Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions
Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions
复制标题
DOI：
10.1021/bm025527d
复制
发表时间：
2002-07-01
影响因子：
6.2
通讯作者：
Kataoka, K
中科院分区：
文献类型：
--
作者：
Itaka, K;Harada, A;Kataoka, K
利用荧光共振能量转移（FRET）技术，检测荧光素（能量供体）和X-罗丹明（能量受体）同时标记的复合质粒DNA（pDNA）在生理介质中的构象变化，评价复合物型和脂质复合物型非病毒基因载体在生理介质中的稳定性。在与阳离子组分如LipofectAMINE、聚（L-赖氨酸）和聚（乙二醇）-聚（L-赖氨酸）嵌段共聚物（PEG-PLys）混合后，双标记的pDNA的荧光光谱发生了剧烈变化，这是由于通过络合作用在pDNA上的供体-受体对之间发生了FRET，从而形成球状构象（凝聚态）。在20%血清的存在下也进行测量，在该条件下清楚地监测来自浓缩的pDNA的FRET，而不受培养基中共存组分的干扰，允许在生理条件下评价非病毒基因载体中pDNA的浓缩状态。加入血清后立即诱导LipofectAMINE/pDNA（lipoplex）系统的FRET急剧下降，这与含血清培养基中lipoplex系统转染效率的急剧下降一致。相比之下，PEG-PLys/pDNA复合物（聚离子复合物胶束）系统即使在含血清的培养基中也保持了可观的转染效率，并且FRET效率保持恒定长达12 h，表明聚离子复合物胶束在生理条件下的高稳定性。
The stability in physiological medium of polyplex- and lipoplex-type nonviral gene vectors was evaluated by detecting the conformational change of complexed plasmid DNA (pDNA) labeled simultaneously with fluorescein (energy donor) and X-rhodamine (energy acceptor) through fluorescence resonance energy transfer (FRET). Upon mixing with cationic components, such as LipofectAMINE, poly(L-lysine), and poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLys), the fluorescence spectrum of doubly labeled pDNA underwent a drastic change due to the occurrence of FRET between the donor-acceptor pair on pDNA taking a globular conformation (condensed state) through complexation. The measurement was carried out also in the presence of 20% serum, under which conditions FRET from condensed pDNA was clearly monitored without interference from coexisting components in the medium, allowing evaluation of the condensed state of pDNA in nonviral gene vectors under physiological conditions. Serum addition immediately induced a sharp decrease in FRET for the LipofectAMINE/pDNA (lipoplex) system, which was consistent with the sharp decrease in the transfection efficiency of the lipoplex system in serum-containing medium. In contrast, the PEG-PLys/pDNA polyplex (polyion complex micelle) system maintained appreciable transfection efficiency even in serum-containing medium, and FRET efficiency remained constant for up to 12 h, indicating the high stability of the polyion complex micelle under physiological conditions.