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Supporting Information
Thiol chitosan-wrapped gold nanoshells for near-infrared laser-induced
photothermal destruction of antibiotic-resistant bacteria
Panchanathan Manivasagana, Fazlurrahman Khana, Giang Hoanga, Sudip Mondala, Hyehyun
Kima, Vu Hoang Minh Doanc, Young-Mog Kima,b, Junghwan Oha,c,*
a Marine-Integrated Bionics Research Center, Pukyong National University, Busan 48513,
Republic of Korea.
b Department of Food Science and Technology, Pukyong National University, Busan 48513,
Republic of Korea.
c Department of Biomedical Engineering and Center for Marine-Integrated Biotechnology (BK21
Plus), Pukyong National University, Busan 48513, Republic of Korea.
* Corresponding author: Prof. Junghwan Oha,c,*
Email: [email protected] ; Tel: +82-51-629-5771, Fax: +82-51-629-5779.
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2. Materials and methods
2.1. Materials
Low-molecular-weight chitosan (M W 50,000–190,000 Da, deacetylation degree: 75–
85%), LA, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), N-
hydroxysulfosuccinimide sodium (sulfo-NHS), 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), propidium iodide (PI), 2,4,6-trinitrobenzene sulfonic acid
(TNBS), and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and
used without any further purification. SYTO 9 was obtained from Molecular Probes, Life
Technologies (Invitrogen, Carlsbad, CA, USA). S. aureus (Korean Collection for Type Cultures
(KCTC) 1916), P. aeruginosa (KCTC 1637), and E. coli (KCTC 1682) were obtained from the
KCTC. Tryptic soy agar (TSA) and tryptic soy broth (TSB) were obtained from Difco
Laboratories Inc. (Detroit, MI, USA). Luria-Bertani broth (LB) was obtained from Acumedia
(Neogen Lansing, MI, USA). A human embryonic kidney cell line (HEK 293), a human cervix
adenocarcinoma cell line (HeLa cells), and a human breast cancer cell line (MDA-MB-231 cells)
were purchased from the American Type Culture Collection (Manassas, VA, USA). Phosphate-
buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), antibiotics (1%
penicillin-streptomycin), and 10% fetal bovine serum (FBS) were obtained from Hyclone
Laboratories (Logan, UT, USA).
2.2. Synthesis of TC-AuNSs
AuNSs were synthesized according to the previously reported methods of thin gold
colloid coating on the surface of silica cores (Oldenburg, Averitt, Westcott & Halas, 1998; Pham,
Jackson, Halas & Lee, 2002). 120 nm silica cores were first prepared by a modified Stöber
reaction according to an earlier reported method (Stöber, Fink & Bohn, 1968). Then, 10 nm gold
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colloids were prepared according to an earlier reported method (Duff, Baiker & Edwards, 1993),
and thin seed gold colloids were layered on the surface of silica cores. 5 g of chitosan (C) was
completely dissolved in 500 mL of 2% acetic acid solution, and then C was synthesized
according to previously reported methods (Wang, Chang & Peng, 2011; Zhu et al., 2012) and
low-molecular-weight C was lyophilized. C-LA (TC) conjugate was prepared by covalent
coupling of the free amino groups of C and the carboxyl group of LA in the presence of
EDC/NHS (Manivasagan et al., 2018; Zhou et al., 2016). Briefly, 1.0 g of C, 0.247 g of LA (2.4
mM), 0.288 g of EDC (3 mM), and 0.651 g of NHS (6 mM) were completely dissolved in
distilled water (DW, 25 mL), and this mixed solution was stirred to react for 24 h. The solution
was then dialyzed, filtered, and lyophilized, and the product C-LA (thiol chitosan (TC)) was
obtained. For the surface modification of AuNSs by the covalent gold-thiol linkages, 10 mL of
TC (0.5 mg/mL) was added to the purified AuNS solution (20 mL) and stirred for 24 h. The
surface-modified AuNSs (TC-AuNSs) were purified by centrifugation and then lyophilized.
2.3. Characterization
2.3.1 Ultraviolet–Visible (UV–Vis)
The UV–Vis absorption spectra of the nanomaterials were observed using a Genesys 30S
UV–Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).
2.3.2. Field emission transmission electron microscopy (FETEM) and energy-dispersive X-ray
spectroscopy (EDX)
FETEM images were obtained using a JEM-2100F microscope (JEOL, Tokyo, Japan)
operating at an accelerating voltage of 200 kV in combination with EDX.
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2.3.3. Dynamic light scattering (DLS) and zeta potential (ZP)
The particle size distribution and ZP of the samples were measured using a DLS-8000
light scattering system (Otsuka Electronics Co., Ltd., Osaka, Japan). The ZP for different pH
values were measured using a Zetasizer 3000HSA (Malvern Instruments, Malvern, UK).
2.3.4. Fourier transform infrared (FTIR) spectroscopy
FTIR spectroscopy was performed at room temperature (25°C) on a PerkinElmer
Spectrum 100 FTIR spectrometer (PerkinElmer, Waltham, MA, USA).
2.3.5. Nuclear magnetic resonance spectra (1H NMR)
The samples were determined by 1H NMR (JNM ECP-400 spectrometer operating at 400
MHz; JEOL) using D2O as the solvent.
2.3.6. TNBS methods
The substitution degree of amino groups (SD, %) of TC was determined using a TNBS
reaction (Hu, Liu, Du & Yuan, 2009).
2.3.7. NIR laser and IR camera
An NIR laser with a wavelength of 808 nm was used for all experiments. Thermograph
images were recorded using an FLIR i5 IR camera (FLIR Systems Inc., Portland, OR, USA).
2.3.8. Field emission scanning electron microscopy (FESEM)
FESEM (JSM-6700F; JEOL) images were used to observe the morphological changes of
bacteria.
2.3.9. Stability of nanomaterials
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For the stability of TC-AuNSs under various biological conditions, TC-AuNSs were
added to DW, PBS, and DMEM with 10% FBS for 15 days. The stability of TC-AuNSs was
characterized using a UV–Vis spectrophotometer, FETEM, DLS, and ZP.
2.4. Measurement of the photothermal effect of TC-AuNSs
In order to measure the photothermal effect of TC-AuNSs, the UV–Vis absorption
spectra of the TC-AuNSs at various concentrations (95, 105, and 115 µg/mL) were recorded
using a Genesys 30S UV–Vis spectrophotometer. 1 mL of TC-AuNSs at various concentrations
in 12-well plates was exposed to an 808 nm laser with an intensity of 0.45 and 0.95 W/cm 2 for 5
min. Changes in temperature were detected with a thermocouple, and thermal IR images were
also recorded with an IR camera. For the photothermal stability of TC-AuNSs, 1 mL of TC-
AuNSs (115 µg/mL) was exposed for 5 min to an NIR laser with an intensity of 0.95 W/cm 2,
followed by cooling six times for 15 min at ambient temperature without NIR laser irradiation.
Then, the photothermal stability of TC-AuNSs was characterized using a UV–Vis
spectrophotometer, FETEM, DLS, and ZP after six cycles of laser irradiation.
2.5. Microorganisms and growth conditions
S. aureus and P. aeruginosa were cultured in TSB, whereas E. coli were cultured in LB
overnight (12 h) at 37 °C under shaking at 200 rpm (Dasagrandhi, Park, Jung & Kim, 2018;
Khan, Manivasagan, Lee, Pham, Oh & Kim, 2019; Murugan et al., 2013). The preculture of all
bacteria was diluted in a ratio of 1:100 in fresh TSB or LB and then allowed to continue
incubation for another 6 h to achieve an optical density (OD600 nm) of 0.4–0.7. Bacterial cultures
were centrifuged at 15,000 ×g for 3 min and washed three times with PBS (100 mM, pH 7.2).
The bacterial pellet was suspended in PBS, and the turbidity of the cell suspension was measured
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at 600 nm using a spectrophotometer, corresponding to a bacterial concentration of
approximately 106 colony forming units (CFU) per milliliter (CFU/mL).
2.6. Photothermal ablation of bacteria
S. aureus, and P. aeruginosa were cultured in TSB, and E. coli were cultured in LB and
incubated at 37 °C under shaking (200 rpm) overnight (12 h) (Murugan et al., 2013). The
overnight (12 h) grown cell culture of all bacteria was then 100-fold-diluted in TSB and LB and
incubated for another 6 h until the optical density (OD600 nm) reached 0.4–0.7. Then, the whole
bacterial culture was diluted to 106 CFU/mL and added to 12-well plates. All bacterial cultures
were treated with TC-AuNSs at various concentrations (95, 105, and 115 µg/mL), and control
bacterial cultures were treated with PBS for 5 h. These wells were either irradiated or not
irradiated using an 808 nm laser with an intensity of 0.95 W/cm2 for 5 min. After irradiation, all
bacterial cultures were serially diluted up to a 10-6 dilution factor in fresh TSB media, and the
diluted cell culture (100 µL) was spread plated on a TSA-agar plate and incubated overnight (24
h). Finally, all bacterial colonies that appeared on the TSA plate were counted.
2.7. Minimum inhibitory concentration (MIC) determination
The MIC of TC-AuNSs against P. aeruginosa, S. aureus, and E. coli was assessed
according to an earlier described procedure (Khan, Manivasagan, Lee, Pham, Oh & Kim, 2019).
Briefly, the overnight (12 h) grown cell culture (250 µL) of all strains at 1:100-fold dilution was
added to a 96-well microtiter plate. These cultures were treated with different concentrations of
TC-AuNSs, ranging from 16 to 4,096 µg/mL. The cell culture in the respective media without
TC-AuNSs was taken as the control. The TSB and LB growth media (250 µL) were also added
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to the titer plate as a negative control. The plate was then incubated at 37 °C under shaking (120
rpm) in titer plate reader for 24 h. The optical density of the cell growth was measured at 600
nm.
2.8. Bacterial morphology determination using FESEM
The morphology of S. aureus, P. aeruginosa, and E. coli was examined using FESEM
according to a previously reported protocol with slight modifications (Kim, Kang, Jeong,
Sharker, In & Park, 2015). All bacterial cells (with an initial OD of 0.4–0.7 at 600 nm) were
added to 12-well plates, with a nylon membrane (0.5 × 0.5 cm) kept at the bottom of the plates.
All bacterial cells were then treated with TC-AuNSs (115 µg/mL) for 5 h at 37 °C, and the cells
were exposed to an 808 nm laser for 5 min with an intensity of 0.95 W/cm 2. Then, all bacterial
cells were fixed with glutaraldehyde (2.5%) overnight (12 h) at 4 °C and then washed with PBS
(100 mM, pH 7.2). The samples were then dehydrated with a sequential treatment with an
increasing concentration (50%–100%) of alcohol solution for 20 min. A freeze-dried membrane
was attached to FESEM stubs, coated with gold, and imaged using FESEM (Khantamat et al.,
2015).
2.9. Fluorescence microscopy
For fluorescence imaging of bacterial cells, 1 mL of TC-AuNSs (115 µg/mL) solution
was mixed with 1 mL of each of S. aureus, P. aeruginosa, and E. coli suspensions (106
CFU/mL). After 5 h of incubation, all bacterial cells were irradiated using an 808 nm laser with
an intensity of 0.95 W/cm2 for 5 min. After irradiation, all bacterial cells were stained with
SYTO 9 (green) and PI (red) to indicate the live and dead cell populations (Kim, Kang, Jeong,
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Sharker, In & Park, 2015). Finally, 10 µL of the sample was placed on a glass side and
visualized using an inverted microscope (DMI300B; Leica Microsystems GmbH, Wetzlar,
Germany).
2.10. Cell viability assay
HEK 293 cells, HeLa cells, and MDA-MB-231 cells (10,000 cells per well) were seeded
on a 96-well plate and incubated overnight (24 h). A culture medium containing TC-AuNSs at
various concentrations (5, 20, 40, 70, 95, 105, 115, 135, and 150 µg/mL) was added to the cells
and cultured for 24 h. After incubation, all cells were irradiated using an 808 nm laser with an
intensity of 0.95 W/cm2 for 5 min and incubated for another 2 h. Then, the cell viability of TC-
AuNSs was assessed by an MTT assay, where absorbance at 570 nm was quantified using a
microplate reader (Tecan Infinite F50).
2.11. Statistical analysis
Data were expressed as the mean ± SD and analyzed using one-way analysis of variance.
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Fig. S1. The synthetic scheme of TC-AuNSs.
Fig. S2. The scheme showing the synthetic preparation of TC-AuNSs.
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Fig. S3. EDX analysis of TC-AuNSs.
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Fig. S4. Zeta potentials in different pH values for the AuNSs and TC-AuNSs.
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Fig. S5. FETEM image of TC-AuNSs in FBS after 15 days.
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Fig. S6. Thermal IR images of TC-AuNSs at various concentrations with the 808 nm laser
irradiation with an intensity of 0.95 W/cm2 for 5 min.
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Fig. S7. FETEM image of TC-AuNSs after six cycles of the laser on/off.
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Fig. S8. Minimum inhibitory concentration determination of TC-AuNSs against P.
aeruginosa, S. aureus and E. coli. The absorbance of the negative controls (blank broth
medium) was found to 0.097, hence on the basis of the negative control, the absorbance
>0.2 were considered as the positive growth.
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Fig. S9. Cell viability of HEK 293 cells, HeLa cells, and MDA-MB-231 cells after treatment
with different concentrations of TC-AuNSs with or without laser irradiation at 0.95 W/cm2
for 5 min.
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