Propylene glycol and vegetable glycerin e-cigarette aerosols impact mucociliary function and cause cytotoxicity in human airway epithelium

Abstract

Electronic cigarettes (e-cigarettes) have emerged as a “healthier alternative” to conventional cigarettes, gaining popularity due to their perceived reduced harm and modern appeal. Nicotine, a common psychoactive substance found in e-cigarettes, has been shown to cause irritation and inflammation in the airways. However, the effects of e-cigarette aerosols, particularly the base components such as propylene glycol (PG) and vegetable glycerin (VG), on the airways, are less well understood. We hypothesized that PG and VG aerosol exposure would negatively impact mucociliary clearance and induce an inflammatory response in airway epithelial cells. To test this hypothesis, cultured human airway epithelial cells isolated from the inferior turbinate of healthy donors were exposed to an e-cigarette aerosol containing PG and VG. Exposure led to an increase in ciliary beat frequency (CBF), a key factor in mucociliary clearance. An LDH cytotoxicity assay revealed an increase in cell damage following exposure. Taken together, this work suggests that e-cigarette aerosol generated from carrier substances is not innocuous.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-33023-5.

Introduction

The increasing prevalence of electronic cigarettes (e-cigarettes) has sparked significant public health discussion concerning their safety and overall physiological impact. Marketed as a modern and customizable alternative for nicotine delivery, e-cigarettes have rapidly gained popularity worldwide, particularly among young adults and former smokers^1,2. Central to this rise is the perception that e-cigarettes deliver fewer harmful substances than combustible cigarettes^3–5. However, the long-term impacts of e-cigarette use on human health remain poorly understood, especially at the cellular level.

The airway epithelium serves as a primary defense mechanism, protecting the respiratory tract from environmental threats, pathogens, and particulate matter^6,7. Key to its protective role is mucociliary clearance and the regulation of inflammation⁸. The airway epithelium is composed of different cell types, and in vivo, ciliated cells work with various secretory cells to generate a mucociliary escalator- a mechanism where mucus traps inhaled particles and debris, and coordinated ciliary motion propels them out of the airways^8,9. Disruptions in these processes, often associated with the inhalation of harmful substances, can increase the risk of respiratory diseases such as chronic obstructive pulmonary disease (COPD), asthma, and bronchitis⁸. With the rising prevalence of e-cigarette use, it is crucial to comprehend how repeated exposure to their aerosolized components influences epithelial cell function.

Among the key components of e-cigarette aerosols are propylene glycol (PG) and vegetable glycerin (VG), substances generally regarded as safe for ingestion but less well understood when inhaled in aerosolized forms¹⁰. These ingredients, often combined with nicotine and flavoring agents, produce distinct exposure profiles that are markedly different from those of conventional cigarette smoke. Existing literature highlights the potential cytotoxic, pro-inflammatory, and oxidative stress-related impacts of these components on airway epithelial cells, which play a crucial role in maintaining respiratory homeostasis^9,11–13. However, most studies to date have focused on acute or single-exposure models, leaving the sub-acute effects of repeated PG/VG aerosol exposure, with and without nicotine, less well characterized^14,15. Given the rising prevalence of e-cigarette use and the frequency of daily exposure among users, it is essential to understand how repeated, short-term exposures influence epithelial cell function and mucociliary defense mechanisms.

To address this gap, the present study investigates the impact of sub-acute (7-day) exposure to PG/VG aerosols, with and without nicotine, on human airway epithelial cells cultured at the air–liquid interface. Using a controlled in vitro exposure system, we assessed changes in ciliary beat frequency (CBF), cell viability, and inflammatory cytokine production to evaluate how repeated e-cigarette aerosol exposure affects epithelial physiology. By characterizing these cellular and molecular responses, this work contributes to a more comprehensive understanding of the biological effects of e-cigarette aerosols and provides context for interpreting their potential respiratory health implications.

Materials and methods

Primary nasal cell culture

Human Nasal epithelial cells (HNECs) were obtained from nasal brushings of healthy individuals (ages 24 to 58 years; 5 female and 3 male) and cultured as previously described^16–18. Cells were obtained and studied under local REB-approved protocols (SickKids), and donors or substitute decision makers signed consent forms. Briefly, frozen passage number 1 nasal cells were seeded onto a collagen-coated flask and expanded to 70%–80% confluence at 37 °C in a 5% CO2 setting. Passage number 3 (P3) cells were subsequently seeded on collagen-coated transwells (6.5 mm diameter, 0.4 μm pore size, Corning) at a density of 10⁵ per insert. P3 cells allow for cell expansion (i.e. maximizes donor cell numbers) but no further as in our experience the cells lose their phenotype and ability to expand with further population doublings. Cells were maintained in PneumaCult EX + media until confluent and changed to a basal differentiation media (PneumaCult ALI; StemCell Tech) at air-liquid interface (ALI). Media was changed every other day in the basolateral compartment until 28 days, after which the cells were well-differentiated and had a ciliated phenotype. All donors were non-smokers.

In vitro vaping exposure system

For in vitro exposure experiments, the Vaping Product Exposure System (VaPES) was obtained through a collaboration with Dr. Ilona Jaspers, Dr. Phillip Clapp, and Kevin Schichlein¹⁹. VaPES is an e-cigarette aerosol exposure platform that uses episodic, vacuum-derived airflow to activate an e-cigarette and deliver uniform, automated deposition of aerosolized e-liquid onto cultured airway epithelial cells within an incubator. Epithelial cells were exposed to a 50:50 (v/v) mixture of propylene glycol (PG) and vegetable glycerin (VG), both with and without nicotine, using the VaPES system to produce a 6.5-second puff every 5 min, totaling 40 puffs per day for seven consecutive days. Aerosols were generated using a commercially available “flavorless” STLTH device (Device Type C; STLTH Vape, Canada). Both the PG/VG-only and the 2% (20 mg/mL) nicotine conditions used manufacturer-supplied, flavorless STLTH pods to ensure consistency across exposures. A vacuum was applied at a rate of 10 L/min to achieve consistent activation and aerosol flow to the air–liquid interface (ALI) culture chamber. In terms of rationale, we picked the duration to allow for the vapor to clear, the flow rate of each exposure chamber was maintained at ~ 2.8 L/min, as defined by our collaborators who designed the device. Puff duration of 6.5 s was selected to ensure even distribution across all chambers. To minimize the risk of device overheating, sufficient intervals between puffs were maintained. No signs of overheating (such as device malfunction, visible coil damage, or altered aerosol characteristics) were observed throughout the experiments. The device temperature was not measured. Between exposure sessions, an afternoon interval was included to allow outcome measures to be collected within a standard workday. Vaping behaviors in the real world are highly heterogeneous, influenced by user, device, and e-liquid factors. The goal of this study was not to model this variability but rather to assess the impact of standardized e-liquid aerosol exposures on airway epithelial cell function under controlled and reproducible conditions. The control group was exposed to vacuum airflow only. Basolateral media was changed each day after exposure, and fresh media (~ 750uL) was added, while the apical surface remained unwashed throughout the exposure period.

Ciliary beat frequency measurements

All experiments were conducted with cells at room temperature (RT) for imaging and incubated at 37 °C with 5% CO2 pre- and post-treatment. Immediately after exposure, ciliary beat frequency (CBF) was measured at 40x magnification using oil immersion on the inverted epifluorescence microscope Nikon Eclipse Ti2-E with a high-speed monochromatic digital camera (Hamamatsu ORCA-Fusion BT) at a sampling rate of > 120 frames per second and a resolution of 430 × 430 pixels of the ciliary layer from an en face viewpoint. Immediately after completion of exposure on day 7, 40X magnification videos were taken again, focusing on the ciliary layer. Five videos were taken- in the center of the insert, the north, south, east, and west quadrants. Upon completion of each set of experiments, the ciliary layer of each video was enhanced via a contrast enhancement tool and exported as TIFF files in Volocity 6.3. Video images were analyzed using MatLab application program Cilia-X developed by Nicolas Simonnet at Epithelix. Analysis was given as the raw Hz values generated.

Cytokine measurements

Basolateral media was collected every 24 h post-exposure for cytokine analysis. Following collection, an equal volume of fresh, pre-warmed media was added to maintain consistent basolateral volume and nutrient availability. Media collections were stored at -80 °C until analysis. Cytokine expression was quantified using Luminex bead-based multiplex assay (Sigma-Aldrich, Milwaukee, WI, USA) according to the manufacturer’s protocol.

Immunofluorescence and confocal microscopy

After the 7-day aerosol exposure, cells were fixed with 4% formaldehyde for 5 min at room temperature. After washing cells 3 times in PBS with 0.1% Triton X-100, cells were blocked with 3% BSA for 30 min. Cells were incubated with primary antibody (MUC5AC, β-tubulin, or ZO-1) for 1 h at room temperature. Following washes with PBS 0.1% Triton X-100, cells were incubated with a secondary antibody for 1 h at room temperature. Following washes with PBS 0.1% Triton X-100, cells were stained with DAPI (Sigma) and visualized by confocal microscopy (Nikon).

LDH cytotoxicity assay

Cytotoxicity was assessed by quantifying lactate dehydrogenase (LDH) levels in apical wash samples obtained following the 7-days aerosol exposure using the CyQUANT™ LDH Cytotoxicity Assay kit following manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA, USA, Cat# C20300).

Western blotting

Cell lysates were collected after exposure with RIPA lysis buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 10 mM DTT) and separated using 4–20% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, blocked for 1 h in 5% milk in TBS,

and probed overnight with primary antibody at 4 °C to detect target proteins. After washing, blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, washed, and then visualized by advanced chemiluminescence (Bio-Rad). The following antibodies were used: ZO-1 (ThermoFisher; 40-2200, 1:1000), Occludin (Invitrogen; 33-1500, 1:500), Claudin (Invitrogen; 51-9000, 1:500), β-actin (Abcepta; AM1021B, 1:10000). Following background correction, band intensities was quantified using Image Lab software and normalized to the loading control. Full-length blots are unavailable because the membranes were sectioned prior to antibody hybridization.

Statistical analysis

The presented data are mean ± SEM and were analyzed using GraphPad Prism software (Version 10). One-way ANOVA with Tukey’s Multiple Comparison post hoc tests were performed on all data with more than two datasets for comparison. All data passed the D’Agostino & Pearson normality test. One-way repeated measures ANOVA with Geisser-Greenhouse correction was performed to analyze the data. A P value of less than 0.05 was considered statistically significant. Additional information on statistical analyses is described in the figure legends.

Results

The effect of PG/VG aerosol exposure on the ciliary beat frequency

We exposed primary HNECs to PG/VG e-cigarette aerosols for 7 days to study sub-acute response to repeated exposure, with cells receiving 40 puffs per day. HNECs exposed to PG/VG aerosols (with or without nicotine) exhibited a significant increase in CBF compared to the control group (Fig. 1).

Fig. 1.

PG/VG aerosol exposure influences CBF in primary human nasal epithelial cells. Ciliary beat frequency (CBF) is significantly increased after seven days of exposure to PG/VG (with or without 2% nicotine) when compared to air-exposed controls. One-way ANOVA with Tukey’s multiple comparison test was performed (**P < 0.01, N = 8).

The effect of PG/VG aerosol exposure on nasal epithelial cell cytotoxicity

Apical wash collections were assessed for LDH activity after 7 days of exposure as a measure of cytotoxicity (cell lysis). The PG/VG exposed group showed significantly higher LDH when compared to the control group. There was no significant difference in LDH between the PG/VG and PG/VG + nicotine groups (Fig. 2).

Fig. 2.

Exposure to PG/VG e-cigarette aerosol caused cytotoxicity in HNECs. Seven-days exposure to PG/VG (with and without 2% nicotine) led to increased cytotoxicity as assessed by lactate dehydrogenase (LDH) release into the apical space. One-way ANOVA with Tukey’s multiple comparison test was performed (*P < 0.05, N = 8).

The effect of PG/VG aerosol exposure on the inflammatory response

IL-8, used as a marker for inflammation, was measured in the basal media following a 7-day exposure to PG/VG alone and PG/VG + nicotine (Fig. 3). The data show that there was no statistically significant difference in IL-8 between the control and PG/VG groups. However, a significant difference in IL-8 was observed between the PG/VG + nicotine group compared to the control group.

Fig. 3.

Exposure to PG/VG aerosol alone does not lead to a significant increase in IL-8 in HNECs. A significant change in IL-8 was not detected after sub-acute exposure to PG/VG aerosols (with or without 2% nicotine). One-way ANOVA with Tukey’s multiple comparison test was performed (*P < 0.05, N = 8).

The effect of PG/VG aerosol exposure on cell morphology and tight junction marker expression

Figure 4 provides a visual representation of the cell morphology of HNECs following exposure.

Fig. 4.

Exposure to PG/VG aerosol does not alter cell morphology or the expression of tight junction proteins. (a) Following differentiation, cells were fixed and immunostained post-exposure. Cells were incubated against top: MUC5AC (red; goblet cells), B-tubulin (green; ciliated cells), and DAPI (cyan; nuclei); bottom: ZO-1 (red; tight junctions) and DAPI and imaged using a Nikon A1R Confocal Microscope at 40X. (b) Western blots of Claudin, b-actin, Occludin, and ZO-1 after exposure to PG/VG and PG/VG + 2% nicotine. These data were from a representative immunoblot undertaken on two separate donors. (c) Quantification of ZO-1. (d) Quantification of Claudin. (E) Quantification of Occludin. One-way ANOVA with Tukey’s multiple comparison test was performed (N = 6).

to different treatments: control, PG/VG, and PG/VG with nicotine. The cells were stained for goblet cells, ciliated, and tight junctions. The ZO-1 marker highlights tight junctions, which play a crucial role in maintaining epithelial integrity and barrier function by regulating permeability and intercellular adhesion. Disruptions in these junctions can compromise epithelial defense mechanisms, potentially leading to increased susceptibility to environmental insults. To further assess potential changes in tight junction integrity, western blot analysis was performed to examine the expression of ZO-1, Claudin, and Occludin. These proteins collectively contribute to epithelial barrier function, and alterations in their expression can be indicative of changes in cellular structure or permeability. We did not see a significant difference in these markers.

Discussion

This study investigated the effects of PG/VG e-cigarette aerosols on human nasal epithelial cells (HNECs), focusing on mucociliary function, cytotoxicity, and inflammation. Our results demonstrate that exposure to PG/VG aerosols leads to a significant increase in ciliary beat frequency (CBF) after 7 days. Additionally, we observed increased cytotoxicity, as indicated by elevated LDH release, suggesting potential cellular stress or damage. While PG/VG alone did not significantly alter IL-8 secretion, the presence of nicotine in the aerosol led to a significant increase in IL-8 levels, indicating an inflammatory response. Notably, there were no detectable changes in cell morphology or tight junction protein expression (ZO-1, Claudin, and Occludin) following exposure. However, it is possible that their subcellular localization or function was altered. Future work will focus on clarifying this.

To evaluate the effects of PG/VG aerosol exposure on the mucociliary function of HNECs, CBF was measured as a determinant of mucociliary clearance rate. The results showed that both the PG/VG alone (nicotine-free) and the nicotine-containing groups exhibited a significant increase in CBF. This finding suggests that PG/VG aerosol exposure, regardless of nicotine content, can enhance CBF in HNECs. The increased CBF may represent a protective mechanism to maintain mucociliary clearance in response to an exogenous stress²⁰. Mucociliary clearance is a critical defense mechanism of the respiratory system, and the observed increase in CBF might be an attempt by the cilia to counteract the potential negative effects of aerosol exposure and ensure that mucus and trapped particles are effectively removed from the respiratory tract^7,21. Interestingly, similar increases in CBF have been observed with cigarette smoke exposure. Studies have shown that exposure to cigarette smoke initially increases CBF as a short-term adaptive response to clear inhaled toxins and particles^20,21. Additionally, CBF has been studied in models of cystic fibrosis (CF)²². Montoro et al. observed an increase in CBF in Foxi1 knockout mouse epithelia, which results in the loss of CFTR expression and disrupts airway fluid and mucus physiology similar to CF²². This suggests that the compensatory mechanism aimed at enhancing MCC is present even in diseased states. However, while the initial response to PG/VG aerosol exposure is an increase in CBF, the long-term impact may be different. Indeed, there are data to show that a drop in CBF seen immediately after an exposure can return to baseline values after a further period of observation²³. Smoking exposure has also been linked to physical changes in cilia (reduced height or number)²⁴; these outcomes were not studied in our current work but are a focus for future experiments.

Cytotoxicity was assessed by measuring LDH activity in apical wash collections after 7 days of exposure to PG/VG aerosol. The control group exhibited minimal cell damage, while the PG/VG groups demonstrated significantly elevated LDH levels, suggesting that PG/VG exposure induces cytotoxicity. There was no significant difference in LDH release between the PG/VG and PG/VG + nicotine groups suggesting that e-cigarette aerosols containing PG/VG are sufficient to contribute to cell damage. These results align with previous studies that have highlighted the potential for PG and VG to disrupt cellular membranes, thereby increasing membrane stiffness and compromising cellular integrity¹⁵.

While exposure to nicotine-containing group led to a significant increase in IL-8 levels, PG/VG alone did not significantly alter IL-8 levels. IL-8 is a key neutrophil cytokine, and chemoattractant and elevated levels may lead to an enhanced inflammatory milieu, potentially leading to tissue damage and chronic inflammation. Of interest, while IL-8 would serve to attract neutrophils, there are data that e-cigarette aerosols in turn directly limit neutrophil function leading to an impaired innate immune state^8,25. This may predispose to lung injury or damage both through persistent inflammation and a susceptibility to infection. Furthermore, the role of IL-8 in inflammation extends beyond neutrophil recruitment. IL-8 can influence other immune cells, such as T cells and monocytes, and affect processes like angiogenesis and wound healing²⁶. Therefore, monitoring IL-8 levels and understanding its regulation in response to e-cigarette aerosols may provide valuable insights into the potential health risks associated with vaping, particularly with prolonged use.

Despite concerns that PG/VG exposure might compromise epithelial barrier integrity, our results showed no significant changes in cell morphology or tight junction protein expression over the time period and dose we studied. While these data provide insight into molecular changes associated with e-cigarette aerosol exposure, the expression levels of tight junction proteins alone cannot confirm whether overall epithelial integrity was maintained or disrupted. Additional functional measurements, such as transepithelial electrical resistance or permeability assays, would be required to directly evaluate barrier function. It should also be noted that heating PG/VG leads to the generation of thermal degradation byproducts²⁷, and these byproducts will be generated in a heterogenous fashion based on e-liquid parameters, device parameters and host characteristics. Thus, while we excluded flavours in the current study to reduce some heterogeneity and enhance generalizability of findings, we also appreciate that vaping is inherently heterogenous as an exposure, even in the absence of flavours.

Limitations

Several limitations should be considered when interpreting the findings of this study. First, the baseline ciliary beat frequency (CBF) observed in this model was lower than expected, which may reflect the absence of a temperature-controlled (heated) stage during imaging. Because CBF is highly temperature-dependent, lower culture or imaging temperatures could have contributed to reduced baseline measurements. Future experiments will address this by incorporating a heated stage and assessing mucociliary clearance (MCC) more directly using fluorescent bead tracking. Second, our conclusions regarding mucociliary function are limited by the absence of direct MCC measurements. While our findings suggest that e-cigarette aerosol exposure may impact MCC, further studies are needed to confirm whether these changes translate to a reduction in overall clearance capacity. Based on prior literature, including work in cystic fibrosis and related models, we suspect that the changes we observed in CBF would lead to diminished MCC; however, this remains to be experimentally validated. With respect to cell viability, the use of this primary airway epithelial cell model presents challenges as subtle changes in cytotoxicity are difficult to detect. We relied on apical release of LDH as a marker of cell lysis, but future work will explore alternate toxicity outcomes. Additionally, to account for airflow-related variables, future studies will include an incubator control as a baseline. Furthermore, we did aim for a brief and physiologically relevant exposure and so we were not expecting to produce large changes in barrier integrity or cell death, as such effects would be inconsistent with the relatively mild acute responses observed in most e-cigarette users. Nonetheless, the observed increase in LDH release suggests some degree of cellular damage, which warrants further investigation under refined exposure conditions. Fourth, while interleukin-8 (IL-8) is an important pro-inflammatory cytokine, it represents only one facet of the airway inflammatory response. Future studies should include a broader panel of cytokines and chemokines, as well as other inflammatory markers, to provide a more comprehensive understanding of e-cigarette aerosol–induced immune activation. Fifth, the absence of direct aerosol deposition measurements limits our ability to precisely correlate in vitro dosing with human exposure. Finally, although both male and female donors were included, the small and unbalanced sample size limits the detection of sex-specific effects, as reported in previous studies^28,29.

In conclusion, this study demonstrates that the base components of e-cigarette aerosols, propylene glycol (PG) and vegetable glycerin (VG), are not biologically inert when aerosolized and can impact airway epithelial cells. We observed an increase in ciliary beat frequency following PG/VG exposure, a response seen in cell models of smoking and CF, and we observed higher LDH in apical washings suggesting cytotoxic effects. These findings highlight the importance of considering exposure duration and method when evaluating the safety of e-cigarette components. Overall, our results contribute to the growing body of evidence indicating that e-cigarettes are not a risk-free alternative to traditional cigarettes. These findings highlight the need for continued investigation and regulatory oversight to ensure the safety of inhaled vaping components.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

K.M. wrote the main manuscript text and prepared Figs. 1, 2, 3 and 4a-b. W.D. provided guidance on experimental setup and statistical analysis, and prepared figures 4c-e. S.T. provided support with cell culture work. K.S. provided technical guidance on experimental setup. I.L. and P.C. informed the methods. T.J.M supervised all aspects, obtained funding and conceptulized the project. All authors read and approved the final manuscript.

Funding

This work was supported by Physician Services Incorporated (PSI) Foundation (22 − 05).

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary file. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Uncropped western blots can be found in the Supplementary file.

Declarations

Competing interests

The authors declare no competing interests.