Kluge Jonathan A, Kahn Brooke T, Brown Joseph E, Omenetto Fiorenzo G, Kaplan David L
Vaxess Technologies, c/o Lab Central, 700 Main Street, Cambridge Massachusetts 02139, United States.
ACS Biomater Sci Eng. 2016 Apr 11;2(4):595-605. doi: 10.1021/acsbiomaterials.5b00556. Epub 2016 Mar 17.
Storage of silk proteins in liquid form can lead to excessive waste from premature gelation, thus an alternative storage strategy is proposed using lyophilization to generate soluble and shelf-stable powder formats for on-demand use. Initial solution stability studies highlighted instabilities of higher-molecular-weight silks that could not be resolved by solution modifications such as autoclaving, pH increases, dilution, or combinations thereof. Conversely, shelf-stable lyophilized stock powders of silk fibroin of moderate to low molecular weights were developed that could be fully constituted even after 1 year of storage at elevated temperatures. Increasing dried silk powder loading in aqueous solution facilitated increased silk solution concentrations-here up to 80 mg/mL solubility was demonstrated across a range of formulations. Powders generated from silk solutions with higher-molecular-weight distributions were less soluble than moderate or lower-molecular-weight versions, despite no differences in their solution glass-transition temperatures. Instead, the aggregation and β-sheet content of lyophilized higher molecular weight stock solutions were identified as the cause of the reduced powder solubility by circular dichroism and dynamic light scattering analyses. The solubility and molecular weight profiles of all formulations investigated were preserved after storing the lyophilized materials over 1 year, even at 37 °C. No long-term powder stability behaviors were influenced by the addition of a secondary drying step in the lyophilization procedure, suggesting that this protocol could be scaled without the burden of lengthy process times. Taken together, these findings provide a very flexible and potentially cost-saving approach to producing shelf-stable silk fibroin stock materials based on the use of moderate to lower-molecular-weight lyophilized preparations. This utility is demonstrated with the formation of silk material formats from the stored powders, including films, gels, and salt-leached porous scaffolds. In turn, a more efficient system allowing full resolubilization will enable stockpiling powder for on-demand usage and for deployment of dried silks for application demands in field settings.
以液体形式储存丝蛋白可能会因过早凝胶化而导致大量浪费，因此提出了一种替代储存策略，即使用冻干法来生成可按需使用的可溶性且货架稳定的粉末形式。初步的溶液稳定性研究突出了高分子量丝的不稳定性，这些不稳定性无法通过诸如高压灭菌、pH值升高、稀释或它们的组合等溶液改性方法来解决。相反，开发出了中低分子量的丝素蛋白的货架稳定冻干储备粉末，即使在高温下储存1年后仍能完全复溶。增加水溶液中干燥丝粉的负载量有助于提高丝溶液的浓度——在此，一系列配方均显示出高达80mg/mL的溶解度。尽管具有较高分子量分布的丝溶液产生的粉末与中低分子量版本的溶液玻璃化转变温度没有差异，但其溶解度较低。相反，通过圆二色性和动态光散射分析确定，冻干的高分子量储备溶液的聚集和β-折叠含量是粉末溶解度降低的原因。即使在37°C下将冻干材料储存超过1年，所有研究配方的溶解度和分子量分布仍得以保留。冻干过程中添加二次干燥步骤对粉末的长期稳定性行为没有影响，这表明该方案可以扩大规模，而无需承担冗长的处理时间。综上所述，这些发现提供了一种非常灵活且可能节省成本的方法，用于基于中低分子量冻干制剂生产货架稳定的丝素蛋白储备材料。通过由储存粉末形成丝材料形式，包括薄膜、凝胶和盐浸多孔支架，证明了这种实用性。反过来，一个更有效的能够完全再溶解的系统将使粉末能够按需储存，并能根据现场应用需求部署干燥的丝。
