Surface-charge-dependent cell localization and cytotoxicity of cerium oxide nanoparticles

Abstract

Cerium oxide nanoparticles (nanoceria) have shown great potential as antioxidant and radioprotective agents for applications in cancer therapy. Recently, various polymer-coated nanoceria preparations have been developed to improve their aqueous solubility and allow for surface functionalization of these nanoparticles. However, the interaction of polymer-coated nanoceria with cells, their uptake mechanism, and subcellular localization are poorly understood. Herein, we engineered polymer-coated cerium oxide nanoparticles with different surface charges (positive, negative, and neutral) and studied their internalization and toxicity in normal and cancer cell lines. The results showed that nanoceria with a positive or neutral charge enters most of the cell lines studied, while nanoceria with a negative charge internalizes mostly in the cancer cell lines. Moreover, upon entry into the cells, nanoceria is localized to different cell compartments (e.g., cytoplasm and lysosomes) depending on the nanoparticle's surface charge. The internalization and subcellular localization of nanoceria plays a key role in the nanoparticles' cytotoxicity profile, exhibiting significant toxicity when they localize in the lysosomes of the cancer cells. In contrast, minimal toxicity is observed when they localize into the cytoplasm or do not enter the cells. Taken together, these results indicate that the differential surface-charge-dependent localization of nanoceria in normal and cancer cells plays a critical role in the nanoparticles' toxicity profile.

Figure 1

Characterization of cerium oxide nanoparticles. (a) Size of cerium oxide nanocrystal core by TEM. (b) Zeta potential of cerium oxide nanoparticles with different surface functionalities (negative, neutral and positive). (c) FT-IR spectra of the carboxylated (negative) and aminated (positive) surface group on nanoceria. (d) Fluorescence emission spectra of the DiI-encapsulating nanoceria and free dye DiI.

Figure 2

Uptake of cerium oxide nanoparticles (nanoceria) by normal cells via confocal microscopy. PNC(−) is not being significantly uptaken by either cell line (a, d) whereas uptake is observed with the ANC(+) (b, e). DNC(0) exhibits diffused cytoplasmic localization (c, f) with greater internalization in H9c2.

Figure 3

Uptake of cerium oxide nanoparticles (nanoceria) by cancer cells. Confocal images of A549 and MCF-7 carcinoma cells after treatment with PNC(−), ANC(+) and DNC(0) for 3 h. PNC(−) (a,d) are being uptaken by A549 and not by MCF-7 cells, while ANC(+) (b,e) are being uptaken by both cell lines. Meanwhile, DNC(0) internalized in the A549 (c), while they were minimally internalized by MCF-7 (f).

Figure 4

Inhibition of endocytic pathways prevents nanoceria uptake. No internalization of ANC(+) and PNC(−) is seen in both cell lines at 4 °C (a, b, c). Similarly, internalization of the nanoparticles is abrogated in the presence of inhibitors (2-deoxyglucose and sodium azide), which collectively block active endocytosis (d, e, f).

Figure 5

Polymer-coated nanoceria’s intracellular localization in lung carcinoma cells (A549). Upon internalization, both the positively charged [ANC(+), a-c] and negatively charged [PNC(−), d-f] nanoceria co-localized with the lysosome. In contrast, neutral nanoceria [DNC(0), g-i] localized mostly in the cytoplasm.

Figure 6

Polymer-coated nanoceria’s intracellular localization in cardiac myocytes (H9c2). Upon internalization, the positively charged [ANC(+), a-c] nanoceria is found in both the cytoplasm and lysosomes. In contrast, neutral nanoceria [DNC(0), d-f] does not significantly co-localized to the lysosomes with most of the nanoparticles found in the cytoplasm.

Figure 7

Lysosomal isolation and determination of the oxidase-like activity of the entrapped nanoceria. In H9c2 and HEK293 cell lines, the ANC(+) nanoparticles are mostly localized into lysosomes, judged by the presence of significant oxidase activity in the lysosomes isolated from these cell lines after incubation with ANC(+) (a and b). PNC(−) and DNC(0) treated H9c2 and HEK293 cells showed minimal oxidase activity in their lysosomes. In the A549 cell lines, oxidase activity was detected in cells treated with ANC(+) and PNC(−), while minimal activity was present in the DNC(0) treated cells (c). The lysomes isolated from MCF-7 cells did not show significant oxidase activity as the nanoceria did not internalized in these cells (d). Lysosomes isolated from non-treated cells do not show any oxidase activity as expected.

Figure 8

Cytotoxicity of cerium oxide nanoparticles. MTT assays show that the ANC(+) is cytotoxic to all cell lines except for the breast carcinoma cells. PNC(−) is only cytotoxic to A549 lung cancer cells as they internalize and localize into the lysosomes of these cells. DNC(0) does not show any toxicity to any of the cell lines.

Figure 9

Cytotoxicity of cerium oxide nanoparticles in the presence of endocytosis inhibitors. Nanoparticles are not cytotoxic to either lung carcinoma or cardiac myocytes in presence of 2-deoxyglucose and sodium azide.

Figure 10

Uptake of polymer-coated iron oxide nanoparticles [IONP(−)] and aminated polymer coated iron oxide nanoparticles [IONP(+)] by A549 and H9c2 cells. Confocal images of A549 and H9c2 cells after treatment with positively charged IONP(+) for 3 h show significant uptake of the nanoparticles (a and c). Meanwhile, no uptake of the negatively charged IONP(−) was observed in either A549 or H9c2 cells (b and d).

Figure 11

Cytotoxicity of polymer coated iron oxide nanoparticles [IONP(−)] and aminated polymer coated iron oxide nanoparticles [IONP(+)] by A549 and H9c2 cells.

Scheme 1

Surface functionalization of cerium oxide nanoparticles. Cerium oxide nanoparticles with different polymer coatings and surface modifications were synthesized to yield nanoparticles with negative [PNC(−)], positive [ANC(+)] , and neutral [DNC(0)] charge. A fluorescent dye (DiI, red circle) was encapsulated using a modified solvent diffusion method.

Scheme 2

Polymer-coated nanoceria’s cell internalization, localization and proposed toxicity mechanism. Neutral DNC(0) internalized and localized mostly into the cytoplasm of cells and hence it is not cytotoxic. ANC(+) and PNC(−) can localize either into the cytoplasm or the lysosomes, depending on the type of cells. When the nanoceria localized to the lysosome, the low pH of this organelle activates the nanoparticle’s oxidase-like activity, exhibiting toxicity. ANC(−) or PNC(+) that localized into the cytoplasm displayed no cytotoxicity.