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2006 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: San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Center Director: Wexler, Anthony S.
Title: Project 4 -- Transport and Fate Particles
Investigators: Wilson, Dennis , Buckpitt, Alan , Fanucchi, Michelle V. , Kennedy, Ian M. , Louie, Angelique
Current Investigators: Wilson, Dennis , Louie, Angelique
Institution: University of California - Davis
EPA Project Officer: Stacey Katz/Gail Robarge,
Project Period: October 1, 2005 through September 30, 2010
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: Particulate Matter Research Centers (2004)
Research Category: Particulate Matter
Description:
Objective:The objectives are: (1) to characterize the time course, tissue distribution, and mechanisms of PM accumulation in the systemic circulation and target organs; (2) to determine the effects of size and charge on this process; and (3) to evaluate the potential the altered lung structure would effect systemic particle distribution.
Progress Summary:Model Ultrafines For Imaging Particle Distribution in the Rat Model: Synthesis and Characterization
The purpose of these syntheses was to generate nanoparticles bearing positron-emitting radioisotopes for positron emission tomographic imaging in the rat model. Two types of nanoparticles were produced: (1) aminated dextran coated iron oxide; and (2) aminated polystyrene nanobeads.
Both types of nanoparticles are positively charged at neutral pH due to the presence of large numbers of amine groups on their surface. The positron emitting radionuclide 64Cu was coupled to the nanoparticles by chelating the ion with p-isothiocyanatobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bz-DOTA) (figure 1, yellow = Cu). The derivatized DOTA couples to free amines on the nanoparticle surface. In the earliest approaches, DOTA was coupled to the nanoparticles, followed by insertion of 64Cu . As outlined in the methods below, an unexpected interaction of copper with the nanoparticle surfaces prevented insertion of the copper into DOTA. We now insert Cu into DOTA prior to coupling DOTA to the nanoparticle, and by this method have successfully coupled Cu to both types of nanoparticles.
Figure 1. Design of Dual-Mode Dextran Sulfate Coated Nanoparticle
Synthesis of Copper Labeled Nanoparticles
- Coupling of DOTA-Nanoparticles
- Synthesis of 64Cu-DOTA-Conjugated Iron Oxide Nanoparticles (IONP)
- Synthesis of 64Cu -Conjugated Iron Oxide Nanoparticles (IONP)
- Synthesis of 64Cu -DOTA-Conjugated 78 nm Polystyrene Beads: Method I
- Synthesis of Cu -DOTA-Conjugated Polystyrene Beads: Method II
Two types of nanoparticles were employed in these studies. Dextran sulfate iron oxide nanoparticles, synthesized as described in the supplemental section below, and polystyrene nanoparticles were purchased from commercial sources (Molecular Probes). Both types of nanoparticles were amine modified.
Conjugation of DOTA to Aminated Iron Oxide Nanoparticles (IONP). p-SCN-Bz-DOTA (1.09 mg, 1.56 μmol) was dissolved in DMSO (1.0 ml) and 59 μl of this solution was added to a solution of IONP (0.7 ml, ~1.95 × 1015 cores) in water (1.8 ml) and stirred overnight at room temperature. A molar ratio of p-SCN-Bz-DOTA:nanoparticle; ~30:1 was used. Product was purified through a Sephadex G-25 column with 0.05 M ammonium bicarbonate as mobile phase.
Conjugation of DOTA to 78 nm Polystyrene Beads (PS-beads). p-SCN-Bz-DOTA (1.26 mg, 1.81 μmol) was dissolved in DMSO (0.3 ml) and added to a solution of 78 nm polystyrene beads (1 ml) in sodium carbonate/bicarbonate buffer (3 ml, pH 9.1). The solution was stirred for 20 h at RT and pH 9.1. A molar ratio of p-SCN-Bz-DOTA:bead; ~14,300:1 was employed. The product was purified by ultrafiltration (30 kDa) with water three times.
64Cu (2.2 mCi in 75 μl-10 mM HCl), NH4OAc (58 μl, 1.0 M, pH 7) and DOTA-IONP the from above synthesis (100 μl in H2O) were incubated for 40 min at 40°C. EDTA-solution (26 μl, 100 mM) was added and the resulting solution purified on a Penefsky column. Unfortunately, all particles were trapped in the column. The Penefsky column is a Sephadex G-50 column that is packed by centrifugation; we believe that the packing material was too tightly packed to allow passage of the nanoparticles. In addition, centrifugation can cause particle aggregation that results in large clusters of nanoparticles that cannot pass through the column.
64Cu (300 μCi in 13 μl-10 mM HCl), NH4OAc (25 μl, 1.0 M, pH 7) and DOTA-IONP (62 μl in H2O, these were dextran sulfate coated IONP) was combined and incubated for ~30 min at 40°C. Thin layer chromatography (TLC) was run but no labeling of the nanoparticles was seen. EDTA-solution (10 μl, 100 mM) was added (to chelate free copper) and a band corresponding to free Cu-EDTA was observed. We believe that the copper interacted with free sulfate groups on the particles/beads rather than the DOTA chelator and then was removed by addition of the EDTA.
64Cu (1.25 mCi in 20 μl-10 mM HCl) and DOTA-polystyrene beads (180 μl in 0.25 M NH4OAc) was combined and incubated for ~30 min at 40°C. TLC was run both with and without EDTA-solution and again, Cu label migrated with the free EDTA band instead of the nanoparticles. It appears that copper is interacting with surface groups, such as amine, that this prevents insertion to DOTA at the stoichiometries used here.
Based on these results we hypothesized that labeling would be more successful if we pre-inserted Cu2+ into the DOTA moiety prior to coupling to the nanoparticles. We performed a number of syntheses with nonradioactive copper to optimize coupling methods prior to working with 64Cu.
Part I: Synthesis of p-SCN-Bz-Cu-DOTA
p-SCN-Bz-DOTA (5.26 mg; 7.66 μmol) was dissolved in 1.0 ml of ddH2O. 20 μl of 0.47835 M Cu2+ solution was added and stirred for an hour at RT. The solution turned green-turquoise after a few minutes. Molar ratio Cu2+:DOTA was 1.25:1. The product was purified on a Sephadex G-25 column. The bluish fraction was removed and insertion of copper into DOTA was verified by UV-Vis absorbance spectroscopy. No peaks corresponding to pure p-SCN-Bz-DOTA or pure Cu2+ were observed, indicating the absence of unreacted Cu2+ or p-SCN-Bz-DOTA. Product formation was verified by electrospray mass spectrometry which yielded a product peak with the anticipated mass of 613.4 (theoretical MW = 615.2). The product was concentrated to 2.09 mM by vacuum methods.
There is a small risk of hydrolyzation of the isothiocyanate moiety when performing the reaction in water but the coordination reaction with copper is much faster than the hydrolysis. If the copper is inserted and the product then immediately conjugated to the beads this lowers the risk of deactiviation of the isothiocyanate group by hydrolysis. Performing the reaction in DMSO could also reduce risk of hydrolysis and will be explored in future studies.
Part II: Conjugation of p-SCN-Bz-Cu-DOTA to Polystyrene Beads (PS)
Coupling of p-SCN-Bz-Cu-DOTA to the beads was performed at two different stoichiometries as outlined in A and B below:
- 150 μl p-SCN-Bz-(Cu-DOTA) solution from Part I was added to 0.5 ml PS-beads (78 nm) solution in 4.5 ml tetramethylammonium phosphate (TMAP) buffer (pH 8). The solution was stirred at room temperature overnight and then purified by dialysis against 4l ddH2O in 4°C for 4 days with 7 changes. Molar ratio of p-SCN-Bz-(DOTA-Cu):bead was 4968:1. Inductively coupled mass spectrometric (ICP-MS) analysis of elemental content showed that each bead was conjugated with ~150 p-SCN-Bz-Cu-DOTA.
- 15 μl p-SCN-Bz-(Cu-DOTA) solution from Part I was added to 0.3 ml PS-beads (1 μm) solution in 4.7 ml TMAP buffer (pH 8). It was stirred at RT over night and then purified by dialysis against 4 l ddH2O in 4°C for 4 days with 10 changes. Molar ratio of p-SCN-Bz-(Cu-DOTA):bead was ~218,000:1. ICP-MS analysis showed that each bead was conjugated with ~40,000 p-SCN-Bz-Cu-DOTA.
TMAP buffer was employed as it does not contain sodium so there is no risk for blocking the DOTA coordination sites; in addition, the risk of hydrolyzation of the isothiocyanate moiety is eliminated by using a tertiary amine. Clearly, there are still competing reactions preventing 100% coupling of all the DOTA material to the beads, therefore molar excesses of the Cu-DOTA complexes will need to be applied to reach the desired labeling levels. The final amounts of copper incorporated in the above syntheses far exceed that which will be necessary for PET imaging with the radionuclide 64Cu. The high concentrations introduced here were necessary to provide sufficient signal for ICP-MS confirmation of copper conjugation to the nanoparticles. We estimate that the final concentration of copper required for PET imaging is in the picomolar range, which is approximately one 64Cu per thousand nanoparticles.
Summary
Many attempts of conjugating cold copper to polystyrene beads via the p-SCN-Bz-DOTA linker have been performed. The first approach was to conjugate p-SCN-Bz-DOTA to the beads and then coordinate Cu2+ to the DOTA moiety. In later approaches we first pre-incubate the p-SCN-Bz-DOTA with Cu2+ and then conjugate that to the nanoparticles. The second method makes sure that no copper will be loosely bound to amine groups of the surface of the beads. Both of these approaches have been performed on different bead sizes (78 nm and 1 μm) in different buffer systems and with different molar ratios.
Different purification methods for the final product have also been tested; ultrafiltration cell, Sephadex G-25 column and dialysis. The ultrafiltration works but there is a loss of material on the filter. We are currently purifying by dialysis against water; this is effective but quite time consuming. We will be exploring other column purification packing materials to increase the efficiency of purification.
So in summary, nanoparticles can be successfully labeled with copper by pre-incubation of copper with p-SCN-Bz-DOTA followed by immediate conjugation of the p-SCN-Bz-Cu-DOTA to aminated polystyrene beads in a tetramethylammonium phosphate buffer (pH 8). The Cu-DOTA labeling of the 78 nm beads was achieved with 3% labeling efficiency and Cu-DOTA labeling of the 1 μm beads, performed at higher Cu-DOTA: nanoparticle ratio (given the larger surface area of the beads) resulted in 18% conjugation efficiency. The low efficiencies of coupling may be improved by reaction in DMSO, but these values are consistent with efficiencies observed in the literature (Gustafsson, et al., 2006).
Supplement: Synthesis of Dextran Sulfate Coated Iron Oxide Nanoparticles
Dextran was reduced by stirring an aqueous solution of dextran with sodium borohydride (26 equivalents) at room temperature for 12 hours. The reaction was then stopped by the dropwise addition of 6N HCl until pH 7 and then the reduced dextran was dialyzed (Spectra/Por 6, MWCO 3,500) against nanopure water. Then a dextran sulfate, reduced dextran (5%, w/w dextran sulfate/reduced dextran) and FeCl3·6H2O solution was prepared (2.7% polysaccharide, 7.4% iron chloride solutions, w/vol). This mixture was bubbled with nitrogen for 30 minutes and cooled to 4°C in an ice bath. A solution of FeCl2·4H2O (1.47-1.5 molar ratio of Fe3+/Fe2+) was freshly prepared, chilled to 4°C, and added to the polysaccharide/Fe3+ solution. The reaction mixture was further bubbled with nitrogen for 5 minutes on an ice bath and then 8.2 mL ammonium hydroxide (28-30%) was added over 2 minutes with rapid magnetic stirring. The slurry was then heated to 90°C and stirred for 2 hours under nitrogen. The product was cooled to room temperature and ultrafiltered (30kDa membrane) against 150mL nanopure water to remove excess starting material. The solution was then ultrafiltered (300 kDa membrane) to isolate the small nanoparticles and the filtrate collected to yield a brown-black, translucent solution. This synthesis produced particles of 25 - 30nm diameter.
Imaging Studies
Whole animal imaging studies in the first reporting period were limited by the challenges of particle conjugation described above. We have used two alternatives to evaluate sensitivity and establish technical approaches to imaging studies with live animals. Our first approach used PET imaging of a rat instilled with a 64Cu solution. Difficulties with the aerosolization technique prevented adequate deep lung penetration of the instilled material but a high signal for 64Cu was achieved (Figure 2).
Given the difficulties in creating the 64Cu conjugates, we also evaluated the potential for using intravital fluorescence as an imaging modality in these studies. We first titrated aerosolized fluorescein on paper media to calibrate sensitivity and then administered soluble fluorescein by insufflation to a rat. Results of this study showed significant background autofluorescence in the whole animal but that fluorescent signal could be detected in dissected tissues. Unfortunately this initial experiment resulted in intraesophageal instillation (Figure 3) but did offer proof of concept for potential future studies using florescence labeled particles in isolated organs.
![Figure 2. MicroPET Image From Whole Rat Instilled With [64]Cu in Saline](https://webarchive.library.unt.edu/eot2008/20081105225059im_/http://es.epa.gov/ncer/progress/images/R832414C004_06_002.gif)
Figure 2. MicroPET Image From Whole Rat Instilled With 64Cu in Saline
Figure 3. Intravital Florescence Image From Heart and Lungs of a Rat Instilled With 4.0 μM Fluorescein
Mechanisms of Particle Transport Across Endothelial Barriers
The mechanism by which ultrafine particles enter the circulation is unknown. It is also uncertain whether circulating ultrafine particles localize in tissue macrophages or exit the circulation to potentially effect organ function. In order to better understand the potential for translocation across endothelial barriers, we exposed human aortic endothelial cell cultures to laboratory generated iron oxide particles and evaluated the time course and location of particles within cells by electron microscopy. Ten μg/ml of 100 nm iron oxide particles suspended by probe sonication were incubated with primary HAEC cultures for periods up to 4 hours followed by fixation and processing for electron microscopy by standard techniques. Results showed that significant re-aggregation of particles was evident at the endothelial cell surface at early time points. Some suggestion that aggregates formed near caveolar like structures was evident (Figure 4). By 2 hours, aggregates of particles were evident in intracellular membrane bound vacuoles and by 4 hours, particles were present on the abluminal surface of the endothelium. Intercellular junctions remained intact and no particle transport between cells was evident. This study suggests that endothelial cells are capable of transcellular transport of ultrafine particles in a relatively short time span and raises the possibility that this occurs through a facilitated transport in the vesiculo-vacuolar system associated with endothelial caveolae.
Figure 4. Ultrastructural Appearance of HAEC Cultured With 100 nM Iron Oxide Particles for 0.5 (A) or 4 (B) Hours. Initial interaction with caveolar like structures (arrows) is associated with intracytoplasmic vesicles followed by transport of particles to ablumenal surface (arrowhead) by 4 hours.
In the next project year, we plan to focus on the blood kinetics of indium-labeled particles while continuing development of our PET imaging studies. Key to the latter will be consistent labeling of synthetic ultrafine particles with 60Cu as this has been the most significant challenge in our initial year. We also are exploring the option of Xenogen-based fluorescence imaging as a development tool that does not require the use of radiochemicals. We will continue our transcellular transport studies in cultured endothelial cells using fluorescence-tagged particles in concert with dual-labeling for caveolar proteins.
Journal Articles:No journal articles submitted with this report: View all 1 publications for this subproject
Supplemental Keywords:Air, Health, RFA, Risk Assessments, particulate matter, human health risk, toxicology, epidemiological studies, lung disease, long term exposure
Relevant Websites:
Progress and Final Reports:
Original Abstract
2007 Progress Report
2008 Progress Report
Main Center Abstract and Reports:
R832414 San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
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