Preparation And Properties Of Regenerated Silk Fibroin/ε-polylysine Composite Hydrogels

Regenerated silk fibroin hydrogels are soft,water-retaining,shape-stable,and modifiable,and provide a three-dimensional environment that mimics the extracellular matrix to modulate cell behavior and tissue function.In addition,regenerated silk fibroin hydrogel has unique biodegradability and biocompatibility,so it has important application prospects in biomedical fields including wound repair.At present,in the preparation of hydrogel,it is widely used to combine the regenerated silk fibroin with other physiological active substances to achieve functional synergy.ε-polylysine is a homopolymeric polypeptide with antibacterial properties produced by Streptomyces albicans,and its application in biomedical materials is gradually being developed.In this study,EDC/NHS was used as a cross-linking agent to prepare a hydrogel with multiple activities such as hydrophilic,antibacterial,and cell adhesion promotion by compounding regenerated silk fibroin and ε-polylysine.In addition,using the posterior silk gland of the silkworm as a bioreactor,an attempt was made to create a transgenic silkworm line expressing the recombinant silk fibroin light chain protein containing polyarginine/Poly aspartic acid at the N-terminus(both with a degree of polymerization of 25).A new type of silk material is obtained to provide a rich source of raw material for functional regenerated silk fibroin hydrogel.1.Preparation and characterization of regenerated silk fibroin/ε-polylysine composite hydrogelsIn order to successfully prepare regenerated silk fibroin/ε-polylysine composite hydrogels,this study set EDC/NHS as crosslinking agent,PBS as buffer,regenerated silk fibroin and ε-polylysine mass ratio of 15∶0,14∶1,13∶2,11∶4,7∶8 as experimental samples to prepare hydrogels.After being placed overnight,it was found that all the samples in the control group were still in solution state,while the samples in the experimental group except for the sample(ENRP78)with a mass ratio of regenerated silk fibroin and ε-polylysine of 7:8,formed colorless and transparent samples.of hydrogel.To understand the structural features of the composite hydrogels,we performed the following characterizations of the hydrogels.Scanning electron microscopy results showed that the composite hydrogel presented a relatively uniform and porous threedimensional network structure.The frequency scan results showed that EDC/NHS transformed the samples in the experimental group from viscoelastic liquid to viscoelastic solid,and the strength of the hydrogel without ε-polylysine(ENRP150)was very weak at 5.37 Pa,ε-polylysine The addition of ε-polylysine significantly improved the strength of the hydrogel,among which the hydrogel containing a small amount of ε-polylysine(ENRP141)had the highest strength,which was 3848.69 Pa.After that,as the content of ε-polylysine increased,the hydrogel The strength of the glue decreases again.Amplitude scanning results showed that the shear strain at break of all hydrogels was greater than 200%,and the content of ε-polylysine was the highest in the hydrogel(ENRP114)with a mass ratio of regenerated silk fibroin and ε-polylysine of 11:4,which fractured The shear strain is significantly larger than the other samples and exceeds 1000%.Infrared spectroscopy results showed that the chemical cross-linking method with EDC/NHS as cross-linking agent did not regenerate the secondary structure of silk fibroin,and its secondary structure was still dominated by α-helix/random coil.In order to determine that the efficient crosslinking agent EDC/NHS can shorten the hydrogel formation time,we made statistics on the hydrogel formation time of all samples.The results showed that EDC crosslinking significantly shortened the hydrogel formation time.And in the lateral comparison of the samples in the experimental group,it was found that the addition of a small amount of ε-polylysine shortened the formation time of the hydrogel.With the increase of the content of ε-polylysine,the formation time of the hydrogel increased again,but was shorter than that without ε-polylysine hydrogel.2.Properties and functions of regenerated silk fibroin/ε-polylysine composite hydrogelsIn order to understand the properties and biological functions of composite hydrogels,we designed the following experiments to explore.The results of the in vitro enzyme solution degradation experiments showed that all hydrogels degraded rapidly within 2 days,and then slowly degraded,and the hydrogels degraded faster in the enzyme solution than in PBS,under the scanning electron microscope,the hydrogel was observed to be degraded into micron-sized particles.The swelling degree test found that all hydrogels swelled rapidly in the first 1 hour,then the rate slowed down,and reached the swelling equilibrium after the 15 th hour.When the hydrogel contains a small amount of ε-polylysine,the equilibrium swelling degree of the hydrogel decreases,but then with the increase of the content of ε-polylysine,the equilibrium swelling degree of the hydrogel increases.More than 400% swelling was obtained in a hydrogel(ENRP114)with a mass ratio of protein to ε-polylysine of 11:4.The bacteriostatic ability of the hydrogel was tested by the bacteriostatic zone digging method.The experimental results showed that the hydrogels with the mass ratio of regenerated silk fibroin and ε-polylysine were 15:0,14:1,and 13:2,respectively.There is no antibacterial zone around the gel ENRP150,ENRP141,ENRP132,but there is an obvious bacteriostatic zone around the hydrogel ENRP114,indicating that the hydrogel contains only a small amount of ε-polylysine.When the content of ε-polylysine is high enough,the hydrogel has the ability to inhibit the growth and reproduction of Staphylococcus aureus and Escherichia coli DH5α.Nih/3t3 fibroblasts derived from mouse embryos were cultured in the composite hydrogel extraction medium in vitro.The results showed that after 120 hours of culture,the composite hydrogel extraction medium had a slight growth inhibition effect on nih/3t3,but it was non-toxic to cells.Then the cells were inoculated on the surface of the composite hydrogel,and the cells were inoculated on the surface of the TC treated polystyrene plate(TCP)as the control.The staining results of living and dead cells showed that after the cells were inoculated for the same time,With the increase of the ε-polylysine content,more and more cells adhered to the surface of hydrogel,and the cell morphology became more and more flat.The number of cells adhered to the surface of hydrogel ENRP132 and the proportion of cell morphology flattening were similar to TCP,while the number of cells adhered to the surface of hydrogel ENRP114 was higher than TCP,and the cells with a higher proportion appeared spindle or strip shape.Mouse mononuclear macrophage RAW264.7 was inoculated on the surface of the hydrogel,and the cells were also inoculated on TCP as a control,and the secretion of nitric oxide(NO)and the three main pro-inflammatory factors,TNF-α,The expression levels of IL-1β and IL-6 at the transcriptional level were detected.The results showed that 24 hours after cells were exposed to hydrogels ENRP132 and ENRP114,the secretion of NO and the expression levels of three major pro-inflammatory factors at the transcriptional level were consistent with those of cells exposed to TCP,indicating that these two hydrogels did not lead to inflammation.3.Creation of transgenic silkworm strainsIn order to obtain genetically modified functionalized silk as raw material for hydrogel preparation,We used piggy Bac transposon-mediated and microinjection transgenic technology to inject the vector expressing recombinant silk fibroin light chain protein specifically in the posterior silk gland of the silkworm into silkworm eggs,so as to obtain the ability to spit out polyarginine-containing or polytiana Transgenic silkworm strains of aspartate silk.The results of Asc I digestion experiments showed that the specific expression vector for the posterior silk gland of the transgenic silkworm was successfully constructed.The transgenic silkworm positive individuals with red fluorescence in the eyes were screened under the asana-fluorescence microscope.The results of genomic PCR showed that the target bands were successfully amplified in the genomes of positive individuals,indicating that the recombinant silk fibroin light chain protein genes 25 RFibL and 25DFib-L had been inserted into their chromosomes.The quantitative results showed the expression of recombinant silk fibroin light chain proteins(25RFib-L and 25DFib-L)at the transcriptional level,and their expression levels were 0.08% and 0.24% of the total silk fibroin light chain proteins,respectively.The statistics of economic characters showed that the whole cocoon weight and pupa weight of transgenic silkworm were not significantly different from those of wild type silkworm,but the cocoon shell weight and cocoon layer rate were slightly reduced.The results of the cocoon silk tensile test showed that the breaking strain of the two transgenic silkworms did not change significantly compared with the wild type silkworm,and the tensile strength decreased by about 25%.