Optimized Preparation Of PSMA Targeted Lipid Nanobubbles And Its Application In The Diagnosis Of Prostate Cancer

Objective:PSMA targeted nanobubbles were prepared,their basic characteristics were tested,and their ultrasound imaging ability and specific targeting ability with prostate cancer cells expressing PSMA were verified in vitro.In vivo experiments of nude mice were conducted to explore the specific enhanced imaging effect of targeted nanobubbles in vivo,and further verify its specific binding ability with prostate cancer cells expressing PSMA,providing new ideas and research basis for molecular targeted ultrasound imaging and targeted treatment of prostate cancer.Methods:1.Using DPPC and DSPE-PEG2000-Biotin as raw materials of lipid shell,biotinylated nanobubbles with nano-size were prepared directly by thin film dispersion method and ultrasonic water bath combined with mechanical oscillation method,and their physical properties were tested.2.Using biotin-avidin bridging method,PSMA single-chain antibodies were coupled to the surface of biotinylated nanobubbles to prepare targeted nanobubbles and detect their physical properties.Flow cytometry was used to analyze the binding rate of targeted nanobubbles to prostate cancer cells expressing PSMA;nanoparticle tracking analyzer and particle size/potential analyzer were used to analyze the stability of targeted nanobubbles in vitro;5 ml EP tube was used to detect the imaging effect of targeted nanobubbles in vitro.3.The expression of PSMA in three prostate cancer cells(LNcap,22RV1 and PC3)was localized by immunofluorescence assay.To establish a nude mice model of tumor-bearing mice,to detect the ultrasound enhanced imaging effect of biotinylated nanobubbles and targeted nanobubbles in vivo and to analyze the data.Laser confocal microscopy was used to observe the targeting distribution of biotinylated nanobubbles and targeted nanobubbles in three kinds of tumors.Results:1.Biotinized nanobubbles with nano-size(414.6 + 30.5 nm)were prepared directly,and PSMA targeted nanobubbles with average particle size(485.3 + 28.4 nm)and average concentration(21.2 + 5.31)× 108 nanobubbles/ml were further synthesized,with good stability in vitro and excellent contrast-enhanced ultrasound imaging effect.2.Cell immunofluorescence showed high PSMA expression in LNcap and 22RV1 cells,but no PSMA expression in PC3 cells.In vitro experiments confirmed the specific targeting of the targeted nanobubbles on tumor cells with high PSMA expression,no targeting on PSMA(-)tumor cells,and also confirmed that the biotinylated nanobubbles had no targeting on tumor cells with high PSMA expression.3.In vivo contrast-enhanced ultrasound showed that the peak contrast intensity of targeted nanobubbles in LNCa P and 22Rv1 transplanted tumors was significantly higher than that of biotinylated nanobubbles(P < 0.05),but there was no significant difference in PC-3 transplanted tumors(P > 0.05).Frozen sections of tumors showed that targeting nanobubbles could penetrate more into the vessel wall of tumors to reach the surface of PSMA(+)tumors.Conclusion:In this study,we directly prepared targeted nanobubbles with uniform size,small particle size and good stability.In vitro and in vivo experiments have demonstrated that the newly synthesized targeted nanobubbles have the ability to specifically bind to prostate cancer cells with high PSMA expression.Targeted nanobubbles can form a good molecular targeted imaging in PSMA-expressing transplanted tumors,which indicates that the targeted nanobubbles prepared in this study are an excellent ultrasound contrast agent for PSMA(+)prostate cancer imaging,providing a basis for molecular imaging of prostate cancer,and a potential delivery system for targeted therapy.