Carbon Dioxide Induced Silk Protein Gelation for Biomedical Applications

Abstract

We present a novel method to fabricate silk fibroin hydrogels using high pressure carbon dioxide (CO(2)) as a volatile acid without the need for chemical cross-linking agents or surfactants. The simple and efficient recovery of CO(2) post processing results in a remarkably clean production method offering tremendous benefit toward materials processing for biomedical applications. Further, with this novel technique we reveal that silk protein gelation can be considerably expedited under high pressure CO(2) with the formation of extensive β-sheet structures and stable hydrogels at processing times less than 2 h. We report a significant influence of the high pressure CO(2) processing environment on silk hydrogel physical properties such as porosity, sample homogeneity, swelling behavior and compressive properties. Microstructural analysis revealed improved porosity and homogeneous composition among high pressure CO(2) specimens in comparison to the less porous and heterogeneous structures of the citric acid control gels. The swelling ratios of silk hydrogels prepared under high pressure CO(2) were significantly reduced compared to the citric acid control gels, which we attribute to enhanced physical cross-linking. Mechanical properties were found to increase significantly for the silk hydrogels prepared under high pressure CO(2), with a 2- and 3-fold increase in the compressive modulus of the 2 and 4 wt % silk hydrogels over the control gels, respectively. We adopted a semiempirical theoretical model to elucidate the mechanism of silk protein gelation demonstrated here. Mechanistically, the rate of silk protein gelation is believed to be a function of the kinetics of solution acidification from absorbed CO(2) and potentially accelerated by high pressure effects. The attractive features of the method described here include the acceleration of stable silk hydrogel formation, free of residual mineral acids or chemical cross-linkers, reducing processing complexity, and avoiding adverse biological responses, while providing direct manipulation of hydrogel physical properties for tailoring toward specific biomedical applications.

Soluble collagen dissolution and assembling in pressurized carbon dioxide water solutions

“…An increase of CO 2 solubilized in water results in an increased carbonic acid content that lowers solution pH. This principle was previously investigated to process various proteins including soy protein [33], insulin [34] and silk fibroin [35,36].…”

CO2-assisted synthesis of silk fibroin hydrogels and aerogels

“…The fabrication of SF hydrogels using low-pressure, gaseous CO 2 has several advantages over the previously reported method [34,36,37] using high-pressure CO 2 . The low-pressure method is simple, safe, rapid and does not require specialty high-pressure equipment; however, the fundamental principles and the underlying mechanisms of the SF sol-gel transition remain the same in both approaches.…”

“…3B are a direct consequence of the slow rate of molecular diffusion of CO 2 into the stagnant solution [34,35]. Although there is a small amount of gel deposition on the inner surfaces of the beaker at 0 min ( Fig.…”

“…Procedure B. SF hydrogel was fabricated using a high-pressure CO 2 procedure described by Floren et al [34]. Briefly, a glass beaker with a volume of 10 ml and an internal diameter of 19 mm was loaded with 5 ml aqueous SF solution (solution depth of 15 mm and surface area $3 cm 2 ).…”

“…In four different experiments the SF solution was maintained at these conditions for 0, 30, 60 and 120 min, without mixing, and then depressurized slowly over a period of 10 min in all four cases. For these specific high-pressure experiments, gelation is defined as the point where the solution becomes opaque and does not flow from an inverted beaker after depressurizing CO 2 [6,34]. The hydrogel that was formed was recovered from the high-pressure vessel and converted into an alcogel by soaking in a series of aqueous alcohol solutions of increasing alcohol concentration as described elsewhere [33].…”