Functionalized Cationic Polymers For The Efficient Cytosolic Delivery Of Adenosine Triphosphate-modified Proteins

Biomacromolecules-based research,such as proteins,has been rapidly developed in recent years.Therapeutic proteins due to their characteristics such as good biocompatibility,high specificity,and excellent biological activity have played an increasingly important role in the field of disease treatment.However,the majority of clinically available therapeutic proteins are designed for extracellular targets,which cannot directly participate in intracellular biochemical reactions.Thus,therapeutic proteins that act on intracellular targets have broad application prospects.However,native proteins are impermeable to cell membranes.A series of systems such as electroporation,inorganic nanoparticles,liposomes,and polymers have been developed to overcome the limitation.Among them,polymers have been one of the most attractive delivery systems owing to their facile synthesis and easy modification.For efficient intracellular delivery of proteins,polymers have to overcome challenges including effectively binding proteins to form stable nanocomplexes(NCs)for cell internalization,quick endosomal/lysosomal escape,and efficient intracellular release of proteins.To address the above-mentioned issues,this thesis designed functionalized cationic polymers including guanidiniummodified α-helical polypeptide and thymidine-modified polyethyleneimine(PEI),and developed adenosine triphosphate-modified pro-proteins to achieve efficient cytosolic protein delivery.These strategies not only enhanced polymer-protein binding to form stable NCs via multiple interaction forces,but also enabled traceless protein release in the acidic endosomal environment owing to the pH-responsive of pro-proteins.In Chapter 1,the definition and application of proteins were summarized.The significance of cytosolic protein delivery and the challenge of cationic polymer carriers for the intracellular protein delivery were elaborated.Protein modification to improve negative charge density and functionalized cationic polymers for intracellular protein delivery were introduced.In Chapter 2,we proposed a green,rapid,and safe pro-proteins modification strategy,which realized protein negative electrification.It aimed to employ the guanidinium group modified α-helical cationic polypeptide LPP as a carrier to achieve efficient intracellular proteins delivery and traceless proteins release.On one hand,2acetylphenylboricacid(ABA)firstly reacted with lysine residues on proteins via biorthogonal chemistry to form a Schiff base which is pH-sensitive.Then,adenosine triphosphate(ATP)was modified on the proteins via boronate esters bonds between ATP and ABA,which was termed as A-proteins.On the other hand,the guanidinium group was modified on α-helical polypeptide by click chemistry,named as LPP.A-proteins could form stable NCs with LPP via electrostatic interaction,hydrogen bonding,and salt bridging,which enabled efficient cell internalization.In the acidic environment of the endosomes/lysosomes,NCs were dissociated due to the cleavage of the Schiff base,thus releasing proteins and restoring their biological activity.This strategy mediated robust delivery of various proteins with distinct MWs(12.4-430 kDa)and isoelectric points(pIs,4.5-10.3)into cancer cells,including enzymes,toxins,and antibodies.It also efficiently delivered CRISPR-Cas9 RNPs to 293T-EGFP and HeLa cells,which achieved EGFP and PLK1 knockout.In Chapter 3,we further designed thymidine(T)-modified cationic polymer,PEIAZT,which was stably bound to adenosine triphosphate-modified proteins A-proteins developed in Chapter 1 via A-T base pairing,thus achieving the efficient intracellular delivery and traceless release of proteins.The azidothymidine(AZT)was reacted with alkyne-modified PEI by click chemistry to offer PEI-AZT.The stability of the NCs was enhanced by introducing electrostatic interaction and hydrogen bonding between PEIAZT and A-protein.The strategy achieved the intracellular delivery of several proteins including BSA,RNase A,Cyt C,and IgG with distinct molecular weights(MWs)and isoelectric points(pIs),and successfully delivered β-gal with large MWs into cancer cells.In the acidic environment of the endosomes/lysosomes,NCs were dissociated due to the cleavage of the Schiff base,thus releasing proteins and restoring biological activity.This strategy also delivered NCs to normal cell lines(NIH-3T3,H9C2)and tumor cell lines(HeLa,A549).It is a robust and universal intracellular protein delivery strategy.In Chapter 4,we summarized the intracellular protein delivery systems and provided prospects for future work.