The use of lanthanide/rare earth doping of metal oxide nanoparticles has proven to be highly effective as a measure of dose and translocation in vivo. The particles are relatively simple to synthesize and the method is well suited to eventual application to inhalation studies. The very high sensitivity of ICP-MS offers a host of new possibilities for dosimetry and deposition studies. Although the basic approach is appealingly simple, we have found that in practice it is very difficult to avoid cross-contamination of samples at the very low levels that are demanded by ICP-MS detection – extraordinary care is necessary during the excision of organs and also during the digestion process, particularly during the separation of control tissues from exposed tissues. Our overall mass balance, based on the true mass input of lanthanides versus the mass recovered, is 83% ± 10% (Table
1). It is very likely that some of the lanthanide that is unaccounted for is present in the fur or carcass that was not digested. The syringe dead volume, used to instill lanthanide NP, was measured gravimetrically and resulted in a decrease of the delivered dose by 2.7% ± 0.7%; this was accounted for in the mass balance. Feces were collected from the bedding and although extreme care was taken to retrieve almost the entire sample, incomplete recovery could be another source of error. Urine samples studied using metabolism cages (data not shown here) did not indicate the presence of lanthanide ions by ICP-MS. In this study, no assumptions were made regarding the rates of fast clearance; 21.4% of the instilled dose was recovered from the feces and GI tract. A translocation study of 20 and 80 nm radioactive iridium particles instilled in rats and mice showed 95% total mass recovery with typical uncertainties related to radiological measurements
. Semmler-Behnke et al. reported close to 100% mass recovery for 1.4 and 18 nm gold particles intratracheally instilled in rats
; the mass removed from the lung by fast clearance was estimated (up to 25%) and subtracted from the delivered dose. The total mass balance determined in this work is comparable to these previous studies, within experimental error. Clearly, particle size also determines ability to translocate. Kreyling et al.
 found that iridium particles of 15 nm were much more capable of translocating from the lung tissue than 80 nm particles.
The number-weighted mean diameter of our particles in suspension in buffer, as delivered to the animals, was 84 nm with a relatively narrow size distribution as measured by dynamic light scattering. We are able to vary this size in our method by controlling independently two parameters: (1) precursor concentrations in the solutions, the easiest parameter to change; (2) the size of droplets formed in our spray. Inhalation experiments that will follow on from this methodology study will examine this important issue.
As shown in Table
1, 24 hours after nanoparticle instillation, 59% of the initial dose was measured in the lung, 20% was excreted in the feces, 0.2% was detected in the liver and less than 0.1% of the delivered dose was detected in the remaining organs. These results are qualitatively similar to those found by Kreyling et al.
 for 15 and 80 nm iridium particles intratracheally instilled in adult rats; after 7 days, 59% of the delivered dose was found in the lungs, 35% was fecally excreted and less than 0.2% found in extrapulmonary organs. In contrast, studies of 40 and 100 nm gold nanoparticle that were instilled intratracheally did not yield measurable signal in the liver or other organs
. This could be due to different protein coating on gold compared to Eu:Gd2O3 nanoparticles or comparable flame-generated metal oxide particles.
It is possible that metals are not transported in animals as nanoparticles but rather as dissolved ions, which could have a quite different propensity for translocation and clearance. Our particles offer yet another advantage: a straightforward means to estimate the rates of dissolution within physiologically relevant media. Our ability to track dissolved versus bound Eu in a particle via photoluminescence offers a way to determine the importance of nanoparticle dissolution. We have found very little evidence of dissolution after 7 days in either of the simulated fluids that particles may encounter: lung BALF and low pH buffer, the latter designed to simulate a lysosomal condition. Further evidence for this assertion comes from examination of the Gd:Eu ratio in recovered tissues – the ratio of the metal ions in the measured samples remains the same. It is unlikely that these ions would diffuse at precisely the same rate, therefore, the ratio measurements indicate that it was likely that the elements translocated together within the particle and that this is the primary contributor to the observed clearance and translocation processes that we have measured.
This experimental method promises to provide new insights into clearance and deposition, with a quantifiable dose. Dose at the target site is proportional to biologic effect – this is a basic tenet of toxicology. Yet when it comes to inhalation dosimetry, air pollution standards and most animal exposures characterize the concentration of particles in the air on the basis of mass per volume as the “dose”. This does not account for the actual mass deposited into the initial target tissue – the respiratory tract – which can be affected by disease, age, exercise etc. Local toxic effects are of special interest for ultrafine particles because they have a larger total surface area for an equivalent mass of larger particles and a long retention time in the lung. While there has been recent notable progress in using computational models to predict total respiratory tract deposition, confirmation of the models with experimental data has lagged
. If we could inexpensively, reliably, and with great sensitivity, track inhaled particles in the body, it would revolutionize the field of inhalation toxicology and allow the interpretation of biological response in relation to delivered, and retained, dose.
While radionuclides have shown promise for this purpose, with sensitivity in the range of 1 ppm
[9, 28, 29], issues of containment and cleanup have limited their usefulness. Stable isotopes exhibit some promise
. Fluorescent beads
 and quantum dots are useful for short term studies, particularly intracellular tracking studies, but issues of photo-bleaching and toxicity
, respectively, can limit their usefulness. The particles described in the current study are a breakthrough in this respect because they are synthesized as part of a flame pyrolysis process and so lend themselves readily to inhalation exposure studies. Furthermore, they can be fine-tuned into specific size ranges by altering spray droplets sizes and the concentrations of precursors in solution.
We chose to use oropharyngeal aspiration for these initial studies because dose can be readily controlled and the particles delivered as a relatively large bolus, which is desirable for studies of translocation and testing of the range of detection. We acknowledge that this method does not approximate a true inhalation exposure and is not appropriate for studies of short-term clearance, which would be more appropriately addressed using an inhalation model
[6, 33]. Furthermore, some aggregates of particles were detected and this can affect deposition and translocation characteristics, but these aggregates were a small percentage of the total number of particles instilled. This is a limitation of the instillation approach and will be addressed in future studies of aerosols.
Particle clearance can be thought of in two phases. The first phase occurs primarily in the 24 hours after dosing and is dominated by conducting-airway mucociliary clearance of the particles to the larynx and then to the GI tract and feces
. The second phase, which is thought to involve macrophage-mediated clearance from the peripheral lung, occurs after 24 hours and can last for a long time (hundreds or thousands of days) depending on the particle type
. The major issue with particle tracking following respiratory tract deposition is that only a very small percentage of the particles escape the lung and circulate to other organs (this varies based on particle size and has been estimated as < 0.1% for particles in our size range
). This means that for a method to be useful for particle tracking, it needs to be exquisitely sensitive to a very small number of particles.
Our approach using Eu:Gd2O3 particles can detect particle loads in the ppb range. However, simply estimating particle mass in a whole organ will likely not be sufficient; the logical next step for biological investigations will be to define the extracellular and intracellular location of the particles, preferably with the same particles from the same exposure that characterized the local dose. This is the goal of future studies and will be facilitated by the native phosphorescence of these particles that will allow them to be visualized microscopically.