Grantee Research Project Results
2007 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 , Kennedy, Ian M. , Fanucchi, Michelle V. , Buckpitt, Alan , 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: October 1, 2006 through September 30, 2007
RFA: Particulate Matter Research Centers (2004) RFA Text | Recipients Lists
Research Category: Human Health , Air
Objective:
- Characterize the time course, tissue distribution, and mechanisms of PM accumulation in the systemic circulation and target organs.
- Determine the effects of size and charge on this process
- Evaluate the potential the altered lung structure would effect systemic particle distribution.
Progress Summary:
Specific Aims 1, 2
- Characterize time course and distribution of circulating particulates in vivo
- Compare the anatomic site of particulate accumulation in tissues with organ distribution as determined by microimaging techniques
This past year we have been developing methods to determine the fate of deposited model ultrafine particles in the rodent model. To summarize, polystyrene nanoparticles have been modified to carry radioactive tracers and these were introduced to rats by insufflation. Two types of radiotracers were used: In-111 for biodistribution studies using gamma counting of harvested organs, and Cu-64 for Positron Emission Tomography (PET) imaging of the model ultrafines. Overall trends indicate that there is elevated accumulation of particles in the heart in the first 4 hours after exposure and that this elevation disappears by 24 hours. Larger particles (one micron) had less movement out of the lung than smaller particles (78nm).
We overcame a number of technical challenges to perform this work. One of the first challenges was coupling radioactive labels to the particles. We originally proposed to couple derivatized chelators to the particles through amine functional groups at the surface of the particles and then insert the radioactive cations. This uses standard conjugation chemistry that we have used extensively in other systems. However, nanoparticles proved to be recalcitrant to coupling by this method and the degree of labeling was low (~6%). We explored a number of synthetic protocols and finally have been able to increase the degree of labeling to 45% using the scheme below. It is unclear why nanoparticles were so resistant to labeling by the original method. We are currently exploring the mechanism for this resistance as we have since observed it for many other types of nanoparticles, regardless of composition, that are amine-functionalized.
+ 64
The second technical challenge was training personnel to perform the insufflation technique on rats. The insufflation technique uses a spritzer device that delivers a fine mist solution to the lungs of the animals when performed properly. Consistently delivering material to the lung required many hours of training. Training was initially performed using fluorescent material and whole animal optical imaging to prevent risk of exposure to radioactivity while personnel were learning to perform the insufflation. The figure below shows and example of deposition to the lungs (right) and esophagus (left) dissected from a rat. While more consistent lung deposition was achieved over time, we were still unable to reliably avoid deposition to the esophagus. Therefore, we are in the future moving to an endotracheal catheter method of deposition which we have found gives much more reliable deposition exclusively to the lungs. This will be accomplished in future studies. The studies shown below all use the insufflation method and therefore, the results are a bit confounded by variable, small amounts of esophageal deposition.
Figure 1. Xenogen images of esophagus and lung after spritzing.
In-111 studies using the labeled probes and insufflation show a general trend of elevated accumulation in the heart in the first 4 hours after insufflation, which is higher for smaller particles. The plots below show sample plots of organ accumulation at the 4 hour timepoint for 1 micron (Figure 2) and 78 nm (Figure 3) particles. By 24 hours the elevation has disappeared.
Figures 2 and 3. Distribution of 78 nm and I micron particles in organs 4 hours after introduction
Both In-111 and Cu-64 studies were performed for pharmacokinetics and preliminary blood collection studies indicate that lung deposition results in early appearance of particles in the blood that disappears over time (below left). With esophageal deposition the opposite effect is seen, particles increase in the blood over time (below right).
Trachea | Esophagus |
Figures 4 and 5. Blood clearance of tracheal (Fig 4.) versus esophageal (Fig.5) initial deposition of particles
With Cu-64 PET imaging it was quite facile to distinguish esophageal deposition (below left), which appears as a three-dimensional tube, from trachael deposition (below right), which clearly shows deposition to both lobes of the lungs.
Figures 6 and 7. PET images of initial tracheal or esophageal deposition of particles.
Over time, esophageal deposition moved to the stomach and intestines as expected (2 hr timepoint, below left), and tracheal deposition showed liver and kidney accumulation over time (4hr timepoints below right) with some movement through the GI tract, presumably from materials that was initially deposited in the esophagus or that had been swept out of lungs and trachea and swallowed thru normal clearance mechanisms.
Figures 8 and 9: Distribution of particles 2-4 hours after initial esophageal or tracheal deposition
The animals were also imaged by Computed Tomography for anatomical reference as shown in the sample below (coronal section, overlay PET (red)/CT (gray) showing lung deposition at time 0).
Figure 10. CT and PET co-registered image of initial tracheal deposition of particles.
In ongoing work we will explore the utility of a mouse model using catheter deposition techniques, we have found these to consistently deliver desired volumes to precise locations. In addition the smaller size of the mice make it more feasible to increase the number of animals imaged per study. We will also be investigating modification to the liquid spritizing protocol that may improve the deposition profiles with that technique, as well as testing a new device designed by a UCD undergraduate team that automates the “spritzing” process and delivers a fixed volume at a velocity every time. We will continue to collect both In-111 distribution data and Cu-64 imaging data. The question of whether the In or Cu could escape from the probes will be answered through stability assays. However, given the very high stability of macrocyclic compounds we do not believe that there can be release of the ions during the course of these imaging and biodistribution studies.
Specific Aims 3,4
- Evaluate potential mechanisms of PM transport across epithelial and endothelial barriers.
- Characterize the dynamics of interaction between particulates and airways and arterial walls
The mechanism(s) of UFP translocation across cellular barriers remains unclear. We hypothesized that ultra fine particles can be internalized and transported across endothelial cell membranes using a caveolar transport mechanism. We examined particle-plasma membrane interactions in endothelial cell culture. To obtain consistent material sizes, we evaluated methods of resuspending laboratory generated iron oxide particles. Iron Oxide particles were generated in flame exhaust and particle size measured by SMPS. UFPs were deposited on filters and then resuspended in cell culture media. Particles were sonicated in suspension by either water bath or probe and measured by DLS before being used in treatments. Dynamic light scattering studies determined that water bath sonication did not adequately recapitulate the original size distribution while probe sonication recreated the synthesized particle distribution that averaged 0.1 mm.
Human aortic endothelial cells (HAECs) were exposed to iron oxide particles (10 µg/ml solution) for 10, 30, 60 min and 4 hrs. Fe2O3 treated HAECs were fixed in 2.5% gluteraldehyde and 2% paraformaldehyde and secondarily fixed in reduced osmium before being embedded for transition electron microscopy (Figure 11). At 30 min, iron oxide aggregates interacted with plasma membrane structures and were visible within vesicles close to caveolae. By 4 hrs the electron dense particles were visible as aggregates in membrane bound vesicles stratified throughout the cells and on the basilar side of the endothelial cells. Intercellular junctions remained intact and paracellular transport was not evident.
To confirm the caveolar transport process, we treated similar cultures with FITC conjugated silica particles. HAECs were exposed to manufactured silica particles labeled with FITC (10 µg/ml) for 30 min and 4 h. Fluorescence immunostaining with was used for caveolin-1 colocalization by confocal microscopy. Treated cell culture monolayers were stained with Caveolin-1 mouse monoclonal (R&D) and localized with anti-mouse Alexa Fluor 555 (Molecular Probes). Caveolin-1 was predominantly centralized at 30 min with FITC-labeled silica particles isolated to the outer plasma membrane with little co-localization. At 4 hours Caveolin-1 was widely disseminated through the cell and there was a strong co-localization between caveolin-1 and the FITC-labeled particles visible in the cells. Our results suggest that ultra fine particles can be internalized and transported across endothelial cell membranes, potentially using a caveolar transport mechanism similar to the observed transport of other macromolecules and drugs.
Figure 11: Transmission electron microscopy of iron oxide particles in endothelial cells at 0.5 or 4 h post treatment. Note extrusion of PM to basal surface by 4 hours (arrowhead)
30 min
4 hrs
Figure 12: Confocal Z series images of FITC labeled particles (green) and caveolin-1 (red) at 30 min and 4 hours after exposure.
Caveolin label is generally central on the cell surface and PM is limited to the apical portion of the cell at 30 min. By 4 hours, bright spots were yellow at multiple levels demonstrating both co-localization and aggregation of caveolin rich vesicles at all levels of cell cytoplasm.
Future Activities:
Aims 1 and 2
Our efforts for next year, as mentioned, will move to the catheter method for particle deposition as well as exploring the use of the mouse model as an alternative. Many more mice can be processed per experiment and mice seem to handle the anesthesia required for these multiple timepoint studies. We are also developing more controlled methods to perform insufflation using an automated delivery system. We will continue to refine the labeling protocol to further increase yield and well as begin utilizing other types of nanoparticles.
Aims 3 and 4
Next we will evaluate transport in dynamic (flow through culture) as opposed to static conditions and to characterize transport in airway epithelial cell cultures. To address Aim 4, transport in perfused vessels, will also be initiated in year 3.
Journal Articles:
No journal articles submitted with this report: View all 3 publications for this subprojectSupplemental Keywords:
RFA, Health, Air, particulate matter, Risk Assessments, toxicology, long term exposure, lung disease, epidemiological studies, PMProgress 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.
Project Research Results
- Final Report
- 2010 Progress Report
- 2009 Progress Report
- 2008 Progress Report
- 2006 Progress Report
- Original Abstract
2 journal articles for this subproject
Main Center: R832414
128 publications for this center
64 journal articles for this center