Non-Functionalized Ultrasmall Silica Nanoparticles Directly and SizeSelectively Activate T Cells Authors: Bradley Vis†‡§*, Rachel E. Hewitt†‡*, Nuno Faria†‡, Carlos Bastos†‡, Helen Chappell‡Φ, Laetitia Pele†‡, Ravin Jugdaohsingh†‡, Stephen D. Kinrade§, Jonathan J. Powell†‡ † Biomineral Research Group, Department of Veterinary Medicine, University of Cambridge, Madingley Road, Cambridge CB3 0ES, UK. ‡ Biomineral Research Group, Department of Mineral Science and Technology, MRC Elsie Widdowson Laboratory, Fulbourn Road, Cambridge CB1 9NL, UK. § Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario P7B 5E1, Canada. Φ School of Food Science and Nutrition, University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK *Equal contributors Corresponding author: Jonathan Powell. [email protected] Figure S1. Model of a 2.9 nm USSN derived from the manual packing of a previous DFT-optimised model of a (SiO2)24 cluster.26 The original small clusters were simulated in a hydrated environment and represent the lowest energy state. However, no further relaxation was carried out on the larger particle and a hydration shell was not included. (A 0.35 nm hydration shell would increase the hydrated particle size to 3.6 nm for example). Figure S2. IL-1β levels in PBMC supernatants after 3 h treatment challenge followed by a 21 h incubation. PBMC cultures were unstimulated or stimulated with 10 ng/mL lipopolysaccharide for 3 h prior to challenge with silica. Data are reported as means ± standard deviation for 4 replicates. * denotes signifcance against the control (p00.05, one-way ANOVA). Figure S3 Dissolution of 800 μM USSNs in cell-free growth media at 37 °C. The percentage of dissolved silica was determined through ultrafltration (03 kDa). Means ± standard deviation for 6 replicates. Figure S4. The cell distribution in enriched T cell cultures. Cells were frst selected based on generous FSC versus SSC profles that also excluded debris, followed by T cell (CD3), and subsequent monocyte (CD11c), B cell (CD19) and unstained (CD3(-)CD11c(-)(CD19(-)) gates. Figure S5. Gating strategy employed for assessing CD25 and CD69 on CD4 and CD8 T cells in PBMC culture. Cells were frst selected based on a T cell FSC versus SSC profle that also excluded debris, followed by a viable T cell (CD3(+)), and a CD4(+) or CD8(+) gate. Percentage of cells positive for CD25 and CD69 were assessed through quadrant gating, and mean fuorescent intensity for the activation markers was recorded. Figure S6. Gating strategy employed for assessing proliferation of CD4 and CD8 T cells in PBMC culture Cells were frst selected based on a T cell FSC versus SSC profle that also excluded debris, followed by a viable T cell (CD3(+)), and a CD4(+) or CD8(+) gate. Percentage of cells divided was assessed through CFDA-SE LOW gate. Figure S7. Gating strategy employed for assessing CD69 on Jurkat cells Cells were frst selected based on a FSC versus SSC profle that excluded debris, followed by a viable T cell (CD3(+)). Percentage of cells positive for CD69 was plotted against the SSC profle. Table S1. USSN zeta-potential and derived count rate in PBS and RPMI. Although USSN-free PBS and RPMI both contained species with zeta-potentials similar to the USSN containing media, the derived count rate (kcps), which is a measure of particle density based on scattering intensity, of the media containing USSN was higher than the USSN-free media. This indicated that particles were detected in the complex media, and the zeta-potentials correspond to that of the USSN. The decrease in charge magnitude in the complex media versus ultrapure water (UHP) is consistent with trends reported in the literature.69 Zeta-potential /mV kcps PBS -22.4±0.9 222 +/- 22 PBS + USSN -19.0 ± 4.4 1436 ± -519 RPMI -11.2+/-1.6 29.0+/-7.8 RPMI + USSN -16.0 ± 1.9 356 ± 89 Table S2. The 100 gene pathways which are most signifcantly up regulated in PBMC culture by 150 μM USSN. NAME KEGG_LYSOSOME SIZE NES 122 FDR q-val 2.412125 0 TRANSFERRIN.ENDOCYTOSIS.AND.RECYCLING 29 2.34737 0 KEGG_COLLECTING.DUCT.ACID.SECRETION 27 2.332215 0 IRON.UPTAKE.AND.TRANSPORT 43 2.306844 0 112 2.278574 0 WP2700.LATENT.INFECTION.OF.HOMO.SAPIENS.WITH.MYCOBACTERIUM.TUBERCULOSIS 30 2.270007 0 LATENT.INFECTION.OF.HOMO.SAPIENS.WITH.MYCOBACTERIUM.TUBERCULOSIS 33 2.214397 2.09E-04 PHAGOSOMAL.MATURATION.EARLY.ENDOSOMAL.STAGE. 33 2.227816 2.35E-04 INSULIN.REC

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