Zelphati O, Szoka F C
University of California, School of Pharmacy, Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, San Francisço 94143-04461, USA.
Pharm Res. 1996 Sep;13(9):1367-72. doi: 10.1023/a:1016026101195.
To study the parameters influencing the intracellular trafficking of oligonucleotides delivered by cationic 1,2-dioleoyl-3-trimethy- lammonium-propane (DOTAP) lipids and to elucidate the mechanism of uptake.
We have studied the intracellular localization of fluorescently labeled oligonucleotide (F-ODN) delivered by DOTAP using confocal microscopy and measured inhibition of luciferase synthesis. The delivery mechanism of ODN/DOTAP complexes was investigated using inhibitors of the endocytosis pathway.
F-ODN delivered by DOTAP liposomes redistribute from punctate cytoplasmic regions into the nucleus. The nuclear uptake of F-ODN depends on: charge ratio (+/-), time of incubation, temperature and presence of serum. A positively charged complex is required for enhanced uptake. The association of neutral lipids with DOTAP reduced the optimum charge ratio without altering the delivery efficiency. DOTAP lipids increased > 100 fold the antisense activity of a specific anti-luciferase ODN. Inhibitors of the endocytosis pathway show that the majority of F-ODN are introduced through an endocytic pathway mainly involving uncoated vesicles. Nuclear accumulation of oligonucleotides can be decreased by inhibitors of actin microfilaments, energy metabolism and proteins implicated in the fusion of endosomes. Nuclear uptake is independent of acidification of the endosomal vesicles and unaffected by inhibitors of microtubules.
Oligonucleotides are delivered by cationic lipids into the cytoplasm at an early stage of the endocytotic pathway which leads to a marked increase in antisense activity and oligonucleotide nuclear uptake.
研究影响阳离子型1,2 - 二油酰基 - 3 - 三甲基铵丙烷（DOTAP）脂质递送的寡核苷酸细胞内转运的参数，并阐明摄取机制。
我们使用共聚焦显微镜研究了DOTAP递送的荧光标记寡核苷酸（F - ODN）的细胞内定位，并测量了荧光素酶合成的抑制情况。使用内吞途径抑制剂研究了ODN / DOTAP复合物的递送机制。
DOTAP脂质体递送的F - ODN从点状细胞质区域重新分布到细胞核中。F - ODN的核摄取取决于：电荷比（+/-）、孵育时间、温度和血清的存在。增强摄取需要带正电荷的复合物。中性脂质与DOTAP的结合降低了最佳电荷比，但不改变递送效率。DOTAP脂质使特定抗荧光素酶ODN的反义活性提高了100倍以上。内吞途径抑制剂表明，大多数F - ODN是通过主要涉及无包被小泡的内吞途径引入的。肌动蛋白微丝、能量代谢以及参与内体融合的蛋白质的抑制剂可降低寡核苷酸的核积累。核摄取与内体小泡的酸化无关，且不受微管抑制剂的影响。
寡核苷酸在胞吞途径的早期由阳离子脂质递送至细胞质，这导致反义活性和寡核苷酸核摄取显著增加。
