Didier Betbeder

Intranasal immunization with recombinant antigens associated with new cationic particles induces strong mucosal as well as systemic antibody and CTL responses

New cationic nanoparticles (SMBV) were evaluated for use as a nasal vaccine delivery system for two recombinant proteins: HBsAg and beta-galactosidase. Each protein was formulated with SMBV and intranasally administrated to non-anesthetized mice. In each model, the formulated protein induced high levels of specific serum IgG antibodies and cytotoxic T lymphocyte (CTL) responses. Moreover, specific IgA antibodies were found in nasal as well as in vaginal washes of intranasally immunized mice with the protein associated with SMBV. In contrast, no IgG or IgA antibodies and no CTL were detected in mice immunized with free protein. The detection of a CTL response and an increase in both IgG1 and IgG2a antibodies in serum suggest that SMBV amplifies both Th1 and Th2 responses without modifying the Th1/Th2 profile of the immune response induced by the natural protein. These data demonstrate the high potential of SMBV for use as a nasal delivery system for sub-unit vaccines.

Head-to-head comparison of four nonadjuvanted inactivated cell culture-derived influenza vaccines: Effect of composition, spatial organization and immunization route on the immunogenicity in a murine challenge model

In order to study the influence of antigen composition, spatial organization of antigen and the route of administration, four cell culture-derived, inactivated, nonadjuvanted influenza vaccine formulations, i.e. whole inactivated virus (WIV), split, subunit and virosome vaccines were prepared from a single antigen batch. We directly compared the immunogenicity and efficacy of these vaccine formulations after intramuscular (i.m.) or intranasal (i.n.) administration in mice. Prime and boost vaccination were followed by a potentially lethal homologous aerosol challenge. For all vaccines, the i.m. route induced higher serum humoral immune responses as compared to the i.n. route and protected all mice against challenge at a dose of 5 microg. Upon i.n. immunization only WIV and split vaccines induced detectable IgG titers and partial protection against challenge but only very low HI titers were induced in almost all mice. WIV induced mainly IgG2a/c titers via both routes, whereas split vaccine induced exclusively IgG1 titers via both routes. Subunit and virosome vaccines induced exclusively IgG1 via the i.m. route. Mucosal sIgA levels were only detected after i.n. vaccination with WIV. Furthermore, vaccines containing all viral components (WIV and split vaccine) induced higher serum HI titers and serum antibody titers than subunit and virosome vaccines. The differences in magnitude and quality of immune responses of split and WIV, having the same composition, are likely related to their distinct spatial organization. In conclusion, the direct comparison between WIV, split, subunit and virosomes, shows that the differences in immune responses between these well known influenza vaccines can be explained by both the composition and particulate structure of these vaccine formulations.

Characterization of endocytosis and exocytosis of cationic nanoparticles in airway epithelium cells

A major challenge of drug delivery using colloids via the airway is to understand the mechanism implied in their interactions with epithelial cells. The purpose of this work was to characterize the process of endocytosis and exocytosis of cationic nanoparticles (NPs) made of maltodextrin which were developed as a delivery system for antigens in vaccine applications. Confocal microscopy demonstrated that these NP are rapidly endocytosed after as little as 3 min incubation, and that the endocytosis was also faster than NP binding since most of the NPs were found in the middle of the cells around the nuclei. A saturation limit was observed after a 40 min incubation, probably due to an equilibrium becoming established between endocytosis and exocytosis. Endocytosis was dramatically reduced at 4 degrees C compared with 37 degrees C, or by NaN(3) treatment, both results suggesting an energy dependent process. Protamine pretreatment of the cells inhibited NPs uptake and we found that clathrin pathway is implied in their endocytosis. Cholesterol depletion increased NP uptake by 300% and this phenomenon was explained by the fact that cholesterol depletion totally blocked NP exocytosis. These results suggest that these cationic NPs interact with anionic sites, are quickly endocytosed via the clathrin pathway and that their exocytosis is cholesterol dependent, and are similar to those obtained in other studies with viruses such as influenza.

Study of serum interaction with a cationic nanoparticle: Implications for in vitro endocytosis, cytotoxicity and genotoxicity

We used well-characterized and positively charged nanoparticles (NP(+)) to investigate the importance of cell culture conditions, specifically the presence of serum and proteins, on NP(+) physicochemical characteristics, and the consequences for their endocytosis and genotoxicity in bronchial epithelial cells (16HBE14o-). NP(+) surface charge was significantly reduced, proportionally to NP(+)/serum and NP(+)/BSA ratios, while NP(+) size was not modified. Microscopy studies showed high endocytosis of NP(+) in 16HBE14o-, and serum/proteins impaired this internalization in a dose-dependent manner. Toxicity studies showed no cytotoxicity, even for very high doses of NP(+). No genotoxicity was observed with classic comet assay while primary oxidative DNA damage was observed when using the lesion-specific repair enzyme, formamidopyrimidine DNA-glycosylase (FPG). The micronucleus test showed NP(+) genotoxicity only for very high doses that cannot be attained in vivo. The low toxicity of these NP(+) might be explained by their high exocytosis from 16HBE14o- cells. Our results confirm the importance of serum and proteins on nanoparticles endocytosis and genotoxicity.

Airway delivery of peptides and proteins using nanoparticles.

Delivery of peptides and proteins via the airways is one of the most exciting potential applications of nanomedicine. These macromolecules could be used for many therapeutic applications, however due to their poor stability in physiological medium and difficulties in delivering them across biological barriers, they are very difficult to use in therapy. Nanoparticulate drug delivery systems have emerged as one of the most promising technologies to overcome these limitations, owing mainly to their proven capacity to cross biological barriers and to enter cells in high yields, thus improving delivery of macromolecules. In this review, we summarize the current advances in nanoparticle designed for transmucosal delivery of peptides and proteins. Challenges that must be overcome in order to derive clinical benefits are also discussed.

Development of innovative paclitaxel-loaded small PLGA nanoparticles: Study of their antiproliferative activity and their molecular interactions on prostatic cancer cells

Taxanes, including paclitaxel, are anti-cancer drugs approved for the treatment of prostate cancer but which have limited clinical application due to their hydrophobicity, their low therapeutic index and the emergence of chemoresistance. These side effects may be avoided through the use of new drug delivery systems such as nanoparticles, and paclitaxel-loaded PLGA nanoparticles up to 200 nm in size have shown encouraging results. As it is known that size affects the tissular penetration and distribution of tumors via the enhanced permeability and retention effect, so nanoparticles smaller than 100 nm are potentially interesting vehicles for improving paclitaxel delivery and efficacy. In this work, new paclitaxel-loaded small PLGA nanoparticles, between 49 nm and 95 nm in size and with positive or negative surface charges, were prepared without detergent. They were stable in the presence of serum, and HPLC showed that high paclitaxel loading and stability were achieved. Intracellular uptake of these nanoparticles was studied in PC3 cells by flow cytometry. Confocal studies confirmed a high tubulin destructuration at very low dose with these nanoparticles. This study suggests that both positively and negatively charged paclitaxel-loaded small PLGA nanoparticles deliver this drug into PC3 cells, and that this nanoparticle mode of delivery highly improves paclitaxel efficiency by up to two log-increase. These results also highlight the importance of small nanoparticles for drug delivery in cancer applications and are extremely promising for in vivo studies.

A new family of carriers (biovectors) enhances the immunogenicity of rabies antigens

Biovectors (BV) are a new family of protein carriers. They are nanoparticles of polymerized polysaccharides substituted with phosphate residues and surrounded by covalently bound lipid molecules (palmitic acid). The effect of BV was tested on the immunogenicity of rabies antigens. Biovectors enhanced the production of antibody induced by both rabies glycoprotein and ribonucleoprotein. Moreover, they enhanced the protective activity of an experimental rabies vaccine composed of inactivated and purified virus. The isotype profile of antibody produced in vivo was not modified when BV were mixed with rabies antigens. To clarify the mechanism of the adjuvant/ immunostimulation effect of BV, two types of approach were used: (1) analysis of the antibody response when antigen and BV were injected separately; (2) determination of the nature of cells involved in the proliferation in vitro of murine splenocytes in the presence of BV. The enhancing effect of BV on antibody production was highest when mixed with antigens. In vitro BV induced the proliferation of B cells. These findings suggest that BV have immunostimulating properties in addition to their probable depot and/or antigen-presentation effect which explain in part their adjuvant activity.

Stabilization and enhancement of interleukin-2 in vitro bioactivity by new carriers: supramolecular biovectors

Human recombinant interleukin-2 can be associated and released from supramolecular biovectors (SMBVs), consisting of particles made of polymerized polysaccharides. The particles are substituted with phosphate residues and contain bound lipid molecules (palmitic acid) buried near their surfaces. The association of IL-2 with SMBVs modifies its in vitro bioactivity. SMBVs prolong the growth of IL-2-dependent cells, enhance IL-2 proliferative activity and restore the activity of impaired IL-2. These properties mainly depend on the presence of lipids linked to the SMBV and on both the degree of acylation and the SMBV: IL-2 ratio. SMBVs are therefore good candidates for the stabilization and enhancement of the biological activity of IL-2.

Development of a nanoparticulate formulation of diminazene to treat African trypanosomiasis

There is a real need to develop new therapeutic strategies for African trypanosomiasis infections. In our study, we developed a new drug delivery system of diminazene (DMZ), a trypanocidal drug registered for veterinary use. This drug candidate presents a limited efficacy, a poor affinity for brain tissue and instability. The development of colloidal formulations based on a porous cationic nanoparticle with an oily core ((70)DGNP(+)), has potentially two advantages: stabilization of the drug and potential targeting of the parasite. We analyzed two processes of drug loading: in process (DMZ was added during the preparation of (70)DGNP(+) at 80 °C) and post-loading (DMZ was mixed with a (70)DGNP(+) solution at room temperature). Poor stability of the drug was observed using the in process technique. When using the post-loading technique over 80% drug entrapment efficiency was obtained at a ratio of DMZ:phospholipids (wt:wt) < 5%. Moreover, DMZ loaded into (70)DGNP(+) was found to be protected against oxidation and was stable for at least six months at 4 °C. Finally, in vitro tests on T.b. brucei showed an increased efficacy of DMZ loaded in (70)DGNP(+).

Drug delivery systems in the treatment of African trypanosomiasis infections

Introduction: Animal African trypanosomiasis (AT) is treated and controlled with homidium, isometamidium and diminazene, whereas human AT is treated with suramin, pentamidine, melarsoprol and eflornithine (DFMO), or a combination of DFMO and Nifurtimox. Monotherapy can present serious side effects, for example, melarsoprol, the more frequently used drug that is effective for both hemolymphatic and meningoencephalic stages of the disease, is so toxic that it kills 5% of treated patients. These treatments are poorly efficient, have a narrow safety index and drug resistance is a growing concern. No new drug has been developed since the discovery of DFMO in the 1970s. There is a pressing need for an effective, safe drug for both stages of the disease, and recent research is focused on the development of new formulations in order to improve their therapeutic index. Areas covered: This review shows the potential interest of using nanoparticulate formulations of trypanocidal drug to improve parasite targeting, efficacy and, potentially, safety while being cost-effective. Expert opinion: The design of drug formulations relevant to the treatment of AT must include a combination of very specific properties. In summary, the drug delivery system must be compatible with the physicochemical properties of the drug (charge, lipophilicity and molecular mass) in order to allow high drug payloads while being biocompatible for the patient.