Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET.
We demonstrate a highly sensitive method to characterize the structural composition and intracellular fate of polymeric DNA nanocomplexes, formed by condensing plasmid DNA with cationic polymers through electrostatic interactions. Rational design of more efficient polymeric gene carriers will be possible only with mechanistic insights of the rate-limiting steps in the non-viral gene transfer process. To characterize the composition and binding dynamics of nanocomplexes, plasmid and its polymer carrier within nanocomplexes were labeled with quantum dots (QDs) and fluorescent organic dyes, respectively, as a donor and acceptor pair for fluorescence resonance energy transfer (FRET). The high signal-to-noise ratio in QD-mediated FRET enabled precise detection of discrete changes in nanocomplex state at the single-particle level, against various intracellular microenvironments. The distribution and unpacking of individual nanocomplexes within cells could thus be unambiguously followed by fluorescence microscopy. QD-FRET is a highly sensitive and quantitative method to determine the composition and dynamic stability of nanocomplexes during intracellular transport, where barriers to gene delivery may be identified to facilitate gene carrier optimization.
Ho
Yi-Ping
YP
Department of Mechanical Engineering, Johns Hopkins University and School of Medicine, Baltimore, Maryland 21218, USA.
Chen
Hunter H
HH
Leong
Kam W
KW
Wang
Tza-Huei
TH
D045744:Cell Line, Tumor; D048271:Chitosan / Q000737:chemistry; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D018014:Gene Transfer Techniques; D006026:Glycoside Hydrolases / Q000737:chemistry; D006493:Heparin / Q000737:chemistry; D006801:Humans; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D053758:Nanoparticles; D010316:Particle Size; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D006493:Heparin; D004247:DNA; D048271:Chitosan; D006026:Glycoside Hydrolases; C019868:chitosanase