BACKGROUND: BAY55-9837, a potential therapeutic peptide for the treatment of type 2 diabetes mellitus (T2DM), can induce glucose (GLC)-dependent insulin secretion. Our previous study has demonstrated that the use of superparamagnetic iron oxide nanoparticle-decorated exosome (exosome-SPION) and external magnetic force (MF) enables BAY 55-9837 to target pancreatic islets. However, the initial burst release of BAY 55-9837 loaded within exosome-SPION shortens its in vivo half-life and consequently reduces the frequency of GLC responsiveness. Therefore, in our study, the transmembrane hydrophobic structure of the exosome signature protein CD81 was fused with BAY 55-9837 to obtain MBAY with sustained-release capability. METHODS: MBAY was fabricated via genetic engineering, and the dissociation constant (Kd) was determined to assess its affinity for vasoactive intestinal peptide receptor type 2 (VPACII). Subsequently, MABY was incorporated into exosomes through electroporation to obtain MBAY-exosome, and SPOIN was adorned on MBAY-exosome by means of the self-assembly of transferrin (Tf) and the transferrin receptor (TfR). The in vitro release profile and in vivo pharmacokinetic profile of MBAY-Exosome-SPION were detected using high-performance liquid chromatography (HPLC). The L9(3) orthogonal design approach was utilized to optimize the drug administration mode in vivo. The therapeutic effect of MBAY-exosome-SPIONs/MF on T2DM was assessed both in vitro and in vivo. RESULTS: In vitro studies showed that the release rate of MBAY from exosome-SPION was slower compared with BAY 55-9837. Meanwhile, MBAY still maintained high affinity and selectivity for VPAC II and MBAY-exosome-SPIONs/MF could effectively promote insulin secretion in response to elevated GLC as BAY-exosome-SPIONs/MF. In vivo studies indicated that MBAY-exosome-SPIONs had a prolonged half-life and improved pharmacokinetic parameters compared to BAY-exosome-SPIONs, which further alleviated the symptoms of T2DM model mice. CONCLUSION: Thus, the reconstructed MBAY loaded in SPION-exosome realized sustained-release and exosomes-SPIONS achieved pancreatic targeting which led to ideal therapeutic effect in T2DM mice.