| Various dysregulated or mutated genes and related proteins in cancer tissue and its microenvironment,these molecules are both major players in cancer diagnosis and potential targets for personalized medicine.However,whether it is cancer diagnosis or treatment,there is an urgent need for a targeted molecule with high sensitivity,high specificity,and relatively stable chemical properties.As a class of oligonucleotide molecules,aptamers can bind to targets with high specificity and sensitivity through a specific tertiary structure formed by spontaneous folding.Aptamers have high affinity,high specificity,low immunogenicity,and small molecular weight,which can provide a new strategy for the targeted diagnosis and treatment of cancer.This thesis takes breast cancer and liver cancer as the research object,and carries out the research of tumor targeted diagnosis and treatment based on aptamer.In terms of diagnosis,phenotypic screening strategy was used to obtain aptamer S1-4 specifically targeting estrogen receptor-positive breast cancer,which could accurately distinguish ER-positive breast cancer MCF-7 cells from different subtypes of breast cancer cells.The Aptamer AP74-4-β(5aminoindole)-podophyllotoxin(AP74-βIZP)has been designed and synthesized with tumor microenvironment targeted delivery strategy.The exponential enrichment ligand phylogenetic evolution(Cell-SELEX)technique based on phenotypic screening was used with estrogen receptor-positive breast cancer Cell MCF-7 as target cells,HER2-positive breast cancer cell SK-BR-3,triple-negative breast cancer cell MDA-MB-231 and normal breast epithelial cell MCF-10A as control cells.The aptamers were screened.After 10 rounds of screening,the fluorescence intensity of the selected secondary DNA libraries was no longer significantly enhanced by flow cytometry,and the results were analyzed by high-throughput sequencing.It was found that the top 60 aptamer sequences with the highest enrichment had a conserved sequence CTTCAGGGGCACAATATG,which was named X1.The secondary structure prediction results of the aptamer showed that the arm structure containing GGGG fragment formed by folding X1 had better MCF-7 specific targeting ability than the stem ring structure.Therefore,the aptamer S1 with Kd value of 123.3 nM was screened.Then,an aptamer S1-4 with shorter length(48 nt)and higher Kd value(97.6 nM)was obtained by truncating the upstream and downstream primer regions with the retention of X1.Fluorescence imaging results showed that the aptamer S1-4 was able to bind to the surface of MCF-7 cells without recognizing other subtypes of breast cancer cells,and preliminary validation was made that the target of the aptamer S1-4 might be membrane proteins,and the aptamer S1-4 had no non-specific binding ability to other common cells.Finally,the fluorescent imaging group Cy5.5 was coupled to the aptamer S1-4,and three different subtypes of breast cancer cell tumor bearing mouse models were detected through tail vein injection.It was found that the aptamer molecule could accurately identify MCF-7 tumor bearing mice,and had good potential for tumor diagnosis.In the early stage,the research group took podophyllotoxin,the main pharmacodynamic component of podophyllotoxin,as the research object,designed and synthesized a series of lead compounds with anti-tumor activity,and successfully excavated βIZP with nanomolar activity against HepG2 cells,which was three orders of magnitude higher than the micromolar activity of oxaliplatin for liver cancer chemotherapy.However,as a cytotoxic compound,βIZP has the problems of poor water solubility,high toxicity,and poor in vivo targeting,which limits the development of new drugs.By targeting galectin-1 protein overexpressed in liver cancer microenvironment,AP74-βIZP was designed and synthesized by introducing galectin-1 APaptamer AP74 into βIZP via disulfide bond.In terms of water solubility,compared withβIZP(water solubility 0.01 mg L-1),the water solubility of AP74-βIZP increased to more than 100 mg L-1.In vivo anti-tumor activity,AP74-βIZP can target delivery to galectin-1 enriched hepatoma microenvironment,and disfracture sulfur bond under high concentration of GSH,releasing free podophylloidal lead compound βIZP.AP74-βIZP showed better anti-tumor effect than the clinical dose of etoposide(20 mg kg-1)in vivo,and the tumor inhibition rate reached 58.8%,which was about 10%higher than βIZP(46.7%),indicating that AP74-βIZP had stronger anti-tumor activity than βIZP.In terms of toxicity,the concentration of GSH in normal tissues is low,and βIZP is hardly released.Compared with etoposide,βIZP and oxaliplatin,the clinical drug for liver cancer,AP74-βIZP with liver cancer targeting ability significantly reduced the toxicity to liver,kidney and other organs and marrow suppression toxicity,and the overall safety in vivo was high.In addition,AP74-βIZP also has good immunomodulatory function.AP74-βIZP inhibits galectin-1 induced apoptosis in vitro and reduces the killing effect on HepG2 cells.The results of in vivo study showed that the tumor inhibition rate of AP74βIZP on C57 tumor-bearing mice with normal immune function reached 70.2%,which was significantly higher than that of free AP74(35.2%),βIZP(44.8%)and etoposide(47.6%).The pharmacological mechanism results showed that AP74-βIZP promoted immune stress by competitively binding to the glucose-recognition sequence of galectin-1,increased the infiltration ratio of immune T cells CD4/CD8 to tumor tissues by 10%and 20%,respectively,and up-regulated the expression of immune factors TNF-α,IFN-γ and IL-2,thus enhancing anti-tumor activity.This thesis focuses on the research of tumor targeted diagnosis and treatment with aptamers.Based on phenotype,aptamer S1-4 was selected to identify estrogen receptor-positive breast cancer.AP74-βIZP,an APtamer conjugate with strong anti-tumor activity,strong water solubility,low toxicity,and immune regulation,was designed and synthesized to provide theoretical reference for other aptamers in tumor diagnosis and treatment and small molecule drug research in chemotherapy. |
