Polyethylene Glycol-engrafted Graphene Oxide as Biocompatible

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Polyethylene Glycol-engrafted Graphene Oxide as Biocompatible Materials for Peptide Nucleic Acid Delivery into Cells Ahruem Baek, Yu Mi Baek, Hyung-Mo Kim, Bong-Hyun Jun, and Dong-Eun Kim Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00025 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 28, 2018

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Bioconjugate Chemistry

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Polyethylene Glycol-engrafted Graphene Oxide as

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Biocompatible Materials for Peptide Nucleic Acid

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Delivery into Cells

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Ahruem Baek, Yu Mi Baek, Hyung-Mo Kim, Bong-Hyun Jun, and Dong-Eun Kim*

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Department of Bioscience and Biotechnology, Konkuk University

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Neundong-ro 120, Gwangjin-gu, Seoul 05029, Republic of Korea

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E-mail: [email protected]

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ABSTRACT: Graphene oxide (GO) is known to strongly bind single-stranded nucleic acids

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with fluorescence quenching near the GO surface. However, GO exhibits weak

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biocompatibility characteristics, such as low dispersibility in cell culture media and

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significant cytotoxicity. To improve dispersibility in cell culture media and cell viability of

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GO, we prepared nano-sized GO (nGO) constructs and modified the nGO surface using

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polyethylene glycol (PEG-nGO). Single-stranded peptide nucleic acid (PNA) was adsorbed

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onto the PEG-nGO and was readily desorbed by adding complementary RNA or under low

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pH conditions. PNA adsorbed on the PEG-nGO was efficiently delivered into lung cancer

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cells via endocytosis without affecting cell viability. Furthermore, antisense PNA delivered

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using PEG-nGO effectively downregulated the expression of the target gene in cancer cells. 1

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Our results suggest that PEG-nGO is a biocompatible carrier useful for PNA delivery into

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cells and serves as a promising gene delivery tool.

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INTRODUCTION

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Antisense oligonucleotides are gene therapy tools delivered to cells via nucleic acid delivery

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systems for the treatment of genetic disorders, such as Parkinson’s disease and cancers.1-3

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Gene therapy requires simple, safe, and improved transfection efficiency of nucleic acids into

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the cells. Multiple problems arise upon gene delivery using viral or non-viral vectors, which

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include liposomes, positively charged chemicals, and nanomaterials.4, 5 For the past few years,

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graphene and its oxidized derivative, graphene oxide (GO), have demonstrated huge potential

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for their use in practical applications in biotechnology, biomedicine, and electronics,

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including metal ion sensing,6 cell imaging,7 drug delivery,8 and protein enzyme

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immobilization.9 GO is known to preferentially bind single-stranded nucleic acids with

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fluorescence quenching near the GO surface10-14 and thus has been recognized as a promising

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carrier for gene delivery. However, GO has low dispersibility in cell culture media and has

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shown significant cytotoxicity in cells.15 When introduced into cells, GO decreases cell

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viability by activating apoptotic caspase-3 and inducing necrosis.16-18 Furthermore, GO

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delivery has resulted in autophagy induction in several cell lines through the generation of

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reactive oxygen species.19 Therefore, GO poses deleterious problems for human cells if the

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GO surface is not properly functionalized to improve biocompatibility.

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Polyethylene glycol (PEG), which is often used as a biocompatible coating for surface

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modification of nanomaterials, was previously engrafted onto the GO surface to improve the

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solubility and stability of insoluble molecules. PEG-modified GO has been used in protein20

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or drug delivery15, 21, 22 systems not only to enhance solubility or dispersibility, but also to 2


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reduce cytotoxicity. PEG-modified GO has also been used as delivery vehicle of therapeutic

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genes, such as siRNA and plasmid DNA.23, 24 To improve the loading capacity of PEG-

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modified GO for negatively charged genes, PEG-modified GO was further functionalized

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with positively charged molecules, including 1-pyrenemethylamine hydrochloride (Py-NH2)

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or polyethylenimine (PEI).23,

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positively charged molecules create toxic conditions inside cells, leading to cell death during

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the gene delivery process.

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However, when engrafted onto the GO surface, these

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In this study, peptide nucleic acid (PNA) was used as the oligonucleotide for gene delivery.

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PNA is a DNA mimic that contains peptide bonds instead of phosphodiester bonds26 and thus

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form considerably more stable complexes with DNA or RNA than DNA-DNA or DNA-RNA

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complexes.27-29 In previous studies, PNA has been used as a therapeutic antisense

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oligonucleotide that inhibits translation by forming complexes with complementary

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mRNA.30-32 Although PNA can form stable complexes with complementary mRNA,

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difficulties in its delivery into cells have hampered its efficacy as a therapeutic

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oligonucleotide. There have been previous attempts to overcome the challenges of PNA

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delivery into cells; PNA was conjugated with cell-penetrating peptides (CPPs) to enhance

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cellular endocytosis,33 PNA was loaded into nanocomposites, such as nano-sized porous

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silicon functionalized with diblock polymer to facilitate endosomal escape of PNA in the

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cells,34 and poly(lactide-co-glycolide) (PLGA)-based nanoparticle for nontoxic delivery of

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PNA.35

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In the present study, we used PEGylated graphene oxide (PEG-nGO) as biocompatible

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carriers of PNA for gene delivery. Fluorescent PNA was observed to be efficiently adsorbed

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onto PEG-nGO constructs and were readily dissociated from the PEG-nGO using

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complementary RNA but not by non-complementary RNA. Fluorescein-labeled PNAs

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adsorbed on PEG-nGO constructs were efficiently delivered into cells without affecting cell 3



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viability and inducing deleterious autophagy. In addition, the PNAs, but not the PEG-nGO

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constructs, were observed to escape the endosomes. Gene delivery of antisense PNAs using

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PEG-nGO effectively led to the knockdown of the target gene in cancer cells. Our results

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suggest that PEG-nGO constructs are suitable carriers of antisense PNA for gene knockdown

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without exerting cellular toxicity.

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RESULTS AND DISCUSSION

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First, we prepared PEGylated nano-sized graphene oxide (PEG-nGO) constructs36 and

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characterized the nGO and PEG-nGO using atomic force microscopy (AFM), transmission

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electron microscopy (TEM), zeta potential, and dynamic light scattering (DLS) measurement.

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The sizes of the prepared nGO and PEG-nGO particles were determined to be < 200 nm

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using AFM (Figure 1a). The average thickness of the nGO particles was 1.39 nm, which

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corresponds to a single layer of graphene, whereas PEG-nGO particles had a higher thickness

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of 4.5–5.2 nm as a result of PEG conjugation onto the nGO surface. TEM images showed

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that sizes of the nGO and PEG-nGO constructs were less than ~200 nm (Figure 1b). PEG-

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nGO had tiny black spots that potentially correspond to the PEGs conjugated onto the nGO

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surface, whereas nGO showed clear and even surfaces. nGO particles showed a negative

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surface charge (-38 mV) based on zeta potential measurements, whereas the zeta potential of

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PEG-nGO particles were higher by +35 mV and reached up to -1.63 mV (Figure 1c). The

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higher zeta potential of the PEG-nGO particles was obviously due to conjugation of PEGs

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onto the GO surface. PEG molecules contain many amines at their arms, resulting in the

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lower negative charge of the PEG-nGO surface. Similar to the results shown in AFM and

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TEM images (Figure 1a, b), size distributions analyzed by DLS showed that the average sizes

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of nGO and PEG-nGO particles were 133.9 nm and 149.8 nm, respectively (Figure 1d). To

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demonstrate the improved dispersibility of PEG-nGO in aqueous solutions, we dissolved 4


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nGO, HOOC-nGO, and PEG-nGO in various aqueous solutions, namely, distilled water,

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phosphate-buffered saline (PBS), and cell culture media (Figure 1e). nGO and HOOC-nGO

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formed aggregates in each aqueous solution after centrifugation at 10,000 g for 5 min. On the

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other hand, PEG-nGO was highly dispersive in all aqueous solutions and did not show

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aggregation after centrifugation. Taken together, conjugation of PEG onto the nGO surface

Figure 1. Characterization of PEG-nGO. (a) AFM images (upper images) and height profile (bottom graphs) of nano-sized graphene oxide (nGO) and PEG-nGO. (b) TEM images of nGO and PEG-nGO; scale bar=100 nm. (c) Zeta potential and (d) DLS of nGO and PEGnGO. (e) Dispersibilities of nGO, HOOC-nGO, and PEG-nGO in different aqueous solutions (water, PBS, and cell culture media). Images show sedimentation of nGO, HOOC-nGO, and PEG-nGO (100 µg·mL-1 each) in solutions after centrifugation at 10,000 g for 5 min. 6

(i.e. PEG-nGO) resulted in higher dispersibility of PEG-nGO in saline solutions (PBS and

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cell culture media) than nGO or HOOC-nGO.

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Next, we investigated the uptake of PEG-nGO by cancer cells using confocal fluorescence

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microscopy and flow cytometry (Figure 2a, S1a). To monitor the presence of PEG-nGO in

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the cells, we prepared fluorescein-labeled PEG-nGO (FAM-PEG-nGO); FAM fluorescence

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was not affected by fluorescence quenching of the nGO surface because of the PEG layers 5



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separating the nGO surface and FAM.37 FAM-PEG-nGO-treated cancer cells showed green

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fluorescence (Figure 2a), whereas no signals were detected from cancer cells treated with a

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mixture of FAM and PEG-nGO. These results indicate that PEG-nGO constructs were

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effectively delivered into the cancer cells.

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Cell viability assay was performed to evaluate the cytotoxicity of PEG-nGO inside the cells

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(Figure 2b, S1b). Cancer cells were treated with 10 and 100 µg·mL-1 nGO or PEG-nGO for

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24 h. Low-dose treatments of nGO and PEG-nGO (10 µg·mL-1) showed nearly the same level

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of cell viability that is similar to that of the control group. By contrast, treatment with a high

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nGO dose significantly decreased cell viability (~50% cell viability), whereas only a slight

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decrease in cell viability was observed upon PEG-nGO treatment, in which ~90% cell

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viability was retained. We also examined the cytotoxicity of PEG-nGO in the normal cell line,

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retinal pigmented epithelial cells (ARPE-19). PEG-nGOs that were readily delivered into the

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cells little affect cellular viability, which is similar to nontoxic transfection into the lung

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cancer cells (A549) (Figure S1b). GO has been previously reported to induce cellular

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autophagy in macrophages by activating the toll-like receptor pathway, a cellular response

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induced upon accumulation of xenobiotic carbon materials.19 To determine whether the PEG-

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nGO can induce autophagy in cells, we monitored levels of the autophagic marker protein (i.e.

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LC3B-I to LC3B-II conversion) in the cells treated with nGO or PEG-nGO (Figure 2c). nGO-

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treated cells showed the LC3B conversion, which indicates autophagic induction, whereas

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LC3 conversion was not observed in PEG-nGO-treated cells. We next examined autophagic

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vacuole formation in cancer cells after treatment with nGO or PEG-nGO using TEM (Figure

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2d). TEM images showed large vacuoles in the cells treated with LPS (positive control),

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which is known to induce autophagic vacuole formation.38 nGO-treated cells developed large

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autophagic vacuoles (red arrows, Figure 2d). By contrast, autophagic vacuoles were not

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observed in PEG-nGO-treated cells. These results indicate that PEG-nGO is a biocompatible

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carbon material that can be delivered into cells without causing significant cytotoxicity.

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Figure 2. Cellular uptake and in vitro cytotoxicity of PEG-nGO. (a) Fluorescence microscopy images of lung cancer cells (A549) after incubation with PEG-nGO+FAM mixture or FAMlabeled PEG-nGO for 24 h (upper images). Fluorescence microscopy images of A549 cells treated with FAM-labeled PEG-nGO obtained by changing the confocal depth along the zaxis (bottom images); scale bar = 10 µm. (b) Cytotoxicity of nGO and PEG-nGO in A549 cells. Cell viability was measured using WST-1 assay after incubation with nGO or PEGnGO (10 or 100 µg·mL-1) for 24 h. (c) Western blot analysis of autophagy marker proteins (LC3 I to II conversion) in A549 cells treated with nGO and PEG-nGO (10 or 100 µg·mL-1) for 24 h. (d) TEM images of A549 cells incubated with nGO or PEG-nGO (100 µg·mL-1) for 24 h. Cells treated with LPS (1 µg·mL-1) were imaged as a control for autophagic vacuole formation. Red arrows indicate autophagic vacuoles in enlarged TEM images (shown in rectangles); scale bar = 2 µm. 1 8


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To utilize PEG-nGO for gene delivery material into cancer cells, we first examined the

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interactions between various types of oligonucleotides and PEG-nGO (Figure S2). PEG-nGO

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(100 µg·mL-1) was incubated with single-stranded DNA, RNA, and PNA (ssDNA, ssRNA,

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and ssPNA, respectively) or double-stranded RNA as small interfering RNA (siRNA) for 10

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min. After incubation, unadsorbed nucleic acids were detected via non-denaturing PAGE

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analysis (Figure S2a). Band intensities of the ssPNAs were found to decrease with increasing

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concentrations of PEG-nGO due to adsorption of PNA onto the PEG-nGO surface, whereas

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the band intensities of other oligonucleotides (ssDNA, ssRNA, and siRNA) did not change

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upon adsorption. We next performed fluorescence quenching analysis after incubation of

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fluorescein-labeled ssPNA, ssDNA, or siRNA with increasing PEG-nGO concentrations

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(Figure S2b). The fluorescence intensity of PNA decreased in a dose-dependent manner,

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indicating that PNA is readily adsorbed onto PEG-nGO.

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PNA was selected as the appropriate oligonucleotide because it can be readily adsorbed onto

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PEG-nGO. We next investigated the absorption and desorption characteristics of PNA with

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PEG-nGO to evaluate the use of PEG-nGO as a carrier for PNA delivery into cells. We

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designed fluorescently labeled PNA oligonucleotide for PNA tracing and subsequent target

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gene (epidermal growth factor receptor, EGFR gene) knockdown in cells (Figure 3a). FITC-

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labeled PNA (FITC-PNA) was used to observe the interactions between PNA and PEG-nGO.

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Adsorption and desorption of PNA can be measured by detecting the decrease and increase in

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fluorescence, respectively.39, 40 FITC-PNA (1 µM) was incubated with varying concentrations

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of nGO or PEG-nGO (0–100 µg·mL-1) for 10 min at room temperature (Figure 3b). PNA

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fluorescence was quenched with increasing concentrations of nGO or PEG-nGO, indicating

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that PNA was readily adsorbed onto the nGO and PEG-nGO surfaces.

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Figure 3. Adsorption and desorption of PNA from PEG-nGO. (a) Structure of PNA (B, base) and sequences of PNA oligonucleotides used in this study. (b) Schematic illustration showing fluorescence quenching of FITC-labeled PNA corresponding to PNA adsorption onto PEGnGO (Box). FITC-labeled PNA (1 µM) was incubated with varying concentrations of nGO or PEG-nGO (0-100 µg·mL-1) for 10 min. Fluorescence intensity of adsorbed PNA on nGO or PEG-nGO was quenched with increasing concentration of GO particles. (c) Schematic illustration showing the increase in fluorescence intensity of FITC-labeled PNA corresponding to PNA desorption from PEG-nGO upon the addition complementary RNA (Box). FITC-labeled PNA (1 µM) was pre-adsorbed on PEG-nGO (20 µg·mL-1), and singlestranded RNA (complementary or non-complementary to the PNA sequence) was then added to a solution containing the PNA/PEG-nGO complexes. (d) FITC-labeled PNA was desorbed from PEG-nGO by adding complementary or non-complementary RNA. PNA desorption was detected using non-denaturing PAGE (10%). Fluorescent PNA was detected by UV transillumination. 1

Next, we examined whether the PNA absorbed onto PEG-nGO can be released by adding

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RNA complementary to the target PNA sequence (Figure 3c). After pre-adsorption of PNA

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onto PEG-nGO surface, complementary or non-complementary RNA was added to the

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PNA/PEG-nGO complexes. Over 90% of PNA fluorescence intensity was recovered by

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adding complementary RNA to the PNA/PEG-nGO complexes, whereas the addition of non-

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complementary RNA did not increase fluorescence. Thus, PNA adsorbed onto PEG-nGO can 10


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be readily released in the presence of complementary RNA sequence. The pre-adsorbed PNA

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was readily desorbed from PEG-nGO by the addition of complementary RNA in a dose-

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dependent manner, which was observed as the gradual increase in fluorescence intensity of

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the PNA band in the gel (Figure 3d). However, addition of non-complementary RNA did not

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cause the release of the fluorescent PNA from PEG-nGO. By contrast, when PNA is pre-

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adsorbed onto nGO instead of PEG-nGO, sequence-specific desorption of PNA using

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complementary RNA was not observed (Figure S3a), which can be due to the stronger

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affinity of nGO to PNA than that of PEG-nGO. Taken together, these results indicate that

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PNA can be adsorbed onto and readily desorbed from the PEG-nGO surface by adding

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complementary RNA to the PNA, demonstrating the feasibility of PEG-nGO as PNA carriers,

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with effective release of PNA inside the cells upon exposure to target RNA.

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To investigate the cellular uptake of PNA using PEG-nGO, fluorescein-labeled PNA/PEG-

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nGO complexes were administered to cancer cells (Figure 4a). TAMRA-labeled PNA was

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pre-adsorbed onto nGO or PEG-nGO surface for 10 min to allow the formation of the

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PNA/GO complexes. After incubating the cells with PNA/nGO or PNA/PEG-nGO

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complexes for 3 h, fluorescence signals from PNA were monitored using fluorescence

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microscopy. Neither free PNA nor the PNA/nGO complexes showed TAMRA fluorescence

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inside the cells. By contrast, cells treated with the PNA/PEG-nGO complexes showed

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significantly stronger fluorescence inside the cells, which was further dissected for vertical

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distribution for the fluorescence in whole cytosol of the cells (Figure 4a, bottom panel).

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To further confirm the cellular uptake of PNA using PEG-nGO and subsequent release of

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PNA, flow cytometry analysis was performed after the FITC-labeled PNA (FITC-PNA)

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treatment for 3 h without delivery vehicle or with GO materials (nGO or PEG-nGO) in A549

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and ARPE-19 cells (Figure S4). Consistent with the fluorescence microscopy results, cells

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treated with the FITC-PNA/PEG-nGO complexes showed a high delivery efficiency in both 11



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cells, which was shown by significantly shifted fluorescence population in the flow

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cytometry.

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In contrast, cells treated with FITC-PNA/nGO complexes showed slight shift of fluorescence

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Figure 4. Cellular uptake of PNA using PEG-nGO. (a) Fluorescence microscopy images of A549 cells incubated with TAMRA-labeled PNA without delivery vehicle or with GO material (nGO or PEG-nGO). TAMRA-labeled PNA (1 µM) was pre-adsorbed onto the nGO or PEG-nGO surface (20 µg·mL-1 each) by incubation for 10 min at room temperature. Free PNA, PNA/nGO, or PNA/PEG-nGO complexes were administered to A549 cells and incubated for 3 h at 37 oC. Fluorescence microscopy images of A549 cells were taken (upper images), and images show A549 cells treated with PNA/PEG-nGO complexes by changing confocal depth along the z-axis (bottom images); scale bar = 30 µm. (b) Fluorescence microscopy images showing intracellular distribution of FITC-labeled PNA (FITC-PNA), FAM-labeled PEG-nGO (FAM-PEG-nGO), and FITC-labeled PNA/PEG-nGO complexes (FITC-PNA/PEG-nGO) in A549 cells after PNA delivery. Cells were incubated with FITCPNA, FAM-PEG-nGO, or FITC-PNA/PEG-nGO complexes for 4.5 h, and endosomes/lysosomes and nuclei were stained with LysoTracker Red and Topro-3, respectively. Scale bar = 5 µm. (c) Fluorescence microscopy images of the cells that were treated with TAMRA-PNA/FAM-PEG-nGO complex. Nuclei are imaged with pseudo-blue fluorescence (TOPRO-3 staining). The enlarged images (shown in rectangles) show the red fluorescence (left) and merged (right) field of interest. Yellow-colored arrows indicate presence of PNA puncta. Scale bar = 5 µm. 1

peak, which can be inferred by lower PNA delivery efficiency or unfavorable release of

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PNAs adsorbed onto nGO surface albeit cellular uptake; in both cases, fluorescence signals

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are not distinguishably present as free PNA in the cells. In fact, we observed that nGO were

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present in the cytosol (Figure 2d) and PNA can be adsorbed onto nGO (Figure 3b). From

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these results, we supposed that PNA delivered into the cells is not likely released from nGO

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due to strong adsorption affinity with nGO. According to several studies, however, pre-

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adsorbed PNA can be released from nGO by interaction with complementary miRNA or

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lncRNA.41, 42 In these studies, for the miRNA or lncRNA detection using PNA adsorbed onto

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nGO in live cells, cells were exposed to PNA/nGO complexes for a long time (14 h) and

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PNA fluorescence were monitored later on, which is longer than the time for our observation

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of PNA release (3 h). Taken together, we suggest that PEG-nGO/PNA is superior to

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PNA/nGO complexes as target gene knockdown system due to its low cytotoxicity as well as

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facile release of PNA.

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Next, to investigate the cellular distribution of the PNA and PEG-nGO after cellular uptake,

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cancer cells were treated with FITC-PNA, FAM-PEG-nGO, and FITC-PNA/PEG-nGO 14


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complexes, and the corresponding fluorescence intensities inside the cells were measured

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(Figures 4b, S5). Transfected xenobiotic materials tend to be trapped via endocytosis in

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cellular acidic organelles, such as endosomes and lysosomes; thus, the organelles were

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tracked by staining with fluorescent dye (i.e. LysoTracker Red). FITC-PNA-treated cells

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showed weak accumulation of green fluorescence signals due to poor transfection efficiency

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of PNA. The green fluorescence puncta of PNA do not completely coincide with red

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fluorescent puncta, suggesting that cellular uptake of free PNA is not mediated by

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endocytosis. Cells treated with FAM-labeled PEG-nGO showed scattered green fluorescence

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that co-localized with red fluorescent puncta, visible as yellow fluorescence regions in the

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merged image. These results indicate that PEG-nGO is taken up by the cells through

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endocytosis and is subsequently enclosed in lysosomes. By contrast, cells treated with the

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FITC-PNA/PEG-nGO complexes showed green fluorescence that increasingly became

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diffusely distributed in the whole cytoplasm over time without co-localization in lysosomes

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(Figure S5c). These results indicate that transfected PNA is released into the cytosol via

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endosomal escape without lysosomal entrapment. The PNA adsorbed onto PEG-nGO was

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readily released from PEG-nGO at low pH conditions (Figure S3b), which are similar to the

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intracellular environment in acidic organelles (i.e. endosomes and lysosomes) during

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endocytosis.

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Next, to further demonstrate different cellular distributions of the PNA and PEG-nGO after

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cellular uptake of PNA/PEG-nGO composites, cells were treated with TAMRA-PNA/FAM-

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PEG-nGO complexes and monitored for the corresponding fluorescence signals from PNA

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and PEG-nGO (Figure 4c). The green fluorescence indicating PEG-nGO was formed as

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punctate structures and increased in a time-dependent manner without diffusion to the cytosol,

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which was retained as punctate form even after 4.5 h. Similar to the subcellular locations of

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PEG-nGO puncta, the red fluorescence showing PNA presence was initially formed as 15



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punctate structure and co-localized with some of FAM-PEG-nGO puncta after 1 h of

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PNA/PEG-nGO composites treatment. However, PNA traced with red fluorescence was

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widely diffused in the cytosol after 3h, which is consistent with the results shown in Figure

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4b and S5c. Taken together, cellular uptake of PNA/PEG-nGO complexes is mediated by

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endocytosis, after which PNA is released from PEG-nGO surface due to the low pH

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conditions in endosomes/lysosomes. PNA then readily diffuses into the cytoplasm via

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endosomal escape.

Figure 5. Target gene knockdown via PEG-nGO-mediated delivery of antisense PNA into cells. (a) Fluorescence microscopy images of eGFP-expressing A549 cells after treatment with scrambled PNA/PEG-nGO or anti-eGFP PNA/PEG-nGO complexes. (b) Western blot analysis of EGFR protein expression (c) Caspase 3/7 apoptosis activity in A549 cells after treatment with scrambled PNA/PEG-nGO or anti-EGFR PNA/PEG-nGO complexes. Bar graphs in (b) indicate EGFR expression levels normalized against internal control, GAPDH. Data are presented as mean ± SD, n=3. *P < 0.01 vs. control group. 8

Finally, we tested the efficiency of gene knockdown via PEG-nGO-mediated delivery of

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PNA in cancer cells (Figure 5). Antisense PNA oligonucleotides targeting the gene encoding

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enhanced green fluorescent protein (eGFP) were complexed with PNA/PEG-nGO and

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administered to eGFP-transfected lung cancer cells (Figure 5a). Cells treated with anti-eGFP

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PNA/PEG-nGO complexes showed weaker green fluorescence signals, whereas cells treated 16


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with PNA/PEG-nGO complexes containing scrambled sequences of PNA retained strong

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fluorescence intensities. To further determine whether our PNA delivery system using PEG-

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nGO can be used for cancer gene therapy, we examined the efficiency of knockdown of the

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EGFR gene using the anti-EGFR PNA/PEG-nGO complexes in lung cancer cells.

5

Knockdown of EGFR, which is known as a regulator of cell proliferation, activates caspase

6

family leading to apoptosis in cancer cells.43 Lung cancer cells treated with 5 µM anti-EGFR

7

PNA/PEG-nGO complexes showed fivefold lower EGFR expression compared with control

8

(Figure 5b). In addition, cancer cells transfected with anti-EGFR PNA/PEG-nGO complexes

9

showed significantly higher caspase 3/7 activity compared to cells treated with control or

10

control PNA Moreover, cells treated with anti-EGFR PNA/PEG-nGO underwent apoptosis as

11

a result of effective EGFR knockdown (Figure 5c). These results indicate that antisense PNA

12

delivered to cancer cells using PEG-nGO led to effective knockdown of the target oncogene.

13

Therefore, PEG-nGO materials serve as a promising PNA delivery system for target gene

14

knockdown in cells.

15 16

CONCLUSION

17

In summary, we demonstrated an effective PNA gene delivery system using PEG-nGO for

18

targeted gene knockdown in cancer cells. PEGylated nano-sized GO showed lower

19

cytotoxicity and higher aqueous dispersibility than GO alone without aggregation in high salt

20

solutions, such as PBS and cell culture media. PEG-nGO showed preserved GO properties of

21

nucleic acid adsorption and readily adsorbed single-stranded PNA on the surface. Moreover,

22

the adsorbed PNA were readily desorbed from the PEG-nGO surface upon the addition of

23

complementary RNA or under low pH conditions, which are similar to the cellular

24

environments in endosomes and lysosomes. PEG-nGO were taken up by lung cancer cells

25

and more effectively delivered PNA than free PNA or PNA/nGO complexes. Cellular uptake 17



1

of PEG-nGO was mediated via endocytosis, whereas free PNA was not observed to be

2

delivered via endocytosis. As illustrated in scheme 1, PNAs adsorbed onto the PEG-nGO

3

surface are released under acidic conditions of endosomes/lysosomes and eventually diffuse

4

to the cytosol through endosomal escape, while PEG-nGO appears to be trapped in the

5

endosomes/lysosomes. Targeted gene knockdown of green fluorescent protein and EGFR was

6

performed to validate the effectiveness of target gene knockdown via PEG-nGO-mediated

7

PNA delivery. Collectively, the results demonstrated that PEG-engrafted graphene oxide is

8

effective for PNA delivery and serves as a promising gene delivery tool for cancer gene

9

therapy.

18


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Scheme 1. Schematic illustration showing targeted gene knockdown via PEG-nGO-mediated delivery of antisense PNA into cells: a) Cellular uptake of PNA/PEG-nGO complexes; b) Endosomal escape of PNA released from the PEG-nGO surface; c) Binding of antisense PNA to the target mRNA, leading to target gene knockdown. 1 2

EXPERIMENTAL PROCEDURES

3

PEGylated nano-sized graphene oxide (PEG-nGO), FAM-labeled PEG-nGO (FAM-

4

PEG-nGO), and oligonucleotides. Graphene oxide (GO) was purchased from Graphene

5

Laboratories, Inc. (HCGO-W-175, Ronkonkoma, NY, USA), and 6-arm polyethylene glycol-

6

amine (15 kDa) was purchased from SunBio (Seoul, Korea). PEG-nGO was prepared

7

according to a previously described method.36 To synthesize FAM-labeled PEG-nGO, PEG-

8

nGO (1 mg·mL-1) was mixed with 5-carboxyfluorescein (5-FAM; Anaspec, Fremont, CA,

9

USA) (0.1 mg·mL-1), and the resulting mixture was bath sonicated for 5 min. N-(3-

10

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC; Sigma-Aldrich, St. Louis,

11

MO, USA) (5 mM) was added to the mixture with stirring for 12 h, and the reaction was

12

terminated by adding 50 mM 2-mercaptoethanol (Bio Basic Inc., Ontario, Canada). The 19



1

mixture was dialyzed in distilled water for 12 h to remove EDC, 2-mercaptoethanol, or free

2

5-FAM. The PNA oligonucleotides used in this study (sequences are shown in Figure 3a)

3

were chemically synthesized by PANAGENE (Daejeon, Korea). RNA oligonucleotides were

4

chemically synthesized and purified via HPLC and polyacrylamide gel electrophoresis (ST

5

Pharm Co. Ltd. Seoul, Korea).

6

7

Characterization of nGO and PEG-GO constructs. The size and thickness of the graphene

8

materials (nGO and PEG-nGO) were measured via atomic force microscopy (XE-100 AFM;

9

Park Systems, Seoul, Korea). The surface morphology of nGO or PEG-nGO was evaluated

10

via transmission electron microscopy (LIBRA 120; Carl Zeiss, Oberkochen, Germany). The

11

corresponding zeta potentials were recorded using a ELS Z-1000 (Otsuka Electronics, Osaka,

12

Japan). Size of nGO and PEG-nGO was measured by dynamic light scattering

13

spectrophotometer (DLS-7000, Otsuka Electronics) in PBS buffer. To examine the solubility

14

of the different types of graphene materials, nGO, HOOC-nGO, and PEG-nGO were

15

prepared in different solutions (water, PBS, and cell culture media). After centrifugation at

16

10,000 g for 5 min, the nGO, HOOC-nGO, and PEG-nGO suspensions were observed.

17

18

Cell culture and PEG-nGO uptake. A549 cells (non-small lung cancer cell line; purchased

19

from American Type Culture Collection, ATCC CCL-185) were maintained in DMEM

20

supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. ARPE-19 cells

21

(retinal pigment epithelial cell line; purchased from American Type Culture Collection,

22

ATCC CRL-2302) were maintained in DMEM-F12 supplemented with 10% fetal bovine

23

serum and 1% penicillin/streptomycin. Both A549 and ARPE-19 cells were cultured at 37 °C 20


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1

under a humid 5% CO2 atmosphere.

2

A549 and ARPE-19 cells were seeded in a 12-well plate with a cover glass (18 mm ∅) and

3

incubated with Opti-MEM at 37 °C for 2 h. Cells were treated with 100 µg·mL-1 FAM-PEG-

4

nGO for 24 h. After treatment, cells were fixed in 4% paraformaldehyde at room temperature

5

for 1 h. Cells were then mounted on a coverslip using ProLong Gold antifade reagent

6

(Invitrogen Life Technologies) and observed using a confocal microscope (Olympus FV-1000

7

spectral, Tokyo, Japan).

8 9

Cytotoxicity and cell images. A549 and ARPE-19 cells were seeded in 24-well plates and

10

treated with nGO or PEG-nGO (10, 100 µg·mL-1) for 24 h. After incubation, cells were

11

washed thrice with PBS. Cell viability was determined using WST-1 reagent (EZ-Cytox Cell

12

Viability Assay Kit, Daeil Lab Service Co Ltd., Seoul, Korea) according to the

13

manufacturer’s instructions. To assess cell viability, the absorbance at 450 nm was measured

14

by using a VICTOR X3 multilabel plate reader (PerkinElmer, Waltham, MA, USA). To

15

observe the autophagic marker, western blot analysis was performed after nGO or PEG-nGO

16

(10, 100 µg·mL-1) treatment. The following antibodies were used: anti-LC3B (Cell Signaling

17

Technology, Beverly, MA, USA), anti-GAPDH (Abcam, Cambridge, MA, USA), and

18

horseradish peroxidase-conjugated anti-rabbit (Santa Cruz biotechnology).

19

A549 cells were seeded in a 100-mm dish and incubated with 100 µg·mL-1 of nGO, PEG-

20

nGO, or 1 µg·mL-1 lipopolysaccharide (LPS, purchased from Sigma-Aldrich) for 24 h. Cells

21

were fixed with 2% paraformaldehyde and 2% glutaraldehyde in 0.05 M sodium cacodylate

22

buffer (pH 7.2) at 4 °C for 2 h. Cells were washed thrice with 0.05 M sodium cacodylate

23

buffer (pH 7.2) and postfixed with 1% osmium tetroxide and 0.05 M cacodylate buffer at

24

4 °C for 2 h. After postfixation, cells were washed twice with distilled water and stained en

25

bloc in 0.5% uranyl acetate at 4 °C overnight. Cells were then dehydrated with graded 21



1

ethanol series and transitioned into 100% propylene oxide. Dehydrated cells were infiltrated

2

with Spurr resin (Electron Microscopy Sciences, Hatfield, PA, USA) in propylene oxide and

3

polymerized in Spurr resin at 70 °C overnight. Cells were sequentially stained with 2%

4

uranyl acetate and Reynolds’ lead citrate after ultramicrotome sectioning (MTX, RMC,

5

Tucson, AZ, USA). Prepared cells were observed via transmission electron microscopy

6

(LIBRA 120).

7 8

Adsorption of fluorescent single-stranded nucleic acids on PEG-GO. For fluorescence-

9

quenching analysis with increasing concentrations of nGO or PEG-nGO, 1 µM FITC-PNA

10

was incubated with varying concentrations of nGO or PEG-nGO (0, 1, 5, 10, 20, 30, 50, and

11

100 µg·mL−1) for 10 min at room temperature. After incubation, FITC fluorescence intensity

12

was measured on a multilabel plate reader (VICTOR X3) with an excitation wavelength of

13

485 nm, and emission wavelength of 535 nm.

14 15

Desorption of fluorescent single-stranded PNA from PEG-nGO. FITC-PNA (1 µM) was

16

pre-adsorbed onto 100 µg·mL−1 nGO or PEG-nGO. After 10 min of incubation at room

17

temperature, complementary or non-complementary RNA (10 to 1000 nM) was added to a

18

mixture of FITC-PNA/nGO or PEG-nGO and incubated for 2 h. FITC fluorescence was

19

measured on a multilabel plate reader (VICTOR X3) with an excitation wavelength of 485

20

nm and emission wavelength of 535 nm. To measure the amount of PNA released from PEG-

21

nGO under acidic conditions, the cell culture media (DMEM, pH 7.5) were adjusted to low

22

pH (pH 5.0 to pH 7.0) with citric acid. FITC-PNA (5 µM) was incubated with PEG-nGO

23

(500 µg·mL−1) for 10 min at room temperature. The resulting FITC-PNA/PEG-nGO mixture

24

(10 µL) was then added to DMEM with a different pH (40 µL). After 10 min of incubation,

22


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1

fluorescence was measured on a multilabel plate reader (VICTOR X3, λex = 485 nm and λem

2

= 535 nm).

3 4

Cellular uptake and endosomal escape of PNA/PEG-nGO. A549 cells were seeded in a

5

12-well plate with a cover glass (18 mm ∅) and incubated with Opti-MEM at 37 °C for 2 h.

6

To prepare the PNA/nGO or PNA/PEG-nGO complexes, fluorescein-labeled PNA (TAMRA

7

or FITC, 5 µM) was added to nGO or PEG-nGO (100 µg·mL-1) in 200 µL of Opti-MEM.

8

After incubation for 10 min at room temperature, the PNA/nGO or PNA/PEG-nGO mixture

9

was added to A549 cells and incubated for 0.5 to 4.5 h. For staining of endosomes/lysosomes,

10

cells were incubated with 75 nM LysoTracker Red DND-99 (Invitrogen Life Technologies,

11

Carlsbad, CA, USA) for 30 min. Cells were then fixed with 4% paraformaldehyde for 1 h at

12

room temperature. Nuclei were stained with Topro-3 (1:1000 dilution), and cells were

13

mounted on a coverslip with ProLong Gold antifade reagent and observed using a confocal

14

microscope (Olympus FV-1000 spectral).

15 16

Gene knockdown with PNA/PEG-nGO. To prepare A549 cells expressing eGFP, cells were

17

seeded in a 60-mm dish and incubated with Opti-MEM at 37 °C for 2 h. Cells were then

18

transfected with 5 µg of eGFP expression plasmid (peGFP-C2; Clontech, Mountain View, CA,

19

USA) using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA). After

20

6 h, cells were washed with PBS and maintained in complete DMEM containing 50 µg·mL-1

21

kanamycin. eGFP-expressing A549 cells were seeded in 12-well plates with a cover glass (18

22

mm ∅) and incubated in Opti-MEM for 2 h. Anti-eGFP PNA (1 µM) was mixed with 20

23

µg·mL-1 PEG-nGO and incubated for 10 min. Anti-eGFP PNA/PEG-nGO complexes were

24

added to eGFP-expressing A549 cells and incubated for 3 h. Afterwards, cells were washed 23



1

and incubated in complete DMEM for 21 h. Cells were fixed with 4% paraformaldehyde and

2

mounted on the coverslip with ProLong Gold antifade reagent. The eGFP fluorescence of

3

A549 cells was observed using a fluorescence microscope (AxioVert 200, Carl Zeiss,

4

Oberkochen, Germany).

5

For EGFR knockdown, A549 cells were seeded in 6-well plates and incubated with Opti-

6

MEM for 2 h. The Anti-EGFR PNA (1 µM) was incubated with 20 µg·mL-1 PEG-nGO for 10

7

min at room temperature. A549 cells were treated with anti-EGFR PNA/PEG-nGO

8

complexes and incubated for 3 h. Cells were then washed and incubated in complete DMEM

9

for 21 h. Caspase 3/7 activity was measured using Caspase-Glo 3/7 reagent (Promega, Paris,

10

France) according to the manufacturer’s instructions. The luminescence corresponding to

11

caspase 3/7 activity was measured on a VICTOR X3 multilabel plate reader.

12 13

24


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1

ASSOCIATED CONTENT

2

Supporting Information.

3

The Supporting Information is available free of charge on the ACS Publications website at

4

DOI:

5 6

AUTHOR INFORMATION

7

Corresponding Author

8

* E-mail: [email protected]

9

Notes

10

The authors declare no competing financial interests.

11 12

ACKNOWLEDGMENT

13

This work was supported by research funds of Konkuk University in 2016.

14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29

Followings are authors’ ORCID information; Ahruem Baek, Ph.D ORCID ID: https://orcid.org/0000-0001-6672-6339 Yu Mi Baek, MS ORCID ID: https://orcid.org/0000-0002-3049-1237 Hyung-Mo Kim, MS ORCID ID: https://orcid.org/0000-0001-8507-089X Bong-Hyun Jun, Ph.D. ORCID ID: https://orcid.org/0000-0001-6556-7946 Dong-Eun Kim, Ph.D. 25



1 2

ORCID ID: https://orcid.org/0000-0001-6545-8387

3

ABBREVIATIONS GO

graphene oxide

nGO

nano-sized graphene oxide

PEG-nGO

polyethylene glycol-engrafted nano-sized graphene oxide

FAM-PEG-nGO

5-carboxyfluorescein-labeled PEG-nGO

PNA

peptide nucleic acid

FITC-PNA

fluorescein Isothiocyanate -labeled peptide nucleic acid

TAMRA-PNA

5-Carboxytetramethylrhodamine -labeled peptide nucleic acid

eGFP

enhanced green fluorescent protein

EGFR

epidermal growth factor receptor

4 5

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Polyethylene Glycol-engrafted Graphene Oxide as Biocompatible

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