Petersen Holger, Fechner Petra M, Martin Alison L, Kunath Klaus, Stolnik Snjezana, Roberts Clive J, Fischer Dagmar, Davies Martyn C, Kissel Thomas
Department of Pharmaceutics and Biopharmacy, Philipps University, Ketzerbach 63, D-35032 Marburg, Germany.
Bioconjug Chem. 2002 Jul-Aug;13(4):845-54. doi: 10.1021/bc025529v.
For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology: PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 +/- 59 to 61 +/- 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 +/- 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 +/- 60 nm). The zeta-potential of complexes was reduced with increasing degree of PEG grafting if MW >or= 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution: all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.
对于两个系列的聚乙烯亚胺接枝聚乙二醇（PEI-g-PEG）嵌段共聚物，研究了共聚物结构对DNA络合的影响，并将这些络合物的物理化学性质与血液相容性、细胞毒性和转染活性测定结果进行了比较。在第一个系列中，将PEI（25 kDa）以不同的取代度接枝到PEG（5 kDa）上，在第二个系列中，改变PEG的分子量（MW）（550 Da至20 kDa）。使用原子力显微镜，我们发现共聚物嵌段结构强烈影响DNA络合物的大小和形态：5 kDa的PEG显著将球形络合物的直径从142±59 nm减小到61±28 nm。随着PEG接枝度的增加，DNA的络合受到阻碍，络合物失去球形。含有20 kDa PEG的共聚物与DNA形成小而紧密的络合物（51±23 nm），而含有550 Da PEG的共聚物则形成大而分散的结构（130±60 nm）。如果MW≥5 kDa，随着PEG接枝度的增加，络合物的ζ电位降低。550 Da的PEG不能充分屏蔽PEI的正电荷，导致溶血和红细胞聚集。细胞毒性（乳酸脱氢酶测定）与PEG的MW无关，但受PEG取代度的影响：所有具有六个以上PEG嵌段的共聚物形成低毒性的DNA络合物。最后，研究了络合物的转染效率。如含有许多550 Da PEG嵌段的共聚物那样，大颗粒、低毒性和高正表面电荷的组合被证明对体外基因转移最有效。总之，发现PEG化程度和PEG的MW强烈影响PEI的DNA缩合，因此也影响PEI-g-PEG/DNA络合物的生物活性。这些结果为嵌段共聚物基因递送系统的合理设计提供了基础。
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