Fusogenic Peptide Research Articles

Amphipathic KALA fusogenic peptide enhances absorption of insulin and calcitonin by pulmonary membranes of rats.

The aim was to investigate the absorption-enhancing effect (AEE) of lysine-alanine-leucine-alanine (KALA) repeating unit peptide upon pulmonary absorption of peptide and protein medicines among rats. Absorption of insulin and calcitonin in the lung was evaluated using varying concentrations of KALA peptide from 0.1% to 1.0% (w/v). The study also examined the lung damage caused by the KALA peptide. KALA peptide with various concentrations improved the absorption of insulin and calcitonin in the lungs. It also reduced glucose and calcium levels in the blood compared to the control, with the AEE increasing in a concentration-dependent manner due to the KALA peptide. In toxicity assays, test results for protein and lactate dehydrogenase (LDH) in bronchoalveolar lavage fluid (BALF) did not show a significant increase in the presence of KALA peptide at various concentrations. This implies that the KALA peptide did not cause any membrane damage to lung tissues. In transmembrane electrical resistance (TEER) and permeability detection, a decrease in TEER value and an increase in papp value by the addition of KALA peptide indicated that KALA peptide had the ability to aid the drug delivery through epithelial cells via both paracellular and transcellular pathways. KALA peptides are suitable as an absorption enhancer at lower concentrations (below 1.0%, w/v) for improving the absorption of insulin and calcitonin from the lung with no observed toxic impact.

Lipid-Based Nanoparticle Functionalization with Coiled-Coil Peptides for In Vitro and In Vivo Drug Delivery.

For the delivery of drugs, different nanosized drug carriers (e.g., liposomes, lipid nanoparticles, and micelles) have been developed in order to treat diseases that afflict society. Frequently, these vehicles are formed by the self-assembly of small molecules to encapsulate the therapeutic cargo of interest. Over decades, nanoparticles have been optimized to make them more efficient and specific to fulfill tailor-made tasks, such as specific cell targeting or enhanced cellular uptake. In recent years, lipid-based nanoparticles in particular have taken center stage; however, off-targeting side effects and poor endosomal escape remain major challenges since therapies require high efficacy and acceptable toxicity.To overcome these issues, many different approaches have been explored to make drug delivery more specific, resulting in reduced side effects, to achieve an optimal therapeutic effect and a lower required dose. The fate of nanoparticles is largely dependent on size, shape, and surface charge. A common approach to designing drug carriers with targeting capability is surface modification. Different approaches to functionalize nanoparticles have been investigated since the attachment of targeting moieties plays a significant role in whether they can later interact with surface-exposed receptors of cells. To this end, various strategies have been used involving different classes of biomolecules, such as small molecules, nucleic acids, antibodies, aptamers, and peptides.Peptides in particular are often used since there are many receptors overexpressed in different specific cell types. Furthermore, peptides can be produced and modified at a low cost, enabling high therapeutic screening. Cell-penetrating peptides (CPPs) and cell-targeting peptides (CTPs) are frequently used for this purpose. Less studied in this context are fusogenic coiled-coil peptides. Lipid-based nanoparticles functionalized with these peptides are able to avoid the endolysosomal pathway; instead such particles can be taken up by membrane fusion, resulting in increased delivery of payload. Furthermore, they can be used for targeting cells/organs but are not directed at surface-exposed receptors. Instead, they recognize complementary peptide sequences, facilitating their uptake into cells.In this Account, we will discuss peptides as moieties for enhanced cytosolic delivery, targeted uptake, and how they can be attached to lipid-based nanoparticles to alter their properties. We will discuss the properties imparted to the particles by peptides, surface modification approaches, and recent examples showing the power of peptides for in vitro and in vivo drug delivery. The main focus will be on the functionalization of lipid-based nanoparticles by fusogenic coiled-coil peptides, highlighting the relevance of this concept for the development of future therapeutics.

Abstract 7252: Characterization of targeting peptide sequences for peptide-mediated delivery of siRNA in HER2+ breast cancer

Abstract Introduction: This work presents the characterization of two novel HER2 targeting peptides to measure which has the most compatible properties to be used as a siRNA carrier to knock down the CD44 gene in HER2+ breast cancer. Procedures: Targeting peptides were electrostatically complexed with non-targeting siRNAs at different N:P ratios to determine the minimum binding ratios needed to form nanoparticles. Different ratios of peptide-siRNA complexes were tested to determine the minimum molar ratio at which the peptides would protect siRNA from degradation. Cytotoxicity assays were performed to determine cytotoxicity levels at different peptide concentrations. Dynamic light scattering assay was performed to determine peptide-siRNA nanoparticle size and zeta potential. Immunofluorescent microscopy was used to determine the targeting of the peptide to HER2+ cells compared to cells not expressing HER2. Summary of Results: Peptide-siRNA complexes for the P51 and P25 peptides formed at a minimum 20:1 N:P ratio. P25 and P51 peptides both successfully protects the siRNA at N:P ratios from 20:1 to 60:1 for both degradation agents. Cytotoxicity assays show that in the SKBR3 cell line, the P25 and P51 peptides both showed above 80% viability until the 70:1 ratio; however, in the BT474 cell line, the P25 showed above 80% viability to the 50:1 ratio, but the P51 was only viable in the 20:1 ratio. The results of these studies illustrate how both peptides protect siRNA but that the P51 peptide is toxic to some HER2+ breast cancer cell lines. Conclusions: Results show that both peptides target HER2+ breast cancer and are compatible carriers of siRNA, protecting the siRNA well within a cell-like environment. However, from MTS studies, P25 appears less cytotoxic to the cell lines than P51, which shows higher cytotoxicity at high molar ratios. We observed that the P25 protects siRNA from degradation, is cytocompatibility, and promotes efficient internalization into HER2+ cell lines. Future work will include the development of a tandem peptide, including a targeting peptide fused with a fusogenic peptide to increase cancer targeting and efficacy of this targeted treatment. Acknowledgments: This research was supported in part by the Dabo Swinney All-In Foundation as well as Materials Assembly and Design Excellence in South Carolina (MADE in SC) under the National Science Foundation EPSCoR Program under NSF Award #OIA-1655740. Data presented in this material was partially collected using SC BioCRAFT facilities supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number P30 GM131959. Citation Format: Audreanna Miserendino, Sevrina Tekle, James Kalogeros, Angela Alexander-Bryant. Characterization of targeting peptide sequences for peptide-mediated delivery of siRNA in HER2+ breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7252.

Cell-Penetrating Peptides as Keys to Endosomal Escape and Intracellular Trafficking in Nanomedicine Delivery.

This review article discusses the challenges of delivering cargoes to the cytoplasm, for example, proteins, peptides, and nucleic acids, and the mechanisms involved in endosomal escape. Endocytosis, endosomal maturation, and exocytosis pose significant barriers to effective cytoplasmic delivery. The article explores various endosomal escape mechanisms, such as the proton sponge effect, osmotic lysis, membrane fusion, pore formation, membrane destabilization/ disruption, and vesicle budding and collapse. Additionally, it discusses the role of lysosomes, glycocalyx, and molecular crowding in the cytoplasmic delivery process. Despite the recent advances in nonviral delivery systems, there is still a need to improve cytoplasmic delivery. Strategies such as fusogenic peptides, endosomolytic polymers, and cell-penetrating peptides have shown promise in improving endosomal escape and cytoplasmic delivery. More research is needed to refine these strategies and make them safer and more effective. In conclusion, the article highlights the challenges associated with cytoplasmic delivery and the importance of understanding the mechanisms involved in endosomal escape. A better understanding of these processes could result in the creation of greater effectiveness and safe delivery systems for various cargoes, including proteins, peptides, and nucleic acids.

Fusogenic Coiled-Coil Peptides Enhance Lipid Nanoparticle-Mediated mRNA Delivery upon Intramyocardial Administration.

Heart failure is a serious condition that results from the extensive loss of specialized cardiac muscle cells called cardiomyocytes (CMs), typically caused by myocardial infarction (MI). Messenger RNA (mRNA) therapeutics are emerging as a very promising gene medicine for regenerative cardiac therapy. To date, lipid nanoparticles (LNPs) represent the most clinically advanced mRNA delivery platform. Yet, their delivery efficiency has been limited by their endosomal entrapment after endocytosis. Previously, we demonstrated that a pair of complementary coiled-coil peptides (CPE4/CPK4) triggered efficient fusion between liposomes and cells, bypassing endosomal entrapment and resulting in efficient drug delivery. Here, we modified mRNA-LNPs with the fusogenic coiled-coil peptides and demonstrated efficient mRNA delivery to difficult-to-transfect induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs). As proof of in vivo applicability of these fusogenic LNPs, local administration via intramyocardial injection led to significantly enhanced mRNA delivery and concomitant protein expression. This represents the successful application of the fusogenic coiled-coil peptides to improve mRNA-LNPs transfection in the heart and provides the potential for the advanced development of effective regenerative therapies for heart failure.

Investigating the Effects of the POPC-POPG Lipid Bilayer Composition on PAP248-286 Binding Using CG Molecular Dynamics Simulations.

PAP248-286 is a fusogenic peptide derived from prostatic acid phosphatase, commonly found in human semen, and is known to mediate HIV fusion with cell membranes. In this study, we performed 120 independent coarse-grained molecular dynamics simulations to investigate the spontaneous binding of PAP248-286 monomers, considering both charged and neutral histidine (His) residues, to membrane bilayers composed of different lipid compositions: 100% POPC, 70% POPC-30% POPG, and 50% POPC-50% POPG. Our simulations revealed that PAP248-286 displayed spontaneous binding to the membrane, with increased binding observed in the presence of anionic lipid POPG. Specifically, in systems containing 30% and 50% POPG lipids, monomer residues, particularly in the systems containing charged histidine (His) residues, exhibited prolonged binding with the membrane. Furthermore, our simulations indicated that PAP248-286 adopted a parallel orientation with the membrane, exposing its positively charged residues to the lipid bilayer. Interestingly, systems containing charged His residues showed a higher lipid occupancy around the peptide. These findings are consistent with previous experimental data, suggesting that PAP248-286 binding is enhanced in membranes with charged His residues, resembling the conditions found in the acidic vaginal pH environment. The results of our study provide further insights into the molecular mechanisms underlying the membrane binding of PAP248-286, contributing to our understanding of its potential role in HIV fusion and infection.

Engineered mesenchymal stem cell-derived extracellular vesicles constitute a versatile platform for targeted drug delivery

Extracellular vesicles (EVs) are promising therapeutic carriers owing to their ideal size range and intrinsic biocompatibility. However, limited targeting ability has caused major setbacks in the clinical application of EV therapeutics. To overcome this, we genetically engineered natural free streptavidin (SA) on the cellular surface of bone marrow mesenchymal stem cells (BMSCs) and obtained typical EVs from these cells (BMSC-EVs). Biotin-coated gold nanoparticles confirmed the expression of SA on the membrane of EVs, which has a high affinity for biotinylated molecules. Using a squamous cell carcinoma model, we demonstrated that a pH-sensitive fusogenic peptide -modification of BMSC-EVs achieved targetability in the microenvironment of a hypoxic tumor to deliver anti-tumor drugs. Using EGFR+HER2- and EGFR-HER2+ breast cancer models, we demonstrated that anti-EGFR and anti-HER2 modifications of BMSC-EVs were able to specifically deliver drugs to EGFR+ and HER2+ tumors, respectively. Using a collagen-induced arthritis model, we confirmed that anti-IL12/IL23-modified BMSC-EVs specifically accumulated in the arthritic joint and alleviated inflammation. Administration of SA-overexpressing BMSC-EVs has limited immunogenicity and high safety in vivo, suggesting that BMSC-derived EVs are ideal drug delivery vehicle. These representative scenarios of targeting modification suggest that, using different biotinylated molecules, the SA-overexpressing BMSC-EVs could be endowed with different targetabilities, which allows BMSC-EVs to serve as a versatile platform for targeted drug delivery under various situations.

Efficient mRNA delivery using lipid nanoparticles modified with fusogenic coiled-coil peptides.

Gene delivery has great potential in modulating protein expression in specific cells to treat diseases. Such therapeutic gene delivery demands sufficient cellular internalization and endosomal escape. Of various nonviral nucleic acid delivery systems, lipid nanoparticles (LNPs) are the most advanced, but still, are very inefficient as the majority are unable to escape from endosomes/lysosomes. Here, we develop a highly efficient gene delivery system using fusogenic coiled-coil peptides. We modified LNPs, carrying EGFP-mRNA, and cells with complementary coiled-coil lipopeptides. Coiled-coil formation between these lipopeptides induced fast nucleic acid uptake and enhanced GFP expression. The cellular uptake of coiled-coil modified LNPs is likely driven by membrane fusion thereby omitting typical endocytosis pathways. This direct cytosolic delivery circumvents the problems commonly observed with the limited endosomal escape of mRNA. Therefore fusogenic coiled-coil peptide modification of existing LNP formulations to enhance nucleic acid delivery efficiency could be beneficial for several gene therapy applications.

Fusogenic peptide delivery of bioactive siRNAs targeting CSNK2A1 for treatment of ovarian cancer

Ovarian cancer has shown little improvement in survival among advanced-stage patients over the past decade. Current treatment strategies have been largely unsuccessful in treating advanced disease, with many patients experiencing systemic toxicity and drug-resistant metastatic cancer. This study evaluates novel fusogenic peptide carriers delivering short interfering RNA (siRNA) targeting casein kinase II, CSNK2A1, for reducing the aggressiveness of ovarian cancer. The peptides were designed to address two significant barriers to siRNA delivery: insufficient cellular uptake and endosomal entrapment. The three peptide variants developed, DIVA3, DIV3H, and DIV3W, were able to form monodisperse nanoparticle complexes with siRNA and protect siRNAs from serum and RNase degradation. Furthermore, DIV3W demonstrated optimal delivery of bioactive siRNAs into ovarian cancer cells with high cellular uptake efficiency and mediated up to 94% knockdown of CSNK2A1 mRNA compared with non-targeting siRNAs, resulting in decreased cell migration and recolonization in vitro. Intratumoral delivery of DIV3W-siCSNK2A1 complexes to subcutaneous ovarian tumors resulted in reduced CSNK2A1 mRNA and CK2α protein expression after 48 h and reduced tumor growth and migration in a 2-week multi-dosing regimen. These results demonstrate the potential of the DIV3W peptide to deliver bioactive siRNAs and confirms the role of CSNK2A1 in cell-cell communication and proliferation in ovarian cancer.

The interaction of viral fusion peptides with lipid membranes

In this paper, we studied fusogenic peptides of class I-III fusion proteins, which are relevant to membrane fusion for certain enveloped viruses, in contact with model lipid membranes. We resolved the vertical structure and examined the adsorption or penetration behavior of the fusogenic peptides at phospholipid Langmuir monolayers with different initial surface pressures with x-ray reflectometry. We show that the fusion loops of tick-borne encephalitis virus (TBEV) glycoprotein E and vesicular stomatitis virus (VSV) G-protein are not able to insert deeply into model lipid membranes, as they adsorbed mainly underneath the headgroups with only limited penetration depths into the lipid films. In contrast, we observed that the hemagglutinin 2 fusion peptide (HA2-FP) and the VSV-transmembrane domain (VSV-TMD) can penetrate deeply into the membranes. However, in the case of VSV-TMD, the penetration was suppressed already at low surface pressures, whereas HA2-FP was able to insert even into highly compressed films. Membrane fusion is accompanied by drastic changes of the membrane curvature. To investigate how the peptides affect the curvature of model lipid membranes, we examined the effect of the fusogenic peptides on the equilibration of cubic monoolein structures after a phase transition from a lamellar state induced by an abrupt hydrostatic pressure reduction. We monitored this process in presence and absence of the peptides with small-angle x-ray scattering and found that HA2-FP and VSV-TMD drastically accelerate the equilibration, while the fusion loops of TBEV and VSV stabilize the swollen state of the lipid structures. In this work, we show that the class I fusion peptide of HA2 penetrates deeply into the hydrophobic region of membranes and is able to promote and accelerate the formation of negative curvature. In contrast, we found that the class II and III fusion loops of TBEV and VSV tend to counteract negative membrane curvature.

Mechanisms of Influenza Virus HA2 Peptide Interaction with Liposomes Studied by Dual-Wavelength MP-SPR.

A phospholipid-based liposome layer was used as an effective biomimetic membrane model to study the binding of the pH-dependent fusogenic peptide (E4-GGYC) from the influenza virus hemagglutinin HA2 subunit. To this end, a multiparameter surface plasmon resonance approach (MP-SPR) was used for monitoring peptide-liposome interactions at two pH values (4.5 and 8) by means of recording sensorgrams in real time without the need for labeling. Biotinylated liposomes were first immobilized as a monolayer onto the surface of an SPR gold chip coated with a streptavidin layer. Multiple sets of sensorgrams with different HA2 peptide concentrations were generated at both pHs. Dual-wavelength Fresnel layer modeling was applied to calculate the thickness (d) and the refractive index (n) of the liposome layer to monitor the change in its optical parameters upon interaction with the peptide. At acidic pH, the peptide, in its α helix form, entered the lipid bilayer of liposomes, inducing vesicle swelling and increasing membrane robustness. Conversely, a contraction of liposomes was observed at pH 8, associated with noninsertion of the peptide in the double layer of phospholipids. The equilibrium dissociation constant KD = 4.7 × 10-7 M of the peptide/liposome interaction at pH 4.5 was determined by fitting the "OneToOne" model to the experimental sensorgrams using Trace Drawer software. Our experimental approach showed that the HA2 peptide at a concentration up to 100 μM produced no disruption of liposomes at pH 4.5.

Guanine nucleotide exchange factor DOCK11-binding peptide fused with a single chain antibody inhibits hepatitis B virus infection and replication

Hepatitis B virus (HBV) infection is a major global health problem with no established cure. Dedicator of cytokinesis 11 (DOCK11), known as a guanine nucleotide exchange factor (GEF) for Cdc42, is reported to be essential for the maintenance of HBV. However, potential therapeutic strategies targeting DOCK11 have not yet been explored. We have previously developed an in vitro virus method as a more efficient tool for the analysis of proteomics and evolutionary protein engineering. In this study, using the in vitro virus method, we screened and identified a novel antiasialoglycoprotein receptor (ASGR) antibody, ASGR3-10M, and a DOCK11-binding peptide, DCS8-42A, for potential use in HBV infection. We further constructed a fusion protein (10M-D42AN) consisting of ASGR3-10M, DCS8-42A, a fusogenic peptide, and a nuclear localization signal to deliver the peptide inside hepatocytes. We show using immunofluorescence staining that 10M-D42AN was endocytosed into early endosomes and released into the cytoplasm and nucleus. Since DCS8-42A shares homology with activated cdc42-associated kinase 1 (Ack1), which promotes EGFR endocytosis required for HBV infection, we also found that 10M-D42AN inhibited endocytosis of EGFR and Ack1. Furthermore, we show 10M-D42AN suppressed the function of DOCK11 in the host DNA repair system required for covalently closed circular DNA synthesis and suppressed HBV proliferation in mice. In conclusion, this study realizes a novel hepatocyte-specific drug delivery system using an anti-ASGR antibody, a fusogenic peptide, and DOCK11-binding peptide to provide a novel treatment for HBV.

In silico approach to probe the binding affinity between OMVs harboring the ZEGFR affibody and the EGF receptor.

There is a growing interest in designing a nanocarrier containing an EGFR targeting affibody to direct toward cancer cells. Here, cytolysin A was cloned at the N-terminus of ZEGFR:1907 affibody to guarantee its surface presentation on the OMVs while targeting the epidermal growth factor receptors (EGFRs). A separate construct including a fusogenic peptide (GALA) was also designed for the endosomal escape of the nanocarrier. Binding of the two constructs ClyA-affiEGFR and ClyA-affiEGFR-GALA to domain III of EGFR was investigated using molecular docking and molecular dynamic simulations. The higher stability of the ClyA-affiEGFR-GALA/EGFR as compared to the ClyA-affiEGFR/EGFR complex was evident. The ClyA-affiEGFR-GALA structure showed a higher RMSD during the first half of the simulation time implying a much less stable behavior. Plateau state of the radius of gyration plot of ClyA-affiEGFR-GALA confirmed a well-folded structure in the presence of the GALA sequence. Solvent accessible surface area for both proteins was in the same range. The data obtained from hydrogen bond analysis revealed a more equilibrated and stable form of the ClyA-affiEGFR-GALA structure upon interaction with EGFR. The data provided here was a requisite for our biological evaluation of the synthesized constructs as a component of a novel drug delivery system.

Peptide-based delivery of therapeutics in cancer treatment.

Current delivery strategies for cancer therapeutics commonly cause significant systemic side effects due to required high doses of therapeutic, inefficient cellular uptake of drug, and poor cell selectivity. Peptide-based delivery systems have shown the ability to alleviate these issues and can significantly enhance therapeutic loading, delivery, and cancer targetability. Peptide systems can be tailor-made for specific cancer applications. This review describes three peptide classes, targeting, cell penetrating, and fusogenic peptides, as stand-alone nanoparticle systems, conjugations to nanoparticle systems, or as the therapeutic modality. Peptide nanoparticle design, characteristics, and applications are discussed as well as peptide applications in the clinical space.

Polymer Micelles vs Polymer-Lipid Hybrid Vesicles: A Comparison Using RAW 264.7 Cells.

Bottom-up synthetic biology aims to integrate artificial moieties with living cells and tissues. Here, two types of structural scaffolds for artificial organelles were compared in terms of their ability to interact with macrophage-like murine RAW 264.7 cells. The amphiphilic block copolymer poly(cholesteryl methacrylate)-block-poly(2-carboxyethyl acrylate) was used to assemble micelles and polymer–lipid hybrid vesicles together with 1,2-dioleoyl-sn-glycero-3-phosphocholine or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) lipids in the latter case. In addition, the pH-sensitive fusogenic peptide GALA was conjugated to the carriers to improve their lysosomal escape ability. All assemblies had low short-term toxicity toward macrophage-like murine RAW 264.7 cells, and the cells internalized both the micelles and hybrid vesicles within 24 h. Assemblies containing DOPE lipids or GALA in their building blocks could escape the lysosomes. However, the intracellular retention of the building blocks was only a few hours in all the cases. Taken together, the provided comparison between two types of potential scaffolds for artificial organelles lays out the fundamental understanding required to advance soft material-based assemblies as intracellular nanoreactors.

Fusogenic peptide modification to enhance gene delivery by peptide-DNA nano-coassemblies.

Endosomal escape is a major obstacle for non-viral nucleic acids delivery. Here, we attached by click reaction a fusogenic peptide (L17E) onto peptide self-assembled disks (∼17 nm), which mimicked the functional subunits of the virus capsid. These peptide disks then spontaneously co-assembled with DNA to form patterned nanostructures (∼100 nm) as viral mimics. This modification did not affect the cellular uptake but enhanced endosomal escape and led to improved transfection in cell culture.

A pH-responsive, endosomolytic liposome functionalized with membrane-anchoring, comb-like pseudopeptides for enhanced intracellular delivery and cancer treatment.

Low intracellular delivery efficiency and multidrug resistance are among major barriers to effective cancer therapy. Herein, we report a novel, virus-mimicking, endosomolytic liposomal drug-delivery platform to address these two key challenges. The pH-responsive, comb-like pseudopeptides were prepared by grafting relatively long alkyl side chains onto a polyamide, poly(L-lysine isophthalamide), to mimic fusogenic peptides in viral spikes. The cholesterol-containing liposome, which mimics the viral envelope, was readily coated with these pseudopeptides due to their hydrophobic side chains acting as membrane anchors. These endosomolytic pseudopeptides displayed high adsorption onto the liposomal membrane and enabled the significantly higher cellular uptake. The virus-mimicking system showed a pH-triggered content-release profile which could be manipulated by varying the structure and concentration of the adsorbed polymers. The endosomolytic ability of the multifunctional liposome and its use for efficient intracellular delivery of the widely used anticancer drug doxorubicin (DOX) were demonstrated. The virus-mimicking liposomal system with DOX encapsulation exhibited considerably higher potency against HeLa cervical cancer cells, A549 lung cancer cells, MES-SA uterus cancer cells, and MES-SA/DX5 multidrug-resistant cancer cells than DOX-loaded bare liposomes and free DOX. These results suggest its potential applications for enhanced cytoplasmic delivery and cancer treatment.

Design of a new cell penetrating peptide for DNA, siRNA and mRNA delivery.

Delivery systems, including peptide-based ones, that destabilize endosomes in a pH-dependent manner are increasingly used to deliver cargoes of therapeutic interest, such as nucleic acids and proteins into mammalian cells. The negatively charged amphipathic alpha-helicoidal forming peptide named HELP (Helical Erythrocyte Lysing Peptide) is a derivative from the bee venom melittin and was shown to have a pH-dependent activity with the highest lytic activity at pH5.0 at the same time as becoming inactive when the pH is increased. The present study aimed to determine whether replacement in the HELP peptide of the glutamic acid residues by histidines, for which the protonation state is sensitive to the pH changes that occur during endosomal acidification, can transform this fusogenic peptide into a carrier able to deliver different nucleic acids into mammalian cells. The resulting HELP-4H peptide displays high plasmid DNA, small interfering RNA and mRNA delivery capabilities. Importantly, in contrast to other cationic peptides, its transfection activity was only marginally affected by the presence of serum. Using circular dichroism, we found that acidic pH did not induce significant conformational changes for HELP-4H. In summary, we were able to develop a new cationic histidine rich peptide able to efficiently deliver various nucleic acids into cells.

Characterization of the membrane penetration-enhancing peptide S19 derived from human syncytin-1 for the intracellular delivery of TAT-fused proteins

Although cell-penetrating peptides such as the HIV-derived TAT peptide have been used as tools for the intracellular delivery of therapeutic peptides and proteins, a problem persists: the endosomal escape efficiency is low. Previously, we found that the fusogenic peptide S19, derived from the human protein syncytin-1, enhance the endosomal escape efficiency of proteins that incorporated by endocytosis via TAT. In this study, we first performed Ala-scanning mutagenesis of S19, and found that all Ile, Val, Leu and Phe with high β-sheet forming propensities in S19 are important for the intracellular uptake of S19-TAT-fused proteins. In a secondary structure analysis of the mutated S19-TAT peptides in the presence of liposomes mimicking late endosomes (LEs), the CD spectra of V3A and I4A mutants with low uptake activity showed the appearance of an α-helix structure, whereas the mutant G5A retained both the uptake activity and the β-structure. In addition, we investigated the appropriate linking position and order of the S19 and TAT peptides to a cargo protein including an apoptosis-induced peptide and found that both the previous C-terminal S19-TAT tag and the N-terminal TAT-S19 tag promote the cytoplasmic delivery of the fusion protein. These results and previous results suggest that the interaction of TAT with the LE membrane causes a structural change in S19 from a random coil to a β-strand and that the subsequent parallel β-sheet formation between two S19 peptides may promote adjacent TAT dimerization, resulting in endosomal escape from the LE membrane.

Surface Engineering of Escherichia coli–Derived OMVs as Promising Nano-Carriers to Target EGFR-Overexpressing Breast Cancer Cells

Bacterial outer membrane vesicles (OMVs) have recently drawn a great deal of attention due to their therapeutic efficiency and ability to target specific cells. In the present study, we sought to probe engineered OMVs as novel and promising carriers to target breast cancer cells. Following the fusion of the affiEGFR-GALA structure to the C-terminal of ClyA as an anchor protein, the ClyA-affiEGFR-GALA construct was successfully expressed on the surface of ∆msbB/∆pagP E. coli W3110-derived OMVs. Morphological features of the engineered and wild-type OMVs were identical. The engineered OMVs induced no endotoxicity, cytotoxicity, or immunogenicity, indicating the safety of their application. These OMVs could specifically bind to EGF receptors of MDA-MB-468 cells expressing high levels of EGFR and not to those with low levels of EGFR (HEK293T cells). Interestingly, despite a lower binding affinity of the engineered OMVs relative to the positive control Cetuximab, it was strong enough to identify these cells. Moreover, confocal microscopy revealed no uptake of the modified OMVs by the EGFR-overexpressing cells in the presence of EGFR competitors. These results suggest that OMVs might internalize into the cells with EGF receptors, as no OMVs entered the cells with any EGFR expression or those pretreated with EGF or Cetuximab. Regarding the EGFR-binding affinity of the engineered OMVs and their cellular uptake, they are presented here as a potential carrier for cell-specific drug delivery to treat a wide variety of cancer cells. Interestingly, the engineered OMVs are capable of reaching the cytoplasm while escaping the endosome due to the incorporation of a fusogenic GALA peptide in the construct.