Design and Evaluation of Dendrimer/Cyclodextrin Conjugate as a Novel Gene Carrier

为了设计新型的基因转移载体，我们制备了不同取代度的α-环糊精（α-CDE）树枝状大分子偶联物（α-CDE偶联物），并对其化学结构、理化性质、体内外基因转移活性和细胞毒性进行了研究。α-CDE偶联物与质粒DNA（pDNA）形成复合物，导致pDNA复合物的粒径发生变化，但它们的载体/pDNA复合物的理化性质差别很小。随着α-环糊精取代度的增加，钙黄绿素脂质体上α-CDE结合物的破膜能力及对NIH 3 T3和HepG 2细胞的细胞毒性增强。α-CDE偶联物在NIH 3 T3和HepG 2细胞中的体外基因转移活性随着电荷比（vector/pDNA）的增加而增加，在树枝状聚合物（G3，DS 2.4）、三种α-CDE偶联物以及商业转染试剂（如Lipofectin和TransFast）中，α-CDE偶联物（G3，DS 2.4）在较高电荷比下的活性最高。小鼠静脉注射pDNA复合物后，与树枝状聚合物和其他α-CDE偶联物相比，α-CDE偶联物（DS 2.4）在脾、肝和肾中更有效地递送pDNA。α-CDE偶联物（G3，DS 2.4）有望在体内外用作非病毒载体，这些数据可用于设计α-CyD与其他非病毒载体的偶联物。此外，与半乳糖结合的α-CDE偶联物（GalCDE偶联物）在HepG 2和NIH 3 T3细胞中的基因转移活性比亲本α-CDE偶联物更高，表明GalCDE偶联物是优于其他市售转染剂上级基因转移载体。

To design a novel gene transfer carrier, we prepared dendrimer conjugates with α-cyclodextrin (α-CDE conjugates) with its various degree of substitution (DS) and investigated the chemical structure, physicochemical properties, in vitro and in vivo gene transfer activity and cytotoxicity. α-CDE conjugates formed the complexes with plasmid DNA (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-cyclodextrin. 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 (G3, DS 2.4) was the highest at higher charge ratios among dendrimer (G3), the three α-CDE conjugates, and commercial transfection reagents such as Lipofectin 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. 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. Furthermore, α-CDE conjugates attached with galactose (GalCDE conjugates) improved gene transfer activity more than parent α-CDE conjugates in HepG2 and NIH3T3 cells, suggesting that GalCDE conjugates are superior gene transfer carrier to the other commercially-available transfection agents.