Physicochemical Properties Can Be Key Determinants of Mesoporous Silica Nanoparticle Potency In Vitro
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1 1 Physicochemical Properties Can Be Key Determinants of Mesoporous Silica Nanoparticle Potency In Vitro Dalibor Breznan 1*, Dharani D. Das 1, Christine MacKinnon-Roy 1, Stéphane Bernatchez 2, Abdelhamid Sayari, Myriam Hill 2, Renaud Vincent 1 and Prem Kumarathasan 1 1 Environmental Health Science and Research Bureau, Health Canada, Tunney s Pasture, Ottawa, Ontario, K1A K9, Canada 2 New Substances Assessment and Control Bureau, Health Canada, Tunney s Pasture, Ottawa, Ontario, K1A K9, Canada Department of Chemistry and Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, K1N 6N5, Canada Keywords: mesoporous silica nanoparticles, cytotoxicity, cytokines, pathway analysis, size, surface modification * Send correspondence to: dalibor.breznan@canada.ca or premkumari.kumarathasan@canada.ca
2 2 Supporting Information Biostatistical and Bioinformatic Analyses Analysis of variance: Experimental data (FE) from all assays and cytokine/chemokine release were transformed, where normality and equal variance were not met and analyzed by three-way ANOVA (SigmaPlot v12.5; Systat Software Inc, San Jose, CA, USA). The factors for the analysis of the msinp cytotoxicity data included Size (25, 7,, 17, 6 nm); Modification (Pristine, C-COOH, C-11-COOH) and Dose (,, 1,, µg/cm 2 ) as factors. Where appropriate, reference particles were included in the analysis. In such case, a two-way ANOVA was conducted with the factors Particle and Dose. The analysis of cytokine/chemokine release data was conducted similarly, except fewer doses were included, since subsets of samples only were assessed for release of inflammatory mediators; A549 and J774A.1 cells (, 1,, µg/cm 2 ) and (,, µg/cm 2 ) for THP-1 cells. A two-way ANOVA was conducted to assess the statistical significance of the DLS/EM ratio data with Size (12, 25, 7,, 17, 6 nm) and Modification (Pristine, C-COOH, C-11- COOH) as factors. The data points from analysis conducted in water, DMEM and DMEM + 5% FBS media were pooled for each particle size and modification. Hierarchical cluster analysis: A Hierarchical cluster analysis (HCA) of β potency estimates and for cytotoxicity assays and cytokine/chemokine release was conducted using the city-block distance measure between the data points and average linkage measurements between the clusters. Prior to analysis, the cytokine β potency estimates were adjusted for average cytotoxicity (consensus β cytotoxicity) for each particle, to account for overt particle effects on
3 viability. The rationale for adjusting the cytokine release for average viability was to obtain a better estimate of cytokine release by fully competent cell population in response to particles, while compensating for cells lost through overt cytotoxicity. An additional HCA was conducted to assess the (dis)similarity of the SiNPs on the basis of their DLS/EM size ratio as measured in a variety of liquid media. Similarly, city-block distance measure and average linkage algorithms were utilized. Clustering of all data was conducted using GenePattern version.6. (Broad Institute, MIT, Cambridge, MA, USA) webtool. 1 The heatmaps were generated using the Java TreeView plugin version 1.16.r2. 2 Pathway analysis: The accession numbers of normalized (fold-effects) and cytotoxicityadjusted, differentially produced cytokines/chemokines by A549, J774A.1 and THP-1 cells were tabulated using Microsoft Excel 27 and imported into Ingenuity Pathway Analysis (IPA) webtool (Ingenuity Systems, CA, USA) to identify potential canonical pathways and biological functions associated with the interacting cytokine networks on the basis of the scientifically curated knowledgebase of IPA. The IPA core and comparison analyses were conducted only on data with statistical significance (ANOVA, p<.5) and fold change above. Fisher s exact test (p<.5) and the ratio of the number of molecules from the dataset that mapped onto the networks were used as a criteria for pathway selection. Furthermore, downstream canonical pathways identified by IPA based on the overlap of the p-value and activation Z-score (p.5; Z 2.; Z -2.) were presented, including the predicted direction of inhibition or activation of the specific pathway for the specified dose of particles.
4 4 Supplementary references 1. Reich, M.; Liefeld, T.; Gould, J.; Lerner, J.; Tamayo, P.; Mesirov, J. P. GenePattern 2.. Nat. Genet. 26, 8, Saldanha, A. J. Java Treeview--Extensible Visualization of Microarray Data. Bioinformatics 24, 2,
5 5 Table S1: DLS and zeta potential of the particles measured in water and serum-free cell culture media Sample name Surface function Size (nm) in dh 2 O at.5 mg/ml PDI ζ (mv) in dh 2 O at.5 mg/ml Size (nm) in dh 2 O at.16 mg/ml PDI ζ (mv) in dh 2 O at.16 mg/ml Size (nm) in media at.5 mg/ml PDI ζ (mv) in media at.5 mg/ml Size (nm) in media at.16 mg/ml PDI ζ (mv) in media at.16 mg/ml - 46 ± ±.9 1 ± ± ± ±.7 48 ± ± 1 C-COOH 86 ± ± ± ± ± ± ± ± 1.7 C11-COOH ± ± ± ± ± ±.6 18 ± ± ± ± ± ± ± ± ± ± 1 msinp7 C-COOH 219 ± ± ± ± ± ± ± ± 1 C11-COOH 217 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±.8 msinp C-COOH 185 ± ± 2 17 ± ± ± ±.6 54 ± ±.7 C11-COOH 19 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 2.7 msinp17 C-COOH 19 ± ± ± ± ± ± 1.1 ± ± 1.7 C11-COOH 21 ± ± ± ± 41 ± ± ± ±.8-79 ± ± ± ± ± ± ± ± 1 msinp6 C-COOH 768 ± ±.7 74 ± ± ± ± ± ±.9 C11-COOH 612 ± ± ± ± 1 87 ± ± ± ± 1.1 µsio ± ± ± ± ± ± ± ± The hydrodynamic (DLS) size, polydispersity index (PDI) and zeta potential (ζ) values for the msinps in dh 2O only are compiled from Das et al., 214 DLS size, PDI and ζ values for only are compiled from Breznan et al., 216 DLS size, PDI and ζ values are presented at.5 and.16 mg/ml of particles representing and µg/cm 2 of well surface area respectively, in water and serum-free cell culture media The hydrodynamic size measurements with PDI above.5 should be taken as less reliable
6 6 Table S2: Integrated particle potency estimates across the cell lines Cytotoxic Potency Inflammatory Potency Overall Potency Particle ß R CELLS Relative Potency (%) Potency Ranking ß I-V CELLS Relative Potency (%) Potency Ranking Iβ CELLS Relative Potency (%) Potency Ranking C C msinp msinp7-c msinp7-c msinp msinp-c msinp-c msinp msinp17-c msinp17-c msinp msinp6-c msinp6-c μsio Biological reactivity of the particles β R CELLS represents the average of absolute values of the cytotoxic potency β V CELLS Inflammatory potency β I-V CELLS is the average of the absolute values of cytokine and chemokine potency values from each cell line Integrated potency Iβ CELLS represents the average of the potency estimates β R CELL and β I-V CELLS across the cell lines Relative Potency (%) is the percent value of the potency estimate in relation to the most potent particle for that estimate Potency ranking indicates the ranking order of the msinps from the most potent (1) to the least potent (15) and μsio 2 reference particles were not ranked
7 7 Table S: Correlation of the potency estimates in the individual cell lines and the physicochemical properties of the msinps Potency Estimate ß R A549 ß R THP-1 ß R J774A.1 ß I-V A549 ß I-V THP-1 ß I-V J774A.1 Iß A549 Iß THP-1 Iß J774A.1 DLS/EM med DLS/EM hi SA BET < Zeta Water med Zeta Water hi Endotoxin (EU/mg) Top number (R-value), bottom number (p-value); Correlations include msinps Significant correlations (p.5, 2-tailed test) are shown in bold-type, grey-fill; Marginal correlations (.5 < p <.1, 2-tailed test) are shown in grey-fill DLS/EM is the agglomeration size / dry-state size ratio DLS was conducted in DMEM media with FBS at μg/cm 2 (medium) and μg/cm 2 (high) dose of msinps Zeta potential was measured in water at the and μg/cm 2 doses of msinps β R, β I-V and Iβ are the cellular reactivity, inflammatory potency and the integrated particle potency of the msinps, respectively
8 Figure S1: Heatmap with hierarchical cluster analysis of the DLS/EM ratio data for the tested SiNPs subjected to DLS in different liquid media, red color represents ratio >1., green is ratio <1.. 8
9 9 A 2. A549 Size x Mod (P =.1) Dose (P <.1) FE Cytosolic ATP C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c B FE Cellular LDH Release A549 Size x Mod (P =.) Dose (P <.1). -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp msinp6-c msinp6-c11
10 1 C FE Resazurin Reduction A549 Size (P =.4) Dose (P =.1). -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 D 2. A549 Size x Mod x Dose (P <.1) FE BrdU Incorporation C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11
11 Figure S2. Cytotoxic profile of A549 cells exposed to, 1, and µg/cm 2 of the silica particles for 24 h. An integrated bioassay was performed comprising of ATP assay (A), LDH release assay (B), Resazurin assay (C) and BrdU incorporation assay (D). Data were expressed as mean fold-effect (FE) ± SEM for n = independent experiments, with technical replicates within each experiment. 11
12 12 A 2. J774A.1 Size x Mod x Dose (P <.1) FE Cytosolic ATP C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c B FE Cellular LDH Release J774A.1 Size x Mod (P <.1) Size x Dose (P =.5) Mod x Dose (P <.1). -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c msinp6 msinp6-c msinp6-c11
13 1 C 2. J774A.1 Size x Mod x Dose (P <.1) FE Resazurin Reduction C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c D 2. J774A.1 Size x Mod (P <.1) Size x Dose (P =.) FE BrdU Incorporation C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11
14 Figure S. Cytotoxic profile of J774A.1 cells exposed to, 1, and µg/cm 2 of the silica particles for 24 h. An integrated bioassay was performed comprising of ATP assay (A), LDH release assay (B), Resazurin assay (C) and BrdU incorporation assay (D). Data were expressed as mean fold-effect (FE) ± SEM for n = independent experiments, with technical replicates within each experiment. 14
15 15 A 2. THP-1 Size x Mod x Dose (P =.5) FE Cytosolic ATP C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c B 4. THP-1 Mod x Dose (P <.1) FE Cellular LDH Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c msinp6 msinp6-c msinp6-c11
16 16 C 2. THP-1 Size x Mod (P =.2) FE Resazurin Reduction C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp msinp6-c msinp6-c11 Figure S4. Cytotoxic profile of THP-1 cells exposed to, 1, and µg/cm 2 of the silica particles for 24 h. An integrated bioassay was performed comprising of ATP assay (A), LDH release assay (B) and Resazurin assay (C). BrdU ELISA was not applicable for differentiated THP- 1 cells. Data were expressed as mean fold-effect (FE) ± SEM for n = independent experiments, with technical replicates within each experiment.
17 17 A B FE IL-6 Release Size x Mod x Dose (P =.29) A549 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c FE IL-1ra Release A549 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Mod (P =.18) Size (P =.4) Dose (P =.)
18 18 C A549 Size x Mod x Dose (P =.) FE IL-8 Release D -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 4. A549 Size x Mod (P =.4) Mod x Dose (P <.1) FE IL-9 Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11
19 19 E F FE IL-12 Release Size x Mod x Dose (P =.7) A549 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c FE IL-1 Release Size x Mod x Dose (P =.1) A549 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11
20 2 G 5. A549 Size x Mod (P =.8) FE G-CSF Release Size x Dose (P =.2) Mod x Dose (P <.1) H -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 4. A549 Mod x Dose (P =.2) Size (P =.15) FE IFN Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c msinp6 msinp6-c msinp6-c11
21 21 I J FE IP-1 Release A549 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Dose (P =.16) FE MCP-1 Release A549 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Mod x Dose (P =.8) Size (P <.1)
22 22 K 2.5 A549 Size x Mod x Dose (P =.2) FE VEGF Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp msinp6-c msinp6-c11 Figure S5. Secretory cytokine/chemokine release from A549 cells exposed to silica particles for 24 h. The following cytokines were statistically significant by ANOVA (P <.5): Interleukin-1 receptor antagonist, IL-1ra (A), IL-6 (B), IL-8 (C), IL-9 (D), IL-1 (E), IL-12 (F), Granulocyte-colony stimulating factor, G-CSF (G), Interferon gamma, IFNγ (H), IFNγ-Inducible Protein 1, IP-1 (I), Monocyte chemoattractant protein-1, MCP-1 (J) and Vascular endothelial growth factor, VEGF (K). Cytokine/chemokine levels were quantitated in cell culture supernatants using the Bio-Plex assay. Data were presented as mean values ± standard error mean, relative to control values (fold-effect) and adjusted for cell viability on a dose by dose basis, n = independent experiments, with technical replicates within each experiment.
23 2 A. J774A.1 Size x Mod x Dose (P =.2) FE IL-1 Release B -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11. J774A.1 Size x Mod x Dose (P =.9) FE IL-9 Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11
24 24 C D -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c FE IL-1 Release J774A.1 Mod (P =.9) Dose (P =.11) msinp-c11 msinp17-c11 msinp6-c FE IL-15 Release J774A.1 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp17 msinp17-c msinp6 msinp6-c Size x Mod (P =.42) Dose (P =.1)
25 25 E J774A.1 Size x Mod (P <.1) Size x Dose (P <.1) FE IL-17 Release Mod x Dose (P <.1) C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c F J774A.1 Size x Mod x Dose (P <.1) -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c FE IP-1 Release
26 26 G H FE MCP-1 Release J774A.1 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Size x Mod x Dose (P <.1) FE MIP-1 Release J774A.1 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Size x Mod x Dose (P <.1)
27 27 I J FE MIP-1 Release J774A.1 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Size x Mod (P =.1) Size x Dose (P =.1) FE MIP-2 Release J774A.1 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Size x Mod x Dose (P =.)
28 28 K J774A.1 Size x Mod (P =.11) Size x Dose (P =.42) FE RANTES Release Mod x Dose (P <.1) 2.. -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c L. 2. J774A.1 Size x Mod (P =.) Size x Dose (P =.2) FE TNF Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11
29 Figure S6. Secretory cytokine/chemokine release from J774A.1 cells exposed to silica particles for 24 h. The following cytokines were statistically significant by ANOVA (p<.5): Interleukin-1 alpha, IL-1α (A), IL-9 (B), IL-1 (C), IL-15 (D), IL-17 (E), Interferon gamma-inducible protein 1, IP-1 (F), Monocyte chemoattractant protein-1, MCP-1 (G), Macrophage Inflammatory Protein alpha, MIP-1α (H), Macrophage Inflammatory Protein beta, MIP-1β (I), MIP-2 (J), Regulated on activation, normal T cell expressed and secreted, RANTES (K) and Tumor necrosis factor alpha, TNF-α (L). Cytokine/chemokine levels were quantitated in cell culture supernatants using the Bio-Plex assay. Data were presented as mean values ± standard error mean, relative to control values (fold-effect) and adjusted for cell viability on a dose by dose basis, n = independent experiments, with technical replicates within each experiment. 29
30 A 8. THP-1 Size x Dose (P =.26) Mod x Dose (P <.1) FE IL-1 Release B -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c THP-1 Size x Mod x Dose (P =.4) FE IL-1ra Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c 1 msinp17-c11 msinp6 msinp6-c msinp6-c11
31 1 C THP-1 Mod x Dose (P =.5) Size (P =.1) FE IL-4 Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 1 D 1. THP-1 Size x Mod x Dose (P =.1) FE IL-8 Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 1 msinp6 msinp6-c msinp6-c11
32 2 E THP-1 Mod x Dose (P <.1) Size (P <.1) FE IL-9 Release F -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c THP-1 Size x Mod x Dose (P <.1) FE IL-1 Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c 1 msinp17-c11 msinp6 msinp6-c msinp6-c11
33 G. 2.5 THP-1 Size x Mod x Dose (P =.16) FE IL-12 Release H -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c THP-1 Size x Mod x Dose (P =.15) FE IL-17 Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c 1 msinp17-c11 msinp6 msinp6-c msinp6-c11
34 4 I 15. THP-1 Size x Mod x Dose (P =.18) FE FGF-b Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 1 J 4. THP-1 Size x Mod x Dose (P =.29) FE G-CSF Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 1 msinp6 msinp6-c msinp6-c11
35 5 K THP-1 Size x Mod (P =.2) Mod x Dose (P =.5) FE IFN Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 1 L THP-1 Size x Mod x Dose (P <.1) FE IP-1 Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c 1 msinp17-c11 msinp6 msinp6-c msinp6-c11
36 6 M N 1 FE MCP-1 Release THP-1 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Size x Mod x Dose (P =.17) 1 FE MIP-1 Release THP-1 -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c11 Mod x Dose (P <.1)
37 7 O 12. THP-1 Size x Mod x Dose (P =.18) FE MIP-1 Release P -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c msinp17-c11 msinp6 msinp6-c msinp6-c THP-1 Size x Mod (P =.1) Size x Dose (P =.) FE RANTES Release Mod x Dose (P <.1).5. -C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c 1 msinp17-c11 msinp6 msinp6-c msinp6-c11
38 8 Q. THP-1 Size x Mod x Dose (P =.2) 2.5 FE VEGF Release C -C11 msinp7 msinp7-c msinp7-c11 msinp msinp-c msinp-c11 msinp17 msinp17-c 1 msinp17-c11 msinp6 msinp6-c msinp6-c11 Figure S7. Secretory cytokine/chemokine release from THP-1 cells exposed to silica particles for 24 h. The following cytokines were statistically significant by ANOVA (p<.5): Interleukin-1 beta, IL-1β (A), Interleukin-1 receptor antagonist, IL-1ra (B), IL-4 (C), IL-8 (D), IL-9 (E), IL-1 (F), IL-12 (G), IL-17 (H), Fibroblast growth factor-basic, FGF-b (I), Granulocyte-colony stimulating factor, G-CSF (J), Interferon gamma, IFNγ (K), IFNγ-Inducible Protein 1, IP-1 (L), Monocyte chemoattractant protein-1, MCP-1 (M), Macrophage Inflammatory Protein alpha, MIP-1α (N), Macrophage Inflammatory Protein beta, MIP-1β (O), Regulated on activation, normal T cell expressed and secreted, RANTES (P) and Vascular endothelial growth factor, VEGF (Q). Cytokine/chemokine levels were quantitated in cell culture supernatants using the Bio-Plex assay. Data were presented as mean values ± standard error mean, relative to control values (fold-effect) and adjusted for cell viability on a dose by dose basis, n = independent experiments, with technical replicates within each experiment.
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