| RNA replicon (self-replicating RNA) has long been regarded as an important form of vaccines due to its remarkable immunogenicity and superior safety features. In this study, we report the first non-viral delivery of RNA replicons using protein-based PRINT particles. Cylindrical bovine serum albumin (BSA) particles (diameter (d) 1 mum, height (h) 1 mum) fabricated utilizing PRINTRTM technology were rendered transiently insoluble using a novel, reductively labile disulfide-based cross-linker. After being cross-linked, the protein particles retain their integrity in aqueous solution and dissolve preferentially under a reducing environment which is found in cytoplasm, the site of action for RNA replicons. Our data reveal that the cross-linker leaves no chemical residue on the amino group it reacts with after being cleaved, which represents a great advantage over traditionally used protein cross-linkers. The particles were loaded with RNA replicons encoding several different proteins including Chloramphenicol Acetyl Transferase (CAT), green fluorescent protein (GFP) and Luciferase protein. The PRINT process used to fabricate protein-based particles in this study is gentle and avoids the obvious degradation of RNA replicons. The disulfide cross-linker is RNA-friendly and can stabilize the particles without affecting the biological performance of RNA replicons. Delivery of the particles to Vero cells was achieved by coating particles with a TransIT RTM-mRNA transfection reagent (TransIT) and cationic lipids (DOTAP and DOPE). Our data suggest that: 1) it is necessary to use a degradable cross-linker for successful delivery of RNA replicon via albumin-based PRINT particles and 2) this may be a promising system for delivery of RNA replicon-based vaccines. |
