N-terminal modification of an LAH4-derived peptide increases mRNA delivery in the presence of serum.

The recently developed mRNA-based coronavirus SARS-CoV-2 vaccines highlighted the great therapeutic potential of the mRNA technology. Although the lipid nanoparticles used for the delivery of the mRNA are very efficient, they showed, in some cases, the induction of side effects as well as the production of antibodies directed against particle components. Thus, the development of alternative delivery systems is of great interest in the pursuit of more effective mRNA treatments. In the present work, we evaluated the mRNA transfection capacities of a series of cationic histidine-rich amphipathic peptides derived from LAH4. We found that while the LAH4-A1 peptide was an efficient carrier for mRNA, its activity was highly serum sensitive. Interestingly, modification of this cell penetrating peptide at the N-terminus with two tyrosines or with salicylic acid allowed to confer serum resistance to the carrier.

Photoactivatable Liposomes for Blue to Deep Red Light-Activated Surface Drug Release: Application to Controlled Delivery of the Antitumoral Drug Melphalan.

Liposome-based nanoparticles able to release, via a photolytic reaction, a payload anchored at the surface of the phospholipid bilayer were prepared. The liposome formulation strategy uses an original drug-conjugated blue light-sensitive photoactivatable coumarinyl linker. This is based on an efficient blue light-sensitive photolabile protecting group modified by a lipid anchor, which enables its incorporation into liposomes, leading to blue to green light-sensitive nanoparticles. In addition, the formulated liposomes were doped with triplet-triplet annihilation upconverting organic chromophores (red to blue light) in order to prepare red light sensitive liposomes able to release a payload, by upconversion-assisted photolysis. Those light-activatable liposomes were used to demonstrate that direct blue or green light photolysis or red light TTA-UC-assisted drug photolysis can effectively photorelease a drug payload (Melphalan) and kill tumor cells in vitro after photoactivation.

Design of a new cell penetrating peptide for DNA, siRNA and mRNA delivery.

BACKGROUND: Delivery systems, including peptide-based ones, that destabilize endosomes in a pH-dependent manner are increasingly used to deliver cargoes of therapeutic interest, such as nucleic acids and proteins into mammalian cells. METHODS: The negatively charged amphipathic alpha-helicoidal forming peptide named HELP (Helical Erythrocyte Lysing Peptide) is a derivative from the bee venom melittin and was shown to have a pH-dependent activity with the highest lytic activity at pH 5.0 at the same time as becoming inactive when the pH is increased. The present study aimed to determine whether replacement in the HELP peptide of the glutamic acid residues by histidines, for which the protonation state is sensitive to the pH changes that occur during endosomal acidification, can transform this fusogenic peptide into a carrier able to deliver different nucleic acids into mammalian cells. RESULTS: The resulting HELP-4H peptide displays high plasmid DNA, small interfering RNA and mRNA delivery capabilities. Importantly, in contrast to other cationic peptides, its transfection activity was only marginally affected by the presence of serum. Using circular dichroism, we found that acidic pH did not induce significant conformational changes for HELP-4H. CONCLUSIONS: In summary, we were able to develop a new cationic histidine rich peptide able to efficiently deliver various nucleic acids into cells.

Design of Oligourea-Based Foldamers with Antibacterial and Antifungal Activities.

There is an urgent need to develop new therapeutic strategies to fight the emergence of multidrug resistant bacteria. Many antimicrobial peptides (AMPs) have been identified and characterized, but clinical translation has been limited partly due to their structural instability and degradability in physiological environments. The use of unnatural backbones leading to foldamers can generate peptidomimetics with improved properties and conformational stability. We recently reported the successful design of urea-based eukaryotic cell-penetrating foldamers (CPFs). Since cell-penetrating peptides and AMPs generally share many common features, we prepared new sequences derived from CPFs by varying the distribution of histidine- and arginine-type residues at the surface of the oligourea helix, and evaluated their activity on both Gram-positive and Gram-negative bacteria as well as on fungi. In addition, we prepared and tested new amphiphilic block cofoldamers consisting of an oligourea and a peptide segment whereby polar and charged residues are located in the peptide segment and more hydrophobic residues in the oligourea segment. Several foldamer sequences were found to display potent antibacterial activities even in the presence of 50% serum. Importantly, we show that these urea-based foldamers also possess promising antifungal properties.

Hybrid Cell-Penetrating Foldamer with Superior Intracellular Delivery Properties and Serum Stability.

Sequence specific molecules with high folding ability (i.e., foldamers) can be used to precisely control the distribution and projection of side chains in space and have recently been introduced as tailored systems for delivering nucleic acids into cells. Designed oligourea sequences with an amphipathic distribution of Arg- and His-type residues were shown to form tight complexes with plasmid DNA, and to effectively promote the release of DNA from the endosomes. Herein, we report the synthesis of new cell-penetrating foldamer sequences in which the foldamer segment is conjugated via a reducible disulfide bond to a ligand that binds cell-surface expressed nucleoproteins with the idea that this system could facilitate both assemblies with nucleic acids and cell entry. This new system was evaluated for delivery of DNA in several cell lines and was found to compare favorably with all comparators tested (DOTAP and b-PEI as well as a number of known cell penetrating peptides) in various cell lines and particularly in culture medium containing up to 50% of serum. These results suggest that this dual molecular platform which is long lasting and noncytotoxic could be of practical use for in vivo applications.

Molecular Determinants of Vectofusin-1 and Its Derivatives for the Enhancement of Lentivirally Mediated Gene Transfer into Hematopoietic Stem/Progenitor Cells.

Gene delivery into hCD34+ hematopoietic stem/progenitor cells (HSPCs) using human immunodeficiency virus, type 1-derived lentiviral vectors (LVs) has several promising therapeutic applications. Numerous clinical trials are currently underway. However, the efficiency, safety, and cost of LV gene therapy could be ameliorated by enhancing target cell transduction levels and reducing the amount of LV used on the cells. Several transduction enhancers already exist, such as fibronectin fragments or cationic compounds. Recently, we discovered Vectofusin-1, a new transduction enhancer, also called LAH4-A4, a short histidine-rich amphipathic peptide derived from the LAH4 family of DNA transfection agents. Vectofusin-1 enhances the infectivity of lentiviral and γ-retroviral vectors pseudotyped with various envelope glycoproteins. In this study, we compared a family of Vectofusin-1 isomers and showed that Vectofusin-1 remains the lead peptide for HSPC transduction enhancement with LVs pseudotyped with vesicular stomatitis virus glycoproteins and also with modified gibbon ape leukemia virus glycoproteins. By comparing the capacity of numerous Vectofusin-1 variants to promote the modified gibbon ape leukemia virus glycoprotein-pseudotyped lentiviral vector infectivity of HSPCs, the lysine residues on the N-terminal extremity of Vectofusin-1, a hydrophilic angle of 140° formed by the histidine residues in the Schiffer-Edmundson helical wheel representation, hydrophobic residues consisting of leucine were all found to be essential and helped to define a minimal active sequence. The data also show that the critical determinants necessary for lentiviral transduction enhancement are partially different from those necessary for efficient antibiotic or DNA transfection activity of LAH4 derivatives. In conclusion, these results help to decipher the action mechanism of Vectofusin-1 in the context of hCD34+ cell-based gene therapy.

Glycoproteomics Reveals Decorin Peptides With Anti-Myostatin Activity in Human Atrial Fibrillation.

BACKGROUND: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). METHODS: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. RESULTS: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. CONCLUSIONS: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.

Histidine-rich designer peptides of the LAH4 family promote cell delivery of a multitude of cargo.

The histidine-rich designer peptides of the LAH4 family exhibit potent antimicrobial, transfection, transduction and cell-penetrating properties. They form non-covalent complexes with their cargo, which often carry a negative overall charge at pH 7.4 and include a large range of molecules and structures such as oligonucleotides, including siRNA and DNA, peptides, proteins, nanodots and adeno-associated viruses. These complexes are thought to enter the cells through an endosomal pathway where the acidification of the organelle is essential for efficient endosomal escape. Biophysical measurements indicate that, upon acidification, almost half the peptides are released from DNA cargo, leading to the suggestion of a self-promoted uptake mechanism where the liberated peptides lyse the endosomal membranes. LAH4 derivatives also help in cellular transduction using lentiviruses. Here, we compare the DNA transfection activities of LAH4 derivatives, which vary in overall charge and/or the composition in the hydrophobic core region. In addition, LAH4 is shown to mediate the transport of functional ß-galactosidase, a large tetrameric protein of about 0.5 MDa, into the cell interior. Interestingly, the LAH1 peptide efficiently imports this protein, while it is inefficient during DNA transfection assays. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Inhibition of the myostatin/Smad signaling pathway by short decorin-derived peptides.

Myostatin, also known as growth differentiation factor 8, is a member of the transforming growth factor-beta superfamily that has been shown to play a key role in the regulation of the skeletal muscle mass. Indeed, while myostatin deletion or loss of function induces muscle hypertrophy, its overexpression or systemic administration causes muscle atrophy. Since myostatin blockade is effective in increasing skeletal muscle mass, myostatin inhibitors have been actively sought after. Decorin, a member of the small leucine-rich proteoglycan family is a metalloprotein that was previously shown to bind and inactivate myostatin in a zinc-dependent manner. Furthermore, the myostatin-binding site has been shown to be located in the decorin N-terminal domain. In the present study, we investigated the anti-myostatin activity of short and soluble fragments of decorin. Our results indicate that the murine decorin peptides DCN48-71 and 42-65 are sufficient for inactivating myostatin in vitro. Moreover, we show that the interaction of mDCN48-71 to myostatin is strictly zinc-dependent. Binding of myostatin to activin type II receptor results in the phosphorylation of Smad2/3. Addition of the decorin peptide 48-71 decreased in a dose-dependent manner the myostatin-induced phosphorylation of Smad2 demonstrating thereby that the peptide inhibits the activation of the Smad signaling pathway. Finally, we found that mDCN48-71 displays a specificity towards myostatin, since it does not inhibit other members of the transforming growth factor-beta family.

A cell-penetrating foldamer with a bioreducible linkage for intracellular delivery of DNA.

Despite significant advances in foldamer chemistry, tailored delivery systems based on foldamer architectures, which provide a high level of control over secondary structure, are curiously rare among non-viral technologies for transporting nucleic acids into cells. A potent pH-responsive, bioreducible cell-penetrating foldamer (CPF) was developed through covalent dimerization of a short (8-mer) amphipathic oligourea sequence bearing histidine-type units. This CPF exhibits a high capacity to assemble with pDNA and mediates efficient delivery of nucleic acids into the cell. Furthermore, it does not adversely affect cellular viability and was shown to compare favorably with a cognate peptide transfection agent based on His-rich sequences.

Triplet-Triplet Annihilation Upconversion-Based Photolysis: Applications in Photopharmacology.

The emerging field of photopharmacology is a promising chemobiological methodology for optical control of drug activities that could ultimately solve the off-target toxicity outside the disease location of many drugs for the treatment of a given pathology. The use of photolytic reactions looks very attractive for a light-activated drug release but requires to develop photolytic reactions sensitive to red or near-infrared light excitation for better tissue penetration. This review will present the concepts of triplet-triplet annihilation upconversion-based photolysis and their recent in vivo applications for light-induced drug delivery using photoactivatable nanoparticles.

Reduction of Pro-Inflammatory Markers in RAW264.7 Macrophages by Polyethylenimines.

The physiological problem of chronic inflammation and its associated pathologies attract ongoing attention with regard to methods for their control. Current systemic pharmacological treatments present problematic side effects. Thus, the possibility of new anti-inflammatory compounds with differing mechanisms of action or biophysical properties is enticing. Cationic polymers, with their ability to act as carriers for other molecules or to form bio-compatible materials, present one such possibility. Although not well described, several polycations such as chitosan and polyarginine, have displayed anti-inflammatory properties. The present work shows the ubiquitous laboratory transfection reagent, polyethylenimine (PEI) and more specifically low molecular weight branched PEI (B-PEI) as also possessing such properties. Using a RAW264.7 murine cell line macrophage as an inflammation model, it is found the B-PEI 700 Da as being capable of reducing the production of several pro-inflammatory molecules induced by the endotoxin lipopolysaccharide. Although further studies are required for elucidation of its mechanisms, the revelation that such a common lab reagent may present these effects has wide-ranging implications, as well as an abundance of possibilities.

Control of pH responsive peptide self-association during endocytosis is required for effective gene transfer.

Cationic amphipathic histidine rich peptides demonstrate differential nucleic acid binding capabilities at neutral and acidic pH and adopt conformations at acidic pH that enable interaction with endosomal membranes, their subsequent disordering and facilitate entry of cargo to the cell cytosol. To better understand the relative contributions of each stage in the process and consequently the structural requirements of pH responsive peptides for optimal nucleic acid transfer, we used biophysical methods to dissect the series of events that occur during endosomal acidification. Far-UV circular dichroism was used to characterise the solution conformation of a series of peptides, containing either four or six histidine residues, designed to respond at differing pH while a novel application of near-UV circular dichroism was used to determine the binding affinities of the peptides for both DNA and siRNA. The peptide induced disordering of neutral and anionic membranes was investigated using (2)H solid-state NMR. While each of these parameters models key stages in the nucleic acid delivery process and all were affected by increasing the histidine content of the peptide, the effect of a more acidic pH response on peptide self-association was most notable and identified as the most important barrier to further enhancing nucleic acid delivery. Further, the results indicate that Coulombic interactions between the histidine residues modulate protonation and subsequent conformational transitions required for peptide mediated gene transfer activity and are an important factor to consider in future peptide design.

Click Chemistry for Liposome Surface Modification.

Click chemistry, and particularly azide-alkyne cycloaddition, represents one of the principal bioconjugation strategies that can be used to conveniently attach various ligands to the surface of preformed liposomes. This efficient and chemoselective reaction involves a Cu(I)-catalyzed azide-alkyne cycloaddition which can be performed under mild experimental conditions in aqueous media. Here we describe the application of a model click reaction to the conjugation, in a single step, of unprotected α-1-thiomannosyl ligands, functionalized with an azide group, to liposomes containing a terminal alkyne-functionalized lipid anchor. Excellent coupling yields were obtained in the presence of bathophenanthrolinedisulphonate, a water-soluble copper-ion chelator, acting as catalyst. No vesicle leakage was triggered by this conjugation reaction, and the coupled mannose ligands were exposed at the surface of the liposomes. The major limitation of Cu(I)-catalyzed click reactions is that this type of conjugation is restricted to liposomes made of saturated (phospho)lipids. To circumvent this constraint, an example of alternate copper-free azide-alkyne click reaction has been developed, and it was applied to the anchoring of a biotin moiety that was fully functional and could be therefore quantified. Molecular tools and results are presented here.

Red Light-Responsive Upconverting Nanoparticles for Quantitative and Controlled Release of a Coumarin-Based Prodrug.

Photolytic reactions allow the optical control of the liberation of biological effectors by photolabile protecting groups. The development of versatile technologies enabling the use of deep-red or NIR light excitation still represents a challenging issue, in particular for light-induced drug release (e.g., light-induced prodrug activation). Here, light-sensitive biocompatible lipid nanocapsules able to liberate an antitumoral drug through photolysis are presented. It is demonstrated that original photon upconverting nanoparticles (LNC-UCs) chemically conjugated to a coumarin-based photocleavable linker can quantitatively and efficiently release a drug by upconversion luminescence-assisted photolysis using a deep-red excitation wavelength. In addition, it is also able to demonstrate that such nanoparticles are stable in the dark, without any drug leakage in the absence of light. These findings open new avenues to specifically liberate diverse drugs using deep-red or NIR excitations for future therapeutic applications in nanomedicine.

Targeted Anticancer Agent with Original Mode of Action Prepared by Supramolecular Assembly of Antibody Oligonucleotide Conjugates and Cationic Nanoparticles.

Despite their clinical success, Antibody-Drug Conjugates (ADCs) are still limited to the delivery of a handful of cytotoxic small-molecule payloads. Adaptation of this successful format to the delivery of alternative types of cytotoxic payloads is of high interest in the search for novel anticancer treatments. Herein, we considered that the inherent toxicity of cationic nanoparticles (cNP), which limits their use as oligonucleotide delivery systems, could be turned into an opportunity to access a new family of toxic payloads. We complexed anti-HER2 antibody-oligonucleotide conjugates (AOC) with cytotoxic cationic polydiacetylenic micelles to obtain Antibody-Toxic-Nanoparticles Conjugates (ATNPs) and studied their physicochemical properties, as well as their bioactivity in both in vitro and in vivo HER2 models. After optimising their AOC/cNP ratio, the small (73 nm) HER2-targeting ATNPs were found to selectively kill antigen-positive SKBR-2 cells over antigen-negative MDA-MB-231 cells in serum-containing medium. Further in vivo anti-cancer activity was demonstrated in an SKBR-3 tumour xenograft model in BALB/c mice in which stable 60% tumour regression could be observed just after two injections of 45 pmol of ATNP. These results open interesting prospects in the use of such cationic nanoparticles as payloads for ADC-like strategies.

Triphenylphosphonium-functionalized N-heterocyclic carbene platinum complexes [(NHC-TPP+)Pt] induce cell death of human glioblastoma cancer stem cells.

A growing body of experimental and clinical evidence suggests that rare cell populations, known as cancer stem cells (CSCs), play an important role in the development and therapeutic resistance of several cancers, including glioblastoma. Elimination of these cells is therefore of paramount importance. Interestingly, recent results have shown that the use of drugs that specifically disrupt mitochondria or induce mitochondria-dependent apoptosis can efficiently kill cancer stem cells. In this context, a novel series of platinum(II) complexes bearing N-heterocyclic carbene (NHC) of the type [(NHC)PtI2(L)] modified with the mitochondria targeting group triphenylphosphonium were synthesized. After a complete characterization of the platinum complexes, the cytotoxicity against two different cancer cell lines, including a cancer stem cell line, was investigated. The best compound reduced the cell viability of both cell lines by 50% in the low µM range, with an approximately 300-fold higher anticancer activity on the CSC line compared to oxaliplatin. Finally, mechanistic studies showed that the triphenylphosphonium functionalized platinum complexes significantly altered mitochondrial function and also induced atypical cell death.

Smart DNA vectors based on cyclodextrin polymers: compaction and endosomal release.

PURPOSE: Neutral ß-cyclodextrin polymers (polyßCD) associated with cationic adamantyl derivatives (Ada) can be used to deliver plasmid DNA into cells. In absence of an endosomolytic agent, transfection efficiency remains low because most complexes are trapped in the endosomal compartment. We asked whether addition of an imidazole-modified Ada can increase efficiency of polyßCD/cationic Ada-based delivery system. METHODS: We synthesized two adamantyl derivatives: Ada5, which has a spacer arm between the Ada moiety and a bi-cationic polar head group, and Ada6, which presents an imidazole group. Strength of association between polyßCD and Ada derivatives was evaluated by fluorimetric titration. RESULTS: Gel mobility shift assay, zeta potential, and dark field transmission electron microscopy experiments demonstrated the system allowed for efficient DNA compaction. In vitro transfection experiments performed on HepG2 and HEK293 cells revealed the quaternary system polyßCD/Ada5/Ada6/DNA has efficiency comparable to cationic lipid DOTAP. CONCLUSION: We successfully designed fine-tuned DNA vectors based on cyclodextrin polymers combined with two new adamantyl derivatives, leading to significant transfection associated with low toxicity.

Polyarginine as a Simultaneous Antimicrobial, Immunomodulatory, and miRNA Delivery Agent within Polyanionic Hydrogel.

Implantation of biomedical devices is followed by immune response to the implant, as well as occasionally bacterial, yeast, and/or fungal infections. In this context, new implant materials and coatings that deal with medical device-associated complications are required. Antibacterial and anti-inflammatory materials are also required for wound healing applications, especially in diabetic patients with chronic wounds. In this work, hyaluronic acid (HA) hydrogels with triple activity: antimicrobial, immunomodulatory, and miRNA delivery agent, are presented. It is demonstrated that polyarginine with a degree of polymerization of 30 (PAR30), which is previously shown to have a prolonged antibacterial activity, decreases inflammatory response of lipopolysaccharide-stimulated macrophages. In addition, PAR30 accelerates fibroblast migration in macrophage/fibroblast coculture system, suggesting a positive effect on wound healing. Furthermore, PAR30 allows to load miRNA into HA hydrogels, and then to deliver them into the cells. To the authors knowledge, this study is the first describing miRNA-loaded hydrogels with antibacterial effect and anti-inflammatory features. Such system can become a tool for the treatment of infected wounds, e.g., diabetic ulcers, as well as for foreign body response modulation.

An N-heterocyclic carbene iridium(III) complex as a potent anti-cancer stem cell therapeutic.

Cancer stem cells (CSCs) represent a difficult to treat cellular niche within tumours due to their unique characteristics, which give them a high propensity for resistance to classical anti-cancer treatments and the ability to repopulate the tumour mass. An attribute that may be implicated in the high rates of recurrence of certain tumours. However, other characteristics specific to these cells, such as their high dependence on mitochondria, may be exploited for the development of new therapeutic agents that are effective against the niche. As such, a previously described phosphorescent N-heterocyclic carbene iridium(III) compound which showed a high level of cytotoxicity against classical tumour cell lines with mitochondria-specific effects was studied for its potential against CSCs. The results showed a significantly higher level of activity against several CSC lines compared to non-CSCs. Mitochondrial localisation and superoxide production were confirmed. Although the cell death involved caspase activation, their role in cell death was not definitive, with a potential implication of other, non-apoptotic pathways shown. A cytostatic effect of the compound was also displayed at low mortality doses. This study thus provides important insights into the mechanisms and the potential for this class of molecule in the domain of anti-CSC therapeutics.