Developing non-viral transfection vectors is vital in advancing gene therapies to treat a plethora of diseases. One promising class of gene delivery vehicles is polymers, which have high transfection efficiencies, but its delivery mechanism remains unclear. Polymer-DNA complexes are small nanoparticles and existing methods lack the spatiotemporal resolution to monitor polyplex, lipid, and protein receptor interactions in situ. To better understand gene delivery, it is necessary to develop methods that can resolve local interactions of polymers with the cell in real-time. Here, we describe ongoing work to understand polymer-mediated gene delivery through the use of super-resolution stimulated Raman microscopy, a novel, sub-diffraction, label-free imaging method that our group has pioneered. Specifically, we describe the development of our newest generation stimulated Raman microscope, built around the Fidelity HP laser and a photonic crystal fiber (PCF), which is used to generate a stable, broadband Raman probe pulse that spans from 1000 – 2200 nm. The continuum output of the PCF is linearly chirped, which can enable hyperspectral imaging through spectral focusing by changing the delay times between the pump and probe pulses. We also describe our work to understand polymer-DNA complexation using Raman spectroscopy. Specifically, we examine a novel polymer, comprised of hydroxyethyl acrylate co-polymerized with quinine, that displays high transfection efficiency. The strongest Raman band of this polymer, which corresponds to the symmetric ring stretching mode of quinine, shows solvatochromic shifts that are well-described by the vibrational Stark effect and can be used to estimate the local electric field felt by the quinoline ring moiety. This mode also shows characteristic frequency shifts that are diagnostic of π-stacking and hydrogen bonding, which can track DNA complexation of the polymer. We anticipate this mode will serve as a sensitive spectroscopic marker to probe polymer-membrane interactions.

Super-resolution Raman imaging to understand polymer-mediated gene delivery

256th National Meeting (2018)

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