Protection against Clostridium difficile infection in a hamster model by oral vaccination using flagellin FliC-loaded pectin beads.

Clostridium difficile flagellin FliC is a highly immunogenic pathogen-associated molecular pattern playing a key role in C. difficile pathogenesis and gut colonization. Here, we designed an oral vaccine against C. difficile with FliC encapsulated into pectin beads for colonic release. Bead stability and FliC retention was confirmed in vitro using simulated intestinal media (SIM), while bead degradation and FliC release was observed upon incubation in simulated colonic media (SCM). The importance of FliC encapsulation into pectin beads for protection against C. difficile was assessed in a vaccination assay using a lethal hamster model of C. difficile infection. Three groups of hamsters orally received either FliC-loaded beads or unloaded beads in gastro-resistant capsule to limit gastric degradation or free FliC. Two other groups were immunized with free FliC, one intra-rectally and the other intra-peritoneally. Hamsters were then challenged with a lethal dose of C. difficile VPI 10463. Fifty percent of hamsters orally immunized with FliC-loaded beads survived whereas all hamsters orally immunized with free FliC died within 7 days post challenge. No significant protection was observed in the other groups. Only intra-peritoneally immunized hamsters presented anti-FliC IgG antibodies in sera after immunizations. These results suggest that an oral immunization with FliC-loaded beads probably induced a mucosal immune response, therefore providing a protective effect. This study confirms the importance of FliC encapsulation into pectin beads for a protective oral vaccine against C. difficile.

Parameters for Stable Water-in-Oil Lipiodol Emulsion for Liver Trans-Arterial Chemo-Eembolization.

PURPOSE: Water-in-oil type and stability are important properties for Lipiodol emulsions during conventional trans-arterial chemo-embolization. Our purpose is to evaluate the influence of 3 technical parameters on those properties. MATERIALS AND METHODS: The Lipiodol emulsions have been formulated by repetitive back-and-forth pumping of two 10-ml syringes through a 3-way stopcock. Three parameters were compared: Lipiodol/doxorubicin ratio (2/1 vs. 3/1), doxorubicin concentration (10 vs. 20 mg/ml) and speed of incorporation of doxorubicin in Lipiodol (bolus vs. incremental vs. continuous). The percentage of water-in-oil emulsion obtained and the duration until complete coalescence (stability) for water-in-oil emulsions were, respectively, evaluated with the drop-test and static light scattering technique (Turbiscan). RESULTS: Among the 48 emulsions formulated, 32 emulsions (67%) were water-in-oil. The percentage of water-in-oil emulsions obtained was significantly higher for incremental (94%) and for continuous (100%) injections compared to bolus injection (6%) of doxorubicin. Emulsion type was neither influenced by Lipiodol/doxorubicin ratio nor by doxorubicin concentration. The mean stability of water-in-oil emulsions was 215 ± 257 min. The emulsions stability was significantly longer when formulated using continuous compared to incremental injection (326 ± 309 vs. 96 ± 101 min, p = 0.018) and using 3/1 compared to 2/1 ratio of Lipiodol/doxorubicin (372 ± 276 vs. 47 ± 43 min, p = <0.0001). Stability was not influenced by the doxorubicin concentration. CONCLUSION: The continuous and incremental injections of doxorubicin in the Lipiodol result in highly predictable water-in-oil emulsion type. It also demonstrates a significant increase in stability compared to bolus injection. Higher ratio of Lipiodol/doxorubicin is a critical parameter for emulsion stability too.

Beads made of α-cyclodextrin and soybean oil: the drying method influences bead properties and drug release.

Freeze-dried beads made of α-cyclodextrin and soybean oil were reported previously as an efficient system for the oral delivery of lipophilic drugs. In the present study, oven-drying was evaluated as another method for drying beads. Oven-drying was optimised and the properties of the resulting beads were assessed. The behavior of oven-dried beads and the release of indomethacin from these beads were evaluated in vitro in simulated gastrointestinal fluids and compared with those of freeze-dried beads. The stability of freeze-dried and oven-dried unloaded beads stored at 25°C for 12 months and at 40°C for 6 months in closed and open vials was also studied by different techniques. An oven-drying time of 6 hours at 25°C was chosen as optimal conditions. Oven-dried beads exhibited a sticky texture making them difficult to handle. They were harder, less fragile and smaller than the freeze-dried ones. The characteristics of oven-dried beads make them more resistant in vitro even in media containing bile salt. The rate of indomethacin release from oven-dried beads was much slower than that from the freeze-dried ones. Whatever the drying method, beads must be stored at room temperature protected from humidity. However, no products of oil degradation were detected with both kinds of beads. This work clearly emphasized that the drying method of the beads had a strong influence on their properties, behavior in simulated gastrointestinal fluids and drug release.

Formulations based on alpha cyclodextrin and soybean oil: an approach to modulate the oral release of lipophilic drugs.

The purpose of this work was to investigate the potential of α-cyclodextrin combined to soybean oil-based formulations to modulate the release of a model drug, indomethacin. Dry emulsion, naked and coated beads were prepared from the same initial formulation using the same manufacturing process. Dry emulsion was selected to accelerate drug release while beads coated with α-cyclodextrin were designed to sustain it. Indomethacin-loaded systems were prepared, characterised and evaluated in vitro. Pharmacokinetic studies were performed in fasted and fed rats. The presence of the α-cyclodextrin coat was confirmed by confocal microscopy, and an increase of the mass and diameter of the beads. The layer of α-cyclodextrin improved their resistance in simulated gastro-intestinal fluids. In vitro, the dissolution of indomethacin was slower with coated beads than with emulsion and naked beads. Lipid-based formulations showed an increase of relative bioavailability of IND versus Indocid®. Whatever the formulation, greater and faster release of indomethacin was noticed in sodium taurocholate-rich medium and in fed rats. Compared to naked beads, an increased Cp(max) with a shorter T(max) was observed with the emulsion while T(max) and MRT were increased and Cp(max) reduced with the coated beads. Interestingly, formulations based on alpha cyclodextrin and soybean oil can modify the release of a lipophilic drug depending on the system formed.

Beads made of cyclodextrin and oil for the oral delivery of lipophilic drugs: in vitro studies in simulated gastro-intestinal fluids.

The aim of this work was to investigate the stability in vitro, in simulated gastro-intestinal fluids, of beads, made of α-cyclodextrin and soybean oil, and to study the release of progesterone, a model of lipophilic drug. This was evaluated over time by the monitoring of the proportion of intact beads, their volume and the percentage of progesterone dissolved. Their incubation in the simulated gastric fluid provoked a moderate reduction of their number (20%) and a decrease of their volume (50%) after 55 min. Whatever the intestinal medium subsequently introduced, bead number and volume decreased more until bead disintegration that appeared faster in sodium taurocholate rich-medium. In such fluid, the amount of progesterone dissolved increased rapidly between 65 and 180 min, with both beads and emulsion to be equal after 85 min. With soft capsules, the increase was more gradual. In sodium taurocholate free-medium, more progesterone was dissolved from the emulsion than from beads or soft capsules. The release of progesterone from beads resulted from the erosion of their matrix and its partition equilibrium between oily micro-droplets and aqueous phase. The original structure of beads confers to this multiparticulate system interesting properties for the oral delivery of lipophilic drugs.

alpha-Cyclodextrin/oil beads: an innovative self-assembling system.

The aim of this work was to characterise a new type of particulate system, named beads, prepared by a straightforward technique starting from a mixture of alpha-cyclodextrin aqueous solution and soybean oil without the use of any organic solvent or surface-active agent. Mechanisms involved in bead formation were also investigated. Optimal ratio between alpha-cyclodextrin (6%, w/w), soybean oil (19.6%, w/w) and water (74.4%, w/w) led to homogeneous bead size (1.6 mm) with a fabrication yield superior to 80% after a continuous external shaking during 2.5 days. After freeze-drying, oil and alpha-cyclodextrin contents were estimated at 80% (w/w) and 20% (w/w), respectively. X-ray diffraction studies revealed that beads presented a crystalline organisation and microscopic techniques showed that their inner structure was constituted by a matrix containing oily compartments. Beads offer interesting prospects for the microencapsulation of lipophilic and poorly stable molecules. Due to their semi-solid consistency and their ability to be freeze-dried, these beads have great potentialities for pharmaceutical (oral and topical routes) and cosmetic applications.

Protective immunity against Salmonella typhimurium elicited in mice by oral vaccination with phosphorylcholine encapsulated in poly(DL-lactide-co-glycolide) microspheres.

Encapsulation of vaccines in biodegradable microspheres provides excellent mucosal immunogens with a high potential for immunization against bacterial infections. We tested the protective immunity elicited by intragastric vaccination with phosphorylcholine (PC) encapsulated in poly(DL-lactide-co-glycolide) (DL-PLG) microspheres against Salmonella typhimurium in a mouse model of invasive intestinal infection. We chose PC as the antigen because it was found to elicit an immune response after intestinal exposure of mice to PC-bearing S. typhimurium and because anti-PC immunity protects mice against Streptococcus pneumoniae, another PC-bearing microorganism. Mice were primed intragastrically on days 1, 2, and 3 and boosted on days 28, 29, and 30 with PC (280 microg) coupled to porcine thyroglobulin (PC-thyr) encapsulated in DL-PLG microspheres, free PC-thyr, or blank microspheres. A significant rise in anti-PC immunoglobulin A (IgA) titers, as measured by an enzyme-linked immunosorbent assay, was observed in the intestinal secretions after immunization with PC-loaded microspheres, compared to the titers of mice immunized with free PC-thyr or blank microspheres. This antibody response peaked 14 days after the last boost and correlated with a highly significant resistance to oral challenge by S. typhimurium C5 (P < 10(-3)). Control mice were primed intraperitoneally on day 1 with 15 microg of PC in complete Freund's adjuvant and boosted on days 10, 14, and 20 with the same dose without adjuvant but via the same route. In these mice, the levels of anti-PC IgA in intestinal secretions were equivalent to those of the mice intragastrically immunized with PC-loaded microspheres, but protection was significantly weaker, suggesting that either the IgAs were not functional or that other immune mechanisms are important in protection. Taken together, our results highlight the potential of antigen encapsulation in DL-PLG microspheres for eliciting protective immunity against invasive intestinal bacterial diseases and suggest that a similar strategy could be used against diseases caused by other PC-bearing microorganisms.

Stimulation of movement and acrosome reaction of human spermatozoa by PC12 liposomes encapsulating ATP.

The effect of co-incubating human spermatozoa with 8 mmol/L dilauroylphosphatidylcholine (PC12) liposomes containing 6 mmol/L adenosine 5'-triphosphate (LATP) was assessed by CASA and compared to that obtained with blank PC12 liposomes (LB). The aim of this study was to investigate if such treatments can improve sperm movement and sustain sperm motility over time. Significant and similar increases in straight-line velocity and linearity of sperm movement in B2 capacitating medium (both p < 0.01) were obtained with LB and LATP treatments (final concentration: 0.38 mmol/L PC12 and 0.5 mmol/L ATP) while in Tyrode's medium supplemented with 10 mg/mL BSA, these movement parameters were increased significantly only in sperm aliquots treated with LATP. Furthermore, after incubation for 0.5 h in Tyrode's, a bioluminescence assay of intracellular ATP indicated no significant change in ATP concentration for LATP-treated spermatozoa while the ATP content of control and LB-treated spermatozoa decreased significantly during the same period (both p < 0.05). The effect of liposomes on the acrosome reaction was also investigated jointly with CASA. These experiments were performed by fluorescence microscopy, using PSA-FITC and the supravital stain Hoechst 33258. After a precapacitation period of 3 h in BWW medium the spermatozoa were incubated for 1 h with LATP, LB, LB+free ATP and free ATP alone (final concentration 0.5 mmol/L ATP). Under these conditions the percentage of acrosome-reacted spermatozoa was increased similarly after LATP and LB treatments compared to control (respectively from 4.9 to 12%, p < 0.01 and 4.9 to 11.3%, p < 0.05) but the percentage of true acrosome-reacted spermatozoa, and the values for all movement characteristics (except percentage motility) were increased significantly only with LATP treatment. The results indicate the potential of PC12 vesicles for introducing highly hydrophilic compounds into spermatozoa, as well as for modulating membrane structures and functions required for fertilization.

Liposomes, an interesting tool to deliver a bioenergetic substrate (ATP). in vitro and in vivo studies.

Adenosine triphosphate (ATP) was proposed in various medical applications, as a possible bioenergetic substrate. Unfortunately, ATP is very difficult to use at a therapeutic level because of its high sensitivity to enzymatic hydrolysis making this molecule unstable in biological fluids. ATP is also a highly hydrophilic molecule that is unable to cross biological membranes. To try to develop a system able to protect ATP against degradation and to efficiently deliver this bioenergetic substrate, its liposomal encapsulation in multilamellar vesicles was carried out. One of the studies described in this paper deals with the efficiency of liposomal ATP in the treatment of cerebral ischemia. Our results show that encapsulation was able to protect ATP from its degradation by ectonucleotidases and that liposomal ATP was active against experimental brain ischemia. The other study deals with the effect of ATP on the motility and the acrosomal reaction of human spermatozoa. The results show that co-incubating ATP-loaded liposomes with sperm cells was able to induce the process of capacitation in vitro and might therefore be a useful tool in the procedure of in vitro fertilization.