主动靶向微泡（MB）的分子超声成像在临床前研究中被证明是有前景的，但其临床转化有限。为了实现这一目标，必须能够以高批次重复性生产主动靶向的 MB，并在表面上具有可控且确定数量的结合配体。在这方面，基于聚（氰基丙烯酸正丁酯）（PBCA）的聚合物MB已用于US分子成像，然而，配体偶联主要通过水解和碳二亚胺化学来完成，这是一个多步骤的过程，重现性差，并且MB产率低。在此，我们开发了一种单步偶联程序，可实现高 MB 产率和最小的批次间差异。使用氨解方案生成主动靶向的 PBCA-MB，其中使用甲醇锂作为催化剂将含胺的 cRGD 添加到 MB 中。我们证实了 cRGD 在 MB 表面上的成功缀合，同时保留了它们的结构和声学信号。与传统的水解方案相比，氨解可产生更高的 MB 产率和更好的偶联效率重现性。光学成像显示，在流动条件下，氨解产生的 cRGD 和罗丹明标记的 MB 在体外与肿瘤坏死因子-α (TNF-α) 激活的内皮细胞特异性结合。此外，US 分子成像显示，在 TNF-α 激活的小鼠主动脉和小鼠 4T1 肿瘤中，cRGD-MB 的结合明显高于对照 MB。因此，使用基于氨解的缀合方法，可以实现对 cRGD-MB 生产的重要改进，这将有助于生产具有优异结合和超声成像性能的临床规模制剂。© 2024。作者。

Molecular ultrasound imaging with actively targeted microbubbles (MB) proved promising in preclinical studies but its clinical translation is limited. To achieve this, it is essential that the actively targeted MB can be produced with high batch-to-batch reproducibility with a controllable and defined number of binding ligands on the surface. In this regard, poly (n-butyl cyanoacrylate) (PBCA)-based polymeric MB have been used for US molecular imaging, however, ligand coupling was mostly done via hydrolysis and carbodiimide chemistry, which is a multi-step procedure with poor reproducibility and low MB yield. Herein, we developed a single-step coupling procedure resulting in high MB yields with minimal batch-to-batch variation. Actively targeted PBCA-MB were generated using an aminolysis protocol, wherein amine-containing cRGD was added to the MB using lithium methoxide as a catalyst. We confirmed the successful conjugation of cRGD on the MB surface, while preserving their structure and acoustic signal. Compared to the conventional hydrolysis protocol, aminolysis resulted in higher MB yields and better reproducibility of coupling efficiency. Optical imaging revealed that under flow conditions, cRGD- and rhodamine-labelled MB, generated by aminolysis, specifically bind to tumor necrosis factor-alpha (TNF-α) activated endothelial cells in vitro. Furthermore, US molecular imaging demonstrated a markedly higher binding of the cRGD-MB than of control MB in TNF-α activated mouse aortas and 4T1 tumors in mice. Thus, using the aminolysis based conjugation approach, important refinements on the production of cRGD-MB could be achieved that will facilitate the production of clinical-scale formulations with excellent binding and ultrasound imaging performance.© 2024. The Author(s).