Study On The Radioprotective Effect Of Chitosan-Superoxide Dismutase Conjugates

Ionizing radiation is radiation that can ionize substances, such as α-particales,β-particles, X-rays and y-rays. The ionizing radiation has been widely applied in nuclear power, disease treatment and diagnosis, crop breeding, metal exploration and scientific research, etc. However, the increasing exposure to radiation is a serious threat to human health and even life. The exact mechanism of radiation damage remains to be well understood, but radiation-induced reactive oxygen species (ROS) contributes definitely to radiation damage. Accordingly, superoxide dismutase (SOD), the most efficient ROS scavenger in living organisms, is acknowledged as an ideal radioprotective drug candidate. Howerver, native SOD shows limited clinical potential due to its very short half-life in vivo, poor stability, week cell affinity and immunogenicity. Therefore, on the basis of previous studies, we synthesized new 6-0-quaternary ammonium chitosan derivatives,6-0-2’-hydroxypropyl trimethyl ammonium chloride chitosan (O-HTCC), and for the first time to modify the terminal carboxyl group of SOD using EDC/NHS method. The O-HTCC-SOD conjugates were purified by DEAE anion exchange chromatography. Their physico-chemical properties were characterized using SDS-PAGE, SEC-HPLC, circular dichroism (CD), size/potential determination and transmission electron microscope (TEM). We then determined the enzyme activities of O-HTCC-SOD conjugates under different pH, temperatures, or in presence of proteolytic enzymes (pepsin or trypsin) by using pyrogallol autoxidation method. The effect of a-amylase on O-HTCC-SOD enzyme activity was also evaluated due to the degradability of O-HTCC by a amylase. Then the radioprotective effect of O-HTCC- SOD on L929 and 3T3 cells was assessed using optimized MTT method. In order to study the in vivo pharmacokinetic profiles of O-HTCC-SOD, plasma, liver and kidney samples were collected and determined using A T-SOD assay kit after tail intravenous injection of O-HTCC-SOD to mice. The data were calculated with DAS 2.0 to obtain the basic pharmacokinetic parameters. At last, we evaluated the in vivo radioprotective effects of O-HTCC-SOD on irradiated BALB/c mice according to guidelines on pre-clinical research of anti-radiation drugs. The main results of each part in this study are as follows:1. Preparation of O-HTCC2,3-epoxypropyl trimethyl ammonium chloride was grafted to reactive C6-OH of chitosan to obtain water-soluble chitosan derivative,O-(2-hydroxyl)-propyl-3-trimethyl ammonium chitosan chloride (O-HTCC) through a three-step reaction. Ultrafiltration was performed to give purified O-HTCC at a yield of 92.0%. The structure was verified by IR and 1H NMR analysis results. The free amino group of O-HTCC is up to 73.35% based on ninhydrin colorimetry method. Because of its good water-solubility and high content of free amino groups, O-HTCC is more suitable for subsequent SOD modification.2. Synthesis, purification and characterization of O-HTCC-SODO-HTCC was covalently coupled to SOD at an efficiency of 90% via EDC/NHS-mediated reaction, followed by purification on DEAE Sepharose Fast Flow column. SDS-PAGE and SEC-HPLC results demonstrated that O-HTCC-SOD had higher molecular weight than native SOD, and showing a wider molecular weight distribution consistent with O-HTCC. By comparing the secondary structure of SOD and O-HTCC-SOD gained from CD results, we found the preferential conformation of O-HTCC-SOD did not change obviously. Meanwhile the spatial arrangement of O-HTCC-SOD was more regular. Particle size assay and TEM observation showed that both O-HTCC-SOD and SOD are spheroidal, but the size of O-HTCC-SOD is significantly larger than that of SOD. Potential analysis proved that O-HTCC-SOD has more positive charge.3. Enzymatic properties of O-HTCC-SODPyrogallol autoxidation method was used to evaluate the enzyme activity of O-HTCC-SOD under different pH, temperature, and in presence of pepsin or trypsin. The results showed that the enzyme activity retention rate of O-HTCC-SOD was significantly higher than that of native SOD at pH 3 or 11. O-HTCC-SOD had better enzyme activity than that of native SOD at a high temperature ranging from 40℃ to 70℃. Protease degradation experiments showed that in presence of pepsin or trypsin, the native SOD lost its enzyme activity rapidly, while O-HTCC-SOD showed stable and even slightly increased enzyme activity. In addition, based on the slow biodegradability of O-HTCC by a-amylase, we also proved that a-amylase continuously increased the enzyme activity of O-HTCC-SOD for a period of time. Presumably, O-HTCC on the surface of conjugates was slowly hydrolysed by a-amylase. Then the enveloped catalytic sites were fully exposed. Therefore, O-HTCC-SOD in combination with a-amylase showed sustained-release enzymatic activity.4. Radioprotective effect of O-HTCC-SOD in vitroMTT assay was used to determine suitable dose of O-HTCC-SOD in L929 cells, followed by its radioprotective effect on the two cells after X-ray irradiation. The results indicated that O-HTCC-SOD at 250 U/mL showed no significant toxicity to L929 cells. O-HTCC-SOD had certain protective effect on the two cells-especially on L929 cells. Moreover, we have found that O-HTCC-SOD co-incubated with a-amylase significantly enhanced its radioprotective effect for a period of time. Cell targeting assay showed that O-HTCC-SOD can enter L929 cell, but SOD cannot.5. Pharmacokinetic profiles of O-HTCC-SOD in miceAfter a single tail intravenous injection of O-HTCC-SOD or native SOD solutions to BALB/c mice, a T-SOD assay kit was used to determine the enzyme activity of total SOD in the peripheral blood, liver and kidney samples. The results showed that the pharmacokinetic behavior of native SOD conformed to the two-compartment model. But the "bell-shaped" plasma concentration-time curve of O-HTCC-SOD was not consistent with any of the conventional intravenous drug delivery models. Meanwhile, the distribution half-life time of O-HTCC-SOD was 1.25 h, which was significantly longer than that of native SOD (only 0.25 h). The bioavailability of O-HTCC-SOD is also slightly higher than that of native SOD. Comparing the pharmacokinetic parameters (including Tmax and Cmax) of O-HTCC-SOD and native SOD, as well as enzymatic properties above, it was confirmed that O-HTCC-SOD had sustained-release effect in vivo. The concentration-time curves of SOD and O-HTCC-SOD in the liver indicated that SOD was quickly removed in the liver. While O-HTCC-SOD rapidly accumulated at first, then slowly diminished, sugesting that O-HTCC-SOD had liver targeting property. The concentration-time curves of SOD and O-HTCC-SOD in the kidney were similar, indicating that O-HTCC’s modification showed less influence on SOD in the kidney.6. Radioprotective effect of O-HTCC-SOD in vivoThe BALB/c mice, which had been intraperitoneally injected with O-HTCC-SOD or native SOD 2 h early, were irradiated using X-ray at 8 Gy to induce acute radiation damage. In the next day, peripheral blood was collected and analyzed using an automated hematology analyzer. The results showed that exposure to radiation led to decline of white blood cells in mice rapidly, but had no obvious effect on red blood cells count and platelets, suggesting that acute radiation damage mainly impaired the immune system related tightly with the leukocyte function. However, when pretreated with high dose of O-HTCC-SOD, the number of white blood cells was recovered to the normal level. The conjugates at mediun or low dose as well as SOD also showed significant radioprotective effect when compared with negative group (P<0.05). Thymus index and spleen index of mice showed obvious decrease after exposed to radiation. Both the thymus index and spleen index of the groups pretreated with O-HTCC-SOD were significantly increased when compared with the negative group (P<0.01 for high dose group, P<0.05 for mediun dose group). Native SOD showed no obvious protective effect on thymus index and spleen index. MDA induced by lipid peroxidation in liver and lung were measured by a trace malondialdehyde (MDA) assay kit. The results indicated that O-HTCC-SOD dose-dependently decreased MDA in liver and lung, but native SOD did not. Pathological changes of small intestine, liver and lung tissues were detected by HE staining. The results showed that O-HTCC-SOD apparently protected the three organs against radiation damage with a dose-dependent manner. In conclusion, O-HTCC-SOD showed dose-dependently protective effect against acute radiation damage of BALB/c mice induced by X-ray irradiation.In summary, modification by the chitosan derivative, O-HTCC, conferred better physicochemical, enzymatic and pharmacokinetic characterization to SOD, and then O-HTCC-SOD conjugates showed remarkable enhanced protective effect against radiation damage. So O-HTCC-SOD might be a potential and promising radioproteetor candidate.