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Thermodynamic study of the interaction of Bovine Serum Albumin and amino-acids with cellulose nanocrystals Salvatore Lombardo, Samuel Eyley, Christina Schütz, Hans van Gorp, Sabine Rosenfeldt, Guy Van den Mooter, and Wim Thielemans Langmuir, Just Accepted Manuscript • Publication Date (Web): 11 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017
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Thermodynamic study of the interaction of Bovine Serum Albumin and amino-acids with cellulose nanocrystals
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Salvatore Lombardo,1 Samuel Eyley,1*, Christina Schütz,1 Hans van Gorp,3 Sabine
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Rosenfeldt,4 Guy Van den Mooter,2 Wim Thielemans1*
1 2
6 7
1
8
Engineering, KU Leuven, Campus Kulak Kortrijk, Etienne Sabbelaan 53 box 7659, 8500
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Kortrijk, Belgium.
Renewable Materials and Nanotechnology Research Group, Department of Chemical
10 11
2
12
Sciences, KU Leuven, O & N II, Herestraat 49 box 921, 3000 Leuven, Belgium.
Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological
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3
15
Celestijnenlaan, 200 F, 3001 Leuven.
Division of Molecular Imaging and Photonics, Department of Chemistry, KU Leuven
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4
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Universitätsstrasse 30, 95440 Bayreuth, Germany.
Physical
Chemistry
I
and
Bavarian
Polymer
institute,
University
Bayreuth,
19 20 21
*
[email protected] 22
*
[email protected] 23 24
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Abstract
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The interaction of bovine serum albumin with sulfated, carboxylated and pyridinium-grafted
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cellulose nanocrystals was studied as a function of the degree of substitution by determining
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the adsorption isotherm and by directly measuring the thermodynamics of interaction. The
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adsorption of BSA onto positively charged pyridinium-grafted cellulose nanocrystals
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followed Langmuirian adsorption with the maximum amount of adsorbed protein increasing
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linearly with increasing degree of substitution. The binding mechanism between the positively
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charged pyridinum-grafted cellulose nanocrystals and BSA was found to be endothermic and
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based on charge neutralization. A positive entropy of adsorption associated with an increase
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of the degree of disorder upon addition of BSA compensated for the unfavorable endothermic
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enthalpy and enabled formation of pyridinium-g-CNC-BSA complexes. The endothermic
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enthalpy of adsorption was further found to decrease as a function of increasing degree of
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substitution. Negatively charged cellulose nanocrystals bearing sulfate and/or carboxylic
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functionalities were found not to interact significantly with the BSA protein. To investigate in
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more detail the role of single amino acids in the adsorption of proteins onto cellulose
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nanocrystals (CNCs), we also studied the interaction of different types of amino acids with
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CNCs, i.e. charged (lysine, aspartic acid), aromatic (tryptophan, tyrosine), and polar (serine)
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amino acids. We found that none of the single amino acids bound with CNCs irrespective of
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surface charge and that therefore the binding of proteins with CNCs appears to require larger
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amino acid sequences that induce a greater entropic contribution to stabilize binding. Single
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amino acids are thus not adsorbed onto cellulose nanocrystals.
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Introduction
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The self-assembly of nanoparticles into ordered structures has already been investigated
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widely to control the fabrication of materials and devices with new or improved optical,
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electronic, magnetic, mechanical, or active properties.1 Controlling this self-assembly process
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is paramount for the reproducible production of materials with consistent properties, and is
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thus a large focus of current research efforts.2 A more fundamental understanding of the
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thermodynamics of the interactions at play is crucial to develop the insights needed to exert
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the necessary control over these self-assembly processes. For this purpose isothermal titration
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calorimetry (ITC) is a promising technique. It can be used to directly probe the
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thermodynamic interactions in real time during nanoparticle assembly. Pioneering studies
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were reported in 2004, in which ITC was employed to characterize the energetics of
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interaction between gold nanoparticles with biological ligands such as amino acids3 and
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DNA.4 However, the use of this technique in nanotechnology is still in its infancy, with few
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documented studies in the literature to date.5 One reason may be that this technique
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experiences many challenges and more detailed studies are required to make ITC a more
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common technique in nanotechnology. The main difficulty comes from the fact that heat is a
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global parameter, therefore impurities or secondary processes can also give rise to heat
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exchange, which could cover or alter the real heat of interaction. Therefore high purity of the
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samples and high control over the system under investigation is a major requirement.
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In this work we used ITC to investigate the interaction of the protein bovine serum albumin
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(BSA) and different amino acids with cellulose nanocrystals (CNCs). Rod-shaped cellulose
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nanocrystals (CNC) are attractive for new material development because of their abundance,
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renewability, physicochemical properties such as high surface area, low density, mechanical
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strength, and potentially low production cost.6,7,8 A variety of possible applications have been
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reported, such as reinforcing agents, optical materials, electronics supports, paint additives
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and stabilizers, and in security papers.7,9,10 Rod-shaped CNCs with variable dimensions can be
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obtained from different cellulose sources such as cotton, wood, and tunicates through a
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controlled hydrolysis with inorganic acids. When using sulfuric acid, the CNC particles are
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stabilized in aqueous suspension through the repulsion of the deprotonated sulfate ester
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groups, which are grafted on the surface of the nanoparticles during the acid hydrolysis.11
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Sulfated CNCs self-assemble in water to form chiral nematic structures.12 In these structures,
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the cellulose nanoparticles are organized in left-handed helicoids with pitch values typically
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around 1 to 2 µm (just above the reflection wavelength of visible light).6,13 This pitch can be
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reduced by increasing the concentration of CNCs,6 increasing the ionic strength,14 addition of
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glucose13, and ultrasonication.15 Expanding on these ideas further, changing the environment
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through the addition of other nanoparticles, proteins, polymers or small molecules will also
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alter the interactions between CNCs. Understanding how these interactions can be
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manipulated is crucial to generate ordered structures, and thus macroscopic functional
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materials, in a controlled manner.2 Another way to tailor the interactions between
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nanoparticles and of nanoparticles with its environment is by altering their surface
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functionalities. Virtually any functional groups can be inserted on the surface of cellulose
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nanocrystals.16 There is also interest in controlling nanocellulose interactions with biological
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systems for their applications as for example drug delivery carriers.17 To this effect, in vivo
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studies have already highlighted the biocompatibility of CNCs in mice.18 However, the use of
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cellulose nanocrystals as drug carriers or other agents in biological systems requires a
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fundamental understanding of the interactions of cellulose nanoparticles both with simple and
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complex biomolecules. Guo et al. investigated the thermodynamics and the specificity of the
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binding of cellulose nanocrystals with cellulose binding module.19 This same group also
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identified and characterized the minimum peptide sequence composed of seven residues
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which was able to bind cellulose nanocrystals, while also studying the thermodynamics of the
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process using ITC.20
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A better understanding of the interactions between nanocellulose and biomolecules is also
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needed to improve enzymatic degradation processes, which are important for the use of CNCs
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in commercial applications. Recently, it has been shown that the addition of bovine serum
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albumin (BSA) improves the enzymatic degradation of cellulose.21 Formation of a so-called
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protein corona (protein adsorption layer) in biological medium formed by protein-
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nanoparticle interactions has not been reported for CNCs, while it has been widely studied in
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BSA-gold nanoparticle systems.22,23 As the interaction of nanoparticles with living matter is
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dictated by the protein corona it is important to understand its formation and structure in much
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more detail.
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The adsorption of BSA onto ten different cellulose derivatives could be described by
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Langmuirian adsorption and was found to depend on pH, ionic strength, and surface charge.24
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In addition, Lavenson et al. used magnetic resonance imaging combined with UV-
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spectroscopy to investigate the adsorption of BSA onto four different cellulose fibers,
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showing that only a small amount of protein was adsorbed. More specifically the authors
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showed a decrease in the free protein concentration of ~ 5%, however, the authors state that
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this value is smaller than error range of the technique employed (10%).25 This was later
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confirmed by surface plasmon resonance measurements published by Orelma et al., who also
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highlighted that a charge difference between BSA and cellulose would increase the adsorption
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efficiency dramatically.26 In addition, Taajamaa et al. showed that BSA does not adsorb on
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pure cellulose.27 Finally, Zimnitsky et al. then investigated the adsorption of various amino
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acids onto carboxymethyl cellulose fibers, showing multilayer adsorption on oxidized
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cellulose at high concentration of amino acids in the treatment medium.28 The
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thermodynamics of the adsorption of BSA onto CNCs has not yet been reported in the
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literature and could help to explain some of the apparently conflicting results mentioned
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above. The dependence of the interactions as function of the amount of surface functionality
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been also not yet been described.
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In this work, we have studied the interactions of BSA and single amino acids with cellulose
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nanocrystals. To investigate the effect of surface characteristics we used sulfated,
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carboxylated and pyridinium-grafted cellulose nanocrystals with different degrees of
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substitution. We also investigated the effect of addition of free amino acids into the CNC
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suspension to measure interactions between single residues and cellulose nanocrystals.
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Experimental
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Materials and Methods
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Cotton wool, hydrochloric acid (37%, extra pure), pyridine (99%, for synthesis), sodium
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hydroxide (99%, p.a., ISO, in pellets), dichloromethane (99.5%, for synthesis), L-Tryptophan
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(>98.5%), sodium hypochlorite 12%, and sodium bromide were purchased from Carl Roth.
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Bovine serum albumin (>96%) and L-Lysine (>98%) were purchased from Sigma-Aldrich.
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Sulfuric acid (95%), pyridine (AnalaR NORMAPUR, 99.7%), methanol (99.8%) and ethanol
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(absolute) were purchased from VWR international. p-Toluenesulfonyl chloride (98%), 4-(1-
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bromoethyl)benzoic acid (98%) and 4-(bromomethyl)benzoic acid (97%) were purchased
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from Alfa Aesar. L-Tyrosine was purchased from TCI, (2,2,6,6-Tetramethylpiperidin-1-
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yl)oxyl (TEMPO), L-Aspartic acid (>98%) and L-Serine were purchased from Acros
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Organics.
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Synthesis and purification of sulfated cellulose nanocrystals was made by standard sulfuric
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acid hydrolysis as previously described.29 Sulfated CNCs were used as starting materials for
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further surface functionalization. Cationic pyridinium-grafted CNCs were synthesized using a
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previously described one pot reaction, using 4-(1-bromoethyl/bromomethyl)benzoic acid and
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4-toluenesulfonyl chloride as reagents with pyridine as a solvent.30 Different degree of
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substitution were obtained by varying reaction temperature, time and stoichiometry of
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reagents (see supplementary information for details). Carboxylated cellulose nanocrystals
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were prepared via oxidation with (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO), as
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previously described.31 The procedures used for the preparation of the different CNCs
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employed in this work are described in detail in the supplementary information. All the
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nanocrystals used in this work were fully characterized by methods developed in our
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laboratory which employed various techniques.32 Infrared Spectroscopy (IR) was used to
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verify that the modifications was performed successfully; Elemental Analysis (EA) was to
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determine the amount of modification; Thermogravimetric Analysis (TGA) was used to
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estimate the water content; X-ray Photoelectron Spectroscopy (XPS) was employed to
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measure the elemental composition on the surface of the nanoparticles; X-ray Diffraction
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(XRD) was employed to determine the crystallinity of the CNCs; a detailed morphological
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analysis was performed using Atomic Force Microscopy (AFM) for the determination of the
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cellulose nanoparticle lengths and Small Angle X-ray Scattering (SAXS) was used to
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determine the width. Full characterization data are reported in the supplementary information.
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Adsorption isotherm experiments
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Binding constants describing the adsorption of BSA onto cellulose nanocrystals at 25°C were
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determined from adsorption isotherms. All measurements were carried out in glass vials
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containing serial dilutions of BSA mixed with an equal volume of an aqueous suspension of
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CNC to a final cellulose concentration of 1-2 mg/ml. All adsorption experiments were carried
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out at least in duplicate. Both positively charged (pyridinium-grafted) and negatively charged
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(sulfated or carboxylated) cellulose nanocrystals were used. Each suspension was also stirred
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for at least 15 minutes before measurement. Previously reported work investigating CNC-
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protein interactions used centrifugation to remove CNC-bound proteins quantitatively.19 In
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this work, we used filtration with a 0.2 µM syringe-filter to retain the CNCs, with or without
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bound BSA, as tests showed that this method resulted in a more effective separation than
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could be achieved with centrifugation. The filtrate containing unbound BSA was measured
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spectrophotometrically at 280 nm using a Shimadzu UV-1800 spectrophotometer. The
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equilibrium association constants (Ka) describing the adsorption of BSA onto cellulose
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nanocrystals were determined by nonlinear regression of bound versus free protein
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concentrations to a one binding site Langmuir model.19 The equation used was the following:
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B=
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where F is the concentration of free protein, determined from the maximum absorption at 280
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nm; B is the concentration of bound protein per gram of cellulose, calculated by subtracting F
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to the starting protein concentration; and Bmax is the maximum amount of adsorbed protein,
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determined from the experiment. These experiments were also carried out for the aromatic
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amino acids tryptophan and tyrosine as they can be detected in solution by UV-Vis
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spectroscopy.
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Isothermal titration calorimetry
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Calorimetric experiments were performed using a TAM III isothermal calorimeter (TA
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Instruments). The experiments were carried out at neutral pH in aqueous solution. In order to
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minimize the pH change during the experiments, the protein was dialyzed against MilliQ
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water, and the dialysate was used to suspend the nanoparticles and to dilute the protein
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solutions. The measured pH of BSA was in the range 7-7.5. All samples were thoroughly
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degassed by sonicating for 15 minutes under vacuum before the experiments. The system was
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equilibrated at 25°C while stirring at 200 rpm until the baseline shift was
