Development and quality assessment of surface-reactive liposomes using a DoE-based microfluidic approach

Treating placental disorders requires safe and specific drug delivery systems. Active targeting strategies offer a compelling approach to deliver therapeutic agents selectively to the placenta. In this context, functionalized liposomes are particularly promising, as they are among the safest available nanocarriers. However, producing such advanced systems remains challenging because reactive lipids often raise major solubilization and formulation issues. This study addresses these limitations by combining microfluidic technology with a design of experiments (DoE) strategy to optimize the production of PEGylated liposomes bearing maleimide groups. Twenty-five experimental runs were performed to evaluate the effects of total flow rate, flow rate ratio, lipid concentration, and solvent ratio. This approach enabled the identification of conditions to prepare liposomes with size and PDI suitable for our application. Purification efficacy was further assessed by quantifying residual organic solvents using two high-throughput analytical methods: an enzymatic spectrophotometric assay and capillary electrophoresis. Cytotoxicity assays on BeWo trophoblast cells confirmed the absence of toxicity up to a total lipid concentration of 1.25 mM. Stability studies showed that formulations around or above 50 nm remained colloidally stable for at least three weeks at 4 °C. Finally, three stable representative formulations were selected for conjugation with a model peptide via thiol–maleimide reaction, all achieving grafting efficiencies above 80%. Together, these results demonstrate that the combined microfluidic–DoE approach enables the robust production of stable surface-reactive liposomes with controlled size, low polydispersity, and suitability for subsequent peptide conjugation.