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Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships.
Plasmid DNA were covalently biotinylated with guanine-specific biotin labels as described by the manufacturer (Mirus Bio, Madison, WI) but scaled to have ~1–2 biotin labels per DNA. Plasmid DNA (pEGFP-C1, 4.9 kb, Clontech, Mountain View, CA) was labeled in this protocol.
|DNase-free and RNase-free water||75 µL|
|10X Labeling Buffer A||20 µL|
|1µg/µL DNA||100 µL|
|Label/IT reagent||5 µL|
|Total Volume||200 µL|
Chitosan (390 kDa, 83.5% deacetylated, Vanson, Redmond, WA) was used as a model cationic polymer in this study. The free primary amines on the chitosan polymer backbone were labeled with Cy5-NHS (Amersham Biosciences, Piscataway, NJ).
The molar ratio of pDNA to QD was kept in excess (pDNA : QD ≈ 1 : 2) to ensure complete conjugation of QDs to pDNA. The number of QDs labeled onto each pDNA can be estimated through TEM imaging or other equivalent facilities. In our study, the number of QDs per pDNA is estimated to be ~1–3 by TEM and single molecule spectroscopy.1 Use Millipore Milli-Q gradient water (>18.0 MW, 0.2um filtered) during the preparation.
Funding support provided by NIH grant HL89764, NSF grants 0546012, 0730503 and 0725528.