Abstract
Targeting ultrasound contrast agents can be prepared by some specific bio-conjugation techniques. The biotin-avidin complex is an extremely useful noncovalent binding system, but the system might induce immunogenic side effects in human bodies. Previous proposed covalently conjugated systems suffered from low conjugation efficiency and complex procedures. In this study, we purpose a covalently conjugated microbubbles coupling with nucleic acid ligands (i.e., aptamer) providing a higher specific affinity (Kd~0.8nM) for ultrasound targeting studies. The synthesizing aptamer conjugated MBs was achieved by thiol-maleimide crosslinking strategy. The thiol-modified aptamer (sgc8c) could be easily crosslinked with the maleimide functionalized PEG-lipid . The TCEP was used to activate the aptamer by breaking the disulfide bond. Afterward, bubble suspensions were formed with perfluoropropane gas by intense shaking, which yielded bubbles with a mean diameter of 800 nm at a concentration of 108~109 bubbles/mL. Note that purification of aptamer and suitable accelerating sonication are critical procedures to ensure the conjugation efficiency. The efficiency was quantified by calculating the changes of fluorescence intensity using spectrophotometer and the result was 45% at the ratio (functionalized lipid/aptamer) of 80. The cell experiments were performed in two cell lines (CEM for experiment and Ramos for control). The figures (a) and (b) show the optical images in dark field and fluorescence microscopy, respectively. Figure (c) shows the quantitative results of flow cytometry. The GMean value in experiment set was 6.2 folds with respect to control ones. The results indicate that aptamer conjugated microbubbles can be fabricated in one-step synthesis. With in vitro selective screening, aptamers can perform higher affinity for specific molecules than those in traditional antibody system. Potential applications include targeting imaging in vivo and drug delivery/release.