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.

Experimental Methods to Get Polydopamine Films: A Comparative Review on the Synthesis Methods, the Films' Composition and Properties.

In 2007, polydopamine (PDA) films were shown to be formed spontaneously on the surface of all known classes of materials by simply dipping those substrates in an aerated dopamine solution at pH = 8.5 in the presence of Tris(hydroxymethyl) amino methane buffer. This universal deposition method has raised a burst of interest in surface science, owing not only to the universality of this water based one pot deposition method but also to the ease of secondary modifications. Since then, PDA films and particles are shown to have applications in energy conversion, water remediation systems, and last but not least in bioscience. The deposition of PDA films from aerated dopamine solutions is however a slow and inefficient process at ambient temperature with most of the formed material being lost as a precipitate. This incited to explore the possibility to get PDA and related films based on other catecholamines, using other oxidants than dissolved oxygen and other deposition methods. Those alternatives to get PDA and related films are reviewed and compared in this paper. It will appear that many more investigations are required to get better insights in the relationships between the preparation method of PDA and the properties of the obtained coatings.

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.

Mimicking the Chemistry of Natural Eumelanin Synthesis: The KE Sequence in Polypeptides and in Proteins Allows for a Specific Control of Nanosized Functional Polydopamine Formation.

The oxidation of dopamine and of other catecholamines leads to the formation of conformal films on the surface of all known materials and to the formation of a precipitate in solution. In some cases, it has been shown that the addition of additives in the dopamine solution, like certain surfactants or polymers, polyelectrolytes, and certain proteins, allows to get polydopamine nanoparticles of controlled size and the concomitant decrease, in an additive/dopamine dependent manner, in film formation on the surface of the reaction beaker. However, the mechanism behind this controlled oxidation and self-assembly of catecholamines is not known. In this article, it is shown that a specific diad of amino acids in proteins, namely KE, allows for specific control in the oxidation-self-assembly of dopamine to obtain polydopamine@protein core-shell nanoparticles which are biocompatible. The interactions between dopamine and the adjacent KE amino acids potentially responsible for the size control of polydopamine aggregates was investigated by molecular dynamics simulations. The obtained core-shell nanoparticles display the biological activity of the protein used to control the self-assembly of PDA. The photon to heat conversion ability of PDA is conserved in the PDA@protein particles.

N-Heterocyclic Carbene-Polyethylenimine Platinum Complexes with Potent in Vitro and in Vivo Antitumor Efficacy.

The current interest for platinum N-heterocyclic carbene complexes in cancer research stems from their impressive toxicity reported against a range of different human cancer cells. To date, the demonstration of their in vivo efficacy relative to that of established platinum-based drugs has not been specifically addressed. Here, we introduce an innovative approach to increase the NHC-Pt complex potency whereby multiple NHC-Pt(II) complexes are coordinated along a polyethylenimine polymer (PEI) chain. We show that such NHC-Pt(II)-PEI conjugates induce human cancer cell death in vitro and in vivo in a xenograft mouse model with no observable side effects in contrast to oxaliplatin. Additional studies indicate nucleus and mitochondria targeting and suggest various mechanisms of action compared to classical platinum-based anticancer drugs.

Targeted delivery of α-galactosylceramide to CD8α+ dendritic cells optimizes type I NKT cell-based antitumor responses.

Immunotherapy aiming at enhancing innate and acquired host immunity is a promising approach for cancer treatment. The invariant NKT (iNKT) cell ligand α-galactosylceramide (α-GalCer) holds great promise in cancer therapy, although several concerns limit its use in clinics, including the uncontrolled response it promotes when delivered in a nonvectorized form. Therefore, development of delivery systems to in vivo target immune cells might be a valuable option to optimize iNKT cell-based antitumor responses. Using dendritic cell (DC)-depleted mice, DC transfer experiments, and in vivo active cell targeting, we show that presentation of α-GalCer by DCs not only triggers optimal primary iNKT cell stimulation, but also maintains secondary iNKT cell activation after challenge. Furthermore, targeted delivery of α-GalCer to CD8α(+) DCs, by means of anti-DEC205 decorated nanoparticles, enhances iNKT cell-based transactivation of NK cells, DCs, and γδ T cells. We report that codelivery of α-GalCer and protein Ag to CD8α(+) DCs triggers optimal Ag-specific Ab and cytotoxic CD8(+) T cell responses. Finally, we show that targeting nanoparticles containing α-GalCer and Ag to CD8α(+) DCs promotes potent antitumor responses, both in prophylactic and in therapeutic settings. Our data may have important implications in tumor immunotherapy and vaccine development.

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.

MiR-20a regulates ASK1 expression and TLR4-dependent cytokine release in rheumatoid fibroblast-like synoviocytes.

OBJECTIVE: To evaluate whether miR-20a belonging to the cluster miR-17-92 is a negative regulator of inflammation in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) by modulating expression of apoptosis signal-regulating kinase (ASK) 1, a key component of the toll-like receptors 4 pathway, upstream of p38 mitogen-activated protein kinase. METHODS: Evaluation of miR-20a and ASK1 mRNA was performed by RT-qPCR. ASK1 protein expression was assessed by western blotting. Overexpression of miR-20a was performed by transfection of RA FLS and THP-1 cells with miR-20a mimics. Interleukin (IL)-6, CXCL-10, IL-1ß and TNF-α release were measured by ELISA. The role of miR-20a in vivo was assessed by IL-6 release from macrophages obtained from mice injected intraperitoneally with vectorised miR-20a mimics. RESULTS: We showed that stimulation of RA FLS with lipopolysacharide (LPS) and bacterial lipoproteins (BLP) induces a drop in expression of miR-20a and that this decrease is associated with an upregulation of ASK1 expression. Using transfection of Ask1 3'UTR reporters, we demonstrate that Ask1 is a direct target of miR-20a. Overexpression of miR-20a led to a global decrease in ASK1 protein in BLP- and LPS-activated cells indicating that miR-20a regulates the expression of ASK1 at the translational level. Transfection of miR-20a mimics decreases IL-6 and CXCL10 release by RA FLS and IL-1ß and TNF-α by activated THP-1 cells but only in response to LPS. Last, injection of vectorised miR-20a mimics to mice led to a global decrease in ASK1 protein expression and IL-6 secretion in LPS-activated macrophages. CONCLUSIONS: Our data point toward an important role for miR-20a in the regulation of pro-inflammatory cytokines release, by controlling ASK1 expression in RA FLS.

Self-construction of supramolecular polyrotaxane films by an electrotriggered morphogen-driven process.

The design of films using a one-pot process has recently attracted increasing interest in the field of polymer thin film formation. Herein we describe the preparation of one-pot supramolecular polyrotaxane (PRX) films using the morphogen-driven self-construction process. This one-pot buildup strategy where the film growth is triggered by the electrochemical formation and diffusion of a catalyst in close vicinity of the substrate has recently been introduced by our group. A one-pot mixture was used that contained (i) poly(acrylic acid) (PAA) functionalized by azide groups grafted on the polymer chain through oligo(ethylene glycol) (EG) arms, leading to PAA-EG13-N3, (ii) cyclodextrins (α and ß CD), as macrocycles that can be threaded along EG arms, (iii) alkyne-functionalized stoppers (ferrocene or adamantane), to cap the PRX assembly by click chemistry, and (iv) copper sulfate. The one-pot mixture solution was brought into contact with a gold electrode. Cu(I), the morphogen, was generated electrochemically from Cu(II) at the electrode/one-pot solution interface. This electrotriggered click reaction leads to the capping of polypseudorotaxane yielding to PRXs. The PRXs can self-assemble through lateral supramolecular interactions to form aggregates and ensure the cohesion of the film. The film buildup was investigated using different types of CD and alkyne functionalized stoppers. Supramolecular PRX aggregates were characterized by X-ray diffraction measurements. The film topographies were imaged by atomic force microscopy. The influence of the concentration in CD and the presence of a competitor were studied as well. The stability of the resulting film was tested in contact with 8 M urea and during the electrochemical oxidation of ferrocene.

Modified and derived ethanol injection toward liposomes: development of the process.

A modified and derived ethanol injection (MDEI) process was developed to produce liposomes. The aim of the present study was to more efficiently control the vesicle diameter than with the conventional ethanol injection method. A hot ethanolic solution of lipids (60°C) was injected into a hot aqueous buffer (70°C). Then, ethanol was removed by rotary evaporation under reduced pressure. The size of the liposomes could be controlled by the ratio of ethanol to hydroalcoholic solution before evaporation. The concentration of lipids, the charge of lipids, and the type of aqueous phase had little effect on the vesicle diameter when the process involved a ratio of 33% (v/v) ethanol. In addition, it was possible to obtain lipid concentrations 10- to 30-fold higher that the conventional ethanol injection method. The encapsulation of a hydrophilic compound was feasible with this MDEI process. The observation by cryogenic transmission electron microscopy revealed that these liposomes were predominantly unilamellar at a ratio as high as 33 or 50% (v/v) ethanol. Thus, the results showed that MDEI is an appropriate alternative for the manufacture of liposomes with respect to the ethanol injection process.

Key role for respiratory CD103(+) dendritic cells, IFN-γ, and IL-17 in protection against Streptococcus pneumoniae infection in response to α-galactosylceramide.

BACKGROUND: Exogenous activation of pulmonary invariant natural killer T (iNKT) cells, a population of lipid-reactive αß T lymphocytes, with use of mucosal α-galactosylceramide (α-GalCer) administration, is a promising approach to control respiratory bacterial infections. We undertook the present study to characterize mechanisms leading to α-GalCer-mediated protection against lethal infection with Streptococcus pneumoniae serotype 1, a major respiratory pathogen in humans. METHODS AND RESULTS: α-GalCer was administered by the intranasal route before infection with S. pneumoniae. We showed that respiratory dendritic cells (DCs), most likely the CD103(+) subset, play a major role in the activation (IFN-γ and IL-17 release) of pulmonary iNKT cells, whereas alveolar and interstitial macrophages are minor players. After challenge, S. pneumoniae was rapidly (4 hours) eliminated in the alveolar spaces, a phenomenon that depended on respiratory DCs and neutrophils, but not macrophages, and on the early production of both IFN-γ and IL-17. Protection was also associated with the synthesis of various interferon-dependent and IL-17-associated genes as revealed by transcriptomic analysis. CONCLUSIONS: These data imply a new function for pulmonary CD103(+) DCs in mucosal activation of iNKT cells and establish a critical role for both IFN-γ and IL-17 signalling pathways in mediating the innate immune response to S. pneumoniae.

Strongly Metal-Adhesive and Self-Healing Gelatin@Polydopamine-Based Hydrogels with Long-Term Antioxidant Activity.

Bioinspired adhesives have been increasingly developed, especially towards a biomedical application. Therefore, in this study, dopamine (DA) was oxidized into polydopamine (PDA) in a gelatin mixture via titration with NaIO4 as a strong oxidant to easily obtain an adhesive antioxidant and self-healing PDA-gelatin hydrogel. Rheology experiments show a stiffness in the order of kPa and a thermal resistance above 50 °C, much above the gel-sol transition temperature of pristine gelatin. After heating at 55 °C, the gel is self-healing. In addition, just after formulation, it shows strong peeling-rate-dependent adhesion to steel with a tensile work per unit area (W) of up to 100 ± 39 J/m2, which is 2.5 times higher than that of the same gel without PDA at a peeling rate of 1000 µm/s. The increase in W between peeling rates of 10 and 1000 µm/s was studied and interpreted in terms of the gels' viscoelasticity. Moreover, this hydrogel offers significant antioxidant activity (measured by DPPH scavenging) that lasts with storage for at least over 15 days, this being then prolonged for 2 additional days, which seems particularly relevant considering the importance of reactive oxygen species (ROS) in wound healing. To summarize, PDA-gelatin gel is a promising strong and antioxidant adhesive.

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.

Preclinical in vitro evaluation of implantable materials: conventional approaches, new models and future directions.

Nowadays, implants and prostheses are widely used to repair damaged tissues or to treat different diseases, but their use is associated with the risk of infection, inflammation and finally rejection. To address these issues, new antimicrobial and anti-inflammatory materials are being developed. Aforementioned materials require their thorough preclinical testing before clinical applications can be envisaged. Although many researchers are currently working on new in vitro tissues for drug screening and tissue replacement, in vitro models for evaluation of new biomaterials are just emerging and are extremely rare. In this context, there is an increased need for advanced in vitro models, which would best recapitulate the in vivo environment, limiting animal experimentation and adapted to the multitude of these materials. Here, we overview currently available preclinical methods and models for biological in vitro evaluation of new biomaterials. We describe several biological tests used in biocompatibility assessment, which is a primordial step in new material's development, and discuss existing challenges in this field. In the second part, the emphasis is made on the development of new 3D models and approaches for preclinical evaluation of biomaterials. The third part focuses on the main parameters to consider to achieve the optimal conditions for evaluating biocompatibility; we also overview differences in regulations across different geographical regions and regulatory systems. Finally, we discuss future directions for the development of innovative biomaterial-related assays: in silico models, dynamic testing models, complex multicellular and multiple organ systems, as well as patient-specific personalized testing approaches.

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.

Cyto-mechanoresponsive polyelectrolyte multilayer films.

Cell adhesion processes take place through mechanotransduction mechanisms where stretching of proteins results in biological responses. In this work, we present the first cyto-mechanoresponsive surface that mimics such behavior by becoming cell-adhesive through exhibition of arginine-glycine-aspartic acid (RGD) adhesion peptides under stretching. This mechanoresponsive surface is based on polyelectrolyte multilayer films built on a silicone sheet and where RGD-grafted polyelectrolytes are embedded under antifouling phosphorylcholine-grafted polyelectrolytes. The stretching of this film induces an increase in fibroblast cell viability and adhesion.

Manufacture of liposomes by isopropanol injection: characterization of the method.

Unilamellar liposomes are conventionally prepared by rapid injection of an ethanolic solution of lipids into an aqueous medium. The aim of the present study was to control, more efficiently, vesicle diameter by using an alternative solvent. The results show that isopropanol injection is a good alternative to ethanol injection for the manufacture of liposomes. Particle size can be controlled by the variation of process parameters, such as stirring speed of the aqueous phase and injection flow rate of lipid-isopropanol solution. Diameter of vesicles obtained by this method is less affected by the nature of phospholipid, as well as lipid concentration, than in the ethanol-injection process. In addition, the vesicles are generally smaller (approximately 40-210 nm). Accurate characterization of the particles, by fluorescence, (31)P-NMR, and cryo-transmission electron microscopy, showed that particles are formed of a single lipid bilayer around an aqueous cavity. We thus provide the scientific community with a fully characterized alternative method to produce unilamellar vesicles.