PEGylated Nano-Graphene Oxide for Delivery of Insoluble
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1 PEGylated Nano-Graphene Oxide for Delivery of Insoluble Cancer Drugs Zhuang Liu, Joshua T. Robinson, Xiaoming Sun and Hongjie Dai Department of Chemistry, Stanford University, Stanford, CA, 9435, USA Supplementary Information 1, Materials and methods 1) Synthesis of NGO-PEG Graphite oxide was synthesized from expandable graphitic flake (Graftech) using a modified Hummer s method 1. Using mortar and pestle, 1 gram of graphite was ground with 5 grams of NaCl for a period of 1 minutes. NaCl was then dissolved and removed by filtration with water. This causes a small loss in graphite. The ground graphite flakes were then added to 23 ml of H2SO4 (98%) and left stirring for 12 hours. Afterwards, while keeping the temperature less than 2ºC, 6 grams of KMnO4 was added. This was stirred at 4 ºC for 3 minutes, then stirred at 9 ºC for 9 minutes. (When the mixture reaches 7 ºC, temperature increases rapidly). Next, 46 ml distilled water were added, and the heat was increased to 15ºC for 25 min. The reaction was ended by a final addition of 14ml distilled water and 1ml 3% H 2 O 2 solution. For purification, the resulting mixture was washed multiple times, first with 5% HCl solution and then with DI water. 2 ml of water were added to the graphite oxide product. The product was deposited on a substrate and imaged by AFM, which revealed that most of the GO sheets are single to few-layered with topographic height of 1-2nm (Fig.S1a). S1
2 In order to attach PEG, we must introduce carboxylic acid functional groups. This is done by taking 1 ml of graphite oxide in water (~4 mg/ml) and bath-sonicating it for 1 hour. Following this, 1 ml of 3M NaOH was added to the graphite oxide solution and bath-sonicated for 3 hours. The fact that PEG-amine was able to bind to the graphite oxide with EDC chemistry after sonication in very basic conditions whereas before sonication PEG-amine was not able to bond suggests that esters on GO are hydrolyzed to carboxyl groups after base treatment. Afterwards, HCl is added to neutralize and the solution is filtered and rinsed. The final product is carboxylic acid modified graphite oxide (GO-COOH). The modified GO is diluted by water until is ~1 mg/ml (O.D. of.4 at 88 nm with 1mm optical path). It is then bath-sonicated with 1 mg/ml of 6-arm Polyetheylene Glycol-Amine (Sunbio Inc.) for 5 minutes. N-(3-Dimethylaminopropyl-N - ethylcarbodiimide hydrochloride (EDC, from Sigma Inc.) is then added to reach 5 mm and the solution is bath sonicated for another 3 minute, followed by adding enough EDC to reach 2 mm and stirring for 12 hours. The reaction is terminated by adding Mercaptoethanol (Fluka Inc.). After 1 hr. of centrifugation (45k rpm) in 2x phosphate buffer solution (PBS) for the solution, the supernatant is saved. The supernatant is the final product, NGO-PEG. The PEGylation was confirmed by IR spectroscopy of NGO- PEG in which pronounced C-H and C-O vibration bands of the PEG chains were observed (Fig.S1b). The fact that NGO-PEG was stable in serum without aggregation also confirmed successful PEGylation. S2
3 GO sheet counts (a) 1. (b) GO thickness (nm) Transmission.98 C=O.96 GO C-O C-H NGO-PEG Wave number (cm -1 ) Figure S1. Characterization of GO and NGO-PEG. (a) Thickness histogram of GO sheets measured by AFM. The average thickness of GO was measured to be 1.8 nm with a standard deviation of.6 nm. (b) Infrared (IR) spectra of GO and NGO-PEG. The peak at ~16 cm -1 in both GO and NGO-PEG samples was attributed to the vibration of C=O bond. The peaks at ~285 cm -1 and ~11 cm -1 in NGO-PEG sample were the signatures of C-H bond and C-O bond in PEG, respectively. Attenuated total reflection infrared spectroscopy (ATR-IR) is performed on the GO and NGO-PEG solutions in water at 3 mg/ml (Bruker Optics, VERTEX 7/7v FT-IR spectrometer) S3
4 2) SN38 loading on NGO-PEG SN38 was purchased from Guanyu Bio Inc from China. 5ml of.5 mg/ml NGO- PEG in water was mixed with.5ml of 2.5 mm SN38 DMSO solution and stirred overnight at room temperature. Excess SN38 precipitated as solid was removed by centrifuge. The supernatant was filtered through a.45 Dm filter to fully remove any solid. The solution was then filtered through a 3 kda molecular weight cutoff filter (Millpore) to remove the small amount of solubilized free SN38. NGO-PEG-SN38 retained in the filter was washed 4 to 6 times and re-suspended in water. The formed NGO-PEG-SN38 was stored at 4 o C. To determine the SN38 loading, UV-VIS spectra of NGO-PEG and NGO-PEG- SN38 were measured by Cary 6i spectrometer. The extinction coefficient of NGO- PEG at 23 nm was 65 L g -1 cm -1 while that at 88 nm was 8.2 L g -1 cm -1. The SN38 concentration was determined by its absorption at 38 nm (after subtracting the absorption contribution from NGO-PEG) with molar extinction coefficient of 25,5 L mol -1 cm -1. Similar method was reported previously to determine the doxorubicin loading on carbon nanotubes. 1 3) Releasing of loaded SN38 in buffer and serum Fluorescence method was used to measure the retained SN38 concentrations after incubating NGO-PEG-SN38 in buffer and serum. Because the fluorescence was quenched once loaded on NGO-PEG-SN38, SN38 should be detached from NGO-PEG in order to use fluorescence to quantitatively measure its concentration. We found that after addition of isopropanol (IPA) into the NGO-PEG-SN38 water solution, the loaded SN38 S4
5 will be fully released as evidenced by the recovery of SN38 fluorescence at 56 nm (Fig. S2). This allowed us to use fluorescence method to quantify the amount of SN38 in NGO-PEG-SN Fluorescence (a.u.) NGO-PEG-SN38, water NGO-PEG-SN38, IPA SN38, water SN38, IPA Wavelength (nm) Figure S2. Fluorescence spectra of NGO-PEG-SN38 and SN38 in water and isopropanol (IPA). The SN38 concentration was 1DM in all the samples. The SN38 fluorescence at 56 nm in the NGO-PEG-SN38 was quenched in water but recovered after transferring into IPA. The excitation wavelength was 38nm. NGO-PEG-SN38 was incubated in phosphate buffered saline and mouse serum over different periods of time. After centrifuge to remove any possible precipitate, the supernatant was filtered by 3 kda filters to wash away released SN38 in solution. The leftover was collected and dissolved in a certain volume of IPA for fluorescence measurement by a fluolog-3 fluorometer. The fluorescence intensity at 56 nm was used to calibrate the SN38 concentration. Measurements were carried out at time points h, 24 h, 48 h and 72 h. S5
6 4) cell culture and in vitro assay HCT-116 colon cancer cell line, OVCAR-3 ovarian cancer cell line, U87MG glioma cell line, MDA-MB-435, MCF-7 and MDA-MB-468 breast cancer cell lines were all obtained from American Type Culture Collect (ATCC) and cultured in the recommended conditions. For the in vitro cell toxicity assay, cells were plated in 96 well plates and added with desired concentrations of NGO-PEG, NGO-PEG-SN38, free SN38 (dissolve in DMSO and diluted in PBS) and CPT-11 added. After incubation for 72 hours, relative cell viability was measured by standard MTS assay using a CellTiter 96 kit (Promega). For NGO-PEG toxicity test, cell medium containing NGO-PEG was changed to fresh medium at 72 h before cell viability test to avoid the dark color of NGO-PEG at high concentrations which interferes the absorbance reading in MTS assay. 5) Apoptosis assay MCF-7 cells were incubated with NGO-PEG-SN38 at 1 mg/l (NGO-PEG concentration) or NGO-PEG at 1 mg/l, 3 mg/l and 1 mg/l for 2 days. Cells floating in the cell medium were collected by centrifuge while adherent cells were collected by treating with trypsin-edta. After washing with PBS, cells were then stained with FITC-annexin V (Molecular Probe) and Propidium Iodide (Aldrich) following the standard protocol. A Becton-Dickinson flow cytometer was used for the FACS measurement. S6
7 2, AFM images of NGO-PEG and GO-PEG-SN38 a b GO sheet counts c NGO-PEG NGO-PEG-SN GO sheet thickness (nm) Figure S3. Thickness change of NGO-PEG before and after SN38 loading. (a&b) AFM images of NGO-PEG (a) and NGO-PEG-SN38 (b). Scale bar: 1 nm. (c) Thickness histogram of NGO-PEG and NGO-PEG-SN38 measured by AFM. The average thicknesses (± standard deviation) of NGO-PEG and NGO-PEG-SN38 were measured to be 1.8 ±.7 nm and 2.5 ±.8 nm, respectively. The increased thickness was likely due the SN38 loaded on GO sheets via J-J stacking. S7
8 3, In vitro toxicity test for other cell lines Table 1. IC5 values (nm) of different cell lines after 72 h drug incubation. Cell line CPT-11 SN38 NGO-PEG-SN38 HCT-116 (colon) 11, 15 6 OVCAR-3 (ovarian) 3,.3.12 U87MG (glioma) >2, 67 5 MDA-MD-435 (breast) 6, 3 2 S8
9 4, Toxicity Assay of NGO-PEG by Annexin V and Propidium Iodide Staining. (a) Untreated R1 R1 NGO-PEG-SN38, 1mg/L R1 PI PI FITC-Annexin V FITC-Annexin V NGO-PEG, 1mg/L NGO-PEG, 3mg/L NGO-PEG, 1mg/L R1 R1 R1 PI PI PI FITC-Annexin V FITC-Annexin V FITC-Annexin V (b) Percentage of cell counts(%) Untreated NGO-PEG- SN38, 1mg/L NGO-PEG, 1mg/L NGO-PEG, 3mg/L Apoptosis Necrosis NGO-PEG, 1mg/L Figure S4. Apoptosis assay of MCF-7 cells after NGO-PEG treatment. MCF-7 cells were incubated with NGO-PEG-SN38 at 1 mg/l (in terms of GO) or NGO-PEG at 1 mg/ml, 3 mg/l and 1 mg/l for 2 days. Cells were stained by FITC-annexin V and Propidium iodide (PI) to label the apoptotic cells and necrotic cells, respectively, for flow cytometry (FACS) measurement. (a) FACS data of MCF-7 cells after different NGO-PEG treatment. R1 (red) and (green) represent the regions of dead cells and apoptotic cells, respectively. (b) Percentage of apoptotic cells and dead cells after different NGO-PEG treatment. While NGO-PEG-SN38 (1mg/L of NGO-PEG) introduced significant cells death and apoptosis at a low GO concentration, plain NGO-PEG without drug was nontoxic to the cells even at high concentrations up to 1mg/L. S9
10 5, cellular uptake of NGO-PEG (a) 37 o C (b) 4 o C Figure S5. Cellular uptake of NGO-PEG. NGO-PEG was labeled by NHS-fluorescein (an amine reactive dye, Pierce) with excess fluorescent dye thoroughly removed by filtration and extensive washing with PBS and water. MCF-7 cells were incubated with NGO-PEG-FITC for 2 hours at 37 o C (a) or 4 o C (b) and imaged by Zeiss confocal fluorescence microscope after washing with PBS. High uptake of NGO-PEG was observed when cells were incubated at 37 o C but no at 4 o C, suggesting that GO-PEG entered cells likely through energy requiring endocytosis mechanism, similar to functionalized single walled carbon nanotubes. 2 S1
11 6, Loading of other aromatic drugs 1) Loading of other camptothecin analogs Beside SN38, several other hydrophobic CPT analogs including natural 2-(S)- camptothecin (CPT, Aldrich) and 1-hydroxycamptothecin (1-OHCPT, Guanyu Bio Inc) were also loaded on NGO-PEG. CPT and 1-OHCPT were loaded on NGO-PEG using the same protocol described earlier. The successful loading of these molecules was confirmed by UV-VIS spectra. 1.2 NGO-PEG Absorbance.8.4 NGO-PEG-CPT NGO-PEG-1-OHCPT Wavelength (nm) Figure S6. UV-VIS spectra of NGO-PEG loaded with different CPT analogs. It is estimated that 1 gram of NGO-PEG loads.9 gram of CPT and.12 gram of 1- OHCPT. 2) Loading of Iressa on NGO-PEG Iressa or geftinib (GEF) is the first selective inhibitor of epidermal growth factor receptor's (EGFR) tyrosine kinase used in the treatment of non-small cell lung cancer by oral route. 3 It has poor solubility in water and physiological buffers with neutral ph (only S11
12 soluble in acidic environment). To load it on NGO-PEG, GEF (LC laboratories) was dissolved in DMSO at 1 mm as stock solution and added into.1 mg/ml NGO-PEG in acetate buffer (ph=5.5) with a final drug concentration of.5 mm. Excess free GEF was removed by filtration through 3kDa filters and washed away. NGO-PEG-GEF was resuspended in water and stored at 4 o C. The loading of GEF was characterized by UV-VIS and cell toxicity experiment was performed with EGFR positive and negative cell lines (Fig. S4). Absorbance (a) NGO-PEG NGO-PEG-GEF Wavelength (nm) Relative cell viability (%) (b) MCF-7 GEF MCF-7 NGO-PEG-GEF MDA-MB-468 GEF MDA-MB-468 NGO-PEG-GEF [GEF]/uM Figure S7. Geftinib loading on NGO-PEG. (a) UV-VIS spectra of NGO-PEG and NGO- PEG-GEF. 1 gram of NGO-PEG loads.5~.6 gram of GEF. (b) In vitro toxicity data. MCF-7 (low EGFR expression) and MDA-MB-468 (high EGFR expression) cells were incubated with GEF and NGO-PEG-GEF at different concentrations. Cell viability assay was performed after 72 h incubation. While MCF-7 cells with low EGFR expression were resistant to GEF and NGO-PEG-GEF (IC5>1 DM), the two formulations of GEF was very potent to the EGFR up-regulated MDA-MB-468 cells with IC5 values of 1.4 DM for GEF and.8 DM for NGO-PEG-GEF. Error bars were based on triplet samples. S12
13 References: 1. Liu, Z.; Sun, X.; Nakayama, N.; Dai, H., Supramolecular Chemistry on Water- Soluble Carbon Nanotubes for Drug Loading and Delivery. ACS Nano 27, 1, (1), Kam, N. W. S.; Liu, Z. A.; Dai, H. J., Carbon nanotubes as intracellular transporters for proteins and DNA: An investigation of the uptake mechanism and pathway. Angewandte Chemie-International Edition 26, 45, (4), Johnson, D. H., Gefitinib (Iressa) trials in non-small cell lung cancer. Lung Cancer 23, 41, S23-S28. S13
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