Freezing-regulated Silk Fibroin Assembly At Micro/nano Scale And Its Applications

The ice template method is a green and rapid means of preparing biomaterials and has been widely used for the preparation of porous scaffolds and the assembly of colloidal particles.However,the conventional ice template method is difficult to achieve the modulation of macromolecules at the micro or nano structural scale.The size of the solidified interface formed by the ice template is at the micro-nano scale and is very susceptible to structural changes due to fluctuations in micro-environmental changes such as temperature fields.As a natural protein material with good biocompatibility,biodegradability,and low inflammatory response,silk fibroin has been widely used in the field of biomaterials.Regenerated silk fibroin aqueous solutions,formed by dissolving and regenerating silk fibroin,can be prepared into porous materials with different structures after freezing and freeze-drying.In this paper,we investigate the structural evolution of aqueous solutions of silk fibroin under temperature field changes after freezing and propose to modulate the assembly of sericin proteins at the micro-or nanoscale by the ice template method and freeze annealing to achieve different morphologies from fibrous structures to nanoparticles and conduct a series of biological application studies based on the prepared micro and nanoparticles.Firstly,the morphological structure evolution and regulation of the silk fibroin under freezing conditions were investigated.The results show that the morphological transformation at the solidification interface of regenerated silk fibroin solutions is a thermodynamic and kinetic process.During freezing the ice template can squeeze the silk fibroin macromolecules into the gaps between the ice crystals to form a lamellar structure.During warming and annealing,the macromolecular lamellae form a nanofibrous structure due to degradation of the edges due to interfacial instability.As the temperature continues to increase,the nanofibrils gradually transform into a beaded structure and eventually form nanoparticles due to Rayleigh instability.By varying the initial freezing temperature,annealing temperature,annealing time and annealing rate during the freezing process,the morphological transformation of the silk fibroin from fibrils to beads and spheres can be effectively regulated.Ultimately,the large scale preparation of silk fibroin nanoparticles can be achieved after annealing treatment at-4 °C for48 h.Secondly,the structural transformation of silk fibroin nanospheres during freeze annealing and the influence of the silk fibroin molecular structure on the freeze assembly were investigated.During annealing at-4 °C,the structure of the silk fibroin shifts to Silk I structure.This indicates that the crystalline structure of the silk fibroin is transformed during the transition to the nanosphere structure.With increasing annealing time,the crystallinity increases,and after annealing by 48 h,Silk I with a stable structure can be formed,thus obtaining water-insoluble silk fibroin micro/nano particles.Two different solubilization systems,calcium chloride and lithium bromide,were used to prepare the regenerated silk fibroin solutions,and the effects of different molecular weights and different secondary structures of silk fibroin on the freezing assembly were investigated.The results showed that the lithium-bromide-solubilized silk fibroin solutions were more likely to form lamellar and fibrillar structures,probably due to the higher molecular weight of the lithium-bromide-solubilized silk fibroin.By adjusting the molecular conformation and chemical cross-linking treatment,the transition to β-sheet structures and the formation of cross-linked networks could be regulated.The freeze-assembly of silk fibroin with β-sheet structures or intermolecular cross-linking results in fibrillar and bead-like structures,but the transition to nanospheres is difficult by increasing the annealing temperature.Therefore,the structure of silk fibroin has an important influence on the transformation of the micro-nano structures during freeze annealing.Finally,the structure,properties and feasibility of nanoparticles as drug carriers were systematically investigated based on a strategy for the efficient preparation of Silk I structured silk fibroin nanospheres by freezing assembly.It was found that the size of nanoparticles prepared from lithium bromide soluble silk fibroin solution was larger than that of nanoparticles prepared from calcium chloride soluble silk fibroin solution.Water stability tests showed that the silk fibroin micro-nano particles maintained their morphological integrity after 28 days of immersion in deionized water.Degradation experiments showed that in PBS,after 7 days of degradation,the nanospheres began to show significant surface morphological changes and began to disintegrate after 14 days.In collagenase IA,the degradation rate was significantly faster,with the nanoparticles starting to show significant surface morphological changes after5 days of degradation and starting to disintegrate after 7 days.Further,the drug loading and release behavior of the silk fibroin nanoparticles was investigated by using fluorescent albumin drug and the anticancer drug Doxorubicin as models.The results showed that the loading rates of the fluorescent albumin drug and the drug doxorubicin were about 95% and 90%,respectively.The drug release profiles showed that the fluorescent albumin drug and the drug doxorubicin were able to maintain a continuous and rapid release within 24 h.The cumulative release after 24 h reached about 95% and 90%,respectively,and the remaining drug was able to maintain a slow release within 24～168 h.In summary,this study proposes a new green and efficient,simple and controllable strategy for the effective regulation of the micro-nano structure of silk fibroin,which can achieve the preparation of silk fibroin from nanofibrils to nanoparticles structures,and illustrates the mechanism of action during the freezing assembly process.Based on this method,the efficient preparation of Silk I structured silk fibroin nanoparticles based on aqueous solution system was achieved and the feasibility of silk fibroin micro-nano particles as rapid drug release carriers was proposed.