Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood Sonya A. MacParland a,b,, Kim M. Tsoi c,d,, Ben Ouyang c, Xue-Zhong Ma a, Justin Manuel a, Ali Fawaz b, Mario A. Ostrowski b, Benjamin A. Alman e, Anton Zilman f, Warren C.W. Chan c,g,h,i,j, Ian D. McGilvray a, AUTHOR ADDRESSES amulti Organ Traplant Program, Toronto General Research Ititute, University Health Network, 2 Elizabeth Street, Toronto, Ontario, Canada, M5G 2C4 bdepartment of Immunology, University of Toronto, Medical Sciences Building, Room 6271, 1 King's College Circle, Toronto, Ontario, Canada, M5S 1A8 cititute of Biomaterials and Biomedical Engineering, University of Toronto, Rosebrugh Building, Room 47, 164 College Street, Toronto, Ontario, Canada, M5S 3G9 ddivision of Orthopaedic Surgery, University of Toronto, 149 College Street, Toronto, Ontario, Canada, M5T 1P5 edepartment of Orthopaedic Surgery, Duke University, Duke University Medical Center, Room 2888, 2 Trent Drive, Durham, North Carolina, USA, 2771 fdepartment of Physics, University of Toronto, 6 St. George Street, Toronto, Ontario, Canada, M5S 1A7 gterrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, 16 College Street, Room 23, Toronto, Ontario, Canada, M5S 3E1 hdepartment of Chemical Engineering, University of Toronto, 2 College Street, Toronto, Ontario, Canada, M5S 3E5 idepartment of Chemistry, University of Toronto, 8 St George Street, Toronto, Ontario, Canada, M5S 3H6 j Department of Material Science and Engineering, University of Toronto, 16 College Street, Room 45, Toronto, Ontario, Canada, M5S 3E1 KEYWORDS: nanoparticle, macrophage, phenotype, phagocytosis, Kupffer cell, cytokine co- first authors co-corresponding authors
Supplementary Figure 1: Rationale for nanoparticle dose selection. In order to select a dose for the macrophage polarization studies, RAW 264.7 cells were incubated with a range of doses of 1nm fluorescent gold nanoparticles. Based on these findings, a dose of 1.6x1 14 nm 2 /well was selected.
Supplementary Figure 2: Expression of M1 and M2 markers in differentially polarized macrophages and monocytes. Flow cytometry histograms showing mean fluorescence inteity for M1 and M2 surface marker expression. Monocytes/macrophages gated as in Figure 2B. Gates for surface stai set based on background in NP- treated utained controls.
Supplementary Figure 3: Brightfield images of polarized macrophages taken at 4x: scale bar 5μm (Viewed with a Leica DM IL microscope).
Supplementary Figure 4: IFN- γ/lps (M1) and LPS/IL- 1 β (M2b)- polarized cells are producing M1 cytokines TNF- α and IL- 12. Freshly isolated monocytes were incubated with 1nm AuNP, washed and then exposed to the established macrophage polarization conditio. After a 6 hr stimulation with 1μg/ml LPS in the presence of brefeldin- A and monein (1:1), CD68 surface- labeled cells were permeablized and stained using the following anti- human antibodies: anti- IFN- γ- AF7, anti- TNF- α, anti- IL12/23, anti- IL- 6 and anti- IL- 1. Gating as in Figure. 2B. Gating for intracellular cytokine staining was calculated based on fluorescence- minus- one (FMO) controls. FMO MCSF IFN-γ/LPS LPS/IL-1β IL-4/IL-13 IL-4/IL-1 TGF-β/IL-1 TNF-α FSC-A IL-12 FSC-A IFN-γ FSC-A IL-1 FSC-A IL-6 FSC-A
Supplementary Figure 5: Flow cytometry correlates with ICP- MS when assessing for uptake of 6 and 1nm fluorescent gold nanoparticles. A&B) RAW 264.7 cells were incubated with six doses of 15, 6, and 1nm fluorescent gold nanoparticles for 4 hours. C) M2C- polarized primary human macrophages were incubated with 4 doses of 1nm fluorescent gold nanoparticles for 4 hours. Plates were prepared in duplicate and one replicate was analyzed for nanoparticle uptake with flow cytometry and the other with ICP- MS. A correlation could not be established for 15nm nanoparticles as no uptake was detected. Correlation was assessed using linear regression and the slope and R squared value for goodness of fit are reported. Plotted are results from 4 independent replicates. ICP- MS values for the three lowest doses of 6nm nanoparticles were below the limit of detection and are not included. A) Raw 264.7 Cells 6nm AF75-AuNPs B) Raw 264.7 Cells 1nm AF75-AuNPs C) M2C Macrophages 1nm AF75-AuNPs Flow Cytometry (Mean Fluorescence Inteity) 5 4 3 2 1 Slope: 3.7±.8 R 2 :.65 2 4 6 8 ICP-MS (#NP/Cell)
Supplementary Figure 6: All macrophage polarizatio preferentially take up larger nanoparticles. Polarized macrophages were incubated with 15nm, 6nm or 1nm fluorescent gold nanoparticles for four hours. Uptake was assessed by flow cytometry and two parameters were utilized. First, the percentage of cells that were nanoparticle positive (%NP+ Cells) compared with untreated controls is reported (i). Second, the mean fluorescence inteity of the nanoparticle- positive gate is demotrated (ii). As 15nm nanoparticles were not taken up by any of the polarized macrophages, they were excluded from the mean fluorescence inteity analysis. Error bars are representative of four independent replicates. Statistical significance was evaluated using a two- tailed independent Student s t- test (P<.1, P<.1, P<.5, = not significant or P>.5).
Supplementary Figure 7: Nanoparticle uptake depends on macrophage polarization. Fresh monocytes (Fresh mono) and/or polarized macrophages were plated and exposed to A) 1nm and B) 6nm fluorescent gold nanoparticles or C) 1nm fluorescent silica nanoparticles for four hours. Dose was normalized based on total nanoparticle surface area per well. Mean fluorescence inteity for 1nm AuNP uptake was normalized for each experiment. Normalized MFI= [MFI for NP uptake/maximum MFI for NP uptake]1. Percentage and MFI of each monocyte/macrophage type that are nanoparticle- positive following 1nm AuNP exposure. Error bars are representative of at least A) 6 and B) 4 and C) 3 independent experiments, ± s.e.m. Statistical significance was evaluated using an unpaired student s T test (P<.1, P<.1, P<.5 =not significant or P>.5). A. 1nm AuNPs % NP + Cells 15 1 5 Monocytes M1 MCSF M2A M2B M2 M2C Normalized MFI 1 8 6 4 2 Monocytes M1 M-CSF M2A M2B M2 M2C B. 6nm AuNPs 1 8 1 % NP + Cells 6 4 2 Monocytes M1 MCSF M2A M2B M2 M2C Normalized MFI 5 Monocytes M1 M-CSF M2A M2B M2 M2C C. 1nm Silica NPs 4 15 % NP + Cells 3 2 MFI 1 5 1 M1 M2C M1 M2C
Supplementary Figure 8: Early nanoparticle uptake depends on macrophage polarization. M1 and M2C polarized macrophages were plated and exposed to 1nm fluorescent gold nanoparticles for 2, 4, 8 and 24 hours. Experiments were carried out in triplicate. Dose was normalized based on total nanoparticle surface area per well. Error bars are representative of at least 3 independent experiments, ± s.e.m. Statistical significance was evaluated using an unpaired student s T test ( P<.1, P<.5 =not significant or P>.5). 1 1nm 1nm -M1 1nm- M2C 15 1nm 1nm-M1 1nm- M2C % NP+ CD68 Cells % NP+ CD68 Cells 8 6 4 4 3 2 1 2 4 6 8 1 12 14 16 18 2 22 24 Hours 1 8 6 4 4 3 2 1 6nm 2 4 6 8 1 12 14 16 18 2 22 24 Hours 6nm-M1 6nm- M2C NP+ MFI (AF-75) NP+ MFI (AF-75) 1 5 2 4 6 8 1 12 14 16 18 2 22 24 Hours 15 1 5 6nm 6nm-M1 6nm- M2C 2 4 6 8 1 12 14 16 18 2 22 24 Hours 1 15nm 15nm 8 15nm- M1 15nm- M2C 15 15nm- M1 15nm- M2C % NP+ CD68 Cells 6 4 4 3 2 1 NP+ MFI (AF-75) 1 5 2 4 6 8 1 12 14 16 18 2 22 24 Hours 2 4 6 8 1 12 14 16 18 2 22 24 Hours
Supplementary Figure 9: Correlation of M1 and M2 surface marker expression and uptake of 6nm Au NP. Monocytes and polarized macrophages were plated and exposed to 6nm fluorescent gold nanoparticles for four hours. Dose was normalized based on total nanoparticle surface area per well. Cells were then stained with CD68 to identify monocytes/macrophages and with anti- human HLA- DR, CD25 and CD86 (M1 surface markers) and anti- human CD163, CD26 and CD29 (M2 surface markers). Staining determined as described in Fig. 2. A, M2 markers positively correlate with uptake of 6nm AuNP. i) Representative plots showing the correlation between CD26 MFI and uptake of 6nm AuNP and CD163 MFI and uptake of 6nm AuNP for all 7 monocyte/macrophage polarizatio for a single experiment. ii) Cumulative plots showing the correlatio between CD29, CD26 and CD163 MFI and uptake of 6nm AuNP. Plots show all 7 monocyte/macrophage polarizatio for 4 separate experiments. B, M1 markers CD86 and HLA- DR MFI do not correlate with uptake of 6nm AuNP, CD25 expression does correlate with uptake of 6nm AuNP. Cumulative plots showing the correlatio between CD25, CD86 and HLA- DR MFI and uptake of 1nm AuNP. Plots show all 7 monocyte/macrophage polarizatio for 4 separate experiments. All correlatio were described using the Spearman s rank correlation coefficient. P<.5 coidered significant.
Supplementary Figure 1: Nanoparticle pre- treatment does not prevent macrophage polarization. Flow cytometry histograms showing mean fluorescence inteity for M1 and M2 surface marker expression in monocytes pre- treated with 1nm AuNP (1nm pre- treat) or vehicle control (CV pre- treat) prior to polarization. Purified monocytes were exposed to 1nm AuNP or control vehicle for 4 hours prior to polarization with TGF- β/il- 1 as described in Fig. 1. Polarized macrophages gated as in Figure 2B. Gates for surface stai were set based on background in NP- treated utained controls.
Supplementary Figure 11: NP pre- treatment for 7 days does not impact macrophage viability. Macrophage viability was measured using Annexin- V and 7- AAD stai, which reflect early and late apoptosis. Flow cytometry plots showing Annexin- V/7 AAD expression in monocytes pre- treated with 1nm AuNP (1nm AuNP pre- ) or vehicle control (CV treated) prior to polarization. Macrophages were ungated for viability analysis. Quadrants set based on background staining in 1nm AuNP treated, utained controls. Quadrants show live cells in the lower left quadrant (Live), early apoptotic cells in the lower right quadrant (Early), late apoptotic cells in the upper right quadrant (Late) and fragmented or necrotic cells (Nec) in the upper left quadrant. Utained 1nm AuNP Early% 1.4.5 Late% Live% Annexin V 96. 2. Nec% 7 AAD CV treated 1nm AuNP pre-mcsf 1nm AuNP pre-m1 4.3 1. 16.1 1.2 32.4.7 Annexin V 5.3 8.4 7.5 12.2 58.4 8.5 7 AAD 1nm AuNP pre-m2b 1nm AuNP pre-m2 1nm AuNP pre-m2c 21.3.5 34.2.6 31.5.3 Annexin V 74. 4.2 58.1 7.1 64.1 4.1 7 AAD
Supplementary Figure 12: Gold nanoparticles of different sizes are not cytotoxic in primary human macrophages polarized towards an M1 or M2c phenotype. Macrophage viability was measured using Annexin- V and 7- AAD stai, which reflect early and late apoptosis. Quadrants show live cells in the lower left quadrant (Live), early apoptotic cells in the lower right quadrant (Early), late apoptotic cells in the upper right quadrant (Late) and fragmented or necrotic cells (Nec) in the upper left quadrant.
Supplementary Figure 13: Characterization of Silica nanoparticles. i) Schematic of silica particles used in this study. 1 nm silica nanoparticles were grafted with 5 polyethylene glycol (PEG) per nm 2 of nanoparticle surface area (1% 5 kda methoxy- PEG- succinimidyl valerate). Subsequently, AlexaFluor 75 was added at a concentration of 1.8 fluorophores per nm 2 of nanoparticle surface area to render silica particles fluorescent. ii) Hydrodynamic diameter of functionalized silica particles. Hydrodynamic diameter measurements were conducted in water. Abbreviation: Polydispersity Index, PDI.