Preparation and characterization of stable dispersions of carbon black and nanodiamond in culture medium for in vitro toxicity assessment

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Preparation and characterization of stable dispersions of carbon black and nanodiamond in culture medium for in vitro toxicity assessment Haruhisa Kato a,*, Ayako Nakamura a,c, Masanori Horie b, Shigehisa Endoh c, Katsuhide Fujita d, Hitoshi Iwahashi e, Shinichi Kinugasa a a

Polymer Standards Section Japan (PSSJ), Particle Metrology Section (PMS), Nanomaterial Characterization Division (NCD), National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan b Institute of Industrial Ecological Sciences, University of Occupational and Environmental Health, Japan c Technology Research Association for Single Wall Carbon Nanotubes (TASC), Japan d Research Institute of Science for Safety and Sustainability (RISS), AIST, Tsukuba, Japan e Faculty of Applied Biological Sciences, Gifu University, Japan

A R T I C L E I N F O

A B S T R A C T

Article history:

Stable dispersions of carbon black and nanodiamond in culture medium were prepared by

Received 8 April 2011

adding a pre-mixed dispersion of commercial carbon black or nanodiamond in aqueous

Accepted 20 May 2011

bovine serum albumin (BSA) solution to culture medium. Dynamic light scattering revealed

Available online 25 May 2011

that carbon black and nanodiamond dispersions prepared in both NaCl solution and culture medium were highly stable. From DLVO theory and the results of zeta potential measurements, the theoretical effect of the electrostatic interactions between adsorbed BSA molecules was found to be minimal. The asymmetric flow field-flow fractionation measurements revealed that 0.05 or 0.56 mg/mL of BSA molecules were adsorbed on 0.11 or 1.09 mg/mL of carbon black, respectively, indicating 1:2 complexation of BSA with the colloidal particles. In the case of nanodiamond, 0.06 or 0.60 mg/mL of BSA molecules were adsorbed on 0.10 or 1.05 mg/mL of nanodiamond, respectively, indicating 1:2 complexation of BSA, which is the same ratio as in the case of carbon black. The adsorbed BSA molecules served as an effective stabilizing agent for the carbon black and nanodiamond, ensuring dispersion stability for at least 1 week. The preparation of the dispersions can be easily carried out by other researchers for toxicity studies.  2011 Elsevier Ltd. All rights reserved.

1.

Introduction

In recent years, carbon nanomaterials have become one of the most important materials for scientific and technological applications. Carbon black is widely used in chemical and industrial applications, for example, as a component in inks and coatings, a rubber reinforcing agent, a pigment for plas-

tics, and a battery cathode [1–6]. Although carbon black is hydrophobic, it can be dispersed in aqueous solutions by using surfactants or by functionalizing the materials surface [7–9]. Stable colloidal dispersions of carbon black are critical for the aforementioned applications, for which nano-sized carbon black is considered to have high potential. For example, nano-sized carbon black has been shown to improve

* Corresponding author: Fax: +81 29 861 4618. E-mail address: [email protected] (H. Kato). 0008-6223/$ - see front matter  2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.carbon.2011.05.039

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the performance of lithium-ion batteries [10]. Nanodiamond is also a notable industrial material, owing to its hardness, low electrical conductivity, and high thermal conductivity [11]. Nanodiamond is also biocompatible and thus can be used in biomedical applications, for example, as a stable biomarkers [12,13]. While carbon nanomaterials have excellent chemical and physical properties, carbon materials of such small size might have high toxicity and adversely affect human health [14]. Because it is important to clarify this potential toxicity for various carbon nanomaterials, numerous toxicity assessments have been carried out by international organizations and researchers [15–21]. For in vitro toxicity assessments, however, it is difficult to maintain stable dispersions of carbon nanomaterials in culture medium. Carbon nanomaterials tend to aggregate through electrostatic interactions between the particles and proteins, as well as through van der Waals and hydrophobic interactions in culture medium [22–24]. These interactions can easily result in secondary colloidal nanoparticles in the same manner as aggregation of metal oxide nanoparticles; the produced secondary colloidal nanoparticle often become unstable in culture medium [25–27]. The instability of these dispersions leads to sedimentation of the agglomerated materials, which in turn affects the toxicity measurements by altering in nanoparticle uptake by the cells; consequently, conflicting results of toxicity studies on carbon nanomaterials have been reported [28]. The establishment of a practical protocol for the preparation of the stable particle dispersion in culture medium should be a great benefit in toxicology assessments, since a well-defined protocol provides the basis for the reliable comparison of different toxicological assessments. The aim of the present study was to develop a procedure for the preparation of stable dispersions of carbon nanomaterials (carbon black and nanodiamond) in a culture medium. A simple and reproducible method for preparing carbon black dispersions that avoids the need for chemical modifications, centrifugation, ultrasonication, and filtration is desired. We propose a novel and practical approach that will allow researchers to obtain reproducible findings from toxicological assessments. This novel procedure for the preparation of stable carbon black dispersions in high ionic strength solvent could also lead to new applications in chemical and industrial fields. The stability of these dispersions was examined through dynamic light scattering (DLS) and zeta potential measurements. Using the DLVO (Derjaguin, Landau, Verwey, and Overbeek) theory [29,30] and zeta potential measurements, the induction of aggregation of carbon nanoparticles was investigated. The amount of bound BSA molecules on the carbon nanomaterials was also analyzed. We have previously established a practical protocol that uses Asymmetric flow fieldflow fractionation (AFFFF) to determine the bulk quantity of BSA molecules in nanoparticle dispersions for in vitro toxicity assessments [31]. AFFFF is an elution technique where nanoparticles and macromolecules are separated by flow control in an aqueous solution [32,33]. AFFFF can be an effective method for separating free protein molecules from colloidal nanoparticles, such as bound protein and carbon nanomaterials during a short elution time.

2.

Experimental

2.1.

Materials and preparation of carbon-black dispersions

Carbon black aqueous dispersions (AB001, 19.2 wt.%, carboxylate-stabilized carbon black) were obtained from Tokai Carbon Co., Ltd. (Japan). Nanodiamond aqueous dispersions (7.1 wt.%, ligand-free) were obtained from Nippon Kayaku Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Invitrogen Corporation) was supplemented with 10% heatinactivated fetal bovine serum (FBS, CELLect GOLD, MP Biomedicals), 100 U/mL of penicillin, 100 lg/mL of streptomycin, and 250 ng/mL of amphotericin B (Nacalai Tesque Inc.). The DMEM mixture is referred to as DMEM–FBS in this study. Bovine serum albumin (BSA) and NaCl were purchased from Wako Pure Chemical Industries Ltd. (Japan). DMEM–FBS and NaCl aqueous solutions were prepared as follows. An analogous preparation technique was used to prepare the nanodiamond aqueous dispersions.

2.1.1.

Stable carbon black aqueous dispersion

The as-received carbon black aqueous solution (AB001) was diluted with ultrapure water prepared using a Milli-Q system (Nihon Millipore KK, Tokyo, Japan) using 0.1-lm filters.

2.1.2.

Stable carbon black dispersion in DMEM–FBS

As-received carbon black aqueous dispersion (AB001, 300 lL) was pre-mixed in 600 lL of BSA aqueous solution at concentrations of 33 or 66 mg/mL and stirred gently. Each dispersion (900 lL) was added to 5100 lL of DMEM–FBS and gently stirred.

2.1.3. Stable carbon black dispersion in NaCl aqueous solution As-received carbon black aqueous dispersion (300 lL, AB001) was pre-mixed in 600 lL of aqueous BSA solution at concentrations of 33 or 66 mg/mL and stirred gently. Each dispersion (900 lL) was added to 5100 lL of aqueous NaCl solution (6.5 mg/mL) and gently stirred.

2.2.

Dynamic light scattering (DLS) measurements

A fiber-optics particle analyzer (FPAR1000, Otsuka Electronics Co., Ltd., Japan) was used with a 5 mW semiconductor laser operating at a wavelength of 658 nm. Measurements were performed at a scattering angle of 160 with a polyethylene–polypropylene sample cell. The measurement temperature was regulated at 25.0 ± 0.1 C using the Peltier method. The instrument was stored in a clean booth kept at a constant room temperature of 23.0 ± 0.3 C and humidity of 40 ± 3%. The same sample positions were used for all bottles of carbon black dispersions for DLS measurement, which were stored over a week. Measurements were repeated at least three times, and mean values are used in this study. Taking the apparent diffusion coefficients obtained by DLS, the hydrodynamic particle size of the carbon black and nanodiamond colloidal nanoparticles were determined on the basis of the Stokes–Einstein relation: d¼

kB T ; 3pgD

ð1Þ

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where kB is the Boltzmann constant, T is the absolute temperature, g is the viscosity of the solvent (0.89025 cP) [34], and d is the calculated hydrodynamic diameter of the colloidal nanoparticles.

2.3.

AFFFF-MALS measurements

Field-flow fractionation measurements were carried out using an AF2000 system (Postnova, Germany) equipped with a cellulose membrane (Z-MEM-AQU-427N) with a molecular mass cutoff of 10,000 Da and a channel width of 350 lm. A constant cross-flow rate of 4.5 mL/min was used, depending on the sample, and the channel flow was kept constant at 1.0 mL/min. The sample particles eluted at the outlet of the channel were monitored on a DAD UV detector (1200 Series, Agilent Technologies Inc., USA). The elution system used in this study was ultrapure water produced by a Milli-Q system using 0.1-lm filters. The electrical resistance of the ultrapure water was 18.2 MX cm and the total organic carbon contained in the water was less than 3 ppb. In order to determine the amount of bulk BSA molecules, 6.5 mg/mL of aqueous NaCl solution was used as the AFFFF eluent.

2.4.

Electrophoretic mobility measurements

Electrophoretic mobility measurements were performed at 25.0 ± 0.1 C using a zeta potential analyzer (ELSZ, Otsuka Electronics Co., Ltd., Japan). The zeta potentials were estimated using the simplified Smoluchowski equation. It should be noted that although it is dependent on the magnitude of the zeta potential, the spherical Smoluchowski approximation could overestimate the actual zeta potential by up to 20% [35].

3.

Results and discussion

3.1. Preparation of carbon black and nanodiamond dispersion in culture medium The general method for preparing the stable carbon black dispersions in DMEM–FBS was as follows. First, the commercial carbon black aqueous dispersion (19.2 wt.%) was mixed with an aqueous solution of bovine serum albumin (BSA). Then, the pre-mixed carbon black/BSA aqueous dispersions were gently shaken and then immediately added into the DMEM– FBS. Carbon black dispersions in an aqueous NaCl solution (6.5 mg/mL; typical NaCl concentration in DMEM) were also prepared at the same concentration as the DMEM–FBS dispersions. All samples were prepared with an equal weight fraction of carbon black and nanodiamond (approximately 1.0 and 0.1 mg/mL) for all dispersions. Examples of stable carbon black dispersions in water, DMEM–FBS and NaCl aqueous solutions are described in Section 2. Interestingly, the prepared carbon black dispersions were homogeneously dispersed and no aggregates were found, as shown in Fig. 1b. In contrast, when the raw carbon black aqueous dispersion was directly introduced into DMEM–FBS, aggregation and precipitation were clearly observed, as shown in Fig. 1a, for example. In this study, nanodiamond dispersions in various solvents were prepared by the same method as the carbon

Fig. 1 – Photographs of carbon black dispersions: (a) raw aqueous carbon black dispersion added to DMEM–FBS and (b) pre-mixed aqueous carbon black/BSA dispersion added to DMEM–FBS. The obvious precipitation can be observed in (a) whereas homogeneously dispersed sample was obtained in (b).

black dispersions. The prepared carbon black and nanodiamond dispersions are summarized in Table 1.

3.2. Stability of prepared carbon black and nanodiamond suspensions over time Dynamic light scattering (DLS) measurements were carried out for the carbon black and nanodiamond suspensions. We prepared one bottle of the particle dispersion and separated it into three bottles. They were statically kept and the same sample positions were used for all bottles of nanoparticle suspension for the DLS measurements, which were stored over a week. In this stability analysis, we examined the size enlargement and sedimentation in experimental period. The stabilities of the suspension of carbon nanoparticles, not only as an agglomeration but also as sedimentation process [36], should be investigated over the whole period of the toxicity assessment, because in vitro toxicity assessments are typically carried out over long periods, i.e., the assessment takes long time to examine the viability of the examined cells. First, we examined the hydrodynamic diameters as a light scattering intensity-averaged diameter. We used a cumulative method for light scattering data analysis because the raw data was in good agreement with the observed photon correlation function. The results are plotted in Figs. 2 and 3 for carbon black suspensions and nanodiamond suspension, respectively. The standard deviations of the determined diameters in the figures were calculated from three repeated measurements. No obvious changes in particle size were observed over 7 days within experimental error as shown in Figs. 2 and 3 for all carbon black and nanodiamond dispersions. The observed raw photon correlation functions of the dispersions were not changed in the experimental period, indicating that the size distribution in the dispersions was not changed. Interestingly, using our pre-mixed preparation method, carbon black and nanodiamond suspension were stable even in high ionic strength aqueous dispersion, both in NaCl aqueous solution and a culture medium. On the other hand, reliable particle size measurements for the samples containing non-pre-mixed carbon black and nan-

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Table 1 – Parameters of carbon black and nanodiamond dispersions examined in this study. Sample Carbon black C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 C12 Nanodiamond N1 N2 N3 N4 N5 N6 N7 N8 N9 N10 N11 N12

Carbon nanomaterial concentration (mg/mL)

Medium

Pre-mixed in BSA solution

0.1

Water 3.3 mg/mL of BSA solution 6.5 mg/mL of NaCl solution 6.5 mg/mL of NaCl solution DMEM–FBS DMEM–FBS

– – Yes No Yes No

1.0

Water 3.3 mg/mL of BSA solution 6.5 mg/mL of NaCl solution 6.5 mg/mL of NaCl solution DMEM–FBS DMEM–FBS

– – Yes No Yes No

0.1

Water 3.3 mg/mL of BSA solution 6.5 mg/mL of NaCl solution 6.5 mg/mL of NaCl solution DMEM–FBS DMEM–FBS

– – Yes No Yes No

1.0

Water 3.3 mg/mL of BSA solution 6.5 mg/mL of NaCl solution 6.5 mg/mL of NaCl solution DMEM–FBS DMEM–FBS

– – Yes No Yes No

a

b

Fig. 2 – Plots of average hydrodynamic diameters from light scattering intensity versus time (days) for carbon black; the diameter did not change during the measurement, indicating no aggregation. (a) 0.1 mg/mL and (b) 1.0 mg/mL.

odiamond could not be obtained. The particle size of the carbon black and nanodiamond became considerably larger when directly dispersed in aqueous NaCl solutions and DMEM–FBS. The practical upper limit of particle size determination using DLS is approximately 1 lm. These results indicate that when not pre-dispersed, carbon black and nanodiamond easily aggregated in these media. As shown in Fig. 1a the size of the carbon black and nanodiamond became larger when directly dispersed in aqueous NaCl solutions and

DMEM–FBS. These results show that the increasing ionic strength of the medium decreased the Debye length. As a result, the average nearest-neighbor distance between the carbon black particles and between nanodiamond particles decreased and the particles agglomerated. Importantly, the dispersions prepared with carbon black and nanodiamond, pre-mixed in aqueous BSA solutions nonetheless remained stable, in contrast to the dispersions prepared without such pre-mixing.

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a

b

Fig. 3 – Plots of average hydrodynamic diameters from light scattering intensity versus time (days) for nanodiamond; the diameter did not change during the measurement, indicating no aggregation. (a) 0.1 mg/mL and (b) 1.0 mg/mL.

The determined particle sizes of carbon black and nanodiamond in various media are summarized in Table 2. In the table, it can be seen that the average sizes of the carbon nanoparticles using our pre-mixing preparation method were slightly larger than those in the corresponding aqueous dispersions prepared without pre-mixing. This may imply that a slight size enlargement occurred as a result of adsorption of BSA molecules on the carbon nanoparticles. Second, the stability of these colloidal suspensions in the aspect of sedimentation was also examined by light scatter-

ing measurements. In general, the Rayleigh light scattering intensity I obtained by DLS for a uniform size distribution of nanoparticles is given by 2  4  6  2 c 2p d m  1 ; ð2Þ I / I0 2 2r k 2 m2 þ 2 where c is the number of nanoparticles, m is the relative refractive index, r is the distance to the particles, k is the wavelength of the polarized laser, and I0 is the laser intensity. Taking the size of particles to be constant during the

Table 2 – Characterization of various carbon black and nanodiamond dispersions by DLS. Sample

a

Pre-mixed in BSA solution

d (nm)

r(d) (nm)

Stability

D(I/I1)day

Carbon black C1 C2 C3 C4 C5 C6

– – Yes No Yes No

161.1 178.7 183.6 NDa 188.1 NDa

1.6 5.3 7.1 NDa 8.6 NDa

Yes Yes Yes No Yes No

0.00 0.01 0.00 NDa 0.01 NDa

C7 C8 C9 C10 C11 C12

– – Yes No Yes No

158.7 171.5 177.0 NDa 187.3 NDa

2.9 6.0 4.8 NDa 7.6 NDa

Yes Yes Yes No Yes No

0.00 0.00 0.01 NDa 0.01 NDa

Nanodiamond N1 N2 N3 N4 N5 N6

– – Yes No Yes No

55.1 71.1 77.1 NDa 74.3 NDa

1.7 2.6 4.2 NDa 3.3 NDa

Yes Yes Yes No Yes No

0.00 0.00 0.00 NDa 0.00 NDa

N7 N8 N9 N10 N11 N12

– – Yes No Yes No

54.6 63.5 75.8 NDa 83.8 NDa

2.2 2.3 8.1 NDa 4.9 NDa

Yes Yes Yes No Yes No

0.00 0.00 0.01 NDa 0.00 NDa

Values are too large to determine reliably by DLS.

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measurement, the light scattering intensity is an indicator of the variation in the concentration of nanoparticles at an irradiated position in the cell culture medium [22,23]. This means the light scattering intensity is an important factor in estimating the degree of sedimentation of the secondary nanoparticles when the observed size of secondary particles is unchanged. The light scattering intensities were within experimental error for these colloidal suspensions over the measurement time. The ratio describing the change of light scattering intensity per day is calculated using the following equation:  i Ph   1  II1 I i D ¼ P ; I1 day Nday;i

ð3Þ

where Nday is the number of days, I is the light scattering intensity, and I1 is the light scattering intensity of secondary nanoparticles on the first day. The results are also summarized in Table 2. The values were sufficiently small, indicating that gravitational settling did not occur.

3.3. Estimation of origin of the stability of carbon black and nanodiamond dispersions To determine the origin of the high stability of the dispersions, zeta potential measurements of the stable carbon black and nanodiamond dispersions were performed to estimate the changes in the surface potentials after pre-mixing in aqueous BSA solutions. The results are summarized in Table 3. The uncertainties in the table were calculated from the repeatability of measurement of the observed zeta potentials calculated in a day for at least three separate measurements. Unfortunately, the zeta potentials in higher-concentration carbon black and nanodiamond dispersions could not be measured because of the concentration limit of the zeta potential instrument. The zeta potentials of carbon black and nanodiamond in a pure aqueous solution were determined to be between approximately 70 and 60 mV, while those in aqueous BSA solution shifted to between approximately 15 and 10 mV, indicating that the BSA was adsorbed on the carbon black and nanodiamond particles. It was determined, as seen from Table 3, that the amount of the adsorbed BSA molecules did not change because the zeta potential values did not change notably. This estimation was also supported by DLS measurements, the results of which are shown in Figs. 2 and 3.

Table 3 – Zeta potential of various carbon black and nanodiamond dispersions. Sample

f potential (first day)

r (mV)

f potential (after 7 days)

r (mV)

C1 C2 C3 C5 N1 N2 N3 N5

67.0 16.2 15.8 14.2 61.7 14.6 11.3 13.8

4.6 1.4 2.5 0.7 2.9 1.4 2.5 3.0

70.9 15.4 15.2 14.7 69.2 13.6 10.1 16.6

3.5 0.9 2.4 3.0 5.7 3.4 3.4 4.2

Using the results of zeta potential measurements, the stability of the carbon nanoparticle dispersions in simple NaCl aqueous solution can be described using DLVO theory. The stability of carbon nanoparticle dispersions is governed by two different interactions in DLVO theory, namely, repulsive electrostatic interactions and attractive van der Waals interactions. The energy of the repulsive electrostatic interaction results from the overlap between diffuse double layers of the particles and the energy decreases approximately exponentially with the separation distance between particles; at large separation distances, the repulsive interaction is diminished, but still notable. On the other hand, the energy of the van der Waals attraction decreases with the inverse sixth power of the separation distance. According to DLVO theory, the repulsive electrostatic interaction can be controlled through the addition of an ionic surfactant, and thereby the aggregation of particles can be prevented by adsorption of the surfactant on the particle surface. The total inter-particle interaction versus distance relationship can be calculated for two particles using the DLVO theory. At the inter-particle distance where the electrostatic interaction falls significantly below kT and the attractive van der Waals interaction becomes important, the inter-particle van der Waals interaction can be described as follows:

VðAÞ ¼ 

A 2 2 4B þ B2 þ þ ln 2 2 6 4B þ B ð2 þ BÞ ð2 þ BÞ2

!! ;

ð4Þ

where B = 2r/d, d is the diameter of a nanoparticle, r is the distance between the two interacting nanoparticles, and A is the Hamaker constant. The value of A was used for the value of BSA in water and is 0.753 · 1020 J [37], since both carbon nanoparticles were found to be coated by BSA. On the other hand, repulsive interaction between nanoparticles from the electrical double-layer can be described as follows: VðRÞ ¼ pe0 edW2 ejr ; and ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi sP 2 2 1 i q1i e zi ¼ ; j e0 ekB T

ð5Þ

ð6Þ

and 1 0:304 ¼ pffiffiffiffiffiffi : j CS

ð7Þ

In the above expressions, e0 is the permittivity of vacuum, e is the dielectric constant of the electrolyte solution, e is the elemental charge, kB is the Boltzmann constant, T is the absolute temperature, q1i is the ion number density in the bulk electrolyte, z is the valence of ion i, and j is the inverse of the Debye length. Eq. (6) can be written as Eq. (7) at room temperature for a 1:1 electrolyte (NaCl) [38]. The calculated value of the Debye lengths at the NaCl concentration of 6.5 mg/mL in an aqueous solution is 0.91 nm. As shown by these equations, the range and strength of the electrostatic interaction are determined by the surface potential and the Debye length. For simplicity, the value of e was taken to be 78.5 for water under the assumption

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Fig. 4 – Plots of the potential energies (Vtotal,DLVO) of interactions in units of kT versus the separation distance between carbon nanoparticles. The diameter of the nanoparticle used in the simulation is 100 nm.

that e0 is 8.85 · 1012, T is 298.15 K, kB is 1.38 · 1023 J K1, and e is 1.6 · 1019 C, although the Debye length is an important factor in the description of the electrical double-layer repulsion interaction between nanoparticles. Assuming that the zeta potentials are equal to the surface potentials of the carbon nanomaterials, their electrostatic surface potentials depend on the NaCl concentration. In Eq. (5), W is the surface potential that is replaced with the zeta potential (we used 15 mV, which is a typical average value of the zeta potential for both carbon black and nanodiamond), and we calculated the potential energy of the repulsive interaction between the electrical double layers. The total DLVO forces are given as Vtotal;DLVO ¼ VðAÞ þ VðRÞ:

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ð8Þ

In Fig. 4, we plot the interaction forces calculated using the DLVO theory for a NaCl aqueous solution; these plots are given in units of kT versus separation distance between surfaces. Interestingly, the DLVO force profiles of the interactions between carbon nanoparticles were negative, indicating that the dispersions would be quite unstable if only repulsive electrostatic interactions and attractive van der Waals interactions were operative. Because the stability of carbon black and nanodiamond dispersions was already observed experimentally, the results in Fig. 4 suggest that the high stability of these carbon nanomaterials in both the aqueous NaCl solution and DMEM–FBS may result from a steric interaction between BSA molecules adsorbed on the carbon black and nanodiamond. This protective layer may prevent aggregation of the carbon nanoparticles, as has been reported previously for fullerene and carbon nanotubes [39,40].

3.4. Estimation of amount of adsorbed BSA molecules in carbon black and nanodiamond dispersions In order to observe the amount of adsorbed BSA molecules on the carbon nanoparticles AFFFF measurements were carried out. AFFFF is an elution technique where nanoparticles and macromolecules are separated by flow control in an aqueous

Fig. 5 – UV absorption at 210 nm for C2 sample separated by the AFFFF method. Peaks: BSA, 7–12 min; carbon black colloidal particles: 20–25 min.

Table 4 – Estimation of adsorbed BSA molecules by AFFFF method. Sample

Carbon Total BSA Free BSA Bound BSA nanomaterial (mg/mL) (mg/mL) (mg/mL) (mg/mL)

C2 C8 N2 N8

0.11 1.09 0.10 1.05

3.36 3.36 3.28 3.28

3.29 2.79 3.21 2.68

0.05 0.56 0.06 0.60

solution. In AFFFF, the retention time of the nanoparticles can be predicted according to Giddings’ theory, in which the retention time tr of the nanoparticle is given by 2

tr ¼

pgdw VC : 2kT V0

ð9Þ

Here, kT is the thermal energy, g is the carrier elution viscosity, d is the diameter of the nanoparticle, w is the channel width, V0 is the volumetric channel-flow rate, and VC is the cross-flow rate. When the cross-flow and channel-flow rates are constant, the nanoparticles can be separated according to their size, and the retention time is proportional to the size of the nanoparticles and molecules in the AFFFF system. Fig. 5 shows the UV intensity (observed at 210 nm) obtained for C2 sample separated by the AFFFF method. The channel flow rate was 1.00 mL/min and the cross-flow rate was 4.5 mL/min. As eluent for the AFFFF measurement, 6.5 mg/mL NaCl aqueous solution was used. In Fig. 5 , the first elution peak at 7–12 min was BSA, including its monomeric, dimeric, and trimeric forms. The total peak area was proportional to the free BSA concentration in solution [31]. On the other hand, the latter peak at 20–25 min was colloidal carbon black particles. In this study, we carried out AFFFF measurements for four different samples, namely, C2, C8, N2, and N8, because the total amounts of BSA is known for these samples. The observed results are summarized in Table 4. Interestingly, the amounts of bound BSA molecules calculated from those of free and total BSA molecules were determined to be approximately 0.05 and 0.5 mg/mL for both carbon black

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and nanodiamond dispersions with 0.1 and 1.0 mg/mL of the total carbon nanomaterials, respectively. In all cases, the calculated weight ratios between BSA molecules and carbon black/nanodiamond were approximately 1:2. These results indicated that the increase in particle size observed by DLS could be attributed to the adsorption of the BSA molecules on the surface of the carbon nanoparticles. For carbon black and nanodiamond, the increase in particle size corresponds to a thickness of the adsorbed BSA layer of 20–30 nm. The observed ratios did not change depending on the concentration of the carbon nanomaterials, suggesting that the surfaces of carbon black and nanodiamond were fully covered at this ratio. It was estimated that this covered BSA molecules worked as a protective layer and prevented the aggregation of the carbon nanoparticles. The high stability of these carbon nanomaterials in both aqueous NaCl solution and DMEM–FBS could be caused by the steric interaction between the adsorbed BSA molecules on the carbon black and nanodiamond.

4.

Conclusions

A stable dispersion of carbon black and nanodiamond in high ionic strength aqueous solutions, namely, NaCl aqueous solution and culture medium, were successfully prepared by a simple procedure involving addition of carbon black premixed in an aqueous BSA solution to these aqueous solutions. The stability of the carbon black and nanodiamond dispersion arose from the adsorbed BSA molecules, even in high ionic strength solutions. In contrast to the pure carbon black and nanodiamond aqueous dispersions, the observed zeta potentials for both carbon nanoparticles after pre-mixing with BSA solution were clearly reduced. The calculated DLVO force profiles of the interactions between carbon nanoparticles were negative, indicating the dispersions would be quite unstable if only repulsive electrostatic interactions and attractive van der Waals interactions are taken into account. The high stability of these carbon nanomaterials in both aqueous NaCl solution and DMEM–FBS could be caused by the steric interaction between the adsorbed BSA molecules on the carbon black and nanodiamond. In all carbon nanomaterial dispersions, the observed weight ratio between BSA and either carbon black or nanodiamond was approximately 1:2, contributing to the high stability of the suspensions. This result provided significant information that is expected to be useful for toxicity studies on carbon nanomaterials.

Acknowledgments Financial support from the Nanotechnology Material Metrology Project conducted by the New Energy and Industrial Technology Development Organization (NEDO) is gratefully acknowledged. R E F E R E N C E S

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