*3.1. Nanoparticle Characterization*

The aim of the present work was to develop a new nanocarrier enriched in functional lipids, such as Ω-3 fatty acids, to form the lipid matrix of NLCs. For that purpose, we tried to substitute the lipids of the nanoformulation with different kinds of Ω-3 fatty acids. Precirol ATO 5® was maintained as solid lipid. All modifications in the optimization process of the nanoformulation were performed in the liquid lipid, DHA, DHA-TG and DHA-EE, modifying the liquid lipid type and also the ratio of solid:lipid to formulate NLCs, since the main goal of this research was to increase the percentage of these functional lipids up to the maximum to form this new nanocarrier. In order to achieve this aim, we increased the percentage of lipid liquid from the 0.25% that we used in previous studies with Mygliol® [44–46] to 1.25%, as described in Table 1. We used DHA and two different types of DHAH to select the most suitable one to prepare the NLCs, and we worked with Mygliol®, the liquid lipid previously used in our research group for different clinical applications [15,47], as our control.

Between DHAH-EE and DHAH-TG, the ethyl ester form (DHAH-EE) was liquid at room temperature, and the obtained lyophilized NLCs were easy to handle. However, the NLCs prepared with DHAH-TG became a soggy and sticky powder at room temperature, and this was more difficult to handle after the lyophilization process. That is why the nanoformulations developed with DHAH-TG (C1–C4, Figure 4) were discarded to continue working. *Pharmaceutics* **2020**, *12*, x 12 of 23

nanoformulations). All the data regarding size, PDI and Z potential of all the developed nanoformulations have been summarized in Table 2. As seen, the size of NLC decreased when the percentage of liquid lipid was higher. Moreover, the Z potential of all nanoformulations was negative, which was reverted after CS and TAT coating (Table 3). **Table 2.** Characterization of the developed NLCs (*n* = 3 for all the nanoformulations). The data are presented in media ± SD. NLCs formed with DHA and DHAH-EE showed similar pharmaceutical characteristics; however, as we probed in a previous in vivo study, DHA and DHAH functional lipids showed different biological activity [31]. Therefore, taking into account the remarkably beneficial effects of DHAH, we chose DHAH-EE nanoformulations to continue working. Among the different nanoformulations developed with DHAH-EE (D1–D4, Figure 4), we selected D3 (51.40nm ± 11.65 and 0.415 ± 0.082 for PDI values) since it was the formulation with the highest percentage of Ω-3 fatty acid incorporated into the lipid matrix of NLCs with the best resuspension characteristics, qualitatively determined. In order to confirm the therapeutic effect of DHAH-EE in the newly developed NLCs, we also chose Mygliol-based NLCs

In the next step, the CS and TAT coating process was performed. In Table 2, we can see the results from CS-TAT-NLC-DHAH-EE nanoformulation, called DHAH-NLCs, and from Mygliol-

Both formulations were around 100 nm in size, with a PDI value below 0.5, and exhibited positive zeta values, indicating that the CS and TAT coating process has been successfully performed. In the external morphological study made by TEM (transmission electron microscopy), the

based nanoparticles with CS and TAT, called Mygliol-NLCs.

Formulation A2 73.11 ± 19.51 0.364 ± 0.071 −16.0 ± 2.8 Formulation A3 75.02 ± 6.97 0.474 ± 0.023 −14.2 ± 19.2 Formulation A4 59.38 ± 25.39 0.468 ± 0.052 −14.4 ± 4.8 Formulation B1 94.19 ± 18.01 0.350 ± 0.092 −19.4 ± 1.8 Formulation B2 85.20 ± 12.20 0.461 ± 0.174 −19.9 ± 3.1 Formulation B3 51.98 ± 10.70 0.420 ± 0.202 −22.7 ± 3.5 Formulation B4 49.93 ± 5.36 0.587 ± 0.118 −22.8 ± 4.1 Formulation C1 63.34 ± 6.95 0.366 ± 0.073 −21.0 ± 5.8 Formulation C2 67.02 ± 4.10 0.463 ± 0.031 −16.5 ± 2.3 Formulation C3 78.63 ± 24.66 0.467 ± 0.062 −20.1 ± 3.4 Formulation C4 80.87 ± 15.15 0.418 ± 0.056 −20.4 ± 1.5 Formulation D1 68.62 ± 16.70 0.404 ± 0.068 −22.7 ± 2.6 Formulation D2 76.68 ± 20.12 0.425 ± 0.101 −24.2 ± 2.9 Formulation D3 51.40 ± 11.65 0.415 ± 0.082 −24.1 ± 2.9 Formulation D4 39.98 ± 10.39 0.401 ± 0.076 −24.9 ± 2.8

**Figure 4***.* Characterization of the developed nanostructured lipids (NLCs) (*n* = 3 for all the **Figure 4.** Characterization of the developed nanostructured lipids (NLCs) (*n* = 3 for all the nanoformulations).

in the same proportion of solid:liquid lipid (A3, Figure 4), with a particle size of 75.02 ± 6.97 nm and 0.474 ± 0.023 PDI value, as the control formulation.

All the data regarding size, PDI and Z potential of all the developed nanoformulations have been summarized in Table 2. As seen, the size of NLC decreased when the percentage of liquid lipid was higher. Moreover, the Z potential of all nanoformulations was negative, which was reverted after CS and TAT coating (Table 3).

**Table 2.** Characterization of the developed NLCs (*n* = 3 for all the nanoformulations). The data are presented in media ± SD.


**Table 3.** Characterization of the final nanoformulations used for cell culture tests. (*n* = 2 independent experiments).


In the next step, the CS and TAT coating process was performed. In Table 2, we can see the results from CS-TAT-NLC-DHAH-EE nanoformulation, called DHAH-NLCs, and from Mygliol-based nanoparticles with CS and TAT, called Mygliol-NLCs.

Both formulations were around 100 nm in size, with a PDI value below 0.5, and exhibited positive zeta values, indicating that the CS and TAT coating process has been successfully performed. In the external morphological study made by TEM (transmission electron microscopy), the nanoparticles showed a uniform size without irregularities (Figure 5). The DSC thermograms of the different excipients and formulations have been summarized in Appendix A Figure A1.

On the other hand, as seen in Figure A2, FTIR results exhibited a number of characteristic protein transmission bands (cm−<sup>1</sup> ). Mygliol-NLC and DHAH-NLC without CS and TAT coating showed typical peaks of lipid components, such as O–H stretching (3315), aliphatic C–H (2915, CH3 and CH2) asymmetrical stretching, aliphatic C–H (2852, CH3 and CH2) symmetrical stretching and C=O (1736, carboxylic group) stretching in addition to the vibrations associated with C–O and C–C (1150 and from 992 to 843) bonds. Moreover, CH2 and CH3 stretching (1466) and bending (1342) bands can be seen. Regarding nanoformulations with TAT and CS coating (CS-TAT-Mygliol-NLC and CS-TAT-DHAH-NLC), a new peak can be seen, concretely, in amide I (N–H stretching) (1645). This new peak is due to a chemical interaction. Indeed, it is related to the presence of the amide bond formed after TAT peptide conjugation through a cross linking reaction.

experiments)*.* 

nanoparticles showed a uniform size without irregularities (Figure 5). The DSC thermograms of the

**Table 3***.* Characterization of the final nanoformulations used for cell culture tests. (*n* = 2 independent

**Formulation Size after Lyoph (nm) PDI Z Potential (mV)** 

different excipients and formulations have been summarized in Appendix Figure A1.

**Figure 5.** TEM (transmission electron microscopy) photographs of NLC (scale bar 100 nm). (**A**) hydroxylated derivate of docohexaenoic acid nanostructured lipids (DHAH-NLCs) (**B**) Mygliol-NLCs*.*  **Figure 5.** TEM (transmission electron microscopy) photographs of NLC (scale bar 100 nm). (**A**) hydroxylated derivate of docohexaenoic acid nanostructured lipids (DHAH-NLCs) (**B**) Mygliol-NLCs.

#### On the other hand, as seen in Figure A2, FTIR results exhibited a number of characteristic protein *3.2. Cell Cultures*

*3.2. Cell Cultures* 

*3.3. In Vitro Cell Viability Study* 

transmission bands (cm−1). Mygliol-NLC and DHAH-NLC without CS and TAT coating showed typical peaks of lipid components, such as O–H stretching (3315), aliphatic C–H (2915, CH3 and CH2) asymmetrical stretching, aliphatic C–H (2852, CH3 and CH2) symmetrical stretching and C=O (1736, carboxylic group) stretching in addition to the vibrations associated with C–O and C–C (1150 and from 992 to 843) bonds. Moreover, CH2 and CH3 stretching (1466) and bending (1342) bands can be seen. Regarding nanoformulations with TAT and CS coating (CS-TAT-Mygliol-NLC and CS-TAT-DHAH-NLC), a new peak can be seen, concretely, in amide I (N–H stretching) (1645). This new peak is due to a chemical interaction. Indeed, it is related to the presence of the amide bond formed after TAT peptide conjugation through a cross linking reaction. In order to assess the purity of our primary cultures, an immunofluoresce technique was performed in both dopaminergic and microglia cell cultures. The experiments were performed in triplicate and repeated at least three times independently (*n* = 3 biological replicates). Dopaminergic cell cultures were positive for TH dopaminergic marker, as shown in Figure 6A. In the case of the microglia cell culture, we tested both the microglia marker Iba1 and the astroglia maker GFAP. The culture was shown to be specific in a high percentage to microglia marker (Figure 6B. iv). However, it must be noted that, after this isolation method, the culture is not 100% specific for microglia cells, and also some astrocytes can be seen in a non-noteworthy way. *Pharmaceutics* **2020**, *12*, x 14 of 23

**Figure 6.** (**A**) Images of the primary dopaminergic cell culture. i: Immunofluorescence staining: positive for TH dopaminergic marker. (Scale bar 100 µM) ii: (scale bar 100 µM) and iii: (scale bar 50 µM) Bright field images of primary dopaminergic cell cultures. (**B**) Images of the primary microglia cell culture. Iv: Immunofluorescence staining. (Scale bar 50 µM) v: Bright field images of glia mix culture. (Scale bar 50 µM). vi: Bright field images of the primary microglia cell culture after isolation. (Scale bar 50 µM). **Figure 6.** (**A**) Images of the primary dopaminergic cell culture. i: Immunofluorescence staining: positive for TH dopaminergic marker. (Scale bar 100 µM) ii: (scale bar 100 µM) and iii: (scale bar 50 µM) Bright field images of primary dopaminergic cell cultures. (**B**) Images of the primary microglia cell culture. Iv: Immunofluorescence staining. (Scale bar 50 µM) v: Bright field images of glia mix culture. (Scale bar 50 µM). vi: Bright field images of the primary microglia cell culture after isolation. (Scale bar 50 µM).

CCK-8 assay. Figure 7A,B illustrates the results obtained in the CCK-8 assay after incubating NLCs with a dopaminergic cell culture. None of the concentrations for the nanoformulations tested in dopaminergic cell cultures were cytotoxic, showing percentages of cell viability >70%. Indeed, as shown in Figure 7B, at 48 h, cell viability for the cultures treated with DHAH-NLCs was slightly better at any of the tested concentrations compared to Mygliol-NLCs treated cells. The results of the viability assay for the microglia cell culture are, to some extent, different. As shown in Figure 7C,D, the tested concentrations of 100 and 75 µM for the Mygliol-NLC formulation decreased cell viability below 70% at both tested time points; this effect was not seen with DHAH-NLC for any of the tested time points. This beneficial effect for DHAH-NLC was seen in any of the tested conditions, being

more notorious at 50, 25 and 12.5 µM concentrations (Figure 7C,D).
