Mouse serum albumin

All nanotubes were weighed and suspended in dispersion media (DM) (Porter et al., 2008 ), which consisted of mouse serum albumin (Sigma, St. Louis, MO; 1 mg/mL or 0.1%) and 1,2 dipalmitoyl-sn-glycero-3-phosphocholine (DSPC, Sigma, 1 μg/mL or 0.0001%) diluted in phosphate-buffered saline (PBS). Nanotube suspensions were sonicated for 5 min at 1/3 max power in a Qsonica cup-horn sonicator (Q500, Newtown, CT) attached to a circulating water-bath (VWR International, Radnor, PA) at 500 watts and 20 Hz (8000 Joules) at a stock concentration of 1 mg/mL.

Male apoE knockout (KO) mice (B6.129P2-Apoe^tm1Unc/J, Jackson Labs (No. 002052)), 9.5-11 months of age on western diet, were used in these experiments. Healthy mice were used as control groups. For the in vivo imaging study, mice (n=4) were anesthetized with 2% isoflurane and injected retro-orbitally with PLA anti-elastin nanoparticles (EL-NPs) in 0.3% mouse serum albumin (Sigma Aldrich, St.Louis, MO) in a total volume of 250 μl. At 24 hours after tracer injection, the mice were euthanized by CO₂ asphyxiation and perfused with heparinized normal saline at ~100 mm Hg via a left ventricular puncture. The aortic trees were harvested, imaged ex vivo on the IVIS Spectrum, and frozen for histology. Additional organs (brain, heart, blood, muscle skin, liver, kidneys, spleen, and lungs) were harvested at the same time to assess bio-distribution of the injected nanoparticles.

Preparations and characteristics of a highly inflammatory multi-walled carbon nanotube (MWCNT; provided by the National Toxicology Program at NIEHS; referred to as FA21 [15 (link)]) and acid-washed crystalline SiO₂ (SiO₂; Min-U-Sil-5, Pennsylvania Glass Sand Corp.; Pittsburgh, PA, USA) are as previously described [15 (link), 16 (link)]. Briefly, both particles were prepared fresh in dispersion media vehicle (DM; 0.6 mg/mL mouse serum albumin [Sigma-Aldrich; St. Louis, MO, USA] and 0.01 mg/mL 1,2-dipalmitoyl-sn-glycero-3-phosphocholine [Sigma-Aldrich] in PBS). The particle suspensions were sonicated (550 watts, 20 kHz, 5 minutes, 35% amplitude) in a cup-horn sonicator.

Preparations and characteristics of MWCNT and SiO₂ particles are the same as described above and as previously described (Hamilton et al., 2012 (link); Jessop et al., 2017 (link)). Briefly, both particles were prepared fresh in dispersion media vehicle (DM; 0.6 mg/ml mouse serum albumin [Sigma-Aldrich] and 0.01 mg/ml 1,2-dipalmitoyl-sn-glycero-3-phosphocholine [Sigma-Aldrich] in PBS). The particle suspensions were sonicated (550 watts, 20 kHz, 5 min, 35% amplitude) in a cup-horn sonicator.

The MWCNT selected for these studies, designated FA-21 (Sun Innovations, Inc., Fremont, CA; www.nanomaterialstore.com), was selected due to its potent NLRP3 Inflammasome activity and has been characterized elsewhere (Hamilton et al., 2012 (link)). This specific MWCNT is 1 of 24 samples provided by Dr. Nigel Walker and Brad Collins at the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS). Purity and metal content of the MWCNT were determined using thermal gravimetric analysis (TGA) and X-ray fluorescence spectrometry, respectively. Diameter and agglomeration state of the MWCNT were determined by transmission electron microscopy (TEM) and dynamic light scattering (DLS), respectively. This pro-inflammatory MWCNT has high nickel contamination (~5.54%), and forms agglomerates that range from 122 to 469 nm depending on the dispersion media. Additionally, the MWCNT is free from endotoxin contamination, a critical quality for investigating the sterile inflammatory response. Prior to in vivo exposure by instillation or dispersion in vitro, MWCNT were suspended in dispersion medium [DM, 0.6 mg/ml mouse serum albumin (Sigma–Aldrich, Saint Louis, MO) and 0.01 mg/ ml 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (Sigma–Aldrich) in PBS] and suspended using sonication for 1 min (Porter et al., 2008 ).