Molecular Elucidation of Biological Response to Mesoporous Silica

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Molecular Elucidation of Biological Response to Mesoporous Silica Nanoparticles in vitro and in vivo Cheng-Chung Chou, Wei Chen, Yann Hung, and Chung-Yuan Mou ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 13 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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ACS Applied Materials & Interfaces

Molecular Elucidation of Biological Response to Mesoporous Silica Nanoparticles in vitro and in vivo Cheng-Chung Chou,†,* Wei Chen,‡ Yann Hung,‡ Chung-Yuan Mou‡ †Department of Life Science and Institute of Molecular Biology, National Chung Cheng University, Chia-Yi, Taiwan 62102, ROC ‡Department of Chemistry, National Taiwan University, Taipei, Taiwan 10617, ROC KEYWORDS mesoporous silica nanoparticles, toxicity, endocytosis, gene expression microarray, reactive oxygen species (ROS), oxidative stress, inflammation, autophagy

ABSTRACT Biomedical applications of mesoporous silica nanoparticles (MSNs) require efficient cellular uptake and low toxicity. The purpose of this study is to investigate the cellular uptake and toxicity of MSNs with different sizes and charges (50, 100 and 250 nm with a positive surface charge and 100 nm with a negative surface charge) exposed to human monocyte-derived macrophages, lung epithelium BEAS-2B cells and mice using genome-wide gene expression analysis and cellular/animal-level end point tests. We found that MSNs can be taken up into cells through endocytosis in a charge- and size-dependent manner, with positively charged and larger MSNs being more easily taken up into the cells by recruiting more types of endocytotic pathways for more cellular uptake. Moreover, the cytotoxicity of MSNs could be correlated with

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the amount of MSNs taken up by cells, which positively correlates to the particle size and dosage. Therefore, only positively charged and larger MSNs (≥100 nm) during higher treatment doses (≥500 µg mL-1) resulted in a sufficient accumulation of internalized MSNs in cells to induce significant release of reactive oxygen species (ROS) and oxidative stress, inflammatory gene upregulation through NF-κB and AP-1, and eventually autophagy-mediated necrotic cell death. Furthermore, genome-wide gene expression analysis could reflect the above in vitro cellular damages and corresponding in vivo injuries in mice, indicating that specific gene expression footprints may be used for assessing the safety of nanoparticles. The present finding provides some insights into the rational design of effective MSN-based drug/gene delivery systems and biomedical applications.

1. INTRODUCTION Mesoporous silica nanoparticles (MSNs), which possess large surface areas and pore volumes, easy surface functionalization, improved aqueous solubility and biodegradability,1,2 have recently attracted considerable attention for use in biomedical applications,3 such as biosensing4 and bioimaging,5 cancer therapy and targeted drug delivery.6 However, the further clinical use of MSNs requires a safety assessment to understand the potential toxicity mechanisms. The safety and toxicity of nanoparticles are known to significantly contribute to their physicochemical properties. In particular, particle size is a key feature for determining the toxicological potential of nanoparticles.7 For a given mass of nanoparticles, a smaller particle size results in a larger total surface area, allowing more surface atoms to interact with biological systems. Therefore, elucidating the relationship between particle size and biological response would provide important insights into nanotoxicity.8 The size effects of MSNs on cellular uptake


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and cytotoxicity have recently been reported.9-13 However, these previous studies focused only on cellular internalization processes and cell viability following MSN exposure, and they lacked extensive investigations of various biological responses (or toxicological endpoints) to different sizes of MSNs. Therefore, to further explore MSN-induced toxicity mechanisms in a more comprehensive manner, using a genome-wide approach to measure MSN-activated changes in gene expression and cellular pathways is a better way to elucidate the size-dependent relationship between particle size and toxicological endpoints at the molecular level. The majority of previous studies on the potential use of MSNs for drug delivery or other medical applications were performed using various cancer cells.14-17 However, it has been reported that nanoparticles will immediately confront and interact with immune cells during systemic administration to the human body.18 Consequently, evaluating the uptake and putative toxicity of nanoparticles on the immune system must also be considered. Among the various types of immune cells, macrophages, which are present in various organs and differentiate from circulating monocytes in the bloodstream, play the role of first-line defender against pathogens and foreign materials through phagocytosis. Thus, over the past decade, macrophages have been widely used for studying immune responses following nanoparticle exposure in vitro and in vivo.12,19-21 In addition, recent biokinetic studies in animals have shown that nanoparticles can translocate from on-site exposure to the blood circulation by crossing the epithelial barriers and subsequently to target organs.22 Therefore, investigating the uptake and toxicity of nanoparticles using epithelial cells is also of critical importance.23 Based on the aforementioned observations, we performed a risk assessment of MSNs using two types of cells: (1) human lung epithelial cell line BEAS-2B, representing an epithelial barrier of the organs, and (2) human monocyte-derived macrophages, representing a principal immune


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cell type in the immune system. In this study, we investigate the effects of different sizes (50, 100 and 250 nm) and charges of MSNs on cellular uptake and toxicity in vitro and in vivo at the molecular level. First, MSNs of different sizes and charges were synthesized and conjugated with fluorescein isothiocyanate (FITC) or rhodamine B isothiocyanate (RITC) for imaging and quantifying MSN uptake. The different cellular uptake pathways induced by different sizes of MSNs were investigated in detail. Second, the size effects of these MSNs on cell death were evaluated using two different assays for cross-validation. Third, genome-wide gene expression following MSN exposure to the both cells was measured and statistically analyzed to elucidate the molecular cytotoxic mechanisms with subsequent experimental validation. Finally, MSNs were administered to mice to investigate whether MSN-induced in vitro cytotoxicity could reflect corresponding in vivo toxicity in mice. 2. EXPERIMENTAL SECTION 2.1. Synthesis and Characterization of MSNs. Three different sizes (50 nm, 100 nm and 250 nm) of positively charged MSNs (+MSN50, +MSN100 and +MSN250), one size (100 nm) of negatively charged MSNs (-MSN100), postively and negatively charged FITC-conjugated MSNs (+FITC@MSNs and -FITC@MSNs), and postively and negatively charged RITCconjugated MSNs (+RITC@MSNs and -RITC@MSNs) were synthesized by following previous reports.24-26 Briefly, different sizes of MSNs were synthesized based on a sol-gel synthetic strategy in the presence of cationic surfactant templates. In addition, MSNs were modified using N-trimethoxysilylpropyl-N,N,N-trimethylammonium

chloride

(TA)

and

sodium

3-

trihydroxysilylpropylmethylphosphonate (THPMP) to produce positively and negatively charged nanoparticles, respectively. To synthesize FITC@MSNs and RITC@MSNs, the FITC-APTMS


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and RITC-APTMS dye solutions were used. The detailed sample preparations are described in Supporting Information. 2.2. Cell Culture and MSN Treatment. Human monocytic leukemia cell line THP-1 (ATCC) and human lung epithelial BEAS-2B cells (ATCC) were maintained in RPMI 1640 medium (Gibco-BRL) containing 1.5 g L-1 sodium bicarbonate, 4.5 g L-1 glucose, 10 mM HEPES, 0.05 mM 2-mercaptoethanol, 10% fetal bovine serum (Gibco-BRL), 100 U mL-1 penicillin, and 100 g mL-1 streptomycin under 5% CO2 at 37 °C. THP-1 cells were pretreated with 0.32 µM phorbol myristate acetate (PMA; Sigma, MO) for 24 h, washed three times with phosphate-buffered saline (PBS), and incubated in fresh medium for an additional 24 h to collect the conditioned medium, which was used to differentiate other THP-1 cells into macrophages to eliminate the residual effect of PMA. THP-1 cells were then incubated in the collected conditioned medium for 24 h, and then the medium was replaced by fresh culture medium for 3 days to be differentiated into macrophages (THP-1-derived macrophages). These THP-1-derived macrophages and BEAS-2B cells were then incubated with fresh medium containing the required concentrations of MSNs and 0.1% Pluronic F-68 for indicated treatment times.. 2.3. Cellular Uptake of MSNs. To investigate the efficiency of MSN uptake into macrophages and BEAS-2B, the cells were seeded on 12-well plates at a density of 1.2×105 cells well-1, treated with various FITC@MSNs (50 µg mL-1) for 24 h, washed twice with PBS, and then removed with trypsin. The collected cells were resuspended in PBS with 0.4% (w/v) trypan blue to quench the fluorescence from FITC@MSNs bound on the cell surface, followed by flow cytometric analysis using a BD FACSCalibur Flow Cytometer. The number of FITC-labeled cells was expressed as the percentage of total cells counted in each experiment, representing the cellular uptake efficiency.


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2.4. Quantitative Inductively Coupled Plasma Mass (ICP-MS) Measurement. After macrophages and BEAS-2B cells were treated with 50 µg mL-1 MSNs with different sizes for 24 h, the cells were washed three times with PBS, trypsinized, centrifuged, and finally resuspended in 1 ml of 10% HNO3 for acid lysis. The silica concentration in the acid-digested solution was then measured using a SCIEX ELAN 5000 ICP-MS spectrometer (Perkin Elmer). 2.5. Lysosome Detection by Lysotracker. To evaluate the co-localization of MSNs and lysosomes, macrophages and BEAS-2B cells (1×105 cells well-1) were seeded onto a chamber slide and incubated with different sizes of MSNs at a concentration of 200 µg mL-1 for 24 h, washed three times with PBS, and then stained with 75 nM Lysotracker (Molecular Probes/Invitrogen) for 1 h to detect acidic late endosomes and lysosomes. The stained cells were washed twice with PBS, fixed with 0.5 mL of 4% paraformaldehyde for 30 min, rinsed twice with PBS, and finally transferred onto a glass slide for microscopy analysis at excitation/emission wavelengths of 530/590 nm excitation/emission for Lysotracker visualization using a Carl Zeiss LSM 510 laser scanning microscope. To quantitatively measure cellular Lysotracker uptake, Lysotracker-stained cells (2×105 cells well-1) were directly subjected to flow cytometric analysis using a BD FACSCalibur Flow Cytometer. 2.6. Acridine Orange (AO) Staining for Detecting Acidic Cellular Vesicles. Macrophages and BEAS-2B cells (2×105 cells well-1) were seeded onto a 12-well plate and incubated with 250 nm MSNs at a concentration of 200 µg mL-1 for 24 h. The cells were washed three times with PBS, stained with 5 µg mL-1 acridine orange (Molecular Probes/Invitrogen) for 15 min, rinsed twice with PBS, and then viewed under a fluorescence microscope (CKX41, OLYMPUS). To perform the quantitative analysis of lysosomal membrane permeabilization by AO staining, each fluorescent image was split to the respective red, green and blue image components. The average


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intensity of AO fluorescence per cell was quantified using NIH ImageJ software that averaged over the red intensity counts above a threshold to eliminate background signals from all pixels in the green fluorescence within cells. 2.7. FM1-43 Staining. To evaluate the effect of the size of MSNs on endocytosis in cells, macrophages and BEAS-2B cells (2×105 cells well-1) were seeded onto a 12-well plate incubated with various sizes of MSNs at a concentration of 200 µg mL-1 for macrophages and a concentration of 50 µg mL-1 for BEAS-2B cells for 2 h. The cells were washed three times with PBS, stained with 5 µg mL-1 FM1-43 (Molecular Probes/Invitrogen) in cold Hank’s balanced salt solution (HBSS) for 5 min, rinsed twice with PBS, and suspended by trypsinization, followed by flow cytometric analysis using a BD FACSCalibur Flow Cytometer. 2.8. Drug Inhibition of Endocytosis Pathways. Macrophages and BEAS-2B cells (2×105 cells well-1) were seeded onto a 12-well plate and incubated with chlorpromazine (10 µg mL-1), genistein (200 µM), or amiloride (10 µM) in culture medium for 1 h at 37 °C. Subsequently, different sizes of FITC@MSNs at a concentration of 200 µg mL-1 were added and incubated for another 2 h. The cells were then rinsed twice with PBS, resuspended in culture medium containing 0.4% (w/v) trypan blue for quenching extracellular fluorescence, and finally followed by flow cytometric analysis using a BD FACSCalibur Flow Cytometer. 2.9. Cell Viability Assay. Macrophages (4×104 cells well-1) and BEAS-2B (0.7×104 cells well-1) were treated with MSNs at the indicated doses in a 96-well plate for 24 h. Cell viability was analyzed using a Sigma Cell Counting-8 kit according to the manufacturer’s instructions. Briefly, 10 µL of 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt (WST-8) was added to each well, and the reaction was continued for 2 h at 37 °C. The absorbance of the sample at 450 nm was measured using a MRX II


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DYNEX spectrophotometer (DYNEX Technologies). This assay is generally known as the WST-8 assay. FITC annexin-V/PI staining was further used for comparison with the WST-8 assay as a secondary method for determining cell viability. The treated cells were trypsinized and stained with FITC-labeled annexin-V and PI dye (Molecular Probes/Invitrogen), followed by flow cytometric analysis using a BD FACSCalibur Flow Cytometer. The stained cell population should separate into three groups: apoptotic cells show green fluorescence (annexin-V positive), necrotic dead cells show red and green fluorescence (annexin-V positive and PI positive), and live cells show little or no fluorescence (annexin-V negative and PI negative). 2.10. RNA Isolation and DNA Microarray Analysis. The total RNAs of macrophages and BEAS-2Bs cells with or without MSN treatment were extracted using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. RNA quality was assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). To remove possible genomic contamination, total RNA was digested with RNase free DNase I (Invitrogen) for 15 min and purified using an RNeasy MinElute cleanup kit (Qiagen). A human-WG6 Illumina gene chip with 36,160 gene probes was used for the global gene expression measurement. Amplification and labeling of the RNA samples, on-chip hybridization, and reading and normalization of chip signals were performed by Genetech Biotech Co., Ltd (Taipei, Taiwan). After averaging the expression data from the probes that refer to the same gene, 24,927 gene expression data were obtained. The microarray experiments were conducted in duplicate for signal averaging. 2.11. Quantitative Real-Time PCR. One microgram of total RNA was used for cDNA synthesis by random hexamers and a SuperScript III First-Strand Synthesis SuperMix kit (Invitrogen). Quantitative real-time PCR reactions were performed in a 48-well microtiter plate using an ABI StepOne Real-Time PCR System (Applied Biosystems) with a total of 20 µL of a


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mixture containing 10 µL of FastStart Universal SYBR Green Master (Roche Applied Science), 0.4 µL of cDNA and 10 pmole of gene-specific primer pair. The PCR reaction conditions were as follows: 95 °C for 10 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The expression level of each gene was determined from the auto cycle threshold normalized to the human TATA box binding protein (TBP) expression level and expressed as fold change. Each measurement was performed in triplicate, and the results are reported as the mean ± standard deviation. The sequences for all the primer pairs used are listed in Supplementary Table S1. 2.12. Detection of Intracellular Reactive Oxygen Species (ROS). For quantifying the level of ROS in MSN-treated cells relative to the untreated controls, macrophages and BEAS-2B cells in 24-well plates were treated with +MSN250 at 500 µg mL-1 for different time courses, and H2O2-treated cells were used as the positive control. Prior to harvesting, the cells were treated with 50 µM cell-permeant 2,7-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Molecular Probes/Invitrogen) at 37 °C for 30 min, washed once with PBS, and shaken with 200 µL of (dimethyl sulfoxide) DMSO containing 1 mM N-acetylcysteine (NAC) for 5-10 s to quench the reaction. Fifty microliters of supernatant was then transferred to 96-well black plates to monitor the level of fluorescent 2,7-dichlorofluorescein (DCF) (converted from H2DCF-DA) using a Victor3 Multilabel Plate Reader (Perkin Elmer) with an excitation wavelength of 485 nm and emission wavelength of 535 nm. The DCF fluorescent intensity was used to represent intracellular ROS level, and it was expressed as the ratio of MSN-treated vs. control cells. 2.13. Western Blotting and Antibodies. For Western blotting, 20 µg of protein extracts obtained from MSN-treated macrophages or BEAS-2B cells were denatured at 100 °C for 5 min, separated in 10% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked in 5% skim milk in PBS containing 0.1% tween-20;


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blotted with the primary antibodies against β-Actin (Santa Cruz), HO-1 (Santa Cruz), SOD2 (Santa Cruz), Phosphor-mTOR (Cell Signaling), p62 (Cell Signaling), 4-hydroxynonenal (4HNE) (Abcam) or LC-3 (MBL) at 4 °C overnight; and finally incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz) at room temperature for 1 h. Western Lightning® Western Blot Chemiluminescence Reagent Plus (PerkinElmer Life and Analytical Sciences) was used as the detection reagent, and the signal of the protein was detected using a Luminescent Image Analyzer LAS-3000 (Fujifilm). 2.14. Immunofluorescence Staining. The cells were seeded on coverslips in 6-well plates at a density of 105 cells well-1. After treatment with MSNs for 24 h, the cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature and then permeabilized with 0.5% Triton X-100 for 10 min, rinsed twice with PBS, and blocked with 5 % skim milk for 30 min. The cells were then blotted with primary antibodies against NF- κB (sc-372, Santa Cruz) and cJun (Epitomics) for 1 h at room temperature, washed with PBS three times, and incubated with Alexa488-

or

Rhodamine-conjugated

secondary

antibody

(Jackson

ImmunoResearch

Laboratories) for 1 h. After staining, the cells were fixed in 4% paraformaldehyde in PBS for 10 min. Cellular chromosomes were stained with Hoechst 33342 (Invitrogen). Fluorescent images were captured using a Carl Zeiss LSM 510 confocal laser microscope. 2.15. Transfection with EGFP-LC3 Plasmid. BEAS-2B cells grown to 80% confluence were transiently transfected with 1 µg mL-1 EGFP-LC3 plasmid for 24 h using a Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Suspended THP-1 cells were first transfected with 1 µg mL-1 EGFP-LC3 plasmid by electroporation using a pipette-type MicroPorator MP-100 (Digital Bio, Seoul, Korea) according to the manufacturer’s instructions, and then they were transferred to RPMI 1640 medium containing 10% FBS, incubated at 37 °C


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with 5% CO2 for 1 h, and finally treated with PMA for 4 h to be differentiated into attached macrophages. After transfection, the cells were treated with different sizes of RITC@MSNs at the indicated doses for 24 h, followed by fixation with 4% paraformaldehyde. Cellular chromosomes were stained with Hochest 33342 (Invitrogen). Fluorescence images for EGFPLC3 punctate and RITC@MSNs were captured using a Carl Zeiss LSM 510 confocal laser microscope. 2.16. Intratracheal Instillation of MSNs into Mice. Eight-week-old ICR male mice (average weight ± standard deviation, 28 ± 3 g) purchased from BioLASCO (Taiwan) were maintained and handled according to the Animal Care and Use Program guidelines accredited by the National Chung Cheng University in Taiwan. The mice were administered normal saline (control) and +MSN250 at doses of 10, 50, 200 or 500 µg mouse-1 in a 50 µL of PBS to the lungs using a modified intratracheal instillation procedure from a previous study.27 In brief, mice were anesthetized by an intraperitoneal injection of ketamine (60 mg kg-1) and xylazine (12 mg kg-1), and the trachea was surgically exposed for instilling MSNs into the lungs. The mice should fully recover from the anesthesia and surgical procedure after 3 to 5 h. 2.17. Isolation of Murine Alveolar Macrophages. Murine alveolar macrophages were collected as previously described.28 In brief, mice after one-day instillation were anesthetized with ketamine/xylazine intraperitoneally and euthanized by exsanguination. The trachea in the neck was surgically exposed and infused/withdrawn three times using 4 mL of PBS with 0.5 mM EDTA. The collected bronchoalveolar lavage (BAL) fluid was centrifuged at 400 g for 10 min at 25 °C. After washing twice, the cells were cultivated on a 6-well plate in 10% FBS RPMI medium and allowed to attach for 2 h at 37 °C. Nonadherent cells were removed by washing with PBS. Mouse alveolar macrophages are adherent and can be isolated using this procedure.


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3. RESULTS AND DISCUSSION 3.1. Preparation and Physicochemical Characterization of MSNs. We prepared different sizes and charges of MSNs (+MSN50, +MSN100, +MSN250 and –MSN100) to investigate the size- and charge-dependent cellular uptake. Transmission electron microscopy (TEM) images (Figure 1A) indicated that the average sizes of the MSNs were 50 nm, 100 nm and 250 nm, with a well-ordered hexagonal mesoporous structure. The size distribution of the hydrated diameter of the various MSNs was confirmed through dynamic light scattering (DLS) measurements (Figure 1B). The results indicated that the MSNs were well dispersed in H2O. N2 adsorption-desorption isotherms (Figure 1C) revealed that the MSNs have a large surface area, which is a general characteristic of MSNs. The detailed physicochemical properties of the MSNs are listed in Table 1. TEM images, DLS measurements and pH-dependent zeta potential values of various RITC@MSNs and FITC@MSNs are included in the Supporting Information (Figures S1 to S4). Note that the zeta potential values of the various FITC@MSNs and RITC@MSNs were adjusted to be approximately the same as their counterpart MSNs (see Table 1 and Figure S4). 3.2. Positively Charged and Larger MSNs Induced Higher Cellular uptake. To investigate the effects of the surface charge and size of MSNs on cellular uptake efficiency, monocyte-derived macrophages and BEAS-2B cells were treated with FITC@MSNs of different sizes and charges at a concentration of 50 µg mL-1 for 24 h and inspected using flow cytometry and ICP-MS spectrometry. The flow cytometry analysis indicated that the cellular uptake of MSNs was charge and size dependent, with positively charged and larger MSNs resulting in higher cellular uptake (Figure 2A-C). For example, positively charged 100 nm MSNs (+FITC@MSN100) were internalized by the cells much more easily than their counterparts with a negative charge (-FITC@MSN100) (Figure 2A and B). The same experimental observations


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have also been reported29,30 and were attributed to the fact that anionic nanoparticles will be repelled by anionic cell membranes, leading to the inefficient cellular uptake of anionic nanoparticles. In addition, less than 5% of BEAS-2B cells internalized -FITC@MSN100, which is 7.9-fold lower than that of macrophages (Figure 2A). Moreover, the mean fluorescent intensity due to the uptake of -FITC@MSN100 by BEAS-2B cells was 3.7-fold lower than that by macrophages (Figure 2B). These results suggest that the cellular uptake of MSNs is also celltype dependent, with anionic MSNs being more preferentially taken up by phagocytic cells, presumably due to their phagocytosis capability of foreign materials such as nanoparticles. To quantify the aforementioned experimental observations, ICP-MS measurements were performed to estimate the amount of silica internalized by the cells. The ICP-MS analysis revealed a similar charge- and size-dependent cellular uptake of MSNs (Figure 2C), with +MSN250 being most readily taken up by cells, followed by +MSN100, +MSN50, and –MSN100. In particular, the amount of silica internalized due to the uptake of –MSN100 by BEAS-2B cells was 3.8-fold less than that by macrophages, which is consistent with the aforementioned flow cytometry analysis (Figure 2B). To evaluate the dose effect of MSNs on cellular uptake, the two types of cells were treated with +FITC@MSN100 at concentrations ranging from 25 to 1000 µg mL-1 for 24 h. The cellular uptake of MSNs increased with increasing doses, and the dose-dependent effect began to gradually saturate at 500 µg mL-1 in both types of cells (Figure 2D). Our current findings are highly consistent with the study of Vallhov et al., in which larger mesoporous silica microparticles (2.5 µm) and higher concentrations induced higher cellular uptake than smaller nanoparticles (270 nm) and lower concentrations in human dendritic cells.13 However, several conflicting results from our experimental observations can also be found in previous studies in the literature. For example, 50 nm MSNs produced using the same synthetic


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method as that used in this study were reported to enter HeLa cervical cancer cells most efficiently among MSNs with sizes ranging from 30 to 280 nm.9 Moreover, other recent studies indicated that 10 nm and 100 nm MSNs, both synthesized using strategies that differ from ours, caused the best cellular uptake efficiency in the MC3T3-E1 calvarial cell line11 and ovarian cancer cells,10 respectively. These literature results differ from our current findings, and the inconsistencies may result from the different types of cells used and the different intrinsic properties of MSNs prepared using different synthetic methods. There are many differences between cancer cells and normal cells. Cancer cells such as HeLa cells lose the adhesion molecules on the membrane such that they have the ability to metastasize or spread to other areas of the body. This change in membrane property likely caused the different uptake efficiencies of MSNs between cancer cells and non-cancer cells, such as the phagocytic macrophages and BEAS-2B epithelial cells used in this study. In addition, the nanoparticle size of 50 nm was also found to generate the maximum cellular uptake for gold nanoparticles8,31 and quantum dots32 due to the thermodynamic driving force for particle wrapping on the cell membrane.33,34 However, the mesoporous structure of MSNs differs from the smooth surface of gold and quantum dot nanoparticles. It has been reported that the surface structure (topography) of nanoparticles regulates cell membrane penetration35 and that even a small topographical change at the nanoscale could affect cellular behaviors.36 Thus, the membrane wrapping theory may not be applicable for MSNs. 3.3. Intracellular trafficking distribution of MSNs was charge and size dependent. To further explore the intracellular trafficking fate of MSNs, macrophages and BEAS-2B cells were incubated with FITC@MSNs of different charges and sizes and examined using confocal microscopy. All of the different types of MSNs almost gathered around the perinuclear region of


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both types of cells (Figure 3A-B), which is a typical cellular distribution for lysosomes.37 Subsequently, we investigated the colocalization of Lysotracker (a red fluorescent marker dye for late endosomes and lysosomes) with FITC@MSNs. As shown in Figure 3, +FITC@MSN50 in macrophages (Figure 3A) and –FITC@MSN100 in BEAS-2B cells (Figure 3B) exhibited negligible colocalization with Lysotracker. However, different extents of partial colocalization of other types of FITC@MSNs with Lysotracker were observed in both cell types, with positively charged and larger MSNs causing more substantial colocalization. In addition, the resulting lysosome biogenesis (evaluated by Lysotracker uptake using flow cytometry) due to MSN uptake in both types of cells was charge and size dependent, with positively charged and larger MSNs inducing more lysosome formation (Figure 3C). This result further confirms the chargeand size-dependent cellular uptake of MSNs shown in Figure 2A-C since the extent of lysosome biogenesis induced by the uptake of MSNs into the cells would actually reflect the cellular uptake efficiency of MSNs. To determine whether internalized MSNs impair lysosomal membrane stabilization, AO dye (a lysosomal marker) staining was applied to both types of cells exposed to +MSN250 at a concentration of 200 µg mL-1 for 24 h. Figure 3D shows high AO fluorescence intensity inside both types of MSN-treated cells due to AO uptake into intact lysosomes, resulting in the enhanced red fluorescence within the cells. To further validate the aforementioned microscopy analysis, we quantified the lysosomal AO fluorescence intensities within the cells and found that much higher levels of AO-stained intact lysosomes accumulated in the MSN-treated macrophages and BEAS-2B cells (Figure 3E). These results confirm that the MSN-treated cells had intact lysosomes and that the internalized MSNs did not result in the impairment of lysosomes.


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3.4 MSNs induced cell type-, charge- and size-dependent endocytosis. To further explore the mechanism for the cellular uptake of MSNs, the cells were exposed to MSNs for 2 h and then treated with FM1-43 fluorescent dye (an endocytosis marker) for 30 min. Exposure to MSNs resulted in a significant increase in fluorescence relative to the controls (Figure 4A), indicating that the MSNs cause enhanced endocytosis in a charge- and size-dependent manner, with positively charged and larger MSNs stimulating more endocytotic vesicle formation. However, compared to the untreated control, the exposure of BEAS-2B cells to –MSN100 only slightly promoted a 9.9-fold increase in FM1-43-sensitized fluorescence, which is considerably less than the 260-fold enhancement for macrophages. This result implies that the endocytotic entry of anionic MSNs into BEAS-2B cells is extremely inefficient. Most nonviral gene delivery systems38 and nanoparticles, including MSNs,3 enter cells via clathrin-mediated endocytosis and are trafficked to late endosomes and lysosomes. However, as mentioned above (Figure 3A-B), some FITC@MSNs appeared to accumulate in the cytoplasm separated from the Lysotracker-labeled compartments, indicating that other clathrin-independent endocytotic pathways may be stimulated for the uptake of MSNs. To further investigate the specific cellular internalization pathways involved in this process, we measured the cellular uptake of various FITC@MSNs in the presence of different endocytotic inhibitors using flow cytometry. Indeed, none of the known endocytotic pathways were involved in the uptake of – MSN100 by BEAS-2B cells (Figure 4D), which may explain why the uptake of negatively charged MSNs into BEAS-2B cells is inefficient. A similar experimental observation has also been reported for the nonspecific adsorptive endocytosis of negatively charged silica-coated nanoparticles by HeLa cells.39 However, the routes for the internalization of positively charged MSNs are all endocytosis dependent, with larger MSNs recruiting more types of endocytotic


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pathways for cellular uptake. Although +FTIC@MSN50 and +FTIC@MSN100 stimulated the same two types of endocytotic pathways (clathrin- and caveolae-mediated pathways), treatment with chlorpromazine (a clathrin-mediated endocytosis inhibitor) and genistein (a caveolae endocytosis inhibitor) reduced the internalization of +FTIC@MSN100 more markedly (Figure 4B-C), indicating that the endocytosis fluxes induced by +MSN100 were higher than those induced by +MSN50. This result is also consistent with the results of the FM1-43 experiment (Figure 4A). Taken together, these data suggest that the uptake of anionic MSNs into BEAS-2B cells is inefficient due to an unknown endocytotic mechanism involved in the particle internalization process, and a greater amount of positively charged and larger MSNs are taken up into both macrophage and BEAS-2B cells through recruiting more types of endocytotic pathways and more endocytotic fluxes. Recent studies regarding the cellular internalization mechanisms of MSNs are fragmented and mixed. Clathrin-mediated endocytosis has been reported to play an important role in the uptake of 110 nm MSNs into HeLa cells.40 Moreover, caveolae-mediated endocytosis was also found to be responsible for the cellular internalization of naked 10-50 nm MSNs and surfacefunctionalized 150 nm MSNs into ovarian cancer cells10 and HeLa cells,14 respectively. Conversely, rod-shaped MSNs were found to enter HeLa and A549 cells primarily via a macropinocytosis mechanism.41 Comparing our present work with these varied findings in the literature is difficult due to the different cell lines and material syntheses used. Nevertheless, our current study provides a comprehensive understanding of how the charge and size of MSNs influence cellular internalization pathways in a systematic manner. The rationale behind the sizedependent endocytosis in this study remains unclear, and additional studies are required to obtain a better understanding of the entire process.


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3.5 Positively Charged and Larger MSNs Induced Greater Cytotoxicity. To explore the effects of charge, size and dose on MSN-induced cytotoxicity, the viability of MSNs with different charges and sizes at exposure concentrations ranging from 25 to 1000 µg mL-1 for 24 h was evaluated using two different assays: WST-8 and annexin-V/propidium iodide (PI) assays. The WST-8 assay evaluates cytotoxicity based on mitochondrial activity, and the annexin-V/PI assay is a double-staining flow cytometric analysis, in which FITC annexin-V is used for detecting apoptosis and PI dye is used for measuring necrosis cell death. As shown in Figure 5, significant cytotoxicity as measured by WST-8 was not observed for either macrophages (Figure 5A) or BEAS-2B cells (Figure 5B) after 24 h of treatment with different sizes and doses of MSNs, except for BEAS-2B cells exposed to +MSN250 at a concentration of 1000 µg mL-1. To further validate the cytotoxicity results from the WST-8 assay, the annexin-V/PI assay was subsequently used to cross-validate the MSN cytotoxicity results. +MSN100 were selected for cytotoxicity assessment at exposure concentrations from 25 to 1000 µg mL-1 using the annexinV/PI and WST-8 assays. As shown in Figure 5C and D, the cell viabilities resulting from the treatment of 25 to 200 µg mL-1 MSNs for macrophages and 25 to 1000 µg mL-1 for BEAS-2B cells were quite consistent between the two assays. However, only the annexin-V/PI assay showed a slight necrotic cell death (Figure S5, see the Supporting Information) for macrophages exposed to 500 and 1000 µg mL-1 MSNs, thus indicating that annexin/PI appears to be a more sensitive cytotoxicity assay than WST-8. Collectively speaking, negatively charged or smaller MSNs (

Molecular Elucidation of Biological Response to Mesoporous Silica

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