*2.2. Complex Preparation*

The OND and a cationic lipid (DDAB or DOTAP) were dissolved in 1 mL of Bligh–Dyer monophase consisting of chloroform:methanol:water 1:2.2:1, as previously described [33]. The monophase was separated into a two-phase system by the addition of an aliquot of 250 µL of water and chloroform. Subsequently, the mixture was vigorously vortexed for 1 min and centrifuged at 600× *g* for 5 min to achieve complete phase separation. The chloroform phase containing OND complexed with DDAB and DOTAP was collected, and the solvent was evaporated under nitrogen flow. The amount of complexed OND was quantified indirectly from the amount of noncomplexed OND in the aqueous phase. The fluorescence of FAM-labeled OND was measured in the aqueous phase at the excitation and emission wavelengths of 495 nm and 520 nm, respectively, in a microplate reader (FLUOstar Omega, BMG Labtech, Ortenberg, Germany). Complexation efficiency (CE) was calculated as follows:

CE = [(initial amount of OND-amount of OND present in aq.phase)/(initial amount of OND)] × 100% (1)

Various charge ratios (+/−) of the cationic ammonium head of a cationic lipid and the anionic phosphate group of the OND backbone were tested in order to achieve efficient CE (>95%), starting at the ratio 1:1 and increasing the amount of cationic lipid. Prepared complexes were stored at −20 ◦C until further use [22,23].

To evaluate the changes introduced by complexation of a cationic lipid and OND, a physical mixture was prepared at the charge ratio of cationic lipid:OND 3:1. The required amount of a cationic lipid was weighed in a 4 mL screw thread glass vial (Thermo Fisher Scientific (Waltham, MA, USA). It was dissolved in a volume of chloroform sufficient to completely dissolve the lipid. Chloroform was subsequently evaporated under a nitrogen stream. The corresponding amount of OND solution was added into the glass vial, and the solvent was evaporated under the nitrogen stream. Solid lipid and OND were mixed into a physical mixture.
