Fundamental study of Mesoporous Silica Nanoparticles mediated SOD protein delivery: Focus on the protein conformation, protein corona, and molecular mechanisms

Abstract
Proteins are considered a more promising therapeutic approach than the use of small molecules and gene therapy. However, the efficacy of protein delivery is limited by inherent shortcomings, including its large molecular size and 3D conformation, and also by the biological barriers and the protein corona effect. Currently, various nanoparticles (NPs) serving as carriers have been developed, providing an opportunity to overcome delivery limitations. Nonetheless, the protein loading strategies employ multiple chemical bonds engaging the side amine groups of amino acids and are covalently incorporated in most polymeric entrapments, leading to conformation change and loss of activity. Hence, understanding the internalization of NPs and conjugated protein is imperative in developing NPs-mediated protein delivery approaches. The present study focused on the protein corona effects’ exploration, providing insights into developing a protein delivery approach. Superoxide dismutase 1 (SOD1) is a protein with high potential enzyme replacement therapy for many health conditions associated with inflammation. Protein delivery in denaturation form is a promising alternative strategy, achieving high recovery of biological activity. Here, an improved mesoporous silica NPs (MSNs)-mediated cytosolic delivery of denatured SOD1 was reported. The RMSNs were surface-modified by grafting polyethyleneimine (PEI), a cross-linker polyethylene glycol (PEG), and nickel-chelated ligand nitrilotriacetic acid (NTA). The transactivator of transcription (TAT) peptide fusion SOD1 could be conjugated via metal coordination at a distance far away from the RMSNs' surface, thus affording the SOD1 flexibility without a risk of distorting its native conformation. The native SOD1 and denatured SOD1 conjugated RMSNs (RMSN-Ni-TAT-SODn and RMSN-Ni-TAT-SODd) presented hydrodynamic sizes below 125 nm and good dispersion, improved compared to the previous report, and suitable for biomedical application. Both RMSN-Ni-TAT-SODn and RMSN-Ni-TAT-SODd presented irregular adsorption of protein corona in TEM imaging, thus allowing the targeting possibility. LC-MS/MS and Ingenuity pathway analyses of the corona proteomics unveiled an abundance of apolipoproteins (ApoE, ApoH, ApoA4, ApoC3, and ApoA2) onto RMSN-Ni-TAT-SODd, favoring its uptake by cells overexpressing lipoprotein receptors. This study demonstrated that delivery of denatured SOD profits from the selective adsorption of transporter proteins, thus leading to effective delivery.