Inflammatory Changes in Lung Tissues Associated with Altered

Sep 12, 2016 - Department of Chemical & Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore 117585, Singapor...
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Article pubs.acs.org/journal/abseba

Inflammatory Changes in Lung Tissues Associated with Altered Inflammation-Related MicroRNA Expression after Intravenous Administration of Gold Nanoparticles in Vivo Cheng-Teng Ng,† Jia’En Jasmine Li,†,‡ Suresh Kumar Balasubramanian,§ Fang You,‡ Lin-Yue Lanry Yung,*,‡ and Boon-Huat Bay*,† †

Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore Department of Chemical & Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore 117585, Singapore § Department of Civil and Environmental Engineering, National University of Singapore, Singapore 117576, Singapore ‡

S Supporting Information *

ABSTRACT: Potential adverse effects of gold nanoparticles (AuNPs) are gaining attention due to their wide industrial, consumer, and biomedical applications. This may give rise to possible health risks from direct exposure to the NPs. Excessive inflammatory response is known to be one of the main effects induced by NPs. In this study, inflammatory and miRNA expression changes in lung tissues were evaluated in rats following intravenous administration of AuNPs. AuNPs (20 nm) at a mass concentration of 256 μg/mL were intravenously injected into 6−8 week old male Wistar rats at single doses of 0.025, 0.05, 0.1, and 0.2 mg/kg and sacrificed at 1 week, 1 month, and 2 months, respectively. The biodistribution of AuNPs in the lungs of the rats was determined by inductively coupled plasma mass spectrometry. There were no apparent changes observed in the body weight of the experimental rats. Histopathological examination revealed the presence of infiltrating lymphocytes in lung interstitial tissues and enhanced IL-1α immunostaining in the lung tissues. Out of 84 rat microRNAs (miRNAs) analyzed, the expression of three miRNAs in rat lungs were dysregulated by more than 2-fold in the 0.1 and 0.2 mg/kg AuNP-treated rats 1 week after exposure. In particular, miR-327 was significantly down-regulated in both groups of treated rats. Taken together, it would seem that miRNAs may regulate inflammatory changes in the lungs after exposure to AuNPs in vivo. KEYWORDS: nanotoxicology, gold nanoparticles, inflammation, miRNAs

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should be comparable in the different experimental settings,9 especially in preclinical studies that assess the toxicity of NPs using animal models.10 Hence, the dose for AuNPs used in this study was rationalized on the basis of published data from a phase I clinical trial.11 NPs have been reported to trigger inflammation and oxidative stress in biological systems.12,13 Furthermore, miR-155, which has been reported previously to be up-regulated in AuNP-treated MRC-5 lung fibroblasts in vitro,14 is well-known for its roles in modulating inflammation.15,16 miRNAs belong to noncoding RNAs which are endogenously expressed as short and single stranded nucleotides.14,17 They are known to suppress expression of their target genes by acting at the posttranscriptional level. Altered miRNAs have been reported to play potential roles in inflammation induced by silica dust.18 However, there is still a lack of information with regard to the

INTRODUCTION

AuNPs are promising agents for therapeutic applications such as drug delivery.1,2 Hence, there is an increase in propensity for human exposure to AuNPs via intravenous (IV) injection in the clinical setting. As AuNPs are directly administered into the bloodstream as drug carriers, the dose applied should be carefully monitored. Entrapment of NPs in the lungs is common if administration of the NPs is mediated via the IV route,3,4 thereby posing a high risk of possible toxicity to the lungs. Thus, it is important to assess potential adverse pulmonary effects of AuNPs in vivo. However, the extent of toxicity brought about by the use of AuNPs is difficult to determine due to inherent differences in experimental settings and parameters used by different laboratories.5,6 Administration of a physiologically relevant dose for both in vitro and in vivo studies has been challenging. There has been a lack of consensus on the dose metrics especially for in vitro studies.7 Relatively high doses applied in in vitro studies have raised questions on the relevance of the findings in the in vivo environment.8 It has been advocated that the dose administered © XXXX American Chemical Society

Received: June 24, 2016 Accepted: September 12, 2016

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DOI: 10.1021/acsbiomaterials.6b00358 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

2 mL portion of concentrated HNO3 (69%) and 2 mL of HCl (30%) were added to the QI using a glass pipet, before adding 1 mL of H2O2 solution. A 0.5 mL portion of H2O2 solution was also added into the Teflon vessel. The QI was put into the Teflon vessel using plastic tweezers, and placed inside the microwave vessel for digestion of the tissues to take place, before the vessel was taken out and cooled. The clear sample solution inside the QI was diluted to 20 mL with ultrapure water, transferred into a cleaned plastic vial, and kept at ∼4 °C before quantifying with ICP-MS. The tissue concentration of Au is given as nanogram per gram of rat lung tissue (ng/g lung tissue). Enzyme-Linked Immunosorbent (ELISA) Assay. Plasma transforming growth factor beta (TGF-β) and interleukin 6 (IL-6) were used as indicators of inflammation. Plasma TGF-β and IL-6 concentration were analyzed in the control and AuNP groups (6 groups) using rat TGF-β and IL-6 platinum ELISA commercial kits (eBioscience), following the instructions included in the kit. Each well was washed once with wash buffer before adding in the sample (pretreated with 10 μL of HCI and NaOH) in duplicates. Samples were incubated for 2 h, on a slow shaker at room temperature. After that, wells were washed five times with washing buffer before the addition of Biotin-Conjugate. Samples were incubated for another hour, washed again, and finally incubated with streptavidin-HRP for another hour. After being washed, samples were added with TMB substrate solution and incubated for 30 min. The optical density was read at 620 nm to determine the absorbance of the samples tested. Western Blot. Citrate-treated rat blood was centrifuged at 3000 rpm for 15 min to obtain plasma. The pellet was discarded, and protein quantification was done using a Bio-Rad Protein Assay (Bio-Rad). Primary rabbit antirat IL-1α antibody (Santa Cruz) and primary rabbit antirat albumin (Aviva) (as internal loading control) were used. Serum protein was collected and denatured with loading dye at 95 °C. The protein sample was separated by electrophoresis (SDS-PAGE) using 10% sodium dodecyl sulfate−polyacrylamide gel. Subsequently, proteins were transferred onto a PVDF membrane (Bio-Rad). Blocking was carried out with 5% milk for an hour. Following overnight incubation of the primary antibodies at 4 °C, the secondary antibody-HRP conjugate (Amersham Biosciences, NJ) was then added. The bands of the proteins of interest were visualized at their respective protein sizes using the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). Band intensity was quantified by a GS-710 (Biorad) densitometer. Histopathological Analysis. Lung tissues harvested for light microscopic examination were fixed in 10% formalin. Tissues were trimmed, immersed in fresh formalin, dehydrated in alcohol, and cleared in clearene and finally in wax, using the ATP 700 Tissue Processor. Tissues were then embedded in paraffin, sectioned at a thickness of 5 μm, and stained with H&E. Briefly, paraffin sections were deparaffinized and rehydrated with a descending percentage of ethanol. Sections were stained with hematoxylin followed by eosin. Samples were dehydrated, air-dried, and mounted using Permount solution before examination. miRNA Isolation from Lung Tissue for miRNA PCR Array. Total miRNA was isolated from rat lung tissues belonging to the water-only control and AuNP groups, using TRIZOL reagent (Invitrogen) and the Qiagen miRNA extraction kit, according to the manufacturer’s instructions. Lung tissues were first homogenized before mixing with chloroform. Centrifugation was performed to extract the aqueous phase, followed by addition of 100% ethanol. Samples were washed, and total mRNA together with small RNA were eluted with RNase free water. The Inflammatory Response and Autoimmunity miRNA PCR Array (SA Bioscience), comprising 84 miRNAs that are known to regulate the expression of proinflammatory or anti-inflammatory genes (prediction), was used. A set of controls present on this array were used as the calibrator, and data analysis was done using the ΔΔCT method of relative quantification. The average value of the control group was assigned as the calibrator, against which all other samples are expressed as a fold difference. Data analysis of the miRNA PCR array was performed on the basis of the ΔΔCt method, with normalization of the raw data to housekeeping genes using the Qiagen online software. P < 0.05 was considered statistically significant.

modulation of miRNA expression after exposure to AuNPs and their downstream effects on inflammatory response. Hence, in this present study, we evaluated inflammatory changes in rat lung tissues by histopathological analysis and IL-1α cytokine expression by immunohistochemistry, as well as inflammationrelated miRNA expression induced by AuNP exposure in vivo.

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MATERIALS AND METHODS

Synthesis and Characterization of AuNPs. AuNPs used in this study were synthesized using Turkevich’s method, through the reduction of Au-containing tetrachloroauric acid (HAuCl4), by trisodium citrate dehydrate as previously described.13,14 The colloidal AuNP suspension was then washed twice with ultrapure water by centrifugation at 9000 rpm for 20 min. AuNPs were washed and concentrated as previously reported.19 A drop of AuNP solution was mounted on Formvar-coated copper grids and viewed using a transmission electron microscope (TEM). In addition, dynamic light scattering (DLS) and zeta potential measurements (Zetasizer Nano ZS, U.K.) were performed to assess the hydrodynamic size and surface charge of AuNPs in solution. For the animal study, washed AuNPs were further diluted with ultrapure water, resulting in a final mass concentration of 256 μg/mL, based on measurements obtained from inductively coupled plasma mass spectrometry (ICP-MS). The concentrated and purified AuNPs were then used for subsequent administration into rats. AuNP Treatment in Rats. Male Wistar-Kyoto rats aged 6−8 weeks (approximately 250 g body weight), purchased from the Centre for Animal Resources (Singapore), were housed at the Comparative Medicine Vivarium, National University of Singapore (NUS), Singapore. Rats underwent a 1-week acclimatization period before the start of the study. The animals were housed in disposable cages with free access to water and food under controlled temperature, humidity, and lighting (12−12 h light−dark cycle). All procedures were approved by the NUS Institutional Animal Care and Use Committee (IACUC) (Protocol number 118/11). Rats were anesthetized by inhalation of 5% isoflurane during AuNP administration. AuNPs were injected intravenously into the tail vein of Wistar rats. The doses administered are expressed as milligrams of AuNPs per kilogram of rat body weight (mg/ kg). The rats were assigned randomly into 7 groups (n = 6 per group per type of NP for different doses and time points), in which single doses were administered at four different concentrations (0.025, 0.05, 0.1, 0.2 mg/kg) and sacrificed after 1 week. Another two groups of mice were administered as single doses of AuNPs at 0.1 mg/kg body weight and sacrificed 1 month and 2 months postexposure, respectively. The seventh (control) group, which was injected with a single dose of 0.2 mL ultrapure water, was included as an internal control. The Institutional Animal Care and Use Committee consented to only one time point (at 2 months) for the control group. Individual body weight was monitored and recorded 3 times a week. The rats were euthanized at the stated time points mentioned above. Tissues were removed, and blood was collected from euthanized rats via cardiac puncture. Briefly, a 21 gauge needle was inserted directly into the thoracic cavity under the xiphoid cartilage, and ∼5 mL of blood was drawn into a tube containing trisodium citrate as an anticoagulant. The blood was then centrifuged (3000 rpm for 5 min), and plasma was stored at −80 °C for further analysis. Part of the lung tissues collected was fixed in 10% formalin followed by paraffin embedding. The rest of the lung tissues were snap frozen using liquid nitrogen before storage at −80 °C. Quantification of Au in Rat Tissues. The biodistribution of AuNPs was investigated quantitatively by ICP-MS, at various time points after different doses of injection. To perform microwave digestion, quartz inserts (QIs), glassware, and Teflon vessels were cleaned with 1% nitric acid (HNO3), followed by rinsing the glassware with ultrapure water (3−4 times). Lung tissue specimens weighing between 100 and 120 mg were put into each vial. A 200 μL portion of Cd standard solution with a concentration of 10 ppm in the final solutions was used as an internal standard and was added to the sample inside the QI before putting on the glass cap. A 1.5 mL portion of ultrapure water was added into a Teflon vessel, followed by 1 mL of water into the QI. A B

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Figure 1. Characterization of AuNPs by (A) representative TEM micrograph of as-synthesized AuNPs showing AuNPs of 20 nm size. (B) DLS showed a hydrodynamic size of 20.15172 nm for AuNPs used. (C) Zeta potential of AuNPs were −28.9 mV.

Figure 2. Biodistribution of AuNPs in the lung. (A) Lung deposition of AuNPs measured with ICP-MS. In comparison with control rats where Au was below the detection limit, the 20 nm AuNPs showed similar levels of accumulation in the lung of all treatment groups. (B) Body weight of 7 groups of male Wistar rats. The body weights were taken every alternate day for all experimental groups and control groups.

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Target Genes Prediction. Given that miRNAs perform its biological function by regulating the target protein-coding genes, the predicted targets of miRNAs were analyzed in this study. The prediction was done using miRBase algorithms, TargetScan Release 6.2 (http:// www.targetscan.org), to predict microRNA gene targets. Immunohistochemical (IHC) Staining for IL-1α. IHC staining for rat lung tissue sections was carried out by the automated Leica BOND-MAX System (Wetzlar, Hesse, Germany). Primary rabbit antiIL-1α (Acris, Germany) (1:200) was used. After the lung sections were deparaffinized and rehydrated, epitope retrieval at pH 6 was performed for 20 min. Visualization of the IL-1α immunostaining was by the avidin−biotin-complex technique, followed by the addition of 3,3diaminobenzidene (DAB). Subsequently, the brown stained sections were counterstained with Shandon’s Haematoxylin, mounted onto a glass slide with Permount mounting medium, and air-dried, before visualization under a microscope. Immunofluorescence (IF). Antigen retrieval and blocking of endogenous peroxidation were first performed. Next, the lung sections were incubated with primary rabbit anti-CD3 (Abcam, United Kingdom) (1:200) for 2 h, before incubation with secondary goat antirabbit FITC conjugated antibody (1:200). Sections were then washed, and nuclei were counterstained with DAPI (1:1000) for 5 min, mounted with a cover slide using Fluorescent Mounting Medium (Dako, Denmark), and kept in the dark at 4 °C. Immunofluorescence images were taken with an Olympus Fluoview FV1000 confocal laser scanning microscope. Statistical Analysis. The results were expressed as mean ± standard error (SE). The differences among control, AuNPs, and water-only group were analyzed using one-way ANOVA, followed by a posthoc Tukey test (GraphPad Software, CA). P < 0.05 was considered statistically significant.

RESULTS Characterization and Dosimetry of AuNPs. AuNPs were observed as spherical, electron dense particles with an average diameter of 20 nm by TEM (Figure 1A), while the washed AuNPs exhibited a hydrodynamic diameter of 20.15 nm as measured by DLS (Figure 1B). The zeta potential for AuNPs was −28.5 ± 0.21 mV (Figure 1C) (due to citrate-ion capping). ICPMS analysis of the AuNP stock solution gave a concentration of 256 μg/mL; thus, 0.1 mg/kg of the formulation would correspond to ≈20 μg of Au, on the basis that a single IV injection was performed into a 200 g rat (assuming even AuNP distribution inside rat body). In this study, we evaluated AuNPinduced changes in lung tissues using a dose that ranged from 0.025 μg to 0.2 μg/g (same as 0.025 mg to 0.2 mg/kg rat body weight) which is equivalent to the dose used in a reported phase I clinical trial,11 so as to achieve a physiologically relevant dose. The highest dose in this present study was 2 times the maximum tolerated dose used in the clinical trial, and the doses used which range from 0.01 to 2700 μg/g are considered as on the lower side of doses that have been reported in the published literature.5 Biodistribution of AuNPs in the Lungs. The lung was selected as the target organ for further investigation as this organ is known to be a target site for AuNP retention through the systemic blood circulation after IV injection.9 Au was detected at levels exceeding those of the control rats, and consistently found in the lungs for all treatment group (Figure 2A). The highest and lowest average concentrations were 100.8 ng/g lung tissue and 28.02 ng/g lung tissue, respectively, in the AuNP-injected rats, as compared with controls. However, it was observed that the level of Au in the lung showed a decrease with time (from 64.61 ng/g lung tissue at post-1 week to 28.02 ng/g lung tissue at post-2 C

DOI: 10.1021/acsbiomaterials.6b00358 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 3. Anti- and proinflammatory cytokine expression in rat plasma. (A) TGF-β and (B) IL-6 in rat plasma. There was a significant change in inflammatory cytokines, especially for rats exposed to AuNP for 1 month and 2 months. Error bar = SEM; *p < 0.05; ***p < 0.001.

Figure 4. (A) Western blot analysis of rat serum from 0.1 mg/kg/week, 0.2 mg/kg/week, and control rats. IL-1α expression in serum was found to increase significantly in AuNP treatment groups compared with control rats. Bottom panel represents albumin as loading control. (B) Bar chart showing optical densitometry values of the IL-1 α bands with albumin as the normalizer. Results represent mean values ± SEM from three biological replicate blots.

compared to controls (Figure 3A). However, at a low dose of AuNPs (0.05 mg/kg; pe 1 week), there was no significant inflammation observed. Proinflammatory cytokine IL-6 expression was also significantly higher in 0.1 mg/kg AuNPtreated rats and persisted up to 2 months postexposure (Figure 3B). Expression of serum IL-1α, protein as analyzed by Western blot, was also observed to be significantly higher in the AuNPtreated group (Figure 4). Effects of AuNP Exposure on Inflammation in Lung Tissues. Histopathological examination revealed signs of focal inflammation with the presence of aggregates of lymphocytes in the rat lung tissues, 1 week after the single IV injection of 0.1 and 0.2 mg/kg AuNPs (Figure 5). The control rats showed no pathological changes in the lung tissues. IF staining for CD3, a lymphocytes marker, showed the presence of positively stained CD3 cells in the lung sections (Figure 6), thereby verifying the influx of lymphocytes in the lung tissues. Inflammatory and Autoimmune Response-Related miRNA Expression in the Lungs of AuNP-Exposed Rat. As expected, the miRNA expression profile was found to be significantly altered in rats which were injected with single doses of 0.1 and 0.2 mg/kg of AuNPs and sacrificed after 1 week as compared with control rats. Out of 84 screened miRNAs, three significantly dysregulated miRNAs in rat lung tissues were miR-

months) with the same dose of 0.1 mg of AuNPs/kg body weight, suggesting clearance of AuNPs from the respiratory organ. As expected, the animals administered with ultrapure water showed negligible Au accumulation. Furthermore, no traces of Au were detected in the blood of all AuNP-injected rats following injection (Supporting Information Figure 1), implying that the Au detected in the lung tissues was present in the parenchyma and not in the blood vessels. Gross Observations of AuNP-Treated Rats. The body weight of the rats was monitored and weighed before necropsy. Exposure of AuNPs did not lead to stress-related clinical signs of toxicity and mortality. Rats behaved normally with no loss of appetite. At the dosage used, AuNP-injected rats did not display any significant gross effects or apparent dysfunctions. The mean body weight in AuNP-injected rats was not statistically different from that of control rats (Figure 2B). Serum Cytokine Levels. Serum levels of inflammatory cytokines, viz., transforming growth factor beta (TGF-β) and interleukin 6 (IL-6), were investigated. Rats that were administered single injections of 0.1 and 0.2 mg/kg of AuNPs showed mild systemic inflammation after 1 week, 1 month, and 2 months postexposure. A significant difference was observed in serum TGF-β expression in single dosed 0.1 mg/kg AuNPtreated rats postexposed for 1 week, 1 month, and 2 months, as D

DOI: 10.1021/acsbiomaterials.6b00358 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 5. Light micrographs of lung tissues of AuNP-treated and control rats. Presence of lymphocytic infiltration in the lungs of both AuNP-treated groups. High-power magnification (on the right) of the boxed area showing lymphocytes in lung tissues of treated rats. Scale bar: 200 μm (left), 100 μm (right).

treated rats as shown above (Figure 4), was also identified as one of the predicted targets of miR-327. To verify the association of miR-327 with IL-1α, we performed IL-1α immunohistochemical staining in the rat lung tissues. IL1α immunostaining was observed in the bronchioles of the lung tissues from the 0.1 and 0.2 mg/kg groups (Figure 7).

140-5p, miR-29b-3p, and miR-327. Compared with the control group, qRT-PCR results identified significant dysregulation of miR-140-5p and miR-327 in 0.1 mg/kg/week rats, and miR-29b3p and miR-327 in 0.2 mg/kg/week rats (Table 1). miR-327 Target Prediction. miR-327, the only miRNA that was observed to be down-regulated in both treatment groups, appears to be linked with the inflammatory response. Next, using TargetScan v6.2, a list of target genes predicted on the basis of seed region match of miR-327 was compiled (Supporting Information Table 1). There were 119 genes predicted to be targets for miR-327, including thioredoxin reductase 1, calnexin, synapsin I, and nicotinamide phosphoribosyltransferase. Interestingly, IL-1α, which was increased in the serum of AuNP-

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DISCUSSION This present study evaluated exposure of different doses of AuNPs on inflammatory changes, particularly in the lung. IV injection was selected as the route of exposure to mimic the clinical setting. Concurrent with other reports,20,21 we have demonstrated here that AuNPs could reach secondary target E

DOI: 10.1021/acsbiomaterials.6b00358 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 6. CD3 immunofluorescence staining in rat lung tissues. Positive staining for CD3, a lymphocyte marker, is depicted by green fluorescence in the lung tissues of AuNP-treated rats (middle panel). Control rat lung sections showed absence of CD3 staining. Scale bar: 20 μm.

injection,20,23,27,28 making the lung a relevant model to study pulmonary toxicity. Acute exposures were conducted at four different single doses (0.025, 0.05, 0.1, and 0.2 mg/kg), and rats were sacrificed after 1 week postexposure. The rats did not suffer from significant weight loss as compared to the control group at 1 week postAuNP-exposure. However, there was the presence of systemic inflammation as evidenced by raised levels of serum TGF-β, IL-6, and IL-1α. In a comparison with the unexposed rats, infiltration of lymphocytes was apparent in the lung tissues of single dose 0.1 and 0.2 mg/kg treatment groups, suggesting pulmonary inflammation. Rattanapinyopituk et al. had previously reported that translocation of AuNPs to lung tissues caused acute inflammation, accompanied by multifocal infiltration of neutrophils, destruction of alveolar wall, increased cytokine (IL-6 and TNF-α), and oxidative stress.28 Exposure to other types of NMs has also been reported to cause inflammation in vivo. For example, exposure of TiO2 has been reported to induce

organs via systemic blood circulation in healthy rats. The levels of Au detected in the lungs are consistent with an earlier study by Balasubramanian et al.,22 although the dose used in the present study was 10 times higher. However, as the amount of Au present in the residual amount of blood in the lungs was found to be negligible, no correction factor was adopted in the present study.23 The IV administered AuNPs which had translocated to the lungs were observed to be retained by the lung tissues after 1 week, 1 month, and 2 month postexposure, with the higher dose showing a faster clearance and decreasing at 2 month postexposure. Several possible clearance pathways have been proposed for inhaled AuNPs, including mucociliary clearance and pulmonary surfactant protein D modulated clearance,21,24,25 alveolar elimination by macrophages in the alveolar region (which does not apply to large agglomerates),26 and hepatobiliary clearance from the liver.23 Moreover, several other studies have demonstrated the presence of AuNPs in the lung after IV F

DOI: 10.1021/acsbiomaterials.6b00358 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Table 1. Dysregulated miRNAs in Rat Lung Tissues

miRNAs

fold regulation in 0.1 mg/kg; pe 1 week

rno-let-7a-5p rno-let-7b-5p rno-let-7c-5p rno-let-7d-5p rno-let-7e-5p rno-let-7f-5p rno-let-7i-5p rno-miR-101a-3p rno-miR-101b-3p rno-miR-106b-5p rno-miR-125a-5p rno-miR-125b-5p rno-miR-128-3p rno-miR-136-5p rno-miR-140-5p rno-miR-141-3p rno-miR-142-3p rno-miR-144-3p rno-miR-145-5p rno-miR-148b-3p rno-miR-152-3p rno-miR-15b-5p rno-miR-16-5p rno-miR-17-5p rno-miR-181a-5p rno-miR-181b-5p rno-miR-181c-5p rno-miR-181d-5p rno-miR-182 rno-miR-183-5p rno-miR-186-5p rno-miR-195-5p rno-miR-19a-3p rno-miR-19b-3p rno-miR-200a-3p rno-miR-200c-3p rno-miR-203a-3p rno-miR-205 rno-miR-20a-5p rno-miR-20b-5p rno-miR-21-5p rno-miR-221-3p rno-miR-222-3p

−1.5987 1.1375 −1.0537 1.321 −1.0912 −1.0766 −1.7808 1.8065 2.148 1.0698 1.1507 −1.286 1.8421 −10.6111 2.3299 2.5632 1.6901 1.7774 1.0514 1.0083 1.1783 1.2942 −1.2472 1.0733 5.4806 2.8706 2.4091 3.2303 1.2914 3.9643 2.2634 −1.2782 −1.1392 1.9278 −1.0134 1.5166 −1.2557 1.2821 1.4012 1.4612 1.0393 1.2042 1.4622

p-value

fold regulation in 0.2 mg/kg; pe 1 week

p-value

0.377392 0.748189 0.746078 0.409699 0.669534 0.84545 0.423329 0.725296 0.656941 0.74723 0.879843 0.389319 0.928627 0.138094 0.040413 0.152477 0.260848 0.26639 0.435742 0.525642 0.419153 0.703103 0.527782 0.885538 0.315947 0.183425 0.353894 0.272705 0.808115 0.240197 0.172856 0.436874 0.347601 0.922583 0.385322 0.82188 0.557842 0.745461 0.594993 0.304098 0.859986 0.800468 0.822264

−1.518 −1.4408 −1.1065 1.2435 −1.4926 −1.0693 −1.3422 −1.1982 1.0318 −1.2157 −1.1681 −1.2464 2.0044 −4.7738 1.4865 −1.0025 2.1199 −3.3497 −1.2831 −1.6989 −1.1074 1.426 −1.7009 −1.2616 2.9008 1.9726 1.4664 1.3028 −1.1651 2.2358 −1.3532 −1.8332 −1.2906 1.596 −1.887 1.1599 −1.3006 −1.5056 1.5676 −1.0181 −1.0002 −1.5474 1.401

0.28784 0.303721 0.409145 0.532552 0.283137 0.92495 0.320175 0.356135 0.439434 0.277403 0.170959 0.194409 0.355946 0.090587 0.217244 0.851235 0.118308 0.081302 0.30982 0.14627 0.940519 0.698452 0.118086 0.239769 0.888551 0.514787 0.936107 0.830934 0.40767 0.530359 0.577474 0.144182 0.241939 0.849849 0.211852 0.414088 0.365274 0.169321 0.352609 0.782896 0.997611 0.074098 0.853995

miRNAs

fold regulation in 0.1 mg/kg; pe 1 week

rno-miR-23a-3p rno-miR-23b-3p rno-miR-26a-5p rno-miR-26b-5p rno-miR-27a-3p rno-miR-27b-3p rno-miR-291a-3p rno-miR-29a-3p rno-miR-29b-3p rno-miR-29c-3p rno-miR-30a-5p rno-miR-30b-5p rno-miR-30c-5p rno-miR-30d-5p rno-miR-30e-5p rno-miR-320-3p rno-miR-322-5p rno-miR-323-3p rno-miR-325-3p rno-miR-327 rno-miR-34a-5p rno-miR-34c-5p rno-miR-351-5p rno-miR-369-3p rno-miR-374-5p rno-miR-381-3p rno-miR-384-5p rno-miR-410-3p rno-miR-429 rno-miR-448-3p rno-miR-449a-5p rno-miR-495 rno-miR-497-5p rno-miR-539-5p rno-miR-664-3p rno-miR-673−5p rno-miR-743b-3p rno-miR-878 rno-miR-9a-5p rno-miR-93-5p rno-miR-98-5p cel-miR-39-3p cel-miR-39-3p

1.0371 −1.1086 1.197 −5.5197 1.8343 −1.0017 −12.3147 −1.7434 −2.3051 −1.9427 −1.0166 1.1494 1.135 −1.1318 −1.3488 1.2408 −1.4606 −11.2582 −9.9159 −5.881 3.0584 1.3411 −1.5468 −11.9486 −1.3261 −5.7765 −6.0376 −7.5761 −1.6002 −11.9854 3.3143 −8.7793 −1.1652 −11.7053 −1.4581 −8.4445 −13.3007 −11.5563 −2.0776 1.5653 3.8687 −18.4395 −20.9224

p-value

fold regulation in 0.2 mg/kg; pe 1 week

p-value

0.938106 0.987966 0.487675 0.856543 0.159152 0.932404 0.084167 0.11738 0.191506 0.180824 0.702256 0.567398 0.592135 0.547435 0.258921 0.531505 0.441302 0.289463 0.1702 0.02188 0.272134 0.370652 0.301245 0.174489 0.82026 0.15977 0.178463 0.10439 0.240994 0.174491 0.144003 0.179737 0.618521 0.174876 0.281782 0.223204 0.233447 0.166953 0.632612 0.744229 0.142509 0.184699 0.173605

−1.5491 −1.2251 −1.0976 −1.024 1.0787 −1.1636 −7.5864 −1.8018 -4.5094 −2.6236 −1.7904 −1.7255 −1.9289 −1.8733 −2.1174 −1.6922 −1.4653 −6.8182 −6.9777 −8.4062 1.8963 1.8404 −1.0805 −8.9264 −1.6879 −11.5601 −3.323 −8.7947 −1.9909 −11.7942 1.6855 −5.6918 −1.9703 −9.4508 −1.5736 −6.9911 −7.5757 −9.6476 −1.3245 1.308 1.7355 −28.3843 −13.4429

0.162065 0.572609 0.731339 0.935448 0.908668 0.441435 0.068704 0.750108 0.022434 0.068012 0.139807 0.090116 0.097851 0.217862 0.082282 0.343749 0.487905 0.202823 0.110941 0.013541 0.937354 0.646683 0.325644 0.115413 0.43963 0.073183 0.123838 0.083526 0.067821 0.095204 0.276309 0.115526 0.193731 0.113622 0.338469 0.165498 0.115296 0.103136 0.509295 0.908042 0.23848 0.112443 0.207856

Figure 7. Immunohistochemical staining for IL-1α in rat lung tissues. Positive staining IL-1α is indicated by brown cytoplasmic staining in the bronchioles of AuNP-treated rats. Scale bar: 25 μm.

G

DOI: 10.1021/acsbiomaterials.6b00358 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

ACS Biomaterials Science & Engineering

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pulmonary airway irritation and inflammation in mice,29 while with single-wall carbon nanotubes (SWCNTs), inflammatory gene expression changes were observed after post-intratracheal instillation in rats.30 The same observation was also made when nickel oxide NPs were intratracheally instilled in rats, causing inflammation and changes in pulmonary cytokine and chemokine expression.31 We adopted the quantitative real-time PCR-based profiling approach using the inflammation and autoimmunity miScript miRNA PCR array to perform a detailed analysis of miRNA expression changes in response to different doses. The results from such profiling analysis could be potentially useful for identifying biomarkers to detect physiopathological conditions32 and for a better understanding of the underlying mechanism associated with inflammation. In our current study, miR-327 was down-regulated following AuNP treatment. Involvement of miRNAs in response to NP exposure has been reported in some studies. For example, miR-183 and let-7a were found to be altered in the lungs and livers of mice treated with PEG-coated AuNPs.33 Recently, Chew et al.32 performed a blood miRNA profiling in AuNP-exposed rats via IV injection and found miR298 up-regulation which is an important regulator for Alzheimer’s disease. miR-26a has been implicated in inflammatory lung diseases in rats exposed to cigarette smoke,34 while miR-181b was reported to be suppressed in the silicosis rat model.18 These studies highlighted the importance of miRNAs in the inflammatory response. It has previously been shown that miRNA induced after exposure to a wide repertoire of inflammatory mediators could interact with their cognate downstream target mRNA via either a canonical or noncanonical manner.35 In this study, decreased miR-327 was repressed resulting in AuNP-induced inflammation, and IL-1α was identified as a potential target of this microRNA. Interestingly, there was an associated increase in the immunohistochemical expression of the IL-1α protein in the AuNP-treated lung tissues. IL-1 α, a proinflammatory cytokine, is known to play a role in resolving infections through the stimulation of immune responses via recruitment of inflammatory cells and production of enzymes.36

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.-Y.L.Y.). *E-mail: [email protected] (B.-H.B.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Mr. Deny Hartono for synthesizing AuNPs used in this study. This work was supported by research funding from the National Research Foundation (NRF), Prime Minister’s Office, Singapore, under its Campus for Research Excellence and Technological Enterprise (CREATE) programme.

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REFERENCES

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CONCLUSION The present study has demonstrated that AuNPs may accumulate in the lungs of rats with administered IV AuNPs. The treated rats showed evidence of inflammation that could be associated with epigenetic alterations. To our knowledge, this is the first report showing that miR-327 is differentially regulated in AuNP-treated rats. Our results indicate that altered miR-327 may be implicated in lung inflammation and may provide some biological insights on AuNP-induced lung pathogenesis. Altered miRNA expression and inflammatory cytokines are potential biomarkers that would be useful tools for the early detection of nanotoxicity.

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.6b00358. List of predicted targets for miR-327, and Au concentration following administration of AuNPs with platinum (Pt) as control in rat blood (PDF) H

DOI: 10.1021/acsbiomaterials.6b00358 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acsbiomaterials.6b00358 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX