Biodistribution of Inhaled Gold Nanoparticles in Mice and the Influence of Surfactant Protein D

Animals

Female C57BL/6 mice (10–12 weeks old, body weight ∼21.5 g) were obtained from Charles River (Sulzfeld, Germany) and housed in the HMGU animal facility in isolated ventilated cages (VentiRack, Biozone, Margate, UK). They received a standard pellet diet and tap water ad libitum. The study was conducted under the guidelines of the Helsinki Convention for the Use and Care of Laboratory Animals and was approved by the Government of the District of Upper Bavaria (approval no. 55.2-1-54-2531-26-10) and the Animal Care and Use Committee of the Helmholtz Center Munich. All chemicals were purchased from Sigma-Aldrich (Deisenhofen, Germany), unless otherwise specified.

Aerosol production and characterization

Ultrafine ¹⁹⁵Au aerosol was produced with a spark ignition generator as previously described.⁽¹⁷⁾ For radiolabeling, the gold electrodes (3.1-mm diameter, 4-mm length) of the spark ignition generator were proton irradiated at one end in the Scanditronix MC40 cyclotron at the Joint Research Center (Ispra, Italy), using the highest proton energy available.

¹⁹⁵Au has several gamma energies below 100 keV and a long half-time of 186 days, allowing gamma spectroscopy for measurement of organ activities.

The ¹⁹⁵Au activity concentration of the aerosol was 1.2 kBq L⁻¹ at the reference date. Aerosol mass concentration was calculated to be 1.6 mg m⁻³ from the radioactivity measurement of an integral filter sample of 0.3 L/min aerosol flow during the entire exposure and the sampled aerosol volume. Size distribution parameters were the following: particle number concentration, 2±0.5×10⁷/cm³; count median diameter (CMD), 22 nm; geometric standard deviation, 1.6; the first being continuously monitored with a condensation particle counter (CPC 3022A; TSI, Aachen, Germany) and the second by a differential particle mobility analyzer (Classifier 3071+CPC 3010; TSI). As the aerosol was heat-treated at 600°C online in a tube furnace immediately after generation and prior to exposure, the agglomerated gold particles were melted to spherical-shaped particles of 22-nm CMD. Morphologically, Au NP were analyzed by TEM collecting Au NP of the freshly generated aerosol after heat treatment with an electrostatic TEM sampler (University of Applied Sciences, Windisch, Switzerland).

Instillation of SP-D

Directly prior to the ¹⁹⁵Au NP inhalation, mice were anesthetized using medetomidine 0.5 mg/kg, midazolam 5 mg/kg, and fentanyl 0.05 mg/kg. Afterwards, the anesthetized mouse was fixed with its incisors to a rubber band on a board at an angle of 60° to the lab bench. A flexible cannula (BD Vasculon Plus; 26 G, 0.75 in) was placed under visual control into the upper third of the trachea. Proper placement in the trachea, and not misplacement into the esophagus, was controlled with a pneumotachograph connected to the cannula. Directly before the start of the Au NP aerosol inhalation, the suspension (50 μl of PBS or 50 μl of PBS containing dissolved 10 μg of SP-D; recombinant human SP-D, R&D Systems, Minneapolis, MN) was gently instilled into the trachea of the mouse during the inspiration phase, followed by 400 μl of air using a 1-ml insulin syringe. The instilled air prevented liquid losses in the syringe and supported deeper penetration of the instillate into the lungs. Intratracheal instillation was aligned with the inspiration of the mouse.

¹⁹⁵Au nanoparticle inhalation

Immediately after SP-D instillation, the intubated mouse was placed in a plethysmograph and connected with the endotracheal cannula to the aerosol line outside of the plethysmograph, as described earlier.^(17,18) Ventilation was computer-controlled by a negative pressure of 1.5 kPa applied to the plethysmograph chamber during 0.25 sec of inspiration followed by 0.25 sec of expiration at ambient air pressure, resulting in a breathing frequency of 120 min⁻¹. This ventilation pattern usually caused an inspiration of >50% of total lung capacity of the mice; therefore, animals were slightly hyperventilated and did not breathe spontaneously, but followed the computer-controlled breathing pattern. Four mice were ventilated simultaneously, each placed in an individual plethysmograph chamber. For radiation protection, the entire aerosol system was maintained at −30 Pa low pressure and additionally installed in a glove box, which was highly ventilated through an absolute particle filter providing an additional low pressure in the glove box of about −20 Pa.

Sample preparation

Directly after inhalation, mice were kept anesthetized (5% isoflurane inhalation) and euthanized by exsanguination via the abdominal aorta. Approximately 70% of the total blood volume was withdrawn. For ¹⁹⁵Au radioanalysis, the following samples were taken by careful necropsy after euthanasia of the animals: blood sample, lungs, esophagus, larynx, stomach, gastrointestinal tract (GIT) (stated as fast clearance via the mucociliary escalator), total skin including fur, bronchoalveolar lavage (BAL), and the remainder (all remaining organs, tissue, muscles, skeleton).

During dissection, no organs were cut in order to avoid any cross contamination.

Afterwards, the organs and tissues were directly frozen and radioanalyzed as quickly as possible without any further preparation step.^(17,19)

By this approach, we quantitatively determined the entire NP dose in the animal by analyzing each organ and tissue in a 100% balance of the biodistribution of the applied NP. As there were no excreta at this early time point immediately after inhalation, no excreta samples were collected.

Bronchoalveolar lavage

A BAL was performed by applying 5×1 ml of PBS without Ca²⁺ or Mg²⁺ under gentle massage of the thorax. The recovered BAL fluid (BALF) (∼90% of instilled saline) was centrifuged at 500 g for 20 min to separate the lavaged cells from the supernatant.

¹⁹⁵Au radioactivityanalysis

The ¹⁹⁵Au radioactivity of all samples was measured by gamma spectroscopy without any further physicochemical preparation in either a lead-shielded 10-mL or a lead-shielded 1-L well-type NaI (Tl) scintillation detector. The count rates were corrected for physical decay and background radiation. In addition, count rates were calibrated to a ¹⁹⁵Au reference source in order to correlate ¹⁹⁵Au radioactivities to the numbers and masses of the Au NP. Samples yielding net counts (i.e., background-corrected counts) less than three standard deviations of the totally measured counts in the photopeak region of interest of the ¹⁹⁵Au gamma spectrum were defined as below the detection limit. For a complete balance of the administered ¹⁹⁵Au radioactivity within each mouse, ¹⁹⁵Au radioactivities of all samples were summed up for each mouse (except the skin and fur sample); this was considered to be the initial lung dose (ILD) and was used as a denominator for the calculation of the ¹⁹⁵Au percentage of each sample. Additionally, all data (except those of mucociliary clearance) were corrected for the fast cleared Au NP determined in the esophagus and GIT. Using this approach, we attempted to normalize the translocated amount of Au NP to the initial peripheral lung dose.

Calculations and statistical analysis

Four animals per group were used. Calculated values are given as a percentage of the relevant integral ¹⁹⁵Au radioactivity (calculated for a reference date) of all samples in each animal with the standard error of the mean (SEM). All radioactivities were correlated with the corresponding mass of gold NP in each animal. Data of animals with and without SP-D were compared using an unpaired t test, and differences were considered to be significant at p<0.05.

Fast clearance via the mucociliary escalator

Immediately after the end of the 2-hr Au NP inhalation period, the fast mucociliary clearance was low and related to only 3.6±1.2% of the deposited Au NP of the control group. However, this is not astonishing, because the animals were intubated during Au NP inhalation, allowing only minor particle transport into the stomach; only NP that deposited in the lung at the very beginning of the inhalation period were able to get cleared and swallowed into the stomach. Hence, we would expect more mucociliary clearance of the Au NP at longer duration after NP inhalation. This is consistent with a former study using inhaled iridium NP.⁽²⁰⁾

Au NP within the BALF

Using centrifugation, we separated the cells of the BALF from the lavage fluid. Hence, we could distinguish the Au NP taken up by macrophages from the Au NP remaining free in the BALF.

Directly after inhalation, more “free” Au NP (29.9±1.8% of lung retained) were found within the BAL supernatant compared with the fraction associated with BAL cells (12.4±1.1% of ILD). As these are the results directly after the 2-hr inhalation period, we believe that this is mainly due to the short time interval between NP deposition and the collection of the BALF. Hence, we hypothesize that at later time points more NP will be taken up by BAL cells compared with free Au NP in the BALF. Similar results were also found with iridium NP,^(21,22) where directly after the inhalation more free NP were found in BALF compared with the amount of NP in cells. Importantly, a rapid increase of NP in the BAL cells within the first day was observed previously.

Au NP associated with the lung tissue

A high fraction of the lung-retained Au NP was associated with the lung tissue (57.5±1.9%) directly after the inhalation period. In this regard, it is important to note that we did not distinguish between NP taken up by lung cells (i.e., epithelium) and NP bound to the lung tissue. However, as it is known that NP can be easily taken up by lung cells in vitro and in vivo,^(23–25) we assume that both uptake and binding occur. Due to the investigated time frame (directly after inhalation), we assume that most of the particles may be attached to the cell surface of the lung tissue.

Au NP translocation via the air–blood barrier

We found a rather high amount of Au NP translocated to the blood and accumulated in secondary organs and tissues directly after inhalation. In this regard, 1.2±0.5% were detected beyond the air–blood barrier. Importantly, we can exclude that uptake via the GIT was responsible for this translocation. On the one hand, there was very limited time for the NP to reach the GIT, and just a limited amount of approximately 3.6% NP was found within these organs. On the other hand, we know from recent gavage and instillation studies that absorption of NP via the GIT is very small.^(19,26,27) Hence, we conclude that the uptake into blood described in the present article was almost exclusively across the air–blood barrier in the lung. A previous publication of our laboratory from Lipka and co-workers described a translocation rate of 2.6% 1 hr after intratracheal instillation of 5-nm Au NP.⁽²⁷⁾ The twice-higher translocation is expected for the smaller 5-nm Au NP compared with the 22-nm Au NP used in this study. However, to compare these results (directly after inhalation and intratracheal instillation) much better, more time points—especially 1 hr after inhalation—need to be performed in future studies.

The influence of SP-D

No significant effect of SP-D on Au NP clearance and translocation was detected—besides the trend of decreased translocation combined with a slightly higher increased cellular binding. This may be due to several reasons.

One possibility is that the instilled SP-D just increased the physiologic pool, which does not have any additional influence compared with the already residing SP-D. Hence, one could speculate that the effect of SP-D on NP in the lungs is already “saturated” in healthy animals. Even if we cannot completely exclude this, we do not believe that this is the case, because it is known that the effect of SP-D is strongly dose-dependent.^(12,14) However, this has to be investigated further, which could be done, for example, by using knockout mice.

A second explanation could be that the early time point of measurement in this proof-of-concept study was too short to detect SP-D modulating effects. We do not expect that the in vivo effect of additional instilled SP-D is very prominent. Hence, later time points would be much more suitable to detect these slight SP-D–modulated influences. This will be considered in a further study.

A third very reasonable explanation is directly linked to the mechanism by which SP-D modulates the clearance of particulates. It is known that SP-D induces agglomeration of various particulates like allergen particles,⁽¹³⁾ E. coli,⁽²⁸⁾ A. fumigatus conidia,⁽²⁹⁾ S. pneumoniae, S. aureus,⁽³⁰⁾ and even Au NP.⁽¹⁴⁾ By this agglomeration, more particles are taken up by macrophages. Importantly, in our present study, SP-D was simply not able to agglomerate the deposited Au NP after inhalation. To become agglomerated, a single Au NP has to come into close contact with another single NP. This can be excluded in our study. By calculation of the total projected area of the deposited Au NP (Table 1) and due to a rather homogeneous Au NP deposition on the entire lung epithelium (due to diffusion), the Au NP coverage of the lung surface is so sparse (<10^–12) with huge free spaces in between that no NP can coagulate—even if SP-D binds to the surface of the NP. Hence, this estimation, as well as our results, is consistent with the assumption that the Au NP modulation by SP-D is only dependent on agglomeration and not on receptor-specific modulation. However, as it is known that SP-D binding to other particulates (e.g., P. aeruginosa) does not induce agglomeration, but enhances phagocytosis by alveolar macrophages,⁽³¹⁾ it has to be proven if this effect is really dependent on the specific type of particulate material or not.

Although there was a trend of decreased translocation from the lungs of SP-D–treated mice to blood, the results are not significant; this interesting tendency needs to be investigated in more detail, for example, by investigating later time points. A more detailed investigation could evaluate whether SP-D mediates binding to resident lung cells like epithelial cells in vivo.