The Hydrogel Based Allergen-Coated Gold Nanoparticles for Topical Administration: A Possible Epicutaneous Immunotherapy in Pollen-Sensitized Mice?

ABSTRACT Background The rapid uptake of antigens by antigen-presenting cells (APCs) and their migration to draining lymph nodes in the initial hours after antigen administration in epicutaneous allergen specific immunotherapy (EPIT) prompted us to investigate whether the topical administration of allergens without patch application could alleviate allergy in pollen-sensitized mice. We evaluated the immunotherapeutic effect of topically administering hydrogel-based Gold nanoparticles (AuNPs) loaded with a total extract of Platanus orientalis pollen (Pla. ext (50 μg)-AuNPs) on intact skin. Methods Mice sensitized to P. orientalis pollen were divided into three groups and treated with Pla. ext (50 μg)-AuNPs: 1) patch with Pla. ext (50 μg)-AuNPs, 2) patch with Pla. ext (50 μg)-AuNPs in combination with hydrogel, and 3) topical application of Pla. ext (50 μg)-AuNPs in combination with hydrogel. The immunotherapeutic effects were evaluated by measuring serum specific and total IgE antibodies, total cell and eosinophil count in nasopharyngeal lavage fluid, cytokines in the supernatants of re-stimulated splenocytes by the total extract, and histological examination of lung and nasal mucosa. Results Topical administration of Pla. ext (50 μg)-AuNPs, like patch-based administration, significantly downregulated specific and total IgE and IL-4 production, promoted secretion of IFN-γ and IL-10, markedly reduced the number of inflammatory cells, particularly eosinophils, in nasopharyngeal lavage fluid (p < .05), and inhibited inflammation and pathological damage in lung and nasal mucosa. Conclusion Our results suggest that topical administration of AuNPs loaded with P. orientalis total pollen extract on intact skin could be a potential application for EPIT in the P. orientalis pollen -sensitized mice.


Introduction
Allergen-specific immunotherapy (AIT) has been found to be beneficial as a potentially disease-modifying treatment for allergic diseases such as asthma, allergic rhinitis (AR), and insect sting hypersensitivity.However, despite the increasing prevalence of allergic diseases, only a low percentage of patients select AIT (Alvaro-Lozano et al., 2020;Asher et al., 2006;L. Cox & Calderon, 2010;L. S. Cox et al., 2006;Hankin et al., 2010;Senti et al., 2014).To improve the attractiveness of AIT, researchers aim to optimize the current AIT methods by considering efficient allergen delivery to professional antigen presenting cells (APCs), prevention of systemic adverse side effects by allergen delivery to non-vascular sites, patient convenience via self or needle-free administration, and application of optimal adjuvants (Senti et al., 2014).Epicutaneous allergen specific immunotherapy (EPIT), which involves the application of allergens to intact skin, is an attractive option as it fulfills these criteria.When allergens are applied to intact skin during EPIT, they are taken up by Langerhans cells (LCs) and dendritic cells (DCs) and transported to draining lymph nodes (LNs).In non-inflammatory conditions, these APCs present antigens to T lymphocytes along with MHC-II, which triggers the production of T regulatory cells (Tregs) and promotes tolerance, resulting in a decrease in the immune system's reactivity to allergens (Dioszeghy et al., 2018;Scheurer & Toda, 2017).Several studies on EPIT have shown that APCs rapidly take up antigens and migrate to LNs in the first few hours after antigen administration to intact skin (Dioszeghy et al., 2011;2018;Tordesillas et al., 2018).In this study, we investigated whether topical allergen administration without patch application would alleviate allergy in a pollen-sensitized mice.
EPIT field studies have pursued innovative approaches to increase efficacy such as nanoparticle (NP) delivery systems, microneedles coated with allergens, and fractional infrared laser ablation on the skin (Korotchenko et al., 2021;Landers et al., 2022;Nesovic et al., 2022).In topical skin drug delivery systems, gold nanoparticles (AuNPs) are an effective option because of their small size, general nontoxicity, easy functionalization, and high surface area (Bessar et al., 2016).Considering that AuNPs in previous our study (Koushki et al., 2020) represent an appropriate delivery system for allergen in EPIT, in the current study, we used AuNPs as a carrier for allergens of the total protein extract of Platanus orientalis pollen in a mouse model.We compared the 48-hour patching and topical hydrogel based application of P. orientalis total pollen extract-loaded AuNPs for EPIT in the P. orientalis pollen-sensitized mice.

Study design
The efficacy of topical administration of P. orientalis total pollen extract-loaded AuNPs in combination with hydrogel for EPIT was tested in a P. orientalis pollen-sensitized mice.Following the sensitization period, 16 female BALB/c mice aged 5-6 weeks were separated into four groups: not treated (sensitized, positive control), and three different treated groups (weekly for 8 weeks duration).The three different treated groups included 1) Pla.ext (50 μg)-AuNPs: 48-hour EPIT with a patch containing 50 μg of the total extract loaded on AuNPs, 2) Pla.ext (50 μg)-AuNPs+GEL: 48-hour EPIT with a patch containing "Pla.ext (50 μg)-AuNPs" in combination with hydrogel, 3) Pla.ext (50 μg)-AuNPs+GEL (Topical): topical administration of Pla.ext (50 μg)-AuNPs in combination with hydrogel (Figure 1).A negative control group, non-sensitized mice (4 female BALB/c mice aged 5-6 weeks), was also included.Blood samples were collected at the start, 25th day, and end of the experiment for IgE measurement.Moreover, at the end of the study, nasopharyngeal lavage fluid was collected for cytological analysis, the spleen was harvested, and splenocytes were isolated for proliferation and cytokine assay, and lung and nasal mucosa was separated for histologic evaluation.

Formulation of P. orientalis total pollen extract-loaded AuNPs
AuNPs with an average particle size of 15 nanometers were synthesized using the method established by Turkevich et al. (1951), where tetrachloroauric acid (HAuCl4) (Sigma, USA) was reduced with sodium citrate (Himedia, India) (Turkevich et al., 1951).To prepare the HAuCl4 3H2O (1 mM) and sodium citrate (38.8 mM) solutions, 9 mg of HAuCl4 3H2O was dissolved in 25 ml of deionized water and 2.8 mg of sodium citrate in 2.5 ml of deionized water, respectively.Next, 2.5 ml of sodium citrate solution was added to a boiling solution of HAuCl4 3H2O, and the mixture was heated to 100°C for 30 minutes.The addition of sodium citrate caused a change in color in the HAuCl4 3H2O solution, and the solution was subsequently cooled to room temperature.
Bioconjugation of AuNPs with a prepared P. orientalis total pollen extract (as previously described (Pazouki et al., 2008)) was carried out according to our prior research (Koushki et al., 2020).We added 200 μg of P. orientalis pollen protein extract to 800 μl of AuNPs solution, which was shaken overnight at room temperature.Then, we used an ultracentrifuge at 13,000 rpm for 20 minutes to remove unbound extract allergens from the solution.The sediment that included the P. orientalis total pollen extract-loaded AuNPs was resuspended in phosphatebuffered saline (PBS) (10 mM, pH 7) and stored at 4°C after three washings with PBS.

Protein loading efficiency
We determined the protein loading efficiency of P. orientalis pollen total protein extract on AuNPs using the bicinchoninic acid (BCA) protein assay kit (Parstous, Iran), and calculated it with the formula: (Amount of attached protein allergens in the solution = initial protein amount in the mixture -supernatant protein amount).To assess the protein loading efficiency, we also conducted Sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein allergens isolated from AuNPs, as per our previous study (Koushki et al., 2020).To detach the attached proteins from the AuNPs surface, we added 20 μl 4X SDS-PAGE loading buffer (containing 5% β-mercaptoethanol, 10% glycerol, 2% SDS, 0.005% bromophenol blue and 0.06 M Tris-HCL pH 6.8) to 60 μl AuNPs-protein complex solution and heated it at 100°C for five minutes.Subsequently, the samples were incubated in a water bath at 42°C for 10 hours.After separating the AuNPs by centrifuge at 13,000 rpm for 5 minutes, we loaded the isolated protein onto 15% SDS-PAGE gels and finally stained the gel with Coomassie Colloidal stain.

Animals and ethics
A total of 20 female BALB/c mice aged 5-6 weeks (weight 16-20 g) were obtained from the Pasteur Institute (Tehran, Iran).The mice were kept in pathogen-free conditions at the Nanotechnology Research Centre (Mashhad University of Medical Sciences, Mashhad, Iran), following animal care guidelines.Throughout the experiment, the mice had access to a standard mouse diet and water and were maintained on a 12-hour light/12-hour dark cycle.The Animal Ethics Committee of Mashhad University of Medical Sciences (IR.MUMS.MEDICAL.REC.1399.727)approved this study.

Mice sensitized to P. orientalis pollen
To sensitize the mice to the total protein extract of P. orientalis pollen, three subcutaneous injections were administered to the back of the mice.The sensitization protocol from Mondoulet, Dioszeghy, Ligouis et al. (2012) was followed, which involved injecting 10 μg of pollen extract from P. orientalis along with 2 mg of aluminum hydroxide.Mice were then exposed to 1% (v/v) P. orientalis total pollen extract for 20 minutes on four consecutive days using an aerosol delivery system (Figure 1).

Sensitization confirmation
To assess IgE levels, a retro-orbital blood sample was collected on the first day of the study and on the 25th day after sensitization.Blood samples were also collected from all mice at the end of the study (day 89) to evaluate the immunotherapy's effect.

Epicutaneous immunotherapy
Twenty-four hours after removing the hair on the backs of the mice with an electric razor, we applied the therapeutic solution.In one treatment group, we applied the therapeutic solution over a 1 cm × 1 cm patch on the backs of the mice for 48 hours.In another group, the therapeutic solution in combination with a prepared hydrogel (1% carbomer, 1% propylene glycol, and 1.2% triethanolamine (Shobeiri et al., 2022)) was patched on the backs of the mice for 48 hours, and in the other group, we applied this combination topically to the back of the mice (without patching).After applying the therapeutic solution topically to the back of the mice, we isolated them in separate cages for two hours.We administered aerosolized P. orientalis total extract 1% (v/v) intranasally for 20 minutes to all mice after completing the immunotherapy phase (Figure 1).

Determination of specific and total IgE in blood sample
To measure specific IgE (sIgE), we utilized an in-house developed enzyme-linked immunosorbent assay (ELISA).Microtiter plates were coated with 50 μg of P. orientalis pollen total protein extract per well and incubated overnight at 4°C.After washing the plates five times with PBS containing 0.5% tween 20, they were blocked with blocking buffer (Parstous, Iran) for one hour at 25°C.Serum samples were added to each well and incubated for 3 hours at room temperature.We used 1:500 dilutions of biotin anti-mouse IgE antibody (BioLegend, USA), 1:3000 dilutions of horseradish peroxidase-conjugated streptavidin (BioLegend, USA), and TMB solution (Parstous, Iran) to detect sIgE.Optical density (OD) at 450 nm and 630 nm as a reference was measured using an ELISA microplate reader (Statfax 2100 Microplate Reader, Awareness Technology, USA) after adding the chromogenic substrate.The tIgE levels in mouse sera were determined using a sandwich ELISA kit according to the manufacturer's instructions (Parstous, Iran).

Cellular immune response assessment by cytokine profile
Three days after the final respiratory challenge with P. orientalis total pollen extract, the mice were euthanized and their spleens were isolated.The spleen was minced and filtered to prepare a single-splenocyte suspension, which was washed three times with red blood cell lysis solution (180 nM NH4Cl, 17 mM Na2EDTA).Finally, splenocytes were resuspended in RPMI (Gibco, UK) supplemented with 10% FCS, 100 U/ml penicillin, and 100 μg/ml streptomycin.Splenocytes were adjusted to 1 × 10 6 cells/ml in each well of a 24-well flat-bottomed culture plate and cultured in the presence of P. orientalis total pollen extract (100 μg/ml) for 72 hours at 37°C and 5% CO2.Cell cultures with PHA (2% v/v) (Gibco, UK) and media were performed as controls.After three days, supernatants were collected and stored at −80°C for cytokine analysis using mouse interferon-gamma (IFN-γ), interleukin-10 (IL-10), interleukin-4 (IL-4), and interleukin-17a (IL-17a) ELISA kits, according to the manufacturer's instructions (Parstous, Iran).

Proliferative spleen cell responses
The proliferation assay of mouse spleen lymphocytes was conducted using previously described methods (Hajavi et al., 2019).Splenocytes (2 × 10 5 cells/well) was added into 96well round-bottom microtiter plate in triplicate and then P. orientalis total pollen extract (20 μg/well), PHA (2% v/v), and sole medium were added to wells.An incubation period of 72 hours in incubator containing 5% CO2 at 37°C was conducted on the plates.In order to calculate the stimulation index (SI), the following formula was used: SI = (OD sample -OD blank)/(OD control -OD blank) (Chen et al., 2020).

Nasopharyngeal lavage fluid and cell differential counting
Seventy-two hours after the mice's latest intranasal exposure to pollen extract, the upper part of their trachea was punctured.Using a 22-gauge catheter inserted into the trachea, we lavaged the nostril with a slow injection of 1 ml PBS.The sample was then centrifuged for seven minutes at 4°C at 2000 rpm, and the resuspended sediment in 500 μl PBS was examined by a blind immunologist for total cell count and cell differentiation.The prepared slides were stained with Giemsa, and 500 white cells per slide were counted for the white cell assessment at 100× magnification.

Histological evaluation
The histological slides were prepared using the following steps: 1) fixation of the lung and head of mice in formaldehyde at 4°C for 24 hours; 2) dehydration in ethanol series, consisting of 1 hour in 70% ethanol, 2 hours in 96% ethanol, and 2 hours in 100% ethanol; 3) clarification in xylene; 4) embedding in paraffin using an automated machine, which involved two one-hour incubations in melting paraffin at 60°C.A 5 µm thick section was prepared, mounted on glass slides, and dried at 25°C.Dewaxing was performed with xylene, followed by rehydration in an ethanol series consisting of three incubations for 5 minutes in 100%, two for 5 minutes in 96%, 5 minutes in 70%, 5 minutes in 50% ethanol, and 5 minutes in distilled water.Tissue sections were finally stained in hematoxylin for 3 minutes, rinsed in tap water for 5 minutes, incubated with eosin for 1 minute, and dehydrated with an ethanol series (three times for 5 minutes in 96% ethanol and three times for 5 minutes in 100% ethanol) and xylene (three times for 15 minutes), then cover-slipped.A blind, expert veterinary pathologist assessed the inflammation and pathological damage of the lung and nasal mucosa on slides under a light microscope, with five randomly selected sections from each slide.

Statistical analysis
Analysis of the significance between the experiments was performed using GraphPad Prism version 8.2 software (version 8.4.2,California, USA).The results are presented as mean ± standard error of the mean (SEM).One-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was employed to determine statistical significance.

Characterization of AuNPs and P. orientalis total pollen extract-loaded AuNPs
The size and size distribution of AuNPs and P. orientalis total pollen extract-loaded AuNPs were investigated using DLS.The mean diameter of AuNPs was approximately 15.4 nm, and after loading with protein on the surface, their hydrodynamic size was 27.3 nm, which is an ideal size for skin penetration (Raju et al., 2018;Sonavane et al., 2008).The polydispersity index of 0.208 was observed, indicating a monodisperse size.SEM observations showed that the AuNPs were relatively monodisperse and had a spherical shape (Figure 2(b)).The absorption spectra of AuNPs indicated ƛmax of 520 nm, which confirms the size shown with DLS (Figure 2(a)) (Sonavane et al., 2008).The increased ƛmax in P. orientalis total pollen extract-loaded AuNPs (523 nm) indicates an increase in size, which could be due to protein binding to the surface of AuNPs (Figure 2(a)) (He et al., 2005).SDS-PAGE revealed approximately 60 μg/ml binding proteins allergens on AuNPs (Supplementary.1),which was confirmed by the result of the BCA protein assay test (Parstous, Iran).

Confirmation of mice sensitized to P. orientalis pollen
As evidenced by significantly higher levels of tIgE and sIgE in the serum of sensitized mice than not sensitized, BALB/c mice were successfully sensitized to P. orientalis total pollen extract (P < .001and P < .0001respectively, Figure 3

Total and P. orientalis pollen extract-specific IgE reduced after EPIT
To evaluate the efficacy of EPIT in three different treated groups of mice, we measured tIgE and P. orientalis pollen extract-specific IgE levels by ELISA and compared them with the positive control group.Three groups treated with Pla.ext (50 μg)-AuNPs under different conditions exhibited a decreased immune response in serum sIgE, as evidenced by significantly lower levels of serum sIgE compared to the positive control group (Figure 3(c)).Notably, topical administration of the therapeutic complex also significantly reduced serum sIgE (P < .0001, Figure 3(c)).Serum sIgE levels in the three different treated groups did not vary significantly.As shown in Figure 3, the results of tIgE concentration after EPIT confirm the sIgE findings (Figure 3(d)).

P. orientalis total pollen extract-loaded AuNPs modulated T cell cytokine profile after EPIT
After EPIT, IFN-γ, an indicator of T helper 1, significantly increased in sensitized animals, regardless of treatment type (P < .05, Figure 4(b)).The concentration did not vary significantly among the three treatment groups.Notably, the hydrogelbased treatment groups, both topical and patch, significantly decreased IL-4 (P < .01, Figure 4(a)) as an indicator of T helper 2 and significantly increased the production of IL-10, an anti-inflammatory cytokine indicative of a T regulatory cell (P < .01 and P < .001, Figure 4(c)).Almost all three treatment groups reduced IL-17a to the same extent compared to the positive control group, although this reduction was not statistically significant in the hydrogel-based treatment groups (Figure 4(d)).The ratio of IFN-γ/IL-4 in the Pla.ext (50 μg)-AuNPs complex treated groups, the Pla.ext (50 μg)-AuNPs complex in combination with hydrogel, and topical administration of this complex increased more than 9.3-, 8.9-, and 16-fold, respectively, compared with the positive control group.This increase was due to an increase in IFN-γ rather than a decrease in IL-4 (Figure 4(e)).
The sensitized control groups exhibited higher production of IL-4 and IL-17a compared to the nonsensitized negative control group (Figures 4(a,d)).

Specific splenocytes proliferative response showed relative reduction after EPIT
The MTT assay results revealed that the specific proliferation response of splenocytes to total extract from P. orientalis pollen decreased by more than 1.2-fold in all treatment groups compared to the positive control group, but this reduction was not statistically significant (Figure (f)).

P. orientalis total pollen extract-loaded AuNPs reduced inflammatory cells and eosinophil count after EPIT
The sensitized control groups had significantly higher numbers of total immune cells and eosinophils in the nasopharyngeal lavage fluid compared to the non-sensitized negative control group (P < .0001,Figures 4(g,h)).Cell analysis of nasopharyngeal lavage fluid after 8 weeks EPIT in the three treatment groups showed a significant decrease in total cells and eosinophils compared to the untreated sensitized group (Figures 4(g,h)), which was consistent with our previous study (Pordel et al., 2023).

Histological analysis of the lung and nasal mucosa indicated improved condition after EPIT
Histological assessment of lung and nasal mucosa for allergic airway inflammation after EPIT could be a useful method to evaluate the treatment's effectiveness.The histologic evaluation of the lungs and nasal mucosa in the treated groups showed a reduction in tissue damage and inflammation compared to the positive control group (Figure 5).The pla. ext (50 μg)-AuNPs-treated group showed moderate infiltration of mononuclear inflammatory cells in the alveolar spaces (Figure 5(c)).When combined with hydrogel, pla.ext (50 μg)-AuNPs resulted in mild infiltration of mononuclear inflammatory cells in the alveolar spaces, while topical administration of this combination resulted in moderate infiltration of mononuclear inflammatory cells in the alveolar spaces (Figure 5(d,e)).Also, the inflammation of the nasal  mucosa significantly decreased in all three groups treated with pla.ext (50 μg)-AuNPs, compared to the positive control group.The treated groups showed mild inflammation, while the positive control group had severe inflammation (Figure 6).

Discussion
In this study, we investigated the therapeutic effects of 8-week EPIT via topical hydrogel-based application of P. orientalis total pollen extract-loaded AuNPs in the pollensensitized mice models triggered by P. orientalis total pollen extract.Our aim was to attenuate allergic inflammation by promoting a switch from a Th2 immune response to Treg and Th1 immune responses, decreasing sIgE levels and reducing inflammatory factors associated with allergy.Previous studies have shown that various NPs have been investigated for AIT in different experiments (Pohlit et al., 2017).In our previous research, we found that conjugating AuNPs with protein allergens from P. orientalis total pollen extract reduced the allergen dose for EPIT by half in comparison to conventional EPIT (Pordel et al., 2023).Furthermore, allergen coupled with nanoparticles can reach equal efficacy with a lower allergen dose compared to conventional allergen (Schöll et al., 2004).Additionally, AuNPs could lead to rapid antigen uptake by macrophages and DCs, thus enhancing immunological activity (Bastús et al., 2009;Kang et al., 2017).
Constructed AuNPs and well-designed AuNPs coated with allergens from P. orientalis total pollen extract in this study had an appropriate size for skin penetration and delivery to LNs (Koushki et al., 2020;Raju et al., 2018;Sonavane et al., 2008).Kang et al. found that OVA-AuNPs with a size of 22 nm and 33 nm had high delivery efficiency to draining LNs (2017).
In this study, we demonstrated that AuNPs loaded with P. orientalis total pollen extract in three different treatment groups were effective in attenuating airway inflammation and pathological damage in the lung and nasal mucosa, reducing inflammatory cell counts, particularly eosinophils in nasopharyngeal lavage fluid, and suppressing the production of sIgE and tIgE.Additionally, the P. orientalis total pollen extract-loaded AuNPs were able to suppress the Th2 response and induce the Th1 response, indicating a competent immunotherapy aspect of this complex.The increased production of IL-10 after EPIT with the complex could point to immune tolerance induction by allergen-specific Treg cells (Xiao et al., 2013).These results are a criterion for AIT efficacy and are consistent with studies in the EPIT field (Koushki et al., 2020;Mondoulet, Dioszeghy, Puteaux, Ligouis, Dhelft, Letourneur, et al., 2012;Mondoulet, Dioszeghy, Puteaux, Ligouis, Dhelft, Plaquet, et al., 2015).It is noteworthy that we observed similar results in the group receiving topical treatment, which did not show a significant difference from the groups receiving patched treatment.This could indicate that the topical administration of allergens combined with AuNPs is effective in reducing allergic inflammation.
There is evidence in the literature supporting our findings.Dioszeghy et al. reported efficient uptake of the allergen by dermal CD11b+ DCs and LCs after a 2-hour antigen application to the intact dorsal skin of BALB/c mice (2018).This group also indicated that DCs migrated to draining LNs 6-hours after antigen patching on intact skin in murine models (Dioszeghy et al., 2011).Tordesillas L et al. demonstrated maximal antigen uptake by CD11c+ MHCII+ cells in the epidermis and dermis within 12 hours after antigen application to intact mouse skin by evaluating 12, 24, 48, and 72 hours of kinetic study (2018).Additionally, Raju et al. showed that AuNPs penetrated the deep layers of the epidermis after only three hours of exposure of the hind paws of rats to AuNPs (2018).These documents indicate that antigen penetration into the skin, uptake by APCs, and transfer to LNs occurs within the first hour of antigen administration to the skin.The use of AuNPs as the allergen delivery system in this study is expected to allow for more efficient allergen penetration into the skin and uptake by APCs (Bastús et al., 2009;Kang et al., 2017).APCs play a critical role in coordinating the immune response as they serve as a link between innate and adaptive immunity.Despite the majority of EPIT studies focusing on 48-hour immunotherapy, EPIT can be effective within a shorter period of time (2010;Dupont et al., 2010;Senti et al., 2009Senti et al., , 2012)).Agostinis et al. reported EPIT via 24-hour allergen patching controlled the symptoms of seasonal allergic rhinoconjunctivitis in children (2010).Senti et al. also reported that EPIT via 8-hour allergen patching alleviated the symptoms of grass pollen-induced rhinoconjunctivitis in patients (2012).
Our findings suggest that the topical administration of AuNPs loaded with P. orientalis total pollen extract is a potential application for EPIT in the P. orientalis pollen-sensitized mice.Further studies should investigate different induced Treg subsets, the increase in specific IgG2, and the decrease in specific IgG1 after treatment to validate the efficacy of this method.