ABSTRACT
Using a strategy of macromolecular assembly, a colloidal vaccine delivery system was obtained from chitosan and dextran sulfate and loaded with an antigenic protein (p24, the capsid protein of HIV-1). The colloidal polyelectrolyte complexes (PECs) were obtained by charge neutralization of the polyanion and polycation at a charge ratio (n(+)/n(-)) of 2 (CHDS). The conditions of assembly were tuned to maintain the colloidal properties of the carrier in high salt environment. The relative molar masses of the two polyions and the degree of acetylation (DA) of chitosan were essential parameters to achieve this goal, and this could be related to the nanometric scale organization of the colloids observed by Small Angle X-rays Scattering experiments. The binding of p24 to the colloidal carrier was achieved and the release of the antigen was investigated. Antigen presenting cells [dendritic cells (DCs)], obtained from monocytes, could internalize the colloids. Immature DCs (iDCs) were not matured by the colloidal PECs either loaded or not loaded with p24, as proved by Fluorescent Activated Cell Sorting (FACS) analysis. Despite this lack of in vitro interaction, a specific immune response was observed in mice with a high production of antibodies, after subcutaneous injection. The analysis of the interleukin production shows that both the cellular and the humoral responses were stimulated. This work brings a physico-chemical insight on polysaccharide-based antigen delivery systems and opens up new perspectives for their use as vaccine carriers.
Subject(s)
Antigen-Presenting Cells/cytology , Macromolecular Substances/chemistry , Polysaccharides/chemistry , Vaccination/methods , AIDS Vaccines/chemistry , Animals , Chitosan/chemistry , Electrolytes , Female , Mice , Mice, Inbred BALB C , Nanoparticles/chemistry , Nanotechnology/methods , Particle Size , Scattering, RadiationABSTRACT
This work reports on the interactions of a model protein (p24, the capside protein of HIV-1 virus) with colloids obtained from polyelectrolyte complexes (PECs) involving two polysaccharides: chitosan and dextran sulfate (DS). The PECs were elaborated by a one-shot addition of default amounts of one counterpart to the polymer in excess. Depending on the nature of the excess polyelectrolyte, the submicrometric colloid was either positively or negatively charged. HIV-1 capsid p24 protein was chosen as antigen, the ultrapure form, lipopolysaccharide-free (endotoxin-, vaccine grade) was used in most experiments, as the level of purity of the protein had a great impact on the immobilization process. p24 sorption kinetics, isotherms, and loading capacities were investigated for positively and negatively charged particles of chitosans and dextran sulfates differing in degrees of polymerization (DP) or acetylation (DA). Compared with the positive particles, negatively charged colloids had higher binding capacities, faster kinetics, and a better stability of the adsorbed p24. Capacities up to 600 mg x g(-1) (protein-colloid) were obtained, suggesting that the protein interacted within the shell of the particles. Small-angle X-rays scattering experiments confirmed this hypothesis. Finally, the immunogenicity of the p24-covered particles was assessed for vaccine purposes in mice. The antibody titers obtained with immobilized p24 was dose dependent and in the same range as for Freund's adjuvant, a gold standard for humoral responses.
Subject(s)
AIDS Vaccines/pharmacokinetics , Colloids/pharmacokinetics , Drug Delivery Systems/methods , HIV Core Protein p24/pharmacokinetics , Polymers/pharmacokinetics , Polysaccharides/pharmacokinetics , AIDS Vaccines/administration & dosage , AIDS Vaccines/chemistry , Animals , Biocompatible Materials/administration & dosage , Biocompatible Materials/pharmacokinetics , Colloids/administration & dosage , Colloids/chemistry , Decapodiformes , Female , HIV Core Protein p24/administration & dosage , Mice , Mice, Inbred BALB C , Polymers/administration & dosage , Polymers/chemistry , Polysaccharides/administration & dosage , Polysaccharides/chemistryABSTRACT
Colloids were obtained from non-stoichiometric polyelectrolyte complexes with two polysaccharides of opposite charge: chitosan and dextran sulfate (DS) as the polycation and polyanion, respectively. The complexes were elaborated by a one-shot addition of the polymer in default to the one in excess. The colloids were positively or negatively charged according to the nature of the polymer in excess. Dynamic light scattering (DLS) demonstrated that particles were formed at a very early stage in the complexation process. The consumption of the excess polyelectrolyte was monitored with a dye assay specific for dextran sulfate (toluidine blue) or chitosan (orange II). From these experiments, two different mechanisms of colloidal PEC formation were evidenced, according to the nature of the polymer in excess. On adding chitosan to DS in excess, regular consumption of the polyanion was observed at a constant stoichiometry, in the 1.5 to 1.85 range (sulfate residues for one glucosamine group), according to the molar mass of the polycation. When DS was added to chitosan in excess, the overall stoichiometry varied from ca. 6 (glucosamine residues for one sulfate group) down to 1 as the charge molar mixing ratio R=n+/n- decreased from 20 to 1. The existence of various mechanisms, according to the nature of the polymer in excess, could be attributed to the differences in chemical reactivity (strong vs low) of the ion in excess and the conformation and flexibility of the macromolecular chains related to their electrostatic potential.
