Microfluidic-assisted preparation of ARV 825 and Osimertinib loaded liposomal formulation as a potential system for colorectal cancer therapy

Colorectal Cancer (CRC) remains the second leading cause of cancer-related deaths globally. Current treatments, including surgery, chemotherapy, and radiotherapy, are often limited by significant off-target toxicity and drug resistance. Overexpression of Epidermal Growth Factor Receptor (EGFR) and Bromodomain-containing protein 4 (BRD4) in CRC contributes to tumor progression and therapeutic resistance. Therefore, targeting EGFR and BRD4 through third-generation EGFR Inhibitor Osimertinib (OSI) and BRD4-targeting PROTAC degrader ARV 825 (ARV) offers a promising strategy; however, their therapeutic efficacy is limited by poor aqueous solubility and bioavailability. In this study, we developed liposomal nanoformulation (AROSIB) encapsulating OSI and ARV using 3D printed microfluidic T-junction chips to improve co-delivery and therapeutic performance. The physicochemical characteristics of AROSIB were assessed using dynamic light scattering and high-performance liquid chromatography, while stability was assessed over 28 days at 4 °C. Cryo-transmission electron microscopy confirmed AROSIB morphology and size. AROSIB exhibited uniform particle size (∼200 nm), optimal polydispersity index (∼0.150), high encapsulation efficiency for ARV (∼75 %) and OSI (∼98 %), and maintained stability during storage. Enhanced in vitro Anticancer efficacy was demonstrated in HCT 116 and HT 29, showing significant cytotoxic effects, Apoptosis induction, and substantial inhibition of migration and clonogenic survival. Moreover, AROSIB demonstrated potent Anticancer activity in 3D tumor spheroid model and favorable hemocompatibility. 3D printed T-junction chips could be a promising alternative for the preparation of liposomes. The microfluidic-assisted co-delivery of ARV and OSI in liposomal formulations represents a compelling approach to overcoming drug resistance and enhancing CRC therapy by concurrently targeting EGFR and BRD4.

3D tumor spheroid model; BRD4 PROTAC; Colorectal cancer; Liposomes; Microfluidic technology; Tyrosine kinase inhibitor.