Effect of Size-Dependent Photodestructive Efficacy by Gold Nanomaterials with Multiphoton Laser

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1 Supporting information Effect of Size-Dependent Photodestructive Efficacy by Gold Nanomaterials with Multiphoton Laser Wen-Tsan Chang,,Δ Shean-Jen Chen,,,,Δ Chia-Yuan Chang,,,Δ Yi-Hsien Liu, Chang-Hsin Chen, Chen-Han Yang, # Lawrence Chao-Shan Chou,, Jui-Cheng Chang,, Li-Chung Cheng, Wen-Shuo Kuo, *,,,, and Jiu-Yao Wang *,,, Department of Biochemistry and Molecular Biology, National Cheng Kung University, Tainan 7, Taiwan Center for Micro/Nano Science and Technology, National Cheng Kung University, Tainan 7, Taiwan Department of Engineering Science, National Cheng Kung University, Tainan 7, Taiwan Advanced Optoelectronic Technology Center, National Cheng Kung University, Tainan 7, Taiwan Antagene, Inc., Santa Clara, California 9554, United States #Institute of Polymer Science and Engineering, National Taiwan University, Taipei 6, Taiwan Department of Chemical Engineering, National Cheng Kung University, Tainan 7, Taiwan Instrument Center, National Cheng Kung University, Tainan 7, Taiwan Allergy & Clinical Immunology Research Center, National Cheng Kung University, Tainan 7, Taiwan Department of Pediatrics, National Cheng Kung University, Tainan 7, Taiwan * To whom correspondence should be addressed. wenshuokuo@ncku.edu.tw; a@mail.ncku.edu.tw Δ Contributed equally to this work.

2 Intensity (AU) Intensity (AU) a Wavelength (nm) b Wavelength (nm) Figure S. Two-photon fluorescence (TPF) spectra of (a) fluorescein (in water, ph ) and (b) Rhodamine B (in methanol) (Sigma Aldrich Co. USA), which were exposed to the. Excitation wavelength: 75 nm.

3 Diameter (mn) Fluorescence (AU) Fluorescence (AU) a.e+8 b.e+7.e+7.e+6.e+5.e+4.e+.e+ Slope.99.E+.E+ Excitation intensity (mw).e+6.e+5.e+4.e+.e+ Slope..E+.E+ Excitation intensity (mw) Figure S. Plots of dependence of two-photon excited fluorescence on excitation intensity for (a) fluorescein (in water, ph ) and (b) Rhodamine B (in methanol), which were exposed to the from to 8 mw. Excitation wavelength: 75 nm.. The slope is indicated in each figure. R > nm Au-PEI-ICG 99nm Au-PEI-ICG 5 48nm Au-PEI-ICG nm Au-PEI-ICG 4 Time (day) Figure S. Size stability test of all sizes of Au-PEI-ICG in aqueous solution for 4 days. The diameter of Au nanomaterials was determined by TEM (n=).

4 a d 5 4 b e c f Wavenumber (cm - ) Wavenumber (cm - ) Figure S4. Fourier transform infrared spectroscopy (FTIR) spectroscopy was used to analyze the exposed functional groups. FTIR spectra of (a) PEI (band C-N stretching 9 cm -, band C-H bending 4 and 4 cm -, band N-H bending 46 cm -, band 4 C-H stretching 86 cm - and band 5 N-H stretching 8 cm - ), (b) ICG (band C-N stretching 8 cm -, band aromatic ring stretching 46 cm - and band N-H stretching 468 cm - ), (c) nm Au-PEI-ICG (band C-N stretching 64 cm -, band aromatic ring stretching 4 cm - and band N-H stretching 56 cm - ), (d) 48 nm Au-PEI-ICG (band C-N stretching cm -, band aromatic ring stretching 458 cm - and band N-H stretching 554 cm - ), (e) 99 nm Au-PEI-ICG (band C-N stretching 68 cm -, band aromatic ring stretching 44 cm - and band N-H stretching 47 cm - ) and (f) 5 nm Au-PEI-ICG (band C-N stretching 75 cm -, band aromatic ring stretching 46 cm - and band N-H stretching 45 cm - ).

5 Bacterial viability (%) Control nm Au-PEI-ICG - Ab protein A 48nm Au-PEI-ICG - Ab protein A 99nm Au-PEI-ICG - Ab protein A E+ E+ E+ Number of Au 5nm Au-PEI-ICG - Ab protein A Figure S5. MRSA were incubated with all sizes of Au-PEI-ICG -antibodies in the dark for 4 h and then their viability was evaluated.. The bacterial viability was converted from the CFU counting method (Figure a) into percent. Delivered dose: ~ Au ;.74 - M of free ICG and ICG coated on the surfaces of Au. Data are means ± SD (n = 6).

6 Viable count (CFU ml-) Bacteria alone with no femtosecond laser exposure Bacteria with 99 nm Au-PEI-ICG exposed to Bacteria with nm Au-PEI-ICG exposed to Bacteria with 5 nm Au-PEI-ICG exposed to Bacteria with 48 nm Au-PEI-ICG exposed to Figure S6. MRSA had been treated with Au-PEI-ICG -Ab protein A, and then exposed to femtosecond laser. The viabilities were evaluated using the CFU counting assay. Results were similar to the quantified viability by LIVE/DEAD kit. Delivered dose: Au ;.74 - M of free ICG and ICG coated on the surfaces of Au. Data are means ± SD (n = 6).

7 Bacterial viability (%) Cell viability (%) a ICG b nm Au d e c * * * * * f nm Au Sample with no Sample with ICG exposed to 99 nm Au 5 nm Au 5 μm mm Sample with nm Au exposed to Sample with 99 nm Au exposed to Sample with 48 nm Au exposed to Sample with 5 nm Au exposed to Figure S7. (a-c) Photodestruction of MRSA shown by fluorescence. (a-b) Images and viabilities of MRSA after they had been treated with ICG-Ab protein A and Au -Ab protein A, and then exposed to. The bacteria were stained with a LIVE/DEAD kit to obtain the fluorescent images, and (c) quantification for viability was estimated. (d-e) ICG and all sizes of Au with conjugated Ab EGFR treated A549 cells, respectively, and were irradiated by femtosecond multiphoton laser. The dotted circles indicate the laser beam area. The cells were stained with calcein AM after irradiation following exposure to laser. The live cells exhibited green fluorescence. (f) The cell viability estimation of ICG-Ab EGFR -treated- and Au -Ab EGFR -treated-a549 cells. Data are means ± SD (n = 6). p < *p value obtained by the Student s t test.

8 Cell Viability (%) After nonlinear laser exposure, Au-PEI-ICG -Ab EGFR still showed a high viability which suggested that the ICG conjugated on the surface of Au were stable and subject to decompose, and may not degrade and produce the laser-induced decomposition products to kill cells even after laser exposure (Figure S8). Control ICG 5 nm Au-PEI- ICG With laser exposure and incubated with cells for h With laser exposure and incubated with cells for day With laser exposure and incubated with cells for 4 days Figure S8. To confirm the stability of Au-PEI-ICG, the MTT assay was conducted. For the long-term stability, ICG-Ab EGFR and 5 nm-au-pei-icg -Ab EGFR were exposed to a femtosecond laser first to test the photostability of ICG and Au nanomaterials, and then added to the confluent cells in a 96-well culture plate and continued to incubate for an additional h, day and 4 days at 7 C in the dark, respectively. And use the MTT assay to estimate the viability. Data are means ± SD (n = 6).

9 More convincing evidence came from EDX measurement and employed to investigate the photostability. We chose 5 nm Au-PEI-ICG for this experiment. After exposure, there was no any content drop of the sulfur from ICG (Figure S9). In other word, no variation of Au nanomaterials was existed and revealed the good photostability. a Element Atomic % C 9.8 O. S.8 Cu.95 Au. b Element Atomic % C 9.6 O.67 S.58 Cu. Au.6 c Figure S9. (a) Representation of ICG structure. EDX analysis of 5 nm Au-PEI-ICG (b) before and (c) after irradiation at a power density of 5. mw for 5 sec, respectively

10 Table S. Two-photon action cross sections of fluorescein (in water, ph ) and Rhodamine B (in methanol). Excitation wavelength: 75 nm. Excitation wavelength at 75 nm Action cross section,ησ (GM, Fluorescein ( in water, ph ) Rhodamine B (in methanol) -5 cm 4 s/photon) Table S. Evaluation of the Au particle number per bacterium or cell of specific targeting or internalization of Au-PEI-ICG for MRSA and A549 cell, respectively. nm Au-PEI-ICG 48nm Au-PEI-ICG 99nm Au-PEI-ICG 5nm Au-PEI-ICG MRSA A549 cell References () Engel, E.; Schraml, R.; Maisch, K.; König, B.; Szeimies, R. M.; Hillenkamp, J.; Bäumler, W.; Vasold, R. Light-Induced Decomposition of Indocyanine Green. Invest. Ophthalmol. Vis. Sci. 8, 49,