Reactive Oxygen Species Regulate The Bioactivation Of Taxane Prodrugs Self-assembled Nanoparticles To Improve Antitumor Efficacy

Recently,a large number of reports have reported that the content of reactive oxygen species in tumor cells is nearly 100 times higher than that in normal cells.Therefore,the design of active oxygen-triggered prodrugs and nano-preparations to specifically release the drug in tumor cells has attracted more and more researchers' attention.However,tumor sites also exhibit heterogeneity,the difference in reactive oxygen species exhibited in different regions of the tumor or in different growth cycles of tumor cells.Therefore,it is far from enough to rely solely on the high ROS environment of the tumor cells,and it is necessary to provide exogenous reactive oxygen species to enhance the anti-tumor effect.Therefore,this project designed three kinds of cabazitaxel-oleic acid prodrugs,forming a prodrug nanosystem in one step after adding DSPE-PEG₂k and encapsulating the photosensitizer?PPa??PCANPs,PCSANPs and PCSeANPs?,it is expected to achieve the combination of chemotherapy and photodynamics,to achieve accurate targeting of tumor sites to quickly release anticancer drugs,thereby improving anti-tumor efficacy.At the same time,many prodrugs have ultra-high reactive oxygen responsi'veness,and it is highly probable that they will be hydrolyzed by the components of the blood before reaching the tumor site,losing the function of the prodrug to selectively release the drug at the tumor site.Therefore,the highly sensitive small molecule prodrug nano preparation needs to be protected,and after it reaches the tumor site,the selective release drug can be realized by means of the change of the active oxygen content of the tumor site.Therefore,in this paper,a reactive oxygen sensitive bond-bridged paclitaxel?PTX?-maleimidocaproic acid?MAL?prodrug nanoformer?Psm?was designed,which was coated with polydopamine.The post-clothing small molecule prodrug nano preparation?PsmDE?selectively releases the drug at the tumor site by means of high reactive oxygen species,thereby improving the antitumor effect.Based on the above principles,we validate our hypothesis by in vitro drug release,cytotoxicity,pharmacokinetics,tissue distribution,and pharmacodynamic experiments.Firstly,the sensitivity of the three prodrug nanopreparations loaded with photosensitizer to the active oxygen was investigated by using phosphate buffered saline?PBS?-anhydrous ethanol with different concentrations of H₂O₂ or different time laser irradiation as the release medium.The results showed that PCSANPs and PCSeANPs can be rapidly and rapidly added in the medium with H₂O₂ release or laser irradiation.The drug is released and shows good responsiveness to both reactive oxygen species.Moreover,the release rate of PCSeANPs under light conditions is faster than that of PCSANPs,indicating that single selenium bond showed better ROS-responsiveness than monosulfide bonds responsiveness.At the same time,different concentrations of H₂O₂ phosphate buffer-anhydrous acetonitrile were used as the release medium to investigate the responsiveness of Psm and PsmDE to reactive oxygen species,and whether dopamine coating would affect the drug release characteristics of the prodrug nanoformulation.The results showed that both Psm and PsmDE showed ultra-high reactivity with reactive oxygen species,and the coating layer did not hinder the rate of drug release.The cytotoxicity of CTX-sol,PPa-sol and three prodrug nanopreparations loaded with photosensitizer on 4T1 breast cancer cells were investigated by MTT.The nano-formulation group containing sensitive bonds after the increase in illumination was twice as large as the non-illumination group IC₅0 value of the corresponding preparation.In addition,the IC₅0 values of PCSANPs were smaller than those of PCSANPs.Subsequently,the HPLS-MS-MS method was used to detect the activation of the drug in the intracellular prodrug after the MTT test,and it was found that the toxicity of the preparation to the cells was positively correlated with the release rate of the intracellular drug.At the same time,the cytotoxicity and intracellular prodrug activation experiments of Psm and PsmDE on 4T1 tumor cells and 3T3 normal cells were investigated.The results showed that both Psm and PsmDE showed high cytotoxicity to tumor cells and less toxicity to normal cells.Subsequently,the results of intracellular prodrug activation experiments showed that the paclitaxel prodrug nanoforms released twice as much drug in tumor cells as normal cells,indicating that the paclitaxel prodrug nanoforms can selectively release drugs according to cell types.The in vivo pharmacokinetic behavior of CTX-sol and three prodrug nanopreparations loaded with photosensitizer in the Sprague-Dawley rat model was investigated by HPLC-MS-MS.The results showed that the AUC of the three prodrug nano-preparations was at least 300 times that of CTX-sol,indicating that the prodrug nanopreparation can greatly improve the time of the drug in the blood circulation.The tissue distribution behavior of PPa-sol and three prodrug nanopreparations loaded with photosensitizer in tumor-bearing mouse model was investigated by small animal in vivo imaging.The results showed that,unlike the in vivo distribution of PPa-sol,the three prodrug nanopreparations loaded with photosensitizers gradually accumulated in the tumor site and peaked after 12 h of administration.It is indicated that the nanopreparation has tumor targeting,and the increase of illumination after 12 hours of administration is the optimal time for chemotherapy combined with photodynamic therapy.At the same time,the tissue distribution of Dir-sol,Psm and PsmDE in tumor-bearing mice was investigated.The results showed that after 24 h of administration,PsmDE showed more tumor accumulation than Dir-sol and Psm.Inhibition of tumors by administration of different preparations was investigated by administering PBS,CTX-sol,PPa-sol and three prodrug nanoprecipitates containing photosensitizers to 4T1 breast cancer-bearing mice.The results showed that the prodrug nanopreparation containing active oxygen sensitive bonds was more effective than the corresponding non-lighting prodrug nanopreparation under the illumination.Therefore,the use of exogenous reactive oxygen species generated by endogenous reactive oxygen species in the tumor combined with photodynamic therapy can significantly improve the antitumor efficacy of the dual-sourced reactive oxygen species.At the same time,the tumor suppressive effect of 4T1 tumor-bearing mice after administration of PBS,Taxol,Psm and PsmDE was investigated.The results showed that the inhibitory effect of PsmDE on tumors was more obvious than that of Taxol and Psm.Therefore,before utilizing the hyperactive oxygen conditions specific to the tumor site,it is first necessary to protect the prodrug nano-preparation with high sensitivity of reactive oxygen species to reach the tumor smoothly.