Construction Of Targeted Activable Prodrugs And Their Applications In Anti-Tumor And Anti-Senescence Therapy

Traditional molecular drugs often cause serious side effects on normal tissues due to the lack of selectivity,which can be attributed to two main reasons:sustained exposure of drug activity and low accumulation of drugs in target sites.To this end,the above two problems can be addressed by activating prodrug strategies and targeted delivery strategies,respectively.The former relates to the conjugation of the active sites of drugs with different chemical structures to reduce or completely abolish drug activity.The blocked active sites can be reactivated by specific exogenous stimuli or endogenous biomarkers and exert the therapeutic effect only after the prodrugs reach the target areas.The latter involves conjugating the drugs with molecules that have targeting functions or making the drugs into nanoparticles to target disease areas actively or passively,increasing the accumulation of the drugs at the target sites.Compared with the individual strategies,the combination of the above two strategies is expected to further improve drug selectivity.Therefore,this thesis aims to address the bottleneck problems in the treatment of diseases and constructs three targeted activable prodrugs respectively,and successfully applied them in anti-tumor and anti-senescence therapy.The specific research contents are as follows:（1）Aiming at addressing the problem of how to improve the drug selectivity and therapeutic effect during tumor chemodynamic therapy（CDT）,an aptamer-prodrug conjugate（Ap Pd C）micelles containing Fe²⁺/heme activable prodrug monomers and hemin is constructed through the solid-phase synthesis and self-assembly process.Experimental results have shown that Ap Pd C micelles could target the upregulated nucleolin protein on the surface of liver cancer cells through the aptamer AS1411.After entering the cells,the prodrug monomers can be activated by Fe²⁺in tumor cells or the heme generated by reducing hemin with GSH to release cytotoxic C and O free radicals,inducing tumor cell apoptosis.Ap Pd C micelles not only improve the drug selectivity but also overcome the limitations of CDT,which can generate free radicals in situ without dependence on either H₂O₂ or p H.Moreover,they can weaken the antioxidant capacity of tumor cells by depleting GSH to obtain a synergistically enhanced CDT effect,which provides a new method for CDT-based drug design.（2）To improve the selectivity of senolytics,we demonstrate for the first time that the L1 cell adhesion molecule（L1CAM）can be used as a senescent endothelial cell-related biomarker,which is upregulated in senescent vascular endothelial cells induced by different stimuli.On this basis,an activable aptamer-senolytic prodrug conjugate(Ap MSPd_β-gal)capable of targeting L1CAM is synthesized.In vitro experiments have demonstrated that Ap MSPd_β-gal was able to distinguish senescent endothelial cells from proliferating or quiescent endothelial cells through the anti-LICAM aptamer.After entering the cells,Ap MSPd_β-gal can be activated by senescence-associatedβ-galactosidase（SA-β-gal）to release the parent drug EF24 and trigger the apoptosis of senescent cells.Ap MSPd_β-gal is an order of magnitude more selective for senescent cells compared to free EF24.In vivo experiments have further shown that Ap MSPd_β-gal can stabilize plaques and inhibit plaques progression by selective clearance of senescent endothelial cells and senescent foamy macrophages accumulated in mouse atherosclerotic plaques,which provides a scientific basis and effective means for the basic research of anti-senescence and targeted intervention of age-related diseases.（3）To further improve the selectivity and broad-spectrum anti-senescence activity of senolytics,a lysosome-targeted activable senolytic prodrug(Lyso-Sph_β-gal)is synthesized,which is based on three common characteristics of lysosomes in senescent cells including the increased lysosomal content,accumulatedβ-galactosidase（β-gal）and increased lysosomal membrane vulnerability.The increased lysosomal content in senescent cells and modification of the lysosome targeting group enable the selective enrichment of Lyso-Sph_β-gal in senescent cells.After cellular internalization,Lyso-Sph_β-gal could be specifically activated by the accumulatedβ-gal in the lysosome of senescent cells through theβ-gal-cleavable linker,releasing lysosome-damaging drug sphingosine and subsequently inducing lysosome-mediated cell apoptosis.In vitro experiments have proved that Lyso-Sph_β-gal could selectively eliminate senescent cells induced by different sources and different stimuli.Moreover,in vivo experiments have proved that Lyso-Sph_β-gal could effectively remove senescent cells induced by chemotherapy drugs in mouse liver and kidney,and restore organ function.This design method utilizing the inherent characteristics of senescent cell organelles is expected to provide new ideas for the development of anti-senescence drugs.