Françoise Pons

Efficient in vitro and in vivo pulmonary delivery of nucleic acid by carbon dot-based nanocarriers

Cationic carbon dots were fabricated by pyrolysis of citric acid and bPEI25k under microwave radiation. Various nanoparticles were produced in a 20-30% yield through straightforward modifications of the reaction parameters (stoichiometry of the reactants and energy supply regime). Particular attention was paid to the purification of the reaction products to ensure satisfactory elimination of the residual starting polyamine. Intrinsic properties of the particles (size, surface charge, photoluminescence and quantum yield) were measured and their ability to form stable complexes with nucleic acid was determined. Their potential to deliver plasmid DNA or small interfering RNA to various cell lines was investigated and compared to that of bPEI25k. The pDNA in vitro transfection efficiency of these carbon dots was similar to that of the parent PEI, as was their cytotoxicity. The higher cytotoxicity of bPEI25k/siRNA complexes when compared to that of the CD/siRNA complexes however had marked consequences on the gene silencing efficiency of the two carriers. These results are not fully consistent with those in some earlier reports on similar nanoparticles, revealing that toxicity of the carbon dots strongly depends on their protocol of fabrication. Finally, these carriers were evaluated for in vivo gene delivery through the non-invasive pulmonary route in mice. High transgene expression was obtained in the lung that was similar to that obtained with the golden standard formulation GL67A, but was associated with significantly lower toxicity. Post-functionalization of these carbon dots with PEG or targeting moieties should significantly broaden their scope and practical implications in improving their in vivo transfection efficiency and biocompatibility.

Liposomes as delivery systems for nasal vaccination: strategies and outcomes

Importance of the field: Among the particulate systems that have been envisaged in vaccine delivery, liposomes are very attractive. These phospholipid vesicles can indeed deliver a wide range of molecules. They have been shown to enhance considerably the immunogenicity of weak protein antigens or synthetic peptides. Also, they offer a wide range of pharmaceutical options for the design of vaccines. In the past decade, the nasal mucosa has emerged as an effective route for vaccine delivery, together with the opportunity to develop non-invasive approaches in vaccination. Areas covered in this review: This review focuses on the recent strategies and outcomes that have been developed around the use of liposomes in nasal vaccination. What the reader will gain: The various formulation parameters, including lipid composition, size, charge and mucoadhesiveness, that have been investigated in the design of liposomal vaccine candidates dedicated to nasal vaccination are outlined. Also, an overview of the immunological and protective responses obtained with the developed formulations is presented. Take home message: This review illustrates the high potential of liposomes as nasal vaccine delivery systems.

Immunomodulatory properties of multi-walled carbon nanotubes in peripheral blood mononuclear cells from healthy subjects and allergic patients

In the present study, we investigated the immunomodulatory activity of multi-walled carbon nanotubes (MWCNTs) in peripheral blood mononuclear cells (PBMCs) from healthy donors and mite-allergic subjects. Freshly prepared PBMCs, stimulated or not with Toll-like receptor (TLR)1-9 agonists, a T cell mitogen (phytohemagglutinin A) or mite allergen extract were cultured in the presence or absence of MWCNTs. Secretion of TNF-α, IL-2, IL-5, IL-6, IL-12/23p40 or IFN-γ was quantified in the culture supernatants by ELISA. Basal secretion of all the cytokines was not altered by MWCNTs in PBMCs from both healthy donors and allergic subjects. In PBMCs from healthy donors, TNF-α, IL-6 and IL-12/23p40 secretion in response to the TLR4 agonist, lipopolysaccharide was however increased in a dose-dependent manner by MWCNTs. Significant increases in the release of these cytokines were also observed in PBMCs stimulated with a TLR2 or TLR3 agonist. MWCNTs also increased the release of IL-2 and IFN-γ by PBMCs stimulated with a T cell mitogen. In contrast, MWCNTs inhibited allergen-induced IL-5 secretion by PBMCs from mite-allergic subjects. As well, MWCNTs altered the capacity of PBMC-derived monocytes to differentiate into functional dendritic cells. All together, our data suggest that according to its immune cell target, MWCNTs may either promote or suppress immune responses in humans. Further investigations are necessary to fully understand the complexity behind interactions of engineered nanoparticles with the immune system.

Lung deposition and toxicological responses evoked by multi-walled carbon nanotubes dispersed in a synthetic lung surfactant in the mouse

In the present work, we elaborated a synthetic lung surfactant composed of dipalmitoyl phosphatidylcholine (DPPC), phosphatidylglycerol, cholesterol and bovine serum albumin (BSA), as a vehicle to study the lung toxicity of pristine multi-walled carbon nanotubes (MWCNT). MWCNT were dispersed in surfactant, saline or saline containing DPPC, BSA, Pluronic® F68 or sodium dodecyl sulfate, for comparison. Dispersions were characterized visually, and by light microscopy, dynamic light scattering and transmission electronic microscopy (TEM). Deposition of surfactant-dispersed MWCNT in the lung of BALB/c mice upon single or repeated administrations was analyzed by histology and TEM. Inflammation and airway remodeling were assessed in bronchoalveolar lavage fluid (BALF) or lung tissue of mice by counting cells and quantifying cytokines, tumor growth factor (TGF)-β1 and collagen, and by histology. We found that the elaborated surfactant is more effective in dispersing MWCNT when compared to the other agents, while being biocompatible. Surfactant-dispersed MWCNT distributed all throughout the mouse airways upon single and repeated administrations and were observed in alveolar macrophages and epithelial cells, and in infiltrated neutrophils. Mice that received a single administration of MWCNT showed neutrophil infiltrate and greater concentrations of tumor necrosis factor (TNF)-α, keratinocyte-derived chemokine (KC) and interleukin (IL)-17 in BALF when compared to controls. After repeated MWCNT administrations, increases in macrophage number, KC and TGF-β1 levels in BALF, and collagen deposition and mucus hyperplasia in lung tissue were observed. Altogether, the elaborated lung surfactant could be a valuable tool to further study the toxicological impact of pristine MWCNT in laboratory animals.

Exposure to multi-walled carbon nanotubes results in aggravation of airway inflammation and remodeling and in increased production of epithelium-derived innate cytokines in a mouse model of asthma

AbstractWith the development of nanotechnologies, the potential adverse effects of nanomaterials such as multi-walled carbon nanotubes (MWCNT) on the respiratory tract of asthmatics are questioned. Furthermore, investigations are necessary to understand how these effects might arise. In the present study, we hypothesized that epithelium-derived innate cytokines that are considered as important promoting factors in allergy may contribute to an aggravating effect of MWCNT on asthma. We investigated in the mouse the effect of MWCNT on systemic immune response and airway inflammation and remodeling induced by the most frequent allergen so far associated with asthma, house dust mite (HDM), and we examined the production of the innate cytokines thymic stromal lymphopoietin (TSLP), IL-25, IL-33, and GM-CSF. Mice exposed to HDM exhibited specific IgG1 in serum and inflammatory cell infiltration, and increased Th2 cytokine production, mucus hyperproduction, and collagen deposition in the airways when compared to naïve animals. Levels of total IgG1 and HDM-specific IgG1, influx of macrophages, eosinophils and neutrophils, production of collagen, TGF-β1, and mucus, as well as levels of IL-13, eotaxin, and TARC, were dose-dependently increased in mice exposed to HDM and MWCNT compared to HDM alone. These effects were associated with an increased production of TSLP, IL-25, IL-33, and GM-CSF in the airways. Our data demonstrate that MWCNT increase in a dose-dependent manner systemic immune response, as well as airway allergic inflammation and remodeling induced by HDM in the mouse. Our data suggest also a role for airway epithelium and innate cytokines in these effects.

Design of a Liposomal Candidate Vaccine Against Pseudomonas aeruginosa and its Evaluation in Triggering Systemic and Lung Mucosal Immunity

PurposeTo design and evaluate liposomal constructs capable of inducing a potent systemic and airway humoral response to Pseudomonas aeruginosaMethodsLiposomes contained a peptide derived from P. aeruginosa pilin protein as B epitope, a peptide derived from Influenza hemagglutinin protein as Th epitope, the TLR agonist Pam3CAG or Pam2CAG as adjuvant, and a mannosylated lipid as dendritic cell targeting agent. These constructions were administered to mice intraperitoneally (i.p.) or intranasally (i.n.). Their immunogenicity was evaluated by measuring B epitope-specific immunoglobulins in the serum and the airways by ELISA.ResultsThe B epitope, in its native form or after substitution of a cysteine by a serine, induced high systemic IgG titers when formulated in the presence of Pam3CAG or Pam2CAG and administered i.p.. No IgA response was observed in the airways upon injection of candidate vaccines by i.p. route, whatever the B epitope or the adjuvant. However, i.n. vaccination resulted in a significant local production of IgA. Finally, the production of IgG was more rapid when mannose was incorporated.ConclusionsAll liposomal candidate vaccines tested induced the production of IgG and/or IgA directed against an immunogenic peptide from P. aeruginosa. Liposomal constructs could be attractive in the vaccination against P. aeruginosa.

Formaldehyde interferes with airway epithelium integrity and functions in a dose- and time-dependent manner

Formaldehyde (HCHO) is a common indoor air pollutant. To assess its potential role and mechanism of action in asthma, we exposed the bronchial epithelial cell lines Calu-3 and 16HBE to HCHO (70-7000 μM) according to two exposure schedules (30 min and 24 h), before measuring cell viability, necrosis and apoptosis, reactive oxygen species production, cytokine release, as well as trans-epithelial electrical resistance (TEER) of cell monolayers. Whereas exposure to HCHO for 30 min had a limited effect on cell viability, exposure for 24h to 1400-7000 μM HCHO induced a pronounced dose-dependent cell death. The important decrease in cell viability observed after 24h exposure to the highest concentrations of HCHO (1400-7000 μM) was accompanied by important LDH release and ROS production, whereas a 4h exposure to lower HCHO concentrations (350 μM) induced cell apoptosis. Also, exposure to HCHO for 30 min dose-dependently inhibited basal and lipopolysaccharide-induced interleukin-6 (IL-6) and IL-8 production by bronchial epithelial cells. As well, HCHO triggered a dose- and time-dependent decrease in TEER of Calu-3 cell monolayers. The present work demonstrates that HCHO interferes with airway epithelium integrity and functions, and may thus modulate the onset and the severity of asthma. However, importantly, conditions of exposure to HCHO, e.g. level and duration, are determinant in the nature of the effects triggered by the pollutant.

A Cationic Phospholipid–Detergent Conjugate as a New Efficient Carrier for siRNA Delivery

Nucleic acids have a huge intrinsic therapeutic potential that, however, relies heavily on the development of methods for their delivery to their intracellular target site. Cationic lipids and polymers were introduced more than twenty years ago for the delivery of DNA molecules to cells. Since then, they have become useful tools for biomedical research and have also been adopted for siRNA delivery. Their mode of action has been roughly determined. It is based on the property of adherent cell lines to easily internalize large quantities of cationic complexes in endocytic compartments, and on the incorporation of membrane-perturbing elements within internalized complexes to trigger subsequent release of the nucleic acid payload into the cytosol. Cationic lipids forming hexagonal phase or formulations of cationic lipids with the fusogenic lipid 1,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE) enable both the formation of cationic nucleic acid complexes (lipoplexes) and rupture of the endosome membrane. Yet, the endosomolytic activity of the current effective synthetic nucleic acid delivery systems remains low and effective delivery is only achieved by using cell entry pathways with a high flow rate. The membrane-disruptive properties of detergents have been considered for improving nucleic acid delivery. However, the use of detergents remains delicate because direct plasma membrane permeation might have an irreversible impact on cell viability. Cationic detergents have been shown to provoke DNA condensation but the resulting lipoplexes have turned out to be inactive or only poorly active, in vitro, when particles are prepared from cationic detergent mixed with DOPE. This is explained by the rapid exchange of the detergent molecules between the complex and the aqueous environment, which results in an irreversible decondensation of the nucleic acid even before entry into the cells. On the other hand, preparation of DNA lipoplexes from cationic lipids in the presence of Tween-80, a non-ionic detergent, has led to systems with enhanced gene transfection properties both in vitro and in vivo. These results led us to explore the consequences on nucleic acid delivery of the conjugation of a detergent molecule to a cationic lipid so that the detergent cannot be depleted from the transfection particle. We selected Triton X-100 (TX100) for its well-known detergent properties and for synthetic convenience. Furthermore, TX100 does not solubilize cholesterol-enriched raft domains, which might be involved in cationic lipoplexes internalization. TX100 was covalently linked to the phosphate group of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a major component of biological membranes (Figure 1). Diacylglycerophosphocholines (PCs) are normal cellular metabolites and offer the opportunity to easily generate cationic lipids by O-alkylation of the phosphate group. Gorman et al. first demonstrated that a PC-derived phosphotriester (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, EDMPC) can mediate efficient gene transfer. Later on, derivatives of DOPC (i.e., ethyl-DOPC, EDOPC, Figure 1) and other PCs were developed by MacDonald et al. , the physical and transfection properties of which were extensively investigated. We hypothesized that the covalent attachment of a detergent molecule and a natural phospholipid to form a cationic phospholipid–detergent conjugate might improve cell uptake and transfection efficiency of lipoplexes prepared thereof. TX100 conjugation to DOPC was realized according to Scheme 1. Detergent activation with trifluoromethanesulfonyl anhydride provided the corresponding sulfonyl ester. This compound is unstable and decomposes in a few hours on standing at room temperature in chloroform. Analysis of degradation products is consistent with a deoligomerization process leading to the formation of dioxane as reported previously with other PEG derivatives. Consequently, the sulfonyl ester (1 equiv) was directly submitted to nucleophilic displacement by the anionic phosphate in DOPC (3 equiv). Conjugate 1 was obtained in 32% yield and unreacted DOPC (61%) was recovered after purification. The use of a larger excess of activated TX100 (7 equiv) improved the DOPC conversion but finally proved detrimental as the purification of the product became difficult (1: 22%; recovered DOPC: 24%). Control compound 2 was prepared similarly, by replacing Triton X-100 by polyethylene glycol mono[a] Dr. P. Pierrat, Dr. G. Creusat, Dr. G. Laverny, Prof. F. Pons, Dr. G. Zuber, Dr. L. Lebeau Laboratoire de Conception et Application de Mol!cules Bioactives CNRS, Universit! de Strasbourg, Facult! de Pharmacie 74 Route du Rhin, BP 60024, Illkirch (France) Fax: (+33)333-6885-4306 E-mail : llebeau@unistra.fr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201103645.

A dynamic system for single and repeated exposure of airway epithelial cells to gaseous pollutants.

In vitro models are promising approaches to investigate the adverse effects and the mode of action of air pollutants on the respiratory tract. We designed a dynamic system that allows the single or repeated exposure of cultured cells to two major indoor air gaseous pollutants, formaldehyde (HCHO) and nitrogen dioxide (NO2), alone or as a mixture. In this system, the Calu-3 human bronchial epithelial cell line was exposed at the air-liquid interface (ALI) or submerged by culture medium to synthetic air or to target concentrations of HCHO and/or NO2 once or on 4 consecutive days before assessment of cell viability and necrosis, IL-6 and IL-8 release and trans-epithelial electrical resistance. Our data showed that whereas the ALI method can be used for single short-term exposures only, the submerged method provides the possibility to expose Calu-3 cells in a repeated manner. As well, we found that repeated exposures of the cells to HCHO and NO2 at concentrations that can be found indoors triggered a significant decrease in cell metabolism and an increase in IL-8 release that were not evoked by a single exposure. Thus, our work highlights the fact that the development of systems and methods that allow repeated exposures of cultured cells to gaseous compounds in mixtures is of major interest to evaluate the impact of air pollution on the respiratory tract.

Phospholipid–Detergent Conjugates as Novel Tools for siRNA Delivery

One of the potential benefits of drug delivery systems in medicine is the creation of nanoparticle-based vectors that deliver a therapeutic cargo in sufficient quantity to a target site to enable a selective effect, width of the therapeutic window depending on the toxicity of the vector and the cargo. In this work, we intended to improve the siRNA delivery efficiency of a new kind of nucleic acid carrier, which is the result of the conjugation of the membrane phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) to the membrane-active species Triton X-100 (TX100). We hypothesized that by improving the biodegradability the cytotoxicity of the conjugate might by reduced, whereas its original transfection potential would be tentatively preserved. DOPC was conjugated to Triton X-100 through spacers displaying various resistance to chemical hydrolysis and enzyme degradation. The results obtained through in vitro siRNA delivery experiments showed that the initial phosphoester bond can be replaced with a phospho(alkyl)enecarbonate group with no loss in the transfection activity, whereas the associated cytotoxicity was significantly decreased, as assessed by metabolic activity and membrane integrity measurements. The toxicity of the conjugates incorporating a phospho(alkyl)enesuccinnate moiety proved even lower but was clearly balanced with a reduction of the siRNA delivery efficiency. Hydrolytic stability and intracellular degradation of the conjugates were investigated by NMR spectroscopy and mass spectrometry. A general trend was that the more readily degraded conjugates were those with the lower toxicity. Otherwise, the phospho(alkyl)enecarbonate conjugates revealed some hemolytic activity, whereas the parent phosphoester did not. The reason why these conjugates behave differently with respect to hemolysis might be a consequence of unusual fusogenic properties and probably reflects the difference in the stability of the conjugates in the intracellular environment.