Study On The Structure Of Succinylated Ovalbumin Gels And Their Application In Wound Dressings

Ovalbumin(OVA)is the predominant protein in egg whites,accounting for 54%of the total protein content in egg whites,and has received widespread attention for its thermogel properties.In addition,OVA has biocompatible,cell adhesion promoting,accelerated neoangiogenesis and antioxidant bioactivities,showing potential for biomedical applications.However,the weak thermal-induced gel structure of OVA itself does not yet meet the requirements for its development as an advanced biomaterial.Therefore,it is important to further improve the thermo-gel properties of OVA and expand the application scope of OVA thermally induced gels to achieve high value utilization of OVA.In this thesis,OVA was used as a research object to investigate the mechanisms related to the regulation of OVA thermally induced gel network structure by succinylation and p H shift,and to construct OVA thermally induced gels with superior performance.On this basis,the OVA composite gel was constructed by introducing the efficacious component,iron tannin(TA@Fe),into the OVA gel through the principle of interpenetrating network,and the potential of the OVA composite gel in wound dressings was initially investigated through in vivo/in vitro experiments.The main research contents and results are as follows:(1)Preparation and physicochemical properties of succinylated OVA.The effects of different amounts of succinic anhydride addition on the modification effect,conformation and properties of OVA were investigated to elucidate the intrinsic relationship between the structure and physicochemical properties of OVA under the regulation of succinylation.FTIR and SDS-PAGE results showed that succinylation of OVA by succinic anhydride occurred through covalent bonding.The degree of succinylation showed an almost linear relationship with the amount of succinic anhydride added at lower amounts of succinic anhydride.When the addition of succinic anhydride was 20%(w/w,protein-based),the degree of succinylation was 69.37%.After succinylation,the structure of OVA underwent drastic changes,with the α-helix in the secondary structure further transformed into a β-sheet and the tertiary structure unfolded.The DSC results showed that the thermal stability of OVA increased by 14.79%after succinylation.The zeta potential and contact angle results indicated that the introduced succinyl groups enhanced the electronegativity and hydrophilicity,resulting in a significant increase in OVA solubility(from 73.9% to 95.4%).(2)The preparation and formation mechanism of succinylated OVA thermally induced gels.The effects of different succinylation levels on the properties,microstructure and intermolecular forces of OVA thermogels were investigated,and the intrinsic link between the gel properties and the network structure under the regulation of succinylation was probed.The results show that with increasing succinylation,the SOVA gels become almost completely transparent(56.7% transmission rate),with 6.5and 2.7 times higher hardness and elasticity,respectively,compared to NOVA gels,and the water-holding capacity increases to over 95%,resulting in a significant improvement in gel properties.The results of micro-rheology and low-field NMR showed that succinylation promoted the formation of a denser network structure,which led to the gradual transformation of free water in the network structure into less mobile water,which was firmly bound in the network structure.The network structure of SOVA gels became more complete and dense with increasing succinylation.Further analysis of the secondary structure content and intermolecular forces in the gel network showed that succinylation increased the intermolecular forces(surface hydrophobicity,hydrogen and disulphide bonding)and β-folding content of the gel network structure.(3)Thermal aggregation behaviour and molecular dynamics of succinylated OVA.The effects of succinylation on the formation of thermal aggregates,structural changes and molecular dynamics of OVA during heating were investigated to elucidate the intrinsic molecular mechanism by which succinylation regulated the formation of fine chain gel network structure.The results showed that NOVA underwent thermal aggregation at 5% protein concentration,with the particle size rapidly increasing from350.2 nm to 28.5 μm within 1 min at 90℃ and forming a completely solid state after30 min,while SOVA remained clear and transparent during thermal denaturation,with the particle size only increasing from 312.3 nm to 523.5 nm within 30 min.The changes in secondary structure content,free sulfhydryl content and surface hydrophobicity of NOVA and SOVA showed similar trends during thermal aggregation,but the changes were more dramatic in NOVA than in SOVA,with the β-sheet,free sulfhydryl content and surface hydrophobicity of SOVA being 0.79,4.85 and 1.29 times higher than those of NOVA at 30 min,respectively.The results of stability tests showed that SOVA thermophores showed higher stability in response to environmental factors(including p H,salt ion concentration and storage time)compared to NOVA thermophores.The results of molecular dynamics simulations show that succinylation is effective in preventing OVA from thermal aggregation too quickly,allowing OVA to unfold sufficiently under heating conditions and improving molecular flexibility.(4)The effect of succinylation combined with p H shift on the thermally induced gel properties of OVA.The results showed that the p H shift resulted in a more extended structure of SOVA,with the surface hydrophobicity and free sulfhydryl groups in S-12 solution being increased by 145.45% and 57.40%,respectively,compared to S-7.The results of the textural study showed that the p H shift further increased the strength of the SOVA gels,with the fracture force and elasticity of S-12 being 2.42 and 1.67 times higher than those of S-7.The SEM results showed that the p H shift resulted in a more dense SOVA gel network and the thickness of the linkage walls decreased with increasing alkaline p H,with the thickness of the linkage walls of S-12 being 15.47% of that of S-7.The results of transparency and water-holding tests showed that S-12 thermogels exhibited high water-holding(99.23%)and high transparency(73.11%).(5)Construction and performance study of SOVA-TA@Fe composite gels.The effects of succinylation-p H shift-addition of TA@Fe co-treatment on the performance of OVA thermogels were investigated,and a multifunctional SOVA-TA@Fe composite gel integrating self-healing,self-adhesion,and p H-responsive release was constructed.the SEM results showed that the network structure of SOVA-TA@Fe composite gel became denser with the increase of TA@Fe addition,and the average pore size of the SOVA-TA@Fe composite gel was 38.70% of that of the control group.The results of the texture showed that the fracture force and adhesion of SOVA-TA@Fe composite gels increased by 2.5 and 15.7 times respectively compared to SOVA gels.The results of the in vitro release of TA showed that the SOVA-TA@Fe composite gels showed p H retardation and that the release of TA was more favorable under acidic conditions.The results of rheology showed that the energy storage modulus of SOVA-TA@Fe composite gel was effectively recovered at both 100% high strain and 1% low strain continuous strain scans,and SOVA-TA@Fe composite gel showed self-repairing properties.The results of cell culture showed that SOVA-TA@Fe complex gel was able to support the growth of L929 cells in a three-dimensional gel network structure,and the cell viability remained above 95% after 48 h of incubation.Cell migration results showed that SOVA-TA@Fe composite gel was able to promote the rate of cell proliferation and migration to the scratch area,and the cells covered almost 100% of the scratch area after 48 h of incubation.(6)Study of SOVA-TA@Fe composite gel for promoting wound healing.The efficacy of SOVA-TA@Fe composite gel in promoting wound repair was evaluated in an acute trauma model on the back of mice,and the molecular mechanism underlying the promotion of wound healing by the composite gel was elucidated.The results of wound healing rate showed that SOVA-TA@Fe composite gel could promote wound tissue repair in mice,and the wound healing rate of SOVA-TA@Fe composite gel treatment reached 96.82% at the 14 th day after surgery,which was significantly higher than that of Control group(61.36%)and Vaseline gauze group(84.77%).The results of total protein and hydroxyproline content showed that SOVA-TA@Fe composite gel could promote the regeneration of granulation tissues,with 37.74% and 43.51%increase in total protein and hydroxyproline content in the SOVA-TA@Fe composite gel-treated wounds compared to the Control group,respectively.The results of ELISA showed that M complex gel was able to downregulate inflammatory factors such as TNF-α and IL-6,which were 79.91% and 68.48% of the Control group(14 d postoperatively),thereby reducing the inflammatory response in the early stages of wound repair.In addition,the SOVA-TA@Fe composite gel did not affect the growth and major organ health of the mice during application,showing a good biosafety profile.