Department of Pharmaceutical Sciences , 428 Church Street , University of Michigan , Ann Arbor , Michigan 48109 , United States.
Department of Biomedical Engineering , 2200 Bonisteel Boulevard , University of Michigan , Ann Arbor , Michigan 48109 , United States.
Bioconjug Chem. 2018 Apr 18;29(4):1251-1260. doi: 10.1021/acs.bioconjchem.8b00033. Epub 2018 Mar 19.
The number and spacing of B-cell epitopes on antigens have a profound impact on the activation of B cells and elicitation of antibody responses, the quantitative aspects of which may be utilized for rational design of vaccines. Ni-chelating liposomes have been widely used as protein carriers in experimental studies of vaccine delivery, owing to the convenience and versatility of this conjugation chemistry. However, the epitope number per particle as well as the stability of protein conjugation on liposomes remain far less characterized. Here we have developed quantitative methods to measure the average spatial density of proteins on liposomes using both ensemble and single-molecule techniques and demonstrated their utility using liposomes conjugated with native proteins of two different sizes. These studies revealed that the initial density of protein conjugation on Ni-chelating liposomes can be finely controlled, but the density can decrease over time upon dilution due to the noncovalent nature of Ni-chelation chemistry. These results indicate that an alternative method other than the Ni-chelation chemistry is needed for stable conjugation of epitopes onto liposomes and also suggest a general strategy that can be used to precisely regulate the epitope density on liposomes for B-cell antigen delivery.
抗原上 B 细胞表位的数量和间距对 B 细胞的激活和抗体反应的产生有深远的影响，其定量方面可用于疫苗的合理设计。由于这种缀合化学的便利性和多功能性，镍螯合脂质体已被广泛用作疫苗传递实验研究中的蛋白质载体。然而，每个颗粒的表位数量以及蛋白质在脂质体上的缀合稳定性仍远未得到充分表征。在这里，我们开发了使用集总和单分子技术来测量脂质体上蛋白质平均空间密度的定量方法，并使用两种不同大小的天然蛋白质缀合的脂质体证明了其用途。这些研究表明，镍螯合脂质体上蛋白质缀合的初始密度可以精细控制，但由于 Ni 螯合化学的非共价性质，在稀释后密度会随时间降低。这些结果表明，需要一种替代 Ni 螯合化学的方法来稳定地将表位缀合到脂质体上，并且还表明了一种通用策略，可用于精确调节脂质体上 B 细胞抗原传递的表位密度。
