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Adenovirus (Ad) is a widely studied viral vector for cancer therapy as it can be engineered to cause selective lysis of cancer cells. However, Ad delivery is limited in treating cancers that do not have coxsackievirus and adenovirus receptors (CAR). To overcome this challenge, Ad-encapsulated liposomes were developed that enhance the delivery of Ads and increase therapeutic efficacy. Cationic empty liposomes were manufactured first, to which an anionic Ad were added, which resulted in encapsulated Ad liposomes through charge interaction. Optimization of the liposome formula was carried out with series of formulation variables experiments using an extrusion process, which is ideal for laboratory-scale small batches. Later, the optimized formulation was manufactured with a homogenization technique—A high shear rotor-stator blending, that is ideal for large-scale manufacturing and is in compliance with Good Manufacturing Practices (GMP). Comparative in vitro transduction, physicochemical characterization, long-term storage stability at different temperature conditions, and in vivo animal studies were performed. Ad encapsulated liposomes transduced CAR deficient cells 100-fold more efficiently than the unencapsulated Ad (p ≤ 0.0001) in vitro, and 4-fold higher in tumors injected in nude mice in vivo. Both extrusion and homogenization performed similarly–with equivalent in vitro and in vivo transduction efficiencies, physicochemical characterization, and long-term storage stability. Thus, two Ad encapsulated liposomes preparation methods used herein, i.e., extrusion vs. homogenization were equivalent in terms of enhanced Ad performance and long-term storage stability; this will, hopefully, facilitate translation to the clinic.