Aptamer Based Targeted Therapy And Its Precise Modification

Cancer is a multifaceted global health issue and the second leading cause of global death with the feature of a low cure rate and short survival,just behind cardiovascular disease.Antibodies and tyrosine kinase inhibitors are the main targeted cancer therapeutics besides the classic cancer therapy strategies of surgery,radiation,and chemotherapy.They conquered the disadvantage of chemotherapeutics that attack both cancerous cells and normal cells and could target the unavailable minimal lesions of surgery and radiation therapy that cause cancer cell metastasis.However,antibodies,owing to their scant targets,heavy mass,and so on,made the development of new antibody drugs difficult,thus restricting their widespread clinical application.The screening of tyrosine kinase inhibitors is also difficult and expensive.In recent years,the rapid development of PCR technology,oligonucleotide solid-phase synthesis techniques,and SELEX,make the “chemical antibodies”aptamer available for scientists.Aptamer’s advantage of time-saving,money-saving and easy synthesis made aptamer hold great potential in cancer precision diagnosis and personalized treatment.Aptamers,selected via SELEX(Systematic Evolution of Ligands by Exponential Enrichment),are regarded as powerful tools to advance molecular medicine owing to their ability of binding to various targets with high specificity and affinity(picomolar to the nanomolar range),for example,its’ targets including metal ions,small molecules,peptides,proteins,bacteria,virus,living cells,and tissues.The first aptamer drug,Pegaptanib,was approved by FDA only after three years of development.Till 2020,there are 11 aptamers have entered the phase I clinical trials.Among them,aptamer-drug conjugate(Ap DCs)that are used to deliver chemotherapeutics are under rapid development.First-class chemotherapy drugs,such as paclitaxel,methotrexate,etc.,have been conjugated with aptamer for targeted drug delivery.Artemisinin and its derivatives not only possess antimalarial function but also exhibit antitumor ability,develop artemisinin-based antitumor drugs could facilitate the development of new types of anticancer drugs.Based on this research background,chapters two and chapter three choose artemisinin derivatives to construct safe and high-specific cancer-targeted drugs.However,aptamer also owns some drawbacks,such as short half-life and the offtarget effect caused by easily serum degradation,and poor tissue permeability restricting its clinical application.Chemical modifications,such as PEG,artificial base,and hydrophobic tags are generally used to improve the properties of the aptamer.However,the current chemical modification groups are still limited,and the problem of aptamer tumor penetration is still unresolved.Therefore,in the fourth chapter and fifth chapter,we explored and synthesized a new chemical modification group,fluorine hydrocarbon chain,to bring new properties to the aptamer.The paper mainly works from the following four parts:(1)Chapter 2 focused on constructing aptamer-drug conjugate and purifying the product by HPLC.Further FACS was used to confirm whether the introduction of small molecular drug artesunate affected the specific recognition ability of aptamer.Subsequent cellular experiments and mice experiments were carried out to verify whether the aptamer-artesunate conjugate still has its specific recognition ability both in vitro and in vivo.(2)Chapter 3 aimed to study the mechanism of the aptamer-artesunate conjugate.This chapter mainly explores the effects of the aptamer-artesunate conjugate on cell cycle,apoptosis,and cellular ROS production,further evaluating targeted cancer therapy efficacy of aptamer-artesunate conjugate to tumor cells by CCK8 assay.(3)Chapter 4 developed a polyfluoroalkyl decoration strategy to regulate the biomedical properties of MUC1 aptamer,including specific recognition,cell internalization,and solid tumor enrichment and penetration,by tuning their hydrophobicity with polyfluoroalkyl tags.(4)Chapter 5 is based on chapter 4,We developed polyfluoroalkyl decorated sgc8 c aptamer to systematically explore the characteristics of the polyfluoroalkyl decoration.Firstly,polyfluoroalkyl phosphoramidite containing 7,11,15,19,and 23 fluorine atoms are synthesized and then attached to the 5’ end of the sgc8 c aptamer(named SFn).After characterization of the retention time of SFn by high-performance liquid chromatography,the binding affinity was tested by flow cytometry,and their performance on the cell was imaged by confocal microscope.Finally,3D tumor spherical and tumor mouse models were constructed to investigate the properties of polyfluoroalkyl modified aptamer both in vitro and in vivo.In conclusion,we have constructed artesunate-sgc8 c aptamer conjugate for cancer targeted therapy and invested in a new chemical modification to enhance the properties of aptamer,aimed to increase the possibility of clinical trials.