Encapsulation of live marine bacteria for use in aquaculture facilities and process evaluation using response surface methodology.

New strategies are being proposed in marine aquaculture to use marine bacteria as alternative to antibiotics, as nutritional additive or as immune-stimulant. These approaches are particularly promising for larval and juvenile cultures. In many cases, the bacteria are released in the seawater, where they have to be at appropriate concentrations. In addition, only low-cost technologies are sustainable for this industry, without any complex requirements for use or storage. In this work, we explore the possibilities of preservation of a potential marine probiotic bacterium (Phaeobacter PP-154) as a product suitable for use in marine aquaculture by addition to the seawater. A method which guaranteed the preservation of the viable marine bacteria in a saline medium and their rapid release in the seawater was searched for. In a previous step, classical procedures (freeze-drying and freezing) had been explored, but undesirable results of the interaction of the products obtained with natural seawater led to investigate alternatives. We report the results of the immobilization of the marine bacteria in calcium alginate beads. The final product complies the salinity which allows the requirements of the bacteria without interference with alginate in the formation of beads, and a balanced hardness to retain the bacteria and to be easily released in the marine aquaculture environment. The process was evaluated using the central composite rotatable design (CCRD), a standard response surface methodology (RSM).

Proteomic Analysis in Morquio A Cells Treated with Immobilized Enzymatic Replacement Therapy on Nanostructured Lipid Systems.

Morquio A syndrome, or mucopolysaccharidosis type IVA (MPS IVA), is a lysosomal storage disease due to mutations in the N-acetylgalactosamine-6-sulfatase (GALNS) gene. Systemic skeletal dysplasia and the related clinical features of MPS IVA are due to disruption of cartilage and its extracellular matrix, leading to an imbalance of growth. Enzyme replacement therapy (ERT) with recombinant human GALNS, alpha elosulfase, provides a systemic treatment. However, this therapy has a limited impact on skeletal dysplasia because the infused enzyme cannot penetrate cartilage and bone. Therefore, an alternative therapeutic approach to reach the cartilage is an unmet challenge. We have developed a new drug delivery system based on a nanostructure lipid carrier with the capacity to immobilize enzymes used for ERT and to target the lysosomes. This study aimed to assess the effect of the encapsulated enzyme in this new delivery system, using in vitro proteomic technology. We found a greater internalization of the enzyme carried by nanoparticles inside the cells and an improvement of cellular protein routes previously impaired by the disease, compared with conventional ERT. This is the first qualitative and quantitative proteomic assay that demonstrates the advantages of a new delivery system to improve the MPS IVA ERT.

Acrylic/cyclodextrin hydrogels with enhanced drug loading and sustained release capability.

The influence of the proportion of acrylamidomethyl-gamma-cyclodextrin (gamma-CD-NMA) on loading and release of the hydrophobic triamcinolone acetonide (TA) and the hydrophilic propranolol (PR) by acrylic acid hydrogels was evaluated. gamma-CD-NMA was synthesized by condensation of gamma-cyclodextrin (gamma-CD) with N-(hydroxymethyl) acrylamide. Hydrogels were prepared with gamma-CD-NMA and sodium acrylate (3 M or 4 M), using N,N'-methylen(bisacrylamide) (BIS) as cross-linker, by free radical polymerization into glass moulds of 2 mm wide and were cut as discs (10 mm diameter). gamma-CD-NMA did not modify the pH-dependent swelling of the hydrogels, but significantly increased the swelling degree in the 40:60 ethanol:water, medium in which TA can be dissolved. Hydrogels prepared with gamma-CD-NMA above 5% (w/w of total monomers) showed a remarkably higher capacity to load TA, e.g., 33 mg/g dry hydrogel versus 0.6 mg/g dry hydrogel without gamma-CD-NMA. This is explained by the formation of 1:1 inclusion complexes of TA with gamma-CD mers that overcomes the lack of interactions with the acrylic groups of the network. The release of TA in water, 0.1 N HCl, or pH 6.8 phosphate buffer was sustained for at least 24 h, whatever the pH and the composition of the medium used. In contrast, loading of PR from the water solutions was greater for hydrogels prepared with 3 M acrylate than with 4 M acrylate, irrespective to their content in gamma-CD-NMA, and in less than 2 h ca. 80% PR was released. The lower affinity of PR for the gamma-CD cavities, compared to the strong intensity of the electrostatic interactions with the acrylic acid groups, explains why the incorporation of gamma-CD-NMA did not increased the loading and control release capacity of the hydrogels of this hydrophilic drug. In summary, the copolymerisation of CD with acrylic monomers can provide highly hydrophilic pH-sensitive networks which load large amounts of hydrophobic drugs and release them in a sustained way.