*3.3. Atomic Force Microscopy (AFM)*

OND and the cationic lipid-OND complexes were imaged by AFM in order to compare their size and morphology. It is important to note that AFM imaging only provides information on surface immobilized structures, and the absolute accuracy is limited by the physical convolution between the tip (radius around 7 nm) and the sample. Nevertheless, AFM topography images are useful for comparing the sizes and for providing a direct observation of the morphology of the OND and complexes on the surfaces.

Figure 3A shows a topographic image of OND molecules dispersed on mica. The molecules were compacted instead of stretched out over the mica substrate, suggesting a potential effect of Mg2+ (a bridging agent used for deposition on the mica substrate) on the molecular conformation [34]. An analysis of height cross-sections indicates a step height of around 1 nm, which is in agreement with the size of a single strand DNA OND, as a double helix of DNA has a diameter of 20 Å (=2 nm) [43,44]. *Pharmaceutics* **2021**, *13*, x 12 of 27

**Figure 3.** Atomic force microscopy (AFM) topography images of structures absorbed in air on mica: (**A**) OND—the height profile corresponds to three colored sections in the graph below the image, (**B**) dimethyldioctadecylammonium bromide (DDAB)-OND complex and (**C**) 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-OND complex, with both complexes prepared at a charge ratio 3:1 (cationic lipid:OND). **Figure 3.** Atomic force microscopy (AFM) topography images of structures absorbed in air on mica: (**A**) OND—the height profile corresponds to three colored sections in the graph below the image, (**B**) dimethyldioctadecylammonium bromide (DDAB)-OND complex and (**C**) 1,2-dioleoyl-3 trimethylammonium propane (DOTAP)-OND complex, with both complexes prepared at a charge ratio 3:1 (cationic lipid:OND).

(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

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

Upon mixing and complexing DOTAP with OND, the melting temperature de-

more fluidity, resulting in a lower melting point [45].

creased to 1.3 and 3.5 °C, respectively (Figure 4B).

physical mixture.

The AFM topographic image of DDAB-OND complexes (Figure 3B) reveals a mixture of nanoparticulate complexes and membrane patches which are presumably the result of collapsed lipid capsules during sample drying. The height of the membrane patches is 4 to 5 nm. In contrast, the topographic image of the DOTAP-OND complexes (Figure 3C) shows mostly homogenous and intact particles. Average height of the particles was 15.1 ± 6.3 nm (*n* = 167) and 20.9 ± 3.5 nm (*n* = 70) for DDAB-OND and DOTAP-OND, respectively. The actual height of both complexes in solution should be higher, as significant deformation is expected during drying and immobilization on the substrate during sample preparation. The spherical lipid capsule collapsed into a shape of a flattened disc, having maintained its volume. Using the reported particle volume from SPIP software, the diameter of the particles was calculated for the original thermodynamically favoured spherical shape. The expected diameters of the spherical complexes are in the range of 31.6–63.4 nm for the DDAB-OND complexes and 75.4–128.2 nm for the DOTAP-OND complexes.
