Solid lipid nanoparticles
As mentioned before, the presence of physiological barriers, particularly the BBB, restricts the accessibility of many therapeutic agents to the brain, which may compromise the therapeutic efficacy in brain diseases. In accordance with Pardridge , more than 98% of potentially therapeutic agents for brain-associated diseases fail in vivo since they cannot passively cross the BBB and reach therapeutic levels. Therefore, SLNs can be used as drug carriers because of their lipophilic properties which lead them to the CNS by an endocytosis mechanism, overcoming the BBB.
On the basis of these considerations, Bondi et al.  developed SLNs composed of biocompatible solid lipid (glyceryl behenate - Compritol 888® ATO) and phosphatidylcholine and taurocholate sodium salt as a surfactant and co-surfactant agents, respectively, for brain delivery of riluzole. Riluzole, a glutamate release inhibitor, is a potent neuroprotective agent which is useful for the treatment of neurodegenerative diseases. This compound represents the first approved disease-modifying treatment for ALS , and its clinical use is linked to limited efficacy due to the abnormal hepatic function history in patients. The SLNs containing riluzole had an average diameter of approximately 88 nm; a highly charged surface, as indicated by the zeta potential of approximately -46 mV; and excellent loading capacity; and the release profile of riluzole was pH- dependent . After i.p. administration to rats, biodistribution studies indicated that the entrapped riluzole crossed the BBB with greater facility than free non-encapsulated riluzole, reaching a significantly higher concentration in the CNS, which resulted in more efficacy in delaying damage clinical signs (i.e. damage of neurons and axons in myelin basic protein of an animal model) using induced experimental allergic encephalomyelitis (EAE) in Sprague-Dawley rats. Additionally, a low biodistribution of riluzole in other organs - liver, spleen, heart, kidneys and lungs - was verified when the drug was loaded in SLNs, which could imply less systemic toxicity. This was proved by the fact that the rats treated with the SLNs developed clinical signs of EAE later than those treated with free riluzole. Moreover, SLNs can be used not only to improve the drug targeting to the brain but also to enhance drug bioavailability. For example, Misra et al.  proposed the development of galantamine hydrobromide (GH)-loaded SLNs formulated with biodegradable and biocompatible components (solid lipid was glyceryl behenate) to overcome some shortcomings of GH, particularly its poor brain penetration which results in low bioavailability to the target site, requiring repeated dosing. Additionally, the cholinergic side effects of GH are also another obstacle to the optimal use of this agent. GH is a reversible and competitive acetylcholinesterase (AchE) inhibitor which has been shown to improve attention and has beneficial effects on cognitive and functional outcomes for AD [135,136] because of its neuroprotective effects [137,138]. The mean size of SLNs was lower than 100 nm (i.e. ideal for brain-targeted therapeutic delivery], and the maximum drug entrapment was 83.42 ± 0.63%. SLN were able to modulate both the in vitro GH release profile and the in vivo time course of the drug. After oral administration, thegroup of rats to which the SLNs were administered showed an increase in bioavailability of approximately twofold and a significant improvement of cognitive impairment in the AD-like preclinical protocol using rats in comparison with the free drug. Similarly, Natarajan et al.  developed SLNs to improve the bioavailability of olanzapine (OLZ), an antipsychotic drug, in the brain by embedding the minimum dose of the drug into SLNs, thereby reducing the side effects of the drug. Despite the high clinical value of OLZ in the treatment of schizophrenia, it has some limitations such as poor bioavailability (57%] due to extensive hepatic first-pass metabolism, requiring the administration of high doses to reach therapeutic levels in the brain and, thus, increasing the likelihood of adverse effects associated with antipsychotic agents, such as extrapyramidal symptoms (EPS) like rigidity, bradykinaesia, tremor, akathisia, and dyskinaesia. OLZ-loaded SLNs were formulated using tripalmitate (TP-SLN) or glyceryl monostearate (GMS-SLN] as a solid lipid and Tween® 80 as a surfactant and SA as a positive charge inducer. Generally, the optimal size range for SLNs in brain targeting ranges from 50 to 300 nm. The mean particle size and zeta potential of GMS-SLNs and TP-SLNs were 165.1 ± 2.2 nm and 110.5 ± 0.5 nm, respectively, and +35.29 ± 1.2 and +66.50 ± 0.7 mV, respectively (i.e. ideal for brain targeting by the AMT route). SLNs revealed a sustained drug release profile. Pharmacokinetics studies were conducted in male Wistar rats through i.v. administration. SLNs enhanced the relative bioavailability of OLZ in the brain up to 23-fold and reduced the clearance compared to pure OLZ suspension. The authors suggest that this may be because of the transport of intact OLZ-loaded SLNs across the BBB via the endocytosis mechanism. These results are promising at reducing the OLZ dose required, which could reduce the EPS.
An additional approach to increase the amount of therapeutic agent which reaches the brain is through active targeting by binding specific ligands to the surface of ONCs which interact with surface proteins expressed constitutively in the BBB (i.e. receptors involved in the RMT pathway). This modification has been proven to enhance the selectivity uptake of therapeutics via RMT . Kuo and Wang explored this strategy using a monoclonal melanotransferrin antibody (mMAb) , since human brain microvascular endothelial cells (HBMECs) expressed a large quantity of membrane- bound melano transferrin . In addition to mMAb, the authors also conjugated tamoxifen (TX), a modulator of selective oestrogen receptor, on etoposide (ETP - chemotherapeutic agent)-loaded SLNs (ETP-SLNs) to target the BBB and glioblastoma multiforme (GBM) . Several chemotherapeutic agents are substrates of the active efflux transporters (e.g. P-glycoprotein [P-gp] and multidrug resistance-related proteins [MRPs]) at the BBB and so the agents do not achieve therapeutic levels [143,144]. Therefore, TX was used to prolong the ETP residence in the brain parenchyma because of its potential ability to suppress the activity of MRPs which limited the efficacy of chemotherapeutic agents . The mMAb- and TX- conjugated ETP-SLNs (mMAb-TX-ETP-SLNs) were used to penetrate the BBB composed of a monolayer of human astrocyte-regulated HBMECs and to inhibitthe growth of malignant U87MG cells (Uppsala 87 Malignant Glioma [U87MG]) . After treating with mMAb-TX- ETP-SLNs, a high viability of HBMECs proved acceptable cytotoxicity for the integrity of the BBB, suggesting high biocompatibility of the developed formulation. Both TX-grafted and mMAb-grafted SLNs significantly increased the BBB permeability coefficient for ETP. mMAb-TX-ETP-SLNs have also been demonstrated to be more effective in both BBB permeability and coupling melano transferrin in GBM than ETP-SLNs. Moreover, the efficiency in antiproliferation against U87MG cells was in the order of mMAb-TX-ETP-SLNs > TX-ETP-SLNs > ETP-SLNs > SLNs. Cationic bovine serum albumin (CBSA) is another example of a targeting ligand which has been investigated to bypass the BBB, maintaining the integrity of the BBB tight junction [146,147]. On the basis of this consideration, Agarwal et al. prepared DOX-loaded SLNs containing tristearin, soya lecithin and stearic acid as a lipid phase stabilised with a Poloxamer 188 (surfactant agent) aqueous phase and conjugated with CBSA . Stearic acid was added for the conjugation of CBSA to the SLNs. CBSA uses the AMT pathway to bypass the BBB, which involves electrostatic interaction between the positively charged protein (CBSA) and negatively charged membrane cells at the BBB [149, 150]. The results demonstrated that CBSA-conjugated SLNs allow sustained and brain-targeted delivery . SLN-DOX and CBSA- SLN-DOX had a mean size of 80.9 ± 1.7 nm (polydispersity index [PDI] of 0.082 ± 0.008) and 95.1 ± 1.8 nm (PDI of 0.073 ± 0.011), respectively. The zeta potential of SLN-DOX was -13.5 ± 0.5 mV and increased to +14.1 ± 0.7 mV upon ligand conjugation. Cellular studies using HNGC-1 cell lines revealed that the targeting ligand enhanced the cellular uptake of SLNs, almost six times, and was more cytotoxic as compared to DOX solution or nonconjugated SLNs. CBSA-anchored SLNs also demonstrated the maximum transcytosis ability across brain capillary endothelial cells. In vivo studies were carried out in mice and confirmed the more efficient delivery of DOX to brain tissues and the least immunogenic effect with a CBSA- conjugated formulation.
In another active brain-targeting study related to AD, Yusuf et al.  assessed the efficacy of brain-targeted piperine (PIP)- loaded SLNs (PIP-SLNs) in an experimentally induced AD model at a low dose of 2 mg/kg. PIP has beneficial effects in AD, including a strong antioxidant effect, inhibits the enzyme AchE and improves the cholinergic system in the brain. The delivery of PIP to the brain is complicated since this molecule undergoes intense first-pass metabolism, pH-mediated metabolism and photo-isomerisation [152, 153]. Therefore, the authors proposed the development of SLNs containing glycerol monostearate as a solid lipid and Epikuron™ 200 (purified phosphatidylcholine) and Tween® 80 or polysorbate-80 (PS-80) as surfactants . The prepared
PIP-SLNs were overcoated with PS-80 to produce PS-80-PIP-SLNs, since PS-80-coated nanoparticles penetrate the BBB by endocytosis (transcytosis) through specific adsorption of ApoE - a lipoprotein fragment of LDL - on its surface, and the nanoparticles mimic the natural LDL particles which could interact with the LDL receptors located in the BMEC, followed by nanoparticle endocytic uptake into the brain [154, 155]. Thus, the coating of nanoparticles with polysorbates can improve the targeting of therapeutics to the brain and, additionally, inhibit P-gp activity, minimising the efflux of the drug [156, 157]. The PS-80-PIP-SLN formulation was administered to animals (albino rats) via the i.p. route . Although the size of PS-80-PIP-SLNs was 312.0 ± 5.1 nm, there was a high concentration of PIP in brain tissues, which can be explained by transcytosis, as previously reported in other studies [158, 159]. The PIP in SLNs demonstrated positive therapeutic results in the AD model via acting on the oxidative cascade (i) by decreasing the superoxide dismutase values by 504 ± 44.24 m units [p < 0.05), (ii) on the cholinergic system by enhancing AchE values by 29.24 ± 4.29 pg/mg (p < 0.01) and (iii) reducing the amyloidal plaque and tangle contents in the brain tissues which were proved by histopathology studies. On the basis of the significant antitumoural activity exhibited by PS-80 nanoparticles in the glioma animal model [160, 161], the coating of cetyl palmitate- based SLNs with PS-80 and PS-60 was also a strategy investigated by Martins et al.  to deliver cytotoxic camptothecin (CPT) into the brain parenchyma by crossing the BBB after i.v. administration. Both polysorbates (i.e. PS-60 and PS-80) are reported to be promising surfactant agents for enhancing drug brain delivery . Although CPT has already revealed antitumour activity in glioblastomas models , its instability in physiological conditions, the limited aqueous solubility and its several toxic effects limit its therapeutic value [164, 165]. The CPT-loaded SLNs had a mean size below 200 nm, a low PDI (<0.25), a negative surface charge (-20 mV) and high encapsulation efficiency (>94%) . Cytotoxicity studies against human glioma cell lines (A172, U251, U373, U87) revealed higher antitumour activity when CPT was encapsulated into SLNs compared to free CPT in solution/suspension or in a physical mixture with SLNs. In vivo biodistribution studies in rats demonstrated superior brain accumulation and lower deposition of CPT in peripheral organs with CPT-loaded SLNs, revealing the positive role of SLNs on brain targeting and, thus, the potential reduction of CPT toxic side effects. Estella-Hermoso de Mendoza et al.  obtained similar results after oral administration of anticancer edelfosin loaded in Compritol® and Precirol® lipid nanoparticles on a xenograft mouse glioma model . The results demonstrated that the high edelfosin accumulation in the brain with lipid nanoparticles is attributed to the inhibition of P-gp by Tween® 80, which was verified using a P-gp drug interaction assay. The authors also proved that after 14 days of treatment, drug-loaded lipid nanoparticles in mice bearing a C6 glioma xenograft tumour exhibited a highly significant decrease in tumour growth after 14 days from the beginning of treatment .
To improve the access of drugs to the brain, the rational use of alternative routes of administration has been triggered, with the i.n. route (non-invasive technique) being an important focus for the transport of neurotherapeutic agents, since the molecules bypass the BBB and directly access the brain through the olfactory region of the nasal mucosa, without previous absorption to the blood circulation [167-169]. Moreover, i.n. administration improves the bioavailability of compounds since it avoids gastrointestinal and first-pass hepatic metabolic processes. In conclusion, the development of nose-to-brain ONCs represents a promising approach for neurotherapeutic agents which have a short circulation half-life (ti/2), a rapid degradation rate, or poor ability to pass through the BBB because of their unsuitable molecular weight, lipophilicity or surface charge. Taking these considerations into account, and to overcome some shortcomings related to risperidone (RSP), Patel etal.  encapsulated this drug in SLNs (solid lipid: glyceryl behenate; surfactant: Pluronic F-127) and explored the brain-targeting efficiency through nose-to-brain delivery. RSP is an antipsychotic agent which has been approved for the management of psychotic disorders. This compound has advantages over conventional antipsychotic agents because of a lower EPS; however, this benefit depends on the dose of the drug, requiring a low-dose therapy. In addition, RSP has some drawbacks which limit its clinical use, such as poor aqueous solubility, poor oral bioavailability since it undergoes extensive first-pass hepatic metabolism and P-gp efflux . SLNs presented an RSP encapsulating efficiency (EE) of 59.65% ± 1.18%, a mean particle size of 148.05 ± 0.85 nm, a PDI of 0.148 ± 0.028 (i.e. a monodisperse stable system) and a zeta potential of-25.35 ± 0.45 mV. SLNs demonstrated a sustained release of RSP . Both pharmacodynamic and pharmacokinetic studies carried out in mice demonstrated that RSP-loaded SLNs (RSLNs) administrated by the i.n. route effectively reach the brain. In vivo biodistribution and gamma scintigraphy imaging studies showed that i.n. administration of RSLN provides higher RSP levels in the brain (with a brain/blood ratio 1 h postadministration of 1.36 ± 0.06) than i.v. administration of the free drug (with a brain/blood ratio of 0.17 ± 0.05) and i.v. administration of RSLN (with a brain/blood ratio of 0.78 ± 0.07), that is, nearly 10- and 5-fold higher, respectively.