| Aptamer,as a novel targeted recognition molecule,has the advantages of high affinity,high specificity,easy synthesis,easy modification,stable property and flexible design.It has great potential and advantages in biomedical imaging,disease detection,targeted therapy and new drug research and development.However,aptamer still has a long way to go for practical clinical application and industrial development.The existing quality aptamers have limited by types and quantities,which are far from meeting the various needs of biomedical and clinical research.In addition,aptamer probes have some shortcomings such as difficult design,high imaging background and poor biological stability,which limit its practical clinical application and industrialization development.In view of the urgent scientific problems such as the lack of specific markers for mesenchymal stem cells and the ineffective chemotherapy caused by multidrug resistance in tumors,this paper aims to develop and expand the novel functional aptamer for the above key problems.A variety of aptamer-based molecular recognition nanoassembly probes are designed and constructed by combining nanotechnology and bioengineering technology.The study of specific tracer imaging,isolation and enrichment of mesenchymal stem cells and multidrug resistance reversal and chemotherapy are systematically carried out.The specific research contents are as follows:1.Screening and tracer imaging of aptamer for mouse bone marrow mesenchymal stem cellsUndifferentiated or not genetically manipulated bone marrow mesenchymal stem cells are an ideal source of seed cells for cell therapy,gene therapy and tissue engineering.However,the lack of highly specific surface antigens makes phenotypic classification of MSCS difficult,making it challenging to track MSCs in vivo by monitoring specific biomarkers.To solve this problem,four Aptamers with high affinity to mouse mesenchymal stem cells(mBMSCs)were screened and obtained by cell-SELEX,which the dissociation equilibrium constant(Kd)were in the extremely low nanomolar range.Then,Aptamer Seq3 had the strongest affinity with the target,which not only recognized the target cells,but also recognized mesenchymal stem cells of different sources.Furthermore,an activatable probe(AAP)was designed based on Seq3 for stem cell tracking,and the distribution of transplanted mBMSCs in mouse chronic kidney disease(CKD)model was monitored in real time.It was expected to provide a new targeted recognition probe for specific imaging and tracing of mesenchymal stem cells in cell therapy.2.Isolation of mesenchymal stem cells using aptamers for mouse bone marrow mesenchymal stem cellsMesenchymal stem cells(MSCs)are difficult to isolate due to the lack of highly specific cell surface markers and the low abundance in the whole bone marrow,which severely limite the biomedical clinical application of MSCs.To solve this problem,we selected Aptamer Seq3,which can specifically bind mBMSCs obtained through cellSELEX in the previous chapter,combining with magnetic separation technology,to construct and design an aptamer-based MSCs sorting platform.To facilitate subsequent application,Aptamer Seq3 was optimized to obtain four truncated sequences,W1,W2,W3 and W4,which bound specifically to mBMSCs and had better affinity than Seq3.Then,Apt-W2,which had excellent affinity for C3H10T1/2 cells,was selected for subsequent application.A magnetic bead sorting method based on Apt-W2 was developed,which successfully achieved rapid,efficient and nondestructive isolation and enrichment of MSCs from the whole bone marrow.The method had good ability to capture MSCs,and the isolation and capture rate of MSCs can reach 88.3%.The sorted MSCs show typical stem cell phenotype and good proliferation ability,which could be induced to differentiate into osteoblasts and adipocyte.Compared with the traditional method,this method had the advantages of high precision,less damage and fast speed.In addition,MSCs can be isolated and enriched without damage,maintaining the proliferation ability and pluripotency of MSCs.This method would provide a new idea for the efficient and rapid isolation of MSCs and a cell source for clinical application and tissue engineering application.3.Reversal of tumor multidrug resistance using aptamers for human multidrug resistant hepatocellular carcinoma cellsMultidrug resistance(MDR)is the main cause of chemotherapy failure in cancer patients.Aptamer could bind to the functional site of target proteins in a manner like small molecule drugs through its specific secondary structure,which has become a potential inhibitor of the active site of target proteins.It provides a new idea and effective treatment for the development of novel,low-toxicity,efficiency and high selectivity reversal agents of tumor multidrug resistance.To solve this problem,in this work,we firstly developed a novel strategy for reversing tumor multidrug resistance based on Aptamer d3,which could bind specifically to hepatocellular carcinoma cells HepG2/Dox.As proof of concept,Aptamer d3 may inhibit the function of multidrug resistant by binding to the surface of drug-resistant proteins,and significantly enhance the sensitivity and uptake of chemotherapy drugs(Dox,VCR,PTX)in tumor multidrug resistant cells.The mortality of chemotherapeutic drugs to HepG2/DOX cells decreased from 85.2%to 64.5%(Dox),54.3%to 39.4%(VCR),53.3%to 40.2%(PTX),respectively.The half-inhibitory concentration(Dox,VCR,PTX)reversed 1.7,1.7,1.4 times,respectively.Further,the animal models of HepG2/Dox xenografts confirmed that Aptamer d3 could improve the killing ability of Dox to tumor tissues and inhibit tumor growth.This work not only likely to develop a new tumor multidrug resistance reversal agent and the potential adjuvant to cancer chemotherapy,which was low toxicity,efficiency and high selectivity,but also expand new research and application of aptamer in the field of medical treatment.Therefore,this is not only an innovative research with important scientific significance for modern biological analysis,but also plays a positive role in promoting the development of cancer medicine.4.A DNA tetrahedron targeted by aptamer for reversal of tumor multidrug resistance and chemotherapy by tumor microenvironment-triggeredSide effects of intravenous chemotherapy drugs is a shortcoming of the previous chapter.Moreover,monomeric aptamer has the insufficient resistance to enzyme digestion of and the high background of fluorescence imaging in vivo.To address there problems,in this work,the ability of Aptamer d3 to target HepG2/Dox cells and reverse tumor multidrug resistance,the ability of I-motif structure of slightly acidic responding,and the ability of DNA tetrahedron to carry Dox and resist enzyme restriction were combined to create an integrated DNA probe for diagnosis and treatment.Therefore,the nanoassembly probe pH-D3-DT-Dox,which based on aptamer targeting and tumor microenvironment triggering,was constructed for low-background accurate imaging of drug resistant tumor,reversing of tumor drug resistance,and effective targeted treating drug resistant tumor.The nanoassembly improved fluorescence imaging speed,reduced imaging background,and enhanced imaging accuracy in vivo through activated fluorescence imaging,targeted recognition,and pH-driven mode.As proof of concept,Aptamer d3 could reverse tumor drug resistance,and improve the intracellular accumulation of the HepG2/Dox cells to Dox,which compared with the drug sensitive cell HepG2.The intracellular accumulation of Dox was increased by 5.36 times,and the IC50 value was reduced from 117.2±2.3μM to 61.5±1.9μM,which the reversal fold was 1.9.Moreover,combined with DNA tetrahedron,the targeted drug delivery therapy of living tumor tissues was realized,which greatly reduced the toxic and side effects on the body.Further,the growth of tumor tissues was significantly inhibited,and the reversal and targeted therapy of drug resistant tumor tissues were realized.Therefore,the pH-d3-DT-Dox nanoassembly probe was expected to develop into a novel and highly effective drug and realize the integration of diagnosis and treatment to tumor multidrug resistance. |
