| Objective:To establish a specific way to preparation of active carbon nanoparticals(ACNP)which could be used as drug carrier, and study the inhibitory effects of ACNP,mitomycin C (MMC) and MMC adsorped on ACNP(CBMC) on human liver cellline Huh-7and cancer stem cells from Huh-7. Compare the results and assess thepotential of using ACNP as an anti-tumor drug carrier for the treatment of livercancer.Methods:1. The activated carbon nanoparticles were prepared by ball milling anddeposited in distilled water to obtain the mixed suspension containing activatedcarbon nanoparticles, in which the nanoparticles with different sizes distributed indifferent layers. Through centrifugation of the suspension in a certain layer, ACNPwith a specific size may be obtained. The size and morphology of ACNP wereexamined through transmission electron microscopy (TEM) and atomic forcemicroscopy (AFM).2. Standard curve of MMC was drawn by using of MMC standard solutions.The concentration of MMC was determined under364nm by a ultravioletspectrophotometer. The adsorption of ACNP for MMC was carried out by allowingthe suspension with ACNP and MMC to standstill for different times underultrasonic condition. The concentration of MMC in the supernatant obtained bycentrifugation of the suspension was measured in different times. The time at whichthe tangent line of the adsorption curve crosses with the horizontal line was selectedas the adsorption equilibrium time. A series of different proportion mixture of MMCand ACNP were prepared in30minutes under ultrasonic condition to obtain the bestratios for adsorption which was determined by absorption equation.3. Seperate the CD133+cells from Human liver cancer Huh-7cell line by flow cytometry using CD133-PE antibodys. The CD133+cells were cultured in serum-freeDMEM/F12medium with the EGF, bFGF and B27. Then using a variety ofidentification experiments in vivo and in vitro to confirm the separated cancer stemcells.4. Huh-7and Huh-7cancer stem cells were exposed to MMC, ACNP andCBMC drug groups and every drug groups involves six concentration of2.5,5,10,20,40and80μg/ml. The concentration of CBMC was calculated in MMC itcontained. CCK-8kit was used to determine the inhibitory effects of drugs on Huh-7and Huh-7cancer stem cells.Results:1. The ACNP made by suspension settlement method has a similar size, aregular spherical shape and a smooth surface, and the average size is89.93±12.63nm as determined by AFM and TEM. The ACNP made by this method in differentbatches has a stable shape and quality.2. The standard curve equation of MMC detected by ultravioletspectrophotometer is D=0.00213+0.06942×C（R=0.9996，P<0.0001); the adsorptionequilibrium time was confirmed as30min; The equal temperature equation wasC/X=1.75048+0.0079×C （R=0.98236，P<0.0001）and the maximum adsorptioncomputed from the equation was126.58mg/g. so the optimal ratio for adsorption ofMMC and ACNP which could be used in the subsequent experiments was1:8.3. The proportion of the sorted CD133+cells was92.81%while the proportionwas just5.18%in the Huh-7cells as determined by flow cytometery. The CD133+cells were cultured in serum-free DMEM/F12medium with the EGF, bFGF and B27.The results of identification experiments in vivo and in vitro suggested that theCD133+cells of Huh-7have a higher proliferation ability, differentiation potentialand tumorigenic ability than Huh-7. So we can confirm that the CD133+cells wesorted by fluorescence activated cell sorting from Huh-7are Huh-7cancer stemcells.4. Huh-7and Huh-7cancer stem cells were exposed to MMC, ACNP and CBMC drug groups and every drug groups involves six concentration of2.5,5,10,20,40and80μg/ml.The half inhibitory concentration (IC50) of MMC and CBMC for Huh-7wererespectively10.28μg/ml and4.822μg/ml while that for Huh-7cancer stem cellswere respectively132.078μg/ml and56.83μg/ml. The results showed that ACNPhas no inhibitory effect for Huh-7and Huh-7cancer stem cells at each doses.Although the inhibitory effects of MMC and CBMC for Huh-7stem cells weresignificantly lower than that for Huh-7at every same dose, the inhibitory effects alsoincreased in a dose-depedent manner and the difference was statistically significant.The inhibitory effect of CBMC for Huh-7and Huh-7cancer stem cells weresignificantly higher than using MMC or ACNP only at each doses. The inhibitoryeffect of MMC was significantly increased by using ACNP as drug carrier. Theresults showed that the difference was statistically significant.Conclusions:1. The ACNP with stable and controllable quality was made in this study andthe average size was89.93±12.63nm.2. The adsorption equilibrium time was30min and the maximum adsorptionwas126.58mg/g which was computed from equal temperature equation. So theoptimal ratio for the adsorption of MMC and ACNP was1:8.3. Successfully separated CD133+cells from Huh-7by fluorescence activatedcell sorting and the proportion of CD133+was92.81%after sorting. The CD133+cells from Huh-7were identified as cancer stem cells of Huh-7through a variety ofidentification experiments in vivo and in vitro.4. The inhibitory effects of MMC and CBMC for Huh-7were significantlylower than Huh-7at the same dose, but the inhibitory effects still increased with theincreasing of dose. The results have statistical significance.5. The inhibitory effects of CBMC for Huh-7and Huh-7cancer stem cellswere significantly higher than using MMC only at each same dose in adose-dependent manner indicating that the inhibitory effect of MMC was significantly increased by using ACNP as drug carrier. The results have statisticalsignificance.6. ACNP has no inhibitory effect for Huh-7and Huh-7cancer stem cells. |
PDF Full Text Request