| Background: Neoplastic targeting therapy is one of the hot spots of neoplastic research. Enhancing anti-cancer drugs'selectivity for tumor cells, cutting down the side effects and increasing therapeutic effect are always an important subject in medicine. The research on nano-size drug carriers has brought hope to solving the problems. Nano-particles targeting to hepatoma not only can selectively transport anti-hepatoma drugs to the hepatoma locum, increase the drug concentration in hepatoma locum, and then dispel drugs slowly to maintain drug concentration to above effective concentration long time comparatively to acquire lasting and effective therapy. But also, nano-particles targeting to hepatoma can carry drugs into viable cells selectively, thus reduce the toxicity of drugs for normal tissues and cells, cut dowm the toxic and side-effects of drugs.Activated carbon nano-particle (ACNP) has strong adsorption and functional desorption of anti-cancer drugs. It is the hepatocytes'active characteristic change of enhanced phagocytosis to nano-particles brought about by the cancerization that makes anti-cancer drugs nano-particles-phored to generate passive and relative cello-targeting to the cancerized hepatocytes. So that ACNP can strengthen the anti-cancer effects and degrade the toxicity of the anti-cancer drugs. There is a favourable precondition for ACNP to be a target-carrier of the anti-cancer drugs, because of the targeting to liver of ACNP. Pt and 5-Fu are general chemotherapeutics of liver cancer. But they kill and wound normal tissues and cells simultaneously when they kill and wound cancer cells, because of the deficiency of selectivity to cancer tissues and cancer cells. So they all bring about serious side effect in clinical application. Frequently, the chems defeat as patients cannot tolerate the side effect. Thus, the drugs'effect is confined.As a kind of model in vitro, cultivation of primary hepatocytes has the prominent fortes:â‘ Its condition conforms with that in vivo, and the density of enzyme and cofactors are normal physio-level. It makes that feasible to study drugs'effects and toxicity in approximate physio-condition.â‘¡It commendably retain and sustain the morphous integrity and the metabolic activity in vitro of hepatocytes, thus, it can reflect metabolism state.â‘¢It deplete the influence from other organs and tissues. Meanwhile, there are some shortages, such as the elaborate and hard to handle isolating procedure and the harsh culturing condition. The technology of isolation and culture primary hepatocytes is always an emphasis in exploitation and research.Objective: The study made ACNP to adsorb two kinds of anti-hepatoma drugs and used it in hepatoma cells and primary hepatocytes to make sure that whether the ACNP can bring strong selectivity to the anti-cancer drugs, enhance their acti-cancer effects and cut down their side effect on normal cells, and offer the experiment foundation for the application of nano-size carrier taken along with anticancer drugs in tumor targeted therapy.Contents: To isolate and culture rat primary hepatocytes with good motility rate and activity; to determine the suitable effect dosage of ACNP and anti-hepatoma drugs, and raise a preparative method of ACNP and anti-hepatoma drugs, and work out the selectivity of anti-hepatoma drugs adsorbed by ACNP; to study the mechanism of ACNP enhancing anti-cancer drugs'selectivity of tumor cells initially through suitable methods.Methods: To isolate and culture rat primary hepatocytes using a method imp- roved from the traditional Seglen's two-step collagenase liver perfusion; to culture, passage and freeze hepatoma cells; in different dosage of ACNP adsorbing two kinds of commonly used anti-hepatoma drugs Pt and 5-Fu was added into the culture medium to observe the effects on primary hep atocytes and hepatoma cells, and observe the cell mitochondria function through detemination of the MTT metabolic rate by UV spectrophotometer to find the suitable action dosage of drugs, to observe the dose-effect relationship of ACNP-anticancer drugs, and then to study the selectivity of the anti-cancer drugs adsorbed by ACNP to tumor cells. Carrying out the flow cytometry by Annexin V-FITC/PI dipl-dye method to assess the apo- ptosis ratio's change of the primary hepatocytes and hepatoma cells; dyeing cells with methyl green and pyronine, to distinguish living cells, dead cells and apoptosis cells by their different expression of mRNA and DNA in the cytoplasm and the caryon; detect- ing the LDH leakage in the culture supernate, to determine the cell membrane intergrali- ty, and then to decide the different cytotoxicity came from different drug. Results: On the found of the traditional Seglen's two-step collagenase liver perfusion, took the experiences and the lessons in literatures'method, and combined the feature in equipment and technique of the laboratory in our own laboratory, finally established a convenient, inexpensive and efficient method for isolation of primary hepatocytes. The method used a"soft-digestion"mode with low concentration collagenase, and an added pre-incubation process. The time for the procedure was shortened, and the collagenase dosage was decreased. Thus the mechanical harm and chronic hypoxia of hepatocytes in perfusion process were diminished as far as possible, and that provided a self-recovery to hepatocytes, obtained with better hepatocytes yield and viability, and settled a good foundation for all the following experiments.The MTT assay demonstrated that the anti-cancer effect of anti-cancer drugs was strengthened and their toxicity on hepatocytes was decreased even after adsorbed by ACNP. This suggested anti-cancer drugs'being adsorbed by ACNP made them have more conspicuous selectivity. ACNP adsorbing two kinds of anti-cancer drugs whose anti-cancer mechanisms are different can strengthen the anti-cancer effects of the both at the same time. It meant that the mechanism of ACNP enhancing the anti-cancer drugs'selectivity lay in ACNP rather than the anti-cancer mechanisms of the anti-cancer drugs.The result of the FCM assay demonstrated that the apoptosis ratios of the anti-cancer drugs, nomater adsorbed by ACNP or being free, were small and there weren't much difference between the two formulation. It told that ACNP adsorbing anti-cancer drugs wasn't worked by influencing the apoptosis.In the methyl green and pyronine dyeing assay, not only the result gained in the FCM was confirmed further, but also the regularity taken from the counting of dead cells was coincident with the result of the MTT assay. The dead heptoma cells'number of anti-cancer drugs adsorbed by ACNP was bigger than that of the anti-cancer free, and it raised as the concentration of ACNP increasing. At the same time, the dead primary cultured hepatocytes cells'number of anti-cancer drugs adsorbed by ACNP was smaller than that of the anti-cancer free, and it lowered as the concentration of ACNP increasing. It told that ACNP adsorbing anti-cancer drugs made the drugs have more conspicuous selectivity through the way of increasing the cell death.The result in LDH leakage assay coincided with the experiment conclusions before. LDH leakage can reflect the integrality of cell membrane. It told that ACNP enhance anti-cancer drugs'selectivity by change the membrane structure. It should have some relationship with the cytology in which cell membrane structure must take a part, such as the phagocytosis.Conclusions: Activated carbon nano-particles adsorbing anti-hepatoma drugs reinforced the restrain effective to hepatoma cells and cutted down the toxic action on normal hepatocytes. The action mechanism of ACNP'synergism and attenuation to anti-cancer drugs, lies in that ACNP could produce effect through regulating the plasma membrane structure and function. Activated carbon nano-particles adsorbing anti-hepatoma drugs could be studied as a new kind of anti-hepatoma drug product. It has a good perspective in medical application with the ability of strengthening the selectivity and lower the side effect of anti-hepatoma drugs.
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