Benoît Frisch

Embedded silver ions-containing liposomes in polyelectrolyte multilayers: Cargos films for antibacterial agents

A new antibacterial coating made of poly(L-lysine)/hyaluronic acid (PLL/HA) multilayer films and liposome aggregates loaded with silver ions was designed. Liposomes filled with an AgNO 3 solution were first aggregated by the addition of PLL in solution. The obtained micrometer-sized aggregates were then deposited on a PLL/HA multilayer film, playing the role of a spacer with the support. Finally, HA/PLL/HA capping layers were deposited on top of the architecture to form a composite AgNO 3 coating. Release of encapsulated AgNO 3 from this composite coating was followed and triggered upon temperature increase over the transition temperature of vesicles, found to be equal to 34 degrees C. After determination of the minimal inhibitory concentration (MIC) of AgNO 3 in solution, the antibacterial activity of the AgNO 3 coating was investigated against Escherichia coli. A 4-log reduction in the number of viable E. coli cells was observed after contact for 120 min with a 120 ng/cm (2) AgNO 3 coating. In comparison, no bactericidal activity was found for PLL/HA films previously dipped in an AgNO 3 solution and for PLL/HA films with liposome aggregates containing no AgNO 3 solution. The strong bactericidal effect could be linked to the diffusion of silver ions out of the AgNO 3 coating, leading to an important bactericidal concentration close to the membrane of the bacteria. A simple method to prepare antibacterial coatings loaded with a high and controlled amount of AgNO 3 is therefore proposed. This procedure is far superior to that soaking AgNO 3 or Ag nanoparticles into a coating. In principle, other small bactericidal chemicals like antibiotics could be encapsulated by this method. This study opens a new route to modify surfaces with small solutes that are not permeating phospholipid membranes below the phase transition temperature.

Protein adsorption on dopamine-melanin films: Role of electrostatic interactions inferred from (ζ-potential measurements versus chemisorption

We recently showed the possibility to build dopamine-melanin films of controlled thickness by successive immersions of a substrate in alkaline solutions of dopamine [F. Bernsmann, A. Ponche, C. Ringwald, J. Hemmerlé, J. Raya, B. Bechinger, J.-C. Voegel, P. Schaaf, V. Ball, J. Phys. Chem. C 113 (2009) 8234-8242]. In this work the structure and properties of such films are further explored. The zeta-potential of dopamine-melanin films is measured as a function of the total immersion time to build the film. It appears that the film bears a constant zeta-potential of (-39+/-3) mV after 12 immersion steps. These data are used to calculate the surface density of charged groups of the dopamine-melanin films at pH 8.5 that are mostly catechol or quinone imine chemical groups. Furthermore the zeta-potential is used to explain the adsorption of three model proteins (lysozyme, myoglobin, alpha-lactalbumin), which is monitored by quartz crystal microbalance. We come to the conclusion that protein adsorption on dopamine-melanin is not only determined by possible covalent binding between amino groups of the proteins and catechol groups of dopamine-melanin but that electrostatic interactions contribute to protein binding. Part of the adsorbed proteins can be desorbed by sodium dodecylsulfate solutions at the critical micellar concentration. The fraction of weakly bound proteins decreases with their isoelectric point. Additionally the number of available sites for covalent binding of amino groups on melanin grains is quantified.

Specific and non-specific phagocytosis of ligand-grafted PLGA microspheres by macrophages.

We evaluated the influence of ligand grafting on the rate and intensity of uptake of poly(d,l-lactide-co-glycolide) microparticles by alveolar macrophages. Microspheres with a mean diameter of 2.5 microm were obtained by spray drying. Three ligands (WGA, an RGD containing peptide and mannose-PEG(3)-NH(2)) and a cationic molecule (PLL) were covalently grafted on the particle surface using the carbodiimide method. Their grafting efficiency was quantified, and WGA grafting was characterized by confocal laser scanning microscopy (CLSM) and by atomic force microscopy (AFM). The uptake by macrophages of surface-modified microspheres was quantified by CLSM. This work showed that the uptake of negatively charged ligand-grafted microspheres (-26 to -51 mV) was increased up to two to four times according to the ligand compared to ungrafted microspheres (-81 mV) and displayed saturation as opposed to the cationic PLL-grafted microspheres. Moreover, a specific receptor-mediated phagocytosis mechanism was suggested based on free ligand, cytochalasin D and +4 degrees C incubation that decreased the microparticle uptake. Furthermore, this work clearly showed that the relative contribution of specific and non-specific processes to the overall uptake varied greatly according to the ligands, and was dependent on the particle-to-cell ratio. In conclusion, this work showed that ligand grafting can enhance the uptake of microparticles, with a variable relative contribution of specific and non-specific uptake mechanism.

Immunogenicity of new heterobifunctional cross-linking reagents used in the conjugation of synthetic peptides to liposomes

We have investigated the immunogenicity of six thiol-reactive heterobifunctional cross-linking reagents that permit the conjugation of cysteine carrying peptides to the surface of liposome containing monophosphoryl lipid A. Such constructs elicit an immune response against short synthetic peptides and our aim was to find the least immunogenic linkers to limit potential carrier-induced epitopic suppression. For that purpose the properties of three new polyoxyethylene linkers of different lengths and thiol-reactive moieties (maleimide, bromoacetyl, dithiopyridine) were compared to known derivatives obtained by reacting the classical reagents SMPB and SPDP or N-succinimidyl bromoacetate with phosphatidylethanolamine. The least immunogenic linkers were the bromoacetate derivatives whereas those containing a maleimide group evoked a significant anti-linker immune response. In addition, using IRGERA as a model peptide, we found that all six liposomal constructs strongly elicited the production of anti-peptide IgG antibodies. This immune response was therefore independent of the length of the linkers (ranging between 0.3 and 1.6 nm) and of the nature of the linkage. between the peptide and the thiol-reactive moieties of the cross-linkers, i.e. stable thioether or bio-reducible disulfide bonds.

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-1β 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-1β 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.

Antitumor activity of liposomal ErbB2/HER2 epitope peptide-based vaccine constructs incorporating TLR agonists and mannose receptor targeting

Synthetic and molecularly defined constructs containing the minimal components to mimic and amplify the physiological immune response are able to induce an efficient cytotoxic response. In the current study this approach was applied to the development of highly versatile liposomal constructs to co-deliver peptide epitopes in combination with TLR agonists in order to induce a specific anti-tumor cellular immune response against ErbB2 protein-expressing tumor cells. Liposomes containing ErbB2 p63-71 cytotoxic T lymphocyte (CTL) and HA307-319 T- helper (Th) peptide epitopes associated to innovative synthetic TLR2/1 (Pam(3)CAG) or TLR2/6 agonists (Pam(2)CAG and Pam(2)CGD), were injected in mice bearing ErbB2 protein-expressing tumor cells. Mannosylated ligands were also incorporated into the constructs to target antigen-presenting cells. We showed that the TLR2/6 agonists were more efficient than the TLR2/1 agonists for the eradication of tumors expressing ErbB2 protein. Furthermore, mannose-targeted liposomes displayed higher therapeutic efficiency against tumor allowing treatment with decreased quantities of both TLR ligands and peptide epitopes. Our results validated that antigen-associated mannosylated liposomes combined with efficient TLR ligands are effective vectors for vaccination against tumor. In this study we developed useful tools to evaluate the vaccination efficiency of various adjuvants and/or targeting molecules and their potential synergy.

Influence of Ligand Valency on the Targeting of Immature Human Dendritic Cells by Mannosylated Liposomes

An important challenge for the development of new generations of vaccines is the efficient delivery of antigens to antigen presenting cells such as dendritic cells. In the present study we compare the interaction of plain and targeted liposomes, containing mono-, di-, and tetraantennary mannosyl lipid derivatives, with human monocyte-derived immature dendritic cells (iDCs). Whereas efficient mannose receptor-mediated endocytosis by iDCs was observed for the mannosylated liposomes, in contrast, only nonspecific interaction with little uptake was observed with plain liposomes. In accordance with the clustering effect, liposomes prepared with multibranched mannosylated lipids displayed higher binding affinity for the mannose receptor than vesicles containing the monomannosylated analogs. Importantly, we have found that dimannosylated ligands present at the surface of the liposomes were as efficient as tetramannosylated ones to engage in multidentate interactions with the mannose receptor of iDCs, resulting in both cases in an effective uptake/endocytosis. This result will greatly facilitate, from a practical standpoint, the design of mannose-targeted vaccination constructs. Moreover, we showed that mannose-mediated uptake of liposomes did not result in an activation of iDCs. Altogether, our results suggest that antigen-associated targeted liposomes containing diantennary mannosylated lipids could be effective vectors for vaccines when combined with additional DC activation signals.

On the Benefits of Rubbing Salt in the Cut: Self‐Healing of Saloplastic PAA/PAH Compact Polyelectrolyte Complexes

The inherent room temperature mending and self-healing properties of saloplastic PAA/PAH CoPECs are studied. After ultracentrifugation of PAA/PAH polyelectrolyte complexes, tough, elastic materials are obtained that undergo self-healing facilitated by salt. At intermediate salt concentrations the CoPECs remain elastic enough to recover their original shape while the chains are mobile enough to repair the cut, thus leading to actual self-healing behavior.

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.

Lectin-Mediated Drug Targeting: Selection of Valency, Sugar Type (Gal/Lac), and Spacer Length for Cluster Glycosides as Parameters to Distinguish Ligand Binding to C-Type Asialoglycoprotein Receptors and Galectins

AbstractPurpose. Common oligosaccharides of cellularglycoconjugates are ligands for more than one type of endogenous lectin.Overlapping specificities to β-galactosides of C-type lectins andgalectins can reduce target selectivity of carbohydrate-ligand-dependentdrug targeting. The purpose of this study is to explore distinct features ofligand presentation and structure for design of cluster glycosides todistinguish between asialoglycoprotein-specific (C-type) lectins andgalectins. Methods. Extent of binding of labeled sugar receptors totwo types of matrix-immobilized (neo)glycoproteins and to cells wasevaluated in the absence and presence of competitive inhibitors. This panelcomprised synthetic mono-, bi-, and trivalent glycosides with two spacerlengths and galactose or lactose as ligand part. Results. In contrast to C-type lectins of hepatocytes andmacrophages, bi- and trivalent glycosides do not yield a notable glycosidecluster effect for galectins-1 and -3. Also, theseCa2+-independent galactoside-binding proteins prefer to homein on lactose-bearing glycosides relative to galactose as ligand, whilespacer length requirements were rather similar. Conclusions. Trivalent cluster glycosides with Gal/GalNAcas ligand markedly distinguish between C-type lectins and galectins.Undesired side reactivities to galectins for C-type lectin drug deliverywill thus be minimal.