Supporting Information

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1 Supporting Information Methods and Materials Synthesis of PEGylated NGS. Graphene oxide (GO) was prepared by a modified Hummers method, using flake expandable graphite as the original material according to our previous protocol 1,2. To prepare NGS-PEG, GO aqueous suspension (5ml) at a concentration of ~3 mg/ml was sonicated for about 30 min to give a clear solution. NaOH (0.12 g/ml) was added to the GO suspension and bath sonicated for about 3 h. The resulting solution was neutralized, purified by repeated rinsing and centrifugation. A solution of 6-arm-polyethylene glycol-amine (Sunbio Inc.) (3 mg/ml) was added to the GO solution (0.5 mg/ml) and the mixture was sonicated for five minutes. N-(3-dimethylaminopropyl-N -ethylcarbodiimide) hydrochloride (EDC, from Fluka Inc.) was then added to the mixture in two equal portions to give a final concentration of 1 mg/ml totally. The reaction was allowed overnight, yielding a NGS-PEG solution which was stored at 4 o C. Fluorescent labeling of NGS-PEG. Excess PEG in the as-synthesized NGS-PEG sample was removed by centrifuge filtration through Amicon centrifugal filters (Millipore) with 100 kda molecular weight cut off (MWCO) and washed with water for 6 times. The purified NGS-PEG (0.5 mg/ml) was reacted with an amine reactive dye, Cy7-SE (Fanbo Biochemicals Co., Ltd. Beijing, China) at 0.1 mg/ml in a ph 7.5 phosphate buffer (0.02 M). The reaction was allowed overnight at room temperature by avoiding light. Excess dye molecules were removed by centrifugation filtration through 100 kda MWCO Amicon filters and washed away with water for S1

2 over 8 times until no noticeable color in the filtrate solution. NGS-PEG-Cy7 was injected into mice in the 0.9 % NaCl saline solution. Determination of NGS and Cy7 concentrations. To determine the NGS concentration, solutions of NGS before PEGylation were measured by a UV-VIS-NIR spectrometer (Cary-6000i), frozen dried and weighed. The weight extinction co-efficient of NGS at 800 nm was determined to be 6.7 L g cm -1. Based on the AFM measurement, we assume that NGS had a sphere shape with an averaged diameter of 30 nm. The molecular weight of NGS is calculated to be 322 kda. The molar extinction co-efficient of Cy7 is 250,000 L mol cm -1 at its absorption peak 3. The estimated number of Cy7 per NGS is calculated by dividing the Cy7 concentration by the estimated NGS molar concentration Xenograft tumor models. 4T1 murine breast cancer cells, KB human epidermoid carcinoma cells and U87MG human glioblastoma cells were cultured in the standard media recommended by American type culture collection (ATCC). Balb/c mice and Athymic nude mice experiments were obtained from Suzhou Belda Bio-Pharmaceutical Co., Ltd. and performed under protocols approved by Soochow University Laboratory Animal Center. The 4T1 and KB tumor models were generated by subcutaneous injection of cells in ~100 µl serum-free RMPI-1640 medium onto the shoulder of female Balb/c mice and nude mice, respectively. More U87MG cells (~ per tumor) were subcutaneously injected into nude mice to generate the U87MG tumor model. The mice were injected with NGS when the tumor volume reached ~100 mm 3. S2

3 In vivo near-infrared fluorescence imaging. Tumor bearing mice were intravenously injected with 200 μl of 2 mg/ml NGS-PEG-Cy7 and imaged using the Maestro in vivo fluorescence imaging system (CRi Inc.). NIR light with a central wavelength at 704 nm was used as the excitation source. In vivo spectral imaging from 740 nm to 950 nm (10 nm step) was carried out with an exposure time of 200 ms for each image frame. Auto-fluorescence (particularly from food residues in the stomach and intestine) was removed by using the spectral unmixing software. Blood circulation. Blood circulation was measured by drawing ~10 µl blood from the tail vein of 4T1 tumor bearing Balb/c mice post injection of NGS-PEG-Cy7. Each blood sample was dissolved in 1ml of lysis buffer (1% SDS, 1% Triton X-100, 40 mm Tris Acetate). The concentration of NGS-PEG-Cy7 in the blood was determined by the fluorescence spectrum of each solubilized blood sample using a FluoroMax 4 fluorometer (HORIBA Jobin Yvon, France). A series of dilutions of the NGS-PEG-Cy7 solution were measured to obtain a standard calibration curve. Blank blood sample without NGS-PEG-Cy7 injection was measured to determine the blood auto-fluorescence level, which was subtracted from the fluorescence intensities of injected samples during the concentration calculation. Photothermal therapy. An optical fiber coupled 808 nm high power laser diode (Hi-Tech Optoelectronics Co., Ltd. Beijing, China) was used to irradiate tumors during our experiments. The tumor on each mouse was radiated by the 808 nm NIR laser at the power density of 2 W/cm 2 for 5 minutes (one minute interval after each minute of irradiation). The tumor sizes were measured by a caliper every the other S3

4 day and calculated as the volume = (tumor length) (tumor width) 2 /2. Relative tumor volumes were calculated as V/V 0 (V 0 was the tumor volume when the treatment was initiated). Histology analysis. Forty days after NGS-PEG injection, 3 mice from the treatment group and 3 age-matched female BALC/c control mice (without any injection of NGS-PEG) were sacrificed by CO 2 asphyxiation for necropsy. Major organs from those mice were harvested, fixed in 10% neutral buffered formalin, processed routinely into paraffin, sectioned at 8 microns, stained with hematoxylin & eosin (H&E) and examined by a digital microscope (Leica QWin). Examined tissues include liver, kidneys, spleen, heart, lung and intestine. Blood analysis. Four healthy Balb/c mice were injected with 200 μl of 2 mg/ml NGS-PEG (a dose of 20 mg/kg). Other four mice were used as the un-treated control. Three months after injection, mice were sacrificed to collect the blood (0.8ml) for blood chemistry test and routine blood analysis. The serum chemistry data and complete blood panel were measured in Shanghai Research Center for Biomodel Organism. S4

5 NGS-PEG NGS Supporting Information Figure S1. A photo of NGS-PEG and NGS (before PEGylation) in 9% NaCl solutions after centrifugation at 21,000 g for 10 minutes. No obvious sediment was observed in the NGS-PEG sample, while the NGS sample precipitated obviously. S5

6 % Transmittance NGS NGS-PEG Wavenumber (cm -1 ) Supporting Information Figure S2. FT-IR spectra of NGS and NGS-PEG. The NGS-PEG sample was filtered over 10 times through a 100 kda MWCO filter to completely remove any un-conjugated PEG (10 kda). The strong C-H stretch peak (~2800cm -1 ) and C-O stretch peaks (1100~1500 cm -1 ) clearly indicate the presence of PEG in the NGS-PEG sample. S6

7 50 40 Sheet Counts Hight / nm Supporting Information Figure S3. Statistics of NGS-PEG thickness measured by AFM. The average thickness of NGS-PEG was measured to be about 1.0 nm. The AFM measured thickness of single layer graphene is reported to be ~0.55 nm 4. The increased measured thickness in our NGS-PEG sample is likely due to the existence of PEG coating on graphene sheets. The majority of our NGS-PEG sample was single or double layered sheets. S7

8 a Absorbance NGS-PEG + Cy7 NGS-PEG-Cy7 NGS-PEG b FL intensity (a.u) Wavelength (nm) Wavelength (nm) Supporting Information Figure S4. (a) UV-VIS-NIR spectra of NGS-PEG, NGS-PEG-Cy7 and NGS-PEG mixed with hydrolyzed Cy7-SE (NGS-PEG + Cy7). NGS-PEG-Cy7 was prepared by reacting NGS-PEG with fresh Cy7-SE (SE: succinimidyl ester) in PBS overnight. For the NGS-PEG + Cy7 control sample, Cy7-SE dye was hydrolyzed in PBS overnight to de-activate the amine reactive SE group before being added into the NGS-PEG sample. Un-conjugated Cy7 was completely removed by filtrations and repeated water washing. UV-VIS-NIR spectra revealed that the majority of Cy7 was conjugated to the amine on NGS-PEG with a minimal degree of physical absorption by π-stacking. (b) A fluorescence spectrum of NGS-PEG-Cy7 recorded by using 704 nm as the excitation wavelength. 704 nm was the longest excitation wavelength available in the Maestro in vivo imaging system. S8

9 a Autofluorescence Cy7 Merged b FL intensity (a.u) NGS-PEG-Cy7 Autofluorescence X Wavelength (nm) Supporting Information Figure S5. Spectral unmixing to remove the mouse auto-fluorescence. (a) Spectral resolved images of an un-injected mouse (right) and a NGS-PEG-Cy7 injected mouse (left). The mouse auto-fluorescence was removed by spectral unmixing using the Maestro software. The green color in the stomach / intestine was from the auto-fluorescence of food residues. (b) Spectra of NGS-PEG-Cy7 fluorescence and auto-fluorescence recorded by using the Maestro in vivo fluorescence imaging system. The auto-fluorescence spectrum shown in (b) was multiplied by five. S9

10 30min 1h 6h 24h KB 4 4 PEG-Cy7 4 4 Cy7 Supporting Information Figure S6. Fluorescence images of KB tumor bearing mice after injection of NGS-PEG-Cy7, PEG-Cy7, or free Cy7 at different time points post injection. All images are presented at the same intensity scale, except for the PEG-Cy7 and Cy7 injected mice at later time points (the intensity was multiplied by 4 times). The same concentration of Cy7 (~80 μm) was injected in all three groups. The majority of PEG-Cy7 and free Cy7 was excreted within 6 h by urine, in marked contrast with NGS-PEG-Cy7 which showed increased brightness in the tumor. S10

11 a KB tumor model SK LI I M SP K T H LU Average FL intensity (a.u.) b skin muscle intestin heart lung liver kidney spleen tumor Supporting Information Figure S7. The biodistribution of NGS-PEG-Cy7 in KB tumor bearing nude mice after intravenous injection. (a) A representative fluorescence image of different organs taken at 24 h p.i. The green color in the stomach was from the auto-fluorescence of food. SK: skin, M: muscle, I: intestine, H: heart, LU: lung, LI: liver, K: kidney, SP: spleen, ST: Stomach, T: tumor. (b) Semi-quantitative biodistribution of NGS-PEG-Cy7 in mice determined by the averaged fluorescence intensity of each organ. Note that the stomach, intestine and skin usually have relatively high auto-fluorescence. Therefore the fluorescence intensities in these organs may not accurately reflect their NGS-PEG levels. Error bars were based on three mice per group. S11

12 a U87MG tumor model L S I M S T K H L Average FL intensity (a.u.) skin b muscle intestin heart lung liver kidney spleen tumor Supporting Information Figure S8. The biodistribution of NGS-PEG-Cy7 in U87MG tumor bearing nude mice after intravenous injection. (a) A representative fluorescence image of different organs taken at 24 h p.i. SK: skin, M: muscle, I: intestine, H: heart, LU: lung, LI: liver, K: kidney, SP: spleen, ST: Stomach, T: tumor. (b) Semi-quantitative biodistribution data of NGS-PEG-Cy7 in U87MG tumor bearing nude mice at 24 h p.i. Error bars were based on three mice per group. S12

13 Supporting Information Table S1. Blood chemistry analysis. Healthy Balb/c mice were injected with NGS-PEG at the dose of 20 mg/kg. The mice will euthanized after 3 months with blood collected for blood chemistry test. Standard deviations were based on four mice per group. GLOB: Globulin, ALB: Albumin, ALP: Alkaline phosphatase, ALT: Alanine transaminase, AST: Aspartate aminotransferase, GGT: Gamma-glutamyl transpeptidase, GLU: Glucose, T-CHO: Total cholesterol, TP: Total protein. Unit Control NGS-PEG Ave. STD Ave. STD GLOB g/l ALB g/l ALB/GLOB Ratio ALP U/L ALT U/L AST U/L GGT U/L GLU mmol/l TCHO mmol/l TP g/l S13

14 Supporting Information Table S2. Complete blood panel. Standard deviations were based on four mice per group. WBC: White blood cell, RBC: Red blood cell, HGB: Hemoglobin, HCT: Hematocrit, MCV: Mean corpuscular volume, MCH: Mean corpuscular hemoglobin, MCHC: Mean corpuscular hemoglobin concentration, PLT: Platelets, W-SCR: Small white blood cell account for the percentage of total of white blood cell, W-MCR: Medium-sized white blood cell account for the percentage of total of white blood cell, W-LCR: Large white blood cell account for the percentage of total of white blood cell, W-SCC: The absolute value of small white blood cells, W-MCC: The absolute value of medium-sized white blood cells, W-LCC: The absolute value of large white blood cells, RDW-SD: Red blood cell distribution width- Standard deviation, RDW-CV: Red blood cell distribution width-coefficient of variation. Unit Control NGS-PEG Ave. STD Ave. STD WBC 10e9/L RBC 10e12/L HGB g/l HCT MCV f L MCH pg MCHC g/l PLT 10e9/L W-SCR W-MCR W-LCR W-SCC 10e9/L W-MCC 10e9/L W-LCC 10e9/L RDW-SD f L RDW-CV Reference: (1) Sun, X.; Liu, Z.; Welsher, K.; Robinson, J. T.; Goodwin, A.; Zaric, S.; Dai, H. Nano Res. 2008, 1, (2) Liu, Z.; Robinson, J. T.; Sun, X. M.; Dai, H. J. J. Am. Chem. Soc. 2008, 130, (3) Watai, Y.; Sase, I.; Shiono, H.; Nakano, Y. FEBS Letters 2001, 488, (4) Niyogi, S.; Bekyarova, E.; Itkis, M. E.; McWilliams, J. L.; Hamon, M. A.; Haddon, R. C. J. Am. Chem. Soc. 2006, 128, S14