Systematic development and optimization of a microfluidic formulation protocol for liposomal azithromycin

Delivery of azithromycin <i>via</i> liposomal formulation (L-AZM) has been shown to improve the therapeutic index and activity of AZM in a preclinical model of cardiac injury, suggesting strong potential for clinical translation to treat inflammation after a myocardial infarction. However, conventional thin film hydration (TFH) utilized to prepare L-AZM limits its clinical development due to scalability and reproducibility concerns. To overcome these manufacturing challenges, we performed a systematic optimization of the L-AZM formulation utilizing microfluidic nanoprecipitation which has been successfully used for large scale manufacturing of lipid-based therapeutics in a reproducible manner. We adjusted the microfluidic operation parameters and evaluated the resultant liposomes for critical quality attributes (CQAs) of size, polydispersity index (PDI), encapsulation efficiency, and leakage. The optimal flow rate ratio (FRR) and total flow rate (TFR) for the lead formulation was determined to be 4 : 1 and 10 mL min^-1, respectively. Utilizing these manufacturing parameters with formulations of different molar ratios resulted in an optimized formulation consisting of DSPC : DSPG : Chol : AZM (1 : 1 : 1 : 0.5) based on the CQAs with decreased size and PDI as compared to TFH. Notably, there is no difference in <i>in vitro</i> macrophage polarization activity between the two formulation methods. Collectively, these data guide continued preclinical development as we advance this formulation toward clinical use.