Small-molecule Modified Aptamer For Targeted Imaging And Tumor Therapy

When nucleic acid aptamers target tumor protein biomarkers on the cell surface by forming three-dimensional structures,they can combine with receptors and enter the target cells by receptor-mediated endocytosis.Aptamers have advantages such as high specificity and affinity comparable to those of antibodies,long shelf life,simple and quick generation,ease of chemical modification,and low immunogenicity.However,most aptamers themselves have no cytotoxicity to tumor cells.Therefore,the modification with various small molecules,such as fluorescence or drug molecules,confer aptamers as diagnosis and treatment reagents,thus receiving significant tumor theranostics applications.Recent researches on aptamers conjugated with small molecule toxins（Aptamer-drug conjugate,Ap DC）has been well developed.It has provided a new molecular targeting strategy for tumor treatment.Ap DC targets and binds to tumor cells with high efficiency and low toxicity to normal tissue.It has the advantages of low cost,easy synthesis,batch stability,and designable structure.Hence,our research focused on improving the serum stability and safety of Ap DC drugs along with their anti-tumor role,which will provide primary research data for Ap DC from the laboratory to its clinical trials.In this thesis,the small molecules of interest were maytansine derivative（DM1）,HSP70targeted peptide and doxorubicin（DOX）,as well as fatty acid chains coupled to DNA aptamers to form three different conjugates.These conjugates were a DNA aptamer-drug conjugate（Ap DC）,an aptamer-peptide conjugate（ApPC as sensitization agent）,and an aptamer-fatty acids complex（ODDA-Sgc8-c）.Ap DC was used to kill HER2-positive tumor cells.ApPC increased the sensitivity of drug-resistant cells to DOX and thus inhibited cell proliferation.ODDA-Sgc8-c improved the serum stability and pharmacokinetic properties of the Sgc8-c aptamer.The binding specificity and the cytotoxicity of Ap DC and ApPC on tumor cells and the significant modification of ODDA-Sgc8-c on the aptamers were demonstrated in cell and animal experiments.The research details are as follows:（1）Ap DC was formed with a HER2 receptor targeting aptamers conjugated to DM1,a tubulin inhibitor,through a stable bifunctional cross-linker（succinimidyl4-（N-maleimidomethyl）cyclohexane-1-carboxylate,SMCC）.In cell experiments,Ap DC targeted HER2 positive cell lines BT474 and SKOV3 with high specificity and affinity,and entered cells through cell receptor-mediated endocytosis.The uptake efficiency and the cytotoxicity of Ap DC to the target cells was very high(the IC₅₀value was very low).Moreover,this Ap DC showed good stability through forming a G4 structure,which allowed the Ap DC a long period of imaging in vivo.In BT474 tumor models,the Ap DC significantly inhibited tumor proliferation compared with the nonmodified drug DM1-SMCC.More importantly,due to its good targeting ability,the toxicity of Ap DC on liver tissues was significantly reduced.（2）In order to explore the effect of HER2-targeted Ap DC,which was synthesized in the first part on a HER2 receptor,a dynamic mathematical model of the receptor cycle was established.When all of the experimental cell data was combined,it was proven that the expression of the HER2 receptor on the cell membrane surface was not affected at a dosage of less than 100 n M of Ap DC.We speculated that the affinity of the aptamer to the HER2receptor is slightly lower than that of antibodies to the HER2 receptor,resulting in different intracellular dissociation levels of ligand-receptor complexes,so the amount of the receptor circulating back to the cell membrane is different.This study is a mutual demonstration between theory and experiments.In terms of mechanism,the Ap DC drug we designed had a lower probability of drug resistance in treatment dose compared to the clinical drug（antibody-drug conjugate,ADC）.As far as we are concerned,on the receptor level,they differed from ADC.（3）In this part,an aptamer targeted the MUC-1 receptor coupled with a targeted HSP70peptide and formed an aptamer-peptide conjugate（ApPC）.Then,DOX bound with GC base of ApPC through non-covalent bonds,resulting in the formation of the chemosensitization system ApPC-DOX.We then evaluated the sensitization effect of ApPC in vitro and in vivo.The results demonstrated that aptamer codelivery of the anti-HSP70 peptide and DOX worked efficiently.ApPC-DOX entered into the drug-resistant breast cancer MCF-7/ADR cells and significantly enhanced the sensitivity of cells to DOX.In the in vivo study,ApPC-DOX killed tumor cells and dramatically reduced the cardiotoxicity of DOX.（4）A well-known aptamer,Sgc8-c,was modified with 1,18-octadecanedioic acid（ODDA）on solid-phase beads and formed ODDA-Sgc8-c through a C-N bond.The recognition ability and high affinity of the modified ODDA-Sgc8-c on HCT116 cells,which highly expressed PTK7,were demonstrated by in vitro experiments.Also,ODDA-Sgc8-c had remarkable resistance toDNaseⅠ nuclease and excellent stability in serum.From the electrophoresis gel,we could confirm that ODDA-Sgc8-c formed a complex with mouse serum albumin.Moreover,ODDA-Sgc8-c was found to have good permeability in the multiple-cell tumorspheres.In vivo studies showed good performance of pharmacokinetic of ODDA-Sgc8-c compared with Sgc8 without ODDA modification.Thus,ODDA modification has been identified as an efficient strategy to improve the aptamer,prolonging its half-life,targeting HCT116 tumor in vivo.Overall,the ODDA-Sgc8-c conjugated with imaging reagent such as gadolinium will be expected to be a valuable tumor diagnostic reagent.In conclusion,we have designed and synthesized Ap DC and ApPC for molecular targeted therapy in cancer research;we have estimated that the Ap DC drug did not affect cell receptors;we found serum stability enhancement of DNA aptamers to improve dynamic parameters by ODDA modification.These implementations of DNA aptamers in this thesis have provided valuable research data for Ap DC application in disease diagnosis and treatment.The developed Ap DC has strong potential as a tumor-targeting agent,and it is worth further study for its possibility in clinical trials.