PEGylation of ribonuclease A and targeting the enzyme by conjugation to anti-EGFR antibody Cetuximab

Abstract

Introduction:RibonucleaseA (RNase A) is a potential anti-cancer agent that degrades single-stranded RNAs leading to alterations in gene expression, biosynthesis of protein, and modulation of several signaling pathways that can result in induction of cell apoptosis. Inhibition of RNase A by ribonuclease inhibitors (RIs) which can be found naturally in the cytosol, is the major problem that limits the therapeutic use of RNases. Nanoformulations such as PEGylation, conjugation/encapsulation with nanostructures can be used to overcome this limitation.The current investigation introduces an efficient and novel tumor therapeutic immunoRNase (PEGylatedRNase A-Cetuximab) for overcoming the intracellular ribonuclease inhibitors as one of the most significant obstacles for the clinical applications of RNases in colorectal cancer (CRC) therapy.Object of this study:aim of this study is to combine polyethylene glycol molecules with RNase A to overcome RIs and furthermore, conjugate the PEGylated enzyme with cetuximab monoclonal antibody in order to target colorectal cancer cells.Materials and methods:First, RNase A was stabilized by polyethylene glycol (PEG), and conjugated to Cetuximab (Cet). The physicochemical properties of engineered immonoRNase (RN-PEG-Cet) was characterized by SDS-PAGE, UV-vis spectrum, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Ribonuclease and antibody activity of ImmunoRNase was confirmed by UV-visible spectrophotometery and western blotting. Its biological impacts, including cell viability, morphological changes of the nucleus, and apoptosis, were evaluated on SW-480 cells. Results: ImmunoRNase bioconjugation was confirmed via SDS-PAGE. SEM and AFM results showed that the size and shape of ImmunoRNasewas about 35 nm and irregular spheres. ImmunoRNase effectively induced significant morphological changes in the nucleus in SW-480 cells and significantly reduced cancer cell viability. Maximum inducing apoptosis in SW-480 cells was obtained after treating cells with an IC50 dose of ImmunoRNase. Conclusion: The engineered ImmunoRNase may be considered a potential candidate for targeted therapy of CRC.