Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus
Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus
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DOI：
10.1021/bc025559y
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发表时间：
2002-09-01
影响因子：
4.7
通讯作者：
Kopecek, J
中科院分区：
文献类型：
--
作者：
Jensen, KD;Kopecková, P;Kopecek, J
研究了游离核苷酸和N-（2-羟丙基）-甲基丙烯酰胺（HPMA）共聚物-硫代寡核苷酸的亚细胞命运和抑制乙型肝炎病毒的活性。用共聚焦显微镜观察它们的内化和亚细胞命运。一部分内化的游离寡核苷酸逃逸到Hep G2细胞的细胞质和细胞核中，但不是有效的抗病毒药物。通过不可降解的二肽GG间隔物将寡核苷酸共价连接到HPMA共聚物上，从而在内化后将寡核苷酸隔离在囊泡中。通过溶酶体可切割的四肽GFLG间隔物将寡核苷酸偶联到HPMA共聚物上，导致溶酶体中的寡核苷酸释放，随后转运到细胞质和细胞核中。HPMA共聚物-寡核苷酸缀合物具有抗病毒活性，表明溶酶体中载体释放的硫代寡核苷酸能够逃逸到细胞质和细胞核中并保持活性。hepg2细胞似乎积极内化硫代寡核苷酸，寡核苷酸- hpma共聚物缀合物的内化程度高于未缀合的聚合物。
The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)-methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.