Two methods of formulating anionic nanocomplexes were evaluated. In both, nanocomplexes were prepared in water at a range of molar charge ratios of L to D while the peptide P to D molar charge ratio was maintained constant at 3:1. Method 1 (L:D:P): DNA was first added to an anionic liposome (LA, LAP1 or LAP2) and incubated for 15min at room temperature and then the peptide was added with rapid mixing and incubated at room temperature for a further 20min; Method 2 (P:D:L): the peptide was added to the DNA and incubated for 15min at room temperature and then liposome was added with rapid mixing and incubated at room temperature for a further 20min. Irrespective of the method of order of mixing, all molar charge ratios in this study refer to L:P:D. Cationic formulations LPD and LCPRGPD were prepared in the order L:P:D as described previously; first, the peptide was added to the liposome DOTMA/DOPE or LCPRG followed by addition of the DNA with rapid mixing and incubated for 30min at room temperature to allow for complex formation [30]. The nanocomplexes prepared were termed LPD (liposome DOTMA/DOPE), LADP and PDLA (liposome LA), PDLAP1 (liposome LAP1), PDLAP2 (liposome LAP2), PDLAPRG (liposome LAPRG) and LCPRGPD (liposome LCPRG).
