The Fluorination Effect Of Fluoroamphiphiles In Cytosolic Protein Delivery

Numerous human diseases arise from mutations or other abnormalities in cytosolic proteins owing to their essential functions in enzyme catalysis,signal transduction,gene regulation,maintaining the delicate balance between cell survival and programmed cell death,etc.During the past decades,protein biotherapeutics including peptide hormones,growth factors,cytokines,and monoclonal antibodies have been discovered or engineered to treat these diseases.Protein therapeutics offer several advantages over small molecule drugs,such as higher specificity,limited adverse effects,and faster clinical development and FDA approval time.However,proteins are generally membrane-impermeable due to their relatively large size,hydrophilicity and limited positive charges,which make them difficult to reach the intracellular targets.Therefore,it is of great importance to develop efficient strategies for the delivery of membrane-impermeable proteins into cells.In retrospect,the most studied protein delivery approach has been fusing proteins with protein transduction domains(PTD)or using nanocarriers.However,the current approaches for cytosolic protein delivery all possess some limitations such as the need of protein modification,complicated synthetic processes,instability of assembled vehicle/protein complexes,and limited transduction efficacy or non-neglectable material toxicity.Recently,fluoroamphiphiles were reported to have promising features during gene delivery,the fluoroamphiphiles possess excellent self-assembly property owing to the low surface energy and lipophobicity of fluoroalkyl ligand.Hence,it is rational to develop fluoroamphiphiles as an efficient tool in cytosolic protein delivery.Protein molecules could be fabricated into nanoparticles via the co-assembly of fluoroamphiphiles and proteins in aqueous solution.The hydrophobic and lipophobic characters of fluorocarbon chains on the amphiphiles could improve the affinity of polymers to cell membranes and facilitate the endocytosis during protein delivery.In addition,the fluorous ligands are generally lipophobic and bio-inert,and this property is beneficial for the avoidance of protein denaturation during the nanoparticle formation and the retention of protein bioactivity after cytosolic delivery.Finally,the replacement of hydrocarbon chains on traditional amphiphiles with fluoroalkyl ligands is responsive for less cytotoxicity and hemolytic activity.As a proof-of-concept,we synthesized a small library of fluoroamphiphiles by grafting various fluoroalkyls to branched PEI for cytosolic protein delivery.Traditional amphiphiles such as alkane-and cycloalkane-grafted PEIs were also included in the library to reveal the effect of fluorination of fluoroamphiphiles during protein delivery and highlight their advantages over other existing amphiphilic materials.Model proteins such as bovine serum albumin(BSA),?-galactosidase(?-Gal),saporin,and peptide(GRKKRRQRRREKIKRPRSSNAETL)with different molecular sizes and charge properties were employed to test the efficacy of developed fluoroamphiphiles.Our results showed that the fluoroamphiphiles exhibit high cytosolic protein transfection efficacy.Fluorous substituents on the amphiphiles play essential roles in the formation of uniform nanoparticles,avoiding protein denaturation,efficient endocytosis,and maintaining low cytotoxicity.Structure-activity relationship studies reveal that longer fluorous chain length and higher fluorination degree contribute to more efficient protein delivery,but excess fluorophilicity on the polymer leads to the pre-assembly of fluoroamphiphiles into stable vesicles,and thus failed protein encapsulation and cytosolic delivery.Our study was the first study revealed that fluoroamphiphiles have high cytosolic protein transfection efficacy,we also revealed the fluorination effect of fluoroamphiphiles in cytosolic protein delivery and meaningful for the further study of fluoroamphiphiles in protein delivey.