Stability of PEI-DNA and DOTAP-DNA complexes:: effect of alkaline pH, heparin and serum
Stability of PEI-DNA and DOTAP-DNA complexes:: effect of alkaline pH, heparin and serum
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DOI：
10.1016/s0168-3659(01)00415-1
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发表时间：
2001-09-11
影响因子：
10.8
通讯作者：
Aliño, SF
中科院分区：
文献类型：
--
作者：
Moret, I;Peris, JE;Aliño, SF
DNA复合物与非病毒载体如聚乙烯亚胺（PEI）或1,2-二酰-3-三甲基氨丙烷（DOTAP）形成广泛应用于基因治疗。这些复合物阻止了DNA与通常用于定量DNA的荧光探针的相互作用。因此，我们研究了DNA复合体的DNA定量程序及其在DNA酶或小鼠、大鼠和人血清中的稳定性。通过增加培养基的pH值（从7.3到13.4）或加入肝素，DNA从复合物中释放出来。在体外，通过在dna酶I和三个物种的血清存在下，在37℃的温度下培养复合物，来测试其抗降解的稳定性。高pH值和肝素都能从复合物中释放DNA。裸DNA与血清蛋白形成聚集体，延迟电泳迁移，这种效果在肝素存在时被逆转。然而，这些聚集体不能保护DNA不被血清DNA酶消化，血清的DNA消化能力为：小鼠>大鼠>人。从复合体的DNA抵抗DNA酶I的降解，虽然从复合体的DNA部分被消化的低比例，由电泳确定。相比之下，PEI-DNA和DOTAP-DNA复合物在所有血清中都是稳定的。肝素和高pH释放DNA复合物。DNA降解顺序为：小鼠>大鼠>人，但DOTAP和PEI避免了血清化合物对DNA的降解。(C) 2001 Elsevier Science B.V.版权所有
DNA complexes formed with nonviral vectors such as polyethylenimine (PEI) or 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) are widely used in gene therapy. These complexes prevent the interaction of DNA with the fluorescent probes usually employed to quantify DNA. We thus studied the procedures for DNA quantification from DNA complexes as well as their stability in the presence of DNase or mouse, rat and human sera. Release of the DNA from its complexes was accomplished by increasing the pH of the medium (from 7.3 to 13.4) or by adding heparin. The stability against degradation was tested in vitro, by incubating the complexes at 37 degreesC in the presence of DNase I and sera from the three species. Both high pH and heparin were able to release DNA from its complexes. Naked DNA formed aggregates with serum proteins that delayed electrophoresis migration, and this effect was reversed in the presence of heparin. However, these aggregates did not protect DNA from digestion by serum DNase, and the DNA digesting ability of serum was: mouse>rat>human. The DNA from the complexes was resistant to degradation by DNase I, although a low proportion of DNA from the complexes was partially digested, as determined by electrophoresis. In contrast, PEI-DNA and DOTAP-DNA complexes were stable in the presence of all sera. Heparin and high pH release DNA from its complexes. The order of DNA degradation is: mouse>rat>human, but DOTAP and PEI avoid degradation of DNA by serum compounds. (C) 2001 Elsevier Science B.V. All rights reserved.