Microfluidic–active loading integration enables rapid two-step manufacturing of antibiotic-loaded liposomes with intravesicular drug nanocrystallization

Liposome manufacturing remains a critical challenge in the translation of nanomedicines due to multi-step preparation procedures and limited process integration. Microfluidic manufacturing enables controlled liposome formation, whereas remote ion-gradient loading provides highly efficient drug encapsulation; however, these processes are typically performed separately. Here, we present a two-step microfluidic-active loading (MF-AL) strategy that integrates Staggered Herringbone Micromixer (SHM) microfluidics with ammonium-sulfate-driven remote loading for rapid production of antibiotic-loaded liposomes. Empty liposomes were first generated in ammonium sulfate using a SHM, followed by a second microfluidic cycle enabling active drug loading without intermediate processing steps. Using moxifloxacin (MOX) as a model amphiphilic antibiotic, liposomes with diameters between 83-117 nm were obtained, having high drug loading efficiencies (D/L ≈ 0.50 mol/mol for most lipid compositions). Importantly, the MF-AL method achieves drug loading and physicochemical properties (size distribution, morphology, crystallinity, and release kinetics) comparable to classical remote loading while eliminating intermediate processing steps, thereby simplifying liposome manufacturing. Cryo-EM revealed granular electron-dense domains within highly loaded vesicles, which, together with the presence of characteristic XRD diffraction peaks, strongly suggests the formation of intravesicular drug nanocrystals produced during ion-gradient-driven loading. Furthermore, MF-AL liposomes showed antimicrobial activity against Staphylococcus epidermidis (planktonic and biofilm models) superior to free drug and comparable to conventionally prepared formulations proving that in addition to structural, also the functional characteristics of MOX-liposomes were preserved. The MF-AL method was further validated using doxorubicin, demonstrating its applicability to other amphiphilic therapeutics. Overall, the MF-AL approach represents a simple, rapid, and scalable strategy for liposome manufacturing, enabling efficient drug loading while preserving the structural and functional characteristics of remotely loaded nanomedicines.