Ultrasound molecular imaging of prostate cancer <i>via</i> PSMA-targeted biosynthetic GVs

<i>Purpose</i>: Early diagnosis of prostate cancer is critical for improving prognosis, but current detection techniques face limitations such as low sensitivity, high cost, and radiation risks. Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in prostate cancer cells and a promising diagnostic and prognostic indicator. This study aims to develop a PSMA-targeted ultrasound contrast agent based on nanobody-modified gas vesicles (GVs) for early diagnosis of prostate cancer. <i>Materials and Methods</i>: GVs were extracted from <i>Halobacterium NRC-1 (Halo)</i>. PSMA-targeting nanobodies (Nb-PSMA) were synthesized by <i>Escherichia coli</i>. PSMA-targeted gas vesicles (PSMA-GVs) were prepared by coupling Nb-PSMA to GVs <i>via</i> the intermediate coupling agent Mal-PEG₂₀₀₀-NHS. Control vesicles were prepared similarly. The targeting specificity of PSMA-GVs towards prostate cancer cells was assessed by flow cytometry and confocal microscopy using PSMA-positive PC-3 cells. <i>In vivo</i> contrast-enhanced ultrasound imaging of PSMA-GVs was performed in prostate cancer-bearing mice at early and advanced stages. The biocompatibility of PSMA-GVs was assessed by hemolysis tests, CCK8 cytotoxicity assays, serum biochemical assays and HE staining. <i>Results</i>: PSMA-GVs exhibited a uniform size, with a hydrodynamic diameter of 267.73 ± 2.86 nm, and showed a high specific binding ability to PC3 cells. <i>In vivo</i> ultrasound imaging of prostate cancer-bearing mice showed that PSMA-GVs had significantly slower tumor signal attenuation than Con-GVs. Our <i>in vitro</i> and <i>in vivo</i> experiments demonstrated that PSMA-GVs could bind to prostate cancer cells with higher specificity, generating stronger and longer-lasting molecular imaging signals in tumors, which presented significant advantages over Con-GVs. Immunofluorescence confirmed that PSMA-GVs crossed the vascular wall, entered the peritumoral vascular space, bound to tumor cells, and enabled PSMA-targeted molecular imaging. Additionally, PSMA-GVs showed good biocompatibility. <i>Conclusion</i>: Our study provides a new strategy for early ultrasound molecular imaging diagnosis of prostate cancer.