| Cancer has become a major killer that threatens human life and health because of the rapid proliferation and metastasis of cancer cells.The incidence of cancer is increasing year by year.Therefore,early diagnosis and treatment of cancer is the greatest challenge for human health.On the one hand,because cancer patients have no abnormal symptoms at the early stage of the disease,it is more difficult to realize the early diagnosis of cancer.Current conventional diagnostic methods are less specific and less sensitive,and are more difficult to detect in the early stage of the tumor.When the tumor cells are diagnosed,the tumor cells have been infiltrated and metastasized,and missed the best time to the treatment of the tumor.To detect and diagnose cancer early,the researchers focused on identifying and detecting tumor cells at the cellular level by tumor markers.On the other hand,the treatment of chemical drugs is the main means of cancer treatment,but there are still many drawbacks for chemotherapy such as the severe side effects of chemotherapy drugs,poor targeted therapy,drug resistance for tumor cells,and so on.So we urgently need develop the drug carrier to control the release of drugs and target the tumor cells to realize drug delivery,reduce the dosage and toxic side-effect,enhance the treatment effect of the drug,thus to improve the therapeutic effect of cancer.Due to the presence of the tumor markers can be ahead of the change of cell or tissue morphology and biology,so in order to achieve early diagnosis and treatment of cancer,we need to find a strong specificity,high sensitivity of tumor markers.In addition,a type of cancer cells can produce a variety of tumor markers,different type of tumor cell or the same of tumor cell in different organization may also produce the same kind of tumor marker.Therefore,the only detection of a type of tumor marker may result in false positive results.Currently,chemotherapy is a main means of cancer treatment,but there are common toxic side effects in clinical treatment.In order to reduce the side effects of drugs and improve their utilization efficiency,new targeted drug delivery systems were constantly researched to improve the specificity of drugs for tumor cells and to solve the intracellular controlled drugs release to achieve precise targeted therapy of tumors.Tumor markers are biomolecules specifically expressed in tumor cells,which are mainly found in biological fluids,circulating tumor cells and tumor tissues.Studies have shown that tumor markers can serve as important analysis indicators for tumor development,progression and prognosis.In general,the target position of the drug in chemotherapy is mainly tumor tissue.Therefore,the tumor markers that specifically expressing on tumor tissues become important drug-loaded system targets and drug release switches.Based on this,the selection of appropriate tumor markers as diagnostic markers,therapeutic targets,and drug release switches is crucial in targeted chemotherapy based on tumor markers.Currently,the majority of the targeting sites and drug release switches on cells are the same biomarker,usually a highly expressed receptor on the cell surface.Although this can effectively distinguish between tumor tissues and normal tissues,most of the drugs are still released outside of cells.The toxic side effects of drug still need to be reduced.The use of different biomarkers to consecutively achieve specific targeting of tumor cells and intracellular drug release process can effectively solve this problem.Based on this difficulty,we selected two tumor markers,MUC1 on cell membrane and telomerase in cells,to detect and target tumor cells.So we have synthesized the corresponding probes.The sequential response of nanoprobes to MUC1 and telomerase enables the simultaneous imaging sensing of both biomarkers with different distributions in the same living cell and enables realizing the visual tracing of the whole process of nanoprobes entering tumor cells based on MUC1 and intracellular fluorescence recovering by telomerase.Another probe is the drug-loaded nanoprobes.The drug-loaded nanoprobe combining tumor targeting based on MUC1 and controllable DOX release by telomerase can realiaze situ fluorescent monitoring of diagnosis and treatment.The research contents of this thesis are as follows:The first chapter introduces the significance and progress of tumor markers and the research progress of tumor markers,and then introduces the significance and the research progress of tumor markers for targeted chemotherapy.The second chapter introduces the composite nanoprobes use AuNPs as carriers and bind nucleic acid probes that respond respectively to MUC1 and telomerase through the Au-S bond.Among them,the nucleic acid probes targeting MUC1 are MUC1-specific aptamers and Alexa Fluor 405 fluorophore is labeled at the 3’ end.The nucleic acid probes that specifically recognize telomerase(telomerase probes)are DNA hybrids formed by hybridization of 3’-notched molecular beacons with shorter telomerase primers(TSP).The 5’ end of the molecular beacon is labeled with a FAM fluorophore.In the initial state,the fluorescence of Alexa Fluor 405 and FAM can be quenched by Au NPs.As the nanoprobe approaches tumor cells,it interacts with MUC1 that is highly expressed on the surface of tumor cells.The hairpin structure of the MUC1 aptamer on the nanoprobes then opens and the fluorescence of Alexa Fluor 405 recovers.This results in the targeted binding to the cancer cells and the detection of surface MUC1 expression.More importantly,with the aid of MUC1-specific binding,the nanoprobes enter cells better through surface receptor-mediated endocytosis.After the probe enters the cell,the telomerase primer starts to generate repeated telomere sequence from 3’ in the presence of telomerase,so that the molecular beacon structure hybridized with it opens and the green fluorescence of FAM recovers.The sequential response of nanoprobes to MUC1 and telomerase enables the simultaneous imaging sensing of both biomarkers with different distributions in the same living cell and enables realizing the visual tracing of the whole process of nanoprobes entering tumor cells based on MUC1 and intracellular fluorescence recovering by telomerase.In the third chapter,we further developed drug-loaded nanoprobes,the drug-loaded nanoprobe combining tumor targeting based on MUC1 and controllable DOX release by telomerase can realiaze situ fluorescent monitoring of diagnosis and treatment.In our experiment,DOX was chosen as a model drug.DOX is a commonly used chemotherapeutic drug that suppresses the synthesis of biomacromolecules by inserting a double-stranded DNA structure,and has an inhibitory effect on various cancers.However,since the drug has strong toxic side effects on normal tissues,and only a small proportion of drugs can reach the lesions to exert its efficacy,the drug is used in a large dosage and causes serious damage to the patient’s body.To achieve such drugs targeting lesions,precise and controlled release is undoubtedly of great significance.As the drug-loaded nanoprobe approaches tumor cells,it interacts with MUC1 that is highly expressed on the surface of tumor cells.This results in the targeted binding to the cancer cells and the detection of surface MUC1 expression.After the drug-loaded nanoprobe enters the cell,accompanied by the structure change of the molecular beacon,the DOX bound to the molecular beacon is released,enabling the in situ fluorescent imaging of telomerase activity and the telomerase-regulated release of drugs.In summary,the developed versatile drug-loaded nanoprobes triggered by two tumor markers can achieve both the specific recognition of tumor cells and the controlled release of drugs within the cells,as well as the in-situ imaging of the entire process of targeted diagnosis and treatment through the response fluorescence of drug-loaded nanoprobes.This study is of great significance for understanding the process of tumor targeted therapy and improving the efficacy of targeted therapy. |
