Design And Synthesis Of Aptamer-Drug Conjugate(ApDC) And Their Applications In Cancer Therapy

Antibody-drug conjugate,in recent years,has gained considerably increased attention in the medicinal field because of their excellent therapeutic effect in cancer targeted therapy.It has specific selection for cancer cells,showing better efficacy and fewer side effects compared to small molecule drugs in cancer chemotherapy.However,antibody-drug conjugate is different to synthesize and purify,thus leading to a heterogeneity issue in different batches.These are challenges for the evaluation of antibody-drug conjugate in efficacy and safety.How to synthesize antibody-drug conjugates conveniently and accurately is still a big problem.Aptamer is a special kind of nucleic acid which similar to antibody,can specifically bind with target by forming a special structure.It has been widely used in bioanalysis,bioimaging,drug delivery,disease diagnosis and treatment,it is a promising molecular recognition tool.The conjugation of aptamer and drug to form aptamer-drug conjugate(ApDC)for targeted cancer therapy has been one of the most popular research areas in aptamer.Ap DC has the advantages of low cost,easy to synthesis,stable batch,and structure designable compared with antibody-drug conjugate.Ap DC is considered to be a potentially effective targeted anti-cancer drug,owning the potential to become one of the clinically use drugs for cancer treatment.In this paper,aptamers were conjugated with triptolide,gemcitabine,clofarabine,and pyropheophytin A to form Ap DCs in different coupling methods.The Ap DCs were used in chemotherapy and photodynamic therapy of cancer.We have tested the specificity and inhibitory efficiency of Ap DCs on cancer cells in vitro and in vivo.The projects are shown as follows.(1)AS1411-triptolide conjugate(ATC)for the treatment of triple-negative breast cancer(TNBC).Triptolide is one of the main active ingredients in the traditional Chinese medicine Thunder God Vine(Tripterygium wilfordii).It has been used to treat rheumatism and immune diseases in clinical.It was reported that triptolide could effectively inhibit the proliferation of a variety of cancer cells.However,the clinical application of triptolide is severely limited by the poor water solubility and high toxicity.Therefore,improving the water solubility of triptolide and reducing its side effects are essential for improving its therapeutic efficacy and accelerating the clinical application of triptolide.Studies have shown that nucleolin,the target of the aptamer AS1411,is highly expressed on various cancer cell membranes.It is a potential biomarker for cancer diagnosis and treatment.The safety and efficacy of aptamer AS1411 in the in vivo treatment of cancers have been proved by numerous experiments and clinical data.In 2014,aptamer AS1411 has been used in phase II clinical trials for the treatment of metastatic kidney cancer.Using triptolide as an anti-cancer drug and aptamer AS1411 as a targeting group,we designed and developed a new Ap DC,AS1411-triptolide conjugate(ATC).It is used to treat the most malignant breast cancer subtype,triple negative breast cancer(TNBC).The ATC was able to specifically recognize TNBC cell line,MDA-MB-231,in vitro and in vivo.Coupling with aptamer significantly enhanced the in vivo anti-tumor activity of triptolide in TNBC treatment.(2)Construction of a molecular cascade reactor based on DNA solid-phase synthesis.Cascade reaction is a type of continuous reaction in which subsequent reactions depend on the previous establishment of chemical functionality.The organism is like a complex molecular reactor,and there is a series of cascade reactions in the life metabolism process of the organism.These reactions lead to the changes of biological characteristics in the organism.In order to understand better of the life process,simulation and mimicking of life process has become hot topic in the field of chemical biology.But,the synthesis of molecular reactors is still a scientifically challenging task to complete.Pyropheophorbide-A(PA)was conjugated with aptamer on DNA solidphase synthesizer.Hemin was inserted into the AS1411-pyropheophorbide-A conjugate to form the molecular cascade reactor AS1411/hemin-PA.Molecular cascade reactor AS1411/hemin-PA can catalyze the generation of oxygen from hydrogen peroxide,and convert oxygen to singlet oxygen under the irridation of light.(3)The molecular cascade reactor AS1411/hemin-PA used to overcome tumor hypoxia for effective photodynamic therapy.Due to the rapid proliferation of tumor cells,the surrounding environment of tumor tissue is characterized by hypoxia,high acidity,and high hydrogen peroxide concentration.Studies have shown that these microenvironment characteristics of tumor tissue reduces the sensitivity of tumor cells to the treatments such as chemotherapy and photodynamic therapy.How to change the microenvironment of tumor tissue is a major problem in cancer treatment.The molecular cascade reactor AS1411/hemin-PA can catalyze the generation of oxygen from hydrogen peroxide in tumor tissues,thereby improving the efficacy of photodynamic therapy.The molecular cascade reactor can generate cascade reaction to inhibit the proliferation of cancer cells in vitro and in vivo.In vivo experiments,the molecular cascade reactor AS1411/hemin-PA overcame the hypoxia issue of tumor tissues,and provided oxygen for photodynamic therapy,resulting in more effectively tumor growth suppression than other groups.(4)The formation of aptamer-nucleoside drug conjugate by a DNA solid-phase synthesizer.Nucleoside drugs are an important strategy for clinical antiviral and anticancer.A lot of nucleoside drugs have been approved by FDA for clinical treatment of cancers,such as clofarabine,gemcitabine,fluorouracil,and decitabine.The clinical use of nucleoside drugs requires high doasges due to the low bioavailability.The high dosage of nucleoside drugs easily to cause side effect on patients.Therefore,we designed and synthesized gemcitabine phosphoramidite and clofarabine phosphoramidite to conjugate with aptamer by a DNA solid-phase synthesizer to improve the bioavailability.The aptamer-nucleoside drug conjugate(DS8)was formed by the mutation of aptamer Sgc8 c with gemcitabine and clofarabine.It was found that the C base at position 14 in Sgc8 c is the most key region for binding.It can cause large decrease of binding affinity when this C base changed to gemcitabine.The DS8 showed high specificity and high cytoxicity on CCRF-CEM cells.