Basic Research On The Application Of Aptamers In Tumor Targeted Diagnosis And Therapy

Biomedical advances depend on the development of molecular tools with high affinity and specificity.Antibodies,with their high affinity(n M-p M)and high specificity,have played a vital role in the biomedical field over the past 40 years.A lot of antibodies or antibody-based conjugates have been approved in oncotherapy.Also,antibodies are widely used in bioassay-related fields such as clinical diagnosis,environmental monitoring.However,these antibodies are usually produced in animals,which makes the antibody production process complex and expensive.Therefore,it is urgent to develop comparable molecules to replace them.Aptamer as a "chemical antibody" has attracted extensive attention.Aptamers can be obtained from a large number of random oligonucleotide libraries by molecular evolutionary biology techniques.Subsequent mass production through chemical synthesis indicates that aptamers are more convenient to obtain than traditional antibodies.In addition,aptamers show greater potential than antibodies,such as longer shelf-life,low batch-to-batch variability,lower immunogenicity,and ease of site-specific modification,thus improving the flexibility in practical applications that antibodies can’t achieve.In the past few decades,aptamers have been considered as effective substitutes for antibodies and have been widely used in various fields.Based on the unique advantages of aptamers and their application in the biomedical field,as well as the preliminary work of our group in the research of aptamers,we developed a series of aptamer for targeted diagnosis and treatment of tumors:(1)Hepatocellular carcinoma(HCC)is a common heterogeneous and refractory malignant tumor with high recurrence rate and mortality.The emergence of multidrug resistance(MDR)is a key factor in the recurrence of HCC.The development of specific ligands for multidrug-resistant HCC will provide useful molecular tools for accurate diagnosis and targeted therapy.In Chapter 2,we obtained the aptamer PS-ZL-7c by cell-SELEX technique and subsequent sequence optimization.PS-ZL-7c could target the multidrug resistant HCC cells(Hep G2/MDR)with equilibrium dissociation constant was in the nanomolar range,which indicated that PS-ZL-7c owned high specificity to Hep G2/MDR cells.In vivo imaging assay showed that PS-ZL-7c was specifically enriched in drug-resistant tumors but not drug-sensitive tumors and normal tissues,indicating PS-ZL-7c owned the in vivo tumor-targeting capacity and could be used as an effective tool for accurate diagnosis and targeted therapy of multidrug-resistant HCC.(2)Since most patients with HCC have entered the advanced stage at the time of diagnosis,chemotherapy is the main treatment method for these patients.However,chemotherapy may destroy normal cells and cause serious side effects.It will also change the ecosystem of cancer cell,which in turn induces cancer cells to mutate or develop drug resistance.Therefore,there is urgent need to develop new therapeutic agents for HCC with strong targeting,less side effects and perfect clinical efficacy.In Chapter 3,we obtained the aptamer ZL-3 which could specifically recognize Hep G2 cells.Further,we found that ZL-3 could inhibit the proliferation of Hep G2 cells,thereby achieving the effect of treating tumors.The discovery of ZL-3 will have the following application potential: a)ZL-3 will bright a new agent for the treatment of HCC;b)ZL-3 will increase new opportunities for clinical application of aptamer agent;c)In addition,ZL-3provides an effective molecular tool for identifying specific biomarkers for hepatocellular carcinoma;d)ZL-3 can be used as a biosensor recognition element to improve the sensitivity of targeted diagnosis of hepatocellular carcinoma.(3)In Chapter 4,in order to clarify the anti-tumor proliferation mechanism of aptamer ZL-3,we identified the target protein of ZL-3 was nucleolin,and found that ZL-3 could specifically promote the ubiquitination of nucleolin to degrade by the proteasome.in Hep G2 cells.However,ZL-3 didn’t affect the expression of nucleolin in L02 cells.In this work,ZL-3 was used as an effective molecular tool to find a potential biomarker of HCC,and provided a new target for the diagnosis and treatment of HCC.It also provided a new idea for the study of aptamer mechanism of aptamer.At the same time,it laid a foundation for promoting the clinical transformation of ZL-3.(4)The degradation of nucleolin affects the growth and survival of tumor cells,which suggests that nucleolin can be used as a protein degradation target in tumor targeted therapy.PROteolysis TArgeting Chimeras(PROTACs)is a novel drug development strategy that hijacks the intracellular ubiquitin proteasome system to induce targeted protein degradation.PROTACs is mainly designed and synthesized based on peptides or small molecules or even antibodies,but there are still huge challenges in the application of PROTACs,such as large molecular weight,poor cell permeability,complicated synthesis process,poor water solubility,cell selective degradation,ect..Therefore,in Chapter 5,we designed and synthesized aptamer-based PROTACs ZL216,which realized tumor-specific degradation of nucleolin by using AS1411 as the target protein ligand to couple with the E3 enzyme VHL ligand by a click chemistry reaction.It has been proved that ZL216 has high water solubility and serum stability.ZL216 was shown to target breast cancer cells,but not normal breast cells.Furthermore,we found that ZL216 promoted the formation of the nucleolin-ZL216-VHL complex in breast cancer cells and effectively induced nucleolin degradation in vitro and in vivo.Therefore,ZL216 inhibited the proliferation and migration of breast cancer cells.These studies suggest that nucleolin,as a protein-targeted degradation target,has the potential to be used in the treatment of various tumors.Furthermore,in addition to peptides and small molecule compounds,aptamers can also be used to construct PROTACs,which broadens the toolbox of PROTACs construction and provides a promising strategy for the development of tumor-selective PROTACs.