Construction And Applications Of Novel Nucleic Acid Drug Delivery Systems

At present,nucleic acid drugs have gradually become a very important tool for the treatment of many diseases.However,their board applications still face a series of challenges,such as poor physiological stability,easy clearance by the immune system,short blood half-life,difficulty in transmembrane caused by high negative charges,and lysosomal degradation.Therefore,the construction of effective nucleic acid drug delivery systems is particularly important.Cationic nucleic acid drug delivery systems can not only efficiently load nucleic acid drugs through the electrostatic interactions and improve their resistance to enzyme digestion,but also realize endosomal escape through the proton sponge effect,leading to broad applications in the delivery of nucleic acid drugs.However,the potential biological toxicities of cationic delivery systems are a major bottleneck restricting their clinical applications.As an endogenous material,extracellular vesicles can effectively avoid immune system recognition and clearance,with good biocompatibility,prolonged blood circulation time,and cell targeti ng capability,making them very attractive as potential delivery systems towards nucleic acid drugs.However,there practical applications for nucleic acid drug delivery still face some issues,such as the low extraction yield,time consuming,high cost,l ow and narrow range of drug loading efficacies.Aiming at addressing these issues,a series of studies have been carried out in this thesis,including development of several novel nucleic acid drug delivery systems,in-depth studies on the cellular internalization mechanisms,and potential applications of these systems for multidrug delivery.The main research contents are as follows:（1）Compared with traditional extracellular vesicles,cell-derived giant membrane vesicles（GMVs）,with a size of about 10μm,are characterized by simple extraction method,high yield,high loading capacities towards different cargoes,good biocompatibility,and low immunogenicity.Therefore,they hold a great promise in the field of drug delivery.However,the cellular intern alization mechanisms of these large vesicles remain unclear.In this chapter,with systematic molecular biology studies,we find that cell uptake of these vesicels were achieved through membrane fusion-mediated internalization.Meanwhile,Sgc8-modified GMVs（GMVs-Sgc8）are successfully prepared by anchoring cholesterol-labeled Sgc8 at the 3’end on the surface of GMVs via hydrophobic interactions.It is found that GMVs-Sgc8 can specifically recognize targeted cells,and the fusion efficiency of GMVs-Sgc8 in target cells is further improved compared with GMVs.（2）Based on the above work,we took aptamer AS1411 as an example to construct a nucleic acid drug delivery system based on GMVs.GMVs are capable of enriching hydrophobic substances and G-quadruplex-containing oligonucleotides.In this regard,we deveop a GMV-derived multifunctional drug delivery system by simultaneously integrating a hydrophobic near-infrared dye（ICG）and G-quadruplex-binding photosensitizer（TMPy P4）in GMVs.The results show that the constructed multi-functional GMVs can significantly improve the physiological stability of AS1411,the photodynamic performance of TMPy P4,and the photothermal conversion efficiency of ICG.In vitro and in vivo experiments demonstrate that the constructed multifunctional GMVs could specifically recognize tumor cells and effectively inhibit tumor growth through multi-mode therapy.（3）Nucleic acid drugs based on natural bases are highly electronegative and easy to be degraded by nuclease in vivo,which are the main reason why nucleic acid drugs have poor physiological stability and are difficult to cross the cell membrane.To solve these problems,this chapter reports the design of an artificial nucleobase-directed amphiphilic artificial nucleic acid drug(Fc₆I₁₄)from the perspective of nucleic acid chemistry.Specifically,Fc ₆I₁₄ is constructed by conjugating two types of artificial nucleobases,hydrophobic ferrocene（Fc）and hydrophilic idoxuridine（I）,via the standard solid-phase synthesis.The hydrophobic Fc nucleobase can trigger assembly of Fc ₆I₁₄ into the micelle structure,which significantly improves the physiological stability of nucleic acids through steric hindrances,and can facilitate cellular uptake without transfection reagents.As an anticancer drug,hydrophilic I bases are phosphorylated by the thymidine kinase（TK）and incorporated into DNA,efficiently interrupting DNA replication and subsequently inducing cancer cell apoptosis.This work provides a new way for broadening the application range of nucleic acid drugs.（4）Developing new nucleic acid delivery technology to minimize the toxicity risks of cationic delivery systems is an effective strategy to promote the clinical applications of nucleic acid drugs.In the above work,Fc ₆I₁₄ can not only function as a new nucleic acid drug,but also has characteristics similar to natural base pair complementation.Therefore,Fc₆I₁₄ can also be used as a cation-independent nucleic acid delivery system.With this in mind,Fc₆I₁₄ has been explored in this chapter as a cation-independent delivery system towards si RNA.Given the fact that thymidylate synthase（TS）silencing would lead to activation of the TK signaling pathway,we constructed an enhanced version of artificial nucleic acid drugs(Fc ₆I₁₄/si TS)by assembling Fc₆I₁₄ with si RNA targeting TS（si TS）through the A-I base pairing interaction.Fc in Fc₆I₁₄/si TS can further promote efficient endosomal escape of the loaded si TS via the Fenton-like reaction under the tumor microenvironment,leading to efficient silencing of TS and significant inhibition of the proliferation and metastasis of cancer cells.What’s more,Fc₆I₁₄/si TS has the synergistic effect of chemotherapy and gene silencing as compared to Fc ₆I₁₄,resulting in a reduction of effective drug concentration by two orders of magnitude.To prove the aforementioned synergistic mechanism,we further design an Fc₆I₁₄-based fluorescent probe to monitor the activity of TK in HCT116 cells after transfection of Fc₆I₁₄/si TS.The synergistic mechanism of si TS and idoxuridine is confirmed in combinationwith q PCR analysis.Such artificial/natural base assembly derived drug design provides a new direction for researchers to prepare advanced nucleic acid drugs.