Protein-polysaccharide complexes, a tool for protein delivery in CaCO3 microparticles

INTRODUCTION
The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated. The success of these carriers has been driven by the ionic interactions between proteins and particles making the encapsulation of proteins highly dependent on the pH of reaction solutions and the isoelectric point of the protein.1 This poses a great limitation on the successful loading of proteins into microparticles. In this study, we explored the use of polysaccharide-protein interactions to strongly enhance the encapsulation of proteins in CaCO3 microparticles.

EXPERIMENTAL METHODS
Previously, Vandevenne and colleagues2 inserted a human chitin binding domain (ChBD) that has intrinsic affinity for hyaluronic acid (HA) into β-lactamase (BlaP). This generated chimeric protein, named BlaPChBD, was shown to be fully bifunctional. In this study this hybrid protein (BlapChBD) was associated to HA and successfully loaded into CaCO3 microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. Furthermore, thrombin cleavage sites were engineered on both sides of the inserted ChBD in the chimeric BlaP so that release of the protein from the microparticles could be easily achieved by protease cleavage. The microparticles were characterised for size, surface charge, poly-morphism and protein loading and in-vitro release assays were performed.

RESULTS AND DISCUSSION
The presence of ChBD inserted into the β-lactamase increased the encapsulation of the protein by 6-fold when complexed with HA (Fig. 1). In addition, we also showed that the encapsulated BlaP remains stable during this process using kinetic reaction of β-lactam hydrolysis. Our data showed that vaterite CaCO3 microparticles of sizes ranging between 4 and 7 µm were produced. We were also able to demonstrate that thrombin cleavage increased the release of the protein from the microparticles within 36 hours from <25% to 87% (Fig. 2). In conclusion, the presence of ChBD successfully improved the encapsulation yield of the protein while retaining up to 81% of its activity.

CONCLUSION
Protein-polysaccharide complexation demonstrates an excellent approach for the delivery of sensitive biomacromolecules which can otherwise be complicated due to electrostatic hindrances. Future prospects include using the methods we have developed for encapsulation of therapeutic proteins and using calcium carbonate as a carrier and scaffold in bone regeneration for example.