Preparation And Biological Evaluation Of Carboxymethyl Chitosan

Chitosan and its derivatives are a new type of bio-amylose, which are widely used in the fields of agriculture, industry and medicine etc. . In the past 10 years, this kind of materials has been paid more and more attentions in the medicine. One of its derivatives, carboxymethyl chitosan (CMC), has been researched much earlier and is also one of the most attracted research fields. The purposes of this paper are to confirm preparation technics of carboxyl substituted CMC on three different positions, and make biological evaluation .N,O- carboxymethyl chitosan (N,O-CMC) was prepared with chloroacetic acid and chitosan in alkaline condition. N-carboxymethyl chitosan(N-CMC) was prepared by two steps: firstly ,chitosan reacted with Glyoxylic acid, then, deoxidized by Sodium Borohydride. Chitin was modified with chloroacetic acid in the presence of sodium hydroxide, the production was carboxymethyl chitin, we can get O-CMC after carboxymethyl chitin was deacetylated.The factors, such as the amounts of reactant, the volumes of isopropyl alcohol, reaction temperature, reaction time etc., influencing the yield, degree of substitution (DS) and intrinsic viscosity were respectively investigated. The CMC products were characterized for DS, average molecular weight, IR spectroscopy, etc., respectively.At last, the preparation conditions of N,O-CMC were obtained as: the amount of chitosan 5g, isopropyl alcohol/chitosan(Volume/mass)=7.5:1, 10mol/L sodium hydroxide solution 62.5ml, basified time 12 hours, basified temperature -10℃, chloroacetic acid/chitosan(mole/mole)=6:1, reaction temperature 60 ℃, reaction time 5 hours.The preparation conditions of N -CMC: the amount of chitosan 1g, distilled water 150ml, Glyoxylic acid/chitosan(mole/mole)=1.2, reaction temperature 20-40℃, reaction time 2 hours, pH 8-10, 10% Sodium borohydride solution 10ml, deoxidization temperature 20-40℃, deoxidization time 2 hours.The preparation conditions of O -CMC: the amount of chitin 5g, the volumes of isopropyl alcohol 50ml, 50% sodium hydroxide solution 30ml, basified time 12 hours, basified temperature -10℃, chloroacetic acid/chitin(mole/mole)=4.5:1,reaction temperature 50°C, reaction time 4 hours, deacetylation temperature 90 °C, deacetylation time 1.5 hours, a bit of sodium borohydride can be dropped in the reactive system.The purification effect of magnetic force mix method and ultrasonic method were compared in the process of CMC purification. The result was that the latter was better than the former and the amount of chlorine ion was less than 0.01% after washing 6 times. CMC intrinsic viscosity was measured by viscosity method including traditional step by step dilution method and one-piont method. The result error between the two methods was less than 5%.So one-piont method can replace traditional step by step dilution method and workload was much reduced. The factors of influencing colloidal titration using to measure degree of substitution (DS), were analyzed and we found that the pH value should be tested under the situation in which CMC can be completely ionized. Titration velocity should be 0.02ml / s or so and the concentration of CMC solution should be within the range of 0.01%~0.02%. CMC isoelectric point (IP) was about 3.5. CMC solution can be observed to be ivory-white colloidal in the situation of deviation from isoelectric point and to be transparent in the situation which pH value was higher than 6. Carboxymethyl chitosan with good water solubility was synthesized.Biological evaluation included cytotoxicity test and hemolysis test. Cytotoxicity test: L-929 mouse fibroblasts were cultured in the medium of solution of materials. Cell viability and proliferation were tested by cell proliferation assay through values of OD at 2, 4 and 7 days respectively after innoculation. The cell relative growth rate (RGR) was calculated and the cytotoxicity was graded by generally accepted standard. Hemolysis test: Hemolysis percentage measurement was used to assay the hemolytic properties of four types of materials through direct contact with rabbit blood.Results of the cytotoxicity of purified chitosan and three types of CMC was grade 0 or 1, up to medical purpose. We found that higher the concentration of solution of materials, higher the cytotoxicity was showed and the cytotoxicity of N-CMC was the lowest. The hemolysis rates of four types of materials were less than 5%, up to medical purpose. N-CMC had the lowest hemolysis rates andO-CMC had the highest. This result was in accord with cytotoxicity test.Vitro cytotoxicity test and hemolysis test had shown: chitosan and its derivatives had good biocompatibility which provided possibilities and foundations for the further research.