*3.1. Screening of Drug and Excipients*

Prior to NLC production, it is mandatory to study the compatibility between solid and liquid lipids and between lipids and the drug to obtain a final formulation with high encapsulation efficiency and long-term stability [50,51]. The components of the NLC formulation were selected from previous work developed by our group [52,53]. Precirol® ATO5 was used as the solid lipid, due to its appropriate melting point (56 ◦C) and ability to form the imperfect lipid matrix of the NLC when mixed with a liquid lipid, which provides a high drug loading capacity [54,55]. Vitamin E was selected as the liquid lipid due to its antioxidant activity, which can delay the neural damage caused by the oxidative stress of Alzheimer's disease, improving the neuroprotective effect of the NLC formulation. In addition, vitamin E decreases the risk of lipid oxidation resulting from the preparation of NLC, increasing the chemical stability of the encapsulated drug; easily solubilizes lipophilic molecules; and has high compatibility with lipids and surfactants [56–58].

The compatibility of different amounts of vitamin E with the solid lipid (Precirol® ATO5) was evaluated in ratios ranging from 50:50 up to 90:10 (solid lipid: liquid lipid, % *w*/*w*). For the experiments, the lipid mixture was heated up to 100 ◦C under stirring at 200 rpm for 1 h and cooled down to room temperature (25 ± 0.5 ◦C). The solidified mixture was then analyzed by passing through a filter paper, where the absence of oil stains indicated the existence of miscibility between the lipids. Afterwards, the best proportion of solid and lipid liquids was selected [59].

To study the compatibility between the drug and lipids, different amounts of the drug were added to the lipid mixture previously selected, using as reference the concentration of a commercial drug solution (2%, *w*/*w*). For the tests, increasing amounts of drug (0.1%, 0.2%, 0.5%, 1%, and 2%) were added to the lipids mixture and heated 10 ◦C above the melting point of the solid lipid (70 ± 0.5 ◦C) under stirring at 500 rpm for 1 h. After solidification by cooling to room temperature, the mixture was placed on a filter paper, where the absence of oil droplets indicated the existence of drug solubility in the lipid mixture [59].

SLN and NLC formulations should include two surfactants that promote steric and electrostatic stabilization, avoiding nanoparticle aggregation and ensuring long-term stability. Surfactants should be selected according to their charge, molecular weight, and adequacy for the desired route of administration for the formulation [22,50,60,61]. Smaller particle sizes have been observed when a higher surfactant/lipid ratio was used [31,62,63]. Accordingly, polysorbate 80 (Tween® 80), a non-ionic surfactant containing a polyoxyethylene chain tetrahydrofuran ring that provides steric stabilization and a hydrophobic tail that prevents particle aggregation, was selected based on previous works that showed its compatibility with the lipids used [46,52,53,59,60,64]. The co-surfactant (Phospholipon® 90G) was selected based on its emulsification capacity for the selected lipid mixture, its non-irritating effect on the nasal mucosa, and its ability to minimize the polymorphic state transitions of lipids. Phospholipon® 90G is a (phosphatidylcholine hydrogenated) biological membrane lipid and an amphoteric surfactant that has a synergic effect with Tween® 80, originating NLCs with smaller particle sizes and high stability [64,65]. Different proportions of surfactant and co-surfactant were used to prepare NLC formulations (Table 1) and the best ratio was selected after analysis of the results of particle size, PDI, ZP, and EE tests (Section 3.5) [32].

Benzalkonium chloride, a quaternary ammonium compound, was used as preservative (0.02%) to prevent microbial proliferation of the NLC formulations due to the high water content. This compound is commonly used in nasal formulations as it exhibits low or no toxicity to the nasal cilia when used in concentrations between 0.01 and 0.02% [66].

Several research studies have described the use of similar components to prepare NLC formulations for nose-to-brain delivery. For instance, Khan et al. developed a hydrogel-containing temozolomide-loaded NLC for nose-to-brain delivery using vitamin E as the liquid lipid [67]. Madane et al. prepared a curcumin-loaded NLC for nose-to-brain delivery using Precirol® ATO5, Tween® 80, and lecithin (a phospholipid similar to Phospholipon® 90G) [33]. Wavikar et al. used Tween® 80 and lecithin to prepare a rivastigmine-loaded NLC for nose-to-brain delivery [68]. Precirol® ATO5 and Tween® 80 were used to prepare a NLC to improve the nose-to-brain transport of a glial cell-derived neurotrophic factor (GDNF) [69]. Tween 80® was used as surfactant olanzapine-loaded NLC [70] and in an asenapine-loaded NLC to promote brain delivery through intranasal administration [71].
