Supporting Information. A Real-Time Surface Enhanced Raman Spectroscopy Study of Plasmonic Photothermal Cell Death Using Targeted Gold Nanoparticles
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1 Supporting Information A Real-Time Surface Enhanced Raman Spectroscopy Study of Plasmonic Photothermal Cell Death Using Targeted Gold Nanoparticles Mena Aioub and Mostafa A. El-Sayed* Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia * melsayed@gatech.edu
2 O.D. (Normalized) Citrate-AuNP PEG-AuNP NT-AuNP (529 nm) (531 nm) (533 nm) Wavelength (nm) Figure S1. Characterization of nuclear-targeted AuNPs (NT-AuNPs). UV-Vis extinction spectrum of AuNPs before (black) and after conjugation with PEG (red), and RGD and NLS peptides (blue) in aqueous solution. The red shift in the plasmon peak indicates successful conjugation. S2
3 Raman Scatting Intensity (a.u.) 40 min 60 min 40 min 60 min 80 min 100 min 80 min 100 min Raman Shift (cm -1 ) S S vibrations C S vibrations Phe vibrations C N vibrations Amide III vibrations Figure S2. Time-dependent SERS spectra collected from a single HSC-3 cell incubated with 0.20 nm NT-AuNPs showing no PPT cell death induced by continuous laser exposure at lower power (2.6 mw). The vertical line at 1225 cm -1 highlights the vibrational peak from the amide III β-confirmation, which does not shift during laser exposure at low power, indicating a lack of cell death. Corresponding dark-field images for each spectrum are also shown. S3
4 Raman Scatting Intensity (a.u.) 40 min 60 min 40 min 60 min 80 min 100 min 80 min 100 min Raman Shift (cm -1 ) S S vibrations C S vibrations Phe vibrations C N vibrations Amide III vibrations Figure S3. Time-dependent SERS spectra collected from a single HSC-3 cell incubated with 0.10 nm NT-AuNPs showing no PPT cell death induced by continuous laser exposure at higher power (9.4 mw). The vertical line at 1225 cm -1 highlights the vibrational peak from the amide III β-confirmation, which does not shift during laser exposure when lower nanoparticle concentration is used, indicating a lack of cell death. Corresponding dark-field images for each spectrum are shown. S4
5 O.D. (Normalized) A Citrate-sAuNP (520 nm) PEG-sAuNP (522 nm) NT-sAuNP (523 nm) B Wavelength (nm) 100 nm Figure S4. Characterization of smaller nuclear-targeted AuNPs (NT-sAuNPs). UV-Vis spectrum of saunps before (black) and after conjugation with PEG (red), and RGD and NLS peptides (blue) in aqueous solution. The red shift in the plasmon peak indicates successful conjugation. (B) A TEM micrograph of the 15 nm citrate stabilized saunps. S5
6 Raman Scatting Intensity (a.u.) 5 min 5 min 10 min 10 min 15 min 15 min 25 min 25 min 30 min 30 min 60 min 60 min 90 min 90 min Raman Shift (cm -1 ) S S vibrations C S vibrations Phe vibrations C N vibrations Amide III vibrations Figure S5. Time-dependent SERS spectra collected from a single HSC-3 cell incubated with 0.20 nm NT-sAuNPs during PPT cell death induced by continuous laser exposure at lower power (6.2 mw). The vertical line at 1225 cm -1 highlights the shift in this vibrational peak from the amide III β-confirmation to the aromatic amino acid peaks at 1209 cm -1, which in conjunction with the disappearance of vibrations around 500 cm -1 and in the 1250 cm cm -1 range signals cell death. Corresponding dark-field images for each spectrum are also shown. S6
7 Raman Scatting Intensity (a.u.) 5 min 5 min 10 min 10 min 15 min 15 min 25 min 25 min 30 min 30 min 60 min 60 min 90 min 90 min Raman Shift (cm -1 ) S S vibrations C S vibrations Phe vibrations C N vibrations Amide III vibrations Figure S6. Time-dependent SERS spectra collected from a single HSC-3 cell incubated 0.20 nm NT-sAuNPs during PPT cell death induced by continuous laser exposure at higher power (9.4 mw). The vertical line at 1225 cm -1 highlights the shift in this vibrational peak from the amide III β-confirmation to the aromatic amino acid peaks at 1209 cm -1, which in conjunction with the disappearance of additional vibrations around 500 cm -1 and in the 1250 cm cm -1 range signals cell death. Corresponding dark-field images for each spectrum are also shown. S7
8 A B C D Figure S7. Representative dark-field scattering images of cells incubated with (A,B) 0.2 nm NT- AuNPs or (C,D) 0.20 nm NT-sAuNPs before (A,C) and after (B,D) continuous laser exposure (2 hours, 9.4 mw). White arrows denote the cell of interest. The images illustrate the single cell specificity of this method, as only the cell of interest is irradiated and displays morphological changes while surrounding cells are relatively unchanged and continue to move freely on the coverglass. The scale bar in each image is 50 μm. S8
9 Comp-PI-A Comp-PI-A Comp-PI-A Comp-PI-A Comp-PI-A Comp-PI-A A B Figure S8. Flow cytometry data showing cells incubated with 0.10 nm NT-AuNPs are over 90% healthy before oven heating (A); after oven heating (B), over 95% of cells exhibit late apoptosis/necrosis as evidenced by the increase in both FITC and PI fluorescence. S9
10 Experimental Section Smaller (15 nm) Gold Nanoparticle Synthesis and Functionalization Smaller citrate capped gold nanoparticles (saunps) were also prepared following the Turkevich/Frens citrate reduction technique. A 100 ml aqueous solution of 0.25 mm chloroauric acid was heated with stirring. Just before the solution reached boiling, 2 ml of 65 mm sodium citrate was added. Stirring and heating were discontinued and the solution was allowed to cool to room temperature when a red-wine color was observed. The citrate stabilized AuNPs (15 ± 1 nm, Figure S4B) were first cleaned by centrifugation at 9500 g for 15 min and redispersed in deionized (DI) water. The AuNPs were then conjugated with PEG to increase stability, enhance biocompatibility, and prevent nonspecific interactions in physiological environments. A 1.0 mm solution of mpeg-sh in deionized (DI) water was added to achieve a ~20% surface coverage. The PEG-AuNP solution was shaken at room temperature overnight and unbound ligands removed by centrifugation (12,500 g, 10 min). PEG-sAuNPs were again dispersed in DI water, followed by conjugation with a 1:10 ratio of RGD and NLS peptides to fill remaining surface sites. The AuNPs were again centrifuged (12,500 g, 10 min) to remove unbound peptides and dispersed in DI water to yield the smaller nuclear targeted gold nanoparticles (NT-sAuNPs). Apoptosis-Necrosis Assay Cells were seeded in culture medium for a 70% final confluence and allowed to adhere overnight. The growth media was then removed and the cells incubated with 0.10 nm NT- AuNPs in DMEM for 24 h. After nanoparticle incubation, the NT-AuNP solution was replaced with DMEM and cells to be heated were placed in a 100 C oven for 7 min to induce cell death. For flow cytometric analysis, cells were trypsinized for 7 min and collected via centrifugation (7 min, 250 g). Cells were washed twice with cold PBS and redispersed in 500μl Annexin V binding buffer. Cells were then incubated with 5 μl of Annexin V-FITC and 2 μl of PI (100μg/ml) for 15 min at room temperature. Following incubation, 493 μl of Annexin V binding buffer was added and the samples were immediately analyzed via flow cytometry. Fluorophores were excited with a 488 nm laser and detected by a 525/30 BP filter for FITC and a 575/30 BP filter for PI. Sample populations consisted of at least 10,000 events and results were analyzed using FlowJo software (TreeStar, Inc.). S10
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