*2.11. Degradation by Nucleases*

In order to assess the ability of the SEDDS to protect the encapsulated OND complexes against degradation by nucleases, formulations were incubated with S1 nuclease (100 U/µL), an enzyme that specifically degrades single-stranded nucleic acids. Stability studies were carried out in the following formulations: aqueous solution of naked OND, aqueous dispersion of complexed DDAB-OND in the SEDDS and DOTAP-OND in the SEDDS and dispersion of the nonloaded SEDDS in an aqueous solution of naked OND. The SEDDS were dispersed immediately before the experiment.

The S1 nuclease assay (Thermo Fisher Scientific, Waltham, MA, USA) was performed according to the manufacturer's instructions. Briefly, equivalents containing 1.2 µg of OND were incubated with 12 U of S1 nuclease in the presence of the reaction buffer pH 4.5 at 25 ◦C. As controls, the same formulations were incubated in an acetate buffer of pH 4.5. The reaction was terminated after 30 min by the addition of 0.5-M EDTA and incubation for 10 min at 70 ◦C. Nondegraded intact OND was precipitated via ethanol precipitation. From each formulation, 60 µL was taken, and 1.3 µL of 5%NH4OH, 30 µL of 7.5-M ammonium acetate and 400 µL of 96% ethanol were added into the samples, which were kept at −80 ◦C for at least one hour. Following this, the samples were centrifuged at 15,000× *g* for 30 min at 4 ◦C. Intact OND was pelleted and dissolved in 500 µL of 100-mM triethylammonium acetate (TEAA). The samples were subjected to HPLC analysis. Additionally, 100 µL of supernatant was diluted with 500 µL of 100-mM TEAA.

Thirty microliters of the samples were analyzed using the Waters Symmetry C18 (3.5 µm; 4.6 × 75 mm) HPLC column (Waters Corporation, Milford, MA, USA). The mobile phase consisted of 2 eluents, eluent A of 100-mM TEAA (pH 7.0) and eluent B of acetonitrile, with a linear gradient starting from 1 min at 10% to 30% B in 4 min. Thirty percent B eluent was maintained for 1 min, then decreased to 10% B in 1 min and maintained at this level for 4 min before analysis of the following sample. The flow rate was 1 mL/min. FAM-labeled OND was detected by fluorescent detection excitation/emission 495 nm/520 nm according to the procedure described previously [42]. Each formulation was tested in triplicate. The percentage of intact OND was calculated as a ratio of area under the curve (AUC) of protected OND found in the pellets after incubation with S1 nuclease and pelleted OND recovered from the respective formulations, as described by Equation (2).

intact OND = [AUC of pelleted OND (formualtation + S1 nuclease)/AUC of pelleted OND (formulation)] × 100% (2)
