Journal of Photochemistry & Photobiology, B: Biology 212 (2020) 112028

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Journal of Photochemistry & Photobiology, B: Biology

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Biocompatible functionalized AuPd bimetallic nanoparticles decorated on T reduced graphene oxide sheets for photothermal therapy of targeted cancer cells Punamshree Dasa,b,1, Sushma V. Mudigundac,1, Gitashree Darabdharaa,b, Purna K. Boruaha,b, ⁎ ⁎⁎ Sachin Gharc, Aravind K. Renganc, , Manash R. Dasa,b, a Materials Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat 785006, Assam, India b Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India c Department of Biomedical Engineering, Indian Institute of Technology Hyderabad, India

ARTICLE INFO ABSTRACT

Keywords: Graphene, which is a unique 2D nanomaterials has received widespread attention for photothermal therapy AuPd bimetallic nanoparticles (PTT) application. Here, we have designed the nanocomposite using polydopamine (PDA) functionalized re­ Reduced graphene oxide duced graphene oxide (rGO) nanosheets and bimetallic AuPd nanoparticles (NPs). The bimetallic AuPd nano­ Polydopamine particles decorated PDA functionalized rGO (AuPd-rGO/PDA) nanocomposite is synthesized by simple chemical Photothermal therapy reduction technique resulting in an average size of AuPd bimetallic nanostructure of 4.18 nm. The photothermal Biocompatibility activity of the AuPd-rGO/PDA nanocomposite is explored under the irradiation of near infrared (NIR) laser sources of wavelength 915 nm. The temperature rises nearly 51 ± 3 °C within 3 min of irradiation NIR laser light resulting in the ablation of MDAMB-231 cancer cells up to concentration of 25 μg mL−1 of AuPd-rGO/PDA nanocomposite. This high performance of the ablation of cancer cells by photothermal therapy technique was facilitated using a low concentration of the nanocomposite by the synergistic effects of the bimetallic AuPd as well as rGO and PDA functionalization. The AuPd-rGO/PDA nanocomposite demonstrated the high bio­ compatibility towards normal healthy cell lines (L929) and exhibits survival efficiency of more than 85%. We also demonstrated the biocompatibility of the AuPd-rGO/PDA nanocomposite materials on the zebrafish em­ bryos (Danio rerio). This work thus illustrates that the AuPd-rGO/PDA nanocomposite could behave as favour­ able nanoplatform for tumor therapeutics.

1. Introduction high photostability, good biocompatibility and low cytotoxicity. A variety of nanomaterials with strong absorbance in the NIR region have Photothermal therapy (PTT) has emerged as a non-invasive cancer been extensively explored for their photothermal transduction efficiency therapy process capable of attracting worldwide attention especially with [3]. The most widely used PTT agents include Au, Pd nanostructures [4], the advent of advanced photothermal agents, providing new hope to carbon nanomaterials (graphene, fullerene and carbon nanotubes), etc. cancer patients [1]. Typically, PTT employs the altering of absorbed [5]. As per previous literature reports, organic NPs have been used as near-infrared (NIR) light energy into thermal energy through photo­ photothermal agents for the ablation of cancer both in vitro and in vivo. thermal agents, leading to thermal ablation of the cancer cells. Ad­ Amongst them, graphene and its derivatives displaying excellent physico- ditionally, it is effective towards all solid tumors including those cancer chemical properties like large surface area [6] high drug loading effi­ cells which do not respond to radiotherapy, chemotherapy and the drug ciency [7], strong NIR absorbance [8], good tumor targeting capability resistant tumors with minimal side effects 2[ ]. Currently PTT focuses on [9] and thus have been proposed as one of the most encouraging PTT the development of nanomaterials possessing high suitable size, uniform agents. shape, photothermal conversion efficiencies, excellent dispersibility, Au nanomaterials with surface plasmon resonance (SPR) and

⁎ Corresponding author. ⁎⁎ Corresponding authors at: Materials Sciences and Technology Division, CSIR-North East Institute of Science and Technology, Jorhat 785006, Assam, India. E-mail addresses: [email protected] (A.K. Rengan), [email protected] (M.R. Das). 1 Contributed equally. https://doi.org/10.1016/j.jphotobiol.2020.112028 Received 8 February 2020; Received in revised form 26 August 2020; Accepted 13 September 2020 Available online 18 September 2020 1011-1344/ © 2020 Elsevier B.V. All rights reserved. P. Das, et al. Journal of Photochemistry & Photobiology, B: Biology 212 (2020) 112028 interband transition absorption have been most extensively used as Zebrafish Danio ( rerio) is used as an ideal experimental vertebrate photothermal agents because of their intensified optical absorption model owing to its high genetic homology with humans (~70%) [27]. which is correlated with the photothermal effects [10]. Au nanoma­ Compared to other vertebrate models (rodents), Zebrafish model is af­ terials in different nanostructures including Nano rods, Nano plates, fordable and offers an unerring understanding of the interactions of Nano shells, Nano boxes, Nano cages, Nano frames and Nano stars nanomedicine with the biological system, attributed to its optical exhibit high photothermal conversion efficiencies11 [ ,12]. Various re­ clarity [28]. search groups have established the tuning of absorbance from visible to Here, we explored the synthesis of bimetallic AuPd-NPs on PDA NIR region upon modification of the synthesis route using ligand or functionalized rGO sheets for the better dispersibility in water adopting surfactants to stabilise the Au NPs. But several research articles have an easy solution chemistry approach. The AuPd-rGO/PDA nano­ also been published on the PTT for cancer cells using Au NPs where the composites exhibited excellent photothermal properties when illumi­ synthesized Au NPs sizes are larger and anisotropic [13]. Later on, re­ nated with NIR laser resulting in an increase of temperature nearly searchers have developed nanocomposite materials containing Au NPs 51 ± 3 °C within 3 min of irradiation with low dose amount of loaded on different matrixes such as silicon nanoparticle 14[ ], layered 25 μg mL−1 resulting in ablation of the MDAMB-231 cells line. double hydroxide (LDH) [15], silicon nanowire [16], etc. which ex­ Additionally, the AuPd-rGO/PDA demonstrated high biocompatibility hibited good PTT effect. Thus, decoration of the Au NPs on a suitable towards L929 cell lines and exhibits high survival efficiency rates in substrate is an efficient method for developing nanocomposite material Zebrafish (Danio rerio embryos), thus paving a way for their use as ef­ for non-invasive PTT for cancer treatment. Apart from Au NPs, Pd NPs ficient PTT agents for cancer theranostics. are also investigated as efficient PTT agent because of the biocompat­ ibility and wide absorption in the UV–Vis -NIR region [17]. Huang et al. 2. Experimental explored the utilization of Pd nanosheets as PTT agent to kill the cancer cells. The promotion of PTT is favoured by the ultrathin Pd nanosheets, 2.1. Materials which inhibits them from inflowing the cancer cells [18]. However, the limit associated with Pd nanostructures as PTT agents lies in proper The materials used in this study are described in SI. All materials control of their size and shape. The applicability of a single component were used as received. metal NPs in PTT is limited [19]. To overcome these limitations monometallic NPs can be modified via addition of a second metal 2.2. Cell lines and maintenance component to its structure that leads to the formation of bimetallic NPs . Through bimetallization, the surface plasmon band can be tuned as Both fibroblast (L929) cell lines and breast cancer cell lines well as the stability and dispersion of the NPs can be enhanced [20, 21]. (MDAMB231) of mouse origin were collected from National Centre for In recent times, AuPd has attracted immense attention amongst the Cell Sciences (NCCS), Pune, India. All the cell lines were sterile cultured various noble bimetallic nanoparticles, as a catalytic material in dif­ in Dulbecco's Modified Eagle's medium (DMEM) containing 1% L-glu­ ferent biological field [21]. tamine, 10% (v/v) Fetal bovine serum (FBS) and 100 U/mL penicillin/ Bimetallic nanoparticles are multifunctional nanomaterials with streptomycin. The cell lines were cultured at 37 °C in a humidified at­ applications in different fields. The basic reason behind multifunctional mosphere containing 5% CO2. behaviour is the synergistic effect that exists between the two metal counterparts. Bimetallic nanoparticles are widely used in comparison to 2.3. Preparation of Catalysts the monometallic counterparts in both technological as well as scien­ tific aspects because bimetallic NPs show better and enhanced chemical Graphite oxide is synthesized adopting the modified Hummers and and physical properties. These NPs mainly offers the tendency of opti­ Offemann's method from graphite powder through potassium perman­ mizing the energy of plasmon absorption band of metallic mix, en­ ganate (KMnO4) and sulphuric acid (H2SO4) as oxidising agents. The hancing the stability and dispersion of NPs and regulates the physico­ synthesized graphite oxide was exfoliated in water under high power chemical properties and provides opportunity as multipurpose tool for ultra-sonication to obtain homogenous graphene oxide (GO) suspension PTT applications. Their properties strongly depend upon the structure with a concentration of 10 mg mL−1. The details synthesis procedure of and composition of the nanomaterials, the preparation of bimetallic GO is discussed in one of our previous publications [29]. The above GO NPs with accurately controlled structures and compositions is a suspension was reduced using an eco-friendly reducing agent, ascorbic worthwhile objective. Moreover, unique advantage appended to PTT, acid via ultra-sonication method to get rGO sheet which was further such as high specificity and minimal invasiveness have made this used as a support for synthesizing AuPd-rGO nanocomposite functio­ technique an excellent potential for treating cancer metastasis. nalized by polydopamine i.e. AuPd-rGO/PDA nanocomposites. The Moreover, owing to using bimetallic nanoparticles, PTT can be guided AuPd-rGO/PDA nanocomposite was obtained in two stages. The first with multimodal imaging or combined with the other available thera­ stage contains the synthesis of AuPd-rGO nanocomposite followed by pies to perform more effective targeting 22[ ]. In this work, we designed functionalization with polydopamine. Typically, PdCl2 (3 mM) was AuPd bimetallic nanoparticles decorated on rGO nanosheets and func­ added to oleylamine solution (6 mL) preheated to 45 °C. Separately, tionalized with polydopamine to form AuPd-rGO/PDA. AuPd-rGO/PDA HAuCl4.3H2O (1 mM) was dissolved in 2 mL oleylamine and 1 mL of 1- NPs exhibits favourable properties of both Au and Pd by providing high octadecene solutions and mixed the above PdCl2 solution in dynamic surface plasmon resonance (SPR) enhanced properties in the NIR region stirring and the entire solution was stirred for 20 mins for homogeneous

[23, 24]. PDA coated nanocomposites improves the stability, dispersion mixing. To the resulting solution, 2 mL of 0.01 M NaBH4 solution was and biocompatibility towards the PTT [25]. added to obtain an immediate change in colour from yellow to wine The stability of the bimetallic NPs is an important matter of concern. red. The obtained product was collected through centrifugation and Upon laser irradiation, there might be certain possibility of the na­ continuously washed with 1:3 toluene: ethanol after which the solid nostructures converting into bulk aggregation which can be resolved by product obtained was re-dispersed in a solution containing 15 mL coating the NPs with polymers like polydopamine (PDA) [25]. The high hexane and 20 mL ethanol. To the above, 200 mg of rGO was added and drug loading ability together with the biocompatibility of the PDA further ultrasonicated for 2 h. The solution mixture was filtered and provides prospects to use AuPd-rGO/PDA as a capping agent for PTT of then dried in an air oven at 65 °C to get the AuPd-rGO nanocomposite. cancer cells [26]. The in vitro technique assists in understanding the In the second stage involving functionalization, dopamine hydro­ biocompatibility of NPs. However, thorough understanding of the me­ chloride (5 mg) was added with 10 mL of the above synthesized AuPd- chanisms of PTT of the cancer cell can be obtained by in vivo models. rGO nanocomposite followed by the addition of 30 mL of tris buffer

2 P. Das, et al. Journal of Photochemistry & Photobiology, B: Biology 212 (2020) 112028 solution (pH 8.5) and stirred further for 12 h at 65 °C. The obtained The number of dead embryos/larvae, the number of deformed em­ functionalized AuPd-rGO/PDA nanocomposite was centrifuged at bryos/larvae and the number of non-hatched or hatched embryos were 12000 rpm for 15 min for the removal of unreacted dopamine. calculated using microscope (Olympus, Japan).

2.4. Characterization Techniques 2.8. In Vitro Photothermal Therapeutic Efficacy

The details characterization using all the equipment in this study is 2.8.1. Live/Dead Assay (Qualitative Analysis) described in SI. To visualize the effect of rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/ PDA to NIR light on the cell death, live-dead assay was performed by 2.5. Photothermal Transduction Efficacy Studies fluorescein diacetate (FDA)/propidium iodide (PI) staining. 10,000 cells were seeded in 96-well plate and incubated at 37 °C in 5% CO2 To understand the photothermal efficacy (i.e. light to heat conver­ overnight. 25 μg mL−1 of nanocomposites was added to each well sion) of rGO nanocomposites with varying concentrations (12.5, 25, 50, containing 100 μL complete medium. The resulting nanocomposites 75, 100 and 150 μg mL−1) were examined upon irradiation of NIR were irradiated for 5 min with NIR laser (915 nm). After completion of Laser (915 nm wavelength, 650 mW power) for 5 min. Further the the incubation time, 1× PBS was used to wash the cells and stain them nanocomposites (200 μL, 25 μg mL−1) were placed in a 96 well plate with FDA+ PI, 0.008 mL FDA (5 mg mL−1) and 0.03 mL PI and irradiated with for regular intervals of time to understand the (1 mg mL−1), respectively, in the culture well followed by incubation photothermal transduction efficiency of the 2D nanomaterials [30]. In for 30 min at 37 °C. Finally, the culture well was washed thrice with 1× this study, Indocyanine green (ICG), Milli-Q water were considered as PBS for the removal of any unbound stain and visualized under a positive and negative controls. The temperature of nanocomposite fluorescent microscope (Olympus, Japan). water suspension was monitored before and after the irradiation of the NIR laser sources at the time of 0, 2, 4, 6, 8 and 10 min with a thermal 2.8.2. MTT Assay (Quantitative Analysis) probe. The thermal camera (SEEK thermal camera, CA, USA) was used MDAMB-231 10,000 cells were seeded in 96-well plate and in­ for the thermal images at above defined time and their respective cubated at 37 °C in 5% CO2 overnight and incubated with rGO, rGO/ temperatures were captured. The variation of the temperature along PDA, AuPd-rGO, AuPd-rGO/PDA at 25 μg mL−1 concentration for de­ with respective time was plotted for the Milli-Q water, ICG rGO, rGO/ finite time. After that, the cells were washed with phosphate buffer PDA, AuPd-rGO, and AuPd-rGO/PDA. saline (PBS) 2 times and supplemented with fresh medium. The cells which were treated with nanocomposites were irradiated by an NIR 2.6. Biocompatibility Studies laser of 915 nm for 5 min. Cells of all treatment groups were kept in an incubator for another 12 h. Cell viability was determined using the Biocompatibility of different concentrations of rGO, rGO/PDA, AuPd- standard MTT assay as described above. rGO, AuPd-rGO/PDA nanocomposites were estimated by methyl thia­ zolyltetrazolium (MTT) assay on L929 cell lines. The L929 cell were 2.9. Reactive Oxygen Species (ROS) Generation Assay seeded in culture flasks containing DMEM medium and 10% FBS, which is incubated for 24 h at 37 °C in 5% CO2 and 95% humidity to attain a In this study, 2,7-dichlorodihydrofluorescein diacetate (H2DCFDA) cell growth of 80% confluency. The cells were trypsinzed and transferred used for the formation of ROS inside the cancer cells. The fluorescent 2′, to a 96-well cell culture plate with density of 1 × 104 cells/well before 7′-dichlorofluorescin (DCF) was formed due to cleavage of the acetate proceed for the experiments. The cells were incubated with different group of H2DCFDA by oxidation of intracellular esterases. From the concentrations of nanocomposites materials (10–150 μg mL−1) for 48 h. fluorescence intensity, we can predict the oxidation of the fluorescent After that of 100 μL MTT (0.5 mg mL−1) was added and further the cells probe [32]. For ROS assay, the MDAMB-231 cells were seeded at a were incubated for 3 h at 37 °C. During this process it is observed that density of 10,000 cells/well in 96 well plate and cultured for 24 h. The Formazan crystals were formed which were dissolved with the addition cells were treated with rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/PDA at −1 of 100 μL of dimethyl sulfoxide (DMSO) per well. Plate reader (Enspire 25 μgmL for definite time. After incubation, the cells were treated multimode plate reader (Perkin Elmer, USA) was used for the measure­ with nanocomposites and irradiated with laser light source of 915 nm ment of absorbance at 570 nm. The cells treated with ICG and DMEM for 5 min. Cells of all treatment groups were kept in an incubator for media were used as positive and negative controls respectively. The another 12 h, after incubation cells were treated with H2DCFDA percentage viability was calculated as: (2.5 μM/well) and incubated for 45 min in dark. The excess amount of H2DCFDA in the cells was removed by washing with PBS solution. The Cell viability (%)= Nt /N c × 100 fluorescence intensity was monitored using fluorescent microplate where, Nt — absorbance of nanocomposite treated cells and Nc — ab­ reader (Enspire multimode plate reader (Perkin Elmer, USA). The ex­ sorbance of untreated cells. citation and emission wavelength were 485 nm and 535 nm, respec­ tively. 2.7. Zebra Fish Culture and Embryonic Toxicity of Nanocomposites 3. Results and Discussions Zebrafish (Danio rerio) were cultured and maintained at IIT, Hyderabad following the standard operational guidelines [31] The 3.1. Characterization of the Nanocomposites adult Zebrafish (male: female ratio of 2:1) were kept in a tank under a 14 h light/dark cycle for fertilization. The fertilized eggs from Zebrafish The formation of the AuPd-rGO/PDA nanocomposite is illustrated in were collected and washed with copious amount of oxygenated water. Scheme 1. Bimetallic AuPd nanoparticles decorated on rGO sheets were After that 20 healthy Zebrafish embryos were incubated in presence of prepared through reduction process of the metal ions using oleylamine, rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/PDA nanocomposites solution at which behave as reducing as well as dispersing agent. The formation of 25 μg mL−1 in a 24-well culture plate. Development of Zebrafish em­ alloyed AuPd nanostructure on the polydopamine functionalized rGO bryos and larvae were continuously monitored, from 4 h post-fertili­ sheets was confirmed by XRD, XPS and TEM analysis. In this process, zation (hpf) to 96 hpf, and the photos were captured at the indicated we have used tris base (pH 8.5) solutions which contained dopamine time points using microscope (Olympus, Japan). The survival rate and hydrochloride precursor which contain amine and catechol groups at hatching time were investigated at time point of 24, 48, 72 and 96 hpf. the edges and can be self-polymerized to produce PDA at room

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Scheme 1. Schematic illustration of the AuPd-rGO/PDA

Fig. 1. (A) UV–Vis spectra of rGO, rGO/PDA, AuPd-rGO, AuPd–rGO/PDA nanocomposites with concentration of (50 μg/ mL), (B) Solubility of (1) rGO, (2) rGO/PDA, (3) AuPd rGO, (4) AuPd–rGO/PDA nanocomposites and (C) XRD patterns of rGO/PDA, AuPd-rGO, AuPd-rGO/PDA nanocomposites. temperature. PDA reacts with AuPd-rGO nanocomposite through hy­ nanocomposites are represented in Fig. 1A. The aqueous rGO solution ⁎ drogen bonding and resulted AuPd-rGO/PDA nanocomposite. The op­ shows at 230 nm absorption peak which corresponds to the π–π tical absorbance spectra of rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/PDA transition. In AuPd-rGO and AuPd-rGO/PDA nanocomposites, the peak

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Fig. 2. (a,b) Low and (c) medium magnified TEM images, (d) particle size distribution (e) HRTEM image (f) SAED pattern of AuPd-rGO/PDA nanocomposite. shifts to 250 nm due to the increase in size of the NPs that generally PDA nanocomposites which are presented in supporting information leads to red shifting of the UV peak [21] . In AuPd-rGO/PDA nano­ (Fig. S1). composite, there was substantial increase in absorption at NIR The size, crystallinity and morphology of the as synthesized nano­ (800 nm). This improved absorbance of AuPd-rGO/PDA is expected to composites were studied using TEM analysis. The TEM and HRTEM affect the photothermal transduction efficiency. The solubility ofthe images of the AuPd-rGO and AuPd-rGO/PDA nanocomposites are rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/PDA nanocomposites in aqueous shown in Fig. S2 and Fig. 2, respectively. The TEM images of AuPd-rGO medium are depicted in Fig. 1B. The XRD patterns of rGO/PDA, AuPd- nanocomposites displays high density distribution of AuPd NPs on the rGO, AuPd–rGO/PDA nanocomposites are presented in Fig. 1C. The rGO sheets as shown in Fig. S2(a-c) with an average particle size dis­ diffraction patterns of rGO/PDA appear at the 2θ value of 23.14o cor­ tribution of 4.08 nm as obtained from the size distribution histogram responding to the fcc (002) plane. For AuPd-rGO, the diffraction peaks calculated using Image J software (Fig. S2d). The HRTEM image of appear at 2θ value of 38.9o, 43.25o, 65.59o and 78.20o corresponding to AuPd-rGO nanocomposite shows the presence of clear lattice fringe the fcc AuPd (111), AuPd (200), AuPd (220) and AuPd (311) planes, corresponding to fcc (111), (200) and (220) plane of AuPd (Fig. S2e). respectively. For AuPd-rGO/PDA nanocomposite, the diffraction peaks The selected area electron diffraction (SAED) pattern corresponding to appear at 2θ value of 23.14o and 38.79o. The diffraction peak at 23.14o the fcc (111), (200), (220) crystallographic planes, which reflects the corresponds to the fcc (002) plane of rGO and the diffraction peak at polycrystalline nature of the AuPd-rGO (Fig. S2f) [9]. The TEM images 38.79o corresponds to the fcc (111) plane of AuPd. In comparison to of AuPd-rGO/PDA nanocomposite (Fig. 2(a-c)) also shows the presence rGO/PDA and AuPd-rGO, the diffraction patterns for dopamine func­ of AuPd NPs on functionalized rGO sheets with an average size of tionalized AuPd-rGO/PDA nanocomposite are obtained at same dif­ 4.13 nm as achieved from the size distribution histogram (Fig. 2d). The fraction values similar with AuPd-rGO. No significant change is ob­ TEM image (in Fig. 2c) shows the spherical shape of the AuPd bime­ served in the diffraction pattern for the AuPd-rGO/PDA as tallic NPs. The HRTEM image (Fig. 2e) shows the presence of clear functionalization with PDA does not lead towards any change in the lattice fringes distance of 0.22 nm and 0.25 nm of (002) and (111) crystal structure of the nanocomposites. After functionalization of the planes, respectively. The SAED pattern also confirms the polycrystalline AuPd-rGO nanocomposite with PDA, the diffraction peaks appear at 2θ behaviour of AuPd-rGO/PDA nanocomposite and presence of different value of 23.14° and 38.79°. The diffraction at 23.14° corresponds to the planes corresponding to the (002) and (111) plane of Au and Pd [37]. fcc (002) plane of rGO and the diffraction at 38.79° corresponds to the The TEM images of AuPd-rGO and functionalized AuPd-rGO/PDA na­ fcc (111) plane of AuPd. PDA is produced by self-polymerization of its nocomposite do not show any major difference in the morphology of monomer dopamine [33] The diffraction peak at 23.14° demonstrates the AuPd bimetallic NPs even after functionalization with dopamine. that the dopamine has undergone successfully self-polymerized poly­ The TEM images of monometallic Au-rGO/PDA and Pd-rGO/PDA na­ merization on GO due to the stacking of benzene rings of PDA [34,35]. nocomposites are shown in Fig. S3 and Fig. S4 with an average particle The broad peak observed at 38.79° confirms the formation of fcc (111) diameter of 5.10 nm and 4.92 nm, respectively. The XPS analysis was plane of AuPd. However, after PDA modification of the AuPd-rGO na­ performed to study the surface properties, compositions and oxidation nocomposite, the intensity of the peak decreases due to shielding by the state of AuPd-rGO nanocomposite (shown in Fig. S5). The XPS survey PDA layers [36]. The presence of the polymerized dopamine might also spectrum of AuPd-rGO nanocomposite is shown in Fig. S5a. The survey lead to the loss in the crystallinity of the nanocomposites leading to the spectrum exhibited four different prominent binding energy peaks decreased intensity of the XRD peaks. The XRD patterns of the corre­ corresponding to C, O, Au and Pd thus signifying the existence of these sponding monometallic Au-rGO, Au-rGO/PDA, Pd-rGO and Pd-rGO/ respective elements in AuPd-rGO nanocomposite. In Fig. S5b, the

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Fig. 3. (a) XPS survey spectrum and high-resolution spectra of (b) Au4f (c) Pd3d (d) C1s (e) N1s and (f) O1s of AuPd-rGO/PDA.

Table 1 Pd3d3/2 at 335.30 eV and Pd3d5/2 at 340.70 eV, which confirm the Binding energy and atomic percentage of characteristic peaks of XPS spectra of existence of Pd(0) (Fig. S5c). The existence of these Au and Pd indicates AuPd-rGO and AuPd-rGO/PDA nanocomposites. the presence of both Au and Pd in bimetallic Au-Pd/rGO nanocomposite Sl. no. Core level Binding energy (eV) Atomic % [8]. In Fig. S5d, the high-resolution C1s spectrum is fitted with three different binding energy peaks at 284.80, 286.80 and 288.10 eV cor­ AuPd-rGO AuPd-rGO/PDA AuPd-rGO AuPd-rGO/PDA responding to the C=C, C-O and C=O of rGO in AuPd-rGO nano­ composite. 1 Au4f5/2 83.90 84.00 0.44 0.02

2 Au4f7/2 87.60 87.80 0.61 0.01 The survey scans XPS spectrum of AuPd-rGO/PDA nanocomposite is 3 Pd3d3/2 335.30 335.30 0.42 0.00 shown in Fig. 3a. The survey spectrum shows the presence of C, N, O, 4 Pd3d5/2 340.70 340.70 1.94 0.02 Au and Pd at binding energy values of 284.30, 399.20, 531.30, 83.90 5 C1s (C=C) 284.80 284.80 93.69 65.52 and 336 eV, respectively. The relative atomic percentages of C, N, O, Au 6 C1s e(C-O) 286.80 286.90 7 C1s (C=O) 288.10 288.10 and Pd were found to be 65.52, 26.16, 8.26, 0.03 and 0.02%, respec­ 8 C1s e(C-N) – 285.90 tively. The high-resolution Au4f spectrum was obtained with two pro­ 9 O1s 532.78 531.26 2.90 26.16 minent binding energy peaks assigned as Au4f5/2 at 84.0 eV and Au4f7/ 10 N1s – 400.00 – 8.27 2 at 87.80 eV (Fig. 3b), which can be attributed to the Au(0) in AuPd- rGO/PDA nanocomposite. The high-resolution Pd3d spectrum also

shows two binding energy peaks assigned as Pd3d3/2 at 335.30 eV and deconvoluted high-resolution Au4f XPS spectrum was obtained with Pd3d5/2 at 340.70 eV, signifying the presence of Pd(0) in AuPd-rGO/ two different binding energy peaks assigned to Au4f at 83.90 eV and 5/2 PDA nanocomposite. The high-resolution Au4f and Pd3d spectra con­ Au4f at 87.60 eV. These are the typical binding energy values of Au 7/2 firmed the successful formation of bimetallic AuPd on rGO/PDA sup­ (0) species, which confirms the existence of Au in (0) oxidation state in port [9]. In Fig. 3d, the high-resolution C1s spectrum shows the pre­ the AuPd-rGO nanocomposite. Similarly, the high-resolution Pd3d sence of C=C (284.80 eV), C-N, (285.90 eV), C-O (286.90 eV) and spectrum was obtained with two binding energy peaks assigned to

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doping of other metal NPs [38]. The Raman spectroscopy measurement of AuPd-rGO and AuPd-rGO/PDA were obtained with two main peaks relating to D (1272.7 and 1275.4 cm−1) and G (1452.1 and 1452.8 cm−1) bands as presented in Fig. S7. The G band measures the

crystallinity of carbon, which is obtained from the Raman active t2g mode. The D band is initiated from the breathing mode of the sp2 hy­ bridized carbon atom of the rGO layers. The D band is sensitive to various kinds of structure and heteroatom doping effects [38]. The defect density of carbon-based nanostructure materials is generally

measured from the intensity ratio between the D band and G band (ID/ IG) of the Raman spectrum. The ID/IG value of 0.876 was obtained for AuPd-rGO, which is slightly smaller than the ID/IG value of AuPd-rGO/ PDA (0.878), indicating the sp2 network restoration of rGO after functionalization by PDA in AuPd-rGO/PDA. The leading monolayers to bilayer structures of rGO were confirmed from a 2D band of both AuPd- rGO and AuPd-rGO/PDA that appears at 2717 cm−1 with an additional shoulder at a higher wavelength [21]. The corresponding Raman spectra for Au-rGO, Au-rGO/PDA, Pd-rGO and Pd-rGO/PDA nano­ composites are presented in Fig. S8. From Fig. S8, it is seen that all the nanocomposites acquiring nearly similar peaks corresponding to D (1272.67 cm−1) and G (1452.07 cm−1) bands.

3.2. Photothermal Transduction Efficiency

AuPd-rGO/PDA nanocomposite showed significant photothermal Fig. 4. A) Photothermal transduction efficacy of rGO, rGO/PDA, AuPd-rGO, activity achieved a temperature of above 43 °C using 25 μg mL−1 which AuPd rGO/PDA nanocomposites (25 μg mL−1, (n = 3) with 915 nm NIR Laser. was considered as high enough temperature to photothermal ablation B) FLIR thermal images of rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/PDA upon of the cancer cells. However, in case of other nanocomposites (rGO, NIR laser irradiation. rGO/PDA, AuPd-rGO), an amount of 100 μg mL−1 concentration of the PTT agents was required to achieved the temperature above 43 °C. It C=O (288.10 eV) bond formation on rGO/PDA surface. The occurrence was also observed from the Fig. S9A that the light to heat conversion of N in AuPd-rGO/PDA is due to the PDA functionalization as confirmed efficiency is continuously increased with increasing the concentration by the high-resolution N1s spectrum (Fig. 3e). The high-resolution N1s of the AuPd-rGO/PDA and temperature recorded was around 68 °C peak appears at 400 eV, which can be attributed to the C–N–H bond on which is more than required for the intended study. Therefore, we had PDA surface [21]. The high-resolution O1s peak was obtained at considered 25 μg mL−1 as an optimum concentration of the AuPd-rGO/ 531.26 eV due to oxygen functionalities present on the rGO surface PDA nanocomposite to conducted further PTT studies. Further to un­ (Fig. 3f). The details core level, binding energy and atomic % of AuPd- derstand the effect of time on the photothermal efficiency, photo­ rGO and AuPd-rGO/PDA nanocomposites are inserted in Table 1. The thermal property of the all the nanocomposites (rGO, rGO/PDA, AuPd- survey spectrum of rGO/PDA, Au-rGO/PDA and Pd-rGO/PDA nano­ rGO) were irradiated with NIR laser 915 nm at different time interval composites are shown in supporting information (Fig. S6). From the (2, 4, 6, 8, 10 min). Photothermal efficiency of the AuPd-rGO/PDA survey spectrum of rGO/PDA nanocomposite corresponding C, O and N nanocomposites with various concentrations at different time intervals peaks are clearly evident which signifies the formation of functiona­ was analyzed using NIR laser (915 nm, 650 mw power), as shown in lized rGO nanocomposite. Similarly, in Au-rGO/PDA and Pd-rGO/PDA Fig. S9B. As given in Fig. 4A, the temperature is risen to 55 ± 2 °C in nanocomposites corresponding Au, C, O, N and Pd, C, N, O signifies the presence of rGO, rGO/PDA and AuPd-rGO nanocomposites within presence of these elements in aforementioned nanocomposites. 10 min of irradiation. However, in case of AuPd-rGO/PDA nano­ Raman spectroscopy is a highly potential characterization tool for composite, the temperature is risen to 51 ± 3 °C within 3 min of ir­ the carbon-based materials to evaluate the degree of graphitic layer and radiation of NIR laser. This temperature is considered high enough for

Fig. 5. Biocompatibility of rGO nanocomposites in L929 cell lines.

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Fig. 6. A) Optical images of developmental stages of zebrafish embryos incubated with rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/PDA nanocomposites. B) & C) survival rate and hatching rate of zebrfish embryos embryos treated with all nanocomposites compared with untreated control. Data are represented as mean ± SEM. the photothermal ablation of the tumor cells [30]. We also observed 150 μg mL−1 of rGO, rGO/PDA, AuPd-rGO and AuPd-rGO/PDA nano­ that AuPd-rGO/PDA nanocomposite maintained good thermal stability composites. Whereas in the case of Indocyanine green (ICG) control, (Fig. 4B). Whereas, in the Indocyanine green (ICG) control, though the cell viability was reduced with increasing concentration. Thus the rGO temperature reached 50 ± 2 °C in 5 min, it eventually declined due to nanocomposites showed excellent biocompatibility when compared the degradation of the dye. In the case of Milli-Q water, the temperature with controls. increment was saturating at 38 °C under the same conditions. 3.4. Zebra Fish Culture and Embryonic Toxicity of Nanocomposites 3.3. The Biocompatibility Assay Embryonic development is a suitable model for studying the toxi­ The biocompatibility studies of the nanocomposites are a matter of city. The embryonic development result is demonstrated in the litera­ concern for most of the in vitro application . As shown in Fig. 5, no ture based on the dose-dependent biocompatibility, adverse effects of significant cytotoxicity was observed for L929 cell lines upon incuba­ nanomaterials and drug screening [39]. To evaluate the developmental tion with nanocomposites for 48 h at concentrations lower than toxicity of the rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/PDA, the Zebra­ 150 μg mL−1 and the cell viabilities remained more than 85% even at fish embryos were exposed with the nanocomposites starting from4

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Fig. 7. MDAMB231 cells were incubated with rGO, rGO/PDA, AuPd-rGO, AuPd-rGO/PDA and irradiated for 5 min with a NIR laser operating at 915 nm. Cells were then stained with fluorescein diacetate and PI and examined by fluorescence microscopy. Live cells appear green and dead cells appear red. Scale bar, 50 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

hpf after exposure also showed normal phenotypic development. The survival rate (Fig. 6B) and hatching rate (Fig. 6C) at 48 and 72 hpf were not affected by exposure to all nanocomposites at the highest­ con centration (100 μg mL−1).

3.5. In Vitro Photothermal Therapeutic Efficacy

3.5.1. Live/Dead Assay (Qualitative Analysis) Fluorescence based live/dead assay was performed in MDAMB-231 cells to evaluate the effect of photothermal heat by rGO, rGO/PDA, AuPd-rGO and AuPd-rGO/PDA nanocomposites. Live cells convert the non-fluorescent fluorescein di acetate (FDA) to green fluorescent­ me tabolite fluorescein when taken by cells. Propidium iodide (PI), an in­ tercalating dye, only passes through the membranes of dead or dying cells. As shown in Fig. 7, majority of the irradiated cells with rGO, rGO/ PDA, Au-Pd-rGO are found to be alive (green fluorescence) but the cells Fig. 8. PTT mediated cytotoxicity of nanoparticles in MDAMB-231 cell line. irradiated with AuPd-rGO/PDA were dead cells (red fluorescence). Data are represented as mean ± SEM, n = 3(***p < 0.001). These in vitro results suggested that PTT based on illumination of NIR laser of 915 nm using AuPd-rGO/PDA can efficiently kill cancer cells hpf. Fig. 6A shows the embryonic developmental stages of zebra fishes within 5 min. When rGO, rGO/PDA, AuPd-rGO nanocomposites were incubated with different nanocomposites at 25 μg mL−1. It is noticed interacted with the culture media (DMEM) aggregates were formed due that the Zebrafish embryos as control group showed normal develop­ to various factors such as molecule/protein adsorption or loss of surface mental stages, from cleavage (4 hpf), segmentation (24 hpf), and functionality but AuPd-rGO/PDA nanocomposites though interacted hatching (48 hpf), to fully developed larvae (96 hpf). Survival rate and with the media exhibited high stability which could be a reason for the hatching rates were examined at each time point of 24, 48, 72 and 96 efficient cell death (Fig. S10).

9 P. Das, et al. Journal of Photochemistry & Photobiology, B: Biology 212 (2020) 112028

3.5.2. MTT Assay (Quantitative Analysis) PR16740/NER/95/270/2015, NEIST Project No. GPP 316). PKB is To investigate the photothermal mediated cytotoxicity of the rGO, thankful to the CSIR, New Delhi, India, for CSIR-SRF Fellowship. rGO/PDA, AuPd-rGO, AuPd-rGO/PDA towards MDAMB 231 cells, at Author SG would like to thank DST, Govt. of India for Inspire fellowship first the cells were treated with 25 μg mL−1 concentration followed by (IF180295). Authors are also recognizing the sophisticated analytical NIR laser irradiation (915 nm) for 5 min as shown in the bright field facility of CSIR-NEIST, Jorhat, Assam, India. images. It is observed from Fig. S11 that the cell death upon laser ir­ radiation in presence of AuPd-rGO/PDA which was comparatively Appendix A. Supplementary data higher than the control. In Fig. 8, MTT assay result shows the photo­ thermal cytotoxicity quantitatively in MDAMB 231 cells. 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