Nano-Sized Graphene Oxide as Biocompatible Gene Delivery Carrier for Peptide Nucleic Acid

Oxidized form of graphene such as graphene oxide (GO) attracts lots of interests in metal ion sensing, cell imaging, and adsorption of protein enzyme. GO is known to preferentially bind single-stranded nucleic acids with fluorescence quenching near the GO surface and thus has been recognized as a promising carrier for gene delivery. However, GO has low dispersibility in cell culture media and has shown significant cytotoxicity in cells. To improve the solubility in cell culture media and cytotoxicity of GO, I prepared nano-sized GO (nGO) and modified the nGO surface with polyethylene glycol (PEG), which is often used as a biocompatible coating for surface modification of nanomaterials.1 In the present study, I used PEGylated graphene oxide (PEG-nGO) as biocompatible carriers of antisense peptide nucleic acid (PNA) oligonucleotides for gene delivery. GO was cracked into nGO by tip sonication. For PEGylation, –OH groups on the nGO surface were converted to –COOH groups via conjugation of acetic acid moieties. Carboxylated nGO (HOOC-nGO) was conjugated with 6-arm PEG-amine by EDC coupling. The characterization of PEG-nGO were measured via atomic force microscopy, transmission electron microscopy, and dynamic light scattering spectrophotometer. To observe the interactions between PNA and PEG-nGO, I used fluorescein-labeled PNA. Cellular uptake and distribution of PNA or PEG-nGO were examined by flow cytometry and confocal microscopy. PEG-nGO showed lower cytotoxicity and higher solubility than GO. PEG-nGO were taken up by cells and more effectively delivered PNA than free PNA or PNA/nGO complexes. PEG-nGO readily adsorbed single-stranded PNA on the surface. Moreover, the adsorbed PNA were readily desorbed from the PEG-nGO surface by adding complementary RNA or under low pH conditions, which are similar to the cellular environments in endosomes and lysosomes. PEG-nGO were taken up by lung cancer cells and more effectively delivered PNA than free PNA or PNA/nGO complexes. Cellular uptake of PEG-nGO was mediated via endocytosis, whereas free PNA was not observed to be delivered via endocytosis. As illustrated in schematic illustration, PNAs adsorbed onto the PEG-nGO surface are released under acidic conditions of endosomes/lysosomes and eventually diffuse to the cytosol through endosomal escape, while PEG-nGO appears to be trapped in the endosomes/lysosomes. Gene knockdown of green fluorescent protein and EGFR was performed to validate the effectiveness of target gene knockdown via PEG-nGO-mediated PNA delivery. I suggest that PEG-nGO is a biocompatible carrier useful for PNA delivery into cells and serves as a promising gene delivery tool.