Journal of Biomaterials and Nanobiotechnology, 2011, 2, 347-352 doi:10.4236/jbnb.2011.24043 Published Online October 2011 (http://www.SciRP.org/journal/jbnb) Copyright © 2011 SciRes. JBNB 347 Chitosan Sub-Micron Particles Prepared Using Sulfate Ion Salt as Bacteriostatic Materials in Neutral pH Condition Kanako Saita 1,2 , Shoji Nagaoka 2,3* , Maki Horikawa 2,3 , Tomohiro Shirosaki 2,3 , Shigeki Matsuda 2 , Hirotaka Ihara 1,3* 1 Department of Applied Chemistry and Biochemistry, Kumamoto University, Kumamoto, Japan; 2 Kumamoto Industrial Research Institute, Kumamoto, Japan; 3 Kumamoto Institute for Photo-Electro Organics, Kumamoto, Japan. Email: * ihara@kumamoto-u.ac.jp, nagaoka@kmt-iri.go.jp Received June 4 th , 2011; revised July 22 nd , 2011; accepted September 5 th , 2011. ABSTRACT In this paper, the newly developed ion exchange phase separation method to create chitosan sub-micron particles is introduced: 1) chitosan was dissolved in a lactic acid aqueous solution; 2) the obtained chitosan solution was added stepwise in a sodium sulfate aqueous solution and cooled down to 5˚C to become slightly turbid through agglutination; 3) desalinating and deacidifying of the mixture was carried out by a dialyzing tube method. IR spectroscopy and ele- mental analysis indicated that the agglutination of chitosan was induced by crosslinking effect with an electrostatic interaction between sulfate anions and amino groups in the glucosamine unit although large excess of Na 2 SO 4 caused undesirable further agglutination of the resultant chitosan particles. As a result, the proper amount of Na 2 SO 4 was ap- proximately 1.0 - 10.0 equivalent for the amino group to create the chitosan particles with a sub-micron size. In addi- tion, we investigated an antibacterial activity test for Escherichia coli of the obtained chitosan particles. The significant antibacterial activity was observed in incubation even at neutral pH condition while the chitosan microbeads (size: ca 200 m) prepared by the conventional method and chitosan granules (size: ca 600 m) as starting materials showed almost no antibacterial activity in the same condition. Keywords: Chitosan, Particle, Crosslinking, Microbeads, E. coli 1. Introduction Chitosan is a cationic biopolymer obtained from N- deacetylation of chitin, β-(1,4)-N-acetyl-D-glycan [1]. The non-toxic, biocompatible and biodegradable proper- ties of chitosan provide potential for many types of ap- plications [2-4]. Chitosan and derivatives have become useful polysaccharides in the biomedical field. Especially, these microparticles have been utilized as chroma- tographic packings [5,6], enzyme-immobilized support [7,8], affinity adsorbents for proteins [9], endotoxin ad- sorbents [10] and drug carriers [11,12]. Generally, it was popular to use chitosan as antibacterial compounds in agriculture, as elicitors of plant defense responses [13], as additives in the food industry, as flocculating agents for wastewater [14] and as pharmaceutical agents in biomedicine [15,16]. In this content, environmentally antibacterial activity of chitosan has received consider- able attention recently. However, these activities are li- mited to acidic conditions because of its poor solubility above pH 6.5, where chitosan starts to lose its cationic nature [17-20]. Chitosan is generally insoluble under a neutral pH conditions because of a strong hydrogen bonding and lower pKa (ca. 6) of a residual amino group. Thus, the molding, investigation and application of chitosan have been restricted. The methods for producing porous and spherical chitosan microbeads, such as the “suspension evaporation method” [6] and the “suspension crosslink- ing technique” [9,21] using chitosan acid aqueous solu- tions, have been reported. These methods require the use of organic solvents and emulsifier. Also a sphering me- thod by spray drying [22,23] is known and widely appli- cable. However, these methods have some disadvantages: for example, the particle size control is difficult, espe- cially in below tens of micron but also a heating process as a cost up factor is necessary. Recently, the “rapid ex- pansion of supercritical fluid technology” [24] has been