Enzyme nanocontainers - Coacervate protein carriers with application-dependent stability and delivery properties

Introduction

Encapsulation of proteins is important in many applications, such as controlled delivery of functional ingredients in foods and medical formulations, enzymes used in industrial processes, and storage of proteins. The design of appropriate protein carriers therefore requires detailed understanding of the dynamics and stability under different (industrial, physiological or gastrointestinal) conditions, release mechanisms, and the (bio)activity and bioavailability of the encapsulated product.

Encapsulation of proteins and enzymes can be realized by generating micelles composed of polyelectrolyte complexes (complex coacervate core micelles, C3Ms). This approach has many advantages, but practical application is hampered by the low stability and high exchange dynamics of protein-containing C3Ms, which is attributed to the generally low charge density of protein molecules.

C3Ms are polyelectrolyte complexes that are colloidally stabilized by neutral hydrophilic polymer chains. Here, the formation of a C3M from green fluorescent protein (negatively charged) and the positive-neutral block-copolymer P2MVP-b-PEO is shown. This micelle has a radius of 35 nm and contains about 450 protein molecules. From Nolles et al.
C3Ms are polyelectrolyte complexes that are colloidally stabilized by neutral hydrophilic polymer chains. Here, the formation of a C3M from green fluorescent protein (negatively charged) and the positive-neutral block-copolymer P2MVP-b-PEO is shown. This micelle has a radius of 35 nm and contains about 450 protein molecules. From Nolles et al.

In this project, we will take concrete steps towards application of C3Ms as protein carriers and delivery systems. We will use biocompatible block copolymers for encapsulation of two model proteins, i.e. the enzymes proline dehydrogenase and laccase, which are representative for a broad spectrum of enzyme types. To enhance and tune the stability of the protein-polymer complexes, we will make use of state-of-the-art bio-conjugation chemistry tools to provide the enzymes with additional charged polypeptide sequences of variable length. The encapsulation efficiency, formation and decomposition kinetics, exchange dynamics with relevant media (e.g., physiological solutions) and the effect of packaging on the structure and activity of the enzymes will be determined. In addition, we will quantify the strength of the electrostatic interactions responsible for the stability of the micelles for different media conditions. Finally, we will explore a number of release mechanisms. On the basis of the obtained results, it will be possible to design C3M protein carriers of which the stability and dynamics are tuned to specific applications.

We will develop C3Ms to a practically applicable protein carrier system with tuneable stability and delivery properties. For this, we need to tackle a number of scientific challenges: how to obtain high encapsulation efficiencies combined with low exchange rates between encapsulated and free protein molecules, while maintaining biofunctionality. In addition, for controlled delivery purposes suitable release mechanisms are needed. To ensure the broad applicability, we utilize two enzymes that have different biochemical characteristics, i.e. proline dehydrogenase and laccase.

Approach

We will use the following biophysical methods:

  • Formation and decomposition kinetics of C3Ms, their stability, enzyme encapsulation efficiency and exchange dynamics in relevant media (e.g., physiological solutions) will be measured using dynamic light scattering (DLS), fluorescence correlation spectroscopy (FCS) and different spectroscopic analyses for the observation of Förster resonance energy transfer (FRET). For fluorescence measurements, enzymes will be labelled with appropriate fluorescent dyes. Initially, we will label N-termini, lysine or cysteine residues using standard protocols.
  • The effect of packaging on the structure of the enzymes will be measured by circular dichroism (CD).
  • To quantify the strength of the interactions between the conjugated polypeptide chains and the charged blocks of the copolymers as a function of chain length, charge density and salt concentration, we will perform atomic force microscopy (AFM) measurements. This will provide information on the tuneability of the relevant C3M properties.