Research On Aptamer Mediated Targeted Therapy Of Cancer

Aptamer is an oligonucleotide fragment of DNA(deoxyribonucleic acid),RNA(ribonucleic acid),or XNA(nucleic acid analogue)that can bind to its target specifically.In general,it is obtained from nucleic acid molecular library through in vitro screening technique(Systematic Evolution of Ligands by Exponential Enrichment,SELEX).Aptamer is often referred to as “chemical antibody” and has the same level of specificity and affinity as antibody or even higher.In addition,aptamer possesses its own merits,including shorter screening period,smaller size,easy penetrating into tumor tissues,easy to production and modify,much higher chemical stability,and much lower immunogenicity.Aptamer thus has been widely utilized in various research fields studied since the 1990 s,and exhibits prominent clinical application potential.Chemotherapy for tumors refers to treating cancer with small molecule antitumor drugs that can kill cancer cells.One of therapeutic mechanism is to inhibit the growth of cancer cells by interfering with the process of cell division.Chemotherapy drugs do not have specificity,leading to the unwanted side effects where they could simultaneously kill normal cells undergoing cell division.Aptamers can be used as an efficient molecule recognition tool based on their attractive biological properties,thus endowing small molecule antitumor drugs with targeting ability while they are loaded onto aptamers by controllable chemical conjugation approach.Through the highly specific recognition and rapid endocytosis of aptamers when interacted with target proteins that highly expressed on tumor cells,drugs can be selectively delivered to tumor cells to improve the anti-tumor effect and reduce the toxicity and side effects against normal tissues.This thesis is committed to studying how to use chemical modification technology to better construct aptamer-drug conjugates and achieve controllable drug release function,improving the antitumor efficacy of the engineered conjugates.We selected small molecules antitumor drugs,including: Mitomycin C(MMC),paclitaxel and 7-ethyl-10-hydroxycamptothecin(SN-38)to prepare aptamer-drug conjugates with different chemical links.This strategy allowed us to improve serum stability and pharmacokinetic properties of aptamers.The explorations of anti-tumor ability at the cellular level and the targeting ability in vivo were conducted in this thesis.More specifically,(1)In Chapter 2,we linked MMC to XQ-2d aptamer using three different chemical linkers and investigated the influence of these linkers upon the cytotoxicity of the produced conjugates.The result of cytotoxicity determination experiments showed that the two conjugates possessing two reduction environment-responsive linkers exhibited much higher toxicity than the one with non-responsive linker.Interestingly,conjugate with much faster drug release rate exhibited better apoptosis induction ability against targeted cancer cells.These results indicated that we were able to improve the bioavailability of MMC through the design of controlled release chemical linkers,thus enhancing the anti-tumor effect of the synthesized conjugates.(2)In the third chapter,we explore the mechanism of toxicity enhancement.In Chapter 2,the results showed that Ap DC had a significantly higher killing ability on tumor cells than drugs alone.We chose different aptamers(XQ-2d,Sgc8 c and AS1411)and modified them with MMC respectively.Then we tested the cytotoxicity to their corresponding target cells.The obtained results showed that the cytotoxicity of all three drugs was significantly higher than that of MMC alone.We then utilized XQ-2d-MMC as an example to study the rationalities behind this cytotoxic enhancement effect.Our observations suggested that the specific recognition,high affinity binding and receptor-mediated internalization function of the aptamer were essential for the effective inhibition of cancer cell growth.(3)In the fourth chapter,in order to explore the potential of using aptamer-MMC conjugate in targeted therapy of pancreatic cancer,we modified aptamer by chemical modification technique to improve its stability against nuclease degradation and drug delivery efficiency.Phosphorothioate(PS)backbone was used for the synthesis of fully thio-substituted XQ-2d(S-XQ-2d)and its conjugate,thio-aptamer-drug conjugate(MMC Functionalized S-XQ-2d,MFSX).We found that the cytotoxicity of MFSX against target cells was similar to that of MMC.Targeted binding and endocytosis experiments proved that both S-XQ-2d and MFSX had stronger endocytosis ability than XQ-2d and MFX(MMC Functionalized XQ-2d).In addition,the serum stability of the aptamers was significantly improved after modification,and the half-life of the blood circulation was increased by about 5 times.(4)In Chapter 5,we designed and synthesized an aptamer-polyprodrug conjugate(APDC)to elevate the drug loading and enhance their serum stability.Different number of anticancer drugs,including hydroxycamptocamptin,and paclitaxel,could be modified on the backbone of polymer POEGMA.After that,we bridged the polymer with aptamer to form APDC with high drug loading.Our newly developed APDC exhibited enhanced resistance to nuclease degradation,prolonged in vivo circulation half-life and improved selective binding and cellular uptake capacities.Meanwhile,they also performed the behavior of reductive species-triggered and the synergistic treatment of two anticancer drugs can also be realized.The APDC would provide a better platform for the design of a new generation of multifunctional Ap DC with great application potential.In conclusion,we have designed and synthesized aptamer-MMC conjugates with different linkers and studied their influence upon the cytotoxicity of against the target cancer cells.Then the investigations about plausible mechanism behind the cytotoxicity enhancement events were performed.To improve the stability of and drug delivery efficiency of the aptamer-drug conjugates,aptamers were modified with PS backbone,and their biological properties were investigated.Finally,APDC were designed and synthesized to improve the drug loading and its therapeutic effect.Our studies provided strong support for the development of innovative Ap DC,and might promote the further applications of aptamers in clinical diagnosis and treatment.