| Silk fibroin is a natural polymer protein with unique chemical composition and molecular structure,excellent mechanical properties and good biological properties.In recent decades,besides the traditional textile applications,it has been widely developed and applied in the fields of daily chemical products,health food,drug/enzyme sustained release and delivery,especially medical biomaterials and tissue engineering.Generally,the degummed silk fibroin fiber can be dissolved in high-concentration salt solution and then purified to obtain regenerated silk fibroin solution.The regenerated silk fibroin materials prepared from regenerated silk fibroin solution by many methods including vacuum freeze drying,spray drying,and evaporation drying are often dominated by amorphous structure,which has poor water stability and mechanical properties,requires chemical or physical treatment or "annealing" to endow the fibroin materials with excellent mechanical strength and hydrophobicity.At present,the annealing concept of green and environment-friendly is more popular.Here,the author developed a new green processing technology-unidirectional nanopore dehydration(UND),to directly construct a super-mechanical and water-stable ultra-thick silk fibroin membrane(SFM)from regenerated silk fibroin solution.The physicochemical properties,mechanical properties,microscopic morphology,protein structure,degradability,and biocompatibility of UND-based silk fibroin membrane(SFMu)were characterized in detail.The silk fibroin membrane prepared based on the UND technology was stretched to prepare a silk fibroin bioplastic membrane with strong mechanical properties under dry and wet states.Through this technology,a series of multifunctional materials have been developed,such as crosslinked silk fibroin membrane with excellent mechanical properties,immobilized urease-silk fibroin membrane and conductive polypyrrole-silk fibroin membrane formed by in-situ polymerization.The related physical and chemical characteristics,morphological structure,enzyme activity,biological stability and electrical conductivity were also analyzed in detail.The research content and results of this dissertation mainly include the following six aspects.1)In this paper,the UND technology is proposed for the first time.Using the dialysis membrane that intercepts 8-14 kDa as the bottom of the UND mold,the regenerated silk fibroin solution is unidirectionally dehydrated through the dialysis membrane,a transparent,soft,water-insoluble and ultra-thick silk fibroin membrane is prepared.The thickness and tensile force of SFMu were almost twice that of ordinary silk fibroin membranes cast on flat sheets and annealed with methanol.The tensile strength and elongation at break were as high as 15.82 MPa and 665.23%,respectively.Infrared spectroscopy,X-ray diffraction and Raman spectroscopy showed that SFMu was rich in type Ⅱ β-turn(Silk I)structure,and its crystal structure was as high as 30.75%.The results of small-angle X-ray scattering showed that SFMu had a significant ordered structure.The scanning electron microscope with 20,000×magnification showed that the phenomenon of orderly arrangement of microstructures due to directional dehydration during the formation of SFMu existed,and the fracture surface of silk fibroin membrane showed Silk I crystals structural arrangement,with typical nanofibrous structure.SFMu was almost completely degraded in protease XIV for 2 weeks.L-929 cells adhered,grew and proliferated well on it,and after implantation in vivo for three weeks,related inflammatory factors and skin tissue growth showed the same state as normal rats.In addition,during the UND process,the secondary structure composition could be changed by adjusting the temperature and relative humidity,so as to control its mechanical properties and biodegradation rate,which is suitable for more medical application scenarios.The above results indicate that UND induces the self-assembly and directional alignment of silk fibroin molecules,resulting in the formation of ultra-thick silk fibroin membranes with super-stretching behavior dominated by the Silk I structure.The high-performance silk fibroin material has great potential application prospect in the fields of medical biomaterials,biomimetic materials,sustained drug release,and membrane separation.2)To explore the influence of environmental factors on SFMu,the author firstly analyzed the influence of methanol aqueous solution concentration and annealing treatment time on the physicochemical properties and protein structure of silk fibroin membranes.The results showed that the low concentration and short time methanol annealing hardly affected the secondary structure of SFMu,while the methanol water solution higher than 40%induce the formation of a part of the β-sheet structure,but even after a long annealing for 24 h,the content of Silk I did not decrease significantly either.Secondly,SFMu was subjected to mechanical compression and annealing in a high temperature water environment.It was found that with the increase of compression degree,heat treatment temperature(>50℃)and heat treatment time,the silk fibroin membrane underwent a structural transformation to Silk II and the content of Silk II gradually increased.Due to the increase of β-sheet structure,the swelling rate of SFMu started to decrease after treatment.The sufficient swelling of the structure during high temperature water annealing increases the corresponding degradation rate.SFMu exhibits anisotropic mechanical properties and structural stability under different environmental factors.Taking advantage of this feature,the annealing method of this silk fibroin membrane can be selected to meet the functional diversity of materials in different scenarios.3)To further study the properties and protein structure of the ultra-thick silk fibroin membrane dominated by the Silk I crystal structure prepared by directional dehydration,the membrane was subjected to an annealing treatment of axial mechanical stretching up to 5 times.A flexible regenerated silk fibroin material-silk fibroin bioplastic membrane(SFBM)was developed,which was flexible and firm but not brittle in both dry and wet states.The SFBM had a tensile strength of up to 51.05 MPa and a high toughness of 2 × 105 kJ/m3,and could also be processed into different forms of silk biomaterials in the dry state.After 10 days of incubation in neutral protease,86%of the mass remained,which fully demonstrated that the bioplastic could achieve slow degradation of biomaterials.Through the characterization of infrared spectroscopy,X-ray diffraction and Raman spectroscopy,it was found that the secondary structure of SFBM was mainly β-sheet crystalline structure,and its content increased with the increase of the stretching ratio,reaching a maximum of 52%.In addition,small-angle X-ray scattering analysis showed that the silk fibroin bioplastic membrane had a significant oriented structure,which changed with the strengthening degree of tensile annealing.Compared with SFMu,the scanning electron microscope images showed that its microstructural morphology was axially stretched and also exhibited oriented nanostructures.The L-929 cell culture and in vivo implantation experiments also showed that the silk fibroin bioplastic membrane had good biocompatibility.This anisotropic silk fibroin bioplastic membrane with β-sheet crystalline structure has extremely high mechanical strength,slow degradation rate and good biocompatibility,toughness and processability in dry state,which is a potential biomaterial suitable for various biomedical scenarios.4)Based on the amino acid composition of silk fibroin and the successful case of cross-linking with reactive groups,we deeply explored that adding cross-linking agent-polyethylene glycol diglycidyl ether(PEGDE)into regenerated silk fibroin solution and observed whether the cross-linking reaction could be carried out in the dehydration process and a cross-linked silk fibroin membrane with stronger mechanical properties could be formed.The results showed that the mechanical properties of the silk fibroin-PEGDE cross-linked membrane formed by UND were significantly improved,especially the tensile strength of the cross-linked membrane formed by adding a cross-linking agent at a concentration of 167μL/g was significantly improved by 20%,reaching 18.91±0.96 MPa,while the tractility and swelling rate decreased,indicating that the microstructure of the silk fibroin membrane changed in density after cross-linking.Structural analysis showed that the cross-linked membrane also had a crystalline structure dominated byβ-sheets,and its thermal stability was significantly improved,the enzyme degradation rate was greatly slowed down in neutral protease aqueous solution.Small angle X-ray scattering analysis showed that the original ordered structure of SFMu gradually disappeared due to cross-linking.In vitro and in vivo experiments proved that the silk fibroin cross-linked membrane still has good biocompatibility.The silk fibroin-PEGDE cross-linked membrane prepared by the UND method can effectively improve the mechanical properties of the silk fibroin membrane,and the crystalline structure is dominated by Silk II,which shows slow enzymolysis rate and good biocompatibility,thus providing a new idea for high-strength and slow-degradation biomaterials implanted in vivo.5)It is well known that silk fibroin is a good carrier for immobilized enzymes,therefore,the urease model enzyme was added to the regenerated silk fibroin solution,and an ultra-thick silk fibroin membrane with urease embedded was also prepared by the UND method.Its appearance and mechanical properties are indistinguishable from the aforementioned enzyme-free SFMu.When silk fibroin and urease were prepared at a ratio of 1:4(W/U),the activity recovery rate of the immobilized enzyme could reach 74.53±6.72%,and the relative enzymatic activity recovery rate could be maintained at above 50%after repeating the enzymatic reaction ten times;the relative activity of the enzyme membrane stored in solution at 4℃ for 30 days lost less than 10%,and the activity under dry storage was more stable.After incubated with high temperature at 80℃ for 10 min,the relative enzymatic activity recovery rate could maintain 50%.The above results indicate that the immobilization of urease in the UND-based silk fibroin membrane has high recovery rate and stability of enzyme activity,while not affecting the morphology and structure of silk fibroin membrane.To improve the activity stability of the immobilized urease silk fibroin membrane,a more stable immobilized urease silk fibroin plastic membrane was prepared by stretching the enzyme membrane,and another immobilized urease silk fibroin cross-linked membrane was prepared by blending silk fibroin,enzyme and PEGDE.The reuse and storage stability of the two enzyme membranes were significantly improved,the relative recovery rate of enzyme activity reached 90%,in addition,the cross-linked enzyme membrane was more stable at high temperature.These results fully indicate that PEGDE cross-linking during the preparation of UND or post-stretching treatment can make the immobilized urease activity in the silk fibroin membrane more stable,which opens up a new way for the application of UND technology in urease immobilization.6)Since the development and application of biomaterials in flexible electronic devices has received extensive attention,the author also preliminarily explored the conductive post-treatment of silk fibroin membrane based on UND method,a novel conductive silk fibroin membrane was prepared by in-situ polymerization to form polypyrrole.The conductive silk fibroin membrane formed by in-situ polymerization of pyrrole monomer with a concentration of 4.5 wt%had excellent electrical conductivity,the electrical conductivity reached 218.28 S/m,and could light up 25 parallel LED lights under the driving of 4.5 V DC power supply.The electrical conductivity of the conductive silk fibroin membrane increased gradually with the increase of the polypyrrole content,the swelling rate and ductility decreased and the mechanical strength increased.Infrared spectroscopy,wide-angle X-ray diffraction and small-angle X-ray scattering analysis showed that a polypyrrole membrane was formed on the surface of the silk fibroin membrane,which affected the observation of the corresponding infrared spectrum and directional structure,but the crystalline structure of Silk I did not change.Scanning electron microscope further observed that polypyrrole particles with a particle size of about 150 nm were evenly distributed on the surface and inside of the conductive silk fibroin membrane,indicating that the pyrrole monomer could enter the membrane,and the polymerization reaction could be carried out simultaneously inside and outside the silk fibroin material.In addition,the UND-based cross-linked silk fibroin membranes and silk fibroin bioplastic membranes were also carried out the in-situ polymerization of polypyrrole.The conductivity of the cross-linked conductive membrane is increased to 4 times to 889.96 S/m.To explore the application in flexible wearable electronic components,this paper conducted multi-angle and multi-time bending tests on the conductive silk fibroin membrane,and found that the membrane can output corresponding electrical signals stably and sensitively.In conclusion,the conductive silk fibroin membranes synthesized by in-situ polymerization,all conductive membranes show good electrical conductivity,and can also reflect the signal changes of dynamic deformation stably,efficiently and sensitively,indicating that the conductive composite membrane material prepared from UND-based silk fibroin membrane have great application potential in flexible wearable electronic devices,biological electrodes,electronic sensing elements and other application scenarios.In summary,the unidirectional nanopore dehydration technology is proposed for the first time,and the silk fibroin membrane material with high mechanical strength especially super tensile properties dominated by the Silk I structure is prepared,as well as blending with other active substances during UND,annealing treatment after membrane formation or in situ polymerization,a series of multi-purpose functional silk fibroin membranes have been derived,including high-strength silk fibroin bioplastic membranes that are flexible in dry state,cross-linked silk fibroin membranes with stronger mechanical properties,and immobilized urease silk fibroin membrane,cross-linked silk fibroin membrane or silk fibroin bioplastic membrane,conductive silk fibroin membrane,cross-linked silk fibroin membrane or silk fibroin plastic membrane,etc.The UND method proposed in this experiment is a brand-new preparation method that can lead to the self-assembly and directional arrangement of silk fibroin molecules to form silk super-performance materials.This method is not only simple,low-carbon,controllable,and environmentally friendly,and can form anisotropic super-mechanical properties,especially super-thick silk fibroin membranes with excellent ductility.The physical and chemical properties and crystal structure of silk fibroin membrane can be controlled by temperature,relative humidity and crosslinking reaction during the process of unidirectional nanopores dehydration,or by the above post-treatment such as methanol impregnation,axial stretching and in-situ polymerization,so that the mechanical strength,biological activity,biodegradability and biocompatibility of silk biomaterials can be adjusted to meet the requirements of biomaterials,drug enzyme slow-release systems,medical tissue engineering materials and wearable device materials.This preparation method for unidirectional dehydration using nano-pore is expected to be developed and utilized in other natural macromolecular proteins and new artificial macromolecular functional materials. |
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