Tumor Theranostics Research Based On Nanomaterials And Nucleic Acid Nanotechnology

With the threat of tumor towards human life and health become even more violent,the exploration for deepening our understanding of tumor i s gradually launched.In order to acquire the information of proteins,nucleic acids and biological small molecules that closely related to the occurrence and development of tumor,it is necessary to develop accurate,efficient and sensitive analysis strat egies to meet the current needs for cancer diagnosis and treatment.In recent years,because of the excellent biophysical and biochemical properties,biocompatibility,molecular recognition performance and highly controllable programming of nanomaterials a nd DNA self-assembled nanostructures,they have become hot spots in biomedical detection,clinical diagnosis,environmental monitoring and other research fields,providing a new research guidline and platform for the development of novel biosensors.In this thesis,we combined nanomaterials or DNA self-assembly nanotechnology with tumor theranostics,aiming at the current research focus in the field of biological nanomedicine,which committed to solving the problems and challenges of signal activation,signal amplification,targeted recognition and specific therapy,several theranostic nanoplatforms have been established for the detection of hydrogen peroxide(H₂O₂)in tumor microenvironment,glutathione(GSH)in living cells and tissues,and micro RNA in different cell lines,realizing detection of tumor markers,imaging diagnosis of living cells and tissues as well as chemodynamic therapy of tumors.The detailed research contents are summarized as follows:Chemodynamic therapy(CDT),which utilized Fenton-like reaction to specifically convert hydrogen peroxide(H₂O₂)into highly toxic hydroxyl radical(·OH),playing an important role in guiding the new generation of precise tumor theranostics.However,the current reported CDT methods often have problems such as difficulty in real-time monitoring of CDT process or poor stability in complex biological systems.In chapter2,we proposed for the first time that a novel catalytic nanosystem based on silver nanoparticles(Ag NPs)mediated Fenton-like reaction for tumor theranostics.The synthesis of Ag NPs was directly guided by fluorescent labeled partially complementary double strands DNA(TAMRA-DNA)that adsorbed on the surface of graphene oxide(GO).Due to the dual quenching effect of GO and Ag NPs,the fluorescence of TAMRA-DNA was greatly quenched.When the catalytic nanosystem entered into the cells,because of the high abundance of H ₂O₂ in tumor cells,Ag NPs in the nanosystem catalyzed Fenton-like reaction,which mediated H₂O₂-based etching of Ag NPs and generated·OH and Ag⁺,which effectively etched Ag NPs and cut TAMRA-DNA into small DNA fragments,resulting in restoring the fluorescence of the naosystem.Furthermore,the produced·OH and Ag⁺simultaneously mediated cytotoxicity,thereby inducing apoptosis and death of tumor cells.Therefore,the catalytic nanosystem can achieve activated fluorescence imaging and highly effective CDT and Ag⁺-mediated synergistic therapy,providing a valuable platform for tumor theranostics.Quantum dots(QDs)with large two-photon absorption(TPA)behavior endowed them important application value in biomolecular detection and biomedical imaging,which could be used directly as the efficient tracker for the cellular uptake and labeling of typical organelle.Nevertheless,a further surface functionalization for QDs with bioactive species to endow them the targeting ability or biological function was often necessary for specific bioimaging applications.In most cases,tethering of motifs on QDs was achieved through a post-grafting bioconjugation strategy,which has the limitation of cumbersome multistep modify,low coupling efficiency and side reaction of cross-linking.In chapter 3,the phenylboronic acid-contained aminophenylboronic acid quantum dots(APBA QDs)were synthesized thro ugh self-polymerization reaction by using APBA as the monomer.The APBA QDs were not only used for two-photon fluorescence reporter but also for targeting overexpressed SA on cancer cells membrane.The APBA QDs@Mn O₂ nanocomposites were prepared by one-step in situ synthesis of Mn O₂ nanosheets on the surface of APBA QDs via a redox reaction.Owing to the excellent quenching ability of Mn O₂ nanosheets,the fluorescence of APBA QDs were efficiently quenched via fluorescence resonance energy transfer(FRET).Then,the nanocomposites were internalized into cancer cells because of specific targeting recognization between phenylboronic acid ligand and SA receptors on cell surface,the abundant GSH in cytoplasm would reduce Mn O ₂ nanosheets into Mn²⁺,resulting in decomposing of Mn O₂ nanosheets and recovering the fluorescence of APBA QDs.This nanocomposites might serve as a useful and promising platform for highly selective and sensitive imaging of GSH and related cancer diagnosis.Framework nucleic acids(FNAs),a variety of self-assembled DNA nanoscaffolds including pyramids,tetrahedrons and origami,exhibited unique biophysical and biochemical properties that have shown tremendous advantages and potentials in the field of intracellular molecular detection and imag ing.In particular,the highly integrated DNA probes that can be able to address the robustness,sensitivity and consistency issues in a single assay system were highly desired but remained largely unsolved challenge.In chapter 4,we reported for the first time that the development of the novel DNA nanomachines that split-DNAzyme motif was highly integrated in a single DNA triangular prism(DTP)reactor,and the inert DNAzyme motif would undergo conformation rearrangement in response to endogenous micro RNA-21(mi R-21)and convert into active catalytic structure,leading to the cleavage of substrate strand with FAM fluorophore tag quenched by BHQ1 label and recovery the fluorescence.Meanwhile,the target mi R-21 was released to activate another inert DNA nanomachines and further executing the catalytic cleavage circuits,thereby producing enzymatically amplified fluorescence signal.With the advantages of facile modular design and assembly,high biostability,low cytotoxicity and excellent cellular internalization,the highly integrated DNA nanomachines enabled accurate and effective monitoring of mi R-21 expression levels in living cells.Therefore,our developed strategy may afford a reliable and robust nanoplatform for tumor diagnosis and for related biological research.As a kind of small,noncoding RNA molecules,micro RNAs(mi RNAs)functioned with regulating gene expression which played vital roles in many biological processes.Reported that aberrant expression of mi RNAs were correlated with occurrence and development of many diseases,highlighting their importance as disease biomarkers for diagnosis,therapy and prognosis.DNA molecular nanodevices have attracted much attentions owing to high specificity of Watson-Crick base pairing and predictability of sequence-controlled self-assembly,the DNA nanostructure based biosensor could significantly improve the kinetics and efficiency of related DNA displacement reactions,which beneficial for quantitative detection and imaging of low-abundance tumor-associated mi RNAs.Consider the inherent advantages of DNA molecular nanomachines,in chapter 5,by hybridizing hairpin probes on self-assembled three-dimensional DNA nanostructure,we developed a simple and low-cost DNA nanoamplifier,the local catalytic hairpin cascade reaction performed on the DNA nanostructures with accelerated reaction kinetics and improved efficiency,which enabled efficient and sensitive amplification imaging of tumor-related mi RNA in living cells.Compared with traditional catalytic hairpin as sembly probes,the developed nanoamplifier sped up the target-triggered reaction rate as well as enhanced the reaction efficiency.In addition,the DNA nanoamplifier possessed excellent cell internalization capability,commendable biostability and remarkab le biocompatibility that ensured high fidelity intracellular mi RNA monitoring,creating an enzyme-free platform for diagnosis and research of tumor-related disease.