The biological effects of nanoparticles depend not just on the properties of the material in standardized conditions, but also on the biological system in which the nanoparticles are active [
42,
43,
44,
45,
46,
47]. There is increasing evidence that immortalized cells (i.e., differentiated cancer cells) have unique redox profiles that are different from their native, healthy counterparts [
48,
49]. Selective cytoprotection has been reported following administration of nanoceria in normal, healthy cells, but not in cancer cells [
50,
51]. Often, cancer cells rely more on glycolysis for energy production, and consequently they maintain more acidic intracellular pH values [
52]. Where additional protons are present (i.e., lactate accumulation or localization in acidic organelles), Ce
3+ reacts with a H
+ and O
2•− to produce Ce
4+ and H
2O
2, leading to net oxidation [
38,
53]. Moreover, in a comparison of immortalized colorectal cells (HCT 116) and human embryonic kidney (HEK 293) cells, CeOx NPs increased the ROS load and subsequently induced apoptosis in colon cancer cells but not in the embryonic kidney cells, suggesting that differences in either cellular localization or baseline pH existed in these cell types [
54]. The accumulation of CeOx NPs in this study was not evaluated, so it is possible that the amount of material taken up by these two cell types could have differed and impacted ROS formation. In a study of three different MnOx NPs (MnO
2, Mn
3O
2, Mn
3O
4) with different valance states, the biological implications of valence switching were examined in a cell-free system. Each MnOx NP exhibited both pro- and anti-oxidant activities simultaneously, including oxidase-, catalase-, and superoxide dismutase (SOD)-like activities. These MnOx NPs decreased cell viability in a dose-dependent manner in colorectal adenocarcinoma cells (Caco-2) regardless of valence, and the largest reduction in viability was associated with Mn
3O
4 > Mn
3O
2 > MnO
2. While the MnOx NPs were all cytotoxic, they protected cells when the cells were challenged with peroxide—suggesting that catalase mimetic activity was protective [
55]. Unlike many other metal oxides, the MnOx NPs were devoid of peroxidase or hydroxyl radical scavenging activity in cell-free assays, but when studied in cells, the MnOx NPs were located in the cytosol, which has a higher pH than most other organelles in the cell, and the local pH may have biased the enzyme mimetic activities of the different valences and allowed the particles to provide cytoprotective activity when the cells were challenged with peroxide. Consistent with these findings, MnOx nanoparticles increased catalase and SOD activities, while they also decreased glutathione levels in cell culture [
56]. The decreases in cell viability caused by MnOx NPs were associated with mitochondrial dysfunction and apoptosis, presumably secondary to the reduction in glutathione levels. Glutathione is critical to maintain mitochondrial function and cell viability, and loss of sufficient glutathione levels in mitochondria increased oxidative stress [
57]. Most often, MnOx NPs are cytotoxic in immortalized cell cultures, but the outcome of administration of these materials in whole animals is variable, and some studies show that they are safe (Xiao et al., 2013) but not others [
58]. Hence, these nanoparticles may be protective in certain redox states and certain cell types but not others.
The variable redox effects of metal oxide NPs, which may be either pro-or antioxidant, have been vexing. Beyond the effects of the cells studied and the impact of pH in these test systems, redox activity of NPs may be related to the manner of synthesis (valence ratio), the size of the particles, the complement of adsorbed proteins, and the cellular localization of the material. The redox activity of metal oxide NPs is not easily predicted since local environments may vary so much. Moreover, findings in cell-free systems are not fully recapitulated in more representative biological environments like cell culture or intact animals. The biological impact of these materials seems to be tied to the baseline redox status of the cells being studied, which adds yet another source of variability when trying to characterize the likely therapeutic effect of nanoparticles. While many disease states elevate oxidative stress in tissues, not all tissues will have the same redox changes driven by the disease state. Thus, even within a single organism, the redox activity of a nanoparticle may differ organ by organ or even organelle by organelle. Since the delivery of metal oxides occurs passively, these materials distribute widely throughout the body including healthy cells, and healthy cells may be negatively impacted by NPs while the benefit of these materials as antioxidants may be observed only in cells that have a high oxidative load [
48,
49]. Understanding how these factors modify redox reactivity will be critical to the future development of therapeutic nanoparticles [
59,
60].