The preparation of streptoavidin conjugated ultrasound contrast agent:an experimental study

Objective:The purpose of this study is to prepare streptoavidin(SA) conjugated anionic lipid microbubbles and to assess the physiochemical properties. Methods: The first step was to direct binding of indocarbocyanin(Cy3) labeled SA to microbubbles by mechanical vibration, sonication, surface bind after mechanical vibration or sonication which were divided into four groups. Half of all resulted suspensions were used for repeated washings, trying to remove any free Cy3-SA using floatation method. Then, the size, shape, bubble concentration and fluorescent intensity were measured and analyzed under light and fluorescent microscopy. The binding rate of microbubbles with Cy3-SA was calculated using flow cytometry. Results: Compared with mechanical vibration groups, anionic lipid microbubbles with Cy3-SA in sonication groups appeared to be slightly larger in size. The microbubbles with Cy3-SA delaminated quicker than the control group. All Cy3-SA bubbles could give a bright red fluorescence except the control group. Though microbubbles concentration dropped with washings, it was still fluorescent visible and no significant binding rates differences were found in different preparation methods(P0.05), it was above 98%. Conclusions: Streptoavidin can be conjugated with lipid microbubbles through mechanical vibration or sonication during preparation procedure. The conjugation of MB and SA were firmly high.