Therapeutic Peptides to Treat Myocardial Ischemia-Reperfusion Injury.

Cardiovascular diseases (CVD) including acute myocardial infarction (AMI) rank first in worldwide mortality and according to the World Health Organization (WHO), they will stay at this rank until 2030. Prompt revascularization of the occluded artery to reperfuse the myocardium is the only recommended treatment (by angioplasty or thrombolysis) to decrease infarct size (IS). However, despite beneficial effects on ischemic lesions, reperfusion leads to ischemia-reperfusion (IR) injury related mainly to apoptosis. Improvement of revascularization techniques and patient care has decreased myocardial infarction (MI) mortality however heart failure (HF) morbidity is increasing, contributing to the cost-intense worldwide HF epidemic. Currently, there is no treatment for reperfusion injury despite promising results in animal models. There is now an obvious need to develop new cardioprotective strategies to decrease morbidity/mortality of CVD, which is increasing due to the aging of the population and the rising prevalence rates of diabetes and obesity. In this review, we will summarize the different therapeutic peptides developed or used focused on the treatment of myocardial IR injury (MIRI). Therapeutic peptides will be presented depending on their interacting mechanisms (apoptosis, necroptosis, and inflammation) reported as playing an important role in reperfusion injury following myocardial ischemia. The search and development of therapeutic peptides have become very active, with increasing numbers of candidates entering clinical trials. Their optimization and their potential application in the treatment of patients with AMI will be discussed.

Tips and Tools to Understand Direct Membrane Translocation of siRNA-Loaded WRAP-Based Nanoparticles.

Cell-penetrating peptide (CPP)-based approaches are excellent method for delivering cell-impermeable compounds/therapeutics such as proteins, antibodies, antisense oligonucleotides, siRNAs, plasmids, and drugs, as covalently or noncovalently conjugated cargo into cells. Nowadays, it is generally accepted that cellular internalization of these CPP-cargoes or CPP-nanoparticles occur via endocytosis-dependent mechanisms or by direct cell translocation.Here, we describe a subset of biophysical and biological methods which can be used to dissect the internalization mechanism of CPPs. Presented protocols and results were shown for the recently developed siRNA-loaded WRAP-based nanoparticles. The rapid and efficient cell delivery of WRAP encapsulated siRNA could be attributed to the main direct cellular translocation of the nanoparticles even if, to some extent, endocytosis-dependent internalization occurred.Deciphering the internalization mechanism is still an important requirement to understand and to optimize the action mode of CPPs or CPP-based nanoparticles as transfection reagents.

WRAP-based nanoparticles for siRNA delivery: a SAR study and a comparison with lipid-based transfection reagents.

Recently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure-activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.

Peptide-Based Nanoparticles for Therapeutic Nucleic Acid Delivery.

Gene therapy offers the possibility to skip, repair, or silence faulty genes or to stimulate the immune system to fight against disease by delivering therapeutic nucleic acids (NAs) to a patient. Compared to other drugs or protein treatments, NA-based therapies have the advantage of being a more universal approach to designing therapies because of the versatility of NA design. NAs (siRNA, pDNA, or mRNA) have great potential for therapeutic applications for an immense number of indications. However, the delivery of these exogenous NAs is still challenging and requires a specific delivery system. In this context, beside other non-viral vectors, cell-penetrating peptides (CPPs) gain more and more interest as delivery systems by forming a variety of nanocomplexes depending on the formulation conditions and the properties of the used CPPs/NAs. In this review, we attempt to cover the most important biophysical and biological aspects of non-viral peptide-based nanoparticles (PBNs) for therapeutic nucleic acid formulations as a delivery system. The most relevant peptides or peptide families forming PBNs in the presence of NAs described since 2015 will be presented. All these PBNs able to deliver NAs in vitro and in vivo have common features, which are characterized by defined formulation conditions in order to obtain PBNs from 60 nm to 150 nm with a homogeneous dispersity (PdI lower than 0.3) and a positive charge between +10 mV and +40 mV.

In Vivo Follow-Up of Gene Inhibition in Solid Tumors Using Peptide-Based Nanoparticles for siRNA Delivery.

Small interfering RNA (siRNA) exhibits a high degree of specificity for targeting selected genes. They are efficient on cells in vitro, but in vivo siRNA therapy remains a challenge for solid tumor treatment as siRNAs display difficulty reaching their intracellular target. The present study was designed to show the in vivo efficiency of a new peptide (WRAP5), able to form peptide-based nanoparticles (PBN) that can deliver siRNA to cancer cells in solid tumors. WRAP5:siRNA nanoparticles targeting firefly luciferase (Fluc) were formulated and assayed on Fluc-expressing U87 glioblastoma cells. The mode of action of WRAP5:siRNA by RNA interference was first confirmed in vitro and then investigated in vivo using a combination of bioluminescent reporter genes. Finally, histological analyses were performed to elucidate the cell specificity of this PBN in the context of brain tumors. In vitro and in vivo results showed efficient knock-down of Fluc expression with no toxicity. WRAP5:siFluc remained in the tumor for at least 10 days in vivo. Messenger RNA (mRNA) analyses indicated a specific decrease in Fluc mRNA without affecting tumor growth. Histological studies identified PBN accumulation in the cytoplasm of tumor cells but also in glial and neuronal cells. Through in vivo molecular imaging, our findings established the proof of concept for specific gene silencing in solid tumors. The evidence generated could be translated into therapy for any specific gene in different types of tumors without cell type specificity but with high molecular specificity.

Deciphering the internalization mechanism of WRAP:siRNA nanoparticles.

Gene silencing mediated by double-stranded small interfering RNA (siRNA) has been widely investigated as a potential therapeutic approach for a variety of diseases and, indeed, the first therapeutic siRNA was approved by the FDA in 2018. As an alternative to the traditional delivery systems for nucleic acids, peptide-based nanoparticles (PBNs) have been applied successfully for siRNA delivery. Recently, we have developed amphipathic cell-penetrating peptides (CPPs), called WRAP allowing a rapid and efficient siRNA delivery into several cell lines at low doses (20 to 50 nM). In this study, using a highly specific gene silencing system, we aimed to elucidate the cellular uptake mechanism of WRAP:siRNA nanoparticles by combining biophysical, biological, confocal and electron microscopy approaches. We demonstrated that WRAP:siRNA complexes remain fully active in the presence of chemical inhibitors of different endosomal pathways suggesting a direct cell membrane translocation mechanism. Leakage studies on lipid vesicles indicated membrane destabilization properties of the nanoparticles and this was supported by the measurement of WRAP:siRNA internalization in dynamin triple-KO cells. However, we also observed some evidences for an endocytosis-dependent cellular internalization. Indeed, nanoparticles co-localized with transferrin, siRNA silencing was inhibited by the scavenger receptor A inhibitor Poly I and nanoparticles encapsulated in vesicles were observed by electron microscopy in U87 cells. In conclusion, we demonstrate here that the efficiency of WRAP:siRNA nanoparticles is mainly based on the use of multiple internalization mechanisms including direct translocation as well as endocytosis-dependent pathways.

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide.

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.

Optimization of peptide-plasmid DNA vectors formulation for gene delivery in cancer therapy exploring design of experiments.

The field of gene therapy still attracts great interest due to its potential therapeutic effect towards the most deadly diseases, such as cancer. For cancer gene therapy to be feasible and viable in a clinical setting, the design and development of a suitable gene delivery system is imperative. Peptide based vectors, in particular, reveal to be promising for therapeutic gene release. Following this, two different peptides, RALA and WRAP5, have been investigated mainly regarding their ability to form complexes with a p53 encoding plasmid (pDNA) with suitable properties for gene delivery. To address this issue, and after an initial screening study focused on the dependence of pDNA complexation capacity with the nitrogen to phosphate groups (N/P) ratio, a design of experiments (DoE) tool has been employed. For each peptide/pDNA system, parameters such as, the buffer pH and the N/P ratio were considered the DoE inputs and the vector size, zeta potential and pDNA complexation capacity (CC) were monitored as DoE outputs. The main goal was to find the optimal experimental conditions to minimize particle sizes, as well as, to maximize the positive surface charges of the formulated nanosystems and maximize the pDNA CC. Through the DoE method applied, the optimal RALA/pDNA and WRAP5/pDNA formulations were revealed and show interesting features related to peptide structure and pDNA complexation ability. This work illustrates the great utility of experimental design tools in optimizing the formulation of peptide/pDNA vectors in a minimum number of experiments providing relevant knowledge for the development of more suitable and efficient gene delivery systems. The new insights achieved on these carriers clearly instigate deeper research on gene therapy.

Peptide-Based Nanoparticles to Rapidly and Efficiently "Wrap 'n Roll" siRNA into Cells.

Delivery of small interfering RNA (siRNA) as a therapeutic tool is limited due to critical obstacles such as the cellular barrier, the negative charges of the siRNA molecule, and its instability in serum. Several siRNA delivery systems have been constructed using cell-penetrating peptides (CPPs) since the CPPs have shown a high potential for oligonucleotide delivery into the cells, especially by forming nanoparticles. In this study, we have developed a new family of short (15mer or 16mer) tryptophan-(W) and arginine-(R) rich Amphipathic Peptides (WRAP) able to form stable nanoparticles and to enroll siRNA molecules into cells. The lead peptides, WRAP1 and WRAP5, form defined nanoparticles smaller than 100 nm as characterized by biophysical methods. Furthermore, they have several benefits as oligonucleotide delivery tools such as the rapid encapsulation of the siRNA, the efficient siRNA delivery in several cell types, and the high gene silencing activity, even in the presence of serum. In conclusion, we have designed a new family of CPPs specifically dedicated for siRNA delivery through nanoparticle formation. Our results indicate that the WRAP family has significant potential for the safe, efficient, and rapid delivery of siRNA for diverse applications.

A retro-inverso cell-penetrating peptide for siRNA delivery.

BACKGROUND: Small interfering RNAs (siRNAs) are powerful tools to control gene expression. However, due to their poor cellular permeability and stability, their therapeutic development requires a specific delivery system. Among them, cell-penetrating peptides (CPP) have been shown to transfer efficiently siRNA inside the cells. Recently we developed amphipathic peptides able to self-assemble with siRNAs as peptide-based nanoparticles and to transfect them into cells. However, despite the great potential of these drug delivery systems, most of them display a low resistance to proteases. RESULTS: Here, we report the development and characterization of a new CPP named RICK corresponding to the retro-inverso form of the CADY-K peptide. We show that RICK conserves the main biophysical features of its L-parental homologue and keeps the ability to associate with siRNA in stable peptide-based nanoparticles. Moreover the RICK:siRNA self-assembly prevents siRNA degradation and induces inhibition of gene expression. CONCLUSIONS: This new approach consists in a promising strategy for future in vivo application, especially for targeted anticancer treatment (e.g. knock-down of cell cycle proteins). Graphical abstract RICK-based nanoparticles: RICK peptides and siRNA self-assemble in peptide-based nanoparticles to penetrate into the cells and to induce target protein knock-down.

PEGylation rate influences peptide-based nanoparticles mediated siRNA delivery in vitro and in vivo.

Small interfering RNAs (siRNAs) present a strong therapeutic potential because of their ability to inhibit the expression of any desired protein. Recently, we developed the retro-inverso amphipathic RICK peptide as novel non-covalent siRNA carrier. This peptide is able to form nanoparticles (NPs) by self-assembling with the siRNA resulting in the fully siRNA protection based on its protease resistant peptide sequence. With regard to an in vivo application, we investigated here the influence of the polyethylene glycol (PEG) grafting to RICK NPs on their in vitro and in vivo siRNA delivery properties. A detailed structural study shows that PEGylation did not alter the NP formation (only decrease in zeta potential) regardless of the used PEGylation rates. Compared to the native RICK:siRNA NPs, low PEGylation rates (≤20%) of the NPs did not influence their cellular internalization capacity as well as their knock-down specificity (over-expressed or endogenous system) in vitro. Because the behavior of PEGylated NPs could differ in their in vivo application, we analyzed the repartition of fluorescent labeled NPs injected at the one-cell stage in zebrafish embryos as well as their pharmacokinetic (PK) profile after administration to mice. After an intra-cardiac injection of the PEGylated NPs, we could clearly determine that 20% PEG-RICK NPs reduce significantly liver and kidney accumulation. NPs with 20% PEGylation constitutes a modular, easy-to-handle drug delivery system which could be adapted to other types of functional moieties to develop safe and biocompatible delivery systems for the clinical application of RNAi-based cancer therapeutics.

Optimisation of vectorisation property: A comparative study for a secondary amphipathic peptide.

RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation and intracellular delivery. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. In this context, the secondary amphipathic peptide CADY has shown to form stable siRNA complexes and to improve their cellular uptake independent of the endosomal pathway. In the present work, we have described the parameters influencing CADY nanoparticle formation (buffers, excipients, presence of serum, etc.), and have followed in details the CPP:siRNA self-assembly. Once optimal conditions were determined, we have compared the ability of seven different CADY analogues to form siRNA-loaded nanoparticles compared to CADY:siRNA. First of all, we were able to show by biophysical methods that structural polymorphism (α-helix) is an important prerequisite for stable nanoparticle formation independently of occurring sequence mutations. Luciferase assays revealed that siRNA complexed to CADY-K (shorter version) shows better knock-down efficiency on Neuro2a-Luc(+) and B16-F10-Luc(+) cells compared to CADY:siRNA. Altogether, CADY-K is an ideal candidate for further application especially with regards to ex vivo or in vivo applications.

Everything you always wanted to know about CADY-mediated siRNA delivery* (* but afraid to ask).

Although siRNA consist in very promising therapeutics, their clinical development is limited by several biological barriers including low cellular permeability, poor stability and lack of tissue specificity. Therefore the Achilles' heel for siRNA-based therapy is directly related to the lack of efficient system to promote their delivery. During the last two decades, cell-penetrating peptides (CPPs) have been widely developed to enhance the cellular delivery of therapeutics. In this context we have elaborated a new strategy based on self-assembling peptide-based nanoparticles. The CADY peptide is a 20-residue secondary amphipathic peptide which is able to spontaneously self associate with siRNA with a strong affinity, by combining both electrostatic and hydrophobic interactions, to form stable nanoparticles. Investigations of both physico-chemical properties and cellular siRNA delivery revealed that the CADY/siRNA complexes were able to enter a wide variety of cell lines by a mechanism independent of any endocytotic pathway. In addition a deeper understanding of the self assembly of CADY molecules around siRNA leads to a "raspberry"-like nanoparticle architecture which provides new perspectives for the CADY/siRNA formulations. Finally the robustness of the biological response infers that peptide-based nanoparticle technology holds a strong promise for therapeutic applications. The present review deals with most of the biophysical characteristics as well as the cellular mechanism and cellular applications of CADY/siRNA nanoparticles.

Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles.

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.

Malaria vaccine candidate: design of a multivalent subunit α-helical coiled coil poly-epitope.

A new strategy for the rapid identification of new malaria antigens based on protein structural motifs was previously described. We identified and evaluated the malaria vaccine potential of fragments of several malaria antigens containing α-helical coiled coil protein motifs. By taking advantage of the relatively short size of these structural fragments, we constructed different poly-epitopes in which 3 or 4 of these segments were joined together via a non-immunogenic linker. Only peptides that are targets of human antibodies with anti-parasite in vitro biological activities were incorporated. One of the constructs, P181, was well recognized by sera and peripheral blood mononuclear cells (PBMC) of adults living in malaria-endemic areas. Affinity purified antigen-specific human antibodies and sera from P181-immunized mice recognised native proteins on malaria-infected erythrocytes in both immunofluorescence and western blot assays. In addition, specific antibodies inhibited parasite development in an antibody dependent cellular inhibition (ADCI) assay. Naturally induced antigen-specific human antibodies were at high titers and associated with clinical protection from malaria in longitudinal follow-up studies in Senegal.

Interactions of amphipathic CPPs with model membranes.

Due to the poor permeability of the plasma membrane, several strategies are designed to enhance the transfer of therapeutics into cells. Over the last 20 years, small peptides called Cell-Penetrating Peptides (CPPs) have been widely developed to improve the cellular delivery of biomolecules. These small peptides derive from protein transduction domains, chimerical constructs, or model sequences. Several CPPs are primary or secondary amphipathic peptides, depending on whether the distribution of their hydrophobic and hydrophilic domains occurs from their amino-acid sequence or through α-helical folding. Most of the CPPs are able to deliver different therapeutics such as nucleic acids or proteins in vitro and in vivo. Although their mechanisms of internalization are varied and controversial, the understanding of the intrinsic features of CPPs is essential for future developments. This chapter describes several protocols for the investigation of biophysical properties of amphipathic CPPs. Surface physics approaches are specifically applied to characterize the interactions of amphipathic peptides with model membranes. Circular dichroism and infra-red spectroscopy allow the identification of their structural state. These methods are exemplified by the analyses of the main biophysical features of the cell-penetrating peptides MPG, Pep-1, and CADY.

Structural polymorphism of non-covalent peptide-based delivery systems: highway to cellular uptake.

During the last two decades, delivery has become a major challenge for the development of new therapeutic molecules for the clinic. Although, several strategies either viral or non viral have been proposed to favor cellular uptake and targeting of therapeutics, only few of them have reach preclinical evaluation. Amongst them, cell-penetrating peptide (CPP) constitutes one of the most promising strategy and has applied for systemic in vivo delivery of a variety of therapeutic molecules. Two CPP-strategies have been described; using peptide carriers either covalently-linked to the cargo or forming non-covalent stable complexes with cargo. Peptide-based nanoparticle delivery system corresponds to small amphipathic peptides able to form stable nanoparticles with either proteins/peptides or nucleic acids and to enter the cell independently of the endosomal pathway. Three families of peptide-based nanoparticle systems; MPG, PEP and CADY have been successfully used for the delivery of various biologically active cargoes both ex vivo and in vivo in several animal models. This review will focus on the mechanism of the peptide-based nanoparticles; PEP, MPG and CADY in a structural and biophysical context. It will also highlight the major parameters associated to particle formation/stabilization and the impact of the carrier structural polymorphism in triggering cellular uptake.

Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells.

Human immunodeficiency virus type 1 (HIV-1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV-1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV-1-infected primary CD4(+) T-cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P(2) molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane-embedded PI(4,5)P(2) only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P(2)-mediated recruitment of cellular proteins. Tat-PI(4,5)P(2) interaction is strictly required for Tat secretion, a process that is very efficient, as approximately 2/3 of Tat are exported by HIV-1-infected cells during their lifespan. The function of extracellular Tat in HIV-1 infection might thus be more significant than earlier thought.

Secondary structure of cell-penetrating peptides controls membrane interaction and insertion.

The clinical use of efficient therapeutic agents is often limited by the poor permeability of the biological membranes. In order to enhance their cell delivery, short amphipathic peptides called cell-penetrating peptides (CPPs) have been intensively developed for the last two decades. CPPs are based either on protein transduction domains, model peptide or chimeric constructs and have been used to deliver cargoes into cells through either covalent or non-covalent strategies. Although several parameters are simultaneously involved in their internalization mechanism, recent focuses on CPPs suggested that structural properties and interactions with membrane phospholipids could play a major role in the cellular uptake mechanism. In the present work, we report a comparative analysis of the structural plasticity of 10 well-known CPPs as well as their ability to interact with phospholipid membranes. We propose a new classification of CPPs based on their structural properties, affinity for phospholipids and internalization pathways already reported in the literature.