Nucleic acids are used as the template for protein synthesis, and this nature allows a wide range of biomedical applications. Developing new tools for efficient delivery of the exogenous nucleic acids into cells would expand the range of possible targets beyond what is generally accessible by conventional pharmaceutics. However, the clinical promise of nucleic acid is hindered by the poor cytosol delivery. For mRNA, the intact and functional mRNA is expected to be delivered into cytosol; for DNA, successful cytosol delivery is a pre-requisite for efficient transfection. Delivery difficulties drive the design of various delivering vehicles, categorized as viral and non-viral. The non-viral vectors appear to be the next generation tools for efficient delivery, but there is still much room...[ Read more ]

Nucleic acids are used as the template for protein synthesis, and this nature allows a wide range of biomedical applications. Developing new tools for efficient delivery of the exogenous nucleic acids into cells would expand the range of possible targets beyond what is generally accessible by conventional pharmaceutics. However, the clinical promise of nucleic acid is hindered by the poor cytosol delivery. For mRNA, the intact and functional mRNA is expected to be delivered into cytosol; for DNA, successful cytosol delivery is a pre-requisite for efficient transfection. Delivery difficulties drive the design of various delivering vehicles, categorized as viral and non-viral. The non-viral vectors appear to be the next generation tools for efficient delivery, but there is still much room for improvement in transfection efficiency compared to viruses. Therefore, it has been considered to take advantage of both systems by incorporating some features of the virus into non-viral delivery systems. Our lab has previously designed a platform to mimic viral capsid based on oligopeptide. Compared with the dominant lipid- and polymer-based systems, the peptide-based systems enable automated synthesis, allow singlestep formulation with nucleic acid, and have good biocompatibility. However, limited endosomal escape and poor cytosol delivery were the bottle neck for peptide-based systems, as in other non-viral systems.

This thesis explored multiple ways for intracellular delivery in peptide-based systems, especially overcoming the endosomal escape bottle neck. Different methods have been incorporated, including peptide sequence modification, co-assembling procedure optimization, and surface modification of the assemblies. By understanding of the inter- and intra-molecular interactions within the assemblies’ structure, we discovered the significant alteration resulted by the subtle changes within the peptide sequence. In particular, the side-chain length and the neighboring atom (carbon vs sulfur) of the diphenylalanine analogues had significant impact on the morphologies of the peptide self-assemblies and peptide/DNA co-assemblies. This study demonstrates the significance of subtle alterations in aromatic interactions and contributes to a deeper understanding of the sequence modification. Therefore, to improve endosomal escape, we firstly considered directly incorporating fusogenic peptides that have been shown to improve endosomal escape in other delivery systems. For fusogenic peptide sequence incorporation, we reported the improvement by attaching the fusogenic peptide, L17E, onto the peptide self-assembled nanodisks structure. This modification achieved 2-fold increase in DNA transfection efficiency. However, the final efficiency was less than that of commercially available reagents, and complex preparation procedures might hinder the single-step formulation advantage of the peptide-based delivery system. Therefore, for subsequent mRNA delivery, direct sequence modification was used. The peptide for mRNA delivery was named pepMAX, which contains 1) a positively charged N-terminal to strengthen mRNA binding, 2) a hydrophobic segment with redox trigger to promote self-assembly and cytosol disassembly, 3) hydrophilic N-terminal for aqueous dispersion and 4) more histidines to increase the proton sponge effect. PepMAX was capable of co-assembling with mRNA into 100-150 nm nanostructures for efficient transfection of multiple cell lines. In HeLa, Hek293 and SKNMC, transfection attained (>80%) was comparable with commercially available vectors specific for mRNA delivery (LipoMMAX). The pepMAX efficiency was further improved by manipulating the peptide/mRNA co-assembling procedure. Micron-sized co-assemblies were obtained by addition of salt during pre-incubation. Here, pepMAX2 differs from the pepMAX in its N-terminal, with peptide sequence the same while no Fmoc at the end, which might lead to a higher proportion of charge-charge interactions in regulating co-assembling process. Due to ionic electrical screening, the size of the co-assemblies could be adjusted by salt concentrations. These micron-sized co-assemblies showed 90% transfected cell percentage and 2-fold protein expression level compared to the LipoMMAX. Mechanism study and live-cell confocal intracellular tracking demonstrated the non-classical endocytosis pathways by these particles. Micron-sized particles enter cells via an energy-dependent lipid-raft pathway and then release mRNA into cytosol, which might act as a reservoir in the cytosol to avoid enzymatic degradation while maintain a more consistent release rate of mRNA.

In general, this thesis reported the design methods within the peptide-based delivery system for improving the transfection efficiency, including changes in peptide sequence, on peptide self-assemblies’ surface, and during co-assembling procedures. Staring with single cell line DNA delivery, we eventually achieved with multiple cell line, efficient mRNA delivery and even 2-fold higher protein expression than commercially available reagent. (LipoMMAX) We successfully achieved intracellular delivery, especially overcoming the endosomal escape bottle neck.