Lanthanum Strontium Manganite Nanoparticles: Airway Epithelial Cell Toxicity and Mucus Secretion Impact

Introduction

Lanthanum strontium manganite (LSM) possesses a perovskite crystal structure with the general formula La_1−xSr_xMnO₃, where x reflects the strontium doping level. This material can be processed as a powder for tape casting, air/thermal/plasma spray, and fuel‑cell applications. LSM’s high surface area and electrochemical performance make it a leading electrode candidate for SOFCs, which are central to the emerging hydrogen economy. Despite extensive studies on LSM’s mechanical and electrical properties, its biological interactions remain underexplored. Many nanoparticles are known to elevate mucus secretion and contribute to respiratory disorders. Emerging evidence suggests LSM may also serve as a drug carrier or imaging contrast agent, but its potential toxicity must be clarified before any biomedical deployment. This investigation evaluates LSM’s cytotoxic profile in primary tracheal epithelial cells, assessing ROS generation, mitochondrial integrity, apoptosis, and mucus secretion.

Results and Discussions

Nanoparticle Characterization and Cell Viability

Surface morphology was examined by scanning electron microscopy (SEM). LSM nanoparticles displayed a coarse surface and aggregated into clusters ranging from ~35 nm to >200 nm (Fig. 1). Cell viability was measured with the CCK‑8 assay. Viability sharply decreased between 50 and 100 µg mL⁻¹ but plateaued thereafter, remaining stable at higher concentrations (Fig. 2).

ROS Production and Mucin Release

ROS levels were quantified using CM‑H₂DCFDA fluorescence. Increasing LSM concentration did not significantly elevate ROS; the 250 µg mL⁻¹ point showed the largest deviation, yet remained statistically insignificant (Fig. 3). Mucin secretion, measured by enzyme‑linked lectin assay (ELLA), decreased progressively with higher LSM doses, dropping to ~40 % of control at 500 µg mL⁻¹ (Fig. 4).

Mitochondrial Integrity and Apoptosis

JC‑1 staining revealed mitochondrial depolarization at concentrations ≥100 µg mL⁻¹ (Fig. 5). Despite this, cytochrome C release and caspase‑3 activation only showed modest reductions, indicating limited apoptosis induction (Fig. 6).

Conclusions

This first assessment of LSM toxicity in airway epithelial cells demonstrates negligible cytotoxicity and apoptosis, but a pronounced inhibition of mucus secretion. While ROS generation and mitochondrial damage were minimal, the reduced mucus release may impair airway clearance and warrants careful evaluation for future applications in energy and biomedical fields.

Materials and Methods

Culture of Tracheal Primary Cells

Primary bovine tracheal epithelial cells were isolated per established protocols, cultured in serum‑free medium (SFM) supplemented with EGF 1–53 and bovine pituitary extract (BPE), and maintained on collagen‑coated plates at 37 °C, 5 % CO₂. Cell density was monitored via trypan blue exclusion.

Cell Preparation and Viability Assay

Cells were seeded in collagen‑coated 96‑well plates (5 × 10⁴ cells/well) for viability assays and in 4‑well plates (5 × 10⁵ cells/well) for calcium, ROS, and mitochondrial studies. After 24 h incubation, media were replaced with PBS, then with LSM suspensions. Cell viability was quantified with the CCK‑8 assay (WST‑8 conversion to formazan) and expressed as a percentage of untreated control.

Lanthanum Strontium Manganite Nanoparticles

LSM (La_0.15Sr_0.85MnO₃, 35 nm, 99.5 %) was sonicated and tested at 500, 250, 100, and 50 µg mL⁻¹. Concentrations were chosen to match typical TiO₂ nanoparticle exposures reported in the literature.

Scanning Electron Microscopy

Particles were drop‑cast on silicon wafers, air‑dried, and imaged with a Zeiss Gemini SEM to confirm size distribution.

Reactive Oxygen Species Assay

ROS production was assessed using CM‑H₂DCFDA fluorescence after 15 min LSM exposure. Fluorescence intensity was compared to untreated controls.

Mitochondrial Damage Measurement

JC‑1 dye was used to evaluate mitochondrial membrane potential; red/green fluorescence ratios were measured by fluorescence microscopy at 10‑min intervals.

Mucin Secretion and ELLA

After 15 min LSM exposure, supernatants were collected, centrifuged, and assayed for mucin via ELLA using WGA‑HRP and TMB substrate. Absorbance was read at 450 nm.

Apoptosis Marker Assays

Cell lysates were prepared with RIPA buffer and probed for cytochrome C and active caspase‑3 using ELISA protocols. Secondary HRP‑conjugated antibodies facilitated detection.

Statistical Analysis

Data are presented as mean ± SD from at least three independent experiments. One‑way ANOVA determined significance (p < 0.05).

Abbreviations

BPE

Bovine pituitary extract

ELISA

Enzyme‑linked immunosorbent assay

LSM

Lanthanum strontium manganite

ROS

Reactive oxygen species

SFM

Serum‑free medium

SOFCs

Solar oxidized fuel cells