Inorganic Nanomaterials as Highly Efficient Inhibitors of Cellular

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Biological and Medical Applications of Materials and Interfaces

Inorganic Nanomaterials as Highly Efficient Inhibitors of Cellular Hepatic Fibrosis Fei Peng, Jie Kai Tee, Magdiel Inggrid Setyawati, Xianguang Ding, Hui Ling Angie Yeo, Yeong Lan Tan, David Tai Leong, and Han Kiat Ho ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10527 • Publication Date (Web): 29 Aug 2018 Downloaded from http://pubs.acs.org on August 30, 2018

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ACS Applied Materials & Interfaces

Inorganic Nanomaterials as Highly Efficient Inhibitors of Cellular Hepatic Fibrosis Fei Peng,†,‡ Jie Kai Tee,†,#,‡ Magdiel Inggrid Setyawati,§ Xianguang Ding,§ Hui Ling Angie Yeo,† Yeong Lan Tan,†,# David Tai Leong,§,* and Han Kiat Ho†,* †

Department of Pharmacy, Faculty of Science, National University of Singapore, 18 Science

Drive 4, Singapore 117543, Singapore §

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, Singapore 117585, Singapore #

NUS Graduate School for Integrative Sciences & Engineering, Centre for Life Sciences, 28

Medical Drive, Singapore 117456, Singapore KEYWORDS: nanoparticles, titanium dioxide, silicon dioxide, hepatic stellate cells, fibrosis

ACS Paragon Plus Environment

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ABSTRACT

Chronic liver dysfunction usually begins with hepatic fibrosis. To date, there are still no effective anti-fibrotic drugs approved for clinical use in humans. In the current work, titanium dioxide (TiO2) nanoparticles (NPs) and silicon dioxide (SiO2) NPs are used as active inhibitors with intrinsic chemico-physico properties to block fibrosis and the associated phenotypes through acting on hepatic stellate cells (HSCs, the liver machinery for depositing scar tissues seen in fibrosis). Using LX-2 cells as the HSC model, internalized nanomaterials are found to suppress classical outcomes of cellular fibrosis, e.g., inhibit the expression of collagen I (Col-I) and alpha smooth muscle actin (α-SMA), initiated by transforming growth factor β (TGF-β)-activated HSCs in both a concentration-dependent and a time-dependent manner. Biochemically, these nanomaterials could also facilitate the proteolytic breakdown of collagen by up-regulation of matrix metalloproteinases (MMPs) and down-regulation of tissue inhibitors of matrix metalloproteinases (TIMPs). Furthermore, through regulating epithelial-mesenchymal transition (EMT) genes (e.g., E-cadherin (E-Cad) and N-cadherin (N-Cad)), the adhesion and migration profiles of TGF-β-activated LX-2 cells treated with nanomaterials were further inhibited, reverting them to a more quiescent state. Thus, the collective evidences pave the new way that nanomaterials can be used as potential therapeutic inhibitors for the treatment of in vivo fibrosis.


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INTRODUCTION Hepatic fibrosis, commonly caused by chronic alcohol consumption, viral hepatitis or metabolic syndrome, is mediated by the activation of HSCs, accompanied with the overproduction and accumulation of abnormal extracellular matrix (ECM, mainly collagen I (Col-I)).1-5 When this process becomes persistent and dysregulated, it can lead to more severe consequences including cirrhosis, end-stage liver failure, or hepatic carcinoma, which account for the high morbidity and mortality in many countries.6,7 Despite remarkable achievements in understanding the molecular mechanisms underlying liver fibrosis, there are still no effective anti-fibrotic drugs approved for clinical use.8,9 Hitherto potential drug targets identified to play critical roles in HSC activation include fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), innervated via an interaction with their corresponding receptor tyrosine kinases (RTKs).10-12 Many small molecule drugs focused on inhibiting these proteins function (e.g., PDGFR inhibitors, VEGF inhibitor, TGF-β inhibitors, etc.) have been developed and evaluated in animal models.8,13-15 For example, tyrosine kinase inhibitors (TKIs), such as Sunitinib, Sorafenib, and Nintedanib (BIBF1120, have been approved for use in pulmonary fibrosis), have been shown to ameliorate fibrosis in cirrohotic rats or liver fibrogenesis mouse model.8,16,17 However, another TKI, Imatinib, has been reported to only reduce early fibrogenesis instead of blocking fibrosis progression in the long term.11 Furthermore, the EGFR inhibitor, Gefitinib, could induce lethal chronic pulmonary fibrosis when they are used to treat non-small-cell lung cancer patients.18 These mixed findings underscored the multi-targeting nature of even supposedly specific TKIs, presenting the reality that the use of such small molecule drugs has to content with the inconsistent anti-fibrotic actions and side effects that pose major obstacles to successful therapeutic application.6,11,18 Therefore,


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developing effective therapies to attenuate fibrosis/cirrhosis through arresting the activation of HSCs remains an unfulfilled goal. In recent years, the emergence of nanotechnology, especially nanomedicine, offers several advantages and provides new insights to revisit the inhibition of hepatic fibrosis.9,19-23 Firstly, the abundance of blood supply and rich resident phagocytic cells in liver allows the physiological accumulation of administered NPs (30-99%).24 The sequestration of NPs by the liver may solve the problem of insufficient drug concentrations in the target sites for treating liver disease. In 2015, Dong and coworkers developed corona retinol binding protein 4 (RBP) coated drug-laden nanocarriers for target liver fibrosis therapy.25 Secondly, several studies have demonstrated that even pure nanomaterials exposure (e.g., silver NPs (Ag NPs), Au NPs, SiO2 NPs, etc.) could effectively inhibit VEGF-induced cellular migration or angiogenesis.26-28 In 2009, Gurunathan et al. demonstrated that Ag NPs could inhibit activation of PI3K/Akt for subsequent blocking of VEGF-induced bovine retinal endothelial cells' migration.26 Mukherjee and coworkers indicated that Au NPs could suppress VEGF-induced angiogenesis in a size-dependent manner.27 More recent work also showed that SiO2 NPs could be utilized as highly effective inhibitor to treat angiogenesis-related blindness induced by VEGF.28 Most importantly, these efforts have utilized pure nanomaterials as inhibitors to suppress the VEGF-induced angiogenesis-related blindness, indicating that some possible intrinsic roles of undecorated nanomaterials may have been overlooked in previous nanocarrier applications. Due to their ultrasmall sizes and charged nature, NPs can behave similarly like nanosized drugs. Although the specificity of particles towards certain proteins should pale in comparison to small molecular drugs that have certain morphological features, this promiscuity of particles arising from their sizes, charges and shapes could also act as broad-based strategy for desired tissue-specific outcomes. In order to


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investigate these intrinsic functions, pure nanomaterials of distinct sizes and properties are selected for an in vitro study in order to determine their specific actions on critical processes that drive liver fibrosis in TGF-β-activated HSCs. A better understanding of the biological functions of nanomaterials in HSCs could provide stronger justification for designing therapeutic NPs to treat liver fibrosis/cirrhosis in the future, thus promoting their use as carrier systems to synergize with the pharmacological activity of its payload. Therefore, we systematically study the effects of TiO2 NPs with diameters around 20 nm (TiO2-20) and 200 nm (TiO2-200), and SiO2 NPs on the proliferation, fibrosis, adhesion, and migration of LX-2 cells as a model of HSC activation. We aim to verify that NPs of various diameters or types could display novel characteristics of inhibitors that suppress the liver fibrosis in in vitro experimental conditions. Most notably, the reduction of the overall Col-I deposition and aggravation of migratory phenotype could in turn decrease the adhesion and migration of TGF-β-activated LX-2 cells for added therapeutic gains. RESULTS Characterization of Nanomaterials. In this exploratory work, TiO2 NPs of two different diameters and SiO2 NPs were investigated for their effects on the inhibition of hepatic fibrosis (Figure 1). The diameters of two different TiO2 NPs and SiO2 NPs measured by TEM were ~21 (TiO2-20), ~203 (TiO2-200), and ~21 nm (SiO2 NPs) respectively. Furthermore, the hydrodynamic diameters of these nanomaterials detected by DLS were 69 (TiO2-20), 293 (TiO2200), and 71 nm (SiO2 NPs) respectively. Zeta potentials revealed a consistently negatively charged surface at ~-10 mV for all nanomaterials tested (Supporting Information, Table S1).


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Biocompatibility of Nanomaterials. Before investigating the effects of NPs on the inhibition of hepatic fibrosis, we systematically examined the biocompatibility of these nanomaterials on LX2 cells. The calcein-AM/PI staining results qualitatively indicated strong green fluorescence and weak red fluorescence for the NP-treated LX-2 cells at concentration lower than 20 µg mL-1, suggesting that most of the LX-2 cells were viable after treatment with NPs for 24 h (Figure S1). In contrast, more red fluorescent pigmentations were observed in LX-2 cells treated with 40 µg mL-1 TiO2-20 NPs, demonstrating slight cytotoxicity. Using MTT viability assay, results corroborated the earlier findings where all three NPs showed high biocompatibility to LX-2 cells (Figure 2a-c), with cellular viabilities higher than 80% after 24-h exposure at concentrations lower than 20 µg mL-1. In addition, after treatment with 40 µg mL-1 TiO2-20 or TiO2-200 for 24 h, the viabilities of LX-2 cells were slightly decreased to 79.0% or 79.5% respectively (Figure 2d). Taken together, 20 µg mL-1 was assessed as the highest concentration without observable cytotoxicity in this study, hence used for subsequent investigations. Internalization and Subcellular Localization of TiO2-FITC NPs. To evaluate the cellular response to NPs, LX-2 cells incubated with TiO2-FITC NPs for different time periods were examined by LSCM (Figure 3). The cellular internalization results exhibited a feeble green fluorescence of TiO2-FITC NPs could be detected after 0.5-h treatment, and the green signals were detectable during 3 h incubation. Increasing fluorescent intensity was observed with incubation time extending to 6, 12, and 24 h, suggesting that TiO2-FITC NPs were progressively taken up by the cells. Additionally, to determine the subcellular distribution of the TiO2-FITC NPs, LX-2 cells treated with TiO2-FITC NPs were further labeled with Hoechst 33242 and LysoTracker Red for LSCM imaging (Figure S2). Compared with the control group, intense


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green fluorescence consistent with internalization study could be observed in LX-2 cells, most of which were co-localized with the lysosomes (i.e., red fluorescence). NPs Suppress the Expression of Fibrosis Markers in LX-2 Cells and TGF-β-activated LX-2 Cells. We next attempted to uncover the impact of NPs treatment on hepatic fibrosis. After treatment with NPs for 24 h, the expression of fibrotic markers (e.g., Col-I and α-SMA) in LX-2 cells (Figure S3) and TGF-β-activated LX-2 cells (Figure 4) were respectively analyzed through immunofluorescence imaging, western blotting, and RT-PCR assay. Compared with control group without any treatment, the expression of Col-I and α-SMA were significantly increased following TGF-β activation (Figure 4), and the quantitative mRNA expression assay of Col-I and α-SMA showed ~2.79 and ~2.32 fold increase respectively. Importantly, the fluorescent signals of both markers were markedly suppressed in Sunitinib-, TiO2-20-, TiO2-200 - and SiO2 NPstreated groups (Figure S3a). Consistent with immunofluorescence images, the expressions of protein and mRNA levels were also correspondingly decreased (Figure S3b and c). RT-PCR results demonstrated that these expressions were respectively decreased to ~0.19, ~0.33, ~0.71, and ~0.71 for Col-I, and ~0.74, ~0.20, ~0.45, and ~0.42 for α-SMA respectively. Furthermore, treatment with TiO2-20, TiO2-200 or SiO2 NPs induced significant dose-dependent and timedependent reduction in Col-I and α-SMA protein expressions in LX-2 cells (Figure S4 and S5). Compared with the control group without any treatment, positive control (TGF-β-activated group), and negative control (TGF-β-activated group treated with Sunitinib), TiO2-20, TiO2-200 and SiO2 NPs also contributed to the suppression of TGF-β activation as depicted by attenuation of fluorescent intensity (Figure 4a), protein levels (Figure 4b), and mRNA levels (~0.42, ~1.2, and ~1.45 for Col-I, and ~0.53, ~0.57, and ~1.00 for α-SMA) (Figure 4c) of Col-I and α-SMA,


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demonstrating that these nanomaterials counter-acted the biochemical stimulus of TGF-β and suppressed the phenotype of hepatic fibrosis. It is well established that MMPs and their endogenous inhibitors, TIMPs, play a critical role in sustaining the homeostasis of ECM in healthy liver.10,29 To assess if NPs inhibit hepatic fibrosis via a perturbation of this homeostasis, we measured the effects of NPs on key mRNA expression levels of MMP-13 and TIMP-1 (Figure 4d). Significantly, the expression of MMP-13 was decreased to ~0.48 and the expression of TIMP-1 was increased to ~2.26 in TGF-β-activated LX-2 cells. However, the expressions of MMP-13 and TIMP-1 were reversed by Sunitinib and NPs. Notably, the expression of MMP-13 was significantly up-regulated (~2.43, ~1.95, ~1.28, and ~1.18) with a reciprocal suppression of TIMP-1 (~0.92, ~0.99, ~0.98, and ~1.03) to normal levels in Sunitinib-, TiO2-20-, TiO2-200- and SiO2 NPs-treated groups. NPs Reduce the Adhesion and Migration of TGF-β-activated LX-2 Cells. The ECM, mainly Col-I, are the products of fibrosis and essential matrix for adhesion and migration of LX-2 cells during hepatic fibrosis.16,30 We postulated that the adhesion and migration of TGF-β-activated LX-2 cells could be reduced by NPs (Figure 5a-d). As shown in the fluorescent images (Figure 5a and c), compared with control group, much more LX-2 cell signals were detected following TGF-β activation, suggesting the enhanced adhesion and migration of TGF-β-activated LX-2 cells. In sharp contrast, TGF-β-activated LX-2 cells incubated with Sunitinib showed less adhesion and migration properties. Moreover, fewer CellTracker-labeled LX-2 cells were found after TiO2-20, TiO2-200 or SiO2 NPs exposure. The results quantitatively indicated that the attachment and migration of LX-2 cells activated by TGF-β could significantly increase by ~3.71 and ~1.47 fold respectively. However, the fold-change in Sunitinib-, TiO2-20-, TiO2-200- and


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SiO2 NPs-treated groups were ~0.93, ~1.22, ~1.47, and 1.38 for adhesion assay, and ~0.53, ~0.72, ~0.85, and 0.72 for migration assay, respectively (Figure 5b and d). To ascertain if the inhibition of cell migration was mediated by EMT modulation, we studied the gene expression levels of E-Cad and N-Cad (Figure 5e and f). A 0.60 fold down-regulation of E-Cad and 2.28 fold up-regulation of N-Cad could be detected in TGF-β-activated LX-2 cells. Following treatment with Sunitinib, TiO2-20, TiO2-200 or SiO2 NPs, we observed significant increase of E-Cad expression to ~3.50, ~1.51, 1.20, and ~1.17 fold respectively, and noticeable reduction of N-Cad expression to ~1.04, ~0.81, ~0.99, and ~1.03 fold respectively. More importantly, the calculated ratio of N-Cad and E-Cad showed a reduction from ~3.89 in TGF-βactivated LX-2 cells to ~0.31, ~0.54, ~0.82, and ~0.89 in TGF-β-activated LX-2 treated with Sunitinib, TiO2-20, TiO2-200 and SiO2 NPs respectively. This further demonstrated that EMT was modulated and could impose an effect on LX-2 cell migration. DISCUSSION A number of nanomaterials are now presented as new frontiers in supporting therapeutics, particularly as nanocarriers for cancer and other disease treatment.31-37 Among them, the applications of TiO2 NPs and SiO2 NPs are well established in the nutrition, cosmetics and medical industries, largely due to their favorable reactivity, conductivity, and optical sensitivity.38,39 While 30-99% of the administered nanomedicine were accumulated in liver,24 they have not shown obvious long-term toxic effects in rodents.40,41 Of relevance to this work, Elgrabli and co-workers demonstrated that there were no tissue damage nor toxicological effects found in liver after treatment with 7.7-9.4 mg kg-1 TiO2 NPs in rats.42 Similar results were also observed in mice after exposure to a significantly high concentration (15 mg kg-1) of platinum


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nanoparticles.43 More importantly, Yen and co-workers reported that naringenin-loaded nanoparticles showed hepatoprotective effects in CCl4-induced acute rat liver failure.44 These independent evidences alleviate some of the safety concerns that such therapeutic agents on hepatic exposure, and provide new opportunities to exploit nanomaterials-based methods for chronic hepatic diseases like fibrosis. More specifically, recent studies on inorganic NPs (e.g., iron oxide magnetic NPs, Au NPs, cerium oxide (CeO2) NPs), which exhibit low toxicity profile, have also shown promising therapeutic effects upon conjugation with drugs for liver fibrosis.40,4548

Given that the cellular response and nanobiological effects of nanomaterials are particularly

sensitive to their sizes and the types of nanomaterials used,39,49-51 the diversity of their biological effect should be appropriately considered in any nanomedicine investigation. In choosing the appropriate nanoparticles for our investigation, we considered that the positively charged nanoparticles exhibit a greater propensity to be adsorbed/aggregated onto the cellular surfaces or proteins in the blood which carry slightly negative charge.52-55 This could increase the risk of non-specific binding and induce cytotoxicity and other unintended effects that hinder their application in vivo. The use of negatively charged nanoparticles could circumvent this potential risk. Riding on the threshold of these development and rationale, we take a targeted approach to probe more deeply on three different negatively charged NPs (TiO2-20, TiO2-200 and SiO2 NPs), to evaluate if they could exhibit intrinsic pharmacological activity to bring about anti-fibrotic effects in TGF-β-activated LX-2 cells, and henceforth, be utilized as novel inhibitors for either stand-alone or adjuvant therapy against liver fibrosis. Firstly, a noteworthy characteristic is the low toxicity of the administered NPs as opposed to conventional anti-fibrotic drugs, demonstrated in the current LX-2 cell model (Figure 2). While all three nanomaterials showed similar profile at increasing concentrations up to 40 µg mL-1,


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endocytosis of TiO2-FITC NPs was confirmed with NPs detected in the lysosomes at 24 hours treatment (Figure 3 and Figure S2). This finding is of dual significance: on one hand, it demonstrates the uptake of TiO2-FITC NPs into the cells, thereby highlighting the possibility that the mechanism of action could occur intracellularly. At the same time, it unveils a route for NP elimination as a safeguard against excessive accumulation, though the kinetics of this process and the impact of long-term exposure will require further investigation. Hepatic fibrogenesis is a dynamic response of the liver driven by different cells and mediators to resolve injury.56 Upon trigger and activation, signals converge onto HSCs, resulting in the increase in well-characterized fibrotic markers. Therefore, we aimed to investigate whether the experimental NPs could exert anti-fibrotic effect via reducing the principal phenotype of activated HSCs. Accordingly, our results have displayed an effective inhibition of the cellular phenotypes exhibited by TGF-β-activated HSCs. Specifically, we demonstrated a reduction in the biochemical features of suppressing mRNA and protein expression of fibrotic markers such as Col-I (ECM molecules) and α-SMA (HSCs activation marker) (Figure 4). Even at 10 µg mL-1, the expression of these fibrotic markers did not revert to its activated levels (Figure S4). This may suggest a sustained effect of the NPs which could be aided by their cumulative intracellular uptake. We postulate a potentially novel mechanism where TiO2 NPs and SiO2 NPs could inhibit fibrotic markers via hindering the transcription of mRNA, thus resulting in the overall suppression of the pro-fibrotic proteins expressed. Since matrix production has been one of the most prominent features of fibrogenesis on a TGFβ-induced fibrotic model,57,58 we further challenged our hypothesis to determine if these inorganic NPs could alter matrix stability and further affect cellular morphology and activity via the regulation of EMT. Interestingly, both TiO2 NPs and SiO2 NPs could stimulate expression of


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MMP-13 and reduce the expression of corresponding endogenous inhibitor, TIMP-1, which shifts the balance in favour of ECM (mainly Col-I) degradation (Figure 4d). Presumably, the suppression of Col-I deposition could de-stabilize the ECM and further reduce the adhesion of TGF-β-activated LX-2 cells (Figure 5a and b). Such effect on cellular adhesion could also mediate a subsequent reduction in cellular migration. As reported in literature, HSCs are stimulated to migrate towards injury sites as part the phenotype in liver fibrosis.2,59 Therefore, the loss of adhesion and migration capabilities with the treatment of NPs could support the recovery from this pathological process. While reduction in adhesion and migration could be a passive consequence to a change in ECM instability, we consider the possibility that the NPs may be exerting a more direct mechanotransduction effect on the LX-2 cells. It has been recently reported that Col-I was implicated in N-Cad-mediated cell-cell adhesion by Okada and co-workers.60 Therefore, EMT was considered as a key event after treatment with these nanomaterials. Our results showed that these NPs could also increase E-Cad expression and suppress the expression of N-Cad in TGF-β-activated LX-2 cells, albeit not as effective as conventional TKI drugs such as Sunitinib (Figure 5e and f). Similarly, Cho and co-workers had also indicated that the overexpression of E-Cad could inhibit TGFβ1 gene inductions, and decrease α-SMA expression level, which further suppressed hepatic fibrosis.61 Since EMT involves a complex cellular reorganization and microenvironmental changes, we postulated that inorganic NPs have the potential to suppress the cells from transformation through imposing physical stimulation and constraints. From this perspective, these inorganic NPs could potentially be an alternative nanomedicine treatment with a direct therapeutic action on liver fibrosis, or could also enhance the delivery and uptake TKIs in the form of a carrier and adjuvant therapy.


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CONCLUSION Taken together, we report the anti-fibrotic activities of TiO2 NPs and SiO2 NPs even as standalone entities (Figure 6). Their intrinsic pharmacological activity could be bolstered by antiadhesive and anti-migrative effects coupled with favorable biocompatibility to support longer term evaluation. Our results demonstrate that these nanomaterials could be performed as highly efficient inhibitors without obvious cytotoxicity in LX-2 cells, which uncover new insights into treating hepatic fibrosis. Moreover, by systematically comparing the results induced by TiO2 NPs with different diameters and SiO2 NPs, we could speculate the size-dependent and typedependent therapeutic efficacy in this work. We conclude that NPs are efficient inhibitors which could potentially attenuate TGF-β activation of LX-2 cells, and thereby enhance the therapeutic efficacy to combat hepatic fibrosis with convenience and safety. EXPERIMENTAL SECTION Characterization of Nanoparticles. TiO2-20, TiO2-200, and SiO2 NPs were purchased from Sigma Aldrich (USA). The shapes and primary sizes of these NPs were characterized by field emission transmission electronic microscopy (FE-TEM, JEOL 2100-F). Briefly, the ultrapure water-dispersed NPs were firstly dropped on carbon coated TEM grid, and then determined under accelerating voltage of 200 kV. The primary size of the NPs was randomly calculated by counting at least 50 selected nanoparticles with ImageJ software. Their hydrodynamic size and zeta potential were measured by utilizing dynamic light scattering (DLS) analysis with Zetasizer (Malvern, UK). The samples were dispersed in the ultrapure water (pH = 7.2) with probe sonication (Qsonica, USA) for 1 min before the DLS analysis. To understand the NPs behavior during treatment, the NPs were dispersed in complete cell culture medium with sonication and


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were incubated for another 30 min. Thereafter, the NPs were collected with centrifugation, redispersed back in ultrapure water prior to DLS analysis. Cell Lines. Human microvascular endothelial cells (HMVEC, Thermo Scientific, USA) were maintained with EndoGRO-MV-VEGF medium supplemented (Merck Millipore) containing 50 µg mL-1 of Gentamicin (Sigma Aldrich, USA) and 50 ng mL-1 of Amphotericin-B (Sigma Aldrich, USA). Human hepatic stellate cells (HSCs, LX-2 cells, received as a kind gift from Prof. Scott Friedman) were cultured with Dulbecco’s modified Eagle medium (DMEM) containing 1% fetal bovine serum (FBS).5 Both cell lines were incubated at 37 oC in a humidified atmosphere containing 5% CO2. Cell Viability Assays. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and propidium iodode (PI) staining assay were conducted to quantitatively study the biocompatibility of TiO2-20, TiO2-200, and SiO2 NPs. For MTT assay, LX-2 cells were seeded in 96-well cell culture plates at a density of 8000 cells per well with fresh medium at 37 oC in the humid atmosphere for 12 h. Thereafter, the cells were respectively treated with 100 µL medium containing various concentrations (10, 20, and 40 µg mL-1) of TiO2-20, TiO2-200, and SiO2 NPs for different time periods (0.5, 3, 6, 12, and 24 h), and LX-2 cells without any treatment were performed as control group. Then, the well established MTT assay was taken to measure the cell viabilities. To further confirm the cytotoxicity of these NPs, LX-2 cells were plated in 6-well cell culture plates at a density of 2×105 cells per well overnight and treated with various concentrations (2.5, 5, 10, 20, and 40 µg mL-1) of NPs at 37 oC for another 24 h for PI staining assay. Following treatment, both floating and adherent cells were harvested, washed with phosphate-buffered


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saline (PBS) for three times, and then stained with PI solution (2 µg mL-1) for Tali analysis (Life Technologies, USA). Calcein-AM/PI Staining Assay. Calcein-AM/PI staining assay was performed to qualitatively study the biocompatibility of TiO2-20, TiO2-200, and SiO2 NPs. Live/dead cells staining were performed using calcein-AM (2 µM) and PI (2 µM) solution, respectively. Briefly, LX-2 cell were seeded in 6-well cell culture plates overnight. After the similar treatment described in PI staining assay, LX-2 cell were incubated in 200 µL PBS containing calcein-AM and PI solution for 30 min at room temperature. Finally, 20 µL samples were dropped on cover glass slides for laser scanning confocal microscopy (LSCM, Olympus, Fluoview, FV10i Japan) imaging. The excitation wavelength was set as 488 nm for calcein-AM investigation, and 543 nm for PI investigation. All images were captured and analyzed under the same experiment condition. TiO2-FITC NPs Internalization and Subcellular Distribution Study. Fluorescein isothiocyanate (FITC)-conjugated TiO2-20 (TiO2-FITC NPs) were prepared as reported previously.21 LX-2 cells were grown (37 oC, 5% CO2) in 24-well cell culture plates containing cover slips at a density of approximately 1×105 cells per well overnight. Then, the cells were incubated with TiO2-FITC NPs for various durations (0.5, 3, 6, 12, and 24 h). Before LSCM imaging, the cells were washed with PBS for three times to eliminate non-specific adsorption TiO2-FITC NPs, and stained with Hoechst 33242 at 37 oC for 30 min. To determine subcellular distribution of TiO2-FITC NPs, LX-2 cells treated with TiO2-FITC NPs for 12 h were stained with Hoechst 33242 and 100 nM LysoTracker Red DND-99 (30 min). All images were captured under the same instrument setting to ensure no background cellular fluorescence. Three independent assays were performed for all measurements.


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Immunofluorescence Staining. The expression of fibrosis markers (e.g., Col-I and α-SMA) in both LX-2 cells and TGF-β-activated LX-2 cells were visualized by immunofluorescence staining. LX-2 cells were seeded on glass coverslips in 24-well cell culture plates at a density of 0.8×105 cells per well over night. Cells were exposed with TGF-β (2 ng mL-1), Sunitinib (10 µM), TiO2-20 (20 µg mL-1), TiO2-200 (20 µg mL-1) or SiO2 NPs (20 µg mL-1) for 24 h. To establish TGF-β-activated LX-2 cells, LX-2 cells were serum starved for 4 h and then incubated with serum-free DMEM containing 2 ng mL-1 TGF-β for another 4 h. Following activation, TGF-β-activated LX-2 cells were respectively treated with Sunitinib (10 µM), TiO2-20 (20 µg mL-1), TiO2-200 (20 µg mL-1) or SiO2 NPs (20 µg mL-1) combined with 2 ng mL-1 TGF-β for 24 h. After that, cells were fixed with PBS containing 4% paraformaldehyde for 20 min at room temperature, and permeabilized with 0.1% Triton X 100 and 4% bovine serum albumin (BSA) in PBS (blocking solution) for 40 min. Thereafter, LX-2 cells were incubated with primary antibodies (diluted 1:300 in blocking solution) at 4 oC overnight. Following that, the cells were rinsed with PBST (PBS containing 0.1% Tween 20) for three times, and cultured with Alex 488labeled secondary antibodies in blocking solution for 1 h at 37 oC. Finally, the cells were washed with PBST, stained with Hoechst 33242, and viewed with LSCM. Immunoblot Assay. LX-2 cells plated in 6-cm cell culture dishes overnight were treated with the same method described in immunofluorescence staining part. Following treatment, cells were lysed with RIPA lysis buffer (Sigma Aldrich, USA) containing protease and phosphatase inhibitors (1%; Sigma Aldrich, USA). After centrifugation, the supernatant was measured by bicinchoninic acid (BCA, Thermo Scientific) assay to determine protein concentration. Protein samples were adjusted to same concentration, 20 µL of the samples were loaded into 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred


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onto a PVDF membrane (Thermo Scientific). The membranes were incubated in Tris-buffered saline (TBS) containing 5% BSA for 1 h at room temperature and then in primary antibody solution at 4 oC overnight. Thereafter, appropriate horseradish peroxidase (HRP)-labeled secondary antibody solutions were incubated with the membranes for 1 h. Finally, Western Lightning Plus-ECL reagent (Perkin Elmer) was added for band visualization. RNA Extraction and Quantitative Real-time Polymerase Chain Reaction (RT-PCR). TGFβ-activated LX-2 cells were exposed to Sunitinib (10 µM), TiO2-20 (20 µg mL-1), TiO2-200 (20 µg mL-1) or SiO2 NPs (20 µg mL-1) for 24 h, and total RNA was isolated with RNeasy Mini kit (Qiagen) according to manufacturer’s instructions. 600 ng of extracted RNA was converted to complementary DNA using qScript cDNA Supermix (Quanta). Gene expression of target genes levels were determined using SYBR Green Supermix (Bio-Rad). The housekeeping gene human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used for normalization of expression using a comparative CT method. The primer pairs are as follows: GAPDH: 5′-ACT TTG GTA TCG TGG AAG GAC T-3′ (forward) and 5′-GTA GAG GCA GGG ATG ATG TTC T-3′ (reverse); Col-I: 5′-CCT GGA TGC CAT CAA AGT CT-3′ (forward) and 5′-CGC CAT ACT CGA ACT GGA AT-3′ (reverse); α-SMA: 5′-CCG GGA GAA AAT GAC TCA AA-3′ (forward) and 5′-GCA AGG CAT AGC CCT CAT AG-3′ (reverse); MMPs-13: 5′-TGT GAT CCC TTG AGA TAT GGA A-3′ (forward) and 5′-CAA TAA GTG CCA AGC ACC CT-3′ (reverse); TIMPs-1: 5′-AAG GCT CTG AAA AGG GCT TC-3′ (forward) and 5′-GAA AGA TGG GAG TGG GAA CA-3′ (reverse); E-Cad: 5′- AAT TCC TGC CAT TCT GGG GA-3′ (forward) and 5′- TCT TCT CCG CCT CCT TCT TC-3′ (reverse); and N-Cad: 5′-TGA GCC TGA AGC CAA CCT TA-3′ (forward) and 5′-AGG TCC CCT GGA GTT TTC TG-3′ (reverse).


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Cell Adhesion Assay. Cell adhesion assay was performed to quantify LX-2 cell adhesion to HMVEC. Briefly, the TGF-β-activated LX-2 cells treated with Sunitinib (10 µM), TiO2-20 (20 µg mL-1), TiO2-200 (20 µg mL-1) or SiO2 NPs (20 µg mL-1) for 24 h were collected and stained with CellTracker Green, and then plated at a density of 0.8×105 cells per well into CellTracker Red-stained HMVEC monolayers for 2 h. Thereafter, the nonadherent LX-2 cells were removed by washing three times with PBS. The HMVEC monolayer and adherent LX-2 cells were then fixed and mounted for fluorescence imaging. The adhesion of LX-2 cells to HMVEC was counted from fluorescent images by ImageJ (open source software avaliable at https://imagej.nih.gov/ij/), and the extent of adhesion was expressed as normalized cell number compared with control group. Cell Migration Assay. Migration assay was performed with the use of transwell inserts (8.0 µm pore, Corning Costar, USA). Briefly, CellTracker Green-labeled TGF-β-activated LX-2 cells treated with Sunitinib (10 µM), TiO2-20 (20 µg mL-1), TiO2-200 (20 µg mL-1) or SiO2 NPs (20 µg mL-1) for 24 h were resuspended in serum-free medium, and 40,000 cells per well were seeded onto the upper transwell chamber. Next day, cells remaining in the upper chamber were scrapped off with cotton buds. The migrated cells in the lower chamber were monitored with Leica epifluorescence microscope (DMI6000) for fluorescent imaging. Cells were enumerated by counting at least three microscopic fields per well, and the extent of cell migration was expressed as normalized cell number compared with control group. Statistical Analysis. Each experiment was performed with three replications. Origin® software was used to calculate the statistical significance of the experimental results. Statistical significance was determined by One-way Analysis of Variance (ANOVA) based on P value less than 0.05.


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ASSOCIATED CONTENT Supporting Information. Figure S1-S5 and Table S1 could be found in the supplementary information. This material is available free of charge on ACS Publication website at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mails: [email protected] (D.T. Leong) *E-mails: [email protected] (H.K. Ho) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. Funding Sources This work was supported by Ministry of Education Academic Research Grants (R148-000-217112 and R-279-000-418-112). Notes The authors declare no competing finical interests. REFERENCES (1)

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Figure 1. Nanoparticles characterization. Representative TEM image of (a) TiO2-20, (b) TiO2-200, and (c) SiO2 NPs. Scale bar = 50 nm. 85x28mm (299 x 299 DPI)


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Figure 2. TiO2-20, TiO2-200 and SiO2 NPs showed no obvious cytotoxicity on LX-2 cells. MTT assay indicated the high cell viabilities of LX-2 cells exposed to different dose of (a) TiO2-20, (b) TiO2-200 and (c) SiO2 NPs for different time periods. (d) Percentage of PI staining assay showed little damage of LX-2 cell membrane after exposing to different dose of TiO2-20, TiO2-200 and SiO2 NPs for 24 h. 85x73mm (300 x 300 DPI)



Figure 3. Progressive uptake and intracellular distribution of TiO2-FITC NPs in LX-2 cells. LSCM images showed a significant uptake and intracellular distribution of TiO2-FITC NPs (green) in LX-2 cells. After treatment with 20 µg mL-1 TiO2-FITC NPs for different time periods, the LX-2 cells were stained with Hoechst 33242 (light blue) for 30 min. Scale bar = 20 µm. 85x56mm (300 x 300 DPI)


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Figure 4. NPs suppressed the expressions of Col-I and α-SMA in TGF-β-activated LX-2 cells, coupled with the up-regulation of MMP-13, and down-regulation of TIMP-1. (a) Representative immunofluorescence imaging and (b) western blot analysis of Col-I and α-SMA in TGF-β-activated LX-2 cells. Scale bar = 20 µm. Quantitative analysis of (c) Col-I and α-SMA, and (d) MMPs and TIMPs (normalized with GAPDH) mRNA expression in TGF-β-activated LX-2 cells. Data are mean ± SD, n=3, Student’s t-test, compared with control group *P

Inorganic Nanomaterials as Highly Efficient Inhibitors of Cellular

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