Study of Bleb-derived Nanovesicles Containing Fusion Protein for Targeting Prostate Cancer

Abstract

Existing methods for targeting and treating prostate cancer have limitations. Conventional techniques often lead to physical discomfort and do not always target the cancerous region. Therefore, there is an urgent need for the development of more precise and noninvasive methods for the targeting and treatment of prostate cancer. In this thesis, the design of fusion protein expressed in Bleb-derived Nanovesicles (BNVs) for targeting and treating prostate cancer is demonstrated. Prostate-specific membrane antigen (PSMA), a transmembrane protein, is recognized as a critical marker of prostate cancer. It is overexpressed in prostate cancerous cells, establishing it as a compelling target for diagnostics and therapeutics. A fusion protein, which can target specific membrane antigens, is included. This fusion protein incorporates a VHH, a single-domain antibody fragment that is renowned for its superior binding ability in comparison to conventional antibodies. Additionally, the design of the fusion protein encompasses the integration of the transmembrane domain sequence of the Integrin Alpha 1 (ITGA1) protein. ITGA1, a membrane protein, is noted for not influencing the PSMA target. Additionally, a leader sequence was incorporated into the fusion protein's design to guarantee the exterior expression of the VHH on the cell membrane for targeting purposes. In this thesis, BNVs, a platform designed to overcome the challenge of low production yield associated with exosomes, are introduced as an alternative method. Owing to the sole presence of membrane elements in BNVs, minimal influence is exerted by intracellular elements on the targeting or treatment. To confirm that BNVs with fusion proteins are targetable to PSMA proteins, western blotting and immunofluorescence were performed. In these results, the potential of BNVs expressing fusion proteins as a platform for targeting and treating prostate cancer is demonstrated by confirming their ability to target PSMA proteins. This information will be useful to guide advanced targeting and treatment of prostate cancer by using BNVs.