Studies On Preparation And Immunoenhancing Effect Of Chitosan Nanoparticles Loading CpG-ODN

Many functional genes emerge prominently one after another along with the never-ending recognition and study on genes. Bacterial DNA and synthetic oligdeoxynucleotides (ODN) with motifs consisting of a central unmethylated CpG dinucleotides are immunostimulatory and can activate many kinds of immunological cells, and trigger Th1-polarized immune response. Owing to their strong immunostimulatory activity, a number of CpG-ODN are at various stages of preclinical and clinical evaluation as anti-tumor, antiviral, anti-anaphylaxis agents and as adjuvant in immunotherapy. To elicit immunostimulatory activity CpG-ODN need to be internalized by cells and act on Toll-like receptor-9. However, nucleic acid inclines to degradation in face of nuclease in vivo, besides, electronegative nucleic acid appears to be hard to bind to cell surface loading negative charge, so delivery of CpG-ODN is the critical step for CpG-ODN activity. Chitosan(CS), a kind of biodegradable, biocompatible and innoxious cationic polymer, has been popularly investigated as gene carrier. It is known that chitosan gene nanoparticles can be good for delivering genes and bind to effector cells, and enhance transfection efficiency of genes. Little is known as to the delivery of CpG-ODN using chitosan nanoparticles so far, in the current study, we therefore prepared CS-CpG-ODN nanoparticles, initially investigated their physico-chemical properties and other characteristics, then mice were immunized to research the immunocompetence in vivo, we hypothesized that the'protected and controlled'gene delivery of CpG-ODN would enhance the immunostimulating effect of CpG-ODN in mice. The main contents are as follows in this paper:1. CS-CpG-ODN nanoparcles were prepared by complex coacervation. The nanoparcles entrapment efficiency and loading capacity were detected by UV and ninhydrin assay respectively. Meanwhile, some factors of effecting entrapment efficiency were investigated in preparation process. The results obtained indicated that the nanoparticles showed 90.67% of mean entrapment efficiency and 38.78% of mean loading capacity respectively. To some extent, there was less effect of each factor to entrapment efficiency.2. Size distribution and surface morphology of the particles were identified by transmission electron microscopy (TEM) measurements. The mean diameter, polydispersity, as well as mean zeta potential of nanoparticles were determined by a particle size / zeta potential analyzer. Gel retardation assay of 3% agarose was used to test the nature of surface charge and formed complexes, enzyme protection assay was carried out to investigate the inhibition of degradation by degrading enzymes. L-02 cells and HepG2 cells were used as model cells to study the transfection activity of nanoparcles. As seen in the electron microscope figure, The morphology of nanoparticles exhibited spherical shape, and the size was uniform with a size distribution range of 75-100nm on the whole. The mean diameter was 93.7 nm, the mean zeta potential was +20.2mV. The complexes could effectively bind CpG-ODN and protect it from nuclease degradation, the result of cellular transfection showed that these gene nanoparticles could be taken in by L-02 cells and HepG2 cells.3. All mice were randomized into four groups: control group, CS group, CpG-ODN group and CS-CpG-ODN group,injected into posterior thigh muscles, then were given another shot after 2 weeks using the same formulation. Mice were killed two weeks later after secondary immunization, spleens were collected for cellular immunity analysis, blood serum was harvested and assayed for IL-2, IL-12 and IgG, cellular supernatant was extracted and assayed for IL-4 and IFN-γ, heparinized blood was gained for T lymphocyte subsets analysis. After that, Splenic lymphocytes proliferation was detected by MTT method, levels of IL-4, IFN-γ, IL-2, IL-12 and IgG were measured by ELISA. T lymphocyte subsets CD4+CD8+ from peripheral blood were analyzed by flow cytometry. After in vivo immunization, CS group and CpG-ODN group showed enhanced proliferative effects of T lymphocytes, increased percentages of CD4+ T cells and CD4+/CD8+ ratios, high levels of IFN-γin cellular supernatant and IL-2, IL-12, IgG in serum. These cases in CS-CpG-ODN group appeared more remarkable, in addition, evidently reduced level of IL-4 was observed in cellular supernatant.The delivery system of CS-CpG-ODN nanoparcles was successfully made in this paper, and favourable entrapment efficiency, loading capacity and effect of enzyme protection were obtained. Furthermore, the nanoparcles result in effective cell uptake. Increased amounts of IL-2, IL-12 and IFN-γTh1 cytokines compared to decreased amount of IL-4 Th1 cytokine are believed to support the strongly biased towards Th1 response.The immunoenhancing potency could be related to availably re-establishing immunologic balance of T cells by adjusting the percentages of CD4+CD8+ T cells. The results in our work demonstrate for the first time the delivery system of CS-CpG-ODN nanoparcles may be associated with the strengthened immunostimulating effect of CpG-ODN, and then have an important base on CpG-ODN used for gene therapy.