Design Of Functional Nanostructures And Their Applications For Detection,Imaging And Drug Delivery In Tumor Cells

Cancer,also known as malignant tumor,is the transformation of certain normal cells under the action of endogenous or exogenous carcinogens,the formation of abnormal cells with biological characteristics such as differentiation and proliferation abnormalities,infiltration and metastasis,and then developed into disease of tumor tissue.One of the main causes of cancer patients’ death is delayed diagnosis and treatment,because symptoms usually do not appear until late cancer.Therefore,early,accurate detection and accurate diagnosis of cancer are the prerequisite and key to successful treatment.Conventional diagnostic methods have low specificity and sensitivity,and are difficult to detect in the early stages of tumor development,while cancer detection based on tumor markers can significantly improve the effectiveness of early diagnosis and subsequent treatment.Chemotherapy,as one of the commonly used tumor treatment methods,has the problem of non-specific administration.That is means while exerting a killing effect on tumor cells,it also has serious side effects on normal cells.In addition,with repeated administration,it is easy for tumor cells to develop drug resistance and reduce therapeutic effect.Therefore,there is an urgent need to develop nanostructures with targeted functions to achieve early,accurate diagnosis and precise treatment of cancer.This thesis is dedicated to the design of functionalized nanostructures and their applications in tumor cell detection,imaging,and drug delivery.By summarizing the currently reported functionalized nanostructures,it is found that with the in-depth application of functionalized nanostructures,some deficiencies and new challenges are gradually exposed:(1)How to construct simple,easy to carry,visual assay to detect tumor markers?(2)In the face of drug resistance caused by chemotherapy,how to reduce the anti-apoptotic defense of tumor cells and restore the sensitivity of tumor cells to chemotherapeutics while avoid the efflux?(3)In the face of drug resistance caused by chemotherapy,how to avoid the re-efflux before chemotherapeutics entering nucleus of tumor in time?Based on the above problems and challenges,this thesis developed a series of functionalized nanostructures and used for visual detection of tumor marker telomerase,imaging of tumor cell membrane proteins,drug delivery and intracellular controlled release for tumor cells.The thesis is mainly divided into the following five chapters:The first chapter is an introduction.It mainly summarizes and outlines the design principles of functionalized nanostructures and the application of tumor cell detection,imaging and drug delivery.In addition,the existing limits and challenges of the functionalized nanostructures are also summarized.In the second chapter,convertible DNA ends-based silver nanoprobes was constructed for sensitive and specific colorimetric detection telomerase activity.The design is to combine the elongation characteristics of telomerase-bound substrate under the action of telomerase and the plasma effect of silver nanoparticles.Under the action of telomerase,TS on the silver nanoprobes are elongated with telomeric repeats,converting DNA stiffblunt ends to flexible single-stranded dangling ends.The dangling ends enhance the stability of nanoprobes and relieve their saltinduced aggregation,and the solution shows a yellow color.When telomerase is inactive,the blunt end-terminated nanoprobes cannot resist salt-induced aggregation,resulting in a gray color of solution.Based on telomerase-regulated DNA "blunt-dangling",ends conversion-induced AgNPs’ dispersity and color change,colorimetric detection of the endogenous telomerase with AgNPs is realized.The detection limit is equivalent to 1 cell/μL of telomerase activity,and extracts from cancer cells and normal cells are visually distinguished through color difference.The proposed strategy will offer a new approach for reliable,convenient quantification of telomerase activity in biochemical research and clinical diagnosis.In the third chapter,DNA-gold nanoprobe based lateral flow biosensor was constructed for rapid and visual detection of telomerase activity.This design combines the extension characteristics of telomerase-bound substrate under the action of telomerase,plasma effect of gold nanoparticles and lateral flow device.It mainly includes preparation of lateral flow device and visual detection research.The telomerase binding substrate undergoes chain extension under action of telomerase,and then the sample containing telomerase extension sequence is dropped on the sample pad,and flows backwards along the device under capillary action.The telomerase extension product can interact with DNA modified on the surface of gold nanoparticles coated on the hydration binding pad complements each other and then flows to the rear end,which can be combined with test DNA in the test zone.The red color appears in the test zone,indicating that telomerase in the sample to be tested has activity.That is,after the sample is dropped on the test strip,a series of reactions such as DNA hybridization and color development can occur spontaneously during the flow process,which realizes rapid and automated detection of telomerase activity.In Chapter four,pH-responsive DNA nanofirework is constructed,which is used for imaging of drug-resistant breast cancer cells and pH-responsive release of chemotherapeutic drug doxorubicin,which reversing the resistance of tumor cells to a certain extent and improving therapeutic effect of chemotherapy.PH-responsive DNA nano-fireworks are based on "activatable" aptamers and i-motif structures as basic functional elements.The outermost sequence of the outermost Y-shaped structure of nano-fireworks is designed to be "activatable" MUC1 aptamer,and fluorescent group Cy5 and complementary sequence labeling quenching group BHQ-2 are labeled at the end of the MUC1 aptamer sequence.Specific identification and imaging of drug-resistant breast cancer cells is achieved.I-motif structure is designed as a pH-responsive DNA nano-firework layer-to-layer cross-linking point.The conformation changes in lysosome realizes the disintegration of pH-responsive DNA nano-firework and rapid release of doxorubicin.The concentration of doxorubicin exceeds pumping capacity of efflux pump,so that more doxorubicin enters the nucleus to play a lethal effect.The results show that designed pH-responsive nanocarrier has certain application potential in the identification,imaging and reversal of drug resistance of tumor cells.In Chapter five,aptamer-conjugated DNA tetrahedral nanocarrier is constructed for synergistic drug delivery in drug-resistant breast cancer cells,reversing drug resistance and enhancing chemotherapeutic effect.This design combines targeting of MUC1 aptamer with synergistic effect of chemotherapeutics doxorubicin and gene drug siRNA.MUC1 aptamer can achieve targeted delivery of drug carriers to MCF-7/ADR cells.As an anti-apoptotic gene inhibitor,Bcl-2 siRNA down-regulates expression level of Bcl-2 mRNA to 24%,restoring sensitivity of tumor cells to doxorubicin,and thus increases killing effect of doxorubicin in tumor cells.Compared with free doxorubicin,doxorubicin and Bcl-2 siRNA loaded nanocarrier shows an enhanced chemotherapy effect.These results indicate that anocarrier has potential application in reducing side effects of chemotherapy drugs and reversing drug resistance of tumor cells.Chapter six is a conclusion section,and it mainly summarizes the innovation of the thesis.