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IRON OXIDE NANOPARTICLE-INDUCED OXIDATIVE STRESS AND INFLAMMATION
The manufacture of nanoparticles (<100 nm diameter) of varying shapes and compositions has exploded over the last several years with applications ranging from diagnostic imaging to nanoscale molecular construction. Given the vast potential use of these materials, both intentional and unintentional human exposures are likely. Although much has recently been learned about their synthesis, very little is known about cellular or organ responses upon contact with nanoparticles. A defining feature of nanoparticles is their large specific surface area; thus, it is possible that current concepts of dose expressed as mass concentration, which is very low for nanoparticles, may fail in predicting exposure outcomes if this feature is not taken into account. We hypothesize that the small size of nanoparticles contributes to their evasion of normal particle clearance mechanisms, increases the likelihood of contact with cells of many types, particularly epithelial cells, and allows their translocation to sites distant from the original exposure. We hypothesize further that this contact results in inflammation and oxidant stress and that the large surface area of the nanoparticles potentiates their effects. We will address these hypotheses with the following objectives to determine if nanoparticles: 1) induce oxidative stress and toxicity in cultured epithelial and endothelial cells; 2) cause lung inflammation or extrapulmonary effects after in vivo exposure; and 3) are translocated to extrapulmonary sites.
- Nanoparticle Physicochemical Characterizations
- In Vitro Assessment of Nanoparticle-Induced Cytotoxicity and Oxidative Stress
- In Vitro Assessment of Nanoparticle Uptake
- In Vivo Effects of Pt Nanoparticles
- Tissue Distribution of Semiconductor Nanocrystals (Quantum Dots)
- Significance of Findings
We first focused on creating NP systems that could be used to test our hypotheses and assessing their stability in aqueous media. The iron oxide NP systems were not stable in cell culture medium or saline (rapid and severe agglomeration), the carriers required for in vitro and in vivo exposures. We switched to a platinum-based system to study cellular uptake and oxidative stress in epithelial and endothelial cells. The Pt NP shapes (flowers, multipods, flower spheres, and pod spheres; 11-35 nm; 1-27 m2/g) are synthesized in the liquid phase from the same starting materials and the final shape is determined by reaction temperature and time (Maksimuk et al., 2006; Maksimuk et al., 2007; Teng et al., 2005; Teng et al., 2006; Figure 1). This system allowed us to test the effects of surface area and shape on uptake and effects. We first used an acellular assay of fluorescein oxidation in the presence of horseradish peroxidase (electron acceptor) to assess the inherent oxidative capacity of the Pt NPs (with activity expressed as H2O2 equivalents). The Pt shapes demonstrated a range of oxidant activity, with the highest activity being found for the flowers and the spheres made from flowers (Figure 2). These two shapes also have the largest surface area of the group of NPs, which may explain the difference in reactivity. In comparison to other metal NPs such as Cu, the activity of the Pt nanoshapes is low, but similar in magnitude to that for TiO2, Ag, Au, and Al oxide NPs. The results of these investigations suggest that the Pt NPs may be useful as negative controls, along with TiO2 and colloidal Au. However, recent studies using electron spin resonance with and without cells suggests that very different results can be obtained when cells are present. Nevertheless, the fluorescein-based assay is still adequately predictive of in vivo responses to intratracheally instilled NP (Rushton et al., submitted).
Figure 1. Pt nanoparticles. A, Pt flower-shaped nanoparticles (~ 35 nm; 27 m2/g); B, Pt spheres (~ 22 nm; 12 m2/g) formed from the flower-shaped nanoparticles; C, Pt multipods (~ 20 nm; 2 m2/g); and D, Pt spheres made from multipods (~ 11 nm; 1 m2/g).
Figure 2. ROS-generating capacity, expressed in terms of particle mass (left) and surface area (right), of several metal and metal oxide nanoparticles, including the platinum shapes.
We further examined the Pt flowers and multipods suspended in culture medium with serum via electron microscopy (EM) to assess their agglomerate state and found a mixture of singlets and agglomerates. We also assessed the agglomerate state of the Pt NPs in suspension using a Malvern nano-zeta sizer (NNIN). The hydrodynamic sizes of the Pt flowers and multipods were measured in water, phosphate buffer, 0.9% saline, and culture medium with and without serum. For the two shapes we analyzed, agglomerate sizes were between 80 and 150 nm regardless of the medium. Interestingly, agglomerate sizes were smaller in culture medium with serum than without serum, which could be due to the presence of globular proteins that approach the size of NPs. Such characterizations of particle surface oxidative activity and agglomeration state are important for interpreting results from either in vitro or in vivo studies.
In our studies of NP toxicity, we used A549 type II-like alveolar epithelial cells that are stably transfected with a luciferase reporter that is located in the promoter region (NF-κB, AP-1 responsive) for the interleukin (IL)-8 gene. Luciferase reporter activity is, thus, used as an indicator of oxidative stress in these cells. We measured reporter activity and LDH release in A549 cells exposed to aggregates of DPPC-coated Fe2O3, ultrafine TiO2, or Pt NPs and observed no significant increases in response. The Pt shapes (flowers, multipods), induced increases in reporter activity only at the highest dose (500 μg/well; 132 μg/cm2) (Figure 3); this was not related to an increase in LDH release (not shown).
Figure 3. Activation of the IL-8 gene (luciferase reporter activity) in stably transfected A549 lung epithelial cells.
Figure 4. Exposure of HUVECs to a wide range of Pt nanoshapes doses does not induce and increase in IL-6 release or in LDH activity (latter not shown). Results from a time course study had similar findings.
We also hypothesized that NPs can breech the lung alveolar epithelial barrier and come into contact with the endothelial cells lining the vasculature. We conducted studies with the Pt shapes using primary human umbilical vein endothelial cells (HUVEC) as a model of the endothelium over a wide range of doses (0.01-500 μg/well; 0.003-132 μg/m2) and times (0-48 hrs). The results showed that Pt NP agglomerates induce oxidative stress, as assessed by interleukin (IL)-6 release, only at the highest doses (Figure 4); LDH was not increased as a result of exposure (not shown). This was similar to what was observed with epithelial cells. The effects of the nanoshapes in endothelial cells on LDH and IL-6 release are similar to those caused by nanosized TiO2. It is possible, however, that NP agglomeration in culture medium could mask the effects of smaller singlets. To the extent that agglomeration is partly dependent on concentration, our exposures at the lowest dose (0.01 μg/well) suggest that large and small agglomerates have similar in vitro effects for the same material.
Another open question was whether or not endothelial cells took up the Pt shapes; this was a particularly important question to answer given the largely negative results from the effects studies. EM analyses of exposed HUVECs revealed intracellular accumulations of Pt flowers and multipods (Figure 5). Both singlets and agglomerates, even very large ones, were found inside the cells. This brief time-course study showed that the NPs were diffusely distributed immediately after exposure, but were later sequestered, possibly in lysosomal structures.
We also quantitated the uptake of the Pt shapes (flowers and multipods) by HUVECs by analyzing the Pt content in cell pellet and culture supernatant samples via inductively-coupled plasma mass spectrometry (ICPMS) at four dose levels (0-250 μg/ml; 0.7-66 μg/cm2; 0-525 x 109 particles) after a 24 hr exposure. The results (Figure 6) showed that the Pt nanoflowers were taken up by the cells at about twice the rate of the multipods. In addition, at the lower doses, more of the Pt was intracellular than extracellular; the shift at the highest dose probably represents a smaller loss of materials from particle adherence to the stock tubes prior to exposure (i.e. the mass balance is better at the highest dose), but could also indicate a saturation of cellular uptake. Another motivation for studying Pt NP uptake by HUVECs was the assessment of a realistic lower limit of detection in biological samples. Given the losses of Pt via particle adherence to surfaces prior to exposure, the lowest dose is estimated to have really been ~0.4 μg/ml (according to the mass balance). This level of exposure produced signals for Pt that were ~2 orders of magnitude higher than background levels, suggesting that the use of Pt coupled with ICPMS detection offers an extremely sensitive system for quantitating NPs in cells and tissues.
Figure 5. Pt NPs are taken up by cells during in vitro exposure. Example EM image is from a sectioned HUVEC exposed for 24 hrs to Pt multipods. Both agglomerates and singlets can be seen in this image.
Figure 6. Quantitation of Pt NP uptake by HUVEC via ICPMS.
Since in vitro systems cannot replicate the complexity of cellular interactions or transport of NPs throughout the body, data from in vivo studies is essential for hazard identification. We exposed groups of rats to the Pt flowers or multipods suspended in 0.9% saline via intratracheal instillation (100 μg instilled). Large agglomerates of the NPs were present in the saline suspensions. The NP agglomeration state in the lung lining fluid, however, is unknown. In comparison to saline-exposed controls, both the flowers and the multipods produced small, but significant increases in the percentage of neutrophils in bronchoalveolar lavage fluid. We found 4.3% ± 2.2 (SD) neutrophils in lavage fluid following exposure to Pt flowers (p = 0.06), while Pt multipod-exposed rats had 5.3% ± 2.7 (p = 0.03). In comparison, saline-exposed controls had 1.8% ± 1.0 neutrophils. Neutrophil recruitment to the alveolar space is a sensitive marker of lung inflammation. As the percentage of neutrophils in lavage fluid is typically ~1% in a healthy rat, these data indicate that the inflammatory response resulting from Pt NP exposure was mild in nature. Similar responses have been observed in rats exposed to ultrafine TiO2 using the same method and similar doses. We did not observe any increases in total cell numbers or in lavage fluid protein concentration or LDH/β-glucuronidase activities that would suggest particle-induced cytotoxicity or disruption/injury to the epithelial barrier of the lungs. The neutrophil chemoattractant signal likely arose from contact of the particles with the epithelium and from those macrophages that engulfed NP agglomerates.
From the instilled dose, we also predicted the inhaled deposited dose that would result in the same lung burden of Pt so that we could calculate an inhaled dose in humans that would result in the same lung burden. This was done using a predictive lung deposition model (MPPD v. 2; CIIT, RTP, NCA and RIVM, Bilthoven, The Netherlands) to obtain the human to rat ratio for total inhaled mass deposited in the alveoli. A human would have to inhale a NP-containing aerosol at a mass concentration of 4.6 mg/m3 over an 8-h period in order to deposit a similar mass in the lungs as what was delivered to the rats via instillation at the dose of 100 μg Pt NPs. This is a high aerosol concentration, but is similar to the 3 mg/m3 occupational exposure limit of the American Conference of Governmental Industrial Hygienists for “particles not otherwise categorized”.
In order to evaluate the compartmentalized retention of the Pt NPs in the lung, we quantified the Pt content in lavaged lung parenchymal tissues with trachea and large airways removed, lavage cells and the lavage supernatant fraction. We found that the Pt flowers were retained to a greater degree than the Pt multipods. Twenty-one percent of delivered dose remained in these tissues after 24 h for the flowers, while only 12% could be detected for the multipods. The NPs were found in the lung parenchyma, presumably associated with or inside the epithelial cells or in the interstitium, while larger amounts were associated with the lavage cell pellet. Interestingly, no detectable Pt was found in the lavage supernatant, suggesting that the majority of NPs might be cell-associated. The parenchymal tissue retained 8.6 ± 1.6 μg Pt after exposure to the nanoflowers and 3.1 ± 0.6 μg Pt after exposure to the multipods. We found that the lavaged cells retained 12.0 ± 6.7 μg Pt for the flowers and 8.5 ± 4.3 μg Pt for the multipods. The remaining 79-88% of the instilled dose was likely cleared rapidly from the lungs via the mucociliary escalator or by passing through the interstitium to the local lymph nodes or blood. We did not evaluate other tissues in this experiment. The details of these in vitro and in vivo studies using Pt nanosized particles were reported in a recent paper (Elder et al., 2007).
It is important to understand the behavior of NPs following in vivo exposures in terms of distribution to tissues over time. We conducted a biokinetics study in rats of the tissue distribution over time of semiconductor quantum dots (QDs) with three different surface coatings (polyethylene glycol (PEG), PEG-amine, and carboxylic acid) following intratracheal microspray (10 μg QDs as Cd) or intravenous injection (5 μg QDs as Cd) exposures. The QDs (Invitrogen; TOP/O-capped CdSe-ZnS nanocrystals, 4.6 nm core size) are the same ones that have been used by Montiero-Riviere’s group to examine QD penetration through skin (Ryman-Rasmussen et al., 2006). EM analyses of dried QD suspensions that were microsprayed onto grids revealed mixtures of singlets and chain agglomerates (not shown). The three QDs differed not only in their surface coating, but also in hydrodynamic diameter (in saline, PEG: 23 nm; PEG-amine: 17 nm; carboxyl: 14 nm) and surface charge (in saline, PEG: -1.5 mV; PEG-amine: -0.3 mV; carboxyl: -40.0 mV). We also measured the dissolution rates of the PEG and PEG-amine QDs in model physiological buffer systems (pH 4.5, 7.4) via microdialysis using a 3500 MW cut-off 25 μm regenerated cellulose membrane. We found that about 0.6% of the Cd in the starting material was dissolved in the first 24 hrs and also that the rate increased over time (please note that we have not yet completed the dissolution measurements for the carboxylated QDs).
Because inflammatory responses can affect epithelial and endothelial permeability, the influx of neutrophils into the alveolar space was assessed. Twenty-four hrs after intratracheal exposure, only the rats exposed to the carboxylated QDs had a significant increase in the percentage of neutrophils in bronchoalveolar lavage fluid (9.8 % ± 1.5); at 7 days post-exposure, significant and large increases in lavage neutrophils (20-45%, depending on surface coating) were found in rats exposed to all three QD type. The carboxylated QDs had the highest inflammatory potential. These changes are likely due, in part, to the bioavailability of Cd. Using scanning confocal microscopy, we were able to demonstrate that the QDs were taken up by alveolar macrophages and not just associated with the cell surface; thus, it is likely that the nanoparticles were exposed to the low pH environment of the phagolysosomes. Intravenous exposure did not induce an influx of neutrophils into the lung.
Tissue distribution of QDs was determined by quantitating Cd in samples, including urine and feces, that were obtained 24 hrs and 7 days following exposure (also immediately post-exposure for rats exposed via the intratracheal route) via atomic absorption spectroscopy. First, we did not find detectable Cd in any tissues from unexposed controls except for kidney and liver. Furthermore, we did not find Cd in any of the brain regions examined or in cerebrospinal fluid in any of the exposed rats. Immediately following intratracheal microspray exposure, most of the QD dose was retained in the lungs, which is not surprising. However, as shown in Figure 7a, we found significant increases of Cd in kidney tissue from rats exposed to PEG (~0.4% of initial lung burden, ILB, calculated as the sum of lung tissue, lavage fluid supernate, lavage cell, and trachea Cd levels) and PEG-amine QDs (~0.1% of ILB). By 24 hrs post-exposure, all three QD types were found in kidney tissue. This is unlike what is known about the transport of soluble Cd, where the metal is first absorbed in the proximal small intestine, then taken to the liver and then the kidney. We did not find any changes in liver Cd content until 7 days after exposure, when all three QD types (or the Cd) were found (Figure 7b). The major organs that still retained Cd from the QDs 7 days after exposure were: lung >> kidney > liver >> spleen. Except for liver tissue, which retained more Cd from PEG-amine QD-exposed rats, the other tissues retained similar amounts of Cd when comparing the three surface coatings. This may have to do with the proteins associated with the nanoparticle surface as the QDs were transported out of the lungs to extrapulmonary tissues. No Cd was detected in blood or urine from any of the rats exposed to QDs via the respiratory tract, but there was significant fecal excretion of Cd (some of which probably came from a portion of the intratracheal dose that was immediately swallowed).
Figure 7a. Tissue distribution of Cd (background-corrected) in tissues immediately following intratracheal microspray exposure of rats to surface-functionalized QDs (10 μg as Cd).
Figure 7b. Tissue distribution of Cd (background-corrected) in tissues 7 days after intratracheal microspray exposure of rats to surface-functionalized QDs (10 μg as Cd).
When the QDs were injected via the tail vein, they were rapidly cleared from the blood as a function of the surface coating. The carboxylated QDs were completely cleared within 24 hrs and the aminated ones by day 7. Only the PEGylated QDs remained in the circulation, albeit at a small fraction (1%) of injected dose, after 7 days. The liver was the organ that retained most of the injected QD dose and there were significant differences in retention based on QD surface coating, the carboxylated dots having the highest liver Cd burden and the PEGylated ones the lowest (Figure 8a). Conventional fluorescence microscopy images of liver tissue revealed punctae in putative Kupffer cells and, possibly, in hepatocytes at 1 day post-exposure. No such punctae were observed at 7 days; however, the hepatocytes from this animal appear to be swollen (Figure 8b). The spleen and kidney were also significant sinks for QDs, with spleen retaining more Cd. The PEGylated QDs were not cleared by the liver as efficiently as the other QD types and, therefore, remained in circulation for a longer period of time. They also accumulated to a greater extent in heart and kidney as compared to the other QD types. In addition to these organs, significant accumulations of QDs as Cd were found in bone marrow, spleen, and lymph nods (lung-associated, axillary, inguinal). In addition, there was accumulation of Cd in lung tissue after 7 days, although the exact location of the QDs is not yet clear. The pattern of organ Cd retention was similar at 24 hrs and 7 days post-exposure. Interestingly, there was no significant fecal Cd excretion except in rats exposed to the carboxylated dots (comparisons of cumulative fecal Cd levels). This suggests that the fecal Cd excretion found in rats exposed via the intratracheal route may be largely accounted for by the amount swallowed immediately after exposure and during the respiratory tact clearance of the QDs.
Two important issues that will need to be addressed in future studies include whether or not 1) the Cd signal in tissues is associated with fluorescent particles (i.e. do our results reflect QD or Cd transport?) and 2) any pathological changes are associated with exposure. The details of these studies in rats will be presented in a paper that is currently in preparation.
Figure 8a. Tissue distribution of Cd (background-corrected) in tissues 24 hrs after intravenous exposure of rats to surface-functionalized QDs (5 μg as Cd).
Figure 8b. Retention of QDs in liver tissue after intravenous injection (10 μm frozen sections, 60x magnification).
Our studies with low-toxicity Pt nanoparticles, through comparison with other metal NPs, help to define some of the surface characteristics that affect cellular uptake and effects (namely shape, surface area). Although we found that the particles were taken up by cells following in vitro exposures, they did not induce changes in cell viability or oxidative stress. This correlated with the inherent oxidative capacity of the particles (i.e. DCF oxidation), a correlation which appears to hold for other nanoparticle types. Given the fairly good predictive power of the acellular oxidation assay, this could be used as one in a set of assays to screen the toxic potential of nanomaterials of unknown activity.
Another issue that is highlighted by our studies, and which should be obvious given the state of the current literature, is the need for thorough characterization of the nanoparticles as they are used in safety assessments. Particle agglomeration state and hydrodynamic diameters in different aqueous carriers were addressed in this report, as were shape and size. Characterization of the particles in liquid or gaseous media not only provides insight into changes in size or shape, but also about the true exposure doses. Several organizations have published lists of the necessary characterizations, so that will not be repeated here other than to stress its importance.
Knowledge regarding nanoparticle disposition after exposure is a critical element to the prediction of outcomes following human exposures in environmental, occupational, and therapeutic settings. Our studies with QDs demonstrated the importance of particle surface characteristics in determining the fate of nanoparticles. Such data is essential for determining internal or target organ dose and should be evaluated for nanosized materials that are intended for therapeutic uses or those to which humans and animals might be exposed in the environment or the workplace.
A final point is that recent studies have highlighted the dynamic nature of the protein corona that is associated with the nanoparticle surface (Cedervall et al., 2007 a,b; Ehrenberg and McGrath, 2005; Linse et al, 2007; Lynch et al., 2007). This corona will ultimately determine the fate of those particles that are taken up by cells or into biological fluids. Our future work is directed toward this line of investigation.
Quality Assurance Report
Our laboratory has a collection of standard operating procedures (SOP) that were followed for all experiments in this project. Details of the experimental set-up and objectives as well as all notes, data, results from data analyses, and other records were collected in binders. Any deviations from SOPs were noted therein. Data is also stored, with few exceptions, in electronic format on disks and backed up on a regular basis. The disks and their locations are identified in the notebooks. The binders are kept in the laboratory and will stay there for 7 years, according to auditing procedures.
Cedervall T, Lynch I, Lindman S, Berggård T, Thulin E, Nilsson H, Dawson KA, Linse S. Understanding the Nanoparticle-Protein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles. Proc Natl Acad Sci USA 104: 2050-2055, 2007.
Cedervall T, Lynch I, Foy M, Berggård T, Donnely SC, Cagney G, Linse S, Dawson KA. Detailed Identification of Plasma Proteins Adsorbed on Copolymer Nanoparticles. Angew Chem Int Ed 46: 5754-5756, 2007.
Ehrenberg M, McGrath JL. Binding Between Particles and Proteins in Extracts: Implications for Microrheology and Toxicity. Acta Biomater 1: 305-315, 2005.
Linse S, Cabaleiro-Lago C, Xue WF, Lynch I, Lindman S, Thulin E, Radford SE, Dawson KA. Nucleation of Protein Fibrillation by Nanoparticles. Proc Natl Acad Sci USA 104: 8691-8696, 2007.
Lynch I, Cedervall T, Lundqvist M, Cabaleiro-Lago C, Linse S, Dawson KA. The Nanoparticle-Protein Complex as a Biological Entity: A Complex Fluids and Surface Science Challenge for the 21st Century. Adv Colloid Inter Sci 134-135: 167-174, 2007.
Ryman-Rasmussen, J.P., Riviere, J.E., Monteiro-Riviere, N.A. Penetration of Intact Skin by Quantum Dots with Diverse Physicochemical Properties. Toxicol. Sci. 91(1): 159-165, 2006.
Teng, X. and H. Yang. Synthesis of Platinum Multipods: An Induced Anisotropic Growth. Nano Lettr. 5(5): 885-891, 2005.
Teng, X., Y. Liang, S. Maksimuk, and H. Yang. Synthesis of Platinum Porous Nanoparticles. Small 2(2): 249-253, 2006.
Record Details:Record Type: PROJECT (ABSTRACT)
Organization:U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
NATIONAL CENTER FOR ENVIRONMENTAL RESEARCH