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The purpose of the present study is to optimize the structure of the polyamidoamine starburst dendrimer (dendrimer) conjugate with α-cyclodextrin (α-CDE conjugate) as a nonviral vector. α-CDE conjugates of dendrimer (generation 3, G3) with various average degrees of substitution (DS) of α-CyD of 1.1, 2.4, and 5.4 were prepared. α-CDE conjugates formed the complexes with pDNA, resulting in a change of the particle sizes of pDNA complexes, but the distinction of physicochemical properties among their vector/pDNA complexes was only very slight. The membrane-disruptive ability of α-CDE conjugates on liposomes encapsulating calcein and their cytotoxicity to NIH3T3 and HepG2 increased with an increase in the DS value of α-CyD. In vitro gene transfer activity of α-CDE conjugates in both NIH3T3 and HepG2 cells augmented as the charge ratio (vector/pDNA) increased, and the activity of α-CDE conjugate (DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three α-CDE conjugates, and TransFast. After intravenous administration of pDNA complexes in mice, α-CDE conjugate (DS 2.4) delivered pDNA more efficiently in spleen, liver, and kidney, compared with dendrimer and other α-CDE conjugates (DS 1.1 and 5.4). The potential use of α-CDE conjugate (G3, DS 2.4) could be expected as a nonviral vector in vitro and in vivo, and these data may be useful for design of α-CyD conjugates with other nonviral vectors.

To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with α-cyclodextrin (α-CDE conjugate (G2)), we prepared α-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of α-CDE conjugate (G2) and TransFast, a novel transfection reagent. α-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 α-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, α-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 α-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. α-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 α-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.

The hemolytic behavior of a novel cytoprotective agent, DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate) was investigated using rabbit erythrocytes. Further, the effects of water-soluble cyclodextrin derivatives, such as 2-hydroxypropyl-β-cyclodextrin (HP-β-CyD) and sulfobutyl ether of β-cyclodextrin (SBE-β-CyD), on the hemolytic activity of DY-9760e were studied. DY-9760e induced hemolysis at concentrations >0.2–0.3 mM in phosphate buffered saline (PBS) of pH 4.0 and 6.0, where DY-9760e is predominantly in dicationic and monocationic forms, respectively. The hemolytic activity of the monocationic DY-9760e was higher than that of the dicationic species, and the hemolysis at pH 4.0 involved the formation of methemoglobin. DY9760e induced the morphological change of erythrocytes towards membrane invagination at both pH 4.0 and 6.0. SBE7-β-CyD significantly suppressed the DY-9760e-induced hemolysis and morphological change at both pH 4.0 and 6.0, as well as the formation of methemoglobin at pH 4.0. On the other hand, HP-β-CyD suppressed only the hemolysis, but neither the morphological change nor the formation of methemoglobin. In addition, the inhibitory effect of SBE7-β-CyD on the hemolysis was greater than that of HP-β-CyD. The superior inhibitory effect of SBE7-β-CyD on the DY-9760-induced hemolysis, the morphological change, and the formation of methemoglobin may be attributable to the formation of a stable inclusion complex with DY-9760e and to the weaker hemolytic activity of SBE7β-CyD than HP-β-CyD. These results suggest potential use of SBE7-β-CyD as a parenteral carrier for DY-9760e. © 2002 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 91:2382–2389, 2002

To improve the transfection efficiency of nonviral vector, we synthesized the starburst polyamidoamine dendrimer conjugates with α-, β-, and γ-cyclodextrins (CDE conjugates), expecting the synergistic effect of dendrimer and cyclodextrins (CyDs). The ¹H NMR spectroscopic data indicated that α-, 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 α-CyD (α-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 α-CyD) in NIH3T3 and RAW264.7 cells. In addition, the gene transfer activity of α-CDE conjugate was superior to that of Lipofectin. The enhancing gene transfer effect of α-CDE conjugate may be attributable to not only increasing the cellular association, but also changing the intracellular trafficking of pDNA. These findings suggest that α-CDE conjugate could be a new preferable nonviral vector of pDNA.

6^A-O-(n-Butanoyl)-β-cyclodextrin was prepared and its hydrolysis behavior in aqueous solutions and in rat intestinal fluids was investigated. Furthermore, the enzymatic hydrolyses of the n-butyric acid–β-cyclodextrin conjugate using α-amylase and esterase were studied to gain insight into the release behavior of n-butyric acid from the conjugate. The hydrolysis of the conjugate proceeded according to a first-order kinetics in aqueous solution, and gave a V-shaped pH profile, indicating a specific acid-base-catalyzed hydrolysis at acidic and neutral-alkaline regions, respectively. The half-lives (t1/2) of the conjugate at pH 4.4, 6.8, and 7.4 at 37 °C were ~580, 43, and 6 days, respectively, indicating that the conjugate is stable in aqueous solution. No appreciable release of n-butyric acid from the conjugate was observed in the stomach and small intestinal contents of rats, or in the small and large intestinal homogenates of rats. On the other hand, a fast disappearance of the conjugate and an appearance of n-butyric acid were observed in the cecal and colonic contents of rats. The t1/2 values of the disappearance were ~4, 1, and 6 h in 10 and 15% cecal contents and 10% colonic contents, respectively, and the appearance of n-butyric acid after 6 h was ~10% in the 15% cecal contents. Aspergillus oryzae α-amylase hydrolyzed the conjugate to small saccharide conjugates, such as the triose and maltose conjugates, but there was no appreciable release of n-butyric acid. The conjugate was less susceptible to carboxylic esterase (from porcine live), thus releasing no appreciable amounts of n-butyric acid. On the other hand, a fast release of n-butyric acid was observed when the esterase was employed after amylase hydrolysis, suggesting that two types of enzymes, sugar-degrading and ester-hydrolyzing enzymes, are necessary for the release of n-butyric acid from the conjugate in large intestinal contents. © 2000 Wiley-Liss Inc. and the American Pharmaceutical Association J Pharm Sci 89: 1486–1495, 2000