| Chemotherapy plays an important role on treating the irresectable hepatocellular carcinoma and eliminating the tiny carcinoma focus. But both the system chemotherapy and transcatheter hepatic arterial chemotherapy cann't achieve the ideal effect and produce serious side-effect. Magnetic drug nanoparticles are composed of chemotherapeutic agent, superparamagnetic materials and coating polymers, and possesses the long circulating ability. When it is injected into body, it can accumulate at the target region under the magnetic field, and then drug is released smoothly to kill the tumor cell avoiding systematic toxicity. In this study the carboplatin-Fe@C nanocage-loaded chitosan nanoparticles (C-Fe@CN-CN) were prepared by reverse microemulsion method. The effect of C-Fe@CN-CN on the proliferation and cell cycle of human hepatoma cell line HepG2 in vitro was investigated. The effects of targeted treatment of C-Fe@CN-CN combining with external magnetic field in rats with the transplanted liver cancer and the toxic side effects of the targeted treatment were also observed.Article I : Effect of the carboplatin-Fe@C nanocage-loaded chitosan nanoparticles on human hepatoma cell line HepG2Objective: To investigate the effect of the carboplatin-Fe@C nanocage-loaded chitosan nanoparticles (C-Fe@CN-CN) on the proliferation and cell cycle of human hepatoma cell line HepG2 in vitro. Methods: The C-Fe@CN-CN were prepared by the reverse microemulsion method. The inhibitive effect of C-Fe@CN-CN on the proliferation of the HepG2 cell was measured by MTT colorimetry at four levels ofdrug concentration (20, 40, 80 and 160 mg/L) in 24, 48 and 72 hour. The distribution of cell cycle was analysed by flow cytometry at two levels of drug concentration (8 and 16 mg/L) in 48 hour. Results: C-Fe@CN-CN could apparently inhibit the proliferation of HepG2 cell in the dose-dependent and time-dependent manner. On the increase of drug concentration the inhibitive effect raised from 18.8% to 53.2% in 24 hour. On the increase of time the inhibitive effect raised from 53.2% to 98.7%. The inhibitive effect of C-Fe@CN-CN in 24 hour was lower than that of original carboplatin, and were equal to original carboplatin in 48 hour and 72 hour. Cell cycle analysis indicated that C-Fe@CN-CN or original carboplatin blocked HepG2 cell in S phase in dose-dependent manner from 47.2% to 59.3% in 48 hour. Fe@CN-CN had no effect on the proliferation and cell cycle of HepG2 cell. Conclusion: C-Fe@CN-CN can effectively inhibit the proliferation of HepG2 cell through the similar mechanism as original carboplatin inducing the arrest of S phase. Fe@CN-CN indicates favourable biocompatibility.Article II: Effects of targeted treatment of the carboplatin-Fe@C nanocage-loaded chitosan nanoparticles on rats with transplanted liver cancerObective: To investigate the effects of targeted treatment of the carboplatin-Fe@C nanocage-loaded chitosan nanoparticles combining external magnetic field on rats with transplanted liver cancer. Methods: Twenty-four model rats with transplanted liver cancer were established and divided into four groups randomly(n=6). Abdominal exposure was carried out through a midline abdominal incision, and a cannula was inserted into the hepatic artery and fixed. A group: saline water was injected to serve as the control. B group: saline water with 10mg/kg free carboplatin was given. C group: saline water with carboplatin-Fe@C nanocage-loaded chitosan nanoparticles(equivalent dose of free carboplatin 10mg/kg) was injected in absence ofmagnetic field. D group: saline water with carboplatin-Fe@C nanocage-loaded chitosan nanoparticles(equivalent dose of free carboplatin 10mg/kg) was injected in presence of magnetic field in 30 min. All the animals were sacrificed and abdominal exposure was done again after 7 days, tumors were resected, the tumor weight and tumor volume were measured, the tumor inhibiting rate of tumor weight and tumor volume were counted, tumor and liver tissues were examined for histological changes and distribution of nanoparticles. Results: The growth speed of tumor was significantly inhibited after different dosage forms of carboplatin were given. There is significant difference in the tumor weight of A,B,C,D groups(0.85±0.12, 0.61±0.10, 0.48 ± 0.09, 0.33±0.06 respectively), the tumor inhibiting rate of tumor weight of B,C,D groups were 28.9%, 43.4%, 61.7% respectively , D group was 2.1 times as high as B group. There is also significant difference in the tumor volume of A,B,C,D groups( 1.06± 0.24, 0.72 ± 0.10, 0.50 ± 0.07, 0.28 ± 0.05 respectively), the tumor inhibiting rate of tumor volume of B,C,D groups were 28.9%, 43.4%, 61.7% respectively. A group was 3.8 times as high as D group. In the pathological feature of liver tumor, there were a large number of concentration of nanoparticles the intervals of the tumor cells, and tumor tissue present server necrosis in D group, there were no significant concentration of nanoparticles the intervals of the tumor cells in C group, and tumor tissues present middle necrosis in C group and B group, tumor tissues present mild necrosis because of overgrowth of tumor cells. Conclusion: C-Fe@CN-C can significantly increase the therapeutic effects of carboplatin by hepatic artery injection combining with an external magnetic field on the tumor.Article III: The side effects of targeted treatment on the rats with transplanted liver cancer with the carboplatin-Fe@C nanocage-loaded chitosan nanoparticlesObjective: To observe the side effects of targeted treatment on the rats withtransplanted liver cancer with the carboplatin-Fe@C nanocage-loaded chitosan nanoparticles combining external magnetic field. Methods: Twenty-four model rats with transplanted liver cancer were established and divided into four groups randomly(n=6). Abdominal exposure was canied out through a midline abdominal incision, and a cannula was inserted into the hepatic artery and fixed. A group: saline water was injected to serve as the control. B group: saline water with 10mg/kg free carboplatin was given. C group : saline water with carboplatin-Fe@C nanocage-loaded chitosan nanoparticles(equivalent dose of free carboplatin 10mg/kg) was injected in absence of magnetic field. D group: saline water with carboplatin-Fe@C nanocage-loaded chitosan nanoparticles(equivalent dose of free carboplatin 10mg/kg) was injected in presence of magnetic field in 30 min. the general condition of rats were observed after drugs were given and the weight of rats were recorded. All the animals were sacrificed and abdominal exposure was done again after 7 days, abdominal aorta blood were collected, the numbers of RBC, WBC, PLT were counted, ALT, AST, TP, ALB, BUN and Cr of serum were detected, bone marrow was collected to observe granulocyte/macrophage progenitors(GFU-GM). The tissues of liver, kidney, spleen, lung, heart were examined for histological changes and the distribution of nanoparticles. Results: In B group, the metal status of rats were very poor, food intake and activities were lessened and coat color were lackluster after drugs were given, the weight of rats of B group were obviously lower than those of other 3 groups, there is no significant difference between C,D and A in numbers of blood cells and GFU-GM, the numbers of white blood cells and GFU-GM of B group were 7.8 ± 1.7 × 10~9/L, 82.5 ± 12.18/10~4 respectively ,which were obviously lower than those of A group(10.4 ± 2.3 × 10~9/L, 129.67± 17.47/10~4 respectively).There were no significant difference in every groups of ALT, AST, TP, ALB, BUN and Cr of serum . In the pathological feature , A group: there were no obvious pathological changes in every organs beside tumor tissue present mild necrosis because of overgrowth of tumor cells. B group: the epithelial cells of nephric tubule deformed and present mild cloudy swelling beside the pathological change of liver tumor. C and D groups: the there were no obvious pathological changes andobvious concentration of nanoparticles in every organs beside the pathological change of liver tumor. Conclusion: C-Fe@CN-CN can effectively reduce the side effects of carboplatin , and exhibit ideal biocompatibility in the rats with transplanted liver cancer as a safe magnetic nanoparticle to delivery chemotherapeutic agent. |
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