Mechanistic Studies of Antimicrobial Peptide Encapsulation by Lipid Nanocarriers
Creators: name: Lakic, Biserka
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 340307
subject: Structure and dynamics of materials
classificationCode: 310607
subject: Nanobiotechnology

description: Antimicrobial resistance (AMR) poses a significant health challenge globally, threatening the effectiveness of antimicrobial agents. A promising class of new antibiotics are antimicrobial peptides (AMPs) which are part of the innate immune system of many organisms including bacteria, fungi, plants, animals and humans. AMPs have gained considerable interest due to their broad-spectrum activity against various pathogens, including those that have developed resistance to conventional antibiotics. While AMPs offer advantages over conventional antibiotics, they also exhibit several disadvantages that limit their clinical use, such as high production costs, potential cytotoxicity, and susceptibility to proteolytic degradation. Recently, research has also focused on ultra-short AMPs which are short derivatives of AMPs, typically ranging from 3 – 10 amino acids in length. Ultra-short AMPs are promising as they have been shown to retain antimicrobial activity but can have easier synthetic pathways and much lower production costs compared to natural AMPs.
Several recent research studies have shown that lipid nanocarriers (NCs) such as cubosomes and hexosomes are highly prospective as drug delivery candidates for antimicrobial agents, such as AMPs. However, the interactions of lipid NCs with bacteria remain poorly understood. To date, the antimicrobial efficacy of AMPs loaded in cubosomes has displayed very high levels of variability and there is no coherent understanding as to why cubosome encapsulation works for some AMPs and not others. A relatively small number of research studies focus on the use of hexosomes as delivery vehicles for AMPs. Additionally, ultra-short AMPs have not been investigated with lipid NCs such as cubosomes and hexosomes as drug delivery systems targeting bacteria. Therefore, research in this thesis initially investigated the interactions of lipid NCs, including cubosomes and hexosomes, with E. coli mimicking supported lipid bilayers (SLBs). The encapsulation of a range of AMPs, including the natural AMP indolicidin and ultra-short peptide fragments derived from indolicidin, in cubosomes was also investigated using both experimental techniques and molecular dynamics simulations. Finally, the effect of a range of parameters including the lipid composition and the lipid NC internal structure on the peptide encapsulation efficiency and the eventual antimicrobial efficacy was assessed against both gram-negative and gram-positive bacterial strains. The results are interpreted with reference to recent models describing the interactions between lipid NCs and bacteria and the known mechanism of action of the peptides.
The interaction of lipid NCs (cubosomes and hexosomes) with E. coli mimicking SLBs was investigated using a combination of experimental techniques including total internal reflection fluorescence microscopy (TIRF-M), quartz crystal microbalance with dissipation (QCM-D), and synchrotron small angle X-ray scattering (SR-SAXS). Incorporation of the fusogenic lipid di-oleoyl phosphatidylethanolamine (DOPE) to the lipid NCs increased the interactions with the bacterial SLB, whereas the addition of a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) significantly augmented these interactions. The lipid mesophase of cubosomes was disrupted upon interacting with the E. coli model membranes, while hexosomes remained structurally interact. These findings indicate that cubosomes primarily interact with bacterial model membranes through fusion, while hexosomes presumably interact via a different mechanism which preserves their structural integrity, potentially similar to the ‘phase through’ or ‘hemi-fusion’ mechanism observed in eukaryotic membranes.
Encapsulation of the ultra-short AMPs, RW5 and Fmoc-RW5, in cubosomes and the impact on the cubosome mesostructure were investigated using a combination of liquid chromatography-mass spectrometry (LC-MS), SR-SAXS, cryogenic transmission electron microscopy (cryo-TEM), dynamic light scattering (DLS) and molecular dynamics (MD) simulations. Incorporation of RW5 in both MO and PT cubosomes resulted in very poor encapsulation efficiency. The addition of a hydrophobic Fmoc moiety to this peptide significantly augmented the encapsulation efficiency. This effect was further enhanced in the presence of 150 mM NaCl, which screens repulsion between the cationic peptides. MD simulations confirmed the higher encapsulation efficiency observed for Fmoc-RW5, due to stronger and deeper interactions with the MO bilayer primarily facilitated by the Fmoc group. This study suggests that the addition of a Fmoc group to an AMP is a successful strategy to improve the encapsulation efficiency.
Incorporation and delivery of the AMP indolicidin and its ultra-short derivative priscilicidin in cubosomes were investigated using LC-MS, SR-SAXS, cryo-TEM and DLS. Priscilicidin demonstrated higher encapsulation efficiency in MO cubosomes than indolicidin, presumably due to the fmoc group. Encapsulation of either peptide significantly disrupted the cubic nanostructure in water and effected a conformational change in the peptide. The presence of salt improved the encapsulation efficiency and promoted retention of the cubic mesophase. For both indolicidin and priscilicidin, encapsulation in MO cubosomes generally reduced the antimicrobial activity against gram-negative and gram-positive bacteria. This may be explained by the mechanism of action of these peptides, involving some level of self-assembly which is presumably hindered by encapsulation. These findings are consistent with a recent diffusion to capture model, suggesting that antimicrobial compounds of high permeation, such as AMPs, may not be suitable for cubosome encapsulation.
The addition of the fusogenic lipid DOPE on lipid NC-mediated delivery of both indolicidin and priscilicidin was investigated. The addition of DOPE to MO cubosomes resulted in a phase transition to hexosomes, allowing for a direct comparison between cubosomes and hexosomes for peptide delivery to bacteria. The peptide loading of both indolicidin and priscilicidin increased with the addition of DOPE; a conformational change in both peptides was observed following encapsulation. In the presence of salt, which significantly increased peptide loading, the addition of peptide effected a phase transition towards lipid mesophases of less negative interfacial curvature. These changes are correlated with hydrophobic mismatch between the peptides and the MO bilayer and the depth of AMP insertion into the lipid bilayer. While the addition of DOPE to MO cubosomes was shown to both increase peptide loading and increased interactions with bacterial model membranes, the antimicrobial efficacy of cubosome-loaded peptides was still not higher than that of the free peptide. However, addition of 30 mol% DOPE (and the formation of hexosomes) led to significantly higher antimicrobial activity than MO cubosomes suggesting that the use of DOPE may be an effective strategy, particularly for antimicrobial compounds of low permeation. Findings presented herein emphasise the importance of carefully selecting appropriate AMPs for cubosome encapsulation, as an effective strategy to treat bacterial infections.