5.1 Preparation and Characterization of ZnONPs
The preparation of amine-modified zinc oxide nanoparticles (NH2-ZnONPs) was followed by previously published method [14]. Briefly, 3.35 mM zinc acetate dihydrate (Zn(CH3COO)2(H2O)) in 31.25 ml ethanol was mixed with potassium hydroxide (KOH) in 16.25 ml methanol and stirred at 60°C. Then, KOH was added into the mixture and reacted for 1.5 hrs until the solution became turbid. The precipitate was collected by centrifugation at 10,000 rpm for 10 min, and then the pellet (ZnONPs) was washed twice with ethanol. Next, 0.25 ml APTES ((3-aminopropyl) triethoxysilane), 0.05 ml 25 wt% ammonia and 0.5 ml distilled water were added to the ZnONPs solution and stirred for 20 hrs at room temperature. The precipitate was collected by centrifugation at 10,000 rpm for 15 min. The resulting solution was the NH2-ZnONPs solution.
The size and morphology of ZnONPs was observed by using transmission electron microscope (TEM, JEOL JEM- 2100F transmission electron microscope). More than 100 particles were measured to determine the mean size. The nanoparticles’ element is analysis by using Energy Dispersive X-ray Spectrometer (EDS) function of TEM. The hydrodynamic diameters, polydispersity index and zeta potentials of ZnONPs were measured by DelsaNano C Particle Size and Zeta Potential Analyzer (Beckman Coulter, USA). The fluorescence spectrum of ZnONPs were measured by SpectraMax iD5 multi-mode microplate reader (Molecular devices, CA, USA).
5.2 Preparation of R6G-ZnONPs
R6G-ZnO NP synthesis was performed by the following steps. First, 1 ml of 1000 µg/ml ZnONPs was mixed with 5 µl of 1 mM rhodamine 6G (R6G, Sigma, #252433). The mixture was sonicated for 30 min to conjugate ZnONPs and R6G. After sonication, the precipitate was collected by centrifugation at 13,500 rpm for 30 min. Then, the R6G-ZnONP solution was prepared by resuspending the pellet in distilled water. The chemical bonding and surface chemistry of R6G-ZnONPs were examined by using X-ray photoelectron spectroscopy (XPS). The XPS spectra of ZnONPs and R6G-ZnONPs were recorded by using an X-ray photoelectron spectrometer (PHI VersaProbe 4, Physical Electronics, Inc. USA) with an aluminum mono Kα source. To obtain information on each element, a high-resolution survey (pass energy 117.40 eV) was performed at spectral regions relating to zinc, carbon, and oxygen.
5.3 In Vivo Experiment (Acute UVB Irradiation)
The in vivo acute UVB irradiation model was established as described in a previous study [70]. The minimal erythemal dose (MED) of hairless mice was approximately 60 mJ/cm2. Therefore, to induce acute damage from UVB irradiation, the irradiation intensity was set at 2.5 MED (150 mJ/cm2) [9, 14]. ZnONPs was administered at doses of 2 mg/cm2 (low dose) and 16 mg/cm2 (high dose) based on the standard protocol for determining the sun protection factor (SPF) of sunscreen products (ISO24444:2019) [8, 9]. First, Mice were exposed with a single dose of UVB (150 mJ/cm2) by using UV Crosslinker (UVP CL-1000). The animal experiment was divided into eight groups, including UVB, ZnONPs alone exposure, and combined exposure. One of the combined exposure group was simultaneous exposure, in which mice skin were topically treated with ZnONPs (2 or 16 mg/cm2) then exposed to UVB and the other one was 24 hrs after UVB exposure, the mice were topically treated with ZnONPs (2 or 16 mg/cm2) in the same area. More detailed experimental processes were shown in Fig. 2A. The transepidermal water loss (TEWL) was measured at 24, 48 and 72 hrs by Tewameter® TM 300 probe (Courage & Khazaka GmbH, Cologne, Germany) after the UVB treatment. Mice were sacrificed at the time points described in the results, the skin was immediately harvested for further analysis.
For histopathological analysis, skin samples were fixed with 4% paraformaldehyde in PBS, then dehydrated and paraffin-embedded for further analysis. The other skin samples were stored at -80°C before protein for other analysis.
5.4 Skin Thickness Measurement
The detail skin thickness measurement was described in previous study [14]. Briefly, the microscope image of Hematoxylin and eosin stained skin samples were used to determine the epidermis thickness then analyzed with open software ImageJ.
5.5 Nanoparticle distribution in skin
Fluorescence microscopy was applied to visualize process of R6G-ZnONPs penetrate in skin. Mice were treated with the same process of in vivo study. After the exposure process, mice were sacrificed, the skin samples were fixed and dehydrated with 30% sucrose solution. The skin samples were embedded and freeze in OCT gel for the cryosection. The image of skin section was obtained with fluorescence microscope (Nikon H600L, Japan) and analyzed using the Nikon microscope software NIS-Elements.
In addition to detecting R6G-labeled ZnONPs in skin samples, direct detection of ZnONPs in skin samples was also performed by using multiphoton microscopy. According to a previous study, ZnONPs exhibit various fluorescence emissions due to their band structure (2.38–3.54 eV), and ZnONPs can be identified by green fluorescence emission [71]. Multiphoton microscopy was performed using a homemade system (Core Facility Center, National Cheng Kung University, Taiwan) that included a Chameleon Vision II titanium sapphire femtosecond laser source with a tuning range from 680 to 1600 nm (Chameleon, PA, USA). Fluorescence emission was detected using a Zeiss Plan-APOCHROMAT 20×/0.8 objective lens with 420/10 nm (blue) and 531/40 nm (green) low-pass filters. Photon signals were collected by photomultiplier tubes (PMTs, H10721P-110, Hamamatsu, Japan). The image was mapped in a 200 × 200 µm (256 × 256 pixel) area. The image of the skin section was stitched together from four images by using ImageJ software.
5.6 Acridine Orange (AO) Staining
Acridine orange (AO) (Invitrogen, Carlsbad, CA, USA) was used to detect the acidic vesicular such as autophagolysosomes in cytosol. The detail staining protocol was followed previous study [14]. Briefly, cells were stained with AO solution (1 µg/ml) for 20 min at 37°C after the treatment and washed once with PBS. After the incubation, cells were trypsinized and resuspended in PBS. The fluorescence was detected by using FACS Calibur flow cytometry. Ten thousand cells in each sample. The raw data was analyzed by FlowJo 7.6.1 software.
5.7 Immunofluorescence Staining of M1/M2 Macrophages
The paraffin-embedded skin samples were first deparaffinization. Then the samples were recovered antigenicity by using the Tris-EDTA buffer and heated to 100°C for 20–40 minutes. To block the autofluorescence, slides were incubated with 0.1% Sudan Black in 70% ethanol solution for 10 minutes at room temperature. The samples were stained with the indicated primary antibody against CD86 (Novus, NBP2-25208) (1:200) and CD206 (abcam, ab64693) (1:200) for 1 hour at 37°C and incubated with Alexa Fluor 488- or Alexa Fluor 594-conjugated secondary antibody (1:200) for 1 hour at 37°C. The nuclei were stained by DAPI staining. Images were obtained with fluorescence microscope (Nikon H600L, Japan) and analyzed using the Nikon microscope software NIS-Elements.
5.8 Immunohistochemistry staining for cryopreservation of skin tissue
The OCT compound-embedded skin samples were cut into cryostat sections at 5–10 µm and mounted on gelatin-coated histological slides. Then the samples were rehydrated, treated with 3% hydrogen peroxide for 5 mins, and blocked the section with 1–3 drops of serum blocking reagent (Millipore, DAB500) for 1 h at room temperature. The tissue sections were incubated with primary antibodies against IL-1β (16806-1-AP, Proteintech Group, Chicago, IL, USA) at 4°C overnight. After washing, added aliquots of biotinylated secondary antibodies. The reaction was visualized by streptavidin HRP and DAB chromogen reagent. Then, the slides were stained with hematoxylin and were observed under a light microscope.
5.9 Immunofluorescence staining for detecting autophagic flux blockage
Cells were seeded on black clear bottom 96-well plate. After treatment, the cells were fixed with 4% paraformaldehyde and stained for nuclei (DAPI), autophagosomes (LC3) and lysosomes (LAMP-1), as described under “Immunofluorescence Staining and High-Throughput Screen” (Supplementary information). The plates were assayed using ImageXpress® Micro Confocal High-Content Imaging System (Molecular Devices, San Jose, CA, USA). LC3 immunostaining was captured using a FITC filter and LAMP1 immunostaining using a TRITC filter. Images were subjected to segmentation and quantification using the custom module editor in MetaXpress analysis software.
5.10 M1/M2 Macrophages detection
THP-1 cells were first differentiated with 100 ng/ml PMA (phorbol-12-myristate-13-acetate). After incubation with or without 100 ng/ml LPS and exosome for 24 hrs, cells were trypsinized and resuspended in PBS. Cells were centrifuged 5 min at 200 × g, 4°C. The pellet was resuspended in 100 µl PBS with 20 µl of primary APC-anti-CD206 (BD Biosciences, #550889) and 20 µl of PE-anti-CD86 (BD Biosciences, #560957) antibody and incubated for 30 min at 37°C. Cells were also incubated with the control isotype corresponding to each primary antibody (PE Rat IgG2a, κ Isotype Control, #553930) (Alexa Fluor® 647 Rat IgG2a, κ Isotype Control, #557690). After incubation, cells were washed with PBS and recollected by centrifugation at 200 × g, 4°C for 5 min. Three washes with PBS were next performed. Cells were analyzed by flow cytometry with CytoFLEX Flow Cytometer (Beckman Coulter, USA).
5.11 Exosome isolation
The exosome isolation protocol was followed by previous study [14]. THP-1 cells and HaCaT cells were cultured in Ultra CULTURE™ Serum-free Cell Culture Medium (Lonza, #12-725F) to avoid contamination of EVs from fetal bovine serum. After exposure, the culture medium was collected and exosomes were isolated via differential centrifugation. Briefly, the medium was centrifuged for 10 min at 3000 × g to eliminate cell debris, then the supernatants was centrifuged for 30 min at 10,000 × g to eliminate microvesicles. The exosomes were collected by using ultracentrifuge for 120 min at 100,000 × g. (Optima L-100XP Ultracentrifuge (Beckman), SW28 rotor (Beckman)), the exosome pellets were resuspended in 0.2 µm filtered particle free PBS.
5.12 NanoSight detection
Exosome samples were measured by using nanoparticle tracking analysis (NTA) (NanoSight, Amesbury, UK) to obtain the concentration and size distribution of exosomes [72]. Samples were diluted with 1,000 µl PBS. Three hundred µl sample was acquired into the chamber of NTA unit (NanoSight, Amesbury, UK). The image data was analyzed with NTA 2.3 software (NanoSight, Amesbury, UK).
5.13 Transwell Experiment
To analysis exosome interaction between cells, a coculture system was used to mimic the cellular environment, the detailed protocol was described previously [73]. 4×104 HaCaT cells were first cultured in the upper chamber of the 0.4 µm pores Transwell (Corning #3413). Then cells membrane was labeled with DioC18 dye (5 µg/ml; Invitrogen, D275) at 37°C, 30 min. After staining, the cells were washed three times with PBS to remove excess dye. After the indication treatment of HaCaT cells. The HaCaT cells were cocultured with DioC18 unstained THP-1 cells (4×105 cells/well, 100 ng/ml PMA differentiated) in two separate compartments of the system. DioC18-labeled exosomes from upper compartment cells were uptaken by the THP-1 cells in the lower compartment, the exosomes uptake rate was observed by fluorescence microscopy and quantified by flow cytometry analysis.
5.14 Real-Time Quantitative Polymerase Chain Reaction (qPCR)
THP-1 cells were exposed to 10 or 12.5 µg/ml ZnO NPs. After 18 hrs exposure, the RNA was isolated using TOOLSharp RNA Extractor (TTD-NRNA200, BIOTOOLS Co., Ltd., Taipei, Taiwan). RNA concentrations and 260/280 nm ratios were measured by NanoDrop 2000 (Thermo Scientific™, Waltham, MA, USA). Reverse transcription (RT) was performed using TOOLSQuant II Fast RT Kit (KRT-BA06-2, BIOTOOLS Co., Ltd., Taipei, Taiwan). Finally, a mixture of the synthesized cDNA, primers and TOOLS 2 × SYBR qPCR Mix (FPT-BB05, BIOTOOLS Co., Ltd., Taipei, Taiwan) was carried out in a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster, CA, USA) using SYBR Green Master Mix (Thermo Fisher Scientific). The average of the ΔΔC(t) of triplicate samples was calculated using β-actin as a housekeeping gene. The sequences of forward primers and reverse primers were summarized as described under Supplementary information.
5.15 Western Blot Analysis
Cell or tissue lysates were extracted in lysis buffer. The protein concentration was determined by BCA assay. About 20 µg of protein extract was separated on 6–15% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Merck Millipore, Darmstadt, Germany). After 1 hr blocking procedure, the target proteins were probed by corresponding primary antibodies in 1:1000 dilution. The information and resources of antibodies are provided in supplementary information. The membrane was washed with 1×TBST after primary antibody hybridization, target proteins were reacted with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies in 1:10000 dilution. The immunoreactive proteins were visualized with chemiluminescence HRP substrate (Merck, USA) and filmed with BioMax LightFilm (Eastman Kodak Company, New Heaven, CT, USA). The band intensities were quantified by ImageJ software.
5.16 Immunoprecipitation
After treatment with ZnONPs for indicated time points, THP-1-derived macrophage were lysed and the soluble fraction was incubated overnight at 4°C with an antibody to anti-ASC (ABclonal Inc., Boston, MA, USA) and anti-NLRP3 (Cell Signaling, Beverly, MA, USA); then, A/G agarose beads (Merck Millipore, MA, USA) were mixed with the solution for further incubation for 1 hr at 4°C. The beads/pellets were washed three times with the protein extraction buffer, boiled in SDS sample buffer for 5 min, and centrifuged. The supernatant was subjected to western blot analysis using the indicated antibodies performed as described above.
5.17 Detecting cytosolic release of lysosomal proteases
The HaCaT cells were exposed to ZnONPs (12.5 µg/ml) or UVB (68 mJ/cm2) for indicated times. To separate the cytosolic fraction (lysosome-free) from HaCaT cells, we used proper volume cold hypotonic buffer (10 mM HEPES − NaOH (pH 7.9), containing 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT, 1× protease inhibitor, 1× phosphatase inhibitor) to dissolve the HaCaT cell pellets. The cell suspensions were incubated on ice for 30 min, mixing for 10 sec every 5 min. After centrifugation at 400 × g for 10 min at 4°C, the supernatants were collected as cytosolic fractions. To extract the membrane fractions, (containing lysosomes and mitochondria) the cold MSC-RIPA buffer (20 mM Tris − HCl (pH 7.4) containing 100 mM NaCl, 6.05 M EG, 0.5% SDC, 0.05% SLS, 1× protease inhibitor, 1× phosphatase inhibitor) was added to the pellets and 4°C incubation for 30 min. After centrifugation at 400 × g for 10 min at 4°C, the supernatants were collected as membrane fractions. Then, the samples were subjected to western blot analysis using the Cathepsin B (1:1000), COX IV (1:5000), and LAMP-1 (1:1000) antibodies performed as described above.
5.18 Statistical analysis
SigmaPlot 10.0 (Systat Software, Inc., USA) was used to analyze the data and the results were presented as mean ± standard deviations (SD). The differences between two groups or multiple groups were evaluated using a two-sample t-test or one-way analysis of variance with a post hoc Dunnett’s multiple comparison test, respectively. P value < 0.05 was considered statistically significant difference.
Additional materials and methods are available in Supporting information.