Cell Targeting Peptide Conjugation to siRNA Polyplexes for Effective

Center for Controlled Chemical Delivery (CCCD), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 841...
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Cell Targeting Peptide Conjugation to siRNA Polyplexes for Effective Gene Silencing in Cardiomyocytes Hye Yeong Nam,† Jaesung Kim,† Sung Wan Kim,† and David A. Bull*,‡ †

Center for Controlled Chemical Delivery (CCCD), Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, United States ‡ Division of Cardiothoracic Surgery, School of Medicine, University of Utah, Salt Lake City, Utah 84132, United States ABSTRACT: To deliver siRNA specifically to cardiomyocytes with a high transfection efficiency, primary cardiomyocytetargeting (PCM) and/or cell-penetrating (Tat) peptides were incorporated into the siRNA. With the addition of plasmid DNA, these peptide-conjugated siRNAs were able to form compact and stable nanosized polyplex particles with bioreducible poly(CBA-DAH). The peptide-modified siRNA polyplexes enhanced the cellular uptake and the gene-silencing capacity of the siRNA in cardiomyocytes without significant immunogenicity or cytotoxicity. These findings demonstrate that the cell-targeting peptide and/or cell-penetrating peptide conjugation of siRNA may be a potentially important strategy for cell-specific gene therapy in gene-mediated disease states. KEYWORDS: siRNA, targeting, bioreducible polymer, gene therapy silencing effects of the siRNA.29 To overcome this problem, a variety of strategies have been proposed, including: (1) modification of the siRNAs with sticky overhangs or multimerization via chemical linkages; and (2) formulation with other polyanionic additives to stabilize electrostatic complexation.30−33 Long pDNA may be used to effectively form nanosized siRNA particles and enhance gene silencing.34 This codelivery system of siRNA and pDNA may aid in the formation of compact particles for the treatment of a variety of disease states. In this study, we report the development of a novel celltargeting siRNA delivery system using peptide conjugation with the PCM and Tat peptides for cell targeting and cell penetration, respectively, and plasmid DNA for effective polyplex formation. Each peptide is conjugated to the siRNA via cleavable disulfide linkages, which facilitates siRNA release in the reductive conditions of the cytoplasm.35,36 To investigate the targeting and transfection efficiencies of this novel construct, we used a bioreducible polymer, (poly(CBADAH)) (CD), synthesized by polymerization between 1,6diaminohexane and cystamine bis-acrylamide, as an siRNA carrier. Reducible cationic copolymers, such as disulfidecontaining poly(amido amine)s, (SS-PAAs), increase gene transfection efficiency and cell viability.37−41 A reporter pDNA was used to make a compact polyplex, and these complexes were utilized for further study. We performed cellular uptake

1. INTRODUCTION Cardiovascular disease (CVD) is one of the leading causes of death worldwide. While conventional therapies, such as percutaneous coronary interventions (PCI) and coronary artery bypass grafting (CABG), can improve blood flow to the myocardium, they cannot restore or regenerate myocardium lost to ischemia-reperfusion injury or infarction (MI).1−3 Recently, siRNA has emerged as a promising therapeutic strategy to inhibit specific mRNA expression in clinical disease states such as myocardial ischemia and infarction.4−6 The inherent instability and poor cellular uptake of siRNA, however, have limited its clinical application.7−10 The ability to deliver siRNA targeted to cardiomyocytes, therefore, would greatly enhance the therapeutic potential of siRNA for the treatment of cardiovascular disease.11−14 The conjugation of specific ligands is a promising technique to selectively deliver therapeutic genes to specific target cells.15−18 We recently demonstrated that the primary cardiomyocyte (PCM) targeting peptide, (CWLSEAGPVVTVRALRGTGSW), conjugated to a bioreducible polymer, can markedly increase the efficiency of gene delivery targeted to cardiomyocytes. The additional conjugation of a cell-penetrating peptide (CPP), Tat (C-47YGRKKRRQRRR57), to a bioreducible polymer, further enhances cellular uptake and transfection efficiency.19−28 Using PCM and Tat peptideconjugated polymeric carriers for targeted gene delivery is a novel biologic strategy for the treatment of cardiovascular disease. The modification of a short and rigid siRNA with a peptide can result in large and loose nanoparticles which may be easily attacked and degraded by RNases, thereby reducing the © 2012 American Chemical Society

Received: Revised: Accepted: Published: 1302

November 18, 2011 February 26, 2012 March 27, 2012 March 27, 2012 dx.doi.org/10.1021/mp200589z | Mol. Pharmaceutics 2012, 9, 1302−1309

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The average hydrodynamic sizes and zeta-potential measurements of the polyplexes were determined after the desired dilution of formed polyplex with filtered DW using Nano ZS (ZEN3600, Malvern Instruments) with a He−Ne ion laser (633 nm). The results are reported as the mean values ± standard error of the mean (SEM). To compare the stability of the polyplexes, the release of siRNA and pDNA from the polyplex was measured by picogreen assay after 1 h of incubation of various heparin concentrations. 2.4. In Vitro Transfection. For the measurement of gene silencing and transfection efficiency, cells were plated at a number of 5 × 104 cells/well in 24-well plates in 500 μL media containing 10% FBS and incubated for 24 h before transfection. Polyplexes with siRNAs (0.35 μg, equivalent to 50 nM unmodified-siRNA per well) were prepared at different weight ratios in HEPES buffer, with the total amount of siRNAs in the polyplexes being the same. For the codelivery system, the same amount of plasmid DNA (0.35 μg) with siRNA (0.35 μg) was used. After 30 min incubation, polyplexes were added to the cells in the absence of serum for 4 h at 37 °C. The media was then replaced with fresh 10% serum-containing medium. After incubation for 24 h, the media was collected, and the amount of secreted VEGF from the cells was measured using a VEGF immunoassay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's protocol. The result is presented as the relative percentage of VEGF expression. GFP expression from the cells was detected using an EVOS fluorescence microscope (Advanced Microscopy Group, Bothell, WA), and the level of expression was measured using a Qubit fluorometer (Molecular Probes Inc.) and FACS analysis. 2.5. In Vitro Cellular Uptake. To investigate cellular uptake, cells were transfected with the polyplex (at a weight ratio of 20) with the mixture of pDNA (0.7 μg/mL), labeled with YOYO-1 iodide (1 molecule of the dye per 100 base pairs of the nucleotide), and siRNAs (0.7 μg/mL). The cells were treated with the polyplex solutions for 4 h at 37 °C, washed with cold DPBS, and then trypsinized. The collected cells were suspended in 0.5 mL of DPBS. The cellular uptake was examined with a FACScan analyzer (Becton Dickinson, San Jose, CA) using Cell Quest software. A total of 10 000 cells were acquired for each analysis. 2.6. Cytotoxicity and Immune Response. The cells were seeded in 24-well plates for the MTT assays and grown to reach 70−80% confluence for 24 h. After 1 day of transfection with the polyplexes in 10% serum DMEM, 25 μL of MTT stock solution (2 mg/mL in PBS) was added to each well. After incubation at 37 °C for 4 h, 150 μL of DMSO was added to dissolve the formazan crystal after removing the media with the MTT solution. The absorbance was measured using a microplate reader (model 680, Bio-Rad Lab, Hercules, CA) at 570 nm, and the cell viability was recorded as a percentage relative to control cells in quadruplicate. To determine the immune response of cells induced by the polyplexes, peripheral blood mononuclear cells (PBMCs), supplied by SeraCare Life Sciences Inc. (Gaithersburg, MD), were seeded in 96-well plates and cultured in RPMI 1640 medium with 10% FBS for 24 h. The cells were treated with various siRNA formulations at a final concentration of 3 μg/mL. Synthetic poly(I:C) (SigmaAldrich) was used to induce an immune response in PBMCs as a positive control. After 24 h of incubation, the culture supernatants were collected and assayed using an INF-α sandwich enzyme-linked immunosorbent assay (ELISA, In-

and transfection experiments with the peptide-modified siRNA/CD complex using FACS analysis and a reporter gene expression assay. We evaluated the biodistribution of the novel complex using real-time RT-PCR. The innate immune response was determined by the INF-α response in human peripheral blood mononuclear cells (PBMCs).

2. MATERIALS AND METHODS 2.1. Materials. N,N′-Cystaminebisacrylamide (CBA) was purchased from PolySciences, Inc. (Warrington, PA). tert-ButylN-(6-aminohexyl) carbamate (N-Boc-1,6-diaminohexane, NBoc-DAH), trifluoroacetic acid (TFA), triisobutylsilane (TIS), N,N-diisopropylethylamine (DIPEA), N,N,N′,N′-tetramethylazodicarboxamide (TMAD), and 3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS), Dulbecco's phosphate buffered saline (DPBS), Roswell Park Memorial Institute 1640 medium (RPMI 1640), Dulbecco's modified Eagle's medium (DMEM), TRIzol reagent, and YOYO-1 dye were obtained from Invitrogen (Carlsbad, CA). Spectrapor dialysis membrane was purchased from Spectrum Laboratories, Inc. (Rancho Dominguez, CA). Plasmid DNA (pDNA) encoded with the GFP reporter gene (gWiz-GFP) was purchased from Aldevron, Inc. (Fargo, ND). 5′-Thiol-modified siRNAs (the 5′-ends of the sense strands modified with thiol group) were synthesized by Bioneer (Daejon, Korea). The sequences of siRNA used for these experiments are as follows: antihuman-VEGF siRNA: 5′-GGAGUACCCUGAUGAGAUCdTdT-3′ (sense strand), 5′-GAUCUCAUC AGGGAUCUCC dTdT-3′ (antisense strand); rat SHP-1 siRNA: 5′GGACAUUUCUUGUG CGUGAdTdT-3′ (sense strand), 5′UCACGCACAAGAAAUGUC C-3′ (antisense strand). The Taq-Man gene expression assays for real-time PCR were purchased from Applied Biosystems (Carlsbad, CA). 2.2. Synthesis of Peptide-Conjugated siRNA and Polymer. siRNA was modified with a peptide bearing a bioreducible disulfide bond between the siRNA and peptide as shown in Figure 1. Thiol-modified siRNA dissolved in N-(2hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES) buffer (10 mM, pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA)) was reacted with TMAD at room temperature for 1 day under dark conditions. After the reaction, the solution was purified using a PD-10 desalting column (GE Healthcare) and lyophilized. The synthesis was confirmed by polyacrylamide gel electrophoresis (PAGE) analysis. Poly(CBA-DAH) was synthesized as previously published by our group.38,39 2.3. Polyplex Formation and Characterization. Polyplex formation was characterized using an agarose gel electrophoresis assay. Size and surface charges were measured with dyanmic light scattering (DLS). Polyplexes were formed with a known amount of siRNAs and polymer in HEPES buffer (10 mM HEPES, 1 mM NaCl, pH 7.4) at various weight ratios. For the siRNA-pDNA/polymer complexes, the same amount of pDNA was added to the siRNA solution. The resulting solution was mixed with various amounts of polymer in HEPES buffer solution. Each solution was combined, lightly vortexed, and incubated for 30 min at room temperature. After complex formation, the samples were loaded onto an agarose gel (0.8%, w/v) containing an SYBR gel staining solution and electrophoresed in TAE buffer (10 mM Tris/HCl, 1% v/v acetic acid, 1 mM EDTA) at 110 V for 20 min. The nucleic acid bands were visualized with a UV illuminator (Gel Documentation Systems, Bio-Rad, Hercules, CA). 1303

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vitrogen, Carlsbad, CA) according to the manufacturer's instructions. 2.7. Biodistribution. For the biodistribution assay, fiveweek-old female BALB/c mice were purchased from Charles River (Wilmington, MA), used at 7 weeks of age and cared for in the Animal Core Facility in the BioPolymers Research Building at the University of Utah. Mice were injected via the tail vein with various formulations containing 20 μg of genes (pGFP/siRNA = 1:1) in 150 μL of 5% glucose solution. At 36 h postinjection, the mice were sacrificed, and the heart, liver, spleen, lung and kidney were collected and washed with PBS. The harvested tissues were weighed and then homogenized. Genomic DNA isolation from the homogenate was carried out using a Maxwell 16 Tissue DNA Purification Kit (Promega) according to the manufacturer's guidelines. The amount of DNA isolated was determined using Nanodrop. The copy number of the reporter gene, GFP, in various tissues was measured by quantitative real-time PCR using Taq-Man GFP primers and a fluorescently labeled probe with FAM (Applied Biosystem). The reactions were set up using 50 ng of isolated DNA and a Taq-Man universal PCR master mix according to the manufacturer's instructions. Real-time PCR was performed using a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). For a standard curve, the plasmid GFP used in the polyplex formation was serially diluted and analyzed. 2.8. Statistical Analysis. Results are expressed as the mean ± SEM. The statistical differences between groups were determined using one-way analysis of variance (ANOVA) followed by Turkey post hoc analysis, with P < 0.05 considered significant.

Figure 1. Schematic diagram of the preparation of peptide-modified siRNA and the behavior of the combined siRNA, C-siRNA/polymer complex under reductive conditions.

modified siRNA/polymer nanoparticles demonstrated SYBR intensity in a retarded band at a weight ratio of 5, indicating the formation of loosely condensed complexes. These loose nanoparticles were also demonstrated by average size measurement. The average hydrodynamic diameter of the PCM-, Tat-, or combined siRNA polyplexes with the CD polymer was found to range from 210 to 260 nm at a weight ratio of 5 and to be approximately 200 nm at a weight ratio over 5 (Figure 2B). In comparison, the unmodified siRNA/CD complexes had a hydrodynamic diameter less than 180 nm at a weight ratio of 5 and above (data not shown). The formation of loose complexes was associated with a decrease in transfection efficiency. In this study, a high molecular weight and long pDNA was used to overcome this problem by helping to form compact complexes of the peptide-conjugated siRNA. Figure 2B summarizes the average hydrodynamic size and zetapotential values of the polyplex particles. The hydrodynamic diameters (Z average) of the C-siRNA-pDNA/CD were 150 and 145 nm, while the C-siRNA/CD hydrodynamic diameters were 212 and 194 nm, at a weight ratio of 10 and 20, respectively. The zeta-potential values of the C-siRNA-pDNA/ CD were kept similar or at a more positive value at a weight ratio above 10. This allowed the positively charged CD polymer to condense the negatively charged pDNA and siRNA more effectively, resulting in fewer genes exposed on the surface of the particle. The polyplex stability was determined by measuring heparin-induced decomplexation at a weight ratio of 5. The results shown in Figure 2C demonstrate slower decomplexation, that is, that the C-siRNA/pDNA polyplex is more stable compared to the siRNA-only system. These results indicate that the pDNA introduced into the polyplexes can help to form compact nanoparticles with a positive surface charge. 3.3. Cellular Uptake and in Vitro Transfection. To investigate the effects of the peptide-modified siRNAs and added pDNA on gene silencing, siRNA/CD and siRNA-

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of PeptideModified siRNA and Bioreducible Polymer. The conjugation of the PCM or Tat peptide to siRNA was performed by disulfide-mediated conjugation under basic and mild oxidation conditions using N,N,N′,N′-tetramethylazodicarboxamide in HEPES buffer according to the synthetic scheme in Figure 1. The synthesis of peptide conjugated siRNA, siRNAPCM, or siRNA-Tat was confirmed through the change in the surface charge of siRNA with PAGE electrophoresis. The zetapotential of the siRNA was −25 mV, while the zeta potential of the peptide-conjugated siRNA was −5 to 10 mV due to the positive charge of the added peptide. In the PAGE analysis, a slight shift of the band was observed as shown in Figure 1. The bioreducible CD polymer as a gene carrier was synthesized as previously described,35,36 and the reaction was confirmed with proton NMR and size exclusion chromatography (SEC). The molecular weight of the synthesized CD was 3.5 kDa. 3.2. Physicochemical Properties of the siRNA/Polymer and siRNA/pDNA/Polymer Complexes. To find an optimal weight ratio of the CD polymer to the siRNAs, a gel retardation assay and hydrodynamic size and surface charge measurements were performed. Polyplexes were first formed in four types: siRNA/CD, siRNA-PCM/CD, siRNA-Tat/CD, and C-siRNA (combination of siRNA-PCM and siRNA-Tat, 1:1)/CD. Agarose gel electrophoresis showed that the unmodified siRNA and the siRNA-PCM, siRNA-Tat, and C-siRNA polyplexes all had a weight ratio of greater than 5, confirming that all of the siRNAs were condensed into the polyplex (Figure 2A). As shown by gel electrophoresis, the unmodified siRNA formed a compact complex with the polymer, while the 1304

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Figure 2. Physical characterization of siRNA/polyplex complexes. (A) Gel retardation assay, (B) particle size and zeta-potential measurement at various weight ratios, and (C) heparin-induced decomplexation assay of polyplexes (WR = 5) at various concentrations of haparin. C-siRNA refers to the siRNA combined with PCM-siRNA and Tat-siRNA (1:1). C-siRNA-pDNA refers to a 1:1 mixture of the C-siRNA and plasmid DNA.

pDNA/CD polyplexes were delivered to hepato-cellular carcinoma cells (Huh 7) and human alveolar epithelial cells (A549) to determine the degree of VEGF gene silencing. The combined siRNA modified with both PCM-siRNA and TatsiRNA reduced VEGF gene expression to a similar degree when the total amount of siRNA present was the same (0.35 μg per well, 24 well). The transfection efficiency of this combined system may be reduced, however, by the chemical conjugation leading to lose complexation. This may result in a decrease in the gene silencing efficacy of the siRNA compared to the unmodified siRNA, despite the enhanced cell penetration resulting from the Tat modification. Interestingly, the efficacy of the C-siRNA/CD was restored with the addition of plasmid DNA to the complex (Figure 3). Presumably, the addition of pDNA to the C-siRNA/CD complexes enhances the transfection efficiency by helping to form more compact polyplexes for the delivery of the siRNA.34 The cellular uptake of the cell-specific peptide-conjugated siRNA polyplexes by noncardiomyocytes, specifically NIH 3T3 cells, and cardiomyocytes, specifically H9C2 cells, was evaluated by flow cytometry using a codelivery system with YOYO-1 intercalated pDNA. The cells treated with the PCM or Tat modified siRNA-pDNA/CD polyplexes for 5 h demonstrated an increase in the uptake of the siRNA polyplexes at a weight ratio of 20. The combined system (C-siRNA) had a significantly stronger fluorescent histogram than PCM- or Tat-modified siRNA alone in H9C2 cells. This finding suggests a synergy between the targeting ability of the PCM modification for cardiomyocytes and the cell-penetrating ability

Figure 3. VEGF gene silencing by peptide modified-VEGF siRNA/ CD polyplexes at a weight ratio of 20 in Huh7 and A549 cells. *p < 0.05 versus the C-siRNA group.

of the Tat conjugation (Figure 4). In NIH 3T3 cells, the PCM peptide modification did not result in an increase in cellular uptake. To further confirm the enhanced targeting ability and transfection efficiency of the combined PCM and Tat modification, in vitro transfection experiments were carried out using the pCMV-GFP reporter gene combined with the siRNAs. As described above, polyplexes containing both pDNA and siRNA formed compact complexes due to the long and flexible pDNA with higher molecular weights. The siRNA-pGFP was 1305

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Figure 4. Cellular uptake of siRNA-pYOYO/polymer complexes (at a weight ratio of 20) in NIH 3T3 and H9C2 cells. Black line, control siRNA; blue line, PCM-siRNA; red line, Tat-siRNA and green line, C-siRNA.

Figure 5. GFP expression of siRNA-pGFP polyplexes at a weight ratio of 20 after 24 h trasfection in NIH 3T3 and H9C2 cells; (A) fluorescence images, (B) the level of GFP expression by Qubit fluorometer, and (C) FACS histogram.

siRNA and the combined-siRNA (C-siRNA) in H9C2 cells compared with NIH 3T3 cells. These results are consistent with the measurements of cellular uptake by flow cytometric analysis. 3.4. In Vitro Cytotoxicity and Immune Response. The cytotoxicity of cells treated with unmodified siRNA, peptide modified siRNA, or siRNA-pDNA polyplexes was measured by MTT assay under the same conditions as the transfection study. As shown in Figure 6A, the polyplexes did not differ with regard to cellular toxicity, with a viability above 80% in both cell types.

transfected into both NIH 3T3 and H9C2 cells using the CD polymer at a weight ratio of 20. The level of GFP expression was assessed using fluorescent microscopy after transfection for 24 h (Figure 5A). There was no difference in GFP fluorescence in either cell type treated with siRNA-pGFP. PCM-siRNA and C-siRNA transfected H9C2 cells, however, demonstrated highly increased GFP expression. The level of GFP expression was also quantified using a fluorometer (Figure 5B)42 and analyzed by flow cytometry (Figure 5B). Transfection and expression of GFP was increased with the PCM-modified 1306

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with the CD polymer strongly induced the production of IFNα, there was no significant production of IFN-α in the PBMCs treated with the unmodified siRNA, modified siRNA or siRNApDNA complexes. 3.5. Biodistribution. In previous reports, we demonstrated that peptide modification can enhance the targeting and cellpenetrating ability of polymer delivery systems in vitro.24,25 Modification with PCM and Tat peptides can also enhance the ability of siRNA to target and penetrate cardiomyocytes in vitro. To study the biodistribution of the peptide-modified siRNAs and the codelivery system with pDNA in vivo, we intravenously injected siRNA-pDNA/CD polyplex solutions into mice. Each organ including the lung, heart, kidney, liver, and spleen was harvested at 24 h post injection. The gene delivery to the various organs was quantified with real-time PCR. Within the heart, the highest relative level of gene delivery occurred with the combined C-siRNA (PCM-siRNA and Tat-siRNA; Figure 7a). These results confirm efficient targeting to the heart with the combined peptide modified C-siRNA system in vivo. Based on the PCR results, an in vivo biodistribution profile demonstrated the expected subsequent late redistribution, in rank order, to the spleen, liver, kidney, and lung (Figure 7b).

4. CONCLUSION RNAi-mediated gene silencing is an effective therapeutic strategy for the treatment of gene-mediated disease states. This study was designed to deliver siRNA effectively to a specific target, cardiomyocytes, with a high transfection efficiency, by modifying siRNA with primary cardiomyocytetargeting (PCM) or cell-penetrating (Tat) peptides. These peptide-conjugated siRNAs did not initially produce dense nanoparticles with the bioreducible polymer, CD, but the addition of plasmid DNA resulted in a more stable and compact polyplex formation. The resulting compact C-siRNApDNA/CD polyplexes promoted high levels of cellular uptake and effective gene silencing in cardiomyocytes without significant immunogenicity. In addition, the combined siRNA polyplexes, that is, C-siRNA with PCM-siRNA and Tat-siRNA, were delivered to the heart at significantly higher levels compared to the unmodified siRNA following systemic administration. Taken in aggregate, these findings indicate that these siRNA-pDNA/CD complexes may be potentially

Figure 6. (A) Cytotoxicity and (B) interferon-α production in peripheral blood mononuclear cells (PBMC) 24 h after treatment with the polyplexes (at a weight ratio of 20).

The immunogenicity of the siRNAs or siRNAs/CD complex systems was evaluated in peripheral blood mononuclear cells (PBMCs) by measuring IFN-α induction by ELISA (Figure 6B). As a positive control, the immune-stimulant, synthetic double poly(I:C) was used to produce IFN-α. While poly (I:C)

Figure 7. Biodistribution of siRNA-pDNA/CD complexes in mice. At 24 h postinjection, the amount of delivered genomic DNA in different tissues (A: heart, B: spleen, liver, kidney, and lung) was determined using real-time PCR and described as a relative distribution level. 1307

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(16) Park, I. K.; Lasiene, J.; Chou, S. H.; Horner, P. J.; Pun, S. H. Neuron-specific delivery of nucleic acids mediated by Tet1-modified poly(ethylenimine). J. Gene Med. 2007, 9 (8), 691−702. (17) Barry, M. A.; Dower, W. J.; Johnston, S. A. Toward cell-targeting gene therapy vectors: selection of cell-binding peptides from random peptide-presenting phage libraries. Nat. Med. 1996, 2 (3), 299−305. (18) Kwon, E. J.; Bergen, J. M.; Park, I. K.; Pun, S. H. Peptidemodified vectors for nucleic acid delivery to neurons. J. Controlled Release 2008, 132 (3), 230−5. (19) McGuire, M. J.; Samli, K. N.; Johnston, S. A.; Brown, K. C. In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo. J. Mol. Biol. 2004, 342 (1), 171−82. (20) Ko, Y. T.; Hartner, W. C.; Kale, A.; Torchilin, V. P. Gene delivery into ischemic myocardium by double-targeted lipoplexes with anti-myosin antibody and TAT peptide. Gene Ther. 2008, 16 (1), 52. (21) Torchilin, V. P.; Levchenko, T. S.; Rammohan, R.; Volodina, N.; Papahadjopoulos-Sternberg, B.; D'Souza, G. G. Cell transfection in vitro and in vivo with nontoxic TAT peptide-liposome-DNA complexes. Proc. Natl. Acad. Sci. U.S.A. 2003, 100 (4), 1972−7. (22) Tunnemann, G.; Karczewski, P.; Haase, H.; Cardoso, M. C.; Morano, I. Modulation of muscle contraction by a cell-permeable peptide. J. Mol. Med. (Berlin) 2007, 85 (12), 1405−12. (23) Wei, B.; Wei, Y.; Zhang, K.; Wang, J.; Xu, R.; Zhan, S.; Lin, G.; Wang, W.; Liu, M.; Wang, L.; Zhang, R.; Li, J. Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell-penetrating peptide. Biomed. Pharmacother. 2009, 63 (4), 313−8. (24) Endoh, T.; Ohtsuki, T. Cellular siRNA delivery using cellpenetrating peptides modified for endosomal escape. Adv. Drug Delivery Rev. 2009, 61 (9), 704−9. (25) Lundberg, P.; Langel, U. A brief introduction to cell-penetrating peptides. J. Mol. Recognit. 2003, 16 (5), 227−33. (26) Nam, H. Y.; McGinn, A.; Kim, P. H.; Kim, S. W.; Bull, D. A. Primary cardiomyocyte-targeted bioreducible polymer for efficient gene delivery to the myocardium. Biomaterials 2010, 31 (31), 8081−7. (27) Nam, H. Y.; Kim, J.; Kim, S.; Yockman, J. W.; Kim, S. W.; Bull, D. A. Cell penetrating peptide conjugated bioreducible polymer for siRNA delivery. Biomaterials 2011, 32 (22), 5213−22. (28) Jeong, J. H.; Kim, S. W.; Park, T. G. Molecular design of functional polymers for gene therapy. Prog. Polym. Sci. 2007, 32 (11), 1239−74. (29) Gary, D. J.; Puri, N.; Won, Y. Y. Polymer-based siRNA delivery: perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery. J. Controlled Release 2007, 121 (1− 2), 64−73. (30) Bolcato-Bellemin, A. L.; Bonnet, M. E.; Creusat, G.; Erbacher, P.; Behr, J. P. Sticky overhangs enhance siRNA-mediated gene silencing. Proc. Natl. Acad. Sci. U.S.A. 2007, 104 (41), 16050−5. (31) Mok, H.; Lee, S. H.; Park, J. W.; Park, T. G. Multimeric small interfering ribonucleic acid for highly efficient sequence-specific gene silencing. Nat. Mater. 2010, 9 (3), 272−8. (32) Takemoto, H.; Ishii, A.; Miyata, K.; Nakanishi, M.; Oba, M.; Ishii, T.; Yamasaki, Y.; Nishiyama, N.; Kataoka, K. Polyion complex stability and gene silencing efficiency with a siRNA-grafted polymer delivery system. Biomaterials 2010, 31 (31), 8097−105. (33) Lee, S. Y.; Huh, M. S.; Lee, S.; Lee, S. J.; Chung, H.; Park, J. H.; Oh, Y. K.; Choi, K.; Kim, K.; Kwon, I. C. Stability and cellular uptake of polymerized siRNA (poly-siRNA)/polyethylenimine (PEI) complexes for efficient gene silencing. J. Controlled Release 2010, 141 (3), 339−46. (34) Kang, H. C.; Bae, Y. H. Co-delivery of small interfering RNA and plasmid DNA using a polymeric vector incorporating endosomolytic oligomeric sulfonamide. Biomaterials 2011, 32 (21), 4914− 24. (35) Kang, H. C.; Kang, H. J.; Bae, Y. H. A reducible polycationic gene vector derived from thiolated low molecular weight branched polyethyleneimine linked by 2-iminothiolane. Biomaterials 2011, 32 (4), 1193−203.

useful therapeutic tools for cardiomyocyte specific gene therapy. Cell-targeting peptide and/or cell-penetrating peptide conjugation of siRNA may be a potentially important method for cell-specific gene therapy in gene-mediated disease states.

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AUTHOR INFORMATION

Corresponding Author

*University of Utah, School of Medicine, Division of Cardiothoracic Surgery, Salt Lake City, UT, 84132. E-mail: [email protected]. Phone: 801−581−5311. Fax: 801− 585−3936. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by NIH grants HL HL071541 (D.A.B.) and 065477 (S.W.K.).

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