| Artesunate(ART)is a sesquiterpene structure compound and one of the semi-synthetic derivatives of artemisinin,has been recognized as a safe and highly effective anti-malaria drug.It has been widely reported that artesunate not only has good antimalarial activity,but also can inhibit the proliferation of tumor cells,regulate immune function and significantly inhibit the release of pro-inflammatory factors from macrophages induced by a variety of inflammatory factors.However,the applications of artesunate in clinic are still limited due to its almost insoluble in water,the concentration of ART in blood drops rapidly after intravenous injection and the half-life is only about 30 minutes,low bioavailability as well as poor stability.Colon cancer is considered as one of the most common malignancies with high incidence and mortality worldwide,with estimated 1.1 million new cases and 551,269 deaths in 2018 worldwide,which post a great challenge to human life and health.Currently,the mainstream clinical treatments for cancer are surgery,chemotherapy,and radiotherapy,etc.Among which chemotherapy isregardedas one of the most common therapies for colon cancer,bring more survival benefits to patients.However,the prognosis of chemotherapy is hardly idealistic due to the non-targeted anti-cancer drug delivery to the tumor site and the severe toxic effects on normal tissues,leading to limited therapeutic efficacy as well as drug resistance or migration of the tumor cells to other organs,thus limiting 5-year survival rates of patients.Hence,many researchers employed the advantages of new drug delivery systems to improve the stability and bioavailability of artesunate,with subsequent improved efficacy,for example,nanoparticles,liposomes,dendrimers,and nanomicelles and so on.In our study,we designed and synthesized pH responsive artesunate prodrug(PBAE-ART),artesunate(ART)was conjugated onto poly(2-ethyl-(2-oxazoline)-poly(lactic acid)-poly(β-aminoester)(PEOz-PLA-PBAE).The physical and chemical character,in vitro and in vivo antitumor activity,as well as the anti-tumor mechanism of the as-prepared prodrugs were investigated,to explore the potential application of ART prodrugs in adjuvant treatment of colon cancer.The synthesis of the ART prodrugs were summarizedas follows:firstly,Boc-OTs was employed as an initiator and was synthesized by substitution reaction of N-Boc-ethanolamine and p-toluenesulfonyl chloride;PEOz was synthesized by ring-opening polymerization of 2-ethyl-2-oxazoline as initiated byBoc-OTs;Poly(2-ethyl-2-oxazoline)-b-poly(lactide)(PEOz-PLA)was synthesized by ring opening polymerization of D,L-lactide initiated by PEOz.After the end hydroxyl groups of PEOz-PLA copolymer were activated by acryloyl chloride,1,4-butanediol diacrylate(or 1,6-hexanediol diacrylate,or 1,9-bis(acryloyloxy)nonane)and 2-amino-1,3-proranediol(or 6-amino-1-hexanol)were added to synthesize six different pH responsive triblock copolymers of PEOz-PLA-PBAE.Characterization of polymers:firstly,the structure and molecular weight of polymers were confirmed by 1H-NMR;secondly,the alkali dissociation constant(pKb)of polymers was determined by acid-base titration to reflect the pH response of polymer materials;finally,pyrene was introduced as a fluorescence probe to determine the critical micelle concentration(CMC)of the polymers by fluorescence spectrophotometry.The IH-NMR spectrum demonstrated the copolymers were successfully synthesized,the molecular weight of the six copolymers of PEOz-PLA-PBAE were 7249Da,8293Da,7153Da,7782Da,6985Da,repectively.The pKb values of the polymer materials are all about 6.5,indicating that the polymer materials had a certain pH response.And the CMC of the six polymer materials was 2.37mg·L-1,2.38mg·L-1,1.79mg·L-1,2.05mg·L-1,5.01mg·L-1,1.74mg·L-1,respectively,this shows that polymer materials can form micelles spontaneously at lower concentrationsThe prodrugs were prepared by conjugation betweenthe hydroxyl group on the polymer PBAE segment and the carboxyl group of ART.The polymer prodrugs were purified by dialysis against DI water and freeze drying.Laser light scattering(LLS)was used to measure the size and size distribution of the polymer prodrugs.The drug conjugation efficiency of the prodrug micelles were determined by HPLC.Evaluation of prodrug micelles stability by measurement of the size changes at 37℃ for 96h.Based on the above experimental results,the prodrug micelles were screened with better stabilities.The pH-responsive drug release behavior of the micelles was determined by dialysis method.The experimental results show that the particle size distribution of the artesunate prodrug micelles is uniform,but the size difference of the prodrug micelles was large.Compared with the blank micelles,the size of ART prodrug micelles increased to some extent due to the increased content of the hydrophobic core.pH sensitive properties were observed in the as-prepared polymer prodrugs.No significant increase in particle size within 96 hours was observed,demonstrating good stability of the polymer micelles.Different drug conjugation content was determined by using different polymer chain segments.When 1,4-butanediol diacrylate was used as PBAE segment,the drug loading was large,but the particle size of the micelles was large,which was 5.31%%±0.25%,190.31±4.5nm,9.57%±1.24%,197.25±6.4nm,respectively.When 1,9-bis(acryloyloxy)nonane)was used as PBAE segment,the drug loading was small and the particle size was small,3.98%±0.59%,33.45±0.8nm,2.14%±1.08%,97.43±2.2nm,respectively.Therefore,PBAE-ART4 and PBAE-ART2 were selected for subsequent study.The release profiles of PBAE-ART4 and PBAE-ART2 were studied under pH7.4 and pH6.0,respectively.Similar release profiles were observed for PBAE-ART4 and PBAE-ART2 at the current research conditions.The cumulative release of ART from PBAE-ART4 and PBAE-ART2micelles at pH7.4 in 48h was 57.2%and 55.9%respectively.However,the cumulative drug release at pH6.0 for 48h was 91.4%and 92.8%,respectively,demonstrating the accerlerated drug release at mild acidic conditions.No significant difference was observed in the release profiles of the two prodrug micelles under the same conditions.Mouse CT-26 colon cancer cells were used for cytotoxicity studies.MTT assay was used to detect the inhibitory effect of free ART and prodrug micelles of PBAE-ART4 and PBAE-ART2 on the proliferation of the cells.Flow cytometry was used to detect the effect of artesunate prodrugs on apoptosis.The mitochondrial membrane potential of cells after treatment by ART prodrug micelles was monitored by fluorescence microscopy with JC-1 as a probe.ELISA was used to detect intracellular reactive oxygen species after incubation of the cells with different ART formulations.Western blot experiments were used to further verify the enhanced apoptosis by the nanodrug formulations.It was demonstrated that the half cellular inhibitory concentrations of ART,PBAE-ART4,and PBAE-ART2 decreased with the increase of incubation time as IC50(24h)>IC50(48h)>IC50(72h),respectively,with the strongest inhibitory effect of PBAE-ART2 group.It should be noted that the prodrug groups showed stronger inhibition on mouse CT-26 colon cancer cells than the free drug ART.After the cells were treated for 72 hours,the apoptosis rate of the cells in free ART,PBAE-ART4,and PBAE-ART2 groups reached 18.41%,22.49%,and 26.85%,indicating the enhanced inhibition of the prodrugs on the colon cancer cells.It was also found that the polymer prodrugs could promote the depolarization of mitochondrial membrane potential of the cancer cells,as a comparison to the free drug.On the other hand,the intracellular ROS level was significantly increased in the prodrug groups than that treated by free ART.The artesunate formulationscould promote the expression of cytochrome c and apoptosis-related proteins such as Caspase 3 and Caspase 9,with the strongest impact in the PBAE-ART2 group.Therefore,the polymer prodrugs could significantly promote the apoptosis of CT-26 cells,as compared with the free drug ART.For animal studies,BALB/c mice were used to establish a subcutaneous xenograft model of mouse CT-26 cells.After the volume of the xenograft tumor reached to～100 mm3,the tumor bearing mice were administrated with free ART,PBAE-ART4 and PBAE-ART2 formulations through tail vein injection.The tumor volume and body weight of mice were monitored thrice a week.H&E staining was used to detect the cell morphology in the tumor tissue of the various treatment groups,demonstrating the therapeutic efficacy of the prodrugs on the tumor bearing mice.Our study demonstrated that the polymer prodrugs can effectively inhibit the tumor growth,as compared to that of saline group,with the most effective inhibition for the PBAE-ART2 group.Morover,the body weight of the mice in the treatment groups steadily increased upon the observation period,demonstrating the good biocompatibility of the current nano formulations.After the mice were sacrificed,the tumor tissue was removed and weighed.In the histopathology studies,the morphological structure of the cells in each administration group was observed.Compared with the normal saline group,the cell morphology of the administration group changed to different degrees,especially in the PBAE-ART2 group,the tumor cells were heterogeneously distributed and irregular sized tumor cells were observed,which indicates that the PBAE-ART2 prodrug micelles have better tumor suppressive effect. |
