**4. Discussion**

NDs are one of the main problems of the public health system in the 21st century. Although they have different clinical manifestations and symptoms, they all share a complex mechanism of neurodegeneration, with aging as the main risk factor, and they are without an effective treatment [1,48]. In order to obtain an effective treatment for NDs, in the last few years, different therapeutic molecules have been raised as new clinical candidates. Among others, the use of functional lipids such as PUFAs and, more concretely, DHA and DHAH have been raised as a useful tool to treat NDs since they exhibit beneficial effects, decreasing neuroinflammation and protein deposit or increasing the release of neuroprotective agents [30,49]. However, no matter the treatment, one of the main issues with treating NDs is reaching the brain and passing through the BBB. That is why the use of nanotechnology and, more concretely, the NLCs, has exhibited promising results for targeting the brain, increasing bioavailability, protecting from oxidation and, therefore, maintaining the bioactivity of different molecules [19].

The main goal of this work was to combine both strategies to develop a new therapeutic nanocarrier enriched with PUFAs, more concretely, with DHAH, to generate a new type of NP that could target the brain and exhibit neuroprotective and antiinflammatory effects, therefore becoming a functional nanocarrier that could be combined with different molecules in the future to promote a synergistic therapy.

All the developed nanoformulations were 50–90 nm in size, with a PDI value below 0.5 indicating a homogenous suspension (Figure 4). The increment of the liquid lipid ratio led to a decrease in the size of the nanoparticles, as shown in previous publications (Table 2) [50,51]. Moreover, as seen in DSC thermograms (Figure A1), the addition of the liquid lipid to the solid lipid led to a slight reduction of the melting point of Precirol of 3–4 ◦C in all NLC thermograms, which is similar to previously conducted studies [50,52,53]. Anyway, the selected solid:liquid lipid ratio for the two different lipids, Mygliol and DHAH, was in the ratio normally used for NLC preparation [24]. After selecting the ratio and the lipid to constitute the new nanoformulation, the NPs' surface was modified with the addition of CS and TAT, resulting in two different formulations, named DHAH-NLCs and Mygliol-NLCs. The addition of CS and TAT peptide led to the conversion of Z potential from negative values to positive values (Tables 2 and 3), indicating that the undergoing process was successfully performed. Moreover, the presence of amide I in FTIR spectra (Figure A2) confirmed that the coating process with TAT was successfully performed. As we have previously demonstrated, the addition of CS and TAT increased in vivo brain targeting after intranasal administration [16,24]. Moreover, TAT is a well-known CPP, usually used to enhance the delivery of different cargos into different types of cells, such as neurons [54,55]. The resulting NPs were similar in size (around 100 nm) and exhibited positive zeta values due to the successful CS coating process (Table 3).

Among the different NDs, in this research paper, we aim to study the effect of our newly developed nanocarriers in a cell model of PD by adding 6-OHDA neurotoxin, one of the most widely used neurotoxins, to a dopaminergic cell culture to mimic the destruction of catecholaminergic neurons and the degeneration of the nigrostriatal pathway [56]. The addition of 25 µM 6-0HDA for 24 h led to a decrease in cell viability of 50%, as shown in Figure 2B. The dose and incubation time for this neurotoxin is variable, according to the scientific data available, varying from 5 to 100 µM and from 30 min to 48 h [57–60]. That is why we incubated the neuronal cells with different doses for 24 h, an intermediate time point in the published articles, and selected the dose that generated 50% cell death so we could really see the effect of the neurotoxin on cell viability. In order to check the safety and effectiveness of the DHAH incorporated into our NLCs, we incubated the nanoformulations for 24 and 48 h. All tested concentrations were shown to be safe (Figure 7A,B), with a viability up to 70% and showing better values for DHAH-NLCs at 48 h. Moreover, the effectiveness of DHAH-NLCs was also evaluated in dopaminergic cell cultures after the incubation with 6-OHDA. DHAH-NLCs were shown to be effective at protecting the cells from the neurotoxin compared to Mygliol-NLCs as the control group (Figure 2C,D). These data are in line with previous publications regarding the DHA neuroprotective effect shown in neuron cell cultures [61,62]. Moreover, it demonstrates the effectiveness of the DHAH functional lipid incorporated into the newly developed NLCs.

On the other hand, the neuroinflammatory process undergoing NDs and, more specifically, in PD is well known, being the consequence or the cause of the disease. Whatever the origin of the neuroinflammation, it is a fact that a therapeutic intervention downregulating this process could be great at halting the progression of the disease [63]. Although different cell types and molecules are involved in the neuroinflammation, microglia cells are the primary initiators of the central inflammatory response to acute and chronic disorders related to NDs [64]. In order to treat this inflammatory response, PUFAs and, more concretely, DHA and DHAH have been raised as emerging candidates to downregulate this process and become a new therapeutic approach [9,31]. The DHAH-NLCs developed in this study were shown to be safe at any of the tested concentrations, with a viability up to 70%, in contrast to Mygliol-NLCs, where higher concentrations decreased cell viability (Figure 7C,D). Thus, these results demonstrated that the newly developed formulation with DHAH can be used in high concentrations without affecting cell viability.

In order to mimic the neuroinflammatory process in primary microglia cells, different molecules can be used. Among them, the gold standard stimuli for generating reactive gliosis and activating the neuroinflammation cascade is LPS. LPS has been widely used to generate animal models of neuroinflammation or induce it in cell culture, both primary and microglia cell lines [65–67]. The addition of LPS at 50 ng/mL concentration in this study generated the production of proinflammatory cytokines in similar levels to those in previously published scientific articles for microglia primary cell cultures isolated after the shaking method [66]. Regarding the potential antiinflammatory effect of our NPs, we showed that the ability of DHAH to decrease neuroinflammation was maintained in DHAH-NLCs, obtaining proinflammatory cytokine levels similar to C− in IL-6 and IL-1β, and decreasing TNF-α almost by half for any of the tested concentrations (Figure 3B–D). The ability of PUFAs to decrease the cytokine proinflammatory levels has previously been described [68]; thus, this study enforces the use of these kinds of PUFAs formulated in NLCs as an emerging tool to treat the undergoing inflammatory process in NDs.
