Preparation Of A Lipopeptide-Based Alpha(v)Beta(3) Integrin-targeted Ultrasound Contrast Agent And Its Efficacy

In 1968, Gramiak first reported the ultrasonic identification of the cardiac chambers based on intracardiac injection of indocyanine green dye, and also reported the application of ultrasound microbubble (UM). In recent years, many researchers have achieved great progress in UM research. Nowadays, targeted ultrasound microbubble contrast agents become a hot issue of ultrasound-contrast agent research and attracts attention from many other medical areas. An increasing number of researchers find that targeted ultrasound microbubble contrast agents can distinguish from the level of molecular in lesions; it can produce special imaging in the targeted site point, which will obviously improve the ultrasound diagnosis ability of early pathological changes.In particular, avP3 integrin is highly expressed on activated angiogenic endothelial cells in tumor vessels, resulting in clinical value that are to be a potential tumor targeted receptor. Therefore, preparation of avP3 integrin targete d ultrasound microbubble contrast agent can directly realize in enhancing the imaging of the tumor angiogenesis. In this study, we successfully couple of thio-carry iRGD ligand to DSPE-PEG2000 Maleimides in covalent method, which design the novel a lipopeptide-based alpha (v) beta (3) integrin-targeted ultrasound contrast agent. And then demonstrate its capacity of specially binding to αvβ3 integrin expressed in mouse with endothelial cells (bEend.3) in vitro. Subsequently, to investigate the feaibility of tumor neovascularization specifical ly strengthen the ultrasound molecular imaging and targeted effect in vivo 4T1 breast cancer model, indicating its usefulness as ultrasonic molecular probe in monitoring integrin αvβ3 expression during tumor angiogenesis, and looking for new targets for diagnosing and treating cancer at early stages.1. The purpose of our study is to develop and characterize a novel ultrasound microbubble agent. The microbubbles were characterized by Accusizer 780 and inverted fluorescence microscope and ultrasonic imaging device. Coupling of thio-carry iRGD to maleimide-carrying phospholipid materials (DSPE-PEG2000-Maleimide), the synthetic product DSPE-PEG2000-iRGD was detected by mass spectrometry (MS). The amount of the iRGD peptide on the microbubble surface was then calculated by G-250 method.Results suggested iRGD peptide was successfully coupled on DSPE-PEG 2000 Maleimide and iRGD-targeted microbubble. iRGD-targeted microbubble showed a similar characters with the control microbubble in partical size, concentration, ultrasonic imaging in vitro and optical morphology. DSPC:DSPE-PEG2000:DSPE-PEG-iRGD was 9:0.5:0.5 (by molar ratio), while the number of iRGD peptide molecules in tagrated into MBs had reached max and therefore satisfied specific requirement ultrasound microbubbles.2. The binding effect and specificity of iRGD-MBs with vascular endothelial cells is determined in vitro. The stastic experiments showed that iRGD-MBs could be more and tightly binding with bEnd.3 cell.This function could be blocked by adding intoanti-integrin avβ3 monoclonal antibody, which did not observed iRGD-MBs adhereing on bEnd.3 cell. Parallel plate flow chamber revealed that iRGD-MBs had the ability in binding on bEnd.3 cell at 1 dyne/cm2. However, the preblocking of the integrin αvβ3 on bEnd.3 cell with antibody and the control groups, the attachment of MBs was not obvious.3. In this study, we assessed the potential of Ultrasound molecular imaging with real time and non-invasively using vascular endothelial integrin avβ3 receptor-targeted microbubble on mouse 4T1 tumor angiogenesis model. Ultrasound imaging signal was 10-fold using iRGD-MBs (10.70±2.75) than that of control MBs (1.13±0.18), with statistical significance (P<0.05). Immunofluorescence showed co-localization of the alpha (v) (green colour) with CD31 (red colour) on mose 4T1 tumor angiogenic endothelial cells. These results suggests that iRGD-MBs can obviously targeted tumor angiogenesis compared with control MBs, indicating its usefulness as ultrasonic molecular probe in intravascular, and to realize the ultrasonic molecular imaging of tumor angiogenesis, which may be a promising approach for early detection and for the sensitive assessment of therapy effects in tumor.