Journal of Medicinal Chemistry

Engineered Brain Organoids on a 3D Scaffold Unveil Synaptic Toxicity of Benzophenone-3 Mediated by Wnt/βcatenin Dysregulation

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Abstract

Benzophenone-3 (BP-3), a common Ultraviolet (UV) filter used in sunscreens, has poorly understood neurotoxic effects, and early  life exposure may disrupt normal neurodevelopment. In this study, we fabricated a 3D inverse opal scaffold using Polyethylene   Glycol Diacrylate (PEGDA) hydrogel. The scaffold structure was characterized using Scanning Electron Microscopy (SEM), and its   biocompatibility was validated. The organoids were exposed to BP-3 and the results demonstrate that BP-3 induces concentration-   and time-dependent toxicity, disrupts neural activity, promotes oxidative stress, and compromises cell membrane integrity. Prenatal   BP-3 exposure may impair early brain development via dysregulation of the Wnt/β-catenin pathway, and synaptic integrity is   proposed as a potential early biomarker for BP-3-induced neurodevelopmental toxicity. This study successfully established a uniform   and biocompatible 3D scaffold supporting brain organoid formation, providing a robust platform for assessing BP-3-mediated   neurotoxicity. These findings enhance the understanding of BP-3’s neurotoxic mechanisms and contribute to the development of   preventive and therapeutic strategies.

Keywords

Brain organoid; Inverse opal scaffold; Benzophenone-3; Neurotoxicology; Wnt/β-catenin signaling

INTRODUCTION

Benzophenone-3 (BP-3) is widely utilized as an Ultraviolet (UV) protective ingredient in sunscreens due to its broad-spectrum light absorption, chemical stability, and cost-effectiveness [1,2]. Studies have detected that BP-3 is widely present in maternal biological samples, including maternal serum (limit of detection [LOD] -25.1 ng/mL), umbilical cord serum (LOD -56.6 ng/ mL), breast milk (0.12 to 8.2 ng/mL), placental tissue (limit of detection -5.3 ng/mL), amniotic fluid (limit of detection -11.6 ng/mL) and maternal urine (0.46 1162 ng/mL) [3-6]. Accumulating evidence indicates that BP-3 exhibits endocrine disrupting effects, reproductive and developmental toxicity, and potential for neurotoxicity and carcinogenic effects [7-9]. The selection of BP-3 concentrations was informed by established human exposure data and standardized in vitro toxicological protocols. Epidemiological investigations have demonstrated marked national and age-related disparities in urinary total BP-3 concentrations, with BP-3 molecular weight calculated as 228.25 g/mol. To ensure unit consistency, all concentration data were converted to μM and ng/mL. Specifically, BP-3 concentrations in Chinese children ranged from 1.095×10-3 μM (0.250 ng/mL) to 2.81×10-2 μM (6.41 ng/mL), whereas Chinese adults exhibited concentrations between 7.45×10-4 μM (0.17 ng/mL) and 4.12×10-2 μM (9.39 ng/mL). In contrast, urinary BP-3 levels in American children extended from 7.45×10-4 μM (0.17 ng/mL) up to 3.12 μM (713 ng/mL), while those in adults varied from 2.15×10-3 μM (0.491 ng/mL) to 1.81 μM (413 ng/mL) [10]. Notably, BP-3 has been detected in various environmental matrices, including water, soil, sediments, sludge, and biota. Maximum concentrations of 125 ng/L in freshwater, 577.5 ng/L in seawater, and 10,400 ng/L in wastewater influent have been reported, providing valuable contextual reference for the current experimental design. These findings also emphasize the need to employ relatively higher concentrations in in vitro assays. Such an approach allows subtle toxicological effects to be amplified, thereby providing clearer evidence for elucidating the mechanistic actions of BP-3 [11].

The development of the nervous system is an intricate and finely regulated process, particularly vulnerable to environmental insults during embryonic stages. Studies have demonstrated that BP-3 can cross the blood-brain and placental barriers, enter the central nervous system, accumulating in the prefrontal cortex and hippocampus of rats -brain regions critical for cognition, memory, and emotional regulation [12,13]. Consequently, BP-3 exposure may interfere with normal neural development, leading to long-term deficits in behavior, learning, and memory. Epidemiological and model organism studies (e.g., zebrafish and rodents) have linked BP-3 exposure to impairments in spatial memory, motor function, and social behavior [14-17].

While most existing studies focus primarily on neuronal cells, the role of synaptic structures in BP-3-induced neurodevelopmental disorders remains unclear. Previous studies have shown that developing synapses are sensitive to environmental chemicals, such as citronellol, bisphenol A (BPA), and ammonia (NH3) [18-20]. However, the potential impact of BP-3 exposure on the development of synaptogenesis during fetal development warrants further investigated.

Conventional studies on BP-3 have mainly focused on two dimensional (2D) cell cultures and animal experiments, both of which present significant limitations: animal models raise ethical and cost concerns, while 2D cultures lack physiological spatial organization. In contrast, three-dimensional (3D) cell culture system better mimic the highly complex architecture of neural network and allow for more realistic modeling of brain inflammation [21]. Brain organoids are derived from pluripotent stem cells, can recapitulate multiple brain regions in vitro, including the cerebrum, midbrain, and striatum [22,23]. They have emerged as valuable tools for studying brain development and disease mechanisms [22-24]. As well as for evaluating the impact of environmental factors on the brain. For instance, brain organoids exposed to Zika virus show increased apoptosis and reduced neurogenesis [25,26]. Qin’s team established a series of brain organoid models to investigate the effects of exposure to environmental factors, including heavy metals (e.g., Cadmium [Cd]), alcohol, and Valproic Acid (VPA), on the neural functions [27,28].

Current traditional brain organoid culture methods rely on matrix gel as the core support, which still holds advantages in promoting differentiation maturity and maintaining stable cellular physiological functions. However, these traditional approaches face significant limitations—the high cost of matrix gel itself hinders widespread adoption in resource-constrained research environments and impedes the establishment of standardized culture systems. We first fabricated a biocompatible 3D scaffold using microfluidic technology to support neural cell growth. This novel three-dimensional scaffold culture method effectively addresses the core challenges of traditional matrix gel culture: it eliminates reliance on expensive matrix gels, simplifies operational procedures, significantly reduces cultivation costs and technical barriers, and facilitates broader application across diverse research settings. Preliminary evidence suggests that BP-3 may induce neurological damage via the Wnt/β-catenin signaling pathway. To elucidate the early biological responses and mechanistic basis of BP-3 exposure, this study investigated the role of synaptic structures in BP-3 induced impairments during synaptogenesis. In the present study, brain organoids were then generated on the 3D scaffold and exposed to BP-3 to assess its neurotoxic effects. Our results indicate that BP-3 disrupts the Wnt/β-catenin signaling pathway at environmentally relevant concentrations. This study underscores synaptic damage as an early indicator of BP-3-induced neurodevelopmental toxicity and suggests that synapse-related markers may serve as biomarkers for risk assessment. Firstly, a 3D scaffold was fabricated with good biocompatibility that could support neural cell seeding using a microfluidic device. Subsequently, brain organoids were generated on this 3D scaffold, and the brain organoids were treated with the environmental contaminant BP-3 to explore its neurotoxicity. Our results indicate that BP-3 exposure at concentrations beyond the normal physiological range, particularly those higher than typical environmental or biological levels, disrupts the Wnt/β-catenin signaling pathway. The experimental framework relied on an in vitro high-dose exposure model. The rationale for using this model is that daily low-dose exposure produces cellular effects that are typically subtle and not easily detectable through conventional analytical techniques. By contrast, elevated concentrations enhance toxicological responses, rendering phenomena such as metabolic disturbances, signaling abnormalities, and gene expression imbalances more readily observable. This enables clearer elucidation of the molecular mechanisms underlying its toxicity. In human exposure scenarios, BP-3 contact usually occurs in a chronic, low-dose, and repetitive manner, such as through daily application of sunscreens containing the compound. Nevertheless, assessing the risks of such exposure through short term epidemiological studies remains challenging. Potential health effects may take decades to manifest, while longitudinal studies are constrained by high financial and temporal demands. In this context, the in vitro high-dose exposure model is of considerable value. Accordingly, this strategy offers critical insights into the potential long-term health risks associated with BP-3.

MATERIALS AND METHODS

Materials

Poly (Ethylene Glycol) Diacrylate (PEGDA) was purchased from Sigma-Aldrich, USA. 2-Hydroxy-2-Methylpropiophenone (HMPP) and F108 were purchased from Shanghai Sangong Bioengineering Co., Ltd. Sodium Dodecyl Sulfate (SDS) and BP-3 were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The live-dead staining reagent, TUNEL assay reagent, Hoechst stain, ROS assay reagent, and Fluo-3AM assay reagent were purchased from Biyuntian Biotechnology Co., Ltd. LDH assay reagent was purchased from Wuhan Sevier Biotechnology Co., Ltd. The primary antibodies used in the present study were as follows: rabbit anti-SOX2 (1:200; Sangong Biotech Co., Ltd., Shanghai, China), rabbit anti-TBR1 and anti CTIP2 (1:400; Boster Biological Technology, Wuhan, China), rabbit anti-Wnt5a, anti-β-catenin, anti-TCF7L2, anti-GSK-3β, anti-Synapsin (anti-Syn), and anti-PSD95 (1:500; ProteinTech Group, Hubei, China). The secondary antibodies used in this study were those labeled with Cy3 (red fluorescence) and FITC (green fluorescence) (1:800; ProteinTech Group, Hubei, China). Cell culture reagents were purchased from Vicente Biotechnology Co. HT22 cells were obtained from the Cell Bank of the Henan University of Science and Technology, Luoyang, China.

Preparation of the microfluidic device

Glass capillaries (0.58 mm inner diameter) were sharpened using a P-2000 microelectrode puller (Sutter Instrument, Novato, CA, USA) and polished to the target diameter (≤100 μm) with abrasive paper. Microscope slides were trimmed, lint-free cleaned, and then used as substrates for adhesive bonding of the conduits. Capillaries of identical specifications were inserted from both ends for laminated assembly. Excess capillary segments were excised, the interfaces were reinforced with needles, and the f inal devices were stored in a dry, well-ventilated environment.

Fabrication of the hydrogel scaffolds

A capillary microfluidic system was used to generate single emulsion templates, with methyl silicone oil (50 cSt; inner phase, flow rate: 1 mL/h) and a 60% PEGDA premix (outer phase, flow rate: 12 mL/h) as the dispersed and continuous phases, respectively. Both phases were delivered using Harvard PHD 2000 syringe pumps. The emulsions were transferred to centrifuge tubes for 1 minute of self-assembly to form a pre-gel, followed by 10 seconds of UV curing to solidify the hydrogels. Porous PEGDA scaffolds were obtained by sequentially immersing the hydrogels in graded ethanol solutions to remove emulsion droplets; the resulting scaffolds were then washed with deionizedwater and stored at 4 °C.

Isolation of primary neural cells

Sprague-Dawley (SD) rat suckling pups (postnatal day 0, P0) born within 24 hours were selected. Under ice-cold conditions, whole brain tissues were rapidly isolated; subsequently, the brain cortex was carefully dissected, and brain vascular tissues were completely removed. The cortical tissues were then minced into 1 mm³ tissue blocks. The digested tissue suspension was filtered through a 70 μm cell strainer, and the filtrate was collected and centrifuged at 1000 rpm for 5 minutes. After discarding the supernatant, the cell pellet was resuspended in complete medium to obtain dissociated cortical neuronal cells. These neurons were seeded onto 3D scaffolds at a density of 3×10⁶ cells/mL (with DMEM/F12 used as the seeding medium). The medium was replaced with fresh medium 12 hours later.

Cell culture

DMEM/F12 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics (penicillin-streptomycin) was used for cell culture. For the generation of 3D cellular spheroids, the fabricated PEGDA scaffolds were sequentially cleaned with alcohol and PBS, followed by overnight UV irradiation to support surface sterilization. HT22 cells were enzymatically detached, centrifuged, and resuspended to a density of 3 × 10⁶ cells/mL. Each PEGDA scaffold was transferred to a 24-well culture plate, and 1 mL of the cell suspension was carefully pipetted into each well. The plate was gently agitated to ensure uniform cell distribution across the scaffolds. The culture system was then maintained in a controlled environment (37 °C, 5% CO₂) to support cellular proliferation.

Biocompatibility of 3D scaffolds

3D scaffolds seeded with HT22 cells were cultured at 37 °C in a 5% CO2 atmosphere for 7 days, with periodic observation of cell growth and spheroid formation in the wells. Prior to staining, the culture medium was aspirated, and a freshly prepared Calcein-AM/PI double-staining working solution was added. The working solution was prepared by mixing 5 μL Calcein-AM solution and 5 μL of PI solution into 5 mL PBS. The samples were incubated at 37 °C for 20 minutes under light protection. Subsequently, the staining solution was removed, and the scaffolds were rinsed twice with pre-cooled PBS buffer; finally, an appropriate volume of PBS was added to maintain scaffold hydration. Throughout the procedure, tin foil was used for light protection to prevent fluorescence quenching. Immediately after staining, the growth status and viability of cell spheroids were observed and documented using a fluorescence microscope.

Immunofluorescence

The sample preparation and immunofluorescence staining procedures were performed as follows. First, the culture medium in the well plate was discarded, and the samples were gently rinsed once with PBS. Then, pre-cooled 4% paraformaldehyde solution was added, and the samples were fixed at 4 ℃ for 2 hours. The sample were then rinsed three times with PBS (5 minutes per rinse), with thorough removal of residual liquid after each wash. Subsequently, permeabilization was performed using 0.25% Triton X-100 at room temperature for 10 minutes. After three additional PBS rinses, 5% Bovine Serum Albumin (BSA) blocking solution was added, and the samples were incubated at room temperature for 2 hours to achieve blocking. The samples were then incubated with the primary antibody at 4 ℃ overnight. Following this, the samples were rinsed three times with PBS and incubated with the fluorescent secondary antibody at room temperature for 2 hours. After three additional PBS rinses, the cell nuclei were stained with Hoechst 33258 for 15 minutes. Finally, the samples were rinsed three times with PBS, and observed and imaged using a confocal laser scanning microscope (CLSM, Nikon C2+, Nikon, Janpan). Notably, all the above operations were performed under light-protected conditions.

Cell viability determination

To assess the cell viability status of brain organoids across different experimental groups, Calcein-AM/PI live-dead cell staining assay was performed. After 7 days of culture, the brain organoids were grouped according to different concentrations of BP-3 and treatment durations, including 6-, 12-, 24-, 36-, 48-, and 72-hour groups. Each time-point group was further subdivided into 0 μM, 10 μM, 25 μM, 50 μM, and 100 μM BP-3 subgroups. The low-dose groups (10 μM and 25 μM) were defined in reference to documented human exposure levels and standardized guidelines for in vitro toxicological research. This design accounted for the possibility of local accumulation in target tissues, where concentrations could surpass those in systemic circulation. The high-dose groups (50 μM and 100 μM) were set to simulate potential biological effects of prolonged low-dose exposure within the temporal limitations of short-term in vitro models. Following the addition of BP-3 at specified concentrations, the organoids were cultured at 37 °C in a 5% CO₂ atmosphere. Upon reaching the target treatment duration, the medium was aspirated, and the organoids were rinsed three times with PBS. After removing residual PBS, the prepared Calcein-AM/PI live-dead double-staining working solution was added, and the samples were incubated for 20 minutes at 37 °C in a 5% CO₂ incubator under light protection. The staining solution was then removed, and the organoids were rinsed three times with PBS. A small volume of PBS was added to maintain hydration of the 3D scaffolds before observation using a laser confocal microscope.

TUNEL staining

To detect cell apoptosis in 3D scaffolds, this study used the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method. The specific steps are as follows: The medium was aspirated, and the samples were rinsed three times with PBS, followed by fixation with 4% paraformaldehyde for 30 minutes. After removing the paraformaldehyde solution, the samples were rinsed three times with PBS, then 0.25% Triton X-100 was added for a 5-minute incubation at room temperature. The Triton X-100 solution was discarded, and after three additional PBS rinses, the prepared TUNEL solution was added. The samples were incubated at 37 °C for 30 minutes under light protection. Following removal of the TUNEL solution and three PBS rinses, Hoechst 33258 was added for a further 10-minute light-protected incubation. Upon completion of staining, the solution was discarded, and the samples were rinsed twice with PBS. An appropriate volume of PBS was then added to maintain hydration of the 3D scaffolds before observation using a laser confocal microscope.

Calcium content detection

The calcium ion fluorescent probe Fluo-3 AM was used to detect changes in calcium levels in brain organoids across different groups, aiming to evaluate the effect of BP-3 on calcium content in these organoids. The results of the cell viability experiment showed that 50 μM and 100 μM BP-3 induced significant differences in cell viability compared to the control group, so these two concentrations were selected for subsequent experiments. After 7 days of culture, the brain organoids were divided into three groups (0 μM, 50 μM, and 100 μM BP-3) and treated for 72 hours. The culture medium was aspirated, and the Fluo-3 AM probe was diluted 1:1000 in PBS to a final concentration of 5 μM. Each sample was incubated with 300 μL of the prepared Fluo-3 AM solution in a 37 °C, 5% CO₂ incubator for 10 minutes. Following incubation, the working solution was removed, and the samples were rinsed three times with PBS. An appropriate volume of PBS was added to maintain hydration of the 3D scaffolds. Observations were then performed using a laser confocal microscope with a 488 nm excitation wavelength and 525 nm emission wavelength.

Reactive Oxygen Species (ROS) assay

After 7 days of culture, the brain organoids were divided into three groups and treated with 0 μM, 50 μM, and 100 μM BP-3 respectively, followed by incubation in a 37 ℃, 5% CO₂ incubator for 72 hours. The DCFH-DA fluorescent probe was used to detect ROS production in brain organoids across groups, aiming to verify the damaging effects of BP-3 on these organoids. DCFH-DA was diluted 1:1000 in PBS to a final concentration of 10 μM. Each sample was incubated with 300 μL of the prepared DCFH-DA solution at 37 °C in a 5% CO₂ incubator for 20 minutes. Following incubation, the working solution was removed, and the samples were rinsed three times with PBS. An appropriate volume of PBS was added to maintain hydration of the 3D scaffolds before observation using a laser confocal microscope with a 488 nm excitation wavelength and 525 nm emission wavelength.

Lactate Dehydrogenase (LDH) release assay

After 7 days of culture, the brain organoids were divided into three groups, which were treated with different concentrations (0 μM, 50 μM, and 100 μM) for a predetermined duration of 72 hours. Upon reaching the preset treatment time, the original medium was removed, and the brain organoids were rinsed once with PBS. Subsequently, 120 μL of cell lysate was added to each group, and the organoids were incubated in a 37 °C, 5% CO₂ incubator for 60 minutes. After incubation, the lysate supernatant was aspirated into a centrifuge tube and centrifuged at 1000 r/ min for 5 minutes. Thereafter, 80 μL of the centrifuged lysate supernatant was transferred to each well of a 96-well plate, and 80 μL of LDH detection solution was added to each sample well, resulting in a 1:1 volume ratio between the sample and the LDH assay working solution. After adding the detection solution, the mixture was thoroughly mixed and incubated in the dark for 30 minutes. Finally, an enzyme-labeled instrument (Model: EPOCH, BioTek Instruments, Inc. [Boteng Instrument Co., Ltd.], Winooski, VT, USA) was used to measure the absorbance of each well at a wavelength of 490 nm.

Western blotting

Western blotting was employed to determine the expression levels of key proteins in the Wnt/β-catenin signaling pathway (PSD95, GSK-3β, Syn, TCF7L2, Wnt5a, and β-catenin) in HT22 cells. The experiment consisted of three groups, with the 0 μM BP-3 group serving as the control, and the other two groups treated with BP-3 at concentrations of 50 μM and 100 μM, respectively. HT22 cells were seeded in 6-well plates at a density of 3×10⁵ cells/well. Following treatment with BP-3 at the aforementioned concentrations for 72 hours, protein extraction was performed. To ensure result reliability, each group underwent 3 independent protein extractions (biological replicates), and each protein sample was subjected to 3 technical replicate assays. After protein extraction, the protein samples were mixed with loading buffer at a 4:1 ratio and boiled at 100 °C for 10 minutes to induce protein denaturation. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) was then conducted for 1.5 hours, after which the proteins were transferred onto a nitrocellulose membrane. The membrane was first blocked, then incubated overnight at 4°C with specific primary antibodies. On the subsequent day, the membrane was washed with TBST buffer and incubated with the corresponding secondary antibodies at room temperature for 1 hour. Finally, images were captured using an automatic chemiluminescence image analyzer (Shanghai Tanno Technology Co., Ltd.), and consistent trends were observed across three independent replicate experiments.

Statistical assay

Image data were processed and analyzed using ImageJ software. All experimental data are presented as mean ± standard deviation (x̄±s). Statistical analyses were performed using GraphPad Prism 5.4.1 software. Multiple group comparisons were analyzed by one-way analysis of variance (ANOVA), and graphs were generated using GraphPad Prism 5.4.1. Statistical significance was defined as follows: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, and non-significant (ns) as P>0.05.

RESULTS

Preparation, characterization and biocompatibility of the PEGDA hydrogel scaffolds

The PEGDA scaffold was prepared with a microfluidic device according to our group’s previously reported method [29]. By optimizing critical microfluidic operating parameters, including the flow rates of the aqueous Polyethylene Glycol Diacrylate (PEGDA) precursor phase and the oil phase (for droplet generation), the temperature of the chip channel (regulated to stabilize fluid viscosity), and the inlet pressure (controlled to ensure consistent fluid shear forces), the continuous production of monodisperse droplets was successfully realized within the microchannels of the microfluidic chip. Owing to their uniform size, the droplets were capable of self-assembling into a multilayer structure in the PEGDA precursor solution. Taking advantage of this property, the PEGDA scaffold with an inverse-opal structure was obtained by crosslinking the PEGDA precursor solution under ultraviolet radiation and subsequently removing the oil droplets via washing (Figure 1A). As observed under Scanning Electron Microscopy (SEM), the PEGDA scaffold had a highly ordered 3D porous structure with a uniform pore size distribution. (Figure 1B), and the pore diameter of the scaffold was approximately 250 μm (Figure 1B), which facilitating cell growth and formation of cell spheres. To evaluate the biocompatibility of the PEGDA scaffold, HT22 cells were seeded in the PEGDA scaffolds. The viability of cell spheroids was dynamically monitored over a 9-day in vitro culture period. The results showed that HT22 cells exhibited typical 3D growth characteristics on the day 3 and day 9 (Figure 1C). The above results confirmed that the scaffold possessed excellent biocompatibility, which provided a reliable platform for subsequent studies (Figure 1).

Figure 1: Fabrication, characterization, and biocompatibility of the inverse-opal structured scaffold. (A) Micrograph of the self-assembly process of droplet templates, including three key stages: (a) polymerization, (b) solidification, and (c) elution. (B) SEM image and pore size analysis of the dehydrated inverse-opal structured scaffold: (a) Low-magnification SEM image showing the overall morphology of the scaffold; (b) High-magnification SEM image displaying the partial microstructural details; (c) Pore diameter distribution diagram of the 3D scaffold. (C) Evaluation of cell viability and survival rate during in vitro culture at different time points: (a) Cell viability status at 3 days of culture; (b) Cell viability status at 9 days of culture; (c) Quantitative analysis of cell survival rate at different culture times based on immunofluorescence assay, with the results expressed as the absolute value of fluorescence intensity.

Characterization of brain organoids on 3D scaffolds

The constructed brain organoids were identified with the neuroepithelial developmental marker SOX2. Immunofluorescence staining showed that SOX2+ neural precursor cells exhibited regional enrichment in the intermediate zone of the organoid’s central axis region (Figure 2A). This spatially specific expression pattern is consistent with the characteristics of the dorsal cortex in early telencephalic development, suggesting that the in vitro 3D culture system has successfully recapitulated the radial lamellar organization process of the neuroepithelium. Notably, the morphogenesis of cortical structures is the most significant cross-species differentiating feature in the evolutionary history of the mammalian brain. In order to resolve this key node of evolution, the forebrain specific marker TBR1 and cortical deep neuron marker CTIP2 were used to systematically evaluate the cortical developmental features of brain organoids. As shown in Figure 2B and 2C, in brain organoids cultured for up to day 14, the TBR1+ anterior plate region and CTIP2+ neuron-enriched region were clearly observed, indicating that the cortical plate had already developed initial hierarchical structural characteristics at this stage (Figure 2).

Figure 2: Identification of specific proteins in brain organoids. (A) SOX2 was used to detect the neuroepithelial development marker (specific for neural progenitor cells) in cellular structures (red). Hoechst 33258 was used to label the nuclei (blue). (B) TBR1 was utilized to identify the forebrain-specific marker in cellular structures (red). Hoechst 33258 was used to label the nucleus (blue). (C) CTIP2 was employed to detect the cortical deep-layer neuron marker in cellular structures (red). Hoechst 33258 was used to label the nucleus (blue). “Merge” represents the superimposed images of the respective protein staining and nuclear staining. Scale bar: 100 μm.

Cell viability determination

In order to systematically evaluate the neurotoxicity characteristics of BP-3 exposure on brain organoids as well as select the appropriate doses and exposure time, Calcein-AM/ PI staining was performed. Gradient concentrations (0, 10, 25, 50, 100 μM) with multiple time points (6, 12, 24, 36, 48, 72 h) were set to conduct dynamic monitoring of viability of HT22 cell (See the Supporting Information for more details). Notably, when the exposure time was extended to 72 h, the high concentration group (50/100 μM) exhibited dose-dependent growth inhibition (Figure 3A, 3B). The data indicated that as the concentration of BP-3 increased and the exposure time was prolonged, the survival rate of brain organoids gradually decreased. At low concentrations (10 μM, 25 μM) and shorter treatment times (6-48 h), the decrease in survival rate was not significant; however, at high concentrations (50 μM, 100 μM) and with an action time of 72 h, the survival rate significantly decreased, indicating that the toxicity of BP-3 to the organoids was both concentration and time-dependent. Therefore, we selected 50/100 μM-72 h as the standard exposure parameter to provide a reliable toxicological model for subsequent research on the mechanism of neurodevelopmental disorders. We further constructed a brain organoid model using a 3D culture system of primary neural cells and conducted validation experiments using the established neurotoxicity exposure parameter (50/100 μM-72 h). Calcein-AM/PI staining showed that the viable cell density in the experimental group was significantly lower than that in the control group, a result consistent with the toxicity response observed in the neurosphere model (Figure 3C, 3D). To further confirm the neurotoxicity of BP-3 and validate the above candidate parameter, we performed TUNEL assay to detect apoptosis. Following treatment with the 50/100 μM-72 h parameter, TUNEL analysis showed a significant, dose-dependent increase in the proportion of apoptotic cells in the experimental group, which was correlated with the decreased trend of cell viability observed in the Calcein-AM/PI assay (Figure 3E, 3F). The results of this multimodal assessment (combining viability staining and apoptosis detection) confirmed the rationality of our dose and time selection for toxicity evaluation. Thus, 50/100 μM-72 h was finally determined as the standard intervention parameter for subsequent studies (Figure 3).

Figure 3: Changes in the survival rate of brain organoids. (A) Live-dead staining results of brain organoids (derived from HT22 cells) exposed to different concentrations of BP-3. (B) Quantitative analysis of immunofluorescence corresponding to Figure 3A, showing concentration- and time-dependent decrease in survival rate of HT22 cell-derived brain organoids. (C) Live-dead staining results of brain organoids (derived from primary neural cells) exposed to different concentrations of BP-3. (D) Quantitative analysis of immunofluorescence corresponding to Figure 3C, verifying reduced live cell density in primary neural cell-derived organoids under standard exposure parameters. (E) TUNEL staining results of brain organoids (primary neural cells) exposed to different concentrations of BP-3. (F) Quantitative analysis of immunofluorescence corresponding to Figure 3E, demonstrating dose-dependent increase in apoptotic cells correlating with reduced cell viability. The scale bar is 100 µm.

Calcium content Determination

Changes in Ca²⁺ concentration are closely related to neuronal activity. To detect neural excitability, Fluo-3 AM calcium ion probe combined with laser confocal microscopy imaging technology was used to monitor calcium dynamics in brain organoids constructed from the HT22 cell line and primary neural cells. Following standardized exposure (50/100 μM-72h), the results revealed that the intracellular Ca²⁺ fluorescence intensity in the experimental groups of the two models was decreased compared with the control group, showing a trend of decreasing gradient with the dose increasing (Figure 4A, 4B). This suggested that BP-3 may interfere with action potential conduction. Notably, the attenuation of calcium signals in both models confirmed that this neuroelectrophysiological toxicity exhibited a cell type independent feature, providing key experimental evidence for establishing a standardized biomarker system for the assessment of neurofunctional toxicity of chemical substances.

Figure 4: Effects of BP-3 on brain organoid. (A) ROS detection results of brain organoids treated with different concentrations of BP-3 for 72 hours. (B) Quantitative analysis of grayscale values corresponding to ROS detection results (Figure 4A) of brain organoids treated with different concentrations of BP-3 for 72 hours. (C) Changes in calcium ion content of brain organoids treated with different concentrations of BP-3 for 72 hours. (D) Quantitative analysis of grayscale values corresponding to calcium ion content detection results (Figure 4C) of brain organoids treated with different concentrations of BP-3 for 72 hours. LDH release of brain organoids derived from HT22 cells (E) and primary neuronal cells (F) treated with different concentrations of BP-3 for 72 hours.

ROS analysis

Intracellular ROS can oxidize non-fluorescent DCFH to generate f luorescent DCF, and the detection of DCF fluorescence enables the assessment of intracellular ROS levels. Here, the oxidative stress effects of BP-3 on brain organoids were systematically evaluated using the DCFH-DA fluorescence probe method. After a 72 h exposure period in HT22 cell line-derived and primary neural cell-derived brain organoids, the intracellular ROS levels in the 50/100 μM BP-3 treatment group were significantly higher than in control group (Figure 4C, 4D). This cross-model consistency revealed that its neurotoxic effects were associated with a cascade response of oxidative stress driven by mitochondrial dysfunction.

LDH release assay

In order to systematically assess the damaging effects of BP-3 on the integrity of nerve cells membrane, LDH release assay was performed. Based on the standardized 72-hour exposure model, the LDH activity in the organoid culture supernatant of different concentration treatment groups (0-100 μM) were determined using a colorimetric method with a cytotoxicity assay kit. The results showed that the LDH release in the 100 μM treatment group was significantly increased compared with the control group (Figure 4E, 4F). Together with the previous live-dead staining results, this finding confirmed that BP-3 at specific concentrations of 50 μM and 100 μM could disrupt membrane stability and induce secondary necrosis (Figure 4).

Wnt/β-catenin signaling protein expression

The Wnt signaling pathway, an evolutionarily conserved morphogenetic system, plays a multidimensional regulatory role in synaptogenesis and functional maintenance in the nervous system (Figure 5). GSK-3β is one of the key downstream molecules of the Wnt pathway, and as one of the core members in the molecular regulatory network of neural regeneration, it regulates axon regeneration by coordinating different biological processes, such as protein translation, gene transcription, and cytoskeleton organization [30]. TCF7L2 is an important Wnt/ β-catenin signaling mediator that plays an important role in a variety of cellular functions from early development to mature tissue homeostasis. Throughout early development, TCF7L2 expression in neurons is critical for synaptic transmission [31]. In the present study, the regulatory effect of BP-3 exposure on the Wnt/β-catenin signaling pathway was systematically assessed. Under standardized toxicity parameters (using primary neuronal cells derived organoids, treated with 50/100 μM BP-3 for 72 h), the expression levels of Wnt5a, β-catenin, GSK 3β and TCF7L2 were measured using immunofluorescence staining (Figure 6A-6H). Compared with the control group, the expression levels of Wnt5a, β-catenin, GSK-3β and TCF7L2 in the BP-3 treatment group were significantly decreased, indicating that after the treatment of brain organoids with BP-3, the Wnt/β-catenin signaling pathway is inhibited. This inhibition drives the epigenetic silencing of synapse-related genes Syn and PSD95 via the aberrant activation of the TCF7L2/β-catenin complex, ultimately leading to the decreased expression of synapse-associated proteins PSD95 and Syn (Figure 6I-6L), thus regulating the induction of synaptic structural and functional plasticity (Figure 6). We also evaluated the Wnt pathway-related proteins in HT22 cells after BP-3 treatment. As shown in Figure 7, BP-3 treatment inhibited the expression of Wnt5a, β-catenin, GSK-3β, TCF7L2, PSD95 and Syn, when compared with the levels in the control group (Figure 7) (Figure 8).

Figure 5: Illustrations of brain organoids used to investigate the effects of BP-3 on early brain neurodevelopmental mechanisms. BP-3 exposure acts on brain organoids, and intracellularly, it inhibits the expression of Wnt5a. As Wnt5a is involved in regulating relevant signaling pathways, its downregulation triggers a series of subsequent molecular events: it reduces the level of β-catenin, and simultaneously alters the states of TCF7L2 and Gsk-3β, thus inhibiting cell proliferation. Additionally, in terms of synaptogenesis, the aforementioned signaling pathway disruptions lead to decreased levels of PSD95 and Syn, ultimately resulting in abnormal neuronal development in brain organoid.

Figure 6: Effect of BP-3 on the expression profile of different proteins in brain organoids and their quantitative analysis. (A) Representative immunofluorescence image of Wnt5a in brain organoids after 72 h BP-3 exposure; (B) Quantitative analysis of Wnt5a fluorescent intensity corresponding to (A). (C) Representative immunofluorescence image of β-catenin in brain organoids after 72 h BP-3 exposure; (D) Quantitative analysis of β-catenin fluorescent intensity corresponding to (C). (E) Representative immunofluorescence image of TCF7L2 in brain organoids after 72 h BP-3 exposure; (F) Quantitative analysis of TCF7L2 fluorescent intensity corresponding to (E). (G) Representative immunofluorescence image of GSK-3β in brain organoids after 72 h BP-3 exposure; (H) Quantitative analysis of GSK-3β fluorescent intensity corresponding to (G). (I) Representative immunofluorescence image of Syn in brain organoids after 72 h BP-3 exposure; (J) Quantitative analysis of Syn fluorescent intensity corresponding to (I). (K) Representative immunofluorescence image of PSD95 in brain organoids after 72 h BP-3 exposure; (L) Quantitative analysis of PSD95 fluorescent intensity corresponding to (K).

Figure 7: Expression and grayscale quantification of Wnt pathway-related proteins and synaptic proteins in HT22 cells following BP-3 stimulation. (A) Representative Western Blot images showing the protein bands of Wnt pathway-related proteins in HT22 cells: GSK-3β (46 kDa), Wnt5a (42 kDa), β-catenin (92 kDa), TCF7L2 (68 kDa), with GAPDH used as the internal reference. (B) Grayscale quantitative analysis of the relative expression levels of GSK-3β, Wnt5a, β-catenin, and TCF7L2 in HT22 cells (corresponding to the Western Blot results in (A)), normalized to GAPDH. (C) Representative Western Blot images showing the protein bands of synaptic proteins in HT22 cells: PSD95 (95 kDa), Syn (38 kDa), with Actin used as the internal reference. (D) Grayscale quantitative analysis of the relative expression levels of PSD95 and Syn in HT22 cells (corresponding to the Western Blot results in (C)), normalized to Actin. Statistical significance was set at p < 0.05; *p < 0.05, **p < 0.01, ns = not significant.

Figure 8: Survival of different concentrations of BP-3 effects on organoids at different times. (A) and (B) Live-dead staining and quantification of different concentrations of BP-3 after 6 hours of action on brain-organoids (HT22 cells). (C) and (D) Live-dead staining and quantification of different concentrations of BP-3 after 12 hours of action on brain-organoids (HT22 cells). (E) and (F) Live-dead staining and quantification of different concentrations of BP-3 after 24 hours of action on brain-organoids (HT22 cells). (G) and (H) Live-dead staining and quantification of different concentrations of BP-3 after 36 hours of action on brain-organoids (HT22 cells). (I) and (J) Live-dead staining and quantification of different concentrations of BP-3 after 48 hours of action on brain-organoids (HT22 cells).

DISCUSSION

In this study, we developed a novel 3D inverse-opal PEGDA scaffold that supports the formation of structured brain organoids. Using this model, we demonstrated that BP-3 induces neurotoxicity in a dose- and time-dependent manner, with significant effects observed at 50 μM and 100 μM after 72 h of exposure. Mechanistic studies revealed that BP-3 triggers oxidative stress, disrupts calcium homeostasis, compromises membrane integrity, and suppresses the Wnt/β-catenin signaling pathway, ultimately leading to impaired synaptic function.

Traditional 3D culture primarily relies on Matrigel®, which suffers from high cost, undefined composition, and batch variability. However, due to the high cost of matrix gel, its unclear composition and inherent batch-to-batch variations, a novel 3D scaffolding material has been developed for 3D cell culture [32-35]. Based on the established 3D culture system, a systematic evaluation of the neurotoxicity of BP-3 was further conducted: a dose-time gradient experiment (0-100 μM, 6-72 h) was performed in combination with the Calcein-AM/PI double staining method, which led to the determination of 50/100 μM for 72 h as the standardized exposure condition [36]. And it was found that with the increase of concentration and exposure time, the integrity of the organoid was impaired, and the cell spheres exhibited a tendency toward disintegration.

Our PEGDA-based scaffold provides a reproducible and biocompatible alternative, enabling high-fidelity modeling of neurodevelopmental processes. The observed radial organization and cortical layer-specific marker expression in our organoids confirm their physiological relevance.

The experimental results showed that when the concentration of BP-3 reached 50 μM and the exposure time exceeded 72 h, the activity of brain organoids was significantly inhibited. Through calcium ion fluorescent probe detection, ROS level determination, and LDH release assay, it was found that BP-3 could induce a cascade of toxic pathways to cause neurological damage, specifically including oxidative stress, calcium homeostasis imbalance, and membrane integrity disruption, which ultimately led to neurofunctional impairment [37,38]. Studies have demonstrated that under normal conditions, ROS regulate redox homeostasis and function as important messengers in cell signalling pathways [39,40]. Alterations in ROS concentration can modulate synaptic plasticity through pathways such as N-methyl-D-aspartate (NMDA) receptor pathway and suppress neuronal excitability by influencing the activity of ion channels such as K+ channels [41]. BP-3 exposure induced a cascade of toxic effects. The observed increase in ROS levels is consistent with previous studies linking BP-3 to oxidative stress [37]. Excessive ROS can disrupt neuronal function by promoting lipid peroxidation, altering ion channel activity, and triggering mitochondrial-dependent apoptosis [42,43]. Concurrently, the disruption of calcium homeostasis likely contributes to reduced neuronal excitability and impaired synaptic transmission [40]. The observed LDH release at high concentrations further confirms BP-3’s damaging effects on membrane integrity. Increased ROS generation can trigger neuronal apoptosis through mitochondria-dependent pathways in organoids derived from different cellular sources; notably, there was a significant increase in ROS production following BP-3 exposure [37]. ROS can induce lipid peroxidation in the neuronal cell membranes, disrupting the integrity and fluidity of the cell membrane, which in turn impairs neuronal signal transmission and induces the activation of apoptotic signaling pathways, and ultimately leading to neural death [42]. For instance, in Parkinson's disease, mitochondrial dysfunction in nigrostriatal dopaminergic neurons leads to excessive ROS production. This excess ROS promotes the aggregation of neuronal proteins into Lewy bodies, which further exacerbates neuronal degeneration and death [43].

Calcium ions (Ca²⁺) are important second messengers, and their concentration exerts a profound influence on synaptic function. Specifically, Ca²⁺ modulates neuronal depolarization and regulates neural excitability; it also plays a key role in mediating synaptic communication and information transmission. Abnormal transmission of synaptic information is often associated with various neurological diseases such as dyskinesia [40]. After exposing brain organoids to BP-3, the intracellular Ca²⁺ level in the experimental group was significantly lower than that in the control group. Analysis of the experimental results revealed that BP-3 induces neurotoxicity mainly through the production of excessive ROS, and the elevated ROS levels not only affect the redox microenvironment required for the activity of nerve excitatory impulses but also directly interfere with the activity of K+ ion channels, thereby reducing the excitability of the neural networks. On the other hand, owing to its lipid solubility, BP-3 can directly interact with the phospholipid bilayer of the cell membranes. This interaction impairs the normal physiological function of ion channels and induces fluctuations in intracellular Ca²⁺ levels, which plays a central role in neuronal communication and cellular physiology. Notably, existing studies have shown that disturbances in Ca2+ homeostasis may play a key role in neural network dysfunction and neuronal apoptosis [44]. When the brain organoids were treated with BP-3, the production of ROS was elevated. When the cellular detoxification mechanisms are unable to scavenge excess ROS, ROS overload occurs, which induces oxidative stress and causes cellular damage. ROS overload also interferes with the normal redox microenvironment required for the regulation of neural excitability, which further exacerbates neuronal apoptosis. It also directly interferes with the activity of ion channels (e.g., inhibiting Na⁺ channel activity), and such inhibition of Na⁺ channels may play a key role in neural network dysfunction and apoptosis ultimately leading to reduced excitability of neural networks [44]. Many studies have shown that oxidative stress is implicated in many neurological diseases. For example, mutations in genes such as PINK1 and PARK2 impair mitochondrial function, which in turn elevates ROS levels and increases cellular susceptibility to oxidative stress.

In both of these aspects (ROS and Ca²⁺), significant changes were observed prior to the increase in LDH release, which may be due to the fact that the higher sensitivity of intracellular small molecules to BP-3 interference. The potential molecular mechanism underlying BP-3-induced damage to brain organoids are as follows: on the one hand, BP-3 directly induces oxidative stress, which leads to the rapid accumulation of ROS and further exacerbates cellular injury [45]. On the other hand, BP-3 disrupts intracellular Ca²⁺ homeostasis, which in turn inhibits the excitability of neural networks [46].

A growing body of evidence links prenatal exposure to environmental chemicals including arsenic and lead, and perfluoro octane sulfonate, to deficits in neurological structure and function [47,48]. In addition to its effects on the fine structure of neural tissue, BP-3 exposure has been associated with the induction of behavioural toxicity and cognitive impairments in rodent models. Several lines of evidence suggest that abnormalities in synaptic structure may be associated with the pathogenesis of various neurological disorders, including anxiety, depression, autism, and others.

Notably, our results provide new evidence that BP-3 impairs the Wnt/β-catenin signaling pathway—a critical regulator of neurodevelopment and synaptogenesis. The downregulation of Wnt5a, β-catenin, GSK-3β, and TCF7L2, along with the reduction in PSD95 and Syn levels, suggests that BP-3 disrupts synaptic formation and function through this pathway. This f inding is particularly significant given the role of Wnt signaling in axonal guidance, synaptic plasticity, and neural circuit formation [49]. The Wnt/β-catenin signaling pathway plays a critical role in regulating synaptogenesis. In the present study, immunofluorescence assays demonstrated that BP-3 exposure significantly downregulated the expression of Wnt/β-catenin, its downstream effector proteins, and the synaptic proteins PSD95 and Syn [49]. Beyond its effects on synapses, BP-3 has also been reported to impair neural differentiation by disrupting early cerebrovascular development, and behavioural studies further showed that this may lead to depressive-like behaviours in offspring mice. However, the exact mechanisms underlying the neurodevelopmental outcome after BP exposure, whether mediated by synaptic or cerebrovascular injury, remain to be further elucidated. These findings suggested that BP-3-induced synaptic disruption may be mediated via the Wnt/β-catenin signaling pathway, thus warranting further investigation into this mechanism.

The sample size of this study was small due to the limited resources, and the generalizability of this conclusion can be verified in the future by realizing high-throughput experiments on brain organoids with the help of automated cultures. In addition, this study mainly focuses on the effect of BP-3 on the structure of organoids, and the functional investigation of brain organoids can be further improved [26]. In the future, we can deeply investigate the effect of BP-3 on the function of brain organoids with the help of perfusion-enabled organoids, the use of multi-electrode arrays, and the use of artificial intelligence and other advanced technologies [50]. The effects of BP-3 on multiple organs throughout the body can also be investigated with the help of multi-organ co-culture technology. Neurology is closely related to cognition and behavior, so it is necessary to further investigate the effects of BP-3 on cognition as well as behavior in the future. More work is needed in the future to explore the interactions between the brain and other organs to better model the innervation of the nervous system.

Although this study offers important insights, certain limitations remain. The in vitro exposure concentrations, while informative, may not fully reflect environmental or physiological levels. Future studies should incorporate metabolic competence and blood-brain barrier models to enhance physiological relevance. In addition, while we identified Wnt/β-catenin signaling as a key mediator, functional rescue experiments using pathway agonists or genetic approaches would strengthen causal inference.

Additionally, co-culture of brain organoids with immune cells such as microglia and vascular endothelial cells (to mimic the structure of the blood-brain barrier) can be achieved with the aid of a perfusion system to recapitulate the overall impact of BP-3 on the in vivo microenvironment. It is even possible to co-culture multiple organoids in a Micro Physiological System (MPS) by co-culturing multiple distinct organoids. Studies have demonstrated that by culturing organoids on an MPS platform not only enhances their viability but also enables in situ assessment of microenvironmental parameters. In conclusion, brain organoid research is still in its infancy. Addressing these challenges requires multidisciplinary collaboration, and integrating various engineering tools, such as biomaterials, 3D printing, and organ-on-a-chip, is expected to accelerate the translation of organoids research into clinical applications.

Looking forward, the integration of advanced technologies such as multi-electrode arrays, perfusion systems, and multicellular co-cultures (e.g., with microglia or vascular endothelial cells) could further enhance the functional relevance of brain organoid models. Such platforms may enable real-time assessment of neural activity, metabolic responses, and complex cell-cell interactions under BP-3 exposure.

CONCLUSION

The core breakthrough of this study resides in the establishment of an in vitro brain organoid culture method based on a novel 3D scaffold. By precisely regulating the pore size uniformity and biocompatibility of the scaffold, this method creates a practical, stable in vitro microenvironment that supports the growth of brain organoids. To validate the applicability of this novel method for investigating the neurotoxicity of environmental pollutants, we exposed brain organoids cultured on the new scaffold to BP-3. Utilizing conventional techniques including calcium imaging and ROS detection, we made preliminary observations: BP-3 at a specific concentration (50 μM) and exposure duration (72 h) impaired the neural firing activity of the organoids, and may also induce oxidative stress and cell membrane damage. Furthermore, immunofluorescence experiments suggested that the toxic effects of BP-3 might be associated with the Wnt/β catenin signaling pathway, which collectively demonstrates that this novel brain organoid culture system can effectively capture the neurotoxic responses of organoids to pollutants. This method exhibits flexibility for application in neurotoxicity assessment of environmental pollutants, laying a methodological foundation for subsequent mechanistic studies on the neurotoxicity of a broader range of pollutants.

CONFLICTS OF INTEREST

The authors declare no conflict of interests.

ACKNOWLEDGEMENTS

This work was financially supported by the National Key R&D Program of China (2022YFE132800), Joint Fund of Henan Province Science and Technology R&D Program (225200810020) and Scientific and Technological Research Project of Henan Province (252102310373).

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Author Info

Received: 20-Sep-2025, Manuscript No. JMC-25-165637; Editor Assigned: 23-Sep-2025, Pre QC No. JMC-25-165637 (PQ); Reviewed: 08-Oct-2025, QC No. JMC-25-165637; Revised: 14-Oct-2025, Manuscript No. JMC-25-165637 (R); Published: 21-Oct-2025

Citation: Wen P (2025) Engineered Brain Organoids on a 3D Scaffold Unveil Synaptic Toxicity of Benzophenone-3 Mediated by Wnt/β-catenin Dysregulation. J Med Chem.1:1.

Copyright: © 2025 Wen P, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.