Synthesis and Size Control of ZnO Nanoparticles
To evaluate the relationship between NP size and cytotoxic properties on various types of immune cells, ZnO NPs (4–20 nm) were synthesized by modifying the hydrolysis molar ratio of water to zinc acetate. Transmission electron microscopy (TEM) measurements shown in Fig. a–d demonstrate that the use of hydrolysis ratios (water:zinc acetate) of 2.4, 6.1, 12.2, and 24.4 yields NPs with average diameters of 4–8, 13, and 20 nm, respectively. The 4-nm NP produced by this synthesis method is roughly spherical in morphology, while NPs ≥8 nm acquire a rod-shaped morphology. The corresponding particle size histogram (Fig. e) shows that all of the ZnO samples have a narrow size distribution. The X-ray diffraction (XRD) patterns shown in Fig. indicate that all of the ZnO samples are well-indexed to the pure wurtzite crystallite phase of ZnO, demonstrating that the sample is comprised of ZnO nanocrystals. Furthermore, the average crystallite sizes estimated using the peak widths (full width at half maximum) of the XRD patterns agree well with TEM results for both size and shape, where the average aspect ratio of NPs >8 nm increases up to 2 (Fig. inset).
Figure 1 TEM images of ZnO nanoparticle samples made using the hydrolysis molar ratio (water:zinc acetate) of 2.4 (panel a), 6.1 (panel b), 12.2 (panel c), and 24.4 (panel d).Panel(e) shows the corresponding size distribution of the samples shown inpanel a(4 nm), (more ...)
Figure 2 X-ray diffraction θ-2θ scans of powder samples of various sizes of ZnO nanoparticles recorded in air at room temperature. The X-ray source used was Cu Kαwith an effective wavelengthλ = 1.5418 Å. The peak widths (more ...)
T and B Lymphocytes are More Resistant to NP Toxicity Compared to Monocytes and NK Cells
Previous studies from our laboratory have determined that rapidly dividing cancerous T cells are more susceptible to ZnO NP toxicity than normal quiescent T cells [13
]. These findings were extended to determine whether NP toxicity might also vary between different types of normal cells comprising the human immune system including T cells, B cells, natural killer (NK) cells, and monocytes. For these studies, freshly isolated peripheral blood mononuclear cell (PBMC) preparations were used, since all of the desired cell types for study are present in the same culture allowing for a well-controlled and uniform NP exposure. Following 24 h NP (8-nm) treatment, the various cell types were identified by staining with antibodies specific for T, B, NK or monocyte surface markers, and NP-induced cytotoxicity was assessed using propidium iodide (PI), which is a red fluorescent nuclear stain that selectively enters cells with disrupted plasma membranes. As shown in Fig. a, differences in NP-induced cytotoxicity were apparent with lymphocytes (CD3+
T cells, CD4+
T cells, and B cells) displaying the most resistance. All of these lymphocyte populations displayed similar IC50
values (~5.0 mM), with no significant difference observed between them at any NP concentration evaluated. In contrast, NK cells were substantially more sensitive to NP-induced cytotoxicity, with an IC50
of ~1.0 mM. Statistically significant differences were detected between NK cells and B and T lymphocytes at 1–5 mM NP concentrations (e.g., p
003 at 1 mM, p
0002 at 2.5 mM, p
002 at 5 mM, and p
= 0.05; NK cells vs. CD3+
T cells at 10 mM NP). Most striking is the increased NP-induced cytotoxicity observed in monocytes, with >50% of the monocytes killed at the lowest NP concentration tested (0.5 mM). Statistically significant differences were observed between monocytes and NK cells (0.5 mM and 1 mM; p
= 0.0002 and p
= 0.0008, respectively), and monocytes compared to T and B lymphocytes (p
< 0.0001 for both cell types at both concentrations tested).
Because adherent monocytes appear considerably more susceptible to NP-induced cytotoxicity compared to other immune cell subsets, additional experiments were performed using purified monocytes and lower concentrations of NP, to more accurately determine the IC50
value. In these experiments, monocytes were treated with varying concentrations of ZnO NPs, and viability was evaluated using the fluorogenic redox Alamar Blue cytotoxicity assay. In agreement with monocyte data obtained from PBMC cultures, an IC50
of ~0.30 mM was observed (Fig. b). The Alamar Blue cytotoxicity assay was also used to confirm the IC50
using purified CD4+
T cells, and a similar IC50
value of ~5.4 was observed (data not shown). As previously reported by our laboratory [13
], control experiments using bulk micron-sized ZnO powder showed no appreciable toxicity effect at any of the concentrations tested (e.g., viability with bulk ZnO: 96 ± 3% at 1 mM, 93 ± 3% at 10 mM), demonstrating that toxicity is limited to nanoscale ZnO. In addition, no appreciable toxicity was observed using NP-free supernatants (e.g., 98% viability with NP-free supernatant equivalent to 1–10 mM), indicating that the toxicity is likely not due to dissolved Zn ions from NP preparations.
Although these results indicate that monocytes are considerably more susceptible to NP-induced cytotoxicity than other immune cell types tested, it is important to note that these differences may be related to distinctions in cell-culture conditions. While T cells, B cells, and NK cells grow as suspension cultures, cultured monocytes grow as an adherent monolayer. These differences in growth characteristics may act to increase the effective ZnO NP concentration in adherent cultures. It is also plausible that the inherently greater capacity of adherent monocytes to phagocytose foreign materials, including NPs, may underlie their greater sensitivity, and the inherent cytolytic activity of NK cells against foreign pathogens, and altered self-cells may contribute to their greater sensitivity compared to lymphocyte populations. To address these possibilities, future experiments involving three-dimensional cell culture systems and those evaluating the extent to which phagocytic/endocytic mechanisms contribute to cell-type differences are needed. Nevertheless, given the variable susceptibilities of different immune cell types to ZnO NPs, it seems clear that careful analysis of in vitro cellular systems, followed by appropriate in vivo studies, is necessary to provide thorough and possibly predictive screening data regarding the relative toxicity and immunomodulatory effects of ZnO NP.
Memory and Naive T cells Differ in Cytotoxic Response to ZnO NP
Our previous findings that ZnO NP toxicity is dependent on the cell activation status, with quiescent T cells being more resistant to ZnO NP-induced cytotoxicity than identical cells activated to divide via stimulation through the T cell receptor [13
], led us to evaluate whether “memory” T cells display greater sensitivity to ZnO NPs than “naive” T cells. During an immune response, the activation of T cells to a specific antigen found on a pathogen results in a cascade of intracellular signaling events, and to the differentiation of naive T cells into memory cells. Once memory cells have formed, they can become activated to proliferate much more readily upon subsequent exposure to the original antigen [24
]. This occurs because memory T cells require lower activation signals/thresholds to proliferate, which is due, at least in part, to alterations in intracellular calcium mobilization and calcium-dependent signaling processes [25
To assess potential susceptibilities of naive and memory T cell populations to ZnO NPs in a well-controlled environment, PBMC (which contained both naive and memory T cells) were treated with 8-nm ZnO NPs for 22–24 h, with the viability assessed by PI uptake using flow cytometry. Naive CD3+
T cells (CD45RA+
) were identified based on the expression of the CD45RA surface marker, while memory T cells (CD45RO+
) were identified based on expression of the distinguishing CD45RO surface marker [26
]. Cytotoxic responses were then compared to bulk cultures of CD3+
T cells containing both naive and memory cells from the same blood donor. As shown in Fig. , memory T cells displayed significantly greater sensitivity to NP toxicity than either naive T cells or bulk cultures of CD3+
T cells (p
= 0.0044 and p
= 0.0244) at 10 mM ZnO NP concentration. Naive T cells appeared more resistant than CD3+
T cells, although statistically significant differences were not observed (p
= 0.08). These results demonstrate that even subsets of T cells show measureable differences in cytotoxic response to ZnO NP, which appears related to their activation threshold and/or proliferation potential.
ZnO NP Induce ROS Production in Monocytes and T Cells
Reactive oxygen species (ROS) are produced by cells as part of normal metabolic processes. However, in situations where ROS production exceeds the cell’s antioxidant capability, cell death can occur by interfering with normal physiological processes and the modification of cellular biomolecules [28
]. Recently, several types of nanomaterials including quantum dots and metal-oxide NPs have been shown to induce intracellular generation of ROS [13
], although only limited studies have evaluated the ability of ZnO NPs to induce ROS in normal/nontransformed human cells. To investigate oxidative stress as a mechanism of ZnO NP-induced cellular toxicity in normal immune cell populations, studies compared ROS production between primary monocytes and T cells. Based on the differing sensitivities of these two cell populations, two different concentrations of 8-nm-sized ZnO NPs were used (i.e., 1 mM and 5 mM). ROS generation was evaluated using the widely used cell permeable DCFH-DA dye to measure oxidative stress in cells. Following diffusion across the plasma membrane, this dye can be subsequently oxidatively modified into a highly fluorescent derivative by ROS, including superoxide anion and hydrogen peroxide in cellular environments containing cofactors [30
]. Studies were performed using mixed PBMC cultures to allow for identical NP-treatment conditions, and cell identification was determined by staining with fluorescently labeled antibodies directed toward the CD3 and CD14 surface markers. Flow cytometry was then used to simultaneously identify monocytes and T cells, as well as their corresponding level of ROS production at both early and extended exposure times. As shown in Fig. , there was a modest amount of ROS produced in monocytes treated with 1 mM NP (19% ROS producing cells) as early as 6 h post ZnO NP exposure, yet no detectable ROS was observed in T cells at the corresponding NP concentration and time point. However, at 20 h of treatment, appreciable ROS production was observable in T cells (~38% ROS producing cells at 5 mM ZnO NP), yet no residual cell-associated ROS signal was observed in monocytes as nearly complete cell death was noted. These results indicate that ZnO NPs are capable of inducing intracellular ROS in both cell types, although ROS production in monocytes occurs considerably earlier than for T cells, which may mechanistically underlie their greater sensitivity to ZnO NPs.
ROS Quenchers Rescue Primary Monocytes and T Cells from ZnO NP-Induced Cytotoxicity
Experiments were then performed to determine the causal role of NP-induced ROS as a major mechanism of toxicity. Purified human CD14+
monocytes and CD4+
T cells were pretreated with N
-acetyl cysteine (NAC), a well-known ROS quenching agent [34
], prior to ZnO NP exposure. Following 24 h of ZnO NP treatment, cell viability was assessed using the Alamar Blue cytotoxicity assay. Figure reveals that 5 mM NAC significantly protects both monocytes and CD4+
T cells against cell death (p
= 0.0001 and p
= 0.0481, respectively), and implicates ROS formation as a major mechanism of ZnO NP-induced toxicity in primary immune cells.
ZnO NP Preferentially Associate with Monocytes Compared to Lymphocytes
Experiments were performed to gain insights into the mechanisms underlying the greater susceptibility of monocytes to NP-induced cytotoxicity, by evaluating the extent to which NPs preferentially associate with these cells. FITC-encapsulated ZnO NPs (FITC-ZnO NPs) were prepared as previously described by our group [23
], and their fluorescence properties were used to monitor the extent to which they physically and stably associate with cells. Freshly isolated PBMC were treated with 5 mM FITC-ZnO NP, or left untreated, and multi-color flow cytometry was used to simultaneous identify monocytes and lymphocyte populations present in the PBMC culture, as well as the relative increase in the FITC-NP signal. As shown in Table , all immune cell types evaluated showed a strong association with NPs, with 78–98% of cells displaying at least some level of positive FITC fluorescence compared to control cells cultured in the absence of NPs. However, a 9.3–13.7-fold increase in the number of NP associating with any given monocyte compared to individual T or B lymphocytes was observed, as indicated by changes in mean fluorescence intensity (MFI) values. These results demonstrate that ZnO NPs preferentially associate with monocytes (MFI: 131.2), compared to lymphocyte subpopulations (MFI: 9.84 for CD3+
T cells, MFI: 14.1 for CD4+
T cells and MFI: 9.61 for B cells). The greater NP association with monocytes may arise through either extracellular membrane-NP interactions or intracellular NP uptake, as we have previously reported the ability of these same FITC-encapsulated NPs to be intracellularly localized in the human Jurkat T cell line using confocal microscopy [23
NP association with immune cell subsets
Effect of ZnO NP Size on Cytotoxicity and ROS Production
To evaluate the relationship between ZnO NP size and its toxic potential, three different sizes of ZnO NPs (4, 13, and 20 nm) were concurrently evaluated using primary human CD4+T cells as a model system. Experiments shown in Fig. a were performed using a NP concentration of 5 mM, given the observed IC50value for T cells observed above. These experiments demonstrate that significantly greater cytotoxicity was observed with 4-nm NPs (80.0% ± 2.0%), compared to either 13- or 20-nm-sized NPs (p = 0.04 andp = 0.01, respectively). In addition, significantly more cytotoxicity (p = 0.05) was observed for 13 nm NPs (70.1% ± 8.0%), compared to 20-nm NPs (44.0% ± 6.8%). To further verify that cytotoxicity increases with decreasing NP size, experiments were performed over a range of NP concentrations. As shown in Fig. b, significantly greater toxicity was observed using 4-nm NPs compared to 20-nm NP at all concentrations tested (p = 0.03 at 1 mM,p = 0.0001 at 5 mM andp = 0.01 at 10 mM NP concentration). In this paper, we have focused on the size-dependence cytotoxicity of NPs. However, as shown in the inset to Fig. , the aspect ratio of NPs increases with size. Therefore, the role of shape morphology on NP cytotoxic response will be the subject of future investigations.
To further evaluate the mechanism of nanotoxicity, studies were performed to investigate the relationship between size dependence and ZnO NP-induced ROS production using PBMC cell cultures. Following a 3 h treatment with different-sized ZnO NPs (4, 13 and 20 nm), a size-dependent induction of ROS was observed at both NP concentrations, with 4-nm-sized NPs consistently inducing higher levels of ROS compared to 13- or 20-nm-sized NPs (Fig. ). At 5 mM NP concentrations, significantly higher levels of ROS were observed for 4 and 13-nm NPs compared to 20-nm NPs (~4-fold relative increase (p = 0.005) and ~3-fold increase (p = 0.01), respectively). Similarly, 10 mM NP treatment resulted in significant differences in ROS production between all sizes of NP with a 3.2-fold greater induction of ROS observed between 4 and 20-nm NPs (p = 0.001), and a 1.9-fold increase observed between 13 and 20-nm NPs (p = 0.0021). These findings indicate that the generation of ROS is dependent on NP size.
The increased nanotoxicity with decreasing NP size may be due in part to, the larger surface area/volume ratio of smaller NPs, which provides them with a greater area to associate with cellular membranes and proteins, as well as greater surface reactivity. In addition, ZnO particles prepared in a nonaqueous medium may have oxygen deficient/zinc rich surface chemistries [10
] that exhibit strong electrostatic interactions with the negatively charged cell membrane [35
], with smaller particles predicted to have a greater positive surface charge to volume ratio. Thus, greater initial cell membrane-NP association would be expected for smaller NP, leading to potentially greater intracellular uptake.
Following initial NP-cell electrostatic interactions, mechanisms of toxicity likely proceed via the formation of highly reactive oxygen species, such as hydrogen peroxide, hydroxyl radical, and superoxide anion [16
]. The ability of smaller-sized ZnO NPs to promote greater levels of ROS may occur because as ZnO NP size decreases, so does the nanocrystal quality, which results in increased interstitial zinc ions and oxygen vacancies [37
]. These crystal defects lead to a large number of electron-hole pairs (e−
), which are typically activated by both UV and visible light. However, for nanoscale ZnO, large numbers of valence band holes and/or conduction band electrons are thought to be available to serve in redox reactions even in the absence of UV irradiation [17
]. The holes can split water molecules derived from the ZnO suspension into H+
. The resulting electrons react with dissolved oxygen molecules to generate superoxide radical anions (·
), which in turn react with H+
to generate (HO2·
) radicals. These HO2·
molecules can then produce hydrogen peroxide anions (HO2−
) following a subsequent encounter with electrons. Hydrogen peroxide anions can then react with hydrogen ions to produce hydrogen peroxide (H2
All of the various ROS molecules produced in this fashion can trigger redox-cycling cascades in the cell, or on adjacent cell membranes. This can then lead to the depletion of endogenous cellular reserves of antioxidants such that irreparable oxidative damage occurs to cellular biomolecules and eventually results in cell death.
ZnO NP Induce Proinflammatory Cytokine Production in Primary Human Immune Cells
An important task of nanobiotechnology is to understand the effect these nanomaterials have to modulate expression of cytokines, which are soluble biological protein messengers that regulate the immune system. Published studies have demonstrated the ability of certain nanomaterials to induce cytokine production, although this appears heavily dependent on a variety of factors, including material composition, size, and method of delivery [39
]. Much remains to be learned, however, regarding the pro-inflammatory potential of ZnO NPs. To address this gap in knowledge, studies were performed to evaluate the ability of ZnO NPs to modulate IFN-γ, TNF-α, and IL-12 cytokine production in primary human immune cells. These particular cytokines were chosen because they represent critical pathways involved in the inflammatory response and differentiation processes. Freshly isolated PBMC were treated with varying concentrations of 8-nm ZnO NPs for 38 h, and cell-free supernatants were used to quantify cytokine levels using an ELISA. For IL-12 production, cell samples were first pretreated with IFN-γ (1,000 U/mL) before addition of NPs to provide a priming signal for IL-12 [41
]. Results demonstrate significant dose-dependent increases in IFN-γ and TNF-α at all NP concentrations tested (0.05 mM, 0.1 mM and 0.2 mM) (Fig. ). ZnO NPs had no effect on IL-12 production in unprimed control cultures, but pretreatment with low level IFN-γ prior to NP exposure resulted in appreciable amounts of IL-12 in a concentration-dependent manner. The inability of ZnO NPs to induce IL-12 in resting cells was not altogether unexpected, as expression of this cytokine typically occurs in cells that have first received a priming signal, such as IFN-γ, which is locally produced by other cells participating in the immune response [41
]. These results suggest that a synergistic relationship between ZnO NPs and IFN-γ may occur in in vivo settings employing ZnO NPs, and demonstrate that ZnO NPs are capable of inducing at least some key components of inflammation.
Figure 9 ZnO NPs increase proinflammatory cytokine production in primary human peripheral blood cells. PBMC were left untreated or treated with varying concentrations (0, 0.05, 0.1, and 0.2 mM) of 8-nm ZnO NPs, both alone and with the addition of exogenous IFN-γ (more ...)
The ability of ZnO NPs to induce proinflammatory cytokine expression in human primary immune cells is consistent with the recognized relationship between oxidative stress and inflammation, which is partially mediated by induction of the NF-κB transcription factor [42
]. To date, only limited studies have evaluated the effects of ZnO NPs on cytokine production, and most of these studies have been conducted in nonhematological cell types or in immortalized cell lines, which frequently display alterations in signal transduction pathways, leading to unpredictable changes in protein expression. In one report, ZnO NPs were shown to increase IL-8 and MCP-1 cytokine mRNA expression in human aortic endothelial cells, although no information was provided regarding changes in corresponding protein levels [43
]. In two other studies conducted in immortalized rodent lung epithelial cells, alveolar macrophage cell lines and primary alveolar macrophages, ZnO NPs fail to induce TNF-α at concentrations exceeding those used in this study [40
]. In addition, no changes in other cytokines and chemokines including IL-6, G-CSF, MIP-2, CXCL10, and CCL2 were detected. The ability of ZnO NPs to induce high levels of TNF-α in our studies may be due to differing responses observed between cell populations studied (i.e., PBMC vs. alveolar macrophages), or reflect the longer NP treatment exposure period (i.e., 38 h vs. 24 h). Although, to the best of our knowledge, no published studies have demonstrated the ability of ZnO NPs to induce TNF-α or IL-6 production in purified primary cell cultures or in toxicological evaluations, it is interesting to note that inhalation of ultrafine ZnO particles in occupational settings can increase the expression of these cytokines, which is symptomatically recognized as metal fume fever in welders (e.g., fatigue, fever, chills, myalgias, cough, and leukocytosis) [16
The ability of ZnO NPs to induce IL-12, IFN-γ, and TNF-α at NP concentrations below those causing appreciable cytotoxicity indicates immunomodulatory effects that may function to bias the immune response toward Th1-mediated immunity. It is the cytokine profile that directs the development and differentiation of T helper cells into the two different subsets, called type 1 (Th1) and type 2 (Th2) [45
]. Th1 cells are recognized to play an essential role in promoting innate and cell-mediated immunity, while Th2 cells promote antibody-based humoral responses [45
]. Relevant to our findings, IL-12 and IFN-γ play critical roles in Th1 development, and help set-up a perpetuating loop whereby more Th1 development is favored, Th2 development is suppressed, and the cytotoxicity activity of both NK cells and T cytotoxic cells against cancerous cells, virally infected cells, or intracellular pathogens is enhanced [46
]. Thus, our findings indicate that careful titration of ZnO NP-based therapeutic interventions may be successful in elevating a group of cytokines important for eliciting a Th1-mediated immune response with effective anti-cancer actions. These results, combined with our previous observations demonstrating that immortalized hematopoietic cancer cells are preferentially killed (~33-fold) by ZnO NPs compared to normal cells of identical lineage [13
], suggest that ZnO NPs may function via a two-fold mechanism to eliminate cancer cells by direct and preferential cytotoxic actions, and by enhancing the type of immunity most effective at eliciting an in vivo anti-cancer response.
The ability of ZnO NPs to induce TNF-α may also help to promote Th1 differentiation [45
] as well as functioning as a regulator of acute inflammation [48
]. Notably, this cytokine received its name based on its potent in vitro and in vivo anti-tumor activity. However, high level and/or chronic exposure to TNF-α has been shown to produce serious detrimental effects on the host, including septic shock or symptoms associated with autoimmune disease [48
]. Our results demonstrate significant dose-dependent increases in TNF-α over a somewhat narrow range of ZnO NP concentrations. The magnitude of TNF-α induction, as well as other proinflammatory cytokines, and their local–regional delivery to tumor sites or other desired areas, will undoubtedly be important parameters when considering ZnO NP for biomedical purposes to achieve the desired therapeutic response without eliciting potential systemic damaging effects from these cytokines.