ability to inhibit the development and the maintenance of pathologic retina neovascularizations and strongly suggest that the CNPs can be therapeutically effective in the treatment of pathologic neovascularizations in human diseases such as diabetic retinopathy and age related macular degeneration.

19.9Toxicity and Environmental Impacts

In addition to their antioxidant and protection effects, CeO2 nanoparticles must be shown to be safe to use without any negative environmental effects. In all our experiments with CNP treatment of wild-type and mutant rats or mice, no inßammatory or other adverse side effects have been found. Other research groups have also demonstrated that the CNPs do not exhibit any toxicity in cultured cells and mouse tissues [12, 30, 34]. Studies [104] demonstrated that uncoated nanoceria exhibit high afÞnity for nucleic acids including human-pathogenic viruses, such as adenovirus, adenoassociated virus and human immunodeÞciency virus type 1 and can clear such pathogens from aqueous solution. Nanoceria at 5Ð10 mg/mL did not cause any genotoxic effects (DNA damage or sister chromatid exchanges) in human lens epithelial cells [105]. Nanoceria labeled with ßuorescein were taken up into caveolin-1 and LAMP-1 positive endosomal compartments in BEAS-2B (human lung epithelial cell) and RAW 264.7 cell lines and continued to suppress ROS production and make cells resistant to exogenous oxidative stress without inßammation or toxicity [106].

It has been reported that the surface area, charge, and structures as well as the cellular localization of the nanoparticles are determinate factors closely related to the toxicity of nanoparticles [12, 107]. Since cancer cells display an acidic microenvironment [46] the positively and neutrally charged nanoparticles are preferentially taken up by cancer cells [12, 14]. When the nanoceria are internalized in the acidic lysosomes in the cancer cells which have signiÞcant levels of oxidase activity, they exhibit signiÞcant cellular toxicity. However, the nanoparticles internalized in the cytoplasm showed no toxicity independent of their surface charges [12]. Van HoeckeÕs group exposed green algae, crustaceans and embryos of Danio rerio (zebraÞsh) to three different sized nanoceria in standard media, which caused nanoparticle aggregation to 400 nm. This resulted in signiÞcant chronic toxicity to green algae even at a very low dose (2.6 mg/L) which does not produce acute toxicity in other species. Surprisingly, the increase in toxicity is related to the decreasing size of the nanoparticles [108]. Park et al. reported that nanoceria of 30 nm in concentrations of 5, 10, 20, 40 mg/mL led to BEAS-2B cell death, ROS increase, and oxidative stress-associated gene expression. However, nanoceria did not cause signiÞcant cytotoxicity in cultured T98G (derived from human brain) or H9C2 (derived from rat cardiomyocyte) cells [109]. Furthermore nanoceria increased ROS concentration subsequently leading to the induction of heme oxygenase-1 (HO-1) via P38-Nrf-2 signaling pathway [110]. But there is no difference in the cell viability among the various sized nanoceria suggesting that there is no correlation between nanoparticle size and toxicity [109, 111, 112]. Exposure of human lung epithelial cells, A549

carcinoma cells and L-132 normal cells, to the nanoceria had a slight adverse effect on cell proliferation and cell viability over short time periods but caused membrane damage and colony formation in long-term cultures [113]. Nanoceria in 20 nm size caused oxidative stress in human lung cancer cells as indicated by lipid peroxidation and cell membrane damage indicators, and the toxic effects are dose and time dependent [114]. These contrary reports are possibly because of the differences in nanoceria synthesis, dispersion in media, presence of other surface species, nanoceria sizes, dosing, cell types, and subcellular localizations. Therefore, it is very important that consistency be maintained in the preparation, particle size, and dosing, when considering target applications.

Potential environmental health hazards were tested and signiÞcant uptake was found in the liver of zebraÞsh [115]. Gradual accumulation of CeO2 by intratracheal instillation in the male albino rats caused lung inßammation and injury which may lead to Þbrosis [116]. Furthermore, Wistar rats retained 63.9% of the instilled CeO2 nanoparticles in the lung by 28 days postexposure and the elimination half-life was 103 days. At the end of testing, only 1/8Ð1/3 of the daily dose to the lung was removed. This implied that reposition and redistribution of CeO2 nanoparticles could result in transport by the systematic circulation and accumulation in the extrapulmonary organs [117].

19.10Conclusion and Future Directions

Medical application of nanoparticles (referred to as nanomedicine) is very different from the traditional drug delivery. A drug molecule with a size less than 10 nm can go almost anywhere inside the body without being impeded by normal barriers [83]. Nanomedicines with unique properties, such as small size with enlarged surface area, enhanced regenerative and catalytic enzyme activities, and subsequent biological effects, etc., brought us to a new research level beyond the traditional concepts of cellular and/or organ systems. Most importantly, the CeO2 nanoparticles can act as direct antioxidants and prolong the survival of cells in vitro and in vivo. A distinct advantage of the nanoceria is that they can retain their effectiveness because of their self-regenerative properties and thereby avoid the side effects arising from repeated dosing. This property of the nanoceria should be especially beneÞcial for the treatment of ocular diseases because with fewer injections, the possibility of damage and/or infection is greatly reduced. The nanoceria should be able to decrease the symptoms of ocular diseases caused by production of excessive amounts of ROS, but they will not cure the original defect. For this reason, we think of the nanoceria as aspirin for blindness.

Acknowledgments The preparation of this review was supported in part by grants from NIH (P30-EY12190, COBRE-P20 RR017703, R21EY018306, and R01EY018724), FFB (C-NP-0707- 0404-UOK08; NSF: CBET-0708172, and OCAST: HR06-075), and unrestricted funds from Presbyterian Health Foundation and Research to Prevent Blindness (RPB). JFM is a recipient of an RPB Senior ScientiÞc Investigator Award.

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