Once the lipid nanoparticles (SLN and NLC) loaded with CBD were produced, the mean particle size (Z-Ave) and polydispersity index (PDI) of all developed formulations were determined directly after production and up to 30 days. Results on the day of production (day 0) were obtained for the empty-SLN and empty-NLC (both considered as standard samples) and reached about 201.5 ± 3.80 nm with PDI 0.281 ± 0.01 [n.u.] and 173.3 ± 4.40 nm with PDI 0.235 ± 0.02 [n.u.], respectively.

CBD-cSLN suspensions, containing 4% (w/w) of solid lipid (Compritol^® 888 ATO), 1.5% (w/w) of surfactant (Poloxamer^® 188) and 1% (w/w) of active compound (CBD extract), were produced using a probe sonication and stored at room temperature (25 °C) for 30 days. Then, Z-Ave, PDI, and zeta potential (ZP) were measured 1, 14, and 30 days after production. The results are presented in Figure 2. As a result, the Z-Ave for CBD-cSLN suspensions varied from 217.2 ± 6.50 nm (day 1) with a PDI of 0.273 ± 0.02 [n.u.]. Furthermore, after two weeks of storage at 25 °C, the particle size of cSLN suspensions loaded with CBD extract increased to 227.7 ± 11.10 nm (day 14) with a PDI of 0.301 ± 0.02 [n.u.]. It can indicate a slight agglomeration process. Finally, four weeks after the production, the CBD-cSLN suspensions gained 222 ± 5.00 nm (day 30) with a PDI of 0.320 ± 0.01 [n.u.].

Zeta potential (ZP) of empty cSLN was −18.09 ± 0.81 [mV], whereas of CBD-cSLN was −12.99 ± 0.90 [mV] (day 0) to −12.42 ± 2.14 [mV] (day 30). All of the results are presented in Table 2. It may indicate that the non-ionic nature of the surfactant has formed a spherically stabilizing adsorbed polymer layer in the SLN surface [34].

CBD-cNLC suspensions, consisting of 3% (w/w) of solid lipid (Compritol^® 888 ATO), 1% (w/w) of liquid lipid (Miglyol^® 812), 1.5% (w/w) of surfactant (Poloxamer^® 188) and 1% (w/w) of active compound (CBD extract), were also produced using a probe sonication and stored at room temperature for 30 days. The Z-Ave and PDI were measured 1, 14, and 30 days after production. Figure 3 shows that the Z-Ave for CBD-cSLN suspensions varied from 158.3 ± 6.60 nm (day 1), with a PDI of 0.325 ± 0.02 [n.u.]. Two weeks later, the particle size of cSLN suspensions loaded with CBD extract stored at 25 °C decreased to 128.6 ± 3.10 nm (day 14) with a PDI of 0.327 ± 0.01 [n.u.]. After one month after the production, the CBD-cSLN suspensions amounted to 146.2 ± 2.10 nm (day 30) with a PDI of 0.340 ± 0.01 [n.u.].

Zeta potential (ZP) of empty cNLC was recorded at −13.63 ± 0.89 [mV], whereas of CBD-cNLC was recorded at −5.51 ± 1.58 mV (day 0) to −12.06 ± 1.50 mV (day 30). Results are also presented in Table 3. Similar to empty-cSLN and CBD-cSLN, empty-cNLC and CBD-cNLC also showed that the presence of active compounds decreased the stability of the samples.

Zeta potential is a physical property exhibited by any particle in suspension, macromolecule, or material surface. A significant positive or negative value of the zeta potential of nanoparticles indicates good physical stability due to electrostatic repulsion. The dissociation of acidic groups on the surface of a particle will give rise to a negatively charged surface. Conversely, a primary surface will take on a positive charge. In both cases, the magnitude of the surface charge depends on the acidic or basic strengths of the surface groups and the pH of the solution. The surface charge can be reduced to zero by suppressing the surface ionization by decreasing the pH of negatively charged particles or increasing the pH of positively charged particles. The general dividing line between stable and unstable suspensions is generally taken at either +30 or −30 mV. Particles with zeta potentials more positive than +30 mV or more negative than −30 mV usually are considered stable. In our present case, our particles were slightly negative, showing that it is not the surface electrical charge that contributes to the physical stability of the particles, but rather the stereochemical hindrance promoted by the surfactants surrounding the particles.

The final size may depend on various factors, such as the chemical structure of the lipids and surfactants composing the systems and their chemical interaction. It should be pointed out that the largest particle size on day 14 corresponded to CBD-cSLN containing Compritol^® 888 ATO as solid lipid. NLC suspensions either consisted of one solid lipid or liquid lipid and showed significantly smaller particle size than cSLN. This could be attributed to the structural differences between lipid nanoparticles and the inclusion of Miglyol^® 812 in the cNLC formulations. In addition to that, PDI was similar in all CBD-loaded nanoparticles and varied in the optimal range from 0.2 to 0.3 [n.u.]. The stereochemical stability of lipid nanoparticles was monitored for 30 days. Additionally, previous research has revealed that the concentration of Miglyol^® 812 remarkably impacts the nanoemulsions’ stability, which is a crucial factor influencing particle size [35]. The slight reduction in particle size with the loading of CBD was attributed to the bonds between the CBD molecule and solid and liquid lipids, resulting in an imperfect crystal-type of NLC. It involves mixing spatially different lipids, consisting of many fatty acids, which introduce imperfections in the order of the crystals. The drug loading can further increase the molecular interactions, resulting in a smaller nanoparticle size.