Construction Of Cancer Marker Detection And Cancer Drug Delivery Platform Based On Hybridization Chain Reaction

The realization of highly sensitive cancer marker detection and the development of intelligent specific drug delivery system are two major themes of cancer diagnosis and treatment.In terms of detection,micro RNA（mi RNA）,as an important biomarker for cancer diagnosis,plays an indispensable role in regulating the gene expression of various types of cells.Therefore,the realization of its high sensitivity and selectivity detection is of great importance for the early diagnosis and prognosis of tumors and other related diseases.However,due to the characteristics of small size,low expression,easy degradation and high sequence homology of mi RNA,the conventional methods to achieve highly sensitive detection of mi RNA face many challenges.In terms of drug treatment,the conventional drug delivery strategies have limited drug payload capacity,and cannot achieve specific targeted and on-site active drug release in complex cell environment,so the treatment effect is insufficient.Therefore,it is still a great challenge to develop an intelligent drug delivery system that can achieve customized treatment functions,efficient drug loading,specific tumor identification,controlled drug release,and reduce the damage to normal tissues while improving the treatment effect.In recent years,With the development of nanomaterials and nucleic acid nanotechnology,nucleic acid-based biosensors and drug delivery systems have become increasingly important emerging strategies.In this paper,DNA molecules are used as building blocks to combine nanomaterials with signal amplification reaction and emerging detection technologies to achieve mi RNA sensitive detection and intelligent drug delivery system development,and it is hoped to provide new design ideas and strategies for applications such as biosensor detection and drug delivery therapy based on self-assembly of nucleic acid nanostructures.This study mainly includes the following two parts:（1）Construction of DNA Nanohydrogel based on gold nanoparticles for specific targeting of tumor cells and ATP responsive drug disassemblyIn this part of work,we first developed a generic DNA Nanohydrogel based on gold nanoparticles（Au NPs）,which mainly uses hybridization chain reaction（HCR）products as the gel network framework,si RNA as the cross-linked chain,and forms a nucleic acid cross-linked network structure through DNA self-assembly and connects to the Au NPs carrier finally.In this structure,HCR can improve the loading rate of si RNA,and si RNA can help to form cross-linked structure and improve the stability of nanohydrogel.On this basis,we have developed a cancer cell drug delivery system with customized treatment function,specific targeted recognition and controlled drug release.It can be designed to contain three different therapeutic components:gold nanorods（Au NRs）,si RNA and chemotherapeutic drugs（Dox）,which can achieve the goal of photothermal,immune and chemical synergistic treatment in the future;The introduction of dual aptamers（AS1411 and anti-VEGF）can synergistically improve Au NRs@DNA Nanohydrogel specific targeting and endocytosis;ATP aptamer in the gel framework can respond to trigger the disassembly of DNA Nanohydrogel and achieve the goal of controllable release of drug molecules in gel through specific binding of ATP in cancer cells.We have successfully confirmed the formation of nanohydrogel and ATP responsive disassembly by means of polyacrylamide gel electrophoresis（PAGE）,agarose gel electrophoresis（AGE）,transmission electron microscopy（TEM）,ultraviolet-visible spectrum（UV-Vis）,Zeta potential,dynamic light scattering（DLS）,fluorescence（FL）and other characterization methods.Finally,we verified the Au NRs@DNA Nanohydrogel can enter cells through endocytosis mediated by double aptamers and successfully achieve controlled release by fluorescence microscope.（2）Construction of a surface-enhanced Raman scattering biosensor platform based on multi-layer“core-satellite”nanostructure for the detection of cancer marker Let-7aIn this part of work,we first synthesize two kinds of gold nanoparticles with different sizes for assembling the“core-satellite”nanostructure:45 nm core-Au NPs and 16 nm satellite-Au NPs.Then,two types of Au NPs were triggered by the target（Let-7a）and successfully self-assembled into a single-layer“core-satellite”nanostructure through DNA hybridization.Furthermore,through HCR reaction,a large number of continuous sites that can connect 16nm satellite-Au NPs can be formed on the surface of 45 nm core-Au NPs,thereby forming multi-layer“core-satellite”nanoassembly structures,which was confirmed through characterization by TEM,UV-Vis,etc.In addition,because the multi-layer“core-satellite”structure has more“hot spots”than the single-layer structure,the SERS signal of Raman reporting molecules（DTNB）attached to the satellite nanoparticles is enhanced more significantly.The results showed that as the concentration of Let-7a increased,the SERS characteristic peak(1334 cm^-1)intensity of DTNB gradually increased,and there was a linear relationship between them in the concentration range of 0.1 to 1000 n M,with a detection limit of 4 p M.At the same target concentration,the SERS signal triggered by single-layer“core-satellite”is significantly weaker than that of multi-layer“core-satellite”,confirming the advantages of multi-layer“core-satellite”assembly.We found that there is a certain linear relationship between the UV-Vis characteristic peak intensity of the solution phase and substrate phase of the multi-layer“core-satellite”nanostructure and the concentration of the target chain,which can be used as a kind of auxiliary detection method outside of SERS.The final experimental results confirm that the multi-layer“core-satellite”biosensor has good target selectivity and detection ability in actual serum samples.