Construction Of PH-sensitive Dual-drug Delivery Targeted Micelle System And Evaluation Of Anti-liver Cancer

Objective:Through the screening of carrier ratio and drug dosage,the active targeting nanoparticles（THCD-NPs）co-loaded Curcumin（Cur）and Dastinib（DAS）was prepared.The particle size and morphology of THCD-NPs were investigated,meanwhile,the drug loading and encapsulation efficiency of the two drugs were determined.The in vitro stability and drug release of THCD-NPs were investigated as well.In vitro,tumour cells were cultured to investigate the pharmacodynamics and targeting properties of THCD-NPs.The tumor-bearing nude mouse model was established to investigate the in vivo targeting and tumor suppressive effects of THCD-NPs.Methods:1.Preparation and characterization of THCD-NPs.The HC was prepared by linking HA with Cur through esterification reaction,then its structure was confirmed by UV,DSC and ~1H-NMR.The THCD-NPs were prepared by thin film dispersion method,the particle size and polydispersity coefficient（PDI）were measured by Malvern particle size potentiometer,the particle morphology was investigated by transmission electron microscopy（TEM）.The drug loading（DL）and encapsulation rate（EE）of Cur/DAS in THCD-NPs were determined using HPLC.Taking the particle size and PDI of THCD-NPs,EE and DL of DAS,DL of Cur as evaluation indicators,the carrier ratio and dosage ratio were investigated to determine the optimal preparation formula of THCD-NPs.2.Stability,drug release and in vitro pharmacodynamics investigation of THCD-NP_S.In vitro stability and dilution stability of THCD-NPs were investigated by measuring changes in particle size.In vitro,simulating physiological conditions（p H≈7）,tumour tissue microenvironment（p H≈6-7）and lysosomal microenvironment（p H≈4-6）,to investigating the drug release of THCD-NPs by dialysis method.Hep G2cells were cultured in vitro,and the proliferation inhibition effect of THCD-NPs on Hep G2 cells was examined using MTT assay,cell viability assay kit and flow cytometry.The cell cycle inhibition effect of THCD-NPs on Hep G2 cells was examined using flow cytometry.The uptake of THCD-NPs by Hep G2 cells was examined using fluorescence microscopy and flow cytometry.3.The targeting,in vivo antitumor effect and safety evaluation of THCD-NPs.The biosafety of THCD-NPs was preliminarly investigated by hemolysis assay,and the tumor-bearing nude mouse model was established to evaluate the in vivo targeting,in vivo tumour suppression and biosafety of THCD-NPs.Results:1.Preparation and characterization of THCD-NPs.UV spectrum,DSC spectrum and ~1H-NMR spectrum all indicated the successful preparation of HC.The optimal formulation of THCD-NPs was HC:TPGS=6:1,the DAS:carrier was 1:10.The particle size of THCD-NPs was 66.14±4.02 nm,and the PDI was 0.22±0.01.The TEM image showed that the particles were spherical and evenly distributed.The DL of DAS was 5.94±0.23%,the EE was 67.09±3.33%,and the DL of Cur was 2.20±0.04%.2.Stability,drug release and in vitro pharmacodynamics investigation of THCD-NP_S.The size of THCD-NPs did not change significantly within seven days and after dilution of 1000 times,which showed good stability.In vitro drug release results indicated that THCD-NPs could accelerate the release of drugs at low p H.MTT assay,cell viability assay and flow cytometry results demonstrated that THCD-NPs had more significant cell proliferation inhibition and could induce more apoptosis.The results of fluorescence microscopy observation and flow cytometry assay all indicated that THCD-NPs treated group had more drug fluorescence.3.The targeting,in vivo antitumor effect and safety evaluation of THCD-NPs.Hemolysis test results indicated that THCD-NPs did not cause hemolysis.In vivo experiments showed that the nanoparticles can prolong the drug circulation time in vivo and target to the tumor site.The results of tumor inhibition experiment showed that THCD-NPs can effectively inhibit tumor growth with good biosafety.Conclusion:THCD-NPs has good stability,can responds to the low p H of tumor microenvironment to achieve selective drug release,and has significant inhibitory effect on tumor growth in vitro and in vivo.As a nano drug delivery system,THCD-NPs has potential clinical significance.