Polyamidoamine-Grafted Multiwalled Carbon Nanotubes for Gene

Oct 13, 2011 - 50% confluence after incubation for 24 h, 100 μL aliquots of. PAA-g-MWNT solutions in DMEM at different concentrations were added to a...
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Polyamidoamine-Grafted Multiwalled Carbon Nanotubes for Gene Delivery: Synthesis, Transfection and Intracellular Trafficking Min Liu,† Biao Chen,† Yanan Xue,‡ Jie Huang,† Liming Zhang,† Shiwen Huang,‡ Qingwen Li,† and Zhijun Zhang*,† †

Division of Nanobiomedicine, and Division of Nano-Devices and Materials, Suzhou Institute of Nano-tech and Nano-bionics, Chinese Academy of Sciences, Suzhou, 215123, P. R. China ‡ Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China S Supporting Information *

ABSTRACT: Functionalized multiwalled carbon nanotubes (f-MWNTs) are of great interest and designed as a novel gene delivery system. In this paper, we presented synthesis of polyamidoamine-functionalized multiwalled carbon nanotubes (PAA-g-MWNTs) and their application as a novel gene delivery system. The PAA-g-MWNTs, obtained from amide formation between PAA and chemically oxidized MWNTs, were stable in aqueous solution and much less toxic to cells than PAA and PEI 25KDa. More importantly, PAA-g-MWNTs showed comparable or even higher transfection efficiency than PAA and PEI at optimal w/w ratio. Intracellular trafficking of Cy3-labeled pGL-3 indicated that a large number of Cy3-labeled pGL-3 were attached to nucleus membrane, the majority of which was localized in nucleus after incubation with cells for 24 h. We have demonstrated that PAA modification of MWNTs facilitate higher DNA uptake and gene expression in vitro. All these facts suggest potential application of PAA-g-MWNTs as a novel gene vector with high transfection efficiency and low cytotoxicity.

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However, polymers with primary amino groups often show higher stability in saline solution and better transfection activity.21 To date, there are few reports on PAA with pendant primary amine groups for gene delivery.22 Herein, we report a novel gene delivery system based on MWNTs grafted with PAA. In our strategy, PAA with pendant primary amine groups was synthesized via Michael polyaddition of N-Boc-protected diamine to N,N-methylenebis(acrylamide). Next, PAA was conjugated to MWNTs (PAA-g-MWNTs) through EDC chemistry. The composition, morphology, surface properties, and cytotoxicity of PAA-g-MWNTs were characterized by FT-IR spectroscopy, thermogravimetric analysis (TGA), scanning electron microscopy (SEM), zeta potential, and WST measurement. Finally, using pGL-3 as a reporter gene, we investigated the ability of PAA-g-MWNTs to deliver foreign luciferase gene, including transfection efficiency and intracellular trafficking.

INTRODUCTION Since application of viral vectors was restricted by their drawbacks such as high immunogenicity, high toxicity, low gene loading rate, and so on, researchers in this field have developed many types of nonviral gene vectors.1−4 Recently, multiwalled carbon nanotubes (MWNTs) have been explored as an efficient novel gene delivery system due to their unique structure and properties. However, the as-prepared carbon nanotubes are water-insoluble, chemical modification of MWNTs is desired for their application in gene delivery.5−12 Dendrimer-grafted MWNTs have been developed as gene delivery vectors.13−15 Dendrimers containing large numbers of surface primary amine groups can effectively improve water solubility of MWNTs; meanwhile, the surface primary amines can condense DNA via electrostatic interaction and then deliver foreign DNA into cells. Yao group16,17 designed and synthesized several kinds of dendrimer-functionalized MWNTs, the obtained PAMAM-MWCNT possessed much lower cytotoxicity and higher transfection efficiency. However, the properties of dendrimers are mainly dependent on the number of their generations and the synthesis is usually complicated. 18 Recently, linear polyamidoamine (PAA), which can be easily synthesized via one-step polyaddition of primary monoamines or bis(secondary amine) to bisacrylamines, has attracted much attention from researchers.19 In a previous work, secondary and tertiary amines were introduced to the side chains to improve water solubility and transfection efficiency. 20 © 2011 American Chemical Society

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EXPERIMENTAL PROCEDURES Materials. MWNTs and EDAC were obtained from domestic suppliers. PAA (Mw = 1.8 × 104) was synthesized as described previously.22 Branched PEI 25KDa was purchased from Sigma-Aldrich and used as received. N-Hydroxysuccinimide Received: April 13, 2011 Revised: September 14, 2011 Published: October 13, 2011 2237

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(NHS) was purchased from Alfa Aesar. Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), 0.25% Trypsin-EDTA, Opti-MEM, and Hoechst 33258 were purchased from Gibco BRL (Grand Island, NY). Label IT Cy3 Nucleic Acid Labeling Kit was obtained from Mirus Bio Corporation. Luciferase assay system was obtained from Promega Corporation. COS-7 and HeLa cell lines were purchased from China Center for Type Culture Collection (CCTCC) (Wuhan University) and cultured in DMEM supplemented with 10% FBS at 37 °C in a humidified air atmosphere containing 5% CO2. Amplification and Purification of Plasmid DNA. pGL-3 was used as the luciferase reporter gene and transformed in E. coli JM109. pEGFP-N1 was used as the green fluorescent protein gene and transformed in E. coli DH5R. Both plasmids were amplified in LB at 37 °C for 12−16 h at 250 rpm, and purified by QIAfilter plasmid Giga kit according to the manufacturer’s protocol. The quantity and quality of plasmid DNA was analyzed by spectrophotometric analysis at 260 and 280 nm and by electrophoresis in 0.7% (w/v) agarose gel. The purified plasmid was dissolved in TE buffer and stored at −20 °C. Synthesis and Characterization of PAA-g-MWNTs. Carboxylic acid groups were introduced onto MWNTs by acid treatment following a previous protocol.23 Next, 10 mg of MWNTs-COOH obtained from the above step was dispersed into 10 mL of aqueous solution (pH = 6) of EDC (9.6 mg) and NHS (5.7 mg) via sonication. After sonication for 3 h, 40 mg of PAA was added to the above solution and changed the solution pH to 9. The mixture was then sonicated for further 4 h and stirred at room temperature overnight. Finally, the solution was dialyzed against distilled water (MWCO = 50 000) to remove the unreacted PAA. Next, the solution was centrifuged at 12 000 rpm for 30 min, the supernatant was sterile-filtered (Millipore 220 nm) and stored as a stock solution. The structure and composition of the purified PAA-g MWNTs were characterized by FTIR and TGA. TGA was performed by scanning from 100 to 800 °C under nitrogen at a heating rate of 20 °C min−1 by using TG/DTA 6200. Morphology of the PAA-g-MWNTs/pEGFP-N1 complexes was characterized by SEM (FEI Quanta 400). The complexes at optimal w/w ratio of 15 were prepared as described above, followed by vortexing for 5 s and incubating for 30 min at room temperature. Afterward, a drop of the solution was deposited onto a glass slide and stored in a desiccator overnight. Cytotoxicity Assay. The cytotoxicity of PAA-g-MWNTs to HeLa and COS-7 cells was evaluated by WST assay, in which PAA was also tested as a control. HeLa and COS-7 cells were seeded into 96 well plate at a density of 6000 and 5000 cells/ well in 100 μL complete DMEM. When cells achieved about 50% confluence after incubation for 24 h, 100 μL aliquots of PAA-g-MWNT solutions in DMEM at different concentrations were added to a 96 well plate and incubated for another 24 h. Then, the DMEM was replaced with 100 μL fresh DMEM, and 10 μL of WST solution was added to each well except for the background wells and incubated for further 2 h. The absorbance at 450 nm was measured using a microplate reader (Perkin-Elmer Victor X4). The percent relative viability in reference to control wells containing complete DMEM without the added PAA-g-MWNTs was calculated by the following equation:

where A is absorbance at 450 nm, A0 represents absorbance of the solution containing cells and complete DMEM without WST and PAA-g-MWNTs. Preparation of PAA-g-MWNTs/DNA Complexes and Agarose Gel Retardation Assay. PAA-g -MWNTs/DNA complexes were freshly prepared in 150 mmol L−1 NaCl by mixing PAA-g-MWNTs with pEGFP-N1 at desired w/w ratios, followed by vortexing for 5 s and incubation for 30 min at room temperature. The complexes for agarose gel retardation assay were prepared at w/w ratios of 1.5:1, 3:1, 4.5:1, 6:1, 9:1, 12:1, and 15:1 as described above. Gel electrophoresis was carried out in TAE buffer in 0.7% (w/v) agarose gel at 80 V for 80 min. The gel was visualized with a Fujifilm LAS-4000 image analyzer. Zeta Potential Measurement. The zeta potential of samples was measured with Zetasizer Nano-ZS (Malvern Instruments) at 25 °C. The PAA-g-MWNTs/pEGFP-N1 complexes at various w/w ratios were freshly prepared as described above. After incubating for 30 min, the solution was diluted to 1 mL with pure water for zeta potential measurement. In Vitro Transfection. In vitro transfection efficiency of complexes was evaluated on HeLa and COS-7 cells, pGL-3 was used as the report gene. Branched PEI 25KDa and PAA were used as a control. HeLa and COS-7 cells were separately seeded into 24 well plates at an appropriate density (6 × 104 for HeLa cells, and 5 × 104 for COS-7 cells) with 1 mL of complete DMEM. When the cells were incubated to 50−60% confluence in the 24 well plate, the medium was replaced with 400 μL of Opti MEM, and then, 100 μL aliquots of PAA-g-MWNT/DNA complexes at various w/w ratios were added to each well. After incubation for 4 h at 37 °C, the culture media was then replaced with 1 mL of complete DMEM, and the cells were incubated for further 48 h. All of the transfections were performed in duplicate. For luciferase expression, after 48 h of incubation, the medium was replaced with 200 μL of cell lysis buffer and freeze−thawed twice. The lysate was centrifuged at 12 000 rpm for 3 min, 20 μL of supernatant was mixed with 100 μL of the luciferase assay substrate (Promega), and light emission was measured with a Lumat 9507 luminometer (Berthold Germany) for 10 s. The relative light units (RLU/mg protein) were normalized against the total protein concentration in the cell extracts, which was determined using a BCA assay kit (Pierce). To evaluate the effect of serum on gene delivery, we used Opti MEM containing 10% FBS for transfection of cells with PAA-g-MWNTs/DNA complexes. For transfection in the presence of serum, when the cells achieved 50−60% confluence, the medium was replaced with fresh Opti MEM containing 10% FBS, and then, complexes at various w/w ratios were added to each well. The other procedure was the same as that described above. Intracellular Trafficking. pGL-3 was labeled by Label IT Cy3 Nucleic Acid Labeling Kit according to the manufacturer’s protocol. Then, PAA-g-MWNTs at optimal w/w ratio (w/w ratio = 15) was screened through the evaluation by luciferase expression. PEI 25KDa at optimal w/w ratio (w/w = 1.3) was used as a control. COS-7 cells were seeded onto a coverslip in a 24 well plate at a density of 4 × 104. After incubation for 24 h, the cells achieved 40−50% confluence, the medium was replaced with 400 μL of Opti MEM, and then PAA-g-MWNTs with labeled pGL-3 at w/w ratio of 15 were added to each well. After incubating for 4 h, the medium was replaced with 1 mL 2238

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Scheme 1. Synthesis Route of PAA-Functionlized MWNTs

Cell Viability Assay. The relative cellular viabilities of PAA-g-MWNTs and PAA are presented in Figure 2. The

complete DMEM. At the end of each incubation period (4 h and 24 h), the medium was removed, and the coverslip was taken out and fixed with 4% formalin for 10 min. Then, the cells were rinsed with PBS twice, and 100 μL of Hoechst 33258 (5 μg/mL) was added onto the coverslip and incubated for 10 min. Finally, the coverslip was washed with PBS and mounted on a microscopic slide. Intracellular localization of Cy3-labeled DNA was visualized by Nikon confocal system with excitation at 405 and 543 nm for Hoechst and Cy3, respectively. Image analysis was used to determine whether Cy3-labeled DNA was localized inside or over the nucleus, 5−10 of Z-series images at 1 μm interval containing blue fluorescence (nucleus) were selected. DNA nucleus localization was confirmed by evidence of the colocalization of blue and red fluorescence.

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RESULTS

Synthesis and Characterization of PAA-g-MWNTs. PAA was conjugated to the carboxyl groups on the surface of MWNTs, which is shown in Scheme 1. After purification, the obtained PAA-g-MWNTs were then characterized by FT-IR. For comparison purpose, IR spectra of PAA, MWNTs-COOH, and PAA-g-MWNTs are shown in Figure S1 (see Supporting Information). The CONH group formed between PAA and MWNTs-COOH shows IR bands at about 3400, 1650, and 1535 cm−1, suggesting successful formation of PAA-g-MWNTs complexes. The relative amount of PAA grafted onto the surface of MWNTs was evaluated by thermogravimetric analysis (TGA). As shown in Figure 1, MWNT-COOH was thermally stable up

Figure 2. In vitro cytotoxicity of PAA-g-MWCNTs in COS-7 (A) and HeLa (B) cells. PAA was used as a control.

cytotoxicity of PAA-g-MWNTs was found to be concentration dependent, which meant that the cell viability decreased with increasing concentration of PAA-g-MWNTs. Figure 2A shows the cytotoxicity of PAA-g-MWNTs against COS-7 cells; the viability of COS-7 cells incubated with the solution of PAA was about 40% at a concentration of 80 μg/mL, while the viability of COS-7 with PAA-g-MWNTs was about 80% at the same concentration, suggesting much lower cytotoxicity of PAA-gMWNTs than that of PAA. Figure 2B gives the cytotoxicity of PAA-g-MWNTs against HeLa cells. The viability of HeLa cells against PAA-g-MWNTs and PAA at a concentration of 80 μg/ mL was about 50% and 20%, respectively. The result is similar to that observed for COS-7 cells. Agarose Gel Retardation. Agarose gel retardation assays were performed to study the condensation ability of PAA-gMWCNTs. As shown in Figure 3, DNA was entirely retained in

Figure 1. TGA curves of MWNTs, PAA, and PAA-g-MWNTs.

to 600 °C, while PAA and PAA-g-MWNTs began to degrade at about 300 °C. At 500 °C, PAA and PAA-g-MWNTs showed about 75.4% and 72.2% weight losses, respectively; thus, PAAg-MWNTs contained about 95.7% PAA. 2239

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Figure 3. Agarose gel electrophoresis retardation assay of PAA-gMWNTs/DNA complexes at various w/w ratios. Lane 1, naked DNA; lanes 2−8, w/w ratios of 1.5:1, 3:1, 4.5:1, 6:1, 9:1, 12:1, and 15:1, respectively.

the wells at w/w ratio above 1.5, suggesting high DNA binding ability at low w/w ratios. Zeta Potential Measurement. It is well-known that positive surface charge of polymer/DNA complexes can facilitate the nonspecific attachment of complex nanoparticles to the negatively charged cellular membranes and the uptake of polycation/DNA complexes by cells. The zeta potential of PAA-g-MWNTs/DNA complexes was measured to assess their surface charge. As shown in Figure 4, at w/w ratios below 3, the

Figure 5. Transfection efficiencies of PAA-g-MWNTs/pGL-3 complexes in COS-7 (A) and HeLa (B) cells. PAA and branched PEI 25KDa were used as positive control.

efficiencies of PAA-g-MWNTs should be related to the combinational factors including low cytotoxicity, strong DNA binding ability and small particle size. Similarly, the transfection results (Figure 5B) indicate that PAA-g-MWNTs still express high transfection efficiencies in HeLa cells, and the luciferase expression at optimal w/w ratio was comparable to that of PEI/pGL-3 complexes. Serum protein can hinder cellular uptake and promote aggregation, often leading to reduction of the transfection efficiency. To understand the effect of serum on the DNA transfection, a transfection experiment was then performed with COS-7 cells in the presence of 10% FBS. As shown in Figure 5A, 10% of serum inhibited luciferase expression in COS-7 cells with each polycation tested in this work including PEI and PAA-g-MWNTs. The results illustrated that luciferase expression in COS-7 cells only slightly decreased at low w/w ratios; specifically, with the w/w ratio increasing, the luciferase expression could not be effectively prohibited by serum, indicating that the PAA-g-MWCNTs/DNA complexes were less serum-sensitive for in vitro transfection. Similar results were obtained in the case of HeLa cells. Intracellular Trafficking of Cy3-Labeled pGL-3. Intracellular trafficking of Cy3-labeled pGL-3 was analyzed by CLSM and the result is shown in Figure 6. The optimal w/w ratio for PAA-g-MWNTs and PEI 25KDa was determined to be 15 and 1.3, respectively. In order to distinguish the nucleus and

Figure 4. Zeta potentials of PAA-g -MWNT/DNA complexes at w/w ratios ranging from 0.5 to 30 in 10 mmol L−1 NaCl aqueous solution.

zeta potential of the complexes is negative, indicating incomplete complexation. With increase of the w/w ratio, the zeta potential increased rapidly and reached a plateau of about 35 mV. In Vitro Transfection. In vitro transfection was conducted on COS-7 and HeLa cells to evaluate the ability of the PAA-gMWNTs to deliver foreign DNA into cells. PAA and branched PEI 25KDa at the optimal w/w ratio were used as positive controls, and pGL-3 was used as the report gene. The transfection results in Figure 5A reveal that PAA-gMWNTs showed excellent transfection efficiency in COS-7 cells at w/w ratios from 2 to 30, which is comparable or higher than that of branched PEI 25KDa. Luciferase expression (RLU/ mg protein) was related to the w/w ratio, with the w/w ratio increasing, the luciferase expression increased obviously. Optimized luciferase expression (RLU/mg protein) in COS-7 cells with PAA-g-MWNTs/pGL-3 complexes was a little higher than that with PEI/pGL-3 complexes. The high transfection 2240

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of MWNTs-COOH precipitated rapidly, usually within a few hours. The size and morphology of MWNTs-COOH, PAA-gMWNTs, and PAA-g-MWNTs/DNA were characterized by SEM. Figure 7A,B shows the image of raw MWNTs-COOH and PAA-g-MWNTs. Obviously, after grafting with PAA, the morphology of MWNTs did not change significantly. Figure 7C presents the image of PAA-g-MWNTs/DNA. The SEM analysis did not reveal significant differences in size among the MWNTsCOOH, PAA-g-MWNTs, and PAA-g-MWNTs/DNA. A similar result was also reported by Gao and colleagues.30 Cytotoxicity of carriers is a very important factor in gene delivery. Previous work25,26 demonstrated that polycations first combined with cell membrane via electrostatic interaction between positively charged polycations and negatively charged sites on the cells, which induced polycations to aggregate on the cell membrane and lead to cell death. Functionalization of carbon nanotubes has proven to be very useful for reducing their cytotoxicity.27−29 Our results indicated that PAA-gMWNTs showed no significant cytotoxicity to COS-7 cells at low concentration. However, with the concentration increasing, the cytotoxicity increased slowly. The IC50 values of PAA-gMWNTs and PAA in COS-7 cells were estimated to be 108 and 66 μg/mL, respectively, much lower than that of PEI (IC50 = 10 μg/mL).22 The IC50 values of PAA-g-MWNTs and PAA to HeLa cells are 78 and 52 μg/mL, respectively, which is similar to the case of COS-7 cells. Since PAA contains primary and tertiary amine groups, PAA-g-MWNTs may behave as a polycation molecule, and thus cause certain cytotoxicity. The ability of vectors to condense DNA into small-sized particles is another important factor in gene delivery. To facilitate efficient gene expression, cationic polymers should strongly condense DNA to protect the DNA against digestion by enzymes, and thus improve transfection efficiency. The interaction of PAA-g-MWNTs with the pEGFP-N1 plasmid was investigated by gel retardation assay, zeta potential measurement, and SEM. Gel retardation assay was performed to explore whether PAA-g-MWNTs could effectively bind DNA. The result in Figure 3 reveals that PAA-g-MWNTs could efficiently condense DNA, and entirely retain DNA at w/w ratio above 1.5. The zeta potential reached a plateau of about 35 mV, while the PAA/ DNA complexes reached a plateau of about 20 mV,22 which was much lower than that of PAA-g-MWNTs/DNA complexes. PAA-g-MWNTs with high zeta potential would facilitate electrostatic interaction between cationic PAA-g-MWNTs and polyanionic DNA. Transfection efficiency is the most important factor in gene delivery. In our work, PAA-g-MWNTs could deliver foreign DNA into COS-7 and HeLa cells, with the transfection

Figure 6. Intracellular trafficking of Cy3-labeled pGL-3 (red) when combined with PAA-g-MWNTs and PEI 25KDa at optimal w/w ratio. The localization of fluorescent particles in COS-7 cells was visualized at 4 and 24 h post-transfection. Nucleus (blue) was stained with Hoechst 33258.

DNA, Hoechst 33258 was used to stain the nucleus, which appeared to be blue fluorescence; meanwhile, the Cy3-labeled pGL-3 appeared to be red fluorescence. As shown in Figure 6, after transfection for 4 h, Cy3-labeled pGL-3 was clearly visible. For PAA-g-MWNTs and PEI, a large amount of Cy3-labeled pGL-3 was dispersed in the cytoplasm and only a few red fluorescence particles were found interacting with the nucleus membrane. Meanwhile, very little Cy3-labeled pGL-3 was observed in the nucleus. After 24 h of transfection, significant differences appeared within each group: most of the red fluorescence particles were found aggregating in the nucleus. For PAA-g-MWNTs, lots of red fluorescence particles around the nucleus were observed, and almost all of the cells in the field of view showed DNA nucleus localization. Similar results were observed for PEI: there was no obvious difference with distribution of Cy3-labeled pGL-3 between PAA-gMWNTs and PEI, except that cells transfected by PEI showed relatively lower fluorescence density in nuclear localization.

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DISCUSSION

In our experiment, the carboxyl groups on the surface of MWNTs were first converted to succinimide-terminated (Scheme 1); after the carboxyl groups were activated, the cationic PAA was then added to the solution above, thus leading to the formation of PAA-g-MWNTs. Similar procedures were also reported previously.24 The obtained aqueous solution of PAA-g-MWNTs was stable at room temperature without any precipitation for up to 6 months, as shown in Figure S2 (see Supporting Information); however, the aqueous solution

Figure 7. SEM images of MWNTs-COOH, PAA-g-MWNTs, and PAA-g-MWNTs/DNA. 2241

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efficiencies comparable or even higher than that of PEI 25KDa at optimal w/w ratio. Many factors are considered to affect transfection efficiencies, among which w/w ratio is the most important factor. With increasing of w/w ratio, the transfection efficiency rosed up steeply, then reached a plateau, and finally decreased. With the w/w ratio increasing, the cytotoxicity increased, leading to decrease in the cell viability and transfection efficiency. In addition, changing the w/w ratios would also alter the surface charge of PAA-g-MWNTs/DNA complexes, which could influence the DNA binding ability, and in turn, the transfection efficiency. Previous work has demonstrated that complexes with positive charge and small size are beneficial for cellular uptake and intracellular trafficking. In our study, PAA-g-MWNTs/DNA complexes possess high positive charge (35 mV), the average length is about 300 nm, and the diameter about 30−40 nm. Cellular uptake and nuclei localization of PAA-g-MWNTs were studied by delivery of Cy3-labeled pGL-3 to cells. Besides the extracellular and cellular obstacles which reduced gene expression, nuclear envelope is recognized as one of the most important intracellular barriers. CLSM results in Figure 6 indicate that, for PAA-g-MWNTs and PEI, a majority of red fluorescence particles were attached to nucleus membrane within 4 h incubation, and some of them had already localized in the perinuclear region and localized in nucleus after 24 h incubation. On the basis of the results, intracellular trafficking and cellular uptake of Cy3-labeled pGL3 displayed that PAA-gMWNTs showed superior DNA delivery ability to COS-7 nucleus than others.

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CONCLUSIONS

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ASSOCIATED CONTENT

S Supporting Information *

Additional figures as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

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ACKNOWLEDGMENTS

REFERENCES

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In summary, PAA-grafted multiwalled carbon nanotubes were successfully prepared to deliver foreign DNA to cells. In comparison to PAA and PEI 25 KDa, PAA-g-MWNTs showed significantly low cytotoxicity and strong ability to condense DNA. Transfection efficiency results revealed that PAA-gMWNTs possessed comparable or even higher luciferase expression than PAA and PEI at optimal w/w ratio. Intracellular trafficking of Cy3-labeled pGL-3 also displayed that PAA-gMWNTs showed superior ability to deliver foreign DNA into cell nucleus. The current study may provide useful insight into design of novel nanovectors for efficient and nontoxic gene delivery.

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Corresponding Author *Telephone: 86-0512-62872556; Fax: 86-0512-62603079; E-mail: [email protected].

The financial support of this research from NSFC (No. 20873090, 21073224) and Innovation Project of CAS (KJCX2.YW.M12) is gratefully acknowledged. We thank Characterization and Test Platform at SINANO for assistance with instrumentation. 2242

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Bioconjugate Chemistry

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dx.doi.org/10.1021/bc200189f | BioconjugateChem. 2011, 22, 2237−2243