Protein-Poly(amino acid) NanocoreâShell Mediated Synthesis of

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Protein-poly(amino acid) nanocore-shell mediated synthesis of branched gold nanostructures for CT imaging and photothermal therapy of cancer Sisini Sasidharan, Dhirendra Bahadur, and Rohit Srivastava ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03428 • Publication Date (Web): 31 May 2016 Downloaded from http://pubs.acs.org on June 1, 2016

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ACS Applied Materials & Interfaces

Protein-poly(amino acid) nanocore-shell mediated synthesis of branched gold nanostructures for CT imaging and photothermal therapy of cancer Sisini Sasidharan,a Dhirendra Bahadurb* and Rohit Srivastavaa* a. Department of Bioscience and Bioengineering, IIT Bombay, Powai, Mumbai, 400076, India. b. Department of Metallurgical Engineering and Materials Science, IIT Bombay, Powai, Mumbai, 400076, India. KEYWORDS: albumin nanoparticles, poly-l-arginine, branched gold nanoparticles, CT imaging, photothermal therapy, cancer

ABSTRACT Anisotropic noble metal nanoparticles especially branched gold nanoparticles with large absorption cross-section and high molar extinction coefficient have promising applications in biomedical field. However, sophisticated and cumbersome methodologies of synthesis along with toxic precursors pose serious concern for its use. Herein, we report the synthesis of branched gold nanostructures from protein (albumin) nanoparticles by a simple reduction method. Albumin nanoparticles were synthesized by a modified desolvation technique with polyL-arginine (cationic poly amino acid) substituting the conventional toxic crosslinker

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glutaraldehyde. In silico molecular docking was carried out to study the interaction of poly-Larginine with albumin which revealed its binding to Pocket 1B of A-chain of albumin. The polyL-arginine-albumin core-shell nanoparticles of ~ 100 nm in size served as a base for attachment of gold ions and its reduction to form 140 nm sized branched gold nanostructures conjugated with glutathione. These gold nanostructures exhibited near infrared absorption λmax at 800 nm with extreme compatibility towards non cancerous (NIH 3T3), oral epithelial carcinoma (KB) cell lines and human blood (red blood cells, platelets and coagulation mechanisms) even upto a high concentration of 250 µg/ml. These structures demonstrated superior computed tomographic (CT) contrast ability and marked photothermal cytotoxicity on KB cells. This study reports for the first time a method to develop blood and cell compatible branched gold nanostructures from protein nanoparticles as a dual CT diagnostic and photothermal therapeutic agent.

1. INTRODUCTION Advancement of nanotechnology in the field of medicine, especially in cancer diagnosis and therapy has been tremendous in the past decade. There has been a significant contribution in the field of non invasive imaging such as quantum dots for fluorescence,1,2 superparamagnetic iron oxide nanoparticles (SPIONS) for magnetic resonance3,4 and gold nanoparticles for computed tomographic (CT) imaging.5,6 Similarly, polymeric nanoparticles in drug delivery,7–9 SPIONS for magnetic hyperthermia10–12 and noble metal nanoparticles for photothermal13–16 and radiotherapy17–19 have been promising on the therapeutic front. Noble metal nanostructures especially gold have been immensely explored as CT contrast agent owing to its exceptional stability against oxidation, higher Z-number, absorption coefficient6 and as photothermal agents because of its unique surface plasmon resonance (SPR) property.20 Gold nanoparticles with


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branched morphology widely known as branched gold nanostructures and sometimes named as flowers,21,22 urchins23 or stars24 exhibit excellent photothermal effects due to its larger absorption cross-section and high molar extinction coefficient. The presence of sharp and elongated branches results in efficient heating in comparison to massive nanostructures as the incoming electric field penetrates easily the thin structure resulting in the heating up of entire gold matter.25,26 Additionally, their irregular shape and very high surface to volume ratio drastically enhances heat production.25–27 Because of their peculiar geometry, these structures offer several other applications such as ultrasensitive detection by surface-enhanced Raman scattering (SERS),28–30 two-photon photoluminescence (TPL) imaging24 and photodynamic therapy.31 The branched morphology in gold nanoparticles can be achieved by means of either seed mediated or seedless method of synthesis.32 The growth process of nanoparticles in seedless method is aggregation based and synthesis is highly sensitive to experimental conditions, leading to difficulty in controlling the morphology and size of nanoparticles. Capping agents such as CTAB helps in arresting the growth of nanoparticles to achieve suitable morphology, however toxicity issues prevail.33 Alternatively, in seeded method, preformed seeds form nucleation centres and additional gold is deposited on its surface for particle growth, resulting in monodispersed particles.34 A number of researchers have reported the synthesis of branched gold nanoparticles by a seed mediated approach with noble metal nanoparticles as seed but with toxic materials such as CTAB,35,36 SDS37 as surfactant or N, N-dimethylformamide (DMF)38,39 as an organic solvent and hydroquinone40 as reducing or capping agent. Moreover, these nanoparticles lack stability at physiological conditions and absence of functional end groups for covalent binding to biomolecules hinders their application in active targeting of tumor tissues. To overcome these issues, we have developed a novel method of synthesis for branched gold


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nanoparticles with protein (albumin) nanoparticles as core and functionalized it with biomolecules such as glutathione. Albumin nanoparticles apart from being non-toxic, nonimmunogenic, is cheap and easy to synthesize at a large scale making it an ideal choice for nanoseed preparation.41,42 However for its use, slightly cationic surface is mandatory for gold attachment and further reduction. Hence, we propose for the first time the use of polycationic amino acid poly-L-arginine (PLA) to synthesize albumin nanoparticles which not only provides the much required cationic surface but also avoids toxic glutaraldehyde as crosslinker for formation of stable nanoparticles. The highly cytofriendly and hemocompatible glutathione functionalized branched gold nanoparticles (BGNs) formed from PLA coated albumin nanoparticles (PLA-Alb NPs) are first of its kind and were used as photothermal and CT contrast agent against KB cells. The high photothermal transduction efficiency, biocompatibility and CT contrast ability of BGNs suggest its potential use as multifunctional theragnostic agent for cancer. 2. MATERIALS AND METHODS 2.1 Materials Bovine serum albumin, poly-L-arginine (mol. wt 5000-15000) and adenosine diphosphate (ADP) were purchased from Sigma Aldrich, sodium chloride (NaCl) from Merck, tetrachloroauric acid (HAuCl4) from Spectrochem, ascorbic acid and reduced glutathione from SD Fine, India. Oral epithelial carcinoma cells (KB) and normal mouse embryonic fibroblast cells (NIH 3T3) were procured from the National Centre for Cell Science, Pune, India. All the chemicals were used as procured without any additional processing or purification. Glasswares and magnetic stir bars were washed thoroughly with aqua regia before use.


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2.2 In silico molecular docking The two dimensional structure of PLA (trimer) was obtained from Pubchem substance (CID 439610), modeled into three dimensional structure using Viewerlite 5.0 software and energy minimized using YASARA energy minimization server43. The X-ray crystallographic structure of Bovine Serum Albumin (BSA) was retrieved from Protein Data Bank (PDB- PDB entry: 3V03). The protein structure was curated using AutoDock tools (ADT) by removal of water molecules and addition of polar hydrogen atoms. The resultant protein structure was utilized for docking simulations by keeping the essential solvation parameters of ADT at default. Grid Box was used to set the grid map (40 x 40 x 40) for BSA. The ligand-protein docking was performed using AutoDock Vina 1.1.2 molecular docking software44 and the results were viewed and analyzed using Accelrys Discovery Studio Visualizer 3.5 (Accelrys Software Inc.) as well as LigPlot+ v.1.4.5. 2.3 Synthesis of poly-L-arginine-albumin core shell nanoparticles (PLA-Alb NPs) Poly-L-arginine-albumin core shell nanoparticles were synthesized by a modified coacervation technique using sodium chloride and ethanol. In a typical experiment, 10 mM NaCl solution was added to 10 mg/ml of albumin solution and stirred for 2-3 min. To this solution, ethanol was added drop-wise at a constant rate for phase separation of albumin in a 6:1 ratio. The solution was stirred for 2 h resulting in formation of white turbid solution. Various concentration of polyL-arginine was then added to reactant mixture to optimize the amount of crosslinker required to stabilize the formed nanoparticles. The formed particles were then centrifuged, washed three times and stored at 4 °C.


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2.4 Synthesis of glutathione conjugated branched gold nanoparticles with poly-L-arginine albumin core-shell nanoseed Briefly, 100 µl of PLA-Alb NPs were added to 5 mM of HAuCl4 solution and stirred for 2-3 min. To this solution 100 mM ascorbic acid was added to yield a brownish-blue solution of branched gold nanoparticles. Different concentration of HAuCl4 and ascorbic acid was used to optimize the formation of BGNs with desired absorbance. The solution was centrifuged, washed three times and pellet was redispersed in deionized water. To 3 mM of glutathione solution, the formed branched gold nanoparticles were added and incubated at 37 °C for 12 h. The glutathione conjugated branched gold nanoparticles (BGNs) solution was centrifuged, pellet was washed, redispersed in deionized water and stored at 4 °C for further characterization.

2.5 Characterization The nanoparticles were subjected to the following characterization techniques. The size and stability of PLA-Alb NPs and BGNs dispersed in water and buffer solutions were characterized by Dynamic Light Scattering (DLS) and zeta potential analyzer (Delta Nano C Particle Analyzer, Beckman Coulter, USA). These analyses were carried out in triplicates. The formation and surface plasmon resonance (SPR) characteristics of BGNs were confirmed by spectrophotometric studies with Perkin Elmer Spectrophotometer (Lambda 25) using quartz cuvette of 1ml at a path length of 1cm. Hyperspectral imaging of BGNs were also performed using the CytoViva 150 Unit integrated onto the Olympus BX43 microscope. For the same, a diluted drop of BGNs solution was placed and spread out on a clean microscopic slide. A coverslip was placed on the dried slide before acquisition of images. Dagexcel X16 camera was used to aquire images and hyperspectral Imaging (HSI) was carried out at 60 X magnification


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with the HSI System 1.1 and ENVI software. Size and surface morphology of the samples were evaluated by FEG-SEM (JSM-7600F, JEOL) for which dilute concentrations of samples were mounted on stubs, air-dried and then sputter coated with gold for imaging. Imaging was carried out with acceleration voltage of 10 kV and working distance of 6 mm. The shape, size, lattice structure were also characterized by FEG-TEM (JEM 2100-F, JEOL) operating at 200 kV. The samples were prepared by placing a diluted drop on a carbon coated copper grid. TEM imaging were carried out at various magnifications and Selected Area Electron Diffraction pattern (SAED) was studied. The elemental composition of nanoparticles especially gold was evaluated using EDAX analysis. The phase purity and crystallinity of BGNs were studied from X-ray diffraction (XRD) measurements by recording the spectrum in the range of 5°–85° using a Philips X’PERT PRO powder diffractometer with a Cu-Kα source (λ = 1.54056 Å). Phase identification was carried out using the standard JCPDS database. Fourier transform infrared (FTIR) spectra over a frequency range of 4000 to 400 cm-1 was recorded to analyze functional groups on the samples using Fourier Transform infrared Spectrometer (MAGNA 550, Nicolet Instruments Corporation, USA) with KBr method (2 mg of sample was mixed with 175 mg KBr and compressed into pellets). Photothermal effect of BGNs was evaluated with the help of 808 nm NIR laser (500 mW -PMC, India). Typically, 200 µl of BGNs (100 µg/ml gold concentration) and control solutions (water and PLA-Alb NPs) were added in a 96 well plate in such a way that the solutions are separated from each other to avoid heat transfer. The plate was floated on a water bath at 37 °C and the laser was passed through the wells containing sample and control solutions. The temperature increment was recorded at 0, 1, 2.5, 5, 7.5, 10 and 15 min with the help of a digital thermometer.


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All the experiments were carried out in triplicates and the temperature increment was plotted with 37 °C as baseline. To analyze BGNs as CT contrast, clinically used 64-slice cardiac capable PET-CT Scanner (Biograph mCT-Molecular CT, SIEMENS) was used. BGNs of different concentrations were placed into 96-well plates with Omnipaque (clinically approved CT contrast) as reference and water as negative control. A tube current of 200 µA, tube voltage of 100 kVp, scan time of 6 s, slice thickness of 1 mm, and rotation time of 1 s were used to determine the contrast of gold nanoparticles. Regions of interest of equal diameter were selected manually for each well and Hounsfield units were determined to compare X ray contrast using ImageJ 1.48 software. 3. BIOLOGICAL STUDIES 3.1 Cell culture studies Cell lines were cultured in a T25 flask in Dulbecco’s modified Eagles Medium (DMEM) (HiMedia, India) with 10% fetal bovine serum (FBS, Himedia, India), 50 IU/ml penicillin and 50 µg/ml streptomycin (HiMedia, India). The cells were maintained at 37 °C in a humidified environment of 5% CO2. 3.1.1 Biocompatibility assay A colorimetric MTT assay, based on the reduction of a soluble yellow tetrazolium salt [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)] to purple formazan crystals by viable cells was used to determine the cell viability upon interaction with PLA-Alb NPs. NIH 3T3 and KB cells were seeded at a density of 7 ×103 cells/well into 96-well tissue culture plates and incubated for 24 h in a humidified 5% CO2 incubator at 37 °C. After 24 h of incubation, the


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culture medium was replaced with media containing different concentrations of PLA-Alb NPs (100, 250, 500, 750 and 1000 µg/ml). Cells treated with Triton X-100 (1%) served as positive control, while those maintained in fresh 10% FBS media were used as the negative control. The wells were washed with PBS after 24 h and 10 µl of the MTT stock solution with 90 µl of the medium was added into each well. Upon further incubation for 4 h, 100 µl of the solubilization buffer was added into each well and incubated for an additional 1 h. Percentage cell viability was evaluated by measuring the optical density of the solution using a microplate reader (ThermoScientific, USA) at a wavelength of 570 nm and the cell viability was calculated using the equation:

Percentage Cell viability =

O. D sample X 100 O. D control

where O.D sample and O.D control represent the Optical Density of cells treated with sample and culture medium respectively. To determine the cell viability upon interaction with BGNs, a fluorimetric Alamar blue assay based on the ability of viable cells to reduce resazurin, a weakly fluorescent blue dye to the pink colored and highly red fluorescent resorufin was used. Alamar blue assay was employed to avoid any interference from absorbance by gold nanoparticles. NIH 3T3 and KB cells were trypsinized and seeded into 96-well tissue culture plates at a density of 7 ×103 cells/well. After 24 h of incubation, the culture medium was replaced with basal media containing different concentrations of BGNs (10, 25, 50, 75, 100 and 250 µg/ml). Cells with Triton X-100 (1%) and cells maintained in fresh basal media served as positive and negative control respectively. The wells were washed using PBS after incubation for 24 h and 10% solution of Alamar blue prepared in basal medium was added into each well. After 4 h of incubation, the fluorescence


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intensity was measured using a microplate reader (ThermoScientific, USA) at an excitation of 560 nm and emission of 590 nm and the percentage cell viability was assessed using the following equation:

Percentage Cell viability =

Intensity sample X 100 Intensity control

where, Intensity sample and Intensity control denote the intensity values of cells treated with sample and basal medium respectively. The experiments were carried out in triplicates. 3.1.2 Cell morphology analysis The effect on the morphology of KB cells upon treatment with nanoparticles was visualized using an inverted microscope (Nikon, Japan). Cells seeded in media were treated for 24 h with the highest concentration of nanoparticles (1000 µg/ml of PLA-Alb NPs and 250 µg/ml of BGNs) and microscopical digital live images were recorded for morphological analysis. Cells with only media served as negative control. 3.1.3 Reactive Oxygen Species (ROS) Assay To determine the reactive oxygen species (ROS) levels generated in cells treated with nanoparticles, ROS assay was carried out with 2′,7′-Dichlorodihydrofluorescein diacetate (H2DCFDA), a cell permeable molecule which in the presence of intracellular ROS gets converted to fluorescent 2′,7′-dichlorofluorescein. The levels of ROS generated in cells were analyzed using flow cytometry (BD FACS Aria, USA). Briefly, KB cells were cultured in 6-well plates for 24 h at a density of 105 cells/well. Subsequently, old media was discarded and the cells were treated with media containing 1000 µg/ml of PLA-Alb NPs and 250 µg/ml of BGNs. Cells


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treated with 30 µM of H2O2 served as positive control while media alone was negative control. Following 12 h of treatment, the cells were washed twice with PBS and treated with 10 µM H2DCFDA. The cells were incubated in dark for 30 min, washed with PBS and trypsinized. The cells were resuspended in PBS and analyzed using FACS. 3.1.4 Cell uptake study of BGNs by KB cells KB cells were cultured in 6-well plates at a seeding density of 2 × 105 cells/well for 24 h. These cells were then treated with 250 µg/ml of BGNs for 12 and 24 h in triplicates. Post treatment, the cells were washed twice with PBS to remove unbound particles. The cell pellets obtained by trypsinization were then digested with aqua regia and concentration of gold was analyzed using ICP-AES. Untreated cells and BGNs alone served as controls. The cell pellets were also analyzed by clinically used 64-slice cardiac capable PET-CT Scanner (Biograph mCT-Molecular CT, SIEMENS) with a tube voltage of 100 kVp, tube current of 200 µA, scan time of 6 s, slice thickness of 1 mm, and rotation time of 1 s. 3.1.5 In vitro photothermal therapy on KB cells KB cells were seeded in 96-well plates at a seeding density of 7 ×103 cells/well. Cells were incubated at 37 °C for 24 h. Subsequently, the culture medium was replaced with 100 µl of culture media containing 250 µg/ml BGNs. Cells with Triton X-100 (1%) and those maintained in fresh media were used as the positive and negative control respectively. After incubation for 24 h, the wells were washed using PBS to remove unbound particles. The following treatments were performed on controls; no treatment as negative control, laser alone for 2.5, 5 and 7.5 min. On wells treated with nanoparticles, laser was irradiated for 0, 2.5, 5 and 7.5 min. For qualitative study, cells were then again incubated for 12 h and washed with PBS and treated with propidium


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iodide for dead cell visualization. Cells were observed using a Nikon Eclipse Ti microscope equipped with filters set of excitation/emission wavelengths at 488/617 nm for propidium iodide. For quantitative study, the cells after respective treatment were incubated for 12 h, washed with PBS and Alamar blue assay was carried out to assess the percentage of cell viability. All experiments were conducted in triplicates. 3.2 Blood Compatibility studies The hemocompatibility studies were conducted after institutional ethical clearance and appropriate informed consent. 5 ml of whole blood were drawn from three healthy volunteers (Age: 22-32 yrs) in sodium citrate containing vials. All studies were done immediately after procurement of blood and in triplicates. 3.2.1 Hemolysis assay The hemolytic property of nanoparticles was studied spectrophotometrically by quantifying free hemoglobin (Hb) released into blood plasma after red blood cell (RBC) lysis. 1 ml of freshly drawn blood was treated with 100 µl of different concentration of BGNs (10, 25, 50, 75, 100 and 250 µg/ml) resuspended in 0.9% saline and incubated for 3 h at 37 °C under mild shaking. 1% Triton X-100 and 0.9% saline served as positive and negative controls respectively. Subsequently, the treated blood was centrifuged at 4500 rpm for 10 min and the collected plasma was diluted with 0.01% sodium carbonate. Absorbance of the samples was measured at 415, 380 and 450 nm using a spectrophotometer (M200 Pro Tecan, USA). Amount of plasma hemoglobin was calculated using the following equation,


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Amount of plasma hemoglobin =

mg ! dl

"2 × A%&' ( − "A*+, + A%', ( × 1000 × dilution factor E × 1.655

where, A380, A415, and A450 are the absorbance values at 380, 415, and 450 nm, respectively. A415 is the sorbent band absorption of oxyhemoglobin and A380 and A450 are correction factors applied for uroporphyrin whose absorption falls in the same wavelength range as that of hemoglobin. E is 79.46, the molar absorptivity value of oxyhemoglobin at 415 nm and 1.655 is the correction factor applied due to interference from turbidity of plasma sample. The hemolytic activity of BGNs was plotted as percentage of hemolysis using the following equation:

% Hemolysis =

Plasma hemoglobin value of sample X100 Total hemoglobin value of blood

3.2.2 Platelet aggregation study The NCL Method, ITA-2 Version-1.0 was adopted for the platelet aggregation study. Platelet rich plasma (PRP) was obtained from whole blood by centrifugation for 10 min at 150 g and 22 °C. 800 µl of PRP was treated with different concentration of BGNs at 37 °C for 30 min and platelet count was done in hematology analyser (Sysmex KX-21N, Kobe, Japan). 0.9% saline and 50 mM Adenosine DiPhosphate (ADP) served as negative and positive control respectively. 3.2.3 Plasma coagulation study The effect of BGNs on the extrinsic and intrinsic pathways of blood coagulation is studied by determining the plasma coagulation time measurements such as prothrombin time (PT) and activated partial thromboplastin time (aPTT) respectively. The NCL Method, ITA-12 Version-


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1.0 was followed for the study. Platelet poor plasma (PPP) was obtained from whole blood by centrifugation at 4000 rpm for 15 min at 22 °C. 450 µl of PPP was treated with 50 µl of different concentrations of BGNs for 30 min at 37 °C. 0.9% saline served as negative control and heparin served as positive control for aPTT. The plasma samples were centrifuged at 1000 rpm for 10 min and liquiplastin was added for determining prothrombin time (PT); and activator (phospholipids) and calcium chloride for activated partial thromboplastin time (APTT) studies. Clotting time of plasma samples were measured optically by coagulation analyzer (CoaDATA 501, Labitec, USA). 4.

RESULT AND DISCUSSION

Branched gold nanoparticles with large absorption cross-section and high molar extinction coefficient are ideal choice as photothermal and CT contrast agent. However cumbersome methods of synthesis and toxic precursors are the biggest setbacks. To overcome these issues, albumin nanoparticles were used as template for the synthesis of branched gold nanoparticles. Different methods have been adopted for the synthesis of albumin nanoparticles till date. They can be prepared by desolvation or coacervation,45–47 emulsification,48 thermal gelation,49 nano spray drying,50 nab-technology51 and self-assembly techniques.52 Glutaraldehyde, the conventional cross linker used during synthesis of albumin nanoparticles, though efficient and being widely used, is toxic at high concentrations which calls for the need of an alternative mechanism.53 Additionally, for its use as a template for formation of BGNs, surface of nanoseed needs to be cationic for gold attachment and further reduction. Hence, albumin nanoparticles coated with poly-L-arginine is synthesized. PLA is a cationic poly amino acid whose degradation product is antimicrobial, inhibits tumor growth and enhances drug delivery across membranes.54


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Synthesis of albumin nanoparticle with PLA therefore will not only provide cationic surface and avoid toxicity concern of glutaraldehyde but add medicinal benefits as well. Controlled reduction of gold ions and growth on PLA coated albumin nanoparticles (PLA-Alb NPs) result in the formation of branched gold nanoparticles which is further functionalized with glutathione and used for imaging and photothermal therapy of cancer cells (Figure 1).

Figure 1. Schematic representation of synthesis of glutathione functionalized branched gold nanoparticle (BGN).


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Albumin, owing to its diminished solubility is phase separated on addition of ethanol into the solution; however, the particles not being adequately stabilized can redissolve upon dispersion in water. Controlled dropwise addition of ethanol was mandatory for formation of monodispersed particles of smaller size. PLA addition to the solution provided the much required stability for the particles. The concentration of PLA required for formation of stable nanoparticles was optimized by synthesizing the particles with different concentrations of PLA. Excessive addition of PLA resulted in the formation of large flocculates in the solution. PLA being cationic in nature is hypothesized to react with negatively charged desolvated albumin through its guanidine group (protonated at reaction condition, pKa ≥ 12.5) which was confirmed by in silico docking analysis of PLA with albumin. The ligand protein docking studies performed to analyze the interaction of PLA with bovine serum albumin (BSA) revealed strong interaction with a binding energy of – 9.3 kcal/mol. PLA binds with Pocket 1B of A-chain of BSA by interacting with various amino acids as shown in Figure 2A. PLA shows strong hydrophobic interactions with Glu 125, Glu 140, Lys 132, Lys 136, Phe 133, Leu 122, Ile 141, Leu 115 as shown in supporting information (Figure S1). Additionally, intermolecular interactions and Vander waal’s forces as shown in Figure 2B play a major role in stable interaction of PLA with residues of albumin. The interactions of PLA are further strengthened by formation of classical hydrogen bonds with Asp 129 and Phe 133; non classical hydrogen bonds with Glu 125 and Lys 136; electrostatic bond with Arg 144 and Glu 125 and pi-cation bond with Phe 133 (Figure 2C). The list of complete interaction is shown in supporting information (Table S1).


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Figure 2. A) In silico molecular modeling of interaction between poly-L-arginine (PLA) and bovine serum albumin (BSA), B) Amino acids of BSA and their interaction mechanism with PLA, color of the residues indicates the form of interaction and C) Amino acids of BSA depicting bond formation with PLA. The PLA-Alb NPs were anionic at physiological pH due to the deprotonated state of carboxyl group with zeta potential value of -23 ± 0.3 mV. However, as the carboxyl groups get protonated at acidic pH a charge reversal (40.8 ± 0.45 mV) was observed. The nanoparticles were found to be stable in different media and PBS. PLA-Alb NPs were found to be white opalescent solution (inset of Figure 3) with a hydrodynamic diameter of 142 ± 5 nm as shown by DLS (supporting information Figure S2). The results were further confirmed by FEG-SEM and FEG-TEM images as seen in Figure 3A and B respectively exhibiting particle size of ~ 100 nm with spherical morphology. The TEM image indicates the core-shell structure of the formed nanoparticles with core of ~ 80 nm and shell thickness of 20 nm. These cationic core-shell nanoparticles serve as a base for formation of branched gold nanoparticles.


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Figure 3. A) FEG-SEM (Inset: photographic image and FEG-SEM at lower magnification) and B) FEG-TEM image of PLA-Alb NPs. A proper choice of preformed seeds plays a major role in precise shape and size control of the nanostructures. An increase in size of PLA-Alb NPs results in formation of large sized BGNs, hence the size of the core particle is limited to 100 nm, the smallest possible size which can be obtained by this desolvation technique. Addition of gold chloride solution to PLA-Alb NPs results in charge reversal of particles (negative to positive due to acidic pH) facilitating the attachment of chloroaurate ions (AuCl4¯) to the cationic PLA-Alb NPs. Further, upon addition of reducing agent, viz ascorbic acid, AuCl4¯attached to PLA-Alb NPs is reduced to gold atoms which act as a nucleus for further growth. The formation and growth of gold nanoparticles is reported to be affected by halide ions.55 The presence of chloride ions (released upon reduction of AuCl4¯ to Au) prevents the isotropic growth of gold nanostructures. Hence, the gold coated PLA-Alb NPs grows to give out spikes and result in branched morphology (Figure 1). The factors affecting the formation of BGNs were evaluated by spectrophotometric study and TEM imaging. The formed branched gold nanoparticles were then conjugated to glutathione through its thiol group resulting in formation of well stabilized branched gold nanoparticles (BGNs) which appears brownish-blue in reflected light and bluish black in transmitted light as seen in inset of Figure 4A. The BGNs were subjected to spectrophotometric study and they exhibited a SPR λmax of 800 nm as depicted in Figure 4A. The SPR λmax can be tuned from 600-1000 nm by varying concentration of HAuCl4 (supporting information Figure S3B). A SPR λmax of 800 nm is preferred as it falls in the NIR therapeutic range with less interference from biomolecules present


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in the body. The reduction of gold in the absence of albumin nanoparticles resulted in a purple solution with SPR λmax of 570 nm (supporting information Figure S3A). These particles were very unstable and aggregated immediately which advocates the mandatory requirement of PLAAlb NPs for formation of BGNs. The ratio of HAuCl4 to PLA-Alb NPs (R= [HAuCl4]/[PLAAlb NPs]) plays a crucial role in the formation of gold nanoparticle. Smaller R values resulted in smaller particles with less and shorter spikes as only less number of gold ions are available for reduction and growth. However, at high ratios, ascorbic acid reduces larger number of gold ions and branching develops significantly with formation of more and longer spikes. This is exhibited in the absorbance spectra in supporting information Figure S3B which shows red shift in plasmon spectrum with lower concentration of HAuCl4 attributed to the smaller core size of gold nanostructures.24 The amount of ascorbic acid used in synthesis is crucial for the appropriate reduction and formation of particles with branched morphology. As one molecule of ascorbic acid can donate only two electrons, for complete reduction of Au3+ in AuCl4¯ the ratio of ascorbic acid to HAuCl4 should be higher than 3:2 (ie, 1.5:1). The absorbance profile of gold nanostructures formed by lesser amount of ascorbic acid (ratio < 1.5:1) revealed that very less amount of gold was reduced onto PLA-Alb NPs as absorbance of protein nanoparticles mask that of gold. However, on increasing the amount of ascorbic acid (1.5:1 ratio) more amount of gold was reduced which can be seen as emergence of peak with SPR λmax at 690nm. Further increase in ascorbic acid (2:1 ratio) resulted in accelerated growth of gold nanostructures resulting in red shift with SPR λmax at 800nm (supporting information Figure S3C and 4A). The dark field optical image of BGNs showed a homogeneous dispersion of nanoparticles seen as bright white spots in Figure 4B. The scattering spectra of BGNs as shown in Figure 4C


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display a scattering peak with λmax at 806 nm. The slight change in the spectral profile from the extinction (absorption + scattering) spectra recorded in solution is due to the different dielectric constant of microscopy glass slides and the lack of the absorption component. The hydrodynamic diameter of BGNs were found to be 243 ± 7 nm (supporting information Figure S4) with zeta potential of -24.9 ± 3mV. To evaluate the morphology of anisotropic nanostuctures being formed, FEG-SEM and FEG-TEM imaging were carried out. Synthesis of gold nanostructure in the absence of PLA-Alb NPs resulted in formation of particles with ~ 40 nm in size and spherical-cuboidal morphology as seen in supporting information Figure S5A and S5B. However, anisotropic morphology can be seen in gold nanostructure formed with PLA-Alb NPs and ascorbic acid to HAuCl4 ratio equal to or more than 1.5:1 as shown in supporting information Figure S5C, S5D, S5E and S5F. As shown in supporting information Figure S5E and S5F increase in concentration of ascorbic acid results in reduction of larger number of gold ions and significant development in branching with formation of more and longer spikes. As depicted in Figure 4D and supporting information Figure S5E, S5F, BGNs with SPR λmax at 800 nm formed with 2:1 ratio of ascorbic acid to HAuCl4 exhibited a branched morphology with a size of ~ 140 nm. Sharp and elongated branch of the gold nanostructure is seen in Figure 4E. It is also observed that branching occurs as a result of gradual twinning, hence the nanostructure ultimately comprise a core with many single crystal spikes branching out from the surface. A representative EDAX spectra of the nanostructures as shown in supporting information Figure S6 revealed the presence of gold with no other element other than that associated with copper grid used for imaging. Crystallinity of the gold nanoparticles were analyzed by X-ray diffraction which exhibited all characteristic peaks of metallic gold in accordance to the JCPDS data base as shown in


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supporting information Figure S7. The peak broadening in the XRD pattern implies the small nanocrystallite size of BGNs.


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Figure 4. A) Absorbance spectra of BGNs. Inset shows photographic image of BGNs in i) reflected and ii) transmitted light. B) Dark field optical image of BGNs for hyperspectra, C) Scattering spectra of BGNs obtained by ENVI software, D) FEG-SEM image of BGNs, E) and F) FEG-TEM images of BGNs at various magnifications, Inset of E) shows the SAED pattern of BGNs. The conjugation of GSH to gold nanoparticles was confirmed from the FTIR spectra of reduced glutathione (GSH) and BGNs as depicted in Figure 5A. The spectrum of BGNs exhibited significant change from those of GSH validating the conjugation of glutathione. The characteristic peaks of GSH corresponding to N-H stretch, N-H deformation, S-H and S-S stretch in the region 3000-3300 cm-1, 900-600 cm-1, 2528 cm-1 and 556 cm-1 respectively are absent in the spectra of BGNs suggesting the linking of GSH to gold nanoparticles through its amine and thiol groups.56,57 The broad peak at 3430 cm-1 of BGNs is attributed to H–O–H bending of the residual water molecules physically adsorbed to nanoparticles. The band at 1614 cm-1 corresponding to the carboxylate anion -COO- in GSH was shifted to 1630 cm-1 in BGNs.57,58 Characteristic peaks of the aliphatic region of GSH attributed to C‒H bending vibrations at 2920 cm-1, O–C‒H and C‒O‒H deformation, C–O, C–O–C stretches and C–O–H bends in the region from 1400-1000 cm-1 remained unaffected in the spectra of BGNs.57 The broad peak from 600 to 400 cm−1 is attributed to binding of gold nanoparticles with sulfur from thiol groups of GSH molecules.57 Moderate hyperthermia (41-43 °C) is known to significantly inhibit tumor growth with minimum injury to the surrounding healthy tissues as tumor cells are more thermally sensitive than normal cells because of poor blood supply and irregular vasculature even with sustained angiogenesis.59,60 In order to evaluate BGNs for hyperthermic application, photothermal


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transduction experiments were carried out to study the temperature rise achieved by BGNs upon laser irradiation. It is clearly depicted from the graph seen in Figure 5B that BGNs could achieve the critical temperature of 43 °C for tumor cell death in 7.5 min, however water and PLA-Alb NPs reached only upto 38-39 °C during the same time period. The potential of BGNs as CT contrast agent is evaluated in comparison to polyiodinated aromatic compound Omnipaque, a clinically approved contrast agent as control. Analysis was carried out at equal concentrations of gold and iodine at tube voltage and tube current of 100 kVp and 200 µA respectively. As seen in Figure 5C, visibly brighter contrast is observed for BGNs at concentrations higher than 0.5 mg/ml (gold concentration) in comparison to Omnipaque. This has also been measured quantitatively in Hounsefield units using ImageJ 1.48 software (Figure 5D). The signal intensity of BGNs was found to be approximately 2 times than Omnipaque at same material concentrations owing to the higher Z-number of gold resulting in better attenuation of X rays and enhanced contrast. Superior contrast of BGNs is of great clinical significance as with lesser amount of nanomaterial better contrast can be achieved with limited toxicity issues.


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Figure 5. A) FTIR spectra of glutathione (GSH) and BGNs, B) Graphical representation of temperature increment in photothermal transduction experiment of BGNs, PLA and water (H2O), C) CT contrast images of (A) Omnipaque and (B) BGNs with increasing concentrations, D) Graphical representation of computed hounsefield unit values of Omnipaque and BGNs. To advocate its use for biomedical purpose, PLA-Alb NPs and BGNs need to be non-toxic in nature. Hence these nanoparticles were subjected to biocompatibility analysis to evaluate toxicity and morphological changes to cells. The cell viability analysis (Top panel of Figure 6) of both KB (oral epithelial carcinoma) and NIH 3T3 (normal mouse embryonic fibroblast) cells showed


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more than 80% of cells to be metabolically active and viable even at high concentrations. The cytocompatibility nature of these nanostructures was further confirmed by studying cell morphology after treatment with particles. It is clearly evident from Figure 6 (bottom panel) that KB cells treated with high concentration of particles such as 1000 µg/ml of PLA-Alb NPs and 250 µg/ml of BGNs proliferated well in culture medium and were morphologically intact similar to untreated negative control. These cells did not exhibit any characteristic features of apoptosis proving its cell compatibility nature. The black spots inside cells seen in those treated with BGNs clearly depict its high uptake by tumor cells.

Figure 6. Top panel: Graphical representations of cell viability tests on KB and NIH 3T3 cells treated with A) PLA-Alb NPs and B) BGNs at varying concentration for 24 h. NC represents


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untreated controls, while PC represents cells treated with 1% Triton X-100. Bottom panel: Representative microscopic images of KB cells incubated with media alone (negative control), PLA-Alb NPs (1000 µg/ml) and BGNs (250 µg/ml) for 24 h (magnification: 20X). To further evaluate the cytocompatible nature, generation of intracellular reactive oxygen species was evaluated using ROS assay by flowcytometry with H2DCFDA. Figure 7 shows the percentage of cells with intracellular ROS generation (DCFH-DA +ve). It is clearly evident that when positive control (H2O2 treated cells) showed high ROS activity with 91.3%, the KB cells treated with high concentration of BGNs (250 µg/ml) and PLA-Alb NPs (1000 µg/ml) did not exhibit any significant ROS generation (0.2-0.3%) similar to the untreated cells. This may be attributed to the ROS scavenging property of glutathione linked to branched gold nanoparticles, PLA and proteins. These studies prove the biocompatible nature of PLA-Alb NPs and BGNs on cells with no adverse effects on their metabolic functions.


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Figure 7. Flow cytogram showing intracellular ROS generation using dichlorofluorosceindiacetate (DCFH-DA) assay in KB cells treated with A) media alone (Negative control), B) 30 µM H2O2 (Positive control), C) PLA-Alb NPs (1000 µg/ml) and D) BGNs (250 µg/ml). The significant cellular uptake of BGNs was confirmed by ICP-AES analysis after digestion of KB cells treated with BGNs for 12 and 24 h. The graph as seen in Figure 8A shows around 80% uptake by KB cells in 24 h. Similarly, uptake of BGNs by cells was also evaluated by CT


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imaging and as shown in Figure 8B, cells treated with BGNs exhibited higher contrast in comparison to the untreated cells suggesting high uptake of gold. BGNs were evaluated for its efficacy as photothermal agent in vitro on KB cells with 808nm laser. KB cells were subjected to various modes of treatment and photothermal mediated cytotoxicity were evaluated quantitatively by Alamar blue assay as seen in Figure 8C. Laser irradation after treatment with BGNs results in significant cell death when compared with laser treatment alone. As seen, with increase in duration of laser exposure, the percentage of cells which are viable decreases, hence proving the photothermal efficacy of BGNs in cancer treatment.


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Figure 8. A) Graphical representation of percentage uptake of BGNs by KB cells after 12 and 24 h of incubation. B) CT contrast images (pseudo colored) of KB cells treated with BGNs and media alone (negative control) for 24 h to show uptake of nanoparticles, C) Graphical representations of cell viability on KB cells treated with BGNs (P) and laser (L) for different


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time duration. NC represents untreated controls, while PC represents cells treated with 1% Triton X-100. Similarly, photothermal mediated cytotoxicity on KB cells were analyzed qualitatively by staining with propidium iodide after treatment. Propidium iodide is a membrane impermeant dye and not taken up by the viable cells, however it stains dead cells by binding to DNA and is seen as red under fluorescent microscope. Figure 9 shows the DIC (left column), fluorescent depicting the PI stain (middle column) and merged (right column) images of KB cells with various mode of treatment. Cells serving as negative control (no treatment with BGNs and laser), those treated with BGNs (250 µg/ml) and ones subjected to laser alone for 7.5 min did not exhibit cell death (no cells were stained red with PI), however cells treated with BGNs and irradiated with laser for 7.5 min exhibited marked cell death (cells stained red with PI). These studies prove the photothermal efficacy of BGNs towards cancer cell line.


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Figure 9. Qualitative analysis of photothermal cytotoxicity of KB cells treated with A-C: media alone, D-F: laser alone for 7.5 min, G-I: BGNs and J-L: BGNs +Laser for 7.5 min. Left column depict DIC image, middle images exhibit PI stain (dead cells in red), and right present the merged images of left and middle showing dead cells due to PTT.


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BGNs, a potent photothermal and CT contrast need to be administered either intra-tumoral or intravenous for its potential application. This brings it in contact with blood which warrants the need for hemocompatibility studies. The blood compatibility of nanoparticles was evaluated by analyzing percentage of hemolysis, aggregation of RBC, platelet count and coagulation time as described below. Blood cell morphological analysis too was performed by imaging through optical microscope after Leishman staining. Hemolysis occurs due to rupturing of red blood cells and leakage of hemoglobin. The size, charge, shape as well as composition of nanoparticles play a major role in hemolytic property of RBC. To evaluate the effect of BGNs on RBC, the whole blood was treated with different concentrations (10, 25, 50, 100, 250 µg/ml) of BGNs and percentage hemolysis as well as morphological changes were evaluated. Figure 10A shows blood samples exhibiting no hemolysis even with high concentration (250 µg/ml) of BGNs whereas positive control (1% Triton X-100) exhibited ~ 80% of hemolysis. This is also evident from the photographic image (Inset of Figure 10A) exhibiting clear plasma supernatant and undamaged RBC sediment at the bottom in case of BGNs similar to negative control (0.9% saline) whereas blood treated with triton showed leakage of hemoglobin into plasma supernatant. This was further confirmed with optical microscope imaging of blood with Leishman staining as shown in Figure 10B. RBCs with intact morphology were seen in both negative control and those treated with BGNs, however ruptured and aggregated RBCs were visible in positive control. This illustrates the non hemolytic nature of BGNs. Nanoparticles can trigger coagulation cascade by platelet activation leading to aggregation of platelets. The effect of BGNs on platelets is illustrated in Figure 10C. As can be seen, there is no


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significant platelet aggregation in case of BGNs and negative control, however positive control (ADP) resulted in ~60% aggregated platelets.

Figure 10. A) In vitro hemolysis study on RBC treated with varying concentrations of BGNs. Inset shows photographic image of blood samples treated with varying concentration of BGNs showing no hemolysis whereas, 0.1% Triton X-100 (positive control) treated RBC shows leakage of hemoglobin to the supernatant B) Microscopic images of RBC treated with 0.9 %


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saline (negative control), BGNs (250 µg/ml) and 0.1% Triton X-100 (positive control) and stained with Leishman stain. C) Graphical representation of platelet aggregation (%) of platelet rich plasma treated with varying concentration of BGNs, 0.9% saline serves as negative control and ADP as positive control. To further evaluate the effect of BGNs on intrinsic and extrinsic pathways of coagulation mechanism, prothrombin time (PT) and aPTT were analyzed. Figure 11 shows the effect of BGNs on these coagulation times. As depicted, PT was in the ideal range (10-14 s in case of liquiplastin) for varying concentration of BGNs suggesting its non-thrombogenic nature. Similarly, the INR (International Normalized Ratio) of aPTT for different concentrations of BGNs were in the normal range of 0.8-1.2. Hence BGNs do not interfere with coagulation factors II, V, VII, X, and fibrinogen of extrinsic pathway as well as coagulation factors I, II, V, VIII, IX, X, XI, XII, prekallikrein and high molecular weight kininogen of intrinsic clotting cascade. These studies hence confirm the non-thrombogenic and compatible nature of BGNs towards blood cells.


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Figure 11. Graph depicting A) Prothrombin time analysis and B) Activated partial thromboplastin time (aPTT) ratio of blood samples treated with varying concentrations of BGNs. NC and PC represent negative and positive control respectively and normal range is highlighted with purple colour. A detailed toxicological analysis of BGNs have been carried out at cellular and hematological levels, however, its in vivo interactions, safety and clearance are required to be analyzed for diagnostic as well as therapeutic applications. Size, charge and surface chemistry play a key role in the clearance of any nanostructured material from the body.61 A particle with size below 5 nm would be subjected to rapid renal clearance, limiting its availability at the tumor site.61 Gold nanostructures with a size ranging from 8-37 nm are reported to be toxic on administration in mice and particles above 200 nm are subjected to rapid hepatic as well as splenic filtration.61,62 Size range from 30-200 nm has been considered ideal for accumulation of nanoparticle in tumors through the enhanced permeability and retention (EPR) effect.61,63 BGNs (~140 nm), falling in this ideal size range will be supposedly distributed primarily in liver, spleen and cleared from the body through hepatobiliary mechanism.63,64 However, a detailed investigation to assess its biodistribution, metabolism and elimination is essential to understand its interactions in the body as well as to mitigate any unwanted toxic effects. 5. CONCLUSION Branched gold nanoparticles with uniform size and morphology have been developed till date with noble metal nanoparticles as seed during synthesis. However, lack of functionality and toxicity issues raise huge concern for its use in vivo. This study puts forth a method to synthesize branched gold nanoparticles of ~140 nm in size from poly-L-arginine -albumin nanoparticles.


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The cationic poly amino acid PLA substituted the conventional toxic crosslinker glutaraldehyde in the synthesis of albumin nanoparticles of ~100 nm and additionally provided cationic surface for the development of branched gold nanoparticles. This glutathione functionalized branched gold nanoparticles exhibited a SPR λmax at 800 nm with good photothermal activity and superior CT contrast ability. These gold nanoparticles were extremely compatible towards NIH 3T3, KB cell lines and human blood with marked photothermal cytotoxicity on KB cell line. Thus, this study demonstrated the method to develop branched gold nanostructures from protein nanoparticles and its application as a dual CT diagnostic agent and a photothermal therapeutic agent for biomedical applications. ASSOCIATED CONTENT Supporting Information. Additional interaction data from in silico study, particle size distribution, absorbance spectra, FEG-TEM image, EDAX, XRD of BGNs. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Authors * Rohit Srivastava, Department of Bioscience and Bioengineering, IIT Bombay, Powai, Mumbai, 400076, India. E-mail: [email protected] * Dhirendra Bahadur, Department of Metallurgical Engineering and Materials Science, IIT Bombay, Powai, Mumbai, 400076, India. Email: [email protected] Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes Authors declare no competing financial interest. ACKNOWLEDGMENTS Authors gratefully acknowledge Dev Thacker, IIT Bombay for his help in in silico docking studies. Authors also thank Nanavati Super Speciality Hospital, Mumbai for CT imaging of samples. The facilities provided by sophisticated analytical instrument facility (SAIF), IRCC and Department of Physics, IIT Bombay is also acknowledged. REFERENCES (1)

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