Evaluation by fluorescence resonance energy transfer of the stability of nonviral gene delivery vectors under physiological conditions

被引:84
作者
Itaka, K
Harada, A
Nakamura, K
Kawaguchi, H
Kataoka, K [1 ]
机构
[1] Univ Tokyo, Grad Sch Engn, Dept Mat Sci, Bunkyo Ku, Tokyo 1138656, Japan
[2] Univ Tokyo, Fac Med, Dept Orthopaed Surg, Bunkyo Ku, Tokyo 1138655, Japan
关键词
D O I
10.1021/bm025527d
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:841 / 845
页数:5
相关论文
共 22 条
[1]   Recognition of DNA topology in reactions between plasmid DNA and cationic copolymers [J].
Bronich, TK ;
Nguyen, HK ;
Eisenberg, A ;
Kabanov, AV .
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, 2000, 122 (35) :8339-8343
[2]  
CHONN A, 1992, J BIOL CHEM, V267, P18759
[3]   Cationic polymer based gene delivery systems [J].
De Smedt, SC ;
Demeester, J ;
Hennink, WE .
PHARMACEUTICAL RESEARCH, 2000, 17 (02) :113-126
[4]   Cationic lipid-mediated gene transfer: effect of serum on cellular uptake and intracellular fate of lipopolyamine/DNA complexes [J].
Escriou, V ;
Ciolina, C ;
Lacroix, F ;
Byk, G ;
Scherman, D ;
Wils, P .
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 1998, 1368 (02) :276-288
[5]   Tracking the intracellular path of poly(ethylenimine)/DNA complexes for gene delivery [J].
Godbey, WT ;
Wu, KK ;
Mikos, AG .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1999, 96 (09) :5177-5181
[6]   FORMATION OF POLYION COMPLEX MICELLES IN AN AQUEOUS MILIEU FROM A PAIR OF OPPOSITELY-CHARGED BLOCK-COPOLYMERS WITH POLY(ETHYLENE GLYCOL) SEGMENTS [J].
HARADA, A ;
KATAOKA, K .
MACROMOLECULES, 1995, 28 (15) :5294-5299
[7]  
HARADASHIBA M, IN PRESS GENE THER
[8]  
Haugland RP., 1999, HDB FLUORESCENT PROB
[9]  
HERMAN B, 1989, METHODS CELL BIOL
[10]   Influence of hydrophilicity of cationic polymers on the biophysical properties of polyelectrolyte complexes formed by self-assembly with DNA [J].
Howard, KA ;
Dash, PR ;
Read, ML ;
Ward, K ;
Tomkins, LM ;
Nazarova, O ;
Ulbrich, K ;
Seymour, LW .
BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 2000, 1475 (03) :245-255