Flow rate ratio as a key parameter in the microfluidic synthesis of doxorubicin-loaded liposomes

• FRR is a key parameter for modulating liposome size, yield, and drug loading. • Increasing FRR increases liposome yield and drug encapsulation. • The method of D/L adjustment (via FRR vs. lipid input) affects liposome yield, size and morphology. • 3D-printed microfluidic devices can be applied to lower cost and increase throughput. Microfluidic devices offer scalable, reproducible platforms to produce liposomal drug delivery systems. Yet the relationship between flow rate ratio (FRR), drug-to-lipid (D/L) ratio, and drug encapsulation efficiency (EE%) remains incompletely defined. This study investigated the impact of FRR and D/L ratio on liposome colloidal properties and the passive loading of doxorubicin hydrochloride (DOX·HCl) using a 3D-printed T-junction microfluidic chip. Zwitterionic (DOPC) and anionic (DOPC:DOPA, 75:25 w/w) liposomes were generated at a fixed total flow rate (12 mL/min) and varying FRRs (3:20 to 1:3, organic: aqueous). Increasing FRR led to a decrease in D/L ratio, causing a significant decrease in liposome size (from ∼120 nm to ∼100 nm) and increase particle concentration (>20-fold). EE% of DOX·HCl increased with decreasing D/L ratio, reaching 4.4% in DOPC and 75.5% in DOPC:DOPA liposomes at FRR 1:3. Comparing methods to adjust D/L (adjusting FRR or total lipid concentration injected into the system) showed that while EE% remained similar, FRR adjustment significantly increased liposome concentration and resulted in less variability in liposome size and morphology. These findings demonstrate that modulating FRR in a macro-geometry microfluidic device enables precise control of liposome size, yield, and loading, without the need to increase lipid input. This work highlights the potential of low-cost 3D-printed microfluidic devices for high-throughput production of drug-loaded liposomes and underscores the critical role of FRR in optimizing encapsulation and formulation parameters