Evaluating the intracellular stability and unpacking of DNA nanocomplexes by quantum dots-FRET

Abstract

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.

Polyethylenimine600-β-cyclodextrin: a promising nanopolymer for nonviral gene delivery of primary mesenchymal stem cells

“…26 The transfection medium was discarded after the cells were incubated for 4 hours. Subsequently, the cells were either not stained or just stained with Hoechst 33342.…”

Theranostics: A Way of Modern Medical Diagnostics and the Role of Chitosan

“…QD FRET studies was also studied using confocal microscopy at different time intervals after transfection in Human Embryonic Kidney 293 (HEK293) cells to study unpackaging of chitosan -p DNA aggregates. These analysis indicates that at 24 h after transfection, polyplexes were bounded in the perinuclear domain, whereas at 72 h post-transfection, polyplexes were disengaged and DNA was located in the nucleus without any signal emitted from chitosan (Cy5) [26]. Figure 3, shows the illustration of QD which serves as both a diagnostic agent (imaging) and a nanocomposites to incorporate multiple functional techniques, like a targeting ligand (peptide, antibody, or protein) and a therapeutic carrier.…”

“…The authors' laboratory has developed a highly luminescent chitosan-l-cysteine functionalized CdTe QDs film with the positive result of antibacterial study which reveals that the synthesized QDs based chitosan film is a promising candidate for wide range of biomedical applications [25]. Ho et al [26] displayed the intracellular detachment of polymer DNA complexes which further analysed by QD FRET (Fluorescence Resonance Energy Transfer). The researchers developed streptavidinfunctionalized, Cy5-labeled chitosan which was combined to QD-labeled pDNA through chemical bonding between biotin streptavidin.…”

Quantum dots, lighting up the research and development of nanomedicine

“…Up to now it is still a major challenge to gain a mechanistic understanding of the rate-limiting steps. Ho YP et al 81 made a valuable attempt to clarify this question relying on QD-FRET technique. As shown in Figure 4, the component plasmid DNA and polymeric gene carrier were individually labeled with QDs and Cy5 dyes, respectively, as a donor and acceptor pair for FRET.…”