Synthesis Of Elastin-Like Proteins-Based Adhesives And Its Application In Wound Closure

BACKGROUND:Wound closure is an important aspect of surgical outcomes.Rapid,safe and effective wound closure techniques can reduce patient pain.However,there are still shortcomings in currently approved commercially available wound closure techniques.Secondary trauma and exclusion reactions caused by suture cannot be avoided.The limited application scenarios and trauma limit the development of stapling as an alternate technical tool.The emerging field,the adhesive,is considered to be an effective wound closure technique with limited development.Available commercial tissue adhesives: cyanoacrylate adhesives have inevitable biotoxicity as a breakdown product;Fibrin adhesive is limited by high price and weak adhesive properties.Therefore,there is a need to revolutionize wound closure technology and develop a new generation of clinically relevant tissue adhesives.In order to develop tissue adhesives with excellent properties,researches have developed a series of adhesives inspired by marine adhesion organisms(mussels,sandcastle worms,etc.).Existing research has focused on tissue adhesives based on biological structural proteins and has been devoted to exploring their adhesion mechanisms.Limited by the less understanding of the adhesion mechanism of marine organisms,the properties of natural proteins cannot be fully presented t,resulting in poor adhesion performance of tissue adhesives for wound closure applications.In addition,the inclusion of condition-responsive modules would enhance the performance of adhesives in wet environments,and temperature response might be a better choice.In the process of exploring the mechanism of subsea bioadhesive behavior,we found that the coacervate phenomenon plays an important role.Coacervate is a liquidliquid phase separation phenomenon resulting from the combination of two oppositely charged molecules.Its combination with structural proteins together contribute to the strong bonding of marine organisms to their surroundings.We selected recombinant elastin-like proteins as an important component of adhesive from the adhesion mechanism.Elastin-like protein is a recombinant protein with editable and easy expression,and the modified elastin-like protein carries a large amount of positive charge.Subsequently,we selected negatively charged biomolecules as another component to form tissue adhesive with strong adhesion properties and excellent biocompatibility.In addition,elastin-like proteins have temperature-responsive properties under certain conditions,and we attempted to transfer their properties to tissue adhesives to develop a new generation of condition-responsive adhesives.Aims:1.To prepare the strong tissue adhesive for wound closure and characterize its adhesion properties.And to investigate its in vivo application,including skin incision repair and visceral hemostasis,to lay the foundation for its further clinical application.2.To prepare conditionally responsive tissue adhesives with temperature responsive properties,validate their temperature responsive properties,and propose new concepts for the use,storage,and transportation of tissue adhesives.Methods:1.Recombinant elastin-like protein expression: firstly,arginine,lysine and valine residues were introduced into elastin-like protein,respectively.The recombinant elastin-like genomes with different number of repeats were then constructed using the directed recursive method,and the recombinant proteins were expressed using the prokaryotic expression host E.coli.2.Preparation of tissue adhesives: After obtaining the recombinant elastin-like protein with positive charge,the biomolecules salmon DNA and SDBS(sodium dodecylbenzene sulfonate)carrying negative charge were selected as the components of adhesives to construct tissue adhesives with strong adhesion effect and tissue adhesives with temperature responsive properties,respectively.3.Characterization of adhesion properties of tissue adhesives: After obtaining the tissue adhesives,lap shear tests were performed on hard substrates(iron,copper,ceramic,glass,PVC)and soft substrates(porcine skin,porcine muscle),respectively,to evaluate their adhesion properties.4.Biocompatibility assessment of tissue adhesives: After obtaining tissue adhesives,their cytotoxicity and biodegradability were evaluated using the CCK-8 method(Cell counting kit 8)and in vitro degradation assays,respectively.5.Assessment of visceral hemostatic effect of tissue adhesives: After obtaining tissue adhesives,a rat visceral hemorrhage model was constructed;commercial cyanoacrylate adhesive treatment was added as a control group.The visceral hemostatic effects of tissue adhesives were compared horizontally.6.Evaluation of wound healing effect of tissue adhesives: After obtaining tissue adhesives,a rat dorsal linear wound model was constructed;suture treatment and commercial cyanoacrylate adhesive treatment were added as control groups.The wound healing effect of the tissue adhesive was subsequently compared crosssectionally by quantifying the wound healing area and the local inflammatory factor of the wound.7.Evaluation of temperature response properties of tissue adhesives: After obtaining the tissue adhesives,their performance at different temperatures was verified using turbidity test,shear adhesion test,rheological test,and computer simulation,respectively.Results:1.The recombinant elastin-like proteins R18,R36,R72,R144,K144,V40K72 and V80K72 were expressed,respectively,and the molecular weight as well as the purity were confirmed by SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)and Western-blot(protein blotting)results.The purity of all the proteins was greater than 90%,which was sufficient for subsequent biological applications.2.R144-DNA,R144-SDBS,K144-DNA,K144-SDBS,V40K72-SDBS and V80K72-SDBS tissue binders were prepared,respectively.Cohesive droplet production with liquid-liquid phase separation was observed under optical microscope.3.The tissue adhesives all exhibited excellent adhesion properties on both soft and hard substrates.In particular,the shear adhesion performance of R144-DNA tissue adhesive on hard substrates reached 18-21 MPa,which exceeded most commercially available tissue adhesives,and showed adhesion on isolated skin and muscle comparable to existing studies.4.Tissue adhesives exhibit good biocompatibility.Cytotoxicity assays showed that the viability of cells co-cultured with the tissue adhesive were maintained at more than 90%.Degradation assays showed that the recombinant protein degraded quickly to absorbable amino acids in the presence of elastase and trypsin,indicating its good biodegradability.5.The tissue adhesive showed excellent hemostatic ability on the visceral hemorrhage model.Bleeding stopped within 10 s of application in the tissue adhesive group,and no swelling of the adhesive occurred.In contrast,the commercial cyanoacrylate stopped bleeding only briefly and then started to continue bleeding.6.Wound healing was faster in the tissue adhesive group and scar formation at the wound site was avoided compared to the conventional approach.The results of HE staining,Masson staining,IL-6 and TNF-α immunohistochemical staining of local sections of the wound showed that the tissue adhesive was able to reduce the degree of inflammatory infiltration at the wound site while promoting wound healing.7.V40K72-SDBS adhesive exhibited stable and reversible temperature response properties.The adhesive’s adhesion properties also increased with warming.The shear bond strength of up to 600 k Pa at 25°C was significantly higher than that of other tissue adhesives.Subsequent rheological tests showed that the loss modulus,storage modulus and viscosity of the system changed as the temperature was changed.Computer simulations showed that the distance between the valine molecules in the system became smaller at elevated temperatures,the conformation of the whole system shifted to a spherical shape,and the aggregation of valine groups prompted the bonding system to expel water from the system.8.Visceral hemostasis experiments with V40K72-SDBS adhesive showed that the 25°C adhesive provided rapid hemostasis,while the 4°C adhesive did not provide sufficient adhesion.Conclusion:1.In this study,we developed powerful tissue adhesives R144-DNA and R144-SDBS for the demand of wound closure,which performed excellent in visceral hemostasis and skin wound closure in rats.Specifically,the tissue adhesives exhibited rapid hemostasis,accelerated healing,and reduced inflammation in the above applications,outperforming commercial cyanoacrylate adhesives.In in vitro characterization,tissue adhesives have outperformed existing studies on soft tissue substrates(skin and muscle)versus hard substrates(metal,silicate materials).It also has good biocompatibility.This part of the study proposes a new paradigm for the development of tissue adhesives and contributes to the preliminary basis for the clinical translation of tissue adhesives.2.In this study,the tissue adhesive V40K72-SDBS was prepared from a conditionally responsive perspective with temperature responsiveness.The addition of the responsive module enhanced the adhesion performance of the system in humid environments.Specifically,its adhesion properties increased substantially when the temperature increased and the system converted to less-flowable state;when the temperature was lower,the adhesive was in flowable liquid state.Subsequent characterization confirms its stable and reversible temperature response properties.This method presents a new application scenario for tissue adhesives;at low temperature storage,the adhesive is flowable,can be uniformly sprayed onto the wound surface,and the liquid is able to adhesive the tissue intact;as the temperature increases,the adhesive begins to exhibit adhesion and close the wound.This method provides an innovative concept for the use,storage and transport of adhesives.