Intracellular Delivery of Proteins and Peptides for Therapeutic Purposes

A special issue of Pharmaceutics (ISSN 1999-4923). This special issue belongs to the section "Drug Delivery and Controlled Release".

Deadline for manuscript submissions: closed (31 January 2022) | Viewed by 16273

Special Issue Editor

Department of Biochemistry, Radboud Institute for Molecular Life Sciences (RIMLS), Radboud University Medical Center, Geert Grooteplein 28, 6525 GA Nijmegen, The Netherlands
Interests: intracellular drug delivery; protein engineering; bacterial toxins; tumor-on-a-chip; modeling drug delivery

Special Issue Information

Dear Colleagues,

The molecular targets exploited in targeted therapy have traditionally consisted of two broad categories: extracellular targets, accessible by antibodies and small molecules; and intracellular targets, which until now have only been addressed by small molecules. Intense efforts have been made to make the intracellular target space druggable by peptides and proteins. In recent years, novel delivery strategies for peptides and proteins have been making headway. Through an enhanced specificity profile in comparison to small molecules, many more intracellular targets could potentially be targeted. The nature of the approaches to achieving delivery have been highly diverse, and range from exploiting bacterial-toxin-derived translocation mechanisms to cell-penetrating peptides and nanoparticles. In parallel, approaches that address targets catalytically, either through enzyme delivery or targeted protein degradation, are coming to the fore. Advances in this area could open up numerous therapeutic opportunities in a wide range of disease areas. By making delivery cell-specific, the therapeutic window could be augmented even further.

This Special Issue serves to highlight advances that are being made in the area of intracellular protein and peptide delivery, either by overcoming delivery barriers at the level of the cells and/or the tissue, or by enhancing the effects of delivered peptides or proteins. Strategies that we focus on must show a clear path towards translation into potential drugs. We invite original research papers as well as review articles for this Special Issue.

Dr. Wouter P.R. Verdurmen
Guest Editor

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Keywords

  • targeted therapy
  • protein delivery
  • peptide delivery
  • intracellular delivery
  • enzymes
  • targeted protein degradation
  • protein engineering
  • protein scaffold
  • cell-penetrating peptide
  • bioconjugation

Published Papers (6 papers)

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Research

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22 pages, 4249 KiB  
Article
In Vitro & In Vivo Studies on Identifying and Designing Temporin-1CEh from the Skin Secretion of Rana chensinensis as the Optimised Antibacterial Prototype Drug
by Zhuming Ye, Xiaowei Zhou, Xinping Xi, Yu Zai, Mei Zhou, Xiaoling Chen, Chengbang Ma, Tianbao Chen, Lei Wang and Hang Fai Kwok
Pharmaceutics 2022, 14(3), 604; https://0-doi-org.brum.beds.ac.uk/10.3390/pharmaceutics14030604 - 10 Mar 2022
Cited by 4 | Viewed by 1906
Abstract
Amphibian skin secretion is an ideal source of antimicrobial peptides that are difficult to induce drug resistance to due to their membrane-targeting mechanism as a new treatment scheme. In this study, a natural antimicrobial peptide Temporin-1CEh was identified by molecular cloning and mass [...] Read more.
Amphibian skin secretion is an ideal source of antimicrobial peptides that are difficult to induce drug resistance to due to their membrane-targeting mechanism as a new treatment scheme. In this study, a natural antimicrobial peptide Temporin-1CEh was identified by molecular cloning and mass spectrometry from the skin secretions of the Chinese forest frog (Rana chensinensis). Through the study of the structure and biological activity, it was found that Temporin-1CEh was a helical peptide from the Temporin family, and possessed good anti-Gram-positive bacteria activity through the mechanism of membrane destruction. Seven analogues were further designed to obtain broad-spectrum antimicrobial activity and higher stability in different physiological conditions. The results showed that T1CEh-KKPWW showed potent antibacterial activity with significantly increasing the activity against Gram-negative bacteria in vitro and in vivo with low haemolysis. In addition, T1CEh-KKPWW2 showed high sensitivity to the pH, serum or salts conditions, which applied a branched structure to allow the active units of the peptide to accumulate. Even though the haemolytic activity was increased, the stable antibacterial activity made this novel analogue meet the conditions to become a potential candidate in future antimicrobial and antibiofilm applications. Full article
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11 pages, 1482 KiB  
Article
Spatiotemporal Correlation Spectroscopy Reveals a Protective Effect of Peptide-Based GLP-1 Receptor Agonism against Lipotoxicity on Insulin Granule Dynamics in Primary Human β-Cells
by Gianmarco Ferri, Marta Tesi, Luca Pesce, Marco Bugliani, Francesca Grano, Margherita Occhipinti, Mara Suleiman, Carmela De Luca, Lorella Marselli, Piero Marchetti and Francesco Cardarelli
Pharmaceutics 2021, 13(9), 1403; https://0-doi-org.brum.beds.ac.uk/10.3390/pharmaceutics13091403 - 03 Sep 2021
Cited by 2 | Viewed by 1849
Abstract
Glucagon-like peptide-1 receptor (GLP-1R) agonists are being used for the treatment of type 2 diabetes (T2D) and may have beneficial effects on the pancreatic β-cells. Here, we evaluated the effects of GLP-1R agonism on insulin secretory granule (ISG) dynamics in primary β-cells isolated [...] Read more.
Glucagon-like peptide-1 receptor (GLP-1R) agonists are being used for the treatment of type 2 diabetes (T2D) and may have beneficial effects on the pancreatic β-cells. Here, we evaluated the effects of GLP-1R agonism on insulin secretory granule (ISG) dynamics in primary β-cells isolated from human islets exposed to palmitate-induced lipotoxic stress. Islets cells were exposed for 48 h to 0.5 mM palmitate (hereafter, ‘Palm’) with or without the addition of a GLP-1 agonist, namely 10 nM exendin-4 (hereafter, ‘Ex-4’). Dissociated cells were first transfected with syncollin-EGFP in order to fluorescently mark the ISGs. Then, by applying a recently established spatiotemporal correlation spectroscopy technique, the average structural (i.e., size) and dynamic (i.e., the local diffusivity and mode of motion) properties of ISGs are extracted from a calculated imaging-derived Mean Square Displacement (iMSD) trace. Besides defining the structural/dynamic fingerprint of ISGs in human cells for the first time, iMSD analysis allowed to probe fingerprint variations under selected conditions: namely, it was shown that Palm affects ISGs dynamics in response to acute glucose stimulation by abolishing the ISGs mobilization typically imparted by glucose and, concomitantly, by reducing the extent of ISGs active/directed intracellular movement. By contrast, co-treatment with Ex-4 normalizes ISG dynamics, i.e., re-establish ISG mobilization and ability to perform active transport in response to glucose stimulation. These observations were correlated with standard glucose-stimulated insulin secretion (GSIS), which resulted in being reduced in cells exposed to Palm but preserved in cells concomitantly exposed to 10 nM Ex-4. Our data support the idea that GLP-1R agonism may exert its beneficial effect on human β-cells under metabolic stress by maintaining ISGs’ proper intracellular dynamics. Full article
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13 pages, 4228 KiB  
Article
Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin
by Lukas Becker, Jasleen Singh Badwal, Fabian Brandl, Wouter P. R. Verdurmen and Andreas Plückthun
Pharmaceutics 2021, 13(8), 1285; https://doi.org/10.3390/pharmaceutics13081285 - 18 Aug 2021
Cited by 4 | Viewed by 3772
Abstract
Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously [...] Read more.
Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed. Full article
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22 pages, 4080 KiB  
Article
Experimental Design Based Optimization and Ex Vivo Permeation of Desmopressin Acetate Loaded Elastic Liposomes Using Rat Skin
by Mohammad A. Altamimi, Afzal Hussain, Sultan Alshehri and Syed Sarim Imam
Pharmaceutics 2021, 13(7), 1047; https://0-doi-org.brum.beds.ac.uk/10.3390/pharmaceutics13071047 - 09 Jul 2021
Cited by 6 | Viewed by 2185
Abstract
The study aimed to develop elastic-liposome-based transdermal delivery of desmopressin acetate for enhanced permeation to control enuresis, central diabetes insipidus, and traumatic injury. Elastic liposomes (ELs)-loaded desmopressin acetate was prepared, optimized, and evaluated for improved transdermal permeation profiles using rat skin. Full factorial [...] Read more.
The study aimed to develop elastic-liposome-based transdermal delivery of desmopressin acetate for enhanced permeation to control enuresis, central diabetes insipidus, and traumatic injury. Elastic liposomes (ELs)-loaded desmopressin acetate was prepared, optimized, and evaluated for improved transdermal permeation profiles using rat skin. Full factorial design with independent factors (X1 for lipid and X2 for surfactant) at three levels was used against four responses (Y1, Y2, Y3, and Y4) (dependent variables). Formulations were characterized for vesicle size, polydispersity index (PDI), zeta potential, % entrapment efficiency (% EE), in vitro drug release, in vitro hemolysis potential, ex vivo drug permeation and drug deposition (DD), and ex vivo vesicle–skin interaction using scanning electron microscopy studies. The optimized formulation ODEL1 based on desirability function was found to have vesicle size, % EE, % DR, and permeation flux values of 118.7 nm, 78.9%, 75.1%, and 5.3 µg/h·cm2, respectively, which were close to predicted values. In vitro release profiles indicated slow and sustained delivery. Permeation flux values of ODEL1 and ODEL2 were 5.3 and 3.1 µg/h·cm2, respectively, which are 7.5- and 4.4-fold higher as compared to DS (0.71 µg/h·cm2). The obtained flux was relatively higher than the clinical target value of the drug for therapeutic efficacy. Moreover, the DD value of ODEL1 was significantly higher than ODEL2 and DS. Hemocompatibility study confirmed safety concerns. Finally, vesicle–skin interaction corroborated mechanistic views of permeation through rat skin. Conclusively, the transdermal delivery may be a suitable alternative to oral and nasal delivery to treat nocturnal enuresis, central diabetes insipidus, hemophilia A and von Willebrand’s disease, and any traumatic injuries. Full article
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17 pages, 4716 KiB  
Article
A Computational Investigation of In Vivo Cytosolic Protein Delivery for Cancer Therapy
by Camilo Torres, Simon Dumas, Valentina Palacio-Castañeda, Stéphanie Descroix, Roland Brock and Wouter P. R. Verdurmen
Pharmaceutics 2021, 13(4), 562; https://0-doi-org.brum.beds.ac.uk/10.3390/pharmaceutics13040562 - 15 Apr 2021
Cited by 5 | Viewed by 1955
Abstract
The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing [...] Read more.
The ability to specifically block or degrade cytosolic targets using therapeutic proteins would bring tremendous therapeutic opportunities in cancer therapy. Over the last few years, significant progress has been made with respect to tissue targeting, cytosolic delivery, and catalytic inactivation of targets, placing this aim within reach. Here, we developed a mathematical model specifically built for the evaluation of approaches towards cytosolic protein delivery, involving all steps from systemic administration to translocation into the cytosol and target engagement. Focusing on solid cancer tissues, we utilized the model to investigate the effects of microvascular permeability, receptor affinity, the cellular density of targeted receptors, as well as the mode of activity (blocking/degradation) on therapeutic potential. Our analyses provide guidance for the rational optimization of protein design for enhanced activity and highlight the importance of tuning the receptor affinity as a function of receptor density as well as the receptor internalization rate. Furthermore, we provide quantitative insights into how enzymatic cargoes can enhance the distribution, extent, and duration of therapeutic activity, already at very low catalytic rates. Our results illustrate that with current protein engineering approaches, the goal of delivery of cytosolic delivery of proteins for therapeutic effects is well within reach. Full article
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Review

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32 pages, 4856 KiB  
Review
Delivery of Various Cargos into Cancer Cells and Tissues via Cell-Penetrating Peptides: A Review of the Last Decade
by Alireza Shoari, Raheleh Tooyserkani, Mehdi Tahmasebi and Dennis W. P. M. Löwik
Pharmaceutics 2021, 13(9), 1391; https://0-doi-org.brum.beds.ac.uk/10.3390/pharmaceutics13091391 - 02 Sep 2021
Cited by 25 | Viewed by 3479
Abstract
Cell-penetrating peptides (CPPs), also known as protein transduction domains, are a class of diverse amino acid sequences with the ability to cross cellular membranes. CPPs can deliver several bioactive cargos, including proteins, peptides, nucleic acids and chemotherapeutics, into cells. Ever since their discovery, [...] Read more.
Cell-penetrating peptides (CPPs), also known as protein transduction domains, are a class of diverse amino acid sequences with the ability to cross cellular membranes. CPPs can deliver several bioactive cargos, including proteins, peptides, nucleic acids and chemotherapeutics, into cells. Ever since their discovery, synthetic and natural CPPs have been utilized in therapeutics delivery, gene editing and cell imaging in fundamental research and clinical experiments. Over the years, CPPs have gained significant attention due to their low cytotoxicity and high transduction efficacy. In the last decade, multiple investigations demonstrated the potential of CPPs as carriers for the delivery of therapeutics to treat various types of cancer. Besides their remarkable efficacy owing to fast and efficient delivery, a crucial benefit of CPP-based cancer treatments is delivering anticancer agents selectively, rather than mediating toxicities toward normal tissues. To obtain a higher therapeutic index and to improve cell and tissue selectivity, CPP-cargo constructions can also be complexed with other agents such as nanocarriers and liposomes to obtain encouraging outcomes. This review summarizes various types of CPPs conjugated to anticancer cargos. Furthermore, we present a brief history of CPP utilization as delivery systems for anticancer agents in the last decade and evaluate several reports on the applications of CPPs in basic research and preclinical studies. Full article
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