LIPID NANOPARTICLES PRODUCTION AND CHARACTERIZATION FOR NUCLEIC ACID DELIVERY

Lipid nanoparticles (LNPs) were produced using a microfluidic MicroMixer chip. Formulations are composed of either an ionizable lipid (ALC-0315, 50%) or a cationic lipid (DDA, 43.5%), along with cholesterol, DSPC, and DMG-PEG2000. A fluorescent probe (25-NBD cholesterol) was added, and a model mRNA (mCherry) was encapsulated. Optimization by Design of Experiment identified optimal production parameters: 5 mg/mL lipid concentration, a flow rate ratio of 1:3, and total flow rates of 960 µL/min for ALC-LNPs and 1400 µL/min for DDA-LNPs. Characterization by DLS, NTA, and TRPS analysis for size, concentration, and surface charge confirmed low polydispersity (PDI < 0.3) and a stable particle size of 200 nm. TEM confirmed particle morphology, and fluorescence spectroscopy allowed tracking and quantification. With the Quant-iT RiboGreen assay the encapsulation efficiency of mRNA was assessed (EE ≈ 80%). In vitro studies on HMC-3 and HEK cells confirmed LNPs uptake and mRNA expression. ALC-LNPs exhibit lower toxicity compared to DDA-LNPs. Finally, protein corona formation was examined using LC-MS, highlighting a different protein composition based on LNP components and brain organoids were developed for further internalization studies.