The Construction And Anti-tumor Activities Of Docetaxel And Dihydroartemisin In Nanoconjugates

Objective:To gain possible synergistic antitumor effect of docetaxel?DTX?and dihydroartemisinin?DHA?,in this paper,docetaxel dihydroartemisinin prodrug with the disulfide bond of glutathione reduction response as the link arm was designed,and self-assembled nanoparticles?docetaxel-dihydroartemisinin nanoconjugates,DSDNs?was prepared by nanoprecipitation method.The preparation characteristics,pharmacokinetics in rats,in vitro antitumor activity to 4T1 cells and in vivo antitumor activity for 4T1 tumor bearing mice were evaluated to provide support for synergistic antitumor efficacy of docetaxel and dihydroartemisinin.Methods:1.Synthesis and characterization of docetaxel-dihydroartemisinin prodrug.In this study,docetaxel and dihydroartemisinin prodrug,linked with disulfide,was synthesized using two-step reaction.The structure of compounds was confirmed by HR-MS,¹H-NMR,¹³C-NMR and FT-IR.2.Preparation and optimization of the docetaxel-dihydroartemisinin nanoconjugates.The in vitro analytical method of DTX-S-S-DHA was established by high performance liquid chromatography?HPLC?;DSDNs preparation was optimized with particle size,entrapment efficiency?EE?,polydispersity index?PDI?and zeta potential as evaluation indexes.Transmission electron microscope?TEM?was used to observe the surface morphology of DSDNs.The drug loading?DL?of nanoparticles was determined by ultrafiltration centrifugation.The reduction response release behavior of DSDNs was investigated by small cup method,and the stability of DSDNs was investigated for one month.3.Pharmacokinetics of the docetaxel-dihydroartemisinin nanoconjugates in rats.High performance liquid chromatography-tandem mass spectrometry?HPLC-MS/MS?method for the determination of DTX and DHA in rat plasma was established for evaluation pharmacokinetics profiles of DTX-solution?DTX-sol?,DHA-solution?DHA-sol?and DSDNs in rats.12 male SD rats were randomly divided into three groups?n=4?.DTX-sol,DHA-sol and DSDNs were intravenously injected into the rats.The pharmacokinetic parameters AUC_0-t,AUC_0-?,MRT,CL,t_1/2 and V were used as indexes to evaluate its pharmacokinetics characteristics.4.In vitro anti-tumor activities of docetaxel-dihydroartemisinin nanoconjugates.MTT method was used to evaluate the inhibitory effect of DSDNs on the proliferation of 4T1 cells;Annexin V-FITC/PI double staining were used to study apoptosis;PI staining was used to carry out cell cycle test;scratch test was used to observe the inhibitory effect of DTX-sol,DHA-sol,MIX-sol and DSDNs on the migration of 4T1 cells.5.In vivo anti-tumor activities of docetaxel-dihydroartemisinin nanoconjugates.In vivo anti-tumor activities of DSDNs were investigated 4T1 bearing Balb/c mice model after intravenous injection every 2 days for 5 times,respectively.The body weight change,tumor volume,tumor weight,survival curve and H&E staining histopathological results of mice in the tail vein injection blank group?saline?,docetaxel injection?DTX-inj?,DHA-sol,MIX-sol and DSDNs?DSDNs?were used as evaluation index of in vivo antitumor effect.Results:1.Synthesis and characterization of docetaxel-dihydroartemisinin prodrug.The yield of anhydrides was 75.12%,and the yield of DHA-S-S-DTX was 55.39%.The m/z of DTX-S-S-DHA by HR-MS was 1288.479,¹H-NMR,¹³C-NMR and FT-IR of product indicated that the target compound was correctly synthesized.2.Preparation and optimization of the docetaxel-dihydroartemisinin nanoconjugates.The HPLC methods were used to determinate the concentration of DTX-S-S-DHA in vitro.The nanoparticles optimized by single factor and response surface method were light blue emulsion with particle size of 172.10±1.70 nm,PDI was 0.05±0.01,zeta potential was-22.60±0.50 mV,entrapment efficiency was 84.00%±1.30%and drug loading was75.91%±1.20%.Under the condition of 10 mM DTT,the cumulative release of DTX and DHA from DSDNs in 48 h were 76.86%±3.10%and 82.99%±3.98%,respectively.3.Pharmacokinetics of the docetaxel-dihydroartemisinin nanoconjugates in rats.The result of pharmacokinetics study showed that after intravenous injection of DTX-sol,DHA-sol and DSDNs to rats,AUC_0-t of DTX in DTX-sol and DSDNs were 35.26±6.67and 64.97±9.00 mg/L?h,the t_1/2 of DTX in DTX-sol and DSDNs were 6.08±1.29 and 9.41±3.16 h,respectively.AUC_0-t of DHA in DHA-sol and DSDNs were 0.54±0.21 and 0.30±0.16 mg/L?h,respectively.the t_1/2 of DHA in DHA-sol and DSDNs were 0.39±0.26 and0.22±0.12 h,respectively.4.In vitro anti-tumor activities of docetaxel-dihydroartemisinin nanoconjugates.MTT showed that DSDNs had inhibitory effect on the proliferation of 4T1 cells,and it was time-dependent and concentration dependent.IC₅₀ of DTX-sol,DHA-sol,MIX-sol and DSDNs was 0.2201±0.0341,1.6060±0.1033,0.4419±0.0177,0.5113±0.0121?M at 48h,and IC₅₀ of DTX-sol,DHA-sol,MIX-sol and DSDNs was 0.02811±0.0199,0.3800±0.0213,0.1624±0.0118,0.1613±0.0036?M at 72 h,respectively.The results of apoptosis showed that DSDNs had more significant ability of inducing apoptosis?P<0.05?;cell cycle test showed that DSDNs was mainly blocked in G₀/G₁ phase;compared with the control groups,anti-metastasis ability of DSDNs was more significant?P<0.01?.5.In vivo anti-tumor activities of docetaxel-dihydroartemisinin nanoconjugates.In vivo anti-tumor activities on the last day of the test,tumor volume of saline,MIX-sol,DTX-inj,DHA-sol and DSDNs was:2482±102,1417±148,1241±118,1134±102,989±164 mm³.The average tumor weights of saline,DTX-inj,DHA-sol,MIX-sol and DSDNs were 2.37±0.086,1.40±0.02,1.30±0.18,1.52±0.18,1.02±0.08 g.At the 20th day,the survival fraction of mice treated with DSDNs,DHA-sol,DTX-inj,MIX-sol and saline group were 8/8,7/8,5/8,4/8 and 3/8,respectively;H&E staining showed that liver injury of MIX-sol was found,but there was not found in other groups.Conclusion:1.Synthesis and characterization of docetaxel-dihydroartemisinin prodrug.The relative error between the accurate mass number measured by HR-MS and the theoretical value was within±3 ppm.The results of ¹H-NMR and ¹³C-NMR were consistent with the structure of the corresponding compounds.The results of FI-TR showed that the characteristic peaks of the corresponding compounds were consistent with those of the predicted compounds,indicating the successful synthesis of docetaxel dihydroartemisinin integrated prodrug.2.Preparation and optimization of the docetaxel-dihydroartemisinin nanoconjugates.The morphology,particle size,PDI,Zeta potential,EE,DL,stability and other evaluation indexes of nanoparticles prepared by response surface method meet the expected requirements.The results of in vitro release showed that DSDNs had a reduction response.3.Pharmacokinetics of the docetaxel-dihydroartemisinin nanoconjugates in rats.Pharmacokinetics of the docetaxel-dihydroartemisinin nanoconjugates in rats showed that the prodrug of DTX and DHA were linked by disulfide bond could optimize pharmacokinetic parameters,increasing blood concentration and prolong half-time of DTX.4.In vitro anti-tumor activities of docetaxel-dihydroartemisinin nanoconjugates.MTT test showed that DSDNs could inhibit the proliferation of 4T1 cells and had strong cytotoxicity;cell apoptosis showed that DSDNs could significantly induce apoptosis;cell cycle block showed that the block phase of DSDNs was similar to DHA-sol,which indicated that DHA played a major role in DSDNs;scratch test showed that DSDNs group had strong anti-metastasis ability.5.In vivo anti-tumor activities of docetaxel-dihydroartemisinin nanoconjugates.Compared with DTX-inj,DHA-sol and MIX-sol,DSDNs had strong anti-tumor activity in vivo,which could significantly inhibit the growth of tumor volume,and significantly improve the survival rate of mice.H&E staining results showed that DSDNs could reduce the toxicity to normal tissues.