Rational design and characterisation of an amphipathic cell penetrating peptide for non-viral gene delivery

McErlean, Emma; McCrudden, Cian M.; McBride, J.W.; Cole, Grace; Kett, Vicky L.; Robson, Tracy; Dunne, Nicholas; McCarthy, Helen

doi:10.1016/j.ijpharm.2021.120223

Cited by 19 publications

(14 citation statements)

References 35 publications

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“…Although A2-17 did not induce significant cytotoxicity in our experimental conditions, a previous study has reported that amphipathic ARPs exhibits similar properties to the membrane-active antimicrobial peptides 13 , which can cause stable membrane pores or destroy the lipid membrane barrier. In this regard, the relationship of the amphipathicity of peptides with their membrane interaction and cell membrane penetration ability is not well understood 12 , 14 – 17 .…”

Section: Introductionmentioning

confidence: 99%

Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides

Takechi-Haraya

Ohgita

Kotani

et al. 2022

Sci Rep

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We previously developed an amphipathic arginine-rich peptide, A2-17, which has high ability to directly penetrate across cell membranes. To understand the mechanism of the efficient cell-penetrating ability of the A2-17 peptide, we designed three structural isomers of A2-17 having different values of the hydrophobic moment and compared their membrane interaction and direct cell penetration. Confocal fluorescence microscopy revealed that cell penetration efficiency of peptides tends to increase with their hydrophobic moment, in which A2-17 L14R/R15L, an A2-17 isomer with the highest hydrophobic moment, predominantly remains on plasma cell membranes. Consistently, Trp fluorescence analysis indicated the deepest insertion of A2-17 L14R/R15L into lipid membranes among all A2-17 isomers. Electrophysiological analysis showed that the duration and charge flux of peptide-induced pores in lipid membranes were prominent for A2-17 L14R/R15L, indicating the formation of stable membrane pores. Indeed, the A2-17 L14R/R15L peptide exhibited the strongest membrane damage to CHO-K1 cells. Atomic force microscopy quantitatively defined the peptide-induced membrane perturbation as the decrease in the stiffness of lipid vesicles, which was correlated with the hydrophobic moment of all A2-17 isomers. These results indicate that optimal membrane perturbation by amphipathic A2-17 peptide is critical for its efficient penetration into cells without inducing stabilized membrane pores.

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Section: Introductionmentioning

confidence: 99%

Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides

Takechi-Haraya

Ohgita

Kotani

et al. 2022

Sci Rep

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“…According to previous studies, the replacement of arginine by histidine is capable of increasing the efficacy of escaping from endosome [ 24 , 25 ]. Therefore, we employed chloroquine, a known endosomal disruptor, which is able to damage the formation process of endosome by inhibit its acidification.…”

Section: Resultsmentioning

confidence: 99%

“…Histidine is an amino acid that has an imidazole group on the side chain with the pKa value of 6.0, which is lower than that of lysine (pKa 10.53) or arginine (pKa 12.48). Histidine-rich peptides are usually considered as being endosomolytic in nature, as it can accelerate the endosomal escaping process through the proton sponge phenomenon or “flip-flop” effects [ 24 , 25 ]. One example of histidine application in CPP was the modification of wild-type S4(13)-PV by adding a five-histidine tail to its N-terminus, resulting in the significantly improved efficacy of peptide-mediated gene silencing in human fibrosarcoma HT1080 cells [ 26 ].…”

Section: Introductionmentioning

confidence: 99%

Development and Characterization of High Efficacy Cell-Penetrating Peptide via Modulation of the Histidine and Arginine Ratio for Gene Therapy

et al. 2021

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Cell-penetrating peptides (CPPs), as non-viral gene delivery vectors, are considered with lower immunogenic response, and safer and higher gene capacity than viral systems. In our previous study, a CPP peptide called RALA (arginine rich) presented desirable transfection efficacy and owns a potential clinic use. It is believed that histidine could enhance the endosome escaping ability of CPPs, yet RALA peptide contains only one histidine in each chain. In order to develop novel superior CPPs, by using RALA as a model, we designed a series of peptides named HALA (increased histidine ratio). Both plasmid DNA (pDNA) and siRNA transfection results on three cell lines revealed that the transfection efficacy is better when histidine replacements were on the C-terminal instead of on the N-terminal, and two histidine replacements are superior to three. By investigating the mechanism of endocytosis of the pDNA nanocomplexes, we discovered that there were multiple pathways that led to the process and caveolae played the main role. During the screening, we discovered a novel peptide-HALA2 of high cellular transfection efficacy, which may act as an exciting gene delivery vector for gene therapy. Our findings also bring new insights on the development of novel robust CPPs.

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“…These data can be used to predict the activity of peptide candidates in de novo design strategies as well [97]. Structural parameters, such as sequence length, frequency of certain amino acid residues in the sequence, hydrophilicity, and overall charge of the peptides are linked to antimicrobial activity and used for the prediction of promising candidate sequences [98,99]. The linguistic model, for the first time, considered the linear arrangement of amino acid residues in a peptide sequence as some kind of language that follows physico-chemical "grammar" rules.…”

Section: Optimization Of Membrane Active Peptidesmentioning

confidence: 99%

Design of Membrane Active Peptides Considering Multi-Objective Optimization for Biomedical Application

2022

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A multitude of membrane active peptides exists that divides into subclasses, such as cell penetrating peptides (CPPs) capable to enter eukaryotic cells or antimicrobial peptides (AMPs) able to interact with prokaryotic cell envelops. Peptide membrane interactions arise from unique sequence motifs of the peptides that account for particular physicochemical properties. Membrane active peptides are mainly cationic, often primary or secondary amphipathic, and they interact with membranes depending on the composition of the bilayer lipids. Sequences of these peptides consist of short 5–30 amino acid sections derived from natural proteins or synthetic sources. Membrane active peptides can be designed using computational methods or can be identified in screenings of combinatorial libraries. This review focuses on strategies that were successfully applied to the design and optimization of membrane active peptides with respect to the fact that diverse features of successful peptide candidates are prerequisites for biomedical application. Not only membrane activity but also degradation stability in biological environments, propensity to induce resistances, and advantageous toxicological properties are crucial parameters that have to be considered in attempts to design useful membrane active peptides. Reliable assay systems to access the different biological characteristics of numerous membrane active peptides are essential tools for multi-objective peptide optimization.

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Rational design and characterisation of an amphipathic cell penetrating peptide for non-viral gene delivery

Cited by 19 publications

References 35 publications

Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides

Effect of hydrophobic moment on membrane interaction and cell penetration of apolipoprotein E-derived arginine-rich amphipathic α-helical peptides

Development and Characterization of High Efficacy Cell-Penetrating Peptide via Modulation of the Histidine and Arginine Ratio for Gene Therapy

Design of Membrane Active Peptides Considering Multi-Objective Optimization for Biomedical Application

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