Isabella Radauer-Preiml

1
, Ancuela Andosch
3
, Martin Himly
1
, Jutta Horejs-Hoeck
1
, Matthew S. P. Boyles
1
, Thomas Hawranek
4
, Ursula Luetz-Meindl
3
, Christian Huber
2
,
Albert Duschl
1

a
1
Department of Molecular Biology, Division of Allergy and Immunology, University of Salzburg, Salzburg, Austria
2
Department of Molecular Biology, Division of Chemistry and Bioanalytics, University of Salzburg, Salzburg, Austria
3
Department of Cell Biology, Division of Plant Physiology, University of Salzburg, Salzburg, Austria
4
Department of Dermatology, Paracelsus Medical University, Salzburg, Austria

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).
Interactions between nanomaterials and allergens: characterization of a hard protein corona formation and influence on
allergic effector function
Introduction:
Applications of nanomaterials (NMs) and concerns of possible health and environmental risks are increasing. Possible exposure routes of NMs are known for all stages during development, manufacturing, application and disposal of the NMs. With exposure to the environment during any of these stages, contamination
with allergens can occur. Thus, it is important to understand how allergen contaminated NM conjugates interact with the immune system.
It is known that NMs bind proteins in biological systems as soon as they come into contact with each other
[1]
. The proteins directly interact with the NM surface and form a hard protein corona. Hence, the protein corona defines the biological identity of the NM and could change the way the cell recognizes and reacts to
the NM
[2]
. Additionally, the interaction of NMs with proteins can change the protein conformation and thus change its activity. In the case of allergen NMs interactions this could increase the allergenic potential due to the availability of multiple allergen binding sites located on the particle surface, facilitating cross-linking
of IgE receptors and activation of effector cells, or it may decrease the allergenic potential by obscuring IgE-binding sites.
In this study 50 nm gold nanoparticles (Au NPs) were coupled to the major allergens of birch pollen (Bet v 1), timothy grass pollen (Phl p 5) and house dust mite (Der p 1), which differ in time and place of their occurrence and presence of enzymatic activity. Formation of allergen hard corona was characterized and
confirmed using DLS, protein negative staining, SDS-page and HPLC. To study the allergic responses to Au NP allergen-conjugates a basophil activation assay was used. Although, basophils contribute less than 1 % of leukocytes in peripheral blood they are associated with many human diseases (e.g. allergic diseases,
autoimmunity, inflammatory disorders and cancer)
[3]
. Basophils are activated by antibody crosslinking in the presence of antigen
[4]
, therefore the focus of this study was to determine if AU NPs allergen conjugates modulate this activation, in comparison to allergen alone.

Material and Methods:

Characterization of the NPs:

NPs coupling to allergens:
Results:

The characterization of the Au NPs allergen conjugates was performed as described for the Au NPs.
The results demonstrate that attachment of the allergens to the Au NPs was successful. The size
increase, as observed by DLS, matches the size of the allergen, proving the addition of an allergen
monolayer on the surface of the NP. Additionally, a change in surface charge was also observed.

Conclusion:
The characterization of the Au NPs allergen conjugates via DLS, z-potential and TEM has shown that the allergens formed a monolayer around the Au NPs. The concentrations of bound allergens determined by HPLC measurements were in accordance with the theoretically calculated concentrations. The three Au NPs
allergen conjugates used in this study had the capability to activate the basophils to a higher extend than the allergen alone. To define the exact mechanism of this response further studies are needed to characterize epitope integrity and to determine uptake mechanisms as well as allergic sensitization mechanisms.
Figure 2: The Au NPs were incubated overnight at 4C with the different allergens. Initially, proteins get
attach to the NPs, with proceeding time the proteins become stably attached
[2]
. After the incubation
period the loosely attached and free allergen was removed via centrifugation for 10 min at 14.000 rpm
followed by the resuspension of Au NPs allergen conjugates in 0.1 mM PBS.
Figure 5: TEM images of the Au NPs and Au NPs allergen conjugates. 1: Plain Au NPs. 2: Negative
staining of Au NPs Bet v 1. 3: Negative staining of Au NPs Der p 1. 4: Negative staining of Au NPs Phl p
5.
Table 1: The results of the DLS and z-potential measurements for Au NPs, allergens and different
allergen conjugates.
Figure 1: Characterization of 50 nm gold NPs (Sigma Aldrich Co. LLC., St. Louis, MO) by means of
dynamic light scattering (DLS), z-potential measurement and transmission electron microscopy (TEM)
after the purchase.
Quantification of bound allergen:
Figure 3: The determination of the bound allergen concentration was done by means of high pressure
liquid chromatography (HPLC). Therefore, the peak area of the free allergen was compared to the peak
area of the unbound allergen. Method: C 18 2.1 mm x 150 mm, 10 60 % Acetonitrile in 0.1 %
Trifluoroacetic acid, 500 l/min, 55 C
Sample d [nm] Z-potential [mV]
Au NPs 64.97 - 42.2
Au NPs Bet v 1 68.51 - 39.6
Bet v 1 4.0
Au NPs Der p 1 77.38 - 29.6
Der p 1 5.0
Au NPs Phl p 5 71.33 - 34.5
Phl p 5 6.8
Sample
Theoretical
concentration of
coupled allergen [ng]
HPLC measured
concentration of
coupled allergen
[ng]
Au NPs Bet v 1 490 550
Au NPs Der p 1 430 530
Au NPs Phl p 5 280 250
Figure 4: The determination of bound protein to the Au NPs via SDS-Page and HPLC.
The presence of allergen within the complexes was confirmed with SDS-Page. However, the required
full detachment of protein from the NPs could not be confirmed. Therefore HPLC was performed
because quantification by SDS-Page could not be relied upon. In this experiment the allergen peak
area of the same concentration used for the coupling reaction was compared to the peak area of the
allergen after the coupling reaction. The differences of the peak areas gave the concentration of the
bound allergen. The concentration measured by HPLC matched that of the calculated concentration
further confirming the presence of a protein monolayer surrounding the Au NPs.
1 2 3 4
A negative staining of the Au NP allergen conjugates was carried out to visualize the protein corona
and to confirm the data obtained from the previous experiments. Samples were taken directly after
the conjugation, therefore before the removal of unbound proteins, and were treated with 1 % uranyl
acetate to stain the proteins. The images 2 4 in Figure 5 show that the allergens form a layer around
the Au NPs.
In order to determine the differences in allergic responses of basophils to allergens compared to Au
NPs allergen conjugates whole blood of allergic patients was exposed to different concentrations of
allergens and Au NPs allergen conjugates. The activation of basophils was measured via fluorescence
activated cell sorting (FACS). In response to Au NPs alone the cells did not show an activation higher
than 1 %. However, when basophils were exposed to the different Au NPs allergen conjugates we
observed that there was often a higher basophil activation compared to the exposure of allergen
alone. This was evident in nearly all concentrations of Au NPs coated with Bet v 1 and Phl p 5, and at
the lower concentrations of Au NPs Der p 1 conjugates, at higher concentrations Der p 1 induced a
higher level of activation than the Au NPs conjugate.
Figure 6: Basophil activation assay of allergic patients whole blood exposed to different
concentrations of allergens. Bet v 1: Mean of 9 Donors. Der p 1: Mean of 2 Donors. Phl p 5: Mean of 6
Donors.
References: 1.Rahman, Masoud, et al. "Nanoparticle and protein corona." Protein-Nanoparticle Interactions. Springer Berlin Heidelberg, 2013. 21-44. 2. Monopoli, Marco P., et al. "Physical chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles." Journal of the American Chemical Society 133.8 (2011): 2525-2534. 3. Siracusa, Mark C., et al. "Basophils and allergic inflammation." Journal of Allergy and Clinical Immunology 132.4 (2013): 789-801. 4. Sokol, C. L., and R. Medzhitov. "Emerging functions of basophils in protective and allergic immune responses." Mucosal immunology 3.2 (2010): 129-137.