Cationic Cell-penetrating Peptides Research Articles

Plasma membrane damage limits cytoplasmic delivery by conventional cell penetrating peptides.

Intracellular delivery of large molecule cargo via cell penetrating peptides (CPPs) is an inefficient process and despite intense efforts in past decades, improvements in efficiency have been marginal. Utilizing a standardized and comparative analysis of the delivery efficiency of previously described cationic, anionic, and amphiphilic CPPs, we demonstrate that the delivery ceiling is accompanied by irreparable plasma membrane damage that is part of the uptake mechanism. As a consequence, intracellular delivery correlates with cell toxicity and is more efficient for smaller peptides than for large molecule cargo. The delivery of pharmaceutically relevant cargo quantities with acceptable toxicity thus seems hard to achieve with the CPPs tested in our study. Our results suggest that any engineered intracellular delivery system based on conventional cationic or amphiphilic CPPs, or the design principles underlying them, needs to accept low delivery yields due to toxicity limiting efficient cytoplasmic uptake. Novel peptide designs based on detailed study of uptake mechanisms are required to overcome these limitations.

Cyclization increases bactericidal activity of arginine-rich cationic cell-penetrating peptide for Neisseria gonorrhoeae.

We previously reported that a linear cationic 12-amino acid cell-penetrating peptide (CPP) was bactericidal for Neisseria gonorrhoeae. In this study, our objectives were to determine the effect of cyclization of the linear CPP on its antibacterial activity for N. gonorrhoeae and cytotoxicity for human cells. We compared the bactericidal effect of 4-hour treatment with the linear CPP to that of CPPs cyclized by a thioether or a disulfide bond on human challenge and multi-drug resistant (MDR) strains of N. gonorrhoeae grown in cell culture media with 10% fetal bovine serum (FBS). The effect of lipooligosaccharide (LOS) sialylation on bactericidal activity was analyzed. We determined the ability of the CPPs to treat human cells infected in vitro with N. gonorrhoeae, to reduce the inflammatory response of human monocytic cells to gonococci, to kill strains of three commensal Neisseria species, and to inhibit gonococcal biofilms. The cyclized CPPs killed 100% of gonococci from all strains at 100 µM and >90% at 20 µM and were more potent than the linear form. The thioether-linked but not the disulfide-linked CPP was less cytotoxic for human cervical cells compared to the linear CPP. LOS sialylation had minimal effect on bactericidal activity. In treating infected human cells, the thioether-linked CPP at 20 µM killed >60% of extra- and intracellular bacteria and reduced TNF-α expression by THP-1 cells. The potency of the CPPs for the pathogenic and the commensal Neisseria was similar. The thioether-linked CPP partially eradicated gonococcal biofilms. Future studies will focus on determining efficacy in the female mouse model of gonorrhea.IMPORTANCENeisseria gonorrhoeae remains a major cause of sexually transmitted infections with 82 million cases worldwide in 2020, and 710,151 confirmed cases in the US in 2021, up 25% from 2017. N. gonorrhoeae can infect multiple tissues including the urethra, cervix, rectum, pharynx, and conjunctiva. The most serious sequelae are suffered by infected women as gonococci ascend to the upper reproductive tract and cause pelvic inflammatory disease, chronic pelvic pain, and infertility in 10%-20% of women. Control of gonococcal infection is widely recognized as increasingly challenging due to the lack of any vaccine. N. gonorrhoeae has quickly developed resistance to all but one class of antibiotics and the emergence of multidrug-resistant strains could result in untreatable infections. As such, gonorrhea is classified by the Center for Disease Control (CDC) as an urgent public health threat. The research presented herein on new therapeutics for gonorrhea has identified a cyclic cell-penetrating peptide (CPP) as a potent molecule targeting N. gonorrhoeae.

Abstract 5816: An effective delivery platform for nucleic acid therapeutics: Enhanced cancer cell internalization and endosomal escape

Abstract Nucleic acid therapeutics (NATs), such as siRNA and mRNA, show great potential as cancer medicines. However, translation of these compounds has been held back by their limited bioavailability in target tissues. The aim of this study was to test the use of a novel cell-penetrating peptide (CPP)-based delivery platform to enhance uptake of NAT into cancer cells. This platform consists of a highly efficient CPP that combines the advantages of multimerization and cyclization of the archetypal CPP, TAT peptide1, plus a photochemical (PC) strategy to promote endosomal release of NAT into the cytoplasm2. Negatively charged NATs form complexes with cationic CPPs through charge:charge interaction. NATs (siRNA or mRNA) were incubated with CPP at different molar ratios and assessed in gel retardation assays. Efficiency of internalization of Cy3-NAT:CPP complexes was evaluated using live cell confocal imaging with quantification of intracellular fluorescence. The mechanism of cellular uptake of NAT:CPP was investigated using inhibitors of endocytosis (chlorpromazine, NaN3, amiloride and methyl-β-cyclodextrin). To trigger NAT release from intracellular vesicles, 420 nm/50 mA LED light irradiation was applied following pre-treatment of cells with a photosensitizer, fimaprofin. CPP-PC mediated internalization and endosomal release was used for delivery of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) siRNA. GAPDH enzymatic activity assays (Sigma, USA) were conducted to confirm inhibition of GAPDH in HeLa, HEK293T and MDA-MB-231 cells. Transfection efficiency of mRNA encoding enhanced green fluorescence protein (eGFP) was evaluated by flow cytometry in HeLa cells. NAT:CPP complexes were found to be maximally efficient at a 10:1 molar ratio. Analysis of live cell confocal images indicated that GAPDH Cy3-siRNA:CPP complexes were more efficiently internalized compared to Cy3-siRNA + monomeric TAT or Cy3-siRNA + lipofectamine (****p<0.0001, one-way ANOVA). The combined CPP-PC platform delivers NAT via a clathrin-mediated endocytosis pathway. CPP-PC delivery of GAPDH siRNA caused marked GAPDH enzyme activity downregulation in HeLa (91%), MDA-MB-231 (51%) and HEK293T (44%) cells. These results were similar to the level of downregulation achieved with lipofectamine + GAPDH siRNA. Flow cytometric analysis revealed that 52.4% of HeLa cells expressed eGFP at 1 h following delivery of mRNA-eGFP by CPP-PC. Functional siRNA and mRNA were efficiently delivered into the cytoplasm of cancer cells through combined use of a multimeric/cyclized CPP plus photochemical-mediated endosomal release. This approach holds promise for clinical application, particularly in the treatment of intraluminal tumors. 1. Tietz, O. et al. Nat Chem. 14: 284-93, 2022. 2. Selbo, P. K. et al. J Control Release. 148, 2-12, 2010. Citation Format: Siqi Li, Ole Tietz, Afaf H. El-Sagheer, Tom Brown, Katherine A. Vallis. An effective delivery platform for nucleic acid therapeutics: Enhanced cancer cell internalization and endosomal escape [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 5816.

Transthyretin-Penetratin: A Potent Fusion Protein Inhibitor against Alzheimer's Amyloid-β Fibrillogenesis with High Blood Brain Barrier Crossing Capability.

The design of a potent amyloid-β protein (Aβ) inhibitor plays a pivotal role in the prevention and treatment of Alzheimer's disease (AD). Despite endogenous transthyretin (TTR) being recognized as an Aβ inhibitor, the weak inhibitory and blood brain barrier (BBB) crossing capabilities hinder it for Aβ aggregation inhibition and transport. Therefore, we have herein designed a recombinant TTR by conjugating a cationic cell penetrating peptide (penetratin, Pen), which not only enabled the fusion protein, TTR-Pen (TP), to present high BBB penetration but also greatly enhanced the potency of Aβ inhibition. Namely, the protein fusion made TP positively charged, leading to a potent suppression of Aβ40 fibrillization at a low concentration (1.5 μM), while a TTR concentration as high as 12.5 μM was required to gain a similar function. Moreover, TP could mitigate Aβ-induced neuronal death, increase cultured cell viability from 72% to 92% at 2.5 μM, and extend the lifespan of AD nematodes from 14 to 18 d. Thermodynamic studies revealed that TP, enriched in positive charges, presented extensive electrostatic interactions with Aβ40. Importantly, TP showed excellent BBB penetration performance, with a 10 times higher BBB permeability than TTR, which would allow TP to enter the brain of AD patients and participate in the transport of Aβ species out of the brain. Thus, it is expected that the fusion protein has great potential for drug development in AD treatment.

PH-dependent interactions of coacervate-forming histidine-rich peptide with model lipid membranes

Peptide-based liquid droplets (coacervates) produced by spontaneous liquid-liquid phase separation (LLPS), have emerged as a promising class of drug delivery systems due to their high entrapping efficiency and the simplicity of their formulation. However, the detailed mechanisms governing their interaction with cell membranes and cellular uptake remain poorly understood. In this study, we investigated the interactions of peptide coacervates composed of HBpep—peptide derived from the histidine-rich beak proteins (HBPs) of the Humboldt squid—with model cellular membranes in the form of supported lipid bilayers (SLBs). We employed quartz crystal microbalance with dissipation monitoring (QCM-D), neutron reflectometry (NR) and atomistic molecular dynamics (MD) simulations to reveal the nature of these interactions in the absence of fluorescent labels or tags. HBpep forms small oligomers at pH 6 whereas it forms µm-sized coacervates at physiological pH. Our findings reveal that both HBpep oligomers and HBpep-coacervates adsorb onto SLBs at pH 6 and 7.4, respectively. At pH 6, when the peptide carries a net positive charge, HBpep oligomers insert into the SLB, facilitated by the peptide’s interactions with the charged lipids and cholesterol. Importantly, however, HBpep coacervate adsorption at physiological pH, when it is largely uncharged, is fully reversible, suggesting no significant lipid bilayer rearrangement. HBpep coacervates, previously identified as efficient drug delivery vehicles, do not interact with the lipid membrane in the same manner as traditional cationic drug delivery systems or cell-penetrating peptides. Based on our findings, HBpep coacervates at physiological pH cannot cross the cell membrane by a simple passive mechanism and are thus likely to adopt a non-canonical cell entry pathway.

New Perspective for Using Antimicrobial and Cell-Penetrating Peptides to Increase Efficacy of Antineoplastic 5-FU in Cancer Cells.

This study explores the effectiveness of the antineoplastic agent 5-FU in cancer cells by leveraging the unique properties of cationic antimicrobial peptides (CAMPs) and cell-penetrating peptides (CPPs). Traditional anticancer therapies face substantial limitations, including unfavorable pharmacokinetic profiles and inadequate specificity for tumor sites. These drawbacks often necessitate higher therapeutic agent doses, leading to severe toxicity in normal cells and adverse side effects. Peptides have emerged as promising carriers for targeted drug delivery, with their ability to selectively deliver therapeutics to cells expressing specific receptors. This enhances intracellular drug delivery, minimizes drug resistance, and reduces toxicity. In this research, we comprehensively evaluate the ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties of various AMPs and CPPs to gain insights into their potential as anticancer agents. The peptide synthesis involved a solid-phase synthesis using a Liberty Microwave Peptide Synthesizer. The peptide purity was confirmed via LC-MS and HPLC methods. For the ADMET screening, computational tools were employed, assessing parameters like absorption, distribution, metabolism, excretion, and toxicity. The cell lines A549 and UM-UC-5 were cultured and treated with 5-FU, CAMPs, and CPPs. The cell viability was measured using the MTT assay. The physicochemical properties analysis revealed favorable drug-likeness attributes. The peptides exhibited potential inhibitory activity against CYP3A4. The ADMET predictions indicated variable absorption and distribution characteristics. Furthermore, we assessed the effectiveness of these peptides alone and in combination with 5-FU, a widely used antineoplastic agent, in two distinct cancer cell lines, UM-UC-5 and A549. Our findings indicate that CAMPs can significantly reduce the cell viability in A549 cells, while CPPs exhibit promising results in UM-UC-5 cells. Understanding these multifaceted effects could open new avenues for antiviral and anticancer research. Further, experimental validation is necessary to confirm the mechanism of action of these peptides, especially in combination with 5-FU.

MicroRNA-195 liposomes for therapy of Alzheimer's disease

The complex etiologies and mechanisms of Alzheimer's disease (AD) underscore the importance for devising multitarget drugs to achieve effective therapy. MicroRNAs (miRNAs) are capable of concurrently regulating the expression of multiple proteins by selectively targeting disease- associated genes in a sequence-specific fashion. Nonetheless, as RNA-based drugs, their stability in the circulation and capacity of traversing the blood–brain barrier (BBB) is largely compromised, thereby limiting their potential clinical applications. In this study, we formulated the nanoliposomes encapsulating polyethyleneimine (PEI)/miR-195 complex (DPMT@PEI/miR-195) that was engineered through dual modifications to contain P-aminophenyl-alpha-d-mannopyranoside (MAN) and cationic cell-penetrating peptide (TAT). DPMT@PEI/miR-195 exhibited the enhanced BBB- and cell membrane penetrating capability. As expected, we observed that DPMT@PEI/miR-195 administered through intravenous tail injection of produced greater effectiveness than donepezil and the same range of effect as aducanumab in alleviating the cognitive decline in 7-month-old APP/PS1 mice. Moreover, the combination treatment with DPMT@PEI/miR-195 and donepezil effectively ameliorated the deterioration of cognition in 16-month-old APP/PS1 mice, with enhanced effects than either DPMT@PEI/miR-195 or donepezil alone. Furthermore, DPMT@PEI/miR-195 effectively attenuated the positive signals of Aβ, AT8, and CD68 in APP/PS1 mice without notable side effects. Our findings indicate DPMT@PEI/miR-195 as a promising potentially new agent or approach for the prophylaxis and treatment of early and advanced stages of Alzheimer's disease.

Peptide-Targeted Photosensitisers: Synthesis and Applications in Photobiology

The conjugation of porphyrin derivatives to peptide ligands is a valuable strategy for enhancing their aqueous solubility and tissue selectivity in the photodynamic therapy (PDT) of cancer. To this end, bioorthogonal ligation (“click”) chemistries provide an efficient way to link porphyrins and other tetrapyrrole systems with complementary multifunctional peptides. We will describe some of our work in this area towards the generation of specific targeted conjugates between porphyrin and chlorin derivatives and tumour-homing or cell-penetrating peptide carriers [1, 2]. Linking otherwise hydrophobic photosensitisers to cationic cell-penetrating peptides (e.g. HIV Tat 48-57) provides amphiphilic derivatives with improved water solubility plus enhanced cellular uptake and predictable sub-cellular localisation. Such molecules are very suitable for use in the light-triggered delivery of macromolecular therapeutics by photochemical internalisation (PCI), which will be illustrated for the targeted delivery of the protein toxin, saporin [3].Peptide targeting strategies are also valuable for PDT with 5-aminolevulinic acid (ALA) [4]. In ALA-PDT, administered ALA is converted within cells via the heme biosynthetic pathway to protoporphyrin IX which may then be exploited as a photosensitiser. In this context, we will present some recent chemical and biological data on the application of peptide targeting for the enhanced delivery of multiple ALA units to specific cell types [5].

Development of Hydrophobic Cell-Penetrating Stapled Peptides as Drug Carriers.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of a variety of cargo molecules, including small molecules, peptides, nucleic acids, and proteins. Many cationic and amphiphilic CPPs have been developed; however, there have been few reports regarding hydrophobic CPPs. Herein, we have developed stapled hydrophobic CPPs based on the hydrophobic CPP, TP10, by introducing an aliphatic carbon side chain on the hydrophobic face of TP10. This side chain maintained the hydrophobicity of TP10 and enhanced the helicity and cell penetrating efficiency. We evaluated the preferred secondary structures, and the ability to deliver 5(6)-carboxyfluorescein (CF) as a model small molecule and plasmid DNA (pDNA) as a model nucleotide. The stapled peptide F-3 with CF, in which the stapling structure was introduced at Gly residues, formed a stable α-helical structure and the highest cell-membrane permeability via an endocytosis process. Meanwhile, peptide F-4 demonstrated remarkable stability when forming a complex with pDNA, making it the optimal choice for the efficient intracellular delivery of pDNA. The results showed that stapled hydrophobic CPPs were able to deliver small molecules and pDNA into cells, and that different stapling positions in hydrophobic CPPs can control the efficiency of the cargo delivery.

Engineered Histidine-Rich Peptides Enhance Endosomal Escape for Antibody-Targeted Intracellular Delivery of Functional Proteins.

Currently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome-to-cytosol delivery based on an extension of the "histidine switch" principle. By substituting the Arg/Lys residues in cationic cell-penetrating peptides (CPPs) with histidine, we obtained peptides with pH-dependent membrane-perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16-residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody-targeted delivery of diverse protein cargoes-including the pro-apoptotic protein BID (BH3-interacting domain death agonist) and Cre recombinase-into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab-hsLMWP-BID fusion conferred strong anti-tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.

Engineered Histidine‐Rich Peptides Enhance Endosomal Escape for Antibody‐Targeted Intracellular Delivery of Functional Proteins

AbstractCurrently, the clinical application of protein/peptide therapeutics is mainly limited to the modulation of diseases in extracellular spaces. Intracellular targets are hardly accessed, owing largely to the endosomal entrapment of internalized proteins/peptides. Here, we report a strategy to design and construct peptides that enable endosome‐to‐cytosol delivery based on an extension of the “histidine switch” principle. By substituting the Arg/Lys residues in cationic cell‐penetrating peptides (CPPs) with histidine, we obtained peptides with pH‐dependent membrane‐perturbation activity. These peptides do not randomly penetrate cells like CPPs, but imitate the endosomal escape of CPPs following cellular uptake. Working with one such 16‐residue peptide (hsLMWP) with high endosomal escape capacity, we engineered modular fusion proteins and achieved antibody‐targeted delivery of diverse protein cargoes—including the pro‐apoptotic protein BID (BH3‐interacting domain death agonist) and Cre recombinase—into the cytosol of multiple cancer cell types. After extensive in vitro testing, an in vivo analysis with xenograft mice ultimately demonstrated that a trastuzumab‐hsLMWP‐BID fusion conferred strong anti‐tumor efficacy without apparent side effects. Notably, our fusion protein features a modular design, allowing flexible applications for any antibody/cargo combination of choice. Therefore, the potential applications extend throughout life science and biomedicine, including gene editing, cancer treatment, and immunotherapy.

Leaflet-Dependent Effect of Anionic Lipids on Membrane Insertion by Cationic Cell-Penetrating Peptides.

Cationic membrane-permeating peptides can cross membranes unassisted by transmembrane protein machinery, and there is consensus that anionic lipids facilitate this process. Although membranes are asymmetric in lipid composition, investigations of the impact of anionic lipids on peptide-membrane insertion in model vesicles primarily use symmetric anionic lipid distributions between bilayer leaflets. Here, we investigate the leaflet-specific influence of three anionic lipid headgroups [phosphatidic acid (PA), phosphatidylserine (PS), and phosphatidylglycerol (PG)] on insertion into model membranes by three cationic membrane-permeating peptides (NAF-144-67, R6W3, and WWWK). We report that outer leaflet anionic lipids enhanced peptide-membrane insertion for all peptides while inner leaflet anionic lipids did not have a significant effect except in the case of NAF-144-67 incubated with PA-containing vesicles. The insertion enhancement was headgroup-dependent for arginine-containing peptides but not WWWK. These results provide significant new insight into the potential role of membrane asymmetry in insertion of peptides into model membranes.

Fabrication, characterization and evaluation of a new designed botulinum toxin-cell penetrating peptide nanoparticulate complex.

To have a better and longer effect, botulinum neurotoxin (BoNT) is injected several times in a treatment course, which could increase side effects and cost. Some of the most cutting-edge strategies being investigated for proteins to their physiologic targets involve the reformulation of BoNT based on peptide-based delivery systems. For this purpose, cell-penetrating peptides (CPPs) are of particular interest because of their capacity to cross the biological membranes. A short and simple CPP sequence was used as a carrier to create nanocomplex particles from BoNT/A, with the purpose of increasing toxin entrapment by target cells, reducing diffusion, and increasing the duration of the effect. CPP-BoNT/A nanocomplexes were formed by polyelectrolyte complex (PEC) method, considering the anionic structure of botulinum toxin and the cationic CPP sequence. The cellular toxicity, and absorption profile of the complex nanoparticles were evaluated, and the digit abduction score (DAS) was used to assess the local muscle weakening efficacy of BoNT/A and CPP-BoNT/A. The provided optimized polyelectrolyte complex nanoparticles had a 244 ± 20nm particle size and 0.28 ± 0.04 PdI. In cellular toxicity, CPP-BoNT/A nanocomplexes as extended-release formulations of BoNT/A showed that nanocomplexes had a more toxic effect than BoNT/A. Furthermore, the comparison of weakening effectiveness on muscle was done among nanoparticles and free toxin on mice based on the digit abduction score (DAS) method, and nanocomplexes had a slower onset effect and a longer duration of action than toxin. Using PEC method allowed us to form nanocomplex from proteins, and peptides without a covalent bond and harsh conditions. The muscle-weakening effect of toxin in CPP-BoNT/A nanocomplexes showed acceptable efficacy and extended-release pattern.

Cell penetrating peptides: Highlighting points in cancer therapy.

Cell-penetrating peptides (CPPs), first identified in HIV a few decades ago, deserved great attention in the last two decades; especially to support the penetration of anticancer drug means. In the drug delivery discipline, they have been involved in various approaches from mixing with hydrophobic drugs to the use of genetically conjugated proteins. The early classification as cationic and amphipathic CPPs has been extended to a few more classes such as hydrophobic and cyclic CPPs so far. Developing potential sequences utilized almost all methods of modern science: choosing high-efficiency peptides from natural protein sequences, sequence-based comparison, amino acid substitution, obtaining chemical and/or genetic conjugations, in silico approaches, in vitro analysis, animal experiments, etc. The bottleneck effect in this discipline reveals the complications that modern science faces in drug delivery research. Most CPP-based drug delivery systems (DDSs) efficiently inhibited tumor volume and weight in mice, but only in rare cases reduced their levels and continued further processes. The integration of chemical synthesis into the development of CPPs made a significant contribution and even reached the clinical stage as a diagnostic tool. But constrained efforts still face serious problems in overcoming biobarriers to reach further achievements. In this work, we reviewed the roles of CPPs in anticancer drug delivery, focusing on their amino acid composition and sequences. As the most suitable point, we relied on significant changes in tumor volume in mice resulting from CPPs. We provide a review of individual CPPs and/or their derivatives in a separate subsection.

Studies on the Effect of Lipofectamine and Cell-Penetrating Peptide on the Properties of 10-23 DNAzyme.

Cationic polymeric materials and cell-penetrating peptides (CPPs) were often used as the delivery vectors in the evaluation of nucleic acid therapeutics. 10-23 DNAzyme is a kind of potential antisense therapeutics by catalytic cleavage of the disease-related RNAs. Here, lipofectamine 2000 and Tat peptide were evaluated for their effect on the catalytic activity of 10-23 DNAzyme, with the observed rate constant, thermal stability, CD spectra, and PAGE analysis, with a duplex DNA mimicking DNAzyme-substrate as a control. It was shown that the cationic carriers had a negative effect on the catalytic performance of the 10-23 DNAzyme. Significantly, the destabilizing effect of the cationic carriers on the duplex formation was noteworthy, as a duplex formation is an essential prerequisite in the silencing mechanisms of antisense and RNAi.

BK channel modulation by positively charged peptides and auxiliary γ subunits mediated by the Ca2+-bowl site.

The large-conductance, Ca2+-, and voltage-activated K+ (BK) channel consists of the pore-forming α (BKα) subunit and regulatory β and γ subunits. The γ1-3 subunits facilitate BK channel activation by shifting the voltage-dependence of channel activation toward the hyperpolarization direction by about 50-150 mV in the absence of Ca2+. We previously found that the intracellular C-terminal positively charged regions of the γ subunits play important roles in BK channel modulation. In this study, we found that the intracellular C-terminal region of BKα is indispensable in BK channel modulation by the γ1 subunit. Notably, synthetic peptide mimics of the γ1-3 subunits' C-terminal positively charged regions caused 30-50 mV shifts in BKα channel voltage-gating toward the hyperpolarization direction. The cationic cell-penetrating HIV-1 Tat peptide exerted a similar BK channel-activating effect. The BK channel-activating effects of the synthetic peptides were reduced in the presence of Ca2+ and markedly ablated by both charge neutralization of the Ca2+-bowl site and high ionic strength, suggesting the involvement of electrostatic interactions. The efficacy of the γ subunits in BK channel modulation was reduced by charge neutralization of the Ca2+-bowl site. However, BK channel modulation by the γ1 subunit was little affected by high ionic strength and the positively charged peptide remained effective in BK channel modulation in the presence of the γ1 subunit. These findings identify positively charged peptides as BK channel modulators and reveal a role for the Ca2+-bowl site in BK channel modulation by positively charged peptides and the C-terminal positively charged regions of auxiliary γ subunits.

An Amphipathic Structure of a Dipropylglycine-Containing Helical Peptide with Sufficient Length Enables Safe and Effective Intracellular siRNA Delivery.

Amphipathic peptides composed of cationic amino acids and hydrophobic amino acids have cell-penetrating ability and are often used as a delivery tool for membrane-impermeable compounds. Small interfering RNA (siRNAs) are one of the delivery targets for such cell-penetrating peptides (CPPs). Cationic CPPs can associate with anionic siRNAs by electrostatic interactions resulting in the formation of nano-sized complexes, which can deliver siRNAs intracellularly. CPPs containing unnatural amino acids offer promising tools to siRNA delivery. However, the detailed structure-activity relationship in siRNA delivery has been rarely studied. In the current study, we designed peptides containing dipropylglycine (Dpg) and explored the cellular uptake and cytotoxicity of peptide/siRNA complexes. The amphipathic structure of the peptides played a key role in complexation with siRNAs and intracellular siRNA delivery. In the amphipathic peptides, cellular uptake of siRNA increased with increasing peptide length, but cytotoxicity was reduced. A peptide containing four Dpg exhibited an effective gene-silencing effect with small amounts of peptides without cytotoxicity in medium containing serum. These findings will be helpful for the design of novel CPPs for siRNA delivery.

Hydrophobic modification improves the delivery of cell-penetrating peptides to eliminate intracellular pathogens in animals.

Infections induced by intracellular pathogens are difficult to eradicate due to poor penetration of antimicrobials into cell membranes. It is of great importance to develop a new generation of antibacterial agents with dual functions of efficient cell penetration and bacterial inhibition. In this study, the association between hydrophobicity and cell-penetrating peptide delivery efficiency was investigated by fragment interception and hydrophobicity modification of natural porcine antimicrobial peptide PR-39 and the combination of cationic cell-penetrating peptide (R6) with antimicrobial peptide fragments modified with hydrophobic residues. The chimeric peptides P3I7 and P3L7, obtained through biofunctional screening, exhibited potent broad-spectrum antibacterial activity and low cytotoxicity. Moreover, P3I7 and P3L7 can effectively penetrate cells to eliminate intracellular pathogens mainly through endocytosis. The membrane destruction mechanism makes the peptides fast sterilizers and less prone to developing drug resistance. Finally, their good biocompatibility and antibacterial infection effects were verified in mice and piglets. To conclude, the chimeric peptides P3I7 and P3L7 show great potential as affordable and effective antimicrobial agents and may serve as ideal candidates for the treatment of intracellular bacterial infections. STATEMENT OF SIGNIFICANCE: The low permeability of antibacterial drugs makes infections induced by intracellular bacteria extremely difficult to treat. To address this issue, we designed chimeric peptides with dual cell-penetrating and antibacterial functions. The active peptides P3I7 and P3L7, acquired through functional screening have strong broad-spectrum antibacterial activity and powerful bactericidal effects against intracellular Staphylococcus aureus. The membrane permeation mechanism of P3I7 and P3L7 against bacteria endows fast bactericidal activity with low drug resistance. The biosafety and antibacterial activity of P3I7 and P3L7 were also validated by in vivo trials. This study provides an ideal drug candidate against intracellular bacterial infections.

Choosing an Optimal Solvent Is Crucial for Obtaining Cell-Penetrating Peptide Nanoparticles with Desired Properties and High Activity in Nucleic Acid Delivery.

Cell-penetrating peptides (CPPs) are highly promising transfection agents that can deliver various compounds into living cells, including nucleic acids (NAs). Positively charged CPPs can form non-covalent complexes with negatively charged NAs, enabling simple and time-efficient nanoparticle preparation. However, as CPPs have substantially different chemical and physical properties, their complexation with the cargo and characteristics of the resulting nanoparticles largely depends on the properties of the surrounding environment, i.e., solution. Here, we show that the solvent used for the initial dissolving of a CPP determines the properties of the resulting CPP particles formed in an aqueous solution, including the activity and toxicity of the CPP-NA complexes. Using different biophysical methods such as dynamic light scattering (DLS), atomic force microscopy (AFM), transmission and scanning electron microscopy (TEM and SEM), we show that PepFect14 (PF14), a cationic amphipathic CPP, forms spherical particles of uniform size when dissolved in organic solvents, such as ethanol and DMSO. Water-dissolved PF14, however, tends to form micelles and non-uniform aggregates. When dissolved in organic solvents, PF14 retains its α-helical conformation and biological activity in cell culture conditions without any increase in cytotoxicity. Altogether, our results indicate that by using a solvent that matches the chemical nature of the CPP, the properties of the peptide-cargo particles can be tuned in the desired way. This can be of critical importance for in vivo applications, where CPP particles that are too large, non-uniform, or prone to aggregation may induce severe consequences.

Co-administration of Transportan Peptide Enhances the Cellular Entry of Liposomes in the Bystander Manner Both In Vitro and In Vivo.

Liposomes have been widely used as a drug delivery vector. One way to further improve its therapeutic efficacy is to increase the cell entry efficiency. Covalent conjugation with cell-penetrating peptides (CPPs) and other types of ligands has been the mainstream strategy to tackle this issue. Although efficient, it requires additional chemical modifications on liposomes, which is undesirable for clinical translation. Our previous study showed that the transportan (TP) peptide, an amphiphilic CPP, was able to increase the cellular uptake of co-administered, but not covalently coupled, metallic nanoparticles (NPs). Termed bystander uptake, this process represents a simpler method to increase the cell entry of NPs without chemical modifications. Here, we extended our efforts to liposomes. Our results showed that co-administration with the TP peptide improved the internalization of liposome into a variety of cell lines in vitro. This effect was also observed in primary cells, ex vivo tumor slices, and in vivo tumor tissues. On the other hand, this peptide-assisted liposome internalization did not apply to cationic CPPs, which were the main inducers for bystander uptake in previous studies. We also found that TP-assisted bystander uptake of liposome is receptor dependent, and its activity is more sensitive to the inhibitors of the macropinocytosis pathway, underlining the potential cell entry mechanism. Overall, our study provides a simple strategy based on TP co-administration to increase the cell entry of liposomes, which may open up new avenues to apply TP peptides in nanotherapeutics.