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: jungoh@pknu.ac.kr ; 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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