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Characterisation of the de-agglomeration effects of bovine serum albumin on nanoparticles in aqueous suspension
Colloids and Surfaces B: Biointerfaces 75 (2010) 275–281
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Characterisation of the de-agglomeration effects of bovine serum albumin on nanoparticles in aqueous suspension Ratna Tantra ∗ , Jordan Tompkins, Paul Quincey National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, United Kingdom
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Article history: Received 7 July 2009 Received in revised form 28 August 2009 Accepted 28 August 2009 Available online 6 September 2009 Keywords: Zinc oxide Titanium dioxide Nanoparticles Albumin De-agglomeration Stability
a b s t r a c t This paper describes the use of nanoparticle characterisation tools to evaluate the interaction between bovine serum albumin (BSA) and dispersed nanoparticles in aqueous media. Dynamic light scattering, zeta-potential measurements and scanning electron microscopy were used to probe the state of zinc oxide (ZnO) and titanium dioxide (TiO2 ) nanoparticles in the presence of various concentrations of BSA, throughout a three-day period. BSA was shown to adhere to ZnO but not to TiO2 . The adsorption of BSA led to subsequent de-agglomeration of the sub-micron ZnO clusters into smaller fragments, even breaking them up into individual isolated nanoparticles. We propose that certain factors, such as adsorption kinetics of BSA on to the surface of ZnO, as well as the initial agglomerated state of the ZnO, prior to BSA addition, are responsible for promoting the de-agglomeration process. Hence, in the case of TiO2 we see no de-agglomeration because: (a) the nanoparticles are more highly agglomerated to begin with and (b) BSA does not adsorb effectively on the surface of the nanoparticles. The zeta-potential results show that, for either ZnO or TiO2 , the presence of BSA resulted in enhanced stability. In the case of ZnO, the enhanced stability is limited to BSA concentrations below 0.5 wt.%. Steric and electrostatic repulsion are thought to be responsible for improved stability of the dispersion. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction The ability to characterise the dispersion of nanoparticles in aqueous media is useful for many different applications [1]. For example, in experimental nanotoxicological studies, measuring the state of the dispersed nanoparticles in a biologically relevant media is of great importance for assessing nanoparticle toxicity. Measuring parameters such as particle size and state of aggregation are vital, as these parameters are thought to influence the level of toxicological activity of the nanoparticles [2]. Stabilisers such as the proteins BSA (bovine serum albumin) or HSA (human serum albumin) are often added to the dispersion media, in order to enhance stabilisation by preventing subsequent agglomeration of the nanoparticles [3]. Hence, the ability to characterise nanoparticle–albumin interaction is of significant interest [4]. In mammals, serum albumin is one of the most abundant plasma proteins and has a role in maintaining colloid osmotic pressure (needed for proper distribution of body fluids between intravascular compartments and body tissues), binding (in which it acts as a plasma carrier by non-specifically binding several hydrophobic steroid hormones) and transport (of hemin and fatty acids) [5]. Furthermore, serum albumin from bovine serum concentrate is a
∗ Corresponding author. E-mail address: [email protected] (R. Tantra).
protein commonly used in biological laboratories for immudiagnostic procedures, clinical reagents, cell culture media, and protein chemistry. BSA (of 69,323.4 Da) has a size of 40 Å × 40 Å × 140 Å, contains 607 number of amino acid and has pI (isoelectric point; this is the pH value at which BSA carries no net electrical charge) of 4.7 in water (at 25 ◦ C) [6]. The traditional approach to examining nanoparticle–albumin interactions is through the use of dynamic light scattering (DLS) methods to determine particle size distribution, and zeta-potential measurements, so as to have some indication of relative stability of the dispersion [7]. However, these techniques have their limitations, in that they do not offer sufficient sensitivity and selectivity to allow us to probe into the mechanism of the interaction in great detail. The additional use of imaging tools such as SEM and TEM can play a crucial role in determining the mechanism behind the nanoparticle–albumin interaction. Imaging techniques make it possible to probe the state of individual nanoparticles [8]. SEM has several advantages over TEM–SEM analysis is faster, cheaper, easier to use and thus more suitable for making routine measurements. More importantly, SEM employs relatively low beam energy in comparison to TEM, which is crucial for accurate characterisation of nanoparticles. A recent paper by Wang et al. [9] has underlined the importance of addressing the inherent problems associated with TEM imaging, in that the state of the nanoparticles can be easily modified by the high-energy electron beam. According to
0927-7765/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.08.049
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Wang, there is potential for the high-energy beam to introduce an “electron beam-induced effect” that can result in defect formation, charging of particles and possibly excitation of the surrounding gases. Previous work [4] has used SEM to measure the state of nanoparticles in the dispersed state, but there are questions about the accuracy of such results, in particular if the images are a true representation of the original suspended state of the nanoparticles. The main disadvantage with techniques such as SEM is in the sample preparation step, in particular the need to transfer a suspension of the nanoparticles on to a suitable substrate, letting the liquid dry under ambient conditions and ensuring that changes to the state of the nanoparticles are minimised in the process. It is vital to have good control over this process, to prevent artefacts (such as irreversible aggregation of the nanoparticles or the formation of crystals from the biologically related media) being introduced during the sample preparation stage [10]. The objective of this study therefore was to characterise nanoparticle–albumin interactions by means of several techniques. Two types of nanoparticle, zinc oxide (ZnO) and titanium dioxide (TiO2 ) were employed, as they are of high commercial relevance [11]. The nanoparticles were dispersed in water using an ultrasonic probe. This technique is commonly used to disperse nanoparticles in aqueous media, by generating and transmitting shear waves to de-agglomerate particles in water [12]. The effect of adding BSA (at various concentrations) to the dispersed nanoparticles was evaluated for a period of three days. The state of the nanoparticles was monitored using three main techniques: particle size analysis (using DLS), zeta-potential (using Doppler electrophoresis) and SEM. In order to improve the sample preparation protocols for SEM imaging, commercially available poly-l-lysine coated glass substrates were employed to fix the nanoparticles prior to SEM analysis. The use of commercially available substrates is particularly advantageous, as this will reduce time and inconsistencies associated with any associated surface modification protocols [13]. One feature of the sampling protocol used is the absence of an undesirable evaporative-drying step in the sample preparation, which can be responsible for inducing aggregation of the nanoparticles, a source of potentially misleading results. In theory, the SEM image acquired will be more representative of the corresponding dispersed nanoparticles. The effectiveness of the protocol was tested using NIST-certified latex beads in the first instance. The study aimed to probe the mechanism behind the BSA–nanoparticle interaction by correlating findings obtained from the SEM images with that of DLS particle size and zeta-potential data. Although characterising the effect of BSA on nanoparticle dispersion is not new [14], the availability of SEM images that clarify these effects is very limited. It was hypothesised that the SEM data obtained would not only support observations from the corresponding DLS/zeta-potential measurements, but would provide additional information not obtained from the other techniques.
2. Methods 2.1. Starting materials De-ionised water (DI, Millipore, MilliQ) was used to prepare all aqueous suspensions/solutions. Stock solution of BSA (Sigma–Aldrich, UK) in DI water (final concentration of 10 wt.%, 100 ml) was made. BSA stock solution was filtered through 0.2 m membrane (Nalgene sterile disposable filter units, Fluka). This stock has been used to disperse nanoparticles (protocol described below) and to also make control samples of BSA (BSA in DI water) at a range of concentrations (0.02, 0.1, 0.5 and 2 wt.%). All BSA solutions were
stored in amber-coloured glass containers at 4 ◦ C (when not in use) and discarded two weeks after preparation. The ZnO was purchased (Nanostructured and Amorphous Materials, Inc.), and the TiO2 was donated by Professor Ken Donaldson (University of Edinburgh, Scotland, UK). The nanoparticles were received in dry powder form and used without further purification. 2.2. Aqueous suspension of the nanoparticles ZnO was weighed on analytical mass balance and then suspended in DI water, at a concentration of 1 mg/ml (500 ml). The suspended nanoparticles were ultrasonically irradiated in a 1 l beaker for 30 s. This was shown to be sufficient time to aid mixing and forming a homogeneous suspension. The ultrasonic treatment was applied by Heat Systems Model XL2020 (with a maximum power output of 550 W and operated at a frequency of 20 kHz). Unless stated otherwise, aliquots of 50 ml of the resultant mixture were transferred into centrifuge tubes (50 ml 10×) and the tubes were subjected to centrifugation (20 ◦ C) for 15 min, at 4800 rpm (using Eppendorf Centrifuge 5804 R). The purpose of this centrifugation step was to remove any large aggregates/agglomerates. A small pellet at the bottom of each tube was observed after the centrifugation step. Immediately after centrifugation, the supernatant was pipetted into a large beaker, taking care not to disturb the sample pellet at the bottom of the tube. Suitable aliquots of this were mixed with appropriate amounts of BSA stock (10 wt.%)/DI water, resulting in a final volume of 20 ml, in order to make a range of BSA concentrations (0, 0.02, 0.1, 0.5 and 2 wt.%); following addition of BSA, the mixture was gently shaken to ensure mixing. The resultant mixture was stored in individual screw capped ambercoloured glass bottles (30 ml, Fisherbrand) and stored in a fridge (4 ◦ C) when not in use. An identical sample preparation procedure was employed for the TiO2 powders. 2.3. Characterisation of the suspension All bottles containing the samples for analysis were stored at 4 ◦ C. Prior to use, the bottles were gently shaken and allowed to equilibrate at room temperature (20 ◦ C) for at least 1 h. The prepared samples of suspended nanoparticles, in the absence and presence of BSA at various concentrations, were analysed daily for a period of three days using size particle DLS, zeta-potential and SEM imaging. Control measurements were carried out using BSA solutions (in the absence of nanoparticles), at various concentrations. The instrument employed for particle size analysis and zetapotential measurements was a Zetasizer Nano ZS (Malvern Instruments, UK) with 633 nm red laser. Particle size was obtained from measuring the time dependent fluctuation of scattered light arising from the suspension of the nanoparticles undergoing random Brownian motion. The diffusion coefficient of the particles is deduced and analysed to give their mean hydrodynamic diameter, i.e. the so-called “z-average” diameter that is reported here. For the zeta-potential measurement, the instrument uses laser Doppler electrophoresis to measure the net velocity of the nanoparticles in the liquid that results when an electric field is applied. Here, the fundamental quantity measured is the net electrophoretic mobility () of the particles, which is then is converted to the zeta-potential () using Henry’s approximation [15]; the Smoluchowski approximation was applied in our measurement (this approximation is generally applied to aqueous media), in which the Henry’s function in the equation takes a value of 3/2. Both types of analysis can be carried out using the same sample cell, a disposable folded capillary cell (DTS1060) supplied by Malvern. Prior to its use, the cell was filled with de-ionised water and allowed to equilibrate for 30 min.
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All of the water was emptied and the cell was flushed with the appropriate sample solution. The measurement was carried out on the second filling; sufficient sample volume was used to completely cover the electrodes of the cell. To avoid air bubbles in the cell, the sample was injected slowly; analysis was carried out if no visible air bubble inclusions were present in the cell. The cell was placed into the Zeta-sizer and equilibrated at 20 ◦ C (close to the average temperature of the laboratory) for 2 min prior to taking the particle size measurements (three replicates); the measurement temperature was set and maintained by the Peltier elements in the sample holder of the instrument. The corresponding zeta-potential measurements (three replicates) were taken immediately after this. Both the sample and cell were then discarded after use. Malvern Instrument’s Dispersion Technology software (Version 4.0) was used to control and analyse all data from the instrument. Data collected were then imported to Excel and functions within Excel were used to compute the mean and standard deviations of the set of replicates measured. The results are plotted as mean values with error bars of one standard deviation. Scanning electron microscope images were collected using a Carl Zeiss Supra 40 electron microscope, in which the optimal spatial resolution of the microscope is a few nanometres. Images were acquired with an accelerating voltage of 15 kV, working distance of 2–3 mm, tilting angle 0◦ , and recorded with an in-lens secondary electron detector. Sample preparation for SEM analysis requires the deposition of the appropriate liquid sample (1 ml) on to a poly-l-lysine coated microscope glass slide (purchased from Fisher Scientific, UK) and allowing it to incubate for a period of 5 min at room temperature (∼20 ◦ C). There were no substantial visible signs of evaporation during the 5-min period. After 5 min, the substrate was then washed with DI water in order to dislodge any loosely bound nanoparticles on the surface. The substrate was then subsequently allowed to air dry for 15 min in an open Petri dish in a fume hood. The step of aliquoting the liquid sample on the surface (followed by incubation for 5 min, washing and subsequently drying) was repeated again on the same substrate. However, in the final step, the slides were allowed to dry for a couple of hours before they were thinly sputtered with gold. The device used to deposit the gold was an Edwards S150B sputter coater unit. Sputtering was conducted under vacuum (7 mbar), while passing pure, dry argon into the coating chamber. Typical plate voltage and current were 1200 V and 15 mA, respectively. The sputtering time was approximately 10 s, which resulted in an estimated gold thickness of not more than a few nanometres being deposited on top of the substrate. The appropriateness of this sample preparation protocol, for the trapping nanoparticles on to a poly-l-lysine coated substrate, was evaluated by using NIST-certified 100 nm latex beads (supplied by Agar Scientific, UK).
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Fig. 1. SEM images of the nanoparticles mounted on carbon pad, showing: primary particle sizes and shapes, as well as the degree of aggregation/agglomeration in the “as-received powders”: (a) ZnO and (b) TiO2 .
3.2. Nanoparticle entrapment using poly-l-lysine substrates Fig. 2a shows a typical SEM image of NIST-certified latex beads fixed on poly-l-lysine coated slide, using the deposition protocol described in Section 2. This is a low magnification image (10,000×),
3. Results and discussion 3.1. Initial particle properties Fig. 1 shows the SEM images of both ZnO and TiO2 nanoparticles of the “as–received” powder, prior to dispersing them in aqueous media. Results show that both nanoparticles exist as aggregates and agglomerates (fusing of particles). The presence of agglomerates, which is particularly evident in the case of TiO2 , has meant that to achieve successful dispersion, significant energy is required to break them up. Hence, the particle dispersion protocol employed in this study involved the use of an ultrasonic probe in order to disperse the nanoparticles [16]. From the SEM images, polydispersity in the samples existed and several shape variations were also observed. This is particularly apparent with ZnO, in which diverse shapes ranging from rod-like, hexagon prism and cuboids are apparent.
Fig. 2. SEM images of sputtered (100 nm) latex beads (NIST-certified) to show the potential of the poly-l-lysine substrate to trap nanoparticles. Images shown were taken at low magnification (10,000×) and high magnification (80,000×).
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showing a highly homogeneous distribution of the latex spheres. The corresponding image taken at higher magnification of 80,000× is given in Fig. 2b, which shows spheres with average size of ∼98 nm, with S.D. of 2 nm (the average and standard deviation data were taken from 10 particles, chosen at random); this coincided with the certified values of 97 ± 3 nm (as listed on the NIST Certificate of Analysis). The attachment of the latex spheres on the surface is most likely to be based on very strong electrostatic interaction that exists between the beads and the coated glass surface [17]. The adhesion strength was shown to be strong enough to avoid the removal of the nanoparticles, even after carrying out the multiple washing steps (described in the protocol). Although many of the latex beads were fixed as singly isolated particles, about a quarter of the latex beads existed as small clusters of particles. Such aggregates may be due to hydrophobic interactions that can occur between the beads. We saw no large aggregate clusters on the surface. In summary, we have shown that the use of such poly-l-lysine coated substrates (along with the protocol developed in this study) is suitable to monitor the state of dispersed nanoparticles, and is readily capable of fixing single isolated nanoparticles. 3.3. Effect of BSA on dispersed ZnO nanoparticles Fig. 3 shows the effect on z-average diameter and zeta-potential values, when BSA (within a concentration range of 0–2 wt.%) was added to dispersed ZnO nanoparticles; the effect of BSA on the nanoparticles were monitored over a three-day period. It is apparent that even a small amount of BSA (0.02 wt.%) can have a drastic effect on the particle size (Fig. 3a). DLS results show that increasing BSA concentration resulted in a decrease of particle size, until the nanoparticle size reached ∼6 nm in size (approximately the same size as observed from the BSA control), when the BSA concentration exceeded 0.5 wt.%. Interestingly, the zeta-potential results
(Fig. 3b) followed the same trend as the DLS results, in that the BSA control and the nanoparticle dispersion showed similar results with BSA concentrations above 0.5 wt.%. Zeta-potential plots show a minimum at around 0.1 wt.% BSA, indicating that this is the concentration of BSA that resulted in the highest stability. Apart from the nanoparticle suspensions containing no BSA, data from day 2 and day 3 followed similar trends to day 1. The nanoparticle suspensions without BSA showed a marked increase in particle size from day 1 to day 3, from 333 to 1044 nm, suggesting significant aggregation of the nanoparticles over this period in the absence of BSA. The reduced particle size observed when BSA is present in the ZnO suspension can be explained by the de-agglomeration of ZnO clusters. Below 0.5 wt.% BSA, the zeta-potential results indicated that such de-agglomerated species would remain stable through time. It has been suggested in the past that BSA offers steric stabilisation of nanoparticles [2,14]. Above 0.5 wt.% however, the system is unstable, as indicated by the zeta-potential results, suggesting that in this concentration range there is a strong tendency for such de-agglomerated species to re-agglomerate. This effect would ultimately lead to the nanoparticles sedimenting out of the suspension, leaving the remaining free BSA in suspension. If BSA is completely absent from the ZnO dispersion, then the nanoparticles have an evident tendency to aggregate significantly over a three-day period. Fig. 4 shows SEM images of the dispersed ZnO nanoparticles on a poly-l-lysine substrate, prepared on day 3: (a) with no BSA and (b) in the presence of 0.02 wt.% BSA (scale bars on the images are 200 and 100 nm, respectively). These images were selected to be “typical”, as chosen (out of random 10 images) by the operator. Images show that in the absence of BSA, nanoparticles show polydispersity and shape variation, quite similar to the “as-received” powder (shown in Fig. 1a). The images illustrate the smaller clusters of nanoparticles observed when BSA is added; the image in Fig. 4b shows a cluster size of approximately 444 by 600 nm (in the presence of BSA). This is smaller in comparison to 840 by 1060 nm in the absence of BSA, as shown in Fig. 4a (inside the dotted box). The SEM images seem to correlate well with the findings from the corresponding DLS data (Fig. 3a), in which the DLS data showed an average particle size of 1044 nm (S.D. 156 nm of three replicates) in the absence of BSA, which reduced dramatically to 288 nm (S.D. 27 nm of three replicates) in the presence of BSA 0.02 wt.%. Although it would seem that the SEM images support our hypothesis, in that de-agglomeration has occurred with ZnO in the presence of BSA, it must be remembered that SEM is a “single particle” based technique (thus impractical to achieve a truly representative data), as oppose to the “population” based technique offered by routine tools such as DLS; in this respect, the interpretation given here must therefore be made with caution. Of interest therefore is the presence of BSA-coated ZnO clusters, as observed from the SEM image of Fig. 4b; this apparent coating was not present in the absence of BSA. Such a thick coating on the ZnO clusters was observed with all other concentrations of BSA. Further inspection of the ZnO nanoparticles on poly-lysine substrate in the present of 0.02 wt.% BSA has shown the presence of single isolated nanoparticles (of rods and pyramid shapes) as shown in Fig. 4c. To our knowledge, these are the first SEM images that depict the presence of isolated single nanoparticles from such a de-agglomeration event. 3.4. Effect of BSA on dispersed TiO2 nanoparticles
Fig. 3. Effects of BSA [at various concentrations: 0, 0.02, 0.1, 0.5 and 2 wt.%] on the dispersion of ZnO in DI water, over a three-day period (data compared to control BSA solutions in the absence of nanoparticles): (a) mean particle size (z-average); (b) zeta-potential values.
Fig. 5a shows the particle size plots of TiO2 dispersed in water in the absence and presence of BSA [concentration up to 2 wt.%], as obtained by DLS measurements over a three-day period. The results show an entirely different response to the corresponding ZnO case
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Fig. 4. SEM images of ZnO nanoparticles on poly-l-lysine substrates, prepared on day 3: (a) with no BSA and (b) with BSA (0.02 wt.%), showing the presence of small cluster of nanoparticles and single isolated nanoparticles on the surface.
(Fig. 3a). Overall, the average particle size of TiO2 is not affected by the addition of BSA; this is true apart from one measurement replicate of TiO2 with 0 wt.% BSA on day 1, which may be a possible outlier. This result gives an average particle size of 278 nm (with S.D. of 10 nm) as opposed to an average value of 194 nm (with S.D. of 8 nm) from the rest of the TiO2 DLS data. Unlike ZnO, the TiO2 plots did not intersect with the corresponding BSA control plot at any BSA concentration. Fig. 5b shows the corresponding zeta-potential plots of TiO2 in the absence and presence of BSA (concentration range of up to 2 wt.%). The general effect upon BSA addition resulted in a negative shift of zeta-potential value indicating enhanced stability, quite similar to what was observed with the ZnO case. However, unlike the ZnO case, the zeta-potential of TiO2 in the presence of BSA was shown to give similar values at all BSA concentrations, with average of −27 mV (S.D. of 3 mV). Data from TiO2 samples with no BSA gave average zeta-potential values of 16 mV (with S.D. 6 mV). Unlike the ZnO case, in which we saw zeta-potential plots that first fell rapidly, passed through minima, and then rose again (Fig. 3b) with increasing BSA concentration, the zeta-potential plots here fall rapidly and reach a saturation level. Fig. 6a and b shows SEM images of dispersed TiO2 nanoparticles fixed on to a poly-l-lysine substrate on day 3: (a) with no BSA and (b) in the presence of 2 wt.% BSA (scale bars on the images are 200 nm), respectively. In the absence of BSA, the image shows small clusters (in agglomerates) with the state of the pri-
mary particle (size and shape) being very similar to those observed in “as-received” powder (shown in Fig. 1b). There was very little observable change after addition of BSA into the dispersed TiO2 , even at a concentration of 2 wt.% (Fig. 6b); similar images were obtained with other BSA concentrations. In these images, no blotchy features on the surface of the nanoparticles were observed, unlike the images obtained with the corresponding ZnO case (for example, in Fig. 4c). An obvious difference between the images observed in Fig. 6, is the much reduced number of TiO2 clusters present on the poly-l-lysine surface when BSA was present in solution. Results show no evidence of TiO2 de-agglomeration taking place in the presence of BSA. In addition, the SEM images suggest that the BSA must exist as free entities in solution rather than being adsorbed on the surface of TiO2 nanoparticles. The presence of free BSA in solution will undoubtedly compete with the TiO2 nanoparticle clusters for binding sites on the poly-llysine substrate. This is likely to explain why fewer TiO2 clusters were observed in the SEM images when BSA is present. Nonetheless, as in the case of ZnO, the presence of BSA in solution has resulted in an enhanced stability of the dispersion, irrespective of BSA concentration. The outlier data observed with the TiO2 in the absence of BSA on day 1 may be explained by possible contamination in the sample cell used, which subsequently induced particle aggregation resulting in larger clusters than with the other results.
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Fig. 5. Effects of BSA [at various concentrations: 0, 0.02, 0.1, 0.5 and 2 wt.%] on the dispersion of TiO2 in DI water, over a three-day period (data compared to control BSA solutions in the absence of nanoparticles): (a) mean particle size (z-average); (b) zeta-potential values.
as the effect of the adsorption may be insufficient to break up the much-agglomerated nanoparticles. For de-agglomeration to occur to the point of creating individual isolated nanoparticles, it is important for the BSA to be sufficiently adsorbed in between adjacent nanoparticles. It is postulated that in the case of ZnO, the thickness of the adsorbed BSA layer is sufficient to result in steric repulsion, strong enough for de-agglomeration into isolated individual particles to occur. Overall, the stability of such deagglomerated species will be related to the zeta-potential of the system; if the value is sufficiently negative, the de-agglomerated species will exist as a stable form for a long period of time. However, if the zeta-potential of the system indicates instability, then such de-agglomerated species will re-agglomerate and eventually fall out of suspension [18]. It is clear that nanoparticle–BSA interaction has led to an adsorbed layer of BSA on the surface. If such interactions occurred in living systems, then the instantaneous formation of the BSA–nanoparticle complex would result, and subsequent toxicological activity would be dominated by the BSA–nanoparticle complex, which would largely define the biological identity of the particle, rather than the inherent properties of the nanoparticles. The formation of such nanoparticle–protein complexes has been studied by past workers [19,20] and the complex is often referred to as a “protein corona”. The results presented here clearly show the existence of such a “protein corona”. Previous work on the “protein corona” has demonstrated that both size and surface properties of the nanoparticles have been found to play a very significant role in determining the nanoparticle coronas on different particles of identical material [21]. Thus, it is reasonable to assume that the size and surface properties of the protein itself will also govern the formation of such coronas, as this will influence the:
3.5. A summary of the mechanisms by which BSA affects dispersed nanoparticles This study has provided insight into the interaction between BSA and dispersed nanoparticles and the main aim of this section is to propose a general mechanism underlying the interaction of BSA with nanoparticles. When nanoparticles are in suspension with BSA, the BSA can either adsorb to the surface of the nanoparticles or exist as free entities in solution. If adsorbed, the BSA has the potential to deagglomerate clusters of nanoparticles by changing the surface forces involved. If nanoparticles are not highly agglomerated to start off with, the de-agglomeration can be instantaneous. If they are highly agglomerated, then de-agglomeration may not occur,
(a) adsorption kinetics between BSA and the nanoparticles; (b) subsequent (steric) stability of the nanoparticle-BSA complex.
We envisage therefore that other proteins with similar properties to BSA (particularly size and pI values) would undoubtedly act as stabilisers in a similar manner. However, we have to be aware of the significant differences that may arise due to batch-to-batch variations (e.g. changes in particle size due to the formation of aggregates/agglomerates, through possible degradation of the protein through time), which would subsequently influence the final results.
Fig. 6. SEM images of TiO2 nanoparticles on poly-l-lysine substrates, prepared on day 3: (a) with no BSA and (b) with BSA (2 wt.%).
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4. Conclusion In summary, our study has highlighted the importance of using different techniques in parallel to probe the interaction between BSA and dispersed nanoparticles, and to have careful protocols for preparing SEM samples of nanoparticles from suspension. Single particle techniques, such as SEM, are thus powerful complementary techniques to population-based methods such as DLS. The poly-llysine coated substrate used in the SEM sample preparation here was shown to be effective, to the extent that it was capable of collecting isolated individual nanoparticles that were present in the dispersed state. Our experiments showed that interactions between nanoparticles and BSA can lead to one of three outcomes: (a) stable de-agglomeration of nanoparticle clusters; (b) unstable de-agglomeration of nanoparticle clusters; (c) no significant agglomeration or de-agglomeration effects taking place. The type of outcome that occurs in a given system is mainly dependent on three factors: (a) the initial agglomerated state of the nanoparticles, (b) the adsorption kinetics of BSA on to the surface of nanoparticles and (c) the zeta-potential of the final system. De-agglomeration into isolated nanoparticles is only possible if the nanoparticle clusters are not highly agglomerated in the first place, the kinetics of BSA adsorption onto the surface of the nanoparticles is fast, and the zeta-potential of the resultant system indicates sufficient stability, so as to prevent the agglomeration of the de-agglomerated species. We propose that a combination of the first two factors meant that the deagglomeration effect was observed with ZnO, for which stable de-agglomerations can be obtained with BSA concentrations less than 0.5 wt.%, but not TiO2 . In any case, BSA, whether it was adsorbed on the nanoparticles or existed as free entities in the suspension, was shown from zeta-potential results to have effectively improved the stability of the nanoparticle suspension, within the specified concentration range studied. It is thought that the stability offered by the BSA is due to a combination of electrostatic and steric hindrance effects. In the absence of BSA, the dispersed system was shown to be unstable, in which case particle–particle interaction is likely to lead to increased coalescence or agglomeration. Conflict of interest None. Acknowledgements This work was supported by DIUS [Department for Innovation, Universities and Skills]. Special thanks to Dr. Neil Harrison for his continuing encouragement and support. We are grateful to the
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Biotechnology, Biomaterials and Materials group at NPL, for the use of their laboratory facilities for these experiments. References [1] C. Saltiel, H. Giesche, Needs and opportunities for nanoparticle characterization, J. Nanoparticle Res. 2 (2000) 325–326. [2] J.K. Jiang, G. Oberdorster, P. Biswas, Characterization of size, surface charge, and agglomeration state of nanoparticle dispersions for toxicological studies, J. Nanoparticle Res. 11 (1) (2009) 77–89. [3] V. Richter, A. Potthoff, W. Pompe, M. Gelinsky, H. Ikonomidou, S. Bastian, K. Schirmer, S. Scholz, J. Hofinger, Evaluation of health risks of nano- and microparticles, Powder metallurgy 51 (1) (2008) 8–9. [4] P. Bihari, M. Vippola, S. Schultes, M. Praetner, A.G. Khandoga, C.A. Reichel, C. Coester, T. Tuomi, M. Rehberg, F. Krombach, Optimized dispersion of nanoparticles for biological in vitro and in vivo studies, Particle Fiber Toxicol. 5 (2008) 14. [5] M.Q. Zhang, T. Desai, M. Ferrari, Proteins and cells on PEG immobilized silicon surfaces, Biomaterials 19 (10) (1998) 953–960. [6] P.A. Zunszain, J. Ghuman, T. Komatsu, E. Tsuchida, S. Curry, Crystal structural analysis of human serum albumin complexed with hemin and fatty acid, BMC Struct. Biol. 7 (3) (2003) 6. [7] K.W. Powers, S.C. Brown, V.B. Krishna, S.C. Wasdo, B.M. Moudgil, S.M. Roberts, Research strategies for safety evaluation of nanomaterials. Part VI. Characterization of nanoscale particles for toxicological evaluation, Toxicol. Sci. 90 (2) (2006) 296–303. [8] A.L. Koh, C.M. Shachaf, S. Elchuri, G.P. Nolan, R. Sinclair, Electron microscopy localization and characterization of functionalized composite organic–inorganic SERS nanoparticles on leukemia cells, Ultramicroscopy 109 (1) (2008) 111–121. [9] C.M. Wang, D.R. Baer, J.E. Amonette, M.H. Engelhard, J.J. Antony, Y. Qiang, Electron beam-induced thickening of the protective oxide layer around Fe nanoparticles, Ultramicroscopy 108 (1) (2007) 43–51. [10] I.A. Rahman, P. Vejayakumaran, C.S. Sipaut, J. Ismail, C.K. Chee, Effect of the drying techniques on the morphology of silica nanoparticles synthesized via sol–gel process, Ceram. Int. 34 (8) (2008) 2059–2066. [11] S. Gonzalez, M. Fernandez-Lorente, Y. Gilaberte-Calzada, The latest on skin photoprotection, Clin. Dermatol. 26 (6) (2008) 614–626. [12] M. Pohl, S. Hogekamp, N.Q. Hoffmann, H.P. Schuchmann, Dispersion and deagglomeration of nanoparticles with ultrasound, Chem. Eng. Technol. 76 (4) (2004) 392–396. [13] R. Tantra, R.J.C. Brown, M.J.T. Milton, D. Gohil, A practical method to fabricate gold substrates for surface-enhanced Raman spectroscopy, Appl. Spectrosc. 62 (9) (2008) 992–1000. [14] C. Schulze, A. Kroll, C.-M. Lehr, U. Schfer, F. Becker, K. Schnekenburger, J.C.S. Isfort, R. Landsiedel, W. Wohlleben, Not ready to use—overcoming pitfalls when dispersing nanoparticles in physiological media, Nanotoxicology 2 (2) (2008) 51–61. [15] R.J. Hunter, Foundations of Colloid Science, 2nd ed., Clarendon Press, Oxford, 2001. [16] K. Sato, J.G. Li, H. Kamiya, T. Ishigaki, Ultrasonic dispersion of TiO2 nanoparticles in aqueous suspension, J. Am. Ceram. Soc. 91 (8) (2008) 2481–2487. [17] Y.J. Min, M. Akbulut, K. Kristiansen, Y. Golan, J. Israelachvili, The role of interparticle and external forces in nanoparticle assembly, Nat. Mater. 7 (7) (2008) 527–538. [18] T.F. Tadros (Ed.), Colloid Stability: The Role of Surface Forces—Part I, vol. 1, Wiley, 2006. [19] T. Cedervall, I. Lynch, S. Lindman, T. Berggard, E. Thulin, H. Nilsson, K.A. Dawson, S. Linse, Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles, Proc. Natl. Acad. Sci. U.S.A. 104 (7) (2007) 2050–2055. [20] B. Sahoo, M. Goswami, S. Nag, S. Maiti, Spontaneous formation of a protein corona prevents the loss of quantum dot fluorescence in physiological buffers, Chem. Phys. Lett. 445 (4–6) (2007) 217–220. [21] M. Lundqvist, J. Stigler, G. Elia, I. Lynch, T. Cedervall, K.A. Dawson, Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts, Proc. Natl. Acad. Sci. U.S.A. 105 (38) (2008) 14265–14270.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Characterisation of the de-agglomeration effects of bovine serum albumin on nanoparticles in aqueous suspension Ratna Tantra ∗ , Jordan Tompkins, Paul Quincey National Physical Laboratory, Hampton Road, Teddington, Middlesex TW11 0LW, United Kingdom
a r t i c l e
i n f o
Article history: Received 7 July 2009 Received in revised form 28 August 2009 Accepted 28 August 2009 Available online 6 September 2009 Keywords: Zinc oxide Titanium dioxide Nanoparticles Albumin De-agglomeration Stability
a b s t r a c t This paper describes the use of nanoparticle characterisation tools to evaluate the interaction between bovine serum albumin (BSA) and dispersed nanoparticles in aqueous media. Dynamic light scattering, zeta-potential measurements and scanning electron microscopy were used to probe the state of zinc oxide (ZnO) and titanium dioxide (TiO2 ) nanoparticles in the presence of various concentrations of BSA, throughout a three-day period. BSA was shown to adhere to ZnO but not to TiO2 . The adsorption of BSA led to subsequent de-agglomeration of the sub-micron ZnO clusters into smaller fragments, even breaking them up into individual isolated nanoparticles. We propose that certain factors, such as adsorption kinetics of BSA on to the surface of ZnO, as well as the initial agglomerated state of the ZnO, prior to BSA addition, are responsible for promoting the de-agglomeration process. Hence, in the case of TiO2 we see no de-agglomeration because: (a) the nanoparticles are more highly agglomerated to begin with and (b) BSA does not adsorb effectively on the surface of the nanoparticles. The zeta-potential results show that, for either ZnO or TiO2 , the presence of BSA resulted in enhanced stability. In the case of ZnO, the enhanced stability is limited to BSA concentrations below 0.5 wt.%. Steric and electrostatic repulsion are thought to be responsible for improved stability of the dispersion. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved.
1. Introduction The ability to characterise the dispersion of nanoparticles in aqueous media is useful for many different applications [1]. For example, in experimental nanotoxicological studies, measuring the state of the dispersed nanoparticles in a biologically relevant media is of great importance for assessing nanoparticle toxicity. Measuring parameters such as particle size and state of aggregation are vital, as these parameters are thought to influence the level of toxicological activity of the nanoparticles [2]. Stabilisers such as the proteins BSA (bovine serum albumin) or HSA (human serum albumin) are often added to the dispersion media, in order to enhance stabilisation by preventing subsequent agglomeration of the nanoparticles [3]. Hence, the ability to characterise nanoparticle–albumin interaction is of significant interest [4]. In mammals, serum albumin is one of the most abundant plasma proteins and has a role in maintaining colloid osmotic pressure (needed for proper distribution of body fluids between intravascular compartments and body tissues), binding (in which it acts as a plasma carrier by non-specifically binding several hydrophobic steroid hormones) and transport (of hemin and fatty acids) [5]. Furthermore, serum albumin from bovine serum concentrate is a
∗ Corresponding author. E-mail address: [email protected] (R. Tantra).
protein commonly used in biological laboratories for immudiagnostic procedures, clinical reagents, cell culture media, and protein chemistry. BSA (of 69,323.4 Da) has a size of 40 Å × 40 Å × 140 Å, contains 607 number of amino acid and has pI (isoelectric point; this is the pH value at which BSA carries no net electrical charge) of 4.7 in water (at 25 ◦ C) [6]. The traditional approach to examining nanoparticle–albumin interactions is through the use of dynamic light scattering (DLS) methods to determine particle size distribution, and zeta-potential measurements, so as to have some indication of relative stability of the dispersion [7]. However, these techniques have their limitations, in that they do not offer sufficient sensitivity and selectivity to allow us to probe into the mechanism of the interaction in great detail. The additional use of imaging tools such as SEM and TEM can play a crucial role in determining the mechanism behind the nanoparticle–albumin interaction. Imaging techniques make it possible to probe the state of individual nanoparticles [8]. SEM has several advantages over TEM–SEM analysis is faster, cheaper, easier to use and thus more suitable for making routine measurements. More importantly, SEM employs relatively low beam energy in comparison to TEM, which is crucial for accurate characterisation of nanoparticles. A recent paper by Wang et al. [9] has underlined the importance of addressing the inherent problems associated with TEM imaging, in that the state of the nanoparticles can be easily modified by the high-energy electron beam. According to
0927-7765/$ – see front matter. Crown Copyright © 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2009.08.049
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Wang, there is potential for the high-energy beam to introduce an “electron beam-induced effect” that can result in defect formation, charging of particles and possibly excitation of the surrounding gases. Previous work [4] has used SEM to measure the state of nanoparticles in the dispersed state, but there are questions about the accuracy of such results, in particular if the images are a true representation of the original suspended state of the nanoparticles. The main disadvantage with techniques such as SEM is in the sample preparation step, in particular the need to transfer a suspension of the nanoparticles on to a suitable substrate, letting the liquid dry under ambient conditions and ensuring that changes to the state of the nanoparticles are minimised in the process. It is vital to have good control over this process, to prevent artefacts (such as irreversible aggregation of the nanoparticles or the formation of crystals from the biologically related media) being introduced during the sample preparation stage [10]. The objective of this study therefore was to characterise nanoparticle–albumin interactions by means of several techniques. Two types of nanoparticle, zinc oxide (ZnO) and titanium dioxide (TiO2 ) were employed, as they are of high commercial relevance [11]. The nanoparticles were dispersed in water using an ultrasonic probe. This technique is commonly used to disperse nanoparticles in aqueous media, by generating and transmitting shear waves to de-agglomerate particles in water [12]. The effect of adding BSA (at various concentrations) to the dispersed nanoparticles was evaluated for a period of three days. The state of the nanoparticles was monitored using three main techniques: particle size analysis (using DLS), zeta-potential (using Doppler electrophoresis) and SEM. In order to improve the sample preparation protocols for SEM imaging, commercially available poly-l-lysine coated glass substrates were employed to fix the nanoparticles prior to SEM analysis. The use of commercially available substrates is particularly advantageous, as this will reduce time and inconsistencies associated with any associated surface modification protocols [13]. One feature of the sampling protocol used is the absence of an undesirable evaporative-drying step in the sample preparation, which can be responsible for inducing aggregation of the nanoparticles, a source of potentially misleading results. In theory, the SEM image acquired will be more representative of the corresponding dispersed nanoparticles. The effectiveness of the protocol was tested using NIST-certified latex beads in the first instance. The study aimed to probe the mechanism behind the BSA–nanoparticle interaction by correlating findings obtained from the SEM images with that of DLS particle size and zeta-potential data. Although characterising the effect of BSA on nanoparticle dispersion is not new [14], the availability of SEM images that clarify these effects is very limited. It was hypothesised that the SEM data obtained would not only support observations from the corresponding DLS/zeta-potential measurements, but would provide additional information not obtained from the other techniques.
2. Methods 2.1. Starting materials De-ionised water (DI, Millipore, MilliQ) was used to prepare all aqueous suspensions/solutions. Stock solution of BSA (Sigma–Aldrich, UK) in DI water (final concentration of 10 wt.%, 100 ml) was made. BSA stock solution was filtered through 0.2 m membrane (Nalgene sterile disposable filter units, Fluka). This stock has been used to disperse nanoparticles (protocol described below) and to also make control samples of BSA (BSA in DI water) at a range of concentrations (0.02, 0.1, 0.5 and 2 wt.%). All BSA solutions were
stored in amber-coloured glass containers at 4 ◦ C (when not in use) and discarded two weeks after preparation. The ZnO was purchased (Nanostructured and Amorphous Materials, Inc.), and the TiO2 was donated by Professor Ken Donaldson (University of Edinburgh, Scotland, UK). The nanoparticles were received in dry powder form and used without further purification. 2.2. Aqueous suspension of the nanoparticles ZnO was weighed on analytical mass balance and then suspended in DI water, at a concentration of 1 mg/ml (500 ml). The suspended nanoparticles were ultrasonically irradiated in a 1 l beaker for 30 s. This was shown to be sufficient time to aid mixing and forming a homogeneous suspension. The ultrasonic treatment was applied by Heat Systems Model XL2020 (with a maximum power output of 550 W and operated at a frequency of 20 kHz). Unless stated otherwise, aliquots of 50 ml of the resultant mixture were transferred into centrifuge tubes (50 ml 10×) and the tubes were subjected to centrifugation (20 ◦ C) for 15 min, at 4800 rpm (using Eppendorf Centrifuge 5804 R). The purpose of this centrifugation step was to remove any large aggregates/agglomerates. A small pellet at the bottom of each tube was observed after the centrifugation step. Immediately after centrifugation, the supernatant was pipetted into a large beaker, taking care not to disturb the sample pellet at the bottom of the tube. Suitable aliquots of this were mixed with appropriate amounts of BSA stock (10 wt.%)/DI water, resulting in a final volume of 20 ml, in order to make a range of BSA concentrations (0, 0.02, 0.1, 0.5 and 2 wt.%); following addition of BSA, the mixture was gently shaken to ensure mixing. The resultant mixture was stored in individual screw capped ambercoloured glass bottles (30 ml, Fisherbrand) and stored in a fridge (4 ◦ C) when not in use. An identical sample preparation procedure was employed for the TiO2 powders. 2.3. Characterisation of the suspension All bottles containing the samples for analysis were stored at 4 ◦ C. Prior to use, the bottles were gently shaken and allowed to equilibrate at room temperature (20 ◦ C) for at least 1 h. The prepared samples of suspended nanoparticles, in the absence and presence of BSA at various concentrations, were analysed daily for a period of three days using size particle DLS, zeta-potential and SEM imaging. Control measurements were carried out using BSA solutions (in the absence of nanoparticles), at various concentrations. The instrument employed for particle size analysis and zetapotential measurements was a Zetasizer Nano ZS (Malvern Instruments, UK) with 633 nm red laser. Particle size was obtained from measuring the time dependent fluctuation of scattered light arising from the suspension of the nanoparticles undergoing random Brownian motion. The diffusion coefficient of the particles is deduced and analysed to give their mean hydrodynamic diameter, i.e. the so-called “z-average” diameter that is reported here. For the zeta-potential measurement, the instrument uses laser Doppler electrophoresis to measure the net velocity of the nanoparticles in the liquid that results when an electric field is applied. Here, the fundamental quantity measured is the net electrophoretic mobility () of the particles, which is then is converted to the zeta-potential () using Henry’s approximation [15]; the Smoluchowski approximation was applied in our measurement (this approximation is generally applied to aqueous media), in which the Henry’s function in the equation takes a value of 3/2. Both types of analysis can be carried out using the same sample cell, a disposable folded capillary cell (DTS1060) supplied by Malvern. Prior to its use, the cell was filled with de-ionised water and allowed to equilibrate for 30 min.
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All of the water was emptied and the cell was flushed with the appropriate sample solution. The measurement was carried out on the second filling; sufficient sample volume was used to completely cover the electrodes of the cell. To avoid air bubbles in the cell, the sample was injected slowly; analysis was carried out if no visible air bubble inclusions were present in the cell. The cell was placed into the Zeta-sizer and equilibrated at 20 ◦ C (close to the average temperature of the laboratory) for 2 min prior to taking the particle size measurements (three replicates); the measurement temperature was set and maintained by the Peltier elements in the sample holder of the instrument. The corresponding zeta-potential measurements (three replicates) were taken immediately after this. Both the sample and cell were then discarded after use. Malvern Instrument’s Dispersion Technology software (Version 4.0) was used to control and analyse all data from the instrument. Data collected were then imported to Excel and functions within Excel were used to compute the mean and standard deviations of the set of replicates measured. The results are plotted as mean values with error bars of one standard deviation. Scanning electron microscope images were collected using a Carl Zeiss Supra 40 electron microscope, in which the optimal spatial resolution of the microscope is a few nanometres. Images were acquired with an accelerating voltage of 15 kV, working distance of 2–3 mm, tilting angle 0◦ , and recorded with an in-lens secondary electron detector. Sample preparation for SEM analysis requires the deposition of the appropriate liquid sample (1 ml) on to a poly-l-lysine coated microscope glass slide (purchased from Fisher Scientific, UK) and allowing it to incubate for a period of 5 min at room temperature (∼20 ◦ C). There were no substantial visible signs of evaporation during the 5-min period. After 5 min, the substrate was then washed with DI water in order to dislodge any loosely bound nanoparticles on the surface. The substrate was then subsequently allowed to air dry for 15 min in an open Petri dish in a fume hood. The step of aliquoting the liquid sample on the surface (followed by incubation for 5 min, washing and subsequently drying) was repeated again on the same substrate. However, in the final step, the slides were allowed to dry for a couple of hours before they were thinly sputtered with gold. The device used to deposit the gold was an Edwards S150B sputter coater unit. Sputtering was conducted under vacuum (7 mbar), while passing pure, dry argon into the coating chamber. Typical plate voltage and current were 1200 V and 15 mA, respectively. The sputtering time was approximately 10 s, which resulted in an estimated gold thickness of not more than a few nanometres being deposited on top of the substrate. The appropriateness of this sample preparation protocol, for the trapping nanoparticles on to a poly-l-lysine coated substrate, was evaluated by using NIST-certified 100 nm latex beads (supplied by Agar Scientific, UK).
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Fig. 1. SEM images of the nanoparticles mounted on carbon pad, showing: primary particle sizes and shapes, as well as the degree of aggregation/agglomeration in the “as-received powders”: (a) ZnO and (b) TiO2 .
3.2. Nanoparticle entrapment using poly-l-lysine substrates Fig. 2a shows a typical SEM image of NIST-certified latex beads fixed on poly-l-lysine coated slide, using the deposition protocol described in Section 2. This is a low magnification image (10,000×),
3. Results and discussion 3.1. Initial particle properties Fig. 1 shows the SEM images of both ZnO and TiO2 nanoparticles of the “as–received” powder, prior to dispersing them in aqueous media. Results show that both nanoparticles exist as aggregates and agglomerates (fusing of particles). The presence of agglomerates, which is particularly evident in the case of TiO2 , has meant that to achieve successful dispersion, significant energy is required to break them up. Hence, the particle dispersion protocol employed in this study involved the use of an ultrasonic probe in order to disperse the nanoparticles [16]. From the SEM images, polydispersity in the samples existed and several shape variations were also observed. This is particularly apparent with ZnO, in which diverse shapes ranging from rod-like, hexagon prism and cuboids are apparent.
Fig. 2. SEM images of sputtered (100 nm) latex beads (NIST-certified) to show the potential of the poly-l-lysine substrate to trap nanoparticles. Images shown were taken at low magnification (10,000×) and high magnification (80,000×).
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showing a highly homogeneous distribution of the latex spheres. The corresponding image taken at higher magnification of 80,000× is given in Fig. 2b, which shows spheres with average size of ∼98 nm, with S.D. of 2 nm (the average and standard deviation data were taken from 10 particles, chosen at random); this coincided with the certified values of 97 ± 3 nm (as listed on the NIST Certificate of Analysis). The attachment of the latex spheres on the surface is most likely to be based on very strong electrostatic interaction that exists between the beads and the coated glass surface [17]. The adhesion strength was shown to be strong enough to avoid the removal of the nanoparticles, even after carrying out the multiple washing steps (described in the protocol). Although many of the latex beads were fixed as singly isolated particles, about a quarter of the latex beads existed as small clusters of particles. Such aggregates may be due to hydrophobic interactions that can occur between the beads. We saw no large aggregate clusters on the surface. In summary, we have shown that the use of such poly-l-lysine coated substrates (along with the protocol developed in this study) is suitable to monitor the state of dispersed nanoparticles, and is readily capable of fixing single isolated nanoparticles. 3.3. Effect of BSA on dispersed ZnO nanoparticles Fig. 3 shows the effect on z-average diameter and zeta-potential values, when BSA (within a concentration range of 0–2 wt.%) was added to dispersed ZnO nanoparticles; the effect of BSA on the nanoparticles were monitored over a three-day period. It is apparent that even a small amount of BSA (0.02 wt.%) can have a drastic effect on the particle size (Fig. 3a). DLS results show that increasing BSA concentration resulted in a decrease of particle size, until the nanoparticle size reached ∼6 nm in size (approximately the same size as observed from the BSA control), when the BSA concentration exceeded 0.5 wt.%. Interestingly, the zeta-potential results
(Fig. 3b) followed the same trend as the DLS results, in that the BSA control and the nanoparticle dispersion showed similar results with BSA concentrations above 0.5 wt.%. Zeta-potential plots show a minimum at around 0.1 wt.% BSA, indicating that this is the concentration of BSA that resulted in the highest stability. Apart from the nanoparticle suspensions containing no BSA, data from day 2 and day 3 followed similar trends to day 1. The nanoparticle suspensions without BSA showed a marked increase in particle size from day 1 to day 3, from 333 to 1044 nm, suggesting significant aggregation of the nanoparticles over this period in the absence of BSA. The reduced particle size observed when BSA is present in the ZnO suspension can be explained by the de-agglomeration of ZnO clusters. Below 0.5 wt.% BSA, the zeta-potential results indicated that such de-agglomerated species would remain stable through time. It has been suggested in the past that BSA offers steric stabilisation of nanoparticles [2,14]. Above 0.5 wt.% however, the system is unstable, as indicated by the zeta-potential results, suggesting that in this concentration range there is a strong tendency for such de-agglomerated species to re-agglomerate. This effect would ultimately lead to the nanoparticles sedimenting out of the suspension, leaving the remaining free BSA in suspension. If BSA is completely absent from the ZnO dispersion, then the nanoparticles have an evident tendency to aggregate significantly over a three-day period. Fig. 4 shows SEM images of the dispersed ZnO nanoparticles on a poly-l-lysine substrate, prepared on day 3: (a) with no BSA and (b) in the presence of 0.02 wt.% BSA (scale bars on the images are 200 and 100 nm, respectively). These images were selected to be “typical”, as chosen (out of random 10 images) by the operator. Images show that in the absence of BSA, nanoparticles show polydispersity and shape variation, quite similar to the “as-received” powder (shown in Fig. 1a). The images illustrate the smaller clusters of nanoparticles observed when BSA is added; the image in Fig. 4b shows a cluster size of approximately 444 by 600 nm (in the presence of BSA). This is smaller in comparison to 840 by 1060 nm in the absence of BSA, as shown in Fig. 4a (inside the dotted box). The SEM images seem to correlate well with the findings from the corresponding DLS data (Fig. 3a), in which the DLS data showed an average particle size of 1044 nm (S.D. 156 nm of three replicates) in the absence of BSA, which reduced dramatically to 288 nm (S.D. 27 nm of three replicates) in the presence of BSA 0.02 wt.%. Although it would seem that the SEM images support our hypothesis, in that de-agglomeration has occurred with ZnO in the presence of BSA, it must be remembered that SEM is a “single particle” based technique (thus impractical to achieve a truly representative data), as oppose to the “population” based technique offered by routine tools such as DLS; in this respect, the interpretation given here must therefore be made with caution. Of interest therefore is the presence of BSA-coated ZnO clusters, as observed from the SEM image of Fig. 4b; this apparent coating was not present in the absence of BSA. Such a thick coating on the ZnO clusters was observed with all other concentrations of BSA. Further inspection of the ZnO nanoparticles on poly-lysine substrate in the present of 0.02 wt.% BSA has shown the presence of single isolated nanoparticles (of rods and pyramid shapes) as shown in Fig. 4c. To our knowledge, these are the first SEM images that depict the presence of isolated single nanoparticles from such a de-agglomeration event. 3.4. Effect of BSA on dispersed TiO2 nanoparticles
Fig. 3. Effects of BSA [at various concentrations: 0, 0.02, 0.1, 0.5 and 2 wt.%] on the dispersion of ZnO in DI water, over a three-day period (data compared to control BSA solutions in the absence of nanoparticles): (a) mean particle size (z-average); (b) zeta-potential values.
Fig. 5a shows the particle size plots of TiO2 dispersed in water in the absence and presence of BSA [concentration up to 2 wt.%], as obtained by DLS measurements over a three-day period. The results show an entirely different response to the corresponding ZnO case
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Fig. 4. SEM images of ZnO nanoparticles on poly-l-lysine substrates, prepared on day 3: (a) with no BSA and (b) with BSA (0.02 wt.%), showing the presence of small cluster of nanoparticles and single isolated nanoparticles on the surface.
(Fig. 3a). Overall, the average particle size of TiO2 is not affected by the addition of BSA; this is true apart from one measurement replicate of TiO2 with 0 wt.% BSA on day 1, which may be a possible outlier. This result gives an average particle size of 278 nm (with S.D. of 10 nm) as opposed to an average value of 194 nm (with S.D. of 8 nm) from the rest of the TiO2 DLS data. Unlike ZnO, the TiO2 plots did not intersect with the corresponding BSA control plot at any BSA concentration. Fig. 5b shows the corresponding zeta-potential plots of TiO2 in the absence and presence of BSA (concentration range of up to 2 wt.%). The general effect upon BSA addition resulted in a negative shift of zeta-potential value indicating enhanced stability, quite similar to what was observed with the ZnO case. However, unlike the ZnO case, the zeta-potential of TiO2 in the presence of BSA was shown to give similar values at all BSA concentrations, with average of −27 mV (S.D. of 3 mV). Data from TiO2 samples with no BSA gave average zeta-potential values of 16 mV (with S.D. 6 mV). Unlike the ZnO case, in which we saw zeta-potential plots that first fell rapidly, passed through minima, and then rose again (Fig. 3b) with increasing BSA concentration, the zeta-potential plots here fall rapidly and reach a saturation level. Fig. 6a and b shows SEM images of dispersed TiO2 nanoparticles fixed on to a poly-l-lysine substrate on day 3: (a) with no BSA and (b) in the presence of 2 wt.% BSA (scale bars on the images are 200 nm), respectively. In the absence of BSA, the image shows small clusters (in agglomerates) with the state of the pri-
mary particle (size and shape) being very similar to those observed in “as-received” powder (shown in Fig. 1b). There was very little observable change after addition of BSA into the dispersed TiO2 , even at a concentration of 2 wt.% (Fig. 6b); similar images were obtained with other BSA concentrations. In these images, no blotchy features on the surface of the nanoparticles were observed, unlike the images obtained with the corresponding ZnO case (for example, in Fig. 4c). An obvious difference between the images observed in Fig. 6, is the much reduced number of TiO2 clusters present on the poly-l-lysine surface when BSA was present in solution. Results show no evidence of TiO2 de-agglomeration taking place in the presence of BSA. In addition, the SEM images suggest that the BSA must exist as free entities in solution rather than being adsorbed on the surface of TiO2 nanoparticles. The presence of free BSA in solution will undoubtedly compete with the TiO2 nanoparticle clusters for binding sites on the poly-llysine substrate. This is likely to explain why fewer TiO2 clusters were observed in the SEM images when BSA is present. Nonetheless, as in the case of ZnO, the presence of BSA in solution has resulted in an enhanced stability of the dispersion, irrespective of BSA concentration. The outlier data observed with the TiO2 in the absence of BSA on day 1 may be explained by possible contamination in the sample cell used, which subsequently induced particle aggregation resulting in larger clusters than with the other results.
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Fig. 5. Effects of BSA [at various concentrations: 0, 0.02, 0.1, 0.5 and 2 wt.%] on the dispersion of TiO2 in DI water, over a three-day period (data compared to control BSA solutions in the absence of nanoparticles): (a) mean particle size (z-average); (b) zeta-potential values.
as the effect of the adsorption may be insufficient to break up the much-agglomerated nanoparticles. For de-agglomeration to occur to the point of creating individual isolated nanoparticles, it is important for the BSA to be sufficiently adsorbed in between adjacent nanoparticles. It is postulated that in the case of ZnO, the thickness of the adsorbed BSA layer is sufficient to result in steric repulsion, strong enough for de-agglomeration into isolated individual particles to occur. Overall, the stability of such deagglomerated species will be related to the zeta-potential of the system; if the value is sufficiently negative, the de-agglomerated species will exist as a stable form for a long period of time. However, if the zeta-potential of the system indicates instability, then such de-agglomerated species will re-agglomerate and eventually fall out of suspension [18]. It is clear that nanoparticle–BSA interaction has led to an adsorbed layer of BSA on the surface. If such interactions occurred in living systems, then the instantaneous formation of the BSA–nanoparticle complex would result, and subsequent toxicological activity would be dominated by the BSA–nanoparticle complex, which would largely define the biological identity of the particle, rather than the inherent properties of the nanoparticles. The formation of such nanoparticle–protein complexes has been studied by past workers [19,20] and the complex is often referred to as a “protein corona”. The results presented here clearly show the existence of such a “protein corona”. Previous work on the “protein corona” has demonstrated that both size and surface properties of the nanoparticles have been found to play a very significant role in determining the nanoparticle coronas on different particles of identical material [21]. Thus, it is reasonable to assume that the size and surface properties of the protein itself will also govern the formation of such coronas, as this will influence the:
3.5. A summary of the mechanisms by which BSA affects dispersed nanoparticles This study has provided insight into the interaction between BSA and dispersed nanoparticles and the main aim of this section is to propose a general mechanism underlying the interaction of BSA with nanoparticles. When nanoparticles are in suspension with BSA, the BSA can either adsorb to the surface of the nanoparticles or exist as free entities in solution. If adsorbed, the BSA has the potential to deagglomerate clusters of nanoparticles by changing the surface forces involved. If nanoparticles are not highly agglomerated to start off with, the de-agglomeration can be instantaneous. If they are highly agglomerated, then de-agglomeration may not occur,
(a) adsorption kinetics between BSA and the nanoparticles; (b) subsequent (steric) stability of the nanoparticle-BSA complex.
We envisage therefore that other proteins with similar properties to BSA (particularly size and pI values) would undoubtedly act as stabilisers in a similar manner. However, we have to be aware of the significant differences that may arise due to batch-to-batch variations (e.g. changes in particle size due to the formation of aggregates/agglomerates, through possible degradation of the protein through time), which would subsequently influence the final results.
Fig. 6. SEM images of TiO2 nanoparticles on poly-l-lysine substrates, prepared on day 3: (a) with no BSA and (b) with BSA (2 wt.%).
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4. Conclusion In summary, our study has highlighted the importance of using different techniques in parallel to probe the interaction between BSA and dispersed nanoparticles, and to have careful protocols for preparing SEM samples of nanoparticles from suspension. Single particle techniques, such as SEM, are thus powerful complementary techniques to population-based methods such as DLS. The poly-llysine coated substrate used in the SEM sample preparation here was shown to be effective, to the extent that it was capable of collecting isolated individual nanoparticles that were present in the dispersed state. Our experiments showed that interactions between nanoparticles and BSA can lead to one of three outcomes: (a) stable de-agglomeration of nanoparticle clusters; (b) unstable de-agglomeration of nanoparticle clusters; (c) no significant agglomeration or de-agglomeration effects taking place. The type of outcome that occurs in a given system is mainly dependent on three factors: (a) the initial agglomerated state of the nanoparticles, (b) the adsorption kinetics of BSA on to the surface of nanoparticles and (c) the zeta-potential of the final system. De-agglomeration into isolated nanoparticles is only possible if the nanoparticle clusters are not highly agglomerated in the first place, the kinetics of BSA adsorption onto the surface of the nanoparticles is fast, and the zeta-potential of the resultant system indicates sufficient stability, so as to prevent the agglomeration of the de-agglomerated species. We propose that a combination of the first two factors meant that the deagglomeration effect was observed with ZnO, for which stable de-agglomerations can be obtained with BSA concentrations less than 0.5 wt.%, but not TiO2 . In any case, BSA, whether it was adsorbed on the nanoparticles or existed as free entities in the suspension, was shown from zeta-potential results to have effectively improved the stability of the nanoparticle suspension, within the specified concentration range studied. It is thought that the stability offered by the BSA is due to a combination of electrostatic and steric hindrance effects. In the absence of BSA, the dispersed system was shown to be unstable, in which case particle–particle interaction is likely to lead to increased coalescence or agglomeration. Conflict of interest None. Acknowledgements This work was supported by DIUS [Department for Innovation, Universities and Skills]. Special thanks to Dr. Neil Harrison for his continuing encouragement and support. We are grateful to the
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