Study On The Fabrication And Bio-application Of Zein-based Supramolecular Adhesives

Medical adhesives can be used for a variety of medical applications such as tissue bonding,gas/liquid sealing and hemostasis,which are of great significance in biological and medical field.Apart from biocompatibility,biodegradability,matching mechanical properties to tissue and acceptable swelling,the ideal medical adhesives should also meet the requirement of good wet adhesion due to the interference of water or blood.Inspired by natural marine biological adhesive system,researchers have developed a number of bionic adhesives,including synthetic polymer-based and protein-based adhesives.The covalent cross-linked polymer adhesives exhibit good bonding properties,but majority of currently available polymer glues are difficult to degrade in vivo,and the accumulation of degradation products may arise severe adverse effects,such as formaldehyde as a degradation product of cyanoacrylate can cause cell and tissue toxicity.Therefore,researchers shifted the design goal to protein with good biocompatibility,which can be classified as recombinant and natural protein adhesives according to the type of materials.The design of recombinant protein is flexible and the sequence can be regulated easily,but the expression/purification process of the recombinant protein is complicated,the yield is too low,and it is difficult to prepare in batches,so there is no product that has been clinically promoted at present.Natural proteins have the advantages of being easy to prepare in large quantities and low in price,non-infectious natural plant proteins are an ideal choice for the preparation of medical adhesives.However,there are few researches on plant protein-based medical adhesives,and weak underwater bonding strength and poor cohesion are still the main issues.In this dissertation,a series of zein/surfactant colloid systems were constructed by entropy effect via natural zein as the basic construction unit.On this basis,we prepared a class of supramolecular underwater adhesives by non-covalent cross-linking copolymerization though zein colloids and short peptides,polypeptides or non-covalent cross-linking autopolymerization of zein colloids.We studied the thermodynamic parameters,polymerization mechanism,influencing factors,applicable conditions,swelling and bonding properties,biocompatibility and degradability,hemolytic properties and biological activities of these adhesives in detail,and explored the application potential of these materials in the sealing and repair of dural defect,and arterial hemostasis.The specific contents are as follows:Firstly,sodium dodecyl sulfate（SDS）was used as dispersant and modifier,which was modified on the surface of hydrophobic zein through entropy-driven hydrophobic effect,at the same time,zein was dispersed in aqueous solution to form stable zein/SDS colloid.The surface of zein/SDS is rich in a large number of negative sulfate ions,which can be cross-linked with cationic short peptides through multiple non-covalent bonds to form macroscopical supramolecular polymers with dense network structure.The obtained polymer showed good underwater bonding properties.Isothermal titration calorimetry（ITC）experiments show that the co-assembly process was a spontaneous process driven by enthalpy effect and entropy effect.The basic residues of cationic short peptides play a leading role in crosslinking copolymerization,and the hydrophobic residues can be used to enhance cohesion.Abundant amino acid residues on the short peptide and the sulfuric acid group on the SDS endow good bonding properties,and the adhesion performance of the resulting adhesive could be controlled effectively by customizing the peptide sequences.The bursting pressure of the adhesive is 78.1±3.2 cm H₂O.The biocompatibility test indicated that the adhesive possess good cell/blood compatibility.The dynamic non-covalent bonds in the network structure of the adhesive give the advantages of slow dissociation and anti-swelling.It can be completely dissociated and degraded within two weeks after implantating into rat,and which can match well with the repair period of dural tissue.In vivo experiments showed that the obtained zein/peptide supramolecular adhesive have good effect on sealing and repairing dural defect.Secondly,the underwater bonding strength and the bursting pressure of zein/peptide adhesive are restricted in the previous part of the work,which can not meet the needs of sealing high pressure wound.Therefore,in this part,we propose two ways of improving the cohesion and removing the interfacial water of the wet interface to improve the bonding strength of zein-based adhesives.ε-polylysine with multiple cationic residues and specific secondary structure was selected to form an underwater adhesive by cross-linking with anionic zein/SDS colloid through multiple electrostatic and hydrogen bonds.ITC experiment results showed that the binding constant between zein/SDS colloid and cationic polypeptide was much higher than that of zein/SDS-peptide system.And the underwater bonding strength was significantly improved compared with zein/SDS-peptide system,indicating that the cross-linking density was effectively improved by increasing the crosslinking sites,and further the cohesion of zein-based adhesive was improved.The dynamic non-covalent bonds in the network structure of the adhesive enable it to rebuild underwater adhesive through the dry powder absorb water quickly.This property can remove the interfacial water of the substrate and absorb water to form adhesive concurrently when spraying adhesive powder on the surface of wet substrate.As a result of the above two strategies,the underwater bonding strength of the obtained adhesive was about 2 times higher than that of the zein/peptide adhesive,and its bursting pressure reached to 147.5±15.2 cm H₂O,which was much higher than that of zein/peptide adhesive（78.1±3.2 cm H₂O）.In addition,the increase of crosslinking sites and density also greatly increased the kinetic rate of crosslinking polymerization,delayed the dissociation rate of adhesives,and prolonged the degradation time in animals.Zein/polypeptide adhesive powder was applied to the arterial hemostasis of rats and rabbits,and which showed good hemostasis effect.Thirdly,considering the application of ionic protein-based adhesive system is limited under strong acid conditions,in this part,fatty acids are selected as dispersing agents,and hydrogen bonds formed by carboxyl groups under acidic conditions are used as the main driving force to develop a class of supramolecular cross-linked self-polymerizing protein-based adhesive.Through detailed optimization of material,sodium lauryl sarcosine（SLS）was selected as dispersing agent,and zein/SLS colloid system was obtained at neutral p H.A kind of self-crosslinking underwater protein-based adhesive were prepared,which dominated by mismatched hydrogen bonds via controlling the protonation of carboxyl groups though adjusting the p H value of the solution to strong acidity（p H 1-2）.The systematic study showed that appropriate length of fatty acids can effectively disperse zein to form stable colloid solution.The network formed by mismatched hydrogen of fatty acids can effectively balance the contradictory relationship between interfacial adhesion and cohesion,and achieve effective underwater bonding.The protein-based supramolecular polymer showed good sealing and bonding property in wet and water environment,and showed obvious stability in strong acid environment,which is expected to be used for wound sealing and repair in acidic environment,such as stomach.In short,this dissertation mainly utilized a hydrophobic plant protein（zein）as the building block,cross-linked copolymerized and cross-linked self-polymerized underwater protein-based adhesives were constructed using multiple non-covalent interactions from the starting point of dispersing agent and the bridging element.In this dissertation,the driving force of supramolecular cross-linking was systematically studied,the mechanism and the influence factors of crosslinking polymerization were explained.The applicable conditions and underwater bonding strength of adhesives can be effectively regulated by the types of cross-linking bonds,the hydrophobicity residues of short peptide,the number of cross-linking sites and secondary structure,et al.In addition,the swelling,biocompatibility,in vivo degradation and hemolytic properties of the zein-based supramolecular adhesives were comprehensively evaluated,and the potential application of these adhesives in the sealing and repairing of dural defect and arterial hemostasis were initially explored.