Electronic supplementary material for Evaluation of Cytotoxicity, Biophysics and
Biomechanics of Cells Treated with Functionalized Hybrid Nanomaterials
Ramesh Subbiah,1,2 Subramaniyan Ramasundaram,3 Ping Du,1,2 Kim Hyojin,2 Dongkyung Sung,4
Kwideok Park,1,2 Nae-Eung Lee,3 Kyusik Yun,5,* and Kyoung Jin Choi6,*
1
University of Science and Technology (UST), 113 Gwahangno, Daejon, South Korea
2
Center for Biomaterials, Korea Institute of Science and Technology (KIST) Seoul, South Korea
3
School of Advanced Materials Science and Engineering, Sungkyunkwan University, Suwon,
South Korea
4
Department of Biomedical Engineering, Samsung Hospital, Seoul, South Korea
5
Department of Bionano Technology, Gachon University, Seongnam, South Korea
6
School of Mechanical & Advanced Materials Engineering, Ulsan National Institute of Science
and Technology (UNIST), Ulsan, South Korea
* Author for correspondence (ykyusik@gachon.ac.kr; choi@unist.ac.kr)
1
T2. Materials and Methods
T2.1. Chemicals and Materials
All chemicals were purchased from commercial sources. SWNT, AgNO3, NaBH4, DNA sodium salt from
herring testes type XIV, PVA, and sodium dodecyl sulfate (SDS) were obtained from Sigma Aldrich
Chemical (St Louis, MO, USA). Nitric acid (HNO3) and acetone were obtained from Duksan Pure
Chemicals (Gyeonggi-do, Korea). The water used throughout the experiments was deionized (DI) water
with a resistance > 18 MΩ, purified by a Milli-Q system from Millipore Co. (Milford, MA, USA).
T2.2. Carboxylation of SWNT (c-SWNT)
SWNT (20 mg) was refluxed with 15 mL of 2.6 M HNO3 for 48 h, centrifuged, and the supernatant was
removed. The sediment was washed several times in DI water. During each cleaning step, the sediment
was suspended in a sonication bath and recovered by centrifugation. The separated sediment was dried in
an oven at 40ºC and stored for later attachment of AgNPs.
T2.3. Incorporation of AgNPs on c-SWNT [1]
c-SWNT (10 mg) was dispersed in acetone then mixed with the 1 mM AgNO3 precursor and stirred for 2
h. The reducing agent (6 mM NaBH4) was slowly added to the mixture using a syringe pump at 10 mL/h
during stirring, which continued for 6 h. The AgNPs incorporated c-SWNT was purified by several
washes with DI water and separated by centrifugation. Finally, the separated sediment was dried in an
oven at 40ºC and stored for further use. The same procedure without c-SWNT was employed for the
synthesis of AgNPs. The presence of metal contents in the SWNT and the incorporation of AgNPs on cSWNT were determined by inductively coupled plasma mass spectrometry (ICPMS) and by the energy
dispersive X-ray analysis (EDXA) respectively.
T2.4. Wrapping DNA on the c-SWNT-AgNPs
c-SWNT-AgNPs (2 mg) and 2 mg of SDS were dispersed in 10 mL DI water by ultrasonication for 2 h.
DNA (2 mg in 1 ml DI water) was added to the above c-SWNT-AgNPs dispersion and sonicated for 1 h
and the same experiment was carried out without SDS to evaluate the toxicity of surfactant. The hybrid
was centrifuged and the sediment was washed several times in DI water to completely remove the
unbound DNA. In each cleaning step, sediment was suspended by sonication bath and recovered again
using centrifugation.
T2.5. Functionalization of the hybrid with polymer
PVA (50 mg dissolved in 4 mL) was added slowly to 10 mg of c-SWNT-AgNPs and 11 mL of c-SWNTAgNPs-DNA, solution and sonicated for 1 h to obtain a uniform solution. c-SWNT-AgNPs-PVA, cSWNT-AgNPs-DNA-PVA was concentrated in an oven at 40ºC to make a final volume of 10 mL. The
resulting solution contained 20 µg/mL of c-SWNT-AgNPs, 20 µg/mL DNA, and 50 µg/mL of PVA as
final concentrations. The hybrid was centrifuged and the sediment was washed several times in DI water
2
to completely remove the unbound PVA. In each cleaning step, sediment was suspended by sonication
bath and recovered again using centrifugation.
T2.6. Morphological Characterization
Images of the samples were obtained by high resolution transmission electron microscopy (TEM; JEOL1010), Bio-AFM (Nanowizard II, JPK), and field emission scanning electron microscopy (FE-SEM;
JEOL 7500F). The incorporation of AgNPs on SWNT was determined by EDXA combined with the FESEM. The sample was air dried and sputter coated with platinum before examination by FE-SEM. Cells
seeded on poly-L-lysine coated glass cover slips were incubated with materials and used for bioAFM
imaging. Cells were fixed by incubation in 2% glutaraldehyde for 45 seconds and simultaneous
incubation in 4% paraformaldehyde for 20 minutes. After aspirating paraformaldehyde, the cells were
washed with DI water and stored in PBS until usage.
T2.7. Zeta potential Measurements
Surface charge of materials was evaluated using Malvern Zetasizer Nano ZS series. The sample
concentration was 0.1 mg/mL, and the measurement was performed at pH 7.0.
T2.8. Determination of metal contents by inductively coupled plasma-mass spectroscopy (ICPMS) [2]
The wet digestion method was used for the pretreatment of samples utilizing hot concentrated acid. The
10 mg samples were put into a PTFE vessel, to which we added a 2 mL of H2SO4. The PTFE vessel was
then put on a hot plate at around 300°C for 14 h. When digestion was over, 1 mL of HNO3 was added 4
3
times separately. The remaining acid was driven off and the final solution was adjusted to a fixed volume
using 2% nitric acid and then diluted using DI water to make up 10 mL followed by evaporation. The
final digestion solutions were first screened for the presence of elements using ICPMS (Elan DRC Plus,
Perkin Elmer).
T2.9. Cell culture and cytotoxicity (CCK-8 Assay)
Human alveolar basal epithelial cell (A549 cells) was provided from the Korea Cell Line Bank (Seoul,
South Korea), fibroblast cells (NIH3T3) and human bone marrow stroma cells (hBMSC; HS-5) were
obtained from ATCC (Manassas, VA). The cells were used for the in vitro study of the toxicology of
materials. At day one, 1.0 × 104 cells were placed in each well of a 96-well plate in 100 μL of RPMI 1640
media (A549), Dulbecco’s modified Eagle’s medium (DMEM for NIH3T3 and HS-5 cells) containing
10% fetal bovine serum (FBS, purchased from GIBCO) and cultured for 24 h at 37°C. The final
concentrations of materials ranged from 40 µg/mL to 2.5 µg/mL and the cytotoxicity were evaluated after
the transfection in triplicate using a colorimetric CCK-8 assay kit (Dojindo Molecular Technologies Inc,
Gaithersburg, MD, USA) according to the manufacturer’s protocols. Measurement was performed using a
Magellan ELISA microplate reading spectrophotometer (Tecan) at wavelengths of 450 nm. Relative
viability was calculated using 0% (wells without cells) and 100% (wells with untreated cells) controls.
T2.10. Glutathione (GSH) Assay
The A549 cells were exposed to materials (1.25 ~ 40 ug/ml) for 24 h, followed by 1X106 cells were
collected by centrifugation at 1000g for 10min at 4°C. The collected cells were homogenized in 1ml 50
mM MES buffer. After homogenization cell lysate was centrifuged at 10000g for 15min at 4°C. The
cellular lysate, deproteinized with 5% sulfosalicylic acid, was analyzed for total GSH using the detectX®
Glutathione colorimetric detection kit and following manufacturer’s instructions. GSH concentration is
obtained by subtracting the oxidized GSH (GSSG) level from the total GSH. The total GSH levels were
calculated as nM protein.
T2.11. Evaluation of Lipid peroxidation (LPO)
LPO was estimated by TBARS assay, which detects malondialdehyde (MDA). A549 cells in an
exponential growth phase were seeded into a 6-well plate and incubated for 24 h. The cells were exposed
to materials (1.25 ~ 40 μg/ml) for 24 h, then 1X106 cells were harvested in 200 ul ice-cold PBS and
allowed to sonication for the complete disruption of cells. Flowingly 20 ul cell lysate was removed for
Bradford protein assay and 100 ul cell lysate was replaced into a new 1.5 ml tube separately. After which
200 ul ice cold 10% TCA (trichloroacetic acid) was added to the each sample and incubated for 5 min on
ice. The tube was centrifuged for 5 min at 12000g and clear supernatant was removed in a new labeled
tube. MDA was measured by QuantiChromTM thiobarbituric acid reactive substance assay (TBARS) Kit
and following manufacturer’s instructions. Results were calculated as nM MDA.
4
T2.12. Cell transfection for AFM studies
Cells were cultured in respective medium (Invitrogen, Grand Island, NY, USA) supplemented with 10%
heat-inactivated FBS (Invitrogen), 1% antibiotics (Invitrogen). Cells were routinely cultivated at 37 °C in
a humidified atmosphere of 5% CO2. Cells were seeded on Thermanox coverslips placed in a12 well
culture plate at a density of 2 × 105 cells per well and allowed to attach overnight and then incubation
medium was replaced by fresh media. Materials were prepared as colloidal dispersion solutions (10
µg/mL). The transfection was carried out by adding the prepared complex to the wells in a drop wise
manner. After 4 h incubation, the medium was exchanged again with fresh medium and transfected cells
were incubated for 24 h. After which the medium was removed and washed with PBS buffer and used for
AFM studies.
T2.13. Biomechanics analysis of cells by AFM [3,4]
All data were acquired at room temperature on Nanowizard II AFM (JPK instruments) mounted on an
inverted optical microscope (Nikon). The combined inverted optical stage helped precise lateral
positioning of the AFM tip over the region of interest in cells. AFM studies for cells were carried out in
liquid contact mode using 0 mM HEPES buffer (pH 7.4). AFM studies for materials were carried out air
contact mode. Biomechanics measurements of the materials and cells treated with materials was measured
using nanoindentation method utilizing silicon nitride (Si3N4) conical tip with the spring constant of 0.292
N/m, (tip height: 5.5 μm and tip radius: <8 nm) and coated with gold on back side of cantilever was used
with a tip. For mechanical analysis, tip-sample separation curve were recorded to measure the relative cell
stiffness (Young’s modulus, E) and cell surface adhesion. The Young’s modulus was subsequently
determined using Hertz’s contact model using JPK data processing software (JPK instruments). For
calculation of stiffness, the poison ratio of the cell was considered to be 0.5 and the half opening angle of
the tip was 35°. Importantly the tip deflection was restricted to low force ranges to provide soft
indentations (~700 nm) to prevent cell surface defects and hertz model limitation.
T2.14. Force-distance curve measurement [5]
Force-distance measurements were performed using special probe HYDRA6V-100NG (APP NANO).
Force-distance curve analysis between the probe-bare substrate, probe-fixed cell substrate and probe-fixed
cells transfected with materials on substrate were then measured by approaching the tip to contact the cell
interface. The force was acquired as a function of the tip-sample distance (μm). After the tip has come
into contact with the cell, the separate adhesion force (nN) during the extension and retraction of vertical
deflection of tip from the substrate surface was analyzed respectively. For all the force-distance curve
analysis, the sensitivity and spring constant of the cantilever were kept constant as 25 nm/V and 0.050
N/m, respectively.
T2.15. Surface roughness analysis [6]
5
The data obtained from the Bio-AFM height scale images were used to calculate the surface roughness of
control and materials treated cells. Using JPK image processing software v3.3.25, the cell surface was
estimated through the application of a mean filter to the raw/original data. Subtraction of the polynomial
surface from one or more rectangular regions of raw height image generated a flattened representation of
the cell. The surface roughness of a selected area of this flattened image was then calculated from the
height standard-deviation, i.e., the root-mean square roughness value (Rrms) of the height distribution:
Rrms =
Where, N is the total number of data points, Zi is the height of the ith point and Zm is the mean height.
Roughness value was measured over the particular cell surface areas with a fixed size of 40 х 40 μm2 (for
normal and materials treated cells).
T2.16. Statistics
Each group was respectively compared, using independent Student’s t-test. For all analyses, the
probability of type I error less than or equal to 0.05 was considered as statistically significant. Each test
was repeated for five/seven separate times.
6
Table S1a. Cytotoxicity data of A549 cells (CCK-8 assay)
No.
SWNT
c-SWNT
AgNPs
Hybrid
Hybrid SDS
Hybrid SDSDNA
Hybrid -
Hybrid -
DNA
PVA
HC
Control
100
100
101.4
100.1
100
100.0
100.0
103.6
100.9
40 µg/mL
38.2
74.6
85.2
96.9
55.5
24.3
53.0
76.2
97.7
10 µg/mL
77.7
86.6
102.6
101.4
89.1
90.4
87.9
96.8
100.0
2.5µg/mL
100.3
98.7
102.2
102.0
97.8
96.7
97.5
106.0
102.9
7
Table S1b. Glutathione assay for A549 cells treated with materials
No.
SWNT
c-SWNT
AgNPs
Hybrid
(nM)
(nM)
(nM)
(nM)
Hybrid
-SDS
(nM)
Hybrid
-SDSDNA
(nM)
Hybrid
Hybrid
-DNA
-PVA
(nM)
(nM)
HC
(nM)
NC
2.3
2.3
2.3
2.3
2.3
2.3
2.3
2.3
2.3
40 µg/mL
0.4
1.1
1.5
2.1
0.7
0.3
0.6
1.6
2.9
10 µg/mL
1.1
1.8
1.9
2.1
1.8
1.5
1.7
1.9
2.5
2.5 µg/mL
2.1
2.0
2.1
2.1
2
1.8
1.9
2.2
2.5
Table S1c. Evaluation of LPO A549 cells treated with materials
No.
SWNT
c-SWNT
AgNPs
Hybrid
(nM)
(nM)
(nM)
(nM)
Hybrid
-SDS
(nM)
Hybrid
-SDSDNA
(nM)
Hybrid
Hybrid
-DNA
-PVA
(nM)
(nM)
HC
(nM)
NC
4.9
4.9
4.9
4.9
4.9
4.9
4.9
4.9
4.9
40 µg/mL
15.2
10.5
7.5
6.2
12.7
19.4
11.8
9.5
5.5
10 µg/mL
11.7
8.1
6.2
4.9
10.6
9.7
9.3
6.8
4.9
2.5 µg/mL
6.1
5.8
5.0
4.9
6.5
6.4
6.6
4.8
4.9
8
Table S2. Force spectra parameters of materials and A549 cells Treated with materials
S No
Samples
Adhesion
force (nN)
Jump into
contact
(nm)
Attractive
force (nN)
Glass slide
1.16±0.1
5.57±0.4
0.32±0.23
1.
SWNT
0.14±0.01
6.68±0.8
0.01±0.001
2.
c-SWNT
9.23±1.36
9.21±1.17
0.11±0.05
3.
Hybrid
5.21±0.06
21.4±0.96
2.63±0.08
4.
Hybrid-DNA
3.78±0.27
11.71±2.9
0.62±0.16
5.
Hybrid-PVA
2.16±0.09
10.66±2.3
0.04±0.01
6.
HC
3.26±0.9
7.5±0.4
0.04±0.01
7.
A549
2.54±0.22
7.73±0.6
0.06±0.006
8.
A549-SWNT
13.2±1.36
19.58±1.39
0.2±0.06
9.
A549-c-SWNT
9.28±0.31
11.96±2.7
0.11±0.05
10.
A549-Hybrid
11.89±0.78
10.48±2.69
0.14±0.07
11.
A549- Hybrid-DNA
12.16±0.98
16.6±2.66
0.3±0.09
12.
A549- Hybrid-PVA
5.72±0.88
12.53±2.82
0.14±0.06
13.
A549-HC
2.95±0.24
9.54±4.7
0.08±0.07
9
Figure S3. Total Elemental analysis (ICP-MS)
Figure S4. EDXA of c-SWNT and c-SWMT-AgNPs
10
Figure S5 Roughness, measured on cytoplasm and invadopodia region of A549 cells treated with
materials
11
Figure S6. FS of materials and control
Force spectra (FS) of the samples A) glass slide, B) A549 cells, C) SWNT, D) c-SWNT, E) c-SWNTAgNPs, F) c-SWNT-AgNPs-DNA, G) c-SWNT-AgNPs-PVA, H) c-SWNT-AgNPs-DNA-PVA. The inset
shows respective AFM image used for FS analysis.
12
Figure S7. Force spectra of A549 cells treated with materials
a) A549+SWNT, b) A549+c-SWNT, c) A549+c-SWNT-AgNPs, d) A549+c-SWNT-AgNPs-DNA, e)
A549+c-SWNT-PVA, f) A549+c-SWNT-AgNPs-DNA-PVA (n=10). The inset shows respective AFM
image used for FS analysis. FS of the glass slide, A549 cells, and materials were given in supplementary
materials (Fig. S5).
13
Figure S8. Zeta potential of materials
14
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