Influence of physicochemical properties of silver nanoparticles on mast cell activation and degranulation

Abstract

Silver nanoparticles (AgNPs) are increasingly being incorporated into products for their antimicrobial properties. This has resulted in increased human exposures and the possibility of adverse health effects. Mast cells orchestrate allergic immune responses through degranulation and release of pre-formed mediators. Little data exists on understanding interactions of AgNPs with mast cells and the properties that influence activation and degranulation. Using bone marrow-derived mast cells and AgNPs of varying physicochemical properties we tested the hypothesis that AgNP physicochemical properties influence mast cell degranulation and osteopontin production. AgNPs evaluated included spherical 20 nm and 110 nm suspended in either polyvinylpyrrolidone (PVP) or citrate, Ag plates suspended in PVP of diameters between 40–60 nm or 100–130 nm, and Ag nanowires suspended in PVP with thicknesses <100 nm and length up to 2 μm. Mast cell responses were found to be dependent on the physicochemical properties of the AgNP. Further, we determined a role for scavenger receptor B1 in AgNP-induced mast cell responses. Mast cell degranulation was not dependent on AgNP dissolution but was prevented by tyrosine kinase inhibitor pretreatment. This study suggests that exposure to AgNPs may elicit adverse mast cell responses that could contribute to the initiation or exacerbation of allergic disease.

Figure 1

Representative TEM images demonstrating AgNP shape and size. AgNPs of differing size and shape were evaluated including spherical 20 nm AgNPs suspended in either polyvinylpyrrolidone (P20) or citrate (C20), spherical 110 nm AgNPs suspended in either polyvinylpyrrolidone (P110) or citrate (C110), Ag plates suspended in polyvinylpyrrolidone of either 550 nm resonance (P550) or 850 nm resonance (P850), and Ag nanowires that were suspended in polyvinylpyrrolidone (Wires).

Figure 2

Representative enhanced dark field images of control or AgNP treated bone marrow-derived mast cells (BMMCs). AgNPs of differing sizes and shapes were evaluated including spherical 20 nm AgNPs suspended in either polyvinylpyrrolidone (P20) or citrate (C20), spherical 110 nm AgNPs suspended in either polyvinylpyrrolidone (P110) or citrate (C110), Ag plates suspended in polyvinylpyrrolidone of either 550 nm resonance (P550) or 850 nm resonance (P850), and Ag nanowires that were suspended in polyvinylpyrrolidone (Wires).

Figure 3

ICP-MS measurement of AgNP uptake by bone marrow-derived mast cells (BMMCs). Cells were pretreated with or without the scavenger receptor B1 (SR-B1) inhibitor Blt2 prior to AgNP exposure. Values are expressed as mean ± SEM (n=3/group). * Indicates significant difference from control group (p ≤ 0.05). # Indicates significant difference of Blt2 pretreated group compared to Blt2 untreated group (p ≤ 0.05).

Figure 4

Mast cell degranulation was evaluated by measuring release of β-hexosaminidase into the supernatant 1 h following AgNP exposure. A) Bone marrow derived mast cell (BMMC) degranulation following exposure to spherical polyvinylpyrrolidone (PVP) coated (red) or citrate coated 20 nm (blue) AgNPs. B) BMMC degranulation following exposure to spherical PVP coated (green) or citrate coated 110 nm (purple) AgNPs. C) 550 nm or 850 nm resonant AgNP plates. D) BMMC degranulation following exposure to PVP coated Ag nanowires. E) BMMC degranulation following exposure to Ag⁺ ions.. F) BMMC degranulation of samples pretreated with or without the scavenger receptor B1 (SR-B1) inhibitor Blt2. Values are expressed as mean ± SEM (n=3/group). * Indicates significant difference from controlled group (p ≤ 0.05). # Indicates significant difference of Blt2 pretreated group compared to Blt2 untreated group (p ≤ 0.05).

Figure 5

Lysosome-associated membrane protein 2 (Lamp2) expression (green) in bone marrow derived mast cells (BMMCs) following AgNP exposure. BMMCs were collected 1 h following AgNP exposure, washed, spun onto glass slides and immunofluorescently stained with DAPI to identify the nucleus (blue) and Lamp2 to determine BMMC degranulation (green).

Figure 6

Osteopontin (OPN) levels were measured in supernatants by ELISA. BMMCs were pretreated with or without the scavenger receptor B1 inhibitor Blt2 before AgNPs exposure.. Values are expressed as mean ± SEM (n=3/group). * Indicates significant difference from controlled group (p ≤ 0.05). # Indicates significant difference of Blt2 pretreated group compared to Blt2 untreated group (p ≤ 0.05).

Figure 7

Bone marrow-derived mast cell (BMMC) degranulation was evaluated by measuring release of β-hexosaminidase 1 h following exposure to 50 μg/ml of 20 nm citrate coated AgNPs. Cells were pretreated with or without imatinib for 30 min at concentration of 0.1, 1, 10, 100 μM. * Indicates significant difference from controlled group (p ≤ 0.05). # Indicates significant difference of Blt2 pretreated group compared to Blt2 untreated group (p ≤ 0.05).