Specifically Formed Corona on Silica Nanoparticles Enhances

Specifically Formed Corona on Silica Nanoparticles Enhances Transforming Growth Factor β1 Activity in Triggering Lung Fibrosis Zhenzhen Wang,†,# Chunming Wang,*,‡,# Shang Liu,† Wei He,† Lintao Wang,† JingJing Gan,† Zhen Huang,† Zhenheng Wang,† Haoyang Wei,† Junfeng Zhang,*,†,§ and Lei Dong*,† †

State Key Laboratory of Pharmaceutical Biotechnology, NJU Advanced Institute for Life Sciences (NAILS), School of Life Sciences, Nanjing University, 163 Xianlin Avenue, Nanjing 210093, China ‡ State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau SAR, China § Jiangsu Provincial Laboratory for Nano-Technology, Nanjing University, Nanjing 210093, China S Supporting Information *

ABSTRACT: A corona is a layer of macromolecules formed on a nanoparticle surface in vivo. It can substantially change the biological identity of nanomaterials and possibly trigger adverse responses from the body tissues. Dissecting the role of the corona in the development of a particular disease may provide profound insights for understanding toxicity of nanomaterials in general. In our present study, we explored the capability of different silica nanoparticles (SiNPs) to induce silicosis in the mouse lung and analyzed the composition of coronas formed on these particles. We found that SiNPs of certain size and surface chemistry could specifically recruit transforming growth factor β1 (TGF-β1) into their corona, which subsequently induces the development of lung fibrosis. Once embedded into the corona on SiNPs, TGF-β1 was remarkably more stable than in its free form, and its fibrosis-triggering activity was significantly prolonged. Our study meaningfully demonstrates that a specific corona component on a certain nanoparticle could initiate a particular pathogenic process in a clinically relevant disease model. Our findings may shed light on the understanding of molecular mechanisms of human health risks correlated with exposure to small-scale substances. KEYWORDS: corona, silica nanoparticles, nano−bio interface, lung fibrosis, TGF-β1

T

corona, as demonstrated in our recent study, can be utilized as a feasible strategy for drug delivery for therapy of liver fibrosis by targeting into hepatic stellate cells.12 Nevertheless, the corona on nanoparticles is also likely to cause adverse responses from the tissue,6,13 because it unavoidably contains proteins that are main regulators of pathological events, such as certain cytokines. We postulate that when they are incorporated into corona, these proteins are actually being concentrated at the interface between nanomaterials and the living tissue, which may accelerate their actions in inducing unfavorable response.14 Finding such evidence will enable us to understand the health risks of nanomaterials at a different level. However, little is known about the involvement

he biomolecular corona can dictate the behavior of nanoparticles in vivo.1 The corona is a complex mixture of biomolecules that rapidly covers the surface of nanoparticles when the latter enter the body.2 Its components, mainly proteins from the blood or tissue fluids, orchestrate an important interface and exert their own inherent activities on the biological system.3,4 In doing so, the corona endows nanoparticles with different “biological identities” in vivo, which are difficult to predict in vitro.5 Specifically, the corona can facilitate unexpected interactions between nanoparticles and the circulation system, including hemolysis, thrombocyte activation, nanoparticle uptake, or triggering of endothelial cell death.6−10 Such interactions further pose unexpected influence on the in vivo application of nanoparticles. For example, the corona could disable binding between transferrin as ligands on a nanocarrier and corresponding cell receptors, and thereby weaken the targeting capability of the carrier.11 Meanwhile, recruiting retinol-binding protein from the blood into a drug carrier’s © 2017 American Chemical Society

Received: November 6, 2016 Accepted: January 13, 2017 Published: January 13, 2017 1659

DOI: 10.1021/acsnano.6b07461 ACS Nano 2017, 11, 1659−1672

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Figure 1. Capabilities of SiNPs to induce murine silicosis and analysis of corona formed on the particles. (a) Representative photographs of Masson’s trichrome staining for the lung tissue from animals administrated with saline or SiNPs of different sizes (scale bar 50 μm). (b) Histological score of Masson’s trichrome staining sections in each group. (c) The hydroxyproline content in lungs in each group (n = 7 mice). (d) Total protein amount absorbed on SiNPs or CNPs. (e) Venn diagram illustrating the number of proteins identified in the corona of SiNP100 or CNP-100. (f) Hierarchical cluster analysis of the proteins according to independent ontology. (g) Heat maps showing corona composition clustering analysis performed on summarizing reported fibrosis-relevant proteins. The collective proteins and corona composition are shown in lines and columns, respectively. The green and yellow squares indicate the corresponding protein-terms detectable or undetectable in LC-MS profile, respectively. The detectable protein-term was noted on the right. Results are representatives of three independent results and shown as mean ± SD, *p < 0.05 after ANOVA with Dunnett’s tests.

pathological basis remains unclear. Importantly, silicosis is a typical disease induced by particulate matter,18−22 and this matter, specifically, silica particles, is significantly capable of absorbing multiple types of proteins onto its surface.23 We hypothesized that the silica dust entering the lung induces specific formation of a corona in which the fibrogenic components further trigger the development of lung fibrosis. To validate this, we prepared a series of silica nanoparticles (SiNP) with varying sizes and surface chemistry and administered them into murine lungs to assess their capabilities

of corona in any nanomaterial-induced tissue damage. There is a demand for a proper model that allows for analyzing corona formation under a pathological circumstance and represents a disease closely correlated with tissue exposure to nanoscale materials.15,16 Silicosis, an occupational lung disease, which caused by inhalation of silica dusts, may be remarkably suitable for finding clues of corona-triggered pathogenesis.17 This disease causes massive, irreversible lung fibrosis that eventually leads to the loss of lung function. Despite over a century’s research, its 1660


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ACS Nano Table 1. Top 20 Most-Abundant Corona Proteins on Different Nanoparticlesa

a

Proteins highlighted in the darker blue are in both indicated nanoparticles. TGF-β1 is outlined in a red grid.

to induce tissue fibrosis. Then, we collected the coronas formed on different particles, designed and employed a combination of biochemical and proteomics assays to analyze their composition, and further examined their fibrogenic potentials in both in vitro and in vivo models. Eventually, we meaningfully discovered that the corona on SiNPs relatively specifically recruits transforming growth factor-β 1 (TGF-β1) than other less or nonfibrogenic nanoparticles and effectively protects it from a deactivating mechanism, which could be the main reason for the induced lung fibrosis.

ments, and Brunauer−Emmett−Teller (BET) analysis (Supplementary Table S1 and Supplementary Figure S1). Then, we generated a silicosis model by infusing SiNPs into murine lungs. These SiNPs had passed endotoxin test before use (Supplementary Figure S2) and were given at different doses (1−200 mg/kg). We compared SiNPs of different sizes (SiNP-10, -100, and -1000 with Ø ≈ 10, 100, and 1000 nm, respectively) and found that SiNP-100 was more potent in inducing lung fibrosis than the other particles in all dose batches (Figure 1a−c and Supplementary Figure S3). We therefore focused on SiNP-100 throughout the study. Next, we incubated SiNPs in lung tissue homogenate supernatant (LH) for 2 h and confirmed the formation of a corona with protein separation and protein analysis. Particularly, we employed label-free liquid chromatography mass spectrometry (LC-MS) to analyze the protein profile of coronas from SiNPs of different sizes (SiNP-10, -100, and -1000) and carbon nanoparticles (CNP-100), in parallel with that of the LH (raw data in Supplementary Tables S2− S6). Here, CNPs served as control because they do not induce fibrosis.24 There was no significant difference in the abundance and components (664 vs 458 vs 590) between the corona proteins from SiNPs of different sizes (Supplementary Figure S4A,B). Also, these samples were similar in terms of multiple biological and chemical properties, such as their molecular functions and the types of biological processes in which they

RESULTS AND DISCUSSION The Composition Analysis of Corona Formed on SiNPs. The primary hypothesis of this study is that the biomolecular corona formed on silica nanoparticles (SiNP) activates signaling pathways that promote fibrosis. To validate this, we should first observe how specific the corona components on the fibrosis-triggering SiNPs are, in comparison with those on less or nonfibrogenic nanoparticles. First, we prepared SiNPs of various sizes (Ø ≈ 10, 30, 100, 200, and 1000 nm) and nanoparticles made of other materials, including CaCO3, carbon nanoparticles (CNP), Au, iron−cobalt−nickel alloy (FeCoNi), and Fe3O4. These nanomaterials were thoroughly characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), ζ potential measure1661


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Figure 2. SiNP-100 specifically recruits TGF-β1 to their corona. (a) Representative confocal microscopic photographs showing the colocalization of F-SiNPs and TGF-β1 on the membrane in vivo (scale bar 100 μm). (b) Representative fluorescent photographs of A549 cells treated with F-SiNPs and TGF-β1; triangles indicate significant colocalization of SiNPs and TGF-β1 on the membrane (scale bar 15 μm). (c) Western blotting analysis of TGF-β1 in corona of SiNPs of different sizes. (d) Western blotting analysis of TGF-β1 in corona on different nanoparticles. (e) Western blotting analysis of TGF-β1 at indicated time points to identify the time evolution of SiNP-bound LH proteins. (f) Quantitative Western blotting of TGF-β1 harvested from SiNP corona formed in LH, with reference to recombinant TGF-β1 protein as the standard control. (g) Western blotting analysis of TGF-β1 in the protein corona on differently modified SiNPs. (h) Representative images of Masson’s staining for lung sections from animals treated with PBS, SiNP-100, N-SiNPs, or P-SiNPs (scale bar 50 μm). (i) The hydroxyproline content in lungs of mice administrated as in panel h (n = 7 mice). Images are representative for three independent experiments. Results are shown as mean ± SD *p < 0.05 after ANOVA with Dunnett’s tests.

addition, different proteins from the tissue environment exhibit their specific kinetics, for example, association and dissociation rates, when interacting with the two surfaces, which further decide the longevity of their interactions and collectively the different profiles of the corona components. Furthermore, proteins related to fibrosis were much more abundant on SiNPs than on CNPs (Figure 1g). This result confirms that the corona composition on SiNPs differs from that on CNPs and contains more fibrogenesis-relevant proteins. Specific Recruitment of TGF-β1 on SiNPs. Next, we examined which exact protein(s) in the SiNP corona mediated fibrosis. With the help of the KEGG Pathway software,30,31 we sketched the fibrosis-relevant signaling pathways and proteins that could be orchestrated by SiNP or CNP corona and found that the corona of SiNPs and CNPs could mediate 7 and 6 signaling pathways, respectively, and corresponding enriched proteins in each pathway were significantly different (Supplementary Table S7 and S8). Notably, the TGF-β pathway was exclusively enriched by the SiNP corona proteome, and the

are involved (Supplementary Figure S4C,D). Nevertheless, proteins in the SiNP fraction were detected to have higher abundance (Figure 1d) and more complex proteome (458 vs 129) (Figure 1e) than those of CNP corona, and the proteome composition varied greatly between the two fractions (Figure 1f and Supplementary Figure S5). The different protein profiles on SiNPs and CNPs could be attributed to the different surface chemistry of these nanoparticles. Multiple factors including chemical composition, surface charge, (de)agglomeration, hydrogen bonds, and van der Waals forces may affect the process of absorbance.25,26 Notably, the sp2 carbon structure and inherent hydrophobic nature of CNPs make it difficult for these particles to absorb a high amount of biomolecules in the living system.27 In contrast, the SiNP is more active because silica exists as tetrahedral silicon−oxygen, in which a silicon atom is in the center and each of its four oxygen atoms is shared with a neighboring Si atom. In the tissue fluid, the abundant −OH groups on the SiNP surface with negative charge28,29 help to shape the specific corona formation. In 1662


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Figure 3. TGF-β1 embedded in SiNP corona can be functionally recognized. (a) Schematic diagram of a flow cytometry-based assay for the detection of exposed TGF-β1 in the corona (FCBA). (b) FCBA analysis of SiNPs incubated with different doses of TGF-β1 in the same volume, with SiNPs in PBS as control. Relative florescence median shift of each sample is listed on the left. (c) Relative florescence median shift versus different TGF-β1 dose as shown in panel b. (d) FCBA analysis of the SiNPs incubated in LH. (e, f) FCBA analysis of TGF-β1 in the proteins recovered from coronas on SiNPs of different sizes or modifications. (g) Schematic diagram of coprecipitation of the SiNP/TGFβ1/TβR complex. (h) Coprecipitation analysis of TGF-β1, TβRI, and TβRII to assess the direct binding between cellular TGF-β1 and its receptors after SiNPs were enveloped with TGF-β1 and mixed with A549 cell lysate. SiNPs without TGF-β1 envelope were the control. Results are the representative of three independent experiments.

TGF-β1 detected in the SiNP fraction was within the top 20 most abundant proteins but not in the CNP fraction (Table 1). ELISA tests suggested that the concentrations of TGF-β1 in the homogenate of healthy and fibrosis lung tissues were 342.8 ± 41.23 and 1145 ± 130.8 pg/mL, respectively, indicating that this cytokine is abundantly presented in the lung tissue. The LC-MS analysis further highlighted the different levels of TGFβ1 in different coronas. In terms of abundance, TGF-β1 ranked 11 out of 458 proteins in the corona on SiNP-100, but only ranked 89 out of 590 proteins in the corona on SiNP-1000, which partly explains why the latter was weaker in inducing fibrosis. Meanwhile, despite a considerable level of TGF-β1 in the corona of SiNP-10 (29 out of 664 proteins), the particles in such a small size also showed low silicosis-triggering ability, possibly because their curvature was too high to maintain the

natural structure of the absorbed protein or to expose the epitope properly.32,33 These data suggest that both the abundance and activity of TGF-β1 in corona influence the biological behavior of the nanoparticles in the physiological environment, which adds weight to the existing findings. Given the major role of TGF-β1 in activating fibrosis-related cellular functions such as epithelial−mesenchymal transition (EMT) and the overexpression of collagen,34,35 we singled out TGF-β1 as the molecule that mediates SiNP-induced lung fibrosis and set out to confirm this in the following experiments. We set out to thoroughly assess the interactions between TGF-β1 and SiNPs. We first injected fluorescence-labeled SiNP-100 (F-SiNPs) through the mice trachea into the lungs of normal animals and then detected TGF-β1 in the lung tissue with immunofluorescence staining 6 h after the injection. Clear 1663


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(DMEM) without fetal bovine serum (FBS). As shown in Supplementary Figure S11D, although the different modifications change the dissolution degrees of the SiNPs, such changes did not correlate directly to the pathogenicity of nanoparticles. For example, the N-SiNPs with higher degree of dissolution demonstrated lower silicosis-triggering capacity; while the PSiNPs with lower degree of dissolution proved still less potent in inducing silicosis. Next, we assessed the biopersistence of raw and N- and P-SiNPs after their exposure to animals for 1 month and found no significant difference in the content of silicon detected in the lung between these samples (Supplementary Figure S14). Taken together, in addition to the general importance of physicochemical features for the bioactivity of materials, the influence of absorbed biomolecules from the physiological environment (i.e., corona components) also plays a dominant role in dictating the biological behavior of the nanoparticles. It is essential to take into consideration the significance of such gained activity when analyzing the real biological identities of nanoparticles in the living tissue. Interaction of SiNP Corona-Bound TGF-β1 with Its Cellular Receptors. In order to exert biological functions, corona proteins should properly expose their epitopes to interact with other biomolecules, particularly their receptors.38,39 By developing a method based on flow cytometry (Figure 3a and Supplementary Figure S15), we examined whether a monoclonal antibody that recognizes the epitope of TGF-β1 could recognize SiNP-bound TGF-β1. The results showed that the intensity of the positive fluorescent signal increased dose dependently as the concentration of TGF-β1 rose (Figure 3b,c), which indicated that the particle-bound protein managed to expose its epitope. Using this method, we further confirmed that TGF-β1 could do so in LH as well (Figure 3d). Nevertheless, significantly fewer TGF-β1 were bound to N- or P-SiNPs (Figure 3e) or SiNP-10, -30, or -1000 (Figure 3f). Intriguingly, although Western blotting revealed considerable adsorption of TGF-β1 to SiNP-10 or -30 (Figure 2c), no significant fluorescence shift was detected on these particles by flow cytometry. These contrasting results reflect that TGF-β1 on smaller particles might fail to expose its epitope, which was consistent with our hypothesis that the high curvature of the smaller particle may (i) compromise the protein’s natural structure or (ii) make the epitope of TGF-β1 embedded in the corona and unrecognized.32,33 We continued to test whether SiNP-bound TGF-β1 could be recognized by the cell receptors and exert cellular functions. Theoretically, TGF-β1 must first be recognized by a dimer of type II TGF-β receptor (TβRII). The activated TβRII dimer recruits type I TGF-β receptor (TβRI) dimer and phosphorylates the latter to initiate intracellular signaling cascade.40 By developing an assay in which TβRs were pulled down by TGFβ1 on the SiNPs incubated with cell lysate (Figure 3g), we demonstrated that these TGF-β1 presented on SiNPs could increase the contents of TβRI and -II in the precipitation, indicating effective assembly of the functional TGF-β/TβRs signaling complex in the presence of SiNPs (Figure 3h). The Effect of SiNP Corona-Bound TGF-β1 in Promoting EMT and Triggering Lung Fibrosis. Our further evidence suggested that SiNP-100-corona-bound TGF-β1 exacerbated epithelial−mesenchymal transition (EMT) in lung epithelial cells; EMT is the key step in lung fibrogenesis, and TGF-β1 is a potent mediator.34,35 First, SiNP-100 could promote TGF-β1-induced EMT in A549 cells after incubation with TGF-β1 or LH, whereas a TGF-β1 receptor blocker

colocalization of SiNPs and TGF-β1 confirmed the recruitment of this protein to SiNPs in situ on the cell membrane (Figure 2a). Meanwhile, we preincubated F-SiNPs with TGF-β1, treated A549 cells with these particles, and observed again a solid overlap of F-SiNPs (green) and TGF-β1 on the membrane (red, anti-TGF-β1; Figure 2b). These data confirmed the binding of TGF-β1 to SiNPs both in vivo and in vitro. Furthermore, we incubated SiNPs of varying sizes in parallel with other types (metal or mineral) of less-fibrogenic particles with mouse LH, before separating the corona proteins of each sample and detecting TGF-β1 therein with Western blotting. The results indicated that both size and type of nanoparticles affect their recruitment of TGF-β1. SiNP-100 or -200 recruited much more TGF-β1 than those of other sizes (Figure 2c and Supplementary Figure S6), which is in agreement with their capabilities in triggering silicosis; meanwhile, TGF-β1 was much more abundant on SiO2 than on all other types of particles (Figure 2d and Supplementary Figure S7). Time evolution is a typical process of corona formation, during which the biomolecules with higher binding affinity for a particular particle gradually accumulate into the corona.36 We examined TGF-β1 in SiNP corona incubated in LH for different time courses between 5 min and 2 h. We found that the content of TGF-β1 in the corona increased over the incubation time and peaked at 1 h of incubation (Figure 2e and Supplementary Figure S8). The content of TGF-β1 in corona was further quantified as about 500−1000 ng per milligram of SiNPs, or about 16−32 TGF-β1 molecules on one particle (Figure 2f and Supplementary Figure S9). Additionally, following in vitro incubation, SiNPs could completely retard the migration of TGF-β1 in native-polyacrylamide gel electrophoresis mobility shift assay (Supplementary Figure S10), which further confirmed the binding between them. Taken together, these data suggest that TGF-β1 can be relatively specifically recruited to and stabilized on the SiNPs than other nanoparticles’ surface. We asked how the surface properties of SiNPs can affect these particles in recruiting TGF-β1. We modified SiNP-100 with hydration (H-SiNPs) and amination (N-SiNPs), as well as different polymers including PEI (P-SiNPs), dextran (DSiNPs), and gelatin (G-SiNPs). A series of tests were performed to characterize these particles, including TEM, DLS, ζ-potential analyses, and Fourier transform infrared spectroscopy (FTIR). The outcomes confirmed a dominant dispersion of the nanoparticles as well as successful modification for those differently modified particles (Supplementary Table S9 and Supplementary Figure S11). Interestingly, N- and P-SiNPs completely failed to recruit TGF-β1, while other modified particles preserved this ability well (Figure 2g and Supplementary Figure S12). This finding indicated that a negative surface charge of SiNPs was crucial to facilitate TGFβ1 binding; once it was altered to positive by animation or PEI grafting, much less TGF-β1 would be recruited to the corona. Consequently, N- and P-SiNPs heavily lost the fibrosisinducing capability in murine lungs, in contrast to the unmodified SiNP-100, independent of duration and dose of silica treatment (Figure 2h,I and Supplementary Figure S13). Dissolution and biological persistence of nanoparticles may also influence their biological behavior.21,37 First, we examined the dissolution of SiNPs and the modified particles in two buffering systems, namely, simulated lung interstitial fluid (Gamble’s solution) and Dulbecco’s modified Eagle’s medium 1664


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Figure 4. SiNP/TGF-β1 promotes EMT in A549 cell culture. (a) Representative images of A549 cells treated with PBS, TGF-β1 (5 ng/mL), SiNP-100 (50 μg/mL), or the TGF-β1/SiNP-100 with or without TGF-β1 receptor inhibitor (SB431542) (scale bar 50 μm). (b) Western blotting analysis of the expression of EMT markers, E-cadherin and vimentin, in A549 cells treated as in panel a. (c) Wound healing assay of A549 cells treated as in panel a. (d) The wound gap distance in panel c quantitatively evaluated with the ImageJ program. The relative distance was normalized to the average gap of the A549 cells treated with TGF-β1/SiNP-100, of which the relative gap distance was taken as control (scale bar 100 μm). (e) The EMT-promoting effects of SiNPs of different sizes, as evaluated by the morphological changes imaged by microscope (scale bar 50 μm). (f) The EMT-promoting effects of SiNPs with different modifications, as evaluated by the morphological changes imaged by microscope (scale bar 50 μm). Photographic images are the representatives of three independent results. Data are presented as mean ± SD.

and P-SiNPs (Figure 4f and Supplementary Figure S19), failed to promote EMT or cell migration. Taken together, these data suggest that SiNP-100 effectively promotes cellular EMT via TGF-β signaling. Stabilization and Enhancement of TGF-β Action in SiNP Corona. Perhaps the most striking finding is that SiNPs could enhance the activity of TGF-β1 by inhibiting this protein’s degradation and prolonging its functions. It is known that the TGF-β/TβRs heterotetrameric complex is internalized into the cell while the ligand is degraded.42 Here, we treated the cells with TGF-β1/SiNPs or free TGF-β1 at 4 °C, allowing for ligand−receptor binding while preventing cell internalization. When the receptors were saturated and excessive TGF-β1 and SiNPs removed, the initial amount of TGF-β1 molecules was comparable among different cells. The cell culture was then returned to 37 °C. We observed a continuous reduction of

(SB431542) significantly reduced this effect (Figure 4a and Supplementary Figure S16A). Next, cells treated with SiNP100/TGF-β1 or SiNP-100/LH expressed more vimentin but less E-cadherin; this change is regarded as a typical sign of EMT progression but was effectively abolished by TβR-blocker (Figure 4b, Supplementary Figure S17 and Supplementary Figure S16B,C). Then, we instilled mice with SiNP-100, TGFβ1, and SiNP-100/TGF-β1 to examine the induction of EMT in vivo. Consistently, we observed signs of enhanced EMT in the group treated with the combination of SiNP-100 and TGFβ1 (Supplementary Figure S16 D−F). These results indicate that TGF-β1 or LH adsorbed to SiNP 100 is responsible for more severe EMT. Finally, the enhanced EMT further accelerated the epithelial cell migration in vitro (Figure 4c,d).41 In contrast, SiNPs of other sizes (SiNP-10, -30, and -1000;Figure 4e and Supplementary Figure S18), as well as N1665


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Figure 5. SiNPs enhance and prolong the activity of TGF-β1. (a) Representative confocal microscopic images of TGF-β1 in A549 cells at indicated time points at 37 °C. The cells were preincubated with TGF-β1/SiNPs or free TGF-β1 at 4 °C for 1 h (scale bar 15 μm). (b) Western blotting analysis of TGF-β1 on the membrane of A549 cells treated as in panel a. (c) Western blotting analysis of pSmad2 and total Smad2 of A549 cells treated as in panel a and relative phosphorylation levels calculated by band intensity (right panel). (d) Western blotting analysis of TGF-β1 on the membrane of A549 cells treated with TGF-β1 and SiNPs of different sizes, in the same procedure as in panel a. The samples were cultured at 37 °C for 0.5 h. (e) Representative confocal microscopic images of A549 cells treated with SiNPs of different sizes as in panel d (scale bar 15 μm). Data are representative of three independent experiments, quantified as mean ± SD and statistically analyzed by performing repeated measures ANOVA test with post hoc Bonferroni correction.

which has been widely suspected in the recent years. Further investigations targeting the control of corona formation may inspire promising therapeutic approaches for silicosis and other relevant diseases.

TGF-β1 from the cell membrane (Figure 5a,b) in both free TGF-β1 and TGF-β1/SiNP groups. However, the signal decrease in free TGF-β1 group was much faster than that of TGF-β1/SiNP group, suggesting that SiNPs could prevent TGF-β1 from being internalized into cytosol for degradation. Furthermore, the copresence of SiNPs amplified and prolonged the phosphorylation of Smad2 (Figure 5c), which is downstream of activated TβRI and a central event in fibrogenic pathways.43,44 As expected, both larger (1000 nm) and smaller (10 and 30 nm) nanoparticles, which were demonstrated incapable of recruiting active TGF-β1 into their corona, failed to protect TGF-β1 from degradation (Figure 5d,e) or enhance the phosphorylation of Smad2 (Supplementary Figure S20). During all these treatments, the expression level of TβRI remained constant (Supplementary Figure S21). Taken together, the above data suggested that SiNPs slowed down the normal process of internalization and degradation of their recruited TGF-β1. The particles enhanced and prolonged the activities of TGF-β1 in their corona to induce fibrogenic pathways, as compared with TGF-β1 in free form. This should be the fundamental reason why SiNPs accelerate TGF-β1mediated tissue fibrosis in the lung that eventually develops into silicosis.

MATERIALS AND METHODS Reagents, Cells, and Animals. Dextran, poly(ether imide) (PEI), 3-aminopropyltriethoxysilane (APTES), gelatin, transforming growth factor β1 (TGF-β1) receptor inhibitor SB431542, and the other chemicals were purchased (Sigma-Aldrich, St. Louis, MO, USA). Human lung carcinoma epithelial cells A549 were kindly provided by Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). All cell culture media and bovine serum were obtained from Gibco (Thermo Scientific, MA, USA). Male ICR mice (20 ± 2 g) of the same ground were purchased from Model Animal Research Center of Nanjing University (Nanjing, China). The animals were raised in a ventilated, temperature-controlled room (23 °C) with abundant access to rodent chow and water provided. Animal protocols were reviewed and approved by the Animal Care and Use Committee of the authors’ institutes and conformed to the Guidelines for the Care and Use of Laboratory Animals published by the National Institutes of Health. Nanoparticles. The various nanoparticles used in this study were purchased or synthesized. Fluorescein isothiocyanate (FITC)-labeled silica nanoparticles (F-SiNPs) and silica nanoparticles (SiNPs) including SiNP-10, SiNP-30, SiNP-100, SiNP-200, and SiNP-1000 were obtained from Kisker Biotech (Steinfurt, Germany). Carbon nanoparticles (CNP-100) and nanoparticles made of other materials (CaCO3, Au, iron−cobalt−nickel alloy (FeCoNi), and Fe3O4) were purchased from DK Nano (Beijing, China). SiNP-100 was further modified with hydration (H-SiNPs) or amination (N-SiNPs), as well as with different polymers including PEI (P-SiNPs), dextran (DSiNPs), and gelatin (G-SiNPs) in the following ways: (i) H-SiNPs were obtained by boiling SiNP-100 for 1 h. (ii) N-SiNPs were synthesized according to a published method.45 Briefly, 100 mg of SiNP-100 was suspended in 4 mL of toluene, and 2 mL of APTES was dropped into the suspension. The mixture was stirred at 70 °C for 6 h in a reflux device. Then, the N-SiNP particles were collected, washed

CONCLUSION Our present study demonstrates that SiNPs at the size of 100 nm can specifically recruit and enrich TGF-β1 into their protein corona in the lung. The SiNP corona-bound TGF-β1 not only preserves its biological activities in binding cell receptors and triggering lung fibrosis but also exhibits slower degradation and prolonged activation of the TGF-β/Smad2 pathway, which directly promotes tissue fibrosis. Our findings provide direct evidence that the corona on nanoparticles could induce adverse responses such as pathological changes in the biological system, 1666


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ACS Nano with ethanol, and dried. (iii) P-SiNPs, D-SiNPs, and G-SiNPs were prepared by adding 10 mg of SiNP-100 into 30% PEI or 5 g/mL solution of dextran or gelatin, respectively. The mixtures were stirred for 12 h at room temperature, and then the particles were separated by centrifugation after washing with phosphate buffered saline (PBS). A series of tests were performed to thoroughly characterize the nanoparticles. All the particles were characterized for their ζ potential and particle size using a 90plus Particle Sizer (Brookhaven Instruments, NY, USA). Second, to obtain essential information on the nanoparticles’ size and shape, transmission electron microscopy (TEM) was carried out. After a few drops of deionized water-dispersed nanoparticles were dropped on the 300 mesh carbon-coated copper grid, TEM images of each sample were collected using TEM (JEOL Ltd., Tokyo, JAPAN). In addition, XRD measurements were conducted on a Bruker AXS D8 Advance and D2 Phaser (Karlsruhe, Germany) to study the crystal structures of silica nanoparticles. Fourier transform infrared spectroscopy (FTIR) spectra (Shimadzu Corp., Kyoto, Japan) of the modified SiNPs were obtained (scanning range 4000−400 cm −1 ). To analyze the effect of different modifications on the dissolution of SiNPs, inductively coupled plasma optical emission spectrometry (ICP-OES) analysis was used (Shimadzu, Tokyo, Japan). The suspensions of pristine SiNPs and modified SiNPs were prepared at 10 mg/mL in different solutions, including simulated lung fluid−Gamble’s solution (magnesium chloride 0.095 g/L, sodium chloride 6.019 g/L, potassium phosphate monobasic 0.298 g/L, disodium hydrogen phosphate 0.126 g/L, sodium sulfate 0.063 g/L, calcium chloride dehydrate 0.368 g/L, sodium acetate 0.574 g/L, sodium hydrogen carbonate 2.604 g/L, sodium citrate dehydrate 0.097 g/L) and Dulbecco’s modified Eagle’s medium (DMEM) without fetal bovine serum (FBS) (Life Technologies). All suspensions were incubated at 37 °C in a 5% CO2 atmosphere for 24 h. After centrifugation at 15 000g for 20 min, the Si content of the supernatant of each sample was quantified using an ICP-OES. The biopersistence of nanoparticles is analyzed in a similar way by harvesting the lungs after exposure to SiNPs, N-SiNPs, and P-SiNPs (4 mg per mouse) for 1 month in the animals and employing ICP-OES to analyze the concentration of silicon in the supernatant of each sample after acid digestion. Each sample and the standard were analyzed in triplicate. Each exposure medium in the absence of silica nanoparticles was also detected as a blank reagent. Endotoxin Assay. Three aliquots of the suspension of raw or modified SiNPs were screened for the presence of endotoxin in duplicate, by chromogenic Limulus Amebocyte Lysate Endotoxin Assay Kit, which was commercially available (Genscript, NJ, USA), following the manufacturer’s protocol. Silicosis Model in Mice. Silica-induced lung fibrosis was generated according to a previously published method.46 Animals were randomly divided into eight groups (each group contained at least 7 mice). Mice were anesthetized by intraperitoneal injection of pentobarbital sodium at 45 mg/kg of body weight. After the trachea was exposed by opening the neck skin and blunt dissection, mice received the suspension of silica of different size and modifications (0.02 mg, 0.1 mg, 0.2 mg, 1 mg, 2 mg, 4 mg), TGF-β1 (1 μg), or TGF-β1/SiNP-100 in a total volume of 50 μL of sterile physiological saline by inserting a 7-gauge needle into the trachea transorally. After the site of surgery was sutured and cleaned with penicillin, the mice were allowed to recover until they were sacrificed. As a control, PBS was applied in a similar manner. Before use, silica was boiled in 1 N HCl, washed, dried, suspended in sterile saline, and then sonicated for 10 min. Mice were sacrificed after 20 days (the group of exposure dose of 4 mg) or 56 days post-particle administration. The lungs were collected for subsequent biological and histological examinations. Lung Homogenate Preparation. Lung homogenate (LH) was extracted from the lungs of healthy mice according to institutional bioethics approval. Briefly, the extracted lung tissue samples were homogenized in an equal volume of PBS by a homogenizer (approximately 3 mice/mL of LH) and then centrifuged to remove the debris to obtain LH. Enzyme-Linked Immunosorbent Assay (ELISA) of TGF-β1. The concentration of TGF-β1 in the lung homogenate was measured

by ELISA, according to the manufacturer’s instruction (eBioscience, California, USA). Briefly, the ELISA plate was coated with monoclonal anti-TGF-β1 overnight at 4 °C. After the unbound monoclonal antibody was removed, the standard growth factor in serial dilutions or 100 μL of LH was dropped into the precoated wells for antigen capture for 2 h. Next, each well was washed with buffer (PBS with 0.05% Tween-20) five times and an enzyme-linked anti-TGF-β1 polyclonal antibody was dropped into the wells for 2 h. Then, a substrate solution was added for 15−30 min to allow the color development reaction, following rinses to eliminate unbound secondary antibody. The colorimetric intensity was finally measured at 450 nm (Thermo Scientific). Formation and Separation of the Corona of SiNPs. The protocol was adhered to a published method.47 SiNPs were incubated with LH or TGF-β1 (10 μg/mL) under stirring at 4 °C for indicated time. To be highlighted, the total particle surface area was both theoretically calculated (A = 4πR2) and experimentally estimated by Brunauer−Emmett−Teller (BET) analysis, by which we could control the ratio of this area to the volume of LH or TGF-β1 solution to be constant. This control served to ensure that the protein contents in the coronas of different particles were comparable. Then, the mixture was centrifuged through a 0.7 M sucrose cushion for 20 min at 4 °C at 15 300g, in order to separate the nanoparticle−corona complexes from LH. The pellet was rinsed with PBS (1 mL), and then the pellet solution was further centrifuged through the cushion to reduce the contamination of unbound-protein for 20 min at 4 °C at 15 300g two more times. The proteins in the corona were eluted by adding polyacrylamide gel electrophoresis (PAGE) buffer (62.5 mM TrisHCl, pH 6.8; 2% w/v sodium dodecyl sulfate (SDS), 10% glycerol, 0.01% w/v bromophenol blue) or liquid chromatography mass spectrometry (LC-MS) buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS) to the pellet on the ice for 1 h. After centrifugation (20 min at 15 300g at 4 °C), the supernatant was collected and store at −20 °C. Furthermore, to ensure the complete elution of bound proteins from nanoparticles, the recovered nanoparticles after washing with PBS were detected by SDSPAGE and further Coomassie Brilliant Blue R-250 staining. Proteins in the corona on the recovered nanoparticles should be eluted again until no residual proteins bound on the nanoparticle are detectable in the SDS gel. Protein Concentration Assay. The total concentration of the proteins extracted from the corona was detected using a Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific). Briefly, serial dilutions with a range of 50−500 μg/mL of bovine serum albumin (BSA) standards or the protein sample was mixed with BCA working reagent and incubated at 37 °C for 30 min. Next, the absorbance at 562 nm was recorded (Thermo Scientific). The quantity of the protein concentration could be calculated according to the standard curve of BSA concentration versus the absorbance. SDS-PAGE. According to the standard protocol, the proteins in the corona from the nanoparticles were eluted with equal and adequate PAGE sample buffer containing 1 mM phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich). After heat denaturation at 100 °C for 5 min and centrifugation at 12 000 rpm for 5 min, the same volume of eluted corona proteins was loaded onto a 12% SDS-PAGE gel to separate the proteins. Native PAGE. The binding between TGF-β1 and SiNPs was analyzed by native PAGE. TGF-β1 (50 μg/mL) was incubated with 1 mg of SiNP-100 at 4 °C for 1 h before 20 μL of the mixture was loaded onto a native polyacrylamide gel with low pH (T(%) = 12%, T(%) = [acrylamide + bis-acrylamide/V (mL)] × 100%; C(%) = 3.3%: C(%) = bis/(acr + bis) × 100%; separating buffer was 0.06 M potassium hydroxide, pH 4.3). The loading buffer consisted of 15% glycerol, 0.02% methyl green and 70 mM β-alanine. The running buffer was 0.14 M β-alanine (pH 4.5). Electrophoresis was performed under 70 V on ice for 1 h followed by Coomassie Brilliant Blue R-250 staining. Sample Preparation for LC-MS Analysis. The protocol to analyze protein corona adhered to a method described previously.4,12 Proteins eluted from nanoparticles were quantified using BCA protein 1667


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ACS Nano assay kit; 100 μg of total protein was reduced by adding 1 M DLdithiothreitol (DTT) (Sigma-Aldrich) (60 °C, 1 h), and free cysteines were alkylated with 1 M iodoacetamide (IAA) (Sigma-Aldrich) (room temperature, 10 min in the dark). The alkylated proteins were centrifuged in the 10K ultrafiltration tube (Thermo Scientific), and the proteins were retained in the 10K ultrafiltration tube. The proteins were further washed with 100 mM tetratehylammonium bromide (TEAB) three times at 4 °C for 20 min by centrifugation at 12 000 rpm. Then the protein was digested with 2 μg of porcine sequencing grade trypsin (LC-MS grade, Sigma-Aldrich) overnight at 37 °C. After digestion, the resulted peptides were collected (12 000 rpm, 20 min, 4 °C), desalted with Zeba Spin Desalting Columns (Thermo Scientific), and further enriched on C18 reversed-phase columns (Epoch Life Science, Missouri City, Texas, USA). LC-MS Analysis of Tryptic Digests. The samples were subjected to LC-MS analysis on a Shimadzu UFLC 20ADXR HPLC system inline with an AB Sciex 5600 Triple TOF mass spectrometer (AB SCIEX, Framingham, Massachusetts State, USA). The autosampler temperature was set at 4 °C, and the volume of sample between 1 and 5 μL was injected. LC-MS was performed at 40 °C using Eksigent ChromXP NanoLC 3C18-CLcolumn (3 μm, 75 μm × 150 mm) with gradient system at a flow rate of 300 nL/min, in which the aqueous mobile phase was 98% H2O (LC-MS grade, Fisher Chemical, MA, USA) and 2% acetonitrile (LC-MS grade, Fisher Chemical) with 0.1% formic acid and the organic mobile phase was 0.1% formic acid in 98% acetonitrile. Peptides were eluted from the column with a gradient from 5% to 80% organic mobile phase over 46 min at 300 nL/min, followed by a 5 min rinse of 80% organic mobile phase, and then the column was immediately re-equilibrated at initial conditions (5% organic mobile phase) for 9 min. The mass spectrometer was operated in positive product ion mode. Samples were analyzed in three technical replicates. Database Searches, Hierarchical Cluster Analysis, Heat Map Analysis, and Pathway Analysis. Identification of peptides and proteins from continuum LC-MS data was performed with the ProteinPilot 4.5 software (AB SCIEX). Proteins were analyzed by searching the mouse taxon of the UniProtKB/SwissProt database (release 2011_11). Then with the reference of the measured mass error obtained from processing the raw continuum, the searching parameter values were set by the software. The false discovery rate (FDR) analysis was implemented by using the software PSPEP integrated with the ProteinPilot, and for a valid protein identification, two criteria had to be confirmed: at least one specific high-scoring peptide was detected; a detected protein threshold of 1.3 (unused ProtScore), corresponding to a confidence of 95%, was used to identify the proteins. Proteins from ProteinPilot for the final LC-MS data set were identified at a 1% global FDR and local FDR at the protein level. To elaborate functional annotation about protein class and the subcellular localization of the total proteins in the LC-MS profile of each corona, we used the Gene Ontology (http:// geneontology.org/) and Uniprot bioinformatics database (http:// www.uniprot.org/), respectively. To adequately describe the biological role of the proteins, hierarchical cluster analysis was applied on the proteome retained by each nanoparticle (CNP-100 and SiNP-100). The VENNY online Venn diagram plotter (http://bioinformatics.psb. ugent.be/webtools/Venn/) was used to obtain lists of exclusive and common proteins among SiNP-100 and CNP-100. The proteins were clustered according to gene ontology terms, that is, cellular component, molecular function, biological process, as well as molecular weight, hydrophilicity, and isoelectric point. For further analysis of the potential fibrosis related proteins in LC-MS profiles, we summarized the reported fibrosis-relevant proteins according to proteomics study on fibrosis,48−72 collective protein term of which is 165. Then, we carried on clustering analysis to corona composition performed on summarizing proteins with heat map. The collective proteins are shown in the different lines, and the corona composition is shown in the columns. The corresponding protein-term positively detectable was indicated with the green squares, and yellow was opposite. Heat maps were created using HemI (Heatmap Illustrator, version 1.0) (http://hemi.biocuckoo.org/index.php). The proteome

data was further investigated with KEGG database. Pathways related to lung fibrosis and corresponding proteins with significant enrichment in each fraction were listed. Both “KEGG Pathway analysis” and “GO analysis” were analyzed in the standard enrichment computation method. Western Blotting. According to the standard protocol, different proteins were separated by SDS-PAGE and then transferred onto the poly(vinylidine difluoride) (PVDF) membranes. The membranes were blocked with skim milk and then incubated with primary antibody, TGF-β1, β-tubulin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Abcam, Cambridge, MA), E-cadherin, vimentin, pSmad 2 (Cell Signaling Technology, Beverly, MA, USA), type I TGF-β receptor (TβRI), type II TGF-β receptor (TβRII), or Smad 2 (Santa Cruz, CA, USA), at 4 °C with gentle shaking overnight. After being washed with PBST (PBS with 0. 1% Tween-20) 5 times, the membrane was incubated with horseradish peroxidase-conjugated antirabbit, anti-mouse, or anti-goat IgG (Life Technologies, Grand Island, NY, USA) at room temperature. After rinsing, positive signal was visualized with fluorography using an enhanced chemiluminescence system (Cell Signaling Technology). The band intensity was quantitated using image J software (http://rsb.info.nih.gov/ij/), and the statistical analysis of three independent experiments was performed. Quantitative Western Blotting. Different amounts of TGF-β1 (100 ng, 500 ng, 1000 ng, 2000 ng, and 4000 ng) (PeproTech, Rocky Hill, USA) together with the corona protein samples were separated by SDS-PAGE and analyzed by Western blotting as mentioned above (Method 14). A standard curve of TGF-β1 amount versus the band intensity was generated by comparing the amount of TGF-β1 in each band and the band intensity. The quantity of the corona proteins could be calculated by their band intensity against the standard curve. Absolute Quantification of Protein Absorption Number per Particle (PANPP). According to the result of quantitative Western blotting, we approximately calculated the corresponding amount (in units of mass) of TGF-β1 present in per milligram of SiNP-100. To calculate average absorption numbers of TGF-β1 proteins on individual particles, we calculated the average mass of TGF-β1 on a single nanoparticle (by dividing the amount of nanoparticle-eluted TGF-β1 by the number of particles present in the experimental sample) and divided this value by the total number of detected TGFβ1 protein to obtain the rough protein absorption number per particle (PANPP) (expressed in number per nanoparticle). To be emphasized, we estimated the number of nanoparticles in the experimental sample according to the information about the nanoparticle size, density, and number of particles per milligram provided by the manufacture, and we assumed no aggregates in the nanoparticles. Flow Cytometry Based Particle Analysis (FCBA). To determine if TGF-β1 molecules were exposed on the surface of the SiNP corona, we developed a method based on the flow cytometry assays used in biological studies. As demonstrated in Figure 3A, different coronaenveloped SiNPs were incubated with allophycocyanin (APC) antimouse TGF-β1 antibody (R&D System, Minnesota, USA; 1:20 diluted per mg of nanoparticles in 100 μL volume) for 1 h to ensure the exposed TGF-β1 on the particles to be recognized and bound by the antibody. The particles were then washed 3 times in PBS before they were examined in fluorescence activated cell sorter (FACS) Calibur flow cytometer (BD Biosciences, San Diego, CA, USA). The forwardscatter characteristics (FSC) signal was amplified by E01 voltage and the FL4 voltage was adjusted to appropriate values. Naked SiNPs without protein corona were used as the negative control during the process to eliminate nonspecific absorption of the particle to the antibodies. The positive signal of exposed TGF-β1 on the SiNPs was determined by the relative shift of the medians of the positive fluorescent intensity, which was calculate as [An − A0]/[A0]; where An is the median of the tested corona-enveloped particle’s fluorescence intensity and A0 is the median of the naked SiNP fluorescence intensity. In addition to the fluorescence intensity, the analysis of the FSC provided useful information on the change of particle shape or size.73 With larger FSC median, the corona-enveloped SiNPs tend to have larger size, to some extent, indicating their interaction with 1668


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ACS Nano protein. From the FSC histogram shown in Supplementary Figure S15, we found that the FSC median of SiNPs increased from 154 to 252 after incubation with TGF-β1 protein compared with the control naked particles. Correspondingly, the fluorescent histogram revealed obvious shift in the median fluorescent intensity relative to the control group. In conclusion, the method presented here represents a rapid and reliable tool for the investigation of nanoparticle interactions with proteins. Epithelial−Mesenchymal Transition (EMT) Progress. According to the published methods,74 A549 cells were cultured in DMEM supplemented with 10% (v/v) FBS. For cell association studies, harvested A549 cells were seeded onto 6-well plates and cultured for 24 h at 37 °C to reach 50%−60% confluence. A549 cells were stimulated with PBS, TGF-β1 (5 ng/mL), SiNP-100 (50 μg/mL), TGF-β1/SiNP-100, LH, or LH/SiNP-100 with or without TGF-β1 receptor inhibitor (SB431542) for 48 h. Typical EMT morphological changes including cell−cell contacts and cell shape were assessed by TE2000-U inverted phase-contrast microscope (Nikon, Tokyo, Japan). Wound Scratch Assay. EMT in A549 cells should increase the proliferation and migration of the cell, which could be quantitatively evaluated by the wound scratch assay. According to the published methods,75 A549 cells were seeded on 6-well plates and cultured to 70%−80% confluence. Cell monolayers were scarred with a sterile tip, and the wounds were allowed to heal for 48 h. Migration images were captured under TE2000-U inverted microscope. The distances from the edge to the middle of the scratch were measured using image J software. The relative distance was normalized to the average gap of the A549 cells treated with TGF-β1/SiNP-100, of which the relative gap distance was taken as control. RNA Isolation and Quantitative Real-Time PCR. RNA of A549 cells or lung tissues was extracted by using Trizol reagent (Life Technologies). For mRNA detection, real-time polymerase chain reaction (PCR) was launched in an ABI 7300 Fast real-time PCR system (Applied Biosystems, FosterCity, CA) using the SYBR Prime Script RT-PCR Kit (Takara Bio, Shiga, Japan). Each sample was analyzed in triplicate and repeated for three or four independent assays with β-actin as internal control. Primers of E-cadherin and vimentin used are listed as follows (Invitrogen, Carlsbad, CA, USA): Ecadherin-forward, 5′-GACAGAAACGAGACTGGGTCA-3′; E-cadherin-reverse, 5′-CCGGTGATGCTGTAGAAAACC-3; vimentin-forward, 5′-GATCGATGTGGACGTTTCCAA-3′; vimentin-reverse, 5′GTTGGCAGCCTCAGAGAGGT-3′. TGF-β1 Internalization Assays. According to the published methods,76 A549 cells were seeded on 6-well plates and grown to 50%−60% confluence. After being washed twice with TGF-β1-binding medium (200 mM HEPES-buffered DMEM, pH 7.4, 0.2% BSA), cells were then incubated in binding medium for 30 min at 4 °C to stop most of the energy consuming biological processes including internalization. The cells were then treated with cold binding medium with free TGF-β1 or TGF-β1/SiNPs at 4 °C for 1 h before their medium was replaced by fresh one without TGF-β1 and SiNPs to remove the unbound TGF-β1 to ensure the initial amount of TGF-β1 molecules comparable between the two groups and then immediately transferred to 37 °C for the indicated time periods (0, 0.5, and 1 h) to culture. Membrane Protein Extraction. According to the published methods,77 we separated the membrane proteins to analyze the amount of TGF-β1 on the membrane by Western blotting. Briefly, A549 cells with indicated treatments were collected by centrifugation at 350g and washed twice with KSHM buffer (100 mM potassium acetate, 20 mM HEPES/KOH, pH 7.4, 1 mM magnesium acetate, 85 mM sucrose, phosphatase inhibitor PMSF) after washing with PBS at 4 °C. Following centrifugation at 850g and resuspension in an equivalent volume KSHM buffer, the cells were then quick frozen in liquid nitrogen and thawed in a 37 °C water bath twice. Intact cells and nuclei were removed by centrifugation at 2700g for 10 min, and the resulting supernatant was collected, followed by ultracentrifugation for 30 min at 4 °C and 100 000g. Pellet was redissolved in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, sodium pyrophosphate, β-glycerophosphate, ethylene diamine tetraacetic acid

(EDTA), Na3VO4, leupeptin), incubated on ice for 20 min, and centrifuged for 5 min at 4 °C at 13000 rpm. This supernatant enriched in membrane protein was stored at −20 °C. Coprecipitation Assay. If the TGF-β1 on the surface of SiNPs were bioactive, it would be able to be recognized and combined with its receptor (TβRI and TβRII) on the cell membrane to form a SiNP/ TGF-β1/TβRI/TβRII heterozygous complex, and precipitation of SiNPs would pull down this complex. The coexistence of TGF-β1/ TβRI/TβRII with SiNPs could be determined by Western blotting for the three proteins in the precipitate. A549 cells were collected and washed twice with PBS before resuspension in ice-cold lysis buffer and incubation on ice for 30 min. After that, the cell debris was removed by centrifugation to obtain the cell lysate. To obtain the TGF-β1enveloped SiNPs, 10 mg/mL nanoparticles was incubated with 10 μg/ mL TGF-β1 at 4 °C for 1 h, followed by centrifugation to remove unbound protein. Then, SiNP-100 or SiNP-100/TGF-β1 complex were mixed with the equal cell lysate on a vortex mixer for 30 min. Next, the mixtures were centrifuged at 10 000g to collect the precipitates. Having been washed with PBS for three times, the proteins in precipitates were eluted by SDS-PAGE sample buffer before the examination of the contents of TGF-β1, TβRI, and TβRII by Western blotting. The same amount of SiNPs was used for comparing the contents of TβRI and TβRII. Immunofluorescence Staining. We employed F-SiNPs to determine the location of protein and silica nanoparticles in the lung. The lung tissues were harvested and frozen-sectioned 6 h after intratracheal instillation of F-SiNPs into the mice. The sections were stained with a TGF-β1 antibody at 4 °C overnight. The fluorescent secondary antibody (Life Technology) was incubated at 37 °C for 1 h, followed by the nuclei staining with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich). The sections were imaged by C2 plus confocal microscope (Nikon). In the photographs, the F-SiNPs was shown in green and the TGF-β1 in red fluorescence. Cell membrane was visualized with 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate (Dil) staining (represented in blue) and bright-field observation. Overlap of the green and red fluorescence at the same location would merge into yellow. Meanwhile, internalization assay (Method 21) was applied to the A549 cells cultured on 8-well Millicell EZ slide (Millipore, Darmstadt, Germany), followed by TGF-β1 immunostaining and confocal microscope imaging. Histological Studies. The lung tissue fixed in 2.5% paraformaldehyde (PFA) was embedded in paraffin and cut into sections for the hematoxylin and eosin (H&E) (Abcam) and Masson’s trichrome (Abcam) staining according to the manufacturer’s instructions with slight modification. Stained sections were photographed at 100× magnification under a microscope. Under blindfold conditions with standard light microscopy, the lung tissue was examined and evaluated randomly. The severity of fibrosis was determined based on magnified tissue morphology and following a quantitative grading scale from 0 (normal tissue) to 8 (severe lung fiborsis).78 The detailed grades are as follows: 0, normal lung; 1, partly enlarged and rarefied alveoli with no fibrotic masses; 2, slight fibrotic changes with knot-like formation but without connection with each other; 3, enlarged and contiguous fibrotic walls; 4, 10% or less single fibrotic masses in the lung tissue; 5, confluent fibrotic masses with severely damaged lung structure; 6, about 50% or more fibrotic masses in the microscopic field with mostly destroyed architecture; 7, almost all of fibrous masses with few air bubbles; 8, complete fibrotic masses. The mean score from all fields (magnification ×200, average 30 fields/animal) was generated. Quantitation of Hydroxyproline Content in Lung. The content of hydroxyproline in the lungs was measured using hydroxyproline assay kit (Sigma-Aldrich) to quantify collagen content according to the reported literature79 with modifications. Briefly, the lung parenchyma was mixed with 12 N HCl and hydrolyzed at 100 °C for 5 h. A 100 μL sample of hydrolysate was adjusted with pH 6.0−6.5 citrate−acetate buffer (5% citric acid, 1.2% glacial acetic acid, 7.25% sodium acetate, and 3.4% sodium hydroxide), 100 μL of 0.05 M chloramine-T solution and 3.5 M perchloric acid were added, and the mixture was successively incubated at room temperature about 10 min. Then, the diluted dimethylamine borane (DMAB) reagent was added 1669


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ACS Nano and mixed with each sample. After the mixture was incubated at 60 °C for 15 min, absorbance was measured at 550 nm (Thermo Scientific). The hydroxyproline amount of samples was then determined from the standard curve with the hydroxyproline reagent as a standard. The data are expressed as hydroxyproline (μg)/lung wet weight (g). Statistical Analysis. The results are expressed as mean ± standard deviation (SD). Data were statistically analyzed using Prism software (GraphPad) and assessed for normality or homogeneity of variance with D-test and Levene test. Differences between multiple groups were compared using one-way analysis of variance (ANOVA) with Dunnett’s tests or, if appropriate, repeated measures ANOVA test with post hoc Bonferroni correction. Differences between two groups were evaluated using the unpaired Student’s t test. A value of p < 0.05 was considered significant; ns = not significant.

AUTHOR INFORMATION

ASSOCIATED CONTENT

ACKNOWLEDGMENTS This work was supported by the National Basic Research Program of China (2012CB517603), the National High Technology Research and Development Program of China (2014AA020707), the Program for New Century Excellent Talents in University (NCET-13-0272), the National Natural Science Foundation of China (31271013, 31170751, 31200695, 31400671, 51173076, 91129712, 81102489, and J1103512), Nanjing University State Key Laboratory of Pharmaceutical Biotechnology Open Grant (02ZZYJ-201307), the Graduate Education Innovation Project of Jiangsu Province (KYZZ15_0040), and the Scientific Research Foundation of Graduate School of Nanjing University (2015CL10). C.W. acknowledges the funding support from Macau Science and Technology Fund (FDCT 048/2013/A2) and University of Macau (MYRG2014-00069-ICMS-QRCM). We thank Prof. Xiaoyue Wang (Peking Union Medical College) and Dr. Xin Li (Thermo Fisher, Singapore) for their expert advice on LC-MS data analysis and bioinformatics.

Corresponding Authors

*C. Wang. E-mail: [email protected]. *J. Zhang. E-mail: [email protected]. *L Dong. E-mail: [email protected]. ORCID

Lei Dong: 0000-0002-2013-4191 Author Contributions #

Zhenzhen Wang and Chunming Wang contributed equally.

Notes

The authors declare no competing financial interest.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b07461. Nanoparticle characterization; pathways possibly related to tissue fibrosis; proteins significantly enriched in the corresponding pathways related to tissue fibrosis of each corona (By KEGG analysis); characterization of the modified nanoparticles; characterization of different size and different type nanoparticles; endotoxin content of the SiNPs and modified SiNPs; more severe lung fibrosis induced by SiNP-100 than SiNP-10 and -1000 independent of dose; bioinformatics analysis of corona proteins on the SiNPs of different size; bioinformatics analysis on the molecular weight, hydrophilicity, and isoelectric point of corona proteins on the SiNP-100 and CNP-100; densitometric analysis of relative TGF-β1 absorption to SiNPs of different sizes or different nanoparticles and of TGF-β1 absorption levels at different time points; standard curve for quantitative Western blotting of TGF-β1; migration retardance of TGF-β1 induced by SiNPs; physicochemical property characterization of SiNPs and different modified SiNPs; densitometric analysis of relative TGF-β1 absorption levels on differently modified SiNP-100; impaired fibrosis-inducing capability of N- and P-SiNPs in lungs of mice independent of dose; biopersistence profiles of SiNPs and modified SiNPs; TGF-β1 in the TGF-β1enveloped-SiNPs detected by flow cytometry assays; SiNP-100−TGF-β1/LH corona promoted EMT in vitro and in vivo; effects of different stimulations on the transcriptional levels of E-cadherin and vimentin; effects of the SiNPs of different sizes or the variously modified SiNP-100 on EMT and cell migration; phosphorylation levels of Smad2 of A549 treated with different sizes of SiNPs; and change of total TβRI detected by Western blotting (PDF) List of corona proteins identified by LC-MS for LH (XLS) List of corona proteins identified by LC-MS for SiNP-10 (XLS) List of corona proteins identified by LC-MS for SiNP100 (XLS) List of corona proteins identified by LC-MS for SiNP1000 (XLS) List of corona proteins identified by LC-MS for CNP100 (XLS)

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