Preparation of antibody-immobilized gelatin nanospheres incorporating a molecular beacon to visualize the biological function of macrophages
Preparation of antibody-immobilized gelatin nanospheres incorporating a molecular beacon to visualize the biological function of macrophages
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DOI：
10.1016/j.reth.2019.12.009
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发表时间：
2020-01
影响因子：
4.3
通讯作者：
Y. Yoshimoto;J. Jo;Y. Tabata
中科院分区：
文献类型：
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作者：
Y. Yoshimoto;J. Jo;Y. Tabata
前言炎症反应在疾病的进展和治疗效果中起着重要的作用。在这种情况下，非常需要开发一种可视化炎症反应的技术。本研究以参与炎症反应的巨噬细胞及其microRNA（microRNA，miRNA）为研究对象，设计并制备了一种检测巨噬细胞microRNA的分子信标（molecular beacon，MB）系统。通过几种方法将对巨噬细胞表面受体具有亲和力的抗体固定在明胶纳米球上。设计了用于促炎miRNA 155- 5 p的基于核酸的MB，并将其掺入抗体固定化明胶纳米球（MB-明胶NS）中。将极化为促炎或抗炎表型之前和之后的巨噬细胞与MB-明胶NS一起培养，观察细胞内荧光的变化。结果通过用NaIO 4偶联明胶的氨基与抗体糖链制备的抗体固定化明胶纳米球对细胞受体表现出最高的亲和力。MB复合的细胞穿透（CP）肽被成功地纳入抗体固定明胶纳米球。当与促炎性巨噬细胞一起培养时，MB-明胶NS有效地检测到miRNA 155- 5 p发射fluorescence.ConclusionsBy的NaIO 4方法，抗体被固定到明胶纳米球上，同时MB被掺入到抗体固定的明胶纳米球中，保持高亲和力。掺入的MB允许mRNA可视化巨噬细胞的促炎性质。
IntroductionInflammatory response plays an important role in the disease progress or therapeutic effect. In this context, it is highly required to develop a technology to visualize the inflammatory response. In this study, macrophages and their microRNA (miRNA) which are involved in the inflammatory response, were focused while a system of molecular beacon (MB) to detect the miRNA of macrophages was designed and prepared.MethodsGelatin nanospheres were prepared by the conventional coacervation method. An antibody with an affinity for the surface receptor of macrophages was immobilized onto the gelatin nanospheres by several methods. A nucleic acid-based MB for a pro-inflammatory miRNA 155–5p was designed and incorporated into the antibody-immobilized gelatin nanospheres (MB-gelatin NS). Macrophages before and after the polarization into pro-inflammatory or anti-inflammatory phenotypes were cultured with the MB-gelatin NS and change in the intracellular fluorescence was observed.ResultsThe antibody-immobilized gelatin nanospheres prepared by a coupling between the amino groups of gelatin and the sugar chains of antibody with NaIO4showed the highest affinity for cellular receptor. MB complexed with the cell-penetrating (CP) peptide was successfully incorporated into the antibody-immobilized gelatin nanospheres. When cultured with pro-inflammatory macrophages, MB-gelatin NS efficiently detected the miRNA 155–5p to emit fluorescence.ConclusionsBy the NaIO4method, the antibody was immobilized onto gelatin nanospheres with a high affinity remaining while the MB was incorporated into the antibody-immobilized gelatin nanospheres. The MB incorporated allowed mRNA to visualize the pro-inflammatory nature of macrophages.