Co-delivery Of Sorafenib And Sulforaphane Using Targeting Mesoporous Silica Nanoparticle-supported Lipid Bilayers For Targeting Both Liver Cancer Cells And Cancer Stem Cells

In this study,we studied the effection of Sorafenib tosylate(SO) and Sulforaphane(SF) alone and combination aganist liver cancer cells.After that we developed modified mesoporous silica nanoparticles suPPorted lipid bilayers for co-delivery of Sorafenib tosylate (SO) and Sulforaphane (SF), to target both liver cancer cells and cancer stem cells.First, we established the HPLC method to determine the SO or SF contents. The results showed that in the range of 12.5～400μg·mL-1, SO concentrations had a good correlation with the sample peak area (r = 0.9999). The linear regression equation was A=10061C-203.8. The results also showed that in the range of 3.125～200 μg·mL-1, SF concentrations had a good correlation with the sample peak area (r = 0.9999). The linear regression equation was A=31321C + 44464. The linearity, precision, recovery and specificity of SO and SF met the requirement, and could be used to determine the contents of SO or SF.In the mild alkaline condition, taking CTAB as a positive ion surfactant, TEOS as an inorganic silicon source, at the temperature of 80 ℃, we developed mesoporous silica nanoparticles (MSN).For MSN modified with an amine-containing silane (AEPTMS), the pore diameter was 9.97 nm and the pore volume was 0.724cm3/g as evidenced by analysis of nitrogen adsorption-desorption isotherms. The drug loading of SF was improved after MSN was modified as determined by HPLC analysis.Liposomes composed of DOPC, DOPE, cholesterol, and DSPE-PEG2000 using the ethanol injection method. After then, we developed the liposomes combined with mesoporous silica nanoparticles. We observed the size and morphology of the nanoparticles by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The size and potential of the nanoparticles were evaluated byusing a Zetasizer Nano. The surface area and cumulative pore volume of the nanoparticles were measured by nitrogen adsorption-desorption. The results showed that the nanoparticles have well-formed spherical shapes, as evidenced by TEM and SEM.We used Cell Counting Kit (CCK-8) to detect the cell viability of HepG2 cells after treatment of SO and SF. The synergic effect was reflected by the combination index (CI) values:CI> 1.1, antagonistic; 0.9< CI< 1.1, additive; CI< 0.9, synergic. The CCK-8 assay showed that SO combined with SF at the molar rate of 1:1,1:10 and 1:20 exhibited strong antagonism (CI> 1.1), whereas 20:1,5:1 and 1:5 ratio resulted in synergistic activity (CI< 0.9). Furthermore,10:1 ratio acted as an additive effective (0.9< CI< 1.1). The number of colony formation (14.66 ± 2.08) of the group treated with SO combined with SF at 5:1 synergistic ratio was significantly lower than that of the group treated with SO combined with SF at 1:1 antagonistic ratio (31.33 ± 4.16) (P< 0.01). The early and late apoptosis rate of HepG2 cells (21.71±6.06) of the group treated with SO combined with SF at 5:1 synergistic ratio was significantly higher than that of the group treated with SO combined with SF at 1:1 antagonistic ratio (6.64 ± 0.311) (P<0.01).HepG2 tumor spheres, which showed the characteristics of liver cancer stem cells, were obtained by the culture of HepG2 cells in serum-free medium. We used CD 133 as the liver cancer stem cells marker. Using flow cytometry, 14.66% CD133+ cells were detected in the HepG2-TS. The CCK-8 assay results showed that IC50 of SO and SF towards HepG2-TS were lower than that of HepG2 cells. We used tumor sphere-forming efficiency assay to further evaluate the efficacy of SO and SF. After treatment of SO and SF, tumor sphere-forming efficiency declined as the tumor sphere-forming efficiency of PTX control groups went up. This result showed that SO and SF preferentially kill liver cancer stem cells. Compared with the CI value of SO and SF against HepG2-TS, we found that SO combined with SF at a molar ratio of 20:1 resulted in synergistic activity (CI<0.9), whereas 5:1 ratio acted as an additive effectiveness (0.9< CI<1.1).Drug-free mesoporous silica nanoparticles and mesoporous silica nanoparticles combined with lipid bilayer showed low-cytotoxicity in relative concentrations. The efficacy of SF/LMSN against HepG2 cells was more strong than SF alone. The efficacy of SO/LMSN combined with SF/LMSN at the molar rate of 5:1 was more strong than SO/LMSN or SF/LMSN alone.This study developed MSN,which has the well-formed spherical shapes,well topologies and uniform pore sizes.The drug loading and the drug efficiency of SO or SF were determined by HPLC. All the results showed that MSN can improve the drug loading and the drug efficiency of SO. After modified by AEPTMS, the drug loading of SF was improved. SO combined with SF at the molar rate of 5:1 showed synergistic activity in HepG2 cells whereas shows additive effective in HepG2-TS. The efficiency of SO/LMSN combined with SF/LMSN at the molar rate of 5:1 against HepG2 cells was more strong than SO/LMSN or SF/LMSN alone. Our findings suggested MSN merit further investigation as vehicles for loading therapeutic antitumor drugs, and may be used as an effective delivery strategy for other therapeutic reagents.