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Cells
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Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies
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Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies

A special issue of Cells (ISSN 2073-4409).

Deadline for manuscript submissions: closed (25 March 2023) | Viewed by 19158

Special Issue Editors

Dr. Alberto Vitali

E-Mail Website1 Website2
Guest Editor
Consiglio Nazionale delle Ricerche, Rome, Italy
Interests: biologically active peptides (in motility and tissue regeneration); antimicrobial peptides; peptide–protein interaction; protein–peptide purification and structural; characterization; recombinant protein expression and purification; proteomics and peptidomics; nanodrug delivery systems
Special Issues, Collections and Topics in MDPI journals
Dr. Alessandro Gori

E-Mail Website
Guest Editor
Consiglio Nazionale delle Ricerche, Rome, Italy
Interests: peptide chemistry; bioactive peptides; membrane-binding peptides; supramolecular assemblies; nanodelivery

Special Issue Information

Dear colleagues,

Cell-penetrating peptides (CPPs) are defined as peptides usually containing less than 30 residues and characterized by a cationic and amphipathic character, which show a remarkable capability to penetrate membranes’ lipid bilayer. These peculiar features confer to these molecules the ability to be internalized within prokaryotic and eukaryotic cells without damaging or killing them. Penetratin derived from the Drosophila Antennapedia homeodomain and TAT-HIV represent the first progenitors of this class of peptides; since then, a large plethora of CPPs have been discovered from natural sources or generated by rational design. Thanks to their characteristics, they are employed as powerful tools for delivering diverse molecules across cellular membranes, including natural products, other peptides, proteins, nucleic acids, etc. CPPs are also used to carry many types of drugs and nanosystems (nanoparticles, liposomes etc.) for diagnostic and therapeutic applications. Overall, this field is still rich with opportunities and open to ideas and applications yet to be uncovered by researchers with different backgrounds. An open challenge is, for instance, the full comprehension of the molecular and supramolecular mechanisms underlying the internalization of such peptides, which will be of great importance to broaden the application perspectives of CPPs, for example, toward selective organelle/cell compartment targeting.

The aim of this Special Issue of Cells is to provide a panorama on the recent findings regarding the functional, structural characterization of new CPPs both from natural and rationale design sources, the use of CPPs entailing unusual or non-natural amino acids or with peculiar structures (e.g., cyclic peptides), the application of CPPs in targeted drug delivery and imaging, and mechanisms underlying internalization.

For these reasons, we warmly invite expert scientists with their original unpublished new findings and reviews to contribute to this topic.

Dr. Alberto Vitali
Dr. Alessandro Gori
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • cell-penetrating peptides
  • cargo proteins
  • cell-targeted delivery
  • membrane translocation
  • cellular uptake process
  • endocytosis
  • tumor-penetrating peptide
  • peptidomimetics

Published Papers (7 papers)

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Research

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14 pages, 3791 KiB  
Article
Interaction of Arginine-Rich Cell-Penetrating Peptides with an Artificial Neuronal Membrane
by Piotr Mucha, Emilia Sikorska, Piotr Rekowski and Jarosław Ruczyński
Cells 2022, 11(10), 1638; https://doi.org/10.3390/cells11101638 - 13 May 2022
Cited by 3 | Viewed by 2196
Abstract
Arginine-rich cell-penetrating peptides (RRCPPs) exhibit intrinsic neuroprotective effects on neurons injured by acute ischemic stroke. Conformational properties, interaction, and the ability to penetrate the neural membrane are critical for the neuroprotective effects of RRCCPs. In this study, we applied circular dichroism (CD) spectroscopy [...] Read more.
Arginine-rich cell-penetrating peptides (RRCPPs) exhibit intrinsic neuroprotective effects on neurons injured by acute ischemic stroke. Conformational properties, interaction, and the ability to penetrate the neural membrane are critical for the neuroprotective effects of RRCCPs. In this study, we applied circular dichroism (CD) spectroscopy and coarse-grained molecular dynamics (CG MD) simulations to investigate the interactions of two RRCPPs, Tat(49–57)-NH2 (arginine-rich motif of Tat HIV-1 protein) and PTD4 (a less basic Ala-scan analog of the Tat peptide), with an artificial neuronal membrane (ANM). CD spectra showed that in an aqueous environment, such as phosphate-buffered saline, the peptides mostly adopted a random coil (PTD4) or a polyproline type II helical (Tat(49–57)-NH2) conformation. On the other hand, in the hydrophobic environment of the ANM liposomes, the peptides showed moderate conformational changes, especially around 200 nm, as indicated by CD curves. The changes induced by the liposomes were slightly more significant in the PTD4 peptide. However, the nature of the conformational changes could not be clearly defined. CG MD simulations showed that the peptides are quickly attracted to the neuronal lipid bilayer and bind preferentially to monosialotetrahexosylganglioside (DPG1) molecules. However, the peptides did not penetrate the membrane even at increasing concentrations. This suggests that the energy barrier required to break the strong peptide–lipid electrostatic interactions was not exceeded in the simulated models. The obtained results show a correlation between the potential of mean force parameter and a peptide’s cell membrane-penetrating ability and neuroprotective properties. Full article
(This article belongs to the Special Issue Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies)
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15 pages, 2214 KiB  
Article
How Cargo Identity Alters the Uptake of Cell-Penetrating Peptide (CPP)/Cargo Complexes: A Study on the Effect of Net Cargo Charge and Length
by Hannah C. Hymel, Alireza Rahnama, Olivia M. Sanchez, Dong Liu, Ted J. Gauthier and Adam T. Melvin
Cells 2022, 11(7), 1195; https://doi.org/10.3390/cells11071195 - 01 Apr 2022
Cited by 6 | Viewed by 2602
Abstract
Cell-penetrating peptides (CPPs) have emerged as a powerful tool for the delivery of otherwise impermeable cargoes into intact cells. Recent efforts to improve the delivery capability of peptides have mainly focused on the identity of the CPP; however, there is evidence that the [...] Read more.
Cell-penetrating peptides (CPPs) have emerged as a powerful tool for the delivery of otherwise impermeable cargoes into intact cells. Recent efforts to improve the delivery capability of peptides have mainly focused on the identity of the CPP; however, there is evidence that the identity of the cargo itself affects the uptake. The goal of this work was to investigate how the characteristics of a peptide cargo, including net charge and length, either enhance or diminish the internalization efficiency of the CPP/cargo complex. A small library of CPP/cargo complexes were synthesized consisting of structured and unstructured CPPs with cargoes of net positive, negative, or neutral charge and lengths of 4 or 8 amino acids. Cargoes with a net positive charge were found to enhance the overall uptake of the complexes while net neutral and negatively charged cargoes diminished uptake. Conversely, the net length of the cargo had no significant effect on uptake of the CPP/cargo complexes. Microcopy images confirmed the increased uptake of the positively charged cargoes; however, an increase in punctate regions with the addition of a cargo was also observed. The effects of the net positively charged cargoes were confirmed with both structured and unstructured CPPs, which demonstrated similar trends of an increase in uptake with the addition of positively charged residues. These findings demonstrate that the net charge of cargoes impacts the uptake of the complex, which can be considered in the future when designing peptide-based reporters or therapeutics. Full article
(This article belongs to the Special Issue Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies)
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28 pages, 7387 KiB  
Article
Amphiphilic Cell-Penetrating Peptides Containing Natural and Unnatural Amino Acids as Drug Delivery Agents
by David Salehi, Saghar Mozaffari, Khalid Zoghebi, Sandeep Lohan, Dindyal Mandal, Rakesh K. Tiwari and Keykavous Parang
Cells 2022, 11(7), 1156; https://doi.org/10.3390/cells11071156 - 29 Mar 2022
Cited by 8 | Viewed by 4451
Abstract
A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as [...] Read more.
A series of cyclic peptides, [(DipR)(WR)4], [(DipR)2(WR)3], [(DipR)3(WR)2], [(DipR)4(WR)], and [DipR]5, and their linear counterparts containing arginine (R) as positively charged residues and tryptophan (W) or diphenylalanine (Dip) as hydrophobic residues, were synthesized and evaluated for their molecular transporter efficiency. The in vitro cytotoxicity of the synthesized peptides was determined in human epithelial ovary adenocarcinoma cells (SK-OV-3), human lymphoblast peripheral blood cells (CCRF-CEM), human embryonic epithelial kidney healthy cells (HEK-293), human epithelial mammary gland adenocarcinoma cells (MDA-MB-468), pig epithelial kidney normal cells (LLC-PK1), and human epithelial fibroblast uterine sarcoma cells (MES-SA). A concentration of 5–10 µM and 3 h incubation were selected in uptake studies. The cellular uptake of a fluorescent-labeled phosphopeptide, stavudine, lamivudine, emtricitabine, and siRNA was determined in the presence of peptides via flow cytometry. Among the peptides, [DipR]5 (10 µM) was found to be the most efficient transporter and significantly improved the uptake of F’-GpYEEI, i.e., by approximately 130-fold after 3 h incubation in CCRF-CEM cells. Confocal microscopy further confirmed the improved delivery of fluorescent-labeled [DipR]5 (F’-[K(DipR)5]) alone and F’-GpYEEI in the presence of [DipR]5 in MDA-MB-231 cells. The uptake of fluorescent-labeled siRNA (F’-siRNA) in the presence of [DipR]5 with N/P ratios of 10 and 20 was found to be 30- and 50-fold higher, respectively, compared with the cells exposed to F’-siRNA alone. The presence of endocytosis inhibitors, i.e., nystatin, chlorpromazine, chloroquine, and methyl β-cyclodextrin, did not completely inhibit the cellular uptake of F’-[K(DipR)5] alone or F’-GpYEEI in the presence of [DipR]5, suggesting that a combination of mechanisms contributes to uptake. Circular dichroism was utilized to determine the secondary structure, while transmission electron microscopy was used to evaluate the particle sizes and morphology of the peptides. The data suggest the remarkable membrane transporter property of [DipR]5 for improving the delivery of various small molecules and cell-impermeable negatively charged molecules (e.g., siRNA and phosphopeptide). Full article
(This article belongs to the Special Issue Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies)
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22 pages, 4714 KiB  
Article
[(WR)8WKβA]-Doxorubicin Conjugate: A Delivery System to Overcome Multi-Drug Resistance against Doxorubicin
by Khalid Zoghebi, Hamidreza Montazeri Aliabadi, Rakesh Kumar Tiwari and Keykavous Parang
Cells 2022, 11(2), 301; https://doi.org/10.3390/cells11020301 - 16 Jan 2022
Cited by 9 | Viewed by 2940
Abstract
Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR]9 containing alternate tryptophan (W) and arginine [...] Read more.
Doxorubicin (Dox) is an anthracycline chemotherapeutic agent used to treat breast, leukemia, and lymphoma malignancies. However, cardiotoxicity and inherent acquired resistance are major drawbacks, limiting its clinical application. We have previously shown that cyclic peptide [WR]9 containing alternate tryptophan (W) and arginine (R) residues acts as an efficient molecular transporter. An amphiphilic cyclic peptide containing a lysine (K) residue and alternative W and R was conjugated through a free side chain amino group with Dox via a glutarate linker to afford [(WR)8WKβA]-Dox conjugate. Antiproliferative assays were performed in different cancer cell lines using the conjugate and the corresponding physical mixture of the peptide and Dox to evaluate the effectiveness of synthesized conjugate compared to the parent drug alone. [(WR)8WKβA]-Dox conjugate showed higher antiproliferative activity at 10 µM and 5 µM than Dox alone at 5 μM. The conjugate inhibited the cell viability of ovarian adenocarcinoma (SK-OV-3) by 59% and the triple-negative breast cancer cells MDA-MB-231 and MCF-7 by 71% and 77%, respectively, at a concentration of 5 μM after 72 h of incubation. In contrast, Dox inhibited the proliferation of SK-OV-3, MDA-MB-231, and MCF-7 by 35%, 63%, and 57%, respectively. Furthermore, [(WR)8WKβA]-Dox conjugate (5 µM) inhibited the cell viability of Dox-resistant cells (MES-SA/MX2) by 92%, while the viability of cells incubated with free Dox was only 15% at 5 μM. Confocal microscopy images confirmed the ability of both Dox conjugate and the physical mixture of the peptide with the drug to deliver Dox through an endocytosis-independent pathway, as the uptake was not inhibited in the presence of endocytosis inhibitors. The stability of Dox conjugate was observed at different time intervals using analytical HPLC when the conjugate was incubated with 25% human serum. Half-life (t1/2) for [(WR)8WKβA]-Dox conjugate was (∼6 h), and more than 80% of the conjugate was degraded at 12 h. The release of free Dox was assessed intracellularly using the CCRF-CEM cell line. The experiment demonstrated that approximately 100% of free Dox was released from the conjugate intracellularly within 72 h. These data confirm the ability of the cyclic cell-penetrating peptide containing tryptophan and arginine residues as an efficient tool for delivery of Dox and for overcoming resistance to it. Full article
(This article belongs to the Special Issue Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies)
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Review

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29 pages, 2335 KiB  
Review
Effect of the Lipid Landscape on the Efficacy of Cell-Penetrating Peptides
by Florina Zakany, István M. Mándity, Zoltan Varga, Gyorgy Panyi, Peter Nagy and Tamas Kovacs
Cells 2023, 12(13), 1700; https://doi.org/10.3390/cells12131700 - 23 Jun 2023
Cited by 3 | Viewed by 1788
Abstract
Every cell biological textbook teaches us that the main role of the plasma membrane is to separate cells from their neighborhood to allow for a controlled composition of the intracellular space. The mostly hydrophobic nature of the cell membrane presents an impenetrable barrier [...] Read more.
Every cell biological textbook teaches us that the main role of the plasma membrane is to separate cells from their neighborhood to allow for a controlled composition of the intracellular space. The mostly hydrophobic nature of the cell membrane presents an impenetrable barrier for most hydrophilic molecules larger than 1 kDa. On the other hand, cell-penetrating peptides (CPPs) are capable of traversing this barrier without compromising membrane integrity, and they can do so on their own or coupled to cargos. Coupling biologically and medically relevant cargos to CPPs holds great promise of delivering membrane-impermeable drugs into cells. If the cargo is able to interact with certain cell types, uptake of the CPP–drug complex can be tailored to be cell-type-specific. Besides outlining the major membrane penetration pathways of CPPs, this review is aimed at deciphering how properties of the membrane influence the uptake mechanisms of CPPs. By summarizing an extensive body of experimental evidence, we argue that a more ordered, less flexible membrane structure, often present in the very diseases planned to be treated with CPPs, decreases their cellular uptake. These correlations are not only relevant for understanding the cellular biology of CPPs, but also for rationally improving their value in translational or clinical applications. Full article
(This article belongs to the Special Issue Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies)
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37 pages, 1421 KiB  
Review
Cell-Penetrating Peptides as Valuable Tools for Nose-to-Brain Delivery of Biological Drugs
by Lisa Benedetta De Martini, Claudia Sulmona, Liliana Brambilla and Daniela Rossi
Cells 2023, 12(12), 1643; https://doi.org/10.3390/cells12121643 - 16 Jun 2023
Cited by 6 | Viewed by 2240
Abstract
Due to their high specificity toward the target and their low toxicity, biological drugs have been successfully employed in a wide range of therapeutic areas. It is yet to be mentioned that biologics exhibit unfavorable pharmacokinetic properties, are susceptible to degradation by endogenous [...] Read more.
Due to their high specificity toward the target and their low toxicity, biological drugs have been successfully employed in a wide range of therapeutic areas. It is yet to be mentioned that biologics exhibit unfavorable pharmacokinetic properties, are susceptible to degradation by endogenous enzymes, and cannot penetrate biological barriers such as the blood–brain barrier (i.e., the major impediment to reaching the central nervous system (CNS)). Attempts to overcome these issues have been made by exploiting the intracerebroventricular and intrathecal routes of administration. The invasiveness and impracticality of these procedures has, however, prompted the development of novel drug delivery strategies including the intranasal route of administration. This represents a non-invasive way to achieve the CNS, reducing systemic exposure. Nonetheless, biotherapeutics strive to penetrate the nasal epithelium, raising the possibility that direct delivery to the nervous system may not be straightforward. To maximize the advantages of the intranasal route, new approaches have been proposed including the use of cell-penetrating peptides (CPPs) and CPP-functionalized nanosystems. This review aims at describing the most impactful attempts in using CPPs as carriers for the nose-to-brain delivery of biologics by analyzing their positive and negative aspects. Full article
(This article belongs to the Special Issue Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies)
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Other

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10 pages, 2284 KiB  
Brief Report
Validation of AAC-11-Derived Peptide Anti-Tumor Activity in a Single Graft Sézary Patient-Derived Xenograft Mouse Model
by Justine Habault, Nicolas Thonnart, Caroline Ram-Wolff, Martine Bagot, Armand Bensussan, Jean-Luc Poyet and Anne Marie-Cardine
Cells 2022, 11(19), 2933; https://doi.org/10.3390/cells11192933 - 20 Sep 2022
Cited by 4 | Viewed by 1391
Abstract
Sézary syndrome (SS) is an aggressive cutaneous T cell lymphoma with poor prognosis mainly characterized by the expansion of a tumor CD4+ T cell clone in both skin and blood. So far, the development of new therapeutic strategies has been hindered by [...] Read more.
Sézary syndrome (SS) is an aggressive cutaneous T cell lymphoma with poor prognosis mainly characterized by the expansion of a tumor CD4+ T cell clone in both skin and blood. So far, the development of new therapeutic strategies has been hindered by a lack of reproducible in vivo models closely reflecting patients’ clinical features. We developed an SS murine model consisting of the intravenous injection of Sézary patients’ PBMC, together with a mixture of interleukins, in NOD-SCID-gamma mice. Thirty-four to fifty days after injection, mice showed skin disorders similar to that observed in patients, with the detection of epidermis thickening and dermal tumor T cell infiltrates. Although experimental variability was observed, Sézary cells could be tracked in the blood stream, confirming that our model could efficiently exhibit both skin and blood involvement. Using this model, we evaluated the therapeutic potential of RT39, a cell-penetrating peptide derived from the survival protein anti-apoptosis clone 11 (AAC-11), that we previously characterized as specifically inducing apoptosis of Sézary patients’ malignant clone ex vivo. Systemic administration of RT39 led to cutaneous tumor T cells depletion, demonstrating efficient malignant cells’ targeting and a favorable safety profile. These preclinical data confirmed that RT39 might be an innovative therapeutic tool for Sézary syndrome. Full article
(This article belongs to the Special Issue Cell-Penetrating Peptides: From Basic Research to Application in Human Therapies)
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