PEGylated Nano-Graphene Oxide for Delivery of Water Insoluble Cancer Drugs

PEGylated Nano-Graphene Oxide for Delivery of Water Insoluble Cancer Drugs Zhuang Liu, Joshua T. Robinson, Xiaoming Sun and Hongjie Dai* Department of Chemistry, Stanford University, Stanford, CA, 94305, USA Email: hdai@stanford.edu Graphene has emerged as a 2D material with interesting physical properties.1,2 Intensive research is on-going to investigate the quantum physics in this system and potential applications for nano-electronic devices2, transparent conductors and nano- composite materials3. Thus far, little has been done to explore graphene in biological systems, despite much effort in the area of carbon nanotubes for in vitro and in vivo biological applications.4- 9 Here, we synthesize and functionalize nanoscale graphene oxide (NGO) sheets (<50nm) by branched, biocompatible polyethylene glycol (PEG) to render high aqueous solubility and stability in physiological solutions including serum. We then uncover a unique ability of graphene in attaching and delivery of aromatic, water insoluble drugs. It is known that clinical use of various potent, hydrophobic molecules (many of them aromatic) is often hampered by their poor water solubility. Although synthesis of water soluble pro- drugs may circumvent the problem, the efficacy of the drug decreases. Here, we show that PEGylated NGO (NGO-PEG) readily complexes with a water insoluble aromatic molecule SN38, a camptothecin (CPT) analog,10 via non-covalent van der Waals interaction. The NGO-PEG-SN38 complex exhibits excellent aqueous solubility and retains the high potency of free SN38 dissolved in organic solvents. The toxicity exceeds that of irinotecan (CPT-11, a FDA approved SN38 prodrug for colon cancer treatment) by 2-3 orders of magnitude. We prepared graphene oxide by oxidizing graphite using a modified Hummer’s method.3,11 The resulting GO (single layered and few-layered, Supp Info. Fig.S1) was soluble in water but aggregated in solutions rich in salts or proteins such as cell medium and serum (Fig. 1a). This was likely due to screening of the electrostatic charges and non-specific binding of proteins on the GO.12 To impart aqueous stability and prevent bio-fouling, we sonicated the GO to make them into small pieces and conjugated a 6-armed PEG-amine stars to the carboxylic acid groups3 (Supp Info Fig. S3) on GO via carbodiimide catalyzed amide formation. The resulting PEGylated NGO exhibited excellent stability in all biological solutions tested including serum (Fig. 1b). PEGylation was further confirmed by infrared (IR) spectroscopy (Supp Info. Fig.S1b). The as-made GO sheets were 50-500nm in size (Fig.1c), whereas NGO-PEG was ~5-50 nm (Fig. 1d) due to sonication steps (see Supp Info.). We then investigated the binding of SN38 to NGO-PEG. We chose SN38 as a cargo because SN38 is a potent topoisomerase I inhibitor.10 To be active, CPT-11 currently used in clinic has to be metabolized to SN38 after systematic adminsitration.10,13 However a large amount of CPT-11 is excreted before transforming to SN38 or metabolized to other inactive compounds.14 The water insolubility has prevented the direct use of SN38 in the clinic.10 We found that SN38 was complexed with NGO-PEG (Fig.2a) by simple mixing of SN38 dissolved in DMSO with a NGO-PEG water solution. The excess, uncoupled SN38 precipitated and was GO NGO-PEG Water PBS Cell medium Serum (a) (b) (c) (d) Figure 1. PEGylation of graphene oxide. (a&b), photos of GO (a) and NGO-PEG (b) in different solutions recorded after centrifugation at 10,000 g for 5 minutes. GO crashed out slightly in PBS and completely in cell medium and serum (top panel). NGO-PEG was stable in all solutions. (c&d) AFM images of GO (c) and NGO-PEG (d). Figure 2. SN38 loading on NGO-PEG. (a) schematic draw of SN38 loaded NGO-PEG. Inset: a photo of NGO-PEG-SN38 water solution. (b) UV-VIS absorption spectra of NGO-PEG, NGO-PEG-SN38, SN38 in methanol and difference spectrum of NGO-PEG and NGO-PEG-SN38. The SN38 absorbance at 380 nm was used to determine the loading. (c) Fluorescence spectra of SN38 and NGO-PEG-SN38 at [SN38]=1µM. Significant fluorescence quenching was observed for SN38 adsorbed on NGO. (d) Retained SN38 on NGO-PEG over time incubated in PBS and serum respectively. SN38 loaded on NGO-PEG was stable in PBS and released slowly in serum. Error bars were based on triplet samples.

removed by centrifugation. Repeated washing and filtration were used to remove DMSO and any residual free SN38 (see Supp Info. for details). UV-VIS spectrum of the resulting solution revealed SN38 peaks superimposing with the absorption curve of NGO- PEG (Fig. 2b), suggesting loading of SN38 onto NGO-PEG. Based on the extinction coefficients, we estimated that 1 gram of NGO-PEG loaded ~0.1 gram of SN38 (Supp Info.). An increase in sheet thickness was observed after SN38 loading on NGO-PEG (Supp Info. Fig. S3). A control experiment revealed no loading o

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