Adsorption of Amino Acids, Aspartic Acid, and Lysine onto Iron-Oxide

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Adsorption of Amino Acids, Aspartic Acid and Lysine onto Iron-Oxide Nanoparticles Klementina Pušnik, Mojca Peterlin, Irena Kralj-Cigic, Gregor Marolt, Ksenija Kogej, Alenka Mertelj, Sašo Gyergyek, and Darko Makovec J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b03180 • Publication Date (Web): 10 Jun 2016 Downloaded from http://pubs.acs.org on June 14, 2016

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The Journal of Physical Chemistry

Adsorption of Amino Acids, Aspartic Acid and Lysine onto Iron-Oxide Nanoparticles Klementina Pušnik1,2, Mojca Peterlin1,3, Irena Kralj Cigić3, Gregor Marolt3, Ksenija Kogej3, Alenka Mertelj4, Sašo Gyergyek1, Darko Makovec1,2*

1

Department for Materials Synthesis, Jožef Stefan Institute, Ljubljana, Slovenia

2

Jožef Stefan International Postgraduate School, Ljubljana, Slovenia

3

Faculty of Chemistry and Chemical Technology, Ljubljana, Slovenia

4

Department of Complex Matter, Jožef Stefan Institute, Ljubljana, Slovenia

1 ACS Paragon Plus Environment


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ABSTRACT: Understanding the adsorption of amino acids (AAs) onto magnetic iron-oxide nanoparticles (SPIONs) is important not only for the preparation of the aqueous suspensions, but also for understanding the interactions at the bio-nano interface. In this investigation the adsorption of aspartic acid (Asp) and lysine (Lys) onto SPIONs was studied, based on a characterization of the suspension properties, i.e., measurements of the ξ-potential, the hydrodynamic size and the osmolality, and by direct HPLC analysis of the AA in the supernatants and at the nanoparticles of the ultra-centrifuged suspensions. The results show that the AAs adsorb onto the SPIONs in the form of large molecular associates, which decisively influence the nanoparticles’ surface properties. A measurement of the freezing-point depression using a Knauer osmometer proved that the molecular associates are already formed in the AA aqueous solutions.


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INTRODUCTION Superparamagnetic iron-oxide nanoparticles (SPIONs) are used in a broad range of technological and biomedical applications. For example, their technological uses include ferrofluids1 and magnetic separation.2−12 The use of SPIONs for separation ranges from laboratory-scale technologies, e.g., the separation of cells, viruses, proteins and nucleic acids26

, frequently as part of a biological analysis7 or in vitro medical applications8, to technologies

that require the mass production of stable aqueous suspensions of SPIONs with controlled surface properties. Such mass-production technologies can be applied in water remediation, e.g., the magnetic separation of heavy metals and organic pollutants from water,9-11 or in the food industry, e.g., the separation of microorganisms from beverages.12 Aqueous suspensions of nanoparticles are also often used as starting materials in the synthesis of nanocomposite materials.13 In addition, SPIONs can be used in medicine in vivo for diagnostics, e.g., for contrast enhancement in magnetic resonance imaging (MRI),14 in magnetic particle imaging (MPI),15 and in therapy, e.g., for targeted drug delivery16 and for cancer treatments using magnetically mediated hyperthermia.17 Iron-oxide nanoparticles are considered safe and have been approved by the US Food and Drug Administration (FDA) for in-vivo applications.8 Generally, the surface properties of nanoparticles have to be tuned to meet the requirements for different applications. Organic molecules are normally covalently bound or adsorbed onto the nanoparticle surfaces in order to engineer their surface properties, e.g., surface charge, hydrophilicity/hydrophobicity, or the availability of specific functional groups. As the nanoparticles are usually applied in the form of stable suspensions, the surface modification has to provide compatibility with the liquid medium and the repulsive forces between the nanoparticles preventing the agglomeration. For in vivo medical applications, the organic shell of the molecules should be nontoxic and biocompatible. The surface shell of the nanoparticles also significantly determines the interactions between the nanoparticles and living systems,



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for example, their interaction with blood. It influences the adsorption of plasma proteins onto the nanoparticles, the interactions with cells, the blood-circulation time, and the fate of the nanoparticles after intravenous administration.18 Additionally, it is usually necessary for the surface shell of the organic molecules to provide specific functional groups (functionalization) for the subsequent bonding/(bio)conjugation of different molecules that are needed in a specific application, e.g., targeting ligands, therapy agents, fluorophores, etc.8 One possible type of inexpensive, nontoxic and biocompatible molecule that can be used for the engineering of nanoparticle surface properties and for the stabilization of their aqueous suspensions is the amino acid (AAs). The large number of different AAs provides an opportunity to change the properties, e.g., the surface charge and the availability of different surface functional groups, over a broad range. Different AAs have been applied to prepare stable suspensions of iron-oxide nanoparticles.19-26 It was also proposed that the adsorbed AAs can be used for the functionalization of nanoparticles, providing specific functional groups at the nanoparticle surfaces for the subsequent conjugation of different molecules.19,20,23 As AAs play a very important role in the body, their adsorption onto the magnetic nanoparticles was proposed to aid their targeted delivery.25 Natural AAs also seem to be an ideal choice for the adaptation of surface properties and the preparation of colloidally stable aqueous suspensions of magnetic nanoparticles for applications in food processing.12 For all the mentioned applications the molecules should be bound to the nanoparticle surfaces with strong and stable bonds that prevent the molecules from being detached from the surface or exchanged with other ligands present in the liquid medium. The adsorption of AAs onto the surfaces of different minerals, including iron oxide, has been intensively studied.19-28 Apart from the possible preparation of the nanoparticle suspensions, the adsorption of the AAs is also important for prebiotic chemistry, in the general frame of evaluating Bernal’s hypothesis of prebiotic polymerization in the adsorbed


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state as a step in the organization of molecules in the sequence of organizational events leading to the emergence of life.27 The interactions between AAs and nanoparticles are also very important for understanding the interactions of the nanoparticles with polypeptides and proteins, crucial for understanding their fate after administration to the blood in medical applications or in understanding their potential hazards. It is widely accepted that the AAs adsorb onto the iron-oxide surface by forming a chemical bond of the chelate type, involving a carboxylate group and surface iron ions.19-21,23 However, the mechanisms of the AAs’ adsorption onto the nanoparticle surfaces are not yet clear and the literature results are frequently inconsistent. The mechanisms governing the adsorption of AAs onto the nanoparticles were mainly discussed on the basis of FT-IR or Raman spectroscopy, the nanoparticle properties (e.g., ξ-potential and colloidal stability of their aqueous suspensions), or on indirect measurements of their concentration in the suspension supernatants, e.g., by conductometry or by a colorimetric method with ninhydrin. In this investigation the adsorption of two α-amino acids, aspartic acid (Asp) and lysine (Lys), onto iron-oxide maghemite nanoparticles and the preparation of stable aqueous suspensions have been systematically studied. Under biological conditions, Asp (HOOC-CH2-CH(NH2)COOH) is an AA with a negatively charged carboxyl side group (pK1= 2.09, pK2= 3.86, and pK3= 9.82), whereas Lys (H2N-(CH2)4-CH-(NH2)COOH) has a positively charged amino side group (pK1= 2.2, pK2= 8.5, and pK3= 10.28). We combined direct measurements of the Asp and Lys concentrations in the suspensions using liquid chromatography (HPLC) with measurements of the suspension properties (ξ-potential, hydrodynamic size, osmolality) to reveal the mechanisms governing the adsorption of the AAs onto the SPIONs.

MATERIALS AND METHODS



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Materials. A list of chemicals used for the synthesis of the nanoparticles is given in Supporting Information. The superparamagnetic iron-oxide nanoparticles (SPIONs) were synthesized by the co-precipitation of Fe3+ and Fe2+ ions from an aqueous solution with aqueous ammonia.29 Detailed information about the synthesis of the SPIONs is given in Supporting Information. Preparation of Stable Suspensions. For the preparation of the stable suspension 2.89 g of AA (Asp or Lys) was mixed into 450 mL of aqueous suspension containing 1 g of the ironoxide nanoparticles. The pH was set to ∼2.0 using ammonia (for Asp) or HCl (for Lys) and the suspension was mixed for 5 hours. Next, the pH of the Asp suspension was increased to 11.0, whereas the pH of the Lys suspension was increased to 4.0 using ammonia. The excess, non-adsorbed AAs were washed from the suspension using ultrafiltration in which 100 mL of the suspension was washed with 300 mL of diluted aqueous solution of ammonia or HCl maintaining the pH of the original suspension. Adsorption of Amino Acids. The quantity of adsorbed AAs after equilibration with the nanoparticles in the suspensions was evaluated by determining the AA concentrations in the supernatants. First, a mother suspension at a concentration of 2.22 mg /mL was prepared by mixing the SPIONs in distilled water using ultrasound agitation (Sonics Vibra cell™). The mother suspension was divided into vials with 45 mL of the suspension containing 0.1 g of nanoparticles. Then, different amounts of the AAs were dissolved in the suspensions, while the pH was maintained at a certain value with ammonia or HCl. After mixing the suspensions for a long time (1 week), allowing them to reach equilibrium, the nanoparticles were sedimented using ultra-centrifugation (Thermo Scientifics, Sorvall WX 100). The AA concentrations in the supernatants were determined using HPLC. The whole procedure was repeated twice, and the amount of AA adsorbed onto the nanoparticles as a function of its total concentration and the suspension’s pH is given as an average of the two repetitions.


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Characterization of Nanoparticles. For the transmission electron microscopy (TEM) investigations a drop of the nanoparticles’ suspension was dried on a copper-grid-supported, transparent, carbon foil. A TEM JEOL 2010F was operated at 200 kV. The particle size expressed as an equivalent diameter was estimated using visual measurements conducted on 300 nanoparticles. The area of the nanoparticles was estimated from TEM images using DigitalMicrograph™ Gatan Inc. software. For the characterization using X-ray diffractometry (XRD, Siemens D5000 diffractometer - details given in Supporting Information), Raman spectroscopy (Horiba Yvon Lab RAM spectrometer - details given in Supporting Information), for room-temperature magnetic measurements (Lake Shore 7307 VSM vibrating-sample magnetometer - details given in Supporting Information), and for measurements of the specific surface area, the suspension of as-synthesized nanoparticles was lyophilized. The specific surface area was calculated with the BET equation using the N2adsorption data in the P/P0 range between 0.05 and 0.3 (7-point analysis), which was measured at liquid-nitrogen temperature with a gas-sorption analyzer (Quantachrome, Nova 2000e). The samples were degassed at 20 oC in a vacuum for 16 hours prior to the measurement. Characterization of the Suspensions. The colloidal properties of the suspensions were characterized by measuring the ξ-potential and the hydrodynamic size distribution using the dynamic light-scattering method (DLS). The ξ-potential was measured as a function of the suspension’s pH using a Brookhaven Instruments Corporation, Zeta PALS instrument. If not stated otherwise in the corresponding text, the ξ-potential was measured in suspensions diluted with distilled water to ∼0.4 mg of nanoparticles per mL. The starting pH for the measurement was the intrinsic pH of the diluted suspension. NaOH was titrated into the suspension to measure the ξ-potential at higher pH, and separately, HCl was titrated for



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measurements at the decreased pH values. For the DLS measurements a Fritsch, ANALYSETTE 12 Dynasizer apparatus was applied. The design of the apparatus enables measurements at a constant scattering angle of 135o (laser wavelength 658 nm) in a thin film of the suspension, which significantly reduces the probability of multiple scattering of the detected light and thus enables measurements in relatively concentrated, as-prepared suspensions. HPLC Determination of Amino Acids. HPLC was used to determine the amount of AA in the supernatants of the suspensions and associated with the nanoparticles. The suspensions were ultra-centrifuged to separate the AA-adsorbed nanoparticles from the supernatant. For the determination of the amount of AA on the nanoparticles, the sedimented nanoparticles were dried in vacuum at room temperature. A weighed amount of nanoparticles was then dissolved in 6.2-M HCl. Before the HPLC analysis the iron was removed from the samples using an anionic ion-exchange column (Dowex®1×8,100-200mesh, Serva Electrophoresis Gmbh).30 Under such acidic conditions the [FeCl4]− and part of the AA remain on the column; therefore, the retained AA was completely rinsed from the column using 6.2 M HCl. Prior to the AA determination in the samples using HPLC the pH was increased with NaOH to ∼ 9. A list of chemicals used for the HPLC synthesis is given in Supporting Information. HPLC analyses of the AA 3-MPA/OPA (3-mercapto propionic acid/o-phthaldehyde) derivates31 were performed using the Agilent HPLC system 1100 (Palo Alto, USA), equipped with a degasser, a quaternary pump, an auto-sampler, and a diode-array detector set at 338 nm. A Kromasil Eternity-5-C18 (4.6 × 100 mm2) column was employed. The mobile-phase flow rate was 2 mL/min. The separation was carried out using a gradient of three eluents. Eluent A acetonitrile, B methanol and eluent C 40 mmol L-1 NaCH3COO adjusted to pH 7.2 with 2% CH3COOH. The initial mobile-phase composition was 100% C, which changed linearly from 0.5 min to 1 min to the composition 4% A, 8.5% B and 87.5% C and remained constant to 10


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min. At 10 min the second linear gradient starts until 11 min, where the composition was 15% A, 15% B and 70% C and this composition remained constant until 12.5 min. At 13 min the composition changed to 17.5% A, 17.5% B and 65% C until 15 min and then to 35% A, 10% B and 55% C until 18 min. The column was then equilibrated for 2 min with the initial mobile-phase composition. An automatic injector was used for the derivatization with 3-MPA/OPA, which was utilized in a borate buffer with pH 9.9. The pH of the derivatization reagent 3-MPA/OPA was adjusted to pH 9.3. An internal standard (norvaline) was utilized for AA quantification at a wavelength of 338 nm and the limit of detection was estimated to be 10 mg/L. Formation of Molecular Associates in Solutions and SPION Suspensions of Amino Acids. The adsorption of the AA onto the nanoparticles depends on the eventual formation of molecular associates in the solution. The association of the AA molecules was studied by measuring the osmolality of solutions of the pure AAs and of their suspensions with the ironoxide nanoparticles. Osmolality is a colligative property that is a measure of the number of dissolved particles per unit volume of the solution. As a result of intermolecular association the number of particles and thus the osmolality decreases and this affects the freezing point of the solution. The Knauer Cryoscopic Unit Type 7312400000 osmometer was used to measure the freezing-point depressions of the studied AA solutions. The Knauer instrument is equipped with a Peltier cooling system and the measurements are completely automated. The sample solution is cooled down without stirring. The freezing is initiated by starting the vibrator at a pre-set temperature, after which the temperature in the system reaches the freezing point. The freezing point is measured with a built-in thermistor. The instrument was calibrated using aqueous KCl solutions of known osmolalities (0.02–0.10 osm L-1, where “osm” stands for osmoles) (Figure S3, Supplemental Information). The osmolality of KCl was



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calculated by taking into account complete ionization of KCl in water, meaning that one molecule of KCl produces 2 particles in solution. The concentration of the AA solutions for the freezing-point depression measurements was determined by neutralization potentiometric titration to be 30.1 mmol L-1 and 21.9 mmol L-1 for the Asp and Lys, respectively, and the pH was the intrinsic value at the studied concentrations (pHAsp = 3.2, pHLys = 9.6). Each measurement of the sample’s freezing point was performed five times, always with a fresh solution. The osmolality of the AA solutions was determined from the calibration line obtained with KCl (Figure S4, Supplemental Information). The size of the molecular associates in the AA solutions (3 g/mL) at different pH values was followed using DLS. The solutions were centrifuged before the measurements. The light scattering in the solutions was too weak to be detected by the DLS Fritsch, ANALYSETTE 12 Dynasizer apparatus, which was used for measurements in the nanoparticles suspensions. A standard photon correlation setup with a stronger (75 mW) frequency doubled diode pumped Nd-YAG laser (532 nm), a sensitive detector (two ALV-High Q.E. APDs in pseudo cross-correlation regime) and an ALV-6010/160 correlator was therefore used to obtain the autocorrelation function of the scattered light intensity. The measurements were performed at different scattering angles. The radius distribution functions were calculated using the CONTIN analysis,32 which is part of the ALV-Correlator Software V.3.0.

RESULTS AND DISCUSSION Properties of the As-synthesized Iron-Oxide Nanoparticles. XRD analyses of the assynthesized nanoparticles showed only broad peaks corresponding to a spinel structure (Figure S1 in Supporting Information). From the XRD peak broadening (Scherrer equation) the average nanoparticle size was determined to be 10.2 nm. The TEM revealed that the nanoparticles exhibited a globular shape (Figure 1a). The size distribution measured from the


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TEM images (Figure 1b), fitted by a log-normal distribution, showed a number-weighted equivalent diameter of 9.7 ± 1.5 nm. The specific surface area of the lyophilized nanoparticles was measured using the BET method to be 89.9 m2/g. The measured specific surface area is considerably lower than the area of 129 m2/g estimated from the TEM images (assuming a spherical shape of the nanoparticles and a density of maghemite equal to 4.8 g/cm3). The BET specific surface area is usually smaller than that estimated from the TEM size, because of agglomeration of the nanoparticles during drying, which cannot be (entirely) avoided. As the nanoparticles were synthesized in ambient air, it is reasonable to expect that the Fe2+ was almost completely oxidized and the nanoparticles can be described as maghemite (γFe2O3). Raman spectroscopy confirmed this assumption - the Raman spectrum of the nanoparticles showed features characteristic of maghemite (Figure S1 in Supporting Information). A chemical analysis of the nanoparticles synthesized using a similar procedure showed that less than 3 % of all the Fe contained in the nanoparticles was in the oxidation state 2+.29 Magnetic measurements confirmed the superparamagnetic nature of the SPIONs with a saturation magnetization of 63.9 Am2kg-1 (Figure S3 in Supporting Information).

Figure 1. TEM image (a) and the corresponding size distribution (b) of iron-oxide nanoparticles (scale bar = 50 nm).



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Stable Suspensions of Amino-Acid-Adsorbed Iron-Oxide Nanoparticles. A suspension of as-synthesized SPIONs quickly sediments, independently of the pH. However, stable aqueous suspensions can be prepared using aspartic acid (Asp) and lysine (Lys) as the stabilizers.19,20,24,25 Both AAs were adsorbed onto the nanoparticles in aqueous suspensions at an acidic pH,19,20,28 where the carboxyl groups are almost completely protonated. After the adsorption of the AAs onto the nanoparticles at the acidic pH, the pH of the suspension was increased to pH=11 and pH=4 for the Asp and Lys suspensions, respectively, and the suspensions were washed with ultrafiltration. The properties of the suspensions are listed in Table 1. The suspension of Asp-adsorbed nanoparticles remains colloidally stable at pH values above approximately 9. The hydrodynamic size of the nanoparticles in the suspension was followed with DLS. The number-weighted size distribution (Figure 2 (a)) in the Aspstabilized suspension (2 mg/mL) at pH=11 shows a sharp peak at approximately 37 nm, which was attributed to the average hydrodynamic size of the individual, dispersed, Aspadsorbed nanoparticles. However, some nanoparticles agglomerates with sizes between approximately 100 nm and 150 nm were also present. The size did not change significantly with time. However, after a very long time of approximately one month partial flocculation was observed in the suspension. The results of the DLS measurements did not change significantly upon diluting the Asp-stabilized suspension to 0.1 mg/mL; however, dilution to 0.05 mg/mL resulted in slow flocculation. The suspension of Lys-adsorbed nanoparticles was completely stable in an acidic pH, the nanoparticles quickly sedimented at pH values between 5 and 9, whereas at pH 9 and above they sedimented only partially and after a long time (i.e., after several weeks). DLS measurements showed that the vast majority of the nanoparticles in the stable suspension (5 mg/mL) at pH=4 have a hydrodynamic diameter of approximately 18 nm (Figure 2 (b)). Some agglomerates with diameters between 180 nm and 240 nm were also present. The size


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distribution did not change with time, proving the good colloidal stability. No agglomeration was detected when diluting the suspension.

Figure 2. Number-weighted empirical distribution of the hydrodynamic diameters of the nanoparticles in the aqueous suspension of iron-oxide nanoparticles (2 mg/mL) stabilized with Asp (pH=11) (a) and Lys (pH=4) (b).

Table 1. Properties of the Suspensions of AA-adsorbed SPIONS ξ-potential, hydrodynamic diameter (dDLS), and AA content (concentration in the supernatant and surface concentration at the SPIONs). AA

pH

ξ-potential

dDLS*

AA content

[/]

[mV]

[nm]

supernatant [g L ]

at SPIONs [µmol m ]

Asp

11.0

-28

37

32

28.7

Lys

4.0

+30

18

91

6.4

-1

-2

Figure 3 shows the results of ξ-potential measurements as a function of pH for the suspensions of as-synthesized and AA-adsorbed nanoparticles. The as-synthesized nanoparticles exhibited an isoelectric point (IEP) close to pH = 6. With the Asp adsorption the IEP remains practically unchanged, whereas with the Lys adsorption the IEP increases to pH ≈ 7. It was shown with FTIR and Raman spectroscopy that the amino acids adsorb through a chelate bond of the carboxyl groups with Fe3+ ions at the iron-oxide surface, whereas the amino groups do not form an ionic bond with the surface.19,20,24-27 Mikhaylova et al.20 showed



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on the basis of FT-IR analyses that Asp interacts with the surface of iron-oxide nanoparticles through the side carboxyl group, while neither the amine group nor the α-carboxyl group takes part in this interaction (Scheme 1a). However, ξ-potential measurements showed that the IEP of the suspension of iron-oxide nanoparticles was shifted from neutral to alkaline pH (pHIEP ∼8.5) when the Asp was adsorbed onto the nanoparticles,20 suggesting the adsorption of the Asp with both carboxyl groups onto the surface, leaving the amino group oriented outwards into the aqueous medium (Scheme 1b). Schwaminger et al.26 suggested, based on ATR-FTIR results, that glutamic acid, another amino acid with a carboxyl side group, more likely binds to the magnetite at pH = 6, either through its side carboxyl group or with an alpha carboxyl group rather than with the involvement of the both carboxyl groups at the same time. According to a simple model based on the adsorption of Asp molecules onto the iron-oxide surface in a single layer (monolayer) with both carboxyl groups attached to the surface (see Scheme 1b) a shift in the IEP to more basic pH values is expected, because of the amino group oriented towards the medium. As the adsorption of the Asp onto the nanoparticles caused no significant change in the IEP, this model can be ruled out. The ξ-potential results are consistent with the adsorption of the Asp molecules onto the nanoparticles through bonding of the side carboxyl group with the surface, leaving the α-carboxyl and the amino groups oriented towards the solution. However, no change in the IEP can also be explained by allowing the adsorption of the Asp in several molecular layers or in the form of larger molecular associates. This possibility will be discussed in more detail below.


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Scheme 1. Schematic of aspartic acid (a, b) and lysine (c) proposed adsorption onto ironoxide nanoparticles as a single molecular layer and in the form of molecular associates (d).

The situation seems much simpler in the case of the adsorption of the positively charged amino acid Lys. The Lys adsorbs onto the nanoparticle surfaces by bonding with the αcarboxyl group26 (Scheme 1c), leaving the amino groups oriented outwards, which results in the increased IEP. However, the measurement of the Lys-stabilized suspension only shows a slightly increased IEP. This can be explained by the low surface concentration of adsorbed Lys or, again, by adsorption in the form of larger molecular associates. The absolute value of the ξ-potential is related to the electrostatic repulsive forces that prevent agglomeration of the nanoparticles in aqueous suspensions. Usually, it is assumed that stable suspensions are formed if the ξ-potential exceeds a value ±30 mV.33 Both suspensions of the AA-adsorbed nanoparticles were stable despite the relatively low absolute values of the



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ξ-potential: ξ was approximately −28 mV for the Asp-stabilized suspension at pH=11 and approximately +30 mV for the Lys-stabilized suspension at pH=4. However, the absolute ξpotential value is also influenced by the ionic strength of the medium, which is increased by the dissolved AA and other ions remaining in the suspension after purification with ultrafiltration of the suspension. The suspensions were ultra-centrifuged and the concentration of the AAs was determined in the supernatant and on the sedimented nanoparticles using HPLC. The concentration of the AA in the supernatant was relatively low. In the Asp-stabilized suspension (2 mg SPIONs/mL, pH=11) the concentration of Asp in the supernatant was approximately 32 mg/L. The amount of Asp that remained adsorbed on the nanoparticles was much higher: 0.49 g of Asp was adsorbed per 1 g of SPIONs. This leads to a surface concentration of Asp equal to 28.7 µmol/m2, corresponding to more than 17 Asp molecules per nm2 (the specific surface area of 129 m2/g estimated from the nanoparticles’ average TEM size was used for the calculations of the surface concentrations). This high surface concentration rules out the model of Asp adsorption in the form of a monolayer. The result suggests that the Asp actually adsorbs onto the SPIONs in the form of molecular associates. The adsorption of the Asp in the form of molecular associates explains why there is no change in the IEP and also the large hydrodynamic size of the nanoparticles (∼37 nm) compared to the TEM size (∼10 nm). The hydrodynamic size corresponds to the nanoparticle’s inorganic core plus the surface layer that moves with the core in the suspension, whereas the TEM size measures just the inorganic cores. The large molecular associates at the nanoparticle surfaces can additionally contribute to the steric stabilization of the suspensions.


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Figure 3. Change of the ζ-potential with pH in aqueous suspensions of as-synthesized (SPION), Asp-adsorbed (Asp), and Lys-adsorbed iron-oxide nanoparticles (Lys).

In the case of the Lys-stabilized suspension (2 mg SPIONs/mL, pH=4) the concentration of the Lys in the supernatant was approximately 91 mg/L and only 6.4 µmol Lys/m2 remained adsorbed on the nanoparticles (∼5 Lys molecules per nm2). The result suggests that the Lys probably adsorbs on SPIONs in the form of smaller molecular associates as compared to Asp. As a result, the hydrodynamic size of the Lys-stabilized nanoparticles (∼18 nm) is smaller than that of the Asp-stabilized nanoparticles (∼37 nm). Adsorption of the Amino Acid onto Iron-Oxide Nanoparticles. The nanoparticles in the aqueous medium were equilibrated with different amounts of the AAs and at different pH values of the suspension. Then, the suspensions were ultra-centrifuged to sediment the nanoparticles and the supernatants were analyzed using HPLC. The specific surface area of 129 m2/g estimated from the nanoparticles’ TEM size was used for an estimation of the surface concentrations of the adsorbed AAs (adsorption densities). Figure 4 shows the adsorption density of the two AAs at the SPIONs as a function of their total concentration in the suspension for different pH values. The adsorption density of both the AAs increases almost linearly with the concentration in the suspension and does not show any saturation up to the highest concentration studied. The amount of adsorbed AA is very



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high for both AAs. At the highest concentration of 6.4 mg/mL the Asp adsorbs on the surface with a concentration of ∼60 µmol/m2, corresponding to ∼37 Asp molecules per nm2 of the nanoparticle surface. This highest Asp concentration of 6.4 mg/mL already exceeds the solubility limit for pure Asp in water.34 Nevertheless, the Asp completely dissolved in the SPION suspension. The explanation is that the major part of the Asp is immobilized onto the nanoparticles and only a small part remains free in the aqueous solution, thus not exceeding the solubility limit. The highest concentration shown in the graphs of Figure 4 corresponds to 2.88 g of Asp per g of SPIONs (Asp/SPION = 2.88). To further increase the Asp/SPION mass ratio, the amount of nanoparticles in the suspension was decreased. At the Asp/SPION mass ratio of 28.8 the surface concentration of the adsorbed Asp on the nanoparticles was very high; it exceeded 1500 µmol/m2. However, it has to be noted that the expected error of the measurement in this case is rather high. The pH value had only a minor effect on the adsorption density of the Asp at low pH values, whereas at pH=11 it significantly decreased. However, even at pH=11 approximately 15 Asp molecules adsorbed per nm2 of the nanoparticle surface. The adsorption density of the Lys on the SPIONs was similar to that of Asp only at low pH (pH=2), whereas at higher pH values the Lys adsorption density decreased considerably. At pH=4 and Lys concentration of 6.4 mg/mL (Lys/SPION = 2.88) approximately 15 µmol of the Lys adsorbed per m2 of the SPION surface (corresponding to ∼9 molecules/nm2). Also in this case the surface density of the adsorbed AA further increased with increasing the Lys/SPION ratio and no saturation of the surface was indicated. The high adsorption densities of both amino acids can only be explained by proposing adsorption in the form of large molecular associates (Scheme 1d). We propose that the association is governed by hydrogen bonding at acidic pH and by electrostatic interactions at higher pH values. The adsorption density of the AA at the SPION surfaces significantly


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increased when both AAs were adsorbed simultaneously (Table S1 in Supporting Information). This can be explained by the favorable electrostatic interactions between Asp and Lys. As the molecular associates may be sensitive to the manipulation of the sample, its history could have a considerable effect on the adsorption measurements. The associates can decompose under sheer stress (e.g., mixing, ultracentrifugation), which is reflected in the relatively high scattering of the results. At the AA concentration of 6.4 mg/mL the relative deviations of the adsorption density determination (that includes sample preparation and AA determination in the supernatant using HPLC) was ∼ ± 7%. This is considerably higher than the relative standard deviation of the AA determination after repeated injection of the selected solution of AA, which was determined to be within 4.3%.

Figure 4. Adsorption density of Asp (a) and Lys (b) on the SPIONs as a function of their total concentration in the suspension at different pH values. In contrast to our results, Sousa et al.19 showed that the Asp chemisorbs onto the maghemite surface mostly following a Langmuir adsorption isotherm. Their conductometric measurements showed that the maximum adsorption of Asp onto the surface of the iron oxide occurs at a pH close to 3 and decreases with increasing pH values. However, the surface saturation at pH ∼3 was achieved at a very high value of approximately 150 µmol of Asp/m2 of the oxide surface, corresponding to approximately 90 molecules of Asp/nm2.19



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Viota et al.28 quantified the adsorption of Lys onto the iron-oxide nanoparticles using a colorimetric method with ninhydrin. The amount of adsorbed Lys increased with the Lys concentration in the suspension with a Langmuir-type tendency, although saturation was only suggested at the highest Lys concentration at a low pH. The adsorption density depended on the pH, being the lowest in acidic conditions, the highest at neutral pH, before dropping again in basic conditions. The highest absolute value measured at the Lys concentration in a suspension of 10 mg/mL was ∼0.17 µmol/m2 (pH=7), corresponding to one Lys molecule per 10 nm2 (0.1 Lys molecules/nm2).28 The amount of adsorbed Lys was therefore much lower than the one measured in this investigation. There remains an important question: is the formation of molecular associates governing the adsorption process restricted to high AA concentrations? Could it be possible to adsorb AAs on the nanoparticle surfaces in the form of well-defined monolayers at very low AA concentrations? It is not possible to answer this question using the HPLC determination of the AA in the supernatants because the AA concentrations would be below the detection limit. We therefore used measurements of the ξ-potential as a function of the pH of the suspensions containing 0.01 mg/mL of SPIONs and the AA in different AA/SPION weight ratios to resolve this question. The AA was added to the suspension at pH=2. After a longer time of approximately one day allowed for the AA adsorption onto the nanoparticles the ξ-potential was measured while the pH was increased using NaOH. Figure 5 shows the ξ-potential as a function of pH for different Asp/SPION ratios. The IEP slightly increased with the adsorption of low amounts of Asp, reaching the highest value at the Asp/SPION = 0.04. The Asp/SPION = 0.04 corresponds to ∼1.4 molecule per nm2 of the nanoparticle surface. It is therefore expected that the Asp predominantly adsorbs in the form of a single molecular layer and the adsorbed molecules are in equilibrium with the molecules dissolved in the medium. The increase in IEP suggests bonding of the Asp with the both


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carboxyl groups onto the nanoparticle surfaces and the amino group oriented outwards (Scheme 1 (a)). At higher Asp/SPION values the IEP gradually decreases. In accordance with the HPLC analyses, the decrease in the IEP can be explained by the formation of molecular associates. It is especially interesting that the IEP also keeps decreasing at very high values of the Asp/SPION. This observation can be explained by the increasing amount of Asp molecules in the surface layer of the AA-adsorbed nanoparticles that are oriented with their side carboxyl group towards the solution. The absolute value of the ξ-potential at high pH values increases when small amounts of the Asp are adsorbed onto the SPIONs and reaches a maximum value of ∼ −47 mV at the Asp/SPION ratio of 1.35. At even higher Asp additions, the ξ-potential drops, most probably because the ionic strength increases due to the excess of the dissolved Asp. The Lys adsorption progressively increases the IEP of the suspension. It is expected that the Lys adsorbs in the form of a single molecular layer with bonding through the carboxyl group at low concentrations, whereas at high concentrations molecular associates are present at the nanoparticles with an increased proportion of the amino groups at the surfaces. As similar effect as the adsorption of AAs at very low AA/SPION ratios can also be observed if the AA-adsorbed nanoparticles are washed with excess water (see Supporting Information for details).



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Figure 5. ξ-potential as a function of pH for different suspensions containing 0.01 mg/mL of SPIONs and different additions of Asp (a) and Lys (b). The numbers in the legend give the AA/SPION mass ratio. The discrepancies between our results and the results of others19,28 can be at least partially ascribed to different impurities present in the suspensions. The adsorption process can be considered as a ligand-exchange reaction where the original labile solvent layer is replaced with AAs. The AA adsorption can therefore be significantly influenced by the presence of impurities, which adsorb onto the nanoparticles’ surfaces, thus influencing their surface properties in the aqueous suspension.23,35 Moreover, the impurity ions dissolved in the medium can change the adsorption by influencing the association of the AAs into the molecular associates. In this study nanoparticles were synthesized by the co-precipitation of iron sulfates with ammonia. Thus, SO42- and NH4+ ions can be expected in the suspensions as the impurities. We performed several experiments to test the influence of foreign ions on the adsorption of the two AAs. First, the as-synthesized nanoparticles were washed with excess diluted ammonia at pH=10.5 (10 times more diluted ammonia was used for washing compared to the usual experiments), however, no significant changes can be detected, neither in the ξ-potential vs. pH curves, nor in the content of the AAs in the supernatants of the suspensions after the AAs were adsorbed onto the excess-washed nanoparticles, compared to the nanoparticles washed using the normal procedure. Also, washing with excess diluted NaOH at pH = 10.5 or with distilled water had no significant effect on the adsorption of the AAs. Finally, the influence of nitrate ions on the AAs adsorption was tested. The acidic aqueous suspensions of iron-oxide nanoparticles are frequently prepared by peptization with nitric acid. The surface of the peptized nanoparticles is positively charged by the adsorption of H+. As the NO3- anion is not a sufﬁciently polarizable counter ion to entirely screen the nanoparticles surface charge, the overall charge provides electrostatic repulsions.36,37 To test


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the influence of the NO3- anions on the AA adsorption, HNO3 was used to set the pH of the AA adsorption to pH=2 instead of HCl. Neither the HPLC measurements of the AA concentration in the supernatants of the suspensions, nor the ξ-potential measurements vs. pH, revealed significant changes in the AA adsorption in the presence of NO3- compared to the standard procedure. Formation of Molecular Associates of the Amino Acids. The adsorption of the AAs depends strongly on the formation of molecular associates in the suspension. The association of Asp and Lys in the aqueous solution and in the suspension of SPIONs was studied using measurements of the freezing-point depression. The results are listed in Table 2. The osmolality of the solution at an Asp concentration of 30.1 mmol L-1 (pH=3.2) was read from the calibration line obtained with KCl (Figure S3, Supporting Information) and was 14.0 mosm L-1, whereas the solution containing 21.9 mmol L-1 of Lys (pH=9.7) had an osmolality of 10.1 mosm L-1. The measured osmolality (π) is considerably lower than the osmolality of the ideal solution (πid) of non-associated molecules, which is in the case of AAs at their natural (low) pH equal to their concentration. The designation π, which normally stands for osmotic pressure, is used because the osmolality is directly related to the osmotic pressure of the solution via = (here, c is the number concentration of osmotically active species, R is the gas constant and T is the temperature). Values of π/πid (= ⁄ , cid is the number concentration of particles in an ideal AA solution where no dissociation of COOH groups or association of AA molecules is taking place) lower than 1 are a clear sign that both AAs are strongly associated in aqueous solutions at their intrinsic pH (see the π/πid values reported in Table 2). From the measured osmolality, the association degree was calculated. Assuming that the formation of the molecular dimers alone is responsible for the lowering of the number of particles (and osmolality), the estimated degree of the AA association is unrealistic



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(exceeding 100 % in both the solutions). This suggests that association is more extensive, involving more than two molecules per associate. By allowing individual AA molecules to form higher associates (trimers, tetramers, …, n-mers with a high value of the aggregation number, n), the degree of association in the Asp solution was estimated to be from 82% (only trimers) to 55% (only n-mers). The corresponding degree of association in the Lys solution was between 87% (only trimers) and 56% (only n-meres). These numbers are rough estimates because it is reasonable to expect that AA aggregates of various sizes (aggregation numbers

n) are present in the solution. However, there is no method to determine the eventual distribution of species. In the SPION suspension (2 mg/mL) containing the AAs the measured osmolality was lower than in the corresponding pure AA solutions, i.e., 11.57 mosm L-1 for the suspension containing 30.1 mmol L-1 of Asp (pH=3.2), and 9.2 mosm L-1 for the suspension containing 21.9 mmol L-1 of Lys (pH=9.6). These values are slightly lower than those obtained in solutions of pure AAs. No measurable freezing-point depression could be detected in the suspension of the SPIONs without AAs. In the case of the AA-stabilized suspensions the decrease in osmolality can be predominantly ascribed to the decrease in the AA concentration due to its adsorption onto the nanoparticles. The size of the associates in the AA aqueous solutions (3 g/mL) at pH=3 and pH=11 was followed using DLS. In all cases the number-weighted distribution functions showed two distinctive peaks: one narrow peak at hydrodynamic diameters of the order of 1 nm and another smaller, but much broader peak, at diameters of several tens of nm (see Figures S6 S9 in Supporting Information). In the Asp solution at pH=3 the smaller entities with a hydrodynamic diameter of 0.8 ± 0.3 nm and larger entities with a diameter of 100–300 nm were present, whereas at pH=11 the smaller entities had a hydrodynamic diameter of 8 ± 2 nm and the larger ones a size of 40–80 nm. In the Lys solution at pH 3 the light scattering was


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very weak, indicating that the particles are very small. The measured hydrodynamic diameter was 0.8 ± 0.3 nm; however, very few particles larger than 100 nm were also observed. At pH 11, smaller entities with a hydrodynamic diameter of 0.9 ± 0.3 nm coexisted with the larger ones with a diameter larger than 60 nm. The smaller entities with a size below 1 nm can be ascribed to individual molecules surrounded by a hydration layer of water molecules (the van der Waals diameter of the molecule is 0.56 nm and 0.64 nm for the Asp and the Lys, respectively)38 or to the small molecular associates (dimers, trimers), whereas the entities with larger diameters were ascribed to large molecular associates. Table 2. The Properties of the AA Solutions and the SPION Suspensions (2 mg/mL): Osmolality and the Degree of Association Assuming the Formation of Dimers, Trimers and n-mers (see text). sample

C

pH

osmolality

π/πid

Asp solution Asp susp. Lys solution Lys susp.

[mmol L-1] 31 31 21 21

[/] 3.2 3.2 9.7 9.7

[mosm] 13.98 11.57 10.12 9.15

[/] 0.45 0.37 0.46 0.41

degree of association dimers trimers [%] [%] 110 82 126 94 104 78 118 88.5

n-mers [%] 56 63 56 59

In conclusion, the results of the freezing-point depression measurements show that the Asp and Lys already associate in pure aqueous solutions without added SPIONs. It is therefore not surprising that both the AAs adsorb onto the nanoparticles in the form of the molecular associates, which were also evidenced by the direct HPLC analysis. The association of AAs can also be expected in other AAs with polar side chains, and will therefore significantly influence their adsorption on different surfaces in general, not only the iron oxide nanoparticles considered in this article. Adsorption in the form of (the poorly defined) molecular associates can decrease the possibility of using the adsorption of the AAs for tuning the nanoparticles’ surface properties, such as the surface charge. Bonds, i.e., the relatively weak hydrogen bonds and the strong electrostatic interactions that were proposed to bond the individual AAs into these associates,



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depend strongly on the pH. This can represent a disadvantage for some applications of the nanoparticle suspensions. For example, it is not realistic to expect that the AAs adsorbed onto the nanoparticles in the form of the associates would serve as a functionalization layer for the conjugation of different (bio)molecules that are required for specific biomedical applications, as was suggested by some researchers.19,20,23 The associates at the surfaces are also expected to be quite flexible and can disintegrate in the presence of other (charged) molecules that are present in the medium. To illustrate this problem, we performed a separate experiment. The AA-adsorbed nanoparticles were dispersed in phosphate-buffered saline (PBS), a medium frequently used in biomedicine. After some time in the PBS the nanoparticles were separated from the suspension using ultracentrifugation and re-dispersed in distilled water. The details of the experiment are given in the Supporting Information. The measurements of the ξpotential as a function of pH (see Figure S10 in Supporting Information) showed that the surface properties of the AA-adsorbed nanoparticles significantly changed after they were exposed to the PBS. After dipping the AA-adsorbed nanoparticles in the PBS, the isoelectric point was shifted to an acidic pH of ∼ 4 (IEP of the AA-adsorbed nanoparticles was ∼6.2 and ∼7.2 for Asp and Lys, respectively). The change of the IEP can be explained by the exchange of AAs at the nanoparticle surfaces with phosphates (HPO42- and H2PO4-) from the buffer.39

CONCLUSIONS The amino acids (AAs) aspartic acid (Asp) and lysine (Lys) adsorb onto iron-oxide nanoparticles (SPIONs) in very high surface concentrations. The adsorption density of both the AAs increases almost linearly with their concentration in the suspension and does not show any saturation up to the highest concentration studied. The very high surface concentrations, reaching several tens of the AA molecules per nm2 of the nanoparticle surfaces, are explained by AA adsorption in the form of molecular associates. Measurements


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of the freezing-point depression prove the formation of the molecular associates already in pure AA solutions. The adsorption of the AAs in the form of molecular associates decisively influences the surface properties of the AA-adsorbed SPIONs. These AA-adsorbed nanoparticles formed stable aqueous suspensions despite a relatively moderate ξ-potential. Their hydrodynamic size measured with DLS is significantly larger than the size of their inorganic cores obtained from TEM images, suggesting additional steric stabilization of the suspensions by the molecular associates present at the nanoparticle surfaces.

ACKNOWLEDGEMENTS The financial support of the Ministry of Higher Education, Science and Technology of the Republic of Slovenia within the National Research Program P2-0089 is gratefully acknowledged. The authors also acknowledge the use of equipment in the Center of Excellence on Nanoscience and Nanotechnology – Nanocenter. Prof. Ksenija Kogej acknowledges the support of the Physical Chemistry program P1-0201 and Asst. Prof. Irena Kralj Cigić and Dr. Gregor Marolt, the support of the Research and Development of Analytical Methods and Procedures program P1-0153. The authors would also like to thank Dr. Adolf Jesih for help with Raman spectroscopy and Dr. Andraž Kocjan for help with BET.

ASSOCIATED CONTENT Supporting Information Available: Detailed descriptions of iron-oxide nanoparticles synthesis, XRD spectra, Raman spectra and room-temperature magnetic properties of the as-synthesized nanoparticles, HPLC quantification of the simultaneous adsorption of two amino acids, calibration of the osmolality measurements, the list of chemicals used for the HPLC synthesis, DLS measurements of the



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hydrodynamic size of the molecular associates in the amino-acid aqueous solutions, and measurements of the ξ-potential as a function of pH for amino-acid nanoparticles after they were exposed to phosphate-buffered saline. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Phone: +386 1 4773 579

Notes The authors declare no competing financial interest.


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J. Chem. Technol. Biotechnol. 2016, 91, 1232–1239.



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Page 34 of 34

Adsorption of Amino Acids, Aspartic Acid, and Lysine onto Iron-Oxide

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