Protein-polysaccharide complexes for improved protein delivery within CaCO3 microparticles

Introduction: The controlled delivery of proteins within calcium carbonate (CaCO3) particles is currently widely investigated due to accessibility, low cost, safety, pH-sensitive properties, high surface area and high porosity. 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. This poses a great limitation on the successful loading of proteins into microparticles. In this study we explored the use of polysaccharide-protein complexes to enhance the encapsulation of otherwise poorly encapsulated proteins in CaCO3 microparticles.

Methods: A chitin binding domain (ChBD) was inserted on the protein β-lactamase to form a chimeric protein. A protein-polysaccharide complex was formed between the protein and hyaluronic acid (HA) owing to the intrinsic affinity of the ChBD to HA. The chimeric protein was then loaded into CaCO3 microparticles using super critical CO2 technology aided by the templating effect of HA on CaCO3. The microparticles were characterised for size, surface charge, polymorphism and protein loading. Bioactivity and stability of the encapsulated β-lactamase was characterised by kinetic reaction with nitrocefin. A thrombin cleavage site was inserted onto the gene sequence of the protein to achieve release of the protein from the microparticles by proteases mediation using thrombin.

Results: Vaterite CaCO3 microparticles of sizes ranging between 6 and 8 µm were produced. The presence of the ChBD on the β-lactamase increased the encapsulation of the protein by 6 fold when complexed with HA. Thrombin mediated release increased the release of the protein from the microparticles within 36 hours from <25% to 87%. The protein-polysaccharide complex demonstrated success in encapsulation of the protein while retaining up to 81% activity of the protein and allowing controlled release by proteases.

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 for example in bone regeneration.