Application Of Polyphenol Polymers In Cytosolic Protein Delivery

Proteins play important roles in biocatalysis,signal transduction,cell membrane receptors,cytoskeleton,and biomacromolecule delivery.Since the advent of the first protein drug recombinant human insulin,protein drugs have rapidly transformed the pharmaceutical industry and become the favorite of the drug market.Compared with traditional smallmolecule chemical drugs,protein drugs have the advantages of high specificity,small side effects,and short drug development cycles.Compared with genetic drugs,the effect of protein on the target is more direct,which can avoid the off-target effect and cancer risk caused by permanent gene mutation and continuous gene expression.However,due to its large size and hydrophilic properties,it cannot directly cross the cell membrane,so 75% of current protein drugs are based on extracellular development.For protein drugs with intracellular targets,how to achieve efficient cytosolic delivery is an important issue to be solved in this field.The currently reported cytosolic protein delivery carriers mainly include liposomes,inorganic nanomaterials,cell-derived vesicles,and polymers.Among them,cationic polymers have good water solubility and abundant surface active groups,which are easy to be functionalized and modified,and gradually developed into one of the most promising protein cytosolic delivery carriers.However,there are fewer binding sites on the surface of the protein.Therefore,the key issue of cytosolic protein delivery is how to stably bind the protein to the polymer carrier.Natural polyphenols can bind to biological macromolecules such as proteins and nucleic acids through hydrogen bonding,?-? stacking,and hydrophobic interaction to form nanoparticles.If polyphenol functional groups such as phenol,catechol and pyrogallol are grafted onto the cationic polymer,a polymer carrier with strong protein binding ability can be obtained.Based on this assumption,in the research work of this paper,we introduced catechol and its derivatives(benzene,phenol,3,4,5-trihydroxyphenol)on the surface of the dendrimer to obtain a polyphenol-functional cationic polymer,which is used as an efficient and safe protein cytosolic delivery carrier.The specific research content and results are as follows:(1)Using fluorescein-modified bovine serum albumin as the model protein,the relationship between the structure and performance of several polyphenol polymers in cytosolic delivery was studied.The results showed that the polymers connecting 73 catechol functional groups showed the highest efficiency of endocytosis and delivery.Through the combination of polymers and proteins,the stability of the formation of the delivery complex,and the level of cell intake,etc.,the mechanism of polyphenol polymers cytosolic delivery of proteins was studied.The catechol-modified dendrimer polymer has higher salt stability and protein binding rate than other polyphenol polymers.Flow cytometry and laser confocal experiments have shown that catechol modification significantly enhances the cellular uptake of polymers and protein complexes.(2)A variety of different types of proteins and peptides were selected to verify the universality of cytosolic delivery of polycatechols.a: The polycatechol can efficiently deliver fluorescent proteins,such as green fluorescent protein(GFP)and R-phycoerythrin(R-PE),into the cell and achieve uniform distribution in the cell;b: Polycatechols can efficiently deliver biologically active proteins,such as ?-galactosidase(?-gal),?-chymotrypsin,saporin,etc.,and maintain their biological activity;c: In addition to proteins,polycatechols also show high efficiency in the cytosolic delivery of bioactive peptides(ACSAG;IDWKKLLDAAKQIL-NH2).In this paper,by modifying polyphenol functional groups on the surface of cationic dendrimers to enhance the binding of polymers and proteins,we have achieved a win-win situation in terms of improving protein intracellular delivery efficiency and biocompatibility.Structure and performance studies show that catechol-modified can effectively promote the endocytosis of cationic polymers for protein binding and delivery complexes,and is an effective measure to obtain efficient and safe protein cytosolic delivery carriers.This research developed a new type of highly efficient,universal and low-toxic protein cytosolic delivery carrier,which can efficiently and safely deliver unmodified natural protein into the cell,providing a new idea for the design of protein delivery carrier.