Basic Research On The Design And Application Of Theranostic Nanoprobes For Tumor Diagnosis And Treatment

Early detection,diagnosis and treatment of tumors are of great significance for reducing the burden on patients,increasing patient survival and improving people’s health.Owing to the advantages of easy preparation,modifiability,high specific surface area,excellent biocompatibility and stability,functional nanomaterials have been widely applied in the diagnosis of tumors and the pathogenesis of diseases.However,how to improve the targeting of nanoprobes and how to reduce the signal-to-noise ratio of imaging to improve the accuracy of early diagnosis and treatment of tumors are the current challenges in cancer-related fields.In this thesis,based on the rigid DNA tetrahedral nanostructure and the targeting effect of transferrin receptor（TfR）aptamers-modified brain metastatic tumor cell membrane,two kinds of theranostic nanoprobes for the diagnosis and treatment of liver cancer and glioma were designed.Further,an in vitro blood-brain barrier（BBB）model for glioma was constructed,which provided methodological support for studying BBB penetration and efficacy of functional nanomaterials and drugs.The main research contents are as follows:1.Design and application of theranostic nanoprobes based on rigid DNA tetrahedral nanostructuresIn this work,seven customized single-stranded nucleic acid strands containing sequences recognized by three target miRNAs were self-assembled to form a rigid DNA tetrahedral nanostructure（DTNSs）.In the three vertexes of DTNSs,fluorophores and quenchers were brought into close proximity,inducing fluorescence quenching.Upon entering tumor cells and encountering target miRNAs,the recognition sequences in DTNSs were released from the DNA tetrahedron,resulting in fluorescence recovery.The fluorescence was activated by target miRNAs in tumor cells,which improved the signal-to-noise ratio of imaging.At the same time,Due to the unique rigid tetrahedral spatial structure,DTNSs showed excellent resistance to enzymatic degradation and high cellular uptake efficiency.Moreover,through the simultaneous detection of three kinds of intracellular miRNAs,DTNSs not only effectively distinguished tumor cells from normal cells,but also identified tumor cell subtypes,which avoided false-positive signals and significantly improved the accuracy of tumor diagnosis.Moreover,the DTNSs could also act as an anti-cancer drug.Antagomir-21（one recognition sequence）was detached from DTNSs to silence endogenous miRNA-21 inside cells,which would suppress cancer cell migration and invasion,and finally induce tumor cell apoptosis.The result was demonstrated by experiments in vitro and in vivo.The results showed that the intelligent nanostructure provided a feasible approach for the precision diagnosis and treatment of tumors.2.Construction of theranostic nanoprobes for glioma diagnosis and treatment based on transferrin receptor（TfR）aptamerIn this work,we designed biomimetic nanocomplexes（TMPs M）with core-shell structures for the diagnosis and treatment of glioblastoma multiforme（GBM）.The transferrin receptor（TfR）aptamer-modified brain metastases tumor cell membrane shells endowed TMPs M with dual BBB targeting ability,excellent biocompatibility,and prolonged retention time.Upon TMPs M effectively entering glioma cells across the BBB,the hollow Mn O₂ cores were decomposed by intracellular glutathione to release KKGKGQQ-tetraphenylethene（Pep-TPE）and si RNA.In the presence of intracellular transglutaminase 2（TG2）,non-emitting Pep-TPE would self-aggregate to induce emission.The resulting aggregation-induced emission（AIE）fluorescence imaging with a high signal-to-noise ratio could achieve the precise localization of the tumor and dynamic detection of TG2 activity,thereby allowing the GBM accurate diagnosis.Notably,the TG2 could be silenced by the released si RNA to cause cell apoptosis and increase chemotherapeutic sensitivity,ultimately realizing excellent antitumor efficacy.In vitro and in vivo results demonstrated that the as-prepared TMPs M indeed possess superior BBB penetration,precise diagnosis,and effective therapy of GBM,which provided a new strategy for accurate diagnosis and treatment of brain tumors.3.Construction and application of in vitro blood-brain barrier model for gliomaPorous anodized aluminum oxide（AAO）has the advantages of high permeability,uniform pore distribution,rigidity and easy functionalization,in this work,an in vitro BBB model for glioma was constructed based on AAO.In which channel was modified with streptavidin to capture biotinylated nanodrugs,surface was topologically constructed and modified with RGD peptide to promote cell adhesion and proliferation.Compared with the commercial Transwell model,the cells cultured on the AAO-substrate interface exhibited larger spread area,higher expression of tight junction protein ZO-1,and lower reduced permeability.Moreover,AAO allowed phase-contrast visualization of cells and could be reused multiple times.The successful construction of in vitro BBB model for glioma was confirmed by electrochemical and fluorescent methods.The constructed model was further used to evaluate the ability of functional nanomaterials to enter glioma.Seven kinds of nanomaterials with different modification were synthesized.The ability of these functional nanomaterials to cross the BBB was investigated by electrochemical method,and the results were consistent with the literature reported.This model provided an accurate and convenient way for glioma drug screening.In summary,in this thesis,the DNA tetrahedron nanostructure probes and aptamer/cell membrane coating nanocomplexes were successfully prepared.Based on strategies of multiplexed biomarkers detection,target-activated fluorescence imaging,and precise targeting,the accuracy of the tumor diagnosis and treatment had been significantly improved.Furthermore,the constructed in vitro BBB model for glioma could easily and accurately evaluate the ability of functional nanoprobes entering into BBB.These works will provide new methods and approaches for early diagnosis and treatment of cancer,and promote the research related to human health and environmental safety.