Engineered silica nanoparticles act as adjuvants to enhance allergic airway disease in mice

Background With the increase in production and use of engineered nanoparticles (NP; ≤ 100 nm), safety concerns have risen about the potential health effects of occupational or environmental NP exposure. Results of animal toxicology studies suggest that inhalation of NP may cause pulmonary injury with subsequent acute or chronic inflammation. People with chronic respiratory diseases like asthma or allergic rhinitis may be even more susceptible to toxic effects of inhaled NP. Few studies, however, have investigated adverse effects of inhaled NP that may enhance the development of allergic airway disease. Methods We investigated the potential of polyethylene glycol coated amorphous silica NP (SNP; 90 nm diameter) to promote allergic airway disease when co-exposed during sensitization with an allergen. BALB/c mice were sensitized by intranasal instillation with 0.02% ovalbumin (OVA; allergen) or saline (control), and co-exposed to 0, 10, 100, or 400 μg of SNP. OVA-sensitized mice were then challenged intranasally with 0.5% OVA 14 and 15 days after sensitization, and all animals were sacrificed a day after the last OVA challenge. Blood and bronchoalveolar lavage fluid (BALF) were collected, and pulmonary tissue was processed for histopathology and biochemical and molecular analyses. Results Co-exposure to SNP during OVA sensitization caused a dose-dependent enhancement of allergic airway disease upon challenge with OVA alone. This adjuvant-like effect was manifested by significantly greater OVA-specific serum IgE, airway eosinophil infiltration, mucous cell metaplasia, and Th2 and Th17 cytokine gene and protein expression, as compared to mice that were sensitized to OVA without SNP. In saline controls, SNP exposure did cause a moderate increase in airway neutrophils at the highest doses. Conclusions These results suggest that airway exposure to engineered SNP could enhance allergen sensitization and foster greater manifestation of allergic airway disease upon secondary allergen exposures. Whereas SNP caused innate immune responses at high doses in non-allergic mice, the adjuvant effects of SNP were found at lower doses in allergic mice and were Th2/Th17 related. In conclusion, these findings in mice suggest that individuals exposed to SNP might be more prone to manifest allergic airway disease, due to adjuvant-like properties of SNP.


Instruments and characterization
MHz or VXR-500 MHz instrument. The CDCl 3 resonance was used as the internal standard for 13 C NMR (δ = 77.0 ppm) and residual CHCl 3 for 1 H NMR (δ = 7.24 ppm). Fourier transform infrared (FTIR) spectra were recorded on a Mattson Galaxy series FTIR 3000. Thermogravimetric analyses (TGA) were obtained in air from a Perkin-Elmer TGA 7. Samples were held at 120 °C for 30 min to remove absorbed water from particle surfaces and then heated to 850 °C at a rate of 10 °C/min. All samples for FTIR and TGA were vacuum dried at room temperature for 24 h.
Dynamic Light Scattering (DLS) was performed in a Malvern NanoZS ZetaSizer with a 178 degree backscattering detection. Intensity average diameters were calculated from the autocorrelation function using Malvern's Zetasizer Software 6.12. The samples for DLS analyses were sonicated prior to measuring the particle size (FS20H sonicator, Fischer Scientific). Centrifugation was performed at 6000 rpm on KμPrima-18R (Composite Rotor Inc.).

Surface Modification of Colloidal Silica Nanoparticles
As shown in figure S1, PEG-coated SNP were synthesized from plain SNP (commercially available LUDOX ® TM-40 SNP) in three steps. To avoid aggregation, the modified SNP were purified by several steps of washing the nanoparticles with solvent, following by centrifugation. The intermediate particles (aSNP and aaSNP) were immediately further processed according the protocol and aggressive drying such as drying under vacuum [3] was avoided. Figure S1. Process of SNP PEG coating. PEG-coated SNP were synthesized from commercially available LTM40 silica nanoparticles in three steps. First, aminopropyltriethoxy silane (APTES) was condensed on the plain silica particles, resulting amine-modified SNP (aSNP). Then, particles were reacted with propargyl chloroformate to afford the alkyne-modified particles (aaSNP). Finally, particles were clicked with PEG-N 3 catalyzed by 10% of Cu(PPh 3 )Br diisopropylenamine to achieved PEG-coated SNP.
(1) Synthesis of amine-modified SNP (aSNP:. Commercially available plain SNP (LUDOX ® TM-40 colloidal silica; 4.8 g in H 2 O) were diluted with 100 mL of a 1:1 solution of EtOH/H 2 O (v/v), and then aminopropyltriethoxy silane (APTES) (5 mL, 20 mmol) was drop wise added to the silica suspension. The mixture gradually evolved to a turpid suspension due to the formation of silane oligomers. After stirring for 3 days, the suspension was washed with EtOH and centrifuged (6000 rpm) for 20 -40 min to remove the silane oligomer and any other impurities by. The purified product was kept in ethanol.
(2) Synthesis of alkyne-modified SNP (aaSNP): To remove ethanol and water, aSNP were washed and recovered by centrifugation (30 min) twice with reagent grade DMF, followed by washing three times with anhydrous DMF. The aSNP (30 mg, 1 equiv. of amine) were re-dispersed in a mixture of anhydrous DMF and triethylamine (3:1) by sonication (40 min) to obtain a homogenous solution. After cooling in an ice bath for 30 min, propargyl chloroformate (2 mL, 1000 equiv.) was added drop wise and the solution turned to slight yellow. After addition of propargyl chloroformate, the ice bath was removed and the mixture was stirred for 24 h. The product was purified by several cycles of washing with CH 2 Cl 2 and recovery by centrifugation, until the solvent was colorless and no sign of residual amine salts. The product was stored in CH 2 Cl 2 till further modification.
(3) Synthesis of PEG-coated SNP by "Click" modification of aaSN: estabished aaSNP (38 mg, 1 equiv. of alkyne) were dispersed in 50 mL of DMF, followed by sonication for 30 min. An excess of PEG-N 3 (3.4 equiv.) and 0.2 mL of N,Ndiisopropylethylamine were added to the suspension, followed by three freeze-pump-thaw processes to remove O 2 . Solid Cu(PPh 3 )Br (10% equiv.) was added followed by a final freeze-pump-thaw process. After purging with N 2 , the suspension was stirred at RT for 24 h. The suspension was centrifuged to recover the PEGylated SNP (2x10 mL). The purified particles were kept in distilled water. The nanoparticle concentration was measured by drying fixed amounts of the aqueous solution.
Changes in the SNP surface chemistry were tracked by FTIR and figure S2 shows the normalized FTIR spectra of SNP after each modification step. Since the IR band at 806 cm -1 is characteristic for silica, IR data were normalized allowing semi-quantitative comparison of the nanoparticles during the syntheses. For example, the spectrum of aSNP shows increased absorption at 2900-3300 cm -1 and a new band at 1431 cm -1 , corresponding to the alkyl and amine groups of APTES. After treating aSNP with propargyl chloroformate, new absorption bands are seen at 1700 cm -1 due to C=O stretching, and triple bond stretching at 2127 cm -1 , confirming successful alkyne functionalization on the particle surface (aaSNP) [4]. IR is less useful for determining PEGylation; the characteristic C-O bands are obscured by the strong IR bands of the particles, but bands from C-H bending and stretching increased. The TGA data in figure S3 and table 1 (main manuscript) confirm the step-by-step modification of silica particles. Since the TGA mass losses correspond to oxidation of the organic layer on the nanoparticles, the grafting density on the particle surface can be calculated. Table 1 in the article shows a 3.7% weight loss for amine-modified nanoparticles (aSNP), which corresponds to a grafting density of 2.7 group/nm 2 . After the alkyne modification (aaSNP), the additional mass loss (2.4%) indicate that about half of the amines were converted to the triple bonds, and the pegylation step (3.6%) corresponds to a grafting density of 0.7 PEG chain/nm 2 and successful synthesis of water-soluble nanoparticles (PEG-coated SNP). Figure S3. TGA data for PEG-coated SNP and its precursors. All samples were held at 120 °C for 30 min to remove absorbed water from particle surfaces, and then heated in air to 850 °C at a rate of 10 °C/min.
The DLS analysis shows that all nanoparticles have monomodal ( Figure S4), but their size and size distribution depend on the surface chemistry of the particles, and the solvent used for the analysis. The average diameter of commercially available plain SNP is 30 nm (Table 1, article), showing a narrow particle distribution. Adding the amine to the particle surface increased the average diameter to 103 nm (DMF-NEt 3 1:3) and after alkyne modification (hydrophobic) the average particle size was 126 nm (DMF). PEGylation made the particles water soluble, the average diameter decreased to 90 nm and the distribution narrowed as indicated by figure S4 and table 1. Figure S4. DLS data (size distribution by intensity) for PEG-coated SNP and its precursors. Figure S5. Heat map of gene expression array. Gene expression in lung tissue was analyzed with a 96-gene array. Relative increased gene expression towards control is indicated in red (≥2 fold expression) and relative decreased gene expression in green (≤2 fold). Black labels indicate no differences in gene expression. Increased gene expression in allergic and SNP exposed animals was found for various cytokines, chemokines and immune responsive genes as well as secretory mucus/surfactant genes. No changes greater than 2-fold were found for oxidative stress response, growth factors and different transcription factors. Ccl2