Properties of capsaicinoids for the control of fungi and oomycetes pathogenic to pepper.

Capsaicinoids are pungent compounds found in pepper (Capsicum spp.) fruits. Capsaicin showed antimicrobial activity in plate assays against seven isolates of five species of fungi and nine isolates of two species of oomycetes. The general trend was that oomycetes were more inhibited than fungi. Assays of capsaicin biosynthetic precursors suggest that the lateral chain of capsaicinoids has more inhibitory activity than the phenolic part. In planta tests of capsaicinoids (capsaicin and N-vanillylnonanamide) applied to the roots demonstrated that these compounds conferred protection against the pathogenic fungus Verticillium dahliae and induced both chitinase activity and expression of several defence-related genes, such as CASC1, CACHI2 and CABGLU. N-Vanillylnonanamide infiltrated into cotyledons confers systemic protection to the upper leaves of pepper against the fungal pathogen Botrytis cinerea. In wild-type tomato plants such cotyledon infiltration has no protective effect, but is effective in the Never-ripe tomato mutant impaired in ethylene response. A similar effect was observed in tomato after salicylic acid infiltration.

Chitosan nanocapsules as carriers for oral peptide delivery: effect of chitosan molecular weight and type of salt on the in vitro behaviour and in vivo effectiveness.

We have recently reported preliminary data showing the efficacy of chitosan nanocapsules as carriers for oral peptide delivery. In the present work, our aim was to investigate the influence of some chitosan properties, such as the molecular weight and type of salt, on the interaction of these nanocapsules with the Caco-2 cells and also on their in vivo effectiveness. Chitosan nanocapsules were prepared by the solvent displacement technique using high (450 kDa) and medium (160 kDa) molecular weight chitosan glutamate as well as high molecular weight chitosan hydrochloride (270 kDa). The results indicated that the size of the nanocapsules was dependent on the chitosan molecular weight, whereas the zeta potential and the association efficiency of salmon calcitonin were not affected by the chitosan properties. Upon incubation with the Caco-2 cells, chitosan nanocapsules exhibited a dose-dependent cellular viability, which was hardly affected by, either the chitosan molecular weight or, the type of salt. In addition, it was observed that the transepithelial electrical resistance of the Caco-2 monolayer was not significantly modified upon their exposure to chitosan nanocapsules. The results of the in vivo studies, following oral administration to rats, indicated that chitosan nanocapsules were able to reduce significantly the serum calcium levels, and to prolong this reduction for at least 24 hours, irrespective of the type of chitosan salt and molecular weight of chitosan. Consequently, the performance of chitosan nanocapsules as oral carriers for salmon calcitonin was not affected by the characteristics of chitosan.

Efficacy and mechanism of action of chitosan nanocapsules for oral peptide delivery.

PURPOSE: We have previously shown that high molecular weight (MW > 100 kDa) chitosan nanocapsules are efficient vehicles for improving the oral absorption of salmon calcitonin (sCT). In the present work, our objectives were, first, to investigate the influence of some formulation parameters on the efficacy of chitosan nanocapsules as carriers for the oral administration of sCT and, second, to elucidate the mechanism of interaction of chitosan nanocapsules with intestinal model cell lines. METHODS: sCT-loaded chitosan nanocapsules were prepared by the solvent displacement technique. They were characterized for their size, zeta potential, and sCT loading. The ability of chitosan nanocapsules to enhance the oral absorption of sCT was investigated in rats by monitoring the serum calcium levels. Finally, the mechanism of interaction of chitosan nanocapsules with the Caco-2 cell model or in the coculture of Caco-2 with HT29-M6 cells was investigated by confocal fluorescence microscopy. RESULTS: Chitosan nanocapsules presented a particle size in nanometer range, a positive surface charge, and an efficient encapsulation of sCT. Following oral administration to rats, all formulations of nanocapsules exhibited the ability to reduce calcemia levels; however, the intensity of the response varied depending on the formulation conditions. With regard to the mechanism of interaction of chitosan nanocapsules with cell culture, the xz images evidenced that chitosan nanocapsules interact and remain associated to the apical side of both model cell cultures. In addition, chitosan nanocapsules showed a preferable association to the mucus-secreting cells (HT29-M6). CONCLUSIONS: Chitosan nanocapsules are able to enhance and prolong the intestinal absorption of sCT and this effect could be mainly ascribed to their mucoadhesive character and intimate interaction with the intestinal barrier.

Chitosan-PEG nanocapsules as new carriers for oral peptide delivery. Effect of chitosan pegylation degree.

We have previously reported the ability of chitosan nanocapsules to enhance and prolong the oral absorption of peptides. In the present work, our goal was to design a new type of nanocapsules, using chitosan chemically modified with poly(ethylene glycol) (PEG) (0.5% and 1% pegylation degree) and to investigate the consequences of this modification on the in vitro and in vivo behaviour of the nanocapsules. Chitosan-PEG nanocapsules and the control PEG-coated nanoemulsions were obtained by the solvent displacement technique. Their size was in the range of 160-250 nm. Their zeta potential was greatly affected by the nature of the coating, being positive for chitosan-PEG nanocapsules and negative in the case of PEG-coated nanoemulsions. The presence of PEG, whether alone or grafted to chitosan, improved the stability of the nanocapsules in the gastrointestinal fluids. Using the Caco-2 model cell line it was observed that the pegylation of chitosan reduced the cytotoxicity of the nanocapsules. In addition, these nanocapsules did not cause a significant change in the transepithelial resistance of the monolayer. Finally, the results of the in vivo studies showed the capacity of chitosan-PEG nanocapsules to enhance and prolong the intestinal absorption of salmon calcitonin. Additionally, they indicated that the pegylation degree affected the in vivo performance of the nanocapsules. Therefore, by modulating the pegylation degree of chitosan, it was possible to obtain nanocapsules with a good stability, a low cytotoxicity and with absorption enhancing properties.

A comparative study of the potential of solid triglyceride nanostructures coated with chitosan or poly(ethylene glycol) as carriers for oral calcitonin delivery.

We have previously reported the formation and characterization of poly(ethylene glycol) (PEG)-coated and chitosan (CS)-coated lipid nanoparticles. In the present work our goal was to study the interaction of these surface-modified lipid nanoparticles with Caco-2 cells and to evaluate the potential of these nanostructures as oral delivery systems for salmon calcitonin (sCT). The interaction of rhodamine-loaded nanoparticles with the Caco-2 cell monolayers was evaluated quantitatively and qualitatively by confocal laser scanning microscopy and fluorimetry, respectively. The ability of these nanoparticles to reversibly enhance the transport of hydrophilic macromolecules through the monolayers was investigated by measuring the transepithelial electric resistance and the permeability to Texas Red-dextran. Finally, in vivo studies of the response to sCT-loaded nanoparticles were performed in rats. The results showed that the association of rhodamine-loaded nanoparticles to the Caco-2 cell monolayer was independent of the surface coating of the nanoparticles (CS-coated versus PEG-coated nanoparticles). However, while PEG-coated nanoparticles did not affect the permeability of Caco-2 monolayers, CS-coated nanoparticles produced a dose-dependent reduction in the transepithelial electric resistance, simultaneously to an enhanced dextran transport. The results obtained following oral administration of sCT-loaded CS-coated nanoparticles to rats showed a significant and prolonged reduction in the serum calcium levels as compared to those obtained for control (sCT solution). In contrast, the hypocalcemic response of sCT-loaded PEG-coated nanoparticles was not significantly different of that provided by the control (sCT solution). Therefore, these results indicate that the surface composition of the particles is a key factor in the improvement of the efficiency of oral sCT formulations. Moreover, the encouraging results obtained for CS-coated nanoparticles underline their potential as carriers for peptide delivery.

Transmucosal macromolecular drug delivery.

Mucosal surfaces are the most common and convenient routes for delivering drugs to the body. However, macromolecular drugs such as peptides and proteins are unable to overcome the mucosal barriers and/or are degraded before reaching the blood stream. Among the approaches explored so far in order to optimize the transport of these macromolecules across mucosal barriers, the use of nanoparticulate carriers represents a challenging but promising strategy. The present paper aims to compare the characteristics and potential of nanostructures based on the mucoadhesive polysaccharide chitosan (CS). These are CS nanoparticles, CS-coated oil nanodroplets (nanocapsules) and CS-coated lipid nanoparticles. The characteristics and behavior of CS nanoparticles and CS-coated lipid nanoparticles already reported [A. Vila, A. Sanchez, M. Tobio, P. Calvo, M.J. Alonso, Design of biodegradable particles for protein delivery, J. Control. Rel. 78 (2002) 15-24; R. Fernandez-Urrusuno, P. Calvo, C. Remunan-Lopez, J.L. Vila-Jato, M.J. Alonso, Enhancement of nasal absorption of insulin using chitosan nanoparticles, Pharm. Res. 16 (1999) 1576-1581; M. Garcia-Fuentes, D. Torres, M.J. Alonso, New surface-modified lipid nanoparticles as delivery vehicles for salmon calcitonin (submitted for publication).] are compared with those of CS nanocapsules originally reported here. The three types of systems have a size in the nanometer range and a positive zeta potential that was attributed to the presence of CS on their surface. They showed an important capacity for the association of peptides such as insulin, salmon calcitonin and proteins, such as tetanus toxoid. Their mechanism of interaction with epithelia was investigated using the Caco-2 model cell line. The results showed that CS-coated systems caused a concentration-dependent reduction in the transepithelial resistance of the cell monolayer. Moreover, within the range of concentrations investigated, these systems were internalized in the monolayer in a concentration-dependent manner. This uptake was slightly enhanced by the presence of the CS coating but, as compared with previously published results [M. Garcia-Fuentes, C. Prego, D. Torres, M.J. Alonso, Triglyceride-chitosan nanostructures for oral calcitonin delivery: evaluation in the Caco-2 cell model and in vivo (submitted for publication)], highly dependent on the nature of the lipid core. Nevertheless, these differences in the uptake of the CS-coated systems (solid lipid core or oily core) by the Caco-2 cells did not have a consequence in the in vivo behaviour. Indeed, both CS-coated systems (nanocapsules and CS-coated nanoparticles) showed an important capacity to enhance the intestinal absorption of the model peptide, salmon calcitonin, as shown by the important and long-lasting decrease in the calcemia levels observed in rats.