PhD Dissertation Defense-Anastasia Zakharyuta
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NANOSIZED CROSSLINKED PROTEIN AGGREGATES (NANO-CLPA)

 

Anastasia Zakharyuta
Molecular Biology, Genetics And Bioengineering PhD Dissertation, 2015

 

Thesis Jury

Assit. Prof. Dr Alpay Taralp (Thesis Advisor), Assoc. Prof. Dr Batu Erman,

Assit. Prof. Dr Deniz Sezer, Assoc. Prof. Dr Uğur Sezerman, Assist.Prof. Dr Burcu Kaplan

 

 

Date & Time: 5th, 2015 –  12:45 PM

Place: FENS 1040

Keywords : crosslinked protein aggregates, heterogeneous phase catalyst, nanoparticle 

 

Abstract

 

Limitations arising from 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 laborous 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 lyohpilizates 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 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. 

Nano-CLPA/CLPL described herein, aim to extend the number and efficiency of applications in industrial, analytical and biomedical fields. The preliminary in vitro study is included, addressing potential biomedical applications, raising the discussion of systemic or local theraupeutics.