Abstract

Therapeutic vaccines containing aluminum adjuvants have been widely used in the treatment of tumors due to their powerful immune-enhancing effects. However, the neurotoxicity of aluminum adjuvants with different physicochemical properties has not been completely elucidated. In this study, a library of engineered aluminum oxyhydroxide (EAO) and aluminum hydroxyphosphate (EAHP) nanoparticles was synthesized to determine their neurotoxicity in vitro. It was demonstrated that the surface charge of EAHPs and size of EAOs did not affect the cytotoxicity in N9, bEnd.3, and HT22 cells; however, soluble aluminum ions trigger the cytotoxicity in three different cell lines. Moreover, soluble aluminum ions induce apoptosis in N9 cells, and further mechanistic studies demonstrated that this apoptosis was mediated by mitochondrial reactive oxygen species generation and mitochondrial membrane potential loss. This study identifies the safety profile of aluminum-containing salts adjuvants in the nervous system during therapeutic vaccine use, and provides novel design strategies for their safer applications.

SIGNIFICANCE STATEMENT In this study, it was demonstrated that engineered aluminum oxyhydroxide and aluminum hydroxyphosphate nanoparticles did not induce cytotoxicity in N9, bEnd.3, and HT22 cells. In comparation, soluble aluminum ions triggered significant cytotoxicity in three different cell lines, indicating that the form in which aluminum is presenting may play a crucial role in its safety. Moreover, apoptosis induced by soluble aluminum ions was dependent on mitochondrial damage. This study confirms the safety of engineered aluminum adjuvants in vaccine formulations.

Introduction

Vaccines, as a public health tool, play a vital role in infectious diseases prevention and cancer treatment, which could be divided into prophylactic and therapeutic vaccines (Li et al., 2023). In vaccine formulations, adjuvants were used as immune modulators, which could enhance the body’s immunity to antigens or cancer cells (Pulendran et al., 2021; Hager et al., 2022; Lee et al., 2022). Moreover, there have been various known materials with adjuvant activity (e.g., inorganic nanoparticles, saponins, microbial products, emulsions, and liposomes, etc.) (Kheirollahpour et al., 2020). As an inorganic nanoparticle, aluminum-containing salts adjuvants mainly include three types: aluminum hydroxyphosphate (EAHP), aluminum oxyhydroxide (EAO), and aluminum potassium sulfate, and they have been widely used in human vaccines in the last few decades (Ohlsson et al., 2013; Khandhar et al., 2020).

Owing to induction of the immune activation, therapeutic vaccines have been used against many types of cancers (e.g., melanoma, lung cancer, breast cancer, and gastric cancer) (Li et al., 2023). Gan et al. (2020) demonstrated that CpG-loaded aluminum phosphate nanoparticles coated with cell membrane could enhance the cellular immunity by improving the delivery efficiency to antigen presenting cells and extend the survival time of mice by inhibiting the growth of tumor cells. Hernández and Vázquez (2015) showed that racotumomab–alum vaccine could induce a significant and specific antibody response by mimicking the tumor-related antigen NeuGcGM3 to kill NeuGcGM3+/+ tumor cells and prolong the survival time. Zhang et al. (2022) reported that tumor vaccine containing aluminum adjuvant and Toll-like receptor 7 agonists could elicit strong humoral and cellular immunity by inducing antibody production and the activation of CD4+ and CD8+ T cells to suppress tumor growth in vivo. However, the adverse reactions caused by the use of aluminum-based vaccines have promoted study on their safety of various components, including antigens, preservatives and adjuvants. Couette et al. (2009) demonstrated that macrophagic myofasciitis patients induced by the long-term exposure of aluminum hydroxide had a measurable cognitive dysfunction. Tomljenovic and Shaw (2011) showed that there may be a causal relationship between the increased prevalence of autism spectrum disorders and the volume of aluminum administered to preschoolers by vaccination. Wang et al. (2023) reported that aluminum hydroxide particles could induce developmental toxicity neural progenitor cells in human embryonic stem cells-derived dorsal forebrain organoids, which was dependent on Hippo-YAP1 signaling pathway. Moreover, the physicochemical properties (e.g., size, surface charge, and aspect ratio) of aluminum-containing salts adjuvants have been identified to affect their immunostimulating capacity in vitro and in vivo (Sun et al., 2013, 2016, 2017; Liang et al., 2021, 2022). However, the toxicity profile of aluminum adjuvants with different physicochemical properties has not been clarified.

In this study, engineered EAO and EAHP nanoparticles with well-regulated size and surface charge were prepared. Their neurotoxicity was evaluated in vitro. It was shown that EAO and EAHP nanoparticles with different physicochemical properties did not induce cytotoxicity in N9, bEnd.3, and HT22 cells. In comparison, soluble aluminum ions led to significant cytotoxicity in N9, bEnd.3, and HT22 cells. Detailed mechanistic study showed that the cytotoxicity was due to apoptotic cell death, which was mediated by the generation of mitochondrial reactive oxygen species (mtROS) and the loss of mitochondrial membrane potential (MMP). This study provides the basis for the safe application of aluminum adjuvants.

Materials and MethodsChemicals and Reagents.

Commercial aluminum hydroxide (Alhydrogel) and aluminum phosphate (Adju-Phos) adjuvants were obtained from InvivoGen (San Diego, CA). The JC-1 reagent was purchased from Solarbio (Beijing, China). Annexin V-FITC/PI Apoptosis Detection Kit was purchased from Beyotime Biotechnology (Haimen, China). The MitoSOX Red was purchased from Thermo Fisher Scientific (Eugene, OR).

Synthesis of EAO and EAHP Nanoparticles.

EAO nanoparticles were prepared using a hydrothermal synthesis method (Liang et al., 2022). In brief, ethylenediamine was dropped into the aluminum (III) nitrate nonahydrate solution under continuously stirring at room temperature, this process continued until the reaction mixture pH reached 4–6. The mixture was transferred into a Teflon-lined autoclave, and the hydrothermal process was reached at 200°C for 2 hours, 3 hours, 6 hours, and 24 hours, respectively. Then, the products were washed with water for three times before use. EAHP nanoparticles were synthesized using a previously reported method (Liang et al., 2021). Aluminum (III) chloride hexahydrate (AlCl3·6H2O) and sodium phosphate were dispersed in 50 ml of water, respectively. Sodium phosphate solution was injected into 50 ml of aluminum (III) chloride solution under continuous stirring. When the pH of mixture reached 3.4, 5.0, 6.0, the addition of sodium phosphate solution was stopped. The solution was continuously stirred for 30 minutes and transferred into a 100-ml bottle to be sterilized at 121°C for 30 minutes. The products were washed with water for three times before use.

Characterization of EAO and EAHP Nanoparticles.

Transmission electron microscopy (TEM) (EOL JEM-1200EX) was used to determine the specific morphologies and primary sizes of particles. The hydrodynamic size and ζ potential of particles were determined by a ZetaPALS instrument after dispersed in water (90Plus Zeta, Brookhaven, Holtsville, NY). The crystal structure of EAO and EAHP nanoparticles was analyzed by X-ray diffraction (XRD) analysis (D/Max 2400, Rigaku, Japan). A Flourier-transformed infrared spectrophotometer was used to record the Fourier-transform infrared spectroscopy (FTIR) spectra by a KBr pellet technique (Thermo Fisher, Nicolet 6700).

Cell Culture and Assessment of Cytotoxicity In Vitro.

The cytotoxicity of EAO and EAHP nanoparticles was determined using MTS assays (Sun et al., 2015). Briefly, N9, HT22, and bEnd.3 cells at the density of 1.4 × 104, 1.0 × 104, and 1.2 × 104 cells/well were exposed to particles at different doses (EAOs: 31.25–250 μg/ml; EAHPs: 62.5–500 μg/ml). After 24 hours, the 1.67% MTS working solution was used to incubate with cells for 40 minutes at 37°C. The supernatant was used to determine the absorbance at 490 nm.

Assessment of mtROS Generation.

N9 cells (1.4 × 104 cells/well) were exposed to EAO (31.25 μg/ml) or EAHP (62.5 μg/ml) nanoparticles for 24 hours. N9 cells were incubated with MitoSOX Red (5 μm) in HBSS to determine the generation of mtROS. Then, the fluorescence intensity was recorded at 510/580 nm by a microplate reader. Untreated cells were used as negative control, and commercial aluminum adjuvants and soluble AlCl3·6H2O solutions were used as positive control.

Determination of the Solubility of EAO Nanoparticles.

Inductive coupled plasma optical emission spectrometer (ICP-OES) was used to assess the solubility of EAO nanoparticles. Simulated interstitial fluid (IF) and phagolysosomal simulant fluid (PSF) solution were used for simulated human brain tissue fluid to access the dissolution of EAO nanoparticles. Briefly, 5 mg/ml suspension of EAO nanoparticles and commercial Alhydrogel were prepared, respectively. After the nanoparticles were completely dispersed, the tubes were shaken for 2 hours, 6 hours, and 24 hours at 37°C and 250 rpm/min. The concentration of Al3+ in supernatant was determined by ICP-OES. The commercial aluminum adjuvants were used as positive control.

Assessment of MMP.

The changes of MMP in N9 cells were performed after stimulated with EAO (250 μg/ml) and EAHP (500 μg/ml) nanoparticles. Briefly, N9 cells were stimulated with nanoparticles for 24 hours. Dulbecco’s phosphate-buffered saline buffer was used to wash the cells, and then the JC-1 solution was added to incubate with cells at 2.5 μg/ml. After 30 minutes, the fluorescence intensity of JC-1 was determined at excitation 485 nm/emission 555 nm and excitation 560 nm/emission 595 nm. The calculation method for mitochondrial depolarization was red/green fluorescence. The commercial aluminum adjuvants and soluble AlCl3·6H2O solution were used as positive control.

Assessment of Apoptosis.

For the assessment of apoptosis, N9 cells (2.0 × 105 cells/well) were stimulated to EAO (250 μg/ml) or EAHP (500 μg/ml) nanoparticles for 16 hours. Cells were collected and resuspended with staining buffer (300 μl), and stained with Annexin V-FITC (5 μl) and pyridine iodide (10 μl) at room temperature for 15 minutes. The apoptosis was determined by flow cytometry. The commercial aluminum adjuvants and the soluble hexahydrate aluminum AlCl3·6H2O solution were used as positive control.

Statistical Analysis.

The results were presented as mean ± S.D. The statistical significance was determined by using two-tailed Student’s test for two-group analysis. P < 0.05 was considered statistically significant.

ResultsSynthesis and Physicochemical Characterization of EAO and EAHP Nanoparticles.

The engineered EAO and EAHP nanoparticles were prepared by hydrothermal method and chemical precipitation method, respectively. TEM analysis showed that the morphology of EAO nanoparticles were nanorods, and their morphology had good dimensional homogeneity (Fig. 1). The lengths of EAO nanorods were in the range of 200–400 nm, which were named EAOs-1, EAOs-2, EAOs-3, and EAOs-4 based on the length variation (Table 1). The length of EAO nanorods was gradually decreasing depending on the pH change, probably due to the changes of hydroxide (OH–) supersaturation level determining its growth rate in the longitudinal direction (Liang et al., 2022). In comparison, EAHP nanoparticles exhibited an amorphous structure and their diameters were 30–40 nm (Fig. 1). The formation of this amorphous structure is due to interfering with the crystallization process by the addition of phosphate (Liang et al., 2021). The ζ potential of EAOs nanorods was around 30 mV in water. In contrast, the ζ potential of EAHP nanoparticles was 17.05 mV, –8.75 mV, and –27.73 mV in water, respectively. According to their surface charges being electropositive, electroneutral, and electronegative, they were named EAHPs-posi, EAHPs-neut, and EAHPs-nega (Table 2). Moreover, it was demonstrated that the hydrodynamic sizes of EAO nanoparticles were in the range of 100–500 nm by dynamic light scattering (DLS), and the hydrodynamic sizes of EAHP nanoparticles were around 100 nm (Tables 1 and 2). However, the hydrodynamic sizes of EAHPs-neut were significantly bigger than the hydrodynamic sizes of EAHPs-posi and EAHPs-nega. The reason may be due to the neutral surface charge of EAHPs-neut, where the decrease of surface charge attenuates the particle–particle interactions (Liang et al., 2021). For comparison, commercial aluminum hydroxide (Alhydrogel) and aluminum phosphate (Adju-Phos) adjuvants were applied as control, and their characterizations were shown (Tables 1 and 2). Their hydrodynamic sizes were 237 nm and 109 nm, and ζ potentials were 24.92 mV and –12.46 mV, respectively.

Fig. 1.

TEM analysis of EAO and EAHP nanoparticles. (A–D) Representative TEM images of EAOs with different length. (F–H) Representative TEM images of EAHPs. Alhydrogel and Adju-Phos were used as controls. Scale bar, 100 nm.

TABLE 1

Hydrodynamic size, PDI, and ζ potential of EAOs and Alhydrogel suspended in water determined by DLS

TABLE 2

Hydrodynamic size, PDI, and ζ potential of EAHPs and Adju-Phos suspended in water determined by DLS

XRD analysis demonstrated that EAO nanoparticles showed at least five distinct characteristic diffraction peaks, which were consistent to the characteristic peaks of orthorhombic γ-AlOOH with high purity and high crystallinity (Fig. 2A) (Chen et al., 2008). Commercial Alhydrogel adjuvants exhibit the same characteristic diffraction peaks, however, their full width at half maximum were bigger, indicating that the crystallinity of EAO nanoparticles were significantly higher (Fig. 2B). XRD analysis also demonstrated that EAHP nanoparticles were amorphous, which were similar to commercial Adju-Phos adjuvants (Fig. 2, C and D). Moreover, FTIR analysis showed that the characteristic bands of EAO nanoparticles and commercial Alhydrogel adjuvants were at 1067 cm−1, 1156 cm−1, 1626 cm−1, 3095 cm−1, and 3300 cm−1. The bands at 1156 cm−1 and 1067 cm−1 were because of symmetric Al–OH stretching vibration (νs Al–OH) and asymmetric Al–OH stretching vibration (νas Al–OH). The bands at 1626 cm−1 were attributed to the bending patterns of adsorbed water (Fig. 3, A and B) (Qu et al., 2005). Additionally, FTIR analysis also showed that the characteristic bands of EHAPs nanoparticles and commercial Adju-Phos adjuvants were at 540 cm−1, 1100 cm−1, 1640 cm−1, and 3420 cm−1 (Fig. 3, C and D). The band at 540 cm−1 represented P–O-bending band. The region between 2500 cm−1 and 3700 cm−1 was an asymmetric-shaped band, which were attributed to OH-stretching due to hydroxyl groups and absorbed water (Lu et al., 2009).

Fig. 2.

XRD analysis of different nanoparticles. (A) EAOs. (C) Alhydrogel. (B) EAHPs. (D) Adju-Phos.

Fig. 3.

FTIR spectra of different nanoparticles. (A) EAO. (B) Alhydrogel. (C) EAHP. (D) Adju-Phos.

Cytotoxicity Profiling of Engineered EAO and EAHP nanoparticles.

To more accurately determine the toxicity of aluminum adjuvants on the central nervous system, N9, bEnd.3, and HT22 were used as cell models, which were representative cell lines of microglia, brain-derived endothelial cells, and neurons, respectively. The cytotoxicity of EAO and EAHP nanoparticles was determined by MTS assay. After stimulation with different nanoparticles for 24 hours, it was demonstrated that neither EAO nor EAHP nanoparticles induced cytotoxicity in bEnd.3 nor HT22 cells in a concentration-dependent manners (Fig. 4, A and B). However, EAO and EAHP nanoparticles induced minimal cytotoxicity in N9 cells, and the toxic effect of EAHPs-posi was the most one in all particulate nano-aluminum adjuvants, the reason may be due to the interaction between EAHPs-posi and cell membrane (Fig. 4) (Liang et al., 2021). As controls, commercial Alhydrogel and Adju-Phos adjuvants did not induce cytotoxicity in three different cell lines. Moreover, the physicochemical properties of EAO and EAHP nanoparticles did not have impact on their cytotoxicity profiles. Surprisingly, when AlCl3·6H2O was prepared as soluble aluminum ions, it induced significant cytotoxicity in three different cell lines in a dose-dependent manner (Fig. 4). Among different cell types, AlCl3·6H2O induced more significant cell death in N9 cells. Additionally, heat maps were used to exhibit the cytotoxicity induced by different nanoparticles and commercial aluminum adjuvants in three different cell lines (Fig. 4). It was also shown that AlCl3·6H2O, but not EAO and EAHP nanoparticles, triggered neurotoxicity in all three cell lines.

Fig. 4.

Cytotoxicity profiles of EAO and EAHP nanoparticles. The cell viability of N9, bEnd.3, and HT22 cells was determined after stimulation with (A) EAO and (B) EAHP nanoparticles at dosage of 10–110 μg/ml. The cell viability was relative to the nonstimulated control cells (100%). The cytotoxicity of cells was showed as heat maps.

Due to higher toxicity, N9 cells were then used to determine the mechanism of cell death. It was shown that soluble aluminum ions dramatically triggered the apoptotic cell death, and the proportion of apoptotic cells was dramatically increased, indicating soluble aluminum ions play a crucial role in neurotoxicity of aluminum adjuvants (Fig. 5). In comparison, neither EAO nor EAHP nanoparticles induced remarkable apoptosis in N9 cells. These results demonstrated that soluble aluminum ions could induce significant cytotoxicity in N9 cells, but particulate nano-aluminum adjuvants were non-cytotoxic in all three cell lines.

Fig. 5.

The analysis of cell apoptosis induced by EAO and EAHP nanoparticles. N9 cells were stimulated with (A) EAO (250 μg/ml) and (B) EAHP (500 μg/ml) nanoparticles. After 16 hours, the apoptosis of N9 cells was determined by Annexin V-PI apoptosis detection kit.

The Apoptotic Cell Death in N9 Cells Is Dependent on Mitochondria Damage Induced by Soluble Aluminum Ions.

The mitochondrial pathway is one of the pivotal pathways in apoptotic cell death, and mitochondria play an important role in the process of apoptosis (Zhang et al., 2021; Vringer and Tait, 2023). The mitochondria damage induced by AlCl3·6H2O was performed by determining the changes in mtROS and MMP. It was demonstrated that the engineered EAOs and EAHPs did not generate significant mtROS in N9 cells. In comparison, mtROS generation induced by AlCl3·6H2O and commercial Alhydrogel adjuvants was dramatically increased (Fig. 6). To examine the reason why Alhydrogel adjuvants could induce the generation of mtROS, the solubility analysis of EAO nanoparticles and Alhydrogel in simulated IF and PSF buffer was determined by ICP-OES. IF was used to mimic brain interstitial fluid, whereas PSF was used to mimic the phagolysosomal compartment of N9 cells. It was shown that the solubility of commercial Alhydrogel adjuvants was much higher than EAO nanoparticles in IF, which could induce the production of abundant soluble aluminum ions and further trigger the generation of mtROS (Fig. 7). However, there was no significant difference of their solubility in PSF buffer (Fig. 7). These results suggested that soluble aluminum ions play a crucial role in mtROS generation. Moreover, previous studies have been demonstrated that mitochondrial functional status could be reflected by MMP, which also play an important role in apoptosis. JC-1 probe staining showed that AlCl3·6H2O also triggered the loss of MMP, suggesting that mitochondrial membrane permeabilization was critical in cytotoxicity induced by soluble aluminum ions (Fig. 8). Altogether, these results indicated that mitochondria damage induced by soluble aluminum ions plays an important role in apoptosis in N9 cells.

Fig. 6.

The generation of mitochondrial ROS induced by EAOs and EAHPs nanoparticles. N9 cells were stimulated with (A) EAO and (B) EAHP nanoparticles at 50 μg/ml for 2 hours, and then MitoSOX Red (5 μM) was added and coincubated with cells for 20 minute. *P < 0.05.

Fig. 7.

The solubility analysis of EAO nanoparticles in simulated IF and PSF buffer. EAO nanoparticles were dissolved in (A) IF and (B) PSF buffer to form a 5 mg/ml suspension. The supernatant was used to perform the solubility analysis by ICP-OES.

Fig. 8.

The change of mitochondrial membrane potential induced by EAO and EAHP nanoparticles. After exposure to (A) EAO (250 μg/ml) and (B) EAHP (500 μg/ml) nanoparticles for 24 hours, N9 cells were incubated with JC-1 (2 μm), and the green fluorescence intensity (485 nm/555 nm) and red fluorescence intensity (560 nm/595 nm) were recorded. The depolarization of mitochondrial membrane potential was determined by red–green ratio. *P < 0.05, **P < 0.01.

Discussion

In the present study, a library of engineered EAO and EAHP nanoparticles were synthesized to determine the neurotoxicity in different cell lines. It was demonstrated that all particulate nano-aluminum adjuvants did not exhibit cytotoxicity in N9, bEnd.3, and HT22 cells, however, soluble aluminum ions dramatically induced cytotoxicity in different cell lines in a concentration-dependent manner. Moreover, soluble aluminum ions triggered apoptotic cell death in N9 cells, which was dependent on the generation of mtROS and the loss of MMP.

Currently, aluminum adjuvants have been used in authorized human prophylactic and therapeutic vaccines, and their applications have exhibited the broad-spectrum ability to enhance the immune response (Shi et al., 2019; Liang et al., 2020). However, the safety of aluminum adjuvants in the use of vaccines has always been a concern. In our study, it was demonstrated that particulate nano-aluminum adjuvants (EAOs, EAHPs and commercial aluminum adjuvants) did not exhibit neurotoxicity in N9, bEnd.3, and HT22 cells in the range of 0–500 μg/ml (Fig. 5). In previous studies, Mold et al. (2016) demonstrated that EAO nanoparticles did not immediately induce cytotoxicity in the cytoplasm of THP-1 cells, which may be determined by the form of EAO nanoparticles present in the cytoplasm. Radziun et al. (2011) reported that Al2O3 NPs did not exhibit cytotoxic effects on L929 and BJ cells in the tested doses, which were consistent with our result. However, after exposure to soluble aluminum ions, our results showed that they led to significant cytotoxic effects in three different cell lines, and the cytotoxicity in N9 cells was more significant (Fig. 5). Our results were consistent with reported studies. Cirovic and Cirovic (2022) demonstrated that the deficiency of iron could aggravate circulation aluminum-induced osteotoxicity by increasing the binding of aluminum and transferrin receptor 1. Hao et al. (2021) showed that aluminum chloride (AlCl3) could trigger pyroptosis in BV2 microglia cells and neuroinflammation, which was closely related to the development of neurodegenerative diseases. Thus, it suggested that the safety of aluminum may be related to its formats (i.e., particulates or soluble forms). This indication was due to the fact that soluble aluminum ions were easier to induce the accumulation of massive reactive oxygen species than the particulate nano-aluminum adjuvants (Liu et al., 2021; Zeng et al., 2023).

In this study, it was demonstrated that the surface charge of EAO nanoparticles and the long aspect ratios of EAHP nanoparticles did not affect their neurotoxicity in N9, bEnd.3, and HT22 cells. Mechanistic studies showed that EAO and EAHP nanoparticles with different physicochemical properties did not have impact on mitochondrial ROS production and mitochondrial membrane potential loss. However, many studies have demonstrated that the physicochemical properties of carbon nanotubes or graphene oxide could determine their cytotoxicity, which was dependent on mitochondrial dysfunction. Fujita et al. (2020) demonstrated that multi-walled carbon nanotubes with larger diameter (>50 μm) could induce much higher cytotoxicity than smaller ones (<20 μm) mediated by intracellular ROS generation in rat alveolar macrophages. Wang et al. (2012) showed that multi-walled carbon nanotube treated with acid induced more aggressive cytotoxicity in RAW 264.7 cells than taurine functionalized multi-walled carbon nanotube due to the different mitochondrial membrane potential loss. Georgieva et al. (2020) demonstrated that hydroxylamine-aminated graphene oxide triggered more significant cytotoxicity in HepG2 cells than pristine graphene oxide, which was dependent on mitochondrial dysfunction. Therefore, the effect of physicochemical properties of nanomaterials on their cytotoxicity may be closely related to the type of materials.

Furthermore, our study demonstrated that soluble aluminum ions could induce the apoptotic cell death in N9 cells, whereas particulate nano-aluminum adjuvants did not (Fig. 6). Apoptosis could be divided into the extrinsic and intrinsic pathway of apoptosis, and the intrinsic pathway was mediated by mitochondrial regulation (D’Arcy, 2019). Mitochondria, as the center of cellular energy metabolism, are also one of the important sources of ROS in cells (Du et al., 2019; Bock and Tait, 2020). Our mechanistic findings demonstrated that the generation of mtROS and the loss of MMP induced by soluble aluminum ions attributed to the apoptosis in N9 cells, which was consistent with other studies (Cui et al., 2021; Liu et al., 2021). Lu et al. (2020) demonstrated that AlCl3 could trigger the oxidative stress and apoptotic cell death in PC12 cells through AKT/Nrf2/HO-1 signaling pathway. Moreover, Zhang et al. (2020) reported that AlCl3 could induce apoptosis and necroptosis in hippocampal neural cells dependent on different dose by the interleukin-1β/c-Jun N-terminal kinase pathway. Thus, the follow up study could focus on exploring the effect of aluminum ions on the other type of cell death (e.g., necroptosis, pyroptosis, and autophagy, etc.). In addition, our study only determined the effects of particulate nano-aluminum adjuvants and soluble aluminum ions on different nerve cells in vitro. Their different neurotoxicity needs to be further validated in animal models.

Data Availability

The data that support the findings of this study are available on request from the corresponding author.

Authorship Contributions

Participated in research design: Xue, Bi, Sun, Mao.

Conducted experiments: Chen, Jiang.

Performed data analysis: Chen, Jiang, Sun.

Wrote or contributed to the writing of the manuscript: Chen, Xue, Jiang, Hu, Yu, Sun, Mao.

FootnotesReceived November 19, 2023.Accepted January 16, 2024.

This work was supported by the National Natural Science Foundation of China [Grant U22A20455], National Key Research and Development Program of China [Grant 2022YFC2304305], and Fundamental Research Funds for the Central Universities [Grants DUT21ZD216, DUT22LAB601, and DUT22YG120].

↵1C.C., C.X., and J.J. contributed equally to this work.

The authors declare no competing financial interest.

dx.doi.org/10.1124/jpet.123.002031.

AbbreviationsAlCl3aluminum chlorideAlCl3·6H2Oaluminum (III) chloride hexahydrateDLSdynamic light scatteringEAOengineered aluminum oxyhydroxideEAHPaluminum hydroxyphosphateFTIRFourier-transform infrared spectroscopyICP-OESinductive coupled plasma optical emission spectrometerIFinterstitial fluidmtROSmitochondrial reactive oxygen speciesMMPmitochondrial membrane potentialPSFphagolysosomal simulant fluidTEMtransmission electron microscopyXRDX-ray diffractionCopyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics