The Design Of Novel Nucleic Acid Nanocarriers And Their Application In Cancer Cell Imaging And Drug Delivery

At present,the medical level and diagnostic technologies have been rapidly developed,but the morbidity and mortality of cancer are still at a high level,which is one of the major diseases that threaten human health and life.Early diagnosis and precise treatment of cancer are the key factors that determine whether the cancer can be successfully cured.In recent years,the continuous discovery of tumor markers has provided a guarantee for early diagnosis of cancer.There are many types of intracellular tumor markers,and their expression levels are also different.For some tumor markers with low expression abundance,more sensitive analysis methods for their detection need to be established.Besides,chemotherapy is one of the classic therapies of cancer treatment,but chemotherapy is usually restricted by the low solubility,strong toxic and side effects and drug resistance of chemotherapy drugs.Therefore,it is necessary to develop nanocarriers for delivery of chemotherapy drugs to solve these problems.This dissertation is devoted to the design of novel nucleic acid nanocarriers and their applications in tumor cell imaging and drug delivery.By summarizing the currently reported nucleic acid nanocarriers,it is found that with the deepening of the research of nucleic acid nanocarriers,there are still some shortcomings and challenges:1)Most of the existing nucleic acid nanocarriers deliver chemotherapeutic drugs to the cytoplasm.Overexpressed efflux transporters on membranes of drug-resistant cells will re-pump the drug in the cytoplasm out of the cell,reduce the drug concentration in the cell,and limit the effect of chemotherapy.Therefore,how to construct a nanocarrier targeted the site of drug action is of great significance for improving the effect of the drug;2)In response to the problem of drug tolerance during chemotherapy,a common strategy is to use nanocarriers to avoid efflux transporter-mediated drug efflux,while directly inhibiting the expression of efflux transporters is a more fundamental strategy to deal with drug tolerance;3)The expression abundance of many tumor markers in the cell is low.At present,the output mode of most nucleic acid nanocarriers with dual functions of cell imaging and drug delivery is 1:1,which greatly limits the sensitivity of detection and drug release.Amplifying the characteristics of nucleic acid nanocarriers to achieve the sensitive detection and drug release of tumor markers in living cells is a key issue.Based on the above problems and challenges,three novel nucleic acid nanocarriers were designed for imaging and drug delivery of tumor markers in living cells.This dissertation is divided into the following five chapters:Chapter one is an introduction,which mainly analyzes the severe challenges faced in cancer diagnosis and treatment,introduces the classic nucleic acid structure and functional unit,gold nanoparticles used in the design of nucleic acid nanocarriers,and outlines the nucleic acid nanocarriers in cancer diagnosis and treatment and summarizes the current problems of nucleic acid nanocarriers in cell imaging and drug delivery.In chapter two,an endogenous stimuli-responsive nucleus-targeted nanocarrier have been developed for intracellular mRNA imaging and drug delivery.The nanocarrier(AuNP-mRS-DS)consists of three components:(?)a gold nanoparticle(AuNP)to afford the DNA and switch off the fluorescence;(?)mRNA recognition sequence(mRS),which has been modified on the surface of gold nanoparticles by gold-thiol binding,for the specific recognition of MRP1-mRNA;(?)detachable subunit(DS),hybridized with the Cy5-labeled DNA linker and the nucleolin recognition motif and grafted onto mRS via the DNA linker,in order to load the doxorubicin(Dox),acquire binding affinity to nucleolin and report the signal.First,the nucleolin recognition motif of this nanocarrier targets nucleolin that is overexpressed on the surface of cancer cells,then the entire nanocarrier reaches the cell via an internalization mediated by nucleolin The mRS then specifically recognizes the overexpressed MRP 1 mRNA,which leads to the release of detached DS and subsequent restoration of the Cy5 fluorescence deactivated by AuNP.Finally,by translocating nucleolin from the cytoplasm to the nucleus,DS targets the nucleus to deliver dox.A differentiation between drug-resistant and non-drug-resistant cells could be obtained by intracellular fluorescence imaging.Compared to free dox(IC50>8.00 ?M),the AuNP-mRS-DS(IC50=2.20 ?M)loaded with dox achieved a higher suppression efficiency with regard to drug-resistant cancer cells.Such a nanocarrier offers an effective strategy for synergistic detection and the avoidance of drug resistance.In chapter three,gold nanoparticles(AuNP)modified by multifunctional molecular beacons(MB)as nanocarriers(MB-AuNP)have been developed for synergistic inhibition and in situ imaging of drug-resistant related mRNAs in cells.MBs-AuNP includes:(?)specially designed triple molecular beacons,modified on the surface of AuNP to bind related drug-resistant mRNAs,charge doxorubicin(Dox)and signal a fluorescence signal;(?)AuNP to afford the MBs,insert them into the cells and turn off their fluorescence.Upon absorption by cells,MBs-AuNP hybridizes to three linked drug-resistant mRNAs(MDR1 mRNAs,MRP1 mRNAs,BCRP mRNAs)which could inhibit their translation to reduce the expression of efflux protein and fluorescence recovery deactivated by AuNP to perform in situ imaging.The results of the qRT-PCR and the western blotting showed that the drug resistance mRNA and the expression of efflux proteins both decreased.Dox-loaded MB-AuNPs showed higher suppression efficiency than free Dox against HepG2/ADR(IC50 values of 0.35 ?M vs.1.06 ?M)and MCF-7/ADR(IC50 values of 2.78 ?M vs.>5 ?M).Direct observation of intracellular hybridization events and differentiation between drug-resistant cancer cells or non-drug-resistant cancer cells could be obtained by fluorescence imaging analysis.This nanocarrier is capable of downregulating the expression of several efflux proteins by gene silencing,allows in situ monitoring of silencing events and thus offers a powerful strategy to deal with drug resistance at the gene level.In chapter four,a telomerase-specific DNAzyme-driven DNA walker was developed as a nanocarrier(tsDNA-AuNP)for intracellular telomerase activity analysis and drug delivery.The nanocarrier is composed of three parts:(?)AuNP,for loading DNA and quenching fluorescence;(?)walking strand,hybridized with telomerase primer and hairpin structure containing DNAzyme sequence,for binding telomerase and cleaving substrate hairpin;(?)substrate hairpin containing DNAzyme cleavage site for loading doxorubicin and producing fluorescent signal.After the nanocarrier is internalized into the cell,the telomerase primer is extended under the action of telomerase,opening the hairpin structure containing the DNAzyme sequence,releasing the blocked DNAzyme sequence,and then the walking strand moves along the three-dimensional track to cleave substrate hairpin containing DNAzyme cleavage site,producing fluorescent signal and releasing doxorubicin,and then the next round of cleavage reaction is performed.This method can detect telomerase activity in as few as 10 HeLa cells.The fluorescence imaging method can distinguish between cancer cells and normal cells.In addition,the nanocarrier can also be used for the screening of telomerase inhibitors.Chapter five is the conclusion section,which mainly summarizes the innovation and prospect of this paper.