Nanosized crosslinked protein aggregates (nano-CLPA)
Zakharyuta, Anastasia (2015) Nanosized crosslinked protein aggregates (nano-CLPA). [Thesis]
Kinetic limitations associated with heterogeneous phase reaction for micron size catalyst formulations so far have been addressed by bottom-up approach, which while successful through a number of methods tends to involve laborious procedures, high production costs and is restricted to a limited number of proteins. This study presents the top-down approach, based on physical downsizing of conveniently fabricated crosslinked protein aggregates (CLPA) or herein developed crosslinked protein lyophilizates (CLPL). This method, while retaining both process convenience/generality and stability advantages associated with crosslinking of CLPA leads to nano-particle formulations of adjustable size, applicable on a wide range of proteins, independent of their purity grade. Crosslinked protein micron-sized aggregates were prepared by crash precipitation of soluble proteins, alternatively lyophilizates were prepared. The solid proteins were chemically or dehydrothermally crosslinked, forming insoluble powders. The crosslinked precursors were then nanonized, realized through application of mechanical or hydrodynamic shear, facilitated by an optimal medium. Herein, conventional CLPA synthesis was optimized as to facilitate subsequent downsizing. Alternative CLPA/CLPL formulations were also developed, addressing challenges posed by particular protein types, addressing suboptimal overall synthesis yields and/or catalytic activity. Various parameters, such as precursor crosslinked material properties, shear rate and time and downsizing medium composition were employed in nanonization procedure optimization. Catalytically active nano-particles on the range of 100-900 nm were generated. The nanonized aggregates described herein serve to highlight a number of potential advantages in industrial, analytical and biomedical fields. A preliminary in vitro study showed succesful internalization of nanosized CLPAs in different mammalian cell cultures in the formulation dependant manner, raising hopes for novel systemic and local proteinbased therapeutics.
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