Adsorption of lactoferrin and bovine serum albumin nanoparticles on pellicular two-layer agarose-nickel at reactive blue 4 in affinity chromatography

https://doi.org/10.1016/j.jece.2021.105084Get rights and content

Highlights

  • A novel two-layer AN@RB4 absorbent by three-phase emulsion method was fabricated.

  • Absorptions of Lf and BSA nanoparticles by 2L-AN@RB4 were studied and compared.

  • A pseudo-second-order model was proposed to fit the dynamic behavior of adsorbent.

  • Effects of pH, contact time, and initial concentration on adsorption were studied.

  • The Langmuir isotherm model was found to best describe the adsorption kinetics.

Abstract

In this study, adsorption of lactoferrin (Lf) and bovine serum albumin (BSA) nanoparticles on pellicular two-layer agarose-nickel immobilized by reactive blue 4 dye-ligand (2L-AN@RB4) was investigated. 2L-AN@RB4 was prepared using the three-phase emulsion method. The dynamic light scattering was first employed to quantify size distribution of Lf and BSA nanoparticles. Then, scanning electron microscopy (SEM) and atomic force microscope (AFM), respectively, were used to characterize structures of the two types of nanoparticles and adsorbent beads. SEM and AFM images demonstrate that shapes of the nanoparticles are globular and relatively uniform, where no adhesions were observed. Finally, adsorption behaviors of both Lf and BSA nanoparticles on 2L-AN@RB4 in affinity chromatography were investigated, with focus on the adsorption kinetics. Influences of contact time, pH, and initial concentration were analyzed to investigate the adsorbent behaviors in the expanded bed column. The influence of contact time on adsorption of Lf versus BSA nanoparticles indicates that 4 h is enough to adsorb protein models equal to 45%. Results indicate that Lf nanoparticles have a higher rate of around 83% than that of BSA nanoparticles. The increases in pH have negative effect on adsorption while opposite trend was found for initial concentration. Adsorption isotherm results reveal that the Langmuir and Freundlich isotherm models fit both adsorption kinetics of Lf and BSA very well.

Introduction

Model protein nanoparticles have been increasingly used for a variety of applications, including the delivery of therapeutic and diagnostic agents in medical areas [1]. In those applications, purification of model protein nanoparticles sometimes plays a vital role as purity is the prerequisite for medicines to perform effectively [2]. Among various techniques for purification, adsorption has attractive continuous interest due to its relatively simple operation and post-processing [3]. It has been widely recognized that the efficiency of adsorption depends heavily on the used adsorbent [4], [5]. Therefore, securing and preparation of high-performance adsorbent lies in the core of adsorption studies for model protein nanoparticles.

So far, many adsorbents have been applied for the purification of model protein nanoparticles [6]. The majority of adsorbents developed so far are based on polymers. However, for applications like purification and adsorption of model protein nanoparticles, eco-friendly polymers sometimes cannot grant the necessary structures for mechanical strength [7]. For this reason, researchers have turned to various approaches to remedy this shortcoming. The polymer coating is a promising way to increase mechanical strength while preserving the adsorption performance. The metal core can increase the overall hardness of adsorbents with polymer layers and consequently increase the mechanical strength. As a metal lack of toxicity, nickel has been largely chosen as the metal core [8]. Similarly, as a major choice for polymer layer, agarose has been extensively employed in two-layer absorbent synthesis and its combination with nickel has shown many attractive merits [9]. Thus, this research used agarose-nickel as the adsorbent for adsorption of model protein nanoparticles.

As two of most widely encountered model protein nanoparticles in medical use, lactoferrin (Lf) and bovine serum albumin (BSA) nanoparticles, their purification using adsorption is of particular importance and their adsorption performance using different adsorbents have been largely explored [10], [11], [12], [13]. Among the studied adsorbents, agarose-nickel has separately exhibited promising adsorption performance for these two types of model protein nanoparticles. However, it seems that a comparative study to show the difference of adsorption for these two types of model protein nanoparticles on agarose-nickel has not appeared. Therefore, this work was oriented to close this gap to explicitly show their adsorption difference on agarose-nickel.

As a promising technique to investigate adsorption, expanded bed adsorption in affinity chromatography has been recognized as the mainstream platform for adsorption of nanoparticles [14], [15], [16]. Our previous work about adsorption of Lf and BSA nanoparticles on agarose-nickel adsorbent also shows that expanded bed adsorption in affinity chromatography demonstrates reliable capacity [41]. However, for most adsorbents to function normally in expanded bed within affinity chromatography, usually a dye-ligand is required to support adsorbents. In our previous work, cibacron blue 3GA (CB3) was chosen as the dye-ligand. However, we found that the performance of CB3 is not satisfactory because the sulfonic-acid isomer is at the ortho position. Reactive blue 4 (RB4), a sulfonic-acid ring A positioning either at meta or para, can interact better with proteins through either hydrophobic or electrostatic force [17]. Therefore, in this study, RB4 was used as the dye-ligand.

In this work, a two-layer agarose-nickel@reactive blue 4 (2L-AN@RB4) composite was first fabricated using a three-phase emulsion method and then used to adsorb Lf and BSA nanoparticles. The properties of Lf and BSA nanoparticles, and 2L-AN@RB4 were characterized using scanning electron microscopy (SEM), atomic force microscopy (AFM), and dynamic light scattering (DLS). To investigate the behaviors of adsorbent in the expanded bed column, the effects of contact time (4 h), kinetic adsorption (Pseudo-second order equation), pH (2–12), initial concentration (0.5–4 mg/mL adsorbent), and adsorption isotherms were assessed.

Section snippets

Materials

Pure agarose, acetone, and Lf powder were obtained from Merck (Germany). Epichlorohydrin, diethyl ether, glutaraldehyde, and ethanol were purchased from Amersham Biosciences (England, UK Ltd.). BSA was purchased from Equitech Bio, Inc. (Kerrville, TX, USA). Sodium hydroxide, hydrochloric acid, sodium chloride, and glycerol were obtained from Daejung Chemicals and Metals (Siheung, Korea). Sorbitan monooleate (Span® 80), reactive blue 4 dye-ligand, and silicone oil were supplied by Sigma-Aldrich

Characterization of nanoparticles and adsorbents

The surface morphologies of Lf and BSA nanoparticles are shown in Fig. 1. It can be seen that both Lf and BSA nanoparticles exhibit regular and smooth spherical morphologies, where high uniformity and no adhesion are observed. Besides, thorough inspection of the AFM images of Lf and BSA nanoparticles indicates that they possess irregular and rough surfaces. A comparison between these images shows that the BSA nanoparticles appear to be smaller and more spherical, which may be due to the

Conclusion

In this paper, a novel adsorbent 2L-AN@RB4 was successfully fabricated by three-phase emulsion. Also, Lf nanoparticles and BSA nanoparticles were prepared to investigate the adsorption process and compared with each other in the NBG contactor to meet affinity chromatography. The influence of contact time on the adsorption of Lf versus BSA indicates that 4 h is enough to adsorb protein models equal to 45%. Lf nanoparticles has a higher rate of around 83% than that of BSA nanoparticles. The

CRediT authorship contribution statement

Roozbeh Mofidian: Investigation; Methodology; Formal analysis; Writing - original draft. Qingang Xiong: Conceptualization; Project administration; Supervision; Resources; Writing - review & editing. Ali Mohammad Ranjbar: Methodology. Mohammad Ali Sabbaghi: Data curation. Amin Farhadi: Data curation. Seyed Mehdi Alizadeh: Formal analysis.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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