| Proteins and peptides are the most important pharmaceuticals in recent decades due to their highly specified pharmacological effects and irreplaceability in the treatment of many serious diseases. The micronization of proteins plays a key role and can lead to higher efficacy in non-invasive delivery routes like pulmonary route, where formulation design based on particulate system also brings upon different effects such as enhanced protein absorption and controlled protein release. The favorable tailoring of particle morphology and size distribution is essential for the formulation performance in different drug delivery routes. Moreover, as proteins are biomacromolecules that are extremely sensitive to processing, the demands lay on micronization techniques for proteins are very rigorous.Supercritical assisted atomization with an enhanced mixer (SAA-HCM) process is a green micronization technique which makes use of the high expansion power of supercritical CO2 (SCCO2) to obtain fine particles, and a hydrodynamic cavitation mixer is introduced to intensify the mixng between SCCO2 and liquid solution. This process can be operated not only on organic solvents but also aqueous solutions, with mild conditions and advantageous particle size control. Therefore, SAA-HCM process will be very promising in protein micronization. So far, the development of SAA-HCM technique is still in its initial stage and most of the existing work focuse on small molecular pharmaceuticals and polymers. There are few studies on protein micronization using SAA-HCM process and the application of this novel process in protein/polymer composite particles is still unavailable. In this work, the application of SAA-HCM was successfully expanded to protein particulate systems including model protein, therapeutic protein and protein/polymer composite. By proposal of protein particle formation mechanism, the study shed light to direct the protein micronization processes. The comprehensive characterization of microparticles provided large amount of analytical information about protein structure, thermal behavior and bioactivity, which would help understanding how protein integrity can be preserved during micronization. Finally, the in vivo study of protein/polymer composite micropaticles brought in the potential application of this novel process.Firstly, using lysozyme as a model protein, microparticles with well controlled morphology and size were produced from aqueous solution using SAA-HCM. The influence of pocess parameters including precipitator temperature, mixer pressure and temperature, solution concentration and CO2/solution mass ratio on particle morphology and size were studied in detail. Microparticles could be tailored to have sizes ranging in 1-5μm, which would be useful in pulmonary delivery, or in sub-micron region, which might be used in injection. Lysozyme could well retain its bioactivity to more than 85% except for being operated under severe conditions like high temperature and pressure. The chromatography, spectroscopy and thermal analysis confirmed the molecular integrity and thermal stability of lysozyme postprocessing. Comparative study of lysozyme particles produced by different techniques demonstrated the key effect of SCCO2 in the size control using SAA-HCM. Compared with particles produced by spray drying, lyzozyme microparticles from SAA-HCM exhibited much narrower size distribution and higher activity maintenance and also better stability on storage.Insulin was also successfully micronized by SAA-HCM, and effects of parameters on insulin micronization were studied. Different dissolution conditions, solution concentrations and precipitator temperatures induced microparticles having diverse types of surface morphologies as corrugated, highly folded, partly deflated and smooth spherical. A shell formation mechanism of protein was established, which could be extended to understand particle formation of other proteins. Solution concentration had a striking influence on particle size. The size of microperticles could be controlled in 0.5-5 μm through tuning oprating parameters. Insulin also retained its molecular integrity after SAA-HCM processing, and the in vivo bioactivity was confirmed in SD rats.Subsequently, insulin/chitosan composite microparticles were produced by SAA-HCM process. The composite microparticles presented wrinkled surface, and to a higher extent with increasing protein content. The particle morphology and size could be suitably tailored by tuning process parameters. Combining the spectroscopy and thermal analysis, the molecular interactions between insulin and chitosan were explored. Insulin exhibited decreased helical content upon interaction with chitosan, but refolded to native state upon reconstitution. The solid solution dispersion of insulin molecules into chitosan matrices was confirmed and the chitosan chains might have served to prevent insulin from degradation. Specifically, the spacial distribution of insulin molecules in the composite microparticles was comprehensively investigated and evidences were provided to verifying the protein shell formation mechanism. The protein loading efficiency of composite microparticles reached 93.4% and the protein release profile could be controlled.Finally, the SAA-HCM process on producing insulin particulate system was forwarded into application oriented study. Insulin/chitosan oligomers micro-composite particles were successfully produced, with good protein stability and loading efficiency as high as 87.3%. By optimizing the particle aerodynamic performance, an insulin particulate formulation was obtained with relatively high respirable fraction of 52.3%. The in vivo pharmacological efficacy and insulin absorption enhancement was confirmed through intratracheal administration of the composite microparticles into SD rat lungs, with a pharmacological bioavailability of 48.9%.In summary, SAA-HCM process was successfully applied to produce protein based microparticulate systems with favorable control of particle morphology and size without usage of organic solvents, while protein molecular integrity and bioactivity were guaranted. The produced polymer/protein composite formulations could enhance drug absoption and presented many advantages in non-invasive drug delivery routes, especially in pulmonary route. SAA-HCH was demonstrated to be great potential in developing protein drug delivery systems. |
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