## 3 Results and Discussion
These values suggest that LP2 caused an increase in lipid cooperativity. In addition, in LP2-loaded SLNs' calorimetric curve, there was no evidence of the peak at 57.78 \({}^{\circ}\)C, characteristic of LP2, thus indicating that the compound lost the crystalline form and was present in an amorphous state in the SLNs' structure. From a morphological point of view, TEM analyses showed that both unloaded and LP2-loaded SLNs were roughly spherical, with no evident sign of aggregation (Figure 3). Prior to performing in vitro release experiments, the LP2 content (as a mixture of 2\(R\) and 2\(S\) isomers) of SLNs samples was determined by the high-performance liquid chromatography (HPLC) method developed in this study. More than 99% (99.9 \(\pm\) 0.3%) of the LP2-loaded dose was recovered from SLNs samples, and no significant difference in the LP2 isomers levels was detected. Being LP2-lipophilic, all of the loaded drug was supposed to be incorporated into the lipid core of SLNs; therefore, the loading capacity of such SLNs was considered equal to 1.1% _w/w_. As reported in the literature [28], when poorly water-soluble compounds are incorporated into lipid nanoparticles, if the resulting colloidal dispersion is clear, all of the drug must be located in the lipid phase of the SLN dispersion. Therefore, in a clear colloidal dispersion, drug incorporation could be regarded as approximately 100%. Figure 2: Calorimetric curves of LP2, unloaded solid lipid nanoparticles (SLNs), and LP2-loaded solid lipid nanoparticles (LP2-loaded SLNs). In vitro release profiles of both LP2 diastereoisomers from the SLNs under investigation are illustrated in Figure 4. LP2 was loaded into SLNs as a racemic mixture because previous studies highlighted that LP2, 2R-LP2, and 2S-LP2 exhibited a different profile in modulating acute thermal nociception. In particular, the anti-nociceptive effect of LP2 maintained higher threshold values for longer periods compared to both isomers, a profile especially useful in persistent pain treatment [11]. LP2 diastereoisomers' release was supposed to occur exclusively from the SLNs core owing to their lipophilicity, which prevented their solubilization in the aqueous medium [28]. After 24 h, the percentage of LP2 released, calculated as the mixture of 2R and 2S diastereoisomers, was 50.2%. Both diastereoisomers showed an initial fast release followed by a second phase of a slower release. Similar in vitro release profiles have already been reported by other authors for lipophilic drugs loaded into SLNs [29; 30]. It is interesting to note that, although the release profile of both diastereoisomers was similar, the 2R-LP2 release rate was greater than that of 2S-LP2. As previously mentioned, diastereoisomers share identical molecular formulas, atom-to-atom linkages, and bonding distances, but the different spatial disposition of the chemical groups bonded to the chiral center could affect their ability to interact with the surrounding environment. Calculating Log \(p\) values of  
Figure 4: In vitro release from solid lipid nanoparticles of 2_R_-LP2 and 2S-LP2. Figure 3: Transmission electron microscopy (TEM) images of unloaded lipid nanoparticles (**a**) and LP2-loaded lipid nanoparticles (**b**). 2\(R\)- and 2\(S\)-LP2 using Marvin 20.11.0, ChemAxon ([[https://www.chemaxon.com](https://www.chemaxon.com)]([https://www.chemaxon.com](https://www.chemaxon.com)), accessed on 24 March 2021), the same value (Log P 4.61) for both isomers was obtained. On the contrary, the lipophilic index, which stemmed from the interaction between the analyzed compound and the stationary (lipophilic) and mobile (hydrophilic) phases in the HPLC column, highlighted that 25-LP2 was more lipophilic (Log K 0.66) than 2\(R\)-LP2 (Log K 0.60), thus suggesting a greater affinity of 25-LP2 for the lipid core of SLNs, which, in turn, could lead to a slower release from the nanoparticles.