A microemulsion process was used to synthesize the cerium oxide nanoparticles () and they were characterized for morphology and surface chemistry by high-resolution transmission electron microscopy (HRTEM) and X-ray photoelectron spectroscopy (XPS). HRTEM indicated the formation of uniformly distributed, non-agglomerated nanoparticles of Cerium oxide in the range of 3–5nm as shown in the image in . shows a XPS spectrum that indicates a mixed valence state (Ce3+
) for the synthesized Cerium oxide nanoparticles. These results are similar to our previously published results [1
Figure 1 Synthesis and Characterization of Cerium oxide Nanoparticles. (a) Outline of the microemulsion technique to synthesize the nano-Ceria (b) XPS analysis of synthesized Cerium oxide nanoparticles showing the presence of both Ce3+ and Ce4+ valence states (more ...)
The in vitro
studies with the synthesized nanoparticles were carried out in a serum-free cell culture model of adult rat spinal cord () which has been shown to promote growth and long-term survival of dissociated adult rat spinal cord neurons [2
]. This system consists of a patternable [15
], non-biological, cell growth promoting organosilane substrate, N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine (DETA) [16
], coated on a glass surface combined with an empirically derived, novel serum-free medium and a reproducible cellular isolation and pre-plating methodology. The serum-free medium consisted of neurobasal A supplemented with B27 [23
], GlutaMAX™, acidic fibroblast growth factor, heparin sulphate, neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), brain derived neurotrophic factor, glial derived neurotrophic factor, cardiotrophin-1, vitronectin and an antibiotic-antimycotic (). The quality of the surface modified coverslips used for cell culture was monitored using static contact angle measurements and XPS analysis as previously described [2
]. Stable contact angles (40.64° ± 2.9/mean ± SD) throughout the study indicated high reproducibility and quality of the DETA coatings and were similar to previously published results [2
]. Based on the ratio of the N 1s (401 and 399 eV) and the Si 2p3/2
peaks, XPS measurements indicated that a monolayer of DETA () was formed on the coverslips [2
]. The cell isolation process from dissected adult rat spinal cord is briefly described in the methods.
Adult Rat Spinal Cord Culture (a) Isolation of adult rat spinal cord cells from the whole cord (b) Development of serum-free culture medium using various growth factors (c) Surface modification of the glass cover slips for cell culture.
The outline of the cell isolation and cell plating is shown in and documented in detail in our previous work [2
]. In each experiment, an equal volume of the cell suspension (1000 live cells at a density of 2 cells/mm2
) was plated on each coverslip. Of the total number of coverslips plated with cells, half of the coverslips were used for control cultures and the other half received a single dose of 10nM nano-Ceria at the time of cell plating. At two different time intervals, day 15 and day 30, live-dead assays and neuron-glia immunostaining assays were conducted to quantify cell viability and the surviving cell types in both the control and nano-Ceria treated cultures. A student’s T-test was used for statistical analysis. The results are expressed as mean ± SE, n = 6, where n stands for number of coverslips. The total number of coverslips used for these assays were drawn from six different experiments. Live-dead cell assays () indicated a significantly higher cell survival at day 15 (617 ± 34, n = 6) and at day 30 (472 ± 35, n = 6) in nano-Ceria treated cultures as compared to the control cultures at day 15 (479 ± 37, n = 6) and day 30 (328 ± 32, n = 6). We also observed a significantly lower cell death at day 15 (59 ± 7, n = 6) and day 30 (48 ± 7, n = 6) in nano-Ceria treated cultures as compared to the control cultures on day 15 (110 ± 9, n = 6) and day 30 (72 ± 8, n = 6). Neurons and glial cells were identified by immunoreactivity for neurofilament 150 (neuronal marker) and glial fibrilliary acidic protein (GFAP) (glial marker) antibodies respectively. The neuronal population was significantly higher in nano-Ceria treated cultures at day 15 (191 ± 40, n = 6) and at day 30 (221 ± 12, n = 6) compared to the control cultures on day 15 (71 ± 26, n = 6) and day 30 (148 ± 9, n = 6). There was no significant difference in glial cell population or populations of cells which stained for both neuron and glial markers in treated cultures compared to control cultures at either time interval (). Electrical activities of the nano-Ceria treated cultures were assessed using patch-clamp electrophysiology at day 30 in culture. The treated neurons expressed voltage dependent inward and outward currents () and generated single action potentials (), similar to that observed for the controls and in other adult rat CNS cultures [2
Figure 3 Live-Dead Assay and Neuron-Glial Cell Assay Studies of Control and nano-Ceria Treated Cultures of Adult Rat Spinal Cord (a) Live-dead cell assays indicated that nano-Ceria treated cultures had significantly higher cell survival and significantly less (more ...)
Voltage-clamp recording from a treated culture at day 30 (left) Current clamp recording indicating a single action potential in a nano-Ceria treated culture at day 30 (right).
We propose that the presence of the mixed valence states of Ce3+
on the surface of the nano-Ceria act as an anti-oxidant that allow the nanoparticles to scavenge free radicals from the culture system. Another complex set of surface chemical reactions [26
] between the ions in the cell culture medium and the nano-Ceria then appear to be involved in reversing the oxidation state from Ce4+
. We believe that this is indicative of a cyclical regenerative, or auto-catalytic, reaction of the Ceria nanoparticles. The proposed mechanism is shown in . To demonstrate the auto- catalytic property of the engineered nano-Ceria particles, we carried out a UV-visible spectroscopic study of a nano-Ceria sol treated with 10mM hydrogen peroxide (). The UV-Visible spectrum of a sample of the nano-Ceria solution was used as a control (black trace in the graph). We added hydrogen peroxide to this solution and observed a shift in the spectrum to the right or to the lower energy portion of the spectrum (pink trace). In this reaction, hydrogen peroxide provides a source of hydroxyl radicals to mimic oxidative stress found in vivo
. This shift is postulated to be due to a change in the oxidation state from Ce3+
]. The nano-Ceria sample treated with hydrogen peroxide was then kept in the dark for 30 days. UV-Visible spectra of these samples were then taken at day 15 and day 30 (blue and red traces for the day 15 and day 30 spectra, respectively). A gradual shift in the spectra to the left was seen over time. This gradual higher energy shift reflects the regeneration (Ce4+
) of the cerium oxide nanoparticles. When an additional hydrogen peroxide dose was administered to the solution on day 30, the UV-Visible spectrum again shifted to lower energy (green trace) which was followed by a gradual shift to the lower wavelength, as seen previously. The shift of the UV visible spectrum to a lower energy state on exposure to hydrogen peroxide with a recovery toward a higher energy state (Ce3+
). This indicates that nano-ceria exhibits a mechanism in which the engineered particle provides a new material for life science applications with unprecedented antioxidant activity and pseudo-infinite half-life. The auto-regenerative anti-oxidant property of these nanoparticles appears to be the key to its neuroprotective action.
Schematic Detailing the Proposed Regenerative Properties of nano-Ceria and probable mechanism of Cerium oxide nanoparticles’ free radical scavenging property and auto-catalytic behavior.
UV-visible study of Cerium Oxide nanoparticles treated with hydrogen peroxide at different time intervals.
The auto-catalytic properties of the ceria oxide particles were further demonstrated in a hydrogen peroxide-induced oxidative injury model utilizing the adult spinal cord model system. A 100 mM hydrogen peroxide solution was added for 1h to both a control culture and a nano-Ceria treated culture at day 30. After 1h of hydrogen peroxide treatment, the cell viability was assayed using a live-dead assay kit. The nano-Ceria treated cultures had a significantly higher number of live cells (82 ± 18, n = 6) as compared to the control (29 ± 6, n = 6). We did not observe any significant difference in the number of dead cells between nano-Ceria treated (362 ± 73, n = 6) and control (309 ± 44, n = 6) cultures after hydrogen peroxide treatment. This result indicates that the nano-Ceria treated cultures had a significantly higher peroxide detoxification ability () and this may also be a significant indicator of its potential protection abilities after ischemic insult.
Figure 7 Results After Hydrogen Peroxide-Induced Oxidative Injury in Control and Treated Cultures of Adult Rat Spinal Cord at day 30. Live-dead cell assay after hydrogen peroxide treatment indicates that nano-Ceria treated cultures had a significantly higher number (more ...)
Spinal cord neurons and other CNS neurons are prone to damage due to oxidative stress [28
] both in vitro
] and in vivo
]. To maintain healthy in vitro
cultures of spinal neurons and other CNS neurons, several antioxidants are generally used in culture medium. The major source of anti-oxidant molecules in serum-free neuron culture medium is the B27 supplement [23
]. B27 contains five antioxidants; vitamin E, vitamin E acetate, superoxide dismutase, catalase, and glutathione [23
]. However, the half-life of these antioxidants is limited and they have to be replenished each time the medium is changed to maintain a healthy culture [34
]. In nano-Ceria treated cultures, we observed a significant rise in neuron survival as compared to the control culture, which were supplemented only with B27. The auto-regenerative antioxidant properties of a single dose of the autocatalytic nano-Ceria in this in vitro
model is the most probable explanation of the significant neuroprotective effect observed in the treated culture.