Treatment of Neurodegenerative Disorders with ... - Wiley Online Library

Treatment of Neurodegenerative Disorders with Radical Nanomedicine NEERAJ SINGH, COURTNEY A. COHEN, AND BEVERLY A. RZIGALINSKI Virginia College of Osteopathic Medicine and Virginia Polytechnic & State University, Blacksburg, Virginia, USA

ABSTRACT: In engineering and materials science, nanotechnology has provided many advances that effectively reduce oxidative damage generated by free radical production. Despite such advances, there has been little application to biomedical problems. Increased oxidative stress and free radical production are associated with neurodegenerative conditions, including aging, trauma, Alzheimer’s and Parkinson’s diseases, and many others. The antioxidant properties of cerium oxide nanoparticles show promise in the treatment of such diseases. Here, we summarize the work on the biological antioxidant actions of cerium oxide nanoparticles in extension of cell and organism longevity, protection against free radical insult, and protection against trauma-induced neuronal damage. We discuss establishment of effective dosing parameters, along with the physicochemical properties that regulate the pharmacological action of these new nanomaterials. Taken together, these studies suggest that nanotechnology can take pharmacological treatment to a new level, with a novel generation of nanopharmaceuticals. KEYWORDS: antioxidant nanoparticles; cerium oxide; nanotechnology; neuroprotection

INTRODUCTION Nanotechnology encompasses the process of constructing and manipulating materials on a near-atomic scale, at dimensions in the nanometer range. By definition, nanoparticles are structures smaller than 100 nm. The high surface area and catalytic activity of nanoparticles conveyed by their small size has revolutionized many commercial products and processes, improving catalysis, photoreactivity, glass properties, and fuel combustion. However, the high surface area and reactivity that improve commercial products and processes may also impart similar catalytic activity in cells, tissues, and organisms—an Address for correspondence: Beverly A. Rzigalinski, Ph.D., Virginia College of Osteopathic Medicine, NanoNeuroLab, Corporate Research Center—Res. II Bldg, 1861 Pratt Dr., Blacksburg, VA 24060. Voice: 540-231-1744. [email protected] C 2007 New York Academy of Sciences. Ann. N.Y. Acad. Sci. 1122: 219–230 (2007). doi: 10.1196/annals.1403.015 219

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area that lags behind the study of commercial applications. By controlling nanoparticle catalytic activity through synthesizing specific nanoconstructs, nanotechnology holds promise for a new generation of nanopharmaceutical agents. Here, we will discuss the biological activity of one such nanoparticle, cerium oxide (ceria).

CERIUM OXIDE Cerium is a rare-earth element of the lanthanide series, and its oxides have a fluorite lattice structure. Among the lanthanide series of elements, cerium is somewhat distinctive in that it has two partially filled subshells of electrons, 4f and 5d, with many excited substates, resulting in a valence structure that undergoes significant alterations depending on the chemical environment.1,2 In an oxide nanoparticle, the cerium atom can exist in either the +3 (fully reduced) or +4 (fully oxidized) state and may interchange between the two.3 During a redox event involving cerium oxide, the cerium valence state, oxide lattice size, and bond lengths may change as electrons are shuffled through the lattice, resulting in enhanced redox properties. Cerium oxide also contains oxygen vacancies or “defects” in the lattice structure, caused by loss of oxygen and/or its electrons.4 During reactions of ceria, creation and annihilation of oxygen vacancies occurs in the cerium oxide lattice, improving redox capacity even further. In materials science, cerium oxide nanoparticles are used as metal coatings to reduce oxidation, and in catalytic converters, such nanoparticles enhance oxidation of carbon monoxide and hydrocarbons and reduce nitrogen oxide emissions from combustion of fossil fuels.5 The chemical reactions of cerium oxide have been reviewed previously2,3,6 and include oxygen atom transfer and absorption, oxidation of unsaturated hydrocarbons, electron transfer to hydrocarbon radicals, high catalytic activity in redox reactions, and reduction of NO. Thus, the chemistry of cerium oxide is similar to what a cell biologist would term a free radical scavenger or antioxidant. At the nanometer scale, the free radical scavenging properties of cerium oxide are even further enhanced, because of the dramatic increase in surface area. Oxygen vacancies, and their potential for interactions with free radicals, form more readily at the nanoscale.7 Further, as the size of the cerium oxide nanoparticle decreases, there is a concomitant increase in the amount of Ce3+ , which may further enhance reducing power.8 The radical scavenging ability of cerium oxide appears to be dramatically increased during the reduction to the nanoscale.

OXIDATIVE STRESS The basic chemistry involved in removal of contaminants from combustion exhaust and prevention of metal oxidation is similar to redox reactions

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and antioxidant activities in the biological sense. Both involve the production of highly reactive free radicals, which lack the full complement of electrons necessary for molecular stability, such as superoxide (O 2 − ), hydroxyl (OH– ), nitric oxide (NO), peroxynitrite (ONOO– ), and others. Free radicals, when produced intracellularly, can strip electrons from biomolecules such as proteins and lipids, causing considerable damage to cellular constituents.9–11 Unlike materials, biological organisms have innate mechanisms for coping with free radical production, which are the subject of several excellent reviews.11–14 However, organism aging and disease often overtax the system and free radical generation exceeds the antioxidant capacity, resulting in a state termed oxidative stress. Being the most oxidative organ in the body, the brain and central nervous system are sites of heavy free radical production and hence high oxidative stress.9−14 Most neurodegenerative disease including Alzheimer’s, Huntington’s, and Parkinson’s diseases; trauma; and aging itself are associated with excessive oxidative stress. Yet to date, use of antioxidants in abolishing these pathological conditions has met with only limited success. Our traditional pharmacological antioxidants require multiple (often daily) dosing, because the free radical scavenging of each antioxidant molecule is usually limited to one free radical. Moreover, distribution of antioxidants is often limited to specific cellular sites, which may not necessarily coincide with the localized sites of free radical damage.

BIOLOGICAL EFFECTS OF CERIUM OXIDE NANOPARTICLES Our group15–17 and later others18 have found that cerium oxide nanoparticles may provide a future nanopharmacological approach to diseases associated with oxidative stress. In our prior studies using an in vitro model of traumatic brain injury, our laboratory routinely prepared mixed cultures of cortical brain cells, comprising neurons and glia.19–22 When removed from the rat, most primary mixed cortical brain cell cultures (organotypic cultures) remain healthy and viable for an average of 21 days. In a somewhat serendipitous discovery, we found that one treatment of these mixed cortical cultures with 10 nM cerium oxide nanoparticles (average size, 10 nm) increased their lifespan by up to sixfold.15,17,24,25 A representative photomicrograph is shown in FIGURE 1. Panel A shows an untreated mixed organotypic culture after 37 days in vitro (DIV), showing few live cells and no coherent culture network. Panel B shows a culture from the same preparation batch, treated with 10 nM cerium oxide nanoparticles once, on day 10 in vitro. Note the robust cellular network and many processed neurons. Normal and glutamate-stimulated neuronal calcium signaling in these aged cultures was the same as that in their normal, younger counterparts15,17 —hence normal function appeared to be preserved (high-speed microspectrophotometric videos of calcium signaling in these aged cultures are available at http://www.nanoneuro.vcom.vt.edu). Only one 10 nM dose of particles was administered to the cells on day 10 in vitro, remaining in the media

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FIGURE 1. Light photomicrographs of mixed organotypic brain cell cultures. Top panel: Control culture at 37 DIV. Note few cells remaining and lack of integrated astrocyte monolayer with processed neurons. Bottom panel: 37 DIV culture treated with 10 nM cerium oxide nanoparticles at 10 DIV. Note confluent astrocyte monolayer with abundant processed neurons.

for 48 h until the next medium change. As we have previously shown,15–17 this exposure period resulted in cellular uptake of ceria nanoparticles, as shown by electron microscopy. Hence, it appears that one dose was incorporated intracellularly and provided prolonged effects.

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Given the materials science usage of ceria nanoparticles as free radical scavengers, we reasoned that nanoparticles were acting within the cell in a similar fashion, possibly as antioxidants. In support of this hypothesis, we found that ceria nanoparticles also protected neurons from free radical–mediated insult initiated by UV light, H 2 O 2 , irradiation, and excitotoxicity.15–17,26 One dose delivered on day 10 in vitro continued to protect against free radical insult through at least 2 months in vitro, without repetitive dosing. Micronsized particles or nitrates and chlorides of cerium had no effect. In further support for an antioxidant role for ceria, we found that ceriatreated cultures maintained higher levels of catalase and reduced glutathione during the lifespan, compared with untreated controls,27 suggesting that ceria treatment either preserved or added to innate cellular radical scavenging systems. Recently, we confirmed the radical scavenging activity of cerium oxide nanoparticles in vitro. Using electron paramagnetic resonance, we found that cerium oxide nanoparticles did not produce free radicals in vitro and were excellent scavengers of both superoxide and hydroxyl radicals (Singh, Cohen, and Rzigalinski, unpublished data). In dose–response studies, we found that 10–100 nM concentrations produced optimum radical scavenging and longevity enhancing effects. However, the dose–response curve was bell shaped, with doses lower than 1 nM and higher than 1 M having reduced beneficial effects (no toxicity was observed at any dose range examined). This finding is expected, because recent literature suggests that a basal level of free radical production is necessary for normal cell signaling,28,29 which indicates that excessive or repeated doses of ceria may have negative effects. Schubert et al.18 examined the toxicity and free radical scavenging activity of ceria and yttrium oxide nanoparticles in a neuronal (HT22) and a macrophage (RAW164) cell line. During free radical challenge with 5 mM glutamate, the nanoparticles afforded significant antioxidant protection and increased cell survival. Using the free radical indicator dye dichlorofluorescein diacetate, the group demonstrated that nanoparticles could decrease free radical production. This study differs somewhat from our prior work in that cells were not pretreated with nanoparticles, but nanoparticles were delivered just before free radical challenge. In an in vitro model for brain trauma that our laboratory uses routinely,19–22 ceria nanoparticles protected mixed organotypic brain cell cultures from trauma-associated cell damage and maintained normal neuronal calcium signaling in a high percentage of neurons after trauma.15–17,25 Trauma-induced cell damage, as measured by uptake of propidium iodide, was reduced by more than 75% in ceria-treated cultures, compared with controls. In untreated injured cultures, neuronal calcium homeostasis was substantially disrupted at 24 h postinjury, as previously described.19–22 This disruption in neuronal intracellular free calcium ([Ca2+ ] i ) signaling was partially blocked in ceriatreated cultures. A representative tracing from these experiments is shown in

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FIGURE 2. Treatment with cerium oxide nanoparticles preserves normal neuronal calcium signaling after trauma. Mixed neuronal cultures were prepared as previously described.19–22 Nanotreated cultures were exposed to 10 nM cerium oxide in the culture medium for 48 h at 10 DIV. Cultures were injured using an in vitro trauma model and [Ca2+ ] i was measured in neurons 24 h postinjury, using Fura-2 as previously described. Ceria-treated cultures maintained [Ca2+ ] i signaling similar to that in uninjured controls.

FIGURE 2. In this experiment, neurons were selectively loaded with Fura-2, and [Ca2+ ] i was monitored with high-speed ratiometric imaging, as previously described.19–22 Neurons in control uninjured cultures displayed basal oscillations in [Ca2+ ] i as shown and responded to glutamate with an elevation in [Ca2+ ] i of 160 nM. Twenty-four hours after a moderate trauma to untreated cultures, most of the neurons remaining (86%) fell into two basic categories, with respect to [Ca2+ ] i . The first category of neurons displayed a dramatically elevated [Ca2+ ] i level of more than 300 nM, and basal calcium oscillations were no longer observed (FIG. 2, top tracing). This group showed no response to exogenously added glutamate, indicating that neurons may be irreversibly damaged. A second group of neurons had modestly elevated basal [Ca2+ ] i with no oscillations and exhibited a significantly enhanced response to glutamate stimulation, suggesting dysfunction of calcium signaling (FIG. 2, as indicated). However, in injured cultures treated with ceria nanoparticles, normal neuronal calcium oscillations and responses to glutamate were observed in approximately 60% of the neurons (FIG. 2, lower dotted line; compare with control). Importantly, one dose of nanoparticles delivered up to 3 h postinjury also afforded similar neuroprotection. In vitro trauma induces free radical production23 ; hence, we hypothesize that the protective effect of ceria may be a result of its antioxidant properties.

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At the in vivo level, the biological effects of cerium oxide nanoparticles were examined in the fruit fly, Drosophila melanogaster. Adding 10 nM ceria nanoparticles directly to the food for the entire lifetime of the fly increased median survival by 31% and maximum lifespan by 18%.16 However, the dose– response curve in these studies was also bell shaped, and doses lower than 1 nM or higher than 1 M had reduced, and sometimes negative, effects— further supporting the hypothesis that there may be an optimal level of free radical scavenging above which antioxidant nanoparticles may interfere with the beneficial and necessary roles of free radicals within the cell. During these experiments, we also examined the negative geotactic response of Drosophila. The negative geotactic response measures the ability of Drosophila to ascend the walls of the culture vial after a stimulus that taps them to the bottom of the vial. As flies age, the ability to mount a negative geotactic response declines. Flies fed ceria during their lifespan maintained a negative geotactic response for significantly longer than untreated controls, suggesting that function is preserved with increased lifespan. Taken together, these results indicate that cerium oxide nanoparticles may be extremely effective free radical scavengers. But why? From our observations thus far, we hypothesize that cerium oxide is a regenerative free radical scavenger. In cellular studies, one low dose maintained radical scavenging and protective effects for long durations and multiple insults, suggesting the possibility of regenerative activity. Consistent with this mechanism of action, we have observed alterations in the spectrophotometric properties of ceria during free radical challenge (H 2 O 2 ) under physiological conditions.15–17 High-speed fluorescence microspectrophotometry showed that ceria nanoparticles had an excitation maximum of 451 nm, with a secondary minor peak at 354–356 nm. After addition of H 2 O 2 , there was an increase in the peak of 354–356 nm, with a concomitant decrease in the 451-nm peak, followed by a slow return to the original spectra. The shift to higher excitation energies (shorter wavelengths) during radical challenge suggests oxidation of Ce3+ sites to Ce4+ by free radicals, whereas the slow return to lower excitation energies (longer wavelengths) indicates Ce3+ site regeneration necessary to complete the redox cycle.30–32 These findings, taken together with the results of cellular and in vivo experiments, suggest that ceria nanoparticles are indeed regenerative antioxidants. In our prior studies, other lanthanide oxide nanoparticles did not produce the same protective effects as ceria. Of all the metal oxide nanoparticles, what makes ceria so unique? The chemistry and physics of ceria suggest potential answers to these questions. From the lanthanide series, ceria nanoparticles are reported to have several unique properties that make them highly efficient redox reagents—they can alter the valence state of cerium and create and annihilate oxygen vacancies.16 Because of the decrease in size and increase in surface area, these reactions occur more readily at the nanoscale, which may provide the enhanced reducing power for free radical scavenging at the cellular level. After the scavenging event, the original lattice structure may be regenerated

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by electronic reorganization of the ceria lattice or by interactions with other molecules such as H 2 O. Cerium oxides are unique among the rare-earth elements in that there is a high hydrogen absorbing capacity on the surface, enabling reactions with H 2 , O 2 , or H 2 O, which may enhance its regenerative ability. Further experimentation with ceria nanoparticles containing various oxygen vacancies and valence structures will enable us to decipher the role of oxygen vacancies and valence structure in nanoparticle reactivity.

MORE GROUND TO COVER Cerium oxide nanoparticles appear to hold promise for nanopharmacotherapy of neurodegenerative and other disorders linked to oxidative stress. Despite this promise, there remain hurdles to be overcome in moving from benchtop to bedside, as well as many adaptations to our traditional concepts of dose and delivery. One of the most critical issues is consistency in preparations. As noted for carbon nanotubes, tailing of contaminants from the synthesis method often have the propensity to cause cell damage.32–34 Similarly, we have noted that tailing of docusate sodium during microemulsion synthesis is also problematic for delivery of ceria into tissue culture. Yet when added to Drosophila feeding medium, docusate sodium improves nanoparticle dispersion and prevents agglomeration in the fly food at low doses. Therefore, careful assessment of purity of the starting material and description of any additives is important for future studies. A second pharmacological factor is particle size. We rarely categorize a drug or ascertain a dose by molecular size. However, these measurements will be critical to nanopharmacology. Nanoparticle preparations are suspensions by nature and have a tendency to agglomerate in the biological milieu. Particle size and in vivo agglomeration is further affected by pH, ionic strength, and the presence of proteins and lipids.16 Therefore, we must be attentive to the details of how we prepare nanoparticle solutions for delivery and specify all predelivery treatments. Adherence to a specified sonication protocol before delivery results in an even, minimally agglomerated dispersion of nanoparticles, whereas lack of sonication produces an agglomerated delivery solution.16 Such steps can be critical to assessment of pharmacological action. Last, establishing dosing parameters with nanoparticles is complicated because of the nature of the materials and requires much future development. Traditional dosing parameters based on mass or molar number are useful, but they do not account for the reactive surface area of the nanoparticle.33,34 Inherently, when moving to the nanoscale, surface characteristics such as roughness, porosity, and crystal structure will play an important role in biological reactivity/toxicology. Because the surface area of nanoparticles increases with a

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FIGURE 3. Effect of size on H 2 O 2 -mediated damage in mixed organotypic cortical brain cell cultures. Cell cultures were prepared as previously described and treated with 10 nM cerium oxide nanoparticles of the indicated sizes, at 10 DIV. On days 14–16, cultures were subjected to oxidative stress by 100 M H 2 O 2 for 15 min. Number of injured cells was assessed by uptake of propidium iodide.

decrease in particle size, surface area has been suggested as a dosing parameter. However, using dosing as such may not be optimum for ceria and other catalytic nanoparticles, where the degree of redox activity is the discriminating pharmacological factor. In our studies in mixed neuronal cultures, microglia, and Drosophila, ceria nanoparticles exhibited a bell-shaped dose–response curve, with an optimal dose above which the beneficial effects were decreased. For 10-nm particles, 10–100 nM appeared to be the optimal dose range for most cellular studies. In FIGURE 3, we compare the protection against H 2 O 2 -mediated cell injury afforded by three different sizes of cerium oxide nanoparticles, all dosed at the same concentration (10 nM). Consistent with previous studies, 10-nm particles afforded the most protection. However, an equal molar dose of smaller 7-nm particles, with more surface area, afforded less protection than 10-nm particles. Perhaps the higher surface area of the smaller particles produced excessive free radical scavenging, causing detrimental effects on normal signaling. However, when lower doses (i.e., 1 nM) of the 7-nm particles were assessed for H 2 O 2 protection, there was still no increased protection (data not shown). Hence, lower doses (smaller surface area) of smaller particles do not appear to be comparable to higher doses or larger particles, at least for ceria. Also shown in FIGURE 3 is the lack of effect of 50-nm particles on protection from H 2 O 2 . These results suggest that there is an optimum size for ceria nanoparticles, in relation to their biological effects. There may be several reasons for these observations. Limbach et al. found that cellular incorporation of ceria nanoparticles depended on size in a similar manner.36 Large particles appeared to be excluded from cells, as were particles smaller than 10 nm. They

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hypothesized that large particles were simply too large for cell incorporation, whereas smaller particles were more highly agglomerated, thereby blocking cellular incorporation. Alternatively, the effects of size on radical scavenging may be caused by size-induced alterations in the ceria lattice structure. As discussed in our previous review,16 there may be an optimum Ce3+ /Ce4+ ratio for redox activity. Owing to alterations in lattice structure with particle size, there may be an optimum particle size at which regenerative radical scavenging activity is most effective because of the Ce3+ /Ce4+ ratio. Further investigations into the issue of lattice structural changes with particle size will clarify these findings. Nonetheless, given the apparent biological differences between particle size that may be unrelated to surface area, we are investigating another form of dosing parameter. For ceria, particle reactivity may be the critical dosing factor. Using kinetic modeling of the radical scavenging ability of different-sized ceria nanoparticles, we are investigating dosing by particle reactivity, similar to the use of international units, which has become a standard dosing parameter for drugs such as penicillin or for expression of enzyme activity.

NANOPARTICLES FOR TREATMENT OF NEURODEGENERATIVE DISEASE Nanoparticles such as ceria hold promise for more effective treatment of diseases associated with oxidative stress by virtue of their long-lasting antioxidant properties. However, much work remains. Many nanoparticles, such as carboxyfullerenes and other metal oxides, are reported to be free radical generators. Hence, more complete toxicological assessments of ceria are warranted. Also, as discussed above, a basal level of free radical signaling appears necessary for normal biological function. Interference in the basal free radical signaling by nanoparticles may be deleterious, as suggested by tissue culture and Drosophila studies. With traditional pharmacology, an effective dosing regimen could probably be established. However, given our ability to construct nanodevices, the synthesis of particles with controlled activity is a distinct possibility. The ideal would be to design a nanoconstruct that can self-regulate its radical scavenging activity on the basis of the redox status of the intracellular milieu, using bioresponsive coatings sensitive to oxidative stress. Such constructs could direct radical scavenging activity to specific sites of increased cell or tissue oxidative stress, resulting in radical scavenging at the “right place and right time”—a property lacking in many of our current pharmacological antioxidants. Nanotechnology can significantly enhance our treatment of neurodegenerative disorders. Although further studies are needed, redox-active nanoparticles such as ceria may provide the superior antioxidant activity necessary to effectively relieve oxidative stress and improve neurological function.

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ACKNOWLEDGMENTS

Our research is supported by grants from the NIH, NINDS NS40490 and AG022617.

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