FRET Reveals the Formation and Exchange Dynamics of Protein-Containing Complex Coacervate Core Micelles.

The encapsulation of proteins into complex coacervate core micelles (C3Ms) is of potential interest for a wide range of applications. To address the stability and dynamic properties of these polyelectrolyte complexes, combinations of cyan, yellow, and blue fluorescent proteins were encapsulated with cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)128- b-poly(ethylene-oxide)477. Förster resonance energy transfer (FRET) allowed us to determine the kinetics of C3M formation and of protein exchange between C3Ms. Both processes follow first-order kinetics with relaxation times of ±100 s at low ionic strength ( I = 2.5 mM). Stability studies revealed that 50% of FRET was lost at I = 20 mM, pointing to the disintegration of the C3Ms. On the basis of experimental and theoretical considerations, we propose that C3Ms relax to their final state by association and dissociation of near-neutral soluble protein-polymer complexes. To obtain protein-containing C3Ms suitable for applications, it is necessary to improve the rigidity and salt stability of these complexes.

Colorful Packages: Encapsulation of Fluorescent Proteins in Complex Coacervate Core Micelles.

Encapsulation of proteins can be beneficial for food and biomedical applications. To study their biophysical properties in complex coacervate core micelles (C3Ms), we previously encapsulated enhanced green fluorescent protein (EGFP) and its monomeric variant, mEGFP, with the cationic-neutral diblock copolymer poly(2-methyl-vinyl-pyridinium)n-b-poly(ethylene-oxide)m (P2MVPn-b-PEOm) as enveloping material. C3Ms with high packaging densities of fluorescent proteins (FPs) were obtained, resulting in a restricted orientational freedom of the protein molecules, influencing their structural and spectral properties. To address the generality of this behavior, we encapsulated seven FPs with P2MVP41-b-PEO205 and P2MVP128-b-PEO477. Dynamic light scattering and fluorescence correlation spectroscopy showed lower encapsulation efficiencies for members of the Anthozoa class (anFPs) than for Hydrozoa FPs derived from Aequorea victoria (avFPs). Far-UV CD spectra of the free FPs showed remarkable differences between avFPs and anFPs, caused by rounder barrel structures for avFPs and more elliptic ones for anFPs. These structural differences, along with the differences in charge distribution, might explain the variations in encapsulation efficiency between avFPs and anFPs. Furthermore, the avFPs remain monomeric in C3Ms with minor spectral and structural changes. In contrast, the encapsulation of anFPs gives rise to decreased quantum yields (monomeric Kusabira Orange 2 (mKO2) and Tag red fluorescent protein (TagRFP)) or to a pKa shift of the chromophore (FP variant mCherry).

Encapsulation of GFP in Complex Coacervate Core Micelles.

Protein encapsulation with polymers has a high potential for drug delivery, enzyme protection and stabilization. Formation of such structures can be achieved by the use of polyelectrolytes to generate so-called complex coacervate core micelles (C3Ms). Here, encapsulation of enhanced green fluorescent protein (EGFP) was investigated using a cationic-neutral diblock copolymer of two different sizes: poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205 and poly(2-methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477. Dynamic light scattering and fluorescence correlation spectroscopy (FCS) revealed a preferred micellar composition (PMC) with a positive charge composition of 0.65 for both diblock copolymers and micellar hydrodynamic radii of approximately 34 nm. FCS data show that at the PMC, C3Ms are formed above 100 nM EGFP, independent of polymer length. Mixtures of EGFP and nonfluorescent GFP were used to quantify the amount of GFP molecules per C3M, resulting in approximately 450 GFPs encapsulated per micelle. This study shows that FCS can be successfully applied for the characterization of protein-containing C3Ms.

Revealing heterogeneity in correlation times of EGFP encapsulated in complex coacervate core micelles by analysis of fluorescence anisotropies.

Encapsulation of enhanced green fluorescent protein (EGFP) in complex coacervate core micelles (C3Ms) can be established by mixing EGFP with diblock polymers at equal charge ratio. It has previously been shown that this encapsulation system is highly dynamic, implying existence of different populations; GFP free in solution or complexed with polymers (small complexes) and EGFP encapsulated in C3Ms. We performed time resolved fluorescence anisotropy experiments to determine the relative populations of EGFP encapsulated in C3Ms using three different fluorescence anisotropy decay analysis methods. First, Maximum Entropy Method (MEM) data analysis was employed for five different EGFP concentrations in C3Ms that were mixed with dark fluorescent proteins (10, 20, 30, 40 and 50% EGFP, respectively). In all cases, correlation-time distributions between 0.1 and 100 ns (on a logarithmic timescale) are clearly visible showing bimodal distribution. The distribution between 0.1 and 2.0 ns is due to homo-FRET between EGFP molecules packed in micelles and the distribution between 8 and 30 ns coincides with the correlation-time distribution of free EGFP in solution. The fraction of homo-FRET distribution linearly increases with increase of relative micellar EGFP concentrations. These MEM results were corroborated by two different analysis methods: global population analysis of all five fluorescence anisotropy decays arising from EGFP in micelles together with the one of free EGFP (direct analysis of anisotropies) and global associative population analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components. In contrast to global analyses approaches, the MEM method directly reveals distributions of correlation times without any prior information about the sample. However, global associative analysis of anisotropies by fitting parallel and perpendicular fluorescence decay components is the only method that allows to estimate accurately fractions of free fluorophores in solution and encapsulated fluorophores.