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Encapsulation of GFP in complex coacervate core micelles Antsje Nolles, Adrie H. Westphal, Jacob A. de Hoop, Remco G. Fokkink, J. Mieke Kleijn, Willem J.H. van Berkel, and Jan Willem Borst Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00092 • Publication Date (Web): 09 Apr 2015 Downloaded from http://pubs.acs.org on April 13, 2015
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Biomacromolecules
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Encapsulation of GFP in complex coacervate core micelles
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Antsje Nolles1,2,3, Adrie H. Westphal1,3, Jacob A. de Hoop1, Remco G. Fokkink2, J. Mieke Kleijn2,
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Willem J. H. van Berkel1, Jan Willem Borst1,3
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5 6 7 8
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Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands
Physical Chemistry and Soft Matter, Wageningen University, Wageningen, The Netherlands 3
MicroSpectroscopy Centre Wageningen, Wageningen University, Wageningen, The Netherlands
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Abstract
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Protein encapsulation with polymers has a high potential for drug delivery, enzyme protection
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and stabilization. Formation of such structures can be achieved by the use of polyelectrolytes to
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generate so-called complex coacervate core micelles (C3Ms). Here, encapsulation of enhanced
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green fluorescent protein (EGFP) was investigated using a cationic-neutral diblock copolymer of
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two different sizes: poly(2-methyl-vinyl-pyridinium)41-b-poly(ethylene-oxide)205 and poly(2-
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methyl-vinyl-pyridinium)128-b-poly(ethylene-oxide)477.
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fluorescence correlation spectroscopy (FCS) revealed a preferred micellar composition (PMC)
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with a positive charge composition of 0.65 for both diblock copolymers and micellar
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hydrodynamic radii of approximately 34 nm. FCS data show that at the PMC, C3Ms are formed
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above 100 nM EGFP, independent of polymer length. Mixtures of EGFP and non-fluorescent
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GFP were used to quantify the amount of GFP molecules per C3M, resulting in approximately
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450 GFPs encapsulated per micelle. This study shows that FCS can be successfully applied for
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the characterization of protein-containing C3Ms.
Dynamic
light
scattering
and
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Keywords (4-6 words)
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C3M, dynamic light scattering, fluorescence correlation spectroscopy, green fluorescent protein,
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polyelectrolyte, protein encapsulation.
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Introduction
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Micellar protein-polyelectrolyte complexes are unique soluble structures that can be used for
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biomedical and biological applications.1 Such complexes, further defined as complex coacervate
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core micelles (C3Ms), have great potential to protect, stabilize, enhance biological activity, and
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control delivery of the encapsulated protein.2-4 C3Ms are co-assemblies of a charged-neutral
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diblock copolymer with a countercharged polyelectrolyte or protein. A core-shell structure is
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formed in aqueous solutions of which the shell consists of neutral soluble chains. The formation
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is electrostatically driven and therefore by default responsive to ionic strength, and in the case of
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weak polyelectrolytes and proteins, also to pH.5
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Investigation of protein-polyelectrolyte complexes started already in the 1950s with bovine
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serum albumin (BSA).6 Morawetz and coworkers6 successfully separated BSA from
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oxyhemoglobin by incubation with polymethacrylic acid, thereby keeping BSA in its native state
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after complexation. Research on protein encapsulation using C3Ms has been performed mainly
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on BSA and lysozyme using dynamic light scattering (DLS).7, 8 The overall aim of our work is to
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determine the potential of polyelectrolyte micellar complexes as packaging and delivery systems
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for proteins in food and pharmaceutical applications. In this study, we use enhanced green
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fluorescent protein (EGFP), fluorescence correlation spectroscopy (FCS) and DLS to
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characterize in detail protein-loaded structures and the processes of their formation and
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disintegration.
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EGFP is widely applied in biological studies, because of its high stability and unique
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fluorescence properties.9-11 EGFP has been previously used for encapsulation in AOT reversed
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micelles as pH-indicator12 and in virus particles as an inclusion molecule13.
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DLS can be used to determine the size distribution profile of micelles,14 but does not provide
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direct information on the number of protein molecules that has been incorporated.15 FCS is a
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selective and sensitive technique that can measure single molecules; it can be used to measure
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diluted samples up to the nanomolar range and to investigate diffusion-controlled processes in
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biological and colloidal systems on the microsecond to millisecond time scale.16-20 FCS has
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already been used to determine micelle diffusion coefficients,21,
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encapsulated dye molecules23 and the critical micelle concentration (CMC) of micellar systems.24
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In this study, EGFP (27 kDa, pI = 5.6) has been encapsulated at pH 9 using the polymer
9
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the relative amount of
poly(2-methyl-vinyl-pyridinium)n-b-poly(ethylene-oxide)m (P2MVPn-b-PEOm)25,
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of two
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different sizes: P2MVP41-b-PEO205 and P2MVP128-b-PEO477. P2MVPn-b-PEOm is a diblock
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copolymer consisting of a cationic block and a neutral water-soluble block (Figure 1). Using
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DLS and FCS, the preferred micellar composition (PMC) was determined, i.e., the solution
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composition at which the maximum number of micelles are formed, which is dependent on the
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amount of charge on the polymer relative to the total amount of charged groups of polymer and
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protein. Moreover, FCS allowed determining the critical micelle concentration (CMC) and
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quantifying the amount of GFP molecules per C3M.
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Figure 1. Schematic representation of a complex coacervate core micelle (C3M) formed from
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EGFP and poly(2-methyl-vinyl-pyiridinium)41-b-poly(ethylene-oxide)205.
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Experimental section
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Materials
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Complex coacervate core micelles were prepared in aqueous solution from enhanced green
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fluorescent protein (EGFP), which is an ampholyte with pH-dependent charges, and poly(2-
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methyl-vinyl-pyridinium)-b-poly(ethylene-oxide) (P2MVP-b-PEO), a cationic-neutral diblock
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copolymer with pH-independent charges. Two polymers with different chain lengths were used.
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P2VP41-b-PEO205 (Polymer Source Inc., Canada, Mw/Mn = 1.05, Mn = 13.3 kg/mol) and P2VP128-
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b-PEO477 (Polymer Source Inc., Canada, Mw/Mn = 1.10, Mn = 34.5 kg/mol) were quarternized
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with iodomethane (99%, Sigma-Aldrich), according to a procedure described elsewhere.25 For
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P2MVP41-b-PEO205 (Mn = 19.1 kg/mol) and for P2MVP128-b-PEO477 (Mn = 50.7 kg/mol) a final
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degree of quarternization (DQ) of approximately 80% and 87% was obtained, respectively. All
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other chemicals were from commercial sources and of the purest grade available.
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Protein production
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EGFP (Mw = 26.9 kDa) was cloned into the pTYB11 vector (New England Biolabs, Impact
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vector system27-29) and expressed in Escherichia coli BL21 cells. Induction of EGFP expression
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was performed at 20˚C for correct chromophore formation. The purification of EGFP was
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performed following the protocol in the manual of New England Biolabs, Impact vector
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system.29 On column cleavage of EGFP from the chitin binding domain was performed by
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overnight incubation of the bound fusion protein in 50 mM ß-mercaptoethanol and 50 mM
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dithiothreitol in 25 mM HEPES buffer (25 mM HEPES, 2 M NaCl, pH 8.5). The eluted protein
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sample was collected and the buffer was exchanged into 10 mM boric acid (pH 9.0) using an Page 6 of 34
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ultrafiltration unit (Amicon) with a 10-kDa cut-off PES-membrane (Millipore). Aliquots of 32
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µM EGFP were stored at -80˚C.
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Non-fluorescent EGFP (henceforth denoted as darkGFP, Mw = 26.8 kDa) was obtained by
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mutating the three amino acids TYG, forming the chromophore in EGFP, into GGG.30, 31 The
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primer used to change the DNA sequence coding for the corresponding amino acids was 5’-GCA
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CTG CAC GCC GCC TCC CAG GGT GGT C, and used in a PCR strategy for site-directed
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mutagenesis.32 DarkGFP was produced, purified and stored (30 µM aliquots) as described for
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EGFP. Protein concentrations were determined using BCA protein assay (PierceTM) or using an
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absorption measurement at 488 nm for EGFP (ε = 56 mM-1 cm-1).
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Sample preparation
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Stock solutions of P2MVP41-b-PEO205 (51 µM) and P2MVP128-b-PEO477 (50 µM) were
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prepared by dissolving the polymers in 10 mM boric acid (pH 9.0). All solutions were filtered
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through 0.20 µm polyethersulfone membrane syringe filters (Advanced Microdevices Pvt. Ltd.).
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Encapsulation of EGFP with polymers was achieved by first diluting the EGFP stock solution (or
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mixtures of EGFP and darkGFP) in 10 mM boric acid (pH 9.0) to the desired concentration
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followed by addition of the polymer. After mixing, samples were stored at room temperature for
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24 h before measurements. Angle-dependent static light scattering (SLS) experiments to
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determine the structure factor of the C3Ms, indicated that the micelles are spherical objects. For
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each sample, DLS measurements involved collection of 10 light scattering intensity fluctuation
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traces and in FCS measurements 5 fluorescence intensity fluctuation traces of 30 s each were
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collected.
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Dynamic light scattering
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Dynamic light scattering (DLS) measurements were performed on an ALV instrument
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equipped with a 22 mW Uniphase Model 1145P HeNe-laser operating at 632.8 nm, and an
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ALV/Dual High QE APD Detector Unit with a fibre splitting device for 2 detectors connected to
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an external ALV7004 Multiple Tau Digital Correlator (ALV-Laser Vertriebsgesellschaft m-b.H.,
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Germany). The detection angle was set at 90˚.
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The intensity autocorrelation functions
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=
〈 × 〉 〈 〉
(1)
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were analyzed using the inverse Laplace transformation.33 In the above equation, I is the
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intensity of the scattered light. For non-interacting monodisperse spherical particles, the field
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autocorrelation function is an exponentially decaying function dependent on the
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correlation decay rate Γ, diffusion coefficient of the particles and the wave vector :
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, = , Γ = and =
sin
(2)
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Here, λ0 is the used wavelength, n is the refractive index of the medium and θ is the detection
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angle. In the case of a polydisperse system, the overall decay of the function can be
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written as a weight average of all possible decays:
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= lim→$ ∑'* &' Γ( )
(3)
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Biomacromolecules
where &' Γ' is a weighting function determined by the size and the amount of particles in size
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range +. The hydrodynamic radii of the scattering particles, Rh, were calculated using the Stokes-
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Einstein relation for spherical particles:
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./
,- =
012
(4)
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where k is the Boltzmann constant, T is the absolute temperature and η is the viscosity of the
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solvent. The polydispersity of the mixtures can be calculated or a polydispersity index of a peak
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can be determined. The latter is done in this paper according to the relation:
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width
PDI= 3radius4
(4)
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where the width is at half height of the peak of interest in the particle size distribution and the
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radius is at the maximum of that same peak. Results from DLS composition experiments are
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plotted as a function of the composition 5 , defined by
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5 = 6
67 8
7 8 69 8
(5)
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where 6: 8 = ; < and refers to the total concentration of positively charged groups on the
polymers and 6: 8 = ;
