Cutting-edge work with a new nanoparticle being conducted in Florida, Oklahoma and North Dakota is now showing great promise for treating multiple disease conditions—including ocular diseases such as macular degeneration and glaucoma.
The nanoparticle in question, called nanoceria, is composed of clusters of cerium oxide molecules. Cerium oxide is currently used in applications such as histochemistry, fuel cells, ultraviolet absorbents and automotive catalytic converters. The nanoparticle form, in addition to being highly biocompatible and tiny enough to cross the blood-brain barrier, has shown the capacity to act as a self-regenerating antioxidant, and can also function as a drug delivery system for other compounds.
Nanoceria was first developed by Sudipta Seal, an engineering professor who conducts research at the University of Central Florida's Advanced Materials Processing and Analysis Center (Mechanical, Materials and Aerospace Engineering), and the Nanoscience Technology Center, in Orlando, Fla. Professor Seal says the properties of nanoceria first became evident when he was testing it for biocompatibility. "In our early studies using rat brain cells in vitro, we were testing to make sure nanoceria wasn't toxic," he explains. "To our surprise, we found the brain cells were living longer, and their function was preserved. We've also published a study that showed nanoceria can revive spinal cord cells after injury."1,2
It appears that nanoceria's benefits stem from its ability to scavenge free oxygen radicals inside the cells. "Cells generate a lot of reactive oxygen species—superoxide radicals, hydroxy peroxide radicals, and so forth—which damage the cell," says Professor Seal. "This particle scavenges the radicals, preventing further damage, and then regenerates itself so it continues to scavenge radicals over and over again. And, nanoceria seems to have an inherent tendency to move to the locations where excess reactive oxygen species are present."3,4
Combating Retinal Damage
James F. McGinnis, PhD, professor of cell biology and ophthalmology at the Dean A. McGee Eye Institute in Oklahoma City, Okla., is using nanoceria to treat retinal damage and diseases. "The property that got me interested in nanoceria is its ability to destroy reactive oxygen species, which are extremely reactive with any other molecule," he says. "When they're formed inside a cell, they don't go very far because they react with the nearest molecules. But nanoceria also has another interesting ability: It can convert the target molecule to water and then return to its pre-reaction state, so it's ready to eliminate another molecule.
"These nanoparticles are inorganic, and because of that, they're non-inflammatory and nonimmunogenic, at least at the concentrations we've tested so far," he continues. "They're extremely small—about 3 to 5 nm in diameter. For comparison, a red blood cell has a diameter of 7,500 nm. Also, the effective concentrations used so far have been extremely small—about 5 nanomolar, or five billionths of a mole."
Professor McGinnis notes that photoreceptor cells have an extremely high rate of oxygen metabolism, and they're continuously exposed to the deleterious effects of oxidative stress and constant bombardment by photons of light, making them an obvious choice to benefit from a particle with antioxidant properties. "Since nanoceria are effective inside cells, I first tried them in a cell culture of rat retina neurons," he says. "They extended the life of the cells in culture by 50 to 60 percent. So, I decided to test them in vivo. Under normal conditions, six hours of bright light exposure eliminates 60 to 70 percent of the photoreceptors in the eyes of albino rats. So we intravitreally injected either nanoceria, in different concentrations, or saline as a control; three days later we exposed the albino rats to bright light for six hours. A week after that, histological evaluation showed that the nanoceria were highly protective of the photoreceptor cells.
"However, for in vivo studies, the gold standard is function," he continues. "So, we repeated the experiment, checking the rats' vision using electroretinography. The data were spectacular. All animals who were not exposed to light had a very nice waveform in the electroretinogram. But the ones exposed to the light who were injected with saline, or not injected at all, had a nearly flat ERG. In contrast, the rats injected with nanoceria showed a highly significant, concentration-dependent protection of vision."5
Professor McGinnis says he believes that most diseases go through a common phase in which, regardless of the primary insult or defect, cells respond with a rise in the intracellular concentration of reactive oxygen species. "We think that once a threshold is exceeded, cell-signaling pathways initiate either apoptosis or necrosis," he says. "These nanoparticles won't cure the primary disease, but they do appear to interrupt a crucial part of a nearly universal degenerative disease process. That means this treatment might be protective in many diseases, including Huntington's, Alzheimer's, Parkinson's, cardiac damage, cardiac reperfusion, brain reperfusion and stroke. It might prolong the function of the cells, and that would certainly prolong functional lifespan and improve quality of life.
"Since nanoceria appear to interrupt this destructive biochemical process, we decided they might prevent inherited retinal degeneration," he continues. "One mouse that we sometimes use is genetically altered for studying arteriosclerosis, and it has problems with its retina. Using an assay that allows us to quantify vascular endothelial growth factor in the retina, we detected little VEGF in normal mice, but about 200 times as much in the altered mice. We injected nanoceria into the eyes of the altered mice; a week later we found 60 percent less VEGF. In a second study, VEGF levels were nearly undetectable after three weeks." (Professor McGinnis reported some of these results at the 2008 Association for Research in Vision and Ophthalmology meeting.)
"We also used a fluorescence vascular filling assay that makes it possible to quantify vasculature in the retina," he continues. "The experimental mice had very leaky vasculature with a lot of choroidal neovascular tufts—blood vessels that poked through the retinal pigment epithelium cell layer, damaging the photoreceptors. A single injection of nanoceria prevented about 85 percent of these choroidal neovascular projections and about 85 percent of the vascular leakage. (See image, above.) This was an initial study, but I expect future results to be similar."
Delivering Other Drugs
Sanku Mallik, associate professor of pharmaceutical sciences at North Dakota State University in Fargo, N.D., has been using nanoceria as a drug delivery device. "We've successfully attached carbonic anhydrase inhibitors to the particles, and we've been able to demonstrate that the particle/inhibitor conjugate is still a very potent inhibitor of the enzyme," he says.6 "So far, our work has all been in vitro, but we plan to test this in the eye. First, however, we want to make sure the particle conjugate is very water-soluble, so it won't irritate the cornea and delivery will be effective." He adds that other glaucoma drugs should be candidates for this kind of delivery as well. (Professor Seal says one of his focuses is creating a nanoceria eyewash; that work has already reached the stage of animal research.)
Mr. Mallik says that his group is currently attaching anti-cancer drugs to the particles, along with targeting molecules that will ensure that the particles only enter the targeted cells. "So far, nanoceria appears to be nontoxic, but the drugs we attach to the particle might be toxic, so targeting molecules are necessary," he explains. "These particles can also be used for imaging; we can attach molecules that can be made to glow after they reach targets such as cancer cells."
In fact, nanoceria has demonstrated the ability to protect cells from radiation damage. Even more interesting, the protection seems to be selective to healthy cells. In one study, treatment of normal breast tissue cells with nanoceria conferred almost 99 percent protection from radiation-induced cell death, whereas the same concentration showed almost no protection of tumor cell tissue.7 Radiation is known to produce potentially lethal free radicals; the researchers hypothesize that nanoceria's radical scavenging, antioxidant abilities account for its protective effect.
Professor McGinnis says the nanoparticles may remain in the eye and work indefinitely, but so far no one knows the turnover rate. "We don't know if the cells in the retina have an uptake mechanism for these particles," he says. "We don't know how they get into cells, but we know they must be getting in; they have to get close to the reactive oxygen species in order to eliminate them. But we don't know how long the particles will last. If it turns out they remain indefinitely, that's good news and bad news. The good news is that they'll work forever. The bad news is, how do you determine toxicity on something that sticks around forever? How many years do you have to do experiments before the government is convinced it has no side effects?"
Although all of this research is still in its early stages, there's no question the potential is enormous.
1. Bailey D, Chow L, et al. Engineered oxide nanoparticles increase neuronal lifespan in culture and act as free radical scavengers. Nature Biotechnol 2003;14:112
2. Das M, Patil S, et al. Auto-catalytic ceria nanoparticles offer neuroprotection to adult rat spinal cord neurons. Biomaterials 2007;28:1918–1925.
3. Karakoti A, Reveire NM, et al. Overview—Nanoceria as antioxidants—Synthesis and Biomedical Applications. JOM 2008;60:3:33-37.
4. Korsvik C, Patil S, Seal S, Self W. Vacancy engineered ceria oxide nanoparticles catalyze superoxide dismutase activity. Chemical Communications 2007;10:1056-1058.
5. Chen J, Patil S, Seal S, Mcginnis JF. Rare earth nanoparticles prevent retinal degeneration induced by intracellular peroxides. Nature Nanotechnology, published online Nov 2006. Available at www.nature.com.
6. Patil S, Reshetnikov S, Haldar MK, Seal S, Mallik S. Surface-Derivatized Nanoceria with Human Carbonic Anhydrase II Inhibitors and Fluorophores: A Potential Drug Delivery Device. J Phys. Chem 2007;111:8437-8442.
7. Tarnuzzer RW, Colon J, Patil S, Seal S. Vacancy engineered ceria nanostructures for protection from radiation-induced cellular damage. Nano Letters 2005;5:12:2573-2577.