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Enhancement of Star Vector-Based Gene Delivery to Endothelial Cells by Addition of RGD-Peptide Ayaka Ishikawa,† Yue-Min Zhou,† Nobuaki Kambe,‡ and Yasuhide Nakayama*,† Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, Osaka, Japan and Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Osaka Japan. Received October 17, 2007; Revised Manuscript Received December 1, 2007
This study aimed to investigate the feasibility of using a cationic nonviral gene carrier in endothelial cells for enhancing gene expression by the addition of an integrin-binding RGD peptide. A 4-branched cationic polymer of poly(N,N-dimethylaminopropylacrylamide) (star vector), developed as a gene carrier, could complex with the luciferase-encoding plasmid DNA under a charge ratio of 5 (vector/pDNA) to form polymer/DNA complexes (polyplexes). The addition of the RGD-containing peptide (GRGDNP) to the polyplex solution led to a decrease in the ζ-potential from ca. +30 to +20 mV along with the reduction in the particle size from ca. 300 to 200 nm. Additionally, a marked inhibition of polyplex aggregation was observed, indicating the coating of the polyplex surface with RGD peptides. A transfection study on endothelial cells showed that the luciferase activity increased with the amount of RGD peptides added to the polyplexes and exhibited minimal cellular cytotoxicity. The transfection activity further increased when cyclic RGD peptides (RGDFV) were used; the activity with RGD peptide addition was approximately 8-fold compared to that without RGD peptide addition. Gene delivery to endothelial cells was significantly enhanced by only the addition of RGD peptides to star vector-based polyplexes.
INTRODUCTION Cationic polymers such as poly(ethylenimine) (PEI) (1–4) and poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm) (5–8) can generate nanoparticles by the formation of polyion complexes, i.e., “polyplexes” with DNA. These polymers are highly expected to be among the major carriers in nonviral gene delivery systems due to the many advantages they offer over viral systems (9–13). These advantages include blocking of specific immune responses, no restrictions in DNA size, and ease of large-scale production. We attempted to investigate the usefulness of star-shaped cationic polymers as a base chemical structure for a novel high-performance gene carrier (5–8). These polymers, known as star vectors, were prepared by initiator-transfer agent-terminator (iniferter)-based living radical polymerization (14, 15) of DMAPAAm, using the respective multidithiocarbamate-derivatized benzenes (multifunctional iniferters) (5, 7). The gene expression efficiency was enhanced with the increase in the number of branching in COS-1 cells. Recently, it was shown that grafting of each cationic branch in the star vector with nonionic block chain resulted in a considerable improvement in the gene transfection efficiency (7). On the other hand, it was shown that introducing a ligand to the star vector was effective in delivering the target to specific cells. For example, for targeting resting macrophages, the star vector was incorporated with C45D18 peptide, which is an oligopeptide with an active site derived from Vpr of human immunodeficiency virus type-1 (6). The vitronectin receptor RVβ3 is highly upregulated in endothelial cells of tumors, while it is minimally expressed in resting or normal * To whom correspondence should be addressed: Department of Bioengineering, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishiro-dai, Suita, Osaka 565-8565, Japan. Telephone: (+81) 6-6833-5012 (ex. 2624). Fax: (+81) 6-6872-8090. E-mail:
[email protected]. † National Cardiovascular Center Research Institute. ‡ Osaka University.
blood vessels (16). The tripeptide sequence, arginine (Arg)-glycine (Gly)-aspartic acid (Asp) (RGD) found in the active site of vitronectin binds to RVβ3 and almost half of the 22 known integrins (17). RGD peptides are exploited for cell entry by pathogenic microorganisms such as the foot-and-mouth disease virus (18). A great advantage of integrin targeting is that internalization occurs by a “zippering” mechanism (19) that allows the uptake of relatively large structures such as bacteria. Therefore, several cationic polymer vectors combined with RGD peptides were developed for tumortargeted gene delivery (20–23). For example, RGD-containing peptides were coupled with PEI with or without a PEG spacer. The RGD-modified PEI showed a significant increase in transfection efficiency as compared to only PEI in endothelial cells. Almost all RGD-mediated gene delivery systems have been described with the chemical derivatization of RGD peptides to vector compounds. In this paper, we report the influence of the addition of RGD to star vector-based polyplexes on the physicochemical and biological properties of the polyplexes that comprised plasmid DNA and 4-branched cationic star vector with a molecular weight of ca. 18 000. As a model, an RGD-containing peptide GRGDNP and cyclic RGD peptides were used. We strongly believed that if the coating of the polyplex surface with the RGD peptides occurs only by the addition of these peptides, then the gene transfection efficiency to endothelial cells would be enhanced, similar to that in the case of chemical derivatization of the RGD peptides.
MATERIALS AND METHODS Materials. RGD-containing peptide (GRGDNP) was purchased from Wako Pure Chemical Industry (Osaka, Japan) and cyclic RGD peptide (RGDFV) were from Peptide Institute, Inc. (Osaka, Japan). Synthesis of Cationic 4-Branched Polymer. The cationic 4-branched polymer, 1,2,4,5-tetrakis(N,N-diethyldithiocarbamyl(poly(N-[3-(dimethylamino)propyl]acrylamide)benzene was synthesized by iniferter-based living radical photopolymerization from 1,2,4,5-tetrakis(N,N-diethyldithiocarbamylmethyl)benzene
10.1021/bc700385r CCC: $40.75 2008 American Chemical Society Published on Web 01/19/2008
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Figure 1. Synthesis of PDMAPAAm 4-branched polymer (star vector).
as a multifunctional iniferter with DMAPAAm as cationic monomer, according to the method previously reported by us (5, 7). Brifely, to 6 mL of a benzene solution of 1,2,4,5tetrakis(N,N-diethyldithiocarbamylmethyl)benzene (72 mg, 0.1 mmol) DMAPAAm (72 mg, 0.1 mmol) was added; this was diluted to a final volume of 20 mL with methanol and placed into a 50-mL quartz crystal tube. After bubbling with dry N2 gas for 5 min to exclude air, the solution was irradiated for 90 min with a 200-W high-pressure mercury lamp (SPOT CURE; USHIO, Tokyo, Japan) under an N2 atmosphere at room temperature. The light intensity was set to 1 mW/cm2 at a wavelength of 250 nm (UVR-1; TOPCON, Tokyo, Japan). The reaction mixture was concentrated and adjusted to an appropriate concentration in order to facilitate purification by precipitation in a large volume of ether. Reprecipitation was carried out in a methanol-ether system. The final precipitate was dried under vacuum to yield a PDMAPAAm 4-branched polymer (conversion, 13%). The molecular weight (Mn) as determined by GPC analysis was 18 000 g · mol-1. 1H NMR: δ 1.7–1.5 (br, 3H, -CH2-CH- and -CH2-CH2-CH2-), 2.0–1.8 (br, 1H, -CH-CO), 2.2–2.1 (br, 6H, -N-CH3), 2.4–2.2 (br, 2H, -CH2-N(CH3)2), 3.2–3.0 (br, 2H, -NH-CH2-), 7.8–7.4 (br, 1H, -NH). Preparation of Polyplexes. The cationic 4-branched polymer was dissolved in a saline solution. Aliquots of these solutions (60 µL) were added to the plasmid DNA (pGL3-control) dissolved in 90 µL of Tris-HCl buffer (pH 7.4) to obtain polymer/DNA ratios of 5 or 10, which correspond to cation/ anion (C/A) ratios. The solutions (total volume, 150 µL; plasmid concentration, 20 mg/mL) were mixed using a pipet. After 10 min of complex formation, 25 µL of the complex solutions were added into each well of 24-well plates for transfection (the amount of DNA added to a well, 0.5 µg/well). Biophysical Characterization of Polyplexes. The ζ-potentials and mean diameters of the polyplexes in a saline solution having the same concentration that was used for transfection at a C/A ratio of 5 were determined by employing DLS on an ELS-8000 system (Otuska Co., Osaka, Japan) equipped with a 10-mW He-Ne laser. In Vitro Transfection and Cell Viability Assays. COS-1 cells (approximately 3 × 104 cells per well) were seeded prior to treatment in 24-well plates and grown for 24 h in DMEM (Gibco, Invitrogen Corp., Carlsbad, CA) containing 10% fetal calf serum (Hyclone Laboratories Inc., Logan, UT), penicillin (200 units/mL; ICN Biomedicals Inc., Aurora, OH), and streptomycin (200 mg/mL; ICN) in an atmosphere of 5% CO2 at 37 °C. Transfections were performed with 0.5 µg of the DNA in 24-well plates in 0.2 mL of OPTI-MEM I (Gibco). After 3 h of incubation, the cells were washed once with PBS(-) and cultured in 1 mL of DMEM containing 10% fetal calf serum for an additional 48 h. The medium was removed and the cells were washed twice with PBS(-). The cells were lysed with 0.2 mL of cell lysis buffer (Promega, Madison, WI) and mixed by vortexing. The lysate was centrifuged at 15 000 rpm for 1 min at 4 °C and 5 µL of the supernatant was analyzed for
luciferase activity using a Luminous CT-9000D (Dia-Iatron, Tokyo, Japan) luminometer. The relative light unit/s (RLU) were converted into the amount of luciferase (pg) using a luciferase standard curve, which was obtained by diluting recombinant luciferase (Promega) in lysis buffer. The protein concentrations of cell lysates were measured by performing a Bio-Rad protein assay (BIO-RAD, Hercules, CA) using bovine serum albumin as a standard. The expressed luciferase represented the amount (mole quantity), which is standardized for the total protein content of the cell lysate. The data are presented as means ( SD (n ) 5). Cytotoxicity was assessed by performing a cell viability assay using the WST-8 method (Dojindo, Kumamoto, Japan). COS-1 cells were seeded 24 h prior to treatment in 96-well plates at 5000 cells per well. Cells were treated under the same conditions used for luciferase assay with 6.2 µL of the transfection mixture including 0.124 µg of pDNA added to each well. They were treated under appropriate conditions for 3 h, washed once with PBS, and cultured in 50 µL of DMEM (Gibco) containing 10% fetal calf serum for an additional 24 h. To each well, 10 µL of WST-8 reagent (5 mmol/l) was added. After 2 h of incubation at 37 °C, absorbance at 450 nm was read in a BIO-RAD microplate reader (model 680). The data are presented as means ( SD (n ) 5).
RESULTS AND DISCUSSION Interaction of Polyplex and RGD Peptide. The cationic 4-branched polymer with a molecular weight of ca. 18 000 was prepared by iniferter-based living radical polymerization (5, 7). The polymer had an extremely low polydispersity of less than 1.7. When the saline solution of cationic 4-branched polymer (7.9 µg/60 µL) was mixed with a tris-HCl buffer of luciferaseencoding pDNA (3 µg/90 µL) under a charge ratio of 5 (polymer/DNA), polyplexes in the form of approximately 300nm nanoparticles were formed by electrostatic affinity (Figures 1, 2); this observation was similar to that of our previous study (5, 7). First, we evaluated the interaction between the polyplexes and GRGDNP by measuring the change in the particle size. When the solution of the polyplexes, which were formed by mixing the polymer and the pDNA, was allowed to stand at 37 °C, the particle size significantly increased with time due to the aggregation of the polyplexes (Figure 2). The nanosized particles became microsized ones within 10 min. On the other hand, when the saline solution of RGD peptide was added to the polyplex solution immediately after preparation, the size of polyplexes reduced to approximately 200 nm. When the obtained polyplex solution was allowed to stand at 37 °C, the aggregation of the polyplexes was prevented depending on the amount of RDG peptide added. This indicated the interaction between the RGD peptide and the polyplexes. The saturation of the prevention effect was observed at approximately 30 µg. To examine the surface electric property of the polyplexes, their ζ-potential in the presence or absence of RGD peptide
560 Bioconjugate Chem., Vol. 19, No. 2, 2008
Figure 2. Incubation time-dependent cumulant diameter changes in aqueous solutions of polyplexes prepared by mixing DNA (pGL3control, 3 µg) and PDMAPAAm 4-branched polymer (star vector, 7.9 µg) under a charge ratio of 5 at pH 7.4 in the presence of RGD peptides, whose amounts ranged from 0 to 47.7 µg.
Figure 3. Transfection efficiency to endothelial cells of the polyplexes prepared by mixing DNA (pGL3-control, 3 µg) and PDMAPAAm 4-branched polymer (star vector, 7.9 µg) under a charge ratio of 5 in the presence of GRGDNP peptides (b, c, d) or cyclic RGD peptide (a) under charge ratio of 5 (vector/pDNA) in a, c, and d, or 10 in b. The RGD peptide was added before contact of the polyplexes with the endothelial cells (c).
was measured using DLS. In the RGD peptide-free polyplexes, the ζ-potential was approximately +30 mV, whereas it was decreased to approximately +20 mV after the addition of the RGD peptide (47.4 µg). The reduced surface potential might be due to the coating of the polyplex surface with the peptide. Although the driving force for the binding between the polyplexes with a positive charge and the RGD peptide with a lower electrical potential is still unknown, it was estimated that the surface coating occurred due to a physical interaction such as adsorption of the RGD peptide on the polyplexes without any chemical binding. These particle size and ζ-potential data lead to the conclusion that the aggregation of the polyplexes was prevented by the coating of the polyplexes with the RGD peptide in order to reduce the positive charge of the surface and to use almost all of the 30 µg of the RGD peptide added. The amount was about 4 times of that of cationic polymer used to form the polyplexes completely. In Vitro Transfection Efficiency. The gene transfection efficiency of the polyplexes coated or not coated with the RGD peptide was tested on model cell line of mice vascular endothelial cells (ECs). The result is summarized in Figure 3. The cells transfected with the luciferase-encoding plasmid (pGL3) in the absence of the RGD peptide exhibited a low
Ishikawa et al.
luciferase expression, whereas those transfected with polyplexes + RGD peptide exhibited a higher luciferase expression and this expression increased with the amount of RGD peptide added (curve c in Figure 3). Upon an increase in C/A ratio from 5 to 10 further increase in the luciferase expression was observed (curve b in Figure 3). The highest luciferase expression occurred when approximately 50 µg RGD peptide was added. However, further addition of the RGD peptide led to a decrease in the luciferase expression. The enhancement of luciferase expression by the addition of the RGD peptide was considered to be due to 2 major reasons—the stabilization of the polyplex particles and the RGDmediated internalization of the polyplexes into the cells. The DNA expression observed using polyplexes occurred via the following 5 sequential steps: (1) attachment of the polyplexes onto the cell surface, (2) internalization of the polyplexes into the cell, (3) endosomal escape of polyplexes, (4) DNA release from polyplexes, and (5) internalization of the DNA into the nucleus. Almost all of the cationic polymers, including PEI, can conjugate with DNA to form nanosized polyplexes by an electrostatic affinity immediately after mixing. However, the polyplexes gradually aggregate with the increase in incubation time to grow from nanosized to macrosized particles by a similar electrostatic affinity, as demonstrated in Figure 2. In general, it was reported that the transfection efficiency was low in large particles because such particles could not easily internalize into the cells, which is the above-mentioned second step. In this study, indeed, polyplex particles became stable by the addition of the RGD peptide and synchronously, the transfection efficiency increased. However, the enhancement of gene transfection by the stabilization effect of the polyplex particles was eliminated by the following experiment. When approximately 50 µg of RGD peptide was added to the cultured cells before the addition of the solution containing the RGD peptide-coated polyplexes, little change in the luciferase expression was observed (curve d in Figure 3). This indicated that little internalization of the polyplexes into the cells occurred even by polyplex stabilization. The reason for such an observation was considered to be that integrin receptors on the cellular membrane were capped and as a result little polyplex was recognized by the cells. Therefore, we believed that the enhancement of the gene transfection efficiency was due to the RGD-mediated internalization. The changes in cellular morphology and motility were not noticed even after RGD capping of integrin receptors within our observation period including medium change after 3 h of incubation with the polyplexes, although it was reported that integrins regulate several cellular functions including cell migration, growth, and differentiation (25, 26). On the other hand, when cyclic RGD (cRGD) peptide was added to the polyplexes, a further increase in gene transfection efficiency was observed (curve a in Figure 3). Cyclization of the RGD peptides is known to increase receptor affinity and selectivity by providing a conformational restraint (17). In addition, we observed that compared to the noncyclic RGD peptide, the use of the cRGD peptide increased the gene transfection efficiency by approximately 3 times. For both RGD and cRGD peptides, excessive addition of the peptides led to a decrease in the transfection efficiency. From the result presented in Figure 2, the amount of RGD peptide required for completely coating the polyplexes was approximately 30–50 µg. The surplus amount of RGD peptide might bind to the integrin receptor and prevent binding with the RGD peptide coating the polyplexes. Cytotoxicity of Polyplexes with RGD Peptide. By using the WST-8 assay, the in Vitro cytotoxicity of the RGD-added polyplexes for the EC cells was studied as a function of the amount of the RGD peptide added. The polyplexes with a charge
Gene Delivery to Endothelial Cells
Figure 4. Cytotoxicity of the polyplexes prepared by mixing DNA (pGL3-control, 3 µg) and PDMAPAAm 4-branched polymer (star vector, 7.9 µg) when different amount of linear RGD or cyclic RGD peptides are added. For comparison, the cytotoxicity data of polyplexes of linear PEI (ExGen 500) at the charge ratio of 5 are also provided. Cell viability was determined by the WST assay.
ratio of 5 exhibited little toxicity (Figure 4). The addition of up to approximately 40 µg of RGD peptide also demonstrated little toxicity. At higher amounts of RGD peptide (72 µg), permissible viability, which was higher than that with PEI at a charge ratio of 6, was observed. Very low cytotoxicity was also demonstrated in cRGD peptide (Figure 4). There was little difference in cytotoxicity between RGD and cRGD peptides.
CONCLUSIONS Our study is the first to report that cationic polymer-based gene transfer to EC cells is markedly enhanced in the presence of the RGD peptide. This peptide was only added to the solution of the polyplexes, which was prepared by mixing the cationic polymer and pDNA, without derivatization of the peptide to the polymer by chemical binding, as demonstrated in almost all other reports. The enhancement of gene transfer by the addition of the RGD peptide may be applied even for other cationic polymer vectors because the RGD peptide could coat the cationic surface of the polyplexes. The surface coating ability may be enhanced by the introduction of anionic amino acids such as aspartic acid or glutamic acid to the RGD peptide.
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