New cell transfection methodology

Gene therapy is emerging as an alternative therapy for a number of genetic diseases as well as cancer. The process entails the delivery of the healthy gene into diseased cells or other DNA to help kill cells or stimulate an immune response. The efficient delivery of the DNA into cells is paramount for success.

Cationic lipids are often employed to deliver the DNA into the cells via a charge-mediated interaction. These complexes can also carry surface functionalisation with polyethylene glycol (PEG) and signal peptides to facilitate targeted delivery and internalisation.

The scope of the EU-funded TRANSFECTDNA ("Surface functionalised" cationic liposome-DNA complexes containing peptide-lipids with poly(ethylene glycol) spacers: structure, transfection efficiency and interactions with the cytoskeleton) project was to obtain an in-depth understanding of the formation of cationic liposome-DNA complexes and their interaction with cells. The ultimate goal was to unravel the mechanisms and determine the key parameters of gene delivery and gene release.

For this purpose, they employed innovative methodologies alongside small-angle-X-ray-scattering (SAXS). They built an X-ray device that carried a microfluidic chip and a microfluidic sample holder for in-situ SAXS and simultaneous imaging of the flowing material. To validate the functionality of the novel device, researchers used it to study two liquid crystal model systems.

The microfluidic apparatus coupled with X-ray scattering provided a suitable platform to study in-situ structural transitions, and unravel pathways of complex assembly dynamics. Scientists were able to assess the self-assembly of neurofilament proteins as this novel apparatus recapitulated more closely the natural environment of neuronal axons.

Subsequent preparation of lipid-DNA nanoparticles in the microfluidic instrument indicated that the solvent-shifting technique should be performed for optimal complex formation. Analysis of PEG liposome DNA complexes using SAXS indicated a well-defined size in water, which is altered upon incubation in serum.

Overall, the novel microfluidic device is expected to permit the preparation of lipid-DNA particles with controlled number of layers and added functionalisation. The direct observation of the complexation mechanism should be especially useful for academia and pharmaceutical companies.