Grantee Research Project Results
2009 Progress Report: Project 4 -- Transport and Fate Particles
EPA Grant Number: R832414C004Subproject: this is subproject number 004 , established and managed by the Center Director under grant R832414
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: UC Davis Center for Children's Environmental Health and Disease Prevention
Center Director: Van de Water, Judith
Title: Project 4 -- Transport and Fate Particles
Investigators: Wilson, Dennis , Barakat, Abdul , Louie, Angelique
Current Investigators: Wilson, Dennis , Louie, Angelique
Institution: University of California - Davis
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2011)
Project Period Covered by this Report: July 1, 2008 through July 30,2009
RFA: Particulate Matter Research Centers (2004) RFA Text | Recipients Lists
Research Category: Human Health , Air
Objective:
1) To characterize the time course, tissue distribution, and mechanisms of particulate matter (PM) accumulation in the systemic circulation and target organs.
2) To evaluate the effects of size and surface-fixed charge on this process.
3) To determine how altered lung structure affects systemic particle distribution.
4) To characterize how fluid flow modulates particle interactions with vascular endothelial cells.
Progress Summary:
Specific aim 1: To characterize the time course and distribution of circulating particulates in vivo.
In the last year, Positron Emission Tomography (PET) was used to determine the deposition and translocation of 78nm 64Cu-labeled amine terminated polystyrene nanoparticles in both normal and asthmatic mice. Figure 1.1 shows the bio-distribution of particles in a normal mouse over 48 hours. Mucociliary clearance shows particles leaving the trachea and lungs and clearing primarily through the GI tract. This is supported by Figure 1.2 which shows of interest (ROI) analysis of organs over time.
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Figure 1.1 Bio-distribution of polystyrene nanoparticles over 48 hours using PET (n=4).
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Figure 1.2 ROI analysis of bio-distribution over 48 hours in the normal mouse.
For aim 3, asthmatic mice (OVA exposure model) were studied and Figure 2.1 shows the bio-distribution of nanoparticles over 48 hours.
Figure 2.1 Bio-distribution of nanoparticles over 48 hours in the asthmatic mouse (n=7).
Bio-distribution differs from normal mice; uptake in the bladder and liver at 24 and 48 hour time points is shown. ROI analysis is currently underway.
To test the sensitivity of PET for bio-distribution studies a limit of detection experiment was done for the 64-Cu labeled polystyrene nanoparticles; the results of this are shown in Figure 3.1.
Figure 3.1 Limit of detection for 64-Cu labeled polystyrene for the Focus 120 PET instrument. (R2=0.974)
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For this experiment the limit of detection was 1.55E+10 nps/mL for the given degree of radiolabeling (4.3E-18 Ci/np) This value corresponds to ~0.01% of instilled dose (instilled dose = 1.24E+14 nps/mL).
Compromised Cardiovascular Animals: Do animals with pre-existing CV disease accumulate nanoparticles at lesions?
For the injury model (femoral) uptake was shown both at the injury site and control site after 24 hours (yellow arrows). This injury model shown in Figure 4.1 was a clamp/10% FeCl3 injury to the right femoral artery (n=1).
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Figure 4.1 PET/MRI of iron oxide (CLIO-cross linked iron oxide) uptake in an injury mouse at 24 hours. Yellow arrows show accumulation in both the injured and uninjured artery.
The injury model was repeated and the uptake was not observed. The apoE model was also used for these studies and uptake was not visualized in plaques/vessels. This model is challenging because the plaque locations are near the lungs. Signal from the lungs can drown out plaque accumulation.
Future directions for this project include utilizing a smoke/apoE mouse from the Pinkerton laboratory to see if more particles can leave the lungs and accumulate in plaque sites. Also, new injury techniques are under investigation for these studies.
Specific aim 3: To evaluate potential mechanisms of PM transport across epithelial and endothelial barriers.
Mechanisms of Endothelial and Epithelial transport using synthetic fluorescent and electron dense ultrafine particles.
In previously reported work, we characterized the transport of 30 nm synthetic iron oxide particles across endothelial cell monolayers and demonstrated vesicular transport though caveolar like structures by 4 hours. We further characterized this as vesiculocaveolar transport using fluorescence tagged silica particles with confocal microscopy. We now extended this work to ask whether similar rates of transport occur in cultured human airway epithelial cells. We found, in contrast to studies with endothelial cells, airway epithelium did not allow transport during a 4 hour incubation period.
Progress 2008-2009
We synthesized Alexafluor 680 conjugated silica particles for use in real time transport studies using deconvolution microscopy. In addition, we successfully cloned and transfected a GFP caveolin construct that will allow us to co-localize red Alexa 680 labeled PM with green labeled caveoli. Figure 3-1A illustrates that transfection with GFP alone results in a diffuse cellular stain and that addition of silica does not co-localize with GFP.
Using GFP caveolin transfected human aortic endothelial cells, we showed GFP caveolin associated with a typical arrangement of centrally located surface oriented aggregates of caveolar like structures Figure 3-2A. We confirmed co-localization of 30 nm silica particles in caveolar associated vesicles after 30 min. incubation. Furthermore, we demonstrated rearrangement of caveolar structures from aggregates over the center of the cell to dispersed and larger caveolin associated structures after silica treatment (Figure 3-2B).
To take advantage of transfected cells we performed time course imaging of live cells treated with silica particles using spinning disc confocal microscopy (Figure 3-3). This technique was more challenging as it required transport of live cultures to a distant imaging facility. Particularly challenging was reaggregation of dispersed particles in preparations made before transport. This work is ongoing.
We asked whether size influenced the uptake of silica particles. Our hypothesis was that only particles in the ultrafine range would undergo caveolar transport. As part of the refocus of our group on inflammation at the alveolar capillary barrier, we switched our cell lines for this study to human pulmonary microvascular endothelium (HPMVEC). Our preliminary findings demonstrate that both 30 nm and 500 nm silica particles were internalized in HPMVEC. Ongoing experiments ask whether both sizes of particles are within caveolin associated vesicles.
Figure 3-1: Deconvolution microscopic images of HAEC transfected with GFP protein only. A) untreated cells B) cells treated with 10 ug/ml 30 nm Alexafluor 680 conjugated silica particles
Figure 3-2: Deconvolution microscopic images of HAEC transfected with GFP-caveolin. A) Untreated cells show localization at the cell surface over the center of the cell (similar to findings with caveolin immunostaining –data not shown) B) Cells treated with 30 nm Alexafluor 680 conjugated silica particles have yellow fluorescence indicative of co-localization and dispersion of caveolar structures to the periphery of the cell.
Figure 3-3: Spinning disc confocal microscopic images of live HAEC transfected with GFP-caveolin. GFP-Caveolin transfected cells were treated with 10 ug/ml 30 nm Alexafluor 680 conjugated silica particles and individual cells photographed at indicated times to observe internalization and transport.
Figure 3-4: Standard fluorescence microscopy of HPMVEC treated with either A) 30 nm or B) 500 nm Alexafluor 680 conjugated silica particles. Similar amounts of both sizes were internalized in these preliminary experiments.
Specific aim 4: To characterize how fluid flow modulates particle interactions with vascular endothelial cells.
In vivo, vascular endothelial cells are constantly exposed to flow. Research over the past two decades has demonstrated that fluid flow regulates endothelial cell phenotype. Furthermore, the details of this regulation depend on the type of flow to which the cells are subjected. Endothelial cells exposed to either steady (time-independent) or non-reversing pulsatile flow develop an anti-inflammatory phenotype that is largely protected from atherosclerosis. On the other hand, endothelial cells subjected to oscillatory flow exhibit a pro-inflammatory phenotype and are prone to the development of vascular pathology. Oscillatory flow occurs at arterial branches and bifurcations, and these regions correlate with the development of early atherosclerotic lesions.
We have been investigating how different types of flow modulate PM-induced inflammation in cultures of human aortic endothelial cells (HAECs). The experiments involve pre-shearing HAECs with either steady or oscillatory flow for a period of 24 hrs in order to establish either an anti- or pro-inflammatory cellular phenotype. At the end of the flow period, HAECs are exposed to PM for 4 hrs under static conditions, and the mRNA and protein levels of the three inflammatory markers ICAM-1, IL-8, and MCP-1 are subsequently assessed using real-time PCR, Western blotting, or ELISA. The results are compared to control cells that are not exposed to flow.
Our initial experiments have focused on laboratory nanoparticles, most notably zinc oxide (ZnO). These particles were selected because we had previously shown that they induce HAEC inflammation under static (no flow) conditions. Following completion of the ZnO experiments, similar studies will be conducted using ambient particles. The results to date demonstrate that, consistent with our previous studies, ZnO nanoparticles at a concentration of 50 μg/ml induce inflammation in HAECs that have not been exposed to flow. In HAECs pre-conditioned with steady flow, ZnO nanoparticles induce similar levels of inflammation to that in cells that have not been exposed to flow. In HAECs pre-conditioned with oscillatory flow, however, ZnO nanoparticle-induced inflammation is significantly amplified. An example of these results for ICAM-1 mRNA is depicted in Figure 4.1. These results, if conformed in vivo, suggest that particle-induced inflammation would be exacerbated considerably in arterial regions susceptible to atherosclerosis (where oscillatory flow occurs) than in atherosclerosis-resistant regions.
Figure 4.1: Modulation of ZnO nanoparticle-induced inflammation by different types of flow. Shown are the expression levels of ICAM-1 mRNA. Similar results were obtained for other inflammatory markers including IL-8 and MCP-1. Protein results are largely consistent with the mRNA results.
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Why HAEC pre-conditioning with oscillatory flow leads to amplification of PM-induced inflammation remains unknown and is a question of critical importance. We have performed some transmission electron microscopy (TEM) experiments that may shed some light on this issue. As illustrated in Figure 4.2, ZnO nanoparticles induce considerable vacuole formation in HAECs that have not been pre-exposed to flow. This vaculization appears even more pronounced in cells pre-conditioned with oscillatory flow but is largely absent in cells pre-conditioned with steady flow. In previous studies, we had shown that such vacuoles are involved in particle internalization into and transport within the cells. Therefore, it is possible that the results shown in Figure 4.2 reflect enhanced particle internalization in cells pre-conditioned with oscillatory flow and greatly reduced particle uptake in cells pre-conditioned with steady flow. This hypothesis awaits more direct experimental confirmation.
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Figure 4.2: Transmission electron micrographs demonstrating ZnO nanoparticle-induced vacuole formation in HAECs. Note the prominent vacuoles in cells that were either not pre-sheared or pre-sheared with oscillatory flow. These vacuoles are absent in cells pre-sheared with steady flow.
Particle interactions with cells are affected by the state of the particles as they make contact with the cell surface. Of particular importance in this regard is particle aggregation. In order to understand particle-cell interactions, it is important to establish the extent and dynamics of particle aggregation as well as how aggregation is affected by various parameters including the cell culture media and any proteins present in the media. We have been using dynamic light scattering (DLS) to determine the aggregation dynamics of laboratory nanoparticles, most notably iron oxide (Fe2O3) and ZnO particles. Similar studies will be performed on ambient particles once the ZnO studies are completed.
Figure 4.3 depicts the DLS results for both Fe2O3 and ZnO nanoparticles. Previous studies had established that the primary particle size in both cases is of the order 60-100 nm. Several measures of particle aggregation are shown including the “effective” hydrodynamic diameter (based on the mobility of the aggregates within the measurement volume, a value heavily driven by larger aggreagtes), the “peak” diameter, the “number average” diameter, and the “area average” diameter. These different measures represent different moments of the measured distributions. The results demonstrate that Fe2O3 particles stored in water for a few weeks (the “no sonication” bars in Figure 4.3) aggregate considerably. These aggregates are very effectively broken down after application of probe sonication for 1 minute. Following sonication, the aggregates are slow to re-form. ZnO particles behave in a largely similar way to Fe2O3 particles, but the initial aggregates are significantly smaller.
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Figure 4.3: DLS measurements to assess aggregation for A: Fe2O3 and B: ZnO nanoparticles. Both types of particles aggregate significantly upon storage in water. Probe sonication effectively breaks down the aggregates, which are slow to re-form.
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We have performed DLS measurements to assess the effects of media composition as well as the presence of serum in the media on Fe2O3 and ZnO particle aggregation dynamics following probe sonication. The results, depicted in Figure 4.4, demonstrate that particles aggregate to a greater degree in either PBS or cell culture medium (EBM) than in water. Furthermore, the presence of serum generally reduces aggregation. This effect is especially pronounced in water, where virtually no aggregation occurs. We hypothesize that the increased aggregation in PBS and cell culture media relative to water is attributable to differences in ionic strength. We also suspect that the reduction of aggregation by serum incubation is due to the fact that serum coating renders particle surfaces negatively charged, and the electrostatic repulsion limits the extent of aggregation. These hypotheses await experimental verification.
Figure 4.4: DLS measurements of aggregation dynamics following probe sonication for A: Fe2O3 and B: ZnO nanoparticles in different suspension media. PBS: phosphate-buffered saline. EBM: endothelial basal media. Complete: EBM plus all additives used in routine HAEC culture.
Journal Articles:
No journal articles submitted with this report: View all 3 publications for this subprojectSupplemental Keywords:
ambient air, ozone, exposure, health effects, human health, metabolism, sensitive populations, infants, children, PAH, metals, oxidants, agriculture, transportation, , Air, Health, RFA, Risk Assessments, particulate matter, human health risk, toxicology, epidemiological studies, lung disease, long term exposure, RFA, Health, Air, particulate matter, Risk Assessments, toxicology, long term exposure, lung disease, epidemiological studies, PM
Progress and Final Reports:
Original AbstractMain Center Abstract and Reports:
R832414 UC Davis Center for Children's Environmental Health and Disease Prevention Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832414C001 Project 1 -- Pulmonary Metabolic Response
R832414C002 Endothelial Cell Responses to PM—In Vitro and In Vivo
R832414C003 Project 3 -- Inhalation Exposure Assessment of San Joaquin Valley Aerosol
R832414C004 Project 4 -- Transport and Fate Particles
R832414C005 Project 5 -- Architecture Development and Particle Deposition
The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.