*2.2. NLC Preparation and Optimization*

NLC were prepared based on a previously described melt-emulsification technique [16,24]. Four different formulations were developed, combining solid and liquid lipids. The solid lipid Precirol ATO 5 ® (melting point: 56 ◦C) was used in all formulations; however, different liquid lipids in a different solid:lipid ratio were used, as shown Table 1. All nanoformulations shown in Table 1 were performed in triplicate.


**Table 1.** The composition of the different nanoparticles, using Precirol® ATO 5 and different Ω-3 fatty acids or Mygliol® as a liquid lipid with different solid:liquid lipid ratios.

DHA, its hydroxylated derivate in ethyl ester form (DHAH-EE) and Mygliol® (caprylic/capric glyceride) were liquid at room temperature; however, the hydroxylated derivate of DHA in triglyceride form (DHAH-TG) was slightly more viscous. We used two different variants of DHAH, its hydroxylated derivate in ethyl ester form (DHAH-EE) and triglyceride form (DHAH-TG) in order to select the most suitable one to develop our new nanocarrier.

The lipid phase, containing both the solid and the liquid lipid, was heated 5 ◦C above its melting point until a clear and homogeneous phase was obtained. The lipid phase was formed with a different solid:liquid lipid ratio, as described in Table 1. The aqueous solution was composed of Tween 80 (3% *w*/*v*) and poloxamer 188 (2% *w*/*v*) for a final volume of 4 ml. The aqueous phase was warmed and added to the melted oily phase and sonicated for 60 s at 50 W (Branson® Sonifier 250, Fisher Scientific, Madrid, Spain). The obtained nanoemulsion was maintained under magnetic stirring for 15 min at room temperature and stored at 4 ◦C overnight to allow the recrystallisation of the lipid for NLC formation. On the following day, the nanoparticle dispersion was centrifuged in an Amicon filter (Amicon, "Ultracel-100k", Millipore Sigma Life Sciences, Madrid, Spain) at 2500 rpm (MIXTASEL, JP Selecta, Barcelona, Spain) for 15 min and the nanoparticles were washed three times with milli Q water. Finally, 15% *w*/*w* trehalose was added as cryoprotectant, and the NLCs were lyophilized for 42 h (LyoBeta 15, Telstar, Spain).

Prior to the NLC coating process, TAT was covalently linked to CS by a surface activation method previously described by our research group [16] The CS: TAT employed ratio was 1:0.01 (*w*/*w*). TAT conjugation with CS was prepared through a carbodiimide-mediated coupling reaction [37]. The reaction was made though the EDC/NHS technique, in which the reactive sulfo-NHS ester reacted with the amine functionality of proteins and peptides via the formation of an amide bond [38,39]. Briefly, 250 µL EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) in solution (1 mg/mL) and 250 µL of sulfo-NHS (*N*-hydroxysulfosuccinimide) in 0.02 M phosphate buffered saline (PBS) were added dropwise to a 4 mL CS solution (0.5% *w*/*v* in PBS 0.02 M) under magnetic stirring (2 h at room temperature). For the coupling of TAT, a 250 µL TAT solution (1 mg/mL) in PBS (0.02 M; pH 7.4) was added dropwise to the activated CS under gentle agitation. The TAT-CS solution was maintained under agitation for another 4 h at room temperature and then incubated at 4 ◦C overnight. The next day, the NLC were coated with TAT-CS; for that purpose, an NLC dispersion previously prepared was added dropwise to the TAT-CS solution under continuous agitation for 20 min at room temperature. After the coating process, CS-NLC-TAT nanoformulation was centrifuged in Amicon filters (Amicon, "Ultracel-100k", MiIlipore, Millipore Sigma Life Sciences, Madrid, Spain) at 2500 rpm (MIXTASEL, JP Selecta, Barcelona, Spain) for 15 min and washed three times with Milli Q water. Finally, the nanoformulation was freeze-dried with the cryoprotectant trehalose at a final concentration of 15% (*w*/*w*) of the weighed lipid, and then it was lyophilized for 42 h (LyoBeta 15, Telstar, Terrassa, Spain). This final step, regarding the coating with CS and TAT, was only performed after the election of the lipid type and the solid:lipid ratio, resulting in the formulations named (CS-TAT) DHAH-NLC, or just DHAH-NLC, and (CS-TAT) Mygliol-NLC, or just Mygliol-NLC, used in the following described studies in primary cell cultures.
