Construction And Application Of Multivalent Aptamer Functionalized Nano-theranostic Probes Driven By Tumor Microenvironment

Due to their high affinity and specific binding capabilities,the multivalent aptamer-functionalized nano-theranostic probes,constructed by integrating multiple targeting recognition ligands（aptamers）into one nanostructure,have shown broad application prospects in tumor theranostic.The development of multivalent aptamer functionalized nano-theranostics systems driven at tumor site is more conducive to spatially controlled signal activation and drug release,so as to further promote the application of intelligent responsive tumor theranostic systems in precision medicine.In recent years,the rapid development and continuous cross-fusion of oncology,nanomedicine and nucleic acid nanotechnology have brought new opportunities for the development of biosafe and smart responsive multivalent aptamer functionalized nano-theranostics systems.On the one hand,with the intensive investigation of the tumor microenvironment（TME）,it has been found that TME is closely related to tumor proliferation,invasion,metastasis,etc.TME has been widely used as an important target in tumor imaging and treatment research.On the other hand,as the emerging of more functional nucleic acids with special biological functions,excellent properties,and flexible structural design（such as I-motif,antisense oligonucleic acid（ASO）,deoxyribozyme（DNAzyme）,etc.）,which provides more molecular tools and important guidance for the construction of intelligent multivalent aptamer functionalized nano-theranostics systems integrating multiple functions.In this thesis,focusing on the development of a new type of smart responsive multivalent aptamer functionalized nano-theranostics probes,we will take full advantage of the functional nucleic acid units with special properties including ASO and i-motif,and combine them with the difference between tumor tissue and normal tissue.The tumor extracellular acidic microenvironment,tumor extracellular matrix biomarkers or tumor cell specific recognition will be selected as single or combined stimulus.A series of TME-driven in situ assembly/disassembly of multivalent aptamer functionalized nano-theranostics probes has been developed.As a proof of concept,taking liver cancer and breast cancer as research objects,we systematically evaluate contrast-enhanced tumor activation imaging and therapeutic studies at the cellular and living animal levels.The detailed research content is as follows:1.Construction of multivalent aptamer-functionalized nanoprobes by ATP-driven in situ assembly strategy and activatable imaging of tumor cellsWe developed an extracellular ATP-driven in situ assembly of multivalent aptamer-functionalized nanoprobes using abnormally expressed ATP in the tumor microenvironment as a stimulus,ATP aptamer as a stimulus-responsive element,and ZYsls aptamer as a target recognition motif based on the high-affinity advantage of multivalent aptamer-functionalized nanoprobes.In this strategy,two different Y-shaped assembly modules（Apt-YM_Aand Apt-YM_B）are designed,each of which has a ZYsls aptamer sequence that can specifically recognize tumor cells embedded at one end.Among them,the other Cy5-labeled sticky ends of Apt-YM_Bwere blocked by BHQ2-labeled ATP aptamer to form Apt-YM_B-AtC.At physiological conditions（ATP:10-100 n M）,two monomers existed as fluorescence-quenched and monodispersed monomer structures.Arriving at the surface of tumor cells,the ATP aptamer will switch its conformational to bind overexpression ATP（100-500μM）in the tumor microenvironment,leading to fluorescence recovery and leaving the sticky ends unoccupied.Subsequently,the alternating“hand-in-hand”assembly of Apt-YM_Aand Apt-YM_Bwas activated to form multivalent aptamer-functionalized DNA nanostructures（MApt₂-NPs）on the cell surface.Human hepatoma cell SMMC-7721was used as a model target to systematically investigate the feasibility of this strategy for activated fluorescence imaging of tumor cells.The results show that the MApt₂-NPs assembled from two modules keep high specific recognition performance,which is 1.8 times higher than the Apt-YM_B-AtC.At the same time,this strategy enables highly sensitive detection of SMMC-7721 cells,permitting the actual detection of 19 cells detected in 200μL of binding buffer.By replacing the recognition element ZYsls in the two monomers,this strategy successfully achieves ATP-activated fluorescence imaging of MCF-7 cells and CCRF-CEM cells with improved image contrast,demonstrating the great universality of the MApt₂-NPs-based strategy.2.Acidic microenvironment-driven in situ construction of trivalent aptamer-functionalized nano-theranostic probes for enhanced tumor activatable imaging and therapeuticsThe previous chapter has demonstrated that acidic microenvironment-driven in situ formed MApt₂-NPs enable high affinity.However,we hope that it can introduce therapeutic elements in one nanostructure,which is expected to be used in the integration of cancer diagnosis and treatment research.Moreover,the binding affinity of aptamer-based multivalent nanoprobes is closely related to the spatial orientation and density of aptamer as well as the conformational and size of the probes.Herein,a multivalent aptamer-functionalized nano-theranostics probe with precise controllable spatial positioning,defined size and ligand density,was constructed by acidic tumor microenvironment-driven in situ assembly of three DNA modules（pH-aptDMs）.Each of the designed pH-aptDMs was simultaneously integrated with a stimulus-responsive primitive（split i-motif）,a target recognition primitive（ZYsls aptamer）,a sticky end-based assembly primitive,imaging and therapeutic functional elements（Cy5/BHQ2 and Dox）.In the normal physiological environment（pH 7.2-7.4）,the three pH-aptDMs exist in a monodisperse structure with an unactivated fluorescence signal;In the tumor acidic microenvironment（pH 6.2-6.9）,the three activated DNA monomers assemble into a multivalent aptamer functionalized nano-theranostics probe,called“three-arm aptamer nanoclaw”（TA-aptNC）.We investigated the diagnosis and drug delivery effects using nude mice bearing an SMMC-7721xenograft tumor.In comparison of pH-aptDMs monomer,the affinity of in situ formed TA-aptNC was increased by 2-fold,which allowed enhanced cellular internalization.In xenograft tumor model,an obvious fluorescence contrast was obtained by TA-aptNC,which was 3.8 times that of pH-insensitive group（pH-C）.While the monovalent pH-apt DM was merely 1.8-fold higher than that of pH-C.Notably,Dox-loaded TA-aptNC displayed a high antitumor efficacy with 90%inhibition.The proposed multivalent aptamer strategy achieves an in situ enhanced binding affinity and cell internalization ability in the tumor microenvironment,which is expected to provide a new idea for the development of an accurate and efficient theranostics nanosystem with spatiotemporally selective activation properties.3.Acidic microenvironment cascades-driven programmed disassembly of multivalent aptamer-functionalized nano-theranostics probes for tumor activatable imaging and chemo-gene combination therapeuticsGenerally,small-sized DNA assembly modules remains some challenges,including poor blood stability,undesirable tumor accumulation and retention,pharmacokinetics and so on,while static multivalent aptamer-functionalized nano-theranostics probes with large-sized often suffer from the poor performance on deep penetration inside the dense stroma of tumor.To address this problem,we ingeniously designed two i-motifs as stimuli-responsive elements to specifically respond to the pH differences at tumor sites by taking advantage of the design flexibility and adjustable pH response range of i-motif probes and combining with target recognition primitive（aptamer）,imaging and therapeutic functional elements（Cy5/BHQ2,Dox,and ASO）,an acidic microenvironment cascades-driven programmed disassembly strategy of multivalent aptamer-functionalized nano-theranostics probes was developed.Two similar DNA nanobots（R_Aand R_BI_e）as assembly mudules were rational designed to construct multivalent aptamer-functionalized nano-theranostics probes(AI_eB_mi).Each of R_BI_eis composed of a m _Bcontaining an ASO sequence,an i-motif DNA（i _A）specifically responding to the acidic environment of lysosome,a complementary strand（H_B）,a bivalent DNA（D-AS）and two i-motif DNA（I_e）specifically responding to the tumor acidic environment.Similarly,R_Aconsists of m _B,i _B,H_B,D-AS as well as two sticky ends respectively embedded into two arms.The BHQ2-labeled linker DNA in I_eof R_BI_ewas cross-linked with the Cy5-labeled sticky ends in the two arms of R_A,and a multivalent DNA nanoparticle（AI_eBm_i）with pH-driven disassembly at the tumor site was successfully constructed.Under normal physiological conditions,the nano-theranostics probes are"biologically inert"and can increase the enrichment of tumor sites through the targeting of aptamer and the EPR effect when circulating in the blood.Once at the tumor site,the acidic tumor microenvironment drives the disassembly of AI_eB_miinto small-sized bivalent monomers（R_Aand R_BI_e）.The released R_Aand R_BI_ecould effectively penetrate the deep region of the tumor tissue owing to their small size.After aptamer-mediated endocytosis into cells,R_Aand R_BI_eare disintegrated under the acidic environment of lysosomes,enabling rapid drug delivery at the target site.Compared with the AI_cB_mi,which is insensitive to the tumor acidic microenvironment,the AI_eB_mihas a significantly improved tissue penetration effect.In vivo,AI_eB_miachieves highly specific activated tumor imaging capabilities as expected.The AI_eB_midisplayed high antitumor efficacy with 75%,comparing single stimulus-responsive multivalent aptamer-functionalized nano-theranostics probes.