[Evaluation of the relevance of the leak water test for the control of sterilization containers]. / Évaluation de l'intérêt du test de fuite à l'eau pour le contrôle de fonctionnalité des conteneurs de stérilisation.

OBJECTIVES: The objective is to check the maintaining performance of filter barrier in sterilizations containers with the leakage rupture the junction tank/lid of the container. In order to control the good sealing performance, we use the water leak test as described in the normative document AFNOR FD S98-053. Does the leakage rupture cause the contamination of containers during the storage or transport between two sterilization units? METHOD: We use 5 sets of 3 different containers (the first without barrier system (positive control), the others with barrier system (the second with positive water test and the third with negative water test)). The containers are sterilized after having positioned triptyc soy agar (TSA) plates at the bottom. In a closed chamber test, the containers are exposed with an aerosol of Micrococcus luteus under stress of an overpressure (25, 50 or 75 mbar) simulating atmospheric pressure variations. Each triptyc soy agar (TSA) plates are incubated during 5 days at 37°C for bacterial identification. RESULTS: At 25 mbar, 74% of containers having a leakage with a water test let the microorganisms ingress despite the filter barrier. The results confirm that the overpressure increases the level of bacteria in containers. Furthermore, the containers with positive water test are more contaminated than containers with perfect sealing. The effectiveness of the sterile barrier system is evaluated by the logarithmic reduction value (LRV). CONCLUSIONS: The water test could be a good indication to evaluate the effectiveness of the sterile barrier system of rigid sterilization containers.

Biodistribution and preliminary toxicity studies of nanoparticles made of Biotransesterified ß-cyclodextrins and PEGylated phospholipids.

BACKGROUND: The modification of ß-cyclodextrins (ßCDs) by grafting alkyl chains on the primary and/or secondary face yields derivatives (ßCD-C10) able to self-organize under nanoprecipitating conditions into nanoparticles (ßCD-C10-NP) potentially useful for drug delivery. The co-nanoprecipitation of ßCD-C10 with polyethylene glycol (PEG) chains yields PEGylated NPs (ßCD-C10-PEG-NP) with potentially improved stealthiness. The objectives of the present study were to characterize the in vivo biodistribution of ßCD-C10-PEG-NP with PEG chain length of 2000 and 5000Da using nuclear imaging, and to preliminarily evaluate the in vivo acute and extended acute toxicity of the most suitable system. RESEARCH DESIGN AND METHODS: The in vivo and ex vivo biodistribution features of naked and decorated nanoparticles were investigated over time following intravenous injection of 125I-radiolabeled nanoparticles to mice. The potential toxicity of PEGylated ßCD-C10 nanosuspensions was evaluated in a preliminary in vivo toxicity study involving blood assays and tissue histology following repeated intraperitoneal injections of nanoparticles to healthy mice. RESULTS: The results indicated that ßCD-C10-PEG5000-NP presented increased stealthiness with decreased in vivo elimination and increased blood kinetics without inducing blood, kidney, spleen, and liver acute and extended acute toxicity. CONCLUSIONS: ßCD-C10-PEG5000-NPs are stealth and safe systems with potential for drug delivery.

A colorimetric nanosensor based on a selective target-responsive aptamer kissing complex.

Herein, we report a novel approach for the design of a colorimetric aptasensor based on functionalized gold nanoparticle probes. This approach relies on the conjugation of nanoparticles by two functional DNA and RNA hairpins that engage specific kissing (loop-loop) interactions in response to the addition of a small analyte ligand, leading to particle aggregation and then red-to-purple colour change of the colloidal solution.

Biodistribution of intravenously administered amphiphilic beta-cyclodextrin nanospheres.

Amphiphilic beta-cyclodextrin (betaCDa) nanospheres (mean diameter 90-110 nm) prepared by the solvent displacement method were developed as a colloidal drug delivery system. In order to survey the fate of these nanoparticles, the amphiphilic beta-cyclodextrin was first iodinated by a two-step procedure involving iodination of the primary face followed by an acylation of the secondary face. After radiolabeling of this derivative with (125)I, nanospheres made of betaCDa/betaCDa (125)I were formulated. After a single intravenous injection of labeled nanoparticles in mice, the organ distribution was analyzed from 10 min to 6 days. A rapid clearance of (125)I-labeled betaCDa nanospheres from the blood circulation to the mononuclear phagocyte system was visualized by non-invasive planar imaging study. Radioactivity measurements in organs showed that the nanospheres mainly concentrated in the liver and the spleen where 28 and 24% of the radioactivity per gram of organ was, respectively, found 10 min after injection. At the opposite, the blood activity was low at that time and become negligible thereafter. Finally, the fact that no particular sign of toxicity is observed in injected animals should be emphasized since it is the first report on intravenous administration of betaCDa nanoparticles.

Miscellaneous nanoaggregates made of beta-CD esters synthesised by an enzymatic pathway.

Various beta-cyclodextrin (beta-CD) fatty esters with different chain lengths (C4-C14) were synthesised by transesterification of beta-cyclodextrin by vinyl fatty ester using thermolysin in DMSO. For each cyclodextrin derivatives, two batches of synthesis were realized. The ability of these derivatives to form nano-organized systems was investigated through the solvent displacement technique. During the formulation step, the effects of the initial concentration of beta-CD fatty esters in the organic phase and that of the final volume of the aqueous non-solvent phase were studied. Except for the beta-CD C4 ester, the transesterified beta-CD derivatives led to measurable nanoparticles. Cryo-electron microscopy images showed a significant morphological variability. Spherical, rod-like or more irregularly-shaped nano-objects were observed with either matricial or lamellar structures. A statistical analysis by a two-way ANOVA was computed for each class of beta-cyclodextrin esters in order to determine the effects of batch and formulation on the final size of nanoparticles.

Long-term shelf stability of amphiphilic beta-cyclodextrin nanosphere suspensions monitored by dynamic light scattering and cryo-transmission electron microscopy.

Amphiphilic beta-cyclodextrins (betaCDa) were synthesized by statistically grafting hexanoyl carbon chains on the secondary hydroxyl functions of the betaCD glucopyranosyl units. The obtained derivative was used to prepare submicronic colloidal nanosphere suspensions using a nano-precipitation method. The fresh suspensions contained particles with a diameter ranging from 60-100 nm. Taking into account that the physical stability of colloidal systems remains one of the major problems which can restrict their use in pharmaceutical particulate carrier formulations, the long-term stability of the aqueous nano-dispersions was investigated. Two complementary characterization methods, namely dynamic light scattering and cryo-transmission electron microscopy, were used to control the size distribution and morphology of the nanospheres during storage. The zeta potential was measured as well. An unexpected good physical stability of the suspensions after 3 year storage at room temperature was observed. This behaviour appears to be related to the small size and structural organization of the nanoparticles. The mean diameters determined from light scattering experiments are consistent with those measured from electron micrographs. The slight difference between the values obtained by both methods is discussed.

A simple method for the two-step preparation of two pure haemorphins from a total haemoglobin peptic hydrolysate by conventional low-pressure chromatographies.

The development of a simple purification method for two haemorphins, VV-haemorphin-7 (Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe) and VV-haemorphin-4 (Val-Val-Tyr-Pro-Trp-Thr), from a total peptic haemoglobin hydrolysate is described. Cation-exchange chromatography on CM-Sephadex was used as a pre-fractionation step for the hydrolysate. VV-haemorphin-7 and VV-haemorphin-4 were eluted in two different fractions. The second and final purification step was hydrophobic-interaction chromatography on phenyl-Sepharose, which allowed the isolation of the two pure haemorphins. Haemoglobin and globin hydrolysates were compared as starting materials. For easy recovery of haemorphins and easy adjustment of conditions for final purification, a volatile buffer, ammonium acetate buffer, pH 6.5, was employed. This process, which allowed the preparation of pure haemorphins from a total protein hydrolysate, could be scaled up and used in the food industry.

Combining solvent engineering and thermodynamic modeling to enhance selectivity during monoglyceride synthesis by lipase-catalyzed esterification.

Monoglyceride synthesis by Rhyzomucor miehei lipase was investigated via direct esterification between glycerol (adsorbed onto silica gel) and oleic acid in organic solvents. The main difficulty is to avoid the unwanted production of di- and tri-glycerides. It was demonstrated that an increase in solvent polarity, using mixtures of n-hexane and 2-methyl-2-butanol (2M2B), improves drastically the selectivity toward monoglyceride formation. In pure n-hexane, the monoglyceride represents only 6 molar % of the total products at the thermodynamic equilibrium (34 and 60% for di- and tri-glyceride respectively). Use of an equivolume mixture of n-hexane/2M2B enables a product mixture to be obtained containing 94% of monoglyceride at equilibrium (2.4 and 0% for di- and tri-glyceride respectively). This positive effect is counterbalanced by a decrease both in initial velocities and in substrate conversion at thermodynamic equilibrium.A modeling, able to predict the three thermodynamic equilibria governing the 3 consecutive reactions, based on activity coefficient calculations using the UNIFAC model, is proposed. It takes into account both the partition of water between solvent and immobilized catalyst, and the partition of glycerol between solvent and silica gel. A good correlation with experimental data obtained in n-hexane/2M2B mixtures was observed.

Using an experimental design for the optimization of LVV-haemorphin-7 and VV-haemorphin-7 extraction by an organic solvent mixture in the course of bovine haemoglobin peptic hydrolysis.

Mixture design was used to improve the extraction of two opioid peptides (LVV-haemorphin-7 and VV-haemorphin-7) by water-immiscible solvents in the course of bovine haemoglobin peptic hydrolysis. Because of the loss of the peptic activity, these two haemorphins did not appear in the aqueous phase when the peptic reaction was achieved in the presence of butan-2-ol alone. We have shown that it is possible to use octan-1-ol, as a co-solvent, to recover the peptic activity. However, when the hydrolysis was achieved with octan-1-ol alone, the two haemorphins appeared in the aqueous phase, but were not extracted by the organic phase. We therefore investigated haemorphin extraction in the course of the hydrolysis reaction by a mixture of butan-2-ol and octan-1-ol. We have determined the optimal conditions with respect to the extraction of opioid peptides and the stability of the pepsin. To design a future continuous-stirred-tank reactor, we propose a biphasic system composed of 45% water, 45% butan-2-ol and 10% octan-1-ol for the extraction of the two haemorphins in the course of bovine haemoglobin peptic hydrolysis.