Functions And Regulation Of The Secondary Structure Of Synthetic Polypeptides

Polypeptides,also known as poly（amino acid）s,are important protein analogs which possess backbones similar to protein.Polypeptides have good biocompatibility and biodegradability.Moreover,the intermolecular or intramolecular hydrogen bonding of polypeptide renders them unique secondary structures（such as α-helix andβ-sheet structure）compared with other polymers,which endow important research value and application potential in the biological field.Studies indicated that factors affecting the hydrogen bonding in the backbone of polypeptide will also interfere the stability of secondary structure.Recently,researchers have developed a variety of secondary structure-transformable polypeptides capable of modulating the order-disorder of secondary structure.Based on these unique properties,polypeptide biomaterials have exhibited great advantages in cell membrane penetration,gene/drug delivery,protein modification,antibacterial and anti-fouling effects as well as immunomodulation.In order to further explore the conformational relationships of polypeptides and extend their applications in biomedical fields,herein,a series of polypeptide biomaterials were constructed and their secondary structures were modulated,thus fully realizing the advantages of secondary structures in cell membrane penetration,nucleic acid delivery,optical molecular switch and controlled drug release,and the applications of polypeptide delivery carriers in the treatment of inflammation and tumors were finally investigated.The research content of this paper is as follows:In chapter 1,we first give an overview of polypeptide materials,including the synthesis and side group modification methods of polypeptides.Afterwards,we summarized strategies for the secondary structure modulation of polypeptides and their applications in membrane penetration,gene delivery,antibacterial effects,self-assembly and protein modification.In chapter 2,we synthesized a series of water-soluble cationic polypeptides with different fluorine content based on ring-opening polymerization and click chemistry,aiming to synchronize mucus permeation and cell penetration to realize efficient pulmonary siRNA delivery against acute lung injury（ALI）.Studies have shown that the introduction of an appropriate amount of fluorine chains can promote the cellular uptake of polypeptide/siRNA polyplexes.The most effective P7F7 exhibited an approximately 2.3-fold higher cellular uptake level than the nonfluorinated polypeptide PG1,which was mainly because the fluorination promotes the interaction between the polyplexes and the cell membrane.Thus,fluorinated polyplexes possess excellent in vitro gene silencing efficiency,of P7F7 on TNF-α can reach 72%.Moreover,the fluorinated polyplexes also have excellent mucus permeation ability.The results of in vitro bronchial epithelial cell experiments indicated that the apparent permeability coefficients（Papp）of P3F16 and P7F7 increased by 22 times and 25 times compared with polypeptide PG1,respectively,indicating that fluorination can greatly reduce the interaction between the polyplexes and mucin,which can further prevent it from being captured by the mucus layer.Meanwhile,the in vivo anti-inflammatory results indicated that the P3F16/siTNF-αand P7F7/siTNF-α polyplexes can significantly reduce the expression of pro-inflammatory factors,and they also render promising potentials toward the treatment of pulmonary diseases via noninvasive,localized delivery.In chapter 3,we cleverly designed a series of de nova nano-switches G-PQP-M（M=Cy5,Ce6）through ring-opening polymerization,quaternary ammonium salting and phosphorylation reactions.The nano-switch contained a "core" of GNPs and"shells" of polypeptide with fluorescent molecules pendants.Under physiological conditions,the nano-switch possesses a flexible random coil structure due to electrostatic attraction in the side groups.At this point,the fluorescence is quenched due to the closer proximity of fluorescent molecules to GNPs.Once incubated with phosphatase,the phosphate groups detaches from the side chain,and the polypeptide is transformed into rigid α-helical structure.Simultaneously,the distance between the fluorescent molecule and GNPs also increases,and the fluorescence is restored for imaging or photodynamic therapy.It is worthy to note that the nano-switch G-PQP-Ce6 also possesses excellent ROS generation ability and in vitro anti-tumor efficacy under irradiation.Moreover,G-PQP-Ce6 also renders a good anti-tumor effect in vivo.In chapter 4,we constructed a phosphatase-responsive polypeptide drug delivery system through ring-opening polymerization,quaternary ammonium salting and phosphorylation reactions on the basis of Chapter 3.In this system,the polypeptide acts as a "gate" to achieve the selective release of drugs.Under physiological conditions,the polypeptide showed random coil structure due to the electrostatic attraction.Simultaneously,the hydrophobic drug DOX cannot be released from the pore due to polypeptide coated on the surface of the mesoporous silicon.In addition,the carrier has excellent serum stability.Once it entered the tumor microenvironment,the polypeptide became positively charged due to the detachment of the phosphate groups,which can be efficiently taken up by the tumor cells.At the same time,the polypeptide transformed into rigid α-helical structure,and DOX is released to exert anti-tumor effect since the "gate" on the surface of the mesoporous silicon is opened.Based on the difference in phosphatase content between normal cells and tumor cells,selective release of drugs is thus achieved.In chapter 5,we synthesized an amphiphilic selenopolypeptide PEG-PSe-Bn for drug encapsulation and controlled release.PEG-PSe-Bn micelles have excellent drug loading capacity,and the drug loading capacity of doxorubicin（DOX）can reach 21.3%.Moreover,the ROS response concentration of selenoether bonds is lower than thioether bonds.The results indicated that the selenopolypeptide can be oxidized after treatment with 0.1%H--₂O₂.Due to the increased steric hindrance and hydrophilicity of the side chain after oxidation,the secondary structure of selenopolypeptide transformed from random coils to α-helix,thereby realizing rapid drug release and tumor cells killing effects.In chapter 6,we made a summary and gave a prospect.In this paper,we designed a variety of polypeptide delivery carriers based on the unique secondary structure,aiming to overcome various barriers in gene or drug delivery.This research not only gives us a further understanding of the structure-activity relationship of polypeptides,but also provides guidance and insights for the design of delivery systems.