Enhancement of protective efficacy following intranasal immunization with vaccine plus a nontoxic LTK63 mutant delivered with nanoparticles.

Most vaccines are still given parenterally. Mucosal vaccination would offer different advantages over parenteral immunization, including blocking of the pathogens at the portal of entry. In this paper, nontoxic Escherichia coli heat-labile enterotoxin (LT) mutants and Supramolecular Biovector systems (SMBV) were evaluated in mice as mucosal adjuvants and delivery systems, respectively, for intranasal immunization with the conjugated group C meningococcal vaccine. The conjugated vaccine formulated together with the LT mutants and the SMBV induced very high titers of serum and mucosal antibodies specific for the group C meningococcal polysaccharide. This vaccination strategy also induced high titers of antibodies with bactericidal activity, which is known to correlate with efficacy. Importantly, the mucosal vaccination, but not the conventional parenteral vaccination, induced bactericidal antibodies at the mucosal level. These data strongly support the feasibility of development of intranasal vaccines with an enhanced protective efficacy against meningococci and possibly against other encapsulated bacteria.

Development of a new method to characterize (SMBV) antigen formulations using surface plasmon resonance technology.

Supra Molecular Biovectors (SMBV) are nanoparticles composed by a polysaccharidic core surrounded by a lipid bilayer. They are designed for drug delivery and vaccine and can be administrated by nasal route. The association rate and the stability of association between active principle (AP) or antigens (Ag) with SMBV can be evaluated using the plasmon resonance technology using a BIAcore X system and a HPA hydrophobic sensor chip. AP, Ag and/or adjuvant molecule solutions are injected over SMBV saturated HPA sensor chip surface. Using a very small quantity of material, this technique allows us to quickly have an overview of complex formulations using SMBV. It is also the fastest screening technique to select the best SMBV for each Ag and the best formulation process.

Intracellular distribution of ampicillin in murine macrophages infected with Salmonella typhimurium and treated with (3H)ampicillin-loaded nanoparticles.

The intracellular distribution of (3H)ampicillin-loaded polyisohexylcyanoacrylate nanoparticles was studied in murine macrophages (peritoneal cells and the J774 cell line) infected by Salmonella typhimurium C5, using ultrastructural autoradiography. Ampicillin penetration and retention into the cells obviously increased by means of nanoparticles. After short-term (2-4 h) treatment with the nanoparticle formulation, numerous intracellular bacteria were seen to be in the process of destruction. The tritium labelling was located in the cell cytoplasm and inside vacuoles in which bacteria undergoing degradation were often present. After long-term (12 h) treatment, numerous spherical bodies (d: 100 nm to 500 nm) and larger forms (2 microns) were seen in the vacuoles. Radioactivity was mainly found to be localized on the spherical bodies, indicating marked damaging action of the ampicillin on the bacterial walls. The targeting of ampicillin therefore allowed its penetration into the macrophages and vacuoles infected with S. typhimurium.

A new method to isolate polyalkylcyanoacrylate nanoparticle preparations.

A simple method for the separation of polyalkylcyanoacrylate nanoparticles was developed using polyisohexylcyanoacrylate (PIHCA) as a model. Fluorescein isothiocyanate dextran 70 was used to label the nanoparticles. Ultracentrifugation onto a performed sucrose gradient allowed the easy elimination of the dextran and of the free molecules remaining in the upper phase. After such treatment, the physicochemical characteristics of the PIHCA nanoparticles were not modified. This method could be usefully extended to other types of nanoparticles.

The uptake of ampicillin-loaded nanoparticles by murine macrophages infected with Salmonella typhimurium.

The purpose of this study was to investigate the in-vitro interaction between [3H]ampicillin-loaded polyisohexylcyanoacrylate nanoparticles and murine macrophages (peritoneal and J774) infected with Salmonella typhimurium. The multiplicity of infection was ten bacteria to each macrophage and the mean (+/- S.D.) diameter of the nanoparticles was 220 (+/- 20 nm), corresponding to an ampicillin concentration of 2 g/L. The uptake of nanoparticle-bound [3H]ampicillin by non-infected J774 and peritoneal macrophages was six- and 24-fold greater respectively than that of free [3H]ampicillin. For infected cells, uptake by J774 and peritoneal macrophages was nine- and 20-fold greater respectively. However, there was no difference between nanoparticle-bound ampicillin and free ampicillin in terms of bactericidal activity against intracellular S. typhimurium. This unexpected observation might be accounted for by bacterium-induced inhibition of phagosome-lyosome fusion within the macrophages, thereby preventing contact between the bacteria in the phagosomes and the nanoparticles in the secondary lysosomes.

Intracellular visualization of ampicillin-loaded nanoparticles in peritoneal macrophages infected in vitro with Salmonella typhimurium.

Intracellular targeting of ampicillin by means of polyisohexylcyanoacrylate (PIHCA) nanoparticles was studied in murine peritoneal macrophages infected with Salmonella typhimurium. The intracellular distribution of actively endocytosed nanoparticles was visualized by transmission electron microscopy and confocal microscopy. Nanoparticles were either isolated or closely associated with bacteria within phagosomes or phagolysosomes. Thus the potential of ampicillin-loaded nanoparticles in targeting of intracellular bacteria is demonstrated. Consequently, ampicillin, which usually penetrates into cells at a low level, is directly carried in, when loaded on nanoparticles, and brought into contact with intracellular bacteria.