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Understanding the relationship of structural modifications on the assembly and disassembly of synthetic or non-viral gene delivery is crucial with regard to their rational development. This study describes the use of fluorescence correlation spectroscopy (FCS), as a new tool, to investigate the effect of systematic chemical modifications to dicationic N,N-bis(dimethylalkyl)-α,ω-alkanediammonium surfactants (gemini surfactants) on the self-assembly and physical properties of a series of gemini nanoparticles (gemini NPs). A systematic screening of 27 gemini-plasmid (GP) complexes and gemini NPs showed that their final morphology is governed by the pre-compaction of plasmid by the gemini surfactants. The assembly process of gemini-plasmid intermediate complex (GP) and the final gemini NP (or gemini-plasmid-lipid complex, GPL) was monitored by the tracking of the Cy5-labeled plasmid. Based on diffusion properties, GP complexes were larger than gemini NPs (300-500 nm for GP and 200-300 nm for GPLs). Stoichiometric analysis of the raw intensity histograms showed that both GPs and GPLs particles were composed of multiple plasmids. The final GPLs contain fewer plasmids (2-20 per particle) compared to the intermediate GP (5-35 per particle). The addition of phospholipids dispersed and stabilized GPs to form GPL, but the type of phospholipid (DOPE or DD 1:3) had little effect on the final size of the particles. The FCS data were both validated and complemented by the results of studies of dynamic light scattering (DLS), atomic force microscopy (AFM), X-ray scattering and dye-exclusion assays. A model for gemini NP assembly involving supramolecular aggregate intermediates is proposed.

Impact of phospholipids on plasmid packaging and toxicity of gemini nanoparticles