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.

The antibacterial properties of branched peptides based on poly(l-arginine): In vitro antibacterial evaluation and molecular dynamic simulations.

The emergence of bacterial strains resistant to antibiotics is a major issue in the medical field. Antimicrobial peptides are widely studied as they do not generate as much resistant bacterial strains as conventional antibiotics and present a broad range of activity. Among them, the homopolypeptide poly(l-arginine) presents promising antibacterial properties, especially in the perspective of its use in biomaterials. Linear poly(l-arginine) has been extensively studied but the impact of its 3D structure remains unknown. In this study, the antibacterial properties of newly synthesized branched poly(l-arginine) peptides, belonging to the family of multiple antigenic peptides, are evaluated. First, in vitro activities of the peptides shows that branched poly(l-arginine) is more efficient than linear poly(l-arginine) containing the same number of arginine residues. Surprisingly, peptides with more arms and more residues are not the most effective. To better understand these unexpected results, interactions between these peptides and the membranes of Gram positive and Gram negative bacteria are simulated thanks to molecular dynamic. It is observed that the bacterial membrane is more distorted by the branched structure than by the linear one and by peptides containing smaller arms. This mechanism of action is in full agreement with in vitro results and suggest that our simulations form a robust model to evaluate peptide efficiency towards pathogenic bacteria.

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.

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.

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.

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.

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.

Fluorescent bioinspired albumin/polydopamine nanoparticles and their interactions with Escherichia coli cells.

Inspired by the eumelanin aggregates in human skin, polydopamine nanoparticles (PDA NPs) are promising nanovectors for biomedical applications, especially because of their biocompatibility. We synthesized and characterized fluorescent PDA NPs of 10-25 nm diameter based on a protein containing a lysine-glutamate diad (bovine serum albumin, BSA) and determined whether they can penetrate and accumulate in bacterial cells to serve as a marker or drug nanocarrier. Three fluorescent PDA NPs were designed to allow for tracking in three different wavelength ranges by oxidizing BSA/PDA NPs (Ox-BSA/PDA NPs) or labelling with fluorescein 5-isothiocyanate (FITC-BSA/PDA NPs) or rhodamine B isothiocyanate (RhBITC-BSA/PDA NPs). FITC-BSA/PDA NPs and RhBITC-BSA/PDA NPs penetrated and accumulated in both cell wall and inner compartments of Escherichia coli (E. coli) cells. The fluorescence signals were diffuse or displayed aggregate-like patterns with both labelled NPs and free dyes. RhBITC-BSA/PDA NPs led to the most intense fluorescence in cells. Penetration and accumulation of NPs was not accompanied by a bactericidal or inhibitory effect of growth as demonstrated with the Gram-negative E. coli species and confirmed with a Gram-positive bacterial species (Staphylococcus aureus). Altogether, these results allow us to envisage the use of labelled BSA/PDA NPs to track bacteria and carry drugs in the core of bacterial cells.

Formulation and rheological evaluation of liposomes-loaded carbopol hydrogels based on thermal waters.

The aims of this study were to develop topical liposomal hydrogels based on thermal waters (TWs) acquired in the region of Biskra (Northeast Algeria) and also to investigate their rheological properties. Liposomes containing two highly mineralized thermal waters, Baraka (BTW) and Salhine (STW), were prepared by probe sonication using phosphatidylcholine (PC) and cholesterol (Chol), plain or mixed with phosphatidylglycerol (PG). Based on their lipid composition, obtained liposomes presented vesicle sizes of 60 nm, a low polydispersity index, and various negative zeta potentials. It was noted that with increasing counterions charge in TWs the zeta potential of liposomes decreased toward neutral values.Carbopol (1%, w/w) hydrogels prepared with BTW, STW, and also demineralized water (placebo hydrogel) showed a non-Newtonian behavior, pseudoplastic fluid adjusted to Carreau model. The composition of thermal waters influenced highly the rheological properties of Carbopol hydrogels. Liposomal hydrogels were prepared by dispersing liposomes in hydrogels formulated with the same encapsulated thermal water. Regardless of composition or lipid concentration of added liposomes, the viscosity and viscoelastic parameters of Carbopol hydrogels changed negligibly. Indeed, liposome composition and lipid concentration seemed to have no effect on the rheological properties of Carbopol hydrogel in the presence of an important charge of cations. Hence, hydrogels and liposomal hydrogels based on thermal waters had suitable rheological properties for topical application and delivery of minerals in the skin.

Impact of anti-PDGFRα antibody surface functionalization on LNC uptake by oligodendrocyte progenitor cells.

Impairment of oligodendrocyte progenitor cell (OPC) differentiation into oligodendrocytes and chronic inflammation are key determinants of poor remyelination observed in diseases such as multiple sclerosis. For many pro-myelinating molecules, the therapeutic potential is hindered by poor solubility or limited access to the targeted cells. A promising approach to improve the delivery of those molecules to OPC is to encapsulate them in functionalized Lipid Nanocapsules (LNC). We aimed to develop the first OPC-targeting LNC, by grafting an anti-PDGFRα antibody on the surface of the LNC using several strategies and evaluating the interaction with PDGFRα via ELISA. We found that only site-selective click-chemistry grafting maintained anti-PDGFRα/PDGFRα association, which was confirmed in vitro on primary rat OPC. In conclusion, we demonstrated that it was possible to produce anti-PDGFRα functionalized LNC, we confirmed the antibody's ability to recognize its receptor after grafting and we optimized techniques to characterize antibody functionalized LNC.

Anti-inflammatory effects of free and liposome-encapsulated Algerian thermal waters in RAW 264.7 macrophages.

The main objectives of this work were to formulate liposomes encapsulating highly mineralized thermal waters (TWs) and to study anti-inflammatory effect of free and encapsulated thermal waters on RAW 264.7 macrophage cells stimulated with lipopolysaccharide (LPS). TWs-loaded conventional and deformable liposomes (TWs-Lip and TWs-DLip) were prepared by sonication and extrusion, respectively. They were considered for their vesicle size, zeta potential, entrapment efficiency, physical stability and in vitro anti-inflammatory effect. Formulated liposome suspensions have a low polydispersity and nanometric size range with zeta potential values close to zero. The vesicle size was stable for 30 days. Entrapment efficiency of TWs was above 90% in conventional liposomes and 70% in deformable liposomes. Pretreatment of LPS-stimulated murine macrophages, with free and liposome-encapsulated TWs, resulted in a significant reduction in nitric oxide (NO) production and modulated tumor necrosis factor-α (TNF-α) production suggesting an anti-inflammatory effect which was even more striking with TWs-Lip and TWs-DLip. Liposome formulations may offer a suitable approach for transdermal delivery of TWs, indicated in inflammatory skin diseases.

Evaluation of the Cytotoxicity of Cationic Polymers on Glioblastoma Cancer Stem Cells.

Cationic polymers such as polyethylenimine (PEI) have found a pervasive place in laboratories across the world as gene delivery agents. However, their applications are not limited to this role, having found a place as delivery agents for drugs, in complexes known as polymer-drug conjugates (PDCs). Yet a potentially underexplored domain of research is in their inherent potential as anti-cancer therapeutic agents, which has been indicated by several studies. Even more interesting is the recent observation that certain polycations may present a significantly greater toxicity towards the clinically important cancer stem cell (CSC) niche than towards more differentiated bulk tumour cells. These cells, which possess the stem-like characteristics of self-renewal and differentiation, are highly implicated in cancer drug resistance, tumour recurrence and poor clinical prognosis. The search for compounds which may target and eliminate these cells is thus of great research interest. As such, the observation in our previous study on a PEI-based PDC which showed a considerably higher toxicity of PEI towards glioblastoma CSCs (GSCs) than on more differentiated glioma (U87) cells led us to investigate other cationic polymers for a similar effect. The evaluation of the toxicity of a range of different types of polycations, and an investigation into the potential source of GSC's sensitivity to such compounds is thus described.

Development and characterization of layer-by-layer coated liposomes with poly(L-lysine) and poly(L-glutamic acid) to increase their resistance in biological media.

Multilayered coated liposomes were prepared using the layer-by-layer (LbL) technique in an effort to improve their stability in biological media. The formulation strategy was based on the alternate deposition of two biocompatible and biodegradable polyelectrolytes - poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) - on negatively charged small unilamellar vesicles (SUVs). Some parameters of the formulation process were optimized such as the polyelectrolyte concentration and the purification procedure. This optimized procedure has allowed the development of very homogeneous formulations of liposomes coated with up to 6 layers of polymers (so-called layersomes). The coating was characterized by dynamic light scattering (DLS), zeta potential measurements and Förster resonance energy transfer (FRET) between two fluorescently labeled polyelectrolytes. Studies on the stability of the formulations at 4 °C in a buffered solution have shown that most structures are stable over 1 month without impacting their encapsulation capacity. In addition, fluorophore release experiments have demonstrated a better resistance of the layersomes in the presence of a non-ionic detergent (Triton™ X-100) as well as in the presence of phospholipase A2 and human plasma. In conclusion, new multilayered liposomes have been developed to increase the stability of conventional liposomes in biological environments.

Liposomes as tunable platform to decipher the antitumor immune response triggered by TLR and NLR agonists.

Liposomes are powerful tools for the optimization of peptides and adjuvant composition in cancer vaccines. Here, we take advantage of a liposomal platform versatility to develop three vaccine candidates associating a peptide from HA influenza virus protein as CD4 epitope, a peptide from HPV16 E7 oncoprotein as CD8 epitope and TLR4, TLR2/6 or NOD1 agonists as adjuvant. Liposomal vaccine containing MPLA (TLR4 liposomes), are the most effective treatment against the HPV-transformed orthotopic lung tumor mouse model, TC-1. This vaccine induces a potent Th1-oriented antitumor immunity, which leads to a significant reduction in tumor growth and a prolonged survival of mice, even when injected after tumor appearance. This efficacy is dependent on CD8+ T cells. Subcutaneous injection of this treatment induces the migration of skin DCs to draining lymph nodes. Interestingly, TLR2/6 liposomes trigger a weaker Th1-immune response which is not sufficient for the induction of a prolonged antitumor activity. Although NOD1 liposome treatment results in the control of early tumor growth, it does not extend mice survival. Surprisingly, the antitumor activity of NOD1 vaccine is not associated with a specific adaptive immune response. This study shows that our modulable platform can be used for the strategical development of vaccines.

Optimization of peptide-based cancer vaccine compositions, by sequential screening, using versatile liposomal platform.

Therapeutic cancer vaccines need thoughtful design to efficiently deliver appropriate antigens and adjuvants to the immune system. In the current study, we took advantage of the versatility of a liposomal platform to conceive and customize vaccines containing three elements needed for the induction of efficient antitumor immunity: i) a CD4 epitope peptide able to activate CD4+ T helper cells, ii) a CD8 tumor-specific epitope peptide recognized by CD8+ T cytotoxic cells and iii) Pattern Recognition Receptor (PRR) agonists which stand as adjuvants. Each type of component, conjugated to liposomes, was evaluated individually by comparing their vaccine efficacy after immunization of naïve mice. These screening steps resulted in the optimization of three liposomal constructs bearing a peptide from HA influenza virus protein as CD4 epitope, a peptide from HPV16 E7 oncoprotein as CD8 epitope and TLR4, TLR2/6 or NOD1 agonists as adjuvant, which displayed antitumor efficiency against a mouse model of disseminated tumors transformed by HPV16. Our results validated the interest of our customizable liposomal platform as delivery system for cancer vaccination. We also demonstrated its interest as tool for vaccine design allowing the strategical selection of components, and the evaluation of epitope-adjuvant association.

Enhancing tumor specific immune responses by transcutaneous vaccination.

INTRODUCTION: Our understanding of the involvement of the immune system in cancer control has increased over recent years. However, the development of cancer vaccines intended to reverse tumor-induced immune tolerance remains slow as most current vaccine candidates exhibit limited clinical efficacy. The skin is particularly rich with multiple subsets of dendritic cells (DCs) that are involved to varying degrees in the induction of robust immune responses. Transcutaneous administration of cancer vaccines may therefore harness the immune potential of these DCs, however, this approach is hampered by the impermeability of the stratum corneum. Innovative vaccine formulations including various nanoparticles, such as liposomes, are therefore needed to properly deliver cancer vaccine components to skin DCs. Areas covered: The recent insights into skin DC subsets and their functional specialization, the potential of nanoparticle-based vaccines in transcutaneous cancer vaccination and, finally, the most relevant clinical trial advances in liposomal and in cutaneous cancer vaccines will be discussed. Expert commentary: To define the optimal conditions for mounting protective skin DC-induced anti-tumor immune responses, investigation of the cellular and molecular interplay that controls tumor progression should be pursued in parallel with clinical development. The resulting knowledge will then be translated into improved cancer vaccines that better target the most appropriate immune players.

Solubilization of α-galactosylceramide in aqueous medium: Impact on Natural Killer T cell activation and antitumor responses.

The potent antitumor effect of α-galactosylceramide (α-GalCer) is based on its recognition by invariant Natural Killer T cells (iNKT) after its capture and presentation by antigen presenting cells including dendritic cells (DCs). Synthetic α-GalCer has already been tested in advanced cancer patients but no or only moderate clinical responses were obtained. To optimize α-GalCer efficacy, we have postulated that alternative formulations impacting its molecular organization in aqueous medium could modify DC uptake and iNKT-based immune responses. To this end, we have developed two strategies: (1) the formulation of α-GalCer in non-cationic liposomes and (2) the synthesis of a water-soluble α-GalCer analogue by anchoring a polyethyleneglycol moiety on its sugar head. The biological activities of these new preparations were compared to that induced by the classically used Polysorbate 20 α-GalCer micelles. Both formulations retained their uptake by DCs and activated iNKT cells both in vitro and in vivo. Despite a lower cytokine production, the formulations induced a potent immune response able to control lung murine carcinoma. In conclusion, it is possible to increase α-GalCer solubility in aqueous solution without limiting its antitumor properties.

Exogenous Activation of Invariant Natural Killer T Cells by α-Galactosylceramide Reduces Pneumococcal Outgrowth and Dissemination Postinfluenza.

Influenza A virus infection can predispose to potentially devastating secondary bacterial infections. Invariant natural killer T (iNKT) cells are unconventional, lipid-reactive T lymphocytes that exert potent immunostimulatory functions. Using a mouse model of postinfluenza invasive secondary pneumococcal infection, we sought to establish whether α-galactosylceramide (α-GalCer [a potent iNKT cell agonist that is currently in clinical development]) could limit bacterial superinfection. Our results highlighted the presence of a critical time window during which α-GalCer treatment can trigger iNKT cell activation and influence resistance to postinfluenza secondary pneumococcal infection. Intranasal treatment with α-GalCer during the acute phase (on day 7) of influenza virus H3N2 and H1N1 infection failed to activate (gamma interferon [IFN-γ] and interleukin-17A [IL-17A]) iNKT cells; this effect was associated with a strongly reduced number of conventional CD103+ dendritic cells in the respiratory tract. In contrast, α-GalCer treatment during the early phase (on day 4) or during the resolution phase (day 14) of influenza was associated with lower pneumococcal outgrowth and dissemination. Less intense viral-bacterial pneumonia and a lower morbidity rate were observed in superinfected mice treated with both α-GalCer (day 14) and the corticosteroid dexamethasone. Our results open the way to alternative (nonantiviral/nonantibiotic) iNKT-cell-based approaches for limiting postinfluenza secondary bacterial infections. IMPORTANCE: Despite the application of vaccination programs and antiviral drugs, influenza A virus (IAV) infection is responsible for widespread morbidity and mortality (500,000 deaths/year). Influenza infections can also result in sporadic pandemics that can be devastating: the 1918 pandemic led to the death of 50 million people. Severe bacterial infections are commonly associated with influenza and are significant contributors to the excess morbidity and mortality of influenza. Today's treatments of secondary bacterial (pneumococcal) infections are still not effective enough, and antibiotic resistance is a major issue. Hence, there is an urgent need for novel therapies. In the present study, we set out to evaluate the efficacy of α-galactosylceramide (α-GalCer)-a potent agonist of invariant NKT cells that is currently in clinical development-in a mouse model of postinfluenza, highly invasive pneumococcal pneumonia. Our data indicate that treatment with α-GalCer reduces susceptibility to superinfections and, when combined with the corticosteroid dexamethasone, reduces viral-bacterial pneumonia.

Liposomal constructs for antitumoral vaccination by the nasal route.

Mucosal surfaces are promising routes for vaccination. Among mucosa, airway mucosa provides the opportunity to develop non-invasive approaches for vaccine delivery. In the current study, nasal route was used to investigate the potency of highly versatile di-epitopic liposomal constructs of different size, structure and composition to exhibit antitumor efficiency after prophylactic vaccination in mice. Well-characterized small unilamellar (SUV), multilamellar (MLV), reverse-phase evaporation (REV) and ultraflexible small unilamellar vesicles (Uf-SUV), containing the ErbB2 T-cytotoxic epitope, the influenza-derived HA T-helper epitope and the lipopeptide adjuvant Pam2CAG, were formulated. These vaccines were administered into the nasal cavity of BALB/c mice, followed by i.v. or s.c. implantation of ErbB2-surexpressing cancer cells. Nasal vaccination with the SUV vaccine resulted in an efficient antitumor activity against lung tumors and a non-significant protection against s.c. tumors. Size, structure and flexibility of liposomes did not impact vaccine immunity and antitumoral efficiency against lung tumors, in contrast to total dose of vaccine or dose of adjuvant. These results showed an undeniable interest of liposomes as lipid-based carriers for antitumor vaccine delivery by the nasal route, opening new perspectives for cancer treatment.