| Objective: To prepare water-soluble low molecular weight chitosan (WSC) and WSC-DTPA nanoparticles and evaluate WSC's radiation protective effects.Methods: In order to prepare water-soluble low molecular weight chitosan, chitosan was treated with sodium hydroxide and hydrogen peroxide successively which can elevate the degree of deacetylation and degrade the molecular weight respectively. MTT assay was used to evaluate the effects of WSC on BRL cells survival rates in 24 h and 48 h after irradiation. Flow cytometry (FCM) and propidium iodide (PI) staining was used to measure the effects of WSC on cell cycle phases which exposed to 4 Gy 60Coγirradiation. The apoptosis rates and intracellular Ca2+ of BRL cells were determined by AnnexinⅤ-FITC/PI double staining and Fluo-3AM respectively. Surviving fraction (SF) of BRL cells treated with WSC and irradiation were analyzed by colony formation assay. The radiation protective effects of WSC were evaluated with multi-target one shot model. WSC-DTPA compound was synthesized by N-acylation reaction, and its nanoparticles were made by ionic gelation. Their structures and morphological properties were characterized.Results: After eluting with sodium hydroxide for three hours, the degree of deacetylation of chitosan were increased from 79.3%±0.55% to 94.1%±0.06%, which were determined by linear potentiometric titration. The molecular weight of chitosan measured by HPLC was decreased from 2.00×105 to 3.26×103 after degradation with hydrogen peroxide for 120min. The obtained chitosan was identified to be high degree of deacetylation (D.D) and low molecular weight by FTIR. The results of MTT assay indicate that the WSC has no cytotoxity in the range of 1.56～50μg/mL and can resist the inhibition of irradiation to the BRL cells. After treated with 50μg/mL of WSC and 4 Gy 60Coγirradiation, the 24 h and 48 h survival rates of BRL cells were increased from 77.4%±0.19% to 90.6%±0.15% (p<0.05) and from 66.5%±0.19% to 87.5%±0.16% (p<0.05), respectively. The results of PI staining indicate that the proportion of SubG1 of BRL cells after 4 Gy 60Coγirradiation were markedly higher than that of control group (p<0.05), and it decreases with the higher concentration of chitosan. The proportion of SubG1 was decreased from 51.93%±2.11% in the irradiation treated cells to 16.9%±1.3% in the 50μg/ml of WSC and irradiation treated cells in 24 h(p<0.05), which radiation dose was 4 Gy. The results of apoptosis and intracellular Ca2+ of BRL cells showed that WSC can inhibit the apoptosis and calcium overload which induced by irradiation,after treated with 50μg/mL of WSC and irridation, the apoptosis rates decreased from 16.17%±0.38% to 7.09%±0.48% (p<0.05), the intracellular Ca2+ decreased from 22.83±0.25 to 14.00±0.23 ( p<0.05) compared with irridation only.We found an excellent correlation between the apoptosis rates and intracellular calcium, the linear equation is y=1.03x-7.47, R2=0.9994. The results of colony formation assay showed that the PF was more than 1, which was in accordance with radiation protective effects of WSC on normal liver cells. The novel WSC-DTPA compound was successfully synthesized by N-acylation reaction, which was identified with FTIR and 1H-NMR spectrum. The mean diameter of WSC-DTPA nanoparticles was about 46.5±1.51 nm and its PDI was 1.0. Detailed imaging analysis of the particle morphology showed that the WSC-DTPA nanoparticles were spherical shapes with uniform size.Conclusion: The chitosan with high degree of deacetylation (D.D) and low molecular weight was obtained successfully. The cell experiments demonstrated that WSC has radiation protective effects on normal liver cells. WSC-DTPA nanoparticles were prepared successfully, which is beneficial to further investigation.
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