were presented as mean ± standard deviation. A comparison analysis was performed using one-way analysis variance (OriginPro 8, Northampton, MA, USA) to estimate the statistical parameters. 3. Results 3.1. Preparation and characterization of PPy@BSA-Astx We synthesized PPy@BSA-Astx by making a 1:1 mixture of pyrrole and BSA-Astx. Scheme. 1 represents the systematic diagram for the synthesis of PPy@BSA-Astx. We obtained monodispersed water soluble stable nanoparticles. The pyrrole monomer was oxidized by ferric chloride and the particles formed and stabilized with BSA, which is already linked with astx using EDC and NHS as catalyzers. The particles showed spherical shape morphology (Fig. 1B) and a size range of 23–70 nm with an average size of 41.29 nm (Fig. 2A). The polydispersity index was found to be 0.4. As shown in Fig. 1A, the synthesized particles have a broad absorption range in the NIR region, which can potentially be used for PDT and PTT using NIR light. The PPy@BSA-Astx showed a narrow absorption at 436 nm and another broad absorption with highest absorption intensity at 900 nm (Fig. 1A). We have chosen 808-nm absorption point for further an 808-nm laser based experiments. Fig. 2B shows the FTIR spectra of the synthesized PPy@BSA and PPy@BSA-Astx nanoparticles. In Fig. 2B, the FTIR vibration bands at 2892 and 2922 cm−1 correspond to C-H stretching, and an intense band appears at 1525 cm−1 due to the C=C stretching vibration, indicating

the

existence

of

PPy

NPs.

Furthermore,

the

band

at

1171 cm−1 for the C=O stretching and the vibration spectral regions at 1395, and 1040 cm−1, corresponding to the presence of C=C, and C-O stretching vibrations, respectively, and the

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appeared peaks collectively indicated the presence of coated BSA molecules (Fig. 2B). In addition, PPy@BSA-Astx showed almost same the vibration bands but with considerably increased peak intensities and slight peak shifts at 1655, 1530, and 1166 cm−1 for C=O, C=C, and C-O groups, respectively, for the presence of astx linked PPy@BSA nanoparticles (Fig.2B). In addition, both materials (Fig. 2B) showed new vibration peaks at 3355 cm−1 for O-H bending and 3201 cm−1 for N-H bending. From the FITR spectra of the materials PPy@BSA and PPy@BSA-Asta, it was evidenced that astx was conjugated with PPy NPs through BSA molecule. 3.2. Cellular toxicity Two different cell lines were chosen to assess the in vitro toxic effect of synthesized particles. HEK-293 is a non-cancerous cell model and MBA-MD-231 is a breast cancer cell model and both cells were separately treated with different concentrations of PPy@BSA-Astx for 24 h. MTT assay of 24 h incubated cells showed that the synthesized material did not have any toxic effects on MBA-MD-231cells (Fig. 3) and HEK-293 cells (Fig. S1). The formulated nanoparticles have very good biocompatibility for the concentrations ranging from 10 to 50 µg/ml. 3.3. Determination of singlet oxygen generation Singlet oxygen production efficiency of astx incorporated PPy@BSA-Astx was evaluated by DPBF photo-oxidation assay. Initially, free astx and PPy@BSA-Astx were exposed to 808-nm irradiation at a power density of 0.1 W/cm2 for 20 min and analyzed at 5 min intervals for the decay of DPBF absorbance at 418 nm. Fig. S2 shows that the components did not cause a considerable bleaching effect of DPBF, or, in other words, the singlet oxygen production was not effective at the power of 0.1 W/cm2. The power density of the laser was then increased to 0.3 W/cm2 during the treatment of the second batch. Interestingly, the results showed a time-dependent increase in ROS generation by 11

PPy@BSA-Astx (Fig. 4). The same nanoparticles without laser exposure did not show any ROS induction. The free photosensitizer astx was found to be a weak ROS inducer as it was having weak absorbance in the NIR region. PPy@BSA without astx also did not show any considerable ROS induction under 808-nm laser exposure. DPBF assay concluded that PPy@BSA-Astx can be used for PDT treatment modality (Fig. 4B). 3.4. In vitro photodynamic cell killing effect To determine the photodynamic toxic effect on breast cancer cells, MBA-MD-231 cells were treated with PPy@BSA-Astx and illuminated under an 808-nm laser to induce ROS which can cause a dysfunction the normal cellular mechanism. The cytotoxicity effect of PDT was investigated by an MTT assay. Fig. 3 shows that a considerable decrease in cell viability depends on the concentration of PPy@BSA-Astx. The relative cell viability was compared with that of control cells, which were exposed to laser irradiation without PPy@BSA-Astx. The astx-laser did not induce considerable cytotoxicity to the MBA-MD231 cells. 3.5. Morphology of PDT treated cells Bright field and fluorescence microscopy studies were conducted to investigate morphological alterations induced by PDT treatment (Fig. 5). After 24 h of PPy@BSA-Astx (40 µg/ml) mediated PDT treatment, the bright field microscopy observation (Fig. 5) showed apparent morphological changes such as cell shrinkage, cytoplasmic condensation, and decreased cell density. After 24 h of PDT treatment, many cells were detached and washed away during PBS washing. In the untreated control and laser control experiments, the cells were uniform and appeared to have a normal structure (Fig. 5). To observe the structure of nuclei, cells were stained with Hoechst which can bind to the minor groove region of DNA and emit blue fluorescence. In Fig. 5, it can be observed that the cells maintained their normal round or elliptical shaped nuclei structure, without any

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alterations or breakage in untreated and laser control group. A bright blue fluorescence represented nuclei condensation, a characteristic of apoptosis in PPy@BSA-Astx treatment. The bright blue appeared due to highly accumulated Hoechst stain inside the nuclei with fragmented chromatin. 3.6. Intracellular ROS level Laser-triggered intracellular ROS in PPy@BSA-Astx was investigated using DCFHDA staining. The increased green fluorescence intensity was recorded in nanoparticle treated cells under 808-nm laser exposure. The untreated and laser control cells did not express much green signal (Fig. 5). PPy@BSA-Astx sensitized PDT treated cells produced higher green fluorescence intensity. Fig. S3 shows appropriate green color intensity which was measured in a microplate reader and the results were concordant with microscopic cell images (Fig. 5). Mitochondria are always sensitive to propagated ROS as they regulate the electron transport chain and produce ROS itself during the respiration process (Park et al., 2011). The positively charged rho-123 stain specially accumulated in the mitochondrial membrane was used to analyze mitochondrial membrane potential. The cell micrographs showed depolarized mitochondrial membrane through loss of mitochondrial membrane structure in PPy@BSAAstx treated cell lines (Fig. 5). We can observe the membraned mitochondrial structure in untreated and laser control groups. The rho-123 stain indicated that laser generated ROS attacked the mitochondrial membrane and caused depolarization. 3.7. NIR hyperthermic effect To assess the heating effect of PPy@BSA-Astx under 808-nm laser irradiation, an aqueous solution containing a range of nanoparticle concentrations from 10 to 50 µg/mL was taken and irradiated at a power density of 1 W/cm2 for 5 min. Apparently, the thermal effect was increased depending on the concentration of PPy@BSA-Astx (Fig. 6A). The temperature reached 57 ºC with a concentration of 50 µg/mL, and even at low concentration such as 10

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µg/mL, the temperature curve raised to 46 ºC. The temperature distribution in a 12-well plate was photographed with an IR camera and it showed a uniform thermal distribution in treated 12-well plate (Fig. 6B). Also, PPy@BSA-Astx maintains the stability to reproduce the thermal effect up to five cycles of the heating/cooling experiment (Fig. 6C). Even though photostability of the PPy@BSA-Astx was slightly decreased after 30 min of irradiation (Fig. S4) it reaches the maximum temperature of 55 ºC at the end of fifth cycle in heating/cooling experiment. 3.8. In vitro PTT therapy Next, in vitro PTT treatment was performed with PPy@BSA-Astx treated MDA-MB321 cells under irradiation with an 808-nm laser at a powder density of 1 W/cm2. The cell viability was gradually decreased depending on the concentration of treated nanoparticles (Fig. 3). The result showed that laser induced heat killed the cells effectively. We can observe 60% of cell death at a 40 µg/mL concentration of nanoparticles. The heating ablation of cells was confirmed from this experiment, and the PPy@BSA-Astx can be used for either PDT or PTT treatments. Fig. 7 showed laser eradicated cells in the PPy@BSA-Astx (50 µg/mL) treatment group and cells were detached and washed away by laser treatment. The adverse effect was not seen in the control and laser control of MBA-MD-231 cells. 3.9. Photoacoustic imaging efficiency PAI is an emerging non-invasive imaging modality that can be used for diagnosis and treatment monitoring. Cells incubated with different concentration of nanoparticles were applied in a tissue mimicking phantom (Fig. 8A) and transferred to a PAI system to screen the ability of nanoparticles to act as contrast agents to image the treated cells upon 808-nm illumination. Cells with different concentrations of PPy@BSA-Astx produced signals for imaging the cells in a tissue mimicking phantom. The high-amplitude photoacoustic signals were detected from PPy@BSA-Astx treated cell inclusions. Cells applied without

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nanoparticles did not emit any photoacoustic signal. The image contrast was increased depending on the concentration of treated nanoparticles (Fig. 8B) and the particles have the ability to generate photoacoustic signals even at 10 µg/mL. The data processed using the Matlab program displayed a better 3D image of the scanned cell clump in the phantom (Fig. 8C). 4. Discussion PPy NPs have attracted much attention as a novel candidate in photo-based therapy of cancer as they have strong absorption in the NIR spectrum region. Surface modification of nanoparticles is essential to improve their biocompatibility and stability. In this work we have synthesized PPy NPs using astx conjugated BSA as a stabilizing agent. Albumin is a serum protein abundantly found in human serum (Kratz, 2014). We have adapted the protocol of Song et al. (2015) to fabricate BSA coated PPy NPs and used a 1:1 ratio of pyrrole and BSA to obtain monodispersed nanoparticles as shown in TEM image (Fig. 1B). The UV-Vis absorption spectrum showed a broad NIR absorption window and, we have opted for 808-nm for further experiments. It is a prerequisite for photo-based therapy that materials should have strong absorption in the NIR region (λ = 700–1100) as this region is transparent for biological tissues. The use of materials with absorption of above 980 nm is problematic as water has a significance absorption in this region which leads to non-specific tissue damage by the water heating effect (Wang et al., 2013). In addition, 808-nm radiation is transparent to biological fluid, tissue, and water, and thus, we have opted to use an 808-nm laser for our experiment. Whereas most previous studies have focused only on the photothermal effect of PPy NPs, here we have attempted to conjugate astx as a photosensitizer to utilize the synthesized particles in PDT modality. As expected, the PPy@BSA-Astx produced ROS under 808-nm laser irradiation and there was no measurable ROS production recorded from PPy@BSA nanoparticles without astx. The DPBF assay confirmed that PPy@BSA-Astx can be used for

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PDT with an 808-nm laser. Since the astx has weak absorbance at 808 nm, it could not produce ROS effectively under 808-nm laser excitation. The BSA linked astx has shifted absorbance higher at 808 nm (Fig. S5). Further BSA-astx could add the property of fluorescence to PPy@BSA-Astx, which could attribute to the process of photo-induced electron transfer to produce ROS. The photo-induced energy transfer mechanism is supported by the fluorescence spectrum of PPy@BSA-Astx (Fig. S6). The fluorescent spectrum was recorded by fixing excitation wavelength at 808 nm and emission spectrum was recorded from 808 to 900 nm. The fluorescence emission was higher in PPy@BSA-Astx than PPy@BSA. Different NIR range inorganic nanomaterials like copper sulfide nanoparticles (Wang et al., 2015) and gold nanorods (Vankayala et al., 2014) were reported for ROS induction under NIR laser through fluorescence energy transfer mechanism. A huge number of studies have been reported concerning the conjugation of chlorophyll derived chlorin e6 photosensitizer with different nanoparticles such as polydopamine (Zhang et al., 2015), gold nanorods (Huang et al., 2013), upconversion nanoparticles (Ai et al., 2015; Dou et al., 2015), cobalt-bis (dicarbollide) (Efremenko et al., 2014) and magnetic (Mbakidi et al., 2013) nanoparticles. Most of the available photosensitizers are hydrophobic in nature and get easily aggregated in physiological saline. To overcome these obstructions, nanoparticles could be used as stable carriers for effective PDT therapy. Astx is a natural pigment with antitumor activity and has not previously been studied in the phototherapeutic field. Here, we used this pigment to photosensitize the PPy@BSA-Astx for the purpose of PDT treatment modality.

The albumin coated PPy@BSA-Astx has great biocompatibility and did not exert any toxic effects towards tested cell lines within the studied concentrations, and no adverse effects were found. BSA was already reported for colloidal stability, and it can remain in the blood circulation for a long time (Kalidasan et al., 2016) and it can cross the blood-brain

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barrier (Langiu et al., 2014). We initiated photo-treatment after 6 h incubation of MBA-MD231 cells with PPy@BSA-Astx and found that the treatment was effective. PDT-propagated ROS causes cell death by damaging intracellular biomolecules and disturbing membraned cellular organs inside cells. The mechanistic action of PDT induced cell death is dependent on several parameters like nature of photosensitizer, dose of treatment, cell genotype and availability of oxygen amount in treatment vicinity (Yoo and Ha, 2012).

Based on

microscopic observations we detected apoptotic cell death characteristics such as cell shrinkage and nuclei cleavage. Oxidative stress caused by increased ROS could disturb intracellular redox homeostasis and cause irreversible damage to proteins and lipids (Circu and Aw, 2010). The loss of mitochondrial membrane potential was observed in PDT-treated cells (Fig. 5), and the depolarized membrane could release pro-apoptotic factors into the cytoplasm and initiate a cascade event of apoptosis. Cytochrome c leakage from mitochondria acts as a central event of intrinsic mediated apoptosis and eventually leads to the activation of caspase-3 enzyme to fragment the intracellular micro and macromolecules (Simon et al., 2000) which lead to morphological and biochemical changes. Thus, PDTaccelerated oxidation increased the chance of apoptosis through mitochondrial membrane depolarization (Zhu et al., 2015). Oleinick et al. (2002) reported that PDT causes apoptosis by simultaneously triggering pro-apoptotic factors and photo-damaging anti-apoptotic proteins. The PDT stimulated apoptosis pathway was reported for 5-aminolevulinic, a clinically-used photosensitizer which suppresses anti-apoptotic mRNA bcl-2 and elevates pro-apoptotic mRNA bax levels in cervical cancer cell lines (He et al., 2009).

Next, we studied the photothermal effect of BSA@PPy-Astx under 808-nm irradiation at different power densities. The nanoparticles did not produce a considerable heating effect at 0.5 W/cm2 (Fig. S7), but they did exhibit great heating effects under a power

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density of 1 W/cm2 (Fig. 6). Recently, polypyrrole has received great attention in biomedical application due to its conductive and stability properties. The small sized (30–50 nm) PPy@BSA-Asta nanoparticles responded well to NIR laser irradiation, and the increase in the temperature level depended on its concentration. The temperature was raised to 57 ºC at a concentration of 50 µg/mL, and there was not much difference between the concentrations of 30–50 µg/mL. Wang et al. (2014) reported that smaller PPy NPs have higher thermal conversion efficiencies then larger nanoparticles under NIR laser irradiation. In addition, the PPy@BSA-Astx was stable during the repeated heating/cooling experiment (Fig. 6C), in which the thermal measurement was conducted for 5 cycles (5 min heating, 5 min cooling) at a concentration of 50 µg/ml. The absorbance spectrum was not drastically affected by after 5 cycles of heating/cooling experiment (Fig. S4). This experiment showed that the temperature was raised to 55 ºC in each repeated cycle, and the stability could be attributed by polypyrrole. The minimum temperature level required for killing the cells range from 46 to 60 ºC (Trinidad et al., 2014). Polypyrrole has already been reported for its high thermal transduction efficiency under NIR irradiation, and for this reason it was coated on various metal nanoparticles to improve their heating effect (Li et al., 2013). The cell killing effect of photothermal modality on PPy@BSA-Astx treated cells was slightly higher than PDT therapy because the elevated temperature simply lyses the cells mostly through a necrosis process which is not a programmed cell death like apoptosis. The elevated temperature denatures the protein, cytoskeletal structure, rupture the cell membrane and lyse the DNA and RNA molecules (Hildebrandt et al., 2002). Fig. 7 showed that cells were detached away by heating effect. The photothermal cell killing effect was in consistent with Chandrasekaran et al. (2016) where they also observed detached breast tumor cells during gold nanorod mediated PTT. Even PPy@BSA-Astx generated considerable ROS, which was confirmed by DPBF assay (Fig. S8), the cytotoxic effect was initiated and dominated by the heating effect

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at a power density of 1 W/cm2. The effective cell killing effect was achieved by rapid elevation of temperature by NIR light. The different mode of PPy@BSA-Astx based photo therapy is illustrated in Scheme 1. PPy@BSA-Astx was screened for the PAI effect using an ultrasound detector array to construct a three-dimensional image of scanned cells in a tissue mimicking phantom. Since the synthesized particles exhibit surface plasmon resonance in the NIR region we used the 808-nm laser for PAI system. The nanoparticles greatly extended its application by permitting the imaging of treated breast cancer cells. Gold nanorods are widely used as contrast agents for PAI; however, preparation of GNR is a complicated and expensive process (Yang et al., 2009). The PPy@BSA-Astx can be used as a great photoacoustic contrast agent for imaging of treated tissues. Manivasagan et al. (2016) reported PAI imaging of gold nanoparticles treated MBA-MD-231 cells using tissue mimicking phantom study. The laser employed here is 808-nm laser, which can penetrate deep into the tissue to image the deep tissue cancers. PPy@BSA-Astx can serve as a contrast agent and it make an opportunity to do image guided phototherapy.

5. Conclusion In summary, we synthesized PPy@BSA-Astx nanoparticles, which exhibited advantages in PDT and PTT therapy under an 808-nm laser irradiation. The treatment modality can be tuned by changing the power density of the laser source. PPy@BAS-Astx was stable, uniformly dispersible in water, and does not have any toxic effects on the cells in the absence of laser light. Serum albumin has many biologically friendly effects and has vast potential for different functional modifications due to its chemical properties. Moreover, astx is a natural pigment which was used to sensitize the PPy@BSA-Astx to generate ROS for PDT therapeutic modality. As PPy@BSA-Astx has strong NIR absorption and high thermal 19

conversion efficacy, the cytotoxic effect of the photothermal modality was higher when compared to the photodynamic effect. Additionally, it acts as an optical contrast agent for imaging of treated cells in PAI system. Thus, PPy@BSA-Astx can be used for the development of theranostic agents for different application in cancer-based biomedicine.

Disclosure: The authors declare no conflicts of interest.

Acknowledgments: This work was financially supported by Marine Biotechnology Program (20150220) funded by the Ministry of Oceans and Fisheries, Republic of Korea.

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Figure legends

Fig. 1. (A) UV-Vis absorbance spectrum of PPy@BSA-Astx. (B) TEM morphology of PPy@BSA-Astx nanopaticles.

Fig. 2. (A) Size distribution analysis of PPy@BSA-Astx. (B) FTIR spectra of PPy@BSAAstx (red colored line) and Ppy@BSA (black colored line).

Fig. 3. Bar graph representing cell viability of PPy@BSA-Astx treated MBA-MD-231 cells in different treatment conditions. The cells were irradiated under 808- nm laser at 0.3 W/cm2 for 15 min (PDT) and at 1 W/cm2 for 5 min (PTT). The percentage of cytotoxicity is expressed relative to untreated controls (* significant, p < 0.05).

Fig. 4. DPBF assay of singlet oxygen generation efficacy under 808-nm nm laser at power density of 0.3 W/cm2.

Fig. 5. The bright field morphology of MBA-MD-231 cells in different conditions and fluorescent microscopy images showed Hoechst stained nuclei morphology of MBA-MD-231. Intracellular ROS represented by DCFH-DA stain. Rho-123 staining represented mitochondrial membrane potential of MBA-MD-231. The cells were illuminated under 808nm laser at 0.3 W/cm2 for 15 min.

Fig. 6. Thermal curve of PPy@BSA-Astx in different concentrations under 808-nm laser irradiation at a power density of 1 W/cm2 (A) and corresponding IR camera captured thermal images in 12-well plate (B). Five cycles of heating/cooling experiment under a 808-nm laser on/off experiment (1 W/cm2) with 50 µg/mL of PPy@BSA-Astx (C).

Fig. 7. AO stained MBA-MD-231 cells in different conditions. The cells were irradiated under 808-nm laser at 1 W/cm2 for 5 min. The dashed line in PPy@BSA-Astx (50 µg/ml) treated cells indicate the laser spot.

Fig. 8. (A) Photograph of tissue mimicking phantom loaded with different concentrations of PPy@BSA-Astx treated MBA-MD-231 cells. 2D (B) and 3D (C) images of Ppy@BSA-Asta treated MBA-MD-231 cells acquired under 808-nm laser from a PAI system.

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Fig. 1

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Scheme 1. Schematic representation of PPy@BSA-Astx preparation and a possible cellular uptake activity by MDA-MD-231 cells and phototherapy.

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