The PHarmacology And The Mechanism Of Action Of Activated Carbon Nanoparticles Adsorbing 5Fu

The objective of this experiment is to study the pharmacology of activated carbon nanoparticles(ACNP) adsorbed 5Fu in vivo and vitro and the mechanism of the action of ACNP. Firstly, the capacity of ACNP adsorbing 5Fu was examined by ultraviolet spectrophotometer and the in vitro effects of ACNP adsorbing 5Fu was studied, then the dose- and time-effects relations of ACNP were observed. In vivo, models of mice sarcoma S180 and liver H22 cell cancer ascitic tumor were used to study the pharmacology of ACNP adsorbing 5Fu. Through detecting the cell cycle of BGC-823 by ACNP and the apoptosis by ACNP and ACNP adsorbing 5Fu, the mechanism of action of ACNP was investigated.1. Capacity of ACNP adsorbing 5FuThe average particle diameter of ACNP is 80nm approximately. Ultraviolet spectrophotometer was used to examine the capacity of ACNP adsorbing 5Fu. Under the condition of 25℃, the capacity of ACNP adsorbing 5Fu was measured. The result indicated 5Fu could be adsorbed by ACNP with an equation of A=0.00913+0.03172C(R=1.0000,P<0.0001); An exponential relationship existed between absorption quantity and 5Fu concentration with an empirical formula was M=0.295C1.919(R=0.989,P<0.0001). Linear regression demonstrated that the adsorption capacity of ACNP was 286.53mg/g (R=0.960,P<0.0001).2. Experiment in vitroThe objective was to observe the influence of ACNP on the effects of 5Fu in vitro. Using MTT, the pharmacological influences of serial dose ACNP on the effects of 5Fu and the dose- and time-effect relationship were observed under the condition of 5Fu existence with a concentration of 120μg/ml. Then through accounting the cell survival rate by trypan blue, the pharmacological influence of ACNP on the effects of 5Fu was studied. 1.1 MTT measurementMTT measurement is a method of screening anti-cancer drugs in vitro, of which the fundamental principle is that the succinic dehydrogenases in viable cell can reduce the MTT to ianthinus formazan, which crystallizes in cells. Dissolving the crystallization by DMSO , the optical density in a certain wave length is measured to reflect the relative number of viable cell. The result of MTT measurements indicated that ACNP could reinforce the anti-cancer effect of 5Fu in vitro, and such a reinforce effect was positively correlated with dose and time. The dose-effect concentration of ACNP ranged from 12.5 to 200μg/ml while the concentration of 5Fu was 120μg/ml.1.2 Trypan blue measurementTrypan blue measurement is a classical dyeing method used in the judgement of cell death, of which the fundamental principle is that viable cell can repel trypan blue but dead cell not, so the dead cell shows blue while the viable cell no color presentation. Trypan blue in the experiment indicated that ACNP could reinforce the anti-cancer effect of 5Fu remarkably in vitro, and the action was increased with the increase of ACNP concentration. The anti-cancer of ACNP itself was slight.3. Experiment in vivoThe objective of the experiment was to observe whether ACNP could affect the anti-cancer effect of 5Fu or not in vivo. In general, having anti-cancer effect or not in vivo is critical of deciding the anti-cancer effect of drugs. The whole experiment included two experiments, one of which used the model of mice sarcoma S180 to observe the tumor control rate and the other used the ascitic tumor H22 to observe the life elongation rate.3.1 The effects on mice sarcoma S180Model of mice sarcoma S180 can estimate the effect of anti-cancer drug by calculating the tumor control rate. The result indicated that ACNP could reinforce the anti-cancer effect of 5Fu remarkably in vivo. When 5mg/kg 5Fu was with 200,100,50mg/kg ACNP, the tumor control rates were 70.98%,60.82%,51.15%, while 5mg/kg 5Fu singly was 28.54% merely.3.2 The effects on mice ascitic tumor H22Model of mice ascetic tumor H22 can estimate the effect of anti-cancer drug by calculating the life elongation rate. The result indicated that ACNP could lengthen the live time of the tumor-bearing mice in vivo. When 5mg/kg 5Fu was taken with 200,100,50mg/kg ACNP, the life elongation rates were 73.55%,49.09%,37.42%, while 5mg/kg 5Fu singly was 23.23% merely.4. Effect of ACNP on cell cycleCell cycle can ensure the cell correct proliferation. Through determining the effect of anti-cancer drug to cell cycle may understand the mechanism of action of anti-cancer drug. PI(Propidium Iodide) can combine with DNA and RNA, after RNA is digested under the exisitence of DNA enzyme inhibiter, the fluorescence intensity of PI combined with DNA can be determined by flow cytometry to reflect the content of DNA in cell. According to the discriminatory content of DNA in different phase, the cell is distinguished into G1/ G0, S and G2/ M phases, and the percentage of different phase may be calculated by certain software. The result indicated that ACNP can block BGC-823 cell in S phase, which may be one of the reinforcement mechanisms of ACNP to S-phase-specific 5Fu.5. Effect of ACNP and ACNP adsorbing 5Fu on apoptosisApoptosis is one of the basic cell characteristics and has important function in embryo development, tissue repair and internal environment balence, especially in tumor generation and development. Apoptosis is a form of cell death itself and is one of the hotspots in tumor therapy. In this part of experiment, Methyl-Green-Pyronine and AnnexinⅤ-PI dyeings were employed to determine the effect of ACNP and ACNP adsorbing 5Fu to induce apoptosis.5.1 Methyl-Green-Pyronine dyeingMethyl-Green-Pyronine dyeing is a classic method to observe viable cell, apoptosis cell and dead cell intuitively. The effect of nucleolus can be seen clearly through the method, and the viable cell rate, apoptosis cell rate and dead cell rate can be calculated by cell count. The fundamental principle of the dyeing is that Methyl-Green can dye nucleolus to green or aquamarine blue because of its specificity for DNA, and Pyronine can dye cytoplasm to red because of specificity for RNA. The apoptosis cell shows karyopyknosis and higher mRNA content because of increased mRNA in cytoplasm and concentrated cytoplasm, and expresses apoptosis gene and apoptosis related gene at the same time. So the apoptosis cell shows green or aquamarine blue nucleolus and red cytoplasm; the viable cell shows red cytoplasm merely; and the dead cell just shows green or aquamarine blue nucleolus. The result indicated that ACNP could induce slight apoptosis and no cell death; 5Fu could cause cell death and no apoptosis; but while using ACNP and 5Fu together, the apoptosis and cell death were induced remarkably.5.2 AnnexinⅤ-PI dyeingAnnexinⅤ-PI dyeing is a method along with flow cytometry. AnnexinⅤis a Ca2+ dependent albumin, of which the molecular weight is 35-36KD, able to specificly combine with phosphatidyl serine which backruns to outside of membrane in the process of apoptosis. Using AnnexinⅤfluorescent probe marked FITC can determine apoptosis by flow cytometry. PI is a dye of nucleic acid and cannot permeate the unabroken membrane, but in apoptosis metaphase and advanced stage it can reachthrough the mutilated membrane and dye nucleolus to red. The prophase and advanced stage apoptosis and dead cell can be distinguished by combinding with AnnexinⅤand PI. The result indicated that 200μg/ml ACNP single could induce apoptosis but not cell death, and 5Fu singly could induce cell death but not apoptosis, while ACNP and 5Fu together could induce both of apoptosis and cell death.