High-throughput conventional and stealth cationic liposome synthesis using a chaotic advection-based microfluidic device combined with a centrifugal vacuum concentrator

Abstract

Microfluidics is an emerging technology that allows efficient mixing of fluids on the nanoliter scale and has recently been employed for the production of lipid-based systems. In this study, we investigated the manufacturing of conventional (EPC/DOTAP/DOPE) and stealth (EPC/DOTAP/DOPE/DSPE-PEG(2000)) cationic liposomes (CLs) in a high-throughput chaotic advection-based microfluidic device (CA-MD) using water and ethanol. We assessed the effects of total flow rate (TFR), flow rate ratio (FRR), and flow configuration on CL formation. We compared the results with properties of CLs produced on a diffusion-based microfluidic device (D-MD). FRR (aqueous/solvent ratio) values were found to have a strong correlation with vesicle formation and values close to 1 led to the production of conventional and stealth CLs with diameter and polydispersity indices close to 200 nm and 0.2, respectively. Both liposomes are unilamellar, and stealth CL production could minimize micelle formation and operate at high flow velocities without altering characteristics of CLs. These findings were supported with dynamic light scattering (DLS), cryo-transmission electron microscopy (Cryo-TEM), and synchrotron small angle X-ray scattering (SAXS) techniques. The high solvent content in liposomes after microfluidic synthesis (FRR = 1) was reduced by employing centrifugal vacuum concentrators (CVC) at low pressure and 43 °C as an alternative distillation process for successful ethanol removal without changing the structural properties of these nanoaggregates. Finally, both CLs were able to transfect pDNA into PC-3 cancer cells. The CA-MD coupled to a CVC technique produced conventional and stealth CLs with high productivity, which makes it readily applicable for industrial production.