Functional 2D Nanoplatforms Alleviate Eosinophilic Chronic Rhinosinusitis by Modulating Eosinophil Extracellular Trap Formation

Abstract The therapeutic outcomes of patients with eosinophilic chronic rhinosinusitis (ECRS) remain unsatisfactory, largely because the underlying mechanisms of eosinophilic inflammation are uncertain. Here, it is shown that the nasal secretions of ECRS patients have high eosinophil extracellular trap (EET) and cell‐free DNA (cfDNA) levels. Moreover, the cfDNA induced EET formation by activating toll‐like receptor 9 (TLR9) signaling. After demonstrating that DNase I reduced eosinophilic inflammation by modulating EET formation, linear polyglycerol‐amine (LPGA)‐coated TiS2 nanosheets (TLPGA) as functional 2D nanoplatforms with low cytotoxicity, mild protein adsorption, and increased degradation rate is developed. Due to the more flexible linear architecture, TLPGA exhibited higher cfDNA affinity than the TiS2 nanosheets coated with dendritic polyglycerol‐amine (TDPGA). TLPGA reduced cfDNA levels in the nasal secretions of ECRS patients while suppressing cfDNA‐induced TLR9 activation and EET formation in vitro. TLPGA displayed exceptional biocompatibility, preferential nasal localization, and potent inflammation modulation in mice with eosinophilic inflammation. These results highlight the pivotal feature of the linear molecular architecture and 2D sheet‐like nanostructure in the development of anti‐inflammation nanoplatforms, which can be exploited for ECRS treatment.

Equipment: Nuclear magnetic resonance spectroscopy (NMR) spectra were measured with a Jeol Eclipse (USA) nuclear magnetic resonance spectrometer (500 MHz).Ultraviolet-visible spectrophotometry (UV-vis) absorption spectra were measured with a U-3310 spectrophotometer (Hitachi, Japan).Zeta potential and dynamic light scattering (DLS) data were recorded on a Malvern NANO ZSPO in corresponding conditions.Fourier transform infrared spectroscopy (FTIR) spectra were recorded on a Jasco FT/IR-4100 spectrometer.
Transmission electron microscopy (TEM) was performed with a FEI Tecnai G2 F30 TEM.
Fluorescence was measured by a JASCOFP-6500 Spectrofluorometer.Quanti-Blue and CCK8 assays were performed using a microplate reader (Bio-Tek, Winooski, VT).Flow cytometer was performed using a BD FACS caliber flow cytometer.Quantitative polymerase chain reaction (qPCR) was measured with LightCycler 480 II (Roche, US).Confocal laser scanning microscopy (CLSM) experiments were conducted with a Nikon Ti Eclipse inverted microscope with an A1 scanning confocal unit and quantification of the specified color area was calculated by Image J software.H&E, PAS staining slices were scanned using an automated slice scanning system (AxioScan.Z1, Zeiss).Biodistribution fluorescent images were recorded with an IVIS Spectrum system (PerkinElmer, USA).
Biodegradation tests: 3 mg MoS2, TiS2, WS2, MLPGA, TLPGA, WLPGA, and TDPGA were dispersed in 3 mL PBS (7.4) and then evenly divided into three dialysis tubes (MWCO=2k), respectively.After that, the above solutions were subjected to shaking at 37ºC for 4 weeks, and the UV absorbance (360 nm) was recorded at the beginning, 1 week, 2 weeks, 3 weeks, and 4 weeks after incubation.The degradation of nanosheets was calculated by comparing the absorbance value (360 nm) of the solutions with the initial absorbance value (360 nm) before the experiments.Additionally, the nanoscale morphologies of nanosheets before and after degradation were also recorded by TEM.cfDNA binding assay: cfDNA concentration was determined by pico-green assay, a fluorescent dye that could specifically bind cfDNA. [1,2]The cfDNA binding efficacy of LPGA, DPGA, MLPGA, TLPGA, WLPGA, TDPGA and P-G3 were determined by pico-green competitive binding tests.Firstly, diluted pico-green reagent and cfDNA from calf thymus were mixed in Mili-Q water or FBS (10%) aqueous solution and incubated for 30 min at 37ºC in a dark environment.After that, LPGA, DPGA, MLPGA, TLPGA, WLPGA, TDPGA and P-G3 were added to the mixture with nanomaterials/cfDNA mass ratios from 0.0625 to 32 before the mixture was shaken and incubated for 30 min at 37ºC in dark environment.Finally, the fluorescence intensity was measured with a Multiwall Plate Reader (Excitation: 480 nm; Emission: 520 nm).The binding efficiency of LPGA, DPGA, MLPGA, TLPGA, WLPGA, TDPGA and P-G3 to cfDNA was calculated by comparing the fluorescent intensity after incubation to the initial value.
Protein adsorption determination: The protein adsorption determination was performed according to a reported method. [3]LPGA, DPGA, MLPGA, TLPGA, WLPGA, TDPGA, and P-G3 solutions (1 mL, 25 to 100 µg/mL) were mixed with BSA-FITC (1 mL, 100 µg/mL).The mixed solution was then centrifugated at 11,000 rpm for 10 min after stirring at 37 °C for 30 min.The supernatant was carefully collected, and the fluorescent intensity was measured to calculate the protein concentration.The protein adsorption (PA) was calculated using the following equation: PA = (C1 − C2)/C1´100%.In this equation, C1 and C2 are the initial concentration of BSA and the BSA concentration in the supernatant after centrifugation, respectively.
Cytotoxicity test: BEAS-2B cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin (PS) at 37℃ in a humidified atmosphere with 5% CO2.The cytotoxicities of the LPGA, DPGA, MLPGA, TLPGA, WLPGA and TDPGA against BEAS-2B cells were evaluated with CCK-8 assay.Firstly, BEAS-2B cells (5×10 3 /well) were seeded in a 96-well plate and cultured until the cell density reached 70-80%.After that, the culture medium was replaced by fresh medium with different concentrations of LPGA, DPGA, MLPGA, TLPGA, WLPGA and TDPGA.Subsequently, the medium was replaced again with fresh medium containing 10% CCK-8 reagent after 24 h or 48 h incubation.Finally, the plates were incubated for another 2-3 h at 37°C and then the absorbance (450 nm) of the wells was recorded using a Multiplate Reader.The cytotoxicity of LPGA, DPGA, MLPGA, TLPGA, WLPGA and TDPGA was calculated by comparing the absorbance value to the value of cells treated with medium-only.
TLR9 activation with QUANTI-Blue assay: HEK-blue TM hTLR9 (HEK-TLR9) reporter cell lines were obtained from InvivoGen, San Diego, CA, and were cultured following the manufacturer's protocol.The HEK-TLR9 cells were originated and constructed by cotransfection of HEK 293 cells with the hTLR9 gene and optimized embryo-secreted alkaline phosphatase (SEAP) reporter gene. [4]The SEAP activity was determined using the alkaline phosphatase (ALP) detection reagent, QUANTI-Blue™ and calculated by optical density (OD) at 620 nm.ODN1826 as non-methylated DNA and synthetic analogs of cfDNA, was applied as TLR9 agonists.HEK-TLR9 cells were seeded into 96-well plates (8×10 4 /well) and the cells were attached to the wall after 6-8 h incubation.After that, ODN1826, ODN1826+LPGA, ODN1826+DPGA, ODN1826+MLPGA, ODN1826+TLPGA, ODN1826+WLPGA and ODN1826+TDPGA were added to the wells and incubated for the following 24 h.ODN1826 was set as 1 mg/mL and the concentration of nanomaterials was from 0.5 to 4 μg/mL.The cells incubated with medium-only or ODN1826-only were considered as negative control (NC) and positive control (PC), respectively.Finally, 50 μL supernatants of each well were harvested and mixed with 150 μL QUANTI-Blue TM medium.The new plate was incubated for 2 h and TLR9 activation was determined by the absorbance (620 nm) value measured using a Multiwall Plate Reader.
Inhibition of the nasal secretion-induced TLR9 activation: HEK-TLR9 cells were incubated with nasal secretions from ECRS patients in a 96-well plate, and the concentration of nasal secretions was set as 1 μg/mL (cfDNA concentration).LPGA, MLPGA, TLPGA and WLPGA were added to the wells at 2 μg/mL and 4 μg/mL, respectively and the cells were incubated for 24 h.The cells incubated with medium-only and nasal secretion-only were considered as NC and PC.respectively.Then 50 μL culture supernatants were collected and added to 150 μL QUANTI-Blue TM solution in a new plate.After incubation for another 2 h, TLR9 activation was determined by the absorbance (620 nm) value measured using a Multiwall Plate Reader.
Derp1 and LPS-induced cfDNA release: BEAS-2B cells (5×10 3 /well) were seeded in a 96-well plate and incubated at 37°C until the cell density reached 70-80%.In the next step, Derp1 and LPS (1 μg/mL) were added into the medium and the cells were incubated for another 12 h.Subsequently, the culture medium was replaced by fresh medium with LPGA or TLPGA (2 μg/mL or 4 μg/mL) and the cells were cultured for another 12 h.The cells incubated with medium-only during the whole process were considered as NC.The cells incubated with Derp1 or LPS in the first stage and medium-only in the second stage were considered as PC.Finally, the conditioned medium was collected and cfDNA concentrations were measured with a picogreen assay.
TLR9 activation by conditioned medium: HEK-TLR9 cells were incubated with Derp1 or LPSconditioned medium and cfDNA concentration is 1 μg/mL.Subsequently, LPGA or TLPGA (2 μg/mL or 4 μg/mL) were added into the cells and incubated for another 24 h.The cells incubated with only fresh medium or conditioned medium were considered NC and PC, respectively.Finally, culture supernatants (50 μL) were harvested and added to QUANTI-Blue TM solution (150 μL) in a transparent plate and incubated for another 2 h.TLR9 activation was determined by the absorbance (620 nm) value measured using a Multiwall Plate Reader.

ODN1826-induced EET formation:
Human peripheral blood eosinophils were isolated from ECRS patients with a MACSxpress® eosinophil isolation kit (Stem Cell, Canada). [5]osinophils were cultured using Eosinophils Medium W/Kit in 24-well plates with coverslips on the bottom of wells.The eosinophils were split into two groups: one group of cells was cultured in medium-only and the other group was pretreated with ODN2088 for 1 h.After that, ODN1826 was added to these cells and incubated for another 4 h to induce EET formation.
Subsequently, DAPI staining and eosinophil cationic protein (ECP) immunostaining were applied to identify the EETs on the coverslips.The EET formation was observed using confocal laser scanning microscopy (CLSM) and the EETs area in the images was qualitatively calculated by ImageJ software.
Conditioned medium-induced EET formation: Human eosinophils isolated from ECRS patients were cultured using Eosinophils Medium W/Kit in 24-well plates with coverslips on the bottom of wells.After that, Derp1 or LPS-conditioned medium was added to these cells and LPGA or TLPGA (2 μg/mL or 4 μg/mL) was also added to examine their suppression efficacy for EET formation.After 4 h incubation, EETs on the coverslips were identified by DAPI staining and ECP immunostaining.The EET formation was observed by CLSM and qualified by ImageJ software.
Clinical samples: 20 nasal secretions were collected with an expansive sponge from the ECRS patients and 20 control nasal secretions were donated by healthy volunteers in the Sixth Affiliated Hospital of Sun Yat-sen University and the First Affiliated Hospital of Sun Yat-sen University.Nasal secretions collections were approved by the ethics committee of Sun Yat-sen University (2023ZSLYEC-497).

4 .
Figure S3. 1 H NMR data of TLPGA in D2O.Signals at 4.7 ppm are attributed to the protons of

Figure S4 .
Figure S4.The FTIR spectra of TLPGN3 and TLPGA.The peak around 2100 cm -1 was attributed

Figure S5 .
Figure S5.UV-vis absorbance of LPGA at different concentrations from 0.2 to 1 mg/mL.

Figure S10 .
Figure S10.Viability of BEAS-2B cells treated for 48 hours with various concentrations of

Figure S14 .
Figure S14.Comparison of cfDNA binding efficiency of TLPGA and P-G3 in water or FBS

Figure S24 .
Figure S24.(A) Representative H&E staining images of lungs from experimental mice.Scale

Figure S25 .
Figure S25.(A) Representative ECP staining images of lungs from experimental mice.Scale