Design,Preparation,Structure Properties And Safety Evaluation Of Novel Ionic-complementary Peptide Hydrogels (KXDN)_n

Ionic-complementary peptides are composed of alternating hydrophilic and hydrophobic amino acids.They form β-sheet secondary structure in aqueous solution,one side is a hydrophobic surface formed by hydrophobic amino acids,and the other side is a hydrophilic surface formed by hydrophilic amino acids with alternating positive and negative charge distributions.Ionic-complementary peptides can self-assemble into hydrogels upon addition of salt solution through hydrogen bond and hydrophobic force between molecules.These hydrogels have good biocompatibility and degradability,and can be used as a kind of bio-scaffold material for tissue engineering and regenerative medicine.In this thesis,hydrophilic amino acids K and D,hydrophobic amino acids I,V and F were selected to design novel ionic-complementary peptides.Among which I is the L isomer,V has a strong tendency of β-sheet,F is an aromatic amino acid with π-π stacking effect.Six novel ionic-complementary peptide hydrogels KID12-I,KID16-I,KVD12-I,KVD16-I,KFD12-I,KFD16-I were designed and synthesized,and the preliminary evaluation of their properties and safety were studied.In the first part of this thesis,novel ionic-complementary peptides were designed and synthesized,KID12-I、KID16-I、KVD12-I and KFD12-I hydrogels were prepared.The effects of the concentration of peptide solution,the type and concentration of salt solution,the method of adding the salt solution and the trigger time on the hydrogel formation were exploreded.And the effects of ultrasonic power and ultrasonic time on the formation of these hydrogels were also investigated.It shows that the hydrogel properties after addition of DMEM are better than the addition of PBS and NaCl respectively,by comparing the hydrogel formation and properties among different methods.When the concentration of peptide solution is 10 mg/mL,and DMEM is added to the surface of the viscous peptide solution,the complete hydrogels can be prepared within 12 h.In the second part of this thesis,the structures of KID12-I,KID16-I,KVD12-I and KFD12-I hydrogels were studied.The secondary structures of the ionic-complementary peptides in aqueous solution were detected by circular dichroism spectroscopy.And the internal structures of the ion-complementary peptide hydrogels were observed by scanning electron microscopy.The circular dichroism analysis show that KID12-I,KID16-I,KVD12-I and KFD12-I form β-sheet structure in aqueous solution.Under suitable ultrasonic conditions,the internal structure of KID12-I,KID16-I,KVD12-I and KFD12-I hydrogels are membrane structures,observed by scanning electron microscopy.In the third part of this thesis,the rheological properties of KID12-I,KID16-I,KVD12-I and KFD12-I hydrogels were determined.Dynamic frequency scanning experiment was conducted to measure the storage modulus(G’)and loss modulus(G’’)of hydrogels over a continuously varying frequency range.The results show that the G ’of KID12-I,KID16-I,KVD12-I and KFD12-I hydrogels is much higher than G " respectively,G’ and G" remain relatively constant,showing solid-like hydrogel property.In comparison,the G ’ of KID12-I and KFD12-I hydrogels are significantly higher than those of KID16-I and KVD12-I hydrogels,and also higher than that of KLD-12 hydrogel,indicating that the mechanical properties of KID12-I and KFD12-I hydrogels are better.In the fourth part of this thesis,the safety of KID12-I and KFD12-I hydrogels with good mechanical properties were evaluated.The cytotoxicity and hemolytic activity of the peptide solution were evaluated.Cytotoxicity results show that the toxicities of KID12-I and KFD12-I solution to NIH 3T3 cells are low,the survival rate of cells is still close to 80% at the maximum concentration of 200μM.Besides,the survival rate of cells in different concentration of KID12-I and KFD12-I solution does not differ significantly from that of KLD-12 solution.In the hemolytic activity results,the hemolytic activity of KID12-I solution is low,too.The hemolysis rates are lower than 1.0% at all concentrations.And the hemolytic activity of KFD12-I solution is also low,and the hemolysis rates at most concentrations are lower than 1.0%,except that the hemolysis rate are below 2.0% at partial concentrations.It is noteworthy that KLD-12 has similar hemolytic activity in comparing with the KFD12-I solution.Based on these results,we can conclude that the safety of KID12-I and KFD12-I are better.The novel ionic-complementary peptides we designed in this study are first-generation self-assembled peptides.Which can provide research foundation for designing and synthesizing the second generation of self-assembled peptides.The new hydrogels,especially KID12-I and KFD12-I hydrogels have better mechanical properties,and they are expected to be applied to cell culture and tissue engineering after the further evaluation of their safety,degradability and biocompatibility.