Pitfalls in Size Characterization of Soft Particles by Dynamic Light

Oct 4, 2017 - Pitfalls in Size Characterization of Soft Particles by Dynamic Light Scattering Online Coupled to Asymmetrical Flow Field-Flow Fractiona...
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Pitfalls in Size Characterization of Soft Particles by Dynamic Light Scattering on-line coupled to Asymmetrical Flow Field-Flow Fractionation Simona Sitar, Valerija Vezo#nik, Peter Macek, Ksenija Kogej, David Pahovnik, and Ema Žagar Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03251 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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Analytical Chemistry

Pitfalls in Size Characterization of Soft Particles by Dynamic Light Scattering on-line coupled to Asymmetrical Flow Field-Flow Fractionation Simona Sitar1, Valerija Vezočnik2, Peter Maček2, Ksenija Kogej3, David Pahovnik1, and Ema Žagar1* 1

National Institute of Chemistry, Department of Polymer Chemistry and Technology, Hajdrihova 19, 1000 Ljubljana, Slovenia 2 University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, 1000 Ljubljana, Slovenia 3 University of Ljubljana, Faculty of Chemistry and Chemical Technology, Department of Chemistry and Biochemistry, Večna pot 113, 1000 Ljubljana, Slovenia *Corresponding Author E-mail: [email protected] Phone: 00386 1 47 60 203 Fax: 00386 1 47 60 300 ABSTRACT: Asymmetrical flow field-flow fractionation (AF4) technique coupled to a multi-angle light-scattering (MALS) detector with embedded dynamic light-scattering (DLS) module was introduced to study the size-characteristics and shape of soft particles of various size and type; polystyrene nanosphere size standards, lipid droplets (LD), and large unilamellar vesicles (LUV). A range of flow velocities through the LS detector, at which accurate hydrodynamic size can be extracted from the DLS in flow mode, was studied since the particles subjected to a longitudinal flow exhibit not only the Brownian motion due to diffusion, but also the translational movement. In addition, the impact of the longitudinal flow velocity on the shape of the artificial LUV of two different sizes and two different compositions was studied by MALS. For comparison, the conventional batch DLS and static light-scattering (SLS) experiments without prior sample separation by size were performed. From a combination of batch and flow light-scattering results we concluded that the passage flow velocities at the detector used in this study, 0.2, 0.5, and 1 mL/min, have no significant impact on the shape of spherical vesicles, however, the flow DLS experiments give accurate hydrodynamic radius (Rh) only at the lowest investigated passage flow rate at the detector (0.2 mL/min). With increasing rate of passage flow at the DLS detector, the error in the accuracy of the Rh determination rapidly increases. The error in Rh depends solely on the detector flow rate and particle size, but not on the type of the soft particle.

Dynamic (DLS) and static light-scattering (SLS) are very powerful and useful tools for studying the structural and dynamic properties of macromolecules/particles in solution, emulsion or suspension. Light-scattering (LS) techniques can provide quantitative information on particle size, shape and internal structure of scattering objects.1,2 The major advantage of LS over some alternative techniques of particle sizing is in instantaneously, noninvasively performed measurements which provide an absolute estimate of particle size. However, despite of these advantages one of the major limitation of DLS and SLS methods is their accurate application for heterogeneous and highly polydisperse systems, where much stronger scattering from larger particles may obscure the scattering from the smaller particles. Analysis of LS data of such complex systems becomes complicated and the determined size distribution by DLS often does not represent real situation, and in addition, the resolution is poor. In order to improve performance of the so-called batch LS analysis of heterogeneous or

polydisperse systems a fractionation of samples prior to LS measurements is necessary. On-line coupling of size-based separation technique like asymmetrical flow field-flow fractionation (AF4) to LS detectors is able to improve the performance of LS analysis of heterogeneous samples, since the average size as well as size distribution of the fractionated species can be determined more correctly.3-5 Using AF4 separation method with continuous-flow LS detectors; multi-angle light-scattering (MALS) and/or DLS, was proven to provide better understanding of the investigated heterogeneous systems in comparison to DLS and SLS performed in batch mode.6,7 However, in AF4 analysis particles are subjected to flow in the system. It is well known that flow does not affect the physical properties of particles determined by MALS as long as they retain their conformation, since the extracted information is based solely on angular dependence of the scattered light intensity. On the contrary, the effectiveness of DLS data analysis of particles subjected to flow in the system

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is still relatively less understood. Namely, when a flow is induced in a system of particles exhibiting Brownian motion, additional translational component as a result of longitudinal movement of particles occurs. Fuller and coworkers8 developed a theoretical framework for the interpretation of DLS data for the flowing systems. Chowdhury et al. (1984)9 reformulated theoretical expression for the intensity autocorrelation function of light scattered from a system of particles undergoing both random diffusional motion and uniform translational motion. They first introduced the so-called modified time correlation function. Leung et al.10 studied the impact of the flow conditions in the system on the extracted size of the polystyrene latex particles in suspension by DLS using linear cumulant analysis in comparison to the analysis using the modified time correlation function. The results of the cumulant analysis showed rapid decay in the apparent particle size with increasing flow velocity, which was more pronounced for larger particles. By using the modified correlation function the determined particle size remains relatively unaffected by the flow velocity, especially for small particles. In this work DLS on-line coupled to AF4 was used to study the influence of the longitudinal flow velocity on the effectiveness and accuracy of size-characterization of soft particles of different size and type; polystyrene nanosphere standards, artificial lipid droplets, and lipid vesicles. The main goal was to determine a range of flow rates where the particle sizing by flow DLS is still accurate. As a benchmark for accurate flow DLS performance the suspensions of polystyrene nanosphere standards of different, but known size were studied. Moreover, with MALS the size and shape of artificial lipid vesicles in dependence of the rate of passage flow at the detector (the socalled detector flow rate) were monitored to perceive the possible changes in vesicle conformation as a consequence of particle deformation in longitudinal flow. Namely, due to specific vesicle structure (spherical particles composed of unilamellar or multilamellar phospholipid bilayer, surrounding an aqueous core11), which enables membrane fluidity and bending elasticity,12 lipid vesicles, especially giant ones, are known to change their shape under influence of the flow.12-17 Considering that lipid vesicles are involved in many biological processes,18-20 and are used in wide range of pharmaceutical and medical applications, it is important to understand their behavior in flow conditions. Usually vesicles are highly polydisperse in size and thus their dynamics in flow has mostly been described by theoretical tools and numerical simulations,21-26 and only few experimental studies, most of them performed on vesicles flowing in micro-channel, are available to check the relevance of theoretical models.13,18 In addition to lipid vesicles which are quite flexible due to aqueous core, a more compact system - lipid droplets with hydrophobic core containing mainly neutral lipids - was also studied. Three different detector flow velocities were tested; 0.2, 0.5, and 1 mL/min. For comparison, the conventional DLS and SLS experiments were performed in batch mode, without prior sample separation by size.

EXPERIMENTAL SECTION Materials. Polystyrene (PS) nanosphere size standards suspended in water, with narrow size-distribution and defined size, i.e. radii: 30, 50, 100, and 250 nm (diameter: 60, 100, 200, and 495, respectively), and certified mean diameter: 60 ± 4 nm, 100 ± 3 nm, 203 ± 5 nm, and 496 ± 8 determined by photon correlation spectroscopy and validated by NIST (Na-

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tional Institute of Standards and Technology) were purchased from Thermo Scientific (Massachusetts, U.S.A.). Artificial large unilamellar vesicles (LUV) in two significantly different sizes were prepared from porcine brain sphingomyelin (SM) and wool grass cholesterol (Chol) (Avanti Polar Lipids, Alabaster, U.S.A.) in a SM/Chol molar ratio of 1/1 (LUV11) and 4/1 (LUV41) according to reproducible protocol as described before.27 Further in this study LUV with smaller size are designated as LUV-S (LUV11-S and LUV41S), and LUV with larger size as LUV-L (LUV11-L and LUV41-L). Lipid droplet nanoemulsions (LD) were prepared from trioleoylglycerol (TOG) (≥ 99%, Sigma-Aldrich, Munich, Germany), porcine brain SM, and wool grass Chol (Avanti Polar Lipids, Alabaster, U.S.A.) in a SM/Chol/TOG molar ratio of 1/1/4.7 (LD11) and 4/1/11.7 (LD41) as described in detail before.28 Asymmetrical flow field-flow fractionation (AF4). AF4 separations were performed using an Eclipse3+ system (Wyatt Technology Europe GmbH, Dernbach, Germany) connected to the isocratic pump, on-line vacuum degasser and autosampler (Agilent Technologies 1260 series, U.S.A.). Samples were separated in a trapezoidal-shaped channel equipped with the 350 µm spacer and Nadir Cellulose RC (Regenerated Cellulose) membrane with 10 kDa cut-off. The fractionated particles were detected with the on-line UV detector operating at 280 nm (Agilent Technologies, U.S.A.), multi-angle lightscattering detector (MALS) operating at a wavelength of 658 nm, and quasi-elastic light-scattering/dynamic light-scattering (QUELS/DLS) module embedded in the MALS at 99º (DAWN HELEOS, Wyatt Technology, U.S.A.). 90º MALS detector was calibrated using toluene, whereas other detectors were normalized with bovine serum albumin protein as an isotropic scatterer standard. As a running eluent 10 mM Hepes buffer with pH 8.0 and supplemented with 0.02 % w/v sodium azide (NaN3) as a bactericide was used for all samples, except for the 250 nm PS nanosphere standard for which 0.1 % (wt,v) sodium dodecyl sulfate (SDS) gave much narrower peak in the fractograms and better mass recovery than Hepes buffer where pronounced peak tailing and almost complete retention of the sample on the membrane were observed. The eluents were filtered through a Nylon 66 membrane with the pore-size of 0.45 µm (Supelco Analytical, U.S.A.). Between the HPLC pump and AF4 channel an additional filter with the pore size of 0.1 µm was placed (PEEK Inline Filter Holder). For details on AF4 experiments performance see Supporting Information. The theoretical aspects of AF4 instrumental technique can be found elsewhere.29,30 Batch mode dynamic light-scattering (DLS) and static light-scattering (SLS) measurements. Batch DLS and SLS were conducted using a 3D cross-correlation Spectrometer from LS Instruments GmbH (Fribourg, Switzerland), which is based on 3D technology specially designed to filter out multiple scattering from the total scattering. Two coherent incident light beams are generated with a 20 mV He-Ne laser operating at 632.8 nm. All SLS measurements were performed in cylindrical quarz cuvette at 25 ºC. Samples were filtered directly into the measuring cell through the hydrophilic Millex-HV filter with the pore size and diameter of 0.45 µm and 13 mm, respectively. Before each measurement, the sample was tempered for 15 minutes. Intensity of scattered light was collected in the angular range from 30° to 150°. For determination of

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particle Rg, the Zimm and Guinier fits were employed for the smaller (qRg 2. Thus, in our study it is reasonable to present results in the form of Kratky plot only for the LUV-L. Figure 8 presents AF4-MALS fractogram for LUV11-L together with the Kratky plots at the detector flow rate of 1 mL/min for five different elution times (at the peak apex and two elution times before and behind the apex) taking into account the Rg calculated at the selected elution time. Kratky plots for LUV11-L at detector flow 0.2 mL/min and Kratky plots for LUV41-L at 0.2 and 1 mL/min are shown in Figure S-4. Kratky plots exhibit, irrespective of the flow rate and elution time, a bell-shaped profile with a maximum value of (qRg)2P(θ) at around 1 for the qRg ~ √3, which agrees well with the theoretical curve for the hollow sphere, although for longer elution times at higher angles (above qRg around 4) some deviations from the theoretical curve are observed, which are ascribed to low concentration of the species eluted at the end of the fractionation peak for which the calculation of Rg is not so precise anymore due to rather poor scattering. Figure 8 shows also the theoretical Kratky plots corresponding to two extreme cases – the hollow sphere and rod. As seen, the experimental results presented in the form of Kratky plot show no major deviations in vesicle conformation as a consequence of longitudinal flow in AF4 system to which the particles are subjected to. Namely, if spherical vesicles would change to more elongated form under the flow conditions, a bell-shaped curve would probably be lost and instead a plateau would be formed, resulting in Kratky plot curve intermediate between the two idealized curves for the hollow sphere and rod.

Figure 8. AF4-MALS fractogram (left side figure) of LUV11-L together with corresponding Kratky plots (right side figure) constructed from light-scattering data extracted at five elution times in the fractogram recorded at a detector flow rate of 1 mL/min. Colored symbols represent Kratky plots of sample’s fractions eluted at shorter and longer elution times with respect to peak apex as depicted with the colored dashed lines in the fractograms.

Angular dissymmetry factor, defined as a ratio of light scattering intensities at supplementary angles θ and 180° – θ (zθ = Iθ / I180° – θ), is another parameter that can also be used for characterization of the particle shape. If zθ = 1, the particles are small as compared to the wavelength of light and show no angular dissymmetry, whereas dissymmetry becomes noticed as the Rg of particles exceeds ~1/20 of the wavelength of the incident light.39 From the angular dissymmetry of the scattered

light intensity usually the Rg is deducted. In this work, however, we only show the pattern of angular dissymmetry for lipid vesicles with size comparable to the laser wavelength, where instead of the Rayleigh theory a Mie theory should be used for the description of light scattered by such particles. Figure 9 shows a plot of dissymmetry factor calculated at 43° angle (z43) against particle size expressed by Rg. Usually dissymmetry factor is given at 45° angle, however in our MALS the closest angle is 43°. For all investigated samples, the dissymmetry factors were calculated at each elution time in fractograms obtained by AF4-MALS at the detector flow rates of 0.2 and 1 mL/min. In addition, the dissymmetry factors calculated for spherical monodisperse PS standards of four different sizes are plotted. In Figure 9 PS standards are presented with black symbols and designated as PS 30, PS 50, PS 100, and PS 250. The dissymmetry ratio for PS standards increases rapidly with increasing size of the sphere. The calculated z43 factors are consistent with the dissymmetry method theory for spherical particles, which shows a rapid increase of zθ with the size. On the contrary, more elongated particles, like rods, show much smaller increase in zθ with size. The dependences of z43 on particle size for the fractionated LD and LUVS samples is in excellent agreement with the z43 observed for 30, 50, and 100 nm polystyrene spheres, irrespective of the detector flow rate, thus confirming that LD and LUV-S maintain their spherical shape in the longitudinal flow.

Figure 9. Dissymmetry factors calculated at 43° angle for PS standards with radii 30, 50, 100, and 250 nm, as well as for LUV11-S (magenta); LUV11-L (blue); and LUV41-L (red) at a detector flow rate: a) 0.2 mL/min; and b) 1 mL/min.

At all investigated detector flow rates, the dissymmetry factor for LUV-L initially increases in accordance with the z43 values for the 50 and 100 nm PS standard. Above Rg/λ value for the 100 nm PS standard, the values of z43 factor even more rapidly increase with increasing vesicle size until a maximum at Rg/λ of around 0.23 is reached, above which a decrease in dissymmetry factor is observed. Knowing that the scattering pattern of 250 nm size PS standard can be classified as Mie scattering, it is suggested that the dissymmetry factor for LUV-L increases with the size for vesicles which can be classified as Rayleigh scatterers, whereas for the vesicles classified as Mie scatterers, a decrease in the intensity of dissymmetry factor is observed, although, for the same value of z43 = 17, both LUV-L reach a maximum at lower Rg/λ = 0.23 as compared to 250 nm PS standard with Rg/λ = 0.32 at z43 = 17; the above conclusion was made on the basis of the fact that the light scattering of the cross-linked nanospheres can indeed differ from the light scattering of vesicles with bilayer and fluid interior. This specific shape of dissymmetry factor reflects the fact that each type of scattering is characterized by its special angular distribution as already reported before.36

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CONCLUSIONS

ACKNOWLEDGMENT

In this study, AF4 coupled to DLS and MALS detectors was introduced in order to estimate the range of the detector flow velocities for which accurate size-measurements can be performed by flow DLS on one side, while on the other side, with MALS we followed the possible impact of the flow velocity on the shape of the lipid vesicles, the particles which are known to undergo deformation under the shear stress. In addition, batch DLS and SLS measurements were performed for comparison. As a benchmark for proper working of all instruments, SEC-MALS-DLS measurements were performed with calibrated polystyrene nanosphere standards. From a combination of batch and flow DLS results we concluded that flow DLS gives accurate Rh only at the lowest investigated detector flow velocity of 0.2 mL/min, whereas with increasing detector flow velocity the error in particle Rh determination rapidly increases. Flow DLS results for artificial lipid droplets and lipid vesicles as well as polystyrene size-standards showed that the error in Rh determination depends on the size of the examined particles only, irrespective of the soft particles’ physical properties. Namely, the percent error in Rh determination in dependence on the detector flow velocity for approximately 80 nm (200 nm) vesicles with fluid core and 75 nm (250 nm) PS nanosphere standard with dense core are very similar. Such observations were attributed to incorrect calculation method of the correlation function in flow DLS mode since our instrument uses a conventional cumulant DLS data analysis, which takes into account only the Brownian motion of the particles, whereas the particle translational movement under the flow is not included. SLS and DLS measurements in batch mode confirmed the spherical shape of artificial vesicles. From AF4-MALS, the Rg of vesicles at all used detector flow velocities were calculated as well as the Kratky plots and plots of dissymmetry factor as a function of size were constructed. The results show independence of the vesicle Rg on the detector flow rate, whereas the Kratky plots and dissymmetry factors show very good agreement with the theoretical plots for (hollow) sphere at all flow conditions. Such results reveal that the flow conditions used in this study did not affect the shape of the lipid vesicles. Probably higher flow velocity should be applied in order to achieve deformation of the vesicles studied; however, the maximum longitudinal flow rate through our AF4-MALSDLS instrument is 1 mL/min.

The authors gratefully acknowledge the financial support of the Ministry of Higher Education, Science and Technology of the republic of Slovenia, and the Slovenian Research Agency (Program P2-0145). The authors thank Prof. Dr. Vladimir Aseyev for the 70 nm polystyrene size-standard that was obtained as a gift.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. AF4 conditions, Light scattering theory, 3D plot figure, Kratky plot figure (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: 00386 1 47 60 203. Fax: 00386 1 47 60 300.

Notes The authors declare no competing financial interest.

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Figure captions Figure 1. AF4-MALS fractograms (solid curves represent normalized LS intensities at 90° angle) together with Rh from flow DLS (full symbols) and Rg from MALS (empty symbols) for 100 nm calibrated nanosphere PS standard at three different detector flow rates: 0.2 mL/min (black); 0.5 mL/min (red); and 1 mL/min (blue). For the detector flow rates 0.2, 0.5, and 1 mL/min we applied the following linear cross flow gradients: from 0.2 to 0.09 mL/min in 80 min, from 0.3 to 0.09 mL/min in 80 min, and from 0.5 to 0.09 mL/min in 80 min, respectively. Figure 2. Calculated a) Rh and b) Rg as a function of detector flow rate for calibrated PS nanosphere size standards with radii: 30 nm (■); 50 nm (●); 100 nm (▲); and 250 (♦) nm. The values at the detector flow rate of 0 mL/min correspond to batch DLS and SLS measurements. AF4 separation conditions for the 30, 50, and 100 nm PS standards were the same as those listed in caption of Figure 1. For the 250 nm particles, the cross flow linearly decreased from 0.2 to 0.09 mL/min in 90 min for the detector flow rates of 0.5 and 1 mL/min, whereas a constant cross flow rate of 0.1 mL/min for 90 min was applied for the detector flow rate of 0.2 mL/min. Figure 3. 3D plot representing angular dependence (13 angles) of scattered light as a function of elution time for monodispersed PS nanosphere standards with particle size (radius): a) 100 nm; and b) 250 nm as observed by AF4-MALS at a detector flow rate of 1 mL/min. Figure b is enlarged in order to better see the scattering pattern at larger angles. For 100 nm and 250 nm particles the cross flow linearly decreased from 0.5 to 0.09 mL/min in 80 min and from 0.2 to 0.09 mL/min in 90 min, respectively. Figure 4. Calculated a) Rh and b) Rg as a function of detector flow rate for LD11 (■); LD41 (□); LUV11-S (●); LUV41-S (○); LUV11-L (▲); and LUV41-L (∆). The values at the detector flow rate of 0 mL/min correspond to batch DLS and SLS measurements. Figure 5. AF4-MALS fractograms (solid curves represent normalized LS intensities at 90° angle) together with Rg from MALS (symbols) for: a) LD11; b) LD41; c) LUV11-S; d) LUV41-S; e) LUV11-L; and f) LUV41-L at three different detector flow rates: 0.2 mL/min (black); 0.5 mL/min (red); and 1 mL/min (blue). Figure 6. 3D plot representing angular dependence (13 angles) of scattered light as a function of elution time for fractionated LUV11-S as observed by AF4-MALS at a detector flow rate of 0.2 mL/min. Figure 7. 3D plot representing angular dependence (13 angles) of scattered light as a function of elution time for fractionated LUV11-L as observed by AF4-MALS at a detector flow rate of 0.2 mL/min. The bottom figure shows enlargement at larger angles. Figure 8. AF4-MALS fractogram (left side figure) of LUV11-L together with corresponding Kratky plots (right side figure) constructed from light-scattering data extracted at five elution times in the fractograms recorded at a detector flow rate of 1 mL/min. Colored symbols represent Kratky plots of sample’s fractions eluted at shorter and longer elution times with respect to peak apex as depicted with the colored dashed lines in the fractograms. Figure 9. Dissymmetry factors calculated at 43° angle for PS standards with radii 30, 50, 100, and 250 nm, as well as for LUV11-S (magenta); LUV11-L (blue); and LUV41-L (red) at a detector flow rate: a) 0.2 mL/min; and b) 1 mL/min.

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