Preparation And Antibacterial Activity Of Chitosan Nanospheres In Dispersing State

Chitosan (CS) is a positive polysaccharide with broad-spectrum antimicrobial activity. There has been great improvement in the studies about the antimicrobial activity of CS and its derivatives. However, the mechanism of how CS acts upon bacteria has not been elucidated clearly. In previous studies, most researches focused on the antimicrobial activity of CS solution. In this paper, oleoyl-CS (OCS) nanoparticles (OCNP) as a novel antibacterial dispersion system is prepared and the characteristics are evaluated. A new method of surface contact inhibition is used to investigate the antibacterial activity of CS and biochemical information changes of bacteria.CS samples with different molecular weight (Mw) are prepared by the method of acetic acid hydrolysis. Their Mw are 1100 kDa, 300 kDa and 38 kDa. And their degree of deacetylation is almost the same. OCS is synthesized by grafting oleoyl onto the–NH2 at C-2 in the chitosan molecule. The FTIR spectrum and 1H NMR suggest the oleoyl groups are introduced into the CS moleculars. FTIR spectrum also indicates the degrees of substitution (DS) are 11%, 5% and 2.5%. OCNP is prepared using an O/W emulsification method based on OCS. The nanoparticles formulation has an almost spherical shape and the mean diameters are 275.3 nm (sample C) and 327.4 nm (sample B), respectively. OCNP could be well distributed in nutrient broth for a nice dispersion in the tested concentration range.OCNP shows inhibitory effect against E.coli and S.aureus, but the activity varies against strains. Four factors, concentration, chitosan Mw, DS of OCS and pH, are explored on the antibiacterial activity. The effects of four factors to CS are quite different with each microorganmism. Results indicate that the antibacterial activity increases as the concentration of OCNP increased. The minimum inhibitory concentrations (MICs) of OCNP range from 31.25 mg/L to 125 mg/L against E.coli. For S.aureus, the MIC of all samples is 125 mg/L. OCNP of low chitosan Mw appeares most effective against E.coli. For S.aureus, the effect of CS Mw on the antibacterial activity of OCNP is not pronounced. E.coli is most susceptible to OCNP of OCS with DS 5%, while no marked difference is found among OCNP of OCS with different DS against S.aureus. OCNP shows increased antibacterial activity when pH increases from 4.0 to 6.0 and reaches a maximum at pH 6.0. Bactericidal test shows that OCNP kills more than 90% bacteria in 5 min, still keeps the original bactericidal activity of chitosan.Effect of OCNP on the permeability of bacterial membrane and micro-morphology is significant. A260 increases rapidly as OCNP mixed with bacteria, which indicates that large molecules such as DNA, RNA leach out and the cell membranes are damaged. The addition of Mg2+ sharply reduces the release of intracellular materials and stabilizes the structure of cell membranes. This effect shows that the ability of chelated with Mg2+ on the cell wall is a part of OCNP's antibacterial activity. OCNP also rapidly increases 1-N-phenyl-naphthylamine (NPN) uptake and the release of cytoplasmicβ-galactosidase via increasing the permeability of outer membrane (OM) and inner membrane (IM). SDS-PAGE indicates the content of cellular soluble proteins decreases significantly in OCNP-treated bacteria. It is suggestes that OCNP decreases the content of cellular soluble proteins by permeating and disrupting cell membranes. No marked change of sugar content is found in the medium. Electron microscopy clearly demonstrates that OCNP adheres to the surface of E.coli and S.aureus, inhibites their growth throught interfacial interaction on the cell surface and shows extensive cell surface alterations of OCNP-treated bacteria, which supplies direct evidence that binding of OCNP to the highly negatively charged cell surface polymers is the necessary step.There may be many extracellular or intracellular targets in E.coli and S.aureus for OCNP. The cell surface hydrophobicity of E.coli and S.aureus decreases as the concentration of OCNP increased. The fluorescence experiments indicate that OCNP influences the structure of membrane and speculate to interact with proteins on the cell membrane. SDS-PAGE of E.coli outer membrane protein (OMP) treated with OCNP indicates that OMP may be one of the extracellular targets in E.coli. The lecithin effect suggestes that OCNP binds to cytoplasmic membrane phospholipids of S.aureus, and phosphate groups may be an extracellular target contributing to the interaction between OCNP and the cell surface. Fluorescence microscopy observations demonstrate that the internalization pathway of OCNP is completely different in E.coli and S.aureus. The intense green fluorescence is visualized throughout the tested bacteria as soon as fluorescein isothiocyanate (FITC)-OCS nanoparticles mixed with S.aureus, while for E.coli the probe is internalized in FITC-OCS nanoparticles-treated bacteria after 15 min. The gel-retardation experiment shows that OCNP is capable of binding to intracellular targets such as DNA and RNA. These results indicate that OCNP exerts the antibacterial activity by putative binding to intracellular targets such as DNA and RNA.Based on the above considerations, OCNP is a nanosphere dispersion system with good antibacterial actions. Through investigating the antibacterial activity and biochemical information changes of bacteria, the model for the mechanism on the antibacterial activity of OCNP is drawn out.