Encapsulation of enzymes using polyelectrolytes (MSc Riahna Kembaren)

Mixing a protein with a polymer containing a charged block and a neutral hydrophilic block (diblock copolymer) with or without an oppositely charged homopolymer leads to encapsulation of the protein in so-called complex coacervate core micelles (Figure 1)[1]. This encapsulation can e.g. protect the protein against degradation, stabilize the protein, and release the protein upon a trigger. Next to that, it has been shown that enzymes like lysozyme, lipase, and LysK present in such polyelectrolyte complex micelles are more active compared to enzymes free in solution [1e], [2]. Encapsulation of proteins, therefore, can be beneficial in the field of diagnostics, therapeutics, industry, and food technology. 

Dynamic light scattering (DLS) is commonly used to follow the formation of the micelles and determination of their size and size distribution. Next to DLS, fluorescence correlation spectroscopy (FCS) can be used to characterize the micelles. With FCS it is possible to determine the amount of (fluorescently labeled) protein molecules in one micelle.

Proteins are ampholytes, implying that their charge is dependent on pH. Most enzymes are negatively charged at neutral pH. It may be feasible, therefore, to develop a robust micellar system to encapsulate negatively charged enzymes. Cationic-neutral diblock copolymers, in particular, are of interest to be tested in this project for the packaging of enzymes of different molecular size. In the beginning of this study enhanced green fluorescent protein (EGFP, 28 kDa, pI = 5.8) is used; it will be extended to redox proteins and enzymes (e.g. flavodoxin and alditol oxidase). Different sizes of the cationic-neutral diblock copolymer poly(2-methyl vinyl pyridinium)-b-poly(ethylene oxide) (P2MVP-b-PEO) will be tested for their packaging ability (at PCC). In addition, the stability of the micelles and the activity of enzymes in the micelles and after release will be investigated (at BIC). There is also an idea to try to encapsulate proteins/enzymes with self-assembling protein structures [3].

References

1. (a) Lindhoud, S., Polyelectrolyte complex micelles as wrapping for enzymes. Wageningen Universiteit (Wageningen University): 2009;(b) Harada, A.; Kataoka, K., Novel Polyion Complex Micelles Entrapping Enzyme Molecules in the Core. 2. Characterization of the Micelles Prepared at Nonstoichiometric Mixing Ratios †. In Langmuir, 1999; Vol. 15, pp 4208-4212;(c) Klyachko, N. L.; Manickam, D. S.; Brynskikh, A. M.; Uglanova, S. V.; Li, S.; Higginbotham, S. M.; Bronich, T. K.; Batrakova, E. V.; Kabanov, A. V., Cross-linked antioxidant nanozymes for improved delivery to CNS. Nanomed-Nanotechnol 2012, 8 (1), 119-129;(d) Batrakova, E. V.; Li, S.; Reynolds, A. D.; Mosley, R. L.; Bronich, T. K.; Kabanov, A. V.; Gendelman, H. E., A macrophage-nanozyme delivery system for Parkinson's disease. Bioconjugate Chemistry 2007, 18 (5), 1498-1506;(e) Filatova, L. Y.; Donovan, D. M.; Becker, S. C.; Lebedev, D. N.; Priyma, A. D.; Koudriachova, H. V.; Kabanov, A. V.; Klyachko, N. L., Physicochemical characterization of the staphylolytic LysK enzyme in complexes with polycationic polymers as a potent antimicrobial. Biochimie 2013, 95 (9), 1689-1696.

2. (a) Lindhoud, S.; Norde, W.; Cohen Stuart, M. A., Effects of Polyelectrolyte Complex Micelles and Their Components on the Enzymatic Activity of Lipase. In Langmuir, 2010; Vol. 26, pp 9802-9808; (b) Harada, A.; Kataoka, K., Pronounced activity of enzymes through the incorporation into the core of polyion complex micelles made from charged block copolymers. In Journal of Controlled Release, 2001; Vol. 72, pp 85-91.

3. Hernandez-Garcia, A.; Werten, M. W. T.; Stuart, M. C.; de Wolf, F. A.; de Vries, R., Coating of Single DNA Molecules by Genetically Engineered Protein Diblock Copolymers. Small 2012, 8 (22), 3491-3501.