Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta, Midori, Yokohama 226-8501, Japan.
J Control Release. 2011 Oct 10;155(1):34-9. doi: 10.1016/j.jconrel.2010.10.014. Epub 2010 Oct 15.
In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the asialofetuin-oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37°C for at least 3h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs.
为了使肽核酸（PNA）能够作为治疗剂发挥作用，必须开发细胞递药方法。在这里，我们通过通过去唾液酸糖蛋白受体介导的内吞作用展示了递送至肝细胞的肽核酸（PNA）的自发核定位和反义作用。与目的 PNA 互补的与 DNA 寡核苷酸（AF/DNA）偶联的去唾液酸胎球蛋白设计为细胞特异性递药载体。PNA 与去唾液酸胎球蛋白-寡核苷酸缀合物杂交，通过体外受体介导的内吞作用有效地被内化到小鼠原代肝细胞和人 HepG2 肝癌细胞中。孵育 4 小时后，PNA 主要定位于细胞的核内；其涉及的机制尚不清楚。当使用 AF/DNA 将与人端粒酶 RNA 模板互补的 PNA 递送至 HepG2 细胞时，观察到端粒酶活性超过 70%的抑制。PNA 在 37°C 的 50%血清中至少 3 小时与 AF/DNA 缀合物稳定结合。当互补核酸被添加时，PNA 通过链交换机制从缀合物中自发释放。PNA 与 AF/DNA 缀合物的复合物导致在将 PNA 静脉内注射到小鼠后递送至肝脏。本研究表明，与天然蛋白质配体的缀合可用作寡核苷酸类似物细胞递药的无毒载体。
