2.6. Preparation of Solid Lipid Nanoparticles (SLN)

Solid lipid nanoparticles (SLN) were prepared by the phase inversion temperature (PIT) method, according to a procedure previously described [31]. For the preparation of these carriers, the following compounds were used in the oil phase: CP, oleth-20, GO, IDE and IDEPCA. The percentages (w/w) of each compound used to prepare such SLN are reported in Table 2. IDE was loaded into SLN at the maximum percentage used in topical formulations (1% w/w) and its derivative IDEPCA was used in an equimolar amount (1.33% w/w). The aqueous phase consisted of deionized water containing Acnibio AC^® 0.05% w/w and Kemipur 100 0.35% w/w as preservatives. Unloaded (SLN A) and IDE or IDEPCA loaded SLN were prepared using the same procedure. Briefly, the oil phase and the aqueous phase were separately heated and when both the oil and the water phase were at 90 °C, the aqueous phase was slowly added to the oil phase under stirring. Then, the mixture was allowed to cool to room temperature under continuous mixing and the phase inversion temperature (PIT) was recorded using a conductivity meter (model 525, Crison, Modena, Italy) when the turbid mixture turned into clear. Thin layer chromatography (TLC) analyses were performed to assess IDE and its derivative stability under these conditions, as previously reported [10]. TLC analyses were carried out as described in Section 2.2. Chemistry. TLC analyses pointed out that no degradation of IDE or IDEPCA occurred under these conditions.

Composition of the oil phase of unloaded and drug loaded solid lipid nanoparticles (SLN) ^a.

^a Water phase q.s. 100.00%. IDE = idebenone, IDEPCA = idebenone ester with pyroglutamic acid, GO = glyceryl oleate, CP = cetyl palmitate.

SLN samples were stored in airtight jars at room temperature and in the dark until used.