Improving Targeted Delivery Of Aptamer-Small Molecule Drug Conjugates Based On DNA Technology

Nucleic acid aptamers(hereinafter referred to as aptamers)are known as "chemical antibodies",which can specifically recognize target tumor cells and enter cells through receptor-mediated internalization,but cannot enter nontarget cells.Aptamer-mediated small-molecule drug loading and delivery is a promising tumor-targeted therapy strategy that has emerged in recent years.However,there are still some scientific challenges in aptamer-based smallmolecule drug delivery systems.First of all,most of the conjugation between aptamers and small-molecule drugs is liquid-phase method.There are few reports on solid-phase automated conjugating,and few studies explain the interaction between solid-phase synthesized conjugates and cells and the antitumor studies in vivo.Second,monovalent aptamers-drug system still has great challenges of insufficient biostability and limited cellular selective uptake.Finally,aptamer-based small-molecule drug delivery and therapy may cause "on-target,non-tumor" side effects due to the nonexclusivity and heterogeneity of receptor expression.On the other hand,DNA molecular engineering and nanotechnology from the perspectives of chemistry and materials have been increasingly cared and favored by researchers.The DNA synthesis is modular and are automatically prepared by the base monomers under a DNA synthesizer,which is very beneficial to the programmable design and precise conjugation of drugs.DNA nanotechnology is precisely programmable and dynamically designable,and can carry different kinds of cargos in various ways.Besides,DNA nanostructures have favorable cell internalization capabilities and passive targeting functions,which can help them to construct targeting drug delivery systems.In this dissertation,we utilized DNA-related technology,especially DNA nanotechnology,to carry out some research on aptamer-based targeted delivery systems for small-molecule drugs.These studies contained:construction of modular aptamer-drug conjugates(ApDCs)using DNA solidphase synthesis technology,and the study of their interaction with cells and tumor growth inhibition;load multiple ApDCs by a static DNA nanostructure,and study the effect of their structural stability and pharmacokinetics on the therapeutic effect of cells and animals;carry a variety of aptamers and smallmolecule drugs through a dynamic DNA nanodevice,and study their effects on precise theranostics to various cells lines under logic-gated computing.This DNA nanodevice expanded the related functions of the aptamer-functionalized drug delivery system.The detailed research contents are as follows:(1)In Chapter 2,to solve the modular systhesis of ApDC,Sgc8c-CA4conjugate(“SC” for short)was modularly fabricated by the DNA solid-phase synthesizer with combining the microtubule inhibitor CA4(Combretastatin A4)drug base and four other natural bases.After verifying its good selective cytotoxicity,we studied the main molecular interactions and cell fate events from SC encountering target cells to making target cells apoptotic,which was divided into five steps to exert its efficacy.The specific tumor imaging and tumor growth inhibition ability of SC on tumor-bearing mice were also studied,and favorable therapeutic effect was obtained.(2)In Chapter 3,to solve the limited biostability of ApDC,a hierarchical assembly strategy was designed from single-stranded,four-arm tile to the high-efficiency self-assembly of rigid DNA octahedral wireframe structure.We designed the functionalized arms to load up to 24 monovalent ApDC molecules(CA4-FS)on the DNA wireframe.This assembly formed a threelayer core-shell nanomedicine,CA4-Oct,ranging from aptamer,drug to DNA octahedral scaffold.The nanowireframe drug delivery system could hide the 3’and 5’ terminals of all DNA,and hide the CA4 molecule in the middle of the interlayer,which can effectively prevent the recognition and degradation of nucleases in serum.We compared the serum stability of CA4-Oct and CA4-FS,and studied their pharmacokinetics in vivo,which provided an important guarantee and reference value for subsequent cell testing and antitumor evaluation.(3)In Chapter 4,to solve the problem about limited targted cellular internalization of ApDC,the octahedral nanomedicine CA4-Oct enhanced the specific cell internalization and tumor tissue accumulation effect by integrating multivalent aptamers-mediated active targeting and EPR-mediated passive targeting.Affected by the stability of the nanostructure,CA4-Oct is more toxic to target cells and less toxic to nontarget cells than monovalent CA4-FS.Besides,the nanomedicine delivery system showed more excellent drug delivery ability in deeper tumor sites.By studying the tumor suppressive ability and potential drug toxicity of CA4-Oct to tumor-bearing mice,we found that the programmable assembly and loading of DNA octahedron made the delivery and therapeutic effect of small-molecule drugs more effective and safer than monovalent CA4-FS.(4)In Chapter 5,to eliminate the undesired killing phenomenon of ApDC caused by the expression of the same target on a variety of cells,we constructed aptamer-functionalized DNA logic-gated nanorobot(DLGN)for multiplexed imaging and precise killing of defined tumor cells.In order to eliminate the wrong killing phenomenon caused by the expression of the same target in a variety of cells,we designed a DNA nanorobot integrating 3aptamers and 2 small-molecule drugs to anchor on the living cell membrane for realizing the fast theranostics by second-order logic-gated biocomputing.While using 3 pairs of activatable fluorescent dyes to distinguish 5 kinds of solid tumors and normal cells,DLGN also precisely delivered effector aptamer(Sgc8c)-tethered two synergistic small-molecule drugs,PTX(paclitaxel)and CA4.Although three of the model cells highly expressed Sgc8c’s receptor,PTK7,the DLGN only killed one of the cancer cells,preventing the killing of bystander cells.Fluorescence imaging and apoptosis results could be encoded into binary barcodes,and the design can be extended to other cell models and drug models to achieve universal multi-cellular identification and integrated research of diagnosis and treatment.