ACNP-MET Nano Drug Delivery System And Primary Study On ACNP-MET Targeting To Stem Cells Of Liver Cancer

Objective TO prepare the activated carbon nanoparticals of specific particle sizeand build nano-drug delivery system with metformin and investigate the targetingeffects of ACNP-MET to the CD133⁺cells from human hepatocellular carcinomaHuh-7cells line.Materials and Methods ACNP was prepared by ball milling and deposition indistilled water. The morphology and size of ACNP were examined through laserparticle size analyzer, transmission electron microscopy （TEM） and atomic forcemicroscopy （AFM）. Huh-7cells were treated with ACNP at various concentrations fordifferent durations. The alterations in morphology of cells were observed by lightmicroscopy （LM） and TEM. The lactate dehydrogenase （LDH） leakages in the culturemedium were determined with LDH activity detection kit. LDH leakage reflects theintegrality of cell membrane. Mixed suspension of ACNP and MET was prepared andadsorption was allowed under ultrasonic condition and the concentration of MET inthe fluid was measured. The time when the amount of the MET adsorbed on ACNPreached to the highest was treated as the equilibrium time. A series of differentproportion mixture of ACNP and MET were prepared and absorbed MET wasmeasured to learn the absorption equation. The best ratio of ACNP and MET wasdetermined based on the isothermal adsorption formula. CD133⁺cells were sorted byflow cytometry and the percentage of them in cultured Huh-7cells was analyzed. Theself-renewing and sphere-forming ability of CD133⁺cell were observed by lightmicroscope in vitro in comparison with CD133-cells. Tumor-forming ability ofCD133⁺cells were observed by xenografts of them in NOD/SCID mice. Theinhibitory effects of the ACNP-MET on the proliferation of CD133⁺cells were observed by CCK-8assay. The targeting effects of the ACNP-MET on the percentageof CD133⁺cells in Huh-7cells were measured by flow cytometry （FCM）. Weperformed tumor-sphere assays with CD133⁺cells grown in the presence or absenceof ACNP-MET. The tumor-sphere sizes were measured and the alterations inmorphology were observed under LM. TO employ mammosphere-forming efficiency（MSFE） and sphere size were used as indicators of CD133⁺cells-renewal andprogenitor cell proliferation.Results The ACNP has a similar size, a regular spherical shape and a smoothsurface. The average size is188nm. LM observation showed that ACNP canaccumulate around the cells and on the surface of cells. Neither evenly distributed inthe medium, nor aggregated and settled on the bottom of culture dish. Cells lived ingood condition. Under TEM, the outline of cell was obvious; the membrane ofnucleus was complete; the euchromatin was well distributed in nucleus; the nucleoluswas big and clear. The distribution characteristic of ACNP was advantageous forimproving the drug concentrations in the microenvironment directly contacting thecells. Abdominal cavity injection of ACNP to the mice, after3months, did not lead toedema、 adhesion and inflammatory reaction as examined by histopathologicalobservation. The experiments show that a large number of ACNP exist in lymphoidtissue and exudation and edema, inflammatory cells infiltration from the surroundingtissue were not seen when examining the abdominal wall and the peritoneum,peritoneal lymphoid tissue and lymph nodes in abdominal tissues. There was someACNP in Lymphocyte nuclei but the structure of the cell and sub-cellular structureswere normal.20days and6moths after ACNP were administrated by intravenousinjection, exudation, edema, inflammatory cellsinfiltration was not seen around theACNP in mouse heart, liver, spleen, lung and kidney, and ACNP had not been foundin brain tissue at the same time. After the Huh-7cells were treated with ACNP atvarious concentrations for24,48and72h, the LDH leakage was not higher than thatof the control group（P>0.05）. MET has the maximum absorption in233nm in allwavelength scanning by ultraviolet and visible spectrophotometer. The absorption at233nm was chosen as the determine wavelength of MET. In the concentration rangeof1～10μg/ml, the absorption of MET （A） had direct proportion with theconcentration （C）. The equation was A=0.00⁴79+0.08137C（r²=0.9995，P<0.01）.Under25℃, ACNP adsorption for MET got to stable state at4hours later with theequal temperature equation of C/X=-0.00531+0.00399C（r²=0.995，P<0.01）. ACNP had a maximum adsorption with a value of256.17mg MET in1g ACNP. Theoptimal ratio for adsorption was MET: ACNP=1:4. Flow cytometry analysis indicatedthat the purity of CD133⁺subset cells exceed90%. CD133⁺subset cells were verifiedmultipotent with the ability of forming tumor spheres within3culture days. AndCD133⁺subset cells were higher proliferative in vitro and had higher tumorigeniticability in vivo than CD133-subset cells within21d. And the tumor volumes were57.43,57.20,52.51and53.66mm³. The histopathologic slides of xenografts in mouseshowed that the tissue had capsule outsides, huge cell nucleus, and nucleolus. Themorphological characteristics of cells had significant atypia. The nuclear debris wasblack. The zone of necrosis was pink. ACNP-MET significantly inhibited theproliferation of CD133⁺and CD133-cells in a dose-dependent manner, with a higherinhibition rate compared with MET. The50%inhibition concentrations (IC₅₀) of METand ACNP-MET to CD133⁺cells was64.509and2.847μg/ml, respectively, while toCD133-cells was2.514×10⁴and316.997μg/ml respectively. ACNP groups did nothave a greater killing effect to CD133⁺and CD133-cells than control groups.Compared with MET, ACNP-MET can induced the percentage of CD133⁺cells inHuh-7cells in dose dependent（P<0.01） manner. In the tumor sphere-formingexperiment, the group of200μg/ml MET can reduce the numbers of CD133⁺cellsspheres compared with blank control group and ACNP group. Within50～200μg/ml,ACNP-MET had significant inhibiting effects on tumor sphere-forming indose-dependent manner（P<0.01）.Conclusions ACNP prepared by ball milling and deposition in the distilled waterhad a smaller size, a better quality than that sold in the market and it can be producedwith a big scale. ACNP had good biosafety. CD133⁺cells super marker sortingmethod can enrich CD133⁺cells in high purity, and CD133⁺cells sorted with CD133antibody possess the characteristics of TSC. The best proportion of MET and ACNPwas1/4and the best time was four hours for adsorption. Containing the same dose ofMET, ACNP-MET groups had a more significant inhibiting effect on CD133⁺andCD133-cells than MET groups. ACNP itself could not inhibit the cells but it couldboost up the targeting and inhibiting effect of MET. ACNP-MET had better targetingeffect without combination with other chemotherapy than free MET to TSC.