Activity Of Cell-penetrating Peptides Research Articles

Elucidating the Impact of Payload Conjugation on the Cell-Penetrating Efficiency of the Endosomal Escape Peptide dfTAT: Implications for Future Designs for CPP-Based Delivery Systems.

Cell-penetrating peptides (CPPs) are promising tools for the intracellular delivery of various biological payloads. However, the impact of payload conjugation on the cell-penetrating activity of CPPs is poorly understood. This study focused on dfTAT, a modified version of the HIV-TAT peptide with enhanced endosomal escape activity, to explore how different payloads affect its cell-penetrating activity. We systematically examined dfTAT conjugated with the SnoopTag/SnoopCatcher pair and found that while smaller payloads such as short peptides do not significantly impair dfTAT's cell delivery activity, larger payloads markedly reduce both its endocytic uptake and endosomal escape efficiency. Our results highlight the role of the payload size and bulk in limiting CPP-mediated delivery. While further research is needed to understand the molecular underpinnings of these effects, our findings pave the way for developing more effective CPP-based delivery systems.

Impact of the Endosomal Escape Activity of Cell-Penetrating Peptides on the Endocytic Pathway.

Cell-penetrating peptides (CPPs) are routinely used for the delivery of macromolecules into live human cells. To enter the cytosolic space of cells, CPPs typically permeabilize the membrane of endosomes. In turn, several approaches have been developed to increase the endosomal membrane permeation activity of CPPs so as to improve delivery efficiencies. The endocytic pathway is, however, important in maintaining cellular homeostasis, and understanding how endosomal permeation impacts cells is now critical to define the general utility of CPPs. Herein, we investigate how CPP-based delivery protocols affect the endocytic network. We detect that, in some cases, cell penetration induces the activation of Chmp1b, Galectin-3, and TFEB, which are components of endosomal repair, organelle clearance, and biogenesis pathways, respectively. We also detect that cellular delivery modulates endocytosis and endocytic proteolysis. Remarkably, a multimeric analogue of the prototypical CPP TAT permeabilizes endosomes efficiently without inducing membrane damage responses. These results challenge the notion that reagents that make endosomes leaky are generally toxic. Instead, our data indicates that it is possible to enter cells with minimal deleterious effects.

KELM-CPPpred: Kernel Extreme Learning Machine Based Prediction Model for Cell-Penetrating Peptides.

Cell-penetrating peptides (CPPs) facilitate the transport of pharmacologically active molecules, such as plasmid DNA, short interfering RNA, nanoparticles, and small peptides. The accurate identification of new and unique CPPs is the initial step to gain insight into CPP activity. Experiments can provide detailed insight into the cell-penetration property of CPPs. However, the synthesis and identification of CPPs through wet-lab experiments is both resource- and time-expensive. Therefore, the development of an efficient prediction tool is essential for the identification of unique CPP prior to experiments. To this end, we developed a kernel extreme learning machine (KELM) based CPP prediction model called KELM-CPPpred. The main data set used in this study consists of 408 CPPs and an equal number of non-CPPs. The input features, used to train the proposed prediction model, include amino acid composition, dipeptide amino acid composition, pseudo amino acid composition, and the motif-based hybrid features. We further used an independent data set to validate the proposed model. In addition, we have also tested the prediction accuracy of KELM-CPPpred models with the existing artificial neural network (ANN), random forest (RF), and support vector machine (SVM) approaches on respective benchmark data sets used in the previous studies. Empirical tests showed that KELM-CPPpred outperformed existing prediction approaches based on SVM, RF, and ANN. We developed a web interface named KELM-CPPpred, which is freely available at http://sairam.people.iitgn.ac.in/KELM-CPPpred.html.

β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides.

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

Abstract 3708: Targeted delivery of doxorubicin to glioblastomas by thermally sensitive polypeptides

Abstract Glioblastoma (GBM) ranks among the most common, aggressive, and least curable cancers due to a strong tendency for intracranial dissemination, high proliferation potential and inherent tumor resistance to radiation or chemotherapy. Current treatments face the critical challenge of adverse treatment effects in normal tissue within the brain itself and also when the agent cannot adequately penetrate the blood brain barrier (BBB) to reach the tumor microenvironment. We have developed a system to selectively deliver chemotherapeutic doxorubicin to GBM tumors. This carrier is based on elastin-like polypeptide (ELP), which is soluble at physiological temperatures but undergoes a phase transition and accumulates at tumor sites with externally applied, mild (40-41°C) hyperthermia. The CPP-ELP-Dox conjugate consists of a cell penetrating peptide (CPP), which facilitates transcytosis through the BBB and cell entry, and the 6-maleimidocaproyl hydrazone derivative of doxorubicin (Dox) at the C-terminus of ELP. The acid-sensitive hydrazone linker ensures release of Dox in the lysosomes/endosomes after cellular uptake of the drug conjugate. We have shown that CPP-ELP-Dox effectively inhibits cell proliferation in three GBM cell lines; GBM6, D54, and U251 with IC50 values of 250 nM, 60nM, and 30nM, respectively. Both the free drug and CPP-ELP-Dox conjugate exhibited similar in vitro cytotoxicity, although their subcellular localization was considerably different. The Dox conjugate was mainly dispersed in the cytoplasm, while free drug was largely dispersed in the cytoplasm but also had partial nuclear accumulation. The accumulation of free Dox in GBM cells was measured by flow cytometry to determine whether the cytotoxic activity of free Dox or CPP- ELP-Dox is related to intracellular Dox levels. Intracellular Dox concentration was increased in the CPP-ELP-Dox cells compared to that in the free Dox cells, which positively correlates with cytotoxic activity. Flow cytometry was also used to quantitatively investigate apoptosis of GBM cells. After incubation for 24 h, the total apoptosis percentage of free Dox was 1.5% in GBM6, 6% in D54, and 7% in U251 cells. However, the total apoptosis rates of CPP-ELP-Dox treated cells were 5%, 12% and 5% in GBM6, D54, and U251 cells respectively, which confirms increased apoptosis compared to free Dox treated cells. Cell cycle analysis of GBM cells treated with free Dox showed enhanced arrest of the cells in G1 phase, while there was increased percentage arrest of cells in G2 phase in cells treated with CPP-ELP-Dox.In summary, our findings demonstrate that CPP-ELP-Dox effectively kills GBM cells. Development of such a drug carrier has the potential to greatly improve current therapeutic approaches for GBM by increasing the specificity and efficacy of treatment and reducing cytotoxicity in normal tissues. Citation Format: Sonja Dragojevic, Rebecca B. Mackey, Felix Kratz, Drazen Raucher. Targeted delivery of doxorubicin to glioblastomas by thermally sensitive polypeptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3708.

Cell-penetrating peptides derived from Clostridium difficile TcdB2 and a related large clostridial toxin

Clostridium difficile TcdB (2366 amino acid residues) is an intracellular bacterial toxin that binds to cells and enters the cytosol where it glucosylates small GTPases. In the current study, we examined a putative cell entry region of TcdB (amino acid residues 1753-1851) for short sequences that function as cell-penetrating peptides (CPPs). To screen for TcdB-derived CPPs, a panel of synthetic peptides was tested for the ability to enhance transferrin (Tf) association with cells. Four candidate CPPs were discovered, and further study on one peptide (PepB2) pinpointed an asparagine residue necessary for CPP activity. PepB2 mediated the cell entry of a wide variety of molecules including dextran, streptavidin, microspheres, and lentivirus particles. Of note, this uptake was dramatically reduced in the presence of the Na+/H+ exchange blocker and micropinocytosis inhibitor amiloride, suggesting that PepB2 invokes macropinocytosis. Moreover, we found that PepB2 had more efficient cell-penetrating activity than several other well-known CPPs (TAT, penetratin, Pep-1, and TP10). Finally, Tf assay-based screening of peptides derived from two other large clostridial toxins, TcdA and TcsL, uncovered two new TcdA-derived CPPs. In conclusion, we have identified six CPPs from large clostridial toxins and have demonstrated the ability of PepB2 to promote cell association and entry of several molecules through a putative fluid-phase macropinocytotic mechanism.

Antibacterial properties of Latarcin 1 derived cell-penetrating peptides

Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) share certain physicochemical parameters such as amphipathicity, hydrophobicity, cationicity and pI, due to which these two groups of peptides also exhibit overlapping functional characteristics. In our current work, we have evaluated antimicrobial properties of cell-penetrating peptides derived from Latarcin1. Latarcin derived peptide (LDP) exhibited antimicrobial activity against representative microorganisms tested and bactericidal effect against methicillin resistant Staphylococcus aureus (MRSA), which was used as model organism of study in the present work. However, LDP exhibited cytotoxicity against HeLa cells. Further, nuclear localization sequence (NLS) was fused to LDP and interestingly, LDP-NLS showed antimicrobial effect against bacteria, showed bactericidal effect against MRSA and also did not exhibit cytotoxicity in HeLa cells till the highest concentrations tested. Thus, our results inferred that fusion of NLS to LDP significantly reduced cytotoxicity of LDP against HeLa cells (Ponnappan and Chugh, 2017) and exhibited significantly higher cell-penetrating activity in MRSA in comparison to LDP alone. Consolidated results of uptake assays, time-kill assays and PI membrane damage assays show that LDP killed MRSA mainly by membrane damage, where as LDP-NLS might have intracellular targets. Owing to its cell-penetrating activity in HeLa cells and antimicrobial activity against MRSA, LDP-NLS efficiently inhibited intracellular infection of MRSA in HeLa cells as observed in invasion assays. Hence, our results suggest that LDP-NLS is a dual action peptide with AMP and CPP activity and could be potential candidate as peptide antibiotic and drug delivery vector in both mammalian and bacterial cells.

Cell-Penetrating Peptides: Design Strategies beyond Primary Structure and Amphipathicity.

Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies.

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic-Targeting and Imaging.

Matrix metalloproteinases (MMPs) activatable imaging probe has been explored for tumor detection. However, activation of the probe is mainly done in the extracellular space without intracellular uptake of the probe for more sensitivity. Although cell-penetrating peptides (CPPs) have been demonstrated to enable intracellular delivery of the imaging probe, they nevertheless encounter off-target delivery of the cargos to normal tissues. Herein, we have developed a dual MMP-2-activatable and tumor cell-permeable magnetic nanoprobe to simultaneously achieve selective and intracellular tumor imaging. This novel imaging probe was constructed by self-assembling a hexahistidine-tagged (His-tagged) fluorescent fusion protein chimera and nickel ferrite nanoparticles via a chelation mechanism. The His-tagged fluorescent protein chimera consisted of a red fluorescent protein mCherry that acted as the fluorophore, the low-molecular-weight protamine peptide as the CPP, and the MMP-2 cleavage sequence fused with the hexahistidine tag, whereas the nickel ferrite nanoparticles functioned as the His-tagged protein binder and also the fluorescent quencher. Both in vitro and in vivo results revealed that this imaging probe would not only remain nonpermeable to normal tissues, thereby offsetting the nonselective cellular uptake, but was also suppressed of fluorescent signals during magnetic tumor-targeting in the circulation, primarily because of the masking of the CPP activity and quenching of the fluorophore by the associated NiFe2O4 nanoparticles. However, these properties were recovered or "turned on" by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. In this regard, the presented tumor-activatable and cell-permeable system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. Its impact is therefore significant, far-reaching, and wide-spread.

An unexpected cell-penetrating peptide from Bothrops jararaca venom identified through a novel size exclusion chromatography screening.

Efficient drug delivery systems are currently one of the greatest challenges in pharmacokinetics, and the transposition of the gap between in vitro candidate molecule and in vivo test drug is, sometimes, poles apart. In this sense, the cell-penetrating peptides (CPP) may be the bridge uniting these worlds. Here, we describe a technique to rapidly identify unlabeled CPPs after incubation with liposomes, based on commercial desalting (size exclusion) columns and liquid chromatography-MS/MS, for peptide de novo sequencing. Using this approach, we found it possible to identify one new CPP - interestingly, a classical bradykinin-potentiating peptide - in the peptide-rich low molecular mass fraction of the Bothrops jararaca venom, which was also able to penetrate live cell membranes, as confirmed by classical approaches employing fluorescence-labeled analogues of this CPP. Moreover, both the labeled and unlabeled CPPs caused no metabolic, cell-cycle or morphologic alterations, proving to be unmistakably cargo deliverers and not drugs themselves. In sum, we have developed and validated a method for screening label-free peptides for CPP activity, regardless of their biological origin, which could lead to the identification of new and more efficient drug delivery systems. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Activation of Cell-Penetrating Peptides with Ionpair−π Interactions and Fluorophiles

In this report, we elaborate on two new concepts to activate arginine-rich cell-penetrating peptides (CPPs). Early on, we have argued that repulsion-driven ion-pairing interactions with anionic lipids account for their ability to move across hydrophobic cell membranes and that hydrophobic anions such as pyrenebutyrate can accelerate this process to kinetically outcompete endosomal capture. The original explanation that the high activity of pyrenebutyrate might originate from ionpair-π interactions between CPP and activator implied that replacement of the π-basic pyrene with polarized push-pull aromatics should afford more powerful CPP activators. To elaborate on this hypothesis, we prepared a small collection of anionic amphiphiles that could recognize cations by ionpair-π interactions. Consistent with theoretical predictions, we find that parallel but not antiparallel ionpair-π interactions afford operational CPP activators in model membranes and cells. The alternative suggestion that the high activity of pyrenebutyrate might originate from self-assembly in membranes was explored with perfluorinated fatty acids. Their fluorophilicity was expected to promote self-assembly in membranes, while their high acidity should prevent charge neutralization in response to self-assembly, i.e., generate repulsion-driven ion-pairing interactions. Consistent with these expectations, we find that perfluorinated fatty acids are powerful CPP activators in HeLa cells but not in model membranes. These findings support parallel ionpair-π interactions and repulsion-driven ion pairing with self-assembled fluorophiles as innovative concepts to activate CPPs. These results also add much corroborative support for counterion-mediated uptake as the productive mode of action of arginine-rich CPPs.

Ion Pair-π Interactions.

We report that anion-π and cation-π interactions can occur on the same aromatic surface. Interactions of this type are referred to as ion pair-π interactions. Their existence, nature, and significance are elaborated in the context of spectral tuning, ion binding in solution, and activation of cell-penetrating peptides. The origin of spectral tuning by ion pair-π interactions is unraveled with energy-minimized excited-state structures: The solvent- and pH-independent red shift of absorption and emission of push-pull fluorophores originates from antiparallel ion pair-π attraction to their polarized excited state. In contrast, the complementary parallel ion pair-π repulsion is spectroscopically irrelevant, in part because of charge neutralization by intriguing proton and electron transfers on excited push-pull surfaces. With time-resolved fluorescence measurements, very important differences between antiparallel and parallel ion pair-π interactions are identified and quantitatively dissected from interference by aggregation and ion pair dissociation. Contributions from hydrogen bonding, proton transfer, π-π interactions, chromophore twisting, ion pairing, and self-assembly are systematically addressed and eliminated by concise structural modifications. Ion-exchange studies in solution, activation of cell-penetrating peptides in vesicles, and computational analysis all imply that the situation in the ground state is complementary to spectral tuning in the excited state; i.e., parallel rather than antiparallel ion pair-π interactions are preferred, despite repulsion from the push-pull dipole. The overall quite complete picture of ion pair-π interactions provided by these remarkably coherent yet complex results is expected to attract attention throughout the multiple disciplines of chemistry involved.

Abstract 4391: Evaluation of tumor targeting activity by elastin like polypeptide containing cell penetrating peptide and IL-4 receptor targeting peptide

Abstract The tumor specific internalization of therapeutics drug is been a great importance for cancer therapy. Targeted macromolecular carrier has been used as drug cargos since it improve the cellular uptake of cytotoxic drug in the tumor, while preventing cellular uptake by healthy tissues. Because of favorable pharmokinetic profile, elastin like polypeptide (ELPs) has been used as macromolecular drug carrier for cancer therapy. Fusion with cell-penetrating peptides (CPPs) such as TAT, assist easy penetration and internalization of macromolecular drug conjugates into wide variety of tumor types. For therapeutic peptide to be more penetrable and more effective towards specific tumor tissue, here we have designed a chimeric polypeptide cargo, TAT-AP1-ELP containing TAT, IL-4 receptor (IL4R) targeting peptide AP-1and thermal sensitive elastin-like polypeptides (ELPs). Then it was successfully expressed and purified from E coli using inverse temperature cycling method. Cellular uptake and intracellular localization of TAT-AP1-ELP was monitored through flow cytometry and confocal microscopy. Flow cytometry analysis data shown that both TAT-AP1-ELP and TAT-ELP showed various extends of penetration ability to different cancer cells but not to normal cells. Further a greater cellular uptake was observed in IL4R highly expressing cancer cells when treated with TAT-AP1-ELP compared to TAT-ELP at 37°C in dose-dependent manner. Confocal imaging verified the better cellular accumulation of TAT-AP1-ELP than TAT-ELP in IL4R highly expressing cancer cells. Importantly, the FNR 675-labeled TAT-AP1-ELP polymer showed better tumor accumulation in MDA-MB 231 xenograft mouse model. Thus in this study we demonstrate the improved activity of CPP, fused with targeting modalities in order to enhance intra tumor accumulation of therapeutic peptides. Citation Format: Young-Jin Lee, Vijaya Sarangthem, Yun-Jae Kim, Kuen Hur, Byung-Heon Lee, Rang Woon Park. Evaluation of tumor targeting activity by elastin like polypeptide containing cell penetrating peptide and IL-4 receptor targeting peptide. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4391. doi:10.1158/1538-7445.AM2015-4391

15 years of ATTEMPTS: A macromolecular drug delivery system based on the CPP-mediated intracellular drug delivery and antibody targeting

Traditionally, any drug intended for combating the tumor would distribute profoundly to other organs and tissues as lack of targeting specificity, thus resulting in limited therapeutic effects toward the tumor but severe drug-induced toxic side effects. To prevail over this obstacle of drug-induced systemic toxicity, a novel approach termed “ATTEMPTS” (antibody targeted triggered electrically modified prodrug type strategy) was designed, which directly introduces both of the targeting and prodrug features onto the protein drugs. The ATTEMPTS system is composed of the antibody targeting component consisting of antibodies linked with heparin, and the cell penetrating peptide (CPP) modified drug component. The two components mentioned above self-assembled into a tight complex via the charge to charge interaction between the anionic heparin and cationic CPP. Once accumulated at the targeting site, the CPP modified drug is released from the blockage by a second triggering agent, while remaining inactive in the circulation during tumor targeting thus aborting its effect on normal tissues. We utilized the heparin-induced inhibition on the cell-penetrating activity of CPP to create the prodrug feature, and subsequently the protamine-induced reversal of heparin inhibition to resume cell transduction of the protein drug via the CPP function. Our approach is the first known system to overcome this selectivity issue, enabling CPP-mediated cellular drug delivery to be practically applicable clinically. In this review, we thoroughly discussed the historical and novel progress of the “ATTEMPTS” system.

Exploration of the Design Principles of a Cell-Penetrating Bicylic Peptide Scaffold

Cell-penetrating peptides (CPPs) possess the capacity to induce cell entry of themselves and attached molecular cargo, either by endocytosis or by direct translocation. Conformational constraints have been described as one means to increase the activity of CPPs, especially for direct crossing of the plasma membrane. Here, we explored the structure-activity relationship of bicyclic peptides for cell entry. These peptides may be considered minimal analogues of naturally occurring oligocyclic peptide toxins and are a promising scaffold for the design of bioactive molecules. Increasing numbers of arginine residues that are primarily contributing to cell-penetrating activity were introduced either into the cycles, or as stretches outside the cycles, at both ends or at one end only. In addition, we probed for the impact of negatively charged residues on activity for both patterns of arginine substitution. Uptake was investigated in HeLa cells by flow cytometry and confocal microscopy. Overall, uptake efficiency showed a positive correlation with the number of arginine residues. The subcellular distribution was indicative of endocytic uptake. One linear stretch of arginines coupled outside the bicycle was as effective in promoting uptake as substituting the same number of arginines inside the bicycles. However, the internally substituted analogues were more sensitive to the presence of negatively charged residues. For a given bicyclic peptide, uptake was more effective than for the linear counterpart. Introduction of histidine and tryptophans further increased uptake efficiency to comparable levels as that of nonaarginine despite the larger size of the bicyclic backbone. The results demonstrate that both arginine clustering and spatial constraints are uptake-promoting structural principles, an observation that gives freedom in the introduction of cell-penetrating capacity to structurally constrained scaffolds.

An integrated cross-linking-MS approach to investigate cell penetrating peptides interacting partners

Cell penetrating peptides (CPPs) are attracting attention because of their ability to deliver biologically active molecules into cells. On their way they can interact with membrane and intracellular proteins. To fully understand and improve CPP efficiency as drug delivery tools, their partners need to be identified. To investigate CPP-protein complexes, chemical cross-linking coupled to mass spectrometry is a relevant method. With this aim, we developed an original approach based on two parallel strategies, an intact complex analysis and a bottom-up one, to have a global characterization of the cross-linked complexes composition as well as a detailed mapping of the interaction zones. Biological significanceThe robust and efficient cross-linking-MS workflow presented here can easily be adapted to any CPP-protein interacting system and could thus contribute to a better understanding of CPPs activity as cell-specific drug delivery tools. We validated the relevancy of this cross-linking-MS approach with two biologically active CPPs, (R/W)9 and (R/W)16, and two interacting protein partners, actin and albumin, previously reported using isothermal titration calorimetry (ITC) and NMR. Cross-linking-MS results obtained on these previous studies allowed us to go further by providing a detailed mapping of the interaction zones. The identified interaction zones between actin and CPPs (R/W)9 and (R/W)16 are biologically meaningful. Two cross-linked zones [46–57] and [202–210] of actin are indeed involved in the modulation of its dynamics. Moreover, [46–57] domain has also been described as one interaction domain for thymosin β4 whose actin binding can be displaced by competition with (R/W)16 (NMR experiments).

Activation of cell-penetrating peptides by disulfide bridge formation of truncated precursors

A small library of oligoarginine peptides equipped with terminal cysteines was studied with respect to their cell-penetrating properties. The peptides themselves were inactive but gained the ability to enter cells upon extension of their sequence through disulfide bridge formation.

The enhanced membrane interaction and perturbation of a cell penetrating peptide in the presence of anionic lipids: Toward an understanding of its selectivity for cancer cells

Cell penetrating peptides (CPPs) are usually short, highly cationic peptides that are capable of crossing the cell membrane and transport cargos of varied size and nature in cells by energy- and receptor-independent mechanisms. An additional potential is the newly discovered anti-tumor activity of certain CPPs, including RW16 (RRWRRWWRRWWRRWRR) which is derived from penetratin and is investigated here. The use of CPPs in therapeutics, diagnosis and potential application as anti-tumor agents increases the necessity of understanding their mode of action, a subject yet not totally understood. With this in mind, the membrane interaction and perturbation mechanisms of RW16 with both zwitterionic and anionic lipid model systems (used as representative models of healthy vs tumor cells) were investigated using a large panoply of biophysical techniques. It was shown that RW16 autoassociates and that its oligomerization state highly influences its membrane interaction. Overall a stronger association and perturbation of anionic membranes was observed, especially in the presence of oligomeric peptide, when compared to zwitterionic ones. This might explain, at least in part, the anti-tumor activity and so the selective interaction with cancer cells whose membranes have been shown to be especially anionic. Hydrophobic contacts between the peptide and lipids were also shown to play an important role in the interaction. That probably results from the tryptophan insertion into the fatty acid lipid area following a peptide flip after the first electrostatic recognition. A model is presented that reflects the ensemble of results.

Digital Switching of Local Arginine Density in a Genetically Encoded Self-Assembled Polypeptide Nanoparticle Controls Cellular Uptake

Cell-penetrating peptides (CPPs) are a class of molecules that enable efficient internalization of a wide variety of cargo in diverse cell types, making them desirable for delivery of anticancer drugs to solid tumors. For CPPs to be useful, it is important to be able to turn their function on in response to an external trigger that can be spatially localized in vivo. Here we describe an approach to turning on CPP function by modulation of the local density of arginine (Arg) residues by temperature-triggered micelle assembly of diblock copolymer elastin-like polypeptides (ELP(BC)s). A greater than 8-fold increase in cellular uptake occurs when Arg residues are presented on the corona of ELP(BC) micelles, as compared to the same ELP(BC) at a temperature in which it is a soluble unimer. This approach is the first to demonstrate digital 'off-on' control of CPP activity by an extrinsic thermal trigger in a clinically relevant temperature range by modulation of the interfacial density of Arg residues on the exterior of a nanoparticle.

A Peptide-based Vector for Efficient Gene Transfer In Vitro and In Vivo

Finding suitable nonviral delivery vehicles for nucleic acid-based therapeutics is a landmark goal in gene therapy. Cell-penetrating peptides (CPPs) are one class of delivery vectors that has been exploited for this purpose. However, since CPPs use endocytosis to enter cells, a large fraction of peptides remain trapped in endosomes. We have previously reported that stearylation of amphipathic CPPs, such as transportan 10 (TP10), dramatically increases transfection of oligonucleotides in vitro partially by promoting endosomal escape. Therefore, we aimed to evaluate whether stearyl-TP10 could be used for the delivery of plasmids as well. Our results demonstrate that stearyl-TP10 forms stable nanoparticles with plasmids that efficiently enter different cell-types in a ubiquitous manner, including primary cells, resulting in significantly higher gene expression levels than when using stearyl-Arg9 or unmodified CPPs. In fact, the transfection efficacy of stearyl-TP10 almost reached the levels of Lipofectamine 2000 (LF2000), however, without any of the observed lipofection-associated toxicities. Most importantly, stearyl-TP10/plasmid nanoparticles are nonimmunogenic, mediate efficient gene delivery in vivo, when administrated intramuscularly (i.m.) or intradermally (i.d.) without any associated toxicity in mice.