Display omitted •Optimization of unilamellar liposome manufacturing via microfluidic system;•Comparison of thin-film hydration method and microfluidic system in liposome properties;•Drug co-loaded liposomes as novel formulation for breast cancer treatment;•Effect of prenyloxy coumarin as fluidity moderator in lipid bilayer; Developing more efficient manufacturing methods for nano therapeutic systems is becoming important, not only to better control their physico-chemical characteristics and therapeutic efficacy but also to ensure scale-up is cost-effective. The principle of cross-flow chemistry allows precise control over manufacturing parameters for the fabrication of uniform liposomal formulations, as well as providing reproducible manufacturing scale-up compared to conventional methods. We have herein investigated the use of microfluidics to produce PEGylated DSPC liposomes loaded with doxorubicin and compared their performance against identical formulations prepared by the thin-film method. The isoprenylated coumarin umbelliprenin was selected as a co-therapeutic. Umbelliprenin-loaded and doxorubicin:umbelliprenin co-loaded liposomes were fabricated using the optimised microfluidic set-up. The role of umbelliprenin as lipid bilayer fluidity modulation was characterized, and we investigated its role on liposomes size, size distribution, shape and stability compared to doxorubicin-loaded liposomes. Finally, the toxicity of all liposomal formulations was tested on a panel of human breast cancer cells (MCF-7, MDA-MB 231, BT-474) to identify the most potent formulation by liposomal fabrication method and loaded compound(s). We herein show that the microfluidic system is an alternative method to produce doxorubicin:umbelliprenin co-loaded liposomes, allowing fine control over liposome size (100–250 nm), shape, uniformity and doxorubicin drug loading (>80%). Umbelliprenin was shown to confer fluidity to model lipid biomembranes, which helps to explain the more homogeneous size and shape of co-loaded liposomes compared to liposomes without umbelliprenin. The toxicity of doxorubicin:umbelliprenin co-loaded liposomes was lower than that of free doxorubicin, due to the delayed release of doxorubicin from liposomes. An alternative, rapid and easy manufacturing method for the production of liposomes has been established using microfluidics to effectively produce uniform doxorubicin:umbelliprenin co-loaded liposomal formulations with proven cytotoxicity in human breast cancer cell lines in vitro.