*3.4. Differential Scanning Calorimetry (DSC)*

Figure 4 depicts thermograms of the bulk lipids (DDAB/DOTAP), physical mixtures (mix DDAB/DOTAP+OND) and complexes (DDAB/DOTAP-OND), both at the charge ratio 3:1. The bulk lipids showed melting peaks at 88.5 ◦C and 6.3 ◦C, respectively, for DDAB and DOTAP. This difference is a function of the chemical structure of the cationic lipids. Double bonds in DOTAP's aliphatic C18 chains lead to more loose packing, which provide more fluidity, resulting in a lower melting point [45]. *Pharmaceutics* **2021**, *13*, x 13 of 27

**Figure 4.** Differential scanning calorimetry of the bulk lipids, physical mixtures and complexes of (**A**) DDAB and (**B**) DOTAP. Thermograms were recorded from −50 up to 150 °C at the heating rate 10 °C/min. **Figure 4.** Differential scanning calorimetry of the bulk lipids, physical mixtures and complexes of (**A**) DDAB and (**B**) DOTAP. Thermograms were recorded from −50 up to 150 ◦C at the heating rate 10 ◦C/min.

*3.5. ATR-FTIR Spectroscopy*  In the region of the FTIR spectra depicting changes in base pairing (1800–1500 cm−1), OND bands of 1643 cm−1and 1605 cm−1 were observed (Figure 5A,B). Upon complex formation, these bands moved to the higher wavenumbers of 1659 cm−1 and 1697 cm−1. The positive shift in this FTIR region suggests the disintegration of the hydrogen bonding net-For DDAB, both the physical mixture and the complex revealed several melting peaks, and they melted at lower temperatures (Figure 4A). The melting peaks were found at 56.5, 71.6 and 86.7 ◦C for the physical mixture and at 39.0, 68.3 and 85.0 ◦C for DDAB-OND. Evidently, the complex shows different thermotropic behavior compared to the physical mixture.

work involved in base pairing [24,46]. Although the model OND is single-stranded, partial base pairing could arise. Upon mixing and complexing DOTAP with OND, the melting temperature decreased to 1.3 and 3.5 ◦C, respectively (Figure 4B).

#### The list of complex-specific bands is summarized in Table 5. The OND peak at 1211 *3.5. ATR-FTIR Spectroscopy*

sugar puckering modes.

cm−1 shifted positively to 1257 cm−1 in both complexes but remained at the same position (1211 cm−1) in the mixture. A new band appeared at 1095 cm−1 and 1088–1080 cm−1 in DDAB-OND and DOTAP-OND, respectively. These bands characterize P=O vibrations. The peak at 1018 cm−1, typical for the P-O-C bond, gained more intensity upon complexation. This suggests an increase in the vibrational frequencies of anionic PO2 in the OND In the region of the FTIR spectra depicting changes in base pairing (1800–1500 cm−<sup>1</sup> ), OND bands of 1643 cm−1and 1605 cm−<sup>1</sup> were observed (Figure 5A,B). Upon complex formation, these bands moved to the higher wavenumbers of 1659 cm−<sup>1</sup> and 1697 cm−<sup>1</sup> . The positive shift in this FTIR region suggests the disintegration of the hydrogen bonding

the conformation of deoxyribose (Figure 5A,B). Together with this intense peak, a broader peak at 864 cm−1 in the case of DOTAP-OND suggests an N-type (C3′-endo) sugar puckering mode [46]. For DDAB-OND, no other markers of the N-type puckering mode existed. This could be due to the DDAB peak occurring at a similar wavelength (887 cm−1), resulting in an interference with other N-type marker bands. In the physical mixtures, there were less intensive and broader peaks at 879–887 cm−1 and 840 cm−1 in DDAB-OND and DOTAP-OND, respectively. These bands are characteristic of S-type sugars [46]. In contrast, the bands found in OND showed a rather broad shoulder peak at 818 cm−1. The position of this broad shoulder peak is unspecific and might suggest the presence of both

network involved in base pairing [24,46]. Although the model OND is single-stranded, partial base pairing could arise. *Pharmaceutics* **2021**, *13*, x 14 of 27

**Figure 5.** Attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) absorbance spectra of OND, cationic lipid-OND complexes at various ratios, a cationic lipid-OND physical mixture at the ratio 3:1 and a bulk cationic lipid. Spectra of the OND-specific region (1800–600 cm<sup>−</sup>1) of DDAB-OND (**A**) and DOTAP-OND (**B**), and the spectra of the lipid CH2 stretching vibrations **Figure 5.** Attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) absorbance spectra of OND, cationic lipid-OND complexes at various ratios, a cationic lipid-OND physical mixture at the ratio 3:1 and a bulk cationic lipid. Spectra of the OND-specific region (1800–600 cm−<sup>1</sup> ) of DDAB-OND (**A**) and DOTAP-OND (**B**), and the spectra of the lipid CH2 stretching vibrations (3000–2800 cm−<sup>1</sup> ) of DDAB-OND (**C**) and DOTAP-OND (**D**).

(3000–2800 cm<sup>−</sup>1) of DDAB-OND (**C**) and DOTAP-OND (**D**). The spectra of the cationic lipids carried several specific bands (Figure 5C,D). The ester group of DOTAP gave rise to a peak at 1744 cm−1 [47,48] that also remains in the The list of complex-specific bands is summarized in Table 5. The OND peak at 1211 cm−<sup>1</sup> shifted positively to 1257 cm−<sup>1</sup> in both complexes but remained at the same position (1211 cm−<sup>1</sup> ) in the mixture. A new band appeared at 1095 cm−<sup>1</sup> and 1088–1080 cm−<sup>1</sup> in

lipids. This weak band is likely overlapped by the CH2 scissoring deformation at 1466 cm−<sup>1</sup>

DDAB-OND and DOTAP-OND, respectively. These bands characterize P=O vibrations. The peak at 1018 cm−<sup>1</sup> , typical for the P-O-C bond, gained more intensity upon complexation. This suggests an increase in the vibrational frequencies of anionic PO<sup>2</sup> in the OND backbone as a result of an interaction with the cationic head of a lipid.

An intense peak at 802 cm−<sup>1</sup> was observed in all complexes, suggesting a change in the conformation of deoxyribose (Figure 5A,B). Together with this intense peak, a broader peak at 864 cm−<sup>1</sup> in the case of DOTAP-OND suggests an N-type (C30 -endo) sugar puckering mode [46]. For DDAB-OND, no other markers of the N-type puckering mode existed. This could be due to the DDAB peak occurring at a similar wavelength (887 cm−<sup>1</sup> ), resulting in an interference with other N-type marker bands. In the physical mixtures, there were less intensive and broader peaks at 879–887 cm−<sup>1</sup> and 840 cm−<sup>1</sup> in DDAB-OND and DOTAP-OND, respectively. These bands are characteristic of S-type sugars [46]. In contrast, the bands found in OND showed a rather broad shoulder peak at 818 cm−<sup>1</sup> . The position of this broad shoulder peak is unspecific and might suggest the presence of both sugar puckering modes.

The spectra of the cationic lipids carried several specific bands (Figure 5C,D). The ester group of DOTAP gave rise to a peak at 1744 cm−<sup>1</sup> [47,48] that also remains in the same position after complexation with OND. A weak band of around 1489 cm−<sup>1</sup> characteristic of the trimethylammonium headgroups [47] was not observed in any of the cationic lipids. This weak band is likely overlapped by the CH<sup>2</sup> scissoring deformation at 1466 cm−<sup>1</sup> seen in both lipids. In the formed complexes, this peak did not change its position, which correlates with the behavior of lipid peaks at higher wavelengths in the C-H stretching region. In DDAB, the symmetric (2849 cm−<sup>1</sup> ) and asymmetric (2916 cm−<sup>1</sup> ) CH<sup>2</sup> vibrations and, similarly, the symmetric (2854 cm−<sup>1</sup> ) and asymmetric (2924 cm−<sup>1</sup> ) CH<sup>2</sup> vibrations of DOTAP remained at the same vibrational frequency upon complexation at all tested ratios [47], which indicated no changes in the lipid chain arrangement.

**Table 5.** Bands specific to the complexes of both cationic lipids and OND. Fourier-transform infrared (FTIR) spectra of the respective cationic lipid and OND were subtracted from the FTIR spectra of the complexes to obtain complex-specific bands. Bands were observed in the complexes at all ratios if not otherwise specified.

