Abstract
Ensilication is a novel method of protein thermal stabilisation using silica. It uses a modified sol–gel process which tailor fits a protective silica shell around the solvent accessible protein surface. This, electrostatically attached, shell has been found to protect the protein against thermal influences and retains its native structure and function after release. Here, we report the calorimetric analysis of an ensilicated model protein, hen egg-white lysozyme (HEWL) under several ensilication conditions. DSC, TGA-DTA-MS, CD, were used to determine unfolding temperatures of native, released and ensilicated lysozyme to verify the thermal resilience of the ensilicated material. Our findings indicate that ensilication protects against thermal fluctuations even at low concentrations of silica used for ensilication. Secondly, the thermal stabilisation is comparable to lyophilisation, and in some cases is even greater than lyophilisation. Additionally, we performed a mouse in vivo study using lysozyme to demonstrate the antigenic retention over long-term storage. The results suggest that protein is confined within the ensilicated material, and thus is unable to unfold and denature but is still functional after long-term storage.
Highlights
Protein functionality occurs as a direct result of intramolecular interactions between amino acid side chains of polypeptides.[1]
In contrast with conventional protein encapsulation, where the particles are solidi ed and stored in liquid, our method of ensilication applied in this study, using tetra-ethyl orthosilicate (TEOS) as a precursor, which creates a protein loaded dry silica powder.[8]. Applying this methodology with lysozyme, we found that native protein structure and function were retained following ensilication in our proof of concept study.[8]
An earlier study into the mechanism of ensilication showed a diffusion limited cluster aggregation (DLCA) type process.[10]. This means the more silica added to the reaction, the faster the ensilication will occur because of the abundance of polymeric silica.[33,34]. This was observed by Field emission scanning electron microscopy (FE-SEM) (Fig. 1 and Table 1) when lysozyme was ensilicated at various ratios
Summary
Protein functionality occurs as a direct result of intramolecular interactions between amino acid side chains of polypeptides.[1]. These changes can alter the tertiary structure leading to protein unfolding, aggregation and precipitation in solution.[2,3] Previous strategies employed to increase protein stability in solution included excipients, buffer compositions, ionic liquids and encapsulation.[4,5] Lyophilisation, freeze-drying, is the current gold standard in biomolecule preservation.[6,7]
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