Endosafe pts 2005

Pegylated silica nanoparticles (Si-PEG nps) were prepared by adding polyethylene glycol succinimidyl ester (mPEG-NSH; MW 5000, PDI <1.08, purity >95%, Cat. PG1-SC-5k, Nanocs Inc) into 3.4 mg mL^-1 Si-NH₂ nps in 2:1 (mPEG-NSH:Si-NH₂) molar ratio in an endotoxin-free environment. The mixture was stirred at room temperature for 2 h. Endotoxin level in pegylated silica nps was analyzed using LAL-based assay Endosafe PTS-2005 (Charles River Laboratories, Wilmington, MA) and was found to be < 2 EU mL^-1.

PLGA and PCL nps were prepared by an oil-in-water (o/w) emulsion solvent evaporation method as described previously [34 (link)]. Briefly, 400 μl of mixture of PLGA or PCL (4 mg) and PEGPE (8 mg) in chloroform was added dropwise into 4 ml water. Then the solution was sonicated at 10 W (Branson Sonifier 450 microtip probe ultrasonicator, Danbury, CT, USA) for four 25s bursts interspersed with cooling on an ice bath for 60s. The chloroform was separated from the emulsified solution by using a rotary evaporator (Rotavapor R-215, Büchi, Postfach, Switzerland). Nanoparticles were washed three times by centrifugation (18000 g, 5 mins) using Amicon ultra centrifugal filter devices (Millipore, Billerica, MA, USA) to remove free PEGPE. Endotoxin level in PLGA and PCL nps were analyzed using LAL-based assay Endosafe PTS-2005 (Charles River Laboratories, Wilmington, MA) and were found to be < 2.5 EU mL^-1.

Amine-functionalized silica nanoparticles (Si-NH₂ nps) was prepared according to literature with some modification [35 (link)]. Commercial silica nps as above were dialyzed against Milli Q water at 4°C for 24 h and adjusted to a nominal silica concentration of 30 mg mL^-1 with Milli Q water. Then, nanoparticle solution was subjected to centrifugal wash (18000 g, 20 mins) with absolute ethanol three times, followed by sonication (Branson Ultrasonics Corporation, Danbury, CT) at output 30 for 4 cycles of 20s to re-suspend in ethanol. Nanoparticle solution in absolute ethanol was incubated with 14% (v/v) (3-aminopropyl)triethoxysilane (APTES; Cat. A3648, Sigma-Aldrich, St Louis, MA) for 3h with constant stirring. After incubation, nanoparticle-APTES solution was subjected to centrifugal washing (18000 g, 20 mins) with absolute ethanol three times, followed by sonication to finally re-suspend in absolute ethanol. Then, amine functionalized nps were dialyzed against Milli Q water at 4°C for 24h and adjusted to a concentration of 2 mg mL^-1 with Milli Q water. Endotoxin levels in amine-functionalized silica nps was analyzed using a LAL-based assay Endosafe PTS-2005 (Charles River Laboratories, Wilmington, MA) and was found to be < 2 EU mL^-1.