Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.
Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol.
复制标题
使用通过聚乙二醇与叶酸缀合的脂质体，将针对人表皮生长因子受体的反义寡脱氧核糖核苷酸递送至培养的 KB 细胞中。
DOI：
10.1073/pnas.92.8.3318
复制
发表时间：
1995
影响因子：
11.1
通讯作者：
P. Low
中科院分区：
文献类型：
--
作者：
Susan H. H. Wang;R. J. Lee;G. Cauchon;D. Gorenstein;P. Low
靶向表皮生长因子（EGF）受体的反义寡脱氧核糖核苷酸通过聚乙二醇间隔剂被包裹到与叶酸相连的脂质体中（叶酸- peg -脂质体），并通过叶酸受体介导的内吞作用有效地递送到培养的KB细胞中。寡核苷酸是EGF受体（EGFR）基因终止密码子（AEGFR2）的磷酸二酯15-mer反义序列，每个末端具有三个硫代键的相同序列（AEGFR2S），与AEGFR2 （RC15）相似碱基组成的随机15-mer对照，来自对称大肠杆菌lac操作符（LACM）的14-mer对照，以及上述几种5'-荧光素标记的同源物。叶酸- peg脂质体包裹的AEGFR2的细胞摄取比非靶向脂质体包裹的AEGFR2高9倍，比未包裹的AEGFR2高16倍。在叶酸- peg -脂质体中使用AEGFR2处理KB细胞可导致生长抑制和显著的形态学改变。奇怪的是，包裹在叶酸- peg脂质体中的AEGFR2和AEGFR2S表现出几乎相同的生长抑制作用，在细胞用3微米寡核苷酸处理4小时后，48小时可使KB细胞的增殖减少50%至90%。游离的AEGFR2几乎不引起生长抑制，而游离的AEGFR2S的效力仅为叶酸- peg -脂质体封装的寡核苷酸的五分之一。寡核苷酸处理细胞的生长抑制可能是由于EGFR表达减少，因为用抗EGFR单克隆抗体对细胞进行间接免疫荧光染色显示，用叶酸- peg -脂质体包裹的egfr2处理的细胞中，EGFR几乎定量减少。这些结果表明，反义寡核苷酸包封在叶酸- peg脂质体中有望有效和肿瘤特异性递送，而通过这种途径递送时，硫代寡核苷酸似乎没有比天然磷酸二酯DNA更大的优势。
Antisense oligodeoxyribonucleotides targeted to the epidermal growth factor (EGF) receptor were encapsulated into liposomes linked to folate via a polyethylene glycol spacer (folate-PEG-liposomes) and efficiently delivered into cultured KB cells via folate receptor-mediated endocytosis. The oligonucleotides were a phosphodiester 15-mer antisense to the EGF receptor (EGFR) gene stop codon (AEGFR2), the same sequence with three phosphorothioate linkages at each terminus (AEGFR2S), a randomized 15-mer control of similar base composition to AEGFR2 (RC15), a 14-mer control derived from a symmetrized Escherichia coli lac operator (LACM), and the 5'-fluorescein-labeled homologs of several of the above. Cellular uptake of AEGFR2 encapsulated in folate-PEG-liposomes was nine times higher than AEGFR2 encapsulated in nontargeted liposomes and 16 times higher than unencapsulated AEGFR2. Treatment of KB cells with AEGFR2 in folate-PEG-liposomes resulted in growth inhibition and significant morphological changes. Curiously, AEGFR2 and AEGFR2S encapsulated in folate-PEG-liposomes exhibited virtually identical growth inhibitory effects, reducing KB cell proliferation by > 90% 48 hr after the cells were treated for 4 hr with 3 microM oligonucleotide. Free AEGFR2 caused almost no growth inhibition, whereas free AEGFR2S was only one-fifth as potent as the folate-PEG-liposome-encapsulated oligonucleotide. Growth inhibition of the oligonucleotide-treated cells was probably due to reduced EGFR expression because indirect immunofluorescence staining of the cells with a monoclonal antibody against the EGFR showed an almost quantitative reduction of the EGFR in cells treated with folate-PEG-liposome-entrapped AEGFR2. These results suggest that antisense oligonucleotide encapsulation in folate-PEG-liposomes promise efficient and tumor-specific delivery and that phosphorothioate oligonucleotides appear to offer no major advantage over native phosphodiester DNA when delivered by this route.