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Complex Formation Between Lysozyme and Stabilized Micelles with a Mixed Poly(ethylene oxide)/Poly(acrylic acid) Shell Maria Karayianni,*,†,‡ Valeria Gancheva,‡ Stergios Pispas,† and Petar Petrov*,‡ †

Theoretical and Physical Chemistry Institute, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, 11635 Athens, Greece ‡ Institute of Polymers, Bulgarian Academy of Sciences, Akad. G. Bonchev Str., block 103-A, BG-1113 Sofia, Bulgaria S Supporting Information *

ABSTRACT: The electrostatic complexation between lysozyme and stabilized polymeric micelles (SPMs) with a poly(acrylic acid) (PAA) or a mixed poly(ethylene oxide)/poly(acrylic acid) (PEO/PAA) shell (SPMs with a mixed shell, SPMMS) and a temperature-responsive poly(propylene oxide) (PPO) core was investigated by means of dynamic, static, and electrophoretic light scattering. The SPMs and different types of SPMMS used resulted from the self-assembly of PAA−PPO−PAA triblock copolymer chains, or PAA−PPO−PAA and PEO−PPO−PEO triblock copolymer chain mixtures (with varying chain lengths and molar ratios) in aqueous solutions at pH 10 and the subsequent cross-linking of their PPO cores via loading and photo-cross-linking of pentaerythritol tetraacrylate (PETA). The solution behavior, structure and properties of the formed complexes at pH 7 and 0.01 M ionic strength, were studied as a function of the protein concentration in the solution (the concentration of the stabilized micelles was kept constant) or equivalently the ratio of the two components. The complexation process and properties of the complexes proved to be dependent on the protein concentration, while of particular interest was the effect of the structure of the shell of the SPMs on the stability/solubility of the complexes. Finally, the fluorescence and mid infrared spectroscopic investigation of the structure of the complexed protein showed that, although a small stretching of the protein molecules occurred in some cases, no protein denaturation takes place upon complexation. micelles.10 Another interesting case is that of PEO−PPO−PEO (Pluronic) triblock copolymer micelles, which were effectively stabilized through the formation of an interpenetrating network of the core-forming chains. This process was achieved either by developing a thermoresponsive hydrogel with the aid of poly(N-isopropylacrylamide) and poly(N,N-diethylacrylamide) polymers11,12 or by the UV-induced polymerization of the hydrophobic pentaerythritol tetraacrylate.13,14 In the latter case the resulting stabilized polymeric micelles resisted changes in concentration and solvent quality, while they maintained their original structure and size even when ultrasound irradiated at 20 kHz. The introduction of a third block, either in the form of ABC triblock copolymers or AB and BC diblock copolymers mixtures, provides intriguing new functionalities and higher diversity of micellar organizations. Depending on the solubility of the blocks micelles with a compartmentalized core (two insoluble blocks) or a compartmentalized corona (one insoluble block) can be produced. In the second case, if the insoluble block is located at one extremity of the triblock (A or C block) “core-shell-corona” micelles with a two-layer corona are assembled, while when the insoluble block is located

1. INTRODUCTION The self-assembly process of amphiphilic block copolymers in water has been at the focus of extensive scientific interest over the last decades, mainly due to the numerous potential applications involving medicine, biotechnology, catalysis, nanotechnology, etc.1−3 The most widely studied cases are that of AB- and ABA-type amphiphilic block copolymers, which above the critical micelle concentration (cmc) form core−shell micelles. In such systems polymeric micelles and molecularly dissolved copolymer chains are in a dynamic equilibrium according to the closed association model. Therefore, micelles might dissociate spontaneously upon changes in concentration, temperature, pH, etc. It is thus advantageous to stabilize the micelles when they have to be studied and exploited with the same morphology (aggregation number and shape) under different conditions. The chemical cross-linking of either their core or their shell is an effective strategy in order to lock the originally obtained micellar structure. Representative examples of covalently stabilized micellar structures include the cross-linking of a polybutadiene core reported by Procházka et al.,4,5 or the photo-cross-linking of a poly(cinnamoylethyl methacrylate) core as shown by Liu and co-workers.6,7 Cross-linking of the corona rather than the core of micelles was first examined by Wooley et al. in the specific case of a poly(acrylic acid) corona.8,9 In a similar manner Armes and co-workers synthesized shell cross-linked onion-like © XXXX American Chemical Society

Received: January 18, 2016 Revised: February 16, 2016

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DOI: 10.1021/acs.jpcb.6b00550 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

2. EXPERIMENTAL SECTION 2.1. Materials. Protein. HEWL (dialyzed, lyophilized powder) with a molecular weight of Mr = 14 700 g mol−1 was purchased from Fluka and used without any further purification. Synthesis of the Stabilized Polymeric Micelles. The synthesis of the stabilized polymeric micelles used in this study has been reported elsewhere17 and is described in detail in the SI section. Here we briefly review the main steps of this procedure. First, two poly(tert-butyl acrylate)−poly(propylene oxide)−poly(tert-butyl acrylate), PtBA20−PPO34−PtBA20 and PtBA40−PPO34−PtBA40, triblock copolymers were synthesized by ATRP of tBA initiated by a PPO macroinitiator in acetone. Subsequently, the triblock copolymers were purified by precipitation and elution through a silica gel column in order to remove traces of Cu catalyst. PtBA−PPO−PtBA copolymers were derivatized into PAA−PPO−PAA triblocks by hydrolysis of the outer PtBA blocks with 5 equiv of trifluoroacetic acid with respect to the tBA units. 1H NMR and size exclusion chromatography (SEC) were used for the characterization of the obtained PAA20−PPO34−PAA20 and PAA40−PPO34−PAA40 copolymers (see SI section). Polymeric micelles with a temperature-responsive PPO core and a PAA shell were produced through the self-assembly of PAA40−PPO34−PAA40 triblock copolymer, while those with a mixed PEO/PAA shell were prepared by mixing well-defined amounts of a PEO26−PPO40−PEO26 (Pluronic P85) and the two PAA−PPO−PAA triblock copolymers, in aqueous media with pH 10 (by adding NaOH) in order to ionize the PAA blocks and thus increase their hydrophilicity. The Pluronic P85 triblock copolymer (kindly donated by BASF) has an average molecular weight of 4600 g mol−1 (according to the supplier), and a monomodal and narrow molecular weight distribution.17 Further on, the resulting micellar structures were stabilized by loading the micelles with the hydrophobic acrylate PETA (20 wt % to the copolymers amount). The subsequent UVassisted free radical polymerization of this tetrafunctional monomer resulted in the formation of a tridimensional network of poly(PETA), which entrapped the polyether blocks.13,14 After cross-linking, the micelles were dialyzed against water for 14 days so as to remove the nonstabilized copolymer and the acetone. The cross-linking efficiency (yield) was gravimetrically determined as 74 ± 8%. In a previous study it was confirmed that the compositions of stabilized micelles is very close to the composition of the original copolymer mixture.17 By varying the copolymers molar ratio or PAA chain length different stabilized polymeric micelles with mixed shells were prepared, as shown in Table 1. Preparation of the Complexes. In the first place, a 0.01 M phosphate buffer solution with pH 7 and 200 ppm of NaN3 (in order to avoid bacterial growth) was prepared and used as the

between the two soluble ones (B block) core−shell micelles with a mixed shell are formed (this is also the case for AB and BC mixtures).15 Furthermore, the specific physical and chemical properties of the blocks broaden the scope of potential activities and related applications. Specifically, when one of the soluble blocks is a charged polymer the resulting micelles have a polyelectrolyte shell, which renders them capable of binding oppositely charged ions, organic molecules, metal nanoparticles, surfactants or synthetic and biological macromolecules through electrostatic interactions.16 For instance, spherical and wormlike micelles with a mixed PEO/ polyelectrolyte shell and a stabilized temperature-responsive PPO core, were effectively used for the template-assisted preparation of Ag nanoparticles.17 One particularly interesting category of macromolecular electrostatic interactions concerns protein/polyelectrolyte complexation.18,19 Such systems have been utilized in a vast variety of technological applications involving protein encapsulation, immobilization, purification and separation, as well as in the development of functional nanobiomaterials and bioorganic hybrids with potential use in nanobiotechnology. At the same time their study provides valuable insight into the interactions between charged biomacromolecules that take place in several biological systems. Numerous parameters either concerning the solution conditions or the chemical structure of the two components influence the interaction between the two types of macromolecules and thus determine the solution behavior and structure of the resulting complexes. As far as the solution properties of the complexes are concerned, complexation can lead to the formation of soluble complexes or even cause the liquid−liquid (coacervation) or solid−liquid (precipitation) phase separation of the solution, depending on the characteristics of each system.18,19 Although a large number of experimental studies has been attributed to the electrostatic complexation between proteins and various natural or synthetic polyelectrolytes, as well as double hydrophilic block polyelectrolyte copolymers,20 the interaction between proteins and micelles with a polyelectrolyte shell/corona has been scarcely investigated.21−23 Of course one should acknowledge the studies of Ballauff and co-workers on the interaction of various proteins with spherical polyelectrolyte brushes composed of linear poly(acrylic acid) or poly(styrenesulfonate) chains chemically grafted on the surface of spherical polystyrene colloidal particles,24,25 due to the structural analogies between the colloidal and micellar polymeric nanoparticles. In the present work we investigate the complexation process between hen egg white lysozyme (HEWL), a well-studied globular protein, and the polyelectrolyte shell of stabilized polymeric micelles with a poly(acrylic acid) (PAA) nor a mixed poly(ethylene oxide)/poly(acrylic acid) (PEO/PAA) shell and a temperature-responsive poly(propylene oxide) (PPO) core. Dynamic, static, and electrophoretic light scattering (DLS, SLS, and ELS) techniques were employed in order to examine the solution behavior, structure and properties of the formed complexes at pH 7 and low ionic strength (0.01 M), as a function of the protein concentration in the solution (or equivalently the ratio of the two components). Of particular interest was the evaluation of the effect of the structure of the shell of the SPMs on the stability/solubility of the complexes. Finally, the conformation of HEWL after complexation was monitored by means of fluorescence and mid-infrared spectroscopic measurements.

Table 1. Composition of the Different Types of Stabilized Polymeric Micelles Used in This Studya code

copolymers

molar ratio

SPM-1 SPMMS-1 SPMMS-2 SPMMS-3

PAA40−PPO34−PAA40 PAA20−PPO34−PAA20:P85 PAA40−PPO34−PAA40:P85 PAA40−PPO34−PAA40:P85

1:2 1:2 1:3

a pH 10; reaction temperature = 40 °C; reaction time = 45 min (P85 = PEO26−PPO40−PEO26)

B


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temperature in the mid-infrared range (5000−550 cm−1). Before each measurement a small aliquot of the corresponding solution was placed on the ATR element and dried under N2 flow. For each sample the final spectrum is the average of three 100-scan measurements at 2 cm−1 resolution.

common solvent. Consequently, stock solutions of the stabilized polymeric micelles or the protein at the desired concentration were prepared accordingly and were left to stand overnight for better equilibration. The solutions of the complexes were prepared by appropriate mixing of the solutions of the two components, in a similar manner to analogues previous studies.23 Specifically, different amounts of a 0.5 mg/mL HEWL solution were added under stirring to the same volume of an also 0.5 mg/mL solution of the different SPMs samples. Subsequently, the mixtures were diluted by addition of appropriate volumes of the buffer solution, so as to maintain the same final volume (15 mL) and ionic strength (equal to that of the buffer solution) for all the solutions of the complexes. In this way, the concentration of the different SPMs was kept constant throughout the series of solutions, while that of HEWL varied thus changing the ratio of the two components. Moreover, the concentration of the two components was the same for all the systems involving different types of SPMs, so as to facilitate a direct comparison. After equilibration, coacervation (liquid−liquid phase separation) of the solutions of the complexes was observed at high HEWL concentration values, for all four systems under study. 2.2. Techniques. Dynamic and Static Light Scattering (DLS and SLS). Light scattering measurements were performed in the range from 20 to 150° at 25 °C on an ALV/CGS-3 compact goniometer system (ALVGmbH, Germany), equipped with an ALV-5000/EPP multi tau digital correlator, a He−Ne laser operating at the wavelength of 632.8 nm, and an avalanche photodiode detector. All solutions were filtered through 0.45 μm hydrophilic PTFE Millex syringe filters (Millipore), in order to remove any dust particles or large aggregates, and subsequently loaded into standard 1 cm width Helma quartz dust-free cells. Details about the analysis of the measurements are given in the SI section. Electrophoretic Light Scattering (ELS). A ZetaPlus Analyzer (Brookhaven Instruments) equipped with a 35 mW solid state laser, operating at λ = 660 nm was used. The obtained ζpotential values were determined using the Smolukowski equation relating the ionic mobilities with surface charge, and are reported as averages of ten repeated measurements. Transmission Electron Microscopy (TEM). TEM analysis was conducted with a JEOL 2100 electron microscope at an accelerating voltage of 200 kV, equipped with a digital camera. A drop of sample solution was deposited on a TEM copper grid coated with a carbon film, and the solvent was allowed to evaporate. Fluorescence Spectroscopy. The steady-state fluorescence spectra of the neat and complexed HEWL were recorded at room temperature with a double-grating excitation and a singlegrating emission spectrofluorometer (Fluorolog-3, model FL321, Jobin Yvon-Spex). Excitation wavelength was λ = 290 nm and emission spectra were recorded in the region 350−500 nm, with an increment of 1 nm, using an integration time of 0.5 s, while slit openings of 1 nm were used for both the excitation and the emitted beam. In this way the fluorescence from the tryptophan residues of HEWL is observed, which is directly correlated to the protein conformation. The neat SPMs solutions did not show any fluorescence. Infrared Spectroscopy (IR). A Fourier transform instrument (Bruker Equinox 55), equipped with a single bounce attenuated total reflectance (ATR) diamond accessory (SENS-IR) was used in order to obtain infrared spectra of the protein, stabilized polymeric micelles and complexes in thin film form at room

3. RESULTS AND DISCUSSION 3.1. Properties of the Stabilized Polymeric Micelles. As already mentioned, the polyelectrolyte nanoparticles investigated in this study were a series of stabilized polymeric micelles with a thermoresponsive PPO core and either a PAA shell, or a PAA and PEO mixed shell. The preparation of these micelles involved the self-assembly process of PAA−PPO−PAA triblock copolymer chains or the coassembly of PAA−PPO− PAA and PEO−PPO−PEO triblock copolymers in aqueous solutions at pH 10 and the subsequent cross-linking of their PPO cores via loading and photo-cross-linking of PETA. Four different types of SPMs with regard to their shell composition were used. Specifically, one type with a corona comprising only of PAA chains (SPM-1), one with a mixed shell of PAA and PEO chains with similar chain lengths and a 1:2 molar ratio between the two types of chains (SPMMS-1), and two with a PAA/PEO mixed shell with longer PAA chains (compared to PEO) but with an analogy of 1:2 or 1:3 of PAA to PEO chains (SPMMS-2 and SPMMS-3, respectively). The composition of the different types of micelles is given in Table 1, while Scheme 1 shows a schematic representation of their preparation procedure and structure. Scheme 1. Formation of the Different Types of Stabilized Polymeric Micelles

The solution behavior of the various SPMs in aqueous media at pH 7 and low ionic strength was investigated by means of dynamic and static light scattering measurements (DLS and SLS). The corresponding hydrodynamic radius, Rh, distributions obtained from the CONTIN analysis of DLS measurements at 90°, for representative SPM-1, SPMMS-1, SPMMS-2, and SPMMS-3 solutions of 0.1 mg/mL concentration at pH 7 and 0.01 M ionic strength are shown in Figure 1. In all cases, the distributions exhibit a main peak at Rh values in the range from 59 to 79 nm (Rh2), which denotes the presence of the SPMs. The fact that the Rh2 peaks are quite broad suggests that the micelles have a rather broad size distribution. Moreover, a second peak (Rh1) with values around 15 nm and a significantly smaller relative contribution to the total distribution is observed. This peak possibly indicates the presence of a second population of micelles (since the size is quite large for unimer triblock copolymer chains) in the solutions of the SPMs. Most probably, this population corresponds to PAA−PPO−PAA C


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Figure 1. Hydrodynamic radius, Rh, distributions from DLS measurements at 90°, for (a) SPM-1, (b) SPMMS-1, (c) SPMMS-2, and (d) SPMMS-3 solutions of 0.1 mg/mL concentration at pH 7 and 0.01 M ionic strength.

technique to the species in solution that scatter the most, the obtained results are considered to better reflect the larger population of micelles (Rh2). Furthermore, we should note that the contribution of the poly(PETA) in the Mw values of the SPMs was not taken into account since it is difficult to estimate. As a consequence, the calculated Nagg values are somewhat overestimated but still can be used for an approximate comparison between the different SPMs. Despite the overestimation, the observed quite large Nagg values are in agreement with the low hydrophilic to hydrophobic content of the triblock copolymer chains.26 Moreover, all three types of SPMs with a mixed shell (SPMMS-1, SPMMS-2, and SPMMS3) have significantly higher Nagg values (compared to SPM-1) since the introduction of the PEO chains reduces the electrostatic repulsions between the PAA chains in the shell of the micelles. This effect is even more pronounced in the case of SPMMS-1, where the PEO and PAA chains have similar lengths; hence, these micelles exhibit the highest Nagg value. As far as the morphology of the micelles is concerned, the derived ρ values are in the range of 1.02 to 1.08. These values are closer to the theoretical value of 1 predicted for hollow spheres or vesicles, that the ones reported for core−shell micelles. Core−shell micelles usually exhibit ρ values somewhat smaller than the theoretical prediction of 0.775 for hard spheres, since the density of the core is usually much higher than that of the shell. However, representative TEM images of samples SPMMS-1 and SPMMS-3, shown in Figure 2, confirm the spherical morphology of the stabilized micelles and give no indication of vesicular structures. Moreover, for all the SPMs samples the reduced translational diffusion coefficients (D = Γ/ q2) of both populations in solution exhibit no angular dependence (see the SI section), further confirming that the micelles have an isotropic spherical structure. Therefore, a possible explanation for the observed high ρ values could be that in our case the density of the shell is rather high compared

micelles with a smaller aggregation number and thus size in the case of SPM-1, while for the rest SPMMS to PEO−PPO−PEO micelles since the Pluronic copolymer chains are always in excess. This assumption is further supported by the way this population interacts with lysozyme, as we will discuss in the next section. Moreover, due to the small size of this population we assume that they are nonstabilized micelles formed by the self-assembly of either PAA−PPO−PAA or PEO−PPO−PEO chains that were not incorporated in the formation of the SPMs. Finally, both populations show a linear dependence between the relaxation rate, Γ, and q2 (see the SI section), characteristic of a diffusive behavior. Supplementary SLS measurements provide additional information about the SPMs. Table 2 lists the corresponding Table 2. Static Light Scattering (SLS) Results for SPM-1, SPMMS-1, SPMMS-2, and SPMMS-3 Solutions of 0.1 mg/ mL Concentration at pH 7 and 0.01 M Ionic Strength sample

Mw (106 g mol−1)

Nagg

Rg (nm)

Rh (nm)

ρ = Rg/Rh

SPM-1 SPMMS-1 SPMMS-2 SPMMS-3

5.1 9.1 6.5 5.7

660 1940 1150 1060

64 57 65 76

61 56 60 74

1.05 1.02 1.08 1.03

results regarding the molecular weight, Mw, the aggregation number, Nagg, (regarding the core-forming PPO chains), the radius of gyration, Rg, the (extrapolated at 0° angle) hydrodynamic radius, Rh, and the characteristic ratio ρ = Rg/ Rh. Of course, one should keep in mind that since the DLS measurements revealed the existence of two different populations of micelles in the solutions of the SPMs, the interpretation of the SLS results is not as straightforward (in the sense that they constitute weighted average values of both populations). Nevertheless, due to the higher sensitivity of this D


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of the scattering intensity, I90, which is proportional to the mass of the species in solution, increase gradually as a function of CHEWL, thus proving that complexation takes place. For CHEWL ≥ 0.07 mg/mL coacervation (liquid−liquid phase separation) of the solutions of the complexes occurs (the corresponding I90 values are from the measurement of the supernatant solution). In a similar manner, the apparent Mw of the complexes formed upon the addition of the protein (CHEWL ≈ 0.02 mg/mL) is already two times larger than that of the SPM-1 micelles, and increases further as CHEWL becomes higher (SLS measurements for the coacervated solutions were not possible since the concentration of the supernatant is unknown). Of course we should note that the derived apparent Mw values for the solutions of the complexes are a weighted average of both populations in solution, especially since the relative contribution of the smaller in size population is quite significant as shown by the Rh distributions (Figure 3d). The considerable increase of the relative contribution of the Rh1 peak and the increase of the size of both peaks (Figure 3c) indicate that both populations of stabilized micelles in the original SPM-1 solution (small nonstabilized and large stabilized PAA−PPO-PAA micelles) interact with the protein. Some amount of large complexes is still present in the supernatant of the coacervated solutions. Notably, for CHEWL ≥ 0.1 mg/mL a small peak around 1.5 nm is observed, which denotes the presence of free protein and thus suggests that the maximum degree of interaction/complexation between the micelles and the protein molecules has been reached. Finally, the negative effective charge of the initial micelles is reduced as a result of the interaction with the positively charged protein and the resulting charge neutralization, as shown by the decrease in the absolute value of the ζP (Figure 3e) (again the measurement of the coacervated solutions was not possible because of insufficient signal in the supernatant). The observed changes suggest that upon the addition of the protein to the stabilized micelles solution, the protein molecules interact electrostatically with the PAA chains in the shell of the SPMs (and the small nonstabilized micelles, respectively). This interaction leads to charge neutralization of the polyelectrolyte chains in the micellar shell and consequently reduces the solubility of the formed complexes. As a result a secondary aggregation of the complexed with protein micelles takes place. Another factor that could also contribute to the secondary aggregation of the complexes is that the protein molecules can possibly act as binding sites between PAA chains in the shell of different micelles, since protein molecules are characterized by a random distribution of oppositely charged surface patches. This assumption is mainly based on the fact that the specific SPMs are characterized by a very dense shell conformation, which hinders the incorporation and most probably leads to a peripheral distribution of the protein molecules. As the protein concentration becomes higher the secondary aggregation becomes even more pronounced, finally causing the coacervation of the complexes. Similar results were obtained for the SPMMS-1/HEWL system at pH 7 and 0.01 M ionic strength, as shown in Figure 4. The main difference in this case is that the onset of coacervation is moved to higher CHEWL values (CHEWL ≥ 0.1 mg/mL), while at the same time the secondary aggregation of the complexed with protein stabilized micelles is even more pronounced, as evidenced by the drastic increase of the normalized apparent Mw (Figure 4b). It seems that the introduction of the PEO chains in the shell of the stabilized

Figure 2. TEM images of the (a) SPMMS-1 and (b) SPMMS-3 stabilized micelles.

to that of the core due to the high aggregation number and the architecture of the triblock copolymer chains (note that the number of chains in the shell of the stabilized micelles is equal to 2Nagg). In a similar manner, Yao et al. found that micelles with a low-density core and spherical morphology exhibit ρ values around 1.1.27 Another factor that also leads to high ρ values for core−shell spherical micelles is the highly stretched conformation of the chains in the shell.26 This applies as well for the specific SPMs because of the high aggregation number and the electrostatic repulsions between the PAA chains. 3.2. Complexation of the Stabilized Polymeric Micelles with Lysozyme. The complexation process between the four different types of stabilized polymeric micelles and HEWL at pH 7 and 0.01 M ionic strength was investigated by means of dynamic, static and electrophoretic light scattering. Figure 3 shows the obtained results for the SPM-1/HEWL system regarding: (a) the values of the light scattering intensity, I90, (corrected for the concentration increase) from DLS measurements at 90°, (b) the normalized apparent molecular weight, Mw, (i.e., the ratio of the measured Mw values for the solutions of the complexes to the corresponding Mw value for the solution of the SPMs) from multiangle SLS measurements, (c) the hydrodynamic radius, Rh, values for each population in the solution (the error bars represent the percentage of each peak), along with (d) the corresponding Rh distributions from the CONTIN analysis of DLS measurements at 90°, and finally (e) the zeta potential, ζP, as a function of the protein concentration, CHEWL, in the solutions of the complexes. The concentration of SPM-1 is kept constant at 0.1 mg/mL throughout the series of solutions, while the point at zero protein concentration (CHEWL = 0) denotes the corresponding value of the net SPM-1 solution (these two observations also apply for the rest of the systems). As it can be seen, both the properties (mass, size and effective charge) and the solution behavior of the formed complexes depend on the protein concentration (or equivalently the ratio of the two components). Specifically, the values E


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Figure 3. (a) Light scattering intensity at 90°, I90, (b) normalized apparent molecular weight, Mw, from SLS measurements, (c) hydrodynamic radius, Rh, values for each population in the solution, (d) corresponding Rh distributions from the CONTIN analysis of DLS measurements at 90°, and (e) zeta potential, ζP, as a function of CHEWL for the solutions of the SPM-1/HEWL system at pH 7 and 0.01 M ionic strength.

distribution of the protein and thus favoring intermicellar binding. Eventually, as the protein concentration increases the mass of the aggregates becomes even higher, thus causing the coacervation of the solutions. At CHEWL = 0.2 mg/mL the characteristic peak of free protein (around 1.5 nm) is once again discerned in the Rh distribution of the supernatant solution (Figure 4c), indicating the maximum degree of interaction/complexation between the two components. Another factor that should be noticed is that the size and relative contribution of the Rh1 peak remain practically the same (actually the percentage of the Rh1 peak is smaller compared to

micelles enhances greatly the solubility of the formed complexes. Therefore, even though the secondary aggregates are comprised by a large number of micelles complexed with protein they remain soluble. This pronounced secondary aggregation is possibly linked to the conformation of the specific micellar shell. Since the SPMMS-1 micelles exhibit the highest Nagg number and the lengths of the PAA and PEO chains in the shell are similar, one may assume that they have the most dense and stretched shell. This fact renders the incorporation of the protein molecules in the shell practically impossible, resulting in an almost exclusive peripheral F


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Figure 4. (a) Light scattering intensity at 90°, I90, (b) normalized apparent molecular weight, Mw, from SLS measurements, (c) hydrodynamic radius, Rh, values for each population in the solution, (d) corresponding Rh distributions from the CONTIN analysis of DLS measurements at 90°, and (e) zeta potential, ζP, as a function of CHEWL for the solutions of the SPMMS-1/HEWL system at pH 7 and 0.01 M ionic strength.

the SPMMS-1 distribution because the mass of the aggregated complexes is comparably much larger), which means that this population does not interact with the protein. This is the main reason why we suppose that this population corresponds to Pluronic micelles with a PPO core and a PEO shell. Lastly, the effective charge of the complexes once again decreases in absolute value compared to that of the SPMMS-1 micelles (which in turn is smaller than that of the SPM-1 micelles due to the smaller percentage of PAA chains in the shell), as a consequence of the charge neutralization caused by the

complexation between the protein molecules and the PAA chains. As seen in Figure 5, the complexation process and the properties of the complexes are almost identical in the case of the SPMMS-2/HEWL system at pH 7 and 0.01 M ionic strength. Quite interestingly, a smaller degree of secondary aggregation is derived from the increase of the normalized apparent Mw for this system, in comparison to the previous one (SPMMS-1/HEWL). Most likely the smaller Nagg of the SPMMS-2 micelles and mainly the longer length of the PAA chains in the shell (compared to that of the PEO chains) allow G


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been reached. The difficulty in the distinction of the specific peak can be attributed to the complexity of the system, the great difference in mass between the various populations in solution, and/or the small number of free protein molecules. As far as the effective charge of the complexes is concerned, an analogous to the previous instances transition of the ζP values (Figure 5e) denotes the ongoing micellar charge neutralization, caused by the complexation (note that the effective charge of the SPMMS-2 micelles is larger compared to the SPMMS-1 ones due to the longer PAA chains).

for a larger degree of interaction between the protein molecules and the stabilized micelles, thus rendering the intermicellar binding less favorable. The interaction of each population of micelles with the protein is the same (once again verifying that the Rh1 peak corresponds to Pluronic micelles). A very small peak (less than 1% contribution to the total distribution) corresponding to free protein is detected in the supernatant of the coacervated solutions, and for this reason the corresponding Rh value has not been included in Figure 5c. Nevertheless, this is an indication that the maximum degree of interaction/ complexation between the two components has most likely H


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amount of free protein is also detected in the supernatant (not included in Figure 6c due to its very small intensity). Finally, the observed change in the ζP values (Figure 6e) is similar to the rest of the systems, with the effective charge of the SPMMS-3 micelles being smaller to the corresponding one for the SPMMS-2 sample because of the higher PEO content. In summary, for all four different systems under study the complexation of the SPMs with the protein molecules results in charge neutralization, which in turn causes the reduction of the solubility/stability of the complexes. Consequently, a secondary aggregation of the complexed with protein micelles takes place, eventually leading to coacervation at high protein concentration

As expected the situation is essentially the same for the last SPMMS-3/HEWL system, with the corresponding results given in Figure 6. Here the onset of coacervation is moved to even higher CHEWL values (CHEWL ≥ 0.13 mg/mL) and the secondary aggregation is somewhat less intense. The increased solubility (as to the previous systems) of the resulting aggregates is directly correlated to the higher content of PEO chains in the shell of the stabilized micelles. It should be reminded that the PAA to PEO chain ratio in the shell of the SPMMS-3 micelles is 1:3. A gradual increase of the Rh2 values is once again observed, while Rh1 remains constant, in accordance to the previous system. For the coacervated solutions a very small I


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Scheme 2. Representation of the Complexation Process, as well as the Structure and Properties of the Formed Complexes, for the SPM-1/HEWL, SPMMS-1/HEWL, SPMMS-2/HEWL, and SPMMS-3/HEWL Systems at pH 7 and 0.01 M Ionic Strength

values. Most probably, the occurring secondary aggregation between the initial complexes is further assisted by the peripheral distribution of the complexed protein molecules that act as binding sites between different micelles. The comparison between the different types of SPMs reveals that the presence of the PEO chains in the shell of the micelles enhances significantly the solubility/stability of the resulting complexes and secondary aggregates and this effect is even more pronounced for the cases of higher PEO content, i.e., 1:3 PAA to PEO chains in comparison to 1:2 (SPMMS-3/HEWL to SPMMS-2/HEWL system). Moreover, for the same PEO content (1:2 PAA to PEO) but different PAA chain length, the micelles with the longer PAA chains provide more stable complexes than the ones with the short PAA (SPMMS-2/ HEWL to SPMMS-1/HEWL system). In other words the stability of the formed complexes for the four different systems varies as SPM-1/HEWL < SPMMS-1/HEWL < SPMMS-2/ HEWL < SPMMS-3/HEWL. A schematic representation of the complexation process, along with the structure and properties of the resulting complexes for all the studied systems is shown in Scheme 2. 3.3. Protein Structure within the Complexes. For most potential biotechnological applications involving protein/ polyelectrolyte complexes the preservation of enzymatic activity is of major importance. In practice, the activity of the protein is directly correlated to its conformation. For this reason, the structure of the complexed protein for the four different systems was investigated by means of fluorescent and mid infrared spectroscopic techniques, which respectively probe the tertiary and secondary structure of the protein molecule. At first, fluorescence measurements for the stable solutions of the SPM-1/HEWL, SPMMS-1/HEWL, SPMMS-2/HEWL, and SPMMS-3/HEWL systems at pH 7 and 0.01 M ionic

strength were performed and the obtained spectra are shown in Figure 7a. For each system the corresponding spectrum of a neat HEWL solution at 0.5 mg/mL concentration is also included for comparison. The intensity of the spectra is proportional to the protein concentration in the solutions of the complexes, hence the observed variations. The preservation of the overall spectral characteristics and the position of the maximum around 330 nm between the complexed and the neat protein observed in the cases of the SPM-1/HEWL and SPMMS-2/HEWL systems prove that no protein denaturation occurs upon complexation. For the other two systems, SPMMS-1/HEWL and SPMMS-3/HEWL, a broadening of the peak accompanied by a red shift less than 10 nm of the maximum is observed, mainly evidenced at higher CHEWL values where the degree of interaction/aggregation is greater. This shift may be correlated with some subtle changes of the tryptophan environment, or with the formation of closely packed protein aggregates within the complexes, which causes a slight stretching of the protein molecule.28,29 However, the spectrum of fully denatured lysozyme exhibits a red shift of more than 10 nm.30,31 Therefore, we conclude that although some minor changes in the tertiary structure of the complexed protein molecules take place in these cases, no protein denaturation is observed. Additional mid infrared spectroscopic measurements were conducted for the determination of the complexed protein structure. Figure 7b presents the acquired spectra of representative solutions at CHEWL = 0.03 or 0.07 mg/mL of the SPM-1/HEWL, SPMMS-1/HEWL, SPMMS-2/HEWL, and SPMMS-3/HEWL systems at pH 7 and 0.01 M ionic strength, along with the corresponding spectrum of neat HEWL, in the Amide I and II region. For all systems the spectra have been normalized to the intensity of the Amide I J


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Figure 7. (a) Fluorescence and (b) mid infrared spectra of the solutions of the complexes of the (i) SPM-1/HEWL, (ii) SPMMS-1/HEWL, (iii) SPMMS-2/HEWL, and (iv) SPMMS-3/HEWL systems at pH 7 and 0.01 M ionic strength. The corresponding spectrum of a neat HEWL solution is also included for comparison.

band, after subtraction of the spectral contribution of the neat SPMs samples. Unfortunately, at CHEWL = 0.03 mg/mL the

intensity of the spectra is quite insufficient because of the low protein concentration. Still, the observed constancy of the K



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Amide I and II profiles, peaking at ca. 1655 and 1540 cm−1 respectively, indicates the absence of significant protein configuration changes, such as those observed upon denaturation.32,33 This fact proves that the secondary structure of the protein molecules is preserved upon complexation in all cases.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b00550. Detailed synthesis procedure, 1H NMR spectra, SEC chromatograms, analysis of the light scattering measurements, angular dependence of the DLS measurements for the SPMs, and SLS results regarding Rg, Rh and ρ = Rg/Rh values for the solutions of the complexes for the four different systems. (PDF)

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REFERENCES

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4. CONCLUSIONS We have investigated the electrostatic complexation process between lysozyme and stabilized polymeric micelles with a cross-linked PPO core and a polyelectrolyte shell comprised of PAA chains or a mixed PAA/PEO shell, at pH 7 and 0.01 M ionic strength. Dynamic, static, and electrophoretic light scattering measurements showed that for each system the complexation process as well as the size, structure, effective charge, and solution behavior of the formed complexes depend mainly on the protein concentration in the solution, or in other words on the ratio of the two components. In all cases the complexation of the SPMs with the protein molecules results in charge neutralization and causes the destabilization of the complexes in regard to their solubility. Consequently, a secondary aggregation of the initial complexes takes place, eventually leading to coacervation at high protein concentration values. This secondary aggregation between the complexed micelles is probably further assisted by the peripheral distribution of the complexed protein molecules (imposed by the particularly dense conformation of the shell of the SPMs), which act as binding sites between different micelles. Of course, the presence of the PEO chains in the shell of the stabilized micelles greatly enhances the solubility/stability of the formed aggregates. This effect is even more pronounced as the PEO content increases, while the same applies for the SPMMS that have longer PAA to PEO chains (in the sense that they form more stable complexes). Finally, the spectroscopic investigation of the structure of the complexed protein revealed that although some stretching of the protein molecules takes place in some cases, no protein denaturation occurs due to complexation.

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AUTHOR INFORMATION

Corresponding Authors

*Telephone: +30 210 7273836. Fax: +30 210 7273794. E-mail: [email protected]. *Telephone: +359 2 9796335. Fax: +359 2 8700309. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Authors acknowledge financial support of the work by the FP7 POLINNOVA project (GA No. 316086). P.P. thanks the National Science Fund of Bulgaria (Project T02/7-2014). L


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