Effects of Structure of Polyamidoamine Dendrimer on Gene Transfer

Statistical significance of mean coefficients was performed by analysis of variance followed by Student's t-test. P values for significance were set a...
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Bioconjugate Chem. 2002, 13, 1211−1219

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Effects of Structure of Polyamidoamine Dendrimer on Gene Transfer Efficiency of the Dendrimer Conjugate with r-Cyclodextrin Fumihiro Kihara, Hidetoshi Arima, Toshihito Tsutsumi, Fumitoshi Hirayama, and Kaneto Uekama* Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan. Received June 9, 2002; Revised Manuscript Received September 3, 2002

To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with R-cyclodextrin (R-CDE conjugate (G2)), we prepared R-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of R-CDE conjugate (G2) and TransFast, a novel transfection reagent. R-CDE conjugates (G2, G3, and G4) formed the complexes with pDNA, changing the ζ-potential and particle size of pDNA complexes and the protection of pDNA from DNase I in a charge ratio-dependent manner, although their differences at higher charge ratios (vector/pDNA) were small. The gene transfer activity of R-CDE conjugates (G2, G3, and G4) was higher than that of the corresponding dendrimer alone in NIH3T3 and RAW264.7 cells. Of these CDE conjugates, R-CDE conjugate (G3) had a superior gene transfer activity which was comparable to that of TransFast in NIH3T3 cells. The intracellular distribution of pDNA after application of the pDNA complex with R-CDE conjugate (G3) to NIH3T3 cells was different from that with dendrimer alone (G3), although the cellular association of pDNA was almost comparable among all vectors. R-CDE conjugate (G3) strongly interacted with a fluorescence probe, 2-(p-toluidinyl)naphthalene-6-sulfonate (TNS), suggesting that the conjugate possesses the inclusion ability with biomembrane constituents such as phospholipids after transfection. These results suggest that R-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.

INTRODUCTION

The promising concept of gene therapy has encouraged the improvement of gene transfer techniques (1). At present, the following four methods may be available for gene transfer, i.e., viral vector, nonviral vector and physical (electroporation and gene gun, etc.) methods as well as naked plasmid DNA (pDNA1) method without vectors (2, 3). Nonvirus vectors have been noticed due to easy preparation of vector/pDNA complexes, low cytotoxicity, and lack of immunogeneity of vectors, but the insufficient gene transfer ability should be improved (4, 5). Starburst polyamidoamine (PAMAM) dendrimer (dendrimer) employed in this study is a spherical, highly ordered, dendritic polymer with positively charged primary amino groups on the surface at physiological pH (6) and is reported to be useful as nonviral vectors (711). Although the dendrimers with generations higher than 5 (G5) have sufficient gene transfer activity, cytotoxicity increases concomitantly as the generation increases due to higher positive charges (12). Cyclodextrins (CyDs) are cyclic (R-1,4)-linked oligosaccharides of R-D-glucopyranose containing a hydrophobic central cavity and hydrophilic outer surface (13-15). The most common CyDs are R-, β-, and γ-CyDs, which consist of six, seven, and eight D-glucopyranose units, respectively. CyDs are known to form inclusion complexes with * To whom correspondence should be addressed.. Telephone: (81)-96-371-4160. Fax: (81)-96-371-4420. E-mail: uekama@ gpo.kumamoto-u.ac.jp. 1 Abbreviation: CyDs, cyclodextrins; CDE conjugate, cyclodextrin-dendrimer conjugate; pDNA, plasmid DNA; dendrimer, Starburst polyamidoamine dendrimer; TNS, 2-(p-toluidinyl)naphthalene-6-sulfonate potassium salt; [32P]pDNA, [32P]labeled plasmid DNA; FITC-pDNA, fluorescein isothiocianatelabeled pDNA.

a variety of guest molecules in solution and in a solid state (16, 17). The solubilization of lipophilic compounds by CyDs has many uses in the pharmaceutical field (18, 19), while CyDs at higher concentrations induce hemolysis and decrease the integrity of the nasal and intestinal epithelial cells, leading to an increase in the permeability of water-soluble drugs through the membranes (20-22). Recently, CyDs have been applied to gene transfer (23-25) and oligonucleotides delivery (26-29). Notably, Davis and his colleagues (30, 31) reported that of cationic β-CyD polymers, a water soluble β-CyD polymer with 6A,6D-dideoxy-6A,6D-di-(2-aminoethanethio)-β-CyD and dimethylsuberimidate (βCDP6) gave gene transfer activity much higher than Superfect and poly-L-lysine in BHK21 and CHO-K1 with less cytotoxicity. In addition, most recently they reported that the combination of βCDP6 and poly(ethylene glycol) conjugate with adamantane (PEG-AD) forms stable complexes with pDNA through β-CyD/adamantane host/guest interactions and the ternary complex of βCDP6, galactosylated PEG-AD, and pDNA possesses higher transfection efficiency in hepatoma cells through receptor-mediated endocytosis (32). We reported previously that gene transfer activity was significantly improved when the dendrimer (G2) conjugates with R-CyD, β-CyD, and γ-CyD were employed as vectors, where the activity was most enhanced by the R-CyD conjugate (R-CDE conjugate) almost 100 times, compared with that of dendrimer (G2) (30). Thus, the attachment of a molecule of R-CyD to a terminal amino group of dendrimer (G2) provoked the marked improvement of gene transfer activity, so we expected that the conjugation of R-CyD with dendrimer of higher generation would provide much more gene transfer activity. Therefore, we prepared R-CDE conjugates with dendrimers having different generations (G2, G3, and G4) and

10.1021/bc025557d CCC: $22.00 © 2002 American Chemical Society Published on Web 11/01/2002

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investigated the effects of generation on physicochemical properties such as net charge, ζ-potential, particle size, pDNA degradation by DNase I, gene transfer activity, and cytotoxicity of pDNA complexes with dendrimers (pDNA/dendrimers) and R-CDE conjugates (pDNA/RCDE conjugates). In addition, the enhancing mechanism of gene transfer activity of R-CDE conjugate (G3) was examined. MATERIALS AND METHODS

Materials. R-CyD was donated by Nihon Shokuhin Kako (Tokyo) and recrystallized from water. Starburst PAMAM dendrimers (ethylenediamine core, G2, G3, and G4) were purchased from Aldrich Chemical (Tokyo). p-Toluenesulfonyl chloride was purchased from Nakarai Tesque (Kyoto, Japan). Fetal calf serum (FCS) was purchased from Nichirei (Tokyo). Dulbecco’s modified Eagle’s medium and RPMI1640 medium were purchased from Nissui Pharmaceuticals (Tokyo). TransFast transfection reagent, the plasmid pGL3 control vector, which contains the firefly luciferase gene under the control of the SV40 promoter (pDNA) and DNase I were obtained from Promega (Tokyo). 2-(p-Toluidinyl)naphthalene-6sulfonate potassium salt (TNS) was obtained from Research Organics (Cleveland, OH). [R-32P]CTP was obtained from Amersham Pharmacia Biotech (Tokyo). The purification of pDNA amplified in bacteria was carried out using QIAGEN EndoFree plasmid maxi kit ( dendrimer (G3) > dendrimer (G4) > R-CDE conjugate (G2) . dendrimer (G2) in a deceasing order. In addition, the gene transfer activity of R-CDE conjugate (G3) was higher than approximately 20 and 2 times than that of R-CDE conjugate (G2) and R-CDE conjugate (G4), respectively, and was comparable with that of TransFast in NIH3T3 cells. On the other hand, in RAW264 cells, the greatest gene transfer activity was elicited by the pDNA complexes with R-CDE conjugate (G3), followed in a decreasing order by the complex with R-CDE conjugate (G2) > dendrimer (G3) > dendrimer (G2) > dendrimer (G4) g R-CDE conjugate (G4). These results indicate that of the vectors used here, R-CDE conjugate (G3) possesses highest gene transfer activity

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Figure 6. Transfection efficiency of the complexes of pDNA/dendrimers (G2, G3, and G4) or pDNA/R-CDE conjugates (G2, G3, and G4) at the charge ratio of 200/1 (vector/pDNA) in NIH3T3 cells (A) and RAW264.7 cells (B). The luciferase activity in cell lysates was determined 25 h after incubation with pDNA alone or various complexes. The charge ratio of pDNA and TransFast is 1/1. Open, closed and hatched columns represent the complexes of pDNA/dendrimer, pDNA/R-CDE conjugate and pDNA/TransFast. Each value represents the mean ( SEM of 4-6 (A) or 4 (B). *p < 0.05, compared with dendrimer alone.

in NIH3T3 and RAW264.7 cells. Figure 7A and 7B show the charge ratios of the complexes on gene transfer activity of dendrimers and R-CDE conjugates in NIH3T3 cells and RAW264.7 cells, respectively. In NIH3T3 cells, the transfection efficiency of all dendrimers and R-CDE conjugates employed increased as the charge ratio (vector/pDNA) increased in the region of lower ratios, and especially R-CDE conjugates possessed higher gene transfer activity than the corresponding dendrimer alone. Thus, R-CDE conjugate (G3) conferred the greatest transfection activity in the charge ratios of 5/1-200/1 (vector/pDNA). As shown in Figure 7B, the transfection efficiency of the complexes of pDNA/R-CDE conjugates (G2 and G3) gradually increased as the charge ratio (vector/pDNA) increased in RAW264.7 cells. The transfection efficiency of the complexes of pDNA/dendrimer (G4) and pDNA/R-CDE conjugate (G4) significantly decreased as the charge ratio increased more than 50/1 (vector/pDNA), although the efficiency of the complex of pDNA/R-CDE conjugate (G4) was higher than that of pDNA/R-CDE conjugates (G2 and G3) at lower charge ratios (vector/pDNA). Thus, low gene transfer activity of the pDNA complexes with dendrimers or R-CDE conjugates may be due to the cytotoxicity to NIH3T3 cells (Figure 5). These results clearly indicate that R-CDE conjugate (G3) possesses preferable gene transfer activity among the dendrimers and R-CDE conjugates used here. CyDs are known to induce hemolysis and enhance the permeation of water-soluble drugs through biological membranes via the release of membrane components such as phospholipids and cholesterol as described above (21, 22). This fact allows us to deduce that R-CyD enhances gene transfer activity through membranepermeability of pDNA/R-CDE conjugates. To confirm this possibility, we examined the effects of physical mixture of R-CyD and the pDNA complexes with dendrimers (G3 and G4) at various charge ratios in the gene transfer. As a result, there is no difference in the gene transfer activity in NIH3T3 and RAW264.7 cells between the pDNA complex with dendrimer (G3 or G4) and the simple physical mixture of the pDNA/dendrimer complex and R-CyD (data not shown). The same results were conferred in dendrimer (G2) and R-CDE conjugate (G2) (33). Therefore, the conjugation of dendrimers with R-CyD is crucial for the gene transfer activity. Cellular Association of pDNA. We previously reported that the enhancing effect of R-CDE conjugate (G2)

relative to dendrimer (G2) could not be attributed to the increased cellular association of pDNA. To test whether R-CDE conjugates (G3 and G4) increase the association of pDNA, we measured the extent of pDNA associating to cells using [32P]pDNA. The association of pDNA to the NIH3T3 (Figure 8A) and RAW264.7 (Figure 8B) cells increased as the charge ratio increased up to 5/1 (vector/ pDNA), and above the ratio the association showed a plateau in all of the complexes of pDNA/dendrimers and pDNA/R-CDE conjugates. Here R-CyD showed no influence on the cellular association of pDNA under the present experimental conditions (data not shown), suggesting that R-CyD perturbs the integrity of plasma membrane of cells only very slightly. These results suggest that R-CDE conjugate (G3) may decrease the physical barrier function such as endosomal and nuclear membranes more efficiently than R-CDE conjugate (G2). Intracellular Distribution of pDNA. Cellular uptake of pDNA and its release from endosomes are necessary for gene expression of pDNA (38). Thus, the intracellular distribution of pDNA was observed using FITC-pDNA under confocal laser microscopy. Figure 9A and 9B show the results 25 h after transfection of the complexes of pDNA/dendrimer (G3) and pDNA/R-CDE conjugate (G3) in NIH3T3 cells. The complex of pDNA/ R-CDE conjugate (G3) provided higher fluorescence intensity in cytoplasm than that of pDNA/dendrimer (G3), whereas there was no fluorescence in the cells when pDNA alone was applied (data not shown). These results suggest that R-CDE conjugate (G3) changes the distribution of pDNA in the cells, possibly in terms of increase in the release of pDNA from the endosomes into cytoplasm after endocytosis. Inclusion Ability of r-CDE Conjugate (G3). It is possible that R-CDE conjugate (G3) may interact with membrane constituents. However, the ability of inclusion complexation of R-CDE conjugates still remains unclear. Thus, we examined the ability of inclusion complexation of R-CDE conjugate (G3) spectrophotometrically. Here we used a fluorescent guest molecule, TNS, because the inclusion of TNS into hydrophobic environment such as the R-CyD’s cavity is known to induce prominent fluorescence. As shown in Figure 10, the addition of dendrimer (G3) to TNS solution induced fluorescence corresponding to TNS, and the fluorescence intensity was increased by the addition of R-CyD. Importantly, R-CDE conjugate (G3) increased the fluorescent intensity re-

Gene Transfer by R-CDE Conjugates

Figure 7. Transfection efficiency of the complexes of pDNA/ dendrimers (G2, G3, and G4) or pDNA/R-CDE conjugates (G2, G3, and G4) at the charge ratios of 200/1 (vector/pDNA) in NIH3T3 cells (A) and RAW264.7 cells (B). The luciferase activity in cell lysates was determined 25 h after incubation with pDNA alone or various complexes. The charge ratio of pDNA and TransFast is 1/1. Square, dendrimer (G2) or R-CDE conjugate (G2); circle, dendrimer (G3) or R-CDE conjugate (G3); triangle, dendrimer (G4) or R-CDE conjugate (G4). Open and closed symbols represent the complex of pDNA/dendrimer and the complex of pDNA/R-CDE conjugate, respectively. Each value represents the mean ( SEM of 4-6 (A) or 4 (B). *p < 0.05, compared with dendrimer alone.

markably, compared with that of the dendrimer (G3) system. These results suggest that R-CDE conjugate (G3) possesses the inclusion ability of guest molecules into the cavity of R-CyD and thus it may interact with constituents of endosomal membranes such as phospholipids after endocytosis. DISCUSSION

We previously reported that of three CDE conjugates in which R-, β-, and γ-CyD covalently bound to dendrimer (G2) at a molar ratio of 1:1, R-CDE conjugate (G2) possesses the highest transfection activity (33). To improve the efficacy, we prepared three R-CDE conjugates (G2, G3, and G4) with different generation of dendrimers, and their interaction with pDNA, the protective effect of pDNA against DNase I, the gene transfer activity, and the cytotoxicity were examined. Consequently, we clarified that R-CDE conjugate (G3) possesses the greatest gene transfer activity with lower cytotoxicity. Cationic polymer-mediated transfection should overcome three major barriers for transfection: (1) binding of pDNA to cell surface, (2) endosomal release, and (3) entry of pDNA into the nucleus (39). Likewise, the physicochemical properties of polyplex such as net charge

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Figure 8. Cellular associations of [R-32P] labeled pDNA at various charge ratios in NIH3T3 cells (A) and RAW264.7 cells (B). Square, dendrimer (G2) or R-CDE conjugate (G2); circle, dendrimer (G3) or R-CDE conjugate (G3); triangle, dendrimer (G4) or R-CDE conjugate (G4). Open and closed symbols represent dendrimer and R-CDE conjugate, respectively. Each value represents the mean ( SEM of 4. *p < 0.05, compared with dendrimer alone.

and particle size markedly affect these barriers, eventually changing transfection efficiency (36). In the present study, the complexes of pDNA/R-CDE conjugates (G2, G3, and G4) showed gel mobility rates (Figure 2) and ζ-potentials (Figure 3A) similar to those of pDNA/ dendrimers (G2, G3, and G4) at the higher charge ratios (vector/pDNA). This similarity could be attributed to the two reasons: (1) the structure of R-CDE conjugate and (2) a lack of interaction between R-CyD and pDNA, and R-CyD and the pDNA complexes with dendrimers. The first reason may be explained by the fact that only a molecule of R-CyD binds to an amino group of dendrimers, thereby many intact positively charged amino groups still remains on the surface of these molecules. Second reason may be showed the following data: the addition of R-CyD to the solution containing pDNA or the complex of pDNA/dendrimer (G2, G3, or G4) did not affect their gel mobility rates (data not shown), indicating that R-CyD interacts with pDNA and their complexes only very slightly. Furthermore, a parallel of physicochemical properties between the pDNA complexes with dendrimers and R-CDE conjugate at higher charge ratio may be due to the relatively larger and complicated structure of the complexes because the calculated molar ratio (vector and pDNA) of the pDNA complexes with R-CDE conjugates (G2, G3, and G4) are 701, 339, and 167 in the complexes with R-CDE conjugates (G2, G3, and G4), respectively, even at the charge ratio of 1/1. Therefore, R-CyD changed

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Figure 9. Confocal laser microscopic images for the distribution of FITC-pDNA in NIH3T3 cells after transfection of the complexes of pDNA/dendrimer (G3) (A) and pDNA/R-CDE conjugate (G3) (B). The cells were incubated with the pDNA complexes for 25 h at 37 °C. The charge ratio (vector/pDNA) of the complexes of pDNA/dendrimer (G3) and of pDNA/R-CDE conjugate (G3) was 200/1. Scale bars represent 20 µm. The images shown are typical among similar ones in the several visual fields.

Figure 10. Fluorescence spectra of TNS in the absence and presence of dendrimer (G3) or R-CDE conjugate (G3). The fluorescent spectra of TNS were measured at 25 °C. The concentrations of TNS, dendrimer, R-CyD and R-CDE conjugate (G3) in 10 mM acetate buffer (pH 5.0) were 0.1 mM, 1 mM, 1 mM, and 1 mM, respectively. The excitation wavelength was 333 nm and the fluorescence spectra were measured in the range of 350-550 nm.

the intricate structure of the pDNA complex only very slightly. Thus, physicochemical properties of the pDNA complexes with R-CDE conjugates were changed in the generation-dependent manner, because the number of primary amino groups increases as the generation increases (Figures 2 and 3). It is evident that the stability against enzymatic degradation of pDNA influences gene transfer activity. The DNase I protection assay demonstrated that pDNA is protected from DNase I by the complexation with dendrimers and R-CDE conjugates, and the protective effects were almost identical between dendrimers and R-CDE conjugates regardless of their generation, up to the charge ratio of 100/1 (vector/pDNA, Figure 4). The stabilizing effect of dendrimers and R-CDE conjugates on pDNA degradation could be concerned with gene transfer into the cells under the experimental conditions. However, the stabilizing effect of dendrimer (G2) and

Kihara et al.

R-CDE conjugate (G2) on the degradation of pDNA in our previous paper (33) seems to be somewhat different from the present data: dendrimer (G2) and R-CDE conjugate (G2) prevented the disappearance of the bands by the charge ratio of 20/1 (vector/pDNA), but above the ratios the bands vanished. This inconsistency may be due to difference in the experimental conditions, i.e., the concentration of SDS used for dissociation of pDNA from the vectors in the complexes were 3.8% (w/v) and 4.8% (w/v) in the previous and present studies, respectively. Taken together, the physicochemical properties such as gel mobility shift, ζ-potential, particle size, and enzymatic stability of the complexes of pDNA/dendrimers and pDNA/R-CDE conjugates (G2, G3, and G4) were almost comparable, at least, at the higher charge ratios (vector/ pDNA). Gene transfer activity of dendrimers augmented in the order of G2 < G3 < G4 at the lower charge ratios (Figure 7), probably due to an increase in the proton sponge effect in endosomes (8). However, the transfection efficiency of dendrimer (G4) significantly attenuated at the higher charge ratios (vector/pDNA, Figures 6 and 7). This attenuation is certainly due to cytotoxicity (Figure 5), consistent with the results reported by Roberts et al., i.e., the cytotoxicity of dendrimers (G3, G5, and G7) to Chinese hamster lung fibroblasts increased as the generation increased (40). The similar patterns of gene transfer activity and cytotoxicity were also observed in the R-CDE conjugate system. The severe cytotoxicity of the pDNA complexes with dendrimer (G4) and R-CDE conjugate (G4) observed in RAW264.7 cells may be due to the distinction in the cellular sensitivity to positively charged amino groups. It is of importance that of the three R-CDE conjugates used in this study, gene transfer activity of R-CDE conjugate (G3) was the highest. The superior enhancing effect of R-CDE conjugate (G3) to dendrimer (G3) on gene transfer was observed at various times (4, 6, 12, and 25 h) after transfection in NIH3T3 cells. This greatest gene transfer activity could be attributed to the synergistic effects of the proton sponge effect of dendrimer (G3) and the tentative membrane-disrupting effect of R-CyD on endosomal membranes (Figures 9 and 10) because R-CDE conjugate (G3) possesses inclusion ability with a guest molecule, TNS (Figure 10). In addition, the inclusion ability of R-CDE conjugate (G3) with lipids was confirmed by our preliminary experiments: R-CDE conjugate (G3) is able to disrupt liposomal membranes consisting of phospholipids in vitro (data not shown). Thus, it is noteworthy that the introduction of an R-CyD molecule to dendrimer augmented the gene transfer activity of dendrimer (G2 and G3) than the parent dendrimers, showing no increase in cytotoxicity. Moreover, it should be noted that R-CDE conjugate (G3) possessed the comparable gene transfer activity to TransFast in NIH3T3 cells (Figure 6A). In conclusion, gene transfer activity of R-CDE conjugate (G3) was higher than that of dendrimers (G2, G3, and G4) and R-CDE conjugate (G2), and cytotoxicity of R-CDE conjugate (G3) was lower than that of R-CDE conjugate (G4). Therefore R-CDE conjugate (G3) may be a potent candidate for nonviral vectors. The present data may be useful for design of R-CyD conjugates with other nonviral vectors. ACKNOWLEDGMENT

We thank Dr. Makoto Uemura for assistance with the light scattering experiments Coulter N4 Plus machine at the Kumamoto Industrial Research Institute. We are

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grateful to Dr. Yoko Yamashita for assistance with measurements of ζ potential ELS-800 machine at the Fukuoka Industrial Technology Center. This work was supported by a Grant-in-Aid from Tokyo Biochemical Research Foundation, a Grant-in-Aid from the Research Foundation for Pharmaceutical Sciences and a Grantin-Aid for Encouragement of Young Scientists from the Ministry of Education, Science and Culture of Japan (12771464). 1H

NMR spectra of the R-CDE conjugates. This material is available free of charge via the Internet at http://pubs.acs.org. Supporting Information Available:

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