In vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090 Brussels, Belgium.
J Control Release. 2012 Mar 10;158(2):346-53. doi: 10.1016/j.jconrel.2011.12.007. Epub 2011 Dec 16.
Camelid-derived single-domain antibody-fragments (~15kDa), called nanobodies, are a new class of molecular tracers that are routinely identified with nanomolar affinity for their target and that are easily tailored for molecular imaging and drug delivery applications. We hypothesized that they are well-suited for the design of targeted microbubbles (μBs) and aimed to develop and characterize eGFP- and VCAM-1-targeted μBs. Anti-eGFP (cAbGFP4) and anti-VCAM-1 (cAbVCAM1-5) nanobodies were site-specifically biotinylated in bacteria. This metabolic biotinylation method yielded functional nanobodies with one biotin located at a distant site of the antigen-binding region of the molecule. The biotinylated nanobodies were coupled to biotinylated lipid μBs via streptavidin-biotin bridging. The ability of μB-cAbGFP4 to recognize eGFP was tested as proof-of-principle by fluorescent microscopy and confirmed the specific binding of eGFP to μB-cAbGFP4. Dynamic flow chamber studies demonstrated the ability of μB-cAbVCAM1-5 to bind VCAM-1 in fast flow (up to 5 dynes/cm(2)). In vivo targeting studies were performed in MC38 tumor-bearing mice (n=4). μB-cAbVCAM1-5 or control μB-cAbGFP4 were injected intravenously and imaged using a contrast-specific ultrasound imaging mode. The echo intensity in the tumor was measured 10min post-injection. μB-cAbVCAM1-5 showed an enhanced signal compared to control μBs (p<0.05). Using metabolic and site-specific biotinylation of nanobodies, a method to develop nanobody-coupled μBs was described. The application of VCAM-1-targeted μBs as novel molecular ultrasound contrast agent was demonstrated both in vitro and in vivo.
骆驼科来源的单域抗体片段（~15kDa），称为纳米体，是一类新型的分子示踪剂，其对靶标具有纳摩尔亲和力，并且易于针对分子成像和药物输送应用进行定制。我们假设它们非常适合用于设计靶向性微泡（μBs），并旨在开发和表征 GFP 和 VCAM-1 靶向的μBs。抗 GFP（cAbGFP4）和抗 VCAM-1（cAbVCAM1-5）纳米体在细菌中进行了定点生物素化。这种代谢生物素化方法产生了具有一个生物素位于分子抗原结合区域的远位的功能性纳米体。生物素化的纳米体通过链霉亲和素-生物素桥接与生物素化脂质μB 偶联。通过荧光显微镜测试μB-cAbGFP4 识别 GFP 的能力作为原理验证，并证实了 GFP 与μB-cAbGFP4 的特异性结合。动态流动室研究证明了μB-cAbVCAM1-5 在高速流动（高达 5 达因/厘米 2）下结合 VCAM-1 的能力。在 MC38 荷瘤小鼠（n=4）中进行了体内靶向研究。静脉内注射μB-cAbVCAM1-5 或对照μB-cAbGFP4，并使用对比特异性超声成像模式进行成像。在注射后 10 分钟测量肿瘤中的回波强度。与对照μBs 相比，μB-cAbVCAM1-5 显示出增强的信号（p<0.05）。使用纳米体的代谢和定点生物素化，描述了一种开发纳米体偶联μB 的方法。在体外和体内证明了 VCAM-1 靶向μBs 作为新型分子超声对比剂的应用。
