Particle synthesis
Europium doped gadolinium oxide nanoparticles were synthesized using a flame spray pyrolysis technique and a forced jet atomizer similar to that described by Dosev et al.
[35]. The burner schematic is shown in Figure
7. This technique is well-suited for generating environmentally relevant metal oxide nanoparticles in high concentrations with reasonable control on particle size
[36]. Gadolinium and europium nitrate salts (35.5 mM and 9.3 mM respectively) were dissolved in ethanol and used as the liquid precursor. The precursor was sprayed into a hydrogen-air flame at 40 ml/hr using a syringe pump. The precursor droplets were pyrolyzed to form nanoparticles in the high temperature environment. The Gd:Eu ratio measured by ICP-MS was 4.7 ± 0.1 and in agreement with the initial precursor ratio (4.74) based on nitrate concentrations in the precursor. Particles were collected on a cold finger by thermophoresis; the collected powder was washed in Milli-Qultrapure (MQ, 18.3 MΩ-cm) water to remove any unreacted precursor from the nanoparticles. The nanoparticles were dried and stock solutions of 10 mg/mL were prepared in MQ water for the instillation experiment.
Particle characterization
The nanoparticle morphologies were investigated using a Phillips CM-12 transmission electron microscope (TEM) operated at 120 kV. Nanoparticles were suspended in MQ water and deposited on a 400 mesh copper TEM grid with a carbon/Formvar® film (Ted Pella Inc. Redding CA. Prod # 01754-F); excess liquid was wicked away.
The particle crystalline phase was identified using a Scintag powder x-ray diffractometer (XRD) with Cu Kα radiation operated at 45 kV and 40 mA.The powder was scanned for 2θ = 25° calculated using Scherrer’s formula,
where the shape factor K is 0.9
[37], λ is the incident x-ray wavelength (=1.54 Å), β is the peak full width-half maximum (FWHM) at Bragg angle θ = 32.5°.
The surface area of nanoparticles was measured with the Brunauer-Emmett-Teller (BET) method using an Autosorb-1 instrument (Quantachrome Instruments, Boynton Beach, FL). A sample of 380 mg was measured with nitrogen as the adsorbate. Hydrodynamic particle size measurements were performed using a BIC 90Plus dynamic light scattering instrument
(Brookhaven Instruments, Holtsville, NY). The number-weighted (NW) particle size distributions were calculated by the 90Plus software (Brookhaven Instruments). The particle concentration at the start of the DLS experiment was 20 mg L-1 in MQ water. The solution was bath-sonicated for one minute to disperse the particles. At least five runs were measured and the results were averaged.
Zeta potential was measured by light scattering using a BIC ZetaPlus instrument (Brookhaven Instruments Corporation, NY). Particles were suspended in 1 mM KCl with particle concentration of 100 mg L-1 and the suspension was bath-sonicated for five minutes before each sample measurement. At least five measurements were made for each sample and the data were averaged.
Surface adsorbed protein quantification
Particles were suspended in either PBS (1x) or BSA (100 mg/mL of dry protein in PBS (1x)) with particle concentrations at 200 mg L-1. Particles were washed in PBS (1x) three times. The solutions were bath-sonicated for five minutes, centrifuged at 9500 g ; the supernatant was replaced with PBS between washes to remove any unbound protein. Surface adsorbed proteins were quantified using the bicinchoninic acid (BCA) assay with bovine serum albumin (BSA) as a calibration standard. The absorption measurements (excitation wavelength, λ = 560 nm) were performed on a SpectraMax M2 cuvette/microplate reader (Molecular Devices Inc, Sunnyvale CA) using the SOFTmax PRO software. Samples were bath-sonicated before measurements. The measurements were made with six replicates. A 96-well plate was used with 200 μL in each well. After the reagent was added, the well was incubated at 37°C for 30 minutes. The calibration range extended between 20 to 500 μg/mL of BSA with R2 linearity of 0.998. The adsorbed protein concentration was determined by subtracting the signal of uncoated bare particles from protein-coated particles.
Particle dissolution
We investigated the possibility that Eu and Gd ions are translocated, not as nanoparticles, but as dissolved ions. It is important to estimate the rate of dissolution of these particles under conditions that approxim ate the fluid in the lung and also the intracellular conditions of an endosome. To obtain sufficient lung lavage fluid for in vitro experiments of particle dissolution, lung bronchoalveolar lavage fluid (BALF) was obtained from adult Sprauge-Dawley rats (N = 6), instead of the mice that were used for the translocation studies. Briefly, rats were euthanized with an overdose of pentobarbital given i.p., the trachea cannulated and a single volume of 35 ml kg-1 of buffered saline solution was lavaged into the lung three times. The physiological conditions that are typical of a cellular lysosome were modeled with PBS adjusted to a pH of 5.
The Eu3+ ions in Gd2O3 emit light only when contained within the host matrix. Any dissolution or leaching of Eu3+ ions from the host Gd2O3 nanoparticle will result in a drop in the photo-luminescence (PL). The PL spectra of particle suspensions were obtained using a Varian Cary Eclipse Fluorescence Spectrophotometer equipped with a Xenon lamp as an excitation source. The dry nanoparticles samples were dispersed in solutions of lung serum and PBS at pH5 and sonicated for 15 min to obtain a transparent dispersion of nanoparticles with a concentration of 200 μgml-1. In each case, 4 ml of the nanoparticle dispersion were added to a quartz cuvette and excited at 250 nm to obtain the emission spectra of the nanoparticles over time. To investigate the dissolution of the nanoparticles at different pH, PBS solutions were adjusted to different pH (i.e. 2 to 7) and emission the spectrum were obtained using the same method.
Oropharyngeal aspiration
Animals
All animal experiments were performed under protocols approved by the University of California Davis Institutional Animal Care and Use Committee in accordance with NIH guidelines. For studies of particle translocation in vivo, adult (8 weeks) male NIH Swiss mice with a weight of 25 to 30 grams were purchased from Harlan Laboratories (Livermore, CA). Mice were delivered one week prior to exposure, and housed in AALAC approved facility at the Center for Health and the Environment, University of California Davisand provided with Laboratory Rodent Diet (Purina Mills, St. Louis, MO) and water ad libitum.
Oropharyngeal aspiration of particles
Oropharyngeal aspiration in mice is an attractive alternative to direct tracheal instillation because it results in less variability among animals and gives a more uniform pulmonary distribution of the administered particles
[38]. Mice were anesthetized using a Quantiflex anesthesia machine (Midmark Corp., Versailles, OH) equipped with an isoflurane vaporizer. Mice were placed in a Plexiglass® box connected to anesthesia machine. A mixture of 2.5% isoflurane and oxygen was delivered at a rate of 1 L/min to effect, approximately five minutes. Once anesthetized, a known concentration of particles (40 μL dose of 0.160 ± 0.02 g L-1 Eu/Gd PM suspended in MQ water) was pipetted into the oropharynx and aspirated into the lungs
[39]. Mice were monitored until recovery. The dead volume of the instillation system (Product # RSPSMI, Kent Scientific, CT) was determined gravimetrically. Following recovery from anesthesia, each mouse was placed in an individual cage. N = 4 per group
Necropsies and organ harvest
Tubes were weighed before and after sample was collected. Mice were euthanized an overdose of pentobarbital (150 mg/kg) given i.p and exsanguinated. Blood was collected and the abdominal cavity was opened and the spleen, kidneys and liver were removed. Then the thoracic cavity was opened and the heart and lungs were removed. Finally, the stomach and intestines were removed as a unit. Feces from the cages were collected and the remaining carcass was weighed and frozen at −80°C. Minced, tissues, blood and feces were placed in individual 15 mL polystyrene conical tubes (BD Biosciences, Franklin Lakes, NJ) for processing. Instruments were cleaned in DI water and rinsed in 70% ethanol between uses to prevent cross contamination.
Tissue acid digestion and ICP-MS analysis
Tissue samples were digested in trace metal grade nitric acid and hydrogen peroxide for elemental analysis. Acid digestion occurred at 70°C for 24 hours followed by hydrogen peroxide (70°C) digestion overnight. Tissue samples were diluted with MQ water to an acid concentration of 6%.
The concentrations of elemental gadolinium and europium were quantified using inductively coupled plasma mass spectrometry (ICP-MS). The ICP-MS instrument was calibrated using a NIST traceable standard for Eu and Gd. The standards came in stock solution at 1000 ppb and 100 ppb; serial dilutions of 500, 200 and 100 ppb were used at the high end of the concentration range and 100, 10, 1 and 0.1 ppb for the low concentration standard to generate the calibration curve. The instrument level of detection (LOD) was 1.9 ppt (parts per trillion) for Eu and 2.7 ppt for Gd and the BEC (background equivalent concentrations) is 1.6 ppt for Eu and 3.9 ppt for Gd. The LOD and BEC are determined by instrument rinses with at least n = 5 throughout the experimental run on a particular day. Blank tissues were used as a control; the ICP-MS analysis of Eu indicated a maximum concentration in these tissues of 0.1 ppb, setting a lower limit to our sensitivity in the exposure experiments that translates to a limit of quantification of about 4 ng in the lung samples and about 1 ng in the kidneys.
Quality control of the ICP-MS analysis and the integrity of nanoparticles was checked by comparing the measured Gd:Eu ratio from tissue samples to that of the original nanoparticles, ensuring that the elemental signal was from nanoparticles and not from the background, and that analytical artifacts had not been introduced. Indeed, europium-doped gadolinium oxide is suitable for this experiment as both elements have a low natural abundance abundance. The most common rare earth element is Cerium with a natural abundance in the Earth’s crust of about 43 ppm
[40]; the least common is Thulium at 0.3 ppm. Europium and Gadolinium are present in the Earth’s crust at around the 1 ppm level. Good hygiene in the laboratory ensures that the background levels in our experiments are well below the natural level with our controls exhibiting concentrations at about 0.1 ppb.