Enzymatic ligation of an antibody and arginine 9 peptide for efficient and cell-specific siRNA delivery

Abstract

A fusion protein comprising an antibody and a cationic peptide, such as arginine-9 (R9), is a candidate molecule for efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and R9 with the Q-tag sequence LLQG at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 was synthesized at a maximum binding efficiency of 85.1%, without changing the binding affinity of the nanobody for the antigen. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9–siRNA complex, at a 30:1 ratio, induced an approximately 58.6% reduction in the amount of target protein due to RNAi in mRNA compared to lipofectamine.

Chemical Modification of Cytochrome C for Acid-Responsive Intracellular Apoptotic Protein Delivery for Cancer Eradication

“…Cell-penetrating peptides (CPPs) are short peptides (5–30 amino acids) that possess the unique ability to translocate across the cell membrane while preserving membrane integrity, exhibiting minimal invasiveness, and demonstrating low cytotoxicity [ 7 , 8 , 9 ]. The versatility of CPPs allows them to transport a range of cargo molecules, including peptides, proteins, nucleic acids, small molecules, and nanoparticles [ 10 , 11 , 12 , 13 , 14 , 15 ]. This capability has spearheaded numerous preclinical and clinical studies [ 16 , 17 , 18 ].…”

Phytol Suppresses Osteoclast Differentiation and Oxidative Stress through Nrf2/HO-1 Regulation in RANKL-Induced RAW264.7 Cells

“…In the normal bone state, the cells constantly fight against excess ROS via intracellular antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), and the balance of the intracellular levels of these enzymes is necessary for normal conditions [ 12 ]. Nrf2 deletion increases bone resorption, RANKL expression, and osteoclast count; reduces the mechanical properties of the skeletal system and load-driven bone formation; and impairs fracture healing in mice [ 13 , 14 , 15 , 16 ]. In addition, Nrf2 deletion in osteoclast precursor cells derived from Nrf2-knockdown mice induces oxidative stress and RANKL-induced osteoclast differentiation [ 17 ].…”