Investigation On The Linker Chemistry Of Aptamer-combretastatin A4 Conjugates

Aptamers are oligonucleotide sequences screened via Systematic Evolution of Ligands by Exponential Enrichment（SELEX）,which can specifically recognize and bind to the target molecule.The unique merits of aptamers,including high specificity,high binding affinity,easy cell internalization and rapid tissue penetration ability,made them have great application potential in the field of tumor diagnosis and treatment.Among them,Aptamer-Drug Conjugates（or Ap DCs）constructed by coupling aptamers with drug molecules through various linkers can improve the biocompatibility of drugs,enhance the ability of targeted delivery and reduce the side effects of drugs,which have led Ap DCs to evolve into one of the most attractive strategies for targeted drug delivery purpose.Nevertheless,the critical role of linkers in regulating of anticancer efficacy of these conjugates,especially those engineered by automated modular synthesis techniques,has been rarely explored.Therefore,we synthesized three Ap DCs containing different linkers based on solid-phase synthesis and liquid-phase coupling,and use it as a targeted drug delivery model to conduct a series of studies on drug release mechanism and cytotoxicity,etc.The main contents of this work are shown as follows:（1）The design and synthesis of Ap DCs containing different linkers and the research on the drug release mechanism of Ap DCs.We designed and synthesized Ap DCs with different linkers through solid-phase synthesis and liquid-phase coupling respectively.We directly synthesized Con-1 containing phosphodiester bond linker through solid-phase synthesis.Two aptamers modified with sulfhydryl and amino groups at the 5 ends:NH₂-Sgc8c and SH-Sgc8c,were firstly synthesized by solid-phase synthesizer,and then form Con-2 containing disulfide bond linker and Con-3 containing carbamate linker by liquid-phase coupling.On this basis,the drug release mechanism of Con-1,Con-2,and Con-3 constructed by different linkers was discussed.Studies of drug release mechanism have shown that Con-1 can respond to both phosphodiesterase and reduced glutathione（GSH）;Con-2 can respond to GSH;Con-3 can’t respond to GSH.In addition,we further designed Xq-2d-CA4 conjugates and TCO1-CA4 conjugates contained phosphodiester bonds linker,and successfully verified the versatility of this dual response model.（2）Investigation on the drug release mechanism of Con-1,Con-2 and Con-3 in complex systems.We investigated the effects of experiments on serum stability,targeted endocytosis and cytotoxicity in complex systems such as cells and serum.The results show that compared with the Sgc8c,the chemical modification of drug molecules can effectively limit the recognition and cleavage of terminal drug bases by nucleases,thereby improving the stability of Con-1,Con-2 and Con-3;At the same time,the three Ap DCs can’t affect the targeting ability and cell internalization.In vitro cytotoxicity assay,the results show that Con-1 has a stronger ability to inhibit the proliferation of HCT116 cells than Con-2 and Con-3 because of this dual response.In addition,the understanding of this dual-response model allowed us to further design an Xq-2d-CA4 conjugate and TCO1-CA4 conjugate that can induce K562 and Ramos cells apoptosis in a more efficient manner,respectively,which successfully verified that the difference in target cytotoxicity of Ap DCs contained different linkers is universal.The above-mentioned series of studies on Ap DCs with different linkers constructed by different synthesis methods and connection methods will help us better understand the drug release mechanism of Ap DCs,and design more efficient targeted treatment strategies.