Study On Self-assembly Into Hydrogel Of Short Peptides And Their Application In Cell Culture

Hydrogel is a kind of gel in which water is the dispersion medium, due to poresand three-dimensional network structures can provide with well permeability andmechanical support, therefor, it has wide biomedical applications, for example, drugdelivery, tissue engineering and so on. Hydrogel can be got from the self-assembly ofsome small molecules, and peptide should be a perfect choice without fail. Peptidesself-assembly occur widely in nature, and peptides can form many protein moleculeswith different functions through self-assembly which play an important role in thebiological evolution and maintaining the biological species diversity. Throughdesigning and regulating the peptide sequences and changing external environments,peptides can assemble into aggregation with specific morpho-structure and functionsvia some non-covalent forces, such as hydrophobic interactions, hydrogen bonds,electrostatic interaction, π-π stack and so on. Hydrogel is a kind ofmacro-expressiveness. Structural unit of peptide is the amino acid, and there are20common amino acids in nature. We can get many peptide hydrogel materials withspecific function via designing and synthesizing different peptide sequences.Furthermore, the peptide hydrogels are biocompatible, biodegradable in vivo, andnon-toxic of metabolite, which have broad application prospects in the field ofmaterials, biomedicine and clinical medicine. In recent years, peptide assemblyhydrogels have become a hot spot of scientific research.On the base of available research, there are three different series of short peptidesdesigned via modifying Ac-I3K-NH2in this subject. We investigate the effects ofredox, pH, and enzyme catalysis on peptide assembly into hydrogels respectively, anddetect the property of the hydrogels. According to the hydrogels’ property, theirapplications on drug delivery, tissue engineering and thrombin are studied. Thespecific researches are as follows:(1) Hydrogelation under redox: Ac-I3K-NH2can assemble into long fiber, and canform weak hydrogel when its concentration is above10mM. On the basis ofAc-I3K-NH2, the cysteine and glycine residues are introduced into the peptidesequence, we design and synthesize Ac-I3CGK-NH2. CD and FTIR suggest thatthe secondary structure is still β-sheet. AFM and TEM show that self-assemble morphology are long nanofibers. There are no changes in secondary andself-assemble morphology after the introduction of cysteine. However, the thiolfrom cystein can be oxidized to form disulfide bond, and chemical cross-linkingsoccur between nanofibers which promote the formation of hydrogel. In theexperiment, we find that Ac-I3CGK-NH2can form hydrogel at0.5mM, andrheological experiments prove that strength of the hydrogel can be tuned throughchanging environmental redox. In order to verify the important action of thiol onhydrogelation, the cysteine residue is replaced with methionine, andAc-I3MGK-NH2is designed and synthesized as a control. For Ac-I3MGK-NH2,thiol is methylated, so there is no way to form disulfide bond. Under the sameconditions, Ac-I3MGK-NH2can not assemble into hydrogel. In addition, wedesign and synthesize two other molecules, Ac-I3CCGK-NH2Ac-I3KGCG-NH2,to investigate how the number and location of thiol in the peptide sequences affecttime of hydrogel formation.(2) Hydrogelation under pH trigger: since lysine residue from Ac-I3K-NH2ispH-sensitive, the peptide molecule will charge differently at different pH range.On the basis of Ac-I3K-NH2, the serine and glycine residues are introduced intothe peptide sequence, so we design and synthesize Ac-I3SGK-NH2. Comparedwith Ac-I3K-NH2, Ac-I3SGK-NH2can assemble into relatively transparenthydrogel at a pH range of8.6-10. Through pH titration, we find that there is a pKashift of primary amine of lysine side chain from10.53to8, and confirm thatAc-I3SGK-NH2charges at different pH. CD shows that8mM Ac-I3SGK-NH2canform more β-sheet secondary structures at pH=9than at pH=3. AFM and TEMshow that Ac-I3SGK-NH2can assemble into nanofibers both at pH=3and pH=9,and the chaos of nanofibers at pH=9increase dramatically. In addition, we designpeptides Ac-I3AGK-NH2, Ac-I3TGK-NH2as control to testify the importance ofblance between hydrophilicity and hydrophobicity in hydrogelation. Rheologicalexperiments prove that Ac-I3SGK-NH2hydrogel have well mechanical propertyand recovery performance from destruction. On account of Ac-I3SGK-NH2hydrogel. Base on this point, we further study application of Ac-I3SGK-NH2hydrogel in drug release and tissue engineering.(3) Hydrogel under enzyme catalysis: transglutaminase can catalyze the formation ofa covalent bond between a free amine group (e.g., protein-or peptide-boundlysine) and the gamma-carboxamide group of protein-or peptide-bound glutamine. On base of this mechanism, we introduce glutamine and glycine into theAc-I3K-NH2sequence to design and synthesize Ac-I3QGK-NH2, and studytransglutaminase catalyzes Ac-I3QGK-NH2to assemble into hydroel. From MSdata, we find that cross-links occur between Ac-I3QGK-NH2molecules, and makedimer formed only. Rheological experiments prove that strength ofAc-I3QGK-NH2hydrogel increases dramatically under enzyme catalysis. Giventhat mammal blood containing transglutaminase, we study the application ofAc-I3QGK-NH2hydrogel on thrombin, and find that platelets can adhere on thesurface of Ac-I3QGK-NH2and arise coagulation. Plasma amine oxidase is a keycomponent in the formation and repair of the native extracellular matrix, thisenzyme oxidizes primary amines of lysines to aldehydes. Using this enzyme, westudy how plasma ammonia oxidase promoting Ac-I3SGK-NH2assemble intohydrogel, and the application of Ac-I3SGK-NH2hydrogel on cell scaffoldmaterials.