ovalbumin and triphosphoric-acid

The mechanism by which sodium tripolyphosphate affected the aggregation behavior of ovalbumin-lysozyme complexes was investigated in this work. The highest stability coefficients were detected for natural ovalbumin and lysozyme at pH 9.0 and pH 5.0, with values of 0.981 and 0.931, respectively. The turbidity of the phosphorylated ovalbumin-lysozyme complexes was 1.71-fold to the natural complexes at pH 7.0. This result was related to the fact that the phosphorylated sample had a lower isoelectric point. Besides, both intermolecular forces and SDS-PAGE analysis indicated that the disulfide bond was the most important interaction in the complex. Circular dichroism analysis showed that phosphorylation weakened the unfolding and stretching of the structure caused by heat treatment. Moreover, transmission electron microscopy pictures confirmed that the network structure of phosphorylated ovalbumin-lysozyme complex was broader than natural protein. This study provides information for further understanding the effect of phosphorylation on protein aggregation behavior.

Topics: Hydrogen-Ion Concentration; Muramidase; Ovalbumin; Phosphorylation; Polyphosphates; Protein Aggregates; Protein Binding

Characterisation of chitosan-tripolyphosphate nanoparticles is presented with the aim of correlating particle shape and morphology, size distribution, surface chemistry, and production automatisation with preparation procedure, chitosan molecular weight and loaded protein.. Nanoparticles were prepared by adding drop wise a tripolyphosphate-pentasodium solution to chitosan solutions under stirring. Trehalose, mannitol and polyethylene-glycol as bioprotectants were used to prevent particle aggregation and to reduce mechanical stress during freezing and drying processes.. As a novel result, time evolution of the particle size distribution curve showed the presence of a bimodal population composed of a fraction of small particles and of a second fraction of larger particles attributed to the rearrangement of particles after the addition of tripolyphosphate. Storage for 4 weeks resulted in a slight increase in average size, due to the continuous rearrangement of small particles. Improvement of nanoparticle stability after lyophilisation and spray-drying was observed in the presence of all bioprotectants. Trehalose was the best protectant for both methods. Finally, in vivo tests using chick embryos assessed the biocompatibility of chitosan, tripolyphosphate and the nanoparticles.. The simple ionotropic gelation method with low-MW chitosan was effective in achieving reproducible nanoparticles with the desired physico-chemical and safety characteristics.

Topics: Animals; Chick Embryo; Chitosan; Chorioallantoic Membrane; Cryoprotective Agents; Drug Compounding; Drug Stability; Insulin; Mannitol; Nanoparticles; Ovalbumin; Particle Size; Polyethylene Glycols; Polyphosphates; Serum Albumin, Bovine; Trehalose

Nasal vaccination is a promising, but challenging vaccination strategy. Poor absorption by the nasal epithelium and failure to break nasal tolerance are regarded as important reasons for poor efficacy of nasally applied vaccines. Formulation of the antigen into mucoadhesive nanoparticles, made of N-trimethyl chitosan (TMC) crosslinked with tripolyphosphate (TPP), has been shown to overcome these obstacles. However, although nasally administered antigen loaded TMC/TPP nanoparticles induce a strong humoral response, antibody subtyping indicates a Th2 bias. To design a nasal antigen delivery system capable of inducing stronger Th1 type responses, TPP as a crosslinking agent was replaced by unmethylated CpG DNA, a TLR-9 ligand and a potent inducer of Th1 responses, to prepare ovalbumin (OVA) loaded TMC nanoparticles (TMC/CpG/OVA). Several physicochemical characteristics of TMC/CpG/OVA (size, zetapotential, loading efficiency and antigen release profile) were assessed and compared to TMC nanoparticles prepared by crosslinking with TPP (TMC/TPP/OVA). Mice were nasally administered TMC/TPP/OVA and TMC/CpG/OVA after which antibody responses in serum and nasal washes were assessed and T-cell activation in the spleens determined. TMC/CpG/OVA showed similar physical properties as TMC/TPP/OVA in terms of particle size (380 nm), zetapotential (+21 mV) and antigen release characteristics. Nasal administration of TMC/CpG/OVA and TMC/TPP/OVA to mice resulted in comparable serum IgG levels (ca. 1000 fold higher than those induced by unadjuvanted OVA) and local secretory IgA levels. Moreover, TMC/CpG/OVA induced a 10 fold higher IgG2a response than TMC/TPP/OVA and enhanced the number of OVA specific IFN-gamma-producing T-cells in the spleen. In conclusion, OVA loaded TMC nanoparticles, containing CpG as adjuvant and crosslinker, are capable of provoking strong humoral as well as Th1 type cellular immune responses after nasal vaccination.

Topics: Adjuvants, Immunologic; Administration, Intranasal; Animals; Chitosan; CpG Islands; Cross-Linking Reagents; Female; Mice; Mice, Inbred BALB C; Nanoparticles; Ovalbumin; Polyphosphates; Vaccination