Tumor Acidic Microenvironment Triggered In Situ Reconfiguration Of DNA Nanoprobe For Targeted Theranostics Of Tumor

Developing new strategies for imaging and therapy of tumors are of great importance for increasing the cure rate of patients.As an emerging tumor marker,tumor acidic microenvironment is closely related to the occurrence and development of tumors,which provides a new breakthrough point for tumor imaging and therapy.At present,a series of acid responsive theranostic strategies based on inorganic nanoparticles,polymer nanomaterials and DNA molecules have been developed for tumor imaging and therapy.However,the former two often have some defects,such as complex preparation,long reaction time and poor biocompatibility.Although the acid responsive theranostic strategies based on DNA molecules overcome the above deficiencies,it have low sensitivity and can’t efficiently respond to tumor acidic extracellular microenvironment(p H_e 6.2-6.9),which are difficult to meet the clinical needs of tumor theranostics.Hence,developing acid responsive theranostic strategies with high sensitivity,high specificity,and good biocompatibility are of great significance.In this thesis,we aim to build precise,sensitive and efficient acid responsive theranostic probes by introducing i-motif structure as the acid responsive unit,Aptamer as the target recognition molecule and DNA nanostructure as the scaffold,which could assemble in situ triggered by tumor acidic microenvironment with enhanced fluorescence.By taking human hepatoma cell line as the model cancer cell,two novel nucleic acid nanoprobes based on tumor acidic microenvironment have been developed at the molecular and cellular levels.The detailed description is listed as follows:1.Tumor acidic microenvironment triggered in situ reconfiguration of aptamer-based multivalent DNA nanoprobe for enhanced affinity and drug deliveryAptamers are promising in cancer imaging and therapy,but their poor affinity under physiological conditions is a challenge.In view of the acidic extracellular microenvironment of solid tumors,we developed a multival ent strategy based on extracellular p H regulation to increase the affinity of the Aptamer probes to target cells at physiological temperature.By rational design,a DNA monomer(AptDM)with Aptamer as the targeting unit and the split i-motif fragment as the acid responsive unit could be loaded with the chemothe rapy drug(Dox).At neutral p H,AptDM existed in an independent dispersed form,showing weak cell binding and internalization.Under acidic extracellular conditions of tumors,AptDM s crosslinked to form multivalent DNA assemblies(MDAs)by forming intermol ecular i-motif structures.Due to the multivalent effect,MDAs showed significantly enhanced binding affinity(K_d=9.96±1.06 nm)and stable binding capacity at physiological temperature,which could be used for highly sensitive diagnosis,efficient drug delivery,and enhanced inhibition on target tumor cells,but decreased toxicity to nontarget cells.This multivalent method is expected to facilitate the development of new Aptamer functionalized nanodevices in clinic.2.Tumor acidic microenvironment triggered in situ hybridization chain reaction of DNA nanoprobe for activated detection of tumor cellsIn view of the two key problems of poor specificity and low sensitivity in the process of theranostic process of nanodrugs,a multivalent activated DNA triangu lar prism nanoprobe(DTPI)was developed combined wit h the unique acidic microenvironment of tumors by using DNA triangular prism structure as scaffold,i-motif structure as the acid response unit,Aptamer as the target recognition molecule and HCR as the signal amplification technology.Under acidic extracellular conditions of tumors,i-motif protonated to form a tetramer structure and fell off from DTPI,which led to the exposure of trigger.The exposed trigger turned on H1and H2 one after another,triggering HCR on DTPI to form DTPI-HCR for achieving activated fluorescence signal amplification,whil e DTPI could not be activated to form DTPI-HCR at neutral p H.Compared with ZY11 and DTPI,DTPI-HCR exhibited enhanced target cell recognition performance and highly sensitive detection performance at physiological temperature,with a minimum of 33 cells detected in 150μL of binding buffer.The strategy used multiple HCR as amplification mechanism to trigger in situ self-assembly of probes on the cell surface for achieving activated signal amplification,which may provide a new method for the development of multivalent activated Aptamer nanoprobes.