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Autodynamic cancer therapy possesses tremendous potential for enhancing therapeutic efficacy by initiating the treatment process autonomously within targeted cells. However, challenges related to biocompatibility and targeted delivery have hindered its clinical translation owing to the induction of adverse effects and cytotoxicity in healthy cells. In this study, a novel approach for auto‐initiated dynamic therapy by conjugating zwitterionic near‐infrared fluorophores to a cell‐penetrating peptide is proposed. This enables efficient cellular uptake and specific targeting of therapy to desired cells while avoiding off‐target uptake. The zwitterionic bioconjugate causes cancer‐specific toxicity following its internalization into the targeted cells, triggered by specific intracellular conditions in lysosomes. This innovative approach enables selective targeting of lysosomes in malignant cells while minimizing cytotoxic effects on normal cells. By targeting lysosomes, the method overcomes inherent risks and side effects associated with conventional cancer treatments, offering a selective and effective approach to cancer therapy. [ABSTRACT FROM AUTHOR]

Background/Objectives: Peptide-based treatments represent an expanding area and require innovative approaches to enhance bioavailability. Combination with cell-penetrating peptides (CPPs) is an attractive strategy to improve non-invasive delivery across nasal epithelial barriers for systemic and direct nose-to-brain transport. We previously developed a modified CPP system termed Glycosaminoglycan-binding Enhanced Transduction (GET) that improves insulin delivery across gastrointestinal epithelium. It contains a membrane docking sequence to promote cellular interactions (P21), a cationic polyarginine domain to stimulate uptake (8R) and an endosomal escaping sequence to maximize availability for onward distribution (LK15). It is synthesized as a single 44-residue peptide (P21-LK15-8R; PLR). Methods: The current research used in vitro assays for a novel exploration of PLR's ability to improve the transport of two contrasting peptides, insulin (51 residues, net negative charge) and oxytocin (9 residues, weak positive charge) across an RPMI 2650 human nasal epithelial cell barrier cultured at the air–liquid interface. Results: PLR enhanced insulin transcytosis over a 6 h period by 7.8-fold when used at a 2:1 molar ratio of insulin/PLR (p < 0.0001 versus insulin alone). Enhanced oxytocin transcytosis (5-fold) occurred with a 1:10 ratio of oytocin/PLR (p < 0.01). Importantly, these were independent of any impact on transepithelial electrical resistance (TEER) or cell viability (p > 0.05). Conclusions: We advocate the continued evaluation of insulin–PLR and oxytocin–PLR formulations, including longer-term assessments of ciliotoxicity and cytotoxicity in vitro followed by in vivo assessments of systemic and nose-to-brain delivery. [ABSTRACT FROM AUTHOR]

The synthetic peptide TAT-I24 (GRKKRRQRRRPPQCLAFYACFC) exerts antiviral activity against several double-stranded (ds) DNA viruses, including herpes simplex viruses, cytomegalovirus, some adenoviruses, vaccinia virus and SV40 polyomavirus. In the present study, in vitro profiling of this peptide was performed with the aim of characterizing and improving its properties for further development. As TAT-I24 contains three free cysteine residues, a potential disadvantageous feature, peptide variants with replacements or deletions of specific residues were generated and tested in various cell systems and by biochemical analyses. Some cysteine replacements had no impact on the antiviral activity, such as the deletion of cysteine 14, which also showed improved biochemical properties, while the cyclization of cysteines 14 and 20 had the most detrimental effect on antiviral activity. At concentrations below 20 µM, TAT-I24 and selected variants did not induce hemolysis in red blood cells (RBCs) nor modulated lipopolysaccharide (LPS)-induced release of cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), in human peripheral blood mononuclear cells (PBMCs). These data indicate that TAT-I24 or its peptide variants are not expected to cause unwanted effects on blood cells. [ABSTRACT FROM AUTHOR]

Protein kinase B (AKT1) is a serine/threonine kinase that regulates fundamental cellular processes, including cell survival, proliferation, and metabolism. AKT1 activity is controlled by two regulatory phosphorylation sites (Thr308, Ser473) that stimulate a downstream signaling cascade through phosphorylation of many target proteins. At either or both regulatory sites, hyperphosphorylation is associated with poor survival outcomes in many human cancers. Our previous biochemical and chemoproteomic studies showed that the phosphorylated forms of AKT1 have differential selectivity toward peptide substrates. Here, we investigated AKT1‐dependent activity in human cells, using a cell‐penetrating peptide (transactivator of transcription, TAT) to deliver inactive AKT1 or active phospho‐variants to cells. We used enzyme engineering and genetic code expansion relying on a phosphoseryl‐transfer RNA (tRNA) synthetase (SepRS) and tRNASep pair to produce TAT‐tagged AKT1 with programmed phosphorylation at one or both key regulatory sites. We found that all TAT‐tagged AKT1 variants were efficiently delivered into human embryonic kidney (HEK 293T) cells and that only the phosphorylated AKT1 (pAKT1) variants stimulated downstream signaling. All TAT‐pAKT1 variants induced glycogen synthase kinase (GSK)‐3α phosphorylation, as well as phosphorylation of ribosomal protein S6 at Ser240/244, demonstrating stimulation of downstream AKT1 signaling. Fascinatingly, only the AKT1 variants phosphorylated at S473 (TAT‐pAKT1S473 or TAT‐pAKT1T308,S473) were able to increase phospho‐GSK‐3β levels. Although each TAT‐pAKT1 variant significantly stimulated cell proliferation, cells transduced with TAT‐pAKT1T308 grew significantly faster than with the other pAKT1 variants. The data demonstrate differential activity of the AKT1 phospho‐forms in modulating downstream signaling and proliferation in human cells. [ABSTRACT FROM AUTHOR]

Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly- l -α-lysine consisting of 25–35 l -α-lysine residues (ε-PαL 25-35) and ε-poly- l -β-lysine consisting of l -β-lysine residues (ε-PβL 4-13), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5–14 l -α-lysine residues (ε-PαL 5-14) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL 5-14 and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL 5-14 -FAM, with HeLa cells. Unlike ε-PαL 25-35 -FAM, ε-PαL 5-14 -FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (>50 μM) was required for the internalization events. ε-PαL 5-14 has a chain length almost equal to that of the membrane permeable ε-PβL 4-13 , which can enter cells at low concentrations. Considering that the basicity of the β-amino group is higher than that of α-amino acid at physiological pH, ε-PβL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PβL would be more advantageous for cellular internalization of cargo proteins than ε-PαL 25-35. [Display omitted] [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile. [ABSTRACT FROM AUTHOR]

Abstract Acute myeloid leukemia (AML) is an aggressive blood cancer with frequently poor clinical outcomes. This heterogeneous malignancy encompasses genetically, molecularly, and even clinically different subgroups. This makes it difficult to develop therapeutic agents that are effective for all subtypes of the disease. Therefore, a selective, universal, and adaptable delivery platform capable of carrying various types of anti-neoplastic agents is an unmet requirement in this area. Two multifunctional fusion proteins were designed for the delivery of biotinylated cargoes to human myeloid leukemia cells by fusing an anti-IL-1RAP single-chain antibody with streptavidin (tetramer or monomer), a cell-penetrating peptide (CPP), and an endosomolytic peptide in a single biomacromolecule. The designed fusions were analyzed primarily in silico, and the biofunctionality of the selected fusion was fully characterized via several binding assays, hemolysis assay, confocal microscopy and cell cytotoxicity assay after production via the Escherichia coli (E. coli) system. The refolded protein exhibited desirable binding activity to leukemic cells, pure antigen and biotinylated BSA. Further analyses revealed efficient cellular uptake, endosomolytic activity, and nuclear penetration without any detectable cytotoxicity toward normal epithelial cells. The described platform seems to have great potential for targeted delivery of different therapeutics to malignant myeloid cells.

Ji-Hyun Kang,1,2,&ast; Jin-Hyuk Jeong,1,&ast; Yong-Bin Kwon,1 Young-Jin Kim,1 Dae Hwan Shin,1 Yun-Sang Park,3 Soonsil Hyun,1 Dong-Wook Kim,4 Chun-Woong Park1 1College of Pharmacy, Chungbuk National University, Cheongju, 28160, Republic of Korea; 2School of Pharmacy, Institute of New Drug Development, and Respiratory Drug Development Research Institute, Jeonbuk National University, Jeonju, Republic of Korea; 3Research & Development Center, P2K Bio, Cheongju, Republic of Korea; 4College of Pharmacy, Wonkwang University, Iksan, 54538, Republic of Korea&ast;These authors contributed equally to this workCorrespondence: Chun-Woong Park, College of Pharmacy, Chungbuk National University, Cheongju, 28160, Republic of Korea, Tel +82-43-261-3330, Fax +82-43-268-2732, Email cwpark@cbnu.ac.kr Dong-Wook Kim, College of Pharmacy, Wonkwang University, Iksan, 54538, Republic of Korea, Tel +82-63-229-7130, Fax +82-63-850-7309, Email pharmengin@gmail.comPurpose: Effective mucosal delivery of drugs continues to pose a significant challenge owing to the formidable barrier presented by the respiratory tract mucus, which efficiently traps and clears foreign particulates. The surface characteristics of micelles dictate their ability to penetrate the respiratory tract mucus. In this study, polymeric micelles loaded with insulin (INS) were modified using mucus-penetrative polymers.Methods: We prepared and compared polyethylene glycol (PEG)-coated micelles with micelles where cell-penetrating peptide (CPP) is conjugated to PEG. Systematic investigations of the physicochemical and aerosolization properties, performance, in vitro release, mucus and cell penetration, lung function, and pharmacokinetics/pharmacodynamics (PK/PD) of polymeric micelles were performed to evaluate their interaction with the respiratory tract.Results: The nano-micelles, with a particle size of < 100 nm, exhibited a sustained-release profile. Interestingly, PEG-coated micelles exhibited higher diffusion and deeper penetration across the mucus layer. In addition, CPP-modified micelles showed enhanced in vitro cell penetration. Finally, in the PK/PD studies, the micellar solution demonstrated higher maximum concentration (Cmax) and AUC0-8h values than subcutaneously administered INS solution, along with a sustained blood glucose-lowering effect that lasted for more than 8 h.Conclusion: This study proposes the use of mucus-penetrating micelle formulations as prospective inhalation nano-carriers capable of efficiently transporting peptides to the respiratory tract.Keywords: insulin, polyethylene glycol, cell-penetrating peptide, aerodynamic properties, mucus penetration

[Display omitted] • Host antimicrobial peptides and extracellular vesicles are members of the immune system. • Only limited information is known on the various ways these species may interact with each other. • For a broader overview, 26 AMPs were studied, including cell-penetrating peptides, with red blood cell-derived vesicles. • Based on results arising from spectroscopic methods, eight different modes of interaction mechanisms have been identified Host antimicrobial peptides (AMPs) and extracellular vesicles (EVs) are known to play important roles as part of the immune system, from antimicrobial actions to immune regulation. Recent results also demonstrate that EVs could serve as carriers for AMPs. Related, it was shown that some AMPs can remove the protein corona (PC), the externally adsorbed layer of proteins, from EVs which can be exploited for subtractive proteomics strategies. The interaction of these compounds is thus interesting for multiple reasons from better insight to natural processes to direct applications in EV-based bioengineering. However, we have only limited information on the various ways these species may interact with each other. To reach a broader overview, here we selected twenty-six AMPs, including cell-penetrating peptides (CPPs), and investigated their interactions with red blood cell-derived vesicles (REVs). For this, we employed a complex lipid biophysics including linearly polarized light spectroscopy, flow cytometry, nanoparticle tracking analysis, electron microscopy and also zeta-potential measurements. This enabled the categorization of these peptides into distinct groups. At specific low concentrations, peptides such as LL-37 and lasioglossin-III were effective in PC elimination with minimal disruption of the membrane. In contrast, AMPs like KLA, bradykinin, histatin-5, and most of the tested CPPs (e.g. octa-arginine, penetratin, and buforin II), demonstrate cell-penetrating mechanisms as they could sustain large peptide concentrations with minimal membrane damage. The systematic overview presented here shows the potential mechanism of how AMPs and EVs could interact in vivo, and also how certain peptides may be employed to manipulate EVs for specific applications. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides (AMPs) are the most favorable alternatives in overcoming multidrug resistance, alone or synergistically with conventional antibiotics. Plant-derived AMPs, as cysteine-rich peptides, widely compensate the pharmacokinetic drawbacks of peptide therapeutics. Compared to the putative genes encrypted in the genome, AMPs that are produced under stress are active forms with the ability to combat resistant microbial species. Within this study, plant-derived AMPs, namely, defensins, nodule-specific cysteine-rich peptides, snakins, lipid transfer proteins, hevein-like proteins, α-hairpinins, and aracins, expressed under biotic and abiotic stresses, are classified. We could observe that while α-hairpinins and snakins display a helix-turn-helix structure, conserved motif patterns such as β1αβ2β3 and β1β2β3 exist in plant defensins and hevein-like proteins, respectively. According to the co-expression data, several plant AMPs are expressed together to trigger synergistic effects with membrane disruption mechanisms such as toroidal pore, barrel-stave, and carpet models. The application of AMPs as an eco-friendly strategy in maintaining agricultural productivity through the development of transgenes and bio-pesticides is discussed. These AMPs can be consumed in packaging material, wound-dressing products, coating catheters, implants, and allergology. AMPs with cell-penetrating properties are verified for the clearance of intracellular pathogens. Finally, the dominant pharmacological activities of bioactive peptides derived from the gastrointestinal digestion of plant AMPs, namely, inhibitors of renin and angiotensin-converting enzymes, dipeptidyl peptidase IV and α-glucosidase inhibitors, antioxidants, anti-inflammatory, immunomodulating, and hypolipidemic peptides, are analyzed. Conclusively, as phytopathogens and human pathogens can be affected by plant-derived AMPs, they provide a bright perspective in agriculture, breeding, food, cosmetics, and pharmaceutical industries, translated as farm to bedside. [ABSTRACT FROM AUTHOR]

Cell‐penetrating peptides (CPPs) with better biomolecule delivery properties will expand their clinical applications. Using the MLCPP2.0 machine algorithm, we screened multiple candidate sequences with potential cellular uptake ability from the nuclear localization signal/nuclear export signal database and verified them through cell‐penetrating fluorescent tracing experiments. A peptide (NCR) derived from the Rev protein of the caprine arthritis‐encephalitis virus exhibited efficient cell‐penetrating activity, delivering over four times more EGFP than the classical CPP TAT, allowing it to accumulate in lysosomes. Structural and property analysis revealed that a high hydrophobic moment and an appropriate hydrophobic region contribute to the high delivery activity of NCR. Trastuzumab emtansine (T‐DM1), a HER2‐targeted antibody‐drug conjugate, could improve its anti‐tumor activity by enhancing targeted delivery efficiency and increasing lysosomal drug delivery. This study designed a new NCR vector to non‐covalently bind T‐DM1 by fusing domain Z, which can specifically bind to the Fc region of immunoglobulin G and effectively deliver T‐DM1 to lysosomes. MTT results showed that the domain Z‐NCR vector significantly enhanced the cytotoxicity of T‐DM1 against HER2‐positive tumor cells while maintaining drug specificity. Our results make a useful attempt to explore the potential application of CPP as a lysosome‐targeted delivery tool. [ABSTRACT FROM AUTHOR]

Arginine‐rich cell‐penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona‐arginine (R9)‐mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)‐enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real‐time it is observed that R9 fluidizes PS‐rich liquid‐ordered (Lo) domains into liquid‐disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase‐separated membranes. Collectively, the findings imply that arginine‐rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers. [ABSTRACT FROM AUTHOR]

Delivering nucleic acid therapeutics across cell membranes is a significant challenge. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) show promise for siRNA delivery. To improve siRNA delivery and silence a model STAT3 gene, we hypothesized that oleyl acylation to CPPs, specifically (WRH)n, would enhance STAT3 silencing efficiency in breast and ovarian cancer cells. Using Fmoc/tBu solid-phase peptide chemistry, we synthesized, purified, and characterized the oleyl-conjugated (WRH)n (n = 1–4) peptides. The peptide/siRNA complexes were non-cytotoxic at N/P 40 (~20 μM) against MDA-MB-231, MCF-7, SK-OV-3, and HEK-293 cells after 72 h incubation. All peptide/siRNA complexes showed serum stability at N/P ≥ 40. The synthesized conjugates, with a diameter of <100 nm, formed nano-complexes with siRNA and exhibited a stable range of zeta potential values (13–18 mV at N/P = 40). Confocal microscopy and flow cytometry analysis provided qualitative and quantitative evidence of a successful cellular internalization of siRNA. The peptides oleyl-(WRH)3 and oleyl-(WRH)4 showed ~60% and ~75% cellular uptake of siRNA, respectively, in both MDA-MB-231 and SK-OV-3 cells. Western blot analysis of oleyl-(WRH)4 demonstrated effective silencing of the STAT-3 gene, with ~75% silencing in MDA-MB-231 cells and ~45% in SK-OV-3 cells. [ABSTRACT FROM AUTHOR]

The poliovirus (PV) enters the central nervous system (CNS) via the bloodstream, suggesting the existence of a mechanism to cross the blood–brain barrier. Here, we report that PV capsid proteins (VP1 and VP3) can penetrate cells, with VP3 being more invasive. Two independent parts of VP3 are responsible for this function. Both peptides can penetrate human umbilical cord vascular endothelial cells, and one peptide of VP3 could also penetrate peripheral blood mononuclear cells. In an in vitro blood–brain barrier model using rat‐derived astrocytes, pericytes, and endothelial cells, both peptides were observed to traverse from the blood side to the brain side at 6 h after administration. These results provide insights into the molecular mechanisms underlying PV invasion into the CNS. [ABSTRACT FROM AUTHOR]

Signal transduction and homeostasis are regulated by complex protein interactions in the intracellular environment. Therefore, the transportation of impermeable macromolecules (nucleic acids, proteins, and drugs) that control protein interactions is essential for modulating cell functions and therapeutic applications. However, macromolecule transportation across the cell membrane is not easy because the cell membrane separates the intra/extracellular environments, and the types of molecular transportation are regulated by membrane proteins. Cell-penetrating peptides (CPPs) are expected to be carriers for molecular transport. CPPs can transport macromolecules into cells through endocytosis and direct translocation. The transport mechanism remains largely unclear owing to several possibilities. In this review, we describe the methods for investigating CPP conformation, translocation, and cargo transportation using artificial membranes. We also investigated biomolecular transport across living cell membranes via CPPs. Subsequently, we show not only the biochemical applications but also the synthetic biological applications of CPPs. Finally, recent progress in biomolecule and nanoparticle transportation via CPPs into specific tissues is described from the viewpoint of drug delivery. This review provides the opportunity to discuss the mechanism of biomolecule transportation through these two platforms. [ABSTRACT FROM AUTHOR]

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules. [ABSTRACT FROM AUTHOR]

TP10, a classic cell-penetrating peptide, shows a high degree of similarity to AMPs in structure. Although TP10 has been widely used in drug delivery, the mechanism underlying its cytotoxicity is yet to be elucidated. Herein, we explored the cell-killing mechanism of TP10 against human leukemia Jurkat cells. TP10 induced necrosis in Jurkat cells via rapid disruption of cell membranes, particularly at high concentrations. Although mitochondria in Jurkat cells were damaged by TP10, mitochondria-mediated apoptosis did not occur, possibly due to intracellular ATP depletion. Necroptosis in TP10-treated Jurkat cells became an alternative route of apoptosis. Our results demonstrate that necrosis and necroptosis rather than apoptosis are involved in the cell-killing mechanism of TP10, which contributes to the understanding of its toxicity. [Display omitted] • TP10 can induce necrosis in Jurkat cells via rapid disruption of cell membranes. • TP10 can damage the mitochondria upon entering the cells. • Necroptosis in TP10-treated Jurkat cells becomes an alternative route of apoptosis. [ABSTRACT FROM AUTHOR]

The escalating threat of multidrug-resistant pathogens necessitates innovative approaches to combat infectious diseases. In this study, we examined peptides R23FS*, V31KS*, and R44KS*, which were engineered to include an amyloidogenic fragment sourced from the S1 protein of S. aureus, along with one or two cell-penetrating peptide (CPP) components. We assessed the antimicrobial efficacy of these peptides in a liquid medium against various strains of both Gram-positive bacteria, including S. aureus (209P and 129B strains), MRSA (SA 180 and ATCC 43300 strains), and B. cereus (strain IP 5832), and Gram-negative bacteria such as P. aeruginosa (ATCC 28753 and 2943 strains) and E. coli (MG1655 and K12 strains). Peptides R23FS*, V31KS*, and R44KS* exhibited antimicrobial activity comparable to gentamicin and meropenem against all tested bacteria at concentrations ranging from 24 to 48 μM. The peptides showed a stronger antimicrobial effect against B. cereus. Notably, peptide R44KS* displayed high efficacy compared to peptides R23FS* and V31KS*, particularly evident at lower concentrations, resulting in significant inhibition of bacterial growth. Furthermore, modified peptides V31KS* and R44KS* demonstrated enhanced inhibitory effects on bacterial growth across different strains compared to their unmodified counterparts V31KS and R44KS. These results highlight the potential of integrating cell-penetrating peptides, amyloidogenic fragments, and amino acid residue modifications to advance the innovation in the field of antimicrobial peptides, thereby increasing their effectiveness against a broad spectrum of pathogens. [ABSTRACT FROM AUTHOR]

Abstract Autodynamic cancer therapy possesses tremendous potential for enhancing therapeutic efficacy by initiating the treatment process autonomously within targeted cells. However, challenges related to biocompatibility and targeted delivery have hindered its clinical translation owing to the induction of adverse effects and cytotoxicity in healthy cells. In this study, a novel approach for auto‐initiated dynamic therapy by conjugating zwitterionic near‐infrared fluorophores to a cell‐penetrating peptide is proposed. This enables efficient cellular uptake and specific targeting of therapy to desired cells while avoiding off‐target uptake. The zwitterionic bioconjugate causes cancer‐specific toxicity following its internalization into the targeted cells, triggered by specific intracellular conditions in lysosomes. This innovative approach enables selective targeting of lysosomes in malignant cells while minimizing cytotoxic effects on normal cells. By targeting lysosomes, the method overcomes inherent risks and side effects associated with conventional cancer treatments, offering a selective and effective approach to cancer therapy.

Delivery of antisense oligonucleotides (ASOs) to airway epithelial cells is arduous due to the physiological barriers that protect the lungs and the endosomal entrapment phenomenon, which prevents ASOs from reaching their intracellular targets. Various delivery strategies involving peptide-, lipid-, and polymer-based carriers are being investigated, yet the challenge remains. S10 is a peptide-based delivery agent that enables the intracellular delivery of biomolecules such as GFP, CRISPR-associated nuclease ribonucleoprotein (RNP), base editor RNP, and a fluorescent peptide into lung cells after intranasal or intratracheal administrations to mice, ferrets, and rhesus monkeys. Herein, we demonstrate that covalently attaching S10 to a fluorescently labeled peptide or a functional splice-switching phosphorodiamidate morpholino oligomer improves their intracellular delivery to airway epithelia in mice after a single intranasal instillation. Data reveal a homogeneous delivery from the trachea to the distal region of the lungs, specifically into the cells lining the airway. Quantitative measurements further highlight that conjugation via a disulfide bond through a pegylated (PEG) linker was the most beneficial strategy compared with direct conjugation (without the PEG linker) or conjugation via a permanent thiol-maleimide bond. We believe that S10-based conjugation provides a great strategy to achieve intracellular delivery of peptides and ASOs with therapeutic properties in lungs.

Thermally activated delayed fluorescence (TADF) nanoparticles are used importantly in time‐resolved luminescence imaging for eliminating the background signals from scattering and short‐lived autofluorescence. However, TADF nanoparticles are seldom used for real‐time time‐resolved luminescence imaging, due to their limited luminescence efficiency and lifetimes. To detect delayed fluorescence, multiple excitation cycles with adequate delay times are usually required, leading to long detecting durations and low imaging speed. Herein, highly efficient red TADF nanoparticles are developed through doping a guest molecule, TPAAQ (2,6‐bis[4‐(diphenylamino) phenyl] anthraquinone), in a host (4,4′‐bis(carbazol‐9‐yl)biphenyl, CBP) matrix. With a low doping concentration, the nanoparticles can exhibit obvious TADF with luminescence lifetimes over 0.1 ms and photoluminescence quantum yield up to 35%. A cell‐penetrating peptide is used together with the amphiphilic compound for assembling nanoparticles, which can easily penetrate cells and greatly increase the TADF signals for luminescence lifetime imaging. Thanks to the long‐lived and highly efficient TADF, real‐time in vivo time‐gated luminescence imaging of zebrafish is realized on a chopper‐based wide‐field microscope. This low‐cost time‐resolved luminescence imaging method showed a great potential for real‐time detection of life activities in many organisms with high autofluorescence. [ABSTRACT FROM AUTHOR]

Cell junctions, which are typically associated with dynamic cytoskeletons, are essential for a wide range of cellular activities, including cell migration, cell communication, barrier function and signal transduction. Observing cell junctions in real-time can help us understand the mechanisms by which they regulate these cellular activities. This study examined the binding capacity of a modified tridecapeptide from Connexin 43 (Cx43) to the cell junction protein zonula occludens-1 (ZO-1). The goal was to create a fluorescent peptide that can label cell junctions. A cell-penetrating peptide was linked to the modified tridecapeptide. The heterotrimeric peptide molecule was then synthesized. The binding of the modified tridecapeptide was tested using pulldown and immunoprecipitation assays. The ability of the peptide to label cell junctions was assessed by adding it to fixed or live Caco-2 cells. The testing assays revealed that the Cx43-derived peptide can bind to ZO-1. Additionally, the peptide was able to label cell junctions of fixed cells, although no obvious cell junction labeling was observed clearly in live cells, probably due to the inadequate affinity. These findings suggest that labeling cell junctions using a peptide-based strategy is feasible. Further efforts to improve its affinity are warranted in the future. [ABSTRACT FROM AUTHOR]

Abstract Arginine‐rich cell‐penetrating peptides (CPPs) have emerged as valuable tools for the intracellular delivery of bioactive molecules, but their membrane perturbation during cell penetration is not fully understood. Here, nona‐arginine (R9)‐mediated membrane reorganization that facilitates the translocation of peptides across laterally heterogeneous membranes is directly visualized. The electrostatic binding of cationic R9 to anionic phosphatidylserine (PS)‐enriched domains on a freestanding lipid bilayer induces lateral lipid rearrangements; in particular, in real‐time it is observed that R9 fluidizes PS‐rich liquid‐ordered (Lo) domains into liquid‐disordered (Ld) domains, resulting in the membrane permeabilization. The experiments with giant unilamellar vesicles (GUVs) confirm the preferential translocation of R9 through Ld domains without pore formation, even when Lo domains are more negatively charged. Indeed, whenever R9 comes into contact with negatively charged Lo domains, it dissolves the Lo domains first, promoting translocation across phase‐separated membranes. Collectively, the findings imply that arginine‐rich CPPs modulate lateral membrane heterogeneity, including membrane fluidization, as one of the fundamental processes for their effective cell penetration across densely packed lipid bilayers.

Therapeutic proteins provided new opportunities for patients and high sales volumes. However, they are formulated for extracellular targets. The lipophilic barrier of the plasma membrane renders the vast array of intracellular targets out of reach. Peptide-based delivery systems, namely cell-penetrating peptides (CPPs), have few safety concerns, and low immunogenicity, with control over administered doses. This study investigates CPP-based protein delivery systems by classifying them into CPP-protein “covalent conjugation” and CPP: protein “non-covalent complexation” categories. Covalent conjugates ensure the proximity of the CPP to the cargo, which can improve cellular uptake and endosomal escape. We will discuss various aspects of covalent conjugates through non-cleavable (stable) or cleavable bonds. Non-cleavable CPP-protein conjugates are produced by recombinant DNA technology to express the complete fusion protein in a host cell or by chemical ligation of CPP and protein, which ensures stability during the delivery process. CPP-protein cleavable bonds are classified into pH-sensitive and redox-sensitive bonds, enzyme-cleavable bonds, and physical stimuli cleavable linkers (light radiation, ultrasonic waves, and thermo-responsive). We have highlighted the key characteristics of non-covalent complexes through electrostatic and hydrophobic interactions to preserve the conformational integrity of the CPP and cargo. CPP-mediated protein delivery by non-covalent complexation, such as zippers, CPP adaptor methods, and avidin-biotin technology, are featured. Conclusively, non-covalent complexation methods are appropriate when a high number of CPP or protein samples are to be screened. In contrast, when the high biological activity of the protein is critical in the intracellular compartment, conjugation protocols are preferred.

Abstract Cell-penetrating peptides (CPPs) are short chains of amino acids that have shown remarkable potential to cross the cell membrane and deliver coupled therapeutic cargoes into cells. Designing and testing different CPPs to target specific cells or tissues is crucial to ensure high delivery efficiency and reduced toxicity. However, in vivo/in vitro testing of various CPPs can be both time-consuming and costly, which has led to interest in computational methodologies, such as Machine Learning (ML) approaches, as faster and cheaper methods for CPP design and uptake prediction. However, most ML models developed to date focus on classification rather than regression techniques, because of the lack of informative quantitative uptake values. To address these challenges, we developed POSEIDON, an open-access and up-to-date curated database that provides experimental quantitative uptake values for over 2,300 entries and physicochemical properties of 1,315 peptides. POSEIDON also offers physicochemical properties, such as cell line, cargo, and sequence, among others. By leveraging this database along with cell line genomic features, we processed a dataset of over 1,200 entries to develop an ML regression CPP uptake predictor. Our results demonstrated that POSEIDON accurately predicted peptide cell line uptake, achieving a Pearson correlation of 0.87, Spearman correlation of 0.88, and r2 score of 0.76, on an independent test set. With its comprehensive and novel dataset, along with its potent predictive capabilities, the POSEIDON database and its associated ML predictor signify a significant leap forward in CPP research and development. The POSEIDON database and ML Predictor are available for free and with a user-friendly interface at https://moreiralab.com/resources/poseidon/ , making them valuable resources for advancing research on CPP-related topics. Scientific Contribution Statement: Our research addresses the critical need for more efficient and cost-effective methodologies in Cell-Penetrating Peptide (CPP) research. We introduced POSEIDON, a comprehensive and freely accessible database that delivers quantitative uptake values for over 2,300 entries, along with detailed physicochemical profiles for 1,315 peptides. Recognizing the limitations of current Machine Learning (ML) models for CPP design, our work leveraged the rich dataset provided by POSEIDON to develop a highly accurate ML regression model for predicting CPP uptake. Graphical Abstract

Weinan Hu,&ast; Wenting Cai,&ast; Yan Wu, Chengda Ren, Donghui Yu, Tingting Li, Tianyi Shen, Ding Xu, Jing Yu Department of Ophthalmology, Shanghai Tenth People’s Hospital, Tongji University, Shanghai, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ding Xu; Jing Yu, Email 15821885240@163.com; dryujing@aliyun.comBackground: Age-related macular degeneration (AMD) stands as the foremost cause of irreversible central vision impairment, marked by choroidal neovascularization (CNV). The prevailing clinical approach to AMD treatment relies on intravitreal injections of anti-vascular endothelial growth factor (VEGF) drugs. However, this method is encumbered by diverse complications, prompting exploration of non-invasive alternatives such as ocular administration via eye drops for anti-VEGF therapy.Methods: Two complexes, 5-FITC-CPP-Ranibizumab (5-FCR) and 5-FITC-CPP-Conbercept (5-FCC), were synthesized by incorporating the anti-VEGF drugs Ranibizumab (RBZ) or Conbercept (CBC) with cell-penetrating peptide (CPP). Circular dichroism spectrum (CD) facilitated complexes characterization. Eye drops was utilized to address laser-induced CNV in mice. Fluorescein fundus angiography (FFA) observe the CNV lesion, while FITC-dextran and IB4 dual fluorescent staining, along with hematoxylin-eosin (HE) staining, assessed in lesion size. Tissue immunofluorescence examined CD31 and VEGF expression in choroidal/retinal pigment epithelial (RPE) tissues. Biocompatibility and biosafety of 5-FCR and 5-FCC was evaluated through histological examination of various organs or cell experiments.Results: Both 5-FCR and 5-FCC exhibited favorable biocompatibility and safety profiles. VEGF-induced migration of Human umbilical vein endothelial cells (HUVECs) significantly decreased post-5-FCR/5-FCC treatment. Additionally, both complexes suppressed VEGF-induced tube formation in HUVECs. FFA results revealed a significant improvement in retinal exudation in mice. Histological examination unveiled the lesion areas in the 5-FCR and 5-FCC groups showed a significant reduction compared to the control group. Similar outcomes were observed in histological sections of the RPE-choroid-sclera flat mounts.Conclusion: In this study, utilizing the properties of CPP and two anti-VEGF drugs, we successfully synthesized two complexes, 5-FCR and 5-FCC, through a straightforward approach. Effectively delivering the anti-VEGF drugs to the target area in a non-invasive manner, suppressing the progression of laser-induced CNV. This offers a novel approach for the treatment of wet AMD. Keywords: age-related macular degeneration, choroidal neovascularization, cell-penetrating peptide, ranibizumab, conbercept, drug delivery

Jing Huang,1,&ast; Kaiyu Wang,1,&ast; Xizi Fu,1,&ast; Manfang Zhu,2 Xiaohua Chen,1 Yan Gao,1 Pingchuan Ma,3 Xingmei Duan,2 Ke Men1 1State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China; 2Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China; 3State Key Laboratory of Oral Diseases & National Center for Stomatology & National Clinical Research Center for Oral Diseases & Department of Head and Neck Oncology, West China Hospital of Stomatology, Sichuan University, Chengdu, Sichuan, 610041, People’s Republic of China&ast;These authors contributed equally to this workCorrespondence: Ke Men, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu, 610041, People’s Republic of China, Email mendingbob@hotmail.com Xingmei Duan, Department of Pharmacy, Personalized Drug Therapy Key Laboratory of Sichuan Province Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610072, People’s Republic of China, Email duanxingmei2003@163.comBackground: Messenger ribonucleic acid (mRNA)-based gene therapy has great potential in cancer treatment. However, the application of mRNA-based cancer treatment could be further developed. Elevated delivery ability and enhanced immune response are advantages for expanding the application of mRNA-based cancer therapy. It is crucial that the prepared carrier can cause an immune reaction based on the efficient delivery of mRNA.Methods: We reported DMP nanoparticle previously, which was obtained by the self-assembly of 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and (ethylene glycol)-b-poly (&epsiv;-caprolactone) (mPEG-PCL). Research demonstrated that DMP can deliver mRNA, siRNA, and plasmid. And it is applied to various tumor types. In our work, the tumor cell lysate was introduced to the internal DMP chain, fusing cell-penetrating peptides (CPPs) modification on the surface forming the CLSV system. And then mixed encoded IL-22BP (interleukin-22 binding protein) mRNA and CLSV to form CLSV/IL-22BP complex.Results: The size of the CLSV system was 213.2 nm, and the potential was 45.7 mV. The transfection efficiency of the CLSV system is up to 76.45% in C26 cells via the micropinocytosis pathway. The CLSV system also could induce an immune response and significantly elevate the expression of CD80, CD86, and MHC-II in vivo. Then, by binding with IL-22BP (Interleukin-22 binding protein) mRNA, the CLSV/IL-22BP complex inhibited tumor cell growth, with an inhibition rate of up to 82.3% in vitro. The CLSV/IL-22BP complex also inhibited tumor growth in vivo, the tumor cell growth inhibition up to 75.0% in the subcutaneous tumor model, and 84.9% in the abdominal cavity metastasis tumor model.Conclusion: Our work demonstrates that the CLSV system represents a potent potential for mRNA delivery.Keywords: mRNA, cell-penetrating peptide, gene therapy, tumor cell lysate, immune response

Background/Objectives: Peptide-based treatments represent an expanding area and require innovative approaches to enhance bioavailability. Combination with cell-penetrating peptides (CPPs) is an attractive strategy to improve non-invasive delivery across nasal epithelial barriers for systemic and direct nose-to-brain transport. We previously developed a modified CPP system termed Glycosaminoglycan-binding Enhanced Transduction (GET) that improves insulin delivery across gastrointestinal epithelium. It contains a membrane docking sequence to promote cellular interactions (P21), a cationic polyarginine domain to stimulate uptake (8R) and an endosomal escaping sequence to maximize availability for onward distribution (LK15). It is synthesized as a single 44-residue peptide (P21-LK15-8R; PLR). Methods: The current research used in vitro assays for a novel exploration of PLR’s ability to improve the transport of two contrasting peptides, insulin (51 residues, net negative charge) and oxytocin (9 residues, weak positive charge) across an RPMI 2650 human nasal epithelial cell barrier cultured at the air–liquid interface. Results: PLR enhanced insulin transcytosis over a 6 h period by 7.8-fold when used at a 2:1 molar ratio of insulin/PLR (p < 0.0001 versus insulin alone). Enhanced oxytocin transcytosis (5-fold) occurred with a 1:10 ratio of oytocin/PLR (p < 0.01). Importantly, these were independent of any impact on transepithelial electrical resistance (TEER) or cell viability (p > 0.05). Conclusions: We advocate the continued evaluation of insulin–PLR and oxytocin–PLR formulations, including longer-term assessments of ciliotoxicity and cytotoxicity in vitro followed by in vivo assessments of systemic and nose-to-brain delivery.

Cell-penetrating peptides (CPPs) are short chains of amino acids that have shown remarkable potential to cross the cell membrane and deliver coupled therapeutic cargoes into cells. Designing and testing different CPPs to target specific cells or tissues is crucial to ensure high delivery efficiency and reduced toxicity. However, in vivo/in vitro testing of various CPPs can be both time-consuming and costly, which has led to interest in computational methodologies, such as Machine Learning (ML) approaches, as faster and cheaper methods for CPP design and uptake prediction. However, most ML models developed to date focus on classification rather than regression techniques, because of the lack of informative quantitative uptake values. To address these challenges, we developed POSEIDON, an open-access and up-to-date curated database that provides experimental quantitative uptake values for over 2,300 entries and physicochemical properties of 1,315 peptides. POSEIDON also offers physicochemical properties, such as cell line, cargo, and sequence, among others. By leveraging this database along with cell line genomic features, we processed a dataset of over 1,200 entries to develop an ML regression CPP uptake predictor. Our results demonstrated that POSEIDON accurately predicted peptide cell line uptake, achieving a Pearson correlation of 0.87, Spearman correlation of 0.88, and r2 score of 0.76, on an independent test set. With its comprehensive and novel dataset, along with its potent predictive capabilities, the POSEIDON database and its associated ML predictor signify a significant leap forward in CPP research and development. The POSEIDON database and ML Predictor are available for free and with a user-friendly interface at , making them valuable resources for advancing research on CPP-related topics. Scientific Contribution Statement: Our research addresses the critical need for more efficient and cost-effective methodologies in Cell-Penetrating Peptide (CPP) research. We introduced POSEIDON, a comprehensive and freely accessible database that delivers quantitative uptake values for over 2,300 entries, along with detailed physicochemical profiles for 1,315 peptides. Recognizing the limitations of current Machine Learning (ML) models for CPP design, our work leveraged the rich dataset provided by POSEIDON to develop a highly accurate ML regression model for predicting CPP uptake. [ABSTRACT FROM AUTHOR]

The underdevelopment of adjuvant discovery and diversity, compared to core vaccine technology, is evident. On the other hand, antibiotic resistance is on the list of the top ten threats to global health. Immunomodulatory peptides that target a pathogen and modulate the immune system simultaneously are promising for the development of preventive and therapeutic molecules. Since investigating innate immunity in insects has led to prominent achievements in human immunology, such as toll-like receptor (TLR) discovery, we used the capacity of the immunomodulatory peptides of arthropods with concomitant antimicrobial or antitumor activity. An SVM-based machine learning classifier identified short immunomodulatory sequences encrypted in 643 antimicrobial peptides from 55 foe-to-friend arthropods. The critical features involved in efficacy and safety were calculated. Finally, 76 safe immunomodulators were identified. Then, molecular docking and simulation studies defined the target of the most optimal peptide ligands among all human cell-surface TLRs. SPalf2-453 from a crab is a cell-penetrating immunoadjuvant with antiviral properties. The peptide interacts with the TLR1/2 heterodimer. SBsib-711 from a blackfly is a TLR4/MD2 ligand used as a cancer vaccine immunoadjuvant. In addition, SBsib-711 binds CD47 and PD-L1 on tumor cells, which is applicable in cancer immunotherapy as a checkpoint inhibitor. MRh4-679 from a shrimp is a broad-spectrum or universal immunoadjuvant with a putative Th1/Th2-balanced response. We also implemented a pathway enrichment analysis to define fingerprints or immunological signatures for further in vitro and in vivo immunogenicity and reactogenicity measurements. Conclusively, combinatorial machine learning, molecular docking, and simulation studies, as well as systems biology, open a new opportunity for the discovery and development of multifunctional prophylactic and therapeutic lead peptides. [ABSTRACT FROM AUTHOR]

Background: Heat shock proteins (HSPs) as an adjuvant induce antigen‐specific immunity through facilitating antigen presentation and stimulating T cells. In this study, the immunostimulatory properties of two major fragments of Hsp70 (N‐Hsp70(aa 1–387) with ATPase property and C‐Hsp70 (aa 508–641) with peptide‐binding capacity) and the full length of Hsp27 as vaccine adjuvants were evaluated to boost HIV‐1 Nef antigen‐specific immunity in both in vitro and in vivo experiments. Method s : At first, the nanoparticles harbouring DNA fusion constructs (i.e. N‐Hsp70‐Nef, C‐Hsp70‐Nef and Hsp27‐Nef) complexed with HIV Rev (34–50) cell‐penetrating peptide were generated to deliver DNA into the cells. Then, the recombinant Nef, Hsp27‐Nef, N‐Hsp70‐Nef and C‐Hsp70‐Nef proteins were generated in E.coli expression system. Next, the immunostimulatory properties of these fusion constructs were evaluated in both in vitro and in vivo studies. Finally, the secretion of main cytokines from single‐cycle replicable (SCR) HIV‐1 virion‐exposed splenocytes was investigated. Results: Our data showed that the stable and non‐toxic DNA/Rev nanoparticles could successfully deliver the genes of interest into the cells. Moreover, higher secretion of antibodies and cytokines was detected in mice receiving the Hsp‐Nef constructs than in mice receiving Nef antigen. The C‐Hsp70 was also superior for inducing Nef‐specific Th1 and CTL immunity compared with N‐Hsp70 and Hsp27. The T‐cell activity was maintained in the SCR‐exposed splenocytes, especially the splenocytes of mice receiving the C‐Hsp70‐Nef regimen. Conclusion: Altogether, these findings demonstrate the significance of Hsps as enhancers of antigen‐specific immunity. Notably, the C‐Hsp70 region showed better adjuvant properties for inducing cellular immunity in the improvement of HIV‐1 therapeutic vaccines. [ABSTRACT FROM AUTHOR]

Aim: This study aimed to investigate and verify the effect of cell‐penetrating peptide (CPP)‐conjugated soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) motif of vesicle‐associated membrane protein 2 (VAMP2)‐patterned peptide (INCI name: Acetyl sh‐Oligopeptide‐26 sh‐Oligopeptide‐27 SP, trade name: M.Biome‐BT) on improving skin function in vitro. Methods: The cytotoxicity of CPP‐conjugated SNARE motif of VAMP2‐patterned peptide (CVP) was investigated using the 3‐(4,5‐dimethylthiazol‐2yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay against B16‐F10 cells and human dermal fibroblasts (HDFs) and a reconstructed skin irritation test. The anti‐wrinkle activity of M.Biome‐BT was determined by assessing the release of norepinephrine and dopamine in PC‐12 cells via ELISA. The skin‐whitening effects of CVP were assessed in B16‐F10 cells by measuring the intra‐ and extracellular melanin contents and expression levels of melanin production‐related genes, such as microphthalmia‐associated transcription factor (MITF), tyrosinase (TYR), tyrosinase‐related protein‐1 (TRP‐1), and TRP‐2. Results: CVP is not cytotoxic to B16‐F10 cells and HDFs, and no skin irritation was observed. CVP treatment considerably diminished K+‐induced norepinephrine and dopamine secretion compared with the non‐treated control group (62% and 40%, respectively). Additionally, the inhibition ability of CVP on norepinephrine and dopamine release was comparable to that of botulinum neurotoxin type A (BoNT/A). CVP also increased intracellular melanin content in a dose‐dependent manner, whereas extracellular melanin content decreased (76%–85%). However, CVP treatment did not affect the mRNA expression of MITF, TYR, TRP‐1, and TRP‐2. These results suggest that CVP does not inhibit melanin production; however, it may induce a whitening effect by inhibiting melanin transport. Conclusions: Taken together, our findings indicate that CVP could be used as an active and safe cosmeceutical ingredient for antiaging applications. [ABSTRACT FROM AUTHOR]

Global reports of novel SARS-CoV-2 variants and recurrence cases continue despite substantial vaccination campaigns, raising severe concerns about COVID-19. While repurposed drugs offer some treatment options for COVID-19, notably, nucleoside inhibitors like Remdesivir stand out as curative therapies for COVID-19 that are approved by the US Food and Drug Administration (FDA). The emergence of highly contagious SARS-CoV-2 variants underscores the imperative for antiviral drugs adaptable to evolving viral mutations. RNA-dependent RNA polymerase (RdRp) plays a key role in viral genome replication. Currently, inhibiting viral RdRp function remains a pivotal strategy to tackle the notorious virus. Peptide nucleic acid (PNA) therapy shows promise by effectively targeting specific genome regions, reducing viral replication, and inhibiting infection. In our study, we designed PNA antisense oligomers conjugated with cell-penetrating peptides (CPP) aiming to evaluate their antiviral effects against RdRp target using structure-guided drug design, which involves molecular docking simulations, drug likeliness and pharmacokinetic evaluations, molecular dynamics simulations, and computing binding free energy. The in silico analysis predicts that chemically modified PNAs might act as antisense molecules in order to disrupt ribosome assembly at RdRp's translation start site, and their chemically stable and neutral backbone might enhance sequence-specific RNA binding interaction. Notably, our findings demonstrate that PNA-peptide conjugates might be the most promising inhibitors of SARS-CoV-2 RdRp, with superior binding free energy compared to Remdesivir in the current COVID-19 medication. Specifically, PNA-CPP-1 could bind simultaneously to the active site residues of RdRp protein and sequence-specific RdRp-RNA target in order to control viral replication. [ABSTRACT FROM AUTHOR]

Combining antimicrobial peptides (AMPs) with cell-penetrating peptides (CPPs) has shown promise in boosting antimicrobial potency, especially against Gram-negative bacteria. We examined the CPP-AMP interaction with distinct bacterial types based on cell wall differences. Our investigation focused on AMPs incorporating penetratin CPP and dihybrid peptides containing both cell-penetrating TAT protein fragments from the human immunodeficiency virus and Antennapedia peptide (Antp). Assessment of the peptides TAT-AMP, AMP-Antp, and TAT-AMP-Antp revealed their potential against Gram-positive strains (Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus cereus). Peptides TAT-AMP and AMP-Antp using an amyloidogenic AMP from S1 ribosomal protein Thermus thermophilus, at concentrations ranging from 3 to 12 μM, exhibited enhanced antimicrobial activity against B. cereus. TAT-AMP and TAT-AMP-Antp, using an amyloidogenic AMP from the S1 ribosomal protein Pseudomonas aeruginosa, at a concentration of 12 µM, demonstrated potent antimicrobial activity against S. aureus and MRSA. Notably, the TAT-AMP, at a concentration of 12 µM, effectively inhibited Escherichia coli (E. coli) growth and displayed antimicrobial effects similar to gentamicin after 15 h of incubation. Peptide characteristics determined antimicrobial activity against diverse strains. The study highlights the intricate relationship between peptide properties and antimicrobial potential. Mechanisms of AMP action are closely tied to bacterial cell wall attributes. Peptides with the TAT fragment exhibited enhanced antimicrobial activity against S. aureus, MRSA, and P. aeruginosa. Peptides containing only the Antp fragment displayed lower activity. None of the investigated peptides demonstrated cytotoxic or cytostatic effects on either BT-474 cells or human skin fibroblasts. In conclusion, CPP-AMPs offer promise against various bacterial strains, offering insights for targeted antimicrobial development. [ABSTRACT FROM AUTHOR]

Stroke is a major public health concern worldwide. The lack of effective therapies heightens the need for new therapeutic agents. Previous study identified sirtuin 5 (SIRT5) as a positive regulator of microglia‐induced excessive neuroinflammation following ischemic stroke. Interventions targeting SIRT5 should therefore alleviate neuroinflammation and protect against ischemic stroke. Here, we synthesized a membrane‐permeable peptide specifically bound to SIRT5 through a chaperone‐mediated autophagy targeting motif (Tat‐SIRT5‐CTM) and examined its therapeutic effect in vitro and in vivo. First, in primary microglia, Tat‐SIRT5‐CTM suppressed the binding of SIRT5 with annexin‐A1 (ANXA1), leading to SIRT5 degradation and thus inhibition of SIRT5‐mediated desuccinylation of ANXA1, followed by increased membrane accumulation and secretion of ANXA1. These changes, in turn, alleviated microglia‐induced neuroinflammation. Moreover, following intravenous injection, Tat‐SIRT5‐CTM could efficiently pass through the blood‒brain barrier. Importantly, systemic administration of Tat‐SIRT5‐CTM reduced the brain infarct area and neuronal loss, mitigated neurological deficit scores, and improved long‐term neurological functions in a mouse model of ischemic stroke. Furthermore, no toxicity was observed when high doses Tat‐SIRT5‐CTM were injected into nonischemic mice. Collectively, our study reveals the promising efficacy of the peptide‐directed lysosomal degradation of SIRT5 and suggests it as an effective therapeutic approach for the treatment of ischemic stroke. [ABSTRACT FROM AUTHOR]

This study aimed to prepare a novel multifunctional magnetic nanoparticle (MNP) with a drug-loadable encapsulation and a matrix metalloproteinase (MMP) substrate-modified TAT peptide whose transmembrane ability can be activated in an MMP-rich environment, and to evaluate the uptake of this nanoparticle by cells, as well as its cytotoxicity. Nanoparticles were synthesized and modified with TAT or MMPs-TAT peptides. PC-3 cells and RWPE-1 cells were cultured with these nanoparticles at different concentrations, with or without MMP-2 pretreatment, and their uptake rate and toxicity were determined using flow cytometry and fluorescence microscopy. The nanoparticles were characterized with TEM (transmission electron microscopy) and DLS (dynamic light scattering). The diameter of these nanoparticles ranged from 194.5 ± 69.4 nm to 353.1 ± 103.9 nm, which were in the ideal range for application. The cellular uptake of MMPs-TAT-MNPs appeared similar to MNPs, but significantly increased in the presence of MMP-2 pretreatment. MMPs-TAT-MNPs had no significant cytotoxicity at the concentration of ≤25 μg/ml. MMPs-TAT-MNPs with MMP-2 substrate modified TAT peptide is successfully prepared, possess a low cytotoxicity loadable cargo and can be activated in an MMP-2 rich niche.

Hepatitis B virus (HBV) infection remains a major health threat with limited treatment options. One of various new antiviral strategies is based on a fusion of Staphylococcus aureus nuclease (SN) with the capsid-forming HBV core protein (HBc), termed coreSN. Through co-assembly with wild-type HBc-subunits, the fusion protein is incorporated into HBV nucleocapsids, targeting the nuclease to the encapsidated viral genome. However, coreSN expression was based on transfection of a plasmid vector. Here, we explored whether introducing protein transduction domains (PTDs) into a fluorescent coreSN model could confer cell-penetrating properties for direct protein delivery into cells. Four PTDs were inserted into two different positions of the HBc sequence, comprising the amphiphilic translocation motif (TLM) derived from the HBV surface protein PreS2 domain and three basic PTDs derived from the Tat protein of human immunodeficiency virus-1 (HIV-1), namely Tat4, NP, and NS. To directly monitor the interaction with cells, the SN in coreSN was replaced with the green fluorescent protein (GFP). The fusion proteins were expressed in E. coli, and binding to and potential uptake by human cells was examined through flow cytometry and fluorescence microscopy. The data indicate PTD-dependent interactions with the cells, with evidence of uptake in particular for the basic PTDs. Uptake was enhanced by a triplicated Simian virus 40 (SV40) large T antigen nuclear localization signal (NLS). Interestingly, the basic C terminal domain of the HBV core protein was found to function as a novel PTD. Hence, further developing cell-permeable viral capsid protein fusions appears worthwhile.

Delivering nucleic acid therapeutics across cell membranes is a significant challenge. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) show promise for siRNA delivery. To improve siRNA delivery and silence a model STAT3 gene, we hypothesized that oleyl acylation to CPPs, specifically (WRH)n, would enhance STAT3 silencing efficiency in breast and ovarian cancer cells. Using Fmoc/tBu solid-phase peptide chemistry, we synthesized, purified, and characterized the oleyl-conjugated (WRH)n (n = 1–4) peptides. The peptide/siRNA complexes were non-cytotoxic at N/P 40 (~20 μM) against MDA-MB-231, MCF-7, SK-OV-3, and HEK-293 cells after 72 h incubation. All peptide/siRNA complexes showed serum stability at N/P ≥ 40. The synthesized conjugates, with a diameter of 3 and oleyl-(WRH)4 showed ~60% and ~75% cellular uptake of siRNA, respectively, in both MDA-MB-231 and SK-OV-3 cells. Western blot analysis of oleyl-(WRH)4 demonstrated effective silencing of the STAT-3 gene, with ~75% silencing in MDA-MB-231 cells and ~45% in SK-OV-3 cells.

Signal transduction and homeostasis are regulated by complex protein interactions in the intracellular environment. Therefore, the transportation of impermeable macromolecules (nucleic acids, proteins, and drugs) that control protein interactions is essential for modulating cell functions and therapeutic applications. However, macromolecule transportation across the cell membrane is not easy because the cell membrane separates the intra/extracellular environments, and the types of molecular transportation are regulated by membrane proteins. Cell-penetrating peptides (CPPs) are expected to be carriers for molecular transport. CPPs can transport macromolecules into cells through endocytosis and direct translocation. The transport mechanism remains largely unclear owing to several possibilities. In this review, we describe the methods for investigating CPP conformation, translocation, and cargo transportation using artificial membranes. We also investigated biomolecular transport across living cell membranes via CPPs. Subsequently, we show not only the biochemical applications but also the synthetic biological applications of CPPs. Finally, recent progress in biomolecule and nanoparticle transportation via CPPs into specific tissues is described from the viewpoint of drug delivery. This review provides the opportunity to discuss the mechanism of biomolecule transportation through these two platforms.

Purpose: Aurein 1.2 (Aur) peptide is known for possessing anticancer characteristics devoid of conventional therapeutics side effects. For improving Aur peptide anticancer functionality, different anticancer peptides were constructed based on Aur peptide through targeting two separate strategies, including (1) sequence-based mutations and (2) adding a cell-penetrating peptide linker. Methods: The study was approached by designing three different analogs of Aur, including (a) Aur mutant (Aurm), (b) Aur with N-terminal polyarginine linker (R5-Aur), and (c) Aurm with R5 (R5-Aurm). Computational molecular dynamics simulations clearly showed higher structural stability of R5-Aur and R5-Aurm compared to Aur, solely. The α-helical properties of R5-Aur and R5-Aurm were protected during 500 ns simulations in water solution while no such structural conservation was seen for Aur in silico. Results: The results of the current study highlight response to one of the main challenges of cancer therapy through selective invasion of Aur to cancer cells without significant involvement of normal cells. This issue was confirmed by different assays, including: MTT assay, flow cytometry, qPCR, and nuclei morphological observations. Furthermore, this study intensifies exploiting in silico approaches for adjusting drug delivery. The results of different assessments on designed peptides reveal an anticancer activity pattern rising from Aur toward Aurm, and R5- Aur, consecutively. Conclusion: The designed structure of Aur shows improved anticancer activity through molecular changes which makes it suggestable for anticancer therapies.

Introduction: Tumor-homing peptides have gained great attention as tools for the development of non-invasive and targeting drug delivery systems (DDS) to minimize drug systemic toxicity and enhance bioavailability. This study aims to improve antitumor targeting in prostate cancer via uploading a drug to a DDS comprised of a cell penetrating peptide decorated with a tumor-homing peptide, PL3. Material and Methods: The DDS was constructed via solid-phase peptide synthesis and then characterized via mass spectrum and high performance liquid chromatography. A cell viability assessment to evaluate its cytotoxicity on both tumor (prostate cancer cells) and normal cells was conducted, while a confocal laser scanning microscope and flow-cytometer were employed to investigate internalization. To inspect the effectiveness of the drug-loaded DDS, a biochemical enzyme inhibition assay on the target enzyme dihydrofolate reductase (DHFR) was performed. ‎ Results and Discussion:‏ The findings supported the succeeded synthesis and loading of the drug into this carrier system and demonstrated its high efficacy in cytotoxic effect and inhibiting DHFR with considerable cellular uptake in prostate cancer cells. Conclusion: The drug was delivered to the target prostate cancer cells by the PL3-functionalized DDS, limiting its localization to tumor cells rather than normal cells. Therefore, the study results highlighted the significance of the DDS in tumor therapy interventions.

Abstract Stroke is a major public health concern worldwide. The lack of effective therapies heightens the need for new therapeutic agents. Previous study identified sirtuin 5 (SIRT5) as a positive regulator of microglia‐induced excessive neuroinflammation following ischemic stroke. Interventions targeting SIRT5 should therefore alleviate neuroinflammation and protect against ischemic stroke. Here, we synthesized a membrane‐permeable peptide specifically bound to SIRT5 through a chaperone‐mediated autophagy targeting motif (Tat‐SIRT5‐CTM) and examined its therapeutic effect in vitro and in vivo. First, in primary microglia, Tat‐SIRT5‐CTM suppressed the binding of SIRT5 with annexin‐A1 (ANXA1), leading to SIRT5 degradation and thus inhibition of SIRT5‐mediated desuccinylation of ANXA1, followed by increased membrane accumulation and secretion of ANXA1. These changes, in turn, alleviated microglia‐induced neuroinflammation. Moreover, following intravenous injection, Tat‐SIRT5‐CTM could efficiently pass through the blood‒brain barrier. Importantly, systemic administration of Tat‐SIRT5‐CTM reduced the brain infarct area and neuronal loss, mitigated neurological deficit scores, and improved long‐term neurological functions in a mouse model of ischemic stroke. Furthermore, no toxicity was observed when high doses Tat‐SIRT5‐CTM were injected into nonischemic mice. Collectively, our study reveals the promising efficacy of the peptide‐directed lysosomal degradation of SIRT5 and suggests it as an effective therapeutic approach for the treatment of ischemic stroke.

[Display omitted] The cell-penetrating peptide penetratin and its analogues shuffle and penetramax have been used as carrier peptides for oral delivery of therapeutic peptides such as insulin. Their mechanism of action for this purpose is not fully understood but is believed to depend on the interactions of the peptide with the cell membrane. In the present study, peptide-liposome interactions were investigated using advanced biophysical techniques including small-angle neutron scattering and fluorescence lifetime imaging microscopy. Liposomes were used as a model system for the cell membrane. All the investigated carrier peptides induced liposome clustering at a specific peptide/lipid ratio. However, distinctively different types of membrane interactions were observed, as the liposome clustering was irreversible for penetratin, but fully or partly reversible for shuffle and penetramax, respectively. All three peptides were found to adsorb to the surface of the lipid bilayers, while only shuffle and penetramax led to shape deformation of the liposomes. Importantly, the peptide interactions did not disrupt the liposomes under any of the investigated conditions, which is advantageous for their application in drug delivery. This detailed insight on peptide-membrane interactions is important for understanding the mechanism of peptide-based excipients and the influence of peptide sequence modifications. [ABSTRACT FROM AUTHOR]

This study aimed to prepare a novel multifunctional magnetic nanoparticle (MNP) with a drugloadable encapsulation and a matrix metalloproteinase (MMP) substrate-modified TAT peptide whose transmembrane ability can be activated in an MMP-rich environment, and to evaluate the uptake of this nanoparticle by cells as well as its cytotoxicity. Nanoparticles were synthesized and modified with TAT or MMPs-TAT peptides. PC-3 cells and RWPE-1 cells were cultured with these nanoparticles at different concentrations, with or without MMP-2 pretreatment, and their uptake rate and toxicity were determined using flow cytometry and fluorescence microscopy. The nanoparticles were characterized with TEM (transmission electron microscopy) and DLS (dynamic light scattering). The diameter of these nanoparticles ranged from 194.5 ± 69.4 nm to 353.1 ± 103.9 nm, which were in the ideal range for application. The cellular uptake of MMPs-TAT-MNPs appeared similar to MNPs, but significantly increased in the presence of MMP-2 pretreatment. MMPs-TAT-MNPs had no significant cytotoxicity at a concentration of = 25 µg/mL. MMPs-TAT-MNPs with MMP-2 substrate modified TAT peptide was successfully prepared, possessed low cytotoxicity loadable cargo, and could be activated in an MMP-2 rich niche. [ABSTRACT FROM AUTHOR]

Antisense peptide nucleic acids (PNAs) inhibit bacterial growth in several infection models. Since PNAs are not spontaneously taken up by bacteria, they are often conjugated to carriers such as cell-penetrating peptides (CPPs) in order to improve translocation. Hydrophobic counterions such as pyrenebutyrate (PyB) have been shown to facilitate translocation of peptides over natural and artificial membranes. In this study, the capability of PyB to support translocation of CPP-coupled antisense PNAs into bacteria was investigated in Streptococcus pyogenes and Streptococcus pneumoniae. PyB enhanced the antimicrobial activity of CPP-conjugated antisense PNAs in S. pyogenes. The most significant effect of PyB was observed in combination with K8-conjugated anti-gyrA PNAs. In contrast, no significant effect of PyB on the antimicrobial activity of CPP-conjugated PNAs in S. pneumoniae was detected. Uptake of K8-FITC into S. pyogenes, Escherichia coli, and Klebsiella pneumoniae could be improved by pre-incubation with PyB, indicating that PyB supports the antimicrobial effect of CPP-antisense PNAs in S. pyogenes by facilitating the translocation of peptides across the bacterial membrane. [ABSTRACT FROM AUTHOR]

Abstract Background Topical anticancer drugs offer a potential therapeutic modality with high compliance for treating cutaneous squamous cell carcinoma (cSCC). However, the existing topical treatments for cSCC are associated with limited penetrating ability to achieve the desired outcome. Therefore, there remains an urgent requirement to develop drugs with efficient anticancer activity suitable for treating cSCC and to overcome the skin physiological barrier to improve the efficiency of drug delivery to the tumor. Results We introduced lycorine (LR) into the topical treatment for cSCC and developed a cell-penetrating peptide (CPP)-modified cationic transfersome gel loaded with lycorine-oleic acid ionic complex (LR-OA) (LR@DTFs-CPP Gel) and investigated its topical therapeutic effects on cSCC. The anti-cSCC effects of LR and skin penetration of LR-OA transfersomes were confirmed. Simultaneously, cationic lipids and modification of R5H3 peptide of the transfersomes further enhanced the permeability of the skin and tumor as well as the effective delivery of LR to tumor cells. Conclusions Topical treatment of cSCC-xenografted nude mice with LR@DTFs-CPP Gel showed effective anticancer properties with high safety. This novel formulation provides novel insights into the treatment and pathogenesis of cSCC.

This study describes the development of two methods for delivering exogenous materials into Euglena gracilis, a unicellular flagellate organism. We report that the use of Pep‐1, a short cell‐penetrating peptide (CPP), or dimethyl sulfoxide (DMSO) can mediate fast and efficient intracellular delivery of exogenous materials into E. gracilis, achieving cellular entry efficiency as high as 70–80%. However, compared with human cells, the penetration of this algal cell with CPP requires a much higher concentration of purified proteins. In addition, upon convenient treatment with DMSO, E. gracilis cells can efficiently adsorb exogenous proteins and DNA with 10% DMSO as the optimal concentration for Euglena cells. Our results provide more options for the E. gracilis transformation ‘toolkit box’ and will facilitate future molecular manipulations of this microalgal organism.

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.