The Construction Of Multifunctional Nucleic Acid Nanomicelles Based On Isothermal Signal Amplification Technology And Its Application In Tumor Synergistic Therapy

As an important gene information carriers in life,nucleic acids are promising biomarkers in bioresearch and diagnosis and treatment of diseases.The development of stimulus-responsive nucleic acid nanomaterials has become the focus of research in drug delivery vehicle.Furthermore,nucleic acid nanomaterials constructed based on isothermal amplification technologies are widely used in biosensing,bioimaging and biomedicine.In this paper,click chemistry and amide reaction are applied to conjugate the nucleic acid and polymer,then forming the nucleic acid nanomicelles.Subsequently,the construction of functional assemblies of nucleic acid nanomicelles can be achieved based on rolling circle amplification(RCA)and DNA self-assembly technology.The drugs in the fabricated nucleic acid nanomicelles can be released triggered by tumor microenvironment or biomolecules,which has been successfully applied to synergistic therapy both in vitro and in vivo to improve the therapeutic effect.These techniques provide a new strategy and perspective for DNA self-assembly,signal amplification imaging and synergistic therapy of tumors.This thesis has carried out the following three works:1.Construction of pH-responsive functional assemblies of nucleic acid nanomicelles based on RCAA pH-responsive functional assemblies of nucleic acid nanomicelles has been developed based on RCA in this work.First,the amphiphilic primer-polylactic acid(PLA)conjugates are synthesized via click chemistry and then self-assemble into DNA nanomicelles.The RCA reaction is in situ performed on the primer-PLA nanomicelles.Subsequently,the pH-responsive DNMs can be obtained by crosslinking the RCA products with the duplex/triple helix molecular switch-based probes containing the siRNA to load with anticancer drug doxorubicin(Dox).In the weakly acidic/neutral/basic pH(6.0-9.0),RCA products can combine with the DNA molecular switch-based probes via double helix hybridization,forming stable DNA assemblies of nucleic acid nanomicelles;when exposed to a strong acid microenvironment(pH?5.0),C-G·C~+triple helix formed,resulting in the release of molecular switch-based probes.The proposed DNA nanostructure have the advantages of easy preparation and multifunctionality,which can be applied in the fileds of drug delivery,disease diagnosis and therapy,and so on.2.pH-responsive functional assemblies of DNA nanomicelles for chemo-gene synergetic therapy of anaplastic large cell lymphomaBecause the pH-responsive functional assemblies of DNA nanomicelles have the advanteges of high loading efficiency and good biocompatibility,they have been used as versatile vehicles in co-delivery of anticancer drug Dox and anaplastic lymphoma kinase(ALK)-specific si RNA.The presence of ALK-specific siRNA can enhance the chemosensitivity of Dox,achieving chemo-gene synergetic cancer therapy.Both in vitro and in vivo experiments demonstrate that the proposed functional assemblies of DNA nanomicelles exhibit the significantly enhanced apoptosis of anaplastic large cell lymphoma(ALCL)K299 cells,which further effectively inhibit the tumor growth in vivo.3.Construction of functional assemblies of DNA nanomicelles for loading with dual drugs.In this work,poly(lactic-co-glycolic acid)(PLGA)which encapsulates with hydrophobic drug-curcumin(Cur)can be linked with DNA strands via amide reaction.The above structure in situ hybridizes with other DNA functional molecules such as G-quadruplex and aptamer,generating nucleic acid nanomicelles.Because Dox can intercalate into C-G base pairs,double stranded nucleic acid can load with amphiphilic drug Dox·HCl.Thus,this nucleic acid nanomicelles can efficiently load with two kinds of anticancer drug,providing promising prospect in multi-drug combination chemotherapy.Subsequent research in this work will use G-quadruplex,nucleic acid aptamer and other functional molecules on the nucleic acid nanomicelles,and combine with DNA isothermal signal amplification technology to achieve chemotherapy,photothermal and photodynamic synergetic targeted therapy.This work will provide new research approach for the development of multimodal therapeutic nanoplatform.