476
Bioconjugate Chem. 2001, 12, 476−484
Enhancement of Gene Expression by Polyamidoamine Dendrimer Conjugates with r-, β-, and γ-Cyclodextrins Hidetoshi Arima, Fumihiro Kihara, Fumitoshi Hirayama, and Kaneto Uekama* Faculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan. Received September 11, 2000; Revised Manuscript Received February 23, 2001
To improve the transfection efficiency of nonviral vector, we synthesized the starburst polyamidoamine dendrimer conjugates with R-, β-, and γ-cyclodextrins (CDE conjugates), expecting the synergistic effect of dendrimer and cyclodextrins (CyDs). The 1H NMR spectroscopic data indicated that R-, β-, and γ-CyDs are covalently bound to dendrimer in a molar ratio of 1:1. The agarose gel electrophoretic studies revealed that CDE conjugates formed the complexes with plasmid DNA (pDNA) and protected the degradation of pDNA by DNase I in the same manner as dendrimer. CDE conjugates showed a potent luciferase gene expression, especially in the dendrimer conjugate with R-CyD (R-CDE conjugate) which provided the greatest transfection activity (approximately 100 times higher than those of dendrimer alone and of the physical mixture of dendrimer and R-CyD) in NIH3T3 and RAW264.7 cells. In addition, the gene transfer activity of R-CDE conjugate was superior to that of Lipofectin. The enhancing gene transfer effect of R-CDE conjugate may be attributable to not only increasing the cellular association, but also changing the intracellular trafficking of pDNA. These findings suggest that R-CDE conjugate could be a new preferable nonviral vector of pDNA.
INTRODUCTION
There are two categories of gene therapy vectors, i.e., viral vectors and nonviral vectors. Viral vectors take advantage of being a higher gene expression. However, they have a failing in which the virus replicates and the inflammatory reaction occurs. On the other hand, the nonviral vectors have many advantages over viral vectors, such as easy to manufacture, safety, low immunogenicity, and molecular attachment of targeting ligand (1, 2). However, the problem is that the efficiency of nonviral vector-mediated gene transfer to cells is markedly low, compared to the viral vectors. This is because of the lack of receptor recognition, endosome escape, and nuclear pore targeting (3). To date, several strategies to enhance the gene expression of nonviral vectors are being developed, e.g., the application of helper lipids, pHsensitive lipids, cationic polymers, endosome-disruptive peptides, nuclear proteins, and nuclear localization signal, etc. (3-5). Starburst polyamidoamine dendrimer is a spherical, highly ordered, dendric polymer with positively charged primary amino groups on the surface at physiological pH (6). Recently, their usefulness as nonviral vectors has been reported (7, 8). However, dendrimers with lower generations (generation 1-3) do not have a good gene transfer activity, whereas dendrimers with higher generations may exhibit cytotoxicity (9). Cyclodextrins (CyDs) are cyclic (R-1,4)-linked oligosaccharides of R-D-glucopyranose containing a hydrophobic central cavity and hydrophilic outer surface (10-12). The most common CyDs are R-, β-, and γ-CyDs, which consist * To whom correspondence should be addressed. Phone: (81)96-371-4160. Fax: (81)-96-372-7023. E-mail: uekama@gpo. kumamoto-u.ac.jp. 1 Abbreviation: CyDs, cyclodextrins; CDE conjugate, cyclodextrin-dendrimer conjugate; pDNA, plasmid DNA; dendrimer, Starburst polyamidoamine dendrimer; [32P]pDNA, [32P]-labeled plasmid DNA; FITC-pDNA, Fluorescein isothiocianate labeled pDNA.
of six, seven, and eight D-glucopyranose units, respectively. CyDs are known to form inclusion complexes with a variety of guest molecules in solution and in a solid state. The solubilization of lipophilic compounds by CyDs has many uses in pharmaceutical fields (13, 14), while CyDs at higher concentration induce hemolysis and decrease the integrity of the nasal and intestinal epithelial cells, leading to an increase in the permeability of impermeable drugs through the membrane (15-17). The dissolution of lipids by CyDs from biological membranes may be useful in the study of cellular biology, e.g., caveolae, rafts, and cholesterol transport (18-20). Recently, CyDs have also been applied to the gene transfer and the oligonucleotide delivery (21-24): (1) The cationic β-CyDs such as tertiary amino-β-CyD acted as a viral dispersant, which resulted in an increase in adenoviral transduction in human colon adenocarcinoma Caco-2 cells by enhancing both viral binding and internalization (21). (2) β-CyD increased the gene transfer in the lungs of rats (22). In addition, 2-hydroxypropyl-β-CyD (HP-βCyD) and 2-hydroxyethyl-β-CyD (HE-β-CyD) increased the uptake of the antisense phosphorothioate oligodeoxynucleotides in human T cell leukemia H9 cell line, without cytotoxicity (23). 6-Deoxy-6-S-β-D-galactopyranosyl-6-thio-CyD improved the antiviral activity of the antisense phosphodiester oligonucleotide in human adenocarcinoma cells (24). Unfortunately, these enhancing effects of CyDs were not great. The potential use of the conjugation with CyDs has been studied in the pharmaceutical field, e.g., the sulfonylurea LY237868 conjugate with R-CyD (25) and the epoxysuccinyl peptide conjugate with β-CyD (26) as antitumor agents and the bioactive peptide conjugates with CyDs as protease inhibitors (27). In our laboratory, the potential uses of CyD conjugates with biphenylyl acetic acid and prednisolone for colon-specific drug delivery have been reported. (28, 29). To our knowledge, however, there is only one paper reporting the beneficial
10.1021/bc000111n CCC: $20.00 © 2001 American Chemical Society Published on Web 06/22/2001
Gene Transfer by Dendrimer/Cyclodextrin Conjugates
use of CyD conjugates for gene transfer; a new class of β-CyD-containing polymers was successful in the gene transfer in vitro (30). In the present study, we synthesized the potent new dendrimer conjugates with R-, β-, and γ-CyDs (CDE conjugates), in anticipation of the following synergic effect; i.e., (1) dendrimer has the abilities to complex with plasmid DNA (pDNA) and to enhance the cellular uptake of pDNA and (2) CyDs have a disrupting effect on biological membranes by the complexation with membrane constituents such as phospholipids and cholesterols. Thus, we investigated the effects of CDE conjugates on the gene transfer efficiency in the cells, and the enhancing effects were compared with commercial transfection reagents, Lipofectin and TransFast. EXPERIMENTAL SECTION
Materials. R-, β-, and γ-CyDs were donated by Nihon Shokuhin Kako (Tokyo, Japan) and recrystallized from water. Starburst PAMAM dendrimer (ethylenediamine core, generation 2, the terminal amino groups ) 16, molecular weight ) 3256) was purchased from Aldrich Chemical (Tokyo, Japan). p-Toluenesulfonyl chloride and p-naphthalenesulfonyl chloride were purchased from Nakarai Tesque (Kyoto, Japan). Fetal calf serum (FCS) was purchased from Nichirei (Tokyo, Japan). Dulbecco’s modified Eagle’s medium and RPMI1640 media were obtained from Nissui Pharmaceuticals (Tokyo, Japan). TransFast and Lipofectin transfection reagents were obtained from Promega (Tokyo, Japan) and Life Technologies (Rockville, MD), respectively. The plasmid pGL3 control vector (luciferase reporter vector, pDNA) and DNase I were obtained from Promega (Tokyo, Japan). The purification of pDNA amplified in bacteria was carried out using QIAGEN EndoFree plasmid maxi kit (1/20). Since CyDs alone possessed no protecting ability against the DNase I-catalyzed degradation of pDNA (data not shown), these results suggest that both dendrimer and CDE conjugates protect pDNA, and the optimal charge ratio exists in their protecting effects. The protecting mechanism may be same between dendrimer and CDE conjugates. Transfection. The gene transfer efficiency of dendrimer and CDE conjugates was compared with that of commercial transfection reagents, TransFast and Lipofectin, which were employed as positive controls. Figures 4A and 4B show the transfection efficiency of CDE conjugates in NIH3T3 and RAW264.7 cells, respectively. When pDNA alone with or without CyDs was transfected in both cells, no luciferase activity was observed (data not shown). In NIH3T3 cells, the combination of pDNA/ dendrimer (1/2) gave the luciferase activity of about 104 RLU/µg proteins. However, this activity was only slightly enhanced even when the charge ratio increased to 1/200. On the other hand, the combination of pDNA/CDE conjugates significantly increased the luciferase activity with increasing the charge ratio; especially, R-CDE conjugate enhanced the activity by approximately 100 times that of pDNA/dendrimer. At the charge ratios of 1/100 and 1/200, the luciferase activity of the R-CDE conjugate system was higher than that of the Lipofectin system, although it was lower than that of the TransFast
Gene Transfer by Dendrimer/Cyclodextrin Conjugates
Bioconjugate Chem., Vol. 12, No. 4, 2001 479
Figure 2. Agarose gel electrophoretic analysis of pDNA/dendrimer or pDNA/CDE conjugates using TBE buffer (pH 8.0). The solutions containing pDNA and dendrimer or CDE conjugates were incubated for 15 min at room temperature after slight agitation. The electrophoresis was performed at 100 V for about 50 min. A, dendrimer; B, R-CDE conjugate; C, β-CDE conjugate; D, γ-CDE conjugate.
system. In RAW264.7 cells, the same effect of CDE conjugates was observed, as shown in Figure 4B. Since the luciferase activity was saturated at the charge ratio of 1/100-1/200 (pDNA/CDE conjugate), the complex with the 1/200 charge ratio was employed in the following experiment. Figures 5A and 5B show the time-courses of the gene transfer ability of CDE conjugates in NIH3T3 and RAW264.7 cells, respectively. Of CDE conjugates, the great transfection activities of R-CDE were observed at 7, 13, 25 h after transfection in NIH3T3 cells and at 13 and 25 h in RAW264.7 cells, although their activities were inferior to those of TransFast. These results indicate that R-CDE conjugate has the superior transfection ability than β- and γ-CDE conjugates. Next, we compared the transfection ability of CDE conjugates with that of the simple mixtures of dendrimer and CyDs. As shown in Figure 6, the luciferase activities of the physical mixtures were almost the same as those of dendrimer alone in both NIH3T3 and RAW264.7 cells. These results indicate that the covalent conjugation between dendrimer and CyDs is necessary for the induction of the efficient transfection activity. Cellular Association of pDNA. The enhanced transfection effects of CDE conjugates may be attributable to the increasing cellular association and/or to changing the intracellular trafficking of pDNA. To clarify the mechanism for the higher transfection efficiency of the CDE conjugates, we examined the effects of dendrimer and CDE conjugates on the cellular association of pDNA to NIH3T3 and RAW264.7 cells, using [32P]pDNA. As shown in Figure 7, the association of pDNA to the cells increased as the charge ratio increased up to about 1/2-1/5, and there was a good correlation between the cellular association and the luciferase activity. This suggests that
the cellular entry is a rate-limiting step for gene transfer in lower molar ratios. However, the association was saturated above the charge ratio of about 1/2-1/5 and was almost the same between the dendrimer and the CDE conjugates, which was inconsistent with the results of luciferase activity (Figures 4 and 5). CyDs showed no effects of the cellular association of pDNA under the experimental conditions, implying a lack of perturbing effect of CyDs on the barrier function of plasma membrane (data not shown). These results suggest that CDE conjugates, especially R-CDE conjugate, may change the trafficking of pDNA in the cells. Intracellular Distribution of FITC-Labeled pDNA. To examine the effects of R-CDE conjugate on the distribution of pDNA in the cells, we observed the cells under a confocal laser microscope after transfection of FITC-pDNA for 25 h in NIH3T3 cells. The results are shown in Figure 8. The transfection of pDNA alone gave no fluorescence in the cells (Figure 8A), reflecting a lower cellular uptake of pDNA. The pDNA/dendrimer system gave a moderate fluorescence in cytoplasm (Figure 8B). On the other hand, more intense fluorescence was observed in cytoplasm when R-CDE conjugate was used as a vector (Figure 8C). Separately, we revealed that dendrimer and CDE conjugates entered into the cells in terms of the endocytosis mechanism (data not shown). These results suggest that R-CDE conjugate may change the distribution of pDNA in the cells, possibly in terms of the increase in the release of pDNA from the endosome into cytoplasm after endocytosis. Furthermore, R-CDE conjugate gave the fluorescence similar to that of the dendrimer alone at the low charge ratio (1/5) of the complexes, whereas it gave the stronger fluorescence than that of the dendrimer alone at the higher charge
480 Bioconjugate Chem., Vol. 12, No. 4, 2001
Arima et al.
Figure 3. Effects of dendrimer and CDE conjugates on electrophoretic mobility of pDNA treated with DNase I. The complexes of pDNA/dendrimer or pDNA/CDE conjugates were incubated with 0.1 unit/µL DNase I for 2 h. After DNase I was inactivated at 70 °C for 10 min, 1 µL of EDTA (0.5 M) and SDS (8% w/v) were added to each sample and incubated at 37 °C for 1 h to dissociate pDNA from the complexes. After incubation, gel electorophoresis was carried out at room temperature in TBE (45 mM Tris-Borate and 1 mM EDTA) buffer (pH 8.0) in 1% (w/v) agarose gel (include 0.1 µg/mL ethidium bromide) using Mupid system at 100 V for 50 min. A, dendrimer; B, R-CDE conjugate; C, β-CDE conjugate; D, γ-CDE conjugate.
ratio (1/200), as shown in Figures 8D and 8E. These results support the preceding idea that R-CDE conjugate changes the distribution of pDNA in the cells. DISCUSSION
We prepared three CDE conjugates in which R-, β-, and γ-CyDs were covalently bound to the dendrimer (generation 2) in a molar ratio of 1:1. These CDE conjugates may be unique as gene transfer vectors, because the only conjugates with β-CyD have been published (30). We revealed here that of three CDE conjugates, R-CDE conjugate possessed the greatest transfection activity. This activity was approximately more than 100-fold than that of dendrimer under an optimal condition. There are three major barriers to cationic polymersmediated transfection: (1) interactions between polyplexes and cell surfaces, (2) the endosomal release of polyplexes, and 3) entry of pDNA into the nucleus (35). None of the CyDs alone had the protecting ability of pDNA against DNase I, or the transfection activity at various concentrations, or the enhancing effects of the
cellular association of pDNA (data not shown). In addition, neither dendrimer alone nor the physical mixture of the dendrimer with CyDs showed the significant enhancing effect on the transfection activity (Figures 4-6). On the other hand, the complexes of pDNA with CDE conjugates, especially R-CDE conjugate, possessed a superior transfection activity over the dendrimer and the physical mixtures. The effects of the CDE conjugates on these barriers were investigated to gain an insight into the enhancing mechanism of R-CDE conjugate. From the results of gel mobility shift assay, dendrimer formed the complexes with pDNA (Figure 2). This may be attributed to electrostatic interaction, because terminal amines of dendrimer are only partially protonated because of their low pKa 6.9 (36), thereby interacting with negatively charged pDNA. However, this result was inconsistent with that of Fukowska-Latallo et al. (9), in which the pDNA migration is not retarded in the presence of dendrimers with the generation of 1 to 3. This discrepancy in the complexation of dendrimer with pDNA is not clear; possibly the pH value and components of
Gene Transfer by Dendrimer/Cyclodextrin Conjugates
Figure 4. Transfection efficiency of the complexes of pDNA/ dendrimer or pDNA/CDE conjugates complexes at various charge ratios 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 ratios of pDNA and TransFast and pDNA and Lipofectin were 1:1. open column, dendrimer alone; dark hatched column, R-CDE conjugate; light hatched column, β-CDE conjugate; closed column, γ-CDE conjugate; shade column, TransFast and Lipofectin. Each value represents the mean ( SEM of 5-11. *p < 0.05, compared with dendrimer alone.
buffers might be different. CDE conjugates formed the complex with pDNA in a similar manner as dendrimer (Figure 2), suggesting that the CyD moiety is not involved in the complexation, because the 15 terminal amino groups remain on surface of CDE conjugates. Therefore, it is apparent that the complexation ability of CDE conjugates with pDNA is not necessarily a crucial factor for the enhanced transfection activity of CDE conjugates, although the complexation is needed for the induction of transfection activity. Interestingly, the band driven from pDNA disappeared at the charge ratio of 1/1 (pDNA/CDE conjugates), at which the molar ratio of complexes of pDNA with CDE conjugates was about 1/700 (Figure 2). This suggests that the relatively larger and complicated complexes may be formed at more than 1/1 charge ratios. At the charge ratios of 1/100 and 1/200 (pDNA:CDE conjugates), the conjugates are largely in a free form, although their accurate fractions were unknown. The R-CDE conjugate in a free form may assist in the transfection by increasing the release of pDNA from endosome, due to its endosome-membrane perturbing effect, as shown in Figure 8. To examine the average size of the complex of pDNA with dendrimer or R-CDE conjugate, the diameter of the complex was measured by quasi-elastic (dynamic) light scattering (see Experimental Section). The mean diameters of the complexes of pDNA with dendrimer at the charge ratios of 1/0.5, 1/1, 1/2, 1/10,
Bioconjugate Chem., Vol. 12, No. 4, 2001 481
Figure 5. Time course of transfection efficiency of the complexes of pDNA/dendrimer or pDNA/CDE conjugates complexes at the charge ratio of 1/200 in NIH3T3 cells (A) and RAW264.7 cells (B). The charge ratio of pDNA and TransFast was 1:1. O, dendrimer alone; 4, R-CDE conjugate; 0, β-CDE conjugate; 2, γ-CDE conjugate; 9, TransFast. Each point represents the mean ( SEM of 4. *p < 0.05, compared with dendrimer alone.
1/20, 1/100, and 1/200 (pDNA/vectors) were 794, 594, 737, 731, 804, 717, and 743 nm, respectively, and those with R-CDE conjugate were 781, 753, 718, 735, 761, 747, and 720 nm, respectively. Therefore, the diameter of the complex of pDNA with dendrimer or R-CDE conjugate was not significantly affected by the charge ratios, and there was an insignificant difference in the diameter between the complexes with dendrimer and the conjugate. These results suggest that morphology of the complexes of pDNA with dendrimer or R-CDE conjugate might be independent of the charge ratio, and both dendrimer and R-CDE conjugate may exist as a free form in the solution containing the complexes with the higher molar ratios. The DNase I protection assay indicated that the pDNA/ dendrimer and pDNA/CDE conjugates resist the nuclease-catalyzed degradation (Figure 3). These results were in agreement with the previous reports in which the nuclease-catalyzed degradation is inhibited by the complexation with poly-L-lysine, DC-cholesterol, and Lipofectin (37, 38). However, the optimum charge ratio for the stabilizing effect of CDE conjugates existed, suggesting that factors other than the electrical charge may be associated with the stabilizing effect, e.g., the mode of the complexes and the existence of free fraction of CDE conjugate. Furthermore, there was an insignificant difference in the stabilizing ability between dendrimer and CDE conjugates, indicating that the greater transfection activity of R-CDE conjugate cannot be ex-
482 Bioconjugate Chem., Vol. 12, No. 4, 2001
Figure 6. Transfection efficiency of physical mixtures of pDNA and dendrimer or pDNA and CDE conjugates in NIH3T3 cells (A) and RAW264.7 cells (B). Open column, dendrimer alone; dark hatched column, R-CDE conjugate; light hatched column, β-CDE conjugate; closed column, γ-CDE conjugate. Each value represents the mean ( SEM of 5-11. *p < 0.05, compared with dendrimer alone.
plained simply by the inhibition of the DNase I-catalyzed degradation of pDNA. The entry of the complexes of pDNA with cationic polymers into the cell is the first step of transfection. In the present study, we confirmed a good correlation between cellular association and luciferase activity in lower charge ratios (1/0.2 to 1/2-1/5) of pDNA/CDE conjugates (Figure 7). This suggests that the cellular entry is rate-limiting step for gene transfer. However, no difference in the cellular association of pDNA between dendrimer and CDE conjugates was observed (Figure 7), suggesting that the conjugation of R-CyD with dendrimer, particularly in higher molar ratios, affects intracellular trafficking of pDNA. The fluorescence in cytoplasm was stronger when R-CDE conjugate was used, compared with dendrimer alone (Figure 8), supporting that R-CDE conjugate changes the distribution of pDNA in the cells, possibly in terms of the increase in the release of pDNA from the endosomes into cytoplasm. This hypothesis is based on facts that CyDs induce hemolysis and collapse of liposomes via the release of biological components, such as phospholipids and cholesterols from the membranes (15, 41, 42). It is well recognized that CyDs disrupt the liposomes composed of egg phosphatidylcholine or dipalmytoylphosphatidylcholine in the order of R- > β- > γ-CyD, whereas the incorporation of cholesterol into the liposomes changes the membrane-disrupting ability of CyDs in the order of β- > R- > γ-CyD (41). In addition, CyDs cause hemolysis of human erythrocytes in the order of β- > R- > γ-CyD (15). This difference can be explained by the fact that the species and amounts of released membrane components were dependent upon the cavity
Arima et al.
Figure 7. Cellular associations of [R-32P] labeled pDNA at various charge ratios in NIH3T3 cells (A) and RAW264.7 cells (B). O, dendrimer alone; 4, R-CDE conjugate; 0, β-CDE conjugate; 2, γ-CDE conjugate. Each point represents the mean ( SEM of three experiments.
size of CyDs, i.e., R-CyD extracted mainly phospholipids, β-CyD cholesterol, and γ-CyD both components in smaller amounts (43). However, since endosomal membranes have a relatively higher cholesterol content, the enhanced transfection activity of R-CDE conjugates could not simply be related to the membrane-disrupting effects of CyDs. Furthermore, R-CyD concentration (12 mM) to induce 50% hemolysis (43) is higher than that to enhance the transfection efficiency (for example, about 50 µM in the charge ratio of 1/100). In addition, CDE conjugates at less than micromolar concentrations could not increase the calcein release from the egg phosphatidylcholine liposomes (data not shown). Therefore, the exact mechanism for the enhanced transfection activity of R-CDE conjugate is currently unclear, but it is apparent that the conjugate affects the intracellular trafficking of pDNA. Further studies are necessary to elucidate the enhancing mechanism of CDE conjugates, e.g., whether β-CDE and γ-CDE conjugates change the intracellular trafficking of pDNA, how the complexes of CDE conjugates with pDNA dissociate, and how pDNA enters into the nucleus, compared with those of dendrimer alone. Under the present experimental conditions, no cytotoxicity of CDE conjugates and their complexes with pDNA was observed in NIH3T3 and RAW264.7 cells in WST-1 assay, up to the charge ratio of at least 1/800 (data not shown). Therefore, CDE conjugates, particularly R-CDE conjugate, have great advantages as nonviral vectors, i.e., an easy preparation, a superior transfection efficiency, and less cytotoxicity. In a preliminary study,
Gene Transfer by Dendrimer/Cyclodextrin Conjugates
Bioconjugate Chem., Vol. 12, No. 4, 2001 483
Industrial Research Institute. This research was supported by a Grant-in-Aid from Tokyo Biochemical Research Foundation and a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science and Culture of Japan (12771464). LITERATURE CITED
Figure 8. Confocal laser microscope images for the distribution of FITC-labeled pDNA in NIH3T3 cells after transfection of pDNA alone (A), the complex of pDNA/dendrimer at charge ratio of 1/200 (B), the complex of pDNA/R-CDE conjugate complex at charge ratio of 1/200 (C), the complex of pDNA/dendrimer at charge ratio of 1/5 (D), and the complex of pDNA/R-CDE conjugate complex at charge ratio of 1/5 (E). The cells were incubated with pDNA alone or the complexes for 25 h at 37 °C. Scale bars represent 25 µm. The images shown are typical among similar ones in the several visual fields.
we prepared the R-CDE conjugates containing more than one CyD molecules (average R-CyD/dendrimer molar ratios of 1.8 and 3.2) and then evaluated their transfection activities. The luciferase activities after the transfection of the 1.0, 1.8, and 3.2 R-CDE conjugates in NIH3T3 cells were 3.69 × 105 ( 0.22 × 105, 4.82 × 105 ( 0.27 × 105, and 6.72 × 105 ( 0.31 × 105, respectively, at the charge ratio of 1/200. These results indicated that the transfection activity of pDNA/R-CDE conjugate complex increased with the increasing of molar ratio of R-CyD/dendrimer. In conclusion, the present findings suggest that R-CDE conjugate could be a preferable nonviral vector of plasmid DNA among CDE conjugates. This strategy is applicable to other cationic lipids and polymers such as poly-L-lysine and polyethylenimine, which are now under investigation. ACKNOWLEDGMENT
We are very grateful to Prof. S. H. Lee at the Kunsan National University in Korea for teaching us the method to purify CDE conjugates. We thank Dr. Makoto Uemura for assistance with the light scattering experiments using a Beckman Coulter N4 Plus machine at the Kumamoto
(1) Luo, D., and Saltsmn, W. M. (2000) Synthetic DNA delivery systems. Nature Biotech. 18, 33-37. (2) Rolland, A. P. (1998) From genes to gene medicines: recent advances in nonviral gene delivery. Crit. Rev. Ther. Drug Carrier Syst. 15, 143-198. (3) Subramanian, A., Ranganathan, P., and Diamond, S. L. (2000) Nuclear targeting peptide scaffolds for lipofection of nondividing mammalian cells. Nature Biotech. 17, 873-877. (4) Felbner, P. L., Barenholz, Y., Behr, J. P., Cheng, S. H., Cullis, P., Huang, L., Jesse, J. A., Seymour, L., Szoka, F., Thierry, A. R., Wagner, E., and Wu, G. (1997) Nomenclature for synthetic gene delivery systems. Hum. Gene Ther. 8, 511512. (5) Ledly, F. D. (1995) Nonviral gene therapy: the promise of genes as pharmaceutical products. Human Gene Ther. 6, 1129-1144. (6) Tomalia, D. A., Naylor, A. M., and Goddard, W. A., III. (1990) Starburst dendrimers: Molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angew. Chem., Int. Ed. Engl. 29, 138175. (7) Bielinska, A., Kukowska-Latallo, J. F., Johnson, J., Tomalia, D. A., and Baker, Jr., J. R. (1996) Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers. Nucleic Acid Res. 24, 2176-2182. (8) Qin, L., Pahud, D. R., Ding, Y., Bielinska, A. U., KukowskaLatallo, J. F., Baker, J. R., Jr., and Bromberg, J. S. (1998) Efficient transfer of genes into murine cardiac grafts by starburst polyamidoamine dendrimers. Human Gene Ther. 9, 553-560. (9) Kukowska-Latallo, J. F., Bielinska, A. U., Johnson, J., Splindler, R., Tomalia, D. A., and Baker, J. R., Jr. (1996) Efficient transfer of genetic material into mammalian cells using Starburst polyamidoamine dendrimers. Proc. Natl. Acad. Sci. U.S.A. 93, 4897-4902. (10) Bender, M., and Komiyama, M. (1978) Cyclodextrin Chemistry, Springer-Verlag, Berlin. (11) Saenger, W. (1980) Cyclodextrin inclusion compound as research and industry. Angrew. Chem., Int. Ed. Engl. 19, 344-362. (12) Szejtli, J. (1982) Cyclodextrins and their inclusion complexes, Akade´miai Kiado´, Bupapest. (13) Szejtli, J. (1988) Cyclodextrin technology, Kluwer, Dordrecht, The Netherlands. (14) Uekama, K., Hirayama, F., and Irie, T. (1998) Cyclodextrin drug carrier systems. Chem. Rev. 5, 2045-2076. (15) Irie, T., and Uekama, K. (1997) Pharmaceutical application of cyclodextrin. III. Toxicological issues and safety evaluation. J. Pharm. Sci. 86, 147-162. (16) Irie, T., and Uekama, K. (1999) Cyclodextrins in peptide and protein delivery, Adv. Drug Deliv. Rev. 36, 101-123. (17) Marttin, E., Verhoef, J. C., and Merkus, F. W. (1998) Efficacy, safety and mechanism of cyclodextrins as absorption enhancers in nasal delivery of peptide and protein drugs. J. Drug Target. 6, 17-36. (18) Sternberg, P. W., and Schmid, S. L. (1999) Caveolin, cholesterol and ras signaling. Nature Cell Biol. 1, E35-E37. (19) Ilangumaran, S., and Hoessli, D. C. (1998) Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane. Biochem. J. 335 (Pt 2), 433-40. (20) Rodal, S. K., Skretting, G., Garred, O., Vilhardt, F., vanDeurs, B., and Sandvig, K. (1999) Extraction of cholesterol with methyl-β-cyclodextrin perturbs formation of clathrincoated endocytic vesicles. Mol. Biol. Cell 10, 961-974 (1999).
484 Bioconjugate Chem., Vol. 12, No. 4, 2001 (21) Croyle, M. A., Rosseler, B. J., Hsu, C. P., Sun, R., and Amidon, G. L. (1998) β-cyclodextrins enhance adenoviralmediated gene delivery to the intestine. Pharm. Res. 15, 1348-1355. (22) Freean, D. J., and Niven, R. W. (1996) The influence of sodium glycholate and other additives on the in vivo transfection of plasmid DNA in the lungs. Pharm. Res. 13, 202209. (23) Zhao, Q., Temsamani, J., and Agrawal, S. (1995) Use of cyclodextrin and its derivatives as carriers for oligonucleotide delivery. Antisense Res. Dev. 5, 185-92. (24) Abdou, S., Collomb, J., Sallas, F., Marsura, A., and Finance, C. (1997) β-cyclodextrin derivatives as carriers to enhance the antiviral activity of an antisense oligonucleotide directed toward a coronavirus intergenetic consensus sequence. Arch. Virol. 142, 1585-1602. (25) Kim, C. K., MacKellar, W. C., Cho, N., Byrn, S. R., and Morre´, D. J. (1997) Impermeant antitumor sulfonyluea conjugates that inhibit plasma membrane NADH oxidase and growth of HeLa cells in culture. Identification of binding proteins from sera of cancer patients. Biochim. Biophys. Acta 1324, 171-181. (26) Schaschke, N., Assfang-Machleidt, I., Machleidt, W., Lassleben, T., Sommerhoff, C. P., and Moroder, L. (2000) β-cyclodextrin/epoxysuccinyl peptide conjugates: a new drug targeting system for tumor cells. Bioorg. Med. Chem. Lett. 10, 677-680. (27) Schaschke,N.,Musiol,H.-J.,Assfakg-Machleidt,I.,Machleidt, W., Rudolph-Bo¨hner, S., and Moroder, L. (1996) Cyclodextrins as templates for the presentation of protease inhibitors. FEBS Lett. 391, 297-301. (28) Uekama, K., Minami, K., and Hirayama, F. (1997) 6A-O[(4-biphenylyl)acetyl]-R-, -β-, and -γ-cyclodextrins and 6Adeoxy-6A-[[(4-biphenylyl)acetyl]amino]-R-, -β-, and -γ-cyclodextrins: potential prodrugs for colon-specific delivery. J. Med. Chem. 40, 2755-2761. (29) Minami, K., Hirayama, F., and Uekama, K. (1998) Colonspecific drug delivery based on a cyclodextrin prodrug: release behavior of biphenylylacetic acid from its cyclodextrin conjugates in rat intestinal tracts after oral administration. J. Pharm. Sci. 87, 715-720. (30) Gonzalez, H., Hwang, S. J., and Davis, M. E. (1999) New class of polymers for the delivery of macromolecular therapeutics. Bioconjugate Chem. 10, 1068-1074. (31) Suh, J., Lee, S. H., and Zoh, K. D. (1992) A novel host containing both binding site and nucleophilic prepared by attachment of β-cyclodextrin to poly(ethylenimine). J. Am. Chem. Soc. 114, 7916-7917.
Arima et al. (32) Brown, S. E., Oates, J. H., Coghlan, D. R., Easton, C. J., van Eyk, S. J., Janowski, W., Lepore, A., Lincoln, S. F., Luo, Y., May, B. L., Schiesser, D. S., Wang, P., and Williams, M. L. (1993) Synthesis and properties of 6A-amino-6A-deoxy-Rand β-cyclodextrin. Aust. J. Chem. 46, 953-958. (33) Melton, L. D., and Slessor, K. N. (1971) Synthesis of monosubstituted cyclohexaamyloses, Carbohyd. Res. 18, 29-37. (34) Avall-Lundqvist, E., Economidou-Karaoglou, A., Sjovall, K., Lans, M., Taper, H. S., Roberfroid, M., and Eneroth, P. (1989) Serum alkaline DNase activity in normal or nonhospitalised individuals. Clin. Chim. Acta 185, 35-43. (35) De Smedt, S. C., Demeester, J., and Hennink, W. E. (2000) Cationic polymer based gene delivery systems. Pharm. Res. 17, 113-126. (36) Haensler, J., and Szoka, F. C., Jr. (1993) Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjugate Chem. 7, 703-714. (37) Gao, X., and Huang, L. (1996) Potentiation of cationic liposome-mediated gene delivery by polycations. Biochemistry 35, 1027-1036. (38) Van der Woudem Visser, H. W., ter Beetm M. B. A., Wagenaar, A., Ruiters, M. H. J. Engberts, J. B. F. N., and Hoekstra, D. (1995) Parameters influencing the introduction of plasmid DNA into cells by the use of synthetic amphiphiles as a carrier system. Biochim. Biophys. Acta 1240, 34-40. (39) Schaffer, D. V., and Lauffenburger, D. A. (1998) Optimization of cell surface binding enhances efficinecy and specificity of molecular conjugate gene delivery. J. Biol. Chem. 273, 28004-28009. (40) Matsui, H., Johnson, L. G., Randell, S. H., and Boucherm B. C. (1997) Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells. J. Biol. Chem. 272, 1117-1126. (41) Miyajima, K., Saito, H., and Nakagaki, M. (1987) Interaction of cyclodextrins with lipid membrane, J. Chem. Soc. Jpn. 3, 306-312. (42) Nishijo, J., Shiota, S., Mazima, K., Inoue, Y., Mizuno, H., and Yoshida, J. (2000) Interactions of cyclodextrins with dipalmitoyl, distearoyl, and dimyristoyl phosphatidyl choline liposomes. A study by leakage of carboxyfluorescein in inner aqueous phase of unilammellar liposomes. Chem. Pharm. Bull. 48, 48-52. (43) Irie, T., Otagiri, M., Sunada, M., Uekama, K., Ohtani, Y., Yamada, Y., and Sugiyama, Y. (1982) Cyclodextrin-induced hemolysis and shape changes of human erythrocytes in vitro. J. Pharm. Dyn. 5, 741-744.
BC000111N
