Thierry A R, Dritschilo A
Department of Radiation Medicine, Vincent T. Lombardi Cancer Center, Georgetown University Medical Center, Washington, DC 20007.
Nucleic Acids Res. 1992 Nov 11;20(21):5691-8. doi: 10.1093/nar/20.21.5691.
We have studied factors which may effect the intracellular availability of oligonucleotides to achieve antisense activity. 15-20 mer unmodified, phosphorothioate modified and liposomally encapsulated oligodeoxynucleotides have been tested in leukemia MOLT-3 cells. Phosphorothioate analogs penetrated and accumulated intact in cells in contrast to unmodified oligomers, which showed a high instability in cell culture medium. A slow decrease of intracellular concentration of undegraded phosphorothioate oligodeoxynucleotides was observed after cell treatment and could be predominantly explained by a significant efflux transport. Using laser-assisted confocal microscopy we have observed that fluorescein 5-end-labeled phosphorothioate derivatives predominantly distributed in intracytoplasmic endocytic vesicles following cell treatment. The end-capped version of phosphorothioate oligodeoxynucleotides exhibited greater cellular uptake than fully modified analogues while exhibiting similar biological stability. Liposome encapsulation made possible oligomer protection in serum-containing medium and substantially improved cellular accumulation. Furthermore, the efflux rate of oligomer initially introduced within liposomes is 2-fold lower than that observed in cells which have been incubated with free oligonucleotides. Liposomal preparations of oligodeoxynucleotides facilitate release from endocytic vesicles, and thus, cytoplasmic and nuclear localization are observed following cell treatment. Furthermore, intracellular distribution studies demonstrate that intracellular transport of unmodified oligomers is effectively achieved using the liposomal carrier.
我们研究了可能影响寡核苷酸细胞内可用性以实现反义活性的因素。对15 - 20聚体的未修饰、硫代磷酸酯修饰和脂质体包裹的寡脱氧核苷酸在白血病MOLT - 3细胞中进行了测试。与未修饰的寡聚物相比，硫代磷酸酯类似物完整地穿透并积累在细胞中，未修饰的寡聚物在细胞培养基中表现出高度不稳定性。细胞处理后观察到未降解的硫代磷酸酯寡脱氧核苷酸的细胞内浓度缓慢下降，这主要可以用显著的外流转运来解释。使用激光共聚焦显微镜，我们观察到5'-端荧光素标记的硫代磷酸酯衍生物在细胞处理后主要分布在胞质内吞小泡中。硫代磷酸酯寡脱氧核苷酸的封端版本比完全修饰的类似物表现出更大的细胞摄取，同时表现出相似的生物稳定性。脂质体包裹使寡聚物在含血清培养基中得到保护，并显著改善了细胞内积累。此外，最初引入脂质体中的寡聚物的外排速率比与游离寡核苷酸孵育的细胞中观察到的低2倍。寡脱氧核苷酸的脂质体制剂促进从内吞小泡中释放，因此，细胞处理后可观察到其在细胞质和细胞核中的定位。此外，细胞内分布研究表明，使用脂质体载体可有效实现未修饰寡聚物的细胞内转运。
