Prior Lung Inflammation Impacts on Body Distribution of Gold Nanoparticles

Introduction. Gold- (Au-) based nanomaterials have shown promising potential in nanomedicine. The individual health status is an important determinant of the response to injury/exposure. It is, therefore, critical to evaluate exposure to Au-nanomaterials with varied preexisting health status. Objective. The goal of this research was to determine the extent of extrapulmonary translocation from healthy and inflamed lungs after pulmonary exposure to AuNPs. Male BALB/c mice received a single dose of 0.8 mg · kg−1 AuNPs (40 nm) by oropharyngeal aspiration 24 hours after priming with LPS (0.4 mg · kg−1) through the same route. Metal contents were analyzed in different organs by inductively coupled plasma-mass spectrometry (ICP-MS). Results. Oropharyngeal aspiration resulted in high metal concentrations in lungs (P < 0.001); however, these were much lower after pretreatment with LPS (P < 0.05). Significantly higher concentrations of Au were detected in heart and thymus of healthy animals, whereas higher concentrations of Au NPs were observed in spleen in LPS-primed animals. Conclusions. The distribution of AuNPs from lungs to secondary target organs depends upon the health status, indicating that targeting of distinct secondary organs in nanomedicine needs to be considered carefully under health and inflammatory conditions.


Introduction
Nanotechnologies have shown promising potentials in multiple sectors of everyday life. ese advances span from material science to consumer products. More recently, various nanomaterials have proved themselves as excellent candidates for nanomedical applications. ese range from diagnostics to drug and gene delivery applications. Gold (Au) is one of the major nanomaterials engineered for utilizations in medicine and electronics. e carrier properties of Au NPs make them promising candidates for delivering biological molecules into the cells thus making them an ideal platform for drug and gene delivery [1][2][3][4]. Au-based therapeutic strategies (hyperthermal therapy) mainly imply their role as heat-mediating objects (due to their strong light absorbing properties) to destroy particle-loaded cells/tissues [5,6]. e absorbed light energy is dissipated into the particle surroundings leading to elevated temperatures in their vicinity. is hyperthermal property can further be used as therapeutic strategy to open drug carriers (polymer microcapsules) [7]. Moreover, Au NPs have shown promises as labelling/contrast agents (transmission electron microscopy/X-rays) and sensing agents. Another advantage of these materials is the possibility of utilizing previously mentioned properties at the same time (hyperthermal and photoacoustic imaging) to have combined schemes for the better evaluation of the biological phenomenon. Keeping in view the broad spectrum of in vivo applications of Au NPs, the potential deleterious effects of these NPs might become an issue which needs to be evaluated with care. Presently, there is a need to evaluate the potentials deleterious effects of Au NPs in both in vitro and in vivo conditions as a discrepancy exists in the literature about the cytotoxic effects of Au NPs on different types of cells [8,9].
It has been shown that some NPs can cross physiological barriers, reach to secondary target organs, and may lead to unexpected outcomes [10,11]. ere exists a discrepancy in the existing literature about the extrapulmonary translocation of NPs/ultra�ne particles [10][11][12]; however, the NP size dependence of translocation is an accepted fact [13,14]. Preexisting respiratory disorders (e.g., in�ammation) may modify the effects of NPs on the respiratory tract and can in�uence the amount of translocated material. Under normal conditions, lungs are oen primed with endotoxins from the inhaled air [15]. We hypothesized that preexisting in�ammation may in�uence the ability of Au NPs to pass through the pulmonary barrier and other organs in the body. To verify this hypothesis, we tested Au (40 nm) NPs in LPStreated mice as an airway in�ammation model.

Animals.
Male BALB/c mice (approximately 25 g, 6 weeks old) were obtained from Harlan (e Netherlands). e mice were housed in a conventional animal house with 12 h dark/light cycles. ey received lightly acidi�ed water and pelleted food (Trouw Nutrition, Gent, Belgium) ad libitum. All experimental procedures were approved by the Local Ethical Committee for Animal Experiments (Katholieke Universiteit Leuven, Leuvin, Belgium).

Au NP Characterization
. ese particles were thoroughly characterized for their physicochemical characteristics including morphology, zeta potential ( , size distribution, and hydrodynamic diameters.

Transmission Electron Microscopy (TEM).
Microscope measurements were performed using a Philips CM30 TEM (Philips FEI, Eindhoven, e Netherlands) operating at 300 kV. Small volumes of sample were deposited on copper mesh grids and covered with carbon coating �lms. e samples were then dried under an N 2 atmosphere in a glove box.

Dynamic Light Scattering.
Dynamic light scattering (DLS) measurements were performed with a Brookhaven 90 Plus Nanoparticle Size Distribution Analyzer (scattering angle 90 ∘ , wavelength 659 nm, power 15 mW; Brookhaven Instruments Ltd, Redditch, UK). Correlation functions were analyzed using the Clementine package (maximum entropy method) for Igor Pro 6.02A (WaveMetrics, Portland, OR, USA). is resulted in intensity-weighted distribution functions versus decay times. By converting the decay times with instrument parameters and physical parameters to hydrodynamic diameters, an intensity-weighted size distribution is obtained. A log-normal �t was applied to each population, resulting in the intensity-weighted average hydrodynamic diameter of the population. Mass-and number-weighted distributions were estimated using the Rayleigh scattering approximation and a correction factor for the form factor of spherical particles.

Potential
Measurements. potential measurements were performed on the same NP solutions as used for DLS. Au and potential were measured with a Brookhaven 90Plus/ZetaPlus instrument applying electrophoretic light scattering. A primary and reference beam (659 nm, 35 mW) modulated optics and a dip-in electrode system were used. e frequency shi of scattered light (relative to the reference beam) from a charged particle moving in an electric �eld is related to the electrophoretic mobility of the particle. e Smoluchowski limit was used to calculate the potential from the electrophoretic mobility.

LPS Treatment. LPS (Escherichia coli O55: B5, Sigma
Aldrich) was suspended in HBSS and administered 10 g/ mouse (0.4 mg/kg). e dose of LPS was based on information available in the literature; the amount given induces only local (pulmonary) in�ammation [16].

Experimental Design.
On day, 1 animals were exposed to 10 g (10 L) of LPS or HBSS (vehicle for LPS) by oropharyngeal aspiration (Figure 1). On day 2, animals were administered 40 L of 0.4 mg/mL (0.8 mg/kg) NP suspensions or vehicle through same route. On day 3 (24 hours later), the animals were sacri�ced, blood was collected through retro-orbital plexus, and the organs were removed. Organs (brain, thymus, lung, heart, liver, spleen, kidney, and testis) Organ collection for ICP-MS Body and organ wts LPS/HBSS NPs/vehicle F 1: Experimental design. Animals ( ) were given 10 L of LPS or its vehicle (HBSS) on day 1 by oropharyngeal route. On day 2, animals were administered 40 L of NP suspension or vehicle for NPs (2.5 mM trisodium citrate) by the same route. Twenty four hours later, animals were weighed and sacri�ced, organs were collected, wet organ weights were measured, and samples were processed for ICP-MS analysis.
were weighed to obtain wet weight. Each experimental group comprised of 4-5 animals.

Metal Content Analysis.
Aer weighing, the organs were placed in glass tubes and digested using 2 mL pure 60% nitric acid (Sigma-Aldrich). e tubes were placed in a water bath at 80 ∘ C until all the tissues were solubilized. e samples were then analyzed for metal contents. e analytical determination of Au in samples was performed by inductively coupled plasma-mass spectrometry (ICP-MS) using Agilent 7500cx. Samples were diluted 100 times with a basic diluents (butanol 2%, EDTA 0.05%, NH OH 1%, and triton 0.05%) containing internal standards. e quanti�cation of Au was performed using the unique 197Au isotope and 193Ir as an internal standard in the "nogas mode" (standard mode).

Statistical
Analysis. Data are presented as mean ± SD where -animals per group. All groups were tested for normality using the Kolmogorov-Smirnov normality test. Since our data were normally distributed, we applied an analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons using Graphpad (Graphpad Prism 4.01, Graphpad Soware Inc., San Diego, USA). A level of (two tailed) was considered signi�cant.

Au NP Characteristics.
Au NP suspension optical spectroscopy analysis of Au NP revealed a single Plasmon peak around 520 nm. TEM analysis revealed spherical morphology of Au NPs (Figure 2(a)), and size distribution analysis indicated a single peak of Au NPs with 40 nm hydrodynamic diameter (Figure 2(b)). e potential measurements in 2.5 mM sodium citrate solution (vehicle for animal exposure) indicated that Au NPs had −73 mV showing that electrostatic repulsions are important factor in stabilising the suspensions. is further con�rmed the stabilising effect of citrate solution as these NPs showed lower negative potentials in water (data were not shown).

Animal Study.
Only lung relative weights differed signi�cantly between saline and LPS-treated groups (data were not shown) due to in�ammation and oedema caused by LPS in the lungs. Signi�cant increases in metal concentration (relative to background values in an untreated group) in the lungs of the animals were detected by ICP-MS ( Figure 3). Aer priming with LPS, lower amounts were detected in the lungs of primed animals as compared to those of nonprimed animals ( Figure 3). In nonprimed animals, higher amounts of Au were detected in heart and thymus. However, a signi�cant increase aer LPS priming was observed in spleen (Figure 3 inset).

Discussion
is study was designed to assess the extrapulmonary translocation of Au NPs in a pulmonary in�ammation model. Quantitative data indicated that 81% ± 1 % of the aspirated dose remains in the lungs of the healthy animals, with % ± % and 6% ± 2% being found in heart and thymus, respectively. However, in LPS primed animals, only 2 % ± 8% was detected in lungs, with 7% ± % detected in spleen and % ± 1% detected in thymus. Interestingly, we found that in LPS-primed animals, the amount of Au which reach spleen is 5-6 fold higher than found in healthy animals. We demonstrate here that even in healthy animals, Au NPs reach secondary target organs aer lung exposures. Moreover, striking differences in the target organs (spleen versus heart) between LPS exposed and unexposed animals is of particular interest. A graphical overview of the �ndings is presented in Figure 4. Recently, several reports have emphasized the potential applications of Au NPs in nanomedicine, but the possibilities of such secondary effects have not been illustrated. e mechanisms of translocation through the airblood barrier remains unclear: epithelial uptake may occur and cause damage to epithelial cells; electron microscopic study demonstrated UFPs passage via the cles between the alveolar epithelial cells in healthy conditions. As to the mechanisms of damages of the alveolar wall by LPS, it is suggested that macrophages and neutrophils activated by LPS release free radicals resulting in the degeneration of the air-blood barrier. Translocation of NPs from the air-blood barrier to the capillary lumen may take place through the degenerated structures with acute in�ammatory condition [15,17].
Is has also been observed that NPs potentiate an in�ammatory response in subjects with lung in�ammation. In view of the impact on (innate and adaptive) immunity, NPs in�uence cell populations, such as macrophages�monocytes, neutrophils, dendritic cells, natural killer cells, and lymphocyte [18][19][20]. e preexposure stimulation of the immune system with LPS, resulting in signi�cant changes in the spleen, probably lays on the basis of the different distribution in in�ammatory conditions [21].
An important fact is that these data are obtained using realistic NP doses (doses which do not induce in�ammation in the lungs of healthy animals) contrary to the literature reports which used huge amounts of NPs to observe systemic translocation. Previously, it was demonstrated that the majority of the administered Au NPs are retained within the healthy lung and only small portion reaches systemic circulation aer inhalation exposure [22].   We have recently reported that in the experiments done in parallel with the same dose of Au NPs given through same route did not induced in�ammation (neither an increase in bronchoalveolar lavage �uid cellularity nor cytokines) [23]. e reason for the increase in Au in the spleen aer LPS exposure is not clear. LPS exposure provokes an in�ammatory response, leading to in�ux of in�ammatory cells in the lungs resulting in an increase in phagocytosis of deposited material by macrophages. ese macrophages might play a central role in the transport and clearance of the Au NPs, thus explaining the lower amounts material aer LPS exposure [24]. Moreover, we have recently showed increased numbers of Au laden macrophages in bronchoalveolar lavage �uids of �asthmatic� animals in a mouse model of diisocyanate-induced asthma [23]. It has been demonstrated that macrophages (in particular Kupffer cells) play the most important role in the clearance of intravenously injected Au NPs [25]. We are currently evaluating these mechanisms in detail, but we consider it timely and important to share our preliminary observations with the scienti�c community. However, in another study, it was shown that 15-day inhalation chamber exposure to Au NPs results in accumulation of signi�cant amounts of Au in spleen along with many parts of digestive and cardiovascular system in rats [26]. Recent studies demonstrated the presence of gold nanoparticles and nanorods in spleen aer intravenous exposures [27]. e differential interaction with lung lining �uid (in case of inhalation exposure) and blood proteins (in case of intravenous exposure) were postulated to be the reasons for differential body distributions of Au NPs in inhalation versus intravenous exposures [27]. We show here that Au NPs can reach spleen even in case of lung exposure. It could be speculated that there might exist the possibilities of immunomodulatory/immunotoxic effects of Au NPs. Moreover, it is noteworthy that it has been already shown that Au reduces the antigen presentation and autoimmune reactions in rheumatoid arthritis [28]. However, in depth, mechanistic studies are needed to elucidate whether Au NPs exposure that can also result in similar outcome is unknown.
e shortcomings of the present study did not include directly measuring in�ammatory response in the lungs (relying on the literature data/previous experience with the LPS dose to induce in�ammation) and housing animals in metabolic cages to comprehensively estimate the clearance of NPs from the body and to account for the dose lost from the LPS-primed lungs. Moreover, the role of NP physicochemical characteristics, surface modi�cation/functionalization, and protein corona need in-depth evaluations. Recently, It has been shown that protein corona can signi�cantly modify the responses to biomedical nanoparticles and its complementary factor cell vision should also be considered [29].

Conclusion and Perspectives
In conclusion, our results con�rm the hypothesis of particle translocation from the lungs to secondary organs like spleen, heart, and thymus. e observation that NPs target different organs depending on the health status of the animal warrants further studies to understand the mechanisms involved in this process and possible consequences. ere is an urgent need of both in vivo and in vitro mechanistic studies to better understand the possible differences in interactions of Au NPs with different organ systems in the body. A particular focus must be made to understand the unexpected outcomes of Au NP exposures in animal models of different diseases. Moreover, mechanistic studies are warranted to understand the interaction of Au NPs with immune system.

Executive Summary
(i) Au NPs can cross the air-blood barrier in both healthy and in�amed lungs.
(ii) Preexisting in�ammation alters the body distribution pattern of Au NP.
(iii) Distinct organ targeting in case of in�amed lungs indicates the need to evaluate the consequences of NP administration in diseases status.

Ethical Approval
e authors declare that appropriate institutional review board approval was obtained prior to animal experiments.

Con�ict of �nterests
e authors declare that they have no con�ict of interests.