Targeted Nanotherapy for Cognitive Impairment : Blocking Amyloid-Β-Induced Membrane Damage in Brain Tissue

A frequent co-morbidity of cerebrovascular pathology and Alzheimer's disease pathology has been observed over past decades. Accordingly, much evidence has been reported which indicates that microvascular endothelial dysfunction, due to cerebrovascular risk factors (e.g., atherosclerosis, obesity, diabetes, smoking, hypertension, aging), precedes cognitive decline in Alzheimer's disease and contributes to its pathogenesis. By incorporating appropriate drug(s) into biomimetic (lipid cubic phase) nanocarriers, one obtains a multitasking combination therapeutic which targets certain cell-surface scavenger receptors, mainly class B type I (i.e., SR-BI), and crosses the blood-brain barrier (BBB). Such targeting allows for various Alzheimer's-related cell types to be simultaneously searched out, in vivo, for localized drug treatment. This in vivo targeting advantage may be particularly important for repurposing an FDA-approved drug, especially one which has shown the added ability to restore some cognitive functions in certain animal models of Alzheimer's disease (e.g., the anticancer drug bexarotene); this (candidate repurposing) drug up to now, by itself (i.e, without nanocarrier), displayed poor CNS penetration in human subjects.


LCM/ND Nanoemulsion Type, Lipid Cubic Phases, and Biomimetic Nanocarriers
The self-assembling LCM/ND lipid nanoemulsion class comprises nonionic lipids exclusively (cf.[75,76]) throughout its coated microbubble's and/or related nanoparticle's (i.e., related lipid polymorphs') supramolecular structures(s).This biobased lipid composition of LCM/ND nanoemulsions (i.e., glycerides and cholesterol compounds) is similar to lipids contained in several types of plama lipoproteins; accordingly, when these LCM/ND nanoemulsion particles are injected into the bloodstream, they likely acquire (i.e., bind) plasma apolipoprotein(s) -including notably apoA-I [73].Hence, the molecular composition of the LCM/ND nanoemulsion particles resulted in both microbubble/nanoparticle stability and marked targeting toward tumors and certain hyperproliferative-disease lesions/sites; this very rapid targeting has been demonstrated to occur by an "active uptake" process, i.e., "endocytosis" -which likely involves certain "lipoprotein receptor"-mediated endocytic pathways [2].Importantly, monoglyceride is the largest single-lipid fraction (by wt.%) of the powdered solid lipid surfactants used to produce the (Filmix®) LCM/ND nanoemulsions [73].As a group, monoglycerides exhibit different phase behaviors when they are exposed to water [77] (cf.[78,79]).The ability to exist in several different phases is an important property of pure lipids and lipid mixtures; it depends upon temperature, hydration, and lipid class [77].Although monoglycerides typically have poor water solubility, they have free hydroxyl groups which can hydrogen bond with water, surfactants, cosolvents, etc.As polar lipids, monoglycerides typically: (1) are better solvents for drugs; (2) act as "cosurfactants" which promote mutual solubility between excipients (i.e., inactive ingredients); (3) enhance water uptake; and (4) promote self-dispersibility of lipid formulations [80].The above properties of monoglycerides place them in a lipid class known as "insoluble swelling amphiphiles".These lipid molecules form stable monolayers (at the air/water interface), but also swell in water to form liquid-crystalline phases [81].In their detailed review, Kaasgaard and Drummond [82] explain that these lyotropic (i.e., solvent induced) liquid-crystalline phases of monoglycerides include the one-dimensional lamellar phase, which has been widely studied and employed as a model system for biomembranes and drug-delivery applications.More recently studied are the structurally more complex two-and three-dimensional ordered (lyotropic) liquidcrystalline phases, of which inverse hexagonal and cubic phases are two prominent examples.In agreement with numerous other investigators, Kaasgaard and Drummond also state that all these types of liquid-crystalline phases are frequently stable in excess water, which facilitates the preparation of nanoparticle dispersions and makes them suitable candidates for the encapsulation and controlled release of drugs ( [82]; cf.[83][84][85][86][87][88][89]).
The self-assembly of varied and useful dispersed cubic phases (among other liquidcrystalline phases) depends heavily on the acyl chain length of the glycerides (primarily monoglycerides) placed in contact with water [73].As Yaghmur et al. [89] point out, the significant interest in the formulation and the characterization of these complex and varied, self-assembled, liquid-crystalline cubic phases is driven by both fundamental and practical considerations: They offer many advantages compared to conventional dispersed systems (such as simple emulsions or double emulsions) because of their confined equilibrium nanostructures with high interfacial area, their low viscosity, and their capabilities to solubilize a wide variety of active molecules.Therefore, there is great interest to utilize these dispersed cubic phases for the administration of drugs, or for the formulation of new delivery systems [89].
Besides certain glyceride-based liquid-crystalline systems displaying colloidal stability in excess water, the same important attribute has been documented for cholesterol and cholesterol esters -all of which are present in LCM/ND nanoemulsion formulations [73].
For example, cholesterol and its esters change the packing structure of lipids, and in high concentrations they are known to induce the formation of a liquid-crystal phase [90].In addition, Kuntsche et al. [91,92] have prepared lipid nanoparticles in the (mesomorphic or) liquid-crystalline phase from cholesterol esters with saturated acyl chains.These investigators were motivated by the knowledge that many cholesterol esters are physiologic lipid compounds which can form liquid-crystalline phases (thermotropic mesophases) and, hence, they were interested in their potential for the development of liquid-crystalline nanoparticles as a carrier system for lipophilic drugs [92].In accord with the above observations and considerations, the substantial concentrations of cholesterol esters and cholesterol in the LCM/ND nanoemulsion formulation likely further contribute to the known long-term stability of this nanoemulsion's (liquidcrystalline) lipid nanoparticles in excess water, thereby providing a persistent carrier matrix upon exposure to liquids such as blood plasma [73].
To conclude, self-assembled (colloidal mesophase) lipid nanoemulsions (e.g., [93][94][95][96][97][98]), particularly those predominantly containing dispersed cubic-phase lipid nanoparticles (e.g., [99][100][101][102][103]), continue to receive growing attention in pharmaceutical and/or biological fields.The main reason behind much of this attention is the fact that nonlamellar lipid nanostructures, such as cubic liquid-crystalline phases, have wide potential as delivery systems for numerous drugs, cosmetics, and food applications (e.g., [104][105][106]).Namely, using various lipids and their mixtures to form self-assembled non-lamellar nanostructures, it has continually been reported possible to successfully obtain stable colloidal dispersions of (liquid-crystalline) lipid cubic phases with well-defined particle size and morphology (e.g., [105,106]).In particular, within the range of self-assembled phases in model surfactant-like lipid systems, Yaghmur et al. [107] further emphasized that the monoglyceride-based lyotropic liquid-crystalline phases are relatively unique owing to their rich polymorphism in water and potential application as drug nanocarriers (cf.[108]).A recurring example of a largely monoglyceride-based drug-delivery agent category is the multitasking LCM/ND nanoemulsion formulation (cf.above).In this particular targeted-delivery approach, the self-assembled "lipid particle" structure itself (upon intravenous injection of the LCM/ND nanoemulsion) is apparently successfully utilized as the "active" targeting ligand -which is directed via (adsorption of) plasma lipoproteins toward the appropriate receptors on the target-cell surface.These dispersed liquid-crystalline lipid particles, of the LCM/ND nanoemulsion formulation, are colloidally stable nanocarriers which very likely represent liquid-crystalline inversetopology nanotransporters (nanocarriers), i.e., dispersed lipid cubic phases (cf.[73]).

Calcium Dyshomeostasis, and the Amyloid-β Ion Channel Hypothesis of Alzheimer's Disease
As explained in many reviews (e.g., [109,110]) by different investigators, it has been recognized for over two decades that disturbance of the intracellular calcium homeostasis is central to the pathophysiology of neurodegeneration.In Alzheimer's disease, it is believed by many researchers that enhanced calcium load may be brought about by extracellular accumulation of amyloid-β in the brain.Such studies have laid the foundation for the popular idea that amyloid-β peptides (Aβ; 39-42 amino acid molecules) are, in part, toxic to brain tissue because they form aberrant ion channels in cellular membranes and thereby disrupt Ca 2+ homeostasis in brain tissue and increase intracellular Ca 2+ .More specifically, later studies indicated that soluble forms of Aβ facilitate influx through calcium-conducting ion channels in the plasma membrane, leading to excitotoxic neurodegeneration [109,110].
The precise cellular pathway(s) by which the amyloid-β peptides bring about excitotoxic neurodegeneration has been much debated.A common cellular picture used to explain the disruptive effect of calcium dyshomeostasis within brain tissue, appearing often in the literature (e.g., [111,112]), involves a central role for the tripartite glutamatergic synapse in the pathophysiology of Alzheimer's disease.Under this hypothesis, perturbations in the glutamatergic tripartite synapse (comprised of a presynaptic neuron terminal, a postsynaptic neuron terminal, and an astrocytic process) are believed to underlie the pathogenic mechanisms of Alzheimer's disease.Glutamate is the primary excitatory neurotransmitter in the brain and plays an important role in cognition and memory, but alterations in glutamatergic signaling can lead to excitotoxicity.This "Ca 2+ dyshomeostasis"-induced excitotoxicity occurs when uncontrolled glutamate release surpasses the capacity of astrocytic clearance mechanisms, and is linked to several neurodegenerative disorders including Alzheimer's disease [111](cf.[112]).
Historical support for the above amyloid-β ion channel hypothesis, or so-called "calcium hypothesis", has also been observed at the clinical level [113].Namely, there is little correlation between the amounts of fibrillar (insoluble) deposit at autopsy and the clinical severity of Alzheimer's disease.In contrast, a good correlation exists between early cognitive impairment and levels of soluble forms of Aβ in the brain [114].(Aggregation of Aβ proceeds from formation of soluble (low molecular weight) spherical oligomers toward eventually assuming a final and stable conformation as insoluble fibrils from which amyloid-β plaques are constituted.Neurotoxicity is associated with soluble aggregates (i.e., oligomers) of Aβ rather than with the plaques themselves.)Accordingly, related experimental work has already shown that application of soluble Aβ oligomers (but not monomers or fibrils) to cultured neuroblastoma cells evoked large increases in cytosolic calcium that arise largely through Ca 2+ influx across the plasma membrane [114].
As summarized by Di Scala et al. [113], the structure of amyloid pores has been extensively studied by ultrastructural methods.In particular, one group of investigators recently applied strategies (widely used to examine the structure of membrane proteins) to study the two major Aβ variants, namely, Aβ  and Aβ .Under the optimized detergent micelle conditions: 1) Aβ(1-40) aggregated into amyloid fibrils; 2) contrariwise, Aβ(1-42) assembled into oligomers that inserted into lipid bilayers as welldefined pores [115].(These amyloid pores adopted characteristics of a β-barrel arrangement.)Because Aβ , relative to Aβ , has a more prominent role in Alzheimer's disease, the higher propensity of Aβ  to form β-barrel pore-forming oligomers is an indication of their importance in Alzheimer's disease [115].Very recently, a different research group reported very similar findings [116].As background for their study, these latter authors point out that: elevated Aβ(1-42) plasma levels have been correlated with the progression of late-onset forms of Alzheimer's disease; Aβ(1-42) is significantly more neurotoxic than Aβ(1-40) both in vivo and in neuronal cell culture; and memory impairment is believed to be driven by Aβ(1-42) disruption of long-term (hippocampal) potentiation.In accordance with these considerations, the authors' own detailed experimental data [116] indicated that Aβ(1-42) assemblies in oligomeric preparations form ion channels (in membranes excised from cells of neuronal origin).In contrast, Aβ(1-40) oligomers, fibrils, and monomers did not form channels.Moreover, ion channel conductance results suggested that Aβ(1-42) oligomers, but not monomers and fibrils, formed pore structures.The authors concluded that their findings demonstrate that only Aβ(1-42) contains unique structural features that facilitate membrane insertion and channel formation, now aligning ion channel formation with the neurotoxic effect of Aβ(1-42) compared to Aβ(1-40) in Alzheimer's disease [116].

Humans
The preceding discussion of amyloid pore formation, in the cellular membranes of brain tissue, leads to another important consideration -the role of cholesterol.Namely, cholesterol is required for the assembly of amyloid pores formed by Aβ(1-42) [113].
Therefore, an amphipathic drug (such as bexarotene) which competes with cholesterol for binding to Aβ(1-42) would be capable of preventing oligomeric channel formation (at least in vitro).Such a strategy has already been contemplated for the treatment of Alzheimer's and other neurodegenerative diseases that involve cholesterol-dependent toxic oligomers [117].However, when oral administration of bexarotene was employed subsequently in a Phase Ib (proof of mechanism) clinical trial [118], bexarotene displayed poor CNS penetration in normal human subjects.(Hence, the observed absence of an effect on Aβ metabolism was likely reflective of the low CNS-levels of bexarotene achieved [118](cf.[119])).
Nonetheless, at least two recently published reports (both in 2017) on bexarotene indicate a continuing interest in the ability of this FDA-approved (anticancer) drug to: 1) bind free Aβ peptide as well as 2) bexarotene's previously reported positive effects in Alzheimer'sdisease mouse models [120,121] (cf.[122,123]).Such past studies in animal models of Alzheimer's disease, concerning the beneficial effects of bexarotene, have also motivated a detailed analysis by Fantini et al. [124] that utilized a combination of molecular, physicochemical, and cellular approaches to elucidate the mechanisms underlying the anti-Alzheimer properties of bexarotene in brain cells.These investigators demonstrated that bexarotene shares structural analogy with cholesterol: both bexarotene and cholesterol are amphipathic compounds, with a large apolar part consisting of a succession of hydrocarbon rings and a small polar headgroup (hydroxyl for cholesterol, carboxylate for bexarotene).Molecular dynamics simulations gave structural insights into the role of cholesterol in amyloid channel formation and explained the inhibitory effect of bexarotene.Because it is the first drug that can both inhibit the binding of cholesterol to Aβ  and prevent calcium-permeable amyloid pore formation in the plasma membrane of brain cells, bexarotene might be considered as the prototype of a new class of anti-Alzheimer compounds [124].(Note that because bexarotene shares structural analogy with cholesterol, and the above-described LCM/ND nanoemulsion contains substantial concentrations of cholesterol esters and cholesterol (see Sect. 3), incorporation of the bexarotene molecule into the LCM/ND nanocarrier is expected to represent an uncomplicated, straightforward formulation procedure commercially.)Moreover, Casali et al. [125] have very recently reported that treatment of an Alzheimer's-disease mouse model with (this FDA-approved anticancer drug) bexarotene resulted in enhanced cogniton in the APP/PS1 mice which resembled earlier findings.Strikingly, the authors observed sustained cognitive improvements in the mice even when bexarotene treatment was discontinued for 2 weeks.Also, they observed a sustained reduction in microgliosis and plaque burden, following drug withdrawal, exclusively in the hippocampus.Casali et al. concluded that bexarotene selectively modifies aspects of neuroinflammation in a region-specific manner to reverse hippocampal-dependent cognitive deficits in Alzheimer's-disease (APP/PS1) mice [125].
Additional molecular aspects, concerning the membrane-related mechanisms for the known neuroprotective effect, of bexarotene action on brain tissue continue to be suggested and/or described in the recent literature (cf.[126,127]).In the most recently published study, Kamp et al. [128] show by NMR and CD spectroscopy that bexarotene directly interacts with the transmembrane domain of the amyloid precursor protein (APP) in a region where cholesterol binds.(Note that Aβ peptides are derived from APP, by the sequential action of β-and γ-secretases.γ-Secretase cleavage occurs in the transmembrane domain, of the C-terminal fragment left by β-secretase cleavage, and results in the release of Aβ peptides of various lengths.The longer, neurotoxic, Aβ  peptide is highly aggregation prone and represents the major Aβ species deposited in the brain [128].See also [129][130][131].)These authors argue that their data [128] suggest that bexarotene is a pleiotropic molecule that interferes with Aβ metabolism through multiple mechanisms.The authors point out that one promising strategy is the use of small molecules, that interfere with protein aggregation and the formation of amyloid structures.In reviewing the related literature, Kamp et al. [128] explain that based on molecular modeling, monolayer experiments, and binding assays for bexarotene, it has been hypothesized by some investigators that bexarotene binds to extracellular Aβ peptides and inhibits the cholesterol-driven insertion of these peptides into the cellular membranes of brain tissue, thereby preventing oligomerization and subsequent neurotoxic pore formation [128].

Conclusion
The proposed multitasking combination therapeutic appears likely to display greater efficacy at different stages of Alzheimer's disease (cf.[72]).It is also possible that the effects on various cell types targeted may be additive, multiplicative, or otherwise synergistic [26].As a result, this multitasking (drug-delivery) therapeutic could represent a promising way to treat, delay, or even prevent the disease in the future [1,2].By incorporating the appropriate drug(s) into biomimetic (lipid cubic phase) nanocarriers, one obtains a multitasking combination therapeutic which targets certain cell-surface scavenger receptors, mainly class B type I (SR-BI), and crosses the BBB.Such targeting allows for various Alzheimer's-related cell types to be simultaneously searched out, in vivo, for localized drug treatment.This in vivo targeting advantage may be particularly important for repurposing an FDA-approved drug (such as the anticancer drug bexarotene) which up to now, by itself (i.e., without nanocarrier), has previously displayed poor CNS penetration in human subjects.101.Barauskas, J.; Cervin, C.; Tiberg, F.; Johnsson, M. Structure of lyotropic selfassembled lipid nonlamellar liquid crystals and their nanoparticles in mixtures of phosphatidyl choline and -tocopherol (vitamin E).Phys.Chem.Chem.Phys.2008, 10, 6483-6485.102.Efrat, R.; Aserin, A.; Garti, N. On structural transitions in a discontinuous micellar cubic phase loaded with sodium diclofenac.J. Colloid Interface Sci.2008, 321, 166-176.