6.1.1. Lipid-Based Nanoparticles

These lipid-based nanoparticles have the advantages of being non-toxic and having a high loading capacity for hydrophilic or non-hydrophilic molecules of interest, as well as a capacity to get through the BBB (Figure 3).

**Figure 3.** Representation of different delivery systems for therapeutic molecules. Different systems are used to deliver therapeutic molecules: lipidic nanoparticles, dendrimers and dendrigrafts, quantum dots and metallic nanoparticles, and exosomes. AM: adhesion molecules; TP: transmembrane proteins; APM: antigen presenting molecules; NA: nucleic acid; CP: cytosolic proteins; CyP: cytoskeletal proteins; LR: lipid rafts.

Nanoparticles have been formed from DHA and its hydroxylated derivative (DHAH); these are directly active without the need to load other molecules. These nanoparticles were tested on primary cultures of microglia obtained from rats. The viability of the cells is not affected by the use of these DHA/DHAH nanoparticles; however, an anti-inflammatory action of these nanoparticles is noted when the microglial cells are stimulated by LPS, with a decrease in the release of TNFα, IL-6, and IL-1β [63]. This work allowed us to show in vitro that these DHA/DHAH nanoparticles were not toxic and could reduce the release of proinflammatory cytokines by microglial cells [63]. This approach must still be validated in vivo in animals before testing in humans. This same team had already used lipid nanoparticles coated with chitosan, with the surface modified with a transactivator of transcription (TAT) peptide and loaded with GDNF (glial cell-derived neurotrophic factor) (CS-NLC-TAT-GDNF) [64]. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinson's mouse model, intranasal administration of CS-NLC-TAT-GDNF led to modulation of microglial activation [64]. The use of lipid nanoparticles can induce toxicity and induce activation of brain microglia (involvement of the P2X7/caspase-1/IL-1β pathway). It has been shown that by modifying these lipid nanoparticles with PEGylation, this microglial activation could be reduced [65]. Nanoparticles can pass through the nasal epithelium and reach the brain by two different routes: (a) the extracellular route, the most common mechanism of delivery of therapeutics to the brain, via passive transport through the nasal epithelium, and (b) the intracellular route, involving endocytosis in the branches of the olfactory and trigeminal nerves followed by axonal transport in the brain [66].

Nanoparticles were synthesized from high-density lipoprotein (HDL) associated with apolipoprotein E [67,68]. These nanoparticles are able to enter cerebral vessels and accumulate around Aβ aggregates. First, ApoE-associated nanoparticles were shown to reduce Aβ deposition, attenuate microgliosis, improve neurological changes, and reduce memory deficits in an animal model of AD [67]. In a second step, α-Mangostin (α-M), a polyphenolic agent capable of inhibiting the formation of Aβ oligomers and fibrils and accelerating the cellular degradation of Aβ, was added to these nanoparticles [68]. These α-M-loaded nanoparticles are able to promote the uptake and degradation of Aβ1-42 by microglia more than unloaded nanoparticles [68]. This same system of reconstituted and modified HDL was used with monosialotetrahexosylganglioside (GM1), possessing high Aβ binding affinity [69]. This nanosystem promotes Aβ degradation by microglia following intranasal administration. This structure was used to load a neuroprotective peptide NAP, αNAP-GM1-rHDL, which was able to reduce Aβ deposition more efficiently than the nanostructure alone or α-NAP alone, in mouse models of AD after intranasal administration [69].

Oridonin, a natural diterpenoid compound isolated from the Chinese herb *Rabdosia rubescens*, was loaded into commercial lipid nanocarriers, Lipofundin® (MCT, 10% for infusion, B. Braun AG, Melsungen, Germany), and then given orally or injected into mice constituting an animal model of cerebral amyloidosis for AD, transgenic APP/PS1 mice. Regardless of the mode of injection, oridonin-loaded nanoparticles were able to attenuate microglia activation [66]. In an in vitro model of microglia (line N9) stimulated by LPS, these nanoparticles are able to inhibit the inflammatory response by reducing the NO concentration and decreasing mRNA expression of iNOS, IL-1β, and IL-6 [70].

Nanoparticles with a lipid core (capric/caprylic triglycerides) were loaded with indomethacin (a non-steroidal, anti-inflammatory drug) and their impacts on neuroinflammation were evaluated on organotypic rat hippocampal cultures after treatment with Aβ1-42 peptide, mimicking AD [71]. The use of these nanoparticles allows for the decrease of the TNF-α and the increase of IL-6 induced by Aβ1-42, but also the increase of the release of interleukin-10 [68]. The activation of microglia is reduced by the use of these nanoparticles [71].

The use of lipid nanoparticles seems to be a promising way to deliver molecules of interest to the brain by targeting microglia, while controlling their possible toxicity via surface modifications.

#### 6.1.2. Metal Nanoparticles

Different types of metals can be used to create nanoparticles, such as titanium, gold, or silver (Figure 3).

Regarding titanium nanoparticles, in vitro experiments on BV-2 microglia cells showed that titanium dioxide nanotubes, which were functionalized with 3-aminopropyl triethoxysilane (APTES), allowing the addition of amine functions for drug molecule conjugation, do not induce toxicity or activation of these cells [72]. Titanium dioxide nanoparticles are able to move in the brain by decreasing the transendothelial electrical resistance and by disrupting the tight junctions between the endothelial cells of the brain capillaries [73]. Titanium dioxide (TiO2) nanoparticles are able, without being functionalized by other

molecules, to drive microglia toward the proinflammatory activation phenotype [74]. This change of phenotype would be specific to microglia, as astrocytes do not change their phenotype following the use of these nanoparticles [74].

Gold nanoparticles have also been developed. Some have been tested on primary cultures of microglia. They consist of 18 atoms of gold and are stabilized with glutathione ligands. The authors of this study showed that these Au18 gold nanoclusters (NCs) have, at low concentrations, anti-inflammatory signaling (reduction of (IL1-β) levels, unchanged levels of TNF-α or Ym1/2) but, at higher concentrations, they can have pro-inflammatory activity [75]. The authors suggested that the presence of glutathione could be the source of this anti-inflammatory activity. Gold nanoparticles (AuNCs) were functionalized with dihydrolipoic acid (DHLA-AuNCs), a neuroprotective antioxidant. The BV2 microglial line was used to evaluate their effects on microglial polarization. These nanoparticles induced a polarization toward the M2-like phenotype as well as a decrease of oxidative stress, a reduction of NF-kB signaling, and an increase of cell survival (increase of autophagy, inhibition, of apoptosis) [76]. Microglial changes also impacted neuronal cells by improving neurogenesis and reducing astrogliosis [76]. Another team used the root extract of *Paeonia moutan*, woody trees which are used in traditional Chinese medicine, to functionalize gold nanoparticles. Still, on a BV2 cell model, it was shown that these nanoparticles were able to decrease oxidative stress and inhibit the synthesis of pro-inflammatory cytokines following stimulation by LPS [73]. The effect of these nanoparticles on oxidative stress and inflammation was found in a model of parkinsonian mice, associated with an improvement in motor disorders [77]. An extract of *Ephedra sinica* Stapf was used to functionalize gold nanoparticles. These nanoparticles were able to decrease the production of pro-inflammatory mediators and cytokines (TNF-α, IL-1β, and IL-6) following primary microglia and BV-2 microglial cells induction by LPS (decrease of IκB kinase-α/β, NF-κB, Janus-activated kinase/signal transducers and activators of transcription, mitogen-activated protein kinase, and phospholipase D signaling pathways) [78]. Gold nanoparticles were synthesized with quercetin and then used on BV2 cells stimulated with LPS. The release of pro-inflammatory prostaglandin, prostaglandin E2, NO, upregulation of COX, inducible NO synthase mRNA, and protein levels were strongly inhibited by gold/quercetin nanoparticles [79]. The effects of these nanoparticles are superior to the use of quercetin alone. The use of these goldquercetin nanoparticles could thus decrease the activation of microglia. A gold cluster with a positively charged tridecapeptide Sv (Au25Sv9) (peptide Sv: (H2N-CCYGGPKKKRKVG-COOH)) was produced, and it was shown that these particles were able to attenuate the cytotoxicity of stimulated microglia cells toward neuronal cells [80]. These nanoparticles inhibited IL-6, TNF-α, and NO secretions by suppressing the activation of NF-κB and p38 pathways. The action of these nanoparticles was also observed directly on neuronal cells, indicating that these nanoparticles could target microglia and neurons and could be an effective therapeutic approach [80].

Complex nanoparticles combining a metallic part and a polymer were produced in the following way: diblock polymer Man-PCB-PB was synthesized and then assembled to form nanoparticles to enclose the hydrophobic part fingolimod and zinc [81]. These nanoparticles were named Man-PCB-PB/ZnO/fingolimod NPs or MCPZF NPs. They were then linked to a signal transducer and activator of transcription 3 small interfering RNA (siSTAT3) to give Man-PCB-PB/ZnO/fingolimod/siSTAT3 NPs or MCPZFS NPs [81]. These nanoparticles promote phagocytosis of Aβ by microglia and decrease the release of pro-inflammatory cytokines [81].

Magnetic iron oxide (maghemite, γ-Fe2O3) nanoparticles (high surface-to-volume ratio, diameter 21 ± 3.5 nm, magnetic, biocompatible, relatively non-toxic, biodegradable) were used to deliver the following peptide: fibrin γ377–395 peptide [82]. The effect of these nanoparticles is different depending on the age and the state of AD. Indeed, in the early stages, the reduction of microglial cell activation following the action of these nanoparticles increases the number of neurons with hyperphosphorylated tau in transgenic mice [82]. Abnormal hyperphosphorylation of tau protein in sites that are

not normally phosphorylated leads to the formation of neurofibrillary tangles (NFTs) in neuronal cell bodies and sometimes in glial cells. Hyperphosphorylation and NFT formation induce an inability for tau protein to bind to microtubules, resulting in alterations in axonal trafficking leading to changes in neuronal function and viability. These processes participate in synaptic dysfunction and neurodegeneration. On the other hand, in older mice, the reduction of microglial cell activation reduces the severity of tau pathology [82]. The number of neurons with hyperphosphorylated tau and the number of neurons with tangles are reduced in animals receiving the γ377–395 fibrin peptide-nanoparticle conjugate compared with control animals [82].

### 6.1.3. Polymer Nanoparticles and Mesoporous Silica Nanoparticles

Nanoparticles can be synthesized from synthetic or natural polymers. One team has developed poly(carboxybetaine) (PCB)-based zwitterionic nanoparticles (MCPZFS NP). These nanoparticles decrease microglia priming by lowering the levels of pro-inflammatory mediators and contributing to brain-derived neurotrophic factor (BDNF) secretion [81]. They also enhance Aβ recruitment to microglia, contributing to improved Aβ phagocytosis [81]. Beyond the action on microglia, these nanoparticles can also play on Aβ loading, neuronal damage, memory deficits, and neuroinflammation in APPswe/PS1dE9 mice [81]. Poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and lipid chains as building blocks were used to synthesize 200 nm spherical polymeric nanoconstructs (SPNs) and 1000 nm discoidal polymeric nanoconstructs (DPNs). SPNs are more rapidly absorbed than DPNs and were used to encapsulate curcumin in the PLGA core. These curcumin-loaded nanoparticles decrease the production of proinflammatory cytokines-IL-1β, IL-6, and TNF-α in amyloid-β fiber-stimulated macrophages [83]. The α-M, previously used in a lipid nanoparticle, was encapsulated in the core of poly(ethylene glycol)-poly(llactide) (PEG-PLA) nanoparticles [NP(α-M)] [84]. This nanoformulation reduced Aβ deposition in AD and attenuated neuroinflammatory responses by microglia (using the BV-2 line) [84]. Apart from these effects on microglia, nanoencapsulation has improved the biodistribution and clearance of these molecules [84]. PLGA nanoparticles were used to deliver SurR9-C84A, a survivin mutant belonging to the inhibitors of the apoptosis protein family [85]. For this study, neuron monocultures and co-cultures of neurons and THP-1 (monocytes/macrophages) were used. These cultures were treated with LPS or β-amyloid to mimic the pathological inflammatory conditions of AD. Following this stimulation by LPS or β-amyloid, a decrease in THP-1 secretions was observed by the use of these nanoparticles [85]. This inhibition of secretions decreased the neuronal cell death induced by them.

Amphiphilic sugar-based molecules (AM) derived from mucic acid were synthesized to exhibit high affinity to scavenger receptors, allowing internalization of α-synuclein at the microglia [86]. Internalization of monomeric α-synuclein and formation of intracellular α-synuclein oligomers were decreased in microglial cells treated with these amphiphilic molecules. Following this observation, the antioxidant poly(ferric acid) was added to the core of these amphiphilic molecules by performing nanoprecipitation [86]. Microglial cells treated with these nanoparticles and stimulated by α-synuclein saw a decrease in their activation as well as in the neurotoxicity induced by α-synuclein aggregated at the level of microglia [86]. In vivo, the activation of microglia is also decreased after injection of these nanoparticles in the substantia nigra of mice stimulated by fibrillar α-synuclein. Targeting the receptors responsible for α-synuclein entry into microglia and adding an antioxidant may represent an interesting therapeutic approach via nanotechnology [86].

Polylactic acid (PLA)-coated mesoporous silica nanoparticles were loaded with resveratrol, which exhibits antioxidant activities among others. The PLA coating protects the resveratrol and prevents its systematic release. In the presence of oxidative stress, PLA is degraded and resveratrol can be released. Following this release, resveratrol was able to effectively reduce the activation of microglia cells stimulated by phorbol-myristate-acetate or lipopolysaccharide [87].
