Co-delivery Of Paclitaxel And Tanshinone ⅡA By Loading Into FA-modified Lipid-bilayer Coated Mesoporous Silica Nanoparticles For Tumorsuppression

Objective:The aims of this study were to optimize the combination of active components in traditional Chinese medicine with good therapeutic effect on acute promyelocytic leukemia（APL）,to explore the mechanism of its synergistic effect,to construct active tumor targeted co-delivery system with folic acid（FA）modified lipid-bilayer coated mesoporous silica nanoparticles as carriers,and to systematically evaluate the mechanism of targeting delivery efficiency and anti-tumor attenuation and synergy effect.Methods:We have investigated the proliferation inhibition rate of different active components in traditional Chinese medicine towards acute promyelocytic leukemia cell line（NB₄cell line）by cytotoxicity test in vitro.The drug combination and the optimal ratio were selected for the treatment of APL.Mesoporous silica nanoparticles（MSNs）were prepared by improved St?ber method and the synthesized MSNs were characterized.Ptx and Tan ⅡA loaded folic acid（FA）modified lipid-bilayer coated mesoporous silica nanoparticles（（Ptx+Tan ⅡA）@FA-LB-MSNs）were prepared by film dispersion-ultrasonic method.The NB₄cell uptake study of formulations with different FA modification ratios was used to determine the optimal ratio of FA modification,and the final formulation was characterized.The in vitro release study and stability study were also carried out.The in vitro anti-tumor effect and mechanism in inducing the apoptosis and differentiation of the co-delivery system were investigated by NB₄cells.Cyanine 5.5 NHS ester was selected as the fluorescent probe to investigate the distribution of MSNs,LB-MSNs and FA-LB-MSNs in NB₄tumor cell-bearing nude mice.The tumor growth inhibition effects of different formulations after tail vein injection were investigated with NB₄tumor cell-bearing nude mice model,and the safety of the formulations was also investigated in vivo.Combined with tumor cell apoptosis analysis and detection of related apoptotic proteins,the anti-tumor effect and mechanism of the new drug delivery system were evaluated.Results:The synergistic effect of paclitaxel（Ptx）combined with tanshinone ⅡA（Tan ⅡA）on the proliferation of NB₄cells was stronger than the combination of other active components investigated.When Ptx and Tan ⅡA were applied at a molar ratio of1:1,the CI₅₀value was small and there was obvious synergistic effect.When the FA modification ratio was 0.8%,the uptake of nanoparticles by tumor cells was maximized.The drug loadings of Ptx and Tan ⅡA in（Ptx+Tan ⅡA）@FA-LB-MSNs were 5.5%and 1.8%,respectively.The nanoparticles were ellipsoidal and uniformly dispersed.The results of Fourier transform infrared spectroscopy（FTIR）indicated that the FA modified lipid-bilayer membrane was successfully coated on the surface of MSNs.The drugs were successfully loaded into the pores of MSNs in amorphous or amorphous form.Compared with the uncoated preparation,the lipid-bilayer coated nanoparticles showed a sustained release effect,and Ptx and Tan ⅡA could be released synchronously from the carrier.In the concentration range of 0～500μg·m L^-1,unloaded nanoparticles（MSNs,LB-MSNs,and FA-LB-MSNs）had no obvious toxic effect on NB₄cells.Lipid-bilayer coating could improve the blood compatibility of MSNs,increase the dispersibility of MSNs by avoiding nanoparticle aggregation,and increase the plasma stability of MSNs.The result of pharmacodynamics in vitro showed that（Ptx+Tan ⅡA）@FA-LB-MSNs as nanoparticles targeting NB₄cells could be effectively uptaken by NB₄cells,and Ptx and Tan ⅡA combination with a strong synergistic effect could effectively promote the apoptosis and differentiation of NB₄cells.The result of pharmacodynamics in vivo showed that the fluorescence signals of FA-LB-MSNs group at the tumor site were stronger than those of the LB-MSNs and MSNs group without FA modification at different time points,which was due to the fact that FA-LB-MSNs could exert its targeting effect and concentrate in tumor tissues.The（Ptx+Tan ⅡA）@FA-LB-MSNs group showed significantly better effects on inducing apoptosis and inhibiting tumor growth than the reference groups,and had no significant toxicity to the heart,liver,spleen,lung and kidney of the tumor-bearing nude mice,showing a better attenuation and synergy effect.Conclusion:Ptx and Tan ⅡA had a strong synergistic effect on NB₄tumor cells.FA-LB-MSNs had good drug loading performance and tumor-targeted drug delivery property.Co-delivery of Ptx and Tan ⅡA with FA-LB-MSNs for the treatment of APL could effectively be uptaken by NB₄cells and promote apoptosis and differentiation of NB₄cells,showing a strong tumor inhibition effect.The active tumor-targeting drug co-delivery systems designed in this paper provided a new method for the effective anti-tumor effect of traditional Chinese medicine.