Gene delivery ability of polyethylenimine and polyethylene glycol dual-functionalized nanographene oxide in 11 different cell lines

We recently developed a polyethylenimine (PEI) and polyethylene glycol (PEG) dual-functionalized reduced graphene oxide (GO) (PEG−nrGO−PEI, RGPP) for high-efficient gene delivery in HepG2 and Hela cell lines. To evaluate the feasibility and applicability of RGPP as a gene delivery carrier, we here assessed the transfection efficiency of RGPP on gene plasmids and siRNA in 11 different cell lines. Commercial polyalkyleneimine cation transfection reagent (TR) was used as comparison. In HepG2 cells, RGPP exhibited much stronger delivery ability for siRNA and large size plasmids than TR. For green fluorescent protein (GFP) plasmid, RGPP showed about 47.1% of transfection efficiency in primary rabbit articular chondrocytes, and about 27% of transfection efficiency in both SH-SY5Y and A549 cell lines. RGPP exhibited about 37.2% of GFP plasmid transfection efficiency in EMT6 cells and about 26.0% of GFP plasmid transfection efficiency in LO2 cells, but induced about 33% of cytotoxicity in both cell lines. In 4T1 and H9C2 cell lines, RGPP had less than 10% of GFP plasmid transfection efficiency. Collectively, RGPP is a potential nano-carrier for high-efficiency gene delivery, and needs to be further optimized for different cell lines.

LW, 0000-0001-6265-530X; TC, 0000-0002-0305-2629 We recently developed a polyethylenimine (PEI) and polyethylene glycol (PEG) dual-functionalized reduced graphene oxide (GO) (PEG−nrGO−PEI, RGPP) for highefficient gene delivery in HepG2 and Hela cell lines. To evaluate the feasibility and applicability of RGPP as a gene delivery carrier, we here assessed the transfection efficiency of RGPP on gene plasmids and siRNA in 11 different cell lines. Commercial polyalkyleneimine cation transfection reagent (TR) was used as comparison. In HepG2 cells, RGPP exhibited much stronger delivery ability for siRNA and large size plasmids than TR. For green fluorescent protein (GFP) plasmid, RGPP showed about 47.1% of transfection efficiency in primary rabbit articular chondrocytes, and about 27% of transfection efficiency in both SH-SY5Y and A549 cell lines. RGPP exhibited about 37.2% of GFP plasmid transfection efficiency in EMT6 cells and about 26.0% of GFP plasmid transfection efficiency in LO2 cells, but induced about 33% of cytotoxicity in both cell lines. In 4T1 and H9C2 cell lines, RGPP had less than 10% of GFP plasmid transfection efficiency. Collectively, RGPP is a potential nano-carrier for high-efficiency gene delivery, and needs to be further optimized for different cell lines.
2017 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

Introduction
Gene delivery provides a powerful tool for exploring gene function, generating transgenic organisms and treating gene-related diseases [1][2][3]. A variety of gene functional sensors have been designed to monitor intracellular dynamic processes such as activation of protein kinases, oligomerization of proteins and protein-protein interactions in single living cells [4][5][6][7][8][9][10]. Although RNA interference can only partially silence a target gene and has extensive off-target effects, it has been widely used for genome-wide loss-of-function screens and evaluating the function of genes and proteins [11][12][13][14][15]. CRISPR/Cas9, a versatile genome editing technology, can target nearly any DNA sequence, and the high efficiency of genome editing with Cas9 makes it possible to alter many targets in parallel, thereby enabling unbiased genome-wide functional screens to identify genes that play an important role in a phenotype of interest [15][16][17]. Moreover, CRISPR/Cas9 can generate cellular transgenic models for studying human polygenic diseases, generating transgenic animal models and repressing or activating gene transcription [15,[18][19][20][21][22][23]. With high potential to treat diseases at genetic roots, gene therapy has long fascinated scientists and clinicians. In the past two decades, a series of phase I/II gene-therapy clinical trials have been reported to have significant efficacy and safety for the treatment of various severe inherited diseases of the immune, blood and nervous systems [24][25][26][27][28].
In this report, we evaluate the delivery ability of RGPP for siRNA and plasmids in 11 kinds of cell lines. Commercial polyalkyleneimine cation transfection reagent (TR) was used as comparison. In HepG2 cells, RGPP exhibited better gene delivery ability than TR for siRNA, large size for plasmids such as GFP-Bcl-xL (9.8 kb) and other functional gene plasmids including GFP-Bak, GFP-Puma, GFP-Bax 1-2/L-6 and GFP-NPM NLS1/2D. RGPP could efficiently deliver GFP plasmid into primary rabbit articular chondrocyte, SH-SY5Y, A549, EMT6 and LO2 cell lines. However, RGPP inefficiently delivered GFP plasmid into H9C2 and 4T1 cell lines. chondrocytes were prepared according to our previously reported method and identified by using toluidine blue staining [49,50].

Synthesis of PEG−nGO
GO was prepared by a modified Hummers method using expandable graphite flake [51,52]. GO (10 mg) was sonicated with Ultrasonic Cell Crusher (JY92-2D; Xinzhi Ultrasonic Equipment Co, Ningbo, China) for 1 h in ice bath to obtain nano-GO (nGO) suspension. NaOH (1.2 g) and ClCH 2 COOH (1.0 g) were added to the nGO suspension and sonicated for 30 min in ice bath to obtain carboxylation of nGO (nGO−COOH). The resulting nGO−COOH suspension was washed three to five times with deionized water by using 30 kDa molecular weight cut off (MWCO) ultrafiltration device (Millipore, Bedford, MA, USA) to remove ions.
PEG−nGO was obtained just as described previously [53]. Briefly, 2 ml of EDC aqueous solution (2.0 mg ml −1 ) was added to 10 ml of nGO suspension (1.0 mg ml −1 ), and pH of the mixture was adjusted to 8.0 by 5 mM NaOH and then sonicated for another 10 min. Next, 30 mg of 8-arm PEG-NH 2 was added to the above suspension, and the mixture was sonicated for 10 min and then stirred for 12 h at room temperature to obtain nGO-PEG. The resulting nGO-PEG suspension was washed three to five times with deionized water by using ultrafiltration device (30 kDa MWCO) to remove the unreacted PEG and ions.

Synthesis of PEG−nrGO−PEI
RGPP was prepared as described previously [45]. Briefly, 2 ml PEG-nGO solution (1.0 mg ml −1 ) was mixed with 8 ml PEI (25 kDa) solution (0.75 mg ml −1 ) and then bathed at 80°C for 2 h to obtain RGPP solution. The resulting RGPP solution was dialysed against deionized water in a 30 kDa MWCO dialysis membrane for 2 days. The 630 nm ultraviolet-visible (UV-vis) absorbance of cuprammonium complex formed between PEI and copper(II) ion was measured by UV-vis spectrometer (Lambda 35; PerkinElmer, MA, USA) for determining the amount of modified PEI in RGPP [54].
Cytotoxicity of RGPP and TR as well as plasmid was assessed by CCK-8 assay according to the manufacturer's protocol just as described previously [55]. Cells were collected and seeded in 96-well plates with a density of 1 × 10 4 cells well −1 and incubated for 24 h. Plasmid (0.2 µg) and different amount of RGPP or TR were mixed in 20 µl serum-free media and incubated at room temperature for 20 min. Plasmid/RGPP or plasmid/TR complexes were incubated with cells for 4 h in 100 µl serum-free media. Then, serum-free media were replaced by fresh media containing 10% FBS and the cells were incubated for another 44 h before CCK-8 assay.

Transfection assay of plasmids or siRNA
Cells were seeded in 24-well plates with a density of 1 × 10 5 cells/well and incubated for 24 h before transfection. For siRNA transfection, 0.369 µg of FAM-labelled siRNA and different amount of RGPP or TR were mixed in 100 µl serum-free media and incubated at room temperature for 20 min. Complexes of RGPP/siRNA or TR/siRNA were incubated with cells for 4 h in 500 µl serum-free media, and then siRNA transfection efficiency was determined by fluorescence microscope imaging (Olympus IX73 equipped with a CCD camera, Japan) and flow cytometry analysis (FCM, FACSCCanto II, BD Biosciences, NJ, USA). For plasmid transfection, 1 µg of plasmid and different amount of RGPP or TR were mixed in 100 µl serum-free media and incubated at room temperature for 20 min. After the complexes of RGPP/plasmid or TR/plasmid were incubated with cells for 4 h in 500 µl serum-free media, the serum-free media were replaced by fresh media containing 10% FBS and the cells were cultured for another 44 h before fluorescence microscope imaging or FCM analysis for transfection efficiency.

Statistics
Data were presented as mean ± s.d. from at least three independent experiments. The Student's t-test was used to evaluate the significance of difference between two groups. Statistical and graphic analyses were done using the software SPSS 19.0 (SPSS, Chicago) and Origin 8.5 (OriginLab Corporation). p < 0.05 was defined as statistically significant difference.

Transfection efficiency of RGPP on plasmids and siRNA in HepG2 cells
After exposure of cells to RGPP or TR for 48 h, CCK-8 assay showed that both RGPP and TR exhibited dose-dependent cytotoxicity, and RGPP at N/P ratio of 60 and 0.4% (TR volume/medium volume) of TR had similar cytotoxicity (figure 1a,b). RGPP at N/P ratio of 60 and 0.4% of TR were used for plasmid transfection without indication.
Size of plasmid is one of the elements influencing transfection efficiency during transient transfection assay [56,57]. Transfection efficiency of RGPP for GFP-Bcl-xL plasmid (9.8 kb) was about 18.9%, about threefold higher than TR (about 4.7%; figure 1c), suggesting that RGPP exhibited better delivery ability for large-size plasmid than TR. It was reported that huge aggregates (greater than 1000 nm) of DNA/transferrin-PEI exhibited much higher transfection efficiency than small aggregates in in vitro transfection assay [58]. Diameter of the GFP-Bcl-xL plasmid/RGPP complexes was about 987 nm, and the particle size of DNA/TR was 50-150 nm [59], which might be the reason why RGPP more efficiently delivered large-size plasmid into cells than TR. In addition, RGPP also exhibited better delivery ability for siRNA than TR (figure 1e,f ), which might benefit from the inhibitory effect of RGPP on gene degradation [30].     0%; figure 2c), suggesting that RGPP at N/P ratio of 90 was superior to 0.4% of TR as a gene delivery carrier for SH-SY5Y cells. Fluorescence microscopic images also showed that RGPP at N/P ratio of 90 delivered more GFP plasmid into SH-SY5Y cells than 0.4% of TR (figure 2d).

Conclusion
The transfection efficiency of RGPP for GFP plasmid in 10 different cell lines summarized in table 3 demonstrates that RGPP is a potential gene delivery carrier. Especially, RGPP can efficiently deliver siRNA and large-size plasmids. RGPP exhibits efficient plasmid delivery ability in primary rabbit articular chondrocyte, SH-SY5Y, A549, EMT6 and LO2 cell lines. Moreover, based on the excellent photothermal efficiency of reduced GO (rGO), NIR enhances gene delivery ability of RGPP. Collectively, RGPP is a potential nano-carrier for high-efficiency gene delivery and further studies are needed to optimize its gene delivery ability for different cell lines. Competing interests. We declare we have no competing interests. Funding. This work was supported by the National Natural Science Foundation of China (61527825 and 81471699) and the Guangdong Province Science and Technology Plan Project (2014B090901060).