Itaka Keiji, Yamauchi Kyosuke, Harada Atsushi, Nakamura Kozo, Kawaguchi Hiroshi, Kataoka Kazunori
Department of Materials Science and Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Biomaterials. 2003 Nov;24(24):4495-506. doi: 10.1016/s0142-9612(03)00347-8.
Polyion complex (PIC) micelles composed of the poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) block copolymer and plasmid DNA (pDNA) were investigated in this study from a physicochemical viewpoint to get insight into the structural feature of the PIC micellar vector system to show practical gene transfection efficacy particularly under serum-containing medium. The residual ratio (r) of the lysine units in PEG-PLL to the phosphate units of pDNA in the system significantly affects the size of the PIC micelles evaluated from dynamic light scattering, being decreased from approximately 120 to 80 nm with an increase in the r value for the region with r > or = 1.0. The zeta potential of the complexes slightly increased with r in the same region, yet maintained a very small absolute value and leveled off to a few mV at r approximately 2.0. These results suggest that the micelles are most likely to take the core-shell structure with dense PEG palisades surrounding the PIC core to compartmentalize the condensed pDNA. Furthermore, an increasing r value in the region of r > or = 1 induces a rearrangement of the stoichiometric complex formed at r=1.0 to the non-stoichiometric complex composed of the excess block copolymer. The association number of pDNA and the block copolymer in the micelle was estimated from the apparent micellar molecular weight determined by static light scattering measurements, indicating that a single pDNA molecule was incorporated in each of the micelles prepared from the PEG (Mw=12,000 g/mol)-PLL (polymerization degree of PLL segment: 48) (12-48) block copolymer at r=2.0. These 12-48/pDNA micelles showed a gene expression comparable to the lipofection toward cultured 293 cells, though 100 microM chloroquine was required in the transfection medium. Notably, even in the presence of serum, the PIC micelles achieved appreciable cellular association to attain a high gene expression, which is in sharp contrast with the drastic decrease in the gene expression for lipoplex system in the presence of serum. A virus-comparable size (approximately 100 nm) with a serum-tolerable property of the PIC micelles indeed suggests their promising feasibility as non-viral gene-vector systems used for clinical gene therapy.
本研究从物理化学角度对由聚乙二醇 - 聚（L - 赖氨酸）（PEG - PLL）嵌段共聚物和质粒DNA（pDNA）组成的聚离子复合物（PIC）胶束进行了研究，以深入了解PIC胶束载体系统的结构特征，特别是在含血清培养基中展现出实际的基因转染效果。体系中PEG - PLL中赖氨酸单元与pDNA磷酸单元的残留比（r）显著影响通过动态光散射评估的PIC胶束大小，对于r≥1.0的区域，随着r值增加，胶束大小从约120nm减小至80nm。在同一区域，复合物的zeta电位随r略有增加，但绝对值仍非常小，在r约为2.0时趋于稳定在几毫伏。这些结果表明，胶束很可能具有核壳结构，密集的PEG栅栏围绕PIC核心，将浓缩的pDNA分隔开来。此外，在r≥1的区域，r值增加会导致r = 1.0时形成的化学计量复合物重排为由过量嵌段共聚物组成的非化学计量复合物。通过静态光散射测量确定的表观胶束分子量估算了胶束中pDNA与嵌段共聚物的缔合数，表明在r = 2.0时，由PEG（Mw = 12,000 g/mol）- PLL（PLL链段聚合度：48）（12 - 48）嵌段共聚物制备的每个胶束中掺入了单个pDNA分子。这些12 - 48/pDNA胶束对培养的293细胞的基因表达与脂质体转染相当，不过转染培养基中需要100μM氯喹。值得注意的是，即使存在血清，PIC胶束也能实现可观的细胞结合以获得高基因表达，这与血清存在时脂质体复合物系统基因表达的急剧下降形成鲜明对比。PIC胶束具有与病毒相当的大小（约100nm）且具有血清耐受性，这确实表明它们作为用于临床基因治疗的非病毒基因载体系统具有良好的可行性。
