Division of Biopharmaceutics and Pharmacokinetics, University of Helsinki, Finland.
J Gene Med. 2011 Jul;13(7-8):402-9. doi: 10.1002/jgm.1587.
Polyethylenimine (PEI) polyplexes mediate efficient gene transfer only at high +/- charge ratios at which free noncomplexed PEI is present. The excess of PEI gives polyplexes a positive surface charge that plays a role in polyplex binding on the cell membrane. Although positively charged PEI polyplexes are known to interact with anionic cell-surface glycosaminoglycans (GAGs), the exact role of free PEI in such interactions is unclear.
Chinese hamster ovary wild-type cells and mutants lacking cell-surface GAGs were transfected with marker genes using PEI polyplexes with and without free PEI. The total amount of cell-associated plasmid DNA (pDNA) delivered by polyplexes was determined by quantitative real-time PCR and transgene expression was determined using β-galactosidase and luciferase assays.
Transfection activity of polyplexes without free PEI in cells expressing cell-surface GAGs was low even though pDNA was delivered to cells. In the absence of cell-surface GAGs, polyplexes without free PEI had high transfection efficacy. This indicates that the cell-surface GAGs inhibit transfection by purified polyplexes. PEI polyplexes with free carrier mediated transfection in both normal and GAG-deficient cells because free PEI overcomes the inhibitory effect of cell-surface GAGs on transfection. The intracellular elimination of pDNA was faster in the presence of GAGs and, despite improved transfection, free PEI reduced pDNA association with the cells.
Free PEI is essential for minimizing the undesirable binding of polyplexes to cell-surface GAGs that have a negative impact on transfection. The same mechanism may be important in transfections with other polyplexes that require high charge ratios for transfection.
聚亚乙基亚胺（PEI）多聚物仅在高正/负电荷比下介导有效的基因转移，此时有游离的未复合的 PEI 存在。PEI 的过量赋予多聚物正表面电荷，这在多聚物与细胞膜的结合中起作用。尽管已知带正电荷的 PEI 多聚物与阴离子细胞表面糖胺聚糖（GAG）相互作用，但游离 PEI 在这种相互作用中的确切作用尚不清楚。
使用带和不带游离 PEI 的 PEI 多聚物转染野生型中国仓鼠卵巢细胞和缺乏细胞表面 GAG 的突变体的标记基因。通过定量实时 PCR 测定多聚物递送至细胞的细胞相关质粒 DNA（pDNA）总量，并通过β-半乳糖苷酶和荧光素酶测定测定转导基因的表达。
表达细胞表面 GAG 的细胞中没有游离 PEI 的多聚物的转染活性很低，尽管 pDNA 被递送至细胞。在缺乏细胞表面 GAG 的情况下，没有游离 PEI 的多聚物具有高转染功效。这表明细胞表面 GAG 抑制纯化多聚物的转染。带有游离载体的 PEI 多聚物在正常和 GAG 缺陷细胞中都介导转染，因为游离 PEI 克服了细胞表面 GAG 对转染的抑制作用。在存在 GAG 的情况下，pDNA 的细胞内消除更快，尽管转染得到改善，但游离 PEI 减少了 pDNA 与细胞的结合。
游离 PEI 对于最小化多聚物与具有负向转染影响的细胞表面 GAG 的不需要的结合是必需的。对于需要高电荷比进行转染的其他多聚物，相同的机制可能很重要。
