Tumor Microenvironment Redox-Responsive Prodrug Nansassemblies For Cancer Chemotherapy

Cancer is still the leading cause of death in the world today.Chemotherapy is the most common and effective strategy.However,the current state of chemotherapy is greatly restricted by the inefficient drug delivery,results in serious side effects,narrow therapeutic window and limited clinical efficiency.To address these problems,a wide variety of drug delivery strategies have been developed,including prodrugs and nanoparticulate drug delivery systems(nano-DDS).Furthermore,prodrug-based self-assembled nanoparticles(NPs)integrating multiple drug delivery technologies into one system,has been emerging as a promising platform with impressively high drug loading and low excipient-associated side effects.Moreover,how to realize precise drug release at tumor sites is also of crucial importance for efficient cancer therapy.Compared with normal cells,tumor cells simultaneously overproduce reactive oxygen species(ROS)and glutathione(GSH),leading to a redox-heterogeneous intracellular environment,which has been well studied to achieve on-demand drug release.Our group have developed several thioether bond or disulfide bond-bridged paclitaxel(PTX)prodrug for cancer therapy.The prodrugs could self-assemble into uniform sized NPs.Thioether bond was found to be redox-dual responsive,with impressively high oxidation-responsiveness.In comparison,disulfide bond has excellent reduction-responsiveness.Based on the physicochemical property of disulfide bond,we further hypothesized that disulfide bond might be also used as an oxidation-responsive linkage.Besides,the position of disulfide bond in prodrugs would have great influence on the in vivo fate and pharmacodynamics of prodrug nanoassemblies.To test our hypothesis,three novel prodrugs were synthesized by conjugating PTX with citronellol(CIT),using various lengths of disulfide bond-containing carbon chain as linkages.The sulfur atoms located in the ?-,?-,or?-position of the ester bond,abbreviated as ?-PTX-SS-CIT,?-PTX-SS-CIT,and?-PTX-SS-CIT,respectively.The prodrugs could self-assemble into uniform sized NPs.As expected,the disulfide bond-bridged prodrugs nanoassemblies demonstrated distinct redox dual-responsive capability and we elucidated the responsive mechanism.Moreover,we found that the position of disulfide bond in prodrug significantly affect the redox dual-responsiveness.The oxidation-responsiveness of prodrug nanoassemblies followed the order of a-PTX-SS-CIT NPs>?-PTX-SS-CIT NPs>?-PTX-SS-CIT NPs.The reduction-responsiveness followed the order of?-PTX-SS-CIT NPs>y-PTX-SS-CIT NPs>?-PTX-SS-CIT NPs.a-PTX-SS-CIT prodrug has distinct redox-dual responsiveness.We investigated the cytotoxicity and intracellular drug release of prodrug nanoassemblies.Much more PTX was released from a-PTX-SS-CIT NPs than that from ?-PTX-SS-CIT NPs and y-PTX-SS-CIT NPs,leading to a more potent cytotoxicity.We then evaluate the pharmacokinetic profiles of prodrug nanoassemblies.The position of disulfide bond had an influence on the pharmacokinetic behavior.The AUC of a-PTX-SS-CIT NPs,p-PTX-SS-CIT NPs and ?-PTX-SS-CIT NPs was 8.4-fold,11.9-fold,and 16.6-fold higher than that of Taxol,respectively.But the AUC value of the released PTX from prodrug NPs followed the order of a-PTX-SS-CIT NPs>?-PTX-SS-CIT NPs>?-PTX-SS-CIT NPs.DiR-labeled prodrug nanoassemblies were prepared to investigate the biodistribution of prodrug nanoassemblies in KB tumor bearing nude mice.Compared with DiR solution,DiR-labeled prodrug nanoassemblies demonstrated significantly higher fluorescent intensity in tumor.y-PTX-SS-CIT NPs,with longer circulation time,exhibited better tumor accumulation than a-PTX-SS-CIT NPs and P-PTX-SS-CIT NPs.We then evaluate the antitumor efficiency of prodrug nanoassemblies.Notably,prodrug nanoassemblies showed more potent antitumor activity than Taxol.Interestingly,the superior redox dual-responsive drug release of ?-PTX-SS-CIT NPs in tumor site led to a more potent chemotherapeutic efficacy than ?-PTX-SS-CIT NPs and y-PTX-SS-CIT NPs.The above results indicate that the position of disulfide bond in prodrug has profound impacts on the redox dual-responsiveness,drug release,cytotoxicity,pharmacokinetics,biodistribution and in vivo antitumor efficacy of prodrug nanoassemblies.In the current states,the redox-responsive linkages are mainly focus on sulfur(S)bonds.Selenium(Se),belonging to the same family as S in the periodic table of the elements,has also attracted increasing interest.Therefore,six paclitaxel-citronellol prodrugs were synthesized using sulfur/selenium/carbon bonds as chemical linkages,abbreviated as PTX-S-CIT,PTX-SS-CIT,PTX-Se-CIT,PTX-SeSe-CIT,PTX-C-CIT and PTX-CC-CIT,respect:ively.These hydrophobic prodrugs can self-assemble into uniform size NPs.The chemical linkages impose significant influence on the assembly performance and colloidal stability of prodrug nanoassemblies.Notably,the bond angle/dihedral angle of-SeSe-was closest to 90°,which may provide enough structural flexibility to balance intermolecular forces and establish a favorable conformation during self-assembly.In comparison,the bond angles of carbon linkages were much larger than those of sulfur/selenium bonds,resulting in poor colloidal stability.The sulfur/selenium bond-bridged prodrug nanoassemblies showed redox dual-responsive drug release.The oxidation-responsivity followed the order of selenoether bond>thioether bond>diselenide bond>disulfide bond and the reduction-responsiv:ity were on the contrary.In comparnson,the carbon bond control groups were no redox-responsive,exhibited extremely slow drug release.We investigated the cytotoxicity and intracellular drug release of prodrug nanoassemblies.The cytotoxicity of prodrug nanoassemblies followed the order of PTX-SeSe-CIT NPs>PTX-Se-CIT NPs>PTX-SS-CIT NPs>PTX-S-CIT NPs>PTX-CC-CIT NPs/PTX-C-CIT NPs.Much more PTX was released from PTX-SeSe-CIT NPs and PTX-Se-CIT NPs than from PTX-SS-CIT NPs and PTX-S-CIT NPs.In comparison,negligible PTX was released from PTX-CC-CIT NPs and PTX-C-CIT NPs,explaining the low proliferation inhibition observed.We investigated the influence of prodrug nanoassemblies on the intracellular ROS,GSH and GSSG level in KB cells.Selenium-containing prodrug nanoassemblies,especially PTX-SeSe-CIT NPs,greatly increased the intracellular ROS levels and decreased the intracellular GSH concentration.The increased intracellular oxidative stress could further induce apoptosis of tumor cells.We then evaluate the pharmacokinetic profiles of prodrug nanoassemblies.The prodrug nanoassemblies displayed significantly higher AUC than Taxol.Furthermore,the sulfur/selenium/carbon bonds had a great influence on the pharmacokinetic behavior.Much more PTX was released from PTX-S-CIT NPs/PTX-Se-CIT NPs than from PTX-SeSe-CIT NPs/PTX-SS-CIT NPs,possibly due to the superior oxidation-responsivity and poor stability of thioether/selenoether bonds than disulfide/diselenide bonds.This reason also accounts for the lower AUC values of PTX-Se-CIT NPs/PTX-S-CIT NPs than PTX-SeSe-CIT NPs/PTX-SS-CIT NPs.Further,PTX-CC-CIT NPs and PTX-C-CIT NPs had lower AUCs compared with PTX-SeSe-CIT NPs due to their poor colloidal stability,and thus they could be quickly cleared from the body.DiR-labeled prodrug nanoassemblies were prepared to investigate the biodistribution of prodrug nanoassemblies in KB tumor bearing nude mice.DiR-labeled prodrug nanoassemblies demonstrated significantly higher fluorescent intensity in tumor than DiR solution.In addition,the biodistribution of prodrug nanoassemblies were well in line with their pharmacokinetic behavior.PTX-SeSe-CIT NPs,with better colloidal stability and longer circulation time,showed a distinctly higher concentration than other groups in tumors.KB tumor bearing nude mice were utilized to evaluate the antitumor efficiency of prodrug nanoassemblies.Notably,sulfur/selenium bond-bridged prodrug nanoassemblies had more potent antitumor efficacy than Taxol.In comparison,mice treated with non-sensitive PTX-CC-CIT NPs/PTX-C-CIT NPs exhibited a rapid increase in tumor volume,possibly due to the poor stability of the NPs in vivo and insufficient drug release in tumor sites.Further,PTX-SeSe-CIT NPs/PTX-SS-CIT NPs showed better tumor inhibiting activity than PTX-S-CIT NPs/PTX-Se-CIT NPs,probably due to their better colloidal stability,longer circulation time and higher AUC values.Moreover,PTX-SeSe-CIT NPs exhibited the most potent tumor-inhibiting activity,with significantly reduced tumor volume.The results corroborated that sulfur/selenium/carbon bonds have great influence on the self-assembly,colloidal stability,redox-responsive drug release,cytotoxicity,pharmacokinetics,biodistribution and antitumor efficiency of prodrug nanoassemblies.