Abstract: In this study, a novel nano-drug delivery system, namely hybrid exosome, was constructed via membrane self-assembly of pancreatic cancer cell-derived exosomes with liposomes, which inherits the functionalities of exosomes, including high affinity, good stability and low immunogenicity, but also unites the characteristic of liposomes (e.g., long circulation time, high loading ability) to achieve precise drug navigation and minimum adverse effects. Specifically, two different preparation methods-repeated freeze-thawing and 37℃ incubation were used to fabricate hybrid exosomes at laboratory scale. Comparative analysis and characterization of these synthesized samples were performed based upon size, zeta potential and membrane fusion efficiency. The results showed that the highest exosome yield was attainted after culture for 48 h, with the exosome yield of 0.83 ±0.07 mg/10⁸ cells for HuP-T3 cell line and 0.79 ±0.10 mg/10⁸ cells for Panc0403 cell line. Hybrid exosomes obtained by freeze-thaw method were shown to have higher membrane fusion rate, lower size and polydispersity index (PDI), higher zeta potential and relative more stable, as compared with that made by incubation at 37℃ for 12 h, indicating the former approach is more suitable to construct hybrid exosomes with desirable physicochemical properties. This result may provide a preliminary experimental basis for the subsequent delivery of different anticancer drugs for the treatment of solid tumors such as pancreatic cancer.