pH-Induced Aggregation of Gold Nanoparticles for Photothermal ...
Supporting Information
pH-Induced Aggregation of Gold Nanoparticles for Photothermal Cancer Therapy
Jutaek Nam, Nayoun Won, Ho Jin, Hyokyun Chung, and Sungjee Kim*
Department of Chemistry, Pohang University of Science & Technology, San 31, Hyojadong, Namgu, Pohang 790-784, South Korea
*Sungjee Kim. Tel : 82-54-279-2108,
S1
Fax : 82-54-279-1498,
E-mail :
[email protected]
Experimental details. Methods General (±)-α-Lipoic acid and 1,1-carbonyldiimidazole, sodium borohydride, sodium citrate tribasic dehydrate, trypan blue were purchased from Sigma. Other reagents were obtained from Aldrich and all chemicals w ere used as received without further purification. Water was triply distilled using Millipore filtration syst em. 1H NMR was measured using FT-300MHz Brucker Aspect 3000 and TEM images were recorded us ing JEOL JEM-1011 in POSTECH biotech center. UV-VIS absorption spectra were obtained using Agile nt 8453. Mass spectral data were obtained from the Korea Basic Science Institute (Daegu) on JEOL JMS 700 high resolution mass spectrometer. Dark field images were recorded using Zeiss Axioplan 2 microscope with a highly numerical dark field condenser (0.75-1.0) and 100× / 1.3 oil Iris objective (Zeiss). The dark-field pictures were taken using Zeiss Axiocam HR camera. Confocal microscope images were acquired with laser scanning confocal microscope (Olympus, FV-1000) using a 633 nm cw He-Ne laser as the excitation source. The laser beam was focused by a 60x objective and the images were recorded with a 645-745 nm band pass filter to visualize scatterings by gold nanoparticles. The distributions of aggregates inside of the cells were obtained using z-scan mode.
Synthesis of N-(2-aminoethyl)-5-(1,2-dithiolan-3-yl)pentanamide, 1. (±)-α-Lipoic acid (2.00 g, 9.70 mmol) was dissolved in 12 ml anhydrous chloroform. 1,1carbonyldiimidazole (2.00 g, 12.3 mmol) was added to the lipoic acid solution and stirred for 5 min at room temperature. The resultant solution was added dropwise into ethylenediamine (3.5 ml, 48.4 mmol), and stirred for 40 min in ice bath and for another 30 min at room temperature. The crude product was washed three times with 20 ml of 10% NaCl aqueous solution and once with 20 ml water. It was dried
S2
with sodium sulfate and the solvent was removed using rotary evaporator to obtain yellow liquid (1.9 g). The yield was 78%. 1H-NMR (300 MHz, D2O) : δ 1.35-1.45 (m, 2H), 1.55-1.80 (m, 4H), 1.90-2.05 (m, 1H), 2.29 (t, J = 7.4 Hz, 2H), 2.40-2.55 (m, 1H), 3.10-3.30 (m, 4H), 3.49 (t, J = 6.0 Hz, 2H), 3.65-3.75 (m, 1H).
Synthesis of 4-(2-(5-(1,2-dithiolan-3-yl)pentanamido)ethylamino)-2-methyl-4-oxobut-2-enoic acid, 2. Citraconic anhydride (0. 68 ml, 7.5 mmol) was added dropwise into 15 ml anhydrous chloroform solution of 1 (5.0 mmol). The solution was stirred overnight at room temperature. Precipitate was filtered and washed with anhydrous chloroform to obtain yellow powder (1.3 g). The yield was 72%. 1HNMR (300 MHz, D2O) : δ 1.35-1.45 (m, 2H), 1.55-1.80 (m, 4H), 1.90-2.05 (m, 1H), 1.97 (s, 3H), 2.25 (t, J = 7.2 Hz, 2H), 2.40-2.55 (m, 1H), 3.10-3.30 (m, 2H), 3.32 (s, 4H), 3.65-3.75 (m, 1H), 5.57 (s, 1H). HRMS (m/z) : [M] calculated for C15H25O4N2S2, 360.1177; found, 360.1166, [M]+ calculated for C15H25O4N2S2, 361.1256; found, 361.1260
Synthesis of 4-(2-(6,8-dimercaptooctanamido)ethylamino)-3-methyl-4-oxobut-2-enoic acid, 3. As-synthesized 2 (0.26 g, 0.72 mmol) was dissolved in 5 ml water and the pH was adjusted to 10 using 2 M NaOH aqueous solution. Equal molar amount of sodium borohydride (0.029 g, 0.72 mmol) was added to the solution and stirred at room temperature for 30 min. The color of solution changed from yellow to transparence as the disulfide group being reduced. The resultant mixture was directly used for surface exchange of citrate gold nanoparticles without further purification. 1H-NMR (300 MHz, D2O) : δ 1.30-1.55 (m, 3H), 1.55-1.75 (m, 4H), 1.75-1.95 (m, 1H), 2.00 (s, 3H), 2.28 (t, J = 7.1 Hz, 2H), 2.50-2.70 (m, 2H), 2.85-3.0 (m, 1H), 3.34 (s, 4H), 5.62 (s, 1H).
Synthesis of 2-(5-(1,2-dithiolan-3-yl)pentanamido)-N,N,N-trimethylethanaminium chloride. Lipoic
acid
NHS-ester
(3.0
mmol)
was
S3
dissolved
in
18
ml
of
dioxane.
(2-
aminoethyl)trimethylammonium chloride (4.5 mmol) was dissolved in 18 ml of 0.25 M sodium carbonate buffer, and the pH was adjusted to 7.6 with 2 M NaOH aqueous solution. The solutions were slowly mixed together at -5 ℃ and then stirred for 24 h at room temperature. After insoluble bi-products were removed by vacuum filtration, the solvent was dried using rotary evaporator. The crude product was dissolved by 2 M NaOH aqueous solution, extracted with isopropanol, and dried with sodium sulfate. Final product was obtained as a sticky compound after the solvent removal. 1H NMR (300 MHz, CDCl3): δ 1.72-1.29 (m, 6H), 1.91 (m, 1H), 2.22 (t, J = 7.3 Hz, 2H), 2.41 (m, 1H), 3.91-3.07 (m, 11H), 3.42 (t, J = 6.7 Hz, 2H), 3.70-3.57 (m, 3H). HRMS (m/z) : [M] calculated for C13H27N2OS2, 291.16 ; found 290.99
Synthesis of citrate gold nanoparticles. 100 ml of 5 mM aqueous solution of hydrogen tetrachloroaurate hydrate (99.999%) was refluxed for 30 min. 10 ml of 150 mM aqueous solution of sodium citrate tribasic dihydrate was quickly added. The color of solution changed from yellow to purple and finally became red within 5 min. The mixture was kept boiling for another 2 h and then cooled for 30 min at room temperature. After cooling, the reaction solution was dialyzed three times using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification..
Synthesis of ‘smart’ gold nanoparticles. As-prepared solution of 3 was used directly for this step. 40 ml of citrate gold nanoparticle solution was mixed to the solution of 3 and stirred at room temperature. After 10 h, the reaction solution was dialyzed three times using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification.
Synthesis of 11-mercaptoundecanoic acid (MUA) capped gold nanoparticles. 3 ml of citrate gold nanoparticle solution was adjusted to pH 10.5 using 2 M NaOH aqueous solution,
S4
and 6 mg of MUA was added. The mixture was stirred at room temperature for 19 h and was filtered using 200 nm pore size syringe filter to remove the excess solid MUA. The reaction solution was dialyzed twice using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification.
Synthesis of primary amine-coated gold nanoparticles. N-(2-aminoethyl)-5-(1,2-dithiolan-3-yl)pentanamide (molecule 1 in scheme 1) was used for surface exchange of citrate gold nanoparticles. The chloroform solution of 1 (2 ml, 0.10 mmol) was mixed with 2 ml methanol. Sodium borohydride (0.04 g, 1.1 mmol) was added to the solution and stirred at room temperature for 30 min. The color of solution changed from yellow to transparence as the disulfide group being reduced. The resultant mixture was extracted using 1 M HCl aqueous solution. 10 ml of citrate gold nanoparticle solution was added and stirred at room temperature for 24 h. The reaction solution was dialyzed three times using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification.
Synthesis of quaternary amine-coated gold nanoparticles. 2-(5-(1,2-dithiolan-3-yl)pentanamido)-N,N,N-trimethylethanaminium chloride (0.03 g, 0.10 mmol) was dissolved in 2 ml water. Sodium borohydride (0.015 g, 0.40 mmol) was added to the solution and stirred at room temperature for 30 min. The color of solution changed from yellow to transparence as the disulfide group being reduced. The pH was adjusted to 1.3 using 1 M HCl aqueous solution. 20 ml of citrate gold nanoparticle solution was added and stirred at room temperature for 24 h. The reaction solution was dialyzed three times using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification.
Cell experiments. B16 F10 mouse melanoma cells, NIH 3T3 mouse embryonic fibroblast cells and HeLa human cervical S5
cancer cells were purchased from Korean Cell Line Bank. B16 F10 cells were incubated in Minimum Essential Medium with Earle’s Balanced Salts (MEM/EBSS, HyClone) which was supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillinstreptomycin (PS). HeLa and NIH 3T3 cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM, HyClone) with 10% FBS and 1% PS. Cells were grown onto 12 mm glass coverslips (for dark field imaging or confocal microscope imaging) or directly (for in vitro photothermal therapy) in a 24well plates at a density of 1×105 cells/well at 37 ℃ under 5% CO2. After 3 days, cells were incubated with ‘smart’ gold nanoparticles. Citrate and MUA-capped gold nanoparticles were used as control groups. Cells were rinsed with culture media (MEM/EBSS for B16 F10 and DMEM for HeLa and NIH 3T3) and exposed to laser illumination for in vitro photothermal therapy. Then they were stained with 0.4% trypan blue for 5 min to test cell viability. For dark field imaging, coverslips were fixed using 4% formaldehyde and mounted onto slide glass using aqueous mounting medium with anti-fading agent (biomeda corp.).
Cytotoxicity test To determine the cytotoxicity of ‘smart’ gold nanoparticles, B16 F10 cells were maintained in MEM/EBSS supplemented with 10% FBS and 1% PS. Before the nanoparticle treatment, cell suspension (5000 cells/well) was dispensed in a 96-well plate and incubated for 24 h at 37 °C under 5% CO2. ‘Smart’ gold nanoparticles were added into the growth medium in the plate so as the concentration to reach 5 nM, 50 nM, 100 nM . The plate was incubated for 3 h, 6 h, 12 h and 24 h. 10 µL of Cell Counting Kit-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to each well of the plate. After the further incubation for 2 h, absorbance at 450 nm was measured using a microplate reader. Results are expressed as the ratio of the absorbance of the positive control, no gold nanoparticle treated cells.
S6
Additional data.
Figure S1. 1H-NMR spectrum of 2 in D2O.
Figure S2. High Resolution Mass Spectrum of 2.
S7
Figure S3. TEM images (left side) and size distribution histograms (right side) of citrate gold nanoparticles (a) and ‘smart’ gold nanoparticles (b). The histograms were made from the TEM images by counting more than 100 particles.
S8
11.6 14.4
Figure S4. Hydrodynamic size distributions of citrate gold nanoparticles (black line) and ‘smart’ gold nanoparticles (red line) in D.I. water.
S9
Figure S5. Normalized absorption spectra of citrate gold nanoparticles (black line) and ‘smart’ gold nanoparticles (red line) in D.I. water.
Figure S6. Absorption spectra of citrate gold nanoparticles (a) and ‘smart’ gold nanoparticles (b) in D.I. water (black lines) and in 100 mM NaCl aqueous solution (red lines).
S10
Figure S7. Colloidal stability test of ‘smart’ gold nanoparticles in 100% bovine serum. (a) Hydrodynamic size distributions of ‘smart’ gold nanoparticles dispersed in D.I. water (black line) and in 100% bovine serum (red line); (b) Absorption time evolution of ‘smart’ gold nanoparticles dispersed in 100% bovine serum.
Figure S8. Colloidal stability test of ‘smart’ gold nanoparticles in cell growth medium. (a) Hydrodynamic size distributions of ‘smart’ gold nanoparticles dispersed in D.I. water (black line) and in cell growth medium (red line); (b) Absorption time evolution of ‘smart’ gold nanoparticles dispersed in cell growth medium.
S11
Figure S9. Illustration of the electrostatic attractions between ‘smart’ gold nanoparticles in acidic environment.
S12
Figure S10. Time evolutions of absorption for primary amine-coated gold nanoparticles in pH 4 (left), pH 6 (middle), pH 9 (right) buffer solutions.
Figure S11. Time evolutions of absorption for quaternary amine-coated gold nanoparticles in pH 4 (left), pH 6 (middle), pH 9 (right) buffer solutions.
S13
Figure S12. Z-scanning confocal microscope images of B16 F10 cells incubated with 100 nM ‘smart’ gold nanoparticles (top row) or with MUA-capped gold nanoparticles (bottom row), respectively for 24 hours.
Figure S13. Absorption time evolution of MUA-capped gold nanoparticles in cell growth medium.
S14
Figure S14. Normalized viability of B16 F10 cells treated with 0 nM (black), 5 nM (red), 50 nM (green) and 100 nM (blue) ‘smart’ gold nanoparticles that are incubated for 3, 6, 12, and 24 hours from left to right.
S15
pH-Induced Aggregation of Gold Nanoparticles for Photothermal Cancer Therapy
Jutaek Nam, Nayoun Won, Ho Jin, Hyokyun Chung, and Sungjee Kim*
Department of Chemistry, Pohang University of Science & Technology, San 31, Hyojadong, Namgu, Pohang 790-784, South Korea
*Sungjee Kim. Tel : 82-54-279-2108,
S1
Fax : 82-54-279-1498,
E-mail :
[email protected]
Experimental details. Methods General (±)-α-Lipoic acid and 1,1-carbonyldiimidazole, sodium borohydride, sodium citrate tribasic dehydrate, trypan blue were purchased from Sigma. Other reagents were obtained from Aldrich and all chemicals w ere used as received without further purification. Water was triply distilled using Millipore filtration syst em. 1H NMR was measured using FT-300MHz Brucker Aspect 3000 and TEM images were recorded us ing JEOL JEM-1011 in POSTECH biotech center. UV-VIS absorption spectra were obtained using Agile nt 8453. Mass spectral data were obtained from the Korea Basic Science Institute (Daegu) on JEOL JMS 700 high resolution mass spectrometer. Dark field images were recorded using Zeiss Axioplan 2 microscope with a highly numerical dark field condenser (0.75-1.0) and 100× / 1.3 oil Iris objective (Zeiss). The dark-field pictures were taken using Zeiss Axiocam HR camera. Confocal microscope images were acquired with laser scanning confocal microscope (Olympus, FV-1000) using a 633 nm cw He-Ne laser as the excitation source. The laser beam was focused by a 60x objective and the images were recorded with a 645-745 nm band pass filter to visualize scatterings by gold nanoparticles. The distributions of aggregates inside of the cells were obtained using z-scan mode.
Synthesis of N-(2-aminoethyl)-5-(1,2-dithiolan-3-yl)pentanamide, 1. (±)-α-Lipoic acid (2.00 g, 9.70 mmol) was dissolved in 12 ml anhydrous chloroform. 1,1carbonyldiimidazole (2.00 g, 12.3 mmol) was added to the lipoic acid solution and stirred for 5 min at room temperature. The resultant solution was added dropwise into ethylenediamine (3.5 ml, 48.4 mmol), and stirred for 40 min in ice bath and for another 30 min at room temperature. The crude product was washed three times with 20 ml of 10% NaCl aqueous solution and once with 20 ml water. It was dried
S2
with sodium sulfate and the solvent was removed using rotary evaporator to obtain yellow liquid (1.9 g). The yield was 78%. 1H-NMR (300 MHz, D2O) : δ 1.35-1.45 (m, 2H), 1.55-1.80 (m, 4H), 1.90-2.05 (m, 1H), 2.29 (t, J = 7.4 Hz, 2H), 2.40-2.55 (m, 1H), 3.10-3.30 (m, 4H), 3.49 (t, J = 6.0 Hz, 2H), 3.65-3.75 (m, 1H).
Synthesis of 4-(2-(5-(1,2-dithiolan-3-yl)pentanamido)ethylamino)-2-methyl-4-oxobut-2-enoic acid, 2. Citraconic anhydride (0. 68 ml, 7.5 mmol) was added dropwise into 15 ml anhydrous chloroform solution of 1 (5.0 mmol). The solution was stirred overnight at room temperature. Precipitate was filtered and washed with anhydrous chloroform to obtain yellow powder (1.3 g). The yield was 72%. 1HNMR (300 MHz, D2O) : δ 1.35-1.45 (m, 2H), 1.55-1.80 (m, 4H), 1.90-2.05 (m, 1H), 1.97 (s, 3H), 2.25 (t, J = 7.2 Hz, 2H), 2.40-2.55 (m, 1H), 3.10-3.30 (m, 2H), 3.32 (s, 4H), 3.65-3.75 (m, 1H), 5.57 (s, 1H). HRMS (m/z) : [M] calculated for C15H25O4N2S2, 360.1177; found, 360.1166, [M]+ calculated for C15H25O4N2S2, 361.1256; found, 361.1260
Synthesis of 4-(2-(6,8-dimercaptooctanamido)ethylamino)-3-methyl-4-oxobut-2-enoic acid, 3. As-synthesized 2 (0.26 g, 0.72 mmol) was dissolved in 5 ml water and the pH was adjusted to 10 using 2 M NaOH aqueous solution. Equal molar amount of sodium borohydride (0.029 g, 0.72 mmol) was added to the solution and stirred at room temperature for 30 min. The color of solution changed from yellow to transparence as the disulfide group being reduced. The resultant mixture was directly used for surface exchange of citrate gold nanoparticles without further purification. 1H-NMR (300 MHz, D2O) : δ 1.30-1.55 (m, 3H), 1.55-1.75 (m, 4H), 1.75-1.95 (m, 1H), 2.00 (s, 3H), 2.28 (t, J = 7.1 Hz, 2H), 2.50-2.70 (m, 2H), 2.85-3.0 (m, 1H), 3.34 (s, 4H), 5.62 (s, 1H).
Synthesis of 2-(5-(1,2-dithiolan-3-yl)pentanamido)-N,N,N-trimethylethanaminium chloride. Lipoic
acid
NHS-ester
(3.0
mmol)
was
S3
dissolved
in
18
ml
of
dioxane.
(2-
aminoethyl)trimethylammonium chloride (4.5 mmol) was dissolved in 18 ml of 0.25 M sodium carbonate buffer, and the pH was adjusted to 7.6 with 2 M NaOH aqueous solution. The solutions were slowly mixed together at -5 ℃ and then stirred for 24 h at room temperature. After insoluble bi-products were removed by vacuum filtration, the solvent was dried using rotary evaporator. The crude product was dissolved by 2 M NaOH aqueous solution, extracted with isopropanol, and dried with sodium sulfate. Final product was obtained as a sticky compound after the solvent removal. 1H NMR (300 MHz, CDCl3): δ 1.72-1.29 (m, 6H), 1.91 (m, 1H), 2.22 (t, J = 7.3 Hz, 2H), 2.41 (m, 1H), 3.91-3.07 (m, 11H), 3.42 (t, J = 6.7 Hz, 2H), 3.70-3.57 (m, 3H). HRMS (m/z) : [M] calculated for C13H27N2OS2, 291.16 ; found 290.99
Synthesis of citrate gold nanoparticles. 100 ml of 5 mM aqueous solution of hydrogen tetrachloroaurate hydrate (99.999%) was refluxed for 30 min. 10 ml of 150 mM aqueous solution of sodium citrate tribasic dihydrate was quickly added. The color of solution changed from yellow to purple and finally became red within 5 min. The mixture was kept boiling for another 2 h and then cooled for 30 min at room temperature. After cooling, the reaction solution was dialyzed three times using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification..
Synthesis of ‘smart’ gold nanoparticles. As-prepared solution of 3 was used directly for this step. 40 ml of citrate gold nanoparticle solution was mixed to the solution of 3 and stirred at room temperature. After 10 h, the reaction solution was dialyzed three times using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification.
Synthesis of 11-mercaptoundecanoic acid (MUA) capped gold nanoparticles. 3 ml of citrate gold nanoparticle solution was adjusted to pH 10.5 using 2 M NaOH aqueous solution,
S4
and 6 mg of MUA was added. The mixture was stirred at room temperature for 19 h and was filtered using 200 nm pore size syringe filter to remove the excess solid MUA. The reaction solution was dialyzed twice using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification.
Synthesis of primary amine-coated gold nanoparticles. N-(2-aminoethyl)-5-(1,2-dithiolan-3-yl)pentanamide (molecule 1 in scheme 1) was used for surface exchange of citrate gold nanoparticles. The chloroform solution of 1 (2 ml, 0.10 mmol) was mixed with 2 ml methanol. Sodium borohydride (0.04 g, 1.1 mmol) was added to the solution and stirred at room temperature for 30 min. The color of solution changed from yellow to transparence as the disulfide group being reduced. The resultant mixture was extracted using 1 M HCl aqueous solution. 10 ml of citrate gold nanoparticle solution was added and stirred at room temperature for 24 h. The reaction solution was dialyzed three times using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification.
Synthesis of quaternary amine-coated gold nanoparticles. 2-(5-(1,2-dithiolan-3-yl)pentanamido)-N,N,N-trimethylethanaminium chloride (0.03 g, 0.10 mmol) was dissolved in 2 ml water. Sodium borohydride (0.015 g, 0.40 mmol) was added to the solution and stirred at room temperature for 30 min. The color of solution changed from yellow to transparence as the disulfide group being reduced. The pH was adjusted to 1.3 using 1 M HCl aqueous solution. 20 ml of citrate gold nanoparticle solution was added and stirred at room temperature for 24 h. The reaction solution was dialyzed three times using Amicon ultra 100 KDa Mw cutoff centrifugal filters for purification.
Cell experiments. B16 F10 mouse melanoma cells, NIH 3T3 mouse embryonic fibroblast cells and HeLa human cervical S5
cancer cells were purchased from Korean Cell Line Bank. B16 F10 cells were incubated in Minimum Essential Medium with Earle’s Balanced Salts (MEM/EBSS, HyClone) which was supplemented with 10% fetal bovine serum (FBS, GIBCO) and 1% penicillinstreptomycin (PS). HeLa and NIH 3T3 cells were incubated in Dulbecco’s Modified Eagle Medium (DMEM, HyClone) with 10% FBS and 1% PS. Cells were grown onto 12 mm glass coverslips (for dark field imaging or confocal microscope imaging) or directly (for in vitro photothermal therapy) in a 24well plates at a density of 1×105 cells/well at 37 ℃ under 5% CO2. After 3 days, cells were incubated with ‘smart’ gold nanoparticles. Citrate and MUA-capped gold nanoparticles were used as control groups. Cells were rinsed with culture media (MEM/EBSS for B16 F10 and DMEM for HeLa and NIH 3T3) and exposed to laser illumination for in vitro photothermal therapy. Then they were stained with 0.4% trypan blue for 5 min to test cell viability. For dark field imaging, coverslips were fixed using 4% formaldehyde and mounted onto slide glass using aqueous mounting medium with anti-fading agent (biomeda corp.).
Cytotoxicity test To determine the cytotoxicity of ‘smart’ gold nanoparticles, B16 F10 cells were maintained in MEM/EBSS supplemented with 10% FBS and 1% PS. Before the nanoparticle treatment, cell suspension (5000 cells/well) was dispensed in a 96-well plate and incubated for 24 h at 37 °C under 5% CO2. ‘Smart’ gold nanoparticles were added into the growth medium in the plate so as the concentration to reach 5 nM, 50 nM, 100 nM . The plate was incubated for 3 h, 6 h, 12 h and 24 h. 10 µL of Cell Counting Kit-8 solution (Dojindo Laboratories, Kumamoto, Japan) was added to each well of the plate. After the further incubation for 2 h, absorbance at 450 nm was measured using a microplate reader. Results are expressed as the ratio of the absorbance of the positive control, no gold nanoparticle treated cells.
S6
Additional data.
Figure S1. 1H-NMR spectrum of 2 in D2O.
Figure S2. High Resolution Mass Spectrum of 2.
S7
Figure S3. TEM images (left side) and size distribution histograms (right side) of citrate gold nanoparticles (a) and ‘smart’ gold nanoparticles (b). The histograms were made from the TEM images by counting more than 100 particles.
S8
11.6 14.4
Figure S4. Hydrodynamic size distributions of citrate gold nanoparticles (black line) and ‘smart’ gold nanoparticles (red line) in D.I. water.
S9
Figure S5. Normalized absorption spectra of citrate gold nanoparticles (black line) and ‘smart’ gold nanoparticles (red line) in D.I. water.
Figure S6. Absorption spectra of citrate gold nanoparticles (a) and ‘smart’ gold nanoparticles (b) in D.I. water (black lines) and in 100 mM NaCl aqueous solution (red lines).
S10
Figure S7. Colloidal stability test of ‘smart’ gold nanoparticles in 100% bovine serum. (a) Hydrodynamic size distributions of ‘smart’ gold nanoparticles dispersed in D.I. water (black line) and in 100% bovine serum (red line); (b) Absorption time evolution of ‘smart’ gold nanoparticles dispersed in 100% bovine serum.
Figure S8. Colloidal stability test of ‘smart’ gold nanoparticles in cell growth medium. (a) Hydrodynamic size distributions of ‘smart’ gold nanoparticles dispersed in D.I. water (black line) and in cell growth medium (red line); (b) Absorption time evolution of ‘smart’ gold nanoparticles dispersed in cell growth medium.
S11
Figure S9. Illustration of the electrostatic attractions between ‘smart’ gold nanoparticles in acidic environment.
S12
Figure S10. Time evolutions of absorption for primary amine-coated gold nanoparticles in pH 4 (left), pH 6 (middle), pH 9 (right) buffer solutions.
Figure S11. Time evolutions of absorption for quaternary amine-coated gold nanoparticles in pH 4 (left), pH 6 (middle), pH 9 (right) buffer solutions.
S13
Figure S12. Z-scanning confocal microscope images of B16 F10 cells incubated with 100 nM ‘smart’ gold nanoparticles (top row) or with MUA-capped gold nanoparticles (bottom row), respectively for 24 hours.
Figure S13. Absorption time evolution of MUA-capped gold nanoparticles in cell growth medium.
S14
Figure S14. Normalized viability of B16 F10 cells treated with 0 nM (black), 5 nM (red), 50 nM (green) and 100 nM (blue) ‘smart’ gold nanoparticles that are incubated for 3, 6, 12, and 24 hours from left to right.
S15
