Rapid, One-Pot, Protein-Mediated Green Synthesis of Gold Nanostars

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Rapid, One-Pot, Protein Mediated Green Synthesis of Gold Nanostars for Computed Tomographic Imaging and Photothermal Therapy of Cancer Sisini Sasidharan, Dhirendra Bahadur, and Rohit Srivastava ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02169 • Publication Date (Web): 05 Sep 2017 Downloaded from http://pubs.acs.org on September 9, 2017

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ACS Sustainable Chemistry & Engineering

Rapid, One-Pot, Protein Mediated Green Synthesis of Gold Nanostars for Computed Tomographic Imaging and Photothermal Therapy of Cancer Sisini Sasidharan,a Dhirendra Bahadurb* and Rohit Srivastavaa* a Department of Biosciences and Bioengineering, IIT Bombay, Powai, Mumbai, 400076, India. b Department of Metallurgical Engineering and Materials Science, IIT Bombay, Powai, Mumbai, 400076, India. AUTHOR INFORMATION Sisini Sasidharan- [email protected], [email protected] Corresponding authors Dhirendra Bahadur- [email protected] Rohit Srivastava - [email protected] Keywords: albumin, gold nanostars, gold nanoparticle, photothermal therapy, CT imaging, cancer

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ABSTRACT Plasmonic nanostructures such as gold nanostars have immense prospects in the biomedical field. However, toxic precursors and complex methodologies of synthesis are the biggest setbacks for their application. This study hence makes use of protein macromolecule (albumin) to synthesize gold nanostars by a simple reduction method. The formation of an anisotropic morphology of gold using albumin was determined to be a pH-dependent process. The net positive charge of protein at a pH below its isoelectric point facilitated the attachment of chloroaurate ions which was subsequently reduced to gold atoms. Furthermore, the stretching of α-helices of albumin at low pH and its transformation to β-sheet conformer favored an oriented growth of gold nanostructures to yield a star-shaped morphology. Additionally, albumin being non-toxic and with ligand binding characteristics bestowed stability, functionality as well as biocompatibility to the gold nanostars. The albumin derived gold nanostars exhibited enhanced computed tomographic (CT) contrast, photothermal activity and compatibility towards cells and human blood. This study thus puts forth for the first time, a rapid, one-pot methodology to develop gold nanostars using protein and demonstrates its application as a dual CT diagnostic and photothermal therapeutic agent. INTRODUCTION Anisotropic gold nanostructures have received enormous attention in the past decade owing to its exceptional characteristics.1 Among the number of gold nanostructures such as rods,2 cubes,3 cages,4 prisms,5 plates6 and shells7 which have been investigated, the anisotropicity particularly in the form of branched structures bring about outstanding properties related to photothermal activity. The exceptional photothermal characteristics of branched gold nanostructures, also known as urchins,8 flowers9 or stars10–12 are due to its unique surface plasmon resonance (SPR), 2 ACS Paragon Plus Environment

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larger absorption cross-section and high molar extinction coefficient. Efficient heating of the nanostructures occurs due to the presence of sharp and elongated branches which allow easy penetration of the incoming electric field through the thin structure.13,14 The very high surface area to volume ratio and irregular shape also result in enhanced heat generation.13,14 This anisotropic geometry of gold nanostructures gives rise to several other applications such as photodynamic

therapy,15

surface-enhanced

Raman

scattering

(SERS),16

two-photon

photoluminescence (TPL) imaging10 etc. Additionally, high Z-number and excellent stability against oxidation of these gold nanostructures make it an ideal contrast agent for computed tomographic (CT) imaging applications.17 However, synthesis and bio-application of anisotropic gold nanostructures such as gold nanostars are associated with numerous roadblocks owing to the use of toxic precursors and cumbersome methodologies adopted for its synthesis. The lack of stability of these structures at physiological conditions and absence of functional groups for binding to biomolecules raise concern for its use in vivo. Synthesis of gold nanostars has been reported using toxic reagents namely CTAB,18 SDS,19 N,N-dimethylformamide (DMF),20 hydroquinone21 etc with limited applications in biology. The use of polyethylene glycols,22,23 polyethyleneimines,24–26 dendrimers,27 synthetic copolymers28 and zwitter ionic surfactants29 in the synthesis of gold nanostars has also been reported. However, the use of biogenic reagents is always preferable in place of aforementioned toxic and complex precursors. Hence, we explore the utility of protein namely albumin, a cheap, non-toxic, non-immunogenic, biodegradable,30 with ligand binding characteristics31 for the formation of gold nanostars. This protein, in turn, may also stabilize and functionalize the formed structures. Numerous studies report the conjugation of albumin to gold nanostructures for stabilization and functionalization.32–36 Recently, even our group has used Poly-L-arginine3 ACS Paragon Plus Environment


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albumin core-shell nanoparticles as a seed for the development of branched gold nanoparticles or gold nanostars as well as developed albumin stabilized gold nanostars using a seed-mediated approach.11,12 However herein, albumin molecule as such has been used as a template without using any preformed seeds to form gold nanostars. Hence, a rapid, one-pot method of synthesis with an aqueous route and simple reduction methodology was developed for the first time using protein to synthesize gold nanostars. The mechanism of formation of these gold nanostars, as well as their SPR characteristics, photothermal activity and CT contrast efficiency were evaluated in detail. Subsequently, for its use in biological applications, these albumin derived gold nanostars were also investigated for blood and cell compatibility as well as photothermal cytotoxicity towards oral epithelial carcinoma cell line (KB). EXPERIMENTAL SECTION Materials: All the chemicals namely, tetra chloroauric acid (HAuCl4) [Spectrochem], ascorbic acid [Merck], bovine serum albumin [Sigma Aldrich] and sodium hydroxide (NaOH) [Merck] were used as purchased in the study without any further purification or processing. Mouse fibroblast cells (L929), embryonic fibroblast cells (NIH 3T3) and oral epithelial carcinoma cells (KB) were obtained from the National Centre for Cell Science, Pune, India. The cells were cultured at 37 °C in a T25 flask with Dulbecco’s modified Eagles Medium (DMEM) [HiMedia] supplemented by 10% fetal bovine serum [FBS, Himedia], 50 IU mL-1 penicillin and 50 µg mL-1 streptomycin [HiMedia]. Synthesis of Albumin derived Gold Nanostars (A-GNS): Typically, 80 µl of albumin solution (BSA, 10 mg ml-1) was added to HAuCl4 (5mM) and stirred for 2 min. To this precursor solution, ascorbic acid (100 mM) was added to obtain a greenish blue color solution of gold


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nanostructures. The reaction was carried out at different pH of precursor solutions viz, 2.5, 4 and 5.3 (0.6-0.7 units below and above the isoelectric point of BSA, pH 4.7). The role of albumin in the formation of gold nanostars was determined by carrying out the reaction in the absence of albumin with same parameters. The obtained solution was centrifuged at 12000 rpm for 10 min; the pellet was washed three times, dispersed in deionized water and stored at 4 °C for further studies. Characterization: The formation of gold nanostructures was evaluated and confirmed by its surface plasmon characteristics using U.V–Visible spectrometric studies (Lambda 25, PerkinElmer). The morphology and lattice structure of nanostructures formed at varying reaction conditions were visualized at different magnifications using field emission gun transmission electron microscope -FEG-TEM (JEM 2100-F, JEOL) after drop casting and air drying a diluted solution of the sample on the copper grid. The selected area electron diffraction pattern (SAED) and the elemental composition of nanostructures using EDAX analysis were also studied. The albumin derived gold nanostar with branched morphology and SPR λmax of 800 nm, henceforth known as A-GNS were also analyzed for hyperspectral imaging with CytoViva 150 Unit integrated to Olympus BX43 microscope. Imaging and analysis were carried out at 60 X magnification with HSI system 1.1 along with ENVI software and images were captured by Dagexcel X16 camera. X-ray Diffraction (XRD) measurements of A-GNS were performed with Philips X'PERT PRO diffractometer (Cu-Kα source, λ= 1.54056 Å). The phase identification using standard JCPDS database were done after recording the spectrum from 5°- 85°. The charge on the albumin molecule at different pH (2.5, 4, and 5.3) was analyzed using a zeta potential analyzer (ZetaPALS, Brookhaven Instruments Corporation). The presence of functional groups of albumin on A-GNS was confirmed with Fourier transform infrared (FTIR) spectra (frequency 5 ACS Paragon Plus Environment


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range of 4000 to 400 cm-1) obtained from Fourier transform infrared spectrometer (MAGNA 550, Nicolet Instruments Corporation) using KBr method. The albumin present in A-GNS was measured quantitatively by analyzing the carbon, hydrogen and nitrogen content using CHNS (O) Analyzer (FLASH EA 1112, Thermo Finnigan) and determined using the equation described below: % = % × .

(1)

where, N.F is the nitrogen conversion factor ( N.F for BSA = 6.5). The albumin present in A-GNS was also quantified by thermogravimetric analysis (TGA) using Diamond thermogravimetric analyzer (Perkin Elmer, USA). Albumin (BSA) and A-GNS were heated from ambient temperature to 800 °C at a rate of 10 °C per min on a platinum crucible in the instrument under nitrogen flow and the weight of the samples were recorded for analysis. Circular dichorism (CD) spectroscopic studies were carried out to analyze the changes in the secondary structure of protein during the synthesis of gold nanostars as well as those in A-GNS. Bovine serum albumin, A-GNS and the precursor solutions (BSA along with HAuCl4 at specified pH 2.5, 4 and 5.3) at a protein concentration of 3.41 µM (200 µl) were placed into a quartz cuvette (Hellma, Forest Hills, NY) for carrying out the spectroscopic studies. The CD spectra were recorded using JASCO-810 instrument over a wavelength range of 200-260 nm at 25 °C with a pathlength of 0.1 cm. Deconvolution of the CD spectra (per residue molar absorbance units (∆ε M-1 cm-1) was carried out using the CDPro software package containing three analysis programs (CONTINLL, SELCON3 and CDSSTR) to determine the relative quantities of secondary structures such as α-helix, β-strands, β-turns and random coil. CD pro software contains a large reference set of CD spectra which were generated by consolidating


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reference set of proteins from various sources. A reference set (IBasis7) of 48 proteins and wavelength range of 240-200 nm was selected for analysis. The percentage of each secondary structure was determined by averaging the data obtained from the three different programs. However, in cases where data from one program were not consistent with other two, averaging was performed with data from the two programs which generated similar results. The photothermal efficacy of A-GNS was analyzed by recording the temperature increment of the sample upon irradiation with NIR laser (808 nm, 1.3 Wcm-2 500 mW, PMC, India). Briefly, 200 µl of the sample (A-GNS - 25, 50, 100, 200 µg ml-1) and control (water) were added to the wells (distant to each other to avoid any kind of heat transfer) of 96-well plates maintained at 37 °C with the help of a water bath. The temperature of the solutions was recorded using a digital thermometer after laser irradiation for different time duration such as 0, 0.5, 1, 2.5, 5, 7.5 and 10 min. The temperature increment was plotted against the time duration of laser irradiation. Additionally, photothermal efficiency and specific absorption rate (SAR) or the photothermal conversion efficiency of A-GNS were also calculated based on equations as described in previous studies.12,37–39 For the same, 3 ml of A-GNS solution was added in a quartz cuvette placed at a distance from the base to avoid any heat transfer and a clamped digital thermometer was inserted in the cuvette to record the temperature with its probe away from the path of illumination of laser light. The increment in temperature of the solution was recorded at definite intervals during laser irradiation (500 mW, 1.3 Wcm-2) for 25 min and cooling period as well. Deionized water was used as a control. A-GNS were also subjected to photothermal stability studies by measuring its absorbance both before and after laser irradiation using spectrophotometer (Lambda 25, Perkin Elmer).



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A 64-slice cardiac capable PET-CT Scanner (Biograph mCTMolecular CT, SIEMENS) operating at a tube voltage of 100 kVp and tube current of 200 mA was used to evaluate A-GNS as a CT contrast agent. A-GNS and Omnipaque (clinically used contrast) of varying concentrations were added into 96-well plates and were imaged with a scan time of 6 s, rotation time of 1 s and slice thickness of 1 mm. Deionized water served as the negative control. The Xray contrast of samples was determined in Hounsfield units by manually selecting the regions of interest with equal diameter. Biological Studies: Biocompatibility study: An Alamar blue assay was used to evaluate the biocompatible nature of A-GNS. L929, NIH3T3 and KB cells were seeded at a density of 7 х 103 per well in 96-well plates and cultured for 24 h. Thereafter, old media was discarded and replaced with basal media containing varying concentrations of A-GNS (10, 25, 50, 75, 100 and 200 µg ml-1) and cells were incubated for an additional 24 h. Cells subjected to treatment with only media and Triton X-100 (1%) served as negative and positive control, respectively. Subsequently, the cells were washed twice with PBS to eliminate unbound particles and were treated with 10% Alamar blue solution in basal media and incubated for 4 h. A microplate reader (Thermo-Scientific, USA) was then used to determine the fluorescence intensity at an excitation and emission of 560/590 nm wavelengths and percentage of viable cells were calculated by the following equation:

Cell viability %=

Fluorescence intensity of sample ×100 Fluorescence intensity of negative control (2)


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Cell Uptake Study: KB cells were seeded at a density of 2 х 105 per well in 6-well plates and cultured for 24 h. After discarding the old media, the cells were treated with 100 µg ml-1 of AGNS for 24 h. Subsequently, the cells were trypsinized after washing twice with PBS to remove unbound particles. The amount of A-GNS taken up by the cells were determined qualitatively by CT imaging of the cell pellets and quantitatively by ICP-AES after digestion of the pellet with aqua regia. Untreated cells served as control. Reactive oxygen species assay (ROS assay): Typically, KB cells were seeded at a density of 105 per well in 6-well plates and cultured for 24 h. After discarding the old media, the cells were treated with 100 µg ml-1 of A-GNS for 12 h. Cells subjected to treatment with media alone and H2O2 (30 µM) served as negative and positive control, respectively. Subsequently, cells were washed twice with PBS and incubated in the dark for 30 min after treatment with 10 µM of H2DCFDA. The cells were then washed, trypsinized and resuspended in PBS for analysis by flow cytometry (BD FACS Aria, USA). In-vitro photothermal therapy: In a typical experiment, KB cells were seeded at a density of 7 х 103 per well in 96-well plates and cultured for 24 h. Thereafter, old media was discarded and replaced with basal media containing A-GNS (100 µg ml-1) and cells were incubated for an additional 24 h. Subsequently, the cells were washed twice with PBS to remove unbound particles and were subjected to following treatment where negative control received no treatment; and other controls i.e., those incubated with only media and A-GNS treated cells were irradiated with the laser for 2.5, 5 and 7.5 min. Following incubation for 12 h, the cells were washed with PBS. The photothermal cytotoxicity was then analyzed quantitatively by Alamar blue assay as described previously and qualitatively by staining with propidium iodide dye which stains dead cells as red under Nikon Eclipse Ti fluorescent microscope integrated with filters for 9 ACS Paragon Plus Environment


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excitation/emission at 488/617 nm wavelengths. Cells subjected to treatment with only media and Triton X-100 (1%) served as negative and positive control, respectively. The ROS studies were also carried out with KB cells after following treatment- laser alone of 500 mWcm-2 with multiple pulsed excitations (8 pulses of 2 min) as well as 1.3 Wcm-2 (7.5 min), laser (500 mWcm-2 with multiple pulsed excitations) irradiation after A-GNS (100 µg ml-1) treatment and laser exposure of 1.3 Wcm-2 (7.5 min) after A-GNS (100 µg ml-1) treatment. Blood compatibility studies: The compatibility studies on blood were carried out as per national health guidelines following approval from institutional ethical and biosafety committee of IIT Bombay (Reference No: IITB-IEC/2016/003) as well as informed consent from healthy volunteers (22-32 yrs of age). Experiments were conducted with 5 ml of fresh whole blood withdrawn from each volunteer (3 in number) in vials having sodium citrate. Hemolysis assay -Typically, different concentrations (10, 25, 50, 100 and 200 µg ml-1) of AGNS redispersed in 0.9% saline was added to 1 ml of blood and incubated at 37 °C for 3 h along with mild shaking conditions. Treatment with 0.9% saline and 1% Triton X-100 served as negative and positive controls, respectively. The plasma supernatant obtained after centrifugation of blood at 4500 rpm for 10 min were subjected to spectrophotometric (M200 Pro Tecan, USA) analysis at 380, 415, and 450 nm. The plasma was diluted as required with 0.01% sodium carbonate. The plasma hemoglobin was quantified using the following equation:

()*+) ℎ+(- .

2 × 2345 − 2789 + 2359 × 1000 × ;( + 0= /( ? × 1.655 (3)


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where, absorbance at 380, 415, and 450 nm are denoted by A380, A415, and A450, respectively. The absorption of uroporphyrin in the same wavelength range of hemoglobin is corrected by A380 and A450 and interference due to plasma turbidity by a correction factor of 1.655. A415 and E (i.e., 79.46) refer to the sorbent band absorption and molar absorptivity value (415 nm) of oxyhemoglobin, respectively. The percentage of hemolysis due to A-GNS was quantified from the equation given below: B+(C** % =

()*+) ℎ+(- = *)+D( × 100 E)( ℎ+(- = Fℎ( -(/ (4)

RESULTS AND DISCUSSION Serum albumin with a molecular weight of 66.5 kDa is a single chain protein molecule and accounts for 60% of total protein content in blood serum.40,41 Albumin, being biocompatible and with its numerous functional groups can be used to stabilize gold structures.32–36 However, in addition to its stabilization and functionalization properties, herein, we explore the use of albumin (Bovine serum albumin, BSA) as a template to form gold nanostars in a single step reaction as described in experimental section. During the reaction, addition of gold chloride (HAuCl4) solution to albumin resulted in the reduction of pH of the solution to 2.5 and attachment of chloroaurate ions to the protein structure. These chloroaurate ions get further reduced by ascorbic acid to form greenish blue colored solution (inset of Figure 1) of gold nanostructures. The greenish blue colored solution exhibited an absorption spectrum with SPR λmax at 750 nm as seen in Figure 1A. To understand the mechanism of formation of nanostars, the reaction was also carried out at varying pH, i.e., pH 4 as well as pH 5.3 (0.6-0.7 units below and above the isoelectric point of BSA, pH 4.7) by the addition of NaOH during the synthesis. A 11 ACS Paragon Plus Environment


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greenish blue colored solution was formed even when the reaction was carried out at pH 4 with SPR λmax at 808 nm. However, when pH was raised to 5.3, the color changed to blue along with a blue shift in the absorbance to 650 nm as shown in Figure 1A. The role of albumin in the formation of gold nanostructures was determined by carrying out the reaction in the absence of albumin and at varying pH. In the absence of albumin, a pinkish- red colored solution was formed at all the different pH with SPR λmax ranging from 530-550 nm and no absorbance in the NIR (near infrared) range (Figure 1B) which demonstrates the crucial role played by albumin in the formation of gold nanostructures with NIR absorbance. A NIR wavelength with SPR λmax at 808 nm is preferred for biological applications as there is least interference in this range from biomolecules present in the body. The hyperspectral imaging of these gold nanostructures with SPR λmax at 808 nm revealed a homogeneous dispersion shown as bright white spots in Figure 1C and a scattering spectrum with λmax at 730 nm shown in Figure 1D. The change in the spectral profile of the nanostructures from the extinction spectra observed in the spectrophotometer is due to the absence of the absorption component and different dielectric constant of glass slides used in microscopy. The morphology of the nanostructures formed during different reaction conditions was evaluated by FEG-TEM imaging at various magnifications. A star-shaped branched morphology and size of ~ 150 nm were seen for gold nanostructure synthesized with albumin at pH 2.5 and pH 4 as shown in Figure 2A and 2C, respectively. On the other hand, a quasi-spherical morphology with no definite branching and a size of ~ 67 nm was noted in case of gold nanostructure synthesized with albumin at pH 5.3 as shown in Figure 2E. The highly crystalline lattice structure of gold as well as halo layer of albumin of all the three cases can be seen in high magnification images (Figure 2B, 2D, 2F). However, a quasi-spherical morphology and size of ~ 55-75 nm was 12 ACS Paragon Plus Environment

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observed in gold nanostructure formed in the absence of albumin with not much of a variation in all the three pH conditions as seen in Figure 3. This confirms the crucial role played by pH and albumin in the generation of star-shaped branched morphology in gold nanostructures. Albumin contains both acidic and basic functional groups and the charge on the molecule basically depends on its isoelectric point (pI) as well as pH of the surrounding solution. When pH of the solution is less than pI, protonation of the acidic (R-COO¯ →R-COOH) and basic groups (R-NH2→R-NH3+) result in a net positive charge and deprotonation occurring at pH higher than pI results in a net negative charge on the molecule.41,42 This was further confirmed with zeta potential analysis at different pH, wherein, BSA exhibited a zeta potential value of +12 ± 0.15 mV, +20 ± 0.32 mV and -10 ± 0.42 mV at pH 2.5, pH 4 and pH 5.3 respectively. Gold chloride (HAuCl4) undergoes pH-dependent hydrolysis and results in its speciation. It exists predominantly in the planar form AuCl4¯ at pH ≤ 4, and as [AuCl3(OH)]− from pH above 4 to 6 and is the gold species which is preferred for the reduction.43 BSA with an isoelectric point of 4.7 is positively charged at both pH 2.5 and pH 4 of the reaction solution and hence AuCl4¯ ions get electrostatically attached to albumin. However, at pH 5.3 (above pI), albumin is negatively charged resulting in low binding of chloroaurate ions. Subsequently, the addition of reducing agent (ascorbic acid) reduces the chloroaurate ions attached with albumin to gold atoms which further acts as a nucleus for growth. The isotropic growth of nanostructures is hypothesized to be prevented by chloride ions released during reduction of chloroaurate ions to Au as halide ions are reported to affect the growth and formation of gold nanoparticles.10 The amount of ascorbic acid was also optimized to produce gold nanostructures of desired absorbance and morphology. The ratio of ascorbic acid to HAuCl4 was required to be higher than 3:2 (i.e., 1.5:1) to form branched morphology with absorbance in the NIR region because of donation of two electrons from one 13 ACS Paragon Plus Environment


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molecule of ascorbic acid to Au3+ in AuCl4¯ for its complete reduction as detailed in previous studies.11 Albumin is also reported to undergo pH-dependent structural transitions to N (Normal), F (Fast) and E (Expanded) conformations at pH 4.5 to 7.0, 4, and less than 3, respectively.41 The N form of albumin is globular, whereas in F-state it gets partly opened with a considerable loss in helical content.44 E form results due to further expansion with a loss of intra-domain helices.45 As the pH of the solution decreases, α helices were stretched to a greater extent and transformed into β sheets (analyzed in the ensuing CD spectroscopic studies), which shapes a conformation suited for the oriented growth of gold nanostructures as well as easy access to functional groups for attachment. Hence at pH 5.3, the negatively charged albumin in its globular (N-normal) form prevents the attachment of negatively charged chloroaurate ions and its growth to proper branched morphology as seen in Figure 2E. However, pH 2.5 and pH 4 provide an albumin template structure of cationic in nature for the attachment of AuCl4¯ ions as well as its expanded conformation helps in directional growth to give out spikes and evolve into star morphology as schematically depicted in Figure 4. Though both pH 2.5 and 4 give rise to gold nanostars, the nanostructures formed at pH 4 is preferred for biological application because of the near similar conformation of albumin to physiological conditions in comparison to pH 2.5 and its NIR absorbance with SPR λmax at 808 nm. Therefore, this greenish blue colored albumin derived gold nanostars synthesized at pH 4 with SPR λmax at 808 nm are investigated in detail and are henceforth known as A-GNS. The EDAX spectra of A-GNS as seen in supporting information, Figure S1 depicted the presence of gold as well as carbon and oxygen of albumin along with copper (from copper grid used for imaging). The crystallinity of A-GNS was studied by subjecting it to X-ray diffraction analysis, which displayed all the peaks characteristic of metallic 14 ACS Paragon Plus Environment

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gold as shown in supporting information, Figure S2 in accordance with the JCPDS 01-089-3697 database. The broadening of peaks in the XRD spectra occurs due to the nanocrystallite size of A-GNS. Besides discerning the presence of albumin in TEM images of Figure 2D, the amount of albumin in A-GNS was also quantified to be 16 ± 4 % using CHN analysis. The amount of albumin was also quantified with thermogravimetric analysis (TGA), wherein both BSA and A-GNS showed a similar two step decomposition profile as depicted in Figure 5A. The thermogravimetric profile showed an initial loss of weight from 25 °C to 190 °C due to the bound water, however the decomposition profile of BSA at this stage was found to be more abrupt than A-GNS which may be attributed to the denaturation of protein. The second stage of decomposition from 190 °C showed a slow degradation till 280 °C and thereafter a rapid decline in weight was noted owing to the loss of small molecules. The second stage of decomposition solely attributed to protein content is used to determine the weight loss for quantification of albumin. BSA displayed a weight loss of 69.9 % (88.9-19) during the second stage of decomposition whereas, only a 12 % (90.9-78.9) loss was noted in case of A-GNS which accounted to 17.17 % of albumin in A-GNS. Hence, albumin content of ~ 16-17% was observed in A-GNS as determined by both CHN and thermogravimetric analysis. The mechanism of interaction of albumin with gold in A-GNS was analyzed using FTIR and similarity in bands of both spectra as displayed in Figure 5B confirms the presence of albumin in A-GNS. The spectra revealed strong absorption band ranging from 3000-3500 cm-1 corresponding to O–H bending vibrations in case of both BSA (albumin) and A-GNS. Similarly, peak in the region of 1700-1600 and 1,453 cm-1 corresponding to C=O stretching (amide I) and



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bending CH2 modes, respectively could be seen in both albumin as well as A-GNS.46 The various weak bands of C–O, C–C–O and C–N vibrations of the protein structure were observed in the spectra of both albumin and A-GNS.46 However, absorption band in the region of 3,310, 3,077 cm-1 attributed to symmetric and antisymmetric vibrational modes of primary NH2 and 1600-1500 cm-1 due to N-H bending modes present in albumin were absent in A-GNS. This indicated the interaction of albumin to gold nanostructure through its amine group. The amide I region is able to depict various changes in protein conformation. The varying secondary structure changes the orientation of amide bond and protein backbone bringing about variation in vibrational frequencies. The peak at 1653 cm-1 present in the spectra of albumin indicate the predominant secondary structure, i.e. the α-helix form which is shifted to 1640 cm-1 in A-GNS suggesting a less compact structure which is more random and having open chains due to the effect of lower pH.47 The changes in secondary structure were further evaluated using CD spectroscopic studies as shown in Figure 5C and 5D. BSA showed a predominant α-helix conformation (with two distinct peak minima: 205–208 nm and 222 nm) as seen in Figure 5C. However, the precursor solutions with decrease in pH demonstrated a decrease in peak minima implying a decrease in the helicity. A-GNS displayed a spectrum with the peak minima shifted to 216 nm suggesting a predominance of β-sheet content (Figure 5C and supporting information, Figure S3). The analysis of CD spectra by CDPro software revealed the relative quantity of each secondary structure in samples as tabulated in Figure 5D. BSA depicted a helix rich structure with ~ 57.15%, 17.75%, 25.1% of α-helix, β-sheet (strands + turns) and unordered structure respectively. The precursor solutions with decrease in pH displayed a decrease in α-helix and increase in β-sheet and unordered structures (Figure 5D) demonstrating the effect of pH on 16 ACS Paragon Plus Environment

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protein during the reaction. A marked loss of helicity with predominant β-sheet (56.3%) and unordered (41.2%) structures was noted in A-GNS. The increase in β-sheet and the unordered structure in A-GNS in comparison to its precursor solution (BSA+ HAuCl4, pH 4) are mainly due to the effect of ascorbic acid used further for reduction which results in an additional decrease in pH. This confirms that the protein present in A-GNS are less compact than the helix rich BSA. A-GNS would be an ideal photothermal agent because of its branched morphology with SPR λmax at 808 nm; hence its efficiency was analyzed by recording the temperature increment of solution upon laser irradiation. Figure 5E depicts the temperature increment obtained by A-GNS at different concentration of gold upon laser irradiation for varying time duration. A-GNS (100 µg ml-1 of gold) could raise the temperature up to 43 °C (critical temperature for tumor ablation) within 4 min whereas the temperature of water was 38 °C even after 10 min of laser irradiation as graphically depicted in Figure 5E. The temperature increment was found to increase with increasing gold concentration of A-GNS when exposed to laser for definite time period (7.5 min) as displayed in supporting information, Figure S4. The photothermal transduction efficiency of A-GNS was found to be 66 ± 3% calculated according to equations described in previous studies.12,37 Similarly, the specific adsorption rate of A-GNS was calculated to be 6.2 kW g-1 for 1.3 Wcm-2 of laser power with no change in the absorbance values even after irradiation suggesting its photothermal stability. Additionally, for the potential use of A-GNS as CT contrast agent, imaging was carried out with CT Scanner at 200 mA, 100 KVp and compared with Omnipaque, a clinically approved agent at different concentrations. In comparison to Omnipaque, a brighter contrast was visualized for AGNS at concentrations above 0.5 mg ml-1 which were also quantified in Hounsfield units as seen 17 ACS Paragon Plus Environment


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in Figure 5F and 5G. The signal intensity which is two times higher than Omnipaque was observed in case of A-GNS due to the high Z number of gold which results in enhanced attenuation of X-rays and superior contrast with lesser material thus reducing toxicity issues. The biocompatibility of A-GNS needs to be evaluated for its use in biological applications. Hence, an Alamar blue assay wherein, resazurin, a blue fluorescent dye is reduced to resorufin (a fluorescent red colored compound) by viable cells was performed on L929, NIH3T3 and KB cells. The analysis as depicted in Figure 6A showed more than 80 percentage of cells to be viable even at high concentrations of A-GNS such as 200 µg ml-1. The cells were found to be metabolically active with no change in its morphology. KB cells were found to uptake ~ 85% of A-GNS when treated for 24 h which was evaluated quantitatively using ICP analysis after digestion of cells. The pellet of A-GNS treated cells were also subjected to CT imaging which exhibited a brighter contrast compared to untreated cells (negative control) implying high uptake as shown in Figure 6B. Prior to photothermal evaluation of A-GNS, any interference due to the generation of reactive oxygen species in KB cells upon interaction with A-GNS needs to be evaluated. Hence, an ROS assay was employed wherein a cell permeable molecule namely 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) gets converted to fluorescent 2′,7′-dichlorofluorescein in the presence of ROS and analyzed using flow cytometry. The percentage of cells showing the intracellular generation of ROS can be seen in Figure 7 (A-C) where, no significant levels of ROS was exhibited in cells treated with A-GNS ( 0.3%) similar to the untreated cells (negative control, 0.3%). However, cells treated with H2O2 (positive control) showed very high ROS activity of 98.9%. The absence of ROS generation in cells treated with A-GNS may be attributed to the ROS scavenging property of albumin present in nanostructures. 18 ACS Paragon Plus Environment

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Conventional methods of synthesizing gold nanostars such as those using silver nitrate and surface coated with CTAB or PEG are reported to exhibit increased ROS production resulting in marked cytotoxic reaction in both cancerous (glioblastoma) as well as normal (human dermal fibroblast) cells even when treated for 4 h.48 However, A-GNS were found to be extremely compatible with both normal and cancer cells with no generation of reactive oxygen species even at high concentrations. Hence, protein mediated synthesis of gold nanostars is a better alternative to conventional methods for biological use. The photothermal efficacy of A-GNS on oral epithelial carcinoma cells (KB) was evaluated by subjecting the cells to various modes of treatment and quantified using Alamar blue assay as described in the experimental section. As shown in Figure 7D, cells treated with A-GNS when irradiated with laser exhibited marked cell death and the cytotoxic reaction increased with increasing duration of laser exposure when compared to untreated cells exposed to the laser. This was also confirmed qualitatively by staining the cells after treatment with propidium iodide dye showing the dead cells as red under a fluorescent microscope. The DIC, fluorescent and merged images of KB cells subjected to various modes of treatment are shown in left, middle and right columns in Figure 8, respectively. The untreated cells serving as the negative control and cells treated with A-GNS (100 µg ml-1), and only with the laser for 7.5 min did not show any red colored staining with PI, implying no cell death. However, cells subjected to laser exposure for 7.5 min after treatment with A-GNS (100 µg ml-1) displayed significant cell death with a large number of cells stained red with PI. The red staining of cells due to uptake of propidium iodide implied cell death via necrosis. However, cells treated with A-GNS when subjected to low power (500 mWcm-2) laser irradiation at multiple pulsed excitations displayed generation of reactive oxygen species as shown by H2DCFDA analysis using flow cytometry (supporting information, 19 ACS Paragon Plus Environment


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Figure S5) suggestive of apoptotic mediated cell death. The percentage of cells which showed intracellular generation of ROS upon only laser irradiation (multiple pulsed excitations) was found to be 1.3% (supporting information, Figure S5A) whereas, cells treated with A-GNS and exposed to laser (multiple pulsed excitations) showed very high ROS activity of 83.3% (supporting information, Figure S5B). The necrotic cells formed after treatment of cells with AGNS and high power laser (1.3 Wcm-2, 7.5 min) were found to be merging with debris in the cytogram. These analyses demonstrate the cell compatibility and photothermal efficacy of A-GNS; nevertheless, for its use as an agent for tumor ablation in the body, its interaction with physiologic components such as blood becomes mandatory. Hence, compatibility studies of AGNS on blood were carried out as per national health guidelines. Hemolysis assay was performed to analyze the hemolytic characteristic of nanostructures by estimating the amount of free hemoglobin (Hb) released on lysis of red blood cell (RBC). The percentage hemolysis of blood on treatment with different concentrations (10, 25, 50, 100 and 200 µg ml-1) of A-GNS was analyzed using a spectrophotometer as described in experimental section. Blood treated even with very high concentrations such as 200 µg ml-1 did not exhibit any noticeable hemolysis similar to the negative control (0.9% saline) as shown in Figure 9A which was also substantiated with clear plasma supernatant seen in the photographic image displayed as the inset of Figure 9A. However, cells treated with Triton X-100 (1%) serving as positive control exhibited extensive hemolysis amounting to ~ 87 % which is also evident by the leakage of hemoglobin into plasma supernatant turning it to bright red in color. These results were also confirmed by the morphological evaluation of RBC under an optical microscope after Leishman staining. The blood treated with both 0.9% saline (negative control) and A-GNS exhibited intact morphology 20 ACS Paragon Plus Environment

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of the undamaged RBC, whereas, positive control, Triton X-100 (1%) resulted in ruptured and aggregated form of RBC as shown in Figure 9B. These studies hence demonstrate the nonhemolytic and blood compatible nature of albumin derived gold nanostars. This study utilizes bovine serum albumin as a protein model to synthesize biocompatible gold nanostars, and it opens up possibilities of this kind of green synthetic strategies using other animal and plant proteins as well with due consideration to the isoelectric point of the protein in use. CONCLUSION Anisotropic nanostructures such as gold nanostars with their outstanding photothermal characteristics are usually synthesized by complex methodologies and toxic precursors. This study hence puts forth a rapid one-pot method of synthesis of gold nanostars using albumin molecule as a template. Moreover, the non-toxic albumin with its ligand binding property provided biocompatibility, stability and functionality to the gold nanostars. These albumin derived gold nanostars with SPR λmax at 808 nm exhibited profuse hyperthermia and superior CT contrast characteristics. The extreme compatibility displayed by these gold nanostars towards L929, NIH 3T3, KB cells and human blood as well as pronounced photothermal cytotoxicity on KB cells effectuate an ideal therapeutic agent. Thus, this study reports a simple one-pot methodology to rapidly develop gold nanostars from protein molecule as a template and demonstrate its use as a dual CT diagnostic and photothermal therapeutic agent. Note: All experiments were performed in triplicates unless stated otherwise. ASSOCIATED CONTENT



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Supporting Information EDAX, XRD, CD spectra of A-GNS, temperature change data in photothermal transduction experiment of A-GNS at different gold concentrations and ROS assay flow cytogram of photothermal treatment. ACKNOWLEDGMENTS Authors are grateful to Nanavati Super Speciality Hospital, Mumbai for providing help in CT imaging of samples. Authors also acknowledge sophisticated analytical instrument facility (SAIF), IRCC and Department of Physics, IIT Bombay for all the characterization facilities. REFERENCES (1)

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Figures

Figure 1. Absorbance spectra of gold nanostructures synthesized at different pH (A) with albumin and (B) in the absence of albumin. Inset shows the photograph of respective gold nanostructure suspensions. (C) Hyperspectral dark field optical image and (D) scattering spectrum of gold nanostructures synthesized at pH 4 having SPR λmax at 808 nm.


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Figure 2. FEG-TEM images of gold nanostructures synthesized with albumin at (A-B) pH 2.5, (C-D) pH 4, (E-F) pH 5.3. Right column shows the high magnification images of the respective nanostructures. Inset in the left column shows the low magnification image, size



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distribution histogram and right column shows SAED pattern of respective gold nanostructures.


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Figure 3. FEG-TEM images of gold nanostructures synthesized without albumin at (A-B) pH 2.5, (C-D) pH 4, (E-F) pH 5.3. Right column shows the high magnification images of the respective nanostructures. Inset in the left column shows the low magnification image, size distribution histogram and right column shows SAED pattern of respective gold nanostructures.

Figure 4. Schematic representation of formation of Albumin derived gold nanostars (AGNS). Albumin (BSA) molecules become cationic at a pH lower than its isoelectric point (pI) and AuCl4¯ ions attach to the same and get reduced to form small nuclei which grow to gold nanostars.



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Figure 5. (A) TGA curves of albumin (BSA) and A-GNS showing thermal decomposition, (B) FTIR spectra of albumin (BSA) and A-GNS, (C) CD spectra of BSA, precursor solutions (BSA along with HAuCl4 at specified pH 2.5, 4 and 5.3) and AGNS, (D) Tabulated data specifying the content of different secondary structure of protein on evaluation with 34 ACS Paragon Plus Environment

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CDPro software, (E) graphical representation of temperature increment in photothermal transduction experiment of A-GNS at different gold concentrations and water (H2O) upon exposure to 808 nm laser (1.3 Wcm-2) for different time duration, (F) CT contrast images of a) Omnipaque and b) A-GNS at varying concentrations, (G) graphical representation of computed Hounsfield unit values of Omnipaque and A-GNS from the CT contrast images.

Figure 6. (A) Graphical representations of cell viability tests on NIH 3T3, L929 and KB cells treated with A-GNS at varying concentrations for 24 h. NC and PC represent cells treated with only media and 1% Triton X-100, respectively. (B) CT contrast images (pseudo colored) of cell pellets demonstrating uptake of nanoparticles by KB cells upon treatment for 24 h with media alone (negative control) and A-GNS.



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Figure 7. Flow cytogram depicting generation of ROS with DCFH-DA assay in KB cells treated with (A) media alone (negative control), (B) A-GNS (100 µg ml-1) and (C) 30 µM H2O2 (positive control). (D) Graphical representation of in vitro photothermal study depicting cell viability of KB cells treated with A-GNS (P) and laser (L) for different time duration. NC-negative control (untreated cells) and PC - positive control (cells treated with 1% Triton X-100).


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Figure 8. Microscopic images of KB cells after photothermal experiment, upon treatment with (A−C) only media; (D−F) only laser for 7.5 min; (G−I) A-GNS; and (J−L) A-GNS + laser for 7.5 min. Left and middle column exhibit the DIC and fluorescent (dead cells in red) images of KB cell, respectively. Right column shows the merged images of left and middle columns.



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Figure 9. (A) Graphical representation of hemolytic study of A-GNS using human blood. Inset shows representative photograph of blood samples treated with different concentrations of A-GNS showing no hemolysis (clear plasma supernatant) similar to NC negative control (0.9% saline) whereas, positive control (blood sample treated with 1% Triton X-100) shows leakage of hemoglobin (red colored plasma). (B) Optical microscope images of RBC treated with 0.9% saline (negative control), A-GNS (200 µg ml-1) and 1% Triton X-100 (positive control) and subsequent Leishman staining.


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TABLE OF CONTENTS (TOC) GRAPHIC

Synopsis: A rapid, one-pot method is developed to synthesize gold nanostars using protein which otherwise are synthesized conventionally using toxic precursors and cumbersome methodologies.


Rapid, One-Pot, Protein-Mediated Green Synthesis of Gold Nanostars

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