Automated high-throughput preparation and characterization of oligonucleotide-loaded lipid nanoparticles

Predictive high-throughput screening of PEGylated lipids in oligonucleotide-loaded lipid nanoparticles for neuronal gene silencing

“…S3 † ), which aligns with our previously reported results. 32 Importantly, the scaled-up ASO-LNP formulations also showed comparable mRNA expression in mouse neurons, relative to their corresponding HTS formulations ( Fig. 6C ).…”

“…Previous studies suggest that PEG-lipids play an important role in particle size regulation during self-assembly. 32–35 For the HTS-generated ASO-LNPs, DLS measurements generally indicated there is a negative correlation between particle sizes and molar ratios of PEGylated lipids in the LNP formulations, with mean hydrodynamic diameters ranging between 50–220 nm across the library (Fig. 2A and B).…”

“…LNPs with various PEGylated lipids were prepared using a high-throughput approach reported previously. 32 Briefly, the ASO was dissolved at 93.9 μg mL −1 in citrate buffer (25 mM, pH 4.0) and dispensed at 150 μL per well in a 96-well plate (Greiner Bio One 655101, NC, USA) using a TECAN Freedom EVO robotic liquid handler (Tecan Life Sciences, NC, USA). Using the automation setup, different lipid mixtures composed of MC3, DSPC, cholesterol, and respective PEG-lipid analogs were prepared in ethanol at a molar ratio of 40 : 10 : (50 − X ) : X , where X = 1, 3, or 5; and a total lipid concentration of 4 mM was used to maintain N : P = 2 for the resulting ASO-LNPs.…”

“…A microfluidic mixing method was used for the scale-up preparation of selected ASO-LNP formulations that were identified as either positive or negative hits by the HTS approach. 32 Lipid mixtures that replicate the selected LNP compositions from HTS were manually prepared and dissolved in ethanol at a total lipid concentration of 4 mM. The ethanol stream was rapidly mixed with an aqueous stream containing 93.9 μg mL −1 ASO dissolved in citrate buffer (25 mM, pH 4.0) using a microfluidic laminar mixing device (NanoAssemblr™ Benchtop, Precision NanoSystems, BC, Canada) at a 1 : 3 volume ratio, and a total flow rate of 12 mL min −1 .…”

“…An automated high-throughput screening (HTS) workflow to prepare and characterize LNPs can be used to comprehensively assess how PEG-lipid parameters regulate LNP function by allowing complex LNP screening designs, while saving substantial time and materials. 32 In this study, we generated a library of 54 ASO-LNPs using 18 different PEG-lipids across the phosphoglyceride, diglyceride, and ceramide families, at varying PEG-lipid molar ratios (1, 3, and 5 mol%) using a high-throughput workflow. The impact of PEG-lipid parameters – molecular weight (M.W.…”