Calcium Phosphate Nanoparticles For Protein Delivery

Proteins play diverse roles in the body as they perform significant physiological and biological processes including catalyzing biochemical reactions, forming receptors and channels in membranes, providing intracellular and extracellular scaffolding support. Protein therapeutics have been applied in cancer therapy, vaccination, regenerative medicine, treating loss-of-function genetic diseases and imaging. However, many barriers of delivering target proteins to cells result from intrinsic properties of most proteins, including the large sizes, varying surface charges and fragile tertiary structures. Moreover, the major challenge in all the protein delivery strategies is access of the protein to the cytosol. Most delivery strategies rely on endocytic mechanisms of cellular uptake. Therefore, the delivered proteins are susceptible to degradation in the endosome/lysosome and are also unable to access key subcellular structures and machineries required for most applications. Hence, we utilize a pH-sensitive nanomaterial for efficient endo-lysosomal escape and cytoplasmic protein release. Calcium phosphate (CaP) nanoparticles are a pH-sensitive protein delivery system with excellent biocompatibility and biodegradability. In this dissertation, we develop calcium phosphate nanoparticles as an efficient intracellular protein delivery system.We designed a facile and low-cost approach to prepare CaP nanoparticles. We examined the properties of the nanoparticles, including particle size, particle morphology, loading efficiency, release efficiency, pH-sensitive property and stability in serum. The nanoparticles had the size of approximately 210 nm with the loading efficiency of around 29%. The CaP nanoparticles were well-dispersive and remain stable size. The nanoparticles were pH-sensitive in weakly acidic environments of endosomes and lysosomes and remained stable in physiological conditions (pH= 7.4).Then, we further examined the biological features of protein loaded CaP nanoparticles. On one hand, to investigate the capability of cytosolic delivery of CaP nanoparticles, fluorescent microscopy imaging experiment and flow cytometry analysis were performed. The results suggested that CaP nanoparticles could deliver proteins into cells successfully and efficiently. As endo-lysosomal escape and cytoplasmic drug release were a major challenge, special attention was attached to intracellular delivery of GFP from CaP nanoparticles into cytoplasm. On the other hand, by the approaches of SDS-PAGE analysis and circular dichroism spectroscopy, GFP released from CaP nanoparticles retained its original structure due to the mild encapsulation conditions. In addition, KillerRed released from CaP nanoparticles retained the biological activity. The results mentioned above indicated that CaP nanoparticles had the ability to be used as a suitable protein carrier.