Construction Of DNA Nanomaterials By Rolling Circle Amplification Combined With Silver Nanoclusters For Drug Targeted Delivery And Biosensing

Cancer is a highly lethal disease that endangers people’s lives and health,so there is an urgent need to develop new cancer diagnosis and treatment technologies.As a conventional method for treating cancer,chemotherapy drug DOX has significant side effects and long-term use can lead to drug resistance.How to overcome cancer cell resistance and achieve precise DOX targeted delivery,as well as controllable and rapid drug release,is crucial for cancer treatment.The existing inorganic/organic nanomaterials,as DOX drug carriers,connect DOX loaded by chemical bond into cancer cells,but can’t distinguish normal cells to achieve targeted and accurate delivery;DNA nanomaterials are loaded with DOX through physical intercalation and connected to aptamers through adhesive end hybridization,which can recognize overexpressed proteins on the surface of cancer cells for drug targeted delivery.However,they can only rely on the passive degradation of DNA in the acidic environment of tumor cells to achieve DOX release;The use of chemotherapy alone cannot solve the problem of drug resistance,and it is necessary to introduce gene therapy to overcome drug resistance.Silver nanoclusters have good fluorescence stability and can be used for intracellular localization of drug carriers.Therefore,we are committed to developing a multifunctional drug delivery carrier that can simultaneously deliver chemotherapy drugs and nucleic acid drugs into specific cancer cells.The entire transportation process into cells and the final drug release process can be monitored through fluorescence signals,ultimately achieving chemotherapy and gene synergistic therapy for cancer cells.This study mainly includes the following content:(1)DNA Nanoflower combined with silver nanoclusters for cancer cell imaging and drug targeted deliveryIn the first part,we prepared DNA Nanoflower by rolling circle amplification reaction(RCA)as drug delivery carriers,and sent chemotherapy drugs DOX,antisense nucleic acids of miRNA-21 and MDR1 mRNA into cancer cells to achieve chemotherapy and gene synergistic therapy.First,the size of DNA Nanoflower is controllable and the DNA Nanoflower outer DNA contains Sgc8 aptamer,which can recognize the PTK7 membrane protein on the surface of cancer cells to achieve targeted delivery.Changing the type of aptamer on the surface of Nanoflower can target a variety of cancer cells to make it a universal drug carrier.Secondly,the overexpression of miRNA-21 in drug resistant breast cancer cells is related to the proliferation of cancer cells.MDR1 mRNA regulates the overexpression of P-gp drug resistant protein to make cancer cells resistant to drug.We targeted antisense nucleic acids loaded with miRNA-21 and MDR1 mRNA to achieve gene therapy to reduce the expression of related proteins.We used C1(miRNA-21 ASO)/C2,C3(MDR1mRNA)/C4 and Nanoflower surface DNA to form a three way junction structure with three strands of co-hybridization,which is conducive to DOX embedding into CG base pairs in the structure while loading nucleic acid drugs.DOX loading rate reached 93%.The miRNA-21 and MDR1 mRNA in cancer cells hybridized with C1 and C3,respectively,resulting in the disassembly of the triple stranded structure triggering DOX release,with a DOX release rate of 83%.At the same time,hybridization of miRNA-21 and MDR1 mRNA with their corresponding antisense nucleic acids produces a fluorescence signal change of"green fluorescence on,red fluorescence off".The concentration of target miRNA-21 and MDR1 mRNA in the buffer has a good linear relationship between 0-500 n M.Fluorescence signal switching mediated by miRNA-21 and MDR1 mRNA was observed in cancer cell fluorescence imaging,successfully achieving miRNA-21 and MDR1 mRNA cell imaging.Finally,we organically combine DOX mediated chemotherapy with antisense nucleic acid mediated gene therapy to reduce DOX efflux in cancer cells,which is beneficial for more DOX drug molecules to fully exert chemotherapy effects in the cells.The cytotoxicity experiment showed that the Nanoflower composite drug loading system loaded with miRNA-21/MDR1mRNA ASO and chemotherapy drug DOX had no effect on normal cells,and the killing rate of cancer cells was 60%.The Nanoflower composite drug loading system has realized the precise delivery of drug targeting,controllable and rapid release,and has a good killing effect on cancer cells.(2)Construction of a fluorescent biosensor based on rolling circle amplification reaction for detecting microRNAIn the first part,we detected the tumor marker miRNA-21 in vitro,but did not introduce signal amplification technology,which is not conducive to the sensitive detection of low concentration target miRNA-21.Meanwhile,enzyme free signal amplification techniques such as chain displacement have strong false positive background signals,while enzymes have high catalytic activity and can recognize specific cleavage sites.Therefore,in the second part,we propose the construction of a dual signal amplification fluorescent biosensor using APE1 assisted target cycling combined with rolling circle amplification reaction(RCA)for high sensitivity detection of miRNA-21.The hairpin we designed has an AP site on the HP ring.When HP exists alone,it will not be cleaved by APE1.The target miRNA-21 hybridizes with the HP ring to form a double strand,and the AP site is cleaved by APE1 HP splits into HP1 and HP2 parts and separates.At the same time,the released target miRNA-21 continues to participate in the next cycle reaction to trigger a new HP cleavage.HP1 hybridizes with the dumbbell shaped circular template DP to initiate the RCA reaction,which yields DNA long chains containing multiple G quadruplex repeat units.Subsequently,we first added K~+to the rolling circle amplification product to form a G quadruplex structure,and then added thioflavin T,which enhanced the fluorescence output of thioflavin T and fluorescence detection signal.This detection method has good detection performance in the target miRNA-21 concentration range of 0.1-100 n M,with a detection limit as low as 3.3 p M,good specificity,and precise detection of miRNA-21 in human serum samples.