Integration of Adeno-Associated Virus-Derived Peptides into Nonviral

May 24, 2013 - This study describes a simple, versatile approach for developing a nonviral gene carrier by adopting the highly efficient gene delivery...
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Integration of Adeno-Associated Virus-Derived Peptides into Nonviral Vectors to Synergistically Enhance Cellular Transfection Jung-Suk Kim,† Eunmi Kim,† Ji-Seon Oh, and Jae-Hyung Jang* Department of Chemical and Biomolecular Engineering, Yonsei University, Seoul, Korea, 120-749 S Supporting Information *

ABSTRACT: This study describes a simple, versatile approach for developing a nonviral gene carrier by adopting the highly efficient gene delivery properties of the adenoassociated virus (AAV). Specific viral peptides (r3.45_hepBD) extracted from AAV r3.45, which directly evolved to improve gene delivery capabilities in many cell types, were conjugated onto branched polyethylenimine (PEI) to form hybrid gene carriers. AAV r3.45 carries a sequence insertion (LATQVGQKTA; r3.45) within the heparin-binding domain (LQRGNRQA; hepBD), which ultimately comprises a novel sequence (LQRGNLATQVGQKTARQA; r3.45_hepBD) on the capsid. This sequence is hypothesized to be a crucial cue to enhance gene delivery efficiency. Consequently, the intimate interactions of the conjugated r3.45_hepBD with the glycosaminoglycans, including chondroitin sulfate, resulted in significantly enhanced cellular transfection of DNA/PEIr3.45_hepBD complexes. The successful establishment of a nonviral system that is built with novel peptides will provide a powerful means for developing a substantial number of gene therapy applications.

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higher interactions with known receptors on target cells.13 Among these strategies, adopting principles from viruses into the design of nonviral carriers has provided insight into improving cellular transfection. Integration of virally derived peptides into cationic polymers has been shown to significantly improve performance in gene delivery, including transfection and cell viability. For example, the conjugation of plasma membrane translocating cell penetrating peptides (CPP) into cationic polymers demonstrates that using viral components can be a versatile strategy for improving nonviral gene delivery technologies.14−17 Additionally, various membrane-associating peptides that are originally derived from viruses, such as melittin,18 GALA,19 KALA,20,21 and HIV gp41,22 have been incorporated into nonviral carriers and subsequently employed as key components in the fine-tuning of nonviral-based gene vectors. In this study, gene vectors that integrate specific peptides extracted from adeno-associated viral (AAV) external capsids into a nonviral carrier, polyethylenimine (PEI), were newly designed to significantly enhance gene transfer compared to conventional, nonviral gene vectors, including PEI/DNA, Lipofectamine/DNA or jetPEI/DNA. No previous studies using DNA complexes formed with peptides derived from an AAV vector, which has been regarded as a safe and efficient parvovirus,23 have been reported. In a previous study, a directly

INTRODUCTION Gene therapy research has demonstrated increasing promise in the treatment of inherited or acquired disorders1,2 and cancers,3 as well as in regenerative medicine.4,5 However, the development of efficient and safe gene delivery systems remains a challenge.6 The intrinsic properties of viral vectors that induce higher gene transfer efficiencies than nonviral vectors have enabled their extensive use in the majority of gene therapy applications. However, potential concerns in virus-mediated gene delivery, including pathogenesis, immunogenicity, replication competence, limitations on genomic DNA size, and difficulties in large-scale production, have led to the design of novel nonviral carriers.7 When compared to viral vectors, nonviral vectors, which are based on cationic polymers selfassembling with negatively charged nucleic acids through electrostatic interactions, are superior because of their increased safety and large-scale production capabilities. However, low gene transfer efficiency in clinically valuable cells (e.g., stem or cancer cells) has delayed their extensive use in clinical trials.8 Taken together, the development of versatile gene vectors that can simultaneously supplement the restrictions of both viral and nonviral vectors may be critical to drastically improve gene delivery technologies and facilitate the transition of gene therapies to the clinic. The majority of approaches for enhancing gene delivery have primarily focused on either chemical modifications of synthetic polymers, such as dendrimers,9 liposomes,10 and cationic polymers,11 or the physical inclusion of additives to incorporate desirable properties, including higher cationic charges12 or © XXXX American Chemical Society

Received: February 6, 2013 Revised: May 22, 2013

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Table 1. Sequences and Conjugation Percentages of Each Peptide PEI-peptide

PEI-peptide sequence

peptide conjugation on PEI (%)

PEI-r3.45 PEI_hepBD PEI-r3.45_hepBD

PEI-CGGTQVGQKT PEI-CGGLQRGNRQA PEI-CGGLQRGNLATQVGQKTARQA

0.34 0.40 0.51

Scheme 1. Structures and Synthesis of PEI-Peptidesa

evolved AAV variant, AAV r3.45, which carries an insertion of a selected peptide sequence (LATQVGQKTA) on the surface of the 3-fold spike within the heparin-binding site, significantly enhanced gene delivery to a variety of cell types compared with wild-type AAV2 or AAV5.24 The integration of the peptide moieties (either LATQVGQKTA or combined formulations with the heparin-binding motif) into nonviral carriers, therefore, is hypothesized to result in intimate interactions of the nonviral vectors with cells and to ultimately act as a crucial cue to enhance gene delivery efficiencies. In vitro cellular transfection induced by the nonviral carriers complexed with the peptide-modified PEI and the cytotoxicity of the DNA complexes were investigated in different cell lines (i.e., HEK293T and Panc1), and the results suggested that this approach represents an alternative vector design strategy to further advance nonviral gene delivery technologies.

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MATERIALS AND METHODS

Materials. Branched polyethylenimine (PEI, 25 kDa), Nsuccinimidyl-3-(2-pyridyldithio) propionate (SPDP) (312.4 Da), dimethyl sulfoxide (DMSO), dithiothreitol (DTT), sodium azide, chondroitinase ABC, and heparinase II were purchased from SigmaAldrich (St. Louis, MO, U.S.A.). Peptides used in this study were synthesized and purified to greater than 95% purity by Peptron (Daejeon, Korea). Both pEGFP and pLuc, which encode green fluorescence protein (GFP) and luciferase driven by a cytomegalovirus (CMV) promoter, respectively, were purified using a Qiagen EndoFree Plasmid purification kit (Valencia, CA) according to the manufacturer’s protocol. The luciferase assay kit was purchased from Promega (Madison, WI, U.S.A.). Synthesis and Characterization of PEI-Peptides. Small portions of primary amine groups within branched PEI (25 kDa) were replaced with designated virally derived peptides (i.e., r3.45, hepBD, and r3.45_hepBD), whose sequences are summarized in Table 1. The primary amine groups were premodified with SPDP and, thus, can interact with a thiol group on a cysteine, which was inserted as a linker with two glycine groups at the N-terminal end of each peptide (Scheme 1). Briefly, 50 mg of PEI was dissolved in phosphate-buffered solution (PBS) and mixed with 1.25 mg of SPDP in 0.5 mL of dimethyl sulfoxide (DMSO) for 3 h at room temperature. The SPDPactivated PEI was purified by gel filtration on a PD-10 column (GE Healthcare Life Sciences, Piscataway, NJ) and reduced using an excess amount of dithiothreitol (DTT, Sigma-Aldrich) for 15 min. The release of pyridine-2-thione after reduction with DTT enabled the degree of SPDP conjugation on the PEI to be determined using spectrophotometric analysis at 343 nm. Finally, 1 mg of each peptide dissolved in PBS was added to 1 mL of SPDP-activated PEI to conjugate the peptide into the branched PEI. The mixtures were stirred for 12 h at room temperature, purified using a dialysis membrane (MWCO = 25000 Da), and lyophilized for 3 days. The purified PEI-peptide powders were dissolved in 1 mL of distilled water, and the fluorescence analysis of fluorescamine (Sigma-Aldrich), which reacts with the primary amine groups, was performed to determine the degree of peptide conjugation to the primary amines in PEI.25 The conjugation of peptides into the primary amine groups on PEI, which were dissolved in deuterium oxide (D2O), was confirmed using 1Hnuclear magnetic resonance (1H NMR) spectra (500 MHz FT-NMR spectrometer with CryoProbe, DPX-500, Bruker Biospin, Billerica, MA) and Fourier transform infrared (FT-IR) spectroscopy (Spectrum 1000, Perkin-Elmer, Cambridge, MA).

a

(a) The origin of the r3.45_hepBD peptide. The r3.45_hepBD peptide carries an insertion of LATQVGQKTA on the surface of the 3-fold spike within the heparin-binding site (LQRGNRQA) of wildtype AAV2, to form LQRGN-LATQVGQKTA-RQA (r3.45_hepBD). (b) The synthesis procedures of the PEI-peptides and the chemical structures of each PEI-peptide formulation: PEI-r3.45, PEI-hepBD, and PEI-r3.45_hepBD.

Cell Culture. To measure transfection efficiencies, cell lines derived from human embryonic kidney (HEK293T) and human pancreatic carcinoma (Panc1) cells were used. HEK293T cells, which are highly permissive to nonviral gene vectors, were utilized as cellular models to tune the formulation parameters (e.g., charge ratios, DNA quantity, and incubation time) of DNA complexes for inducing optimal cellular transfection. Panc1 cells were utilized to demonstrate the future potential of the DNA/PEI-r3.45_hepBD complex system, which can be applied for cancer therapy. To examine the cell line specificity of DNA complexes, the human cervical cancer cell line (HeLa) and mouse fibroblast cell line (NIH3T3) were additionally utilized. These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Invitrogen) and 1% penicillin and streptomycin (Invitrogen) at 37 °C and 5% CO2. B

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Characterization of DNA/PEI-Peptide Complexes. Hydrodynamic sizes of DNA complexes formed with each PEI formulation were determined by dynamic light scattering using a Zetasizer (Nano ZS, Malvern Instruments Ltd., Worcestershire, United Kingdom). The electrostatic charges and polydispersity index of DNA complexes were measured using electrophoretic light scattering (ELS Z, Otsuka electronics, Hirakata, Japan). Additionally, morphologies of the peptide-modified PEI/DNA complexes (18 N/P ratio) were visualized using an atomic force microscope (Dimension 3100 system, Digital Instruments, Santa Barbara, CA) at the YONSEI Center for Research Facilities. Finally, the stability of the DNA complexes, which were formed at a ratio of 18 N/P, was determined through measuring the sizes of the DNA complexes over different time periods (specifically, 10, 30, 60, 90, 120, 150, 180, and 210 min) at physiological ionic strength (150 mM NaCl, pH 7.4). Cellular Transfection Assay. To test the cellular transfection of the DNA complexes, each cell type (i.e., the HEK293T and Panc1 cells) was plated onto 48-well tissue culture plates (TCPs) at a density of 2 × 104 cells/well one day prior to transfection. On day 1, 50 μL of the PEI or PEI-peptide solution (0.1 mg/mL in 5% glucose) was gently added to 50 μL of the DNA solution (1 μg) dissolved in 5% glucose, followed by vigorous vortexing for 5 s and incubation for 10 min at room temperature prior to cellular transfection. The N/P ratios utilized for cellular transfection, which are defined as the ratios of the amine groups in PEI to the phosphate groups in DNA molecules, were 2, 4, 6, 12, 18, 24, 30, and 36. The resulting DNA complexes encoding GFP or luciferase driven by the CMV promoter were added to the medium that contains cells attached on the TCP. The cell culture medium was freshly replaced at 6 h post-transfection, and the cells were continuously cultured for additional 36 h before analysis. To examine the capability of cellular transfection by DNA/PEI-peptide vectors, the cells transfected with DNA complexes with PEI alone, Lipofectamine (Life Technologies, Paisley, U.K.) or jetPEI (Polyplustransfection, New York, NY, U.S.A.) were employed as controls. The resulting transfection efficiencies of the DNA complexes were quantified by flow cytometry (Becton Dickinson FACS Caliber, Franklin Lakes, NJ) or luminometer (LB96P, EG & G, Berthold, Germany) at Yonsei University College of Medicine Medical Research Center. To determine the cellular transfection efficiency, both the percentage of GFP-expressing cells out of the total number of cells and the mean fluorescence intensities of the GFP expression were quantified. Additionally, to demonstrate the enhanced capabilities of the DNA/PEI-peptide vectors in a different transgene system, luciferase gene expression was measured on a luminometer using the luciferase assay system (Promega, Madison, WI, U.S.A.), with levels normalized to the initial cell seeding numbers. Cell Viability. To determine the viability of cells transfected with the DNA/PEI-peptide complexes, the metabolic activity of transfected cells was quantified by colorimetric measurement using a WST-1 assay kit (Roche Applied Science, Indianapolis, IN). Briefly, each cell line was seeded on 96-well plates (5000 cells/well), transfected by DNA complexes formulated with different N/P ratios, and harvested at 2 days post-transfection. At the time of harvest, a 0.1 volume of WST-1 solution (1/10 of the original culture medium) was added to each well and incubated for an additional 4 h at 37 °C. Finally, the supernatants were collected, and the colorimetric changes at 440 nm were measured using a spectrophotometer (Nanodrop 2000, Thermo Scientific, West Palm Beach, FL, U.S.A.). Analysis of Vector Delivery Mechanisms. To analyze the delivery mechanisms of the DNA/PEI-peptide hybrid vectors, chlorpromazine (10 μM, Sigma-Aldrich), filipin (4 μM, SigmaAldrich), or amiloride (20 μM, Sigma-Aldrich) was added to inhibit the endocytic cellular uptake of the DNA complexes.26 Each inhibitor was added to the medium containing HEK293T cells and incubated for 30 min at 37 °C. To remove the residual inhibitor, the cells were rinsed twice with PBS and then cultured for an additional 4 h in fresh medium. Subsequently, DNA complexes encoding GFP were added to the cells. At 2 days post-transfection, the percentage of GFP-expressing cells was determined using flow cytometry. To measure cellular transfection, the percentage of GFP-expressing cells in the trans-

fections performed with inhibitors was compared to the percentage of GFP-expressing cells in the transfection performed without inhibitors; these percentages were used as a measure of the inhibitory effects of each drug on endocytic delivery. Additionally, to inhibit the energydependent endocytosis of DNA complexes, HEK293T cells were incubated with sodium azide for 30 min at 4 °C, exposed to each DNA complex formulation, and cultured for additional 1 h at 4 °C. The supernatant was subsequently removed, and the cells were rinsed twice with PBS and cultured for additional 48 h at 37 °C prior to flow cytometry analysis. The percentage of GFP-expressing cells exposed to DNA complexes at 4 °C was compared with that at 37 °C to determine the energy-dependent endocytosis pathway. In addition, to investigate the internalization of DNA complexes within the cytoplasm, plasmid DNA was fluorescently labeled with rhodamine (Mirus, Milwaukee, WI) and complexed with the designated PEI formulations. At 4 h post-transfection, the internalization of fluorescently labeled DNA complexes was visualized using confocal microscopy to assess the capability of peptide-conjugated, PEI-mediated DNA delivery for rapid cellular internalization. Finally, to examine the receptor-mediated delivery mechanism, surface glycosaminoglycans (GAG), such as heparin, heparan sulfate, and chondroitin sulfate, were removed using glycosaminoglycan lyases. Prior to transfection, HEK293T cells were rinsed with PBS twice and incubated for 1 h with either 10 units/mL of chondroitinase ABC (Sigma-Aldrich) or 10 units/mL of heparinase II (Sigma-Aldrich) dissolved in PBS solution supplemented with 10% bovine serum albumin (BSA). Subsequently, cells were rinsed twice with PBS and transfected with each DNA complex formulation using the normal procedure. Both the percentage of GFP-expressing cells and the mean fluorescence intensity measured under the condition with or without the GAG lyases were employed to investigate the GAG-mediated gene delivery mechanism of DNA complexes. Statistics. All of the experiments were performed in triplicate, and the experimental data are illustrated as the mean and the standard deviation (S.D.). A one-way analysis of variance (ANOVA) with a post-hoc Dunnett’s test using the SPSS 18.0 software package (IBM Corporation, Somers, NY, U.S.A.) was used to test for statistical significance.

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RESULTS AND DISCUSSION Prior to assembling the nonviral vectors, small portions of the viral peptides were conjugated onto primary amines within branched PEI (25 kDa), as illustrated in Scheme 1. The exact sequences and characteristic properties of each peptide are summarized in Table 1. Three different peptides, r3.45, hep_BD, and r3.45-hepBD, were employed to engineer the conventional nonviral carrier, PEI. The peptide r3.45 encodes the sequence LATQVGQKTA and was found on the surface of the 3-fold spike of the AAV r3.45 capsid in contrast to the unmodified wild-type AAV2.27 The new peptide was found at amino acid (aa) 587 of the wild-type AAV2 capsid (583LQRGNRQA-), which corresponds to the heparan sulfate proteoglycan (HSPG) binding domain (hepBD). The peptide hepBD, LQRGNRQA, represents one of the HSPG binding domains exposed on the AAV2 capsid. Finally, the peptide r3.45-hepBD, LQRGNLATQVGQKTARQA, is composed of both the r3.45 and hepBD peptides, and r3.45 is inserted between the asparagine (N) and arginine (R) within the hepBD, which is the same peptide architecture found in AAV r3.45 (Scheme 1a). All the peptides were designed to contain one cysteine and two glycines (CGG) at the N-terminal end of each peptide (“GG” worked as a spacer). N-Succinimidyl-3-(2pyridyldithio) propionate (SPDP) was utilized as a linker to form a disulfide bond upon reacting with cysteine-containing peptides (Scheme 1b). Three AAV peptide sequences C

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-CH2CH2SS-: 2.2 ppm and -CH2CH2SS-: 2.9 − 3.0 ppm) indicated the successful grafting of SPDP into branched PEI. Once the modification of PEI with SPDP is completed, a disulfide reducing agent, DTT, is employed to release the pyridine-2-thione, which is quantified using a spectrophotometer at 343 nm (molar absorption coefficient = 8080/M·cm).28 This procedure can confirm the successful coupling of SPDP with PEI chains and estimate the molar ratio of SPDP molecules on PEI molecules. As a result, the degree of SPDP substitution was approximately 9.12 mols of SPDP per 1 mole of PEI. Subsequently, the quantity of primary amines within PEI after SPDP conjugation was determined using a fluorescamine assay. Following the SPDP grafting, approximately 7.9% reduced quantity of primary amines within PEI molecules was observed, which indicates that the SPDP primarily reacted with primary amines. The quantification of the remaining primary amines using a fluorescamine assay was not deviated from the value measured using the UV−vis spectrophotometer. Finally, the terminal −SH group within the modified PEI was then used to couple with the cysteine that is linked at the N-terminal of r3.45_hepBD peptide. The conjugation of the peptides into PEI was confirmed by 1 H NMR spectroscopy and FT-IR (Figure 1c−e). Distinct resonance peaks of methyl groups in valine (V) and leucine (L) within either the r3.45 or the hepBD peptide (i.e., approximately 0.9 ppm) indicated the successful integration of each peptide (i.e., r3.45, hepBD, or r3.45_hepBD) into branched PEI.29 Resonance peaks for other peptides (arginine, ∼1.1 ppm; threonine, ∼1.2 ppm; glycine, δ 3.5−4.0 ppm; asparagine, 2.3, 2.5 ppm) confirmed the successful coupling peptides into PEI. The integration of the peptide into PEI was additionally supported by the presence of amide I bands at 1637 cm−1 in the FT-IR analysis (Figure 1f−i); these bands are characteristic of stretching vibrations of the CO within the peptide group.30 Because the molecular sizes of conjugated peptides (r3.45, 978 g/mol; hepBD, 1159 g/mol; and r3.45_hepBD, 2157 g/mol) were substantially lower than those of 25 kDa PEI molecules, estimating the deviation of absorbances using UV−vis analysis was highly difficult. Instead, the portion of peptide-conjugation onto PEI molecules was calculated using NMR analysis in this study. The degree of r3.45_hepBD peptide conjugation, which was confirmed by calculating the proton integral values of the 1H NMR spectrum at 0.9 ppm (CH3 of valine) and at δ 2.4−3.0 ppm (CH2 of PEI), was approximately 0.51% (PEI-r3.45_hepBD).31 The portions of each peptide on PEI are categorized in Table 1. Because a key moiety sequence within the r3.45_hepBD peptide that specifically interacts with the cellular membrane has not been identified, the linkage site of the peptide to the PEI chains was determined based on the following hypothesis: (i) the presence of hydrophobic peptides at the far end (i.e., the region that does not link to the PEI) may make the peptide translocate gene vectors across the hydrophobic core of the lipid plasma membrane,32 and (ii) the presence of cationic peptides at the far end may enhance the interactions of the peptide with various anionic glycosaminoglycans on the cellular membrane. All of these hypotheses are expected to synergistically increase the delivery efficiency of the gene vectors. Consistently, Boeckle et al. demonstrated that PEI, when covalently coupled with the N-terminus of melittin, significantly enhanced the gene delivery efficiencies, with a substantially reduced cellular toxicity compared to PEI linked with the Cterminus of melittin.33 Although this result is most likely

conjugated onto PEI (i.e., PEI-r3.45, PEI-hepBD, and PEIr3.45_hepBD) are described in Table 1. Prior to confirming the peptide conjugation onto PEI, coupling SPDP linker into primary amines within PEI was initially characterized (Figure 1a,b). Distinct resonance peaks of methylene groups observed in SPDP-conjugated PEI (i.e.,

Figure 1. Structure analysis of each PEI formulation using 1H NMR and FT-IR: (a) 1H NMR (PEI), (b) 1H NMR (PEI-SPDP), (c) 1H NMR (PEI-r3.45), (d) 1H NMR (PEI-hepBD), (e) 1H NMR (PEIr3.45_hepBD), (f) FT-IR (PEI), (g) FT-IR (PEI-r3.45), (h) FT-IR (PEI-hepBD), and (i) FT-IR (PEI-r3.45_hepBD). D

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attributed to the intrinsic properties of melittin (i.e., its membrane-destabilizing property), the cationic C-terminus of melittin (lysine and arginine), which was not coupled to the PEI chains, resulted in an enhanced association of the peptides to the cellular membrane as well as a reduced membrane lysis, thereby apparently promoting gene delivery efficiency and improved cellular viability. Additionally, the hydrophobic face of the peptide may trigger the entrance of the peptide into the membrane interior, which may play a crucial role in mediating lipid−peptide interactions to enhance gene delivery efficiencies.34 To fulfill these hypotheses, one of the hydrophobic peptides (i.e., two alanines) and two cationic peptides (i.e., lysine and arginine), which are present at the near C-terminus of the r3.45_hepBD peptide, were designated toward the far end group, thus, synergistically enhancing the delivery efficiency. Negatively charged plasmid DNA self-assembled with the resulting peptide-conjugated PEIs. As the N/P ratios increased (the N/P ratios are defined as the ratio of moles of amine groups in PEI to those of phosphate groups in DNA), the net charges of the resulting vectors that were complexed with each PEI formulation became highly positive, which indicates that the partial peptide conjugation did not result in a reduction in cationic charges with the addition of the modified PEIs (Figure 2a). Once the net charges of the complexes turned into positive charges (>N/P 6), the average hydrodynamic sizes of the vectors ranged from approximately 30.3 to 86.6 nm (Figure 2b) and were consistently visualized by atomic force microscopy (AFM; Figure 2c). The polydispersity index of DNA complexes that were formed at an N/P ratio of 18 ranged from 0.393 to 0.466 (Table 2). The differences in the vector sizes when complexed with either PEI alone or PEI-peptide conjugates were not significant, which indicates that peptides conjugated to PEI contribute little to the sizes of the DNA complexes. Except for the DNA/PEI-hepBD complexes, the electrophoretic migration of all the DNA complex formulations in an agarose gel was successfully retarded once the complex was formed at N/P ratios greater than 6, confirming the capabilities of each modified PEI to condense the DNA molecules similarly to the unmodified PEI (Figure 2d). Based on the complex characterization shown in Figure 2, it is expected that fully cationic DNA complexes that can be employed as gene carriers should be formed at an N/P ratio of at least 6. Additionally, the stability of the DNA complexes at a physiological ionic strength (i.e., 150 mM NaCl, pH 7.4), which can weaken the electrostatic interaction between cationic polymers and DNA to possibly form aggregates over time, was investigated. Consequently, while the sizes of the DNA/ PEI complexes were approximately four times increased at 1 h postincubation, the sizes of the DNA/PEI-r3.45_hepBD and DNA/PEI-r3.45 complexes were slightly enlarged over the same time periods, confirming the stability of the DNA complexes formed with r3.45_hepBD or r3.45-modified PEI molecules (Figure 2e). Shielding the net positive surface charges of the DNA complexes with the coupled-peptides might prohibit the complexes’ aggregation by reducing the strong interactions with ions that gave a high ionic strength.35,36 The optimal cellular transfection with DNA/PEIr3.45_hepBD in each cell type was determined by considering both the level of GFP expression and the cellular viability, which were dependent on the DNA quantity as well as the DNA complex-residence time with the cells prior to changing the medium (data not shown). In all of the cellular

Figure 2. Characterization of the DNA complexes formed with each PEI formulation as a function of the N/P ratios: (a) Zeta potential analysis, (b) hydrodynamic sizes, (c) the morphologies of DNA complexes were visualized by AFM analysis (N/P ratio: 18), (d) gel retardation analysis of DNA complexes formed with each PEI formulation, and (e) stability of DNA complexes formed at the N/P ratio of 18, which was shown by the time-dependent aggregation of DNA complexes at physiological ionic strength (150 mM NaCl, pH 7.4). E

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expressing cells and the mean fluorescence intensity (Figure 3). Complexation with PEI-SPDP, PEI-r3.45, or PEI-hepBD did not improve the cellular transfection relative to the PEI alone. Importantly, when the r3.45 (LATQVGQKTA) and hepBD (LQRGNRQA) peptide sequences were fused together, a significant enhancement in cellular transfection was observed compared with the other complex formulations, indicating that these peptides possess capabilities that can synergistically increase gene delivery efficiencies. Additionally, complexation with PEI-r3.45_hepBD demonstrated significantly enhanced or comparable gene delivery properties compared to DNA complexes formed with commercially available, representative transfection reagents, Lipofectamine, and jetPEI, which were formulated according to the manufacturer’s protocol. The complexation of DNA with higher N/P ratios resulted in higher cellular transfection for all the formulations. The use of gene vectors with PEI-r3.45_hepBD formed at an N/P ratio of 30 resulted in an approximately 4-fold increase in the percentage of GFP-expressing cells and in 3-fold mean fluorescence intensities relative to nonmodified DNA/PEI complexes, DNA/Lipofectamine or DNA/jetPEI in HEK293T cell transfection (Figure 3a−c). In Panc1 cell transfection, DNA/PEI-r3.45_hepBD formed at an N/P ratio of 24 demonstrated approximately 2−3-fold enhanced gene delivery compared to the control conditions, which further indicates their potential as efficient gene carriers (Figure 3d−f). This enhanced cellular transfection was accompanied by cell line specificity, as DNA/PEI-r3.45_hepBD complexes were not

Table 2. Polydispersity Index of DNA Complexes Formed at an N/P Ratio of 18 PEI-peptide/DNA complexes

polydiversity index of DNA complexes (N/P 18)

PEI/DNA PEI-r3.45/DNA PEI-hepBD/DNA PEI-r3.45_hepBD/DNA

0.428 0.437 0.393 0.466

transfections, the percentage of GFP-expressing cells was typically improved as either the DNA quantity or the complex-incubation time increased. However, the performance of the DNA/PEI-r3.45_hepBD complexes required optimization because higher DNA quantities or longer incubation times typically increased the cellular toxicity (Supporting Information, Figure S1). The amount of free PEI, which is present in solutions that contain complexes formed with larger DNA quantities, or the duration of cellular interactions with free PEI may be critical criteria for determining the cellular viability.37 As a result, the 6 h exposure of PEI-r3.45_hepBD complexes with 0.5 μg DNA resulted in optimal performance in transfected HEK293T cells. Therefore, these designated conditions were employed for further investigations in this study. Two different cell types, HEK293T and Panc1, were transfected by the resulting DNA complexes. Among the DNA complex formulations, the DNA/PEI-r3.45_hepBD vector demonstrated robust cellular transfection capabilities, which were demonstrated by both the percentage of GFP

Figure 3. Comparison of cellular transfection with DNA complexes (0.5 μg in 5% glucose) formed with PEI alone, Lipofectamine, jetPEI, and each PEI-peptide formulation: representative fluorescence images that show GFP-expressing HEK293T cells (a) and Panc1 cells (d). The scale bar indicates 100 μm. To examine the capability of cellular transfection by DNA/PEI-peptide vectors, both the percentage of the GFP-expressing cells out of the total number of cells (HEK293T: (b), Panc1: (e)) and the mean fluorescence intensities of the GFP expression (HEK293T: (c), Panc1: (f)). The symbol * indicates the significant differences in the gene expression for the DNA/PEI and DNA/PEI-r3.45_hepBD complexes (P < 0.05). The symbol ** indicates the significant differences in the gene expression for commercially available reagents, including Lipofectamine or jet PEI, and for DNA/PEI-r3.45_hepBD complexes (P < 0.05). F

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Enhanced cellular viability by transfection with DNA/PEIr3.45_hepBD may be due to lower levels of primary amines compared to the original PEI, which primarily cause detrimental effects on cell metabolism due to the increased cationic charge densities in the PEI chains.39 Importantly, no significant difference was observed in the metabolic activity of the cells transfected with PEI-r3.45_hepBD compared to the complexes with Lipofectamine or jet PEI, which potentially demonstrates the safety of gene vectors complexed with PEIr3.45_hepBD. To investigate the aforementioned gene delivery mechanisms, endocytosis of the DNA complexes across the cellular membrane, which is a normal DNA trafficking pathway by nonviral DNA delivery, was inhibited using filipin, chlorpromazine, and amiloride. Chlorpromazine disrupts clathrindependent cellular uptake by redistributing the clathrin-coated pits to late endosomes, consequently inhibiting clathrinmediated endocytosis.40 The drug filipin is widely utilized to inhibit caveolae-mediated endocytosis and thereby substantially blocks the internalization of cationic nonviral carriers.41 Amiloride can inhibit macropinocytosis through perturbing Na+/H+ exchanges in the cellular membrane.42 Consequently, cellular uptake of all the DNA complex formulations was drastically inhibited in the presence of chlorpromazine and filipin, but no marked decrease in gene expression was observed upon treating cells with amiloride (Figure 5a). Additionally, the entry of DNA/peptide-conjugated PEI complexes, including

efficient in HeLa and 3T3 cell lines, which are representative nonpermissive cell lines to nonviral gene carriers (Supporting Information, Figure S2). In the case of HEK293T cellular transfection with plasmid encoding luciferase, the delivery of DNA/PEI-r3.45_hepBD resulted in an approximately 1.5-fold increment of luciferase gene expression compared to DNA/PEI complexes but slightly reduced luciferase expression relative to DNA/jetPEI complexes (Supporting Information, Figure S3). In Panc1 cell transfection, the level of luciferase gene expression by DNA/PEI r3.45_hepBD complexes was higher than that by DNA/PEI complexes, but the absolute values of gene expression were low compared to those in HEK293T cell transfection, which is consistent with GFP analysis. Compared with transfection with DNA/PEI complexes, the delivery of DNA/PEI-r3.45_hepBD resulted in improved cellular viability in all the cell lines (Figure 4). Transfection

Figure 4. Metabolic activities of HEK293T cells (a) and Panc1 cells (b) at 42 h post-transfection by DNA complexes formed at various N/ P ratios. The metabolic activity of the transfected cells was quantified by a colorimetric measurement at 440 nm using a WST-1 assay kit. The absorbance values were normalized to the absorbance value of the negative control, in which the cells were cultured without DNA complexes.

with the newly conjugated PEIs demonstrated slightly reduced cellular viabilities in all the cell types, as the N/P ratios increased by 36, whereas transfection with the complexes that were formed with PEI alone caused reduced cellular viability compared to transfection with the DNA/PEI-r3.45_hepBD complexes (Figure 4a). High transfection efficiencies with nonviral DNA complexes can typically result in reduced cellular viability, possibly due to the destabilized cellular membrane caused by the penetration of a sufficient quantity of DNA complexes across the membrane.38 Consistently, less permissive properties of DNA/PEI-r3.45_hepBD complexes to Panc1 cells than to HEK293T cells might lead to the absence of severe cellular toxicity in Panc1 cells compared to HEK293T cells (Figure 4b). When the HEK293T cells were transfected with DNA/PEI-r3.45_hepBD complexes formed at an N/P ratio of 30 (the optimal N/P ratio for the highest transfection), the percentage of viable cells remained above 80%, but the delivery of DNA/PEI complexes led to a gradual drop below 55%.

Figure 5. Inhibition of endocytosis for DNA complexes that were formed with each PEI formulation (N/P ratio: 18) using chlorpromazine (10 μM), filipin (4 μM), or amiloride (20 μM) to investigate the delivery mechanism in HEK293T cells (a). Inhibition of the energy-dependent endocytosis of DNA complexes was additionally examined (b). HEK293T cells were incubated with sodium azide for 30 min at 4 °C, exposed to each DNA complex formulation, and cultured for an additional 1 h at 4 °C. The supernatant was subsequently removed, and the cells were rinsed twice with PBS and cultured for an additional 48 h at 37 °C prior to the flow cytometry analysis. The percentage of GFP-expressing cells exposed to DNA complexes at 4 °C was compared with that at 37 °C. The symbol * indicates significant differences (P < 0.05). G

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possibly improving cellular transfection by DNA/KALAplugged PEI complexes.43 Thus, the synergistic effect of both delivery processes, the endocytosis of the DNA complex itself and intimate surface-association of the r3.45_hepBD peptide, might be key contributors to the significantly enhanced cellular transfection of DNA/PEI-r3.45_hepBD. Finally, the role of glycosaminoglycans in PEI-r3.45_hepBDmediated gene transfer was investigated. To evaluate the effect of glycosaminoglycans, such as heparin, heparan sulfate proteoglycan, and chondroitin sulfate proteoglycan, on gene transfer, HEK293T cells were treated with glycosaminoglycan lyases, including chondroitinase ABC or heparinase II, to remove the glycosaminoglycans at the cellular surface prior to cellular transfection.44 As a result, the removal of chondroitin sulfates at the cellular surface resulted in significantly reduced cellular transfection of DNA/PEI-r3.45_hepBD complexes, whereas the removal of heparin/heparan sulfate proteoglycan did not substantially inhibit cellular transfection (Figure 7).

DNA/PEI complexes, was dependent on the energy-mediated endocytosis pathway, as demonstrated by the fact that the percentage of GFP-expressing cells transfected by all of the DNA complexes with sodium azide at 4 °C was significantly lower than that at 37 °C (Figure 5b). These findings indicate that energy-dependent, clathrin- and caveolae-mediated endocytosis can be the primary mechanisms of cell entry for DNA/ peptide-conjugated PEI complexes. The high level of transfection that was observed in Figure 3 could possibly be attributed to the DNA vectors being formulated with PEI-r3.45_hepBD, which were robustly associated with the cellular membrane at the initial stage of cellular transfection (i.e., 4 h) compared with the other DNA complexes (Figure 6). To verify the close interaction of the

Figure 7. Effect of glycosaminoglycans (GAG) on cellular transfection of DNA/peptide-conjugated PEI complexes. Prior to transfection, HEK293T cells were rinsed with PBS twice and incubated for 1 h with either 10 units/mL of chondroitinase ABC or 10 units/mL of heparinase II, to remove chondroitin sulfate proteoglycan or heparin/ heparan sulfate proteoglycans, respectively. Subsequently, cells were transfected with each DNA complex formulation using the normal procedure. Both the percentage of GFP-expressing cells (a) and the mean fluorescence intensity (b) measured under the condition with or without the GAG lyases were employed to investigate the GAGmediated gene delivery of the DNA complexes.

Figure 6. Cellular internalization of DNA complexes (N/P ratio: 18, 4 h post-transfection) across the cellular membrane, as shown by fluorescence images of DNA complexes (red). (a) HEK293T, (b) Panc1. To visualize the cells effectively, bright field images taken to show cell morphologies were merged with the fluorescence images. The scale bar indicates 10 μm.

DNA/PEI-r3.45_hepBD vectors with designated cell types, the presence of fluorescently labeled DNA complexes on or within the cellular membrane was visualized at 4 h post-transfection. Consequently, a dramatically increased presence of fluorescently labeled DNA/PEI-r3.45_hepBD complexes was observed around the cellular membrane or within the cytosol compared with the other complex formulations. This finding indicates that the enhancement of cellular transfection by the DNA/PEI-r3.45_hepBD complexes may be caused by the rapid accumulation of the gene vectors within the cellular membrane, presumably due to the intimate interactions of the r3.45_hepBD peptide with unknown receptors or glycosaminoglycans residing on the phospholipid membrane. Similarly, one of the other virally derived peptides, KALA, has been known to mediate phospholipid membrane fusion, thereby

These findings suggest that chondroitin sulfate proteoglycans can be primary mediators for DNA/PEI-r3.45_hepBD complexes. The significant effect of the less anionic glycosaminoclycan, chondroitin sulfate, on cellular transfection, when compared with the effect of heparin/heparan sulfate, may imply that the structural features of the DNA/peptideconjugated PEI complexes rather than the charges of the complexes can be critical clues to interactions with the plasma membrane that are followed by improved cellular uptake, ultimately working as a key role to enhance cellular transfection. H

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CONCLUSION This study describes a highly efficient, nonviral gene delivery system compared with conventional nonviral gene delivery vectors, such as PEI, Lipofectamine or jetPEI. Small portions of capsid peptides that originate from a highly safe and efficient virus, AAV, were conjugated onto branched PEI, thus, integrating viral components into nonviral constituents. The self-assembled gene vectors, therefore, may integrate advantageous properties adopted from both viral and nonviral aspects, such as efficient and safe gene delivery, respectively. The successful establishment of a novel gene delivery system with enhanced abilities to transfect cells provides a powerful means for numerous gene therapy applications.

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ASSOCIATED CONTENT

S Supporting Information *

Additional data on metabolic activities, cell line specificity, and luciferase gene expression is available. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 82-2-2123-2756. Fax: 82-2-312-6401. E-mail: j-jang@ yonsei.ac.kr. Author Contributions †

These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a National Research Foundation (NRF) grant through the Active Polymer Center for Pattern Integration (No. R11-2007-050-00000-0) and Basic Science Research Program (2012R1A1A1003397) funded by the Ministry of Education, Science and Technology (MEST).

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