Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA
Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA
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DOI：
10.1038/sj.gt.3300485
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发表时间：
1997-09-01
期刊：
影响因子：
5.1
通讯作者：
Huang, L
中科院分区：
文献类型：
--
作者：
Yang, JP;Huang, L
由于阳离子脂质体最初是作为脂转染试剂而开发的，因此注意到一个缺点，即在将血清添加到脂转染介质中后，脂转染的效率急剧下降。这一缺点阻碍了阳离子脂质体在系统递送基因中的应用。在本研究中，我们发现血清对DC-chol脂质体介导的脂转染的影响取决于脂质体与DNA的电荷比。血清在低电荷比下抑制脂质复合物的脂转染活性，而在高电荷比下增强脂质复合物的脂转染活性。使用 DOTAP/DOPE 而非 lipofectamine 观察到了这种现象。 DNA细胞缔合的测量表明，在所有测试的电荷比（即0=10.6）下，血清可以减少脂质复合物与细胞的结合。通过 DEAE-Sephacel 柱从血清中去除带负电荷的蛋白质，消除了血清对脂转染的抑制作用。该级分仅含有带负电荷的血清蛋白，其在低电荷比下强烈抑制脂转染，但在较高电荷比下则不抑制。此外，用带正电荷的聚赖氨酸预孵育血清，可以中和带负电荷的血清蛋白，消除血清对阳离子脂质体的抑制作用是由于带负电荷的血清蛋白造成的，可以通过增加阳离子脂质体-DNA lipoplex的电荷比或用聚赖氨酸中和血清来克服。
Since cationic liposome was first developed as a lipofection reagent, a drawback has been noted in that the efficiency of lipofection decreases dramatically after addition of serum to the lipofection medium. This drawback hampers the application of cationic liposome for systematic delivery of genes. In the present studies, we found that the effect of serum on DC-chol liposome-mediated lipofection is dependent on the charge ratio of liposome to DNA. Serum inhibited lipofection activity of the lipoplex at low charge ratios, whereas it enhanced the lipofection activity at high charge ratios. This phenomenon was observed using DOTAP/DOPE but not lipofectamine. Measurement of cellular association of DNA showed that serum could reduce the binding of lipoplex to cells at all tested charge ratios, ie 0=10.6. Removal of negatively charged proteins from serum by DEAE-Sephacel column abolished the inhibitory effect of serum on lipofection. The fraction contained only negatively charged serum proteins which strongly inhibited lipofection at low charge ratios but not at higher charge ratios. Furthermore, preincubation of serum with positively charged polylysine, which neutralized negatively charged serum proteins, eliminated the inhibitory cationic liposome by serum is due to negatively charged serum proteins and it can be overcome by increasing charge ratio of cationic liposome-DNA lipoplexes or by neutralizing the serum with polylysine.