Lysosomal membrane permeabilization and lysosomal damage induced by silica micro/nanoparticles in murine RAW-Blue macrophages

Abstract

The objective of this study was to examine the mechanisms underlying endocytic uptake, cytotoxicity, oxidative stress, and lysosomal damage caused by silica micro/nanoparticles (SiPs) in murine RAW-Blue macrophages. In parallel studies, we also assessed the effects of NF-κB on lysosomal membrane permeabilization (LMP). Three types of SiPs, 3 μm-plain (3 μm diameter particles without surface modification), 50 nm-plain (50 nm diameter particles without surface modification), and 50 nm-NH₂ (50 nm diameter particles with an amine functional group as surface modification) were tested. A significant decrease in cell proliferation and a significant increase in cell death were evident, when cells were treated with 50 nm-plain. All types of SiPs plus a lysosomal inhibitor caused a significant increase and a significant decrease in cell proliferation and cell death, respectively. When cells were treated with 50 nm-plain, puncta formation was seen. Treatment with 50 nm-plain significantly increased the expression level of inflammatory cytokine/chemokine genes, whereas inhibitors of clathrin-mediated endocytosis, lysosomal function, and cathepsin activity significantly attenuated these responses. Taken together, the present findings suggest that lysosomal dysfunction is closely associated with 50 nm-plain-induced cytotoxic and inflammatory responses. Our findings also provide relevant findings for safe and effective design of SiPs and a framework to maximize their biomedical potential while mitigating lysosomal damage.

INTRODUCTION

The safe integration of silica micro/nanoparticles (SiPs) into biomedical applications requires characterizing their cytotoxicity profile (Huang et al., 2022). This is closely linked to their interaction with cellular components, particularly the lysosomal system, where endocytosis-mediated internalizing SiPs are typically accumulated (Nel et al., 2009). One of the prevailing paradigms in SiP-induced cytotoxicity proposes that NADPH oxidase (NOX)-derived reactive oxygen species (ROS) serve as an initiating signal that triggers the downstream responses (Yu et al., 2015; Inoue et al., 2021). The highly reactive surface silanol groups (Si–OH) on SiPs can directly trigger ROS production independently of this enzymatic association (Joshi et al., 2015; Rubio et al., 2019; Wang et al., 2020).

Beyond the ROS-driven signaling, the accumulated SiPs in the lysosome can lead to lysosomal membrane permeabilization (LMP) (Wang et al., 2020; Syrocheva et al., 2025) and thereby result in cathepsin release into the cytosol (Schütz et al., 2016). These sequential episodes lead to either multiple outcomes including cell death (Kavčič et al., 2020; Ishaq et al., 2022) or activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome (Hornung et al., 2008). The clathrin- and caveolae-mediated endocytosis of folate-conjugated pullulan acetate nanoparticles lead to inflammatory cytokine production in murine Kupffer cells (Tang et al., 2017), while the macropinocytosis of SiPs causes ROS generation and DNA damage in human keratinocytes (Nabeshi et al., 2011). However, evidence linking endocytosis pathways to the regulation of LMP or cathepsin release remains lacking. We have recently demonstrated that blocking clathrin-mediated endocytosis (CME) with chlorpromazine (CPZ) and macropinocytosis with imipramine (IMI) respectively inhibited NF-κB activation and cell death in SiP-treated murine RAW-Blue macrophages (Roy et al., 2025). Although lysosomal dysfunction, cathepsin activation, and inflammatory responses induced by SiPs have been documented in several experimental systems, the factors that determine the severity of lysosomal damage and its relationship to downstream cellular responses remain incompletely understood. In particular, the contribution of particle characteristics such as size and surface modification to lysosomal membrane damage and the extent to which lysosomal dysfunction is associated with inflammatory and cytotoxic responses in macrophages have not been systematically examined. Because macrophages represent a major target cell population for internalized nanoparticles and play essential roles in particle clearance and immune regulation, clarifying these relationships is important for understanding nanoparticle toxicity and for the development of safer silica-based nanomaterials.

Examining the mechanisms governing SiP-induced cytotoxicity by disentangling the roles of endocytic uptake, oxidative stress, and lysosomal damage was the objective of this study. Following an initial screening of ROS and lysosomal inhibitors on cell death and NF-κB activity, we assessed the effects of CME and macropinocytosis on LMP. Eventually, the impact of endocytosis, ROS, and lysosomal inhibition on the mRNA expression levels of Il1b, Mip2, Tnf, and Il6 were evaluated.

MATERIALS AND METHODS

Silica micro/nanoparticles (SiPs)

Three types of FITC-labeled SiPs, 3 μm-P (3 μm diameter particles without surface modification), 50 nm-P (50 nm diameter particles without surface modification) and 50 nm-NH₂ (50 nm diameter particles with amine functional group surface modification) (micromod Partikeltechnologie GmbH, Rostock, Germany) were used for the indicated experiments as described earlier (Roy et al., 2025).

Cells

Murine RAW-Blue macrophages (InvivoGen, San Diego, CA, USA), which are RAW264.7 macrophages stably expressing an NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) reporter, were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% FBS, and 1% penicillin-streptomycin (FUJIFILM WAKO Pure Chemical Corporation). They were grown for 3-4 days at 37°C in a humidified atmosphere of 95% air and 5% CO₂, and were used for the indicated experiments.

Methylthiazole tetrazolium (MTT) assays and lactate dehydrogenase (LDH) assays

Cytotoxicity was determined using MTT assays (MTT assay kit, Roche Diagnostic Corp. Indianapolis, IN, USA) as described previously (Roy et al., 2025). This assay was used to assess viable cell number and metabolic activity, which may reflect both reduced proliferation and loss of viable cells. RAW-Blue macrophages were seeded in 96-well cell culture plates (5 × 10³ cells/well) (Watson, Tokyo, Japan) and grown in DMEM. Cells were pre-treated with the inhibitors for 30 min, 100 μg/mL SiPs were then added to the cells and incubated for additional 24 hr. The remaining cells were used for MTT assays and the supernatant was poured into the other 96-well plates and then used for LDH assays. The inhibitors used were apocynin (APO; Tokyo Chemical Industry, Tokyo, Japan) for NADPH oxidase, deferoxamine mesylate (DFO; Tokyo Chemical Industry) for Fenton reaction, N-acetyl cysteine (NAC; Tokyo Chemical Industry) for ROS, chloroquine (CQ; Tokyo Chemical Industry) for lysosomal function, and aloxistatin (E64d; Tokyo Chemical Industry) for cathepsins. Concentrations applied were 500 μM APO, 50 μM DFO, 3 mM NAC, 10 μM CQ, or 10 μM E64d in a total volume of 200 μL. After incubation of cells with SiPs, 10 μL of MTT reagent was added and incubated for 30 min. All assays were done in triplicate. The absorbance was measured at 535 nm using a SPECTRAmax340 microplate reader (Molecular Devices, San Jose, CA, USA). A viability of 100% corresponds to the control cells. In parallel, the cell death was determined using the LDH-Cytotoxicity Test Wako kit (FUJIFILM Wako Pure Chemical Corporation). Briefly, 50 µL of the coloring solution was added to each well and incubated for 30 min at room temperature. 100 µL of stop solution was added and the absorbance at 560 nm was measured using the microplate reader. Cell death (%) was calculated. The LDH release was used as an indicator of membrane damage-associated cell death. 100% cell death was defined by cells lysed with lysis buffer. All assays were done in triplicate.

Measurement of SEAP activity (SEAP assays)

The SEAP assays were performed as described earlier (Roy et al., 2025). In brief, RAW-Blue macrophages were seeded in 35-mm culture dishes (1.4 × 10⁵ cells/dish) (Corning, Corning, NY, USA) and grown in DMEM plus 10% FBS overnight to allow for cell attachment. Cells were pre-treated with inhibitors (APO, DFO, NAC, CQ, or E64d) for 30 min, and they were treated with 100 μg/mL SiPs for additional 6 hr. Twenty µL of the supernatant was mixed with 180 µL of QUANTI-Blue Solution (InvivoGen) and incubated for 1 hr in a 96-well plate. Absorbance was measured at 620 nm using the microplate reader. Fold change (treated sample absorbance/vehicle absorbance) was calculated.

Observation of puncta formation

To examine whether lysosomal pathway specifically affects puncta formation, galectin-3 (gal-3) staining was performed. RAW-Blue macrophages were seeded at a density of 1.4 × 10⁵ cells/dish and grown for 48 hr on gelatin-coated glass coverslips (Matsunami Glass, Osaka, Japan) placed in 35-mm cell culture dishes containing 2 mL of medium. After pre-incubating in the presence or absence of inhibitors for 30 min, the cells were exposed to 100 μg/mL of 3 μm-P, 50 nm-P or 50 nm-NH₂ for an additional 24 hr. The inhibitors used were CPZ (Tokyo Chemical Industry) for CME, IMI (Tokyo Chemical Industry) for macropinocytosis, and E64d for cathepsins. Concentrations applied were 25 μM CPZ, 5 μM IMI, or 10 μM E64d. Following removal of the supernatant, cells were fixed with 4% paraformaldehyde for 15 min and washed with phosphate buffered saline (PBS). To inhibit non-specific antibody binding and permeabilize the cells, they were then treated for 30 min with 100 μL of blocking buffer (1% bovine serum albumin, and 10% normal goat serum) containing 0.1% Triton X-100 in PBS. Cells were incubated with 100 μL of rat anti-galectin-3 primary antibody (1:250, Thermo Fisher Scientific) for 1 hr at room temperature. After washing out the primary antibodies with PBS, incubation with 100 μL of Alexa350-conjugated goat anti-rat secondary antibody (1:500, Thermo Fisher Scientific) including Hoechst 33342 (1:1000, Sigma-Aldrich) for 30 min at room temperature was performed, and the secondary antibodies were washed out. Gal-3 puncta formation was observed using BZ-X710 fluorescent microscope equipped with a confocal system (Keyence, Osaka, Japan). Cells exhibiting one or more distinct punctate gal-3 structures within the cytoplasm were classified as gal-3-positive cells. Diffuse cytoplasmic staining without puncta formation was not considered positive. Gal-3-positive cells were quantified from two independent experiments. In each experiment, approximately 200 cells were analyzed in 15 randomly selected fields per treatment.

Extraction of total RNA and quantitative reverse transcription PCR (RT-PCR) assays

RAW-Blue macrophages were seeded at a density of 2 × 10⁵ cells/dish and grown for 72 hr in 60-mm cell culture dishes containing 4 mL of medium. After pre-incubating in the presence or absence of inhibitors (25 μM CPZ, 5 μM IMI, 500 μM APO, or 10 μM CQ/E64d) for 30 min, the cells were exposed to SiPs (100 μg/mL) for an additional 6 hr. Total RNA was extracted and cDNA was then synthesized as described previously (Roy et al., 2025). Primers used for amplification were as follows: interleukin 1 beta (Il1b)-specific primer set (sense: CGT GGA CCT TCC AGG ATG AG, antisense: GGA GCC TGT AGT GCA GCT GTC), interleukin 6 (Il6)-specific primer set (sense: ACC ACG GCC TTC CCT ACT TC, antisense: CAC AAC TCT TTT CTC ATT TCC ACG), macrophage inflammatory protein 2 (Mip2)-specific primer set (sense: AGA CAG AAG TCA TAG CCA CTC TCA AG, antisense: CCT CCT TTC CAG GTC AGT TAG C), tumor necrosis factor alpha (Tnf)-specific primer set (sense: GCC GAT GGG TTG TAC CTT GT, antisense: GCT CTT GAC GGC AGA GAG GA), or glyceraldehyde-3-phosphate dehydrogenase (Gapdh)-specific primer set (sense: TGC ACC ACC AAC TGC TTA G, antisense: GAT GCA GGG ATG ATG TTC). PCR was conducted for 40 cycles in a thermal controller (Quant Studio 12K Flex Real-Time PCR System, Thermo Fisher Scientific). Each amplification cycle consisted of 95°C for 5 sec and 60°C for 30 sec. mRNA expression levels were analyzed by the Delta-Delta Ct method (Bubner et al., 2004) and the relative expression levels of these genes to Gapdh was calculated.

Statistical analysis

Statistical analyses were performed using JASP software (Version 0.95.4) (JASP Team, 2025). As regards the data of MTT/LDH/SEAP/immunostaining/quantitative RT-PCR assays, one-way ANOVA with Tukey’s post hoc test was used. Differences were considered statistically significant when the P value was less than 0.05.

RESULTS

Effects of SiPs on cell proliferation, cell death, and SEAP activity in RAW-Blue macrophages

Treatment of cells with 50 nm-P exerted a marked and significant decrease in cell proliferation when compared to that of the other two types of SiPs (Fig. 1A). Also, treatment of cells with 50 nm-P caused a marked and significant increase in cell death when compared to that of the other two types of SiPs (Fig. 1C, D). Inhibitors APO/DFO/NAC failed to affect cell proliferation or cell death (Fig. 1A and 1C). All types of SiPs plus CQ/E64d caused a significant increase in cell proliferation (Fig. 1B). Also, 50 nm-P plus CQ/E64d caused a significant decrease in cell death (Fig. 1D). When cells were treated with 50 nm-P plus APO, the SEAP activity significantly increased (Fig. 1E). In contrast, the SEAP activity significantly decreased when cells were treated with 50 nm-P plus CQ or E64d (Fig. 1F). The results indicate that lysosomal inhibition with CQ or E64d rescues RAW-Blue macrophages from 50 nm-P-induced cytotoxicity.

Fig. 1

Effects of SiPs on cell proliferation, cell death, and SEAP activity in RAW-Blue macrophages. A-F, Cells were treated with SiPs plus APO, DFO, NAC, CQ or E64d as shown + (treated) or − (untreated) for 6 or 24 hr. The results of MTT assays (A and B), LDH assays (C and D), and SEAP assays (E and F) are shown. Box-plot indicates the distribution of data of each assay. The line in the box indicates the median value of the data. Each assay was performed in triplicate. Cell proliferation of 100% corresponds to untreated control cells. One hundred percent cell death was defined using cells lysed with lysis buffer. SEAP activity is shown as fold change. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the untreated control. ^†P < 0.05, ^††P < 0.01, and ^†††P < 0.001 indicate significant differences compared to the corresponding control. APO, apocynin; DFO, deferoxamine mesylate; NAC, N-acetyl cysteine; CQ, chloroquine; E64d, aloxistatin. For additional details, see “Materials and Methods”.

Cellular localization and the effects of SiPs on puncta formation in RAW-Blue macrophages

Treatment of cells with 50 nm-P resulted in puncta formation (Fig. 2A). When cells were treated with 50 nm-P plus CPZ or IMI, puncta formation was significantly decreased (Fig. 2B). Also, when cells were treated with 50 nm-P plus E64d, puncta formation was significantly decreased (Fig. 2C). The results indicate that 50 nm-P induces LMP but this effect is attenuated by endocytosis inhibition with CPZ or IMI.

Fig. 2

Cellular localization and the effects of SiPs on puncta formation in RAW-Blue macrophages. A, representative fluorescence images are shown. Cells were cultured with the indicated SiPs for 24 hr, and gal-3 staining was performed to detect puncta formation. White arrows indicate the cellular localization of 50 nm-P (the first panel), gal-3-positive cell (the second panel), nucleus (the third panel), or merged image (the bottom panel). B-C, the percentages of gal-3-positive cells are shown. Cells were treated with SiPs plus CPZ, IMI, or E64d as shown + (treated) or - (untreated) for 24 hr. The results of the gal-3 staining are shown. Box-plot indicates the distribution of data of each assay. The line in the box indicates the median value of the data. Each assay was performed in duplicate. A total of 15 randomly selected images per treatment and approximately 200 cells per image were analyzed. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the untreated control. ^†P < 0.01, and ^††P < 0.001 indicate significant differences compared to the corresponding control. CPZ, chlorpromazine; IMI, imipramine; E64d, aloxistatin. Scale bars 50 μm, as indicated in Fig. 2A. For additional details, see “Materials and Methods”.

Effects of SiPs on mRNA expression levels of Il1b, Il6, Mip2, and Tnf in RAW-Blue macrophages

To verify whether any of the 3 types of SiPs affect the inflammation status, mRNA expression levels of Il1b, Il6, Mip2, and Tnf were examined. Treatment with 50 nm-P caused a marked and significant increase in the expression levels of all four cytokine/chemokine genes compared with those of untreated cells (Fig. 3A-D). Treatment of cells with 50 nm-P plus CPZ, CQ, or E64d significantly suppressed those of Il1b, Il6, Mip2, and Tnf. In contrast, IMI failed to suppress the expression levels of any of the cytokine/chemokine genes examined, whereas APO showed little effect except for a reduction in Tnf expression level. These findings suggest an important contribution of lysosomal function to the regulation of inflammatory cytokine/chemokine gene expression induced by 50 nm-P.

Fig. 3

Effects of SiPs on mRNA expression levels of Il1b, Il6, Mip2, and Tnf in RAW-Blue macrophages. A-D, Cells were treated with SiPs plus CPZ, IMI, APO, CQ, or E64d as shown + (treated) or - (untreated) for 6 hr. In quantitative RT-PCR assays, the relative mRNA expression levels of four cytokine/chemokine genes are shown. Box-plot indicates the distribution of data of each assay. The line in the box indicates the median value of the data. Each assay was performed in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant differences compared to the untreated control. ^†P < 0.05, ^††P < 0.01, and ^†††P < 0.001 indicate significant differences compared to the corresponding control. CPZ, chlorpromazine; IMI, imipramine; APO, apocynin; CQ, chloroquine; E64d, aloxistatin.

DISCUSSION

Recently we have reported that the size and surface properties of three different types of SiPs played a crucial role in determining their biological effects in RAW-Blue macrophages (Roy et al., 2025). Treatment of cells with 50 nm-P plus CQ or E64d recovered cell proliferation and reduced both cell death and SEAP activity via maintaining lysosomal integrity (Fig. 1B, 1D and 1F). This is presumably due to increases of lysosomal pH by CQ and blockade of autophagosome-lysosome fusion, and also because of inactivation of lysosomal cathepsins by E64d (Mauthe et al., 2018; Ishaq et al., 2022). The failure of inhibitors APO, DFO, or NAC to affect the current results (Fig. 1A, 1C and 1E) suggests that the contribution of ROS-mediated oxidative stress may be limited under the present experimental conditions (Stolk et al., 1994; Wang et al., 2020).

The observation in fluorescence images depicting E64d-mediated decrease in the percentage of gal-3-positive cells (Fig. 2A and 2C), an indicator of LMP, further support the role of cathepsins in lysosomal inhibition induced by 50 nm-P (Ishaq et al., 2022; Chen et al., 2024). CPZ and IMI significantly decreased the percentage of gal-3-positive cells (Fig. 2B), and this is likely to reflect the secondary effect beyond the inhibition of endocytosis by altering membrane lipid composition (Ioffe et al., 2007; Maruoka et al., 2007; Biswas et al., 2017; Li et al., 2023). Notably, SiP uptake was observed in a substantially larger proportion of cells than was gal-3 puncta formation, suggesting that particle internalization alone may not fully explain the induction of detectable lysosomal membrane damage. The downregulation of mRNA expression levels of four cytokine/chemokine genes with CPZ, and together with the absence of such effects with IMI (Fig. 3A-D), may occur independent of LMP inhibition. This interpretation is further supported by the experimental design of the present study, in which inflammatory gene expression was assessed at 6 hr whereas lysosomal membrane damage was evaluated at 24 hr. APO had little effect on NF-κB-related endpoints (Fig. 1E), suggesting a limited contribution of NOX-derived ROS under the present conditions. However, the selective suppression of Tnf expression by APO (Fig. 3D) may indicate involvement of additional ROS-sensitive signaling pathways (Hwang et al., 2016).

Although lysosomal dysfunction, cathepsin activation, and inflammatory responses induced by SiPs have been reported previously, the present study extends these observations by demonstrating that particle size and surface modification substantially influence lysosomal membrane integrity and are closely associated with downstream biological responses. The stronger effects of 50 nm-P compared with 3 μm-P may reflect more efficient cellular uptake and their lysosomal accumulation, and reduced responses with 50 nm-NH₂ suggest the particle–lysosome-derived interactions.

The broad-spectrum suppression of the inflammatory responses by CQ and E64d suggests an important contribution of lysosomal function to silica nanoparticle-induced cellular responses. Together with the differential effects of CPZ and IMI, these findings indicate that inflammatory signaling and cytotoxicity may involve partially distinct downstream mechanisms following silica nanoparticle exposure.

Several limitations of the present study should be acknowledged. First, the present findings support a close association between lysosomal damage and silica nanoparticle-induced cellular responses, however they provide partial causality. Future studies are required to determine the precise contribution of LMP to downstream cellular responses. Second, the inhibitors used in this study may exhibit activities beyond their primary targets. Indeed, CPZ, IMI, CQ, and E64d have been reported to affect multiple cellular processes, including membrane dynamics, lysosomal function, and protease activity. Third, intracellular ROS levels were not directly measured in the present study. Therefore, although APO, DFO, and NAC had limited effects under the present experimental conditions, a contribution of ROS to silica nanoparticle-induced cellular responses cannot be completely excluded. Fourth, a large number of cells were analyzed across multiple microscopic fields; however, the gal-3 imaging analysis was conducted in two independent experiments. Additional biological replicates would further strengthen the robustness of these observations.

In conclusion, the current study demonstrates that 50 nm-P induces marked lysosomal damage in RAW-Blue macrophages and that particle size and surface modification substantially influence lysosomal integrity and subsequent cellular responses. These findings support the concept that lysosomal dysfunction is closely associated with silica nanoparticle-induced cytotoxic and inflammatory responses and provide useful insights for the development of safer silica-based nanomaterials.

ACKNOWLEDGMENTS

We thank Dr. Sachi Sri Kantha (formerly at Gifu University) for valuable comments and editing the manuscript. This study was an outcome of using research equipment shared in MEXT Project for promoting public utilization of advanced research infrastructure (Program for supporting construction of core facilities of Nagoya City University, grant no. JPMXS0441500024).

Funding

This investigation was partially supported by JSPS KAKENHI grant no. JP24K09436, Research Grant Program of the Research Foundation for Opto-Science and Technology, and Grant-in-Aid for Outstanding Research Group Support Program in Nagoya City University Grant Number 2401101.

Conflict of interest

We declare that no conflict of interest.

Data availability

The data presented in this study are included in the body and figures of the paper. Corresponding author will respond to any relevant queries.

Author contributions

Conceptualization: Dipankar Chandra Roy, Kenji Ono, Masumi Suzui

Funding acquisition: Kenji Ono

Investigation: Dipankar Chandra Roy, Kenji Ono, Katsumi Fukamachi

Supervision: Masumi Suzui

Visualization: Dipankar Chandra Roy, Kenji Ono

Writing – original draft: Dipankar Chandra Roy, Kenji Ono, Masumi Suzui

Writing – review & editing: Masumi Suzui

Ethical approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

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