Functionalization of Elongated Tetrahexahedral Au Nanoparticles and

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Functional Inorganic Materials and Devices

Functionalization of elongated tetrahexahedral Au nanoparticles and their antimicrobial activity assay Satya Ranjan Sarker, Shakil Ahmed Polash, Jarryd Boath, Ahmad Esmaielzadeh Kandjani, Arpita Poddar, Chaitali Dekiwadia, Ravi Shukla, Ylias M. Sabri, and Suresh K. Bhargava ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b02279 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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ACS Applied Materials & Interfaces

Functionalization of elongated tetrahexahedral Au nanoparticles and their antimicrobial activity assay Satya Ranjan Sarker,1,2 Shakil Ahmed Polash,2 Jarryd Boath,1 Ahmad E. Kandjani,1 Arpita Poddar,1 Chaitali Dekiwadia,3 Ravi Shukla,1,4 Ylias Sabri1,* and Suresh K. Bhargava1,*

1Centre

for Advanced Materials and Industrial Chemistry (CAMIC), School of Science, RMIT

University, Melbourne 3001, Victoria, Australia 2Department

of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka-

1342, Bangladesh 3RMIT

Microscopy and Microanalysis Facility, RMIT University, Melbourne 3001, Victoria,

Australia 4Ian

Potter NanoBiosensing Facility, NanoBiotechnology Research Laboratory (NBRL), School

of Science, RMIT University, Melbourne 3001, Victoria, Australia

Corresponding author: *E-mail: [email protected]; [email protected]. Phone: +61 3 99252330.

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ABSTRACT Gold nanoparticles are inert for the human body, and therefore, they have been functionalized to provide them with antibacterial properties. Here, elongated tetrahexahedral (ETHH) Au nanoparticles were synthesized, characterized and functionalized with lipoic acid (LA), a natural antioxidant with a terminal carboxylic acid and a dithiolane ring, to generate ETHH-LA Au nanoparticles. The antioxidant activity of Au nanoparticles was investigated in vitro, showing that LA enhances the DPPH free radical scavenging and Fe3+ ion reducing activity of ETHH-LA at higher amounts. The antimicrobial propensities of the nanoparticles were investigated against Gram-positive (B. subtilis) and Gram-negative (E. coli) bacteria through propidium iodide assay as well as disk diffusion assay. ETHH-LA Au nanoparticles showed significantly higher antimicrobial activity against B. subtilis compared to E. coli. Furthermore, ETHH-LA Au nanoparticles also showed significantly better antimicrobial activity against both the bacterial strains when compared to ETHH. ETHH Au nanoparticles also bring about the oxidation of bacterial cell membrane fatty acids and produce lipid peroxides. ETHH-LA showed higher lipid peroxidation potential than that of ETHH against both bacteria tested. The hemolytic potential of Au nanoparticles was investigated using human red blood cells (RBCs) and ETHH-LA showed reduced hemolytic activity than that of ETHH. The cytotoxicity of Au nanoparticles was investigated using human cervical cancer cells, HeLa and ETHH-LA Au nanoparticles showed reduced cytotoxicity than that of ETHH. Taken together, LA enhances the antimicrobial activity of ETHH Au nanoparticles and Au nanoparticles interact with the bacteria through electrostatic interactions as well as hydrophobic interactions and damage the bacterial cell wall followed by oxidation of cell membrane fatty acids. Key words: Au nanoparticles, lipoic acid, antibacterial activity, antioxidant activity, lipid peroxidation. 2 ACS Paragon Plus Environment

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1. INTRODUCTION Gold nanostructures are one of the most extensively studied inorganic nanomaterials in the fields of nanotechnology, nanomedicine, and nanobiotechnology.1-6 Nanoparticles have emerged as an inevitable alternative to antibiotics, since bacteria are known to develop resistance mechanism against all commercially available antibiotics.7 The antibacterial activity of nanoparticles is dependent on their direct interaction with the bacterial cell wall and they can, therefore, act as antibiotics without entry into the cell.7 Other possible modes of antibacterial action for nanoparticles include the generation of reactive oxygen species (ROS), penetration through the cell membrane and interactions with cellular DNA and proteins. As a result of these multiple modes of action, the use of nanoparticles as antibacterial agents would provide a greater barrier to bacteria developing resistance mechanisms that is the case for conventional therapeutics.7 In addition, nanoparticles offer multifaceted platforms for therapeutic applications depending on their physical properties including their size, shape, and functionalization with biomolecules such as proteins and nucleic acids.8 The high surface area to volume ratio of nanoparticles enhances the incorporation of ample functional molecules on the surface of the nanoparticles, paving the way for nanoparticles to be utilized as therapeutic agents.9 The functional molecules on the surface of nanoparticles initiate direct multivalent interactions with the biomolecules and paves the way for nanoparticles to be utilized as self-therapeutic agents.10-11 To make such self-therapeutic nanoparticles, the potentially inert and biocompatible nature of gold makes it a lucrative core material.12 The functionalization of Au nanoparticles with biomolecules (i.e., PEG, lipoic acid, glutathione, antibodies and so on) can protect their core structure, enhance their bioactivity and make them suitable for further functionalization.13 However, the therapeutic applications of Au nanoparticles in clinical trials requires much attention regarding their antimicrobial activity, antioxidant activity, and biocompatibility. 3 ACS Paragon Plus Environment


Au NPs are available in a variety of different sizes and shapes such as rod, cube, sphere, elongated tetrahexahedral, concave cubes, and octahedral morphologies.4, 14-17 In case of noble metal nanoparticles, not only the size but also the shape of particles determine their physical and chemical properties.15 Au nanoparticles with high index facets have high chemical activity and excellent biocompatibility.18-19 Furthermore, they have different binding affinities for various ligand molecules since the binding sites on the surface of nanoparticles are produced by different crystal facets including a large part of edges, terraces, and vertices.20 There are a wide variety of Au NPs which can be utilized for antimicrobial applications (such as rods, cubes, spheres etc.). The cellular uptake efficiency of such NPs depends not only on their size and shape but also their zeta potential and nature of materials used to functionalize the nanoparticles.21 Nanoparticles functionalized with polymers (e.g., PEG) have reduced serum protein adsorption ability, reduced particle size and surface charge, and increased stability.22 Commercially available antibiotics kill bacteria either through interaction with bacterial genetic material or by blocking their cell division rather than making any physical damage on the cell wall. As a result, bacterial morphology remains intact and they have the possibility to develop resistance against the traditional antibiotics.23 On the other hand, nanoparticles kill bacteria through damaging their cell wall and will eventually destroy the development of bacterial resistance mechanism. Nanoparticles with inherent reducing property are preferred because oxidative stress is directly responsible for the onset of many deadly diseases such as cancer, fibrosis, cardiovascular diseases and so on.24 Therefore, treatment of these diseases using functionalized nanoparticles with intrinsic antioxidant activity will enhance the chances of faster recovery.24 Herein, the authors have synthesized elongated tetrahexahedral (ETHH) Au nanoparticles through seed-mediated growth and functionalized them with α-lipoic acid. The antioxidant activity of both the ETHH and α-lipoic acid functionalized ETHH Au nanoparticles were investigated in 4 ACS Paragon Plus Environment

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vitro. Cytotoxic studies of the functionalized nanoparticles were carried out through hemolytic assay

using

human

red

blood

cells

(RBC)

and

3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay using HeLa cells. The antibacterial activities of the nanoparticles were investigated through disk diffusion assays, propidium iodide assays as well as trypan blue dye exclusion assay against a Gram-positive bacterium (i.e., B. subtilis) and a Gramnegative bacterium (i.e., E. coli). 2. MATERIALS AND METHODS 2.1. MATERIALS Gold (III) chloride trihydrate (HAuCl4·3H2O, ACS grade), sodium borohydride (NaBH4, 99%), αlipoic acid and trypan blue were purchased from Alfa Aesar, UK. L-ascorbic acid (AA, 99.5+ %), 2,2-diphenyl-1-picrylhydrazyl (DPPH), silver nitrate (AgNO3), dimethyl sulfoxide (DMSO) and hydrochloric acid (HCl, 37%) were purchased from Sigma-Aldrich, USA. (1-Hexadecyl) trimethylammonium bromide (CTAB) was purchased from BDH, UK. Trichloroacetic acid (TCA), sodium acetate, and glacial acetic acid were purchased from Merck, Germany. Thiobarbituric acid was purchased from JT Baker, USA. FeCl3.6H2O, and 2,4,6-tripyridyl-striazine (TPTZ) were purchased from VEGA, China and Sigma Aldrich, Germany, respectively. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), and penicillinstreptomycin (PS) were purchased from Thermo Fisher Scientific. 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide dye (MTT) was obtained from the School of Science, RMIT University. All reagents were used as received without further purification. Agar powder was purchased from Titan Biotech Ltd., India. Peptone, yeast extract, sodium chloride were collected from Unichem, China. Escherichia coli DH5α and Bacillus subtilis RBW were obtained from the Department of Biotechnology and Genetic Engineering, Jahangirnagar University, Savar, Dhaka



1342, Bangladesh. HeLa cells were obtained from Micro Nano Research Facility (MNRF) at RMIT University. 2.2. METHODS 2.2.1. SYNTHESIS OF GOLD NANOPARTICLES (AuNPs) Elongated tetrahexahedral (ETHH) gold nanoparticles were prepared according to the method reported by Zhang et al14 with slight modification. To synthesize gold seeds, freshly prepared ice cold NaBH4 (0.6 mL, 10 mM) was added into a solution of CTAB (9.75 mL, 0.10 M) and HAuCl4 (0.25 mL, 10 mM) under magnetic stirring (1000 rpm) and was stirred for a further 2 minutes before incubating for 2 h at 28 °C under quiescent conditions. The seed solution was then diluted 50 times using 0.10 M CTAB solution and was used for the seed-mediated growth of elongated tetrahexahedral (ETHH) gold nanoparticles. The growth solution was prepared through the sequential addition of HAuCl4 (0.50 mL, 10 mM), AgNO3 (0.10 mL, 10 mM), HCl (0.20 mL, 1 M), and AA (0.80 mL, 0.10 M) into a CTAB (10 mL, 0.10 M) solution and mixed gently for 30 s. Finally, the growth of elongated tetrahexahedral (ETHH) gold nanoparticles was initiated by the addition of 0.10 mL of the previously diluted gold seed solution. The reaction mixture was gently mixed for 30 s and incubated undisturbed at 28 °C overnight. The formed ETHH gold nanoparticles were washed twice with Milli-Q (MQ) water through centrifugation-redispersion cycles. The ETHH gold nanoparticles were finally redispersed in 1 mL of MQ water. For the synthesis of α-lipoic acid conjugated ETHH gold nanoparticles, 180 μL (1 μg/μL) ETHH gold nanoparticles were mixed with 20 μL (i.e., 10 mM) α-lipoic acid upon stirring and incubated at room temperature overnight. The reaction solution was centrifuged twice to remove unconjugated α-lipoic acid and redispersed in MQ water.


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2.2.2. CHARACTERIZATION OF ETHH Au NANOPARTICLES, AND ETHH Au NANOPARTICLES CONJUGATED WITH α-LIPOIC ACID Elongated tetrahexahedral (ETHH) Au nanoparticles were synthesized and functionalized with αlipoic acid (LA) to prepare ETHH-LA. ETHH Au nanoparticles were characterized using UV-Vis spectroscopy, scanning electron microscopy (SEM) using an FEI Nova NanoSEM instrument operated at an accelerating voltage of 15 kV, transmission electron microscopy (TEM) (JEOL, Japan), and Raman spectroscopy (SERS) (RamanStation 400F, PerkinElmer precisely). ETHH Au nanoparticles functionalized with α-lipoic acid were characterized using UV-Vis spectroscopy and Raman spectroscopy (SERS). The surface potential of all the nanoparticles was characterized using Zetasizer (Zetasizer, Malvern, UK). 2.2.3. ANTIOXIDANT ACTIVITY ASSAY OF AuNPs 2.2.3.1. DPPH FREE RADICAL SCAVENGING ASSAY The antioxidant activity of α-lipoic acid, ETHH AuNPs, and ETHH-LA AuNPs was measured by performing DPPH assay according to Polash et al.25-26 Briefly, 200 μL ascorbic acid of different amounts (6, 12, 30, 60, 120 and 240 μg) were taken in separate test tubes as standard. 200 μL of all the samples at different amounts (6, 12, 30, 60, 120 and 240 μg) were also taken in different test tubes. Both the samples and the standards were taken in triplicates. Then 300 μL methanol and 500 μL DPPH (0.3 mM) were added to all the tubes followed by 30 min incubation at 4 °C in the dark. Finally, the change of DPPH color was measured at 517 nm using a UV-vis spectrophotometer (Optizen POP, Korea) with methanol used as the blank sample. The free radical scavenging activity in percentage (%) was calculated from (Ab – As)/Ab x 100. Here, Ab is the absorbance of the blank, and As is the absorbance of the standard or samples.



2.2.3.2. FERRIC REDUCING/ANTIOXIDANT POWER ASSAY Ferric reducing/antioxidant power (FRAP) assay was performed according to Benzie et al. with slight modifications.27-28 Briefly, freshly prepared FRAP reagent was used for the antioxidant activity assay. FRAP reagent was prepared through mixing 300 mM acetate buffer (pH 3.6) prepared using sodium acetate trihydrate and glacial acetic acid, 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, and 20 mM FeCl3.6H2O at a ratio of 10:1:1. Ascorbic acid was used as a standard. Different amounts (i.e., 15 to 240 μg) of both the standard and Au nanorods (500 μL) were taken in test tubes and incubated at 37 °C in a water bath. Then 1.5 mL of prewarmed (37 °C) FRAP reagent was added to all the test tubes and incubated for 5 mins at 37 °C in a water bath. Only FRAP reagent was used as blank and the final volume of all the tubes was equalized using distilled water. Finally, the absorbance was measured at 593 nm using a UV-Vis spectrophotometer (Optizen POP, Korea). The antioxidant activity of the Au nanorods was expressed as microgram of ascorbic acid (AA) equivalent per milliliter (µg AA/mL). 2.2.4. ANTIMICROBIAL ACTIVITY ASSAY The antibacterial activity of α-lipoic acid (LA), CTAB, gold (III) chloride trihydrate, ETHH AuNPs, and ETHH-LA AuNPs were performed by disk diffusion method according to Mondal et al29 with modifications. Briefly, a Gram-positive strain (Bacillus subtilis) and a Gram-negative strain (Escherichia coli) of bacteria were cultured in Luria Bertani (LB) medium at 37 °C and 120 rpm overnight. 100 μL of each of the bacterial strains were then spreaded uniformly on LB agar plates. Dried and sterile metrical filter paper disks containing samples of known concentrations (20 μL, 1 μg/μL) were placed on the LB agar plates containing uniformly spread bacterial strains and incubated overnight at 37 °C for optimum growth of the bacterial strains. The antibacterial activity of LA, CTAB, gold (III) chloride trihydrate and AuNPs were investigated by determining 8 ACS Paragon Plus Environment

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the presence of clear zones surrounding the disks which confirms the inhibition of the growth of bacteria. After incubation, the antibacterial activities were determined by measuring the diameter of the zone of inhibition with slide calipers. 2.2.5. PROPIDIUM IODIDE ASSAY B. subtilis and E. coli were cultured in LB medium at 37 ºC and 275 rpm until they reached a stationary phase. The optical density of bacteria cultures was measured at 600 nm and fresh LB medium was used to dilute bacteria solutions to fix the concentration at 1 x 107 CFU/mL. A volume of 60 µL of the bacteria solution was added to a 96 well plate, giving a final concentration of 60,000 CFU/well. Cells were then incubated with 40 µL (1 µg/µL) of Au nanorods solutions for 15 minutes at room temperature. After incubation, 20 µL of bacterial solutions were kept aside for SEM slide preparation. Propidium iodide (PI) (1.6 µL; 100 µM) was added to the remaining bacterial culture and incubated at room temperature for 30 minutes in the dark. Furthermore, 20 µL of PI treated bacterial culture from the 96 well plate was kept aside for observing under confocal laser scanning microscopy (EVOS® FL Auto Cell Imaging System, Thermo Fisher Scientific). The fluorescence intensity of the remaining PI treated bacterial cultures was determined using CLARIO star microplate reader at 535 nm. All the treatment and control experiments were carried out in triplicate. Control groups with ETHH-LA, ETHH, LA, cells or media without PI were run simultaneously with the treatment groups. 2.2.6. SCANNING ELECTRON MICROSCOPY The bacterial samples were treated with ETHH and ETHH-LA Au nanorods. The Au nanorods treated bacterial samples were centrifuged at 1,000 rpm for 5 minutes and clean bacterial sample pellets were collected. The cell pellets were then fixed using 2% (v/v) glutaraldehyde/2.5% paraformaldehyde for 30 min at 37 oC followed by three rinses with 0.1 M sodium cacodylate 9 ACS Paragon Plus Environment


buffer. Following fixation, the samples were further fixed in 1% osmium tetroxide for 20 minutes and washed in distilled water. For SEM imaging, the fixed bacterial samples were added to 1% poly-lysine coated coverslips. The samples were further dehydrated in increasing ethanol concentrations from 50% to 100% ethanol. The samples were then dried by adding hexamethyldisilazane (HMDS) and gold sputter coated to ~15 nm thickness using an SPI sputter coater. The images were obtained using FEI Verios SEM instrument under high vacuum mode. 2.2.7. TRYPAN BLUE DYE EXCLUSION ASSAY Trypan blue is a diazo dye that has been frequently used to stain dead cells or tissues. Because of the negative charge of trypan blue it does not interact with cells unless the membrane is damaged or compromised.30 To confirm bacterial cell membrane disruption/damage by the ETHH nanoparticles or gold (III) chloride (HAuCl4), 80 μL (1 × 106 cells/mL) of the respective bacterial strains were mixed with 20 μL (1 μg/μL) of each of the nanoparticles or HAuCl4 and mixed thoroughly. The bacteria-nanoparticle or bacteria-HAuCl4 mixtures were then mixed with 0.4% trypan blue solution at 1:1 ratio, mixed gently and incubated at room temperature for 5 min. After incubation, 15 μL of bacteria-nanoparticle-trypan blue suspension or bacteria-HAuCl4-trypan blue suspension was loaded into a hemocytometer chamber followed by imaging of live and dead cells was performed using a phase contrast microscope (Olympus BX50 Fluorescence Microscope, Olympus, Japan). 2.2.8. LIPID PEROXIDATION ASSAY The lipid peroxidation (LPO) assay of LA, ETHH, and ETHH-LA Au nanorods were performed according to an established protocol with little modifications.31 Briefly, 0.3 mL of each of the bacterial cells (i.e., E. coli, and B. subtilis) was first treated with 200 μL (i.e., 200 μg) of each of the Au nanorods and LA. The bacterial cells were then mixed with 1 mL of trichloroacetic acid 10 ACS Paragon Plus Environment

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(10%) followed by centrifugation at 11,000 rpm for 35 minutes to precipitate the solids. The supernatant was taken out and centrifuged again at 11,000 rpm for 20 min to ensure that AuNPs, LA, cells, and precipitated proteins were removed completely. The supernatant was collected in fresh tubes and mixed with 2 mL freshly prepared 0.67% thiobarbituric acid (TBA) solution. Finally, the samples were incubated in a boiling water bath for 10 min and cooled down at room temperature before taking the absorbance in the range of 200-1000 nm using a UV-Vis spectrophotometer (Specord® 205, Analytik Jena, Germany). 2.2.9. HEMOLYTIC ACTIVITY ASSAY OF AuNPs The hemolytic activity assay of ETHH AuNPs, and AuNPs-LA were performed according to Li et al9 with modifications.32 Briefly, human whole blood was collected in a tube containing 10% EDTA as an anticoagulant and centrifuged at 500 g for 10 min to remove the serum. The red blood cells (RBCs) were then resuspended in 5 mL of phosphate buffered saline (PBS) and washed several times through centrifugation (3,000 g, 3 min each time) until the absorbance (at 570 nm) of the supernatant reached the absorbance of PBS only. Then 0.1 mL of RBC solution was added to 0.4 mL of each type of nanoparticles at different amounts (i.e., 6 to 240 μg). The mixtures were incubated at 37 °C upon stirring at 150 rpm for 30 min followed by centrifugation at 4,000 rpm for 5 min. The absorbance values of the supernatants were measured at 570 nm. RBCs incubated with PBS were used as a negative control and RBCs incubated with water were used as a positive control. All the samples were prepared in triplicates. The percent hemolysis was calculated using the following formula: %Hemolysis=[(A - NCA)/ (PCA-NCA)]*100 Where A represents Sample Absorbance; NCA represents Negative control absorbance and PCA represents Positive control absorbance. 11 ACS Paragon Plus Environment


2.2.10. MAMMALIAN CELL VIABILITY ASSAY The cytotoxicity of the Au nanorods was investigated through MTT assay using HeLa cells. To perform the cell viability assay, HeLa cells were seeded in a 96 well plate at a density of 5 x 103 cells/well and incubated overnight at 37 °C in a 5% CO2 incubator. After overnight incubation, the old media was replaced with 100 μL complete DMEM (DMEM with 10% FBS and 1% PS) containing 6, 12, 30, 60, 120, and 240 μg each of ETHH, and ETHH-LA Au nanorods and incubated overnight in a 5% CO2 incubator at 37 °C. After incubation, the media containing different volumes of nanoparticles was aspirated and 100 µL of culture media (i.e., complete DMEM) containing 0.5 mg/mL MTT (Thiazolyl blue tetrazolium bromide) was added to each well of the 96 well plate and were further incubated for 4 h in an animal cell culture incubator at 37 °C. The media containing MTT was then removed and 100 µL dimethyl sulfoxide (DMSO) was added to each well to solubilize formazan crystal. The plates were read on a micro-plate reader (SpectraMax Paradigm Multi-Mode Microplate Reader, Molecular Devices, USA) at 570 nm with a reference wavelength of 630 nm. The cells cultured with only DMEM medium were used as a control. The percentage of viable cells was calculated as follows: cell viability = OD570 (sample)/OD570 (control) × 100%.33 Cytotoxicity experiment was performed several times to ensure the reproducibility of data. 3. RESULTS AND DISCUSSION 3.1. CHARACTERIZATION OF ETHH Au NANOPARTICLES Through the procedure listed above, elongated tetrahexahedral (ETHH) Au nanoparticles that are highly monodisperse in particle size with uniform shape/morphologies were formed. The length of ETHH Au nanorods is 117±9 nm and width is 58.3±5.5 nm data obtained from TEM images (FIGURE 1). The UV-Vis spectrum confirms the synthesis of ETHH Au nanorods and showed 12 ACS Paragon Plus Environment

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two characteristic peaks at ~525 and ~705 nm (FIGURE 1a). These surface plasmon bands are due to the transverse and longitudinal oscillation modes of the ETHH Au nanoparticles, respectively. The functionalization of ETHH Au nanorods with LA was also confirmed from the UV-Vis spectrum. The UV-Vis spectrum of ETHH-LA showed three characteristic peaks at ~335, ~530, and ~700 nm (FIGURE 1a). The peak at ~335 nm is due to the conjugation of LA with ETHH Au nanorods. The surface plasmon bands at ~530 and ~700 nm are due to the transverse and longitudinal oscillation modes of the ETHH Au nanoparticles. The LA functionalization brings about slight shift for both the transverse (~530 nm) and longitudinal oscillation (~700 nm) modes of the ETHH Au nanoparticles. SEM images show the uniformity and monodispersity of ETHH Au nanoparticles over a large number of particles (FIGURE 1b).

FIGURE 1. Monodispersed ETHH Au nanorods were characterized using UV-Vis Spectroscopy (a), SEM (b), and TEM (c). Raman spectroscopy of the ETHH Au nanoparticles confirms that CTAB acts as a stabilizing agent for the nanoparticles and has been presented in the supplementary information, FIGURE S1. The peaks at 760, 1288, 1438, and 2844 cm-1 are common to both CTAB and ETHH. These peaks are attributed to CN+ stretching, C-C stretching, CH2 twisting, CH2 scissoring and CH3 deformation, and CH2 symmetric stretching, respectively.34 Furthermore, the functionalization of



ETHH with α-lipoic acid (ETHH-LA) has also been confirmed from Raman spectra since ETHHLA showed characteristic LA Raman bands at 2910 and 1434 cm-1 (FIGURE S1). The zeta potentials of the ETHH Au nanorods and ETHH-LA Au nanorods were +16.2±8.8 mV and -14.8±5.8 mV, respectively (TABLE S1). The positive zeta potentials of ETHH Au nanorods were due to the presence of CTAB as stabilizing agent that has a quaternary amine group as hydrophilic head group.35 However, the negative zeta potentials of ETHH-LA Au nanorods were due to the functionalization of ETHH Au nanoparticles with α-lipoic acid. The α-lipoic acid has a terminal carboxylic acid and a terminal dithiolane ring. The dithiolane ring binds with the gold nanoparticles through noncovalent interactions (i.e., chemisorption) and the carboxyl group provides negative zeta potentials to the nanoparticles. The negative zeta potential of ETHH-LA Au nanorods also confirms the functionalization of ETHH with LA. 3.2. ANTIOXIDANT ACTIVITY ASSAY 3.2.1. DPPH FREE RADICAL SCAVENGING ASSAY The antioxidant activity of ETHH Au nanorods, ETHH-LA Au nanorods, and LA was performed through DPPH assay. LA acts as a natural antioxidant because of its dithiolane ring which can be reduced to dithiol that is known to have strong antioxidant activity in the cytosolic environment.36 Therefore, the conjugation of α-lipoic acid with ETHH will act as an antioxidant ligand on gold nanoparticles. The highest free radical scavenging activity was performed by ETHH-LA followed by ETHH and LA (FIGURE 2a). The antioxidant activity performed by ETHH Au nanorods may be due to the reduced state of Au nanorods which has been brought about by the reducing agent sodium borohydride during their synthesis. It could also be due to its metallic nature as gold salt is known to lack any noticeable antioxidant activity.37 On the other hand, the enhanced antioxidant activity of ETHH-LA is due to the direct participation of LA in scavenging DPPH free radicals. 14 ACS Paragon Plus Environment

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Therefore, ETHH-LA Au nanorods can be used as a new functional antioxidant with improved free radical scavenging activity. 3.2.2. FERRIC REDUCING/ANTIOXIDANT POWER ASSAY Ferric reducing/antioxidant power (FRAP) assay was used to investigate the ferric (Fe3+) reducing potential of Au nanorods. Ferric ion (Fe3+) is reduced to ferrous ion (Fe2+) in the presence of various amounts (15 to 240 μg) of LA, ETHH, and ETHH-LA Au nanorods. Both ETHH and ETHH-LA Au nanorods have higher ferric reducing ability than that of LA, a natural antioxidant (FIGURE 2b). The ferric reducing ability increased as the concentration of LA and Au nanorods were increased. ETHH-LA Au nanorods have higher ferric reducing ability than that of ETHH at all the concentrations. This is because of the functionalization of ETHH with LA. It has been reported that the reducing potential increases for antioxidant functionalized gold nanoparticles.38 Therefore, elongated tetrahexahedral Au nanoparticles are capable of scavenging DPPH free radicals (i.e., nitrogen free radicals) as well as reducing ferric to ferrous ions (FIGURE 2a and 2b). More specifically, ETHH-LA Au nanorods have a higher reducing potential than that of ETHH and LA.



FIGURE 2. Antioxidant activity potential for α-lipoic acid (LA), elongated tetrahexahedral (ETHH), and LA functionalized ETHH (ETHH-LA) Au nanorods. (a) DPPH free radical scavenging assay; (b) Ferric reducing/antioxidant power (FRAP) assay. The tests were performed 3 times on three different days. 3.3. ANTIMICROBIAL ACTIVITY ASSAY The antimicrobial activity of LA, CTAB, gold (III) chloride trihydrate, ETHH, and ETHH-LA was investigated using Gram-positive (B. subtilis) and Gram-negative (E. coli) bacteria (FIGURE 3). The highest antibacterial activity was obtained with ETHH-LA against B. subtilis and the zone of inhibition was ~10 mm in diameter. Both ETHH-LA and ETHH showed better antibacterial activity against B. subtilis when compared to that of E.coli while LA and CTAB did not show any antibacterial activity against any of these strains. However, gold (III) chloride trihydrate showed very little antibacterial activity against both B. subtilis and E. coli and the zones of inhibition were 6.92 mm and 6.75 mm, respectively. Gold (III) chloride trihydrate solution showed very little antibacterial activity because of the presence of Au (III) as chloride.39

FIGURE 3. Antimicrobial activity assay. Antimicrobial activity of α-lipoic acid (LA), CTAB, gold (III) chloride trihydrate (HAuCl4.3H2O), elongated tetrahexahedral (ETHH) gold (Au) nanorods, and α-lipoic acid functionalized ETHH (ETHH-LA) Au nanorods against Gram16 ACS Paragon Plus Environment

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positive (B. subtilis) and Gram-negative (E. coli) bacteria. The tests were performed 3 times on three different days. The higher antibacterial activity of ETHH-LA could be due to three reasons: first, the presence of α-lipoic acid on ETHH Au nanorods surface. Alpha-lipoic acid derived from octenoic acid that has a long hydrocarbon chain and the carboxyl group acts as the hydrophilic moiety. The hydrocarbon chain of the α-lipoic acid increases the hydrophobicity of ETHH-LA nanorods and interacts with the cell membrane lipids through noncovalent (hydrophobic) interactions. Therefore, the increased uptake of negatively charged ETHH-LA Au nanorods was due to their higher hydrophobicity compared to ETHH Au nanorods.9,

40

Second, although the zeta potential of

ETHH-LA is negative, the zeta potential of ETHH is positive and the zeta potential of the cell membranes are negative. Therefore, it is postulated that there is still electrostatic interaction between the positively charged ETHH and negatively charged cell membrane (FIGURE S2).41 Third, the interaction between the ETHH-LA Au nanorods and bacteria could also be due to molecular crowding.42 Therefore, hydrophobic and electrostatic interactions as well as molecular crowding are responsible for better interactions between the ETHH-LA Au nanorods and the cell membrane which result in better antibacterial activity. However, the interaction between ETHH Au nanorods and cell membrane is only through electrostatic which provides entry of ETHH Au nanorods to a lesser extent through cell membranes when compared to ETHH-LA Au nanorods. Hence, ETHH Au nanorods have lower antibacterial activity to both Gram-positive and Gramnegative strains of bacteria. Once the Au nanorods reach inside the cell cytoplasm, they are expected to form gold aggregates (also called vacuole formation) which results in cell death. The vacuole formation is due to the increase in the oxidative stress of microbial cells by reactive oxygen species (ROS).43 Both the ETHH and ETHH-LA Au nanorods showed better antibacterial activity to Gram-positive bacteria (B. subtilis) compared to Gram-negative bacteria (E. coli) because of the 17 ACS Paragon Plus Environment


presence of teichoic acid on the cell wall of B. subtilis. Teichoic acid directs the uniform distribution of nanoparticles along the molecular chain of phosphate and, thus prevents the aggregation of nanoparticles.7 In addition, the cell wall of Gram-positive bacteria contains a thin layer of peptidoglycan and abundant pores through which external molecules can enter the cell that brings about membrane damage and cell death.7 On the other hand, the cell wall of Gramnegative bacteria is comprised of lipopolysaccharide (LPS), lipoproteins and phospholipids that form a penetration barrier and allows the movement of macromolecules. The surface potential of Gram-positive bacteria is also more negative than that of Gram-negative bacteria which attracts more nanoparticles.44 The difference in antibacterial activity could also be due to the differences in lipid composition, gross composition of the membranes or even specific protein complexes present on the surface of bacteria.45 3.4. PROPIDIUM IODIDE ASSAY Propidium iodide (PI) is a fluorescent nuclear and chromosomal counterstain and can penetrate bacterial cells with compromised cell membranes.9 Since PI is not permeant to live cells, it is commonly used to detect dead cells. The red fluorescence of Au nanoparticles treated dead bacterial cells was measured quantitatively using a CLARIO star microplate reader at 535 nm. The fluorescence intensity of ETHH-LA Au nanoparticles treated B. subtilis was 2.6 folds higher than that of E. coli (FIGURE 4a). On the other hand, the fluorescence intensity of ETHH-LA Au nanoparticles treated B. subtilis and E. coli were 10 and 3.6 folds higher than that of their respective ETHH treated strains.


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FIGURE 4. Propidium iodide (PI) uptake assay. (a) Fluorescence intensity of bacteria treated with Au nanorods was measured. PI staining of B. subtilis DNA was visualized under Confocal Laser Scanning Microscopy after treatment with ETHH (b), and ETHH-LA (c) Au nanorods. The same experiment was also performed for E. coli DNA after treatment with ETHH (d), and ETHH-LA (e) Au nanorods. Here, ETHH= Elongated tetrahexahedral Au Nanorods; ETHHLA=Elongated tetrahexahedral Au Nanorods functionalized with lipoic acid (LA). The red fluorescence of Au nanoparticle/PI treated dead bacteria cells was also visualized using confocal laser scanning microscopy (CLSM) (FIGURE 4b to 4e and FIGURE S3 and S4). CLSM images confirm the damage of both B. subtilis and E. coli cell membranes and, thereby, fluorescent staining of their DNA. 3.5. SCANNING ELECTRON MICROSCOPY Scanning electron microscopy was performed to examine the antimicrobial effects of ETHH and ETHH-LA nanoparticles on bacteria. The representative SEM images are shown in FIGURE 5 for B. subtilis (FIGURE 5a, b, and c) and E. coli (FIGURE 5d, e, and f), respectively. In the presence of ETHH Au nanoparticles apparently there is cell wall damage leading to partial or complete lysis as indicated by arrows. However, the ETHH-LA Au nanoparticles have a stronger 19 ACS Paragon Plus Environment


effect on the cells indicating thread-like structures, blebs and collapse of bacterial cells as compared to controls that have overall smooth morphology. 3.6. TRYPAN BLUE DYE ASSAY Trypan blue dye exclusion assay was performed to confirm that both ETHH and ETHH-LA Au nanorods were taken up by both the Gram-positive and Gram-negative bacteria. When Au nanorods interact with the cell wall of bacteria through either electrostatic interactions or hydrophobic interactions, nanorods damage the bacterial cell wall. Gold (III) chloride is also known to damage the bacterial cell wall. This facilitates the entry of Trypan blue dye into the bacterial cytosol from their surroundings. Therefore, cell wall compromised or nonviable bacteria look blue under phase contrast light microscope (FIGURE S5 to S7).

FIGURE 5. Scanning electron microscopic (SEM) images showing damage of B. subtilis with ETHH (a), and ETHH-LA (b) Au nanoparticles compared to control (c) and E. coli with ETHH (d), and ETHH-LA (e) compared to control (f).


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3.7. LIPID PEROXIDATION ASSAY A lipid peroxidation assay was performed to investigate the oxidation of bacterial cell membrane fatty acids by Au nanorods. The formation of malondialdehyde adduct was higher when both B. subtilis and E. coli were treated with ETHH-LA when compared to that of ETHH Au nanorods (FIGURE 6) because ETHH-LA Au nanorods have stronger interaction with bacteria. However, there was no malondialdehyde adduct formed when bacteria were treated with LA. The amount of malondialdehyde adduct was more for B. subtilis than that of E. coli when both the bacteria were treated with Au nanorods (FIGURE 6). B. subtilis produces more LPO than that of E. coli because Au nanorods have greater interaction with the Gram-positive bacteria than that of the Gram-negative bacteria. This reveals that lipid peroxide was generated due to the oxidation of bacterial fatty acids by Au nanorods. It has also been reported that nanoparticles derived from transition metals stimulate the production of reactive oxygen species (ROS) leading to oxidative stress for the cells.46 ROS stimulate the oxidation of bacterial cell membrane fatty acids to produce lipid peroxides and the redox balance of cells favor oxidation.7, 47 This creates oxidative stress for the cells which ultimately damages all the individual components (i.e., proteins, DNA and other cellular macromolecules) of the bacterial cells.7, 47 Furthermore, oxidation of membrane lipids changes the permeability of bacterial cells which allows Au nanorods as well as Au to enter into the cells and interact with the cytosolic proteins and DNA to kill the bacteria. ROS also stimulate the expression of apoptotic genes and oxidative proteins to bring about the apoptosis of bacterial cell.7 Therefore, our data did not find any relationship between the antioxidant potential and antimicrobial activity of Au nanorods.



FIGURE 6. Measurement of lipid peroxidation through the release of the malondialdehyde (MDA) adduct with TBA in the supernatant of (a) B. subtilis and (b) E. coli. The λmax for the MDA-TBA pink adduct was measured at 532 nm. 3.8. HEMOLYTIC ACTIVITY ASSAY To investigate the biocompatibility of the synthesized Au nanorods, we performed hemolytic activity assays using human red blood cells. The HC50 value is the concentration required to lyse 50% of RBC.48 The HC50 for ETHH, and ETHH-LA Au nanorods were found to be 56 and 88 μg, respectively (FIGURE 7). The higher HC50 value of ETHH-LA when compared to that of ETHH Au nanorods is due to the negative zeta potential of the former because of its’ functionalization with lipoic acid. The little hemolytic propensity of ETHH Au nanorods at higher amounts (i.e., 60 to 240 μg) could be due to their positive zeta potential (i.e., +16 mV) that brings about strong electrostatic interaction with the negatively charged membrane (i.e., -15.7 mV) of erythrocytes.49 These nanoparticles have low hemolytic activity albeit elevated antimicrobial activity. The low hemolytic activity of antimicrobial gold nanoparticles could be explained by the fact that bacterial cells are more negatively charged than mammalian cells which justifies the higher attraction of ETHH Au nanorods toward bacterial multiplications.50 Furthermore, the abundant presence of cholesterol and lack of acidic phospholipids in the outer layer of erythrocytes make them less sensitive to antimicrobial gold nanoparticles.51 22 ACS Paragon Plus Environment

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FIGURE 7. Hemolytic activity assay using human red blood cells. The hemolytic activity of ETHH and LA functionalized ETHH (ETHH-LA) Au nanorods at different amounts (i.e., 6 to 240 μg). HC50 values for ETHH and ETHH-LA Au nanorods were estimated to be 56 and 88 μg, respectively. Hemolytic activity was performed three times on three different days. 3.9. MAMMALIAN CELL VIABILITY ASSAY MTT assay was carried out to investigate the cytotoxic activity of ETHH and ETHH-LA Au nanorods using human cervical cancer cells, HeLa. The ETHH-LA Au nanorods showed higher percentage of viable cells at all the amounts (6 to 240 μg) when compared to ETHH (FIGURE 8). More specifically, ETHH-LA Au nanorods up to the amount of 60 μg did not produce any significant toxicity to HeLa cells, a dose that was well above the lethal dose (20 μg) for the bacteria used in the study. This is because the zeta potentials of ETHH and ETHH-LA Au nanorods were positive and negative, respectively (TABLE S1). The positive zeta potentials of ETHH Au nanorods are due to the presence of stabilizing agent CTAB, a quaternary ammonium surfactant, while the lipoic acid functionalization is responsible for the negative zeta potential of ETHH-LA Au nanorods. Generally, positively charged nanoparticles show cytotoxicity due to the strong interaction with serum proteins and unspecific binding with the cellular membranes.35 While nanoparticles with negative zeta potentials bring about their reduced unspecific binding to cellular membranes.35 Therefore, the functionalization of ETHH Au nanorods with α-lipoic acid increased 23 ACS Paragon Plus Environment


their biocompatibility. Au (0) nanoparticles usually do not show any cytotoxicity at lower concentrations.52

FIGURE 8. MTT assay. The cytotoxicity of elongated tetrahexahedral (ETHH) Au nanorods, and α-lipoic acid functionalized tetrahexahedral (ETHH-LA) Au nanorods were investigated using cervical cancer cells, HeLa. IC50 for ETHH and ETHH-LA were 186 and 238 μg, respectively. The cytotoxicity tests were performed 3 times on three different days.

4. CONCLUSIONS We synthesized elongated tetrahexahedral Au nanorods, characterized them and evaluated their bioactivity in terms of their antioxidant, biocompatibility and antimicrobial activity assay. The ETHH Au nanorods were then functionalized with α-lipoic acid (LA). Alpha-lipoic acid functionalization of ETHH induces their antioxidant property, lipid peroxidation potential as well as antimicrobial activity. ETHH-LA Au nanorods were also found to possess higher lipid peroxidation potential as well as antimicrobial activity against both Gram-positive and Gramnegative bacteria compared to ETHH Au nanorods. Furthermore, the lipid peroxidation potential as well as antimicrobial activity of both the nanorods (ETHH and ETHH-LA) was improved against Gram-positive bacteria compared to Gram-negative bacteria. ETHH Au nanorods 24 ACS Paragon Plus Environment

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functionalized with LA have reduced cytotoxicity to human red blood cells as well as cervical cancer cells than ETHH alone. It can be concluded that our Au nanorods have high antioxidant potential as well as high antimicrobial propensity and there is no relationship between the antioxidant and antimicrobial potential of Au nanoparticles. Hence, LA functionalized ETHH (ETHH-LA) Au nanorods with multiple bioactive functionalities are potential candidate for future therapeutic applications. Furthermore, the thiol group of LA binds with ETHH nanorods very strongly and the carboxyl group (-COOH) remains available for conjugation with any suitable biomolecules. Therefore, the ETHH-LA Au nanorods presented in this study can be subjected to further functionalization and used for multiple purposes. Conflict of interest The authors declare no conflict of interest.

Acknowledgement The authors acknowledge the RMIT Microscopy and Microanalysis Facility, and Micro Nano Research Facility of RMIT University, and Wazed Miah Science Research Centre of Jahangirnagar University for allowing the use of their comprehensive facilities and services. S.R.S. thanks the Department of Education and Training, Government of Australia for the award of an Endeavor Executive Fellowship. S.R.S also acknowledge the support of Jahangirnagar University Research Grant 2017, Government of Bangladesh. A.E.K. acknowledge RMIT University for Vice Chancellor Fellowship. References (1) Kabir, K. M. M.; Sabri, Y. M.; Kandjani, A. E.; Matthews, G. I.; Field, M.; Jones, L. A.; Nafady, A.; Ippolito, S. J.; Bhargava, S. K., Mercury Sorption and Desorption on Gold: A Comparative Analysis of Surface Acoustic Wave and Quartz Crystal Microbalance-Based Sensors. Langmuir 2015, 31 (30), 8519-8529. 25 ACS Paragon Plus Environment


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Graphical Abstract Fluorescence intensity (Fold change)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7

B. subtilis

E. coli

6 5 4 3 2 1 5 μm

0 ETHH-LA

ETHH

LA

Untreated

Sample

Quantitative antibacterial activity assay

ETHH Au nanorods and their antimicrobial activity assay


Functionalization of Elongated Tetrahexahedral Au Nanoparticles and

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