In vitro evaluation of chondroitin sulphate-chitosan microspheres as carrier for the delivery of proteins.

A novel formulation based on chondroitin sulphate/chitosan microspheres (CS/CH) has been investigated for oral delivery of macromolecules using ovalbumin as the model protein (OVA). The microspheres were prepared by a new emulsion-complex coacervation method. Physico-chemical properties of the polymers constituting microparticulate matrix were investigated by IR, DSC, TGA and X-ray diffraction analyses. In vitro tests were performed to evaluate the drug delivery system degradation and the protein release under conditions simulating the intestinal fluids. The ability of colonic enzymes to degrade the microparticulate systems was simulated employing the chondroitinase ABC enzyme. Results showed that the different CS/CH compositions influenced both microparticles stability and the protein release rate. Only the microspheres composed by 1:1 chondroitin sulphate-chitosan ratio achieved an OVA release profile suitable to a possible colon targeting. These microspheres released approximately 30% of ovalbumin encapsulated in 24 h in the different aqueous media tested, while they released 100% of protein in the presence of chondroitinase. The preliminary results demonstrated that chondroitin sulphate-chitosan microspheres can be a suitable delivery system for protein drug envisaged to oral administration.

Platelet interaction with CNBr peptides from type II collagen via integrin alpha2beta1.

Adhesion of blood platelets to fibrillar collagens plays a crucial role in haemostasis. Collagen type II is a homotrimeric member of the fibrillar collagen family, whose ability to interact with platelets has been poorly investigated. In this work, we analysed platelet adhesion to the whole collagen type II molecule, as well as to its CNBr peptides. We found that collagen type II is as efficient as collagen type I in supporting platelet adhesion. Platelet binding sites on collagen type II were identified in two different CNBr-derived peptides, CB8 and CB11. The ability of these peptides to support platelet adhesion required the triple helical conformation. Interaction of platelets with CB8 and CB11 peptides was totally dependent on the presence of Mg(2+) ions, and was completely inhibited by the anti-integrin alpha(2)beta(1) antibody P1E6. Upon adhesion to CB8 and CB11, a significant increase in intracellular protein tyrosine phosphorylation was observed. The pattern of tyrosine phosphorylated proteins in CB8- and CB11-adherent platelets was very similar to that observed in platelets adherent to the whole collagen molecule. By immunoprecipitation experiments, we identified two substrates that were tyrosine phosphorylated in adherent platelets as the tyrosine kinase Syk and the PLCgamma2 isozyme. By contrast, platelet adhesion to CB8 and CB11 did not promote tyrosine phosphorylation of FcR gamma-chain. Finally, we found that collagen type II, but not the CNBr-derived peptides, was able to induce cell aggregation associated to protein tyrosine phosphorylation when added to a platelet suspension. These results identify the CNBr peptides from collagen type II CB8 and CB11 as ligands for platelet integrin alpha(2)beta(1), and recognise their ability to support platelet adhesion and activation.