Burckbuchler V, Wintgens V, Lecomte S, Percot A, Leborgne C, Danos O, Kichler A, Amiel C
Laboratoire de Recherche sur les Polymères, 2-8 rue Henri Dunant, 94320 Thiais, France.
Biopolymers. 2006 Apr 5;81(5):360-70. doi: 10.1002/bip.20428.
The ability of DNA to bind polycation yielding polyplexes is widely used in nonviral gene delivery. The aim of the present study was to evaluate the DNA compaction with a new DNA vector using Raman spectroscopy. The polyplexes result from an association of a beta-cyclodextrin polymer (polybeta-CD), an amphiphilic cationic connector (DC-Chol or adamantane derivative Ada2), and DNA. The charge of the polymeric vector is effectively controlled by simple addition of cationic connector in the medium. We used surface enhanced Raman spectroscopy (SERS) to characterize this ternary complex, monitoring the accessibility of adenyl residues to silver colloids. The first experiments were performed using model systems based on polyA (polyadenosine monophosphate) well characterized by SERS. This model was then extended to plasmid DNA to study polybeta-CD/Ada2/DNA and polybeta-CD/DC-Chol/DNA polyplexes. The SERS spectra show a decrease of signal intensity when the vector/DNA charge ratio (Z+/-) increases. At the highest ratio (Z+/- = 10) the signal is 6-fold and 3-fold less intense than the DNA reference signal for Ada2 and DC-Chol polyplexes, respectively. Thus adenyl residues have a reduced accessibility as DNA is bound to the vector. Moreover, the SERS intensity variations are in agreement with gel electrophoresis and zeta potential experiments on the same systems. The overall study clearly demonstrates that the cationic charges neutralizing the negative charges of DNA result in the formation of stable polyplexes. In vitro transfection efficiency of those DNA vectors are also presented and compared to the classical DC-Chol lipoplexes (DC-Chol/DNA). The results show an increase of the transfection efficiency 2-fold higher with our vector based on polybeta-CD.
DNA与聚阳离子形成多聚体的能力在非病毒基因递送中被广泛应用。本研究的目的是使用拉曼光谱法评估一种新型DNA载体对DNA的压缩作用。多聚体由β-环糊精聚合物（聚β-CD）、两亲性阳离子连接体（DC-Chol或金刚烷衍生物Ada2）与DNA结合而成。通过在介质中简单添加阳离子连接体，可有效控制聚合物载体的电荷。我们使用表面增强拉曼光谱（SERS）对这种三元复合物进行表征，监测腺苷残基与银胶体的可及性。最初的实验使用基于聚A（聚腺苷酸单磷酸）的模型系统，该系统已通过SERS得到充分表征。然后将该模型扩展到质粒DNA，以研究聚β-CD/Ada2/DNA和聚β-CD/DC-Chol/DNA多聚体。SERS光谱显示，当载体/DNA电荷比（Z+/-）增加时，信号强度降低。在最高比例（Z+/- = 10）时，对于Ada2和DC-Chol多聚体，信号强度分别比DNA参考信号低6倍和3倍。因此，随着DNA与载体结合，腺苷残基的可及性降低。此外，SERS强度变化与相同系统的凝胶电泳和zeta电位实验结果一致。整体研究清楚地表明，中和DNA负电荷的阳离子电荷导致形成稳定的多聚体。还展示了这些DNA载体的体外转染效率，并与经典的DC-Chol脂质体（DC-Chol/DNA）进行了比较。结果表明，基于聚β-CD的载体转染效率提高了2倍。
