Source and Characterization of NPs
Cu NP powders (partially passivated with oxygen by the manufacturer) were purchased and used as received from the manufacturer (Nanostructured and Amorphous Materials, Inc, Houston, TX, USA). The Cu NPs were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and BET techniques as described previously [9
]. The Cu NPs show a core/shell morphology with a metallic Cu core and an oxidized shell consisting of Cu2
O (Cuprite) and CuO (tenorite), with CuO on the surface of the particles. A compilation of some of the properties measured for the commercially manufactured NPs after purchasing are listed in Table .
Table 1 Summary of physicochemical characterization data of Cu NPs  and experimental design of inhalation and instillation studies of pulmonary bacterial clearance
Male 6 week old (22-25 g) C57Bl/6 mice (The Jackson Laboratory, Bar Harbor, ME) were utilized in both the inhalation and instillation studies. Protocols were approved by the Institutional Animal Care and Use Committee and complied with the NIH Guide for the Care and Use of Laboratory Animals. Mice were held in quarantine for 12 days prior to use in AAALAC-accredited vivarium in polypropylene, fiber-covered cages in HEPA-filtered Thoren caging units (Hazelton, PA). They were supplied with food (sterile Teklad 5% stock diet, Harlan, Madison, WI) and water ad libidum and maintained on a 12-hr light-dark cycle.
The experimental design and number of mice used per dose and group is shown in Figure . Briefly, pulmonary responses and the body burden of Cu measured in the tissues and BAL fluids were characterized after Cu NP exposure or sham exposure (filtered lab air for inhalation and optima water for instillation study). We also evaluated lung bacterial clearance following exposures. In the sub-acute inhalation study, additional mice were exposed to Cu NP or filtered lab air for assessment of pulmonary mechanics.
Figure 1 Schematic of the pulmonary bacterial clearance model. We established a murine pulmonary infection model of Klebsiella pneumoniae (K.p.) to determine if pulmonary bacterial clearance is impaired by Cu nanoparticle exposure using two different exposure (more ...)
NP Exposure, Aerosol Generation and Characterization
For the inhalation study, a dynamic whole-body exposure system was used to expose mice to Cu NP aerosols as previously described [8
]. Briefly, mice were placed within sectioned, open-wire cages that were positioned in a whole-body, custom-fabricated aluminum exposure chamber [22
]. A suspension of Cu powder in water (Optima grade, Fisher Scientific, Pittsburgh, PA) was nebulized to generate a Cu NP aerosol. A 1 mg Cu NP/mL suspension was first ultra-sonicated with a high frequency probe set at 30% of the maximum amplitude (20 kHz, model 550, Fisher Scientific, Pittsburgh, PA) for 20 min to minimize the degree of agglomeration. The suspension was then placed in a 6-jet Collison nebulizer (BGI Inc., Waltham, MA) supplied with filtered, pressurized air. The droplets were dried by passing them through a 110°C brass drying column and the dry Cu NPs entered a tube containing a 20 mCi 63
Ni source to remove static charge prior to entering the chamber.
We measured the time-integrated mass concentration of the aerosol in the chamber by gravimetric analysis of 47-mm glass microfiber filters (Whatman, Middlesex, United Kingdom) in line with the 24 L/min exhaust air flow. Sample grids for TEM were placed inside the exposure chamber to measure agglomerate sizes of the NPs. The size distribution of the aerosol in the whole-body exposure chamber during inhalation exposures was measured using a scanning mobility particle sizer (SMPS, model 3080 electrostatic classifier with model 3081 differential mobility analyzer and model 3785 condensation particle counter, TSI Inc., Shoreview, MN) that measured diameters in the range of 7.4 to 289 nm according to the manufacturer's instructions. Geometric mean (GM) and geometric standard deviation (GSD) of aerosol sizes in individual exposures were determined from the SMPS measurements.
Sub-acute Inhalation Exposure
In sub-acute studies, mice were exposed 4 hr/day, 5 d/wk for 2 weeks and necropsied immediately after exposure. The average concentration of Cu NPs was 3.5 ± 0.4 mg/m3
, very close to our previous sub-acute exposure concentration of the same NPs (3.7 mg/m3
]. Sham-exposed controls breathed filtered laboratory air in identical exposure chambers in an adjacent laboratory. The deposited dose was estimated assuming minute volume of 25 mL/min and a deposition fraction of 15% in the tracheobronchial and pulmonary region [23
] yielding an estimated NP dose of 32 μg Cu NP/mouse. This estimation assumes there is no clearance via
the mucociliary escalator to the trachea or gastrointestinal tract and no translocation of NPs to other organs.
Intratracheal Instillation Exposure
Three concentrations of Cu NPs (3, 35, and 100 μg/mouse) were used for intratracheal instillation exposures. These were prepared by suspending particles in Optima water to minimize contaminants found in unprocessed water. This suspension was ultrasonicated as described above and then vortexed immediately before dosing to minimize aggregation of particles. Mice were anesthetized by isoflurane inhalation prior to intratracheal instillation of 50 μL of Cu NP suspensions. Animals were positioned on an inclined restraining stand and a very flexible FEP (Fluorinated ethylene propylene) polymer catheter (24G 3/4", Smiths Medical International Ltd., UK) affixed to a 1-mL syringe was then inserted into the mouth and placed between the vocal cords and into the lumen of the trachea. Illumination of the trachea was provided by a directed fiber optic light source. Mice instilled with 50 μL Optima water alone were used as negative controls. Experimental groups were necropsied 24 hr after the instillation (24 hr post-exposure).
Pulmonary Bacterial Infection and Clearance Model
K.p. was obtained from the American Tissue Culture Collection (ATCC 43816, Rockville, MD). The bacteria were inoculated into 50 mL of tryptic soy broth (TSB) in 250 mL flasks for 18 hr (stationary phase) with shaking (120 rpm) in a gyratory water bath shaker at 37°C (New Brunswick Scientific Co., Edison, NJ). The bacterial suspension was diluted in TSB to obtain an optical density (OD660) of 0.4 by measuring in a Spectra Max microplate reader (Molecular Device, Sunnyvale, CA); 200 μL of this solution was added to 50 mL of TSB for 3 hr to reach mid-log phase of growth (OD660 ~ 0.4, corresponding to ~2 × 108 colony-forming units (CFUs/mL), where bacteria are most virulent.
Immediately following the 10th
sub-acute inhalation or the intratracheal instillation exposure of Cu NPs, mice were anesthetized by isoflurane inhalation and then intratracheally instilled with a bacterial inoculum containing 1.4 ± 0.1 × 105
per mouse incorporated into and mixed with amorphous agar particles (molten 4% Noble Agar, BD, Franklin Lakes, NJ) in a 50 μL volume as described previously [25
]. Twenty-four hr after K.p.
inoculation, mice were euthanized by isoflurane inhalation and the lungs were removed and whole lung tissues were suspended in 1 mL of sterile saline, homogenized and cultured on tryptic soy agar (TSA). The number of bacteria remaining in the lungs was counted after an overnight incubation at 37°C. The concentrations of instilled K.p.
were checked by spread plating the inocula on TSA. The dose and duration of bacterial inoculation were based on preliminary studies showing that this bacterial dose did not cause mortality and could be cleared effectively in control mice.
Evaluation of Bronchoalveolar Lavage (BAL) Fluid
Six mice per group were euthanized by isoflurane inhalation and each lung was lavaged 3 times (total volume, 3 mL) with 0.9% sterile sodium chloride solution (Baxter, Deerfield, IL). BAL fluid was collected, processed and the cell pellet was used for enumeration of total and differential cell counts. The lavage supernatants were analyzed for total protein levels using a Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA), LDH activity measured with a commercial assay (Roche Diagnostics, Penzberg, Germany) and cytokines were quantified by multiplexed fluorescent bead-based immunoassays (Bio-Rad Laboratories, Inc., Hercules, CA). Seven inflammatory cytokines and chemokines were chosen based on our previous study [9
] and included interleukin [IL]-6, IL-12(p40), tumor necrosis factor [TNF]-α, granulocyte macrophage colony stimulating factor [GM-CSF], keratinocyte-derived cytokine [KC], monocyte chemotactic protein [MCP]-1, and macrophage inflammatory protein [MIP]-1α.
Lungs from 3 mice per group that were not lavaged were fixed in 10% formaldehyde-phosphate-buffered saline solution via
the canulated trachea. The tissues were subsequently paraffin-embedded, sectioned at 5 μm, and stained with hematoxylin and eosin (H & E) as previously described [26
]. Tissue sections were evaluated by light microscopy in four categories each employing a five point scale to elucidate abnormalities of the parenchymal architecture (bronchioles, alveoli, pleura, and vasculature); abnormal inflammatory infiltrates; presence or absence of acute lung injury; and presence or absence of fibrosis.
Determination of Cu Concentration in the Tissues and BAL Fluids
To determine the total amount of Cu in the lung, brain, heart, kidney, and splenic tissues, tissues were removed from Cu NP-exposed and sham-exposed mice, frozen (-80°C) and lyophilized for 16 hr at 1.3 × 10-1 mBar and -50°C in a freeze dryer (Labconco Corp., Kansas City, MO) and then weighed. Mixtures of high purity concentrated nitric acid and hydrochloric acid (Fisher Optima® grade) were used to digest the tissues with a HotBlock™ digestion system (Environmental Express, Mt. Pleasant, SC) at 95-98°C. Digestates were diluted to 10 mL with Optima water and Cu analysis was performed using an inductively coupled plasma-mass spectrometer (ICP-MS, X Series, Thermo Scientific). To determine dissolved Cu ion concentration in the lung tissues after NP exposure, the BAL fluid was centrifuged at 44,912 g for 30 min to separate NPs and aggregates that were not dissolved. Cu concentration in the particle-free BAL supernatants was measured by ICP-MS.
Pulmonary Mechanics Measurements
Pulmonary mechanics were measured using the forced oscillation technique. Mice were anesthetized with 90 mg/kg of pentobarbital sodium (Ovation Pharmaceuticals, Inc., Deerfield, IL) by intraperitoneal injection. Tracheotomy was performed using a tracheal cannula with luer adapter (1.3 mm, length 20 mm, Harvard Apparatus, MA) and each mouse was connected to a computer-controlled small animal ventilator (flexiVent, SCIREQ, Montreal, QC, Canada). The mice were ventilated at 150 breaths/min with a tidal volume of 10 mL/kg and positive end-expiratory pressure of 2-3 cm H2O.
To measure airway hyperactivity, mice were challenged with increasing concentrations of methacholine aerosols (1, 3, and 10 mg/mL), generated with an in-line nebulizer (10 sec) directly through the canulated trachea. Response to each methacholine dose was measured every 30 sec for 5 min. Only data with a coefficient of determination greater than 0.95 were included in the final analysis. The lungs were inflated to total lung capacity after each methacholine dose. A sinusoidal (single-frequency) forced oscillation waveform ("snapshot perturbation") maneuver was performed to measure resistance (R), compliance (C), and elastance (E) of the whole respiratory system (airways, lungs and chest wall). A broadband (multi-frequency) forced oscillation waveform was used to produce measurements of airway resistance (Rn), tissue damping (G) and tissue elastance (H). All these parameters were calculated using flexiVent software (version 5). Responses were expressed by computation of the area under the curve (AUC) of each parameter measured versus time for each 5 min methacholine dose monitoring period.
Data from mice exposed to Cu NPs were compared to sham-exposed mice using two-sided t tests for equal or unequal variances and one-way analyses of variance (ANOVA) was done for comparison between groups in the instillation study (SAS Ver. 9.2, SAS, Inc., Cary, NC). Results from pulmonary mechanics measurements were tested for outliers using the stem-and-leaf and normal probability plots produced by the univariate procedure (SAS). We calculated the AUC for each parameter (e.g., R, C and E) versus time plot for all methacholine doses. For the inhalation study, the means and AUCs of pulmonary mechanics parameters for the Cu-exposed group were compared to sham-exposed mice using ANOVA for repeated measures. A p-value less than 0.05 was considered statistically significant. All data are expressed as mean ± standard error (SE) unless otherwise noted.