Development of intracellular controlled release system for optimization of gene therapy

近年来，以纳米球或微球为载体的药物缓释或控释系统在全身药代动力学研究中备受关注。然而，在不久的将来，在药物传递系统的研究中，不仅“全身药代动力学”而且“细胞内药代动力学”似乎也是重要的。将缓释颗粒如纳米球递送到细胞质中的技术对于控制细胞内药代动力学是必不可少的。虽然已经实现了一些系统将可溶性药物递送到细胞质中，但是没有系统成功地将纳米颗粒递送到细胞质中以进一步持续调节细胞内隔室中的药物释放。在这项研究中，我们建立了一个方案，将纳米粒子封装到脂质体中。此外，脂质体与紫外线灭活的仙台病毒进一步融合，以制备融合脂质体。我们证明了融合脂质体可以通过融合依赖的方式而不是胞内方式将包裹的纳米颗粒递送到细胞质中。此外，在用包封纳米颗粒的融合脂质体处理的105个细胞中，94%的细胞引入了纳米颗粒。引入细胞质的纳米颗粒的平均数量为约10个颗粒/细胞11。此外，为了评价药物的细胞内缓释，通过融合脂质体将吸附了FITC标记的寡核苷酸的纳米颗粒引入细胞质中。证实FITC标记的寡核苷酸逐渐释放到细胞质中超过7天。我们的结论是，该技术，可用于任何功能性纳米粒子的封装，是有价值的细胞内药代动力学的调节。

In recent years, sustained or controlled drug-release system using nanospheres or microspheres is noticed in systemic pharmacokinetics. However, in the near future, not only "systemic pharmacokinetics" but also "intracellular pharmacokinetics seems to be important in Drug Delivery System research. Technology for delivering sustained release particles such as nanospheres into cytoplasm is indispensable to control the intracellular pharmacokinetics. Although some systems have been already achieved to deliver soluble drugs into cytoplasm, no system has been successful in delivering nanoparticles into cytoplasm for further sustained regulation of drug release in intracellular compartments. In this study, we established a protocol to encapsulate nanoparticles into liposomes. Additionally, the, liposomes were fur.ther fused with ultraviolet-inactivated Sendai virus in order to make fusogenic liposomes. We demonstrated that fusogenic liposomes could deliver the encapsulated nanoparticless into cytoplasm via fusion-dependent manner rather thanrendocytosis. Also nanoparticles were introduced in 94% of the 105 cells treated with fusogenic liposomes encapsulating nanoparticles. The mean number of nanoparticles introduced into cytoplasm was about 10 particles / ce11. Additionally, to evaluate intracellular slow release of drugs, nanoparticles that had adsorbed FITC-labeled oligonucleotide was introduced into the cytoplasm by fusogenic liposome. FITC-labeled oligonucleotide was confirmed to be released gradually into the cytoplasm for more than 7 days. We conclude that the technology, which can be used for the encapsulation of any functional nanoparticles, is valuable for regulation of intracellular pharmacokinetics.