Preparation Of Enzymatic Frustules-Peptide Composite Hydrogel And Their Application In Cell Culture

Due to peptide self-assembly hydrogels have high water content and class spatial network structure of extracellular matrix,they have good applications in cell culture,drug release,bioscaffold materials and biomedicine.However,the lack of mechanical strength limits their development.Enzymatic reactions have the advantages of high efficiency,specificity and mild conditions,etc.It is also an important means that many scholars use to enhance the gel strength.Glutaminase transaminase?TGase?recognizes the?-amido group of glutamine?Gln,Q?residues and the?-amino group of lysine?Lys,K?residues to form?-??-glutamine?-lysine isopeptide bond.Diatoms are unicellular marine algae that well known for the spectacluar structures of their silica-based cell walls-frustules.Frustules have attracted attention due to their fine micro-nano pores,diverse ribbed bands structure,excellent mechanical properties,and high specific surface area.Based on the enzymatic reaction,frustules are doped into the gel system and acted as a kind of skeleton material,which further increase the mechanical strength of the gel.In this thesis,we successfully extracted biosilica materials from marine diatoms.we also designed and synthesized two peptides I₄K₂?Ac-I₄K₂-NH₂?and Q3?Ac-Q₃GRGDS-COOH?.They effectively bind to construct frustules-peptide composite hydrogel under TGase catalysis.The main contents are as follows:?1?We analyzed the cell density and pH of the environment on the growth of diatom?Thalassiosira rotula?,and purified by SDS/EDTA and H₂O₂/HCl.The research showed that diatoms can collect after culture 6 days that the cell density was stable;the latter method had a higher rate of organic matter removal.The work clearly demonstrated the natural biosilica materials can be obtained by a simple purification method.?2?Based on the work carried out on the formation of hydrogels between I₄K₂ and Q3 by TGase,in order to further enhance its mechanical strength,the frustules were introduced into the enzymatic peptide hydrogel system.The morphology of the gel was characterized,and the influence of different frustules dosage and incubation time on the rheological properties of the gel were systematically investigated.In conclusion,the storage G'of hydrogel without doping frustules was around 1600 Pa.When the concentration of frustules was 2.5 mg/mL,the G'enhanced to about 6 KPa,a 3-4 fold increase.The work showed that the introduction of frustules on the basis of enzymatic reactions can be used as a powerful means to enhance the mechanical strength of peptide hydrogels.?3?We applied the prepared enzymatic composite peptide hydrogel material to cell culture?MC-3T3-E1?and controlled drug release.We performed experimental analysis on cell growth,migration,and differentiation.The results showed that the enzymatic composite hydrogel could be a good scaffold material for MC-3T3-E1 cells and had certain advantages in inducing differentiation.These results can be explained by active ligand RGD?Arg-Gly-Asp?of Q3 to promote cell adhesion and the presence of frustules becoming a potential source of silicon to promote cell differentiation.Also,they acted as a pore-enlarging material to increase the release rate of the drug.