Preparation Of Prostatic Carcinoma-targeted Ultrasound Contrast Agent And Its Experimental Study

Background: Prostatic carcinoma (PCa) is one of the frequent malignant tumors in western countries and accounts for the second place of the male death in USA. The attack rate in our country has been increasing sharply for the past few years. Most of the patients have already suffered from metabasis when the clinical symptoms appears, so early diagnosis is important to the healing and prognosis of PCa. However, all the imaging –modalities haven't discriminated the chronic prostatitis, prostatauxe and PCa sensitively so far, and the differential diagnosis depends on prostatic punctura biopsy that is traumatic. With the progress of ultrasound contrast agent (UCA) and relevant ultrasound techniques, the importance of ultrasound contrast in prostatic carcinoma diagnosis has been further recognized. However, the UCA used in prostatic carcinoma diagnosis are non-targeted up to now. Studies have demonstrated that prostate-specific membrane antigen (PSMA) is a kind of glucoprotein locating on the cell envelope, possessing high prostatic carcinoma tissue specificity and it's expressed highly in prostatic carcinoma tissue. Even more, it is discovered that vascular endothelial growth factor(VEGF) is distributed widely in prostatic carcinoma tissue and it's important in the growth and maintenance of the prostatic carcinoma angiogenesis. To demonstrate if the contrast agents carrying antibody could retain in prostatic carcinoma tissues at a higher concentration and the specific imaging could be achieved, we design to prepare two sorts of targeted ultrasound contrast agents carrying anti-PSMA antibody or anti-VEGF antibody respectively, and apply them to animal experiments. Objective: To prepare two sorts of human prostatic carcinoma-targeted ultrasound contrast agent using anti-PSMA antibody and anti-VEGF antibody as ligand respectively and evaluate their targeting ability in vitro and the specific enhancement effects of these targeted ultrasound contrast agents on human PCa inoculated in nude mice to set up a sensitive early ultrasound diagnosis method of PCa. Methods: Targeted microbubbles carrying anti-PSMA antibody and anti-VEGF antibody were prepared in electrostatic attraction way respectively. The expression of PSMA on prostatic carcinoma cell LNCaP and VEGF on vascular endothelial cell ECV304 was detected with Immunofluorescence. Immunofluorescent straining assay was used to indentify the combination of anti-PSMA antibody or anti-VEGF antibody with liposome microbubbles. Targeted microbubbles carrying anti-PSMA antibody and anti-VEGF antibody were added to prostatic carcinoma cell LNCaP and vascular endothelial cell ECV304 respectively and then observed under the light and fluorescence microscope to evaluate their targeting ability in vitro, while the common microbubbles were as controls. Antibody blocking test was applied to identify the specific binding of the targeted microbubbles to target cells. Tumor cell line LNCaP of human PCa was cultivated, passaged in vitro and inoculated subcutaneously into male nude mice to establish tumor bearing nude mice model of human PCa. Common microbubbles and targeted microbubbles carrying anti-PSMA antibody and anti-VEGF antibody were injected respectively into nude mice bearing tumor via vena caudalis. The continuous change of the grey-scale in the tumor was observed and its intensity was measured, according to which, time-intensity curve (TIC) was drawn and peak intensities and persisting times were compared under different conditions. Twenty four nude mice bearing tumor were divided into 3 groups randomly. Common microbubbles and targeted microbubbles carrying anti-PSMA antibody and anti-VEGF antibody were injected respectively into nude mice bearing tumor via vena caudalis . After a delay of 12 min without scanning, the tumors were examined using power Doppler imaging. The blood flow area ratio (BFAR) was measured and calculated, which was analyzed and compared among the groups. Results: The immunofluorescent straining assay on targeted liposome microbubbles and the expressed PSMA and VEGF were positive. The results of homing test in vitro showed that the targeted microbubbles carrying anti-PSMA antibody and anti-VEGF antibody could actively adhere to prostatic carcinoma cell LNCaP and vascular endothelial cell ECV304 cell respectively, while it was negative in the controls. And the results of antibody blocking test confirmed the specific binding of the targeted microbubbles to target cells.The tumor bearing nude mice model of human PCa was established successfully. The differences of peak intensity and persisting time were not significant after injecting common microbubbles and targeted microbubbles carrying anti-PSMA antibody and anti-VEGF antibody using low MI grey-scale imaging (P>0.05). However, when power Doppler imaging was applied, power Doppler signal in the tumor was enhanced markedly after the injection of the targeted microbubbles carrying anti-VEGF antibody and BFAR in VEGF group was significantly higher than those in common group and PSMA group (P<0.01). Conclusions: The anti-PSMA antibody and anti-VEGF antibody can be binded to the microbubbles successfully by electrostatic attraction. And the microbubbles can be combined with target cells specifically and efficiently in vitro study. The microbubbles carring anti-VEGF antibody can retain in human prostatic cancer tissue specifically and effectively in vivo, which can be applied to diagnose prostatic cancer as a targeted contrast agent. And power Doppler imaging is a good mode to show the enhanced sigal of tumor after targeting contrast.