Confirmation of BMMCs results in RBL-2H3

<p>(A) Cells were pre-treated with Blt-2 (50 μM) 30 min prior to AgNP (50 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed into supernatants. (B) Mast cells degranulation was measured following exposure to AgNPs (50 μg/ml) in the presence and absence of calcium. (C) Cells were pre-treated with the indicated inhibitors 30 min prior to AgNP (50 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed into supernatants. (D) Cells were treated with AgNPs (25 or 50 μg/ml) for 1, 6, and 24 h and cell viability was assessed by measuring the conversion of MTS into formazan. (E) Representative immunoblots for p-PLCγ and p-PI3K of mast cell in the presence or absence of DNP (100 ng/ml) for 5 min or AgNPs (25 μg/ml) for 5 and 30 min. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from AgNP-treated group (p≤0.05)</p

Mast cell degranulation following exposure to AgNPs

<p>Mast cell degranulation was assessed by measuring the release of β-hexosaminidase into supernatants. (A) Cells were pre-treated with the SR-B1 inhibitor Blt-2 (50 μM), SR-B1 specific antibodies (1:100 dilution) 30 min prior to AgNP (25 μg/ml) exposure for 1h and release of β-hexosaminidase was assessed. (B) Cells were sensitized with anti-DNP IgE overnight and then exposed to AgNPs (25 μg/ml) for 1 h and release of β-hexosaminidase was assessed. (C) Cells were pre-treated with Blt-2 (50 μM) for 30 min then activated with either DNP (30 min) or AgNP (1 h) and release of β-hexosaminidase was assessed. (D) Representative immunoblots for global p-Tyr and p-Ser/Thr following DNP (100 ng/ml) or AgNPs (25 μg/ml) exposure. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from AgNP-treated group (p≤0.05)</p

Characterization of 20nm citrate-coated AgNPs in water and HEPES buffer

<p>Characterization of 20nm citrate-coated AgNPs in water and HEPES buffer</p

PLC and PI3K signaling in response to AgNPs

<p>Mast cell degranulation was assessed by measuring the release of β-hexosaminidase into supernatants. Cells were pre-treated with (A) the PLCγ inhibitor U73122 (1 μM) or (B) the PI3K inhibitor wortmannin (100 nM) 30 min prior to AgNP (25 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed. (C) Representative immunoblots for p-PI3K in samples pretreated with or without Blt-2 (50 μM) and followed by AgNP exposure for 1 h. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from AgNP-treated group (p≤0.05)</p

Time Course of Mast Cell Association of AgNPs

<p>(A) Representative Transmission Electron Microcopy (TEM) image demonstrating AgNP shape and size. (B) Representative TEM images of mast cells following exposure to 20 nm AgNPs over time. Mast cells were treated with AgNPs (25 μg/ml) for 10, 20, 30 and 60 min and AgNPs uptake by mast cells was assessed. Arrow indicates AgNPs that were being taken up by a mast cell (inset). A representative image was obtained from at least 5 different images.</p

A schematic representation of proposed signaling pathway involved in activation of mast cells by AgNPs

<p>We propose that AgNPs interact with SR-B1 leading to recruitment of PDZK1 (SR-B1 adaptor protein), which activates downstream signaling cascade involving PI3K and PLCγ. Inositol 1,4,5-triphosphate (IP₃), which is released following activation of PLCγ, interacts with its receptor IP₃R on smooth endoplasmic reticulum (SER) leading to the release of Ca²⁺ from ER stores. As a result of a drop in Ca²⁺ levels in SER, the CRAC Ca²⁺ channels (cell membrane) are activated leading to influx of extracellular Ca²⁺. Increasing intracellular Ca²⁺ levels ([Ca²⁺]_i) is ultimately culminated in mast cell degranulation. PKC: Protein kinase C; IP₃ (InsP₃): inositol triphosphate; PtdIns(4,5)P₂: phosphatidylinositol-4,5-bisphosphate (PIP₂); PtdIns(3,4,5)P₃: phosphatidylinositol-3,4,5-Triphosphate (PIP₃); PIP₂ and PIP₃ are membrane phospholipids; DAG: diacylglycerol. cSrc: cellular sarcoma (protein tyrosine kinase); TRPC: transient receptor potential cation channels.</p

Calcium signal in mast cell following exposure to AgNPs

<p>Mast cell degranulation was assessed by measuring the release of β-hexosaminidase into supernatants. (A) Mast cells degranulation was measured following exposure to AgNPs in the presence and absence of calcium. (B–left panel) Cells were stained with Fluo-4 AM (5 μM) and mean fluorescence intensity was assessed before (baseline NT control, solid line) and after exposure to ionomycin (1 μM) or AgNPs (50 μg/ml) (dotted line) for 2 min. (B–right panel) A representative graph of 3 independent experiments showing fold change of mean fluorescence intensity relative to NT control. (C) Cells were pre-treated with the CRAC calcium channels inhibitor Synta (10 μM) 30 min prior to AgNP (25 μg/ml) exposure for 1 h and release of β-hexosaminidase was assessed. Values are expressed as mean ± SEM of at least 3 independent experiments. *Indicates significant difference from controlled group (p≤0.05). #Indicates significant difference from indicated groups (p≤0.05)</p

Silver Nanoparticle Protein Corona Composition in Cell Culture Media

<div><p>The potential applications of nanomaterials as drug delivery systems and in other products continue to expand. Upon introduction into physiological environments and driven by energetics, nanomaterials readily associate proteins forming a protein corona (PC) on their surface. This PC influences the nanomaterial’s surface characteristics and may impact their interaction with cells. To determine the biological impact of nanomaterial exposure as well as nanotherapeutic applications, it is necessary to understand PC formation. Utilizing a label-free mass spectrometry-based proteomics approach, we examined the composition of the PC for a set of four silver nanoparticles (AgNPs) including citrate-stabilized and polyvinlypyrrolidone-stabilized (PVP) colloidal silver (20 or 110 nm diameter). To simulate cell culture conditions, AgNPs were incubated for 1 h in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, washed, coronal proteins solubilized, and proteins identified and quantified by label-free LC-MS/MS. To determine which attributes influence PC formation, the AgNPs were characterized in both water and cell culture media with 10% FBS. All AgNPs associated a common subset of 11 proteins including albumin, apolipoproteins, keratins, and other serum proteins. 110 nm citrate- and PVP-stabilized AgNPs were found to bind the greatest number of proteins (79 and 85 respectively) compared to 20 nm citrate- and PVP-stabilized AgNPs (45 and 48 respectively), suggesting a difference in PC formation based on surface curvature. While no relationships were found for other protein parameters (isoelectric point or aliphatic index), the PC on 20 nm AgNPs (PVP and citrate) consisted of more hydrophobic proteins compared to 110 nm AgNPs implying that this class of proteins are more receptive to curvature-induced folding and crowding in exchange for an increased hydration in the aqueous environment. These observations demonstrate the significance of electrostatic and hydrophobic interactions in the formation of the PC which may have broad biological and toxicological implications.</p></div

Scanning electron microscopy images and size distribution of A) 20 nm citrate-stabilized AgNP, B) 20 nm PVP-stabilized AgNP, C) 110 nm citrate-stabilized AgNP, and D) 110 nm PVP-stabilized AgNP samples confirming the diameters of all AgNPs used in this study.

<p>Scanning electron microscopy images and size distribution of A) 20 nm citrate-stabilized AgNP, B) 20 nm PVP-stabilized AgNP, C) 110 nm citrate-stabilized AgNP, and D) 110 nm PVP-stabilized AgNP samples confirming the diameters of all AgNPs used in this study.</p

Biomolecular Interactions and Biological Responses of Emerging Two-Dimensional Materials and Aromatic Amino Acid Complexes

The present work experimentally investigates the interaction of aromatic amino acids viz., tyrosine, tryptophan, and phenylalnine with novel two-dimensional (2D) materials including graphene, graphene oxide (GO), and boron nitride (BN). Photoluminescence, micro-Raman spectroscopy, and cyclic voltammetry were employed to investigate the nature of interactions and possible charge transfer between 2D materials and amino acids. Graphene and GO were found to interact strongly with aromatic amino acids through π–π stacking, charge transfer, and H-bonding. Particularly, it was observed that both physi and chemisorption are prominent in the interactions of GO/graphene with phenylalanine and tryptophan while tyrosine exhibited strong chemisorption on graphene and GO. In contrast, BN exhibited little or no interactions, which could be attributed to localized π-electron clouds around N atoms in BN lattice. Lastly, the adsorption of amino acids on 2D materials was observed to considerably change their biological response in terms of reactive oxygen species generation. More importantly, these changes in the biological response followed the same trends observed in the physi and chemisorption measurements