Construction And Characterization Of Collagen Mimetic Peptides

Collagen is a family of proteins with characteristic triple helix structure and is the main component of the extracellular matrix.As the essential structural protein of the human body,collagen provides mechanical strength for tissues and organs;meanwhile,it possesses a variety of biological functions by regulating the interaction with other macromolecules.Collagen has excellent properties such as high biocompatibility,biodegradability and low immunogenicity,therefore it has been widely used in the fields of drug delivery,cell culture and tissue engineering.Collagen is currently mainly extracted from animal tissues.However,animal-derived collagen has severe drawbacks such as potential risks of virus transmission,uneven molecular weight,and difficult quality control,which greatly limit its clinical application.Peptides have attractive advantages of no risk of virus infection,low immunogenicity and convenient quality control;thus,the construction of proper peptides to mimic collagen has always been a hot topic in tissue engineering.This thesis aims to design and develop a variety of self-assembled collagen peptides to better mimic the structure and function of natural collagen.The main contents are as follows:1)Collagen mimetic scaffolds play a pivotal role in regenerative medicine and tissue engineering.We have herein for the first time reported the construction of luminescent lanthanide-collagen peptide hybrid three-dimensional nanofibrous scaffolds,which well mimic the characteristic architectural structure of native collagen.Three collagen peptides composed of the same intermediate repeat sequence and different end group sequences,have been shown to consistently self-assemble to form biocompatible nanofibers under the trigger of a variety of lanthanide ions.Lanthanide-collagen peptide scaffolds show programmable p H-responsive features,which can be finely tuned by the identity of terminal amino acids.Using camptothecin and cefoperazone sodium as two model drugs,the drug-loading and releasing efficiency of the collagen peptide-lanthanide scaffolds are conveniently mediated by p H,demonstrating the efficacy of these nanofibrous scaffolds as p H-responsive drug carriers.These novel luminescent collagen peptide-lanthanide scaffolds provide a facile system for p H-controlled drug delivery,suggesting promising applications in the development of therapies for many diseases.2)Osteogenesis Imperfecta(OI)is a hereditary bone disorder with various phenotypes ranging from mild multiple fractures to perinatal lethal cases,and it mainly results from the substitution of Gly by a bulkier residue in Type I collagen.Triple helical peptide models of Gly mutations have been widely utilized to decipher the etiology of OI,while these studies are mainly limited to characterizing the peptide features such as stability and conformation in the solution state.We have constructed a set of collagen peptides DD(GPO)5ZPO(GPO)4DD(Z=A,R,D,C,E,S,and V)to simulate osteogenesis imperfecta.The terminals of these collagen peptides are designed with special aspartic acid to ensure their assembly to form nanomaterials under the trigger of lanthanide ions.We have for the first time systematically evaluated the effect of different OI mutations on the aggregated state of collagen mimetic peptides.We have revealed that the identity of the Gly substituting residue plays a determinant role in the morphology and secondary structure of the collagen peptide assemblies,while bulkier residues tend to result in disruptive secondary structure and defective morphology,which lead to more severe OI phenotypes.These findings of osteogenesis imperfecta collagen mimetic peptides in the aggregation state provide novel perspectives on the molecular mechanism of Osteogenesis Imperfecta,and may aid the development of new therapeutic strategies.3)A number of self-assembly strategies have been developed to construct biomimetic peptides of collagen,including ?-? stacking,metal ion-ligand interactions,cation-? interactions,and amphiphilic peptides.However,these strategies require the introduction of unnatural amino acids,synthetic polymer materials or metal ions,and their potential toxicity greatly limit their clinical applications.We have herein for the first time constructed self-assembled triblock collagen peptides,whose sequences includes a central triple helical domain as well as N-terminal and C-terminal domains containing oppositely charged amino acids on both sides.The triblock collagen peptides can spontaneously assemble to form fibers with a periodic D-banding structure under mild conditions.A variety of functional sequences such as integrin binding motif GFOGER can be easily incorporated into the middle block without affecting the self-assembly ability of the collagen peptide,which can also endow the peptide with excellent biological functions.This new type of triblock collagen peptides is exclusively composed of biocompatible natural amino acids,and it can well mimic both the structure and function of natural collagen,providing a robust and versatile strategy for the creation of collagen biomimetic materials.4)Collagen mimetic peptide materials are mainly prepared by non-covalent interactions,while the nanostructures of such non-covalent assemblies are very sensitive to the external environment such as temperature and ionic strength,which greatly limits the scope of their applications.We herein for the first time created a covalent photo-crosslinkable collagen peptide system.This series of collagen peptides includes one or more tyrosines in the middle as well as both ends.They can self-assemble to form well-defined nanofibers in the presence of[Ru(bpy)3]2+ and light,and the morphology of the assemblies is precisely regulated by the triple helix structure and the position of tyrosine.The crosslinked peptide scaffold shows excellent biocompatibility and cell adhesion,and promotes the proliferation of fibroblasts.The novel covalent photo-crosslinked collagen peptides can be utilized to construct ultra-stable biomimetic scaffolds,showing promising applications in the fields of tissue engineering and regenerative medicine.5)Type I collagen is the most abundant type of collagen in the human body,and it is the main component of connective tissues such as skin,bones and tendons.It is an AAB heterotrimer composed of two identical ?1 chains and one ?2 chain.Its unique peptide chain composition and structure regulate its biological functions.Collagen biomimetic peptides are currently mainly homotrimers composed of three identical peptide chains,and how to mimic heterotrimer type I collagen remains a huge challenge.For the first time,we have constructed triblock peptides that can form AAB heterotrimers.Each triblock peptide includes a type I collagen domain in the middle and two terminal domains with the same charged residues.Due to the repulsion of the same charges,the triblock peptide cannot form a homotrimer;when two triblock peptides A and B with opposite charges are blended,they will self-assemble to form a stable heterotrimer due to the attraction of opposite charges.The composition of the triblock heterotrimer is completely determined by the concentration of peptide A and peptide B.The sequence of the middle block of the triblock peptide is very flexible,which can handily facilitate the introduction of functional sites of type I collagen.The heterotrimeric triblock peptide system provides a widely applicable strategy to mimic type I collagen with appropriate chain composition and structure,which has great potential in collagen biomimetic materials.