Nanoparticle-allergen interactions mediate human allergic responses: protein corona characterization and cellular responses
© Radauer-Preiml et al. 2016
Received: 28 July 2015
Accepted: 4 January 2016
Published: 16 January 2016
Engineered nanomaterials (ENMs) interact with different biomolecules as soon as they are in contact, resulting in the formation of a biomolecule ‘corona’. Hence, the ‘corona’ defines the biological identity of the ENMs and could affect the response of the immune system to ENM exposure. With up to 40 % of the world population suffering from type I allergy, a possible modulation of allergen effects by binding to ENMs is highly relevant with respect to work place and consumer safety. Therefore, the aim of this present study was to gain an insight into the interactions of gold nanoparticles with different seasonally and perennially occurring outdoor and indoor allergens.
Gold nanoparticles (AuNPs) were conjugated with the major allergens of birch pollen (Bet v 1), timothy grass pollen (Phl p 5) and house dust mite (Der p 1). The AuNP-allergen conjugates were characterized by means of TEM negative staining, dynamic light scattering (DLS), z-potential measurements and hyperspectral imaging. Furthermore, 3D models were constructed, based on the characterization data, to visualize the interaction between the allergens and the AuNPs surface. Differences in the activation of human basophil cells derived from birch/grass pollen- and house dust mite-allergic patients in response to free allergen and AuNP-allergen conjugates were determined using the basophil activation assay (BAT). Potential allergen corona replacement during BAT was controlled for using Western blotting. The protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was assessed, by an enzymatic activity assay and a cellular assay pertaining to lung type II alveolar epithelial cell tight junction integrity.
The formation of a stable corona was found for all three allergens used. Our data suggest, that depending on the allergen, different effects are observed after binding to ENMs, including enhanced allergic responses against Der p 1 and also, for some patients, against Bet v 1. Moreover elevated protease activity of AuNP-Der p 1 conjugates compared to free Der p 1 was found.
In summary, this study presents that conjugation of allergens to ENMs can modulate the human allergic response, and that protease activity can be increased.
KeywordsGold nanoparticles Protein corona Allergy Basophil activation Protease activity A549 cells
With the increasing use of ENMs concerns about the safety upon exposure of workers, consumers and the environment were raised [1–3]. One crucial feature of ENMs is that they possess a higher free energy than the bulk material, which means that the ENMs surface will interact with different biomolecules as soon as they are in contact, resulting in the formation of a ‘corona’ around the ENMs . The biomolecule corona changes the extrinsic properties of the ENMs, but also structures and functions of the biomolecules themselves may be modified upon binding. Thus, if the corona consists of an allergen the overall bio-reactivity of the ENM/corona complex may have the potential to elicit or modulate an allergic response [5–8].
As the overall size of the ENMs would increase only marginally by allergen binding, the ENMs-allergen conjugates could still be inhaled and could translocate from the lung into the bloodstream. Contact with epithelial and/or immune cells and in particular uptake into these cells may result in immune responses [9, 10]. Type I allergy, affecting 30–40 % of the population worldwide, is characterized by the production of immunoglobulin E (IgE) antibodies against allergens. IgE-mediated allergic rhinitis is a risk factor for asthma, a life-long chronic inflammatory disease of the airways, which seriously impacts quality of life and can, when uncontrolled, lead to death . Upon allergic sensitization in the human airways, epithelial cells encountering allergens can release thymic stromal lymphopoietin (TSLP) to attract, activate and polarize dendritic cells (DCs). Activated DCs migrate to lymph nodes, expressing the CC-chemokine ligands (CCL) 17 and CCL22 for T cell attraction [12–14]. In the lymph node, DCs present processed antigens to T cells, and by several mechanisms initiate an immune deviation into a T helper cell 2 (TH2)-type profile characterized by secretion of interleukin (IL)-4, IL-5 and IL-13. The TH2 cells induce a class-switch in B cells resulting in the production of allergen-specific IgE [15, 16]. During the acute phase of allergy (effector phase), IgE molecules on mast cells and basophils crosslink the cells’ high affinity IgE receptors, initiating a signaling cascade that leads to the release of preformed mediators, enzymes and cytokines, resulting in pathological damage and clinical manifestation of allergy [17–19]. Allergic symptoms can occur seasonally, e.g. after exposure to pollen, or perennially during the exposure to indoor allergens . In Europe the most allergenic tree pollen is produced by birch (major allergen Bet v 1). Pollinosis is also caused by grass pollen, a well-known example being timothy grass (major allergens Phl p 1 and 5) . House dust mite, found in 48 % of European homes, represents the major perennial indoor allergen source (major allergens Der p 1 and 2) [22, 23]. Research on co-exposure to allergens and particles has initially focused on diesel exhaust particles (DEPs). It was shown in previous studies that DEPs can bind Lol p 1, a major grass pollen allergen, and that DEPs can increase the production of allergen-specific IgE in Epstein-Barr virus (EBV)-transformed human B-lymphocytes when exposed to different polycyclic aromatic hydrocarbons (PAHs), a main component found in DEPs [24, 25]. When DEPs and different allergens were administered simultaneously, higher specific IgE levels were found in human nasal lavages as well as in blood drawn from mice [24, 26, 27]. Much less is known about effects of co-exposure of allergens with ENMs. Numerous studies have explored options to employ ENMs as carriers in immunotherapy against allergies [28–32], but the possible risk of unintentional co-exposure has been less explored. The studies performed so far have mainly focused on various established mouse models [33–39].
Furthermore, upon release of ENMs into the environment, which can occur by accident but also upon disposal or via abrasion, ENMs can interact with different allergens. Therefore, this study aimed to determine effects of allergens present as corona compounds on ENMs on the allergic response of human cells, focusing on basophils and on alveolar epithelial cells. Bet v 1 for tree and Phl p 5 for grass pollen, both representing outdoor allergens, and Der p 1, the major allergen for house dust mite, representing indoor allergens were selected. The binding of allergens onto AuNPs and investigated differences in the allergic response of free compared to conjugated allergens was determined. AuNPs were chosen as model ENMs as their reactivity is low, allowing well controlled characterization, and they do not cause an allergic response on their own, enabling an investigation solely on modulation of allergen-specific responses upon particle binding. Furthermore, the behavior of these ENM-allergen conjugates may provide insights into the use of AuNPs as candidates in nanotherapeutic applications. Additionally, as the enzymatic function of Der p 1 as a cysteine protease is considered to play a role in the allergic sensitization, we also investigated the influence of the AuNP-allergen interaction on proteolytic activity of Der p 1 [12, 40].
AuNPs (3.5*1010 NPs/ml, 50 nm, stabilized in 0.1 mM phosphate buffered-saline (PBS), Sigma-Aldrich, St. Louis, MO, USA) were incubated with different concentrations of purified recombinant allergens, i.e. 2.5 μg Bet v 1 (courtesy of C. Ackaert as described in 2014 ), 5 μg Der p 1 (Indoor Biotechnologies INC., Cardiff, United Kingdom) or 3 μg Phl p 5 (Allergopharma GmbH, Reinbek, Germany) as described by O. Cromwell et al. , on a test tube rotator (Snijders, Tilburg, Netherlands) at 4 °C overnight. The excess protein was removed by performing three washing steps of 5 min centrifugation at 18,000 rpm followed by resuspension in the original buffer.
Characterization of AuNPs, AuNP-allergen conjugates and allergens
AuNPs were characterized by TEM. The protein corona of AuNP-allergen conjugates was also characterized by TEM with protein negative staining. For these investigations samples were taken directly after the conjugation, before the removal of unbound proteins, and were incubated with 1 % uranyl acetate on coated copper grids to stain the proteins. Images were taken using a LEO 912 AB Omega transmission electron microscope (Carl Zeiss, Oberkochen, Germany) operated with a LaB6 cathode at a voltage of 120 kV. Images were filtered at zero energy loss.
The hydrodynamic radius and surface charge of AuNPs and AuNP-allergen conjugates were determined using a Malvern ZetaSizer Nano ZSP (Malvern Instruments, Malvern, UK). The assessment of the molecular size and potential protein aggregation of the free allergens was performed on the DLS802 (Viscotek, Houston, TX, USA) as previously described by Himly et al. .
For further confirmation of hard corona formation, optical and hyperspectral imaging analysis were conducted on a darkfield-based optical illumination system from CytoViva (CytoViva Inc., Auburn, AL, USA). AuNPs and AuNP-allergen conjugates were applied to a microscope slide and covered with a cover glass (both Carl Roth GmbH CoKG, Karlsruhe Germany). The principle of the measurement is based upon the characteristic scattering profile of AuNPs. Each pixel of the image was recorded at a wavelength of 400–1000 nm, and was automatically compared by the classification algorithm spectral angle mapper (SAM) to the AuNP-allergen conjugates. The data is presented as normalized mean regions of interest (ROI’s) of 1100 AuNP-allergen conjugates normalized to the mean ROI of 1100 AuNPs.
Quantification of the bound allergen
Reversed phase - high performance liquid chromatography (RP-HPLC) was performed to determine the free allergen concentrations before and after the conjugation. Indirect quantification of AuNP-bound allergen was achieved by comparing the obtained peak areas of the UV absorption signals at 214 nm of the original free allergen suspension with the signal obtained from supernatants collected after conjugation with AuNPs, after the above-mentioned washing steps. The measurements were carried out on an UltiMate 3000 system (Thermo-Fisher Scientific Inc., Palo Alto, CA, USA) using a C18 AcclaimTM 300 column (2.1×150 mm, 3 μm, Thermo-Fisher) at a flow rate of 500 μl/min and a column temperature of 50 °C. The gradient of the solvents A (H2O + 0.1 % trifluoroacetic acid (TFA)) and B (acetonitrile (ACN) + 0.1 % TFA, both Sigma-Aldrich) was programmed in the following way: 10–60 % solvent A 10 min, 80 % solvent A 5 min, 10 % solvent A 10 min. A full loop injection of 20 μl with a loop volume of 100 μl was used.
3D model of AuNP-allergen conjugates
Size distribution, PDI and zeta potential measurements of AuNPs, allergens and AuNP-allergen conjugates
Average Diameter (nm)
Difference from AuNPs (nm)
Zeta Potential (mV)
Difference from AuNPs (mV)
50.9 ± 0.6
- 42.2 ± 2.2
Bet v 1
3.92 ± 0.6
Der p 1
4.94 ± 2.4
Phl p 5
6.48 ± 0.6
AuNP-Bet v 1
53.1 ± 1.3
0.101 ± 0.020
2.2 ± 0.7
- 39.2 ± 0.5
3 ± 1.7
AuNP-Der p 1
54.2 ± 2.8
0.141 ± 0.010
3.3 ± 2.2
- 29.6 ± 1.1
12.6 ± 1.1
AuNP-Phl p 5
54.6 ± 1.7
0.103 ± 0.016
3.6 ± 1.1
- 34.2 ± 0.9
8 ± 1.3
Basophil activation test
Whole blood of nine patients allergic to Bet v 1, five patients allergic to Der p 1 and six patients allergic to Phl p 5, displaying rhinitis and conjunctivitis symptoms, diagnosed at the allergy clinic of the Paracelsus Medical University of Salzburg were studied. The study was approved by the local ethics committee and all patients participating gave their written informed consent. Basophil activation was performed from Ethylene diamine tetraacetic acid (EDTA)-whole blood using Flow CAST® (Buehlmann Laboratories, Schoenenbuch, Switzerland) according to the manufacturer’s protocol. Whole blood samples were stimulated with different concentrations of allergen or AuNP-allergen conjugates (Bet v 1: 50 – 0.048 ng, Der p 1: 50 – 0.04 ng, Phl p 5: 50 – 0.078 ng), and the corresponding amounts of free allergen were included as controls. Processed samples were analyzed by flow cytometry (FACSCantoTM II, BD Bioscience, San Jose, CA, USA) using FACSDiva 5.02. Basophils were gated as side scatter (SSC)low/C-C chemokine receptor 3 (CCR3)high and activated basophils were identified as Cluster of differentiation (CD) 63high when cut-off was set with the negative stimulation control. CD63 upregulation was assessed in a minimum of 500 basophils per assay. C50 values (i.e. allergen concentration inducing half-maximal basophil activation) for the AuNP-allergen conjugates were determined by logarithmic approximation (certainty R2 > 0.900) in comparison to the allergen alone.
Enzymatic activity assay
Different concentrations of free Der p 1 (50 and 100 ng) and 30 ng AuNP-Der p 1 conjugates were incubated with 1 mM dithiothreitol (DTT), as reducing agent to activate the cysteine protease, for 10 min at 37 °C. For conjugates the given value refers to the amount of conjugated allergen. After incubation the assay was conducted in 50 mM sodium phosphate buffer, pH 7.0, with 100 μM Boc-Gln-Ala-Arg-AMC (PeptaNova GmbH, Sandhausen, Germany) as substrate according to the manufacturer’s protocol. The fluorescence intensity was recorded at ex/em 380/460 nm using a M200Pro plate reader (Tecan, Groedig, Austria) for 20 min.
Permeability assay and TEER measurements
The human lung alveolar adenocarcinoma cell line A549 (ATCC, Manassas, USA) was cultured in RPMI 1640 medium (Sigma-Aldrich), supplemented with 10 % fetal calf serum (FCS; PAA, Pasching, Austria), 100 U/ml Penicillin, 100 μg/ml Streptomycin, 2 mM L-glutamine (Sigma-Aldrich). The cells were seeded in 24-well inserts (high pore density, 0.33 cm2 growth area, 0.4 μm PET, Merck-Millipore KGaA, Darmstadt, Germany) at a density of 1.5 × 105 cells/ml. Transepithelial electrical resistance (TEER) measurements were performed using a TEER electrode (WPI, Sarasotay, USA) to monitor the cell growth every 24 h. Therefore, the medium was changed and the electrode was washed with RPMI medium between measurements. The TEER values were calculated for the dimension [Ohm*cm2] by subtracting the medium only control from the obtained values followed by multiplication with the surface area of the insert. As soon as no further increase in TEER values could be observed, the medium was changed to growth medium without FCS and cells were exposed to 2.5 mM EDTA as a positive control, 0.01 mM DTT, 3.5 × 1010 AuNPs, 530 ng free Der p 1, and 3.5 × 1010 AuNP-Der p 1 conjugates for 24 h. Thereafter, the medium was changed, and TEER measurements were performed as previously described. For determination of the epithelium permeability A549 cells were incubated with 2.5 μg/insert of Fluorescein (Sigma-Aldrich) at 37 °C for 1 h. To assess the percentage of permeated Fluorescein, 100 μl of the lower compartment was transferred into black culture plates (Greiner Bio-One GmbH, Kremsmuenster, Austria) and the fluorescence at ex/em 485/515 nm was measured using a plate reader (Tecan).
For TEER measurements and permeability experiments, results are expressed as mean value ± standard deviation (SD) of 3 independent experiments; calculated using Microsoft® Office Excel 2007. Statistical analysis was performed using Student’s t-test in GraphPad Prism 5. Due to the donor-to-donor variations expected and observed in the BAT measurements, no statistical analysis was performed for these experiments.
Results and discussion
Preparation and characterization of AuNPs and AuNP-allergen conjugates
DLS and RP-HPLC measurements to determine the amount of conjugated allergen
Calculated concentration from DLS (ng/ml)
Allergen molecules/AuNP determined by DLS
HPLC determined concentration (ng/ml)
Allergen molecules/AuNP determined by HPLC
AuNP-Bet v 1 conjugates
657 ± 13.3
648 ± 16
490 ± 68.3
516 ± 77
AuNP-Der p 1 conjugates
578 ± 11.7
415 ± 10
530 ± 3.78
379 ± 2
AuNP-Phl p 5 conjugates
365 ± 7.4
224 ± 5
250 ± 24.2
138 ± 13
Impact of the conjugation to AuNPs on the effector function of allergens
Donors that did not display a difference in basophil activation when comparing free versus AuNP-allergen conjugates, as similar C50 values were observed. These included examples from each patient subset, with two, and three examples for Bet v 1 and Phl p 5, respectively (Figs. 3, 4, 5, Table 3). This infers that in these donors the allergen-specific IgE antibodies recognized the same epitopes irrespective of whether the allergens were free or conjugated to the AuNPs.
Donors with lower basophil activation and higher C50 values upon stimulation with AuNP-allergen conjugates compared to free allergen, as shown for Bet v 1 and Phl p 5, with three and one examples, respectively (Figs. 3, 5, Table 3). In the case of Der p 1, no reduction in basophil activation was found when allergen was bound to the AuNPs. This observation may be explained by an allergen arrangement on the AuNPs surface, where epitopes are partially hidden or that these patients’ do not display the specific IgE against this epitope [62, 74].
- iii.Donors which displayed an increase in basophil activation and thus lower C50 values when cells were exposed to the AuNP-allergen conjugates compared to free allergen. This was shown in three Bet v 1 donors, four Der p 1 donors, and one Phl p 5 donor (Figs. 3, 4, 5, Table 3). With our hypothesized uniform binding orientation, the conjugated allergens would align in the same direction enabling more IgE molecules to crosslink through an increased localized concentration of the bound allergen .Table 3
Concentrations of half-maximal basophil activation (C50) of free allergen versus AuNP-allergen conjugates for all donors tested. In conjugates the given values refer to the amounts of coupled allergen. Donors displaying X were tested but could not be statistically evaluated
Bet v 1
AuNP-Bet v 1 conjugates
Der p 1
AuNP-Der p 1 conjugates
Phl p 5
AuNP-Phl p 5 conjugates
It was furthermore observed that four donors, two for Bet v 1 (donors 3, 6), one for Der p 1 (donor 1), and one for Phl p 5 (donor 1), displayed a lower basophil sensitivity which manifested in a lower maximal activation response (Figs. 3, 4 and 5). Such examples have been described before and may be explained by either a lower total IgE concentration or less allergen-specific IgE bound to the basophils in those patients compared to the others .
Using the enzymatic function for studying protein-nanoparticle interactions – the protease activity of Der p 1
Additionally, the stability of conjugates, and enzyme activity, upon storage at 4 °C was investigated. Hence, the AuNP-Der p 1 conjugates were analyzed after one, two and three months of storage and compared to the fresh AuNP-Der p 1 conjugates. In Fig. 6b the difference in AMC release for AuNP-Der p 1 conjugates is shown; with a decrease in AMC release of 1.3-fold, 1.5-fold and 2.2-fold after one, two and three months, respectively, when compared to the fresh conjugates. These results gave an insight in the stability of the protein corona of Der p 1 conjugated to AuNPs. Upon storage in 0.1 M PBS without an excess of the protein, the proteins either slowly start to detach from the AuNPs surface or are subject to a limited degree of degradation.
The protease function of Der p 1 has been reported to play a functional role in allergic sensitization, as the protease function may disrupt the epithelial barrier in the lung [40, 76]. Consequently, such a marked increase in the enzymatic activity of AuNP-Der p 1 conjugates may have strong implications for allergic sensitization and lung pathophysiology upon uptake via inhalation. Therefore, permeability assays in A549 cells were carried out to address whether in a more physiological model, compared to the enzyme activity assay, such a high impact of AuNPs-Der p 1 conjugates could be found. A549 are cancer cells derived from human type II alveolar epithelial cells, frequently used in investigating effects of inhaled ENMs, since they can form tight epithelia and reproduce other features of this cell type, for which primary cells cannot be obtained. Figure 6c displays the TEER measurements, as a measure of barrier function, of A549 cells grown on the membrane of an insert until forming a tight cell layer. The TEER values of the control cells on day 4 and 5 did not show significant further increases, indicating the formation of a tight monolayer at this time point, as previously described by Schlinkert et al. . On day 4 the cells were treated for 24 h with free Der p 1, AuNP-Der p 1 conjugates, which were activated with 0.1 mM DTT prior to the exposure, 2.5 M EDTA as a positive control, DTT and plain AuNPs, to determine if any would alter the TEER values, or left untreated (Fig. 6d). The EDTA positive control, and more noteworthy, both allergen preparations, free Der p 1 and AuNP-Der p 1 conjugates significantly decreased barrier function of the lung epithelial cells. Furthermore, when comparing free Der p 1 to AuNP-Der p1 conjugates, AuNP-Der p 1 conjugates had a higher impact on the integrity of the monolayer (P < 0.05). In contrast, neither 0.01 mM DTT (used for activation of the protease function) nor the AuNPs alone had any effect on TEER values. The concentration of fluorescein permeating the tight layer was investigated using the same treatments as described above (Fig. 6e) . These data were in line with the TEER measurements, with a significant increase in permeation when cells were exposed to AuNP-Der p 1 conjugates (P < 0.05) compared to the same amount of free Der p 1. Both experiments verified the significant impact on the cell layer when cells were exposed to AuNP-Der p 1 conjugates, which also correlated well with the data observed in the enzymatic activity assay.
In this study we have shown the successful conjugation of three major outdoor and indoor allergens (Bet v 1, Der p 1 and Phl p 5) as highly purified and well-characterized recombinant molecules to 50 nm AuNPs. The AuNP-allergen conjugates were characterized by TEM negative staining, DLS and hyperspectral imaging. All three characterization methods gave correlating results, namely that the allergens were firmly interacting with the AuNPs’ surface. Indirect quantification by RP-HPLC was performed allowing the determination of the allergen concentration on the AuNPs’ surface. To visualize the interaction between the allergens and the AuNPs at the molecular level a 3D model was established based on crystal and NMR structural information, depicting the arrangement of the allergens on the AuNPs upon electrostatic interaction and the surface exposure of the known IgE-binding epitopes of the allergens. For providing experimental evidence for the generated models, the integrity and accessibility of IgE epitopes was determined by activation assays using human blood basophils derived from a panel of allergic patients. We observed a high donor-to-donor variability, depicting similar, higher or lower basophil responses to free allergen versus the respective amounts of AuNP-allergen conjugates. This led us to the conclusion that, (i) in case of similar results, the IgE epitopes of the allergens can be recognized equally well, (ii) in case of a lower basophil activation, some epitopes may become hidden, and the patients’ specific IgE antibodies are not able to recognize other IgE epitopes present on the allergens’ surface, and (iii) in the case of higher basophil activation, an alignment of the allergens on the AuNPs’ surface takes place which optimally displays the IgE epitopes relevant for the respective patient. This would facilitate a more effective IgE receptor crosslinking on the basophils due to a higher localized concentration of allergens on the AuNPs compared to the free allergen.
AuNP-Der p 1 conjugates were the only AuNP-allergen conjugates affected by replacement of plasma proteins, when determining the “hardness” of the protein corona formed via Western blots as control experiments for the AuNP-allergen conjugate incubation in plasma during BAT (Additional File 1: Supplementary material and Additional File 2: Figure S1). We, furthermore, determined that the enzymatic activity of Der p 1 upon binding to AuNPs was markedly enhanced in a cell free assay, and accordingly, the conjugates reduced the integrity of a tight cell monolayer when compared to free Der p 1. Release of Der p 1 from AuNPs was not fast enough, or to a high enough level, to abolish an allergy-promoting effect as shown by increased basophil activation. Studies with other allergens are needed to show how frequent such effects are for allergens in general.
In summary, this study presents that conjugation of allergens to ENMs can modulate the allergic response. Yet, due to allergic donor variability, this effect was not consistently observed. Furthermore the enzymatic activity of the protease Der p 1 was increased when conjugated to ENMs.
basophil activation test
cluster of differentiation 63
diesel exhaust particles
dynamic light scattering
ethylene diamine tetraacetic acid
fetal calf serum
high affinity receptor for IgE
horse radish peroxidase
nuclear magnetic resonance
region of interest
reversed phase-high performance liquid chromatography
spectral angle mapper
sodium dodecyl sulfate
sodium dodecyl sulfate polyacrylamide gel
transepithelial electrical resistance
transmission electron microscopy
T helper cell 2
thymic stromal lymphopoietin
The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement No: 263147 (NanoValid - Development of reference methods for hazard identification, risk assessment and LCA of engineered nanomaterials). We thank Sabine Flicker, Susanne Vrtala and Rudolf Valenta from the center for pathophysiology, infectiology and immunology at the medical university of Vienna for providing the allergen-specific IgG antibodies; Samuel M. Lawrence, Leslie Krauss from CytoViva Inc. and Hoffmann Goetz from Schaefer Technology GmbH for conducting the hyperspectral images and analysis of AuNPs and AuNP-allergen conjugates, and Quentin Peacock from QxDesign for preparing the abstract graphic.
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