Preparation of antibody-immobilized gelatin nanospheres incorporating a molecular beacon to visualize the biological function of macrophages

• Published in: Regenerative therapy Vol. 14; pp. 11 - 18
• Main Authors: Yoshimoto, Yu, Jo, Jun-ichiro, Tabata, Yasuhiko
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2020; Japanese Society for Regenerative Medicine; Elsevier
• Subjects: Antibody immobilization; Gelatin nanospheres; Inflammatory response; Macrophages; microRNA; Molecular beacon; Original; PBS; BCA; Gelatin nanospheres; IL; DSS; qRT-PCR; Inflammatory response; BHQ; DLS; Macrophages; CP; BSA; WST-8; MB; DDW; FCS; Antibody immobilization; Molecular beacon; microRNA; miRNA; GA; KPB; Ig; BSA, bovine serum albumin; KPB, potassium phosphate-buffered; Ig, immunoglobulin; qRT-PCR, quantitative real time-polymerase chain reaction; BCA, bicinchoninic acid; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; DDW, double-distilled water; DSS, disuccinimidyl suberate; PBS, phosphate buffered-saline; miRNA, microRNA; DLS, dynamic light scattering; MB, molecular beacon; GA, glutaraldehyde; IL, interleukin; CP, cell-penetrating; BHQ, black hole quencher; FCS, fetal calf serum
• ISSN: 2352-3204; 2352-3204
• DOI: 10.1016/j.reth.2019.12.009

Inflammatory 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. Gelatin 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. The antibody-immobilized gelatin nanospheres prepared by a coupling between the amino groups of gelatin and the sugar chains of antibody with NaIO4 showed 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. By the NaIO4 method, 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. •Antibody could be immobilized onto gelatin nanospheres with the affinity remaining.•MB for a pro-inflammatory miRNA was incorporated into gelatin nanospheres.•MB incorporated emitted the fluorescence in the pro-inflammatory macrophages.