Phosphorothioated DNA Functionalized Liposomes For Intracellular Protein Delivery

Intracellular protein delivery is a promising disease therapeutic approach,especially for treating “undruggable” targets.The development of vectors with high safety,excellent specificity and high delivery efficiency is crucial for the clinical application of protein drugs.However,the existing intracellular protein delivery vectors suffer from following problems.Firstly,most vectors rely on the endosomal escape pathway to across cell membranes,which causes a low transmembrane efficiency and protein denaturation.Secondly,traditional vector manufacturing involves pathogen components or cationic materials,resulting in poor biosafety.Thirdly,delivery vectors are easily off-target in vivo,limiting the clinical application potential.Therefore,it is urgent to develop a novel intracellular delivery approach for solving above mentioned problems.Liposomes provide possibilities to address these problems.The composition and structure of liposomes are similar to natural cell membranes,which has the advantages of good biosafety,convenient preparation process,and reduced nonspecific adsorption.Most importantly,liposomes can directly deliver payloads into cytoplasm by membrane fusion,bypassing the traditional endosomal escape pathway and avoiding protein denaturation.However,conventional membrane fusion liposomes require pre-installation of specific receptors on cell membrane surface,making it difficult to apply in vivo.In addition,the unclear targeting relationship between liposome components and cell lines further restricts the application potential of membrane fusion liposomes.In this paper,a novel membrane fusion approach was discovered with phosphorothioated DNA(PS DNA)as functional material.Based on that,a series of intracellular protein delivery approaches were developed.The main contents are as follows:Firstly,the PS DNA was found to facilitate liposome-cell membrane fusion,while the PS DNA-induced membrane fusion mechanism was examined.DNAinduced membrane fusion is a non-endocytosis-dependent transmembrane pathway that enables efficient and safe protein delivery.However,conventional DNA-induced membrane fusion requires the installation of specific ligands on target cell surface,which makes it difficult to be applied in vivo.Therefore,this research utilized the interaction between PS DNA and cell membrane to achieve membrane fusionmediated protein transmembrane delivery.Firstly,content mixing,lipid exchange and particle size analysis results showed that PS DNA efficiently facilitates the membrane fusion between liposomes and CM vesicles.Further mechanistic analysis revealed that the dynamic disulfide bond exchange is pivotal for PS DNA-mediated membrane fusion.This work alternatively employs PS DNA to promote membrane fusion,providing a new avenue for intracellular delivery.Secondly,PS DNA-induced membrane fusion was used for intracellular protein delivery.Intracellular delivery efficiency and biosafety of this delivery method were thoroughly analyzed.Traditional intracellular protein delivery generally relies on the endosomal escape route,which is highly susceptible to protein denaturation.However,membrane fusion enables efficient and safe intracellular protein delivery,bypassing the endosomal escape pathway.Therefore,in this chapter,intracellular protein delivery was achieved with PS DNA-induced membrane fusion.A series of cellular experiments further demonstrated that PS DNA-mediated membrane fusion is responsible for intracellular delivery.It was found that PS DNA-induced membrane fusion could delivery proteins into different cell lines.More importantly,the delivery system does not involve any materials with significant toxicity or positive charge,leading to good biosafety.PS DNA-induced membrane fusion was also used for delivering proteins into primary T cells.Taken together,this method has great potential for clinical application.Thirdly,a stimuli-responsive DNA plug-in unit was designed for spatiotemporalcontrolled and targeted delivery.Virus-like particles(VLPs)could recognize the host cell by glycoproteins,further delivering proteins into target cells via stimuliresponsive membrane fusion.However,the complex preparation process and high immunogenicity of VLPs restrict the application potential of this method in vivo.Therefore,inspired by the conformational function change of hemagglutinin protein,this work utilized PS DNA to design a stimuli-responsive delivery tool(HACKER).By co-assembling the aptamer and HACKER unit on the liposome surface,an approach for fabricating membrane fusion liposome that is simple,secure,usercustomizable,ready to use and stimuli-responsive was developed.This DNA plug-in unit successfully realized spatiotemporally controlled intracellular delivery and targeted protein delivery in vitro and in vivo.Finally,the HACKER unit was utilized for gene editing.In summary,the dynamic disulfide bond-induced membrane fusion was discovered by studying the interaction between PS DNA and cell membranes.Based on that,the interaction between PS DNA and target cell membrane was regulated by PEG maskant,stimuli-responsive motifs and aptamers,which in turn leads to the development of a spatiotemporally controllable intracellular protein delivery strategy.