Preparation and characterization analysis
Chemicals. Cesium bromide (CsBr, 99.999%), lead bromide (PbBr2, 99.999%), tetraethoxysilane (TEOS, 98%), and N, N-dimethylformamide (DMF, 99.9%) were provided by Alfa Aesar. Oleic acid (OA, 90% tech), oleylamine (OAm), and toluene (99.95%) were obtained from Sigma-Aldrich. A 4% paraformaldehyde solution was provided by Boster Biological Technology Co., Ltd. BV-III grade Nitric acid (HNO3, 70%), and ethanol were obtained from Beijing Chemical Corporation. All chemicals were used directly.
Preparation of CsPbBr3and CsPbBr3-SiO2nanoparticles. The 734 mg of PbBr2, 425 mg of CsBr, 3 mL of OAm, and 9 mL of OA were added into 50 mL of DMF, stirring at 1200 rpm and heating at 110 °C. After 3 h, 1 mL ammonia solution (2.8%) was dropwise added to obtain a clear precursor solution. Moreover, 20 mL of the above solution was quickly added into toluene. And the solution turned green, indicating the formation of CsPbBr3 nanoparticles. Additionally, 20 mL of the precursor solution was quickly added into toluene containing 200 µL of TEOS. CsPbBr3-SiO2 nanoparticles were obtained by stirring vigorously for 10 s and stirring at 150 rpm for 2 h. The CsPbBr3 nanoparticles or CsPbBr3-SiO2 nanoparticles were washed three times with toluene, centrifugated at 12,000 rpm, and stored in a vacuum box for further experimentation.
Characterizations. Transmission electron microscope (TEM) images were obtained by a JEM-2100plus TEM. Powder X-ray diffraction (XRD) analysis was carried out by a Bruker D8 advance diffractometer with Cu Kα radiation (λ = 1.5418 Å). Energy-dispersive X-ray spectroscopy (EDS, Horiba 7593-H model) in conjunction with a field-emission scanning electron microscope (FE-SEM, S-4800, Hitachi High Technologies, Japan) was used to determine the composition and morphology of the samples. X-ray photoelectron spectroscopy (XPS) analysis was collected on an ESCALab220i-XL. Fourier transform infrared (FTIR) spectra were recorded with a Bruker Vertex 70 spectrometer.
hESCs culturing and generation of hEROs
The human embryonic stem cell (hESC) line (H9) used was kindly provided by the Stem Cell Bank, Chinese Academy of Sciences (CAS). hESCs were cultured in feeder-free Essential 8™ Culture Medium (A1517001, Gibco). The generation of the retina organoid followed Kuwahara’s protocol [28] with slight modifications as described in our earlier study [29]. Briefly, confluent hESCs were dissociated into a single-cell suspension using TrypLE™ Express (12605028, Gibco). hESCs (1.2 × 104, 100µL) were forced to aggregate into embryoid body (EB) formation in low-adhesion V-shaped bottom 96-well plates (MS-9096VZ, Sumitomo Bakelite) in growth factor-free chemically defined medium (gfCDM) supplemented with 10% KnockOut™ Serum Replacement (KSR) (A3181502, Gibco), 20µM Y-27632 (the ROCK inhibitor) (Sigma), and 1% penicillin-streptomycin (SV30010, Hyclone). gfCDM contains 45% Iscove’s modified Dulbecco’s medium (IMDM) (12440053, Gibco), 45% Ham’s F12-Glutamax (31765035, Gibco), 1% Chemically Defined Lipid Concentrate (Gibco), and monothioglycerol (450 µM) (Sigma). After five-day induction, the culture medium was exchanged for fresh gfCDM (without Y-27632) supplemented with 1.5 nM bone morphogenetic protein 4 (BMP4) (Peprotech, United States). After this, half of the gfCDM was changed every three days. On day 18, hEROs were transferred to an ultra-low attachment dish (752001, NEST) for further culture with long-term culture medium containing Dulbecco’s modified Eagle’s medium (DMEM)/F12 (10565018, Gibco), 1% N2 supplement (A1370701, Gibco), 10% fetal bovine serum (FBS), (35–081, Corning), 0.5 µM retinoic acid (RA) (R2625, Sigma), 0.1 mM taurine (T8691, Sigma), and 1% penicillin-streptomycin (SV30010, Hyclone).
Preparation of exposed solution and determination of actual concentration
Recent studies have shown that the blood lead levels of pregnant women range from 0.023 to 0.348 µg/ml, and umbilical cord blood levels range from 0.021 to 0.45 µg/ml [30]. Furthermore, stored lead in the mother’s body during pregnancy can be released into the bloodstream and passed on to the fetus [31]. Given this, we have prepared the following solution. Pb(AC)2 (CAS: 6080-56-4, L812500, MACKLIN, China), CsPbBr3, and CsPbBr3-SiO2 nanoparticles were prepared at a stock concentration of 100 mg/ml in dimethyl sulfoxide (DMSO, D8371, Solarbio, China). A diluted concentration of 25 µg/ml, 50 µg/ml and 100 µg/ml was added to the medium. Then, we detected the working concentration of Pb2+ in the culture medium using ICP-MS, and the specific data can be found in Supplementary Fig. 2. The ICP-MS data showed that the actual Pb2+ concentration of the preset low-concentration CsPbBr3 nanoparticles group is approximately 0.12 µg/ml, which is at the physiological high value of maternal blood lead levels.
Materials exposure experiments
Following the hEROs were cultured in long-term culture medium (D18), they were assigned to four groups: (i) the control group, (ii) the low-concentration CsPbBr3 nanoparticle group (actual lead content about 0.12 µg/ml) (CPBL), (iii) the middle-concentration CsPbBr3 nanoparticle group (actual lead content about 0.30 µg/ml) (CPBM), (iv) the high- concentration CsPbBr3 nanoparticle group (actual lead content about 0.57 µg/ml) (CPBH) for toxicity assays. In this study, hEROs were also exposed to different chemicals with the same low preset concentration, including CsPbBr3-SiO2 (actual lead content about 0.06 µg/ml), and Pb(AC)2 (actual lead content about 0.23 µg/ml), respectively. Detailed measurement data can be found in the supplementary materials (Fig. S2). To ensure the accuracy of the concentrations used, medium and corresponding concentration nanoparticles and chemicals were refreshed every three days.
hEROs morphological observations and analysis
The morphology and growth of the hEROs were observed and photographed weekly by a Leica DMI3000 B inverted microscope (Leica, Germany). Following the nanoparticle exposure periods, the morphological changes in the neural retina (NR) of hEROs were identified and quantified. We have referenced previously published articles for measuring the area and thickness of the NR [26,32]. The specific method for measuring the area and thickness of the NR is as follows: in the hERO images obtained with the microscope, the area of the NR for each hERO was measured by using ImageJ(64-bit) software (NIH, Bethesda, MD, United States). Moreover, we measured the thickness of the NR at ten points (distributed as evenly as possible) in the hEROs of the control and experimental groups, ensuring that measurements were not taken at the ciliary edge of the NR. Then, the average of these ten measurements was calculated to obtain the thickness of the NR.
Immunofluorescence staining
Immunostaining of hEROs was consistent with the description in our previous study [32]. hEROs were collected and fixed with 4% paraformaldehyde (P0099, Beyotime) at 4 °C for 20 min, followed by dehydration in 30% sucrose (1245GR500, Biofroxx) at 4 °C. hEROs were then embedded in optimal cutting temperature (OCT) compound (Sakura FineTek, Torrance, CA, United States) and transferred into − 80 °C refrigerator. hEROs were cut on a freezing microtome (Leica CM1900UV, Germany) into 12 μm thick sections and mounted onto glass slides. Frozen sections were then stored at -20 °C in a refrigerator until immunostaining. Slides were incubated in PBS at 37 °C for 20 min to remove OCT. Slides were then permeabilized in 0.5% Triton X-100 for 15 min at room temperature. Nonspecific sites were blocked with 3% bovine serum albumin (BSA) in a moist chamber at 37 °C for 30 min. Slides were then sequentially incubated with primary antibodies (see Table S1) at 4 °C overnight. The following day, sections were incubated for 60 min at 37 °C with the appropriate Alexa Fluor dye-conjugated secondary antibodies, including Goat anti-mouse IgG Alexa-Fluor-488 (A11001, Life technologies, 1:400), Goat anti-mouse IgG Alexa-Fluor-568 (A11031, Life technologies, 1:500), Goat anti-rabbit IgG Alexa-Fluor-488 (A11008, Life technologies, 1:400), or Goat anti-rabbit IgG Alexa-Fluor-568 (A11011, Life technologies, 1:500), following washing the sections with 1 × PBS three times every 10 min. Finally, DAPI (C1006, Beyotime) dye was applied for nuclear counterstaining for 10 min at room temperature and coverslips were mounted with anti-fluorescence quenching sealer (P0126, Beyotime). Zeiss inspected and photographed the slides using an LSM880/LSM780 confocal microscope, and the images were analyzed using the Zen 2012 software version.
TUNEL assays
After three weeks of exposure to nanoparticles, samples from both the control and exposure groups were collected and prepared for frozen sections. Apoptosis of neural layer cells was assessed using a TUNEL kit (Roche, Switzerland). The end-labeling enzyme and labeling liquid were mixed at a ratio of 1:9 according to the instructions, after which the sections were incubated with the sections at 37 °C protected from light for one hour, followed by incubation with DAPI for 10 min. Photographs were taken using a laser confocal microscope (Zeiss, LSM880 and LSM780). The images were then analyzed using Zen 2012.
Biological-transmission electron microscopy (Bio-TEM) analysis
hEROs were exposed to Pb(AC)2, CsPbBr3, and CsPbBr3-SiO2 nanoparticles for three weeks. Meanwhile, the normal cultured hEROs served as the control group. On Day 39, the samples from each group were collected. Subsequently, they were washed three times with PBS and then fixed with 2.5% glutaraldehyde. They were further fixed using osmium acid, dehydrated, embedded, sectioned, and stained. The prepared samples were then examined using an electron microscope (Hitachi HT7800) to observe the biodistribution of nanomaterials and the intracellular structure of each group of cells.
Two-dimensional element distribution detection of organoids slices
The LA-ICP-TOFMS technique analyzed the two-dimensional elemental distribution in hERO slices. Following various treatments, the hEROs were embedded in the OCT on a freezing stage, and frozen sections (12 μm) were obtained using a freezing microtome. Next, frozen sections of the hEROs were used for LA-ICP-TOFMS detection. The LA-ICP-TOFMS system consists of a Laser Ablation System (Iridia Bio, Teledyne Photon Machines) and an Inductively Coupled Plasma Time-of-Flight Mass Spectrometry system (icpTOF2R, TOFWERK).
Inductively coupled plasma-mass spectrometry (ICP-MS)
Pb, Br, and Cs concentrations of the day 39 hEROs were determined by ICP-MS. Briefly, the samples were collected and subjected to microwave digestion (Touchwin 2.0) using pure nitric acid, followed by heating at 180 °C for 30 min to drive off the acid. The concentration of the relevant elements was measured on the inductively coupled plasma mass spectrometer (NexION300D, PE, U.S.) after filtering the impurities using a 0.22 μm filter membrane.
RNA sequencing
To comprehensively evaluate the toxic effects of CsPbBr3 on retinal development, we conducted RNA sequencing (RNA-seq) to identify gene profile changes in hEROs after three weeks of CsPbBr3 and Pb(AC)2 treatment. RNA was extracted from hEROs with TRIzol reagent (Invitrogen, CA, United States) and then was quantified using a NanoDrop (DE, United States) and was qualified using Agilent 2100 bioanalyzer (CA, United States). The PrimeScript RT Reagent Kit (Takara, Japan) was used to perform reverse transcription following the manufacturer’s instructions and isolated using Oligo (dT)-attached magnetic beads. After reverse transcription, cDNA fragments were amplified by PCR. The expression of each gene was measured by Fragments Per Kilobase of exon per Million fragments mapped (FPKM). Genes with P-value < 0.05 and fold change > 1.2 or < 0.67 were defined as differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were carried out to annotate the unique biological significance and important pathways of DEGs on the Majorbio platform (https://cloud.majorbio.com). Gene Set Enrichment Analysis (GSEA) pathway analysis after CsPbBr3 nanoparticles exposure was also performed using the Majorbio platform (https://cloud.majorbio.com).
Quantitative real-time PCR
To analyze the mRNA levels of the involved genes, total RNA was extracted from ten hEROs of the various groups as per the instructions of the manufacturer utilizing TRIzol reagent (15596026, Invitrogen). The total RNA of hEROs was extracted as described above and then reverse-transcribed into cDNA using the PrimeScript RT Reagent Kit (RR037A, Takara, Japan) following the manufacturer’s protocol. These cDNAs were then amplified with specific gene primers (see Table S2), and quantitative real-time PCR (qRT-PCR) reactions were executed using SYBR® Premix Ex Taq™ II (Takara, Japan) with a CFX96 Real-Time PCR System (Bio-Rad, United States). The GAPDH gene was used to normalize the expression of various genes.
Statistical analysis
All results were obtained from at least three independent experiments in this study. We conducted all statistical analyses using GraphPad 8.0.432 (San Diego, CA). A One-way analysis of variance (ANOVA) followed by multiple Tukey comparisons, was used to evaluate the differences. Measurement data are presented as the mean ± SEM. Group differences between groups were considered statistically significant at: * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.
