Limulus amebocyte lysate chromogenic endotoxin quantification kit

Microparticles of titanium powder CAS 7740–32-6 (<20 micron, 93%, Alfa Aesar, Ward Hill, MA, USA) and titanium (IV) oxide nanoparticles CAS 13463–67-7 (21 nm, Sigma Aldrich, St Louis, MO, USA) were used in this study. The particles were weighted and separated in 15 mL tubes (Fischer Scientific, Waltham, Massachusetts, USA) containing 25 mg each. A cleaning procedure was required to certify that the particles were not contaminated with amounts of endotoxin (10–20 units/mL) as previously reported (6 ,7 (link),11 ). The particles were cleaned using 25% nitric acid for 2 h followed by 3 washes in phosphate-buffered saline (PBS) (Invitrogen, Waltham, MA, USA) for 5 min each and placed in 95% ethanol with 0.1 N of NAOH for 24 h, followed by 3 washes in PBS (6 ,12 (link)). After the cleaning procedure, the presence of endotoxins was measured using Limulus Amebocyte Lysate Chromogenic Endotoxin Quantification Kit (Thermo Fisher Scientific, Grand Island, NY, USA) following the manufacturer’s protocol. The presence of endotoxins was <0.01 endotoxin units/mL, confirming the effectiveness of the cleaning procedure (11 ). The particles were maintained in PBS 25 mg/mL at 4 °C, and the solution was agitated for 20 min prior to each use (13 ).