Mechanistic Insights into Sphingomyelin Nanoemulsions as Drug Delivery Systems for Non-Small Cell Lung Cancer Therapy

Abstract

Sphingomyelin nanoemulsions (SNs) are promising drug delivery systems with potential for treating challenging tumors, including non-small cell lung cancer (NSCLC), which has a poor prognosis and a 5-year survival rate below 5%. Understanding the toxicity mechanisms and intracellular behavior of SNs is crucial for optimizing their therapeutic application. This study aims to investigate the interaction between SNs and A549 lung adenocarcinoma cells, focusing on their cytotoxic effects and mechanisms of cellular toxicity. SNs were synthesized and characterized for size, surface charge, and stability. A549 cells were treated with varying concentrations of SNs, and cellular uptake pathways were assessed using inhibitors of energy-dependent processes. Cytotoxicity was evaluated through an alamarBlue assay to determine the IC₅₀ value after 24 h. Mechanisms of toxicity, including lysosomal and mitochondrial involvement, were examined using co-localization studies, mitochondrial membrane potential assays, and markers of apoptosis. SNs exhibited rapid cellular uptake via energy-dependent pathways. The IC₅₀ concentration for A549 cells was 0.89 ± 0.15 mg/mL, suggesting favorable cytocompatibility compared to other nanocarriers. At IC₅₀, SNs induced apoptosis characterized by lysosomal damage, mitochondrial membrane permeabilization, and the release of apoptotic factors. These effects disrupted autophagic flux and contributed to cell death, demonstrating potential for overcoming drug resistance. Resveratrol-loaded SNs showed enhanced cytotoxicity, supporting their application as targeted drug delivery vehicles. This study highlights the potential of SNs as efficient drug delivery systems for NSCLC therapy, offering insights into their cellular interactions and toxicity mechanisms. These findings pave the way for the rational design of SN-based therapeutic platforms for cancer and other mitochondria-related diseases.

Keywords: drug delivery; lysosomal escape; mitochondrial targeting; non-small cell lung cancer (NSCLC); sphingomyelin nanosystems; targeted cancer therapy.

Figure 1

A schematic of the formulation method using ethanol injection. SNs are composed of vitamin E and sphingomyelin.

Figure 2

The measurement of the stability of SNs in RPMI medium supplemented with 10% FBS and penicillin/streptomycin. Changes in diameter (nm) and PdI value over time are shown. Results are expressed against negative control (culture medium) as mean ± standard deviation of at least three independent assays.

Figure 3

Percentage of A549 cell viability upon 24 or 72 h incubation with SNs at 2.750–0.165 mg/mL of total lipid. IC₅₀ value at 24 h incubation is 0.89 ± 0.15 mg/mL; at 72 h, incubation is 0.63 ± 0.03 mg/mL. A dashed line indicates the 50% cell viability threshold, serving as a reference point for determining the effective concentration. Data are expressed as mean ± standard deviation of four independent assays. *: p < 0.05. **: p < 0.01.

Figure 4

Percentage of HEK293 cell viability upon 72 h incubation with SNs at 2.750–0.165 mg/mL of total lipid. IC₅₀ value at 72 h incubation is 1.133 ± 0.066 mg/mL. A dashed line indicates the 50% cell viability threshold, serving as a reference point for determining the effective concentration. Data are expressed as mean ± standard deviation of four independent assays.

Figure 5

The percentage of the cell population of A549 after 24 h incubation with RPMI supplemented with 10% FBS and penicillin/streptomycin (C-), SNs at a concentration equivalent to their IC₅₀. (A) Using Annexin V and PI assay by flow cytometry. The early apoptosis (Annexin V⁺, 7-PI⁻), the late apoptosis (Annexin V⁺, PI⁺), the alive cells (Annexin V⁻, PI⁻), the necrosis (Annexin V⁻, PI⁺). The results are expressed against a negative control (culture medium) as mean ± standard deviation of at least three independent assays. (B) Determined with CellEvent Caspase-3/7 Green Detection Reagent and SYTOX AADvanced assay by flow cytometry. The percentage of viable A549 cells in early apoptosis or late apoptosis/necrosis. ·: p < 0.1 *: p < 0.05. ****: p < 0.0001.

Figure 6

A549 incubated with non-toxic concentrations (0.176 mg/mL of total lipid) of the SNs (green) for 30 min (A), 1 h (B), 2 h (C) or 4 h (D). The results of three independent assays.

Figure 7

The effect of SNs on the lysosome of A549 cells after 2 and 24 h incubation. Confocal microscopy images (63×) showing the staining of nuclei in blue (Hoechst 33342) and lysosome in green (LysoTracker Green FM) of A549 cells incubated with the medium upon 2 h (A) and 24 h incubation (D) and with cytotoxic concentrations (IC₅₀) of SNs in red (Cy5) after 2 h (B) and 24 h of incubation (E). Zoom ×3 after 2 h of incubation (C) and zoom ×1.6 after 24 h of incubation (F). The co-localization of lysosome and nanoparticles is marked with white arrows. The results of three independent assays.

Figure 8

(A) The percentage of alive cells with functional lysosomes in A549 cells after 24 h incubation with SNs at a concentration equivalent to their IC₅₀, determined with Lysotracker Green and 7-AAD assay by flow cytometry. The results are expressed against negative control (culture medium) as mean ± standard. **: p < 0.01. The results of five independent assays. (B) The percentage of autophagosome formation on A549 cells after 24 h incubation with SNs at a concentration equivalent to their IC₅₀, determined with Autophagosome Detection Reagent assay by plate reader. The results are expressed against a negative control (culture medium) as mean ± standard. ****: p< 0.0001. The results of five independent assays.

Figure 9

The internalization of SNs on the mitochondria of A549 cells. Confocal microscopy images (63×) showing the staining of nuclei in blue (Hoechst 33342) and mitochondria in green (MitoTracker Green FM) after incubation with culture medium upon 2 h (A) or 24 h (D) and with cytotoxic concentrations of SNs in red (Cy5) upon 2 h (B) and 24 h (E) of incubation. The co-localization of mitochondria and nanoparticles is marked with white arrows. Zoom ×3 after 2 h of incubation (C) and zoom ×1.7 after 24 h of incubation (F). The results of five independent assays.

Figure 10

The effect of SNs on the mitochondrial membrane potential (ΔΨ_m) of A549 cells. The percentage of total cells with damaged and non-damaged ΔΨ_m upon 24 h incubation with cytotoxic concentrations (IC₅₀) of SNs are shown. The results are as mean ± standard deviation of at least three independent assays. *: p < 0.05.

Figure 11

Percentage of A549 cell viability upon 24-h incubation with SNs-RSV and free RSV at 0.003–0.05 mg/mL of total drug. Dash line represents the 50% cell viability threshold, serving as a reference point for determining the IC₅₀ value.