Encapsulation of enzymes using polyelectrolytes: Nanopackaging of enzymes for food-pharma applications

For administration to the human body the encapsulated proteins have to fulfill several requirements:

They should be stable

• at physiological salt
• over a certain pH range
• upon dilution in the gastro-intestinal (GI) tract or in the blood

and there should be a mechanism for release of the enzyme at the desired place

and so on…

Proteins in general are polyampholytes that have a net positive or negative charge at pH-values other than their iso-electric point. Due to this characteristic they can form complexes with oppositely charged polymers, which are called polyelectrolytes. These complexes will remain relatively small and stable in solution if the polyelectrolytes are part of a diblock copolymer, of which the second block is a neutral, water-soluble chain. This is shown schematically in the figure below.

When enzymes are encapsulated in this way, they form so called complex coacervate core micelles (C3Ms). A C3M is defined as a core-shell structure formed in aqueous solution that is stabilized by its shell, consisting of neutral water-soluble units, that surrounds its core that consists of complexed oppositely charged units. In literature other terms for these particles are also used; block ionomer complexes (BIC), polyion complex micelles (PIC micelles), and (inter)polyelectrolyte complexes ((I)PEC) (1).

The particles, or C3Ms, which have been obtained by mixing of the enzyme with the diblock copolymer, can be investigated with several techniques. The main techniques that will be used are dynamic light scattering (DLS) and fluorescence correlation spectroscopy (FCS). With DLS the particle size distribution can be determined, due to the diffusion of the particles. The diffusion and radius of the particles are related to each other according to the equation:

In which D is the diffusion coefficient, kB Boltzmann’s constant, T the absolute temperature, n the viscosity of the surrounding solution and RH the hydrodynamic radius of the particles. Furthermore, the stability of the particles upon salt addition and pH can be determined by DLS.

With FCS the particle sizes can also be determined, according to the same principle (measurement of diffusion rates). Next to that, the efficiency of the encapsulation can be determined when the enzymes have a fluorescent label or are fluorescent themselves.

In this project we will explore the nanopackaging of carbohydrate modifying flavoenzymes. Different polymers will be used for the encapsulation of enzymes. The size and polydispersity of the micelles will be analyzed with Dynamic Light Scattering (DLS). Enzymes will be labeled with a fluorescent tag and the efficiency of encapsulation will be determined with Fluorescence Correlation Spectroscopy (FCS). This project is a collaboration between the Laboratory of Physical Chemistry and Colloid Science (PCC) and the Laboratory of Biochemistry (BIC).

BSc and MSc student projects can focus on the use of different polyelectrolytes (Physical Chemistry) or on the effect of a fluorescent label on the encapsulation of enzymes (Biochemistry).

1. Ilja Voets, ‘Opposites attract?! – On the electrostatically driven co-assembly of polymers in aqueous solution’