Chlorin e6 conjugated copper sulfide nanoparticles for photodynamic combined photothermal therapy

Accepted Manuscript Title: Chlorin e6 conjugated copper sulfide nanoparticles for photodynamic combined photothermal therapy Author: Subramaniyan Bharathiraja Panchanathan Manivasagan Madhappan Santha Moorthy Nhat Quang Bui Kang Dae Lee Junghwan Oh PII: DOI: Reference:

S1572-1000(17)30233-8 http://dx.doi.org/doi:10.1016/j.pdpdt.2017.04.005 PDPDT 940

To appear in:

Photodiagnosis and Photodynamic Therapy

Received date: Revised date: Accepted date:

27-2-2017 28-3-2017 11-4-2017

Please cite this article as: Bharathiraja S, Manivasagan P, Moorthy MS, Bui NQ, Lee KD, Oh J, Chlorin e6 conjugated copper sulfide nanoparticles for photodynamic combined photothermal therapy, Photodiagnosis and Photodynamic Therapy (2017), http://dx.doi.org/10.1016/j.pdpdt.2017.04.005 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.

Chlorin e6 conjugated copper sulfide nanoparticles for photodynamic combined photothermal therapy Subramaniyan Bharathiraja a, Panchanathan Manivasagan a, Madhappan Santha Moorthy a,

ip t

Nhat Quang Bui b, Kang Dae Lee c, Junghwan Oh a,b a

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

cr

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

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M

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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).

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∗

us

Department of Otolaryngology – Head and Neck Surgery, Kosin University College of Medicine, Busan, Republic of Korea.

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Chlorin e6 conjugated copper sulfide nanoparticles for photodynamic combined

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photothermal therapy

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Subramaniyan Bharathiraja a, Panchanathan Manivasagan a, Madhappan Santha Moorthy a,

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Nhat Quang Bui b, Kang Dae Lee c, Junghwan Oh a,b

6

a

Marine-Integrated Bionics Research Center, Pukyong National University, Busan 48513,

Republic of Korea.

cr

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Department of Biomedical Engineering and Center for Marine-Integrated Biotechnology

us

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b

(BK21 Plus), Pukyong National University, Busan 48513, Republic of Korea.

an

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C

Department of Otolaryngology – Head and Neck Surgery, Kosin University College of

Medicine, Busan, Republic of Korea.

∗

Corresponding author at: Marine-Integrated Bionics Research Center, Pukyong National

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University, Busan 48513, Republic of Korea. Fax: +82 51 629 5779.

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E-mail address: [email protected] (J. O).

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Abstract

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The photo-based therapeutic approaches have attracted tremendous attention in recent years

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especially in treatment and management of tumors. Photodynamic and photothermal are two

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major therapeutic modalities which are being applied in clinical therapy. The development of

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nanomaterials for photodynamic combined photothermal therapy has gained significant

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attention for its treatment efficacy. In the present study, we designed chlorin e6 (Ce6)

24

conjugated

copper

sulfide

(CuS)

nanoparticles

(CuS-Ce6 NPs)

through

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functionalization and the synthesized nanoparticles act as a dual-model agent for

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photodynamic therapy and photothermal therapy. CuS-Ce6 NPs showed enhanced

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photodynamic effect through generation of singlet oxygen upon 670 nm laser illumination.

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The same nanoparticles exerted thermal response under an 808 nm laser at 2 W/cm2. The

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fabricated nanoparticles did not show any cytotoxic effect towards breast cancer cells in the

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absence of light. In vitro cell viability assay showed a potent cytotoxicity in photothermal and

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photodynamic treatment. Rather than singular treatment, the photodynamic combined

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photothermal treatment showed an enhanced cytotoxic effect on treated cells. In addition, the

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CuS-Ce6 NPs exert a photoacoustic signal for non-invasive imaging of treated cells in tissue-

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mimicking phantom. In conclusion the CuS-Ce6 NPs act as multimodal agent for photo based

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imaging and therapy.

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Keywords: Copper, amine function, photosensitizer, photothermal, photodynamic

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1. Introduction

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Nanomedicines have tremendous potential in photo-based therapy and imaging due to their

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novel optical, electronic, and structural properties [1]. Recently, copper-based nanomaterials

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have gained significant attention in biomedical applications due to their excellent near-

41

infrared (NIR) absorption and hyperthermic response under NIR irradiation [2, 3]. In addition,

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copper nanoparticles (Cu NPs) are easy to synthesize, biocompatible, and easily degradable,

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and it was a low cost element [4]. So copper was rapidly explored as a promising agent in the

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field of photothermal therapy (PTT). Photodynamic therapy (PDT) is another type of non-

45

invasive phototherapy that has obtained regulatory approval for treating various diseases such

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as psoriasis, age-related macular degeneration, and certain oncological diseases [5]. PDT is

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an important therapeutic option in management of tumor therapy where photosensitizers

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generate cytotoxic, potent reactive oxygen species (ROS) under light exposure [6]. Chlorin e6

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(Ce6), a second-generation photosensitizer with a strong absorbance spectrum in the near

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infrared region (NIR), is used as a photosensitizing drug for PDT [7]. Ce6 is a derivative of a

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naturally occurring chlorophyll pigment that is a promising therapeutic agent for PDT which

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can produce high yield of singlet oxygen under NIR light irradiation [8]. However, Ce6 loses

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its stability in physiological solutions, which significantly diminishes its photosensitizing

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efficiency and lowers the yield of singlet oxygen [9]. The photosensitizers lose their PDT

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efficiency by aggregation in physiological solution and they are appreciably photoactive only

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in monomeric from [10]. The nanoparticle-photosensitizer conjugate could exert effective

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photodynamic action through mediating stability and avoiding the quenching effect. Owing to

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unsatisfactory results by single therapeutic methodology, more attentions have been paid to

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new combination of PDT combined PTT therapy. Generally gold NPs were focused for

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synergetic therapy owing to its novelty [11]. In the present study we have used CuS NPs

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along with Ce6 for photothermal and photodynamic cytotoxic killing of cancer cells

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respectively.

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Photoacoustic tomography (PAT) is an imaging modality that integrates optical

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contrast to produce images in a computer system to clearly visualize the tissue. The NIR

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absorption of Cu NPs can greatly enhance the contrast in deep-tissue imaging. PAT provides

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non-invasive, high-resolution images by detecting acoustic pressure induced by optical

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energy [12]. In the present study, we used polyethylenimine (PEI), a branched polymer with

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high density of amine group, to coat the CuS NPs, on which Ce6 was conjugated for effective

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photodynamic combined photothermal action to enhance cell death. The same nanoparticles

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also provide an opportunity for PAT imaging, which could assist in making a diagnosis.

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2. Materials and methods

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2.1. Synthesis of CuS NPs

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CuS NPs were synthesized according to a previous report [13]. Briefly, 4 mmol of copper (I)

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chloride was dissolved in 24 mL of oleylamine at 100°C. Simultaneously, 2 mmol of sulfur

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solution was prepared using 8 mL octadecene (ODE) by heating it at 180°C under nitrogen

78

gas until the powder dissolved completely. Then, both solutions were allowed to cool down

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to room temperature. The solutions were then mixed together and heated to 180°C at the rate

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of 10°C/min under nitrogen condition. The reaction solution was kept at 180°C for 15 min

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and then cooled down to room temperature. Finally, CuS NPs were precipitated by adding 10

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mL of toluene and excess amount of ethanol. The precipitated nanoparticles were washed one

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more time with ethanol and dispersed in chloroform.

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2.2. Ligand exchange process

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In this process, 5 mL of CuS NPs (2 mg/mL) was diluted with 20 mL of toluene comprising

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0.5 mg/mL of PEI. The mixture was sonicated for 30 sec and added to 10 mL of distilled

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water. The mixed solution was transferred to a separating funnel and allowed to stand until

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two emulsified phases were separated completely. The aqueous phase, which contained PEI-

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modified CuS NPs (CuS-PEI), was separated. This produce was repeated two times to

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transfer the remaining CuS NPs to the aqueous phase. Finally, the amine-functionalized CuS

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NPs were washed with distilled water and collected by centrifugation.

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2.3. Conjugation of Ce6 over CuS-PEI

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To prepare the Ce6-conjugated CuS NPs (CuS-Ce6 NPs), 0.596 mg of Ce6 was dissolved in

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40 µL of dimethyl sulfoxide. An equal molar ratio of 1-Ethyl-3-(3-dimethylaminopropyl)

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carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) was added and then vibrated gently

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for 30 min. After that, the above mixture was added to 10 mL of distilled water containing 10

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mg of amine-functionalized CuS-PEI NPs and stirred overnight. Then, the reaction mixture

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was purified using 10 KDa MWCO filters to remove the unbound Ce6 and excess catalysts.

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The obtained 10 mg of product was dispersed in 5 mL of water. Based on the molecular

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extension co-efficient, it was estimated that 24.22 µg of Ce6 was conjugated with 1 mg CuS-

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PEI NPs.

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2.4. Characterization

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Transmission electron microscopic images were taken using FE-TEM (FETEM; JEM-2100F,

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JEOL, Japan), and absorbance spectrum was measured using Uv-Vis spectroscopy (Beckman

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Coulter, Fullerton, CA, USA). Fourier transform infrared (FITR) spectroscopy was recorded

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by diffuse reflectance mode at a resolution of 4 cm−1, and the wavelength range was fixed at

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4000 to 500 cm−1. The X-ray diffraction (XRD) spectra was analyzed using XRD (X’Pert-

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MPD, Philips, Amsterdam, The Netherlands) with cu-Kα radiation 1.5405 A° over an angular

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range of 5 to 80°.

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2.5. DPBF assay

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A 1, 3-diphenylisobenzofuran (DPBF) assay was followed to measure singlet oxygen

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generation efficiency under 670 nm laser illumination [14]. Briefly, 10 µg/mL of Cus NPs-

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Ce6 and an equal amount of free Ce6 were taken separately in a 1 cm quartz cuvette along

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with 20 µL of DPBF dissolved in ethanol (10 mmol/L). The initial absorbance was measured

115

at 418 nm, and samples were irradiated with an 808/670 nm laser light at 100 mW/cm2 power

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density for 10 min. The absorbance of each sample was measured at 418 nm for every 2 min

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of laser irradiation.

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2.6. Heating experiment

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To measure the photothermal response of CuS-Ce6 NPs, different concentrations of CuS

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NPs-Ce6 (25, 50, 100, 150, and 200 µg/mL) were irradiated with an 808 nm continuous wave

122

laser at 2 W/cm2 power density. The temperature raise was monitored using a thermal fiber,

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which was inserted into the sample medium perpendicularly to the path of laser. The

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temperature was recorded for every second of laser irradiation.

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2.7. Cell culture

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MBA-MD-231 cells were cultured and maintained in Dulbecco’s modified Eagle medium

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(DMEM) supported with 10% fetal bovine serum (FBS), 50 U/mL penicillin, and

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streptomycin. The cells were cultured and maintained in a humidified incubator at 37°C with

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5% carbon dioxide.

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2.8. Cell viability assay

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The biocompatibility of CuS-Ce6 NPs was tested with MBA-MD-231 cells using an MTT

132

assay. Cells were seeded in a 96-well plate at a density of 10,000 cells per well and treated

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with different concentrations of CuS-Ce6 NPs for 24 h. The medium was then removed, and

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100 µL of serum-free medium containing 0.5 mg/mL MTT was added to each well. After 3 h

135

of incubation at 37°C, the medium from the culture plate was aspirated, and 100 µL of

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DMSO was added to each well to dissolve the purple formazan product inside the living cells.

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The intensity of the purple formazan product was then measured at 570 nm.

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2.9. In vitro PDT and PTT cytotoxicity

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Initially, the MBA-MD-231 cells were seeded in a 96-well plate at a concentration of 10000

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cells per well separately for PDT and PTT treatments. The cells were then treated with

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different concentrations (25, 50, 100, 150, 200, 250 µg/mL) of CuS-Ce6 NPs. After 6 h, the

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unbound nanoparticles were rinsed away with phosphate-buffered saline (PBS). The PDT

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therapy was performed on prepared cells with a 670 nm laser light at 100 mW/cm2 for 10 min.

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In another cell culture plate, PTT therapy was performed using an 808 nm laser at a power

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density of 2 W/cm2 for 10 min. After treatments, the cells were incubated in the dark in a CO2

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incubator for 24 h at 37°C. A standard MTT assay was then performed to determine the

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cytotoxic effect of PDT and PTT on the viability of MBA-MD-231 cells.

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2.10. In vitro PDT/PTT synergetic effect

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To evaluate the synergetic effect of PDT combined PDT therapy, MDA-MB-231 cells were

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seeded in a 96-well plate at a concentration of 10, 000 cells/well and treated with different

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concentrations of Cus-Ce6 NPS for 6 h. PDT therapy was then performed for 10 min at 100

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mW/cm2 power density under a 670 nm laser light. Following the PDT therapy, PTT therapy

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was performed using an 808 nm laser at 2 W/cm2 power density for 10 min. The cells were

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then allowed to incubate for 24 h in a CO2 incubator. Finally, a standard MTT assay was

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performed as mentioned previously to determine the percentage of cell viability.

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2.11. Photoacoustic imaging

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CuS-Ce6 NPs were further screened for generating ultrasound signal under an 808 nm laser

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to image the treated cells. In the experiment, pre-seeded MBA-MD-231 cells were treated

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with different concentrations of CuS-Ce6 NPs for 6 h, and cells were then harvested and

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loaded with 2% gelatin on a tissue-mimicking phantom, which was prepared with 8%

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polyvinyl alcohol and 0.4% silica. The cells were then covered with the same 4% gelatin and

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allowed to solidify. The non-invasive photoacoustic imaging (PAI) system previously

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developed and described by Bui et al. [15] was followed to generate an image of the treated

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cells in tissue mimicking. A tunable laser (Surelite OPO Plus, Continuum, CA, USA)

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pumped by a Nd:YAG laser (Surelite III, Continuum, CA, USA) was delivered through a

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multimode optical fiber. The wavelength was fixed at 808 nm to obtain the photoacoustic

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images. The output end of the fiber was integrated with a 10 MHz focused transducer

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(Olympus NDT, Waltham, MA, USA) and aligned so both the incident light and the

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transducer irradiated and imaged the same sample site. The photoacoustic signals were then

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captured, digitized, and stored via a data acquisition (DAQ) system in synchronization with

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the laser system. Finally, the detected photoacoustic signals were converted into

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photoacoustic images via Hilbert transformation.

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3. Results and Discussion

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In the present work photosensitizer is integrated with inorganic CuS NPs for combined

175

phototherapeutic effect. Figure 1 illustrates the strategy for the construction of CuS-Ce6 NPs.

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We synthesized CuS-Ce6 nanoparticles, which can be used as a treatment modality for both

177

PTT and PDT based therapies. The synthesized NPs were capped with oleylamine, and they

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were dispersible in non-polar solvents. The synthesized CuS NPs showed absorption in the

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NIR range, with a maximum absorption of 1000 nm; Ce6 alone showed peaks at 404 nm and

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670 nm (Figure 2a). PEI functionalization shifts the absorption intensity of CuS NPs slightly

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higher in U-Vis and the NIR region, up to 830 nm (Figure 2a). After surface modification

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with PEI, the particles were dispersible in water. The final CuS-Ce6 conjugation shows

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additional peaks at 404 nm and 670 nm, which represents the presence of Ce6. The peak

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intensities for 670 and 808 nm were marked that were used for PDT and PTT respectively.

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The TEM image of CuS NPs is shown in Figure 2b, and the average particle size was found

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to be 6.5 nm. The zeta potential was slightly reduced in CuS-Ce6 compared with CuS NPs (S.

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Figure. a). The Ce6 was attached on amine groups of CuS NPs and thus reduced its zeta

188

potential. The sizes of CuS NPs were not disturbed by surface modification with PEI. The

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Dynamic Light Scattering (DLS) data showed the size distribution of CuS NPs (S. Figure b1-

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b3). The size and shape of the particles were not disturbed, and the particles were mono-

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dispersive after conjugation with Ce6 (Figure 2a2). The XRD pattern represented the main

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phase of the crystalline nature of CuS NPs at 35°C, and other broad at 55°C and 64°C

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appeared broadly (Figure 2c). The process of functionalization with PEI did not affect the

194

structure of nanoparticles, and it introduced amine groups on the surface of the CuS NPs. To

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improve the colloidal stability and biocompatibility, the synthesized CuS NPs were coated

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with PEI on the outer surface of the CuS NP, and the Ce6 was then immobilized onto the PEI

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to produce CuS-Ce6 NPs. The CuS-Ce6 NPs were characterized by FTIR analysis. As

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observed in Figure 2d, the vibration peak at 647 cm-1 indicates the Cu-S stretching modes of

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CuS nanoparticles. An intense band appeared at 1315 cm-1 for the N-H stretching, and the

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peaks at 2853 cm-1 and 2948 cm-1 imply the C-H stretching vibrations for the PEI polymer.

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Furthermore, the strong FTIR stretching peaks appeared at 1077 cm-1, 1479 cm-1, and 1640

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cm-1, respectively, for the C = C, COOH, and C = O groups of immobilized Ce6 molecules.

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The FTIR result confirms the presence of the surface-coated PEI polymer and the successful

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immobilization of Ce6 molecules onto the synthesized CuS NPs [16]. A recent report by Han

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et al. [17] demonstrated conjugation of indocyanine green through amine functionalization of

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Cu NPs with PEI. Recent advances in nanoplatforms pave the way for designing inorganic

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nanomaterials with organic-photosensitizer functionalization to tackle the challenges of

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conventional photosensitizer drugs [18]. In addition, organic-nanomaterials help improve the

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water solubility and stability of conventional photosensitizers. The photosensitizer-integrated

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NPs can achieve passive tumor targeting through leaky tumor vasculature, and they enhance

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the permeability and retention effect [19]. The CuS-Ce6 NPs were stable in different

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physiological solution like water and serum media (Figure 3a) and the medium did not

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influence the absorbance spectrum. The CuS-Ce6 NPs stability in water was observed over a

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period of three months by monitoring UV-Vis absorbance spectrum, which showed the

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stability of the particles remained constant.

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3.1. Singlet oxygen generation and photothermal effect

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To estimate the generation of singlet oxygen, a DPBF assay was performed by monitoring the

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absorbance of DPBF at 418 nm. The production of singlet oxygen could quench the DPBF,

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thus decreasing the absorbance of DPBF at 418 nm. As shown in Figure 4a, singlet oxygen

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induction by CuS-Ce6 NPs under 670 nm laser illumination was sharply increased in a time-

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dependent manner, and it was higher than with free Ce6 alone. However, the 1O2 generation

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efficiency was not efficient when irradiating with an 808 nm laser (Figure 4a). The results

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confirmed that the conjugated Ce6 generated significant singlet oxygen under photon

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illumination with a 670 nm laser. The singlet oxygen production by free Ce6 was less

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effective under a 670 nm laser compared to the CuS-Ce6 NPs. Our results correspond with

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those in Zhang et al. [20], who observed the same phenomena in which Ce6 conjugated

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poly(dopamine) exerted a higher rate of singlet oxygen than free Ce6 in a physiological

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solution. Vieira et al. reported that gold nanoparticle combined Ce6 generated higher

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photodynamic action than free Ce6 [21]. The generation of singlet oxygen was efficient in 10

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min under illumination with a 670 nm laser. Ce6 is a highly used pigment for PDT due to its

231

high yield of singlet oxygen and rapid clearance from the body [22]. However, it becomes

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contemptible in physiological solution where it becomes unstable and loses its fluorescent

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quantum yield. The Ce6 on CuS-Ce6 was anchored covalently to amine group of PEI over

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CuS NPs thus avoid the aggregation and helps to be stable in physiological solution. The Cus

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was act as carrier of Ce6 and present it stably for PDT therapy. Therefore, the CuS-Ce6 NPs

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achieved better results than free Ce6.

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To estimate the photothermal conversion, different concentrations of CuS-Ce6 NPs

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were added to distilled water and irradiated with a continuous wave laser at 808 nm with a

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power density of 2 W/cm2 for 10 min. As shown in Figure 4b, the temperature of the solution

240

reached 53°C at a concentration of 200 µg mL-1 in 10 min, while the temperature level only 10 Page 11 of 28

reached 26°C in 25 µg/mL. The maximum temperature level was reached after 5 min of

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irradiation. This thermal response result corresponds with the findings in Wang et al. [13].

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Traditionally, gold nanoparticles have been investigated for the photothermal ablation of

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cancer cells using NIR light. Recently, Cu NPs with NIR absorption have been applied

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increasingly in PTT therapy [23]. Copper is a semiconductor metal with good optical

246

properties that derives energy from a band-band transition [24]. Due to this band-band

247

transition, the absorption intensity of copper nanoparticles is affected less by the surrounding

248

medium and the size and shape of particles [25]. Li et al. first reported on CuS NPs as a

249

photothermal agent using 808 nm laser irradiation with 24 W/cm2; they observed a lower

250

thermal conversion efficiency [24].

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3.2. Biocompatibility and in vitro PDT and PTT toxicity

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The cellular internalization of CuS-Ce6 NPs was studied using an emission range of Ce6

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under a fluorescence microscope [26]. Figure 5a clearly shows the red color emission from

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the CuS-Ce6 NPs treated cells. The merged image shows the presence of CuS-Ce6 NPs

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inside the cells. The synthesized CuS-Ce6 NPs show cytocompatibility with MBA-MD-231

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cells. The high cell viability of 92.5% was observed at the concentration of 200 µg/mL CuS-

257

Ce6 NPs (Figure 5b). Zhang et al. recorded above 90% of cell viability with 230 µg/mL of

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BSA coated CuS NPs [27]. Since the CuS NPs have a low toxic effect, they are applied in

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biological applications crucially [28]. Various Nps with surface modification shown water

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solubility and biocompatibility and have been explored for image-guided therapy. The MTT

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assay showed that PTT therapy under 808 nm effectively reduced the cell viability of MBA-

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MD-231 cells. The percentage of cell viability was calculated against cells treated under a

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laser without nanoparticles. The cell viability was reduced depended on the concentrations of

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CuS-Ce6 NPs. The photothermal effect of CuS-Ce6 at different concentrations in water was

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correlated in vitro cytotoxicity. The maximum of 60% of cell death was detected at the

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concentration of 200 µg/mL. Tian et al. demonstrated that Cu9S5 nanocrystals as potent laser-

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derived photothermal agents [2]. The larger size of CuS NPs hinders biological applications

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and reduces the PTT efficacy [29]. The formulated small-sized CuS NPs exhibited a potent

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photothermal conversion effect, which drastically reduced cell viability. In another way we used the CuS-Ce6 NPs for PDT using 670 nm laser illumination.

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To confirm the singlet oxygen-induced cell death, CuS-Ce6 NPs-treated MDA-MB-231 cells

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were exposed to a 670 nm laser light for 10 min, and an MTT assay was performed after 24 h

273

of treatment. Figure 5b shows considerable cell death by PDT, with almost 50% of the cells

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killed at a concentration of 150 µg/mL. The PDT modality mechanism induced ROS, which

275

could arrest cell proliferation through cell cycle arrest, apoptosis, or necrosis [30]. The cell

276

death rate was comparatively less in the PDT modality than with PTT therapy. ROS is a well-

277

established mediator that induces oxidative damage to biomolecules and progresses cell death

278

[31]. The DNA and mitochondrial membrane are sensitive to generated ROS, and their

279

damage induces cell death.

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3.3. PDT combined PTT cell toxicity

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The combination of PDT and PTT offers advantages over a single modular treatment. The

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MBA-MD-231 cells were initially treated with 670 nm laser illumination for 10 min at 100

283

mW/cm2 power density; following that, the cells underwent PTT therapy using an 808 nm

284

laser for 10 min at 2 W/cm2. The MTT assay revealed that the PDT/PTT therapy exhibited a

285

synergistic cytotoxic effect on cell viability. The PDT combine PTT therapy reduced cell

286

viability more drastically than either PDT or PTT alone. Almost 84% of cell death was

287

recorded at 200 µg/mL of the CuS-Ce6 NPs treatment. PDT treatment tends to make cells

288

and biomolecules vulnerable to generated ROS [32]. Furthermore, the cells were attacked and

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destroyed by the heating effect, which resulted in more cell death. A higher cell death rate

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was observed in PDT/PTT therapy when compared to PTT and PDT alone. At 200 µg/mL 55%

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and 41% of cell death was observed in PDT and PTT respectively. The cell death rate at the

292

same concentration was 84% in PDT combined PTT therapy model. Shouju Wang reported

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the same phenomena of higher therapeutic index by PDT/PTT synergistic effect where he

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applied Ce6 conjugated gold nanostar [33].

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3.4. Photoacoustic imaging

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Figure 6a shows the top view of the tissue-mimicking phantom with loaded cell inclusion and

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gelatin. The MBA-MD-231 cells treated with 100 µg/mL of CuS-Ce6 NPs produced high-

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amplitude photoacoustic signals for imaging of the treated cells. Figure 6b shows a 2D image

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of treated cells inside the phantom. The cells loaded in the tissue-mimicking phantom without

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CuS-Ce6 NPs do not produce any acoustic signal to generate an image. CuS-Ce6 NPs

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enhance optical absorption and increase the photoacoustic signal under 808 nm laser. The use

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of an 808 nm laser has the advantage of overcoming tissue chromophores, such as

303

hemoglobin, in red blood cells [34]. The homogenous distribution of CuS-ce6 labeled cells in

304

the tissue-mimicking phantom was observed in the 2D image. The non-invasive imaging

305

property of CuS-Ce6 NPs could provide an opportunity for image-guided phototherapy.

306

4. Conclusion

307

In summary, we developed Ce6-sensitized CuS NPs as a multifunctional agent for combined

308

photothermal and photodynamic treatment. The synthesized CuS-Ce6 NPs showed a

309

negligible toxicity at high concentration to breast cancer cells in the absence of light. The

310

Ce6-coated CuS-Ce6 NPs generated cytotoxic, potent ROS under a 670 nm laser, and the Cus

311

generated a thermal effect under 808 nm laser irradiation. The CuS-Ce6 NPs exerted

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photothermal and photodynamic toxicity in treated breast cancer cells, and the PDT

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combined PTT reduced cell viability drastically. The formulated particles generated

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photoacoustic signals to produce images of the treated cells. The combined futures of PTT

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and PDT along with PAT make the CuS-Ce6 NPs a promising theranostic agent for image-

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guided phototherapy in biomedical applications.

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Acknowledgment

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This work was supported by a research grant from Pukyong National University (2015).

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Figure legends

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Figure 1 Representation on preparation of CuS-Ce6 NPS through amine functionalization

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using PEI polymer.

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Figure 2 (a) Uv-Vis absorbance spectrum of Ce6 and different modifications of CuS NPs

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with PEI and Ce6; TEM micrograph of (b1) CuS NP TEM micrograph of (a1) CuS NPs and

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(a2) CuS-PEI; s and (b2) CuS-Ce6; (c) XRD pattern of CuS NPs; (d) FITR spectrum of

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amine-functionalized CuS-PEI and CuS-Ce6 NPs.

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Figure 3 (a) UV-Vis absorbance spectra of CuS-Ce6 NPS in PBS and phenol red free

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DMEM serum medium. (b) Absorbance spectrum of CuS-Ce6 NPs in different period of time.

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Figure 4 (a) Singlet oxygen generation efficiency by free Ce6 and CuS-Ce6 NPs under

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different laser irradiations at 100 mW/cm2 power density; (b) Temperature curve of CuS-Ce6

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NPs under 808 nm laser irradiation at 2 W/cm2 power density.

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Figure 5 (a) Cellular uptake of CuS-Ce6 NPs by MBA-MD-231 cells treated at the

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concentration of 50 µg/mL; (b) In vitro cell viability of MBA-MD-231 cells treated with

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CuS-Ce6 NPs in different conditions; PDT treatment was performed under a 670 nm laser at

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100 mW/cm2; for PTT treatment, cells were exposed to a 808 nm laser at 2 W/cm2 power

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density. The percentage of cytotoxicity is expressed relative to untreated controls (*

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significant, p < 0.05, when compared with PTT or PDT).

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Figure 6 (a) Top view of tissue-mimicking phantom loaded with control and CuS-Ce6 NPs-

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treated MBA-MD-231 cells; (b) Photoacoustic signal-generated 2D image of CuS-Ce6 NPs-

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treated MBA-MD-231 cells.

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Graphiical abstracct

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Highlights  Chlorin e6 conjugated over copper sulfide nanoparticles  PDT efficacy was enhanced  CuS-Ce6 act as dual model agent for PDT and PTT

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 PDT combined PTT efficiency observed along with PAT imaging

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