T. Panagiotou and R. Fisher
Keywords: calcium phosphate, nanoparticles, RNA delivery
Summary:Calcium phosphate (CaP) finds medical applications in a number of areas, including bone and teeth tissue engineering, and drug delivery. It is a well-studied biocompatible, bioactive and bioabsorbable material and has been used extensively in laboratory settings. Calcium phosphate nanoparticles have been successfully used for delivery of nucleic acid therapeutics, antibiotics and anti-inflammatory compounds, as vaccine adjuvants, and in bone regeneration. However, current methods for production of CaP nanoparticles are laboratory based, inefficient, result in low yields, and are unable to produce stable particles. Additionally, such methods are not scalable, robust, or conducive to current Good Manufacturing Practice (cGMP) / aseptic processing, which are key to pharmaceuticals manufacturing. Aseptic processing, in particular, is a key requirement for injectable/implantable formulations, including those for siRNA delivery. Filter sterilization is commonly used to produce sterile formulations. When sensitive active ingredients are present, such as in nucleic acids and biologics containing formulations, filter sterilization may the method of choice or the only option, since it does not affect the integrity of sensitive actives. In contrast, other sterilization methods that are based on high temperatures or exposure to gamma rays may be detrimental to those actives. In particle-based formulations, particles are required to be below 220 nm so they can pass through the pores of the membrane used for filter sterilization. This is generally difficult to achieve using laboratory production methods. Delphi Scientific employed a novel, scalable micro-reaction technology, which is suitable for pharmaceutical processing and for production of CaP particles. Additionally, the microreactor is inherently capable of handling fairly high concentrations of dispersed solids, unlike commonly used methods. CaP nanoparticles were produced as a result of chemical reactions and precipitation that take place inside the microreactor. Based on the operational maps of the microreactor namely pressure, intensity of turbulent field, stoichiometry, residence times, etc., key particle properties may be controlled, including: size and size distribution, particle yield, composition, structure and encapsulation efficiency. Layers that control the functionality of the particles may be added to the particles using variations of this technology. There may be layers used to retain the active ingredient, while others may provide surface modification for enhanced stealthiness and targeting. Calcium phosphate particles were successfully produced as a result of chemical reactions followed by precipitation. Results of this effort indicate that the process is highly tunable and may result in CaP nanoparticles with a variety of properties. Key process and formulation parameters were identified and correlated with rates of the chemical and physical processes present. Based on the processing conditions, particles as low as 70 nm and narrow particle size distribution were produced. Formulations containing such particles were successfully filtered sterilized. By changing the processing conditions, 400 nm particles with also narrow particle size distribution were produced. In some experiments, siRNA was successfully incorporated in the particles. The current process was several orders of magnitude more efficient than existing processes based on the high particle concentration that are produced.