Materials: Methods Synthesis of gold nanoparticles (GNPs): GNPs

Materials:
Chloroauric acid (HAuCl4.3H2O)
and sodium borohydride (NaBH4) were purchased from S.D Fine Chemicals
(Mumbai, India). Tri-sodium citrate dihydrate (Na3C6H5O7.2H2O),
Dulbecco’s Modified Eagle Medium/Nutrient mixture F12 (DMEM/F12), 3-(4,
5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red
dye, 0.25% trypsin EDTA, antibiotic and antimycotic solution, Dulbecco’s
Phosphate buffered saline (DPBS, Ca2+, Mg2+ free) were
purchased from HiMedia (Mumbai, India). Bovine serum albumin (BSA),
Lipopolysachharide (LPS) and 2,7-dichlorofluorescein
diacetate (DCFDA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Griess reagent kit, cDNA synthesis kit and SYBRTM Green PCR Master
mix were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
CpG oligonucleotide (CpG-ODN 2395) was obtained from InvivoGen
(San Diego, CA, USA). Rabbit-polyclonal primary antibodies against inducible
Nitric oxide synthase (iNOS), p-38 and ?-Actin were obtained from Biorbyt (Cambridge, UK). Rabbit polyclonal
antibody against nuclear factor kappa B (NF-?B p65) was obtained from Raybiotech (Norcross, GA, USA). Fetal
Bovine Serum (FBS) was procured from Thermo Fisher Scientific. Human lung
carcinoma cell line (A549) was purchased from National Centre for Cell Science (NCCS)
(Pune, India).

Methods

Synthesis of
gold nanoparticles (GNPs): GNPs were synthesized according
to the method developed by Turkevich et
al (19). All glass wares used for the nanoparticle synthesis were washed
using aqua regia (3:1, HCL: HNO3) and different-sized nanoparticles were
prepared using 18-M?-deionized, 0.22 ?m-?ltered water. Briefly, 1 mL of 38.38 mM tri-sodium citrate
solution was added to 14.85 mL of ice-cold chloroauric acid solution. Next, 0.5
mL of 0.5 M ice-cold solution of sodium borohydride was added to the above mentioned
solution to synthesize 5 nm gold nanoparticles. Reduction of gold salt by
tri-sodium citrate was utilized for the synthesis of 15 and 30 nm gold
nanoparticles. HAuCl4 (150 ?L) was added
to 14.85 mL Milli-Q water and the solution was heated till boiling. Thereafter,
1.8 mL and 1 mL of 38.8 mM trisodium citrate was added drop wise to 14.85 mL of
chloroauric acid solution to synthesize 15 and 30 nm gold nanoparticles,
respectively. The colour of the solution changed from colourless to red
indicating the synthesis of gold nanoparticles. Here, tri-sodium citrate was
used as a capping as well as a reducing agent. All the nanoparticles were
purified and concentrated by centrifugation. The pellet was re-suspended in
appropriate volume of Milli-Q water and stored at 40C until use.

Characterization of GNPs: Size of GNPs was determined with the help of UV-visible
spectrophotometer Biotek Synergy HT (Winooski, VA, USA) by measuring the
absorption maxima in a quartz cuvette with path length of 1 cm. The
hydrodynamic size and zeta potential of GNPs were ascertained by dynamic light
scattering and phase analysis light scattering, respectively, measured using
Zetasizer Nano-ZS (Malvern instruments, Malvern UK). The size and distribution
of gold nanoparticles were confirmed by transmission electron microscopy
assessment. A drop of GNP was placed on the surface of a carbon-coated copper
grid and was allowed to dry. Thereafter, microscopic images were obtained using
a transmission electron microscope (TEM), JEOL JEM1400 (Tokyo, Japan) at an
accelerating voltage of 140 kV.

Cell culture and exposure of A549 cells to gold
nanoparticles: Human lung adenocarcinoma cell line
(A549) was cultured in DMEM/F12 supplemented with 10% FBS and 1% antibiotic and
antimycotic solution at 370C under a humidified atmosphere with 5%
CO2. Cellular uptake and cytotoxicity of GNPs was assessed by
treating A549 cells with different concentrations of 5, 15 and 30 nm GNPs. The
immunomodulatory property of nanoparticles was ascertained by various
experiments which involved treatment of cells with LPS or CpG-ODN only, GNPs
alone or pre-treatment of A549 cells with different-sized GNPs (40 ?g/mL) for 18 hrs and then immunostimulation with LPS and CpG-ODN for
6 hrs (ROS and RNI) and 3 hrs (qPCR and western blot) to activate TLR4 and TLR9
signaling pathways, respectively.

Cellular internalization
of gold nanoparticles: Flow cytometry was used to
assess the internalization of GNPs
by A549 cells utilizing the method developed by Suzuki et al (20). Cells were seeded in 6-well tissue culture plates for 24 hrs and
were treated with different concentrations (20, 40, 80 and 100 ?g/mL) of 5, 15
and 30 nm gold nanoparticles for 24 hrs. After treatment with different-sized GNPs,
cells were harvested by trypsinization and re-suspended in PBS. The internalization
of GNPs was then analyzed using flow cytometer (FACS Calibur BD Biosciences) (San
Jose, CA, USA) by measuring side scatter intensity (SSC). This method is based
on the principle that an increase in the internalization of nanoparticles by
cells results in an increase in the cellular granularity. Increased granularity
can be determined by measuring the increase in the SSC intensity with constant
forward scattered (FSC) light intensity indicating uptake of GNPs by A549 cells.

Cytotoxicity Assessment

MTT assay: Effect of nanoparticle treatment on cell viability was determined
by MTT assay. It involves evaluation of the mitochondrial activity as described
by Mosmann (21). 1×104 cells were seeded in 96-well tissue culture
plates and incubated for 24 hrs. Next, cells were incubated with different concentrations
of GNPs (20, 40, 60, 80 and 100 ?g/mL) for different time periods (6, 24, 48
hrs). After exposure, GNPs containing media was removed and MTT dye (5 mg/mL) in
PBS was added and incubated for 3 hrs. Thereafter, MTT solution was discarded from
each well and the formazan crystals formed by MTT reduction were solubilized by
adding 100 ?L of dimethyl sulfoxide (DMSO), followed by absorbance measurement at
590 nm in the multi-well plate reader.

Neutral Red
Uptake (NRU) Assay: NRU assay was also used to
determine the viability of A549 cells upon GNPs treatment. Neutral red dye
accumulates in the lysosomes of viable cells and is used as a measure of cell
viability (22). 1×104
cells were seeded in 96-well tissue culture plates for 24 hrs and were
incubated with different concentrations of GNPs (20, 40, 60, 80 and 100 ?g/mL) at
three time points (6, 24, 48 hrs). Thereafter, GNPs containing media was
discarded and neutral red dye (50 ?g/mL) in the serum-free medium was added and
incubated for 3 hrs at 370C. The accumulated NR dye was dissolved using
50% ethanol and 1% acetic acid followed by absorbance measurement at 540 nm in the
multi-well plate reader.

ROS Level
Measurement: Intracellular ROS generation by A549
cells upon immunostimulation with LPS and CpG-ODN treatment (10 ng/mL and 1 ?M, respectively) was determined by using 2,7-dichlorofluorescein diacetate
(DCFH-DA) dye (23). 1×104 cells were seeded in a 96-well black bottom cell
culture plates and incubated for 24 hrs. After that cells were pre-treated with
GNPs (18 hrs) followed by exposure to LPS and CpG-ODN (6 hrs) to activate the
TLR4 and TLR9 signaling pathways, respectively. The treatment containing medium
was aspirated from all the wells, followed by wash with 1X PBS. Thereafter cells
were incubated with DCFDA dye (20 ?M) in 1X PBS for 30 mins at 370C.
Next, DCFDA dye was discarded and 100 ?L of PBS was added to each well followed
by the measurement of fluorescence intensity using a multi-well plate reader at
excitation and emission wavelengths of 485 and 528 nm, respectively. ROS
production was also assessed by flow cytometric analysis (24). Briefly, cells were seeded in 6-well plates and were subjected to
treatment conditions as mentioned above (‘Cell culture and exposure of A549 cells to gold
nanoparticles’ section). Next, cells were harvested by
trypsinization and the single cell suspension thus obtained was incubated with DCFDA
(20 ?M) in PBS for 30 mins at 370C.
After incubation, DCFDA dye was discarded and cells were washed with 1X PBS to
remove the remaining dye from the cell suspension. Finally, the cell suspension
was re-suspended in PBS and analysed by flow cytometer (FACS Calibur) using
FL-1 channel. Qualitative analysis of ROS generation by LPS and CpG-ODN stimulation
was also carried out using fluorescence microscope (Leica DMIL LED, Wetzlar,
Germany). Cells were grown on coverslips and treated as mentioned above. Prior
imaging, cells were fixed using 4% paraformaldehyde solution for 15 mins at
room temperature. Cells were washed twice with 1X PBS. Coverslips containing
cells were mounted on slides and were stored at 40C until
fluorescence microscopic imaging.

Nitrite
estimation: Nitric oxide production by A549 cells
was assessed by quantifying stable nitrite using the method developed by Green et al (25). This method involves the use of Griess reagent and is based on the
azo dye formation upon reaction of nitrite with sulfanilic acid and naphthylethylenediamine
dihydrochloride. 1×104 cells were seeded in 96-well plates and
incubated for 24 hrs. Next, cells were pre-incubated with GNPs for 18 hrs,
followed by exposure to LPS and CpG-ODN (6 hrs), respectively. The supernatant
was collected from each well and the absorbance was measured at 540 nm. Nitric
oxide concentration was determined using sodium nitrite as the standard.

Quantitative
RT-PCR: Cells
were seeded in 6-well plates and treated as mentioned above (‘Cell culture and
exposure of A549 cells to gold nanoparticles’ section). Then, cells were harvested by
trypsinization and total RNA was extracted from cells using TRIzol Reagent
(Life Technologies, New Delhi, India). One microgram of total RNA was used as a
template to synthesize cDNA using the Verso cDNA synthesis kit (Thermo Fisher
Scientific). Quantitative RT-PCR was performed on cDNA aliquots by using PowerUp
SYBRTM Green master mix (Thermo Fisher Scientific) in ABI Prism 7000
Real-time PCR system (Thermo Fisher Scientific). RNA expression was normalized
to the expression of the housekeeping gene ?-actin.