Hydrothermal Reduction of Polyethylenimine and Polyethylene Glycol

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Hydrothermal Reduction of Polyethylenimine and Polyethylene Glycol Dual-Functionalized Nanographene Oxide for High-Efficiency Gene Delivery Tan Li,† Liping Wu,† Jiang Zhang,† Gaina Xi,† Yilin Pang,† Xiaoping Wang,*,‡ and Tongsheng Chen*,† †

MOE Key Laboratory of Laser Life Science & College of Biophotonics, South China Normal University, Guangzhou 510631, China Department of Pain Management, The First Affiliated Hospital of Jinan University, Guangzhou, 510632, China

‡

S Supporting Information *

ABSTRACT: In this study, a physiologically stable dual-polymer-functionalized reduced nanographene oxide (nrGO) conjugate (PEG−nrGO−PEI, RGPP) with high efficiency of gene delivery is successfully synthesized through mixing PEGylated nanographene oxide (PEG−nGO, GP) and polyethylenimine (PEI, 25 kDa) solution under 80 °C for 2 h. This hydrothermal reduction of GP during PEIylation promotes the nucleophilic reaction between the amino moieties of PEI and the epoxy groups (or carboxylic groups) in GP and then forms C−NH− groups (or NH−CO groups) to covalently connect PEI and GP, which makes the RGPP nanocomposite more stable in physiological environments and has superior gene transfection efficiency compared with the nonhydrothermally reduced PEG−nGO/PEI conjugate (GPP) obtained by mixing GP and PEI under 20 °C for 2 h. Moreover, 808 nm laser irradiation (2 W/ cm2) for 25 min increases ∼1.5-fold of gene transfection efficiency for RGPP but does not increase the gene transfection efficiency of GPP. Finally, RGPP is also able to efficiently deliver functional plasmid GFP-Bax (pGFP-Bax), exhibiting ∼43% of transfection efficiency in HepG2 cells. Collectively, the RGPP developed here is a highly efficient nanocarrier for gene delivery, and this work encourages further explorations of developing functionalized reduced nano-GO for high-efficiency gene therapy. KEYWORDS: polyethylenimine, polyethylene glycol, hydrothermal reduction, nanographene oxide, photothermal effect, gene delivery

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INTRODUCTION Gene therapy has been explored as a revolutionary therapeutic strategy following chemical, radioactive, and surgical operation methods for the potent potential to cure gene-related diseases in the past two decades. Success of gene therapy requires a safe and high-efficiency gene delivery vector.1 Viral vectors, a highly efficient gene delivery strategy, have been hampered by safety concerns including immune response, toxicity, chromosomal integration, and mutagenesis.2,3 Considering the safety, nonimmunogenicity, and ease of manufacture, nonviral vectors such as polymers,4,5 lipids,6−8 and inorganic nanomaterials (gold nanoparticles, silica nanoparticles, and carbon nanotubes, etc.)9−11 have been intensively investigated for gene delivery with high safety, efficiency, and selectivity. Polyethylenimine (PEI), a cationic polymer material, has been widely used for gene transfection.12 Low molecular weight branched PEI (LMW BPEI) exhibited relatively low cytotoxicity and poor transfection efficiency.13−15 Although high molecular weight branched PEI (HMW BPEI) showed high transfection efficiency, the high cytotoxicity severely limited its application as an effective gene carrier.16−18 Recently, PEI-functionalized nanographene oxide (nGO− PEI) composites through covalent linking or noncovalent adsorption have been developed, and the nGO−PEI showed lower toxicity and improved transfection efficiency compared with PEI alone.19−21 Chen and co-workers successfully © XXXX American Chemical Society

synthesized a GO−PEI composite via EDC chemistry between GO and HMW PEI (25 kDa). 22 Although GO−PEI composites showed a relatively high transfection efficiency and low cytotoxicity, precipitation of the GO−PEI composites in the presence of saline or serum severely limited its further bioapplications.20,23 Polyethylene glycol (PEG), a biocompatible and nontoxic surfactant, is widely used to improve the stability and toxicity of GO composites at physiological conditions.24,25 Feng and co-workers developed PEG and HMW PEI (25 kDa) coconjugated ultrasmall nano-GO carrier (NGO−PEG−PEI) via EDC chemistry for photothermally enhanced gene delivery.23 Compared with the bare PEI and GO−PEI without PEGylation, the NGO−PEG−PEI composites showed excellent stability, lower cytotoxicity, and superior gene transfection efficiency. Based on the higher photothermal efficiency of reduced GO (rGO) over GO,26−28 Kim and colleagues developed a dual-functionalized rGO (PEG−BPEI− rGO) for photothermally triggered cytosolic drug/gene delivery.29,30 Considering the efficient reducing and surface modifying abilities of PEI polymers,31−34 we recently developed a dual-functionalized nanosized rGO (nrGO−PEG/PEI) composite for the synergistic chemo−photothermal therapy Received: August 9, 2016 Accepted: October 26, 2016

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DOI: 10.1021/acsami.6b09915 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

the UV−vis absorbance of cuprammonium complexes of GPP and RGPP at 630 nm. Nitrogen/phosphate ratio (N/P, N: the molar mass of nitrogen element on PEI; P: the molar mass of phosphate element on pDNA) was calculated by the empirical eq 1

of cancers through PEGylation of nGO for PEG−nGO via EDC chemistry and hydrothermal reduction of PEG−nGO with LMW PEI (1.8 kDa) at 80 °C for 2 h.35 Herein, we developed an HMW PEI (25 kDa) and PEG dual-functionalized nanosized rGO (PEG−nrGO−PEI, RGPP) for highly efficient gene delivery through two steps (Scheme 1):

N /P = (A × 23.25)/(B × 3.08) = 7.25 × A /B

where A is the weight mass of PEI (μg) and B is the weight mass of pDNA (μg). Characterizations. X-ray photoelectron spectroscopy (XPS) measurements (AXIS Ultra DLD, Kratos Analytical Ltd., UK) were conducted with a monochromatized Al Ka radiation at 1486.6 eV. Atomic force microscope (AFM) imaging, zeta potentials and diameters analyses, UV−vis and Fourier transfer infrared (FT-IR), as well as Raman spectral analyses of GC, GP, GPP, and RGPP were performed just as described previously.35 Cell Culture and Cytotoxicity Assay. HepG2 and HeLa cells obtained from the Department of Medicine, Jinan University (Guangzhou, China), were cultured in DMEM supplemented with 10% fetal calf serum and maintained in a humidified incubator at 37 °C and 5% CO2. Cytotoxicity of RGPP, GPP, PEI, and Lipofectamine 2000 transfection kit (Lip-2000) was assessed by using a CCK-8 assay.35,37 Briefly, after 24 h incubation of about 1 × 104 of HepG2 cells per well seeded in a 96-well plate, different concentrations of RGPP, GPP, PEI, and Lip-2000 were added to designated wells and incubated for another 24 h before the CCK-8 assay by using a microplate reader as described previously.37 Photothermal Effect Measurements. The test water, GPP, and RGPP (N/P = 60) solutions (100 μL) were introduced in a 96-well plate, respectively, and exposed to the 808 nm laser irradiation (2 W/ cm2) for 30 min, and the corresponding temperatures were recorded by a thermocouple thermometer every other 5 min as described previously.35 To evaluate the safety of NIR laser irradiation, HepG2 cells were seeded in a 96-well plate at a density of 1 × 104 cells per well. After 24 h incubation at 37 °C, the cell medium was replaced with fresh medium containing GPP or RGPP solutions (N/P = 120) and immediately irradiated by 808 nm laser with 2 W/cm2 for different times (0−30 min) in a 37 °C incubator and then recultured in 100 μL of fresh 10% FBS-containing DMEM medium, followed by measuring the relative cell viabilities through CCK-8 assay after 24 h. Agarose Gel Retardation Assay. Complexes of pDNA (GFP) with PEI, GPP, and RGPP and their agarose gel were prepared as described previously by Kim’s group.20 Briefly, 8 μL of sample suspension was added to the pDNA solution (1200 ng, 2 μL) at various N/P ratios and incubated for 30 min at room temperature. The gel was then analyzed on a UV illuminator (WD-9413B, Liuyi, Inc., Beijing, China). Cellular Uptake of the RGPP/pDNA Complex. HepG2 cells were seeded in 24-well plates. pDNA (GFP) was labeled with SYBR Green I to obtain pDNA−(SYBR Green I). RGPP/pDNA−(SYBR Green I) complexes (N/P = 60), GPP/pDNA−(SYBR Green I) complexes (N/P = 60), and PEI/pDNA−(SYBR Green I) complexes (N/P = 30) were diluted with DMEM medium (the mass of pDNA is 600 ng). After the mixtures were incubated with HepG2 cells for 4 h, the cells were rinsed 3 times with PBS. To study cellular uptake of the samples/pDNA, fluorescence images of the cells were captured using a fluorescence microscope (Olympus IX73, Japan). In Vitro GFP and GFP-Bax Plasmid Transfection. About 1 × 105 cells per well were seeded in a 24-well plate for GFP plasmid transfection. Briefly, we diluted 600 ng of GFP plasmid in 25 μL of FBS-free DMEM medium and diluted the designated amount of PEI, GPP, and RGPP, respectively, in 25 μL of FBS-free DMEM medium. Five minutes later, the GFP plasmid and carrier solutions were mixed and incubated together for another 30 min before being added into cells and incubated for 4 h under 500 μL (final volume) of fresh FBSfree DMEM medium, and then the cells were washed with PBS and recultured in 500 μL of fresh 10% FBS-containing DMEM medium for another 44 h. To evaluate GFP expression, cells were imaged by the fluorescence microscope under 488 nm excitation, and then the gene

Scheme 1. Synthesis Scheme of PEG−nrGO−PEI (RGPP) and PEG−nGO/PEI (GPP) Nanocomposites

(1) PEGylation of nGO−COOH (GC) for PEG−nGO (GP) via EDC chemistry and (2) simultaneous hydrothermal reduction and PEIylation of GP by mixing HMW PEI (25 kDa) and GP at 80 °C for 2 h. Compared with the nonhydrothermal PEG−nGO/PEI (GPP) synthesized by mixing PEG−nGO with HMW PEI (25 kDa) at 20 °C for 2 h (Scheme 1), RGPP showed excellent stability and higher transfection efficiency due to the covalent linking between GP and PEI. Meanwhile, RGPP performed photothermally enhanced gene delivery ability due to its sensitive photothermal effect. Our results highlight the great promise of RGPP for highly efficient gene delivery and gene therapy.

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EXPERIMENTAL SECTION

Materials. GO powder was purchased from XF NANO Co., Ltd. (Nanjing, China). Branched polyethylenimine (25 kDa) and N-(3(dimethylamino)propyl-N′-ethylcarbodiimide) hydrochloride (EDC) were purchased from Sigma-Aldrich (Shanghai, China). 8-Armpolyethylene glycol-amine (10 kDa, PEG-NH2) was purchased from Seebio Biotech Inc. (Shanghai, China). Lipofectamine 2000 (Lip2000) transfection kit and Agarose (molecular biology grade) were purchased from Invitrogen (USA). Fetal bovine serum (FBS) was purchased from Sijiqing (Zhejiang, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Thermo Scientific (USA). Green fluorescent protein (GFP) plasmid was provided by Prof. R. J. Youle.36 Synthesis of nGO−COOH (GC) and PEG−nGO (GP). nGO solution was prepared as described previously.35 NaOH (1.2 g) and Cl−CH2−COOH (1.0 g) were added to the nGO suspension and sonicated for 30 min to obtain carboxylation of nGO (nGO−COOH, GC). The resulting GC suspension was neutralized and purified by repeated rinsing and filtration. Next, the PEGylated nGO (PEG− nGO, GP) solution was obtained just as described previously.35 Synthesis of PEG−nGO/PEI (GPP) and PEG−nrGO−PEI (RGPP) Nanocomposites. In a sealed glass bottle, 2 mL of GP solution (approximately 1.0 mg/mL) was mixed with 8 mL of Branched PEI solution (0.75 mg/mL) and then bathed at 80 °C for 2 h to obtain PEG−nrGO−PEI (RGPP) solution or at 20 °C for 2 h to obtain PEG−nGO/PEI (GPP) solution. The resulting GPP and RGPP solutions were purified by a 30 kDa ultracentrifuge tube to remove the unreacted PEI and then stored at 4 °C for further use. The amount of modified PEI in GPP and RGPP was analyzed by measuring B


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ACS Applied Materials & Interfaces delivery efficiency was quantitatively analyzed by counting the bright cells and total cells. In addition, the gene delivery efficiency was also quantitatively analyzed by using flow cytometry (FCM, FACSCantoII, Becton Drive, New Jersey, USA). For FCM quantitative analysis, cells were collected and then resuspended in 500 μL of PBS. The same protocol was used for GPP and PEI transfection. Transfection using Lipofectamine 2000 (Lip-2000) was carried out using the optimal condition recommended by the vendor. To study photothermally enhanced transfection effect, HepG2 cells incubated with RGPP/pDNA or GPP/pDNA were immediately irradiated by an 808 nm laser at power density of 2 W/cm2 for 25 min, and then the cells were washed with PBS and recultured in 500 μL of fresh 10% FBS-containing DMEM medium for another 48 h. For GFP-Bax plasmid transfection, 600 ng of GFP-Bax plasmid was diluted in 25 μL of FBS-free DMEM medium, and the designated amount of GPP or RGPP was diluted in 25 μL of FBS-free DMEM medium, respectively. Five minutes later, the GFP-Bax plasmid and carrier solutions were mixed and incubated together for another 30 min before being added into HepG2 cells and incubated for 24 h under 500 μL (final volume) of fresh 10% FBS-containing DMEM medium, and then the cells were washed with PBS and recultured for another 24 h. Transfection Efficiency Analysis Assay. After pDNA transfection for 48 h in a 24-well plate, HepG2 or HeLa cells were imaged by a fluorescence microscope. FCM analysis was used to quantify the gene transfection efficiency without specific indication, and for each FCM analysis, 10 000 events were recorded. The data in this report indicated the means of at least triplicate measurements.

Figure 1. AFM images of nGO−COOH (GC), PEG−nGO (GP), PEG−nGO/PEI (GPP), and PEG−nrGO−PEI (RGPP).

GO and thus drives more reduction of GO during PEIylation.34,35 Noticeably, GPP and RGPP (200 μg/mL GO) were very stable in water, PBS, and 10% serum-containing medium after centrifuging at 5000g for 10 min (Figure 2A), which might be due to the successful conjunction of PEG.35 Zeta potential analysis was another measurement to verify the PEI and PEG modifications of nGO. As shown in Figure 2B, because of the reaction between negative carboxyl groups of nGO and NH2 groups of PEG,35 the potential (−19.4 mV) of GP was much higher than the −42.2 mV of GC, whereas the zeta potentials of GPP and RGPP in pure water increased to about +35.9 mV due to the cationic PEI decoration (Figure 2B). However, the potential of GPP after PBS elution was reduced to +12.3 mV, which might be due to the separation of partial PEI from GPP under ionic liquid, while the potential of RGPP after PBS elution was still +33.5 mV, indicating that RGPP was very stable against the PBS. In addition, dynamic light scattering (DLS) analysis showed a decreased diameter for GPP after dissolving in PBS solution (Figure 2C). The DLS hydrodynamic sizes of nanoparticles are equivalent to the diameter of the sphere possessing the same translational diffusion coefficient, while the nanoparticle sizes measured by FAM represent the lateral size or basal plane size. Therefore, the DLS hydrodynamic sizes of nanoparticles shown in Figure 2C do not match with the results shown in Figure 1. These results revealed that PEI decoration of RGPP was very stable, while PEI decoration of GPP was not very stable and easily separated from GPP under ionic liquid environment. Characterization of the RGPP Complex. As shown in Figure 3A, the FT-IR spectrum demonstrated the successful synthesis of GP and in situ reduction of GP by PEI under 80 °C for 2 h. The spectra of GC illustrated the presence of C−O−C stretching vibration (at 1225 and 1050 cm−1), C−OH stretching vibration (at 3400 cm−1), and CO stretching vibration (at 1730 cm−1) in carboxylic acid and O−H stretching vibration (at 1381 cm−1) in carboxyl groups.39 The existence of NH−CO (at 1650 cm−1) stretching vibration in GP indicated that GC was covalently conjugated with PEG via an amido bond successfully. The doublet absorption bands at 2841 and

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RESULTS AND DISCUSSION Synthesis and Stability of PEG−nGO/PEI (GPP) and PEG−nrGO−PEI (RGPP). The novel PEI and PEG dualfunctionalized reduced nanographene oxide (PEG−nrGO− PEI, RGPP) was synthesized as shown in Scheme 1. In brief, PEG−NH2 (10 kDa) was covalently conjugated to the carboxylic group of nGO−COOH (GC) using EDC chemistry for 12 h, and the PEGylated nGO (PEG−nGO, GP) was then simultaneously reduced and modified by 25 kDa of HMW PEI under 80 °C for 2 h to obtain PEG−nrGO−PEI (RGPP) or under 20 °C for 2 h to obtain PEG−nGO/PEI (GPP). The estimated conjugation ratios of PEI to GPP and RGPP were 0.714 and 1.066, respectively (Supporting Information, Table S1). AFM images showed that the sizes of GC, GP, GPP, and RGPP were about 121, 105, 109, and 90 nm, respectively (Figure 1), indicating that PEGylation reaction and PEI modification did not affect the size of nGO. The 1.4−2.0 nm thicknesses of GPP and 1.2−1.5 nm thicknesses of GP, slightly larger than the 1.1−1.3 nm of GC, indicated the successful modification of PEG on the GC surface and PEI on the GP surface. However, the 0.9−1.5 nm thickness of RGPP, slightly smaller than that of GP, might be due to the insertion of PEI into GP and the reduction of oxygen-containing groups of GO after reduction reaction. These data firmly confirmed the successful introduction of the PEG and PEI polymers to the nGO surface. The hydrothermal process can provide thermal energy to molecules and accelerate the collision of molecules.38 We recently found that hydrothermal process of nGO under 90 °C for 24 h significantly reduced GO.28 In addition, it was reported that ammonia solution (NH3·H2O) could accelerate the reduction reaction of GO under a hydrothermal process,38 and PEI contains a large number of amino moieties. Therefore, the hydrothermal process promotes the nucleophilic reaction between the amino moieties of PEI and the epoxy groups (or carboxylic groups) in GO to remove the oxygen component at C


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Figure 2. Stability of GPP and RGPP. (A) Photos of GPP and RGPP solutions with approximately 200 μg/mL of GO in different solutions after centrifuging at 5000g for 10 min. (B) Zeta potential of GC, GP, GPP, and RGPP in pure water before and after PBS elution. Note that after PBS elution GC, GP, GPP, and RGPP were resuspended in pure water. (C) Diameters of GC, GP, GPP, and RGPP before and after dissolving in PBS solution (*P < 0.05 and **P < 0.01).

nm) under the same GO concentrations (15.0 μg/mL of GO) (Figure 3C), indicating that bathing the GP with PEI under 80 °C for 2 h strongly reduced GP, while bathing under 20 °C for 2 h only slightly reduced GP, which was further verified by the darkening color of the reduced materials (Figure 2A). These results obviously demonstrated that the hydrothermal processing remarkably enhanced the reduction extent of GP in the presence of PEI. The hydrothermal reduction of GP during PEIylation was further analyzed using X-ray photoelectron spectroscopy (XPS) (Figure 4), and the atomic compositions of GC, GP, GPP, and RGPP are given in Table 1. The PEI-grafting process resulted in a significant increase in N composition, and hydrothermal reduction resulted in a significant decrease in O composition, further demonstrating that the hydrothermal process drives more reduction of GP during PEIylation. The C 1s spectrum of the GC could be quantitatively differentiated into four different carbon species: the sp2-hybridized carbon atoms of graphene at 284.6 eV (C−C), the carbon in hydroxyl and epoxide groups (C−O) at 286.09 eV, the ones in carbonyl groups (CO) at 287.44 eV, and those in carboxyl groups (O−CO) at 288.84 eV (Figure 4A).32−34 After PEG functionalization, an additional component at 286.01 eV corresponding to C−NH− was observed (Figure 4B), indicating that PEG was grafted successfully. The GPP complex showed a slight increase in signals characteristic of the C−NH2 group (Figure 4C),33 indicating that PEI was combined to the surface of GP. After hydrothermal reduction under 80 °C for 2 h, the obtained RGPP showed a decrease in signals characteristic of the C−O group (Figure 4D),32−34 indicating the reduction of GP. Most importantly, the additional component at 287.36 eV (C− NH−) of RGPP (Figure 4D) demonstrated the covalent connection of PEI with GP. Therefore, RGPP is more stable than GPP in physiological conditions (Figure 2). Characterization of the RGPP/pDNA Complex. Agarose gel electrophoresis assay was used to evaluate the binding ability of RGPP with plasmid DNA encoding GFP at different nitrogen/phosphate (N/P) ratios (Figure 5A). Significant retardation of pDNA was observed when PEI or RGPP was mixed with pDNA at N/P ratios above 8, indicating that all pDNA were bound to PEI and RGPP in this circumstance. However, the GPP/pDNA complex at N/P ratios above 15 showed complete pDNA retardation, likely owing to the reduced surface charge of GPP under ionic liquid environment (Figure 2B). As expected, the zeta potential of RGPP/pDNA complexes increased dramatically from −26.2 mV at the N/P ratio of 0 to +18.3 mV at the N/P ratio of 8, while the zeta potential of GPP/pDNA just increased to −10.5 mV (Figure 5B). The diameters of GPP/pDNA and RGPP/pDNA at the

Figure 3. Characterizations of GC, GP, GPP, and RGPP. (A, B, and C) FT-IR spectra (A) and Raman spectra (B) as well as UV−vis absorbance spectra (C) of GC, GP, GPP, and RGPP (15.0 μg/mL of GO).

2943 cm−1 corresponded to symmetric and asymmetric H−C− H of the PEI chains.40,41 Compared with GPP, RGPP had a stronger NH−CO stretching vibration and a decreased C−O (at 1050 cm−1) stretching vibration (Figure 3A), indicating the successful covalent reaction between the amine groups of PEI and the epoxide groups (or carboxylic groups) on GP after hydrothermal reduction of GP during PEIylation. Raman analysis was also provided to estimate the reduction of RGPP (Figure 3B). As shown in Figure 3B, the intensity ratios (ID/IG) of D band to G band of GC, GPP, and RGPP were about 0.881, 0.883, 0.901, and 0.946, respectively. Obviously, GP had a similar ID/IG value to that of GC, indicating that PEGylation did not destroy the aromatic structures of GC.34,35,42 However, the ID/IG value of RGPP was much higher than that of GP, which may be due to the reduction of oxygen-containing functional groups and the introduction of defects into graphene crystal.35,42,43 The slightly increased ID/IG value of GPP might be due to the slight reduction reaction of GP by PEI under room temperature. In addition, the optical absorbance of nanomaterials containing GO at 808 nm was evaluated by UV−vis spectra analysis. UV−vis spectra showed that covalent PEGylation did not significantly influence the NIR absorbance property of GC and that GPP and RGPP presented ∼2.1-fold and ∼11.5-fold increment, respectively, over GP in NIR absorbance (at 808 D


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Figure 4. (A−D) High-resolution XPS C 1s core-level spectra of GC (A), GP (B), GPP (C), and RGPP (D), respectively.

quantitate the cellular uptake efficiency. Microscopic imaging (Figure 6) and the corresponding statistics (Supporting Information, Figure S1) indicated that no noticeable fluorescence of the SYBR-Green I (green) could be observed in HepG2 cells incubated with PEI/pDNA (Figure 6, upper panel), suggesting little pDNA assimilation by cells. After incubation with GPP/pDNA, weak fluorescence was observed, indicating that GPP facilitated intracellular pDNA delivery, though with low efficiency (Figure 6, middle panel). However, strong fluorescence was detected in the cells incubated with the RGPP/pDNA complexes (Figure 6, lower panel), suggesting that RGPP is more efficient at transporting pDNA into cells than GPP and PEI. These results reveal that RGPP has superior pDNA intracellular delivery capability compared with GPP. Cytotoxicity and Gene Transfection Efficiency of RGPP. To evaluate the biocompatibility of RGPP, relative viability of HepG2 cells after being incubated with bare PEI, GPP, and RGPP, respectively, for 24 h was determined by

Table 1. Atomic Compositions of GC, GP, GPP, and RGPP Calculated from XPS Results samples

C (atomic %)

N (atomic %)

O (atomic %)

GC GP GPP RGPP

78.72 68.96 69.65 67.85

0 2.09 12.91 21.31

21.28 28.95 17.44 10.84

N/P ratio of 60 were about 250 nm, which could be suited to endocytosis. These data further confirmed the higher pDNA condensing capability of RGPP over GPP. Intracellular Trafficking Study of the RGPP/pDNA Complex. To image the intracellular distribution of pDNA, pDNA (GFP) was labeled with SYBR-Green I. After incubation of HepG2 cells with PEI/pDNA, GPP/pDNA, and RGPP/ pDNA complexes, respectively, for 4 h, we used a fluorescence microscope to image the cells and also used FCM analysis to

Figure 5. Characterization of RGPP/pDNA complexes. (A) Agarose gel retardation study of the pDNA complexed with PEI, GPP, and RGPP, respectively, at various N/P ratios. (B) Zeta potential of the pDNA complexed with PEI, GPP, and RGPP, respectively, at various N/P ratios and the diameters of GPP/pDNA and RGPP/pDNA at N/P ratio of 60. E


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density. Therefore, the 60 of N/P ratio was the optimal condition for the transfection of RGPP/GPP. In reality, we also evaluated the transfection efficiency of nrGO-PEG/PEI developed recently by using LWM PEI (1.8 kDa)35 and found that this nanocarrier had very low transfection efficiency (data not shown). Therefore, we here developed RGPP by using HWM PEI (25 kDa) for high-efficiency gene delivery. Although RGPP and GPP have a good aqueous stability in both PBS and serum protein solutions (Figure 2 and S3), their ability to deliver gene under the physiological environment containing serum proteins is key for the in vivo gene therapy. We evaluated the GFP transfection efficiencies of RGPP and GPP in the presence of serum in HepG2 cells at their optimal N/P ratios. As shown in Figure 7E and F, bare PEI showed dramatically decreased gene transfection ability when serum was added into the transfection cell culture, which might be due to the adsorption between positive PEI and negative serum protein. 10% of serum showed an increased effect on the transfection efficiencies of RGPP and GPP, likely due to the longer transfection time for 24 h under serum-containing culture instead of the primary transfection time for 4 h under serum-free conditions, indicating that GPP still has a stabilized transfection effect under 10% serum-containing DMEM medium. However, 20% of serum resulted in an ∼10% increase in the gene transfection ability of RGPP but dramatically decreased the gene transfection ability of GPP (Figure S4 (FCM analysis) and the corresponding statistics (Figure 7F)), which might be due to the released PEI from GPP. These results demonstrate that RGPP has not only excellent stability but also superior gene transfection efficiency under a physiological environment containing serum proteins, which is an important advantage for future gene therapy applications. With the stabilized efficient transfection efficiency of RGPP and GPP in HepG2 cells, we also assessed the GFP transfection efficiency of RGPP and GPP in HeLa cells. As shown in Figure S5, the transfection efficiencies of RGPP and GPP with N/P ratio at 60 were ∼83.9% and ∼64.7%, respectively, much higher than the ∼20% transfection efficiency of NGO−PEG−PEI with N/P at 40 reported by Liu and Wang’s group.23 This different transfection efficiency may be due to the different conjugation ratios among nGO, PEG, and PEI, which need to be further confirmed. Because of the covalent connection of GP with PEI, the RGPP complex is very stable in physiological conditions, while noncovalent GPP may be separated into GPP and PEI in FBSfree DMEM medium. Therefore, RGPP is more stable than GPP in physiological conditions (Figure 2). In reality, GPP/ pDNA in FBS-free DMEM medium may contain GPP/pDNA and PEI/pDNA complexes. The PEI/pDNA complex has not only low gene delivery efficiency but also high cytotoxicity,19,20 which may be the reason why RGPP has much higher gene delivery efficiency than GPP. Considering the lower cytotoxicity and high gene delivery ability of RGPP, we here compared the transfection efficiencies of RGPP and Lipofectamine 2000 (Lip-2000), an extensively used commercial transfection reagent, under the same cytotoxicity. Considering the ∼95% of relative cell viability for both RGPP and GPP at N/P = 20 (Figure 7A and 7B), we assessed the cytotoxicity of different volumes of Lip-2000 (Figure S6A), and 0.1 μL for Lip-2000 was chosen to detect the transfection efficiency. Under these safe concentrations of vectors, the transfection efficiency of RGPP was ∼38%, ∼5-fold

Figure 6. Fluorescence images of HepG2 cells treated with PEI/ pDNA (N/P = 30), GPP/pDNA (N/P = 60), and RGPP/pDNA (N/ P = 60) complexes for 4 h, respectively, under FBS-free DMEM medium. Nucleus was stained with Hoechst 33342 (blue), and pDNA was labeled with SYBR Green I (green). Scale bar: 200 μm.

CCK-8 assay (Figure 7A and B). Our results showed that 25 kDa PEI was rather toxic to cells, with the half maximal inhibitory concentration (IC50) value of ∼9 μg/mL (Figure 7A). On the contrary, GPP and RGPP showed very low cytotoxicity at the same PEI concentrations (Figure 7A), likely due to the PEG modifications of nGO. In addition, although both GPP and RGPP with the same N/P ratios showed a similar cytotoxicity, RGPP exhibited higher cytotoxicity over GPP at the same GO concentrations (Figure 7B), which was likely due to the different mass ratios of PEI to GPP and RGPP (Table S1). Gene transfection abilities of GPP, RGPP, and bare PEI were first assessed on HepG2 cells under serum-free conditions by visualizing cells expressing GFP under a fluorescence microscope. HepG2 cells were incubated with RGPP/pDNA or GPP/pDNA complexes with N/P ratios at 15, 30, 60, and 120, corresponding to 2.5, 5.1, 10.1, and 20.2 μg/mL of RGPP or 3.7, 7.4, 14.8, and 29.6 μg/mL of GPP (GO concentration), respectively, in serum-free medium for 4 h, washed with phosphate-buffered saline (PBS), and then reincubated in serum-containing whole cell medium for an additional 44 h before microscopic imaging and FCM analysis. Note that GPP and RGPP solutions were diluted in FBS-free DMEM medium with certain concentrations before transfection. Figure 7C showed the fluorescence microscopic images of HepG2 cells expressing GFP. The transfection efficiencies of RGPP, GPP, and bare PEI were quantitatively analyzed by FCM analysis. RGPP at 60 of N/P ratio showed ∼54.3% gene transfection efficiency, much high than the 29.0% gene transfection efficiency of GPP (Figure S2 (FCM analysis) and the corresponding statistics (Figure 7D)), possibly owing to the high ability of RGPP to transfer much more pDNA into cells. In contrast, bare PEI displayed the lowest transfection efficiency at any N/P ratios, which was likely due to the significant cytotoxicity of bare PEI. Note that cells treated with RGPP/GPP with 120 of N/P ratio showed a decreased cell F


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Figure 7. Gene transfection ability of GPP and RGPP in GepG2 cells. (A and B) Relative cell viability of HepG2 cells treated with different concentrations of RGPP, GPP, and bare PEI, respectively, for 24 h determined by CCK-8 assay. (C and D) Fluorescence microscopic images (C) and the corresponding transfection efficiencies (D) of cells expressing GFP recorded at 48 h after the initiation transfection with RGPP, GPP, and bare PEI, respectively, at different N/P ratios. Note that the transfection efficiency of PEI with 120 of N/P ratio was quantitated by counting the fraction of cells expressing GFP to total cells (n > 1000) due to the high cytotoxicity of PEI. (E and F) Fluorescence microscope images (E) and relative transfection efficiencies (TE) (F) of the cells expressing GFP measured at 48 h after the initiation transfection with RGPP (N/P = 60), GPP (N/P = 60), and bare PEI (N/P = 30) in the absence and presence of FBS. The dose of GFP plasmid was kept at 1.2 μg/mL.

temperatures increased by 8.1 °C for RGPP (N/P = 60) and 2.7 °C for GPP (N/P = 60) as well as 0.5 °C for water (Figure 8A), further demonstrating the higher photothermal conversion ability of RGPP. Moreover, we used CCK-8 assay to assess the cytotoxicity of GPP/RGPP in the presence or absence of NIR irradiation and found that NIR irradiation for 30 min did not significantly induce significant cytotoxicity in the cells cultured with GPP (N/P = 120) or RGPP (N/P = 120) (Figure 8B), indicating the safety of 808 nm laser irradiation. After NIR irradiation for 30 min, the temperature of GRR and RGPP

higher than Lip-2000 (Figure S6B and C), suggesting the great promise of RGPP as a novel gene nanocarrier. Photothermally Controlled Gene Transfection of RGPP. It was reported that NIR irradiation could significantly enhance the gene delivery efficiency of NGO−PEG−PEI by using the photothermal effect of nGO.23 To further confirm the strong NIR optical absorbance of RGPP over GPP at the same GO concentrations (Figure 3C), we here used the 808 nm laser at power densities of 2 W/cm2 to irradiate pure water, GPP, and RGPP solutions for 30 min and found that the G


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Figure 8. Photothermally enhanced GFP transfection of RGPP in HepG 2 cells. (A) Temperature change curves of water, GPP, and RGPP (N/P = 60) after being irradiated by 808 nm laser at 2 W/cm2 for 30 min. (B) Relative cytotoxicity of GPP and RGPP (N/P = 120) after 808 nm laser irradiation for various times. (C and D) Fluorescence microscopic images (C) and transfection efficiencies (D) of cells expressing GFP transfected with GPP or RGPP at different N/P ratios with or without the 808 nm laser irradiation (2 W/cm2) for 25 min.

solutions (N/P = 120) reached ∼39.8 °C and ∼45.7 °C, respectively, which could not significantly induce cell death. It was reported that ∼50 °C was required for significant cell death. We next evaluated the photothermal effect of RGPP or GPP on gene transfection efficiency using GFP as the model plasmid after NIR irradiation. HepG2 cells were incubated with RGPP/ GFP or GPP/GFP complex at different N/P ratios for only 25 min in the presence (laser on) or absence (laser off) of 808 nm laser irradiation (2 W/cm2) under FBS-free DMEM medium and then cultured in fresh 10% FBS-containing DMEM medium for 48 h before microscopic imaging. As shown in Figure 8C and D, NIR laser irradiation could dramatically enhance the transfection efficiency of RGPP with N/P ratio at 60 and 120 by nearly 0.4-fold and 1.5-fold, respectively (Figure S7 (FCM analysis) and the corresponding statistics (Figure 8D)). However, photothermally enhanced transfection efficiency was not observed for GPP (Figure 8C), which was further verified by FCM analysis (Figure S7) and the corresponding statistics (Figure 8D). These observations demonstrate the photothermally controlled gene transfection of RGPP, which may be due to the enhanced intracellular delivery ability of RGPP/pDNA complexes under the mild photothermal heating.23 In Vitro Transfection of Functional Gene Plasmids by RGPP. Plasmid GFP-Bax (pGFP-Bax) was selected as the functional gene to evaluate the transfection efficiency and gene therapy of RGPP and GPP in HepG2 cells. Our recent studies demonstrated that staurosporine (STS), an apoptotic stimulus, induced Bax-mediated apoptosis of HepG2 cells.37,44 Here we chose 30 of N/P ratio for RGPP and GPP to deliver pGFP-Bax, and FCM analysis (Figure S8) with the corresponding statistics (Figure 9A) showed that the transfection efficiency was ∼43% for RGPP and ∼13% for GPP. Furthermore, compared with the

Figure 9. Transfection of GFP-Bax in HepG2 cells with GPP and RGPP. (A) Transfection efficiencies of GPP and RGPP with N/P ratios at 30. (B) Fluorescence microscopic images of cells expressing GFP-Bax with or without STS treatment.

even distribution of Bax-GFP in control cells, the cells treated with 1 μM STS for 3 h exhibited punctate and condensate distribution of GFP-Bax (Figure 9B), indicating that STS induced Bax translocation from cytosol to mitochondria. These results indicate that RGPP is able to efficiently deliver not only model plasmid but also functional plasmid, indicating that RGPP may be a potential commercial transfection reagent.

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CONCLUSIONS In this study we have developed a PEI (25 kDa) and PEG (10 kDa) dual-functionalized nanosized rGO (PEG−nrGO−PEI, RGPP) with high-efficiency gene delivery ability through simultaneous hydrothermal reduction and PEIylation of PEG−nGO (GP) under 80 °C for 2 h. The hydrothermal reduction of GP during PEIylation promotes the nucleophilic reaction between the amino moieties of PEI and the epoxy groups in GP, which not only forms C−NH− groups to covalently connect PEI and GP but also drives more reduction H


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(10) Wang, G.; Zhao, T.; Wang, L.; Hu, B.; Darabi, A.; Lin, J.; Xing, M.; Qiu, X. Studying Different Binding and Intracellular Delivery Efficiency of ssDNA Single-Walled Carbon Nanotubes and Their Effects on LC3-Related Autophagy in Renal Mesangial Cells Via miRNA-382. ACS Appl. Mater. Interfaces 2015, 7, 25733−25740. (11) Kong, L.; Alves, C. S.; Hou, W.; Qiu, J.; Möhwald, H.; Tomás, H.; Shi, X. RGD Peptide-Modified Dendrimer-Entrapped Gold Nanoparticles Enable Highly Efficient and Specific Gene Delivery to Stem Cells. ACS Appl. Mater. Interfaces 2015, 7, 4833−4843. (12) Liu, X.; Ma, D.; Tang, H.; Tan, L.; Xie, Q.; Zhang, Y.; Ma, M.; Yao, S. Polyamidoamine Dendrimer and Oleic Acid-Functionalized Graphene as Biocompatible and Efficient Gene Delivery Vectors. ACS Appl. Mater. Interfaces 2014, 6, 8173−8183. (13) Bieber, T.; Elsasser, H. P. Preparation of a Low Molecular Weight Polyethylenimine for Efficient Cell Transfection. BioTechniques 2001, 30 (74−77), 80−81. (14) Gosselin, M. A.; Guo, W.; Lee, R. J. Efficient Gene Transfer Using Reversibly Cross-Linked Low Molecular Weight Polyethylenimine. Bioconjugate Chem. 2001, 12, 989−994. (15) Xun, M. M.; Xiao, Y. P.; Zhang, J.; Liu, Y. H.; Peng, Q.; Guo, Q.; Wu, W. X.; Xu, Y.; Yu, X. Q. Low Molecular Weight PEI-Based Polycationic Gene Vectors Via Michael Addition Polymerization with Improved Serum-Tolerance. Polymer 2015, 65, 45−54. (16) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.; Scherman, D.; Demeneix, B.; Behr, J. P. A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in Vivo: Polyethylenimine. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 7297−7301. (17) Fischer, D.; Bieber, T.; Li, Y.; Elsasser, H. P.; Kissel, T. A Novel Non-Viral Vector for DNA Delivery Based on Low Molecular Weight, Branched Polyethylenimine: Effect of Molecular Weight on Transfection Efficiency and Cytotoxicity. Pharm. Res. 1999, 16, 1273−1279. (18) Teo, P. Y.; Yang, C.; Hedrick, J. L.; Engler, A. C.; Coady, D. J.; Ghaem-Maghami, S.; George, A. J.; Yang, Y. Y. Hydrophobic Modification of Low Molecular Weight Polyethylenimine for Improved Gene Transfection. Biomaterials 2013, 34, 7971−7979. (19) Feng, L. Z.; Zhang, S. A.; Liu, Z. A. Graphene Based Gene Transfection. Nanoscale 2011, 3, 1252−1257. (20) Kim, H.; Namgung, R.; Singha, K.; Oh, I. K.; Kim, W. J. Graphene Oxide-Polyethylenimine Nanoconstruct as a Gene Delivery Vector and Bioimaging Tool. Bioconjugate Chem. 2011, 22, 2558− 2567. (21) Yan, L.; Chang, Y. N.; Zhao, L. N.; Gu, Z. J.; Liu, X. X.; Tian, G.; Zhou, L. J.; Ren, W. L.; Jin, S.; Yin, W. Y.; Chang, H. Q.; Xing, G. M.; Gao, X. F.; Zhao, Y. L. The Use of Polyethylenimine-Modified Graphene Oxide as a Nanocarrier for Transferring Hydrophobic Nanocrystals into Water to Produce Water-Dispersible Hybrids for Use in Drug Delivery. Carbon 2013, 57, 120−129. (22) Chen, B. A.; Liu, M.; Zhang, L. M.; Huang, J.; Yao, J. L.; Zhang, Z. J. Polyethylenimine-Functionalized Graphene Oxide as an Efficient Gene Delivery Vector. J. Mater. Chem. 2011, 21, 7736−7741. (23) Feng, L. Z.; Yang, X. Z.; Shi, X. Z.; Tan, X. F.; Peng, R.; Wang, J.; Liu, Z. Polyethylene Glycol and Polyethylenimine Dual-Functionalized Nano-Graphene Oxide for Photothermally Enhanced Gene Delivery. Small 2013, 9, 1989−1997. (24) Yang, H. W.; Lu, Y. J.; Lin, K. J.; Hsu, S. C.; Huang, C. Y.; She, S. H.; Liu, H. L.; Lin, C. W.; Xiao, M. C.; Wey, S. P.; Chen, P. Y.; Yen, T. C.; Wei, K. C.; Ma, C. C. M. EGRF Conjugated PEGylated Nanographene Oxide for Targeted Chemotherapy and Photothermal Therapy. Biomaterials 2013, 34, 7204−7214. (25) Bai, J.; Liu, Y. W.; Jiang, X. E. Multifunctional PEG-GO/CuS Nanocomposites for Near-Infrared Chemo-Photothermal Therapy. Biomaterials 2014, 35, 5805−5813. (26) Robinson, J. T.; Tabakman, S. M.; Liang, Y.; Wang, H.; Casalongue, H. S.; Vinh, D.; Dai, H. Ultrasmall Reduced Graphene Oxide with High Near-Infrared Absorbance for Photothermal Therapy. J. Am. Chem. Soc. 2011, 133, 6825−6831. (27) Akhavan, O.; Ghaderi, E.; Aghayee, S.; Fereydooni, Y.; Talebi, A. The Use of a Glucose-Reduced Graphene Oxide Suspension for

of GP. Therefore, RGPP is more stable than GPP in physiological environments and has high-efficiency intracellular gene delivery ability. In addition, RGPP has remarkably enhanced gene transfection efficiency under NIR laser irradiation compared with GPP. Moreover, RGPP is able to efficiently deliver functional plasmid such as GFP-Bax into HepG2 cells. Collectively, our works suggest that RGPP has a great potential as a novel nonviral vector for high-efficiency gene delivery and gene therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b09915. Figures S1−S8 and Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Dr. Tongsheng Chen. E-mail: [email protected] and [email protected]. *Dr. Xiaoping Wang. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (61527825 and 81471699) and the Science and Technology Plan Project of Guangdong Province (2014B090901060).

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Hydrothermal Reduction of Polyethylenimine and Polyethylene Glycol

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