Self-Assembled Terplexes for Targeted Gene Delivery with Improved

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Self-Assembled Terplexes for Targeted Gene Delivery with Improved Transfection Qiao Zhang, Si Chen, Ren-Xi Zhuo, Xian-Zheng Zhang, and Si-Xue Cheng* Key Laboratory of Biomedical Polymers of Ministry of Education, Department of Chemistry, Wuhan University, Wuhan 430072, People’s Republic of China. Received July 7, 2010; Revised Manuscript Received September 20, 2010

To improve transfection efficiency and to incorporate target ligands to the gene delivery systems, heparin and heparin-biotin were introduced to complexes of polyamidoamine dendrimer and DNA (PAMAM/DNA) via electrostatic interactions to form self-assembled PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes, respectively. The self-assembled terplexes were characterized by agarose gel electrophoresis and particle size analysis. The MTT assay indicated that, after incorporation of heparin and heparin-biotin, the terplexes exhibited decreased cytotoxicity. In addition, as compared with PAMAM/DNA and PAMAM/DNA/heparin complexes, the PAMAM/DNA/heparin-biotin complexes exhibited much higher cellular uptake into HeLa cells due to the specific interactions between biotin and biotin receptors on HeLa cells, which led to the enhanced transfection activity. The PAMAM/DNA/heparin-biotin complexes would be a promising targeting gene delivery system.

INTRODUCTION Gene therapy is a promising approach for the treatment of incurable diseases and genetic disorders such as cancers (1, 2). The efficient and safe gene transfer vectors are of critical importance for the applications of gene therapy. Nonviral vectors such as cationic liposomes and polycations have attracted much attention because of their low immune response and safety. However, the low transfection efficiency greatly limits their clinical applications (3). To improve the performance of nonviral vectors and to overcome the barriers in gene delivery, many modification strategies have been developed. Among them, incorporation of various ligands to the polycation vectors could effectively enhance the cellular uptake and achieve improved transfection efficiency for the targeted cells (4). The frequently reported targeting ligands include monoclonal antibodies, peptides (5), carbohydrates such as galactose for hepatocyte targeting (6), and vitamins such as folate (7). Most commonly, the targeting ligands are incorporated to the polymer vectors through covalent bonds. Molecular self-assembly is a rapid and powerful method for fabricating nanosized materials with various supramolecular architectures. In comparison with the chemical reactions, the self-assembly is more convenient, flexible, and favorable for keeping the bioactivity of the ligands. As far as we know, the studies on incorporation of target ligands to gene delivery systems through self-assembly are very limited. In this study, we developed a new strategy to introduce ligands to gene delivery systems through electrostatic interactions; i.e., heparinbiotin was introduced to the complexes of PAMAM/DNA via electrostatic interactions to from PAMAM/DNA/heparin-biotin terplexes. For comparison, PAMAM/DNA/heparin terplexes without the targeting ligand were also prepared through selfassembly. Biotin (vitamin H) is an essential vitamin for the division of all cells, especially for the rapid proliferations of tumor cells. The cancer cells often overexpress biotin receptors on the cell surface. There are many reports on the incorporation * Corresponding author. Tel./fax: +8627 6875 4509. E-mail address: [email protected], [email protected] (S.X. Cheng).

of biotin as cell targeting ligands for targeted drug delivery (8–11). The reported biotin targeted tumor cell lines include L1210FR, Ov2008, M109, 4T1 (8), HeLa (9), JC, MCF-7 (10), and HepG2 (11). Heparin as one of the most potent anticoagulants has been widely used to enhance the blood compatibility of biomaterials (12). The conjugation of heparin to polyethylenimine (PEI) can inhibit the adsorption of serum proteins to the PEI surface and improve the in vivo gene transfection efficiency (13). In addition, the negatively charged heparin can form complexes with polycations such as PEI and PAMAM (14–16). In the current study, heparin was used to improve the biocompatibility and partially shield the positive charge of PMAMA/DNA complexes to reduce the cytotoxicity. Biotin was used as a cell targeting ligand to enhance the cellular uptake for particular cancer cells. The existence of heparin or heparin-biotin lowered the cytotoxicity and enhanced the transfection efficiencies of the terplexes. In addition, the transfection activity of PAMAM/ DNA/heparin-biotin terplexes in particular tumor cells could be further improved due to the increased biotin receptor mediated cell uptake. The terplexes have promising applications in targeting gene delivery to cancer cells.

EXPERIMENTAL PROCEDURES Materials. Poly(amidoamine) (PAMAM) dendrimer (generation 5 with 128 surface amino groups) was purchased from Aldrich. Heparin sodium and heparin-biotin sodium (molecular weight 15 000 Da) were obtained from Sigma. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was from Amresco. Dimethylsulfoxide (DMSO) was from Sigma. The reporter plasmids, pGL3-Luc and pEGFP-C1, were purchased from Promega and Invitrogen, respectively. Plasmids were amplified in Escherichia coli and extracted and purified by E.Z.N.A. Fastfilter Endo-free Plasmid Maxi Kit (Omega). Plasmids were suspended in deionized water and stored at -20 °C. HeLa, HEK293T, and COS-7 cells were obtained from China Center for Typical Culture Collection (Wuhan, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco), supplemented with 10% fetal bovine serum (FBS), 2

10.1021/bc100309e  2010 American Chemical Society Published on Web 10/08/2010

Self-Assembled Terplexes for Gene Delivery

mg/mL NaHCO3, and 100 unit/mL penicillin/streptomycin. Cells were incubated at 37 °C in humidified air/5% CO2. Formation of PAMAM/DNA/Heparin and PAMAM/DNA/ Heparin-Biotin Terplexes. PAMAM was diluted to an appropriate concentration in deionized water and stored at 4 °C until use. PAMAM/DNA complexes were made up by adding the aqueous solution of PAMAM to an equal volume of plasmid DNA in deionized water at a particular N/P ratio, followed by incubation at 37 °C for 30 min. The N/P ratio was based on the calculation of the number of terminal -NH2 groups on the dendrimer versus the number of phosphate groups of the DNA. PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes were prepared by mixing the PAMAM/DNA complexes containing aqueous solution with an aqueous solution of heparin or heparin-biotin of equal volume and then incubating at 37 °C for 30 min. Agarose Gel Electrophoresis. Terplexes with different compositions were evaluated by agarose gel retardation assay. Five microliters of complexes containing solution with 0.1 µg DNA was electrophoresed on the 0.7% (w/v) agarose gel containing GelRed with Tris-acetate-EDTA (TAE) running buffer at 80 V for 80 min. DNA was visualized on a Vilber Lourmat UVtransilluminator. Size and Zeta Potential Measurements. The size and zeta potential of complexes were measured using a Nano-ZS ZEN3600 (Malvern Instruments). The complexes contained solution containing 4 µg pGL3-Luc were diluted by deionized water to 0.8 mL for the size and zeta potential measurements. The data are given as mean ( standard deviation (SD) based on three independent measurements. Cytotoxicity. The cytotoxicities of PAMAM/DNA complexes, PAMAM/DNA/heparin, and PAMAM/DNA/heparinbiotin complexes with the heparin/DNA or heparin-biotin/DNA weight ratio of 2 at various N/P ratios were examined by MTT assay. The HeLa cells were seeded in the 96-well plates at a density of 6 × 103 cells/well in 200 µL of DMEM containing 10% FBS. After incubation for 24 h, the medium was replaced by 200 µL of serum-free DMEM. Then, the complexes containing solution (20 µL containing 0.2 µg DNA) was added into the well. After coincubation at 37 °C for 48 h, the medium was removed and 20 µL of MTT (5 mg/mL) solution was added to each well and further incubated for 4 h. Thereafter, the medium was removed carefully, and 150 µL DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm by a microplate reader (Biorad 550). The cells without coincubation with the complexes were used as the control. The data are given as mean ( standard deviation (SD) based on five measurements. In Vitro Transfections. The transfection activities of various complexes in HeLa, COS-7, and 293T cells were examined. Unless otherwise mentioned, the transfections were carried out in serum-free media. The detailed transfection procedure is as follows. The cells were seeded at a density 5 × 104 cells per well in a 24-well plate with 1 mL of DMEM containing 10% FBS and incubated at 37 °C. After incubation for 24 h, the medium was replaced by 1 mL of serum-free DMEM. Then, the complexes containing solution (100 µL containing 1 µg DNA) was added into the well and incubated at 37 °C for 4 h. After that, the serum-free DMEM was replaced with fresh DMEM containing 10% FBS, and the cells were further incubated for 44 h. (For the transfections carried out in serum containing media, the cells and the complexes were coincubated in DMEM containing 10% FBS for 4 h. Other conditions were the same as that for the transfections carried out in serum-free media.) To assay the expression of pGL3-Luc, the medium was removed and the cells were rinsed gently by phosphate buffered

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saline (PBS, 0.1 M, pH 7.4). After thorough lysis of the cells with reporter lysis buffer (Promega) (200 µL/well), the luciferase activity was determined by detecting the light emission from an aliquot of cell lysate incubated with 100 µL of luciferin substrate (Promega) in a luminometer (Lumat LB9507, Berthold). The protein content of the cell lysate was determined by BCA protein assay kit (Pierce). The optical density (OD) value was determined at 570 nm using a microplate reader (Biorad 550). The data are given as mean ( standard deviation (SD) based on three independent measurements. To assay the expression of pEGFP-C1, the cells expressing green fluorescent proteins were directly observed by an inverted microscope (IX 70, Olympus) with a digital color camera (Roper CoolSnap Color). The micrographs were obtained at the magnification of 100× and recorded using Image-Pro Plus software. Cellular Uptake Study. The uptake of complexes into HeLa cells was studied by confocal microscopy. YOYO-1 was used as molecular probe to label pGL3-Luc. One microgram of pGL3Luc plasmid was mixed with 2.5 µL YOYO-1 solution (10 µM) and incubated for 15 min at 37 °C. The cells were seeded at a density of 5 × 104 cells per well in a 24-well plate with 1 mL of complete growth medium containing 10% FBS and incubated at 37 °C. After incubation for 24 h, the medium was replaced by 1 mL serum-free DMEM. Then, the complexes (100 µL containing 1 µg DNA) were added into the well and incubated at 37 °C for 4 h. After that, the medium was removed, the cells were washed twice with PBS, and then 200 µL DMEM containing 10% FBS and 20 µL Hoechst 33258 solution (10 mg/mL) were added and incubated at 37 °C for 20 min to stain nuclei of the cells. Finally, the solution was removed, cells were washed with PBS twice, and 1 mL of fresh DMEM containing 10% FBS was added. The cells were observed under excitation at 488 nm using a confocal laser scanning microscope (Nikon C1-si TE2000). The micrographs were obtained at the magnification of 800× and recorded using Nikon EZ-C1 FreeViewer software.

RESULTS AND DISCUSSION Formation and Characterization of PAMAM/DNA/Heparin and PAMAM/DNA/Heparin-Biotin Terplexes. PAMAM dendrimers are a class of spherical, nanoscopic, highly ordered polymers with primary amino groups on the surface, which have lower cytotoxicity compared with PEI and have been extensively investigated as gene vectors (17). Heparin is a highly sulfated anionic polysaccharide. It was reported that PAMAM and heparin can form complexes through electrostatic interactions (15, 16). As we know, in order to condense DNA effectively, the complexes of polycation/DNA for gene delivery usually have a positive charge on the surface. In the current study, after mixing the positively charged PAMAM/DNA complexes with the negatively charged heparin and heparin-biotin, PAMAM/ DNA/heparin and PAMAM/DNA/heparin-biotin terplexes were formed, respectively. It was reported that heparin could replace DNA in the polycation/DNA complexes by vigorously mixing. In this study, the stability of different complexes was assessed by the agarose gel retardation assay and the results are shown in Figure 1. For PAMAM/DNA/heparin terplexes with a heparin/DNA weight ratio of 2 (Figure 1a), when the PAMAM/DNA N/P ratios were in the range 5-60, the complexes were able to completely bind DNA. Similarly, for PAMAM/DNA/heparin-biotin terplexes with a heparin-biotin/DNA weight ratio of 2 (Figure 1b), they could efficiently bind DNA at the PAMAM/DNA N/P ratio from 5 to 60. However, for PAMAM/DNA/heparin-biotin terplexes with a PAMAM/DNA N/P ratio of 20 (Figure 1c), PAMAM/ DNA/heparin-biotin could not completely prevent DNA from

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Figure 2. Particle sizes of different complexes with different PAMAM/ DNA N/P ratios. The weight ratio of heparin/DNA and heparin-biotin/ DNA is 2.

Figure 3. Zeta potentials of different complexes with different PAMAM/DNA N/P ratios. The weight ratio of heparin/DNA and heparin-biotin/DNA is 2.

Figure 1. Agarose gel retardation assay of different complexes: (a) PAMAM/DNA/heparin complexes with a heparin/DNA weight ratio of 2 and different PAMAM/DNA N/P ratios, (b) PAMAM/DNA/ heparin-biotin complexes with a heparin-biotin/DNA weight ratio of 2 and different PAMAM/DNA N/P ratios, and (c) PAMAM/DNA/ heparin-biotin complexes with a PAMAM/DNA N/P ratio of 20 and different heparin-biotin/DNA weight ratios.

migrating into the gel when the weight ratio of heparin-biotin/ DNA was 5, indicating that, in order to bind DNA completely, the weight ratio of heparin-biotin/DNA should be less than 5. The particle sizes of different complexes were measured as shown in Figure 2. All sizes of PAMAM/DNA complexes at different N/P ratios were less than 200 nm, which indicated DNA could be effectively condensed by PAMAM at the N/P ratio higher than 5. As compared with the sizes of PAMAM/ DNA complexes, the sizes of PAMAM/DNA/heparin slightly increased. This is due to the fact that the negatively charged heparin was attached to the surface of PAMAM/DNA complexes, leading to the increased sizes. Compared with PAMAM/ DNA complexes, the size of PAMAM/DNA/heparin-biotin also increased. Compared with PAMAM/DNA/heparin terplexes, most commonly the sizes of PAMAM/DNA/heparin-biotin terplexes were smaller. The possible reason for this phenomenon is the hydrophobic nature of biotin moiety, which resulted in a

less extended configuration of heparin-biotin chains on the surface of the terplexes. Nevertheless, the size differences between different complexes were not large, and all complexes had the mean sizes less than 200 nm. The zeta potentials of different complexes are shown in Figure 3. The zeta potentials of all complexes were positive. Because of the positive surface charge caused by the excess PAMAM and the strong electrostatic interactions between PAMAM/DNA complexes and heparin or heparin-biotin, the heparin and heparin-biotin in the systems could form terplexes and the free heparin and heparin-biotin could be ignored. The zeta potentials of the complexes increased with increasing PAMAM/DNA N/P ratio when the N/P ratio was less than 20. Compared with PAMAM/DNA, the zeta potential of PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin decreased significantly, especially at the relatively low N/P ratios, because the negatively charged heparin or heparin-biotin was attached to the surface of PAMAM/DNA complexes. As a result, the positive surface charges of the PAMAM/DNA complexes were partially neutralized. The positive zeta potential values of all complexes were in good agreement with the result of agarose gel retardation assay, which also indicated that PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes with different N/P ratios were stable when the weight ratio of heparin/DNA and heparin-biotin/DNA was 2. In Vitro Cytotoxicity. The cytotoxicity of different complexes in HeLa cells was evaluated by MTT assay. As presented


Figure 4. Cell viability of HeLa cells after coincubation with different complexes with different PAMAM/DNA N/P ratios. The weight ratio of heparin/DNA and heparin-biotin/DNA is 2.

in Figure 4, for PAMAM/DNA complexes, with an increase in N/P ratio, the cell viability decreased. As expected, generally the cytotoxicities of PAMAM/DNA/heparin and PAMAM/ DNA/heparin-biotin terplexes were lower than that of the PAMAM/DNA complexes, especially at the high N/P ratios. As we know, during the gene transfection, the positive charge on the surface of complexes is essential for binding the negatively charged cell membranes and thus being taken up via an endocytic mechanism. However, the higher positive surface charge means higher cytotoxicity of the complexes, and the high cytotoxicity of the polycations limits their clinical applications as gene vectors. One of the strategies to reduce the cytotoxicity is to neutralize or partially shield the positive charge on the polycation/DNA complexes. For example, the PEG chains grafted to polycations could shield the positive surface charge and thus reduce the cytotoxicity while maintaining the efficient transfection ability of the polycations (18–20). In this study, heparin and heparin-biotin were used to partially neutralize the positive charge of PAMAM/DNA complexes. The resultant terplexes showed relatively lower zeta potential as compared with PAMAM/DNA, which resulted in their lowered cytotoxicity. In Vitro Transfection. The luciferase expressions in HeLa cells transfected with different complexes with the heparin/DNA or heparin-biotin/DNA weight ratio of 2 and different PAMAM/ DNA N/P ratios are shown in Figure 5. For all N/P ratios, as compared with PAMAM/DNA complexes, PAMAM/DNA/ heparin terplexes exhibited a much higher gene transfection activity. In addition, the gene expression levels mediated by PAMAM/DNA/heparin-biotin terplexes could obviously be further improved. Both comparisons between the data of PAMAM/DNA and PAMAM/DNA/heparin and the data of PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin showed p < 0.05, indicating that the differences were statistically significant. As we know, heparin is one of the most potent anticoagulants and has been widely used to enhance the blood compatibility of biomaterials. According to previous studies of other researchers, the conjugation of heparin to PEI or physical modification of PEI with heparin via electrostatic interactions canimprovethecytocompatibilityandtransfectionefficiency(13,14). Our results are in accordance with the previous studies. Biotin is an essential vitamin for the division of all cells, especially for the rapid proliferations of tumor cells. The cancer cells often overexpress biotin receptors on the cell surface. The improvement of gene expression mediated by PAMAM/DNA/ heparin-biotin suggested that the presence of biotin moiety


Figure 5. Luciferase expression in HeLa cells transfected with different complexes with different PAMAM/DNA N/P ratios. The weight ratio of heparin/DNA and heparin-biotin/DNA is 2. (* p < 0.05 as compared with the data of PAMAM/DNA complexes at the same N/P ratio, and ** p < 0.05 as compared with the data of PAMAM/DNA/heparin complexes at the same N/P ratio.)

Figure 6. Luciferase expression in HeLa cells transfected with different complexes with different heparin/DNA or heparin-biotin/DNA weight ratios. The PAMAM/DNA N/P ratio is 20. (* p < 0.05 as compared with the data of PAMAM/DNA complexes, ** p < 0.05 as compared with the data of PAMAM/DNA/heparin complexes at the same heparin/ DNA or heparin-biotin/DNA weight ratio.)

facilitated the uptake of terplexes in tumor cells due to the specific interactions between biotin and its receptors on HeLa cells (9) and led to the improved transfection efficiency. Our results are in agreement with the previous literature. As reported, the biotin conjugated PAMAM (G5) exhibited enhanced cellular uptake of HeLa cells, i.e., about 2 times higher than that without incorporation of biotin as determined by the mean fluorescence (9). In our study, the luciferase expressions in HeLa cells transfected with PAMAM/DNA/heparin-biotin were about 2 to 6 times higher than that transfected with PAMAM/DNA/ heparin when the N/P ratios were in the range 5-60 (Figure 5). From Figure 5, the gene expressions increased with increasing N/P ratio at the N/P ratio ranging from 5 to 20. A further increase in N/P ratio did not result in an obvious improvement in the transfection. This is because the complexes with a low N/P ratio could not efficiently mediated gene expressions, while the N/P ratio of more than 20 was too high, which caused the low cell viability after transfection. Thus, we fixed the N/P ratio at 20 for further study. As shown in Figure 6, when fixing the PAMAM/DNA N/P ratio at 20, the gene expressions mediated by PAMAM/DNA/


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Figure 8. Luciferase expression in HeLa cells transfected with (a) PAMAM/DNA, (b) PAMAM/DNA/heparin, and (c) PAMAM/DNA/ heparin-biotin complexes in DMEM containing 10% FBS. The PAMAM/DNA N/P ratio is 20, and the weight ratio of heparin/DNA and heparin-biotin/DNA is 2. (* p < 0.05 as compared with the data of PAMAM/DNA complexes, ** p < 0.05 as compared with the data of PAMAM/DNA/heparin complexes.)

Figure 7. Confocal images of HeLa cells after incubation with (a) PAMAM/DNA, (b) PAMAM/DNA/heparin, and (c) PAMAM/DNA/ heparin-biotin complexes. The PAMAM/DNA N/P ratio is 20, and the weight ratio of heparin/DNA and heparin-biotin/DNA is 2.

heparin and PAMAM/DNA/heparin-biotin terplexes increased with increasing weight ratio of heparin/DNA or heparin-biotin/ DNA, reached maximum values at the heparin/DNA or heparin-biotin/DNA weight ratio of 2, implying that the heparin/DNA or heparin-biotin/DNA weight ratio at 2 was high enough to improve the transfection activity of the terplexes. For the different complexes at each particular heparin/DNA or heparin-biotin/DNA weight ratio, the same trend could be found, i.e., the transfection level increased significantly by binding heparin or heparin-biotin on the surface of PAMAM/ DNA complexes, and the PAMAM/DAN/heparin-biotin complexes exhibited the highest transfection efficiency. To further confirm the mechanism of the improvement of transfection in the presence of biotin moiety, we used confocal microscopy to visualize the cell uptake of different complexes. As shown in Figure 7, after coincubation with PAMAM/DNA complexes for 4 h, only a few green fluorescence dots could be observed in HeLa cells (Figure 7a), while more green fluorescence dots could be observed for the cells treated with PAMAM/ DNA/heparin terplexes (Figure 7b), suggesting that adding

appropriate heparin increased the cellular uptake. According to the earlier reports, anionic dendrimer could result in a markedly improved cellular uptake of oligonucleotide, and the positive surface charge is not the sole factor determining the cell uptake (21). Our result was consistent with the literature. The intensity of fluorescence was highest for the cells treated with PAMAM/ DNA/heparin-biotin terplexes (Figure 7c), indicating that the existence of the biotin moiety played an important role in the cell uptake and the PAMAM/DNA/heparin-biotin terplexes could enter the cells through specific interactions between biotin and its receptors. The confocal microscopic observation clearly indicated that the level of cellular uptake in HeLa cells markedly increased by the presence of heparin and especially heparinbiotin. These results were in good agreement with the results of the in vitro transfection. As it is well know that the presence of serum could reduce the transfection efficiency of polycations, the excess positive charge on the surface of the polycation/DNA complexes results in nonspecific adsorption of plasma proteins in serum. Heparin could inhibit the adsorption of serum proteins to the surfaces of liposomes and PEI (13, 22). In this study, the transfection mediated by different complexes in the serum-contained media was investigated. As shown in Figure 8, the gene expression level increased significantly with the presence of heparin or heparin-biotin in the complexes. However, the expression levels decreased greatly in the serum-containing media (Figure 8) as compared with that in the serum-free media (Figure 5c). This is probably because all the terplexes were still positively charged, and their transfection efficacy was also affected by the presence of serum. To further assess the transfection activities of terplexes in HeLa cells, another plasmid pEGFP-C1 was used to form PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes. Being consistent with gene transfection results using plasmid pGL3-Luc, the images in Figure 9 also showed that the PAMAM/DNA/heparin terplexes exhibited enhanced transfection activity and the PAMAM/DNA/heparin-biotin terplexes had the highest transfection activity, because the weakest fluorescence emission was observed for the HeLa cells transfected by PAMAM/DNA, whereas the highest fluorescent density was observed for the cells transfected with PAMAM/ DNA/heparin-biotin. In order to evaluate the transfection activity of the terplexes, the in vitro transfections were carried out in both 293T cells



Figure 9. Enhanced green fluorescent protein expression in HeLa cells transfected with (a) PAMAM/DNA, (b) PAMAM/DNA/heparin, and (c) PAMAM/ DNA/heparin-biotin complexes. The PAMAM/DNA N/P ratio is 20, and the weight ratio of heparin/DNA and heparin-biotin/DNA is 2.

(Figure 10) and COS-7 cells (Figure 11). The transfection activities of PAMAM/DNA/heparin terplexes and PAMAM/ DNA/heparin-biotin terplexes were also statistically significantly higher than that of PAMAM/DNA complexes (P < 0.05). These results suggested that the presence of heparin or heparin-biotin was favorable for the transfection in these cells. According to the literature (8–11), most tumor cell types overexpress receptors involved in vitamin uptake such as biotin, although some tumor cell types do not overexpress biotin receptors, and normal cells do not overexpress biotin receptors. In our study, the transfection activity of PAMAM/DNA/ heparin-biotin terplexes was almost the same as that of PAMAM/DNA/heparin terplexes in 293T and COS-7 cells. The presence of biotin on the surface of the complexes could not further enhance the transfection, implying that biotin receptors

were not overexpressed in 293T and COS-7 cells, which were nontumor cells.

CONCLUSIONS In this study, heparin and heparin-biotin were introduced to the complexes of PAMAM/DNA via electrostatic interactions. The presence of appropriate amounts of heparin and heparinbiotin did not affect the DNA condense ability of PAMAM and could lead to lowered cytotoxicities and improved gene transfection activities for the self-assembled PAMAM/DNA/heparin and PAMAM/DNA/heparin-biotin terplexes. The PAMAM/ DNA/heparin-biotin terplexes exhibited an enhanced cellular uptake into HeLa cells due to the specific interactions between biotin and biotin receptors on HeLa cells, and thus led to further improvement in the gene transfection in HeLa cells as compared with PAMAM/DNA/heparin. These results indicated that PAM-


Figure 10. Luciferase expression in 293T cells transfected with (a) PAMAM/DNA, (b) PAMAM/DNA/heparin, and (c) PAMAM/DNA/ heparin-biotin complexes. The PAMAM/DNA N/P ratio is 20, and the weight ratio of heparin/DNA and heparin-biotin/DNA is 2. (* p < 0.05 as compared with the data of PAMAM/DNA complexes.)

Figure 11. Luciferase expression in COS-7 cells transfected with (a) PAMAM/DNA, (b) PAMAM/DNA/heparin, and (c) PAMAM/DNA/ heparin-biotin complexes. The PAMAM/DNA N/P ratio is 20, and the weight ratio of heparin/DNA and heparin-biotin/DNA is 2. (* p < 0.05 as compared with the data of PAMAM/DNA complexes.)

AM/DNA/heparin-biotin terplexes would have promising potential applications in targeting gene delivery to cancer cells.

ACKNOWLEDGMENT Financial supports from National Natural Science Foundation of China (20774070 and 21074099) and Ministry of Science and Technology of China (National Basic Research Program of China 2009CB930300) are gratefully acknowledged.

LITERATURE CITED (1) Dominik, E. D., and Nettelbeck, D. M. (2009) Targeting cancer by transcriptional control in cancer gene therapy and viral oncolysis. AdV. Drug DeliVery ReV. 61, 554–571. (2) Waehler, R., Russell, S. J., and Curiel, D. T. (2007) Engineering targeted viral vectors for gene therapy. Nat. ReV. Gene 8, 573– 587. (3) Mintzer, M. A., and Simanek, E. E. (2009) Nonviral vectors for gene delivery. Chem. ReV. 109, 259–302. (4) Zhang, S., Zhao, Y., Zhao, B., and Wang, B. (2010) Hybrids of nonviral vectors for gene delivery. Bioconjugate Chem. 21, 1003–1009. (5) Mendonca, L. S., Firmino, F., Moreira, J. N., Pedroso de Lima, M. C., and Simoes, S. (2010) Transferrin receptor-targeted liposome encapsulating anti-BCR-ABL siRNA or asODN for chronic myeloid leukemia treatment. Bioconjugate Chem. 21, 157–168.

Zhang et al. (6) Zhang, X. Q., Wang, X. L., Zhang, P. C., Liu, Z. L., Zhuo, R. X., Mao, H. Q., and Leong, K. W. (2005) Galactosylated ternary DNA/polyphosphoramidate nanoparticles mediate high gene transfection efficiency in hepatocytes. J. Controlled Release 102, 749–763. (7) Cheng, H., Zhu, J. L., Zeng, X., Jing, Y., Zhang, X. Z., and Zhuo, R. X. (2009) Targeted gene delivery mediated by folatepolyethylenimine-block-poly(ethylene glycol) with receptor selectivity. Bioconjugate Chem. 20, 481–487. (8) Gregory, R. J., McTavish, K., McEwan, J., Rice, J., and Nowotnik, D. (2004) Vitamin-mediated targeting as a potential mechanism to increase drug uptake by tumours. J. Inorg. Biochem. 98, 1625–1633. (9) Yang, W. J., Cheng, Y. Y., Xu, T. W., Wang, X. Y., and Wen, L. P. (2009) Targeting cancer cells with biotin-dendrimer conjugates. Eur. J. Med. Chem. 44, 862–868. (10) Patil, Y., Sadhukha, T., Ma, L., and Panyam, J. (2009) Nanoparticle-mediated simultaneous and targeted delivery of paclitaxel and tariquidar overcomes tumor drug resistance. J. Controlled Release 136, 21–29. (11) Na, K., Lee, T. B., Park, K. H., Shin, E. K., Lee, Y. B., and Choi, H. K. (2003) Self-assembled nanoparticles of hydrophobically-modified polysaccharide bearing vitamin H as a targeted anti-cancer drug delivery system. Eur. J. Pharm. Sci. 18, 165– 173. (12) Andersson, J., Sanchez, J., Ekdahl, K. N., Elgue, G., Nilsson, B., and Larsson, R. (2003) Optimal heparin surface concentration and antithrombin binding capacity as evaluated with human nonanticoagulated blood in vitro. J. Biomed. Mater. Res. A 67, 458– 466. (13) Jeon, O., Yang, H. S., Lee, T. J., and Kim, B. S. (2008) Heparin-conjugated polyethylenimine for gene delivery. J. Controlled Release 132, 236–242. (14) Hu, C. H., Zhang, L., Wu, D. Q., Cheng, S. X., Zhang, X. Z., and Zhuo, R. X. (2009) Heparin-modified PEI encapsulated in thermosensitive hydrogels for efficient gene delivery and expression. J. Mater. Chem. 19, 3189–3197. (15) Bai, S. H., Thomas, C. D., and Ahsan, F. (2007) Dendrimers as a carrier for pulmonary delivery of enoxaparin a low-molecular weight heparin. J. Pharm. Sci. 96, 2090–2106. (16) Bai, S. H., and Ahsan, F. (2009) Synthesis and evaluation of pegylated dendrimeric nanocarrier for pulmonary delivery of low molecular weight heparin. Pharm. Res. 26, 539–548. (17) Dufes, C., Uchegbu, I. F., and Schatzlein, A. G. (2005) Dendrimers in gene delivery. AdV. Drug DeliVery ReV. 57, 2177– 2202. (18) Luo, D., Haverstick, K., Belcheva, N., Han, E., and Saltzman, W. M. (2002) Poly(ethylene glycol)-conjugated PAMAM dendrimer for biocompatible, high-efficiency DNA delivery. Macromolecules 35, 3456–3462. (19) Kojima, C., Kono, K., Maruyama, K., and Takagishi, T. (2000) Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs. Bioconjugate Chem. 11, 910–917. (20) Lee, Y., Miyata, K., Oba, M., Ishii, T., Fukushima, S., Han, M., Koyama, H., Nishiyama, N., and Kataoka, K. (2008) Chargeconversion ternary polyplex with endosome disruption moiety: a technique for efficient and safe gene delivery. Angew. Chem., Int. Ed. 47, 5163–5166. (21) Hussain, M., Shchepinov, M. S., Sohail, M., Benter, I. F., Hollins, A. J., Southern, E. M., and Akhtar, S. (2004) A novel anionic dendrimer for improved cellular delivery of antisense oligonucleotides. J. Controlled Release 99, 139–155. (22) Han, H. D., Lee, A., Song, C. K., Hwang, T., Seong, H., Lee, C. O., and Shin, B. C. (2006) In vivo distribution and antitumor activity of heparin-stabilized doxorubicin-loaded liposomes. Int. J. Pharm. 313, 181–188. BC100309E

Self-Assembled Terplexes for Targeted Gene Delivery with Improved

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