THESIS
2022
1 online resource (xv, 112 pages) : illustrations (some color)
Abstract
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...[
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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.
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