Enhanced Nucleation of Lysozyme Using Inorganic Silica Seed

Article pubs.acs.org/crystal

Enhanced Nucleation of Lysozyme Using Inorganic Silica Seed Particles of Different Sizes Ulrike Weichsel, Doris Segets, Stefanie Janeke, and Wolfgang Peukert* Institute of Particle Technology (LFG), Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), Cauerstraße 4, 91058 Erlangen, Germany S Supporting Information *

ABSTRACT: In this work we investigate the impact of differently sized plain silica nanoparticles (NPs) between 10 and 200 nm on the crystallization of lysozyme (LSZ). In the first part of our work we investigate the electrostatic interactions between LSZ and NPs by zeta potential measurements and place special emphasis on the adsorption behavior of LSZ@SiO2. The determined adsorption isothermsderived from UV−vis spectroscopyindicate that with increasing particle size more LSZ is adsorbed per NP surface area probably due to a size-dependent surface chemistry and the variation of surface curvature. Second, seeded crystallization experiments both at the microliter and milliliter scale and thus close to a technically relevant scale were performed. A clearly extended crystallization window upon the addition of seed particles toward lower protein and salt concentrations was found. Moreover, induction times of crystal formation and crystallization times were considerably reduced. These effects were intensified with the addition of larger seed particles. In general, with the addition of silica seed particles, a shift of the final crystal size distribution to larger structures is observed.

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ities from the mother liquor shall be avoided.6 As a consequence, nucleation is always strongly affected by the smallest impurities which lead to the formation of undefined, highly aggregated solid material of minor quality,7 being disadvantageous for subsequent solid−liquid separation, e.g., by filtration. One approach to avoid these fluctuations in reaction kinetics and product quality is seeding with foreign material. The first reports on the heterogeneous nucleation of proteins were already given in 1988 by McPherson and Shlichta using mineral surfaces. For four different proteins, including lysozyme (LSZ), it was shown that there were material combinations promoting nucleation at lower supersaturations and modifying the crystal habit.8 Tsekova and Rong used flat glass substrates with different surface functionality and topography to crystallize LSZ. For example, Tsekova et al. used hydrophobic glass substrates which show increased nucleation rates compared to bare glass.9 Rong et al. showed that (i) nucleation is promoted at lower supersaturations, (ii) induction times are reduced, and (iii) larger crystals were grown on porous glass. They also found that poly-L-lysine coated microscope slides selectively reduced the number and controlled the orientation of LSZ crystals.10,11 However, during the past decade research was mainly focused on structural biology. Many different material combinations could be used as nucleants for protein

INTRODUCTION Crystallization is an effective separation technique for proteins at the analytical and industrial scale because it enables the combination of product recovery and purification within one step. For structure determination small amounts of a highly crystalline and ultrapure product are required,1 whereas the aim of industrial protein crystallization is a well-defined crystal size and shape distribution.2 “Well-defined” in the context of the latter means to be advantageous for further downstream processing like filtration. Hence, large and compact crystals with a narrow size distribution are preferred.3 In general, the literature on the topic of protein crystallization is dominated by small scale approaches focusing on analytical issues which are realized by high-throughput techniques.4,5 The latter allow a material and time efficient screening for optimal crystallization conditions, but no conclusions on process design and scale up can be drawn. However, a high demand for the development of technical processes which preserve the functionalityin the case of enzymes the activity of a particular protein during downstream processingis clearly identified.2 Technical processes for protein crystallization which are usually based on homogeneous nucleation are often difficult to control and not reproducible. In the case of proteins high supersaturations are needed for nucleation, but at the same time they are an issue during the following, strongly integration controlled crystal growth where high concentrations affect the complex organization in a secondary and tertiary structure.1 Molecules need time to attach in a certain configuration especially when the disadvantageous incorporation of impur© 2015 American Chemical Society

Received: November 17, 2014 Revised: June 17, 2015 Published: June 17, 2015 3582

DOI: 10.1021/cg501681g Cryst. Growth Des. 2015, 15, 3582−3593

Article

Crystal Growth & Design

as an important step toward process control during industrial protein crystallization which is not yet achieved.

crystallization as can be deduced from the excellent review articles of Bergfors and Saridakis.6,12 For our purposes, namely, the deduction of a correlation between particle and conjugate properties and the final outcome of crystallization experiments, mainly three references were identified to be relevant. First, Kallio and co-workers recently showed that the presence of polystyrene nanospheres in LSZ screening experiments produced more crystals compared to control samples without seeding material. Interestingly, the same authors report that seeding with nanospheres can induce both an enhancement and a retardation effect on nucleation, which already implies the complexity of tailored heterogeneous nucleation. In summary, the use of the nanospheres was beneficial in terms of screening for new crystallization conditions to promote nucleation as well as using them as an additive to receive crystals of better quality.13 Second, Ribeiro et al. use small spherical gold NPs with a size of approximately 14 nm as nuclei. The gold NP surface was functionalized with different organic compounds to investigate the impact of surface chemistry on the crystallization of various proteins. For LSZ an impact on crystal morphology, as well as size and number of crystals, was found. In general, the addition of gold NPs prevented excess nucleation resulting in fewer and bigger crystals. However, the focus of this study was clearly set on microscale-scale crystallizations, light microscopy, and X-ray diffraction analysis.14 Third, Delmas and co-workers used colloidal templates as substrates for heterogeneous nucleation of LSZ, namely, silica particles of various sizes (30−700 nm) with different surface functionalities (silanols, NH2, CF3, phenyl, and dodecyl). The particles were either immobilized on glass substrates or suspended in protein solution. They report that nucleation was affected by particle size, surface chemistry, and topography of the templates.15 Because of the various results of the cited authors, we believe that the use of defined seed particles for the crystallization of proteins in larger scales is a promising strategy for process control but needs to be understood in more detail. To the best of our knowledge, there are no studies where milliliter-scale crystallization processes are combined with a detailed investigation on the protein-NP conjugates using silica NPs in the size range from 10 to 200 nm. Noteworthy, with the smallest size we meet the dimensions of a LSZ molecule. Starting from those smallest particles with high surface curvature, the particle size was stepwise increased and the curvature gradually reduced. Accordingly, in the case of the largest seeds we analyze the interaction of LSZ with a nearly flat surface. On the basis of the in-depth understanding of the interactions between LSZ molecules and silica seed particles we find a correlation between the interaction of LSZ and SiO2 and the nucleation process during LSZ crystallization. The in-depth understanding of the interactions between protein and seed particles is an essential aspect for seeded crystallization experiments and tailored crystal properties. Seeding is a promising way for protein crystallization because it enables the induction of nucleation and thus the controlled formation of nuclei. The use of appropriate foreign material with distinct surface properties might selectively guide the crystallization kinetics to achieve desirable product properties. Additionally, heterogeneous nucleants extend the crystallization conditions toward lower supersaturation levels. This prevents spontaneous nucleation and significantly enhances process stability. Thus, the addition of inorganic NPs as heterogeneous nuclei is seen

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MATERIALS AND METHODS

Experimental Section. In this section all the used chemicals and devices for the different experiments are presented. Aqueous dispersions of plain silica particles synthesized by the hydrolysis of orthosilicates were purchased from Micromod GmbH and were used as received. All diluted samples and solutions were prepared with ultrapure water (18.2 MΩ cm−1). For all experiments LSZ (≥90%, Lot# 061M1329 V) and bovine serum albumin BSA (≥98%,Lot#027 K0764) from Sigma-Aldrich were used without further purification. For zeta potential measurements, pH values were adjusted by the addition of HCl and NaOH. Crystallization experiments were performed with sodium acetate buffer solution (≥99%,Carl Roth) and sodium chloride (>99.8%, Carl Roth) as the precipitating agent. Both, the buffer and salt stock solution were filtered with syringe filters of 0.2 μm pore diameter (cellulose acetate, 25 mm, VWR International). The pH values were adjusted by acetic acid (100%, Carl, Roth). The LSZ stock solutions were filtered with sterile syringe filters of 0.2 μm pore diameter (Acrodisc Supor, 13 mm, VWR International) with low protein binding affinity. For crystallization experiments in microliter-scale microwell-plates (V = 200 μL, Carl Roth) and in milliliter-scale microcentrifuge tubes (V = 2 mL, VWR International) were used. For the determination of the depletion of supersaturation, samples were drawn and filtered with sterile syringe filters of 0.2 μm pore diameter (Acrodisc Supor, 13 mm, VWR International). Zeta potential measurements of LSZ and silica particles as well as dynamic light scattering (DLS) measurements were conducted with a Zetasizer Nano ZS (Malvern Instruments) using folded capillary cells (Malvern). The pH values were adjusted using an InLab Micro electrode (Mettler Toledo). To study protein tertiary structure changes a Fluorolog-3-spectrofluorometer was used, and to determine the protein concentration in the supernatants a Cary 100 Scan (Varian) spectrophotometer was used. For crystallization experiments at the microliter- and milliliter-scale a Zeiss Imager.M1m (Carl Zeiss) microscope was used, and crystal size distributions (CSDs) were determined by laser diffraction measurements with a LS 130 (Beckman Coulter). Methodology. In the first part of this work we focused on the systematic investigation of the specific interaction between protein and particle surface because the adsorption step is seen to be crucial for nucleation. Thereby several surface properties have been identified to play a major role such as hydrophobicity, surface charge, nanotopography, and curvature.16 When protein molecules are exposed to inorganic NPs, several events can occur like the formation of a protein corona around the particles, colloidal stabilization, protein-mediated agglomeration, masking of particles’ charge and reactivity, orientation of proteins on the surface, and conformational changes.16 The latter is highly unfavorable in this context as it leads to a modification or even a loss of the protein activity. The careful characterization of the conjugates of silica NPs and LSZ in our work includes the analysis of (i) electrostatic interactions, (ii) adsorption isotherms, (iii) structural changes of the protein, (iv) colloidal stability. To address all those different issues, methods were chosen that indicate both, changes from the particle side and the protein side. Results shown in this article refer to the former aspect. First, zeta potential measurements assess the impact of the addition of LSZ to the silica particles on the net charge of the particles. Second, the adsorbed amount of LSZ per surface area is determined photometrically for the different particle systems. Possible tertiary structure changes induced by the particles are investigated by intrinsic and extrinsic fluorescence spectroscopy and presented in the Supporting Information (SI) S4 because this is a central point for any applicability of the protein after 3583


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Crystal Growth & Design Table 1. Properties of the Silica Seed Particles (Micromod GmbH) in the Initial Suspensionsa x, nm 10 30 50 100 200

z-aveDLS, nm 16.93 25.37 54.11 94.45 193.6

± ± ± ±

0.70 1.15 2.29 4.17

cSiO2, mg/mL 25 25 25 50 50

δ, μmol/g

SiICP, mg/mL

#SiO2, #/mL

85 70 60 40 30

± ± ± ± ±

× × × × ×

9.57 10.95 11.29 21.61 19.61

0.12 0.19 0.18 0.24 0.10

2.4 8.9 1.9 4.8 6.0

16

10 1014 1014 1013 1012

Aparticle, 103nm2

A, nm2/mL

pH

0.3 2.8 7.9 31.4 125.6

× × × × ×

8.3 8.0 8.5 8.5 8.6

7.5 2.5 1.5 1.5 7.5

18

10 1018 1018 1018 1017

Listed are diameter x, z-average from DLS z-aveDLS, concentration of silica cSiO2, surface charge density δ according to the manufacturer, concentration of silicon from ICP-OES SiICP, number of particles per mL #SiO2, total surface area Aparticle, surface area per mL A, pH.

a

crystallization. Only by such an elaborately characterized system it is possible to conduct and understand crystallization experiments. In the second part of our work we combine the detailed characterization of the LSZ−NP conjugates as described before with crystallization experiments at the microliter- and milliliter-scale. For this reason we propose the controlled addition of well-defined seed material to a supersaturated protein solution acting as heterogeneous nuclei to (i) improve process stability, (ii) control the time frame of the overall process, (iii) increase the product quality, and (iv) extend the crystallization conditions. Here, unseeded and seeded crystallization experiments at the microliter-scale are conducted to generate state diagrams. From such state diagrams appropriate conditions for further milliliter-scale experiments are chosen. At the milliliter-scale the depletion of supersaturation with time is monitored, and the resulting CSDs using the different silica seed particles are compared. Especially the latter are not accessible by experiments at the microliter-scale due to the limited product quantity. Seed Particle System Size and Solid Concentration. Table 1 represents characteristic data of the different silica particles obtained from the manufacturer which were confirmed and complemented by our own measurements. For the present work silica NPs with different sizes of 10, 30, 50, 100, and 200 nm were used. All particle sizes determined by DLS are within the specifications of the manufacturer. For TEM and SEM images of the particles as well as particle size distributions measured by DLS the reader is referred to the Supporting Information, S1 and S2. All analyses confirm the mostly spherical particle shape claimed by the manufacturer and expected from the synthesis process (Stöber silica). In addition, the smallest particles of 10 nm are close to the size of a globular LSZ monomer which has geometric dimensions of L × W × H = 4.5 × 3.5 × 3.5 nm17 and a hydrodynamic diameter of 3.5 nm. Inductively coupled plasma optical emission spectrometry (ICP-OES) measurements confirm the specified solid concentrations. The pH values of the delivered dispersions are within a range from pH 8.0 to 8.6. Zeta Potential Analyses. Zeta potentials of silica particles and LSZ in water were determined electrophoretically. For the pH series, the particle stock dispersions were diluted with ultrapure water (dilution factor 10). After the pH adjustment samples were taken at appropriate pH intervals for zeta potential measurements. To further analyze the evolution of zeta potentials during the addition of LSZ, each particle stock dispersion was diluted with ultrapure water (dilution factor 10) and divided into 10 samples of V = 1 mL. The adjustment of five different ratios of LSZ molecules to silica particles in a range of a theoretical monolayer coverage θtheoretical from 0.05 to 1.4 in duplicate was realized by the addition of different quantities of an aqueous LSZ stock solution of 10 g/L. To show that the outcome of the experiments only depends on the ratio of LSZ molecules to the available NP surface, experiments for 10 and 200 nm NPs were repeated with a dilution factor of 20. For the experimental design we assumed a projection area of 10 nm2 for globular LSZ when it is adsorbed to the silica seed particle surface.17 All concentration calculations regarding a theoretical monolayer coverage of LSZ on the particle surface are based on the specifications of mean number weighted particle diameter and the number concentration of particles

per milliliter (see Table 1). Theoretical values for the coverage of silica particles with LSZ molecules were calculated according to θtheoretical =

# of LSZ molecules added # of LSZ molecules needed for monolayer

(1)

For particle sizes of 10, 30, 50, 100, and 200 nm θtheoretical = 1 would be achieved with 31, 280, 785, 3141, and 12 566 LSZ molecules per particle. Zeta potentials were measured directly after the addition of protein to prevent disturbances by the induced agglomeration of the silica particles. Derivation of Adsorption Isotherms. To investigate the adsorption of LSZ on silica particles, diluted dispersions of 10 mL of the different particle systems were placed in vials. Because of the varying original particle concentrations and particle sizes (see Table 1), the provided silica surface [ASiO2/mL] dispersion differs between 7.5 × 1016 nm2 (200 nm) and 7.5 × 1017 nm2 (10 nm). For this reason the added LSZ concentrations were adapted in each case to ensure comparability of all results. The procedure is depicted in Scheme 1. As mentioned, the pH of all silica stock solutions ranged from 8 to 8.6 (see Table 1). Each particle stock dispersion was diluted with ultrapure water (dilution factor 10) and divided into 10 samples of V = 1 mL (microcentrifuge tubes of V = 2 mL, VWR International). The adjustment of five different ratios of LSZ molecules to silica particles (θtheoretical = 0.2−1.6) in duplicate was realized by the addition of

Scheme 1. Procedure for the Determination of Adsorption Isothermsa

a

A constant particle surface is provided in each beaker. Then different amounts of LSZ are added, and the mixtures are agitated for an equilibration time of 3 h. After centrifugation the LSZ concentration in the supernatant is determined photometrically. 3584


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well to reach a stock solution concentration of 100 g/L. To filter the protein stock solution of high concentration, a sterile syringe filter of 0.2 μm pore diameter with low protein binding affinity was used. LSZ solubilities were calculated by third order polynomial fits of solubility data determined by Cacioppo et al.24 Crystallization experiments at the microliter-scale were conducted in microwell-plates with protein concentrations ranging from 10 to 45 g/L and sodium chloride concentrations from 20 to 70 g/L. Microcentrifuge tubes were used in milliliter-scale experiments with a protein concentration of 30 g/L and a salt concentration of 50 g/L throughout all investigations. By mixing the stock solutions of protein and sodium chloride with buffer the requested supersaturation was achieved. All experiments were performed in a way that immediately after the addition of the precipitant a constant volume of seed particle dispersion at different solid concentrations was added to provide a constant particle surface of 1.5 × 1016 nm2/mL. In this way the protein and salt concentrations were kept constant for all experiments. After loading, all microwell plates were sealed with adhesive strips and a lid to prevent evaporation. The plates were stored in an air-conditioned room with a constant temperature of T = 23 °C. The microcentrifuge tubes were put in an end-over-end shaker with constant rotational speed that was placed in a water bath with temperature control (T = 23 °C). This ensured good mixing of the solution and prevented sedimentation of crystalline material. The formed solid material was investigated by light microscopy. Depletion of Supersaturation. To monitor the depletion of supersaturation samples (50 μL) were taken at appropriate time-steps and filtered with a syringe filter of 0.2 μm pore size. By this procedure particulate material >200 nm was removed from the sample to determine the free protein concentration in the supernatant/filtrate. Finally, 10 μL of the filtered supernatant was diluted with buffer (dilution factor 100) prior to absorption measurements from which the free protein concentration was accessible as described above. As a measure of relative supersaturation the following expression was used:45

different quantities of an aqueous LSZ stock solution of 10 g/L. To confirm that the obtained adsorption isotherms only depend on the number ratios of LSZ per particle and not on the absolute concentrations, experiments were repeated with a significantly higher dilution factor of 20 for all particle sizes (3.75 × 1016 nm2 (200 nm) and 3.75 × 1017 nm2 (10 nm)). After LSZ addition, the centrifuge tubes were put for 3 h in an endover-end shaker with constant rotational speed to ensure good mixing and to prevent sedimentation of the particles. To ensure isothermal conditions, the shaker was placed in a water bath for temperature control (T = 23 °C). The equilibration time of 3 h was taken from literature where adsorption of LSZ on silica NPs in the range of 4−100 nm was investigated.17 Also our own previous experiments confirmed that the adsorption equilibrium was already reached within 1 h for all particle sizes investigated during this study (see Supporting Information S5). After equilibration, particles with protein bound to the surface were separated from free, unbound LSZ in the supernatant by centrifugation at 14 000 rpm (11000g) for at least 20 min. For the smallest particles the centrifugation time was increased up to 3 h in maximum until no scattering effects were observed in a UV−vis scan (200−800 nm) of the supernatant anymore (see Supporting Information S5). This ensured that the loss of particles to the supernatant was negligible. To ensure the adequacy of the centrifugation procedure to separate the aggregated particles from the free protein scattering, experiments using ICP-OES measurements with supernatants have been performed (see Supporting Information S5). The amount of protein adsorbed on seed particles was determined via the mass balance by measuring the equilibrium protein concentration in the supernatant according to

c LSZ,adsorbed = c LSZ,added − c LSZ,supernatant

(2)

The concentration of free LSZ was determined from the absorbance at the characteristic wavelength of 280 nm and an extinction coefficient εLSZ of 2.64 mL·mg−1·cm−1.18 Adsorption data were evaluated using the Hill equation which is frequently found in the context of proteins:19,20

S=

1

θ = θmax 1+

(

KD cLSZ,added

c LSZ,filtrate c LSZ,eq

(4)

n

)

In this equation cLSZ,filtrate denotes the free protein concentration after filtration, and cLSZ,eq is the saturation concentration at thermodynamic equilibrium. As mentioned above LSZ saturation concentrations were determined from reference 24. Determination of Crystal Size Distribution. LSZ CSDs in the micrometer range were determined for crystallization experiments at the milliliter scale. The obtained crystals were analyzed by laser diffraction. After a crystallization time at which 60% of the initial protein concentration was depleted, a few drops of the crystal suspension were added to a stirred cell in the laser diffraction device to reach a solid concentration of 8−12%. The cell was immediately filled with 15 mL of a saturated protein solution to prevent dissolution and further growth of the formed crystals. Saturated LSZ solutions were obtained by adjusting the same sodium chloride concentration as in the milliliter experiment and a LSZ concentration of 5 g/L. All CSDs are obtained from the average of three measurements per sample with a measurement time of 90 s, respectively. For conversion of the diffraction data the Fraunhofer model was used.25,26 Offset and background measurements for the saturated protein solution were subtracted from the CSD measurements.

(3)

The equation is mainly used in biochemistry to characterize the binding of a ligand molecule to a macromolecule. It describes the surface coverage θ dependent upon the LSZ concentration in solution cLSZ,added, with θmax being the maximum surface coverage and KD being the dissociation constant as measure for affinity. Low values of KD indicate a high affinity of the ligand to a surface. The parameter n represents the Hill coefficient which is a measure for cooperativity. It is influenced by the protein conformation and by the interactions between protein molecules and between the protein and the surface.21 A Hill coefficient n < 1 (n > 1) denotes negative (positive) cooperativity meaning that the binding affinity effectively decreases (increases) when more protein adsorbs onto the NP surface.22 A coefficient n = 1 indicates that the binding of ligands is independent from already bound molecules and from interactions between ligands. Thus, this case is equivalent to the Langmuir model. Important differences to the Langmuir model in the case of protein adsorption on solid surfaces are multiple-site binding, the heterogeneous nature both of the protein molecule, and a solid surface and lateral interactions between protein molecules. The Hill equation takes into account that interactions between free and adsorbed protein molecules can occur, which alter the probability of further protein adsorption at a surface.23 Crystallization Experiments. The crystallization of LSZ was conducted in sodium acetate buffer solution. The buffer salt was dissolved in ultrapure water, to reach a concentration of 0.1 M, and the pH value of 5 was adjusted by acetic acid. After pH adjustment, the precipitation agent sodium chloride was dissolved in the buffer solution to reach a stock solution concentration of 150 g/L. Both the buffer and salt stock solution were filtered with syringe filters of 0.2 μm pore diameter. The lyophilized LSZ powder was dissolved in buffer as

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RESULTS AND DISCUSSION Electrostatic Interactions between Lysozyme and Silica Nanoparticles. Colloidal silica NPs are typically synthesized by the Stöber−Fink−Bohn wet chemical synthesis route using a reaction mixture composed of an alkoxide precursor, a primary alcohol, water, and ammonia as a catalyst.27 Different particle sizes ranging from 10 to 350 nm are obtained by the variation of the experimental parameters such as reaction temperature, feed rate of reactants, and 3585


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Crystal Growth & Design ammonia concentration.28 Larger particles reaching the micrometer range are usually synthesized by seeded growth techniques.29 The different particle systems employed in this work were all synthesized from identical substances, and thus no fundamental differences in the chemical structure of the formed solid are expected. This is further supported by Zhuravlev who found that the number of OH groups per surface area was constant for 100 different samples of amorphous silica from various sources over a broad range of particles sizes. These results were obtained by thermally pretreated samples to reach a maximum degree of hydroxylation.30 However, a variation of synthesis parameters and the underlying particle formation mechanism are reported to influence the solids properties and the surface chemistry of the particles. For instance, a size-dependent density31 and porosity for water, small ions, and gaseous sorbents was found.32,33 According to de Keizer et al. this particle sizedependent degree of microporosity demands a distinction between surface and volume charge densities caused by internal and external hydroxyl groups.34 In addition, a size-dependent O/Si ratio leading to higher charge densities for larger particles and the occurrence of surface organic groups from precursors are reported.33,34 To account for the last aspect, infrared spectroscopy was performed for the particles used in this work. For all particle sizes the absence of characteristic CH stretching modes which would be caused by remaining ethoxy groupswas confirmed (see Supporting Information S3). To further investigate the surface chemical properties of the employed silica particles zeta potential measurements were conducted. Figure 1a shows the pH-dependent zeta potential for both pure

manufacturer; see Table 1) probably due to different particle formation mechanisms as mentioned in the preceding paragraph and found by Costa.33 The mass specific surface charge density of the manufacturer given in Table 1 is recalculated to a surface specific density with the assumption of SiOH groups being responsible for surface charges. The result shows an increasing charge density from 0.18, 0.44, 0.61, 0.83, to 1.24 SiOH/nm2 with increasing silica particle size from 10, 30, 50, 100, and 200 nm. In agreement with expectations from the aforementioned literature, the isoelectric point (IEP) of silica is situated between pH 3 and 4 and is caused by a high number of silanol groups at the particle surface (for more details see Scheme 2). In contrast, LSZ is positively charged with a zeta potential of 31.6 ± 4.9 mV at pH = 3. It has an IEP of pH 11 (see Figure 1a). Thus, within the observed pH range from pH 5 to pH 9 LSZ and all silica NPs have opposite charges, and the occurrence of strong Coulomb interactions is expected. Figure 1b depicts the evolution of zeta potentials analyzed for differently sized silica particles in water after their exposure to increasing relative amounts of LSZ. The pH of all mixtures is with values between 8.0 and 8.6 still clearly situated above the IEP of silica. Thus, the zeta potential of the particles without adsorbed protein would be negative. As expected, with addition of LSZ the zeta potential is shifted toward positive values because the protein is positively charged under the experimental conditions (see Figure 1b). For all differently sized seed particles a final constant positive zeta potential is reached. These plateau values are in accordance with the measured adsorption behavior for the pure components (see Figure 1a) and emphasize the fact that electrostatic interactions are predominant in the adsorption process. These findings are in a good agreement with the results of Rezwan et al., who correlated the zeta potentials and IEPs of silica with a mean size of 92 nm to the amount of LSZ adsorbed at a pH = 7.5.35 They used electroacoustics and obtained a clear charge reversal of the silica NPs with increasing amount of added LSZ, whereas a constant zeta potential in this case was observed considerably below a theoretical monolayer. These authors also show that an increasing ionic strength in solution is accompanied by a reduction of the adsorbed amount of protein on the particle surface. Thus, with respect to the seeded crystallization experiments performed in the second part of this study, a reduction of electrostatic interactions due to the presence of NaCl as precipitation agent is expected. However, a clear size-dependence is observed in a way that with increasing particle size more LSZ molecules are necessary to mask the negative charge of the particles. Especially in case of the 10, 30, and 50 nm particles with an identical starting point of −60 mV that was determined for the zeta potential at θtheoretical = 0, a clear shift of the IEP is observed. Whereas for a silica particle size of 10 nm only a θtheoretical of 0.16 is needed to reach a net potential of zero; nearly a three times higher amount of θtheoretical = 0.47 of LSZ is needed for the 50 nm NPs. In the case of the larger particles of 100 and 200 nm in size, the zero net charge is achieved for the highest theoretical surface coverage of about θtheoretical = 0.70. The same trend is observed for the monolayer concentration that is needed to adjust a constant positive zeta potential around 25 mV indicating saturation of the particle surface. In the case of the 10 nm NPs it is already achieved at θtheoretical = 0.29, whereas for the particles with a size of 50 nm, θtheoretical is as high as 0.83. Since only minor pH changes for the various added amounts of LSZ to the particle dispersions were measured, we suggest that the

Figure 1. (a) Zeta potentials of LSZ and silica seed particles in water dependent on pH. The vertical line accentuates the different absolute values at pH 8. (b) Zeta potential of silica seed particles with addition of various amounts of LSZ at pH 8.5 in water. The calculation of the theoretical surface coverage of LSZ on silica particles is based on eq 1. Gray data points indicate aggregate formation.

LSZ and the silica seed particles in water. All differently sized plain silica NPs carry a negative net charge with zeta potentials at a pH value of 8.0 ranging from −60.9 ± 1.9 mV for the smallest particles to −90.3 ± 0.43 mV for the 200 nm silica particles. Qualitatively, the evolution of zeta potentials is identical for the different particle sizes; variations in the absolute magnitudes are however an indication of variations with respect to the detailed surface chemistry (cf. corridor in zeta potentials illustrated for pH = 8 by the black vertical line as guide to the eye in Figure 1a). These size-dependent differences are ascribed to a varying surface charge density δ (given by the 3586


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Scheme 2. Schematic Representation of (a) 10 nm Silica Particle with Characteristic Surface Groups According to Zhuravlev30 and a LSZ Molecule (Protein Data Base: 7LYZ; Protein Picture Generator40) and (b) Change in Particle Curvature Proportional to a LSZ Molecule for Particle Sizes of 10, 30, 50, 100 nm, Respectivelya

a

Absolute sizes are not true to scale.

to a monolayer coverage (θ = 1) for the 100 nm particles. For the 200 nm particles the adsorbed amount of LSZ even exceeds the theoretical monolayer (θ > 1). Table 2 contains fit data

surface charge density influences the interactions between LSZ and silica particles. Although electrostatics might not be the only factor influencing the overall adsorption of LSZ at silica surfaces, we believe that this type of interaction is of major importance. For an even better understanding of the interaction between differently sized SiO2 NPs and LSZ the detailed adsorption isotherms will be discussed in the following. Adsorption of Lysozyme on Silica Particles. The further characterization of conjugates includes the determination of the adsorbed amount of LSZ on the silica particle surface. Thus, to complement the finding of a size-dependent charge reversal of the silica particles with addition of LSZ molecules, adsorption experiments at pH 8.0−8.6 were conducted. The resulting adsorption isotherms are shown in Figure 2.

Table 2. Maximum Surface Coverage θmax and Hill Coefficient n Obtained from Fitting the Data Shown in Figure 2 by Equation 3 x/nm 30 50 100 200

θmax/−

KD/μM

± ± ± ±

12.44 ± 0.16 9.67 ± 1.07

0.50 0.84 1.27 1.53

0.04 0.22 0.25 0.06

9.46 ± 0.65

corr R2

n 3.06 1.86 2.17 1.85

± ± ± ±

1.50 0.78 0.66 0.15

0.93 0.86 0.99 0.99

from 30, 50, 100, and 200 nm silica particles using the Hill equation (see eq 3). From these fits the maximum surface coverage θmax, KD and n values are obtained and listed in Table 2 together with the coefficient of determination. The promoted adsorption of LSZ on silica NPs is confirmed by θmax, which increases from a value below 0.25 for 10 nm silica particles (directly extracted from Figure 2) to 0.50 ± 0.04 and 1.53 ± 0.06 for the 30 and 200 nm particles. In addition, values of the dissociation constant KD range from 12.44 ± 0.16 μM to 9.46 ± 0.65 μM and indicate a comparable affinity of LSZ to the silica surface. The identified Hill parameters n which describe the cooperativity of the binding process are (i) all in the same order of magnitude indicating a similar binding mechanism and (ii) >1 for the investigated particle sizes. As described, n > 1 denotes that the binding of LSZ to the particle surface is positively cooperative. Regarding the literature, Jiang et al. investigated the binding of the protein transferrin to polymercoated 5 nm gold particles by fluorescence spectroscopy and found n = 1.7 ± 0.2 using the Hill equation.36 They ascribed this finding to stabilizing interactions between adjacent protein molecules. In addition, Röcker et al. determined values of n = 0.74 ± 0.1 for the binding of human serum albumin to FePt particles.37 They interpreted n < 1 as a steric hindrance effect for the adsorption of further protein molecules to a partially coated particle. Regarding the present system, for all particle sizes adsorption is promoted by the presence of LSZ at the particle surface; however, a significant difference in maximum surface coverage for the differently sized particles was derived. This is seen to be crucial for subsequent crystallization experiments in particular because crystallization clearly takes

Figure 2. Adsorption isotherms for 10, 30, 50, 100, and 200 nm silica particles in water determined at pH 8.5 and an equilibration time of 3 h. Experiments were performed in duplicate for two different initial solid concentrations and are included in the specific error bars.

For all investigated seed particle sizes, a linear region with a slope of 1 is observed at small LSZ concentrations (see black broken line in Figure 2). This means that all added LSZ is adsorbed onto the particles. However, with increased amount of LSZ added to the NPs, the adsorbed amount levels off due to the fact that the surface gets saturated. Noteworthy, this leveling off is found at strongly different values of θtheoretical for the different particle sizes. On the 10 nm silica NPs only a low amount of LSZ referred to the normalized surface area coverage is adsorbed. With increasing particle size and thus a reduced particle curvature the maximum adsorbed amount increases up 3587


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tendency of the association constant to increase with particle size, while the cooperativity (parameter n) was reduced. These findings were attributed to an enhanced protein packing on the larger NPs with an increasing protein layer thickness. Interestingly, the critical particle size at which no further increase in layer thickness was observed was found in this case to be 50 nm.20 Finally and as already mentioned, the sizedependent adsorption behavior is in line with the findings of Vertegel et al., who showed that less LSZ is adsorbed onto smaller silica NPs. At this point it needs to be considered that for our particle system the parameters surface charge density and particle curvature are probably superimposed, which leads to an interpretation of results based on both inseparable effects. It was not our main intention to make a distinction between these parameters. Nevertheless, it would be of great benefit for prospective studies to identify particle systems with identical surface properties upon a variation of particle size and vice versa to prove the impact separately. The first reason why the adsorption of LSZ on larger particles is favored is the higher surface charge density of larger particles, which is confirmed by our zeta potential measurements. As shown above a higher number of OH groups on the silica surface leads to an increased maximum adsorbed amount of LSZ molecules. Second, surface curvature itself is seen to be an important factor. Because of the increasing surface curvature of small particles, less contact points for the LSZ molecules exist, which leads to a suppressed adsorption.17 This effect is illustrated in Scheme 2. Scheme 2a depicts a 10 nm silica particle with specific surface groups and pronounced curvature proportional to a LSZ molecule. As can be seen from 2b the curvature effect is already strongly reduced at a particle size of 50 nm reachingat least from the perspective of a LSZ monomerthe dimensions of a flat surface at a particle size of 100 nm. Although both effectssurface charge density and curvatureare seen to play a role, the important point of this study is the correlation of molecular adsorption data to crystallization experiments. This is so far not reported in the literature and therefore needs to be investigated in more detail. Thus, crystallization experiments at the microliter-scale were performed. Since the initial adsorption step on the foreign surface is crucial for nucleation we expect, based on the results discussed until now, that the crystallization conditions and kinetics will be strongly affected by differently sized seed particles in a way that larger particles are more favorable. Seeded Crystallization at the Microliter-Scale. In the following the influence of the interaction between LSZ and NPs on the protein crystallization shall be investigated. Suitable crystallization conditions for which LSZ crystallizes in its tetragonal crystal system were chosen according to preliminary studies of Wanka and Peukert that were performed without the addition of NPs.41 As described in the experimental section part, immediately after the addition of salt solution to the protein solution a certain amount of NPs were suspended into the supersaturated LSZ solution. The overall surface area in each batch provided by the foreign particles was kept constant. As a reference, experiments without NPs were performed as well. By this method we account for the influence of the container walls and at the same time set a starting point for the seeded crystallization experiments. After 90 min and 22 h state diagrams were generated from microscope images (see Figure 3). State diagrams after a crystallization time of 6 days are included in the Supporting Information S6. Concentration

place at high supersaturations and thus at very high monomer concentrations. To account for reduced attractive electrostatic interactions due to the addition of buffer salt and sodium chloride as the precipitant, adsorption isotherms and zeta potentials were determined at pH = 8.5. The results are shown in the Supporting Information (Figures S5_2 and S5_3). They confirm the trend of a pronounced adsorption of LSZ on larger silica particles. Additionally, zeta potential measurements display reduced absolute values for all silica particles sizes with increasing NaCl concentration with remaining opposite charges between LSZ and silica. To investigate possible structural changes of the protein we demonstrated by fluorescence spectroscopy that the tertiary structure of LSZ remains stable when it is exposed to the different silica particles. This is seen as an important prerequisite for the preservation of the enzyme activity during adsorption and crystal formation. However, both methods revealed a slight impact on the LSZ tertiary structure with increasing seed particle size. For a detailed presentation of results the reader is referred to Supporting Information S4. In addition a brief discussion of CD spectroscopy results for a further investigation of protein structural changes is given in the Supporting Information S4 dealing with the critical point of strongly varying extinction coefficients in the wavelength range of interest. At this point it needs to be mentioned that by the addition of LSZ to the differently sized plain silica NPs a strong destabilizing effect followed by agglomeration/aggregation is observed. Bharti et al. made similar observations and described agglomeration of 20 nm silica particles dependent on the pH value after the addition of protein as LSZ-mediated agglomeration. The pH range of 4−6 is reported as a partial protein binding regime with the formation of compact aggregates. Between pH 7−9 a complete protein binding regime was observed with the formation of loose aggregates leading to a network structure.38 However, even with the assumption of a maximum reduction of the available surface provided by the particles due to ongoing agglomeration, it is not possible to attribute the differences in the adsorbed amount of LSZ in Figure 2 only to an aggregation effect. General Conclusions on the Interaction between LSZ and SiO2 Nanoparticles. Putting all data together, it is concluded that the adsorption of LSZ on silica NPs especially for particles below 100 nm is strongly governed by the particle size. These effects are not only supported by the previously discussed evolution of zeta potentials for different seed particles but also by a critical inspection of the literature. Larsericsdotter and co-workers mention as well a low adsorbed amount of LSZ on small silica particles with a diameter of 11 nm.39 They explained this result by an overestimation of the provided surface area, an effect which is now clearly ruled out in our experiments. Malissek showed in her work that a sizedependent adsorption behavior of BSA exists on citratestabilized silver NPs in the range from 12 to 90 nm. Equilibrium constants were determined by concentrationdependent CD measurements. With increasing particle sizes up to 40 nm, a decreasing equilibrium constant was observed until it remained constant. The lower equilibrium constants were interpreted as higher affinities of the protein for larger particles. Apparently, below a critical particle size the affinity of the protein to the foreign surface is strongly reduced.19 De Paoli Lacerda and co-workers determined Hill parameters for blood proteins and gold NPs in the range from 5 to 100 nm by fluorescence quenching measurements. They found the 3588


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In comparison to samples without NPs, the crystallization window (indicated by blue diamonds in Figure 3) is extended for all particle sizes of 10, 30, 50, 100, and 200 nm to conditions with lower protein (y-axis) and precipitant (x-axis) concentrations. Thus, as already expected from the adsorption studies, the addition of foreign surface has a positive effect on the crystalline product. Consequently, there is a strong hint about the underlying heterogeneous nucleation mechanism based on the interactions between particles and protein molecules and thus based on the adsorption behavior on the particle surface. As nucleation is a transient kinetic process, a comparatively short time scale of 90 min was chosen to carve out the particle size-dependent induction times and crystallization windows. In comparison to the unseeded case, the extension of crystallization conditions is already slightly enhanced for the addition of 10 nm silica particles and clearly enhanced with the addition of larger seed particles. The most developed crystallization window is monitored for largest seed particles of 200 nm. This is explained by the improved adsorption of LSZ on the seed particle surface with lower curvature and higher charge density, which probably enhances nucleation. Already after 22 h and clearly after 6 days (for the latter results the reader is referred to Supporting Information S6), these differences are compensated, probably due to the superposition of nucleation and growth of the protein crystals by agglomeration events. For this reason milliliter-scale crystallization experimentsas presented and discussed in the next chapterare necessary to elaborate time effects more precisely. Remarkably, with the addition of seed particles, well-defined crystal structures are formed. Especially the addition of larger seeds suppresses the formation of “sea urchin” structures at high salt concentrations, which are usually a major issue. These sea urchin structures are described as monoclinic crystal needles around amorphous aggregates.42 In this context two theoretical articles dealing with the heterogeneous nucleation of colloidal hard spheres on flat and curved foreign surfaces have to be mentioned. Cacciutto et al. performed Monte Carlo simulations with smooth spherical seeds of various sizes.43 Their findings show that seed particles need to exceed a minimum size to promote nucleation. For seed particles that are four-times larger than the colloidal particle only homogeneous nucleation in the bulk was observed. Five-times larger seeds lead to nuclei formation, but the height of the free-energy nucleation barrier is hardly affected. This barrier only lowers when the seed particle size is increased further. In addition to energetic considerations the group also states that due to a higher curvature for smaller seeds the formed nuclei detach earlier from the surface. Sandomirski et al. investigated the crystallization of sterically stabilized PMMA and fluorescently labeled particles by confocal microscopy using glass spheres as seeds. They compared their experimental results to simulations by Brownian dynamics.44 They find that an increasing seed size leads to a higher number of ordered layers on the surface. Moreover, a higher seed particle curvature introduces a mismatch in the crystal lattice and consequently leads to the detachment from the surface when the nucleus exceeds a certain maximum size. An additional mechanism to relax the mismatch is probably given by structure deformations for soft spheres and proteins. This fits well with the previously discussed results on LSZ−NP conjugates and the related literature. Vertegel et al. state a stronger deformation of the secondary structure of LSZ molecules on larger particles.17 Our own fluorescence emission measurements (extrinsic with fluorescent dye) show in general

Figure 3. State diagrams generated from microscope images of microliter-scale crystallization experiments after 90 min (blue and red symbols) and after 22 h (gray symbols) (a) without the addition of seed particles, (b) with 10 nm silica NPs, (c) with 30 nm silica NPs, (d) with 50 nm silica NPs, (e) with 100 nm silica NPs, and (f) with 200 nm silica NPs; the foreign particle surface provided throughout all experiments was 1.5 × 1016 nm2/mL. The broken line represents the LSZ solubility calculated from Cacioppo and Pusey.24

analysis of LSZ derived from a parallel experiment confirmed that after this period supersaturation reached a value of 2that means very close to equilibriumin all microwells where solid formation occurred. The results for differently sized seed NPs are summarized in Figure 3. At this point it needs to be mentioned that light microscopy images were used to distinguish between the two states of either no (visible) solids formation or crystal formation at low salt and protein concentrations. At high supersaturations precipitate and crystal formation might overlap, which is the reason for not giving a statement on possible effects of using seed particles in this area of the state diagram. A clear discrimination between “crystals formed” and “amorphous solids formed” or an overlap of both is thus not possible. However, the objective of our study was to investigate crystal formation at comparably low supersaturations rather than to investigate the transition between crystallization and amorphous precipitation at high supersaturations. 3589


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and surface chemistry of the seed particle surface. This adsorption behavior has a large impact on the formation of crystallites at the foreign surface. Protein crystallization can therefore be significantly improved by the addition of wellcontrolled foreign seed particle surfaces. However, so far, crystallization experiments were conducted at the microliter scale. As a final important step to prove the technical applicability of our approach we want to transfer these experiments to the milliliter scale. Seeded Crystallization at the Milliliter Scale. To really compare the different courses of crystallization and to compare the finally obtained crystal quality, we transferred some selected microliter experiments to the milliliter scale. We chose conditions at which crystal formation occurred at the microliter scale (labeled in Figure 3) without as well as with all differently sized seed particles. Again, the provided foreign particle surface was kept constant (1.5 × 1016 nm2/mL). We have to mention here that microliter-scale experiments were performed under static conditions and milliliter-scale experiments were shaken in an end-over-end shaker of constant rotational speed. This means that stirring would probably affect the state diagram and phase transition kinetics might differ between the two different scales. However, the microliter-scale crystallization experiments were essentially used as a screening tool for a transfer of conditions to the milliliter scale and used as a proof of concept of the influence of the addition of foreign seed particles. In Figure 4a the induction times for 2-fold crystallization batches

minor tertiary structure changes but confirm the tendency of a stronger deformation of LSZ molecules on larger seeds. Mechanistically, the results of Figure 3 can be explained by an activated primary nucleation according to Mersmann et al. as a combination of homogeneous (Bhom) and heterogeneous nucleation (Bhet) on a foreign surface as well as surface nucleation events (Bsurf) on the protein crystal surface.45 The prevailing mechanism at low supersaturation is Bsurf on existing crystals, whereas at high supersaturation Bhom in the solution dominates. As mentioned in the beginning, especially the latter causes implications in technical processes. At medium supersaturation, which is the focus of this study with regard to a future scale up to technical processes, the favorable Bhet prevails. According to Mersmann, Bhet mostly depends on the specific surface area of foreign particles, on the contact angle between a nucleus and the foreign particle surface, and on the number of adsorbed units per surface area.45 In the following the influence of these parameters on our particular case is discussed. The heterogeneous nucleation rate Bhet strongly depends on the provided foreign surface area.46 Thus, with an increasing surface area of foreign material in solution the nuclei formation is enhanced. In our experiments the specific surface area of foreign particles was kept constant for comparability of the differently sized NPs. Additionally, the reduction of free energy for the formation of a critical nucleus for heterogeneous nucleation ΔGc,het compared to homogeneous nucleation ΔGc,hom is described by the geometric factor f with values 0 < f < 1:46 ΔGc,het = f ΔGc,hom

(4)

It is assumed that the shape of a cluster on the foreign surface equals a spherical segment. Accordingly, the factor f is calculated by the contact angle θ and thus depends on the wettability of the foreign surface with solution: f=

(2 + cos θ )(1 − cos θ )2 4

(5)

Contact angles approaching 180° would favor homogeneous nucleation events because in this case f equals unity. Regarding this study, the differently sized silica NPs display high wettabilities. A particle size-dependent contact angle and thus the factor f can be derived from geometric aspects and is proven by the work of Viswanadam and Chase. They performed contact angle measurements for a PTFE−water system with PTFE spheres of constant size and variable drop sizes. For a decreasing ratio (drop to particle) the contact angles decrease which denotes higher wettability.47 Noteworthy, the effect of surface charge density that is additionally expected to play a role even strengthens this effect. For this reason heterogeneous nucleation events are expected to depend on an improved wettability of the larger particles which additionally explains the size-dependent maximum adsorbed amount of LSZ molecules on the particle surfaces. The significantly enhanced crystallization window found for the larger silica seed particles is well explained with the previous finding of a promoted LSZ adsorption on larger particles (see Figure 2) with a less pronounced surface curvature and higher surface charge density. Relating the results on the adsorption behavior discussed in the beginning of this work to literature data and to the results obtained from the seeded crystallization experiments, the following conclusions can be drawn: the first adsorption step of molecules strongly depends on the curvature

Figure 4. (a) Induction times (point of visible solid formation) of milliliter-scale crystallization experiments (the red dashed line is introduced as guide to the eye) and (b) corresponding concentration curves with and without the addition of seed particles; cNaCl = 50 g/L; cLSZ* = 3.3 g/L; S0 = 9.0; afor = 1.5 × 1016 nm2/mL.

using the differently sized seed particles are plotted. The induction time is defined as the time that is needed for nuclei to form in a supersaturated solution and to grow up to a detectable size.48 Within this study, they represent the point at which the solid formation was visible to the naked eye. For all investigated particle sizes a clear shift of the induction period to lower values compared to the case without the addition of seed particles was observed. Without the addition of seed particles the first visible solid formation was monitored after 460 ± 40 min (referred to the addition of the salt solution). With the addition of the silica seed particles the solid formation is shifted to significantly shorter induction times within a narrow range. For instance, when using silica particles of 10 and 30 nm in size, the induction times range from 125 ± 21 to 140 min. A further reduction of the induction time to 125 min for the 50 and 100 nm particles down to 80 min for the 200 nm particles is observed. All experiments displayed a high 3590


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Figure 6a shows the corresponding CSDs of the experiments already discussed in the context of Figures 4 and 5. CSDs were

reproducibility. The specific induction times represent in the next step the first data points in the concentration−time curves. Figure 4b depicts the depletion of LSZ dissolved in the supernatant with time. As described in the Methodology section, LSZ concentrations were determined from absorbance analysis of the supernatant after removal of the large crystals by filtration. It can be seen that the supersaturation is depleted within a considerably shorter period of time by the addition of silica seed particles in general. To show the reproducibility of our results all the experiments were conducted by 2-fold crystallization batches. In addition to the 2-fold crystallization batches that were always performed, the experiment on the 200 nm silica seeds was repeated (red circles in Figure 4b) to confirm the high reproducibility of our experiments. Thus, a slight trend toward a faster depletion of supersaturation with increased seed particle size is deduced. It is important to mention that after crystallization the LSZ activity was assayed with no significant loss for the unseeded and seeded batches (see Supporting Information S7). These results indicate a strong dependence of the crystallization process on the seed particle size. With increasing seed size and thus decreasing curvature and increasing surface charge density, stable clusters are formed by heterogeneous nucleation events. This leads to a tremendous reduction of induction and crystallization times. We attribute this effectas already shown for the extension of crystallization conditions in the microliter-scale experimentsto the promoted adsorption of LSZ molecules on the seed particle surface. With increasing seed particle size more LSZ molecules adsorb to the particle surface, probably even in a preferred orientation, acting as a template for crystal formation. The further agglomeration of seed particles with adsorbed protein and a subsequent rearrangement of molecules might enhance the crystal formation and growth steps. However, the original motivation of this study for protein crystallization was to form LSZ crystals with improved filtration properties. Therefore, compact crystals with large diameters are needed. Thus, the obtained CSDs at the end of the crystallization experiments are an important criterion for quality control. Figure 5 exemplarily shows light microscopy

Figure 6. (a) Cumulative CSD after milliliter-scale crystallization experiments and (b) corresponding x50.3 values at t = 200−300 min, cNaCl = 50 g/L, cLSZ* = 3.3 g/L; S0 = 9.0; afor = 1.5 × 1016 nm2/mL. The dashed and broken line is a guide to the eye.

measured at the time where 60% of the initial LSZ concentration was converted to solid material. This corresponds, depending on the induction time and the course of supersaturation depletion, to absolute times of 200−300 min after the addition of NaCl. Figure 6b summarizes the therefrom derived values of x50.3 values in dependence of the added seed particle size. Without the addition of particles an x50.3 value of 34.4 μm was measured. With a seed particle size of 10 nm the x50.3 value shifts up to 47.1 μm. An increase of the seed particle size again leads to smaller x50.3 values down to 37.4 μm for the addition of 200 nm silica NPs. In general and as the most important finding, higher x50.3 values were determined for all samples containing seed particles at significantly reduced total process times. However, we do not think that the operating points analyzed in this study are already optimum conditions for the largest crystals which are influenced by both nucleation and growth. A constant foreign particle surface was provided in our experiments which was realized by different seed number concentrations. Because of the promoted heterogeneous nucleation for larger seed particles, most of the supersaturation is depleted by nucleation and only to a reduced extent by the growth of the crystals. This effect would lead to smaller x50.3 values with a narrow CSD and explains the reciprocal trend of decreasing x50.3 values with increasing seed particle diameter. One further explanation for the differences in the CSDs might be agglomeration of the conjugates or larger building blocks, respectively. A higher collision kernel for larger seed particles might as well lead to shorter induction times, whereas a higher number of small seed particlesdue to a constant foreign particle surfaceincreases the aggregation rates which causes the formation of larger protein crystals. Further crystallization experiments conducted at our institute with a variation of the added amount of seed particles confirm this assumption in a way that a higher amount of seed particles leads to a significant shift of the CSD to larger crystal sizes (and to more defined crystals). Thus, our study can be seen as a starting point for tailored protein crystallization. However, we successfully transferred microliter-scale crystallization experiments to the milliliter scale and received crystals at significantly reduced process times. Together with the results from the characterization of the conjugates it was possible to understand the

Figure 5. Microscope images of LSZ solid material from milliliter-scale crystallization experiments without (left) and with (right) the addition of 200 nm seed particles; arrows indicate well-defined crystal faces; cNaCl = 50 g/L; cLSZ* = 3.3 g/L; S0 = 9.0; afor = 1.5 × 1016 nm2/mL.

images of the formed solid material without (left) and with (right) the addition of 200 nm silica seed particles obtained after 5 h at crystallization conditions of Figure 4. For the seeded experiment a higher quality of solid material with defined crystal faces (tetragonal crystals) and larger diameters is obtained. This is always the case if silica seed particles are present in the supersaturated protein solution and thus strongly indicates a more structured nuclei formation and growth of the protein crystals. 3591


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Materials” at Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU). The authors also gratefully would like to thank Stefan Romeis, Dr. rer. nat. Jochen Schmidt, Paula Hoppe and Patrick Herre (Institute of Particle Technology, FAU ErlangenNürnberg) for their support regarding the characterization of silica particles by SEM, TEM, ICP-OES and infrared spectroscopy (results can be found in the Supporting Information). Moreover, the authors would like to thank Dr. rer. nat. Benedikt Schmid and Prof. Dr. Yves Muller from the Division of Biotechnology (Department of Biology, FAU ErlangenNürnberg) who provided the opportunity to perform CD measurements.

influence of the seed particle curvature and to link molecular interactions with crystal properties.

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CONCLUSIONS In this study we correlate the conjugate properties of LSZ and differently sized silica NPs on a molecular level with their later performance as seed particles for heterogeneous protein crystallization. During the characterization of conjugates, a first analysis of zeta potentials measured upon LSZ addition, revealed a charge reversal of negatively charged silica NPs due to the adsorption of positively charged LSZ molecules. This confirms that electrostatic interactions are of major importance. However, already from the zeta potentials an influence of the different silica particle sizes ranging from 10 to 200 nm was noticed and ascribed to an increasing surface charge density for larger particles. Adsorption experiments performed in the next step reveal that with decreasing surface charge density and particle diameter and thus increasing surface curvature considerably less LSZ is adsorbed. This size effect is not only in agreement with the literature but was also observed for the seeded crystallization of LSZ. First of all, state diagrams generated from microliter-scale experiments show extended crystallization conditions with the addition of silica seed particles in general. Moreover, this extension of conditions toward lower salt and protein concentrations was significantly enhanced with increasing seed particle size. A transfer of crystallization experiments to the milliliter-scale reveals a dramatic reduction of the induction times for the formation of LSZ crystals. With increasing seed particle size this effect was intensified leading to a 4.8-fold reduction of the induction time for the addition of 200 nm seed particles compared to the unseeded case. In general, a higher reproducibility with respect to induction times of crystal formation was found for the seeded batches while preserving enzyme activity. It was also shown that the final crystal size distribution (CSD) was shifted to higher x50.3 values with the addition of the differently sized silica NPs, which is a very important step toward improved crystal properties. Thus, our work paves the way for a reproducible and controllable protein crystallization process aiming at a successful product recovery at the industrial scale.

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ASSOCIATED CONTENT

S Supporting Information *

Characterization of the used silica particles; impact of particles on the structure of LSZ investigated by intrinsic and extrinsic fluorescence spectroscopy; reference experiments for the determination of adsorption isotherms; complementary state diagrams from microliter-scale crystallization; LSZ activity after crystallization using silica seed particles. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/cg501681g.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank the Deutsche Forschungsgesellschaft (DFG) within the framework of its “Excellence Initiative”, the Cluster of Excellence “Engineering of Advanced 3592


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Enhanced Nucleation of Lysozyme Using Inorganic Silica Seed

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