*2.4. Critical Aggregation Concentration (CAC) Determination by Nile Red Encapsulation Assay*

Formation of the DOC micelles was characterized as described in the literature [23], using Nile Red as a fluorescent probe. A 10 mM stock solution of the Nile Red in ethanol was used for all experiments. Briefly, 0.7 μM, 1 μM, 3 μM, 10 μM, 30 μM, and 50 μM of the DOC or control oligonucleotide were incubated in eppendorfs with 100 μM Nile Red in TA or TAM buffer at 25 ◦C for 3 h. After incubation, time samples were transferred in TPP® tissue culture plates (Sigma-Aldrich Chemie GmbH, Buchs, Switzerland). The fluorescence intensity spectra of the Nile Red were obtained at room temperature using a CLARIOStar® Microplate reader (BMG LABTECH GmbH, Ortenberg, Germany). Fluorescent measurements were taken at the excitation wavelength of 550 nm and the emission was monitored from 570 to 740 nm. The oligonucleotide without dodecyl chains was used as a control. The critical aggregation concentration (CAC) could be calculated by tracking the fluorescence intensity of Nile Red as a function of the sample concentration. CAC values were calculated from the plot of the emission intensity at 645 nm (in TA buffer) or 630 nm (in TAM) versus the log of concentrations (M) of dodecyl oligonucleotide conjugates. The CAC was obtained from the intersection of two straight tangents to these regions' lines.
