Corona veo charged aerosol detector

The size and polydispersity index of the nanoparticles was analyzed by dynamic light scattering using a Nanotrac Flex (Microtrac). The peptide content of the NPs was determined by HPLC analysis using a standard dilution of peptides based on net peptide content (15 (link)). All amounts of PLGA-NPs used in this study were calculated according to their net peptide contents except for the particles containing full NY-ESO-1 protein which the content could not be determined due to high amounts of urea and glycine contamination. IMM60 content of the NPs was determined by a Corona Veo Charged Aerosol Detector (CAD) coupled to a DIONEX UltiMate 3000 HPLC system (Thermo Fischer Scientific). The NPs were dissolved in DMSO for a complete dissolution of the components and analyzed by CAD on an XSelect CSH C₁₈ 2.5 μm 3.0 × 150 mm XP column (Waters) with VanGuard Cartridges (Waters) coupled to a column heater (65°C), eluents MeOH-Formic Acid-Triethylamine (99.0/0.05/0.05 vol. %) with isocratic gradient flow rate = 1.0 ml·min⁻¹. The quantity of IMM60 was calculated by interpolation of the standard calibration curves of IMM60 performed in the same way as for the NPs. The endotoxin content of the nanoparticles was analyzed using the gel-clot method by Eurofins PROXY laboratories, Leiden, The Netherlands, and found to be lower than 0.1 EU/mg particles.

The IMM60 content of the NPs was determined by a Corona Veo Charged Aerosol Detector (CAD, Thermo Fisher Scientific, Waltham, MA, USA) coupled to the aforementioned HPLC system. The components of the formulation (PLGA, PVA, IMM60 and peptides) were separated by a XSelect CSH 18 column (130 Å, 2.5 µm, 3 mm × 150 mm) with VanGuard Cartridges (Waters Corp, Milford, MA, USA) coupled to a column heater (65 °C), eluents methanol–formic acid–triethylamine (99.0/0.05/0.05 vol%) with isocratic gradient flow rate at 1.0 mL/min followed by the detection of the components in the CAD system using an electrometer. The injection volume was 20 µL. The quantity of IMM60 was calculated by the interpolation of the standard calibration curves of IMM60 performed in the same way as for the NPs. Working concentrations were >40 µg/mL, while the LOD was determined as 3.7 µg/mL.

The trimyristin content of unloaded nanoemulsions was quantified with a Dionex UltiMate 3000 high performance liquid chromatography (HPLC) system equipped with a LPG-3400SD pump, a WPS-3000TSL autosampler, and a Corona Veo Charged Aerosol detector (Thermo Fisher Scientific, Waltham, MA, USA). The column (Thermo Fisher Scientific Hypersil Gold C18, 2.1 × 150 mm², 1.9 μm) was kept at 25 °C and the flow rate was set to 0.3 mL/min. The mobile phase consisted of acetonitrile/tetrahydrofuran 70/30 (v/v) for trimyristin.
Samples were diluted in tetrahydrofuran/acetonitrile 50/50 (v/v) to an appropriate response; 1 μL was injected and detected at a nebulizer temperature of 50 °C. Every sample was diluted twice and every dilution measured two times (n = 4). Trimyristin amounts were calculated with the Chromeleon 7.2 software (Thermo Fisher Scientific, Waltham, MA, USA) using a calibration curve of trimyristin in different concentrations.