Nanoparticle Delivery of Transition-Metal Chelators to the Brain: Oxidative Stress will Never See it Coming!

Abstract

The pathological lesions typical of Alzheimer disease (AD) are sites of significant and abnormal metal accumulation. Metal chelation therapy, therefore, provides a very attractive therapeutic measure for the neuronal deterioration of AD, though its institution suffers fundamental deficiencies. Namely, chelating agents, which bind to and remove excess transition metals from the body, must penetrate the blood-brain barrier (BBB) to instill any real effect on the oxidative damages caused by the presence of the metals in the brain. Despite many advances in chelation administration, however, this vital requirement remains therapeutically out of reach: the most effective chelators—i.e., those that have high affinity and specificity for transition metals like iron and copper—are bulky and hydrophilic, making it difficult to reach their physiological place of action. Moreover, small, lipophilic chelators, which can pass through the brain’s defensive wall, essentially suffer from their over-effectiveness. That is, they induce toxicity on proliferating cells by removing transition metals from vital RNA enzymes. Fortunately, research has provided a loophole. Nanoparticles, tiny, artificial or natural organic polymers, are capable of transporting metal chelating agents across the BBB regardless of their size and hydrophilicity. The compounds can thereby sufficiently ameliorate the oxidative toxicity of excess metals in an AD brain without inducing any such toxicity themselves. We here discuss the current status of nanoparticle delivery systems as they relate to AD chelation therapy and elaborate on their mechanism of action. An exciting future for AD treatment lies ahead.

Introduction

Alzheimer disease (AD) is a progressive and fatal neurodegenerative condition that afflicts millions of people around the world [1]. The deterioration of cognition and memory that characterizes AD has resisted efforts to develop an effective treatment, though much progress has been made in the last decade. However, current FDA-approved drugs, such as the cholinesterase inhibitors, are a far cry from a full cure: these agents merely attenuate symptoms of disease and, at best, only postpone the inevitable decline in general cognitive functions [2]. Consequently, the economic and societal burden of AD is increasing as the world’s population ages. As an estimated 24 million people suffer from dementia worldwide [3], the associated health-care costs currently exceed $100 billion per year [1, 4], and the emotional burden on the families of the afflicted is immeasurable. There is thus a clear need for an advanced therapeutic strategy for AD that can stop or even reverse its progression. The accumulation of metals such as iron and copper throughout the brain, and specifically within the pathological lesions of AD, provides an untapped target for disease treatment. While there exist many other opportunities for intervention throughout the course of the disease, metal chelation therapy is becoming increasingly attractive.

Oxidative Stress in AD

Although the etiology of AD is incompletely understood, oxidative stress resulting from a variety of intracellular malfunctions undoubtedly plays a key role [5]. There seems to exist a complex cascade whereby free-radical species, inevitably generated from the multitudes of redox reactions in metabolically hyperactive neurons [6], chemically alter vital macromolecules within the cell, such that significant damage is incurred [7–10]. Many in the field assert that it is the irreversible damage to neurons, brought about by this oxidative stress cascade, which elicits the infamous pathologies of AD (amyloid plaques and neurofibrillary tangles) and ultimately neuronal demise [11–13]. Indeed, the levels of oxidatively damaged mitochondrial DNA, proteins, and phospholipids in AD brains are elevated when compared to control cases [8–10]. Furthermore, these damages precede the appearance of the signature pathologies of AD that have long been held responsible for disease initiation [14–16]. It therefore seems likely that the most effective method of treatment of AD, one that would attenuate the root-cause disturbances, is one that targets oxidative stress.

Interestingly, the oxidative reactions in the brain can be catalyzed by transition metals, such as iron and copper [17, 18]. The likelihood that an oxidative reaction will take place in the brain is increased by regional concentrations of these metals, and in fact, some such transition metals, have been found in high concentrations in AD brains, accumulating specifically in the pathological lesions [19–23]. Additionally, amyloid-β (Aβ), a significant, though not causal, pathological factor in AD [24–26], may exert its toxicity through the formation of protease-resistant oligomeric and fibrillar forms of the peptide [27], which is also dependent upon transition-metals [28]. Therefore, metal chelating agents that selectively bind to, remove, and/or “redox-silence” transition metals provide excellent potential for therapeutic intervention in AD. Indeed, such chelators are becoming increasingly scrutinized for just such usage.

Metal Chelation Therapy for AD: Current Biochemical Shortcomings

Currently available metal-chelating compounds have been moderately successful in clinical trials for AD treatment [29]. Notably, while these agents have slowed the progression of AD in some cases [30, 31], they are severely limited in their effectiveness due to fundamental aspects of their biochemistry. The hexadentate iron chelator Desferrioxamine (DFO), for example, which tightly binds iron(III) and moderately binds aluminum, zinc, and copper (Figure 1A, B) [31–34], has impeded the progression of AD in clinical trials, despite its high molecular weight and hydrophillicity [35–37]. Its clearance of transition metals from the brain is the proposed mechanism for its effectiveness, and indeed, it is one of two iron chelators approved by the FDA for iron overload disease [29]. However, its bulky size and hydrophillicity, typical of hexadentate iron chelators (i.e., those that incorporate six atoms into the substrate-ligand bond to form the coordination complex (Figure 1B) [38] limit is capacity to penetrate the blood-brain barrier (BBB)—a vital requirement for any AD therapy. Rather, its success in clinical trials was the result of long, subcutaneous administrations of the compound [34, 39]; something quite impractical and unappealing for widespread AD treatment.

Figure 1.

The hexadentate metal chelator Desferrioxamine (DFO) in its isolated (A) and complexed (B) forms.

Smaller metal chelation compounds, on the other hand, typically have the ability to penetrate the BBB, but are toxic upon administration [37]. In particular, bi- or tri-dentate iron chelators (those that utilize two or three atoms in their substrate-ligand bonds, respectively), such as the α-ketohydroxypyridone derivatives, efficiently bind to and remove iron from intracellular pools; however, in doing so, they eliminate much of the supply of iron necessary for the functioning of human ribonucleotide reductase—a vital enzyme necessary for the reduction of ribonucleotides to deoxyribonucleotides, and thus for DNA synthesis and cell proliferation [37, 40–44]. Furthermore, due to their lipophilicity and small size, such compounds are also thought to penetrate the interior of the large ribonucleotide reductase complexes to chelate and remove the transition metal within them [45]. They thus both directly and indirectly inhibit the vital functioning of ribonucleotide reductase intracellularly and are toxic to proliferating cells. Notably, DFO and other hexadentate iron chelators are somewhat toxic as well, due to their removal of the iron involved in the redox cycling of these RNA enzymes; their toxicity, however, is much less than that of their lipophilic counterparts.

Ultimately, there exists a need to develop metal chelating agents that can effectively penetrate the BBB without inducing toxicity upon neurons. Fortunately, efforts are well underway in such a pursuit, and specifically, nanoparticle delivery of metal chelators has attracted much attention [28, 29]. Ideally, the nanoparticle delivery method will improve drug efficacy while reducing toxicity, thus providing a safe, non-invasive method of treatment.

Nanoparticle Delivery of Metal Chelation Compounds

Nanoparticle delivery systems provide an interesting and rather attractive approach to metal chelation therapy (Figure 2). These polymeric particles, ranging in size from ~10–1000 nm [46], are made from natural or artificial polymers [47], and are capable of binding to drug compounds such that they may be delivered to physiological regions normally prohibitive of their entry. Indeed, nanoparticles present a method of entry into the BBB that AD pathologies will never see coming.

Figure 2.

The hexadentate chelator DFO conjugated to a nanoparticle (shown here) has the potential to enable the hydrophilic molecule’s passage across the blood-brain barrier.

The method of BBB penetration that makes nanoparticle delivery of metal chelators so appealing is not fully known. However, there are a number of possibilities that likely work in combination to achieve the desired effect. These include [47]: i) increased retention of nanoparticles in blood-brain capillaries combined with absorption into capillary walls to create a higher concentration gradient, thus enhancing their transport across endothelial cell layers into the brain; ii) a surfactant effect that would lead to membrane fluidization and enhanced drug permeability; iii) an opening of tight junctions between endothelial cells; iv) endocytosis of the nanoparticle-chelator conjugate; v) transcytosis of said compound through the endothelial cell layer; and vi) an inhibition of the efflux system (i.e., P-glycoprotein) via polysorbate-80 (a nanoparticle coating proven to yield the most effective delivery of drugs across the BBB [48–50]). While each of these mechanisms is quite possible, the most probable one seems to be the endocytosis of the chelator-nanoparticle conjugate. Several studies confirm endocytosis of polysorbate-80 coated nanoparticles [49, 51–54]. Interestingly, absorption of apolipoprotein E (ApoE) on the surface of polysorbate-80 coated nanoparticles coincides with the latter’s BBB penetration [49], providing key insights into the mechanism of action of the endocytosis. Specifically, it is possible that polysorbate-coated nanoparticles mimic low-density lipoprotein (LDL) absorption into the brain: as ApoE facilitates the delivery of LDL across the BBB [47], its absorption onto nanoparticle compounds may act via LDL-receptor-mediated endocytosis (Figure 3), thus granting hydrophilic chelators access to the brain.

Figure 3.

The proposed method of nanoparticle-chelator conjugate entry across BBB. Upon absorption of APOE lipoprotein to the surface of the Polysorbate-80 coated nanoparticle conjugate (1), the particle mimics LDL and binds to LDL-receptor (2) stimulating LDL-mediated endocytosis (3,4).

Incidentally, this approach offers distinct advantages over other methods of chelator administration. First, the chelator needs not be lipophilic to enter the BBB, as the nanoparticle can carry across any compound to which it is covalently bound [29]. This directly eliminates much of the toxicity associated with the bi- or tri-dentate chelators that removed iron supplies from vital enzymes within the cell. Second, as noted above, nanoparticle delivery permits the use of hexadentate iron chelators that were previously too large and hydrophilic to penetrate the BBB [48, 50]. Third, iron-complexed chelators (i.e., those that have complexed with the transition metal within the brain) may be effectively removed from the brain via nanoparticles, thus completing the treatment regimen. As to the latter, it is equally necessary for a metal chelator to be able to exit the brain through the BBB as it is to enter it; a complexed chelator must therefore leave the brain after retaining its substrate. With lipophilic chelators, their removal from the brain becomes problematic once they have complexed with a transition metal, due to their resulting altered lipophilicity [29, 55]; they can no longer exit the BBB and themselves become progenitors of metal-associated oxidative damage [56, 57]. A nanoparticle delivery system, however, theoretically provides an adequate escape route for the chelator [29]. That is, the apolipoprotein system that the nanoparticle conjugates most likely mimic is also responsible for its removal from the brain [47, 57]. Consequently, the nanoparticle-chelator system would enable the full, desired effect of a metal chelator without any toxic side effect.

Recent evidence has confirmed the benefits of nanoparticle metal chelation. As stated above, a large body of literature suggests that the neurotoxicity of Aβ may result from the formation of protease-resistant oligomeric and fibrillar forms of the peptide [27], and that transition metals in the brain are partly responsible for their formation [28]. Correspondingly, reports on the effectiveness of nanoparticle delivery of transition metal chelators have demonstrated an effective prevention of Aβ aggregation and toxicity in vitro. In particular, a co-incubation of Aβ with chelator nanoparticle conjugates completely prevented Aβ precipitation in vitro under physiological conditions [28]. The cells were cultured with either Aβ, chelator nanoparticle conjugates, or both, and ultimately, the latter cases were significantly salvaged from Aβ-induced cell death. Importantly, the chelator-nanoparticle conjugates did not induce toxicity to neurons at the recorded concentrations, and cells treated with Aβ and the conjugates demonstrated similar rates of proliferation as the control cells. Indeed, nanoparticle delivery provided adequate protection of cortical neurons from Aβ-induced toxicity.

CONCLUSIONS

Ideally, nanoparticle chelator conjugate systems can be used to remove transition metals within the brain, prior to any clinical symptoms and possibly in prevention of disease onset. Although these chelators have been demonstrated to effectively stifle the effects of Aβ on cortical neurons in culture, such pathology is present in a brain well on its way through the disease course [2]. That is, as recent evidence suggests, Aβ is merely a secondary pathology of AD that is in fact secreted as a compensatory measure for the oxidative damages that an aging brain accumulates [2, 8–10]. Metal chelation therapies therefore have the potential to deter the formation of oxidative free radicals in the brain, if harnessed appropriately; such would ideally prevent the secretion and aggregation of hallmark AD pathologies that, once accumulated, incur severe damage upon surrounding neurons [58, 59].

Nanoparticle conjugation provides an exciting approach to chelator delivery. Conjugation maximizes effectiveness of metal chelator by granting large, hydrophilic compounds entry to the BBB [48–50]; reduces toxicity of chelation compounds by providing the complexed chelators an escape route [56, 57]; and increases chelator bioavailability [29], all while preserving the chelation capability of the compound [28]. A nanoparticle delivery system thus presents a therapeutic measure with a high potential for success in clinical AD cases; it will certainly yield beneficial results if instituted early enough. Further in vivo and in vitro analyses are warranted.