Fatima
Zia
ab,
Michaela
Kendall
c,
Steve P.
Watson
*bd and
Paula M.
Mendes
*ab
aSchool of Chemical Engineering, College of Engineering and Physical Sciences, University of Birmingham B15 2TT, UK. E-mail: p.m.mendes@bham.ac.uk
bCentre of Membrane Proteins and Receptors (COMPARE), Universities of Birmingham and Nottingham, The Midlands, UK
cAdelan/School of Engineering, Aston University, Birmingham B4 7ET, UK Web: http://www.adelan.co.uk
dInstitute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham B15 2TT, UK. E-mail: S.P.Watson@bham.ac.uk
First published on 12th November 2018
Nanoparticles are key components underlying recent technological advances in various industrial and medical fields, and thus understanding their mode of interaction with biological systems is essential. However, while several nanoparticle systems have been shown to interact with blood platelets, many questions remain concerning the mechanisms of platelet activation and the role that the physicochemical properties of nanoparticles play in inducing platelet aggregation. Here, using negatively charged polystyrene nanoparticles with sizes of 25, 50, 119, 151, 201 nm and negatively charged platinum nanoparticles with sizes of 7 and 73 nm, we show that it is not the size of the nanoparticles but rather the nanoparticle surface area that is critical in mediating the effects on platelet activation. The nanoparticles stimulate platelet aggregation through passive (agglutination) and activation of integrin αIIbβ3 through a pathway regulated by Src and Syk tyrosine kinase.
Several studies have demonstrated that engineered nanoparticles can interact with platelets in different ways causing either no effect,4 inhibition,5 and passive (agglutination) or active (integrin αIIbβ3-dependent) aggregation.6,7 While the mechanisms and critical factors that drive these effects on platelet function are not yet fully elucidated,8 there is increasing evidence that nanoparticle material, shape, size, surface chemistry, surface charge and concentration influence the platelet response. All have been long understood to influence particle mediated health effects.9 For instance, multiwalled and single-walled carbon nanotubes,7 CdSe quantum dots,10 polystyrene nanoparticles11 and dendrimers12 are able to induce platelet aggregation despite variation in surface charge and hydrophilicity. On the other hand, in vitro and in vivo studies have shown that either neutral, positively or negatively charged nanoparticles can promote changes in platelet activity, with the degree of activation not only depending on the presence or not of charge but also the nanoparticle size, shape and composition.11–15 This intricate relationship between platelet response and nanoparticle characteristics is a result of different mechanisms of platelet activation which depend on the specific interactions of platelet membrane receptors with nanoparticles. The nanoparticle–platelet interaction and response may be mediated either via stimulation of platelet surface receptors10 or disruption of the platelet membrane.16
One important question concerning the behaviour of nanoparticles is whether different nanoparticle sizes can exhibit different functional effects on platelets. A number of studies suggest that small particles might act differently from larger particles.12,15,17,18 For instance, exposure of platelets to dendrimers of different generations (G3, G4, G5 and G6) with sizes ranging from 3.1 nm to 7.5 nm led to platelet aggregation only with the larger (G4–G6) dendrimers.12 Another study has suggested that the aggregation effect of 20 nm gold nanoparticles was stronger than that caused by 70 nm nanoparticles.17 This trend is, to a certain extent, in accordance with a study on silica nanoparticles in which platelet aggregation was observed for 10 nm and not 50 nm nanoparticles.18 Since previous size-dependent effect studies have been based on nanoparticle mass and moles concentration as the dose metric, it raises the question on the role that surface area plays in inducing platelet activation. Are the changes in the biological activity of the different size nanoparticles a result of nanoparticle morphology or the greater ratio of surface area and number to mass that occurs as nanoparticle size becomes smaller? Driven by this fundamental question, so critical to understanding the impact of nanoparticles in human health, in this study we investigate the role of nanoparticle surface area mediated effects on platelet activation. Two different nanoparticle materials, namely polystyrene (PS) with sizes of 25, 50, 119, 151, 201 nm and platinum (Pt) with sizes of 7 and 73 nm were used to study active platelet aggregation in a surface area-dependent manner.
These small nanoparticles act as the templates for the growth of the larger nanoparticles in further reaction steps. To 50 ml of water, 100 μl of nanoparticle seeds prepared as described above and 450 μl of chloroplatinic acid hexahydrate (0.4 M) were added and mixed at room temperature. To this solution, 5 ml of reducing agent (1.25% (v/v) ascorbic acid and 1% (v/v) sodium citrate) was added dropwise, after which the mixture was heated to boiling and left to react under stirring for 45 min. The product thus obtained was purified by centrifugation (30 min, 444g) using acetone as the precipitation solvent. Following this, the product was re-dispersed using a mixture of 1:
4 v/v solution of acetone
:
water before centrifuging again. This process was repeated 2 times.
Nanoparticle | Average particle size + SD (nm) | Zeta potential ± SD (mV) | |
---|---|---|---|
Water | Tyrode's-HEPES buffer | ||
PS-25 | 25.2 ± 4.9 | −42.5 ± 2.0 | −39.4 ± 1.9 |
PS-50 | 50.4 ± 4.9 | −50.8 ± 1.6 | −35.8 ± 1.6 |
PS-119 | 119.2 ± 8.5 | −35.9 ± 0.7 | −31.7 ± 1.3 |
PS-151 | 150.6 ± 9.7 | −46.1 ± 1.1 | −25.2 ± 1.8 |
PS-201 | 200.7 ± 10.7 | −56.3 ± 1.2 | −33.7 ± 1.4 |
Having established the physicochemical characteristics of the PS nanoparticles, the effect of size on the functional activity of human platelets in vitro was investigated. Human washed platelets were stimulated with PS nanoparticles and monitored using light transmission aggregometry (LTA). All the nanoparticles induced concentration-dependent platelet aggregation indicated by the increase in light transmission (Fig. 1). There was no difference in the maximal level of aggregation which was similar to that induced by strong platelet agonists such as collagen and thrombin.21
The nanoparticles exhibited bell-shaped dose response curves. Maximum aggregation levels feature at different particle concentrations, with the larger nanoparticles giving rise to significant aggregation at lower nanoparticle concentrations. Importantly, while these results seem supportive of a trend between nanoparticle size and their potency in causing platelet aggregation, they do not take into account the nanoparticle surface area. By considering surface area as a significant nanoparticle characteristic, the data shown in Fig. 1 was re-plotted to illustrate the relationship between surface area and aggregation (Fig. 2).
As demonstrated in Fig. 2, the re-plotted data produced similar bell shaped curves, in which all nanoparticle size trends were super-imposable. These findings imply that the surface area of the PS nanoparticles is a critical factor in inducing platelet activation, independently of nanoparticle size. Thus, if one considers surface area as the most relevant dose metric, higher nanoparticle concentrations of the smallest nanoparticles are required to reach similar impact in platelet aggregation as the largest ones, as supported by the trends shown in Fig. 1. These results thus highlight not only the importance of assessing platelet activity in different dose metrics to understand nanoparticle characteristic effects but also the predominant role of surface area towards platelet activation.
In order to elucidate if similar surface area-dependent trend appears for other nanoparticle types, two different sizes Pt nanoparticles (i.e. 7 nm and 73 nm) were prepared and characterised. Small Pt nanoparticles (7 nm) were synthesized using a sodium borohydride reduction method and these Pt nanoparticles acted as the seeds for the synthesis of larger Pt nanoparticles (73 nm) using ascorbic acid as the reducing agent. Sodium citrate and citric acid were used as surfactants to stabilise the nanoparticles, leading to the formation of nanoparticles coated with citrate ions and citric acid. TEM indicates the formation of monodispersed nanoparticles with spherical shape and narrow size distribution, with small Pt nanoparticles having an average diameter of 7.1 ± 1.1 nm and the larger Pt nanoparticles of 72.9 ± 6.0 nm (Fig. 3).
![]() | ||
Fig. 3 TEM images and particle size distributions for the synthesised Pt nanoparticles with mean diameter + SD of (a) 7.1 ± 1.1 nm and (b) 72.9 ± 6.0 nm. |
Zeta potential values for the small and large Pt nanoparticles when dispersed in both water and Tyrode's-HEPES buffer are reported in Table 2. As expected, the nanoparticles are all negatively charged due the adsorbed monolayers of citrate and citric acid on their surfaces. Within the same medium, the small and large nanoparticles have comparable zeta potential values. In a similar manner as for the PS nanoparticles, the zeta potential values obtained for Tyrode's-HEPES buffer are lower than those in water. However, in both media, the nanoparticles have limited tendency to aggregate since they are highly stable in water (zeta potential ranges between −52.3 to −59.3 mV) and moderately stable in Tyrode's-HEPES medium (zeta potential ranges between −20.8 to −22.4 mV).
Nanoparticle | Zeta potential ± SD (mV) | |
---|---|---|
Water | Tyrode's-HEPES buffer | |
Pt-7 | −59.3 ± 3.2 | −20.8 ± 0.8 |
Pt-73 | −52.3 ± 0.8 | −22.4 ± 1.3 |
Following the characterization of the Pt nanoparticles, attention was turned to the investigation of the effect of surface area on the aggregation of human platelets. Fig. 4(a and b(i–ii)) shows the nanoparticles surface area-based dose response curve for the 7 nm and 73 nm Pt nanoparticles (log101–4 cm2 ml−1). Both particles stimulated rapid aggregation without a noticeable preceding shape change (which manifests as a decrease in light transmission) and produced a similar bell shaped curve, with no measureable aggregation at high doses. Platelet aggregation was observed in the middle of the dose range used. The Pt nanoparticles caused a similar maximal increase in light transmission to that of the PS nanoparticles and this was not statistically different between the two sized particles (Fig. 5).
In order to place the observations above into a physiological context of exposure, Pt nanoparticles were tested in platelet rich plasma (PRP). PRP is highly enriched in clotting factors, growth factors and proteins such as albumin, fibrinogen and globulins. The results in PRP showed that the platinum nanoparticles (7 nm and 73 nm) did not cause aggregation of platelets (not shown). Collagen was used as a positive control in which a typical collagen response was seen, i.e. shape change followed by aggregation (not shown). This result is likely to reflect non-specific binding of platinum nanoparticles to plasma proteins.22–24
To ascertain whether the response to nanoparticle stimulation in washed platelets was mediated by integrin αIIbβ3-dependent aggregation, inhibitory studies were performed using the αIIbβ3 receptor antagonist Integrilin and the Src and Syk tyrosine kinase inhibitors dasatinib and PRT-060318, respectively. The rational for these experiments is that the use of Integrilin will establish whether this is passive (agglutination) or receptor driven aggregation, while the two tyrosine kinase inhibitors will provide information on the mechanism of aggregation. Src and Syk tyrosine kinases mediate activation of platelets by a group of single transmembrane receptors which signal through an immunoreceptor-tyrosine-based-activation-motif (ITAM) namely GPVI, CLEC-2 and FcγRIIA. These three receptors are activated by clustering which is the likely mechanism of activation by the polyvalent nanoparticles.
Prior to the stimulation of platelets by nanoparticles, the platelets were incubated separately with the inhibitors for 2 min. The effect of the inhibitor was compared to vehicle-treated (DMSO) controls. The dose that produced maximal platelet aggregation for both sizes (log102.4 cm2 ml−1) of nanoparticle was used.
All three inhibitors caused a partial (50–70%) inhibition of platelet activation to both sizes of Pt nanoparticles (Fig. 5). The partial inhibition by Integrilin demonstrates that aggregation is composed of two phases, agglutination (which is not blocked by Integrilin) and integrin αIIbβ3-mediated aggregation (which is blocked by Integrilin). The similar inhibitory effect of dasatinib and PRT-060318 demonstrates that activation of integrin αIIbβ3 is likely to be mediated by one or more of the three ITAM receptors described above. The mechanism of agglutination could be through neutralisation of platelet surface charge leading to binding of membrane proteins to each other. Interestingly, a similar profile of results has been reported for activation of human and mouse platelets by diesel exhaust particles, with activation mediated by GPVI with a minor contribution of CLEC-2.25
This journal is © The Royal Society of Chemistry 2018 |