*3.1. Complex Preparation*

First, complexes of a cationic lipid and 20-mer fluorescently labeled OND were prepared using the Bligh–Dyer method [23,33]. Various charge ratios between a quaternary ammonium head group of a cationic lipid (N<sup>+</sup> ) and a nucleotide phosphate group in the backbone of 20-mer OND (PO<sup>2</sup> −) were investigated. Upon complexation, no visible precipitated material in the aqueous phase nor at the interface was observed. The CE of DDAB-OND and DOTAP-OND are depicted in Table 3. Efficient CE (>95%) was achieved for the charge ratio of cationic lipid:OND 3:1, which equals the molar ratio 60:1. Moreover, at this charge ratio, there was no significant difference in CE between DDAB and DOTAP. It is noteworthy that DOTAP also enables a more efficient hydrophobization of OND at a lower charge ratio than DDAB.

**Table 3.** Complexation efficiency (CE, %) of OND with a cationic lipid (DDAB or DOTAP) using the Bligh–Dyer method. Various charge ratios between a quaternary ammonium head group of a cationic lipid (N+) and a nucleotide phosphate group in the backbone of 20-mer OND (PO<sup>2</sup> −) were tested.


Results are presented as mean ± SD (*n* = 3).
