With the increasing use of e-cigarettes, research into their potential pulmonary toxicity has intensified. While an increasing number of studies have focused on the potential cytotoxicity of e-cigarettes, the interactions of e-cigarette vapor with the pulmonary surfactant have received less attention and remain largely unknown. The current study aimed to fill this gap of knowledge and provide an understanding and comparison of the potential disruptive effects of e-cigarette vapor and cigarette smoke on lateral structure and interfacial properties of the pulmonary surfactant.

Studies using calf lung surfactant revealed that e-cigarette vapor does not affect surfactant interfacial properties regardless of the e-liquid flavoring (Fig. 2). The lack of effects from e-cigarette vapor on Infasurf® can be explained by considering the components in e-cigarette vapor. The primary components of e-cigarette vapor are propylene glycol, glycerol, and nicotine [38, 39]. Propylene glycol and glycerol are both hydrophilic and are thus likely to remain in the aqueous subphase and not disturb the lipid film at the air-water interface. On the other hand, nicotine has a very minor effect on surfactant properties as shown in Fig. 5a. Thus, none of the components of e-cigarette smoke are expected to cause significant disruptions to surfactant films. To the best of our knowledge, there has only been one previous study on the interactions of e-cigarette vapor with lung surfactant [38]. In this study, Davies and colleagues used a mixture of dipalmitoyl phosphatidylcholine, phosphatidyl glycerol, and phosphatidic acid (DPPC/POPG/PA, 69/20/11, w/w/w) to mimic lung surfactant, and reported a slight reduction (~10 mN/m) in the surface pressure of this model after exposure to e-cigarette vapor [38]. The potential mechanisms of this detrimental effect were proposed to be nicotine penetration in the surfactant monolayer, lipid peroxidation by free radicals in the vapor, and/or hydrolysis of surfactant phospholipids by nitrosamines in smoke [38]. In the current study, pure nicotine showed a very minor effect on the surface pressure isotherm of Infasurf®. While free radicals [40] and nitrosamines [39] have both been reported in e-cigarette vapor, any potential effects from these components on Infasurf® in the current study was minor. It should be noted, however, that the effects of these chemicals on a complex surfactant such as Infasurf® might be very different compared to the simpler model used by Davies and colleagues [38].

In contrast to e-cigarette vapor, cigarette smoke significantly disrupted surfactant interfacial properties. The disruptive effects of e-cigarette vapor and cigarette smoke correlated with their surface activity. E-cigarette vapor, which did not affect the surface pressure, was not surface active, while cigarette smoke was highly surface-active (Fig. 2e). This correlation between surface activity and surfactant disruption suggests that surfactant inhibition by smoke occurred through the competitive adsorption mechanism [33]. Based on this mechanism, surface active components, in this case from cigarette smoke, are capable of adsorption to the air-water interface and compete with surfactant molecules for space. Adsorption of non-surfactant molecules hinders surfactant adsorption at the air-water interface and interferes with the enrichment of the surface with highly saturated lipids, thereby inhibiting the ability of the surfactant to reduce the surface tension (i.e. increase the surface pressure). A similar mechanism has been shown to be the underlying principle for surfactant inhibition by albumin [34, 41, 42]. Albumin is a surface-active protein and can reach surface pressure values higher than 30 mN/m upon compression [34, 41]. Due to its surface-activity, albumin adsorbs to the air-water interface, interfering with surface adsorption of surfactant molecules and eventually leading to a reduction in the maximum surface pressure achievable by surfactant [34, 41, 42].

This competitive adsorption mechanism is further supported by AFM images. The plateau in Infasurf® surface pressure at ~40 mN/m is the start of the process where unsaturated lipids collapse into multilayers, leaving a surface enriched in highly saturated lipids [25, 29]. This process was observed in the current study where the height of the surfactant structures changed from <1 nm at surface pressure values of 20 and 30 mN/m to 4 and 6 nm at surface pressure values of 40 and 50 mN/m, respectively, due to the presence of the multilayers. Addition of e-cigarette vapor or cigarette smoke did not significantly affect the height of these structures, suggesting that the multilayer formation process was not affected. However, the surface area covered by unsaturated multilayers highly increased as a result of exposure to vapor and smoke. This increase in surface area was quite drastic in the case of cigarette smoke (Fig. 4b). The significant increase in the area of unsaturated lipids consequently hinders the enrichment of the surface by saturated lipids, leaving a surface with a high level of unsaturated lipids that cannot reach high surface pressure values. Particles in cigarette smoke have been reported to have a mass median diameter of 380 nm [43]. Thus, given the average height of the AFM structures, it is unlikely that a large portion of smoke particles have directly penetrated the air-water interface. It appears more likely that water-soluble components from the smoke have adsorbed to the air-water interface, likely partitioning with unsaturated lipids, resulting in an increase in the surface area of unsaturated multilayer phase. A similar phenomenon has been reported for albumin molecules that partition into disordered lipid phases in bovine lipid extract surfactant [34].

While exposure to cigarette smoke is known to alter the level of surfactant lipids and proteins in animals [22, 44] and in humans [45], the effects of cigarette smoke on surfactant interfacial properties remain understudied. Early studies with bronchoalveolar lavage (BAL) have shown that cigarette smoke can affect surfactant function and respreadability [21, 22]. However, as noted by Bringezu and colleagues [23], interfacial studies with BAL are difficult due to the large variability associated with the extraction of BAL and isolation of surfactant and there is a need for more mechanistic studies. To the best of our knowledge, only two detailed mechanistic studies exist on the surfactant inhibitory effects of cigarette smoke [23, 24], both using environmental tobacco smoke (ETS), a combination of smoke from the smoldering cigarette and the smoke inhaled by the smoker (note that only the latter is being examined in the current study) [46]. In one study, ETS was mixed with a (DPPC/POPG/PA, 69/20/11, w/w/w) surfactant model, resulting in slight changes in surfactant respreading and maximum surface pressure values [23]. These effects were attributed to smoke particulates removing the unsaturated POPG to the subphase resulting in a highly saturated surfactant which cannot efficiently respread after compression [23]. While this mechanism seems quite plausible for the DPPC/POPG/PA model, it is less likely to be significant for Infasurf®, as Infasurf® only has 5% POPG [27] compared to 20% in the model of Bringezu and colleagues [23]. In addition, since saturated lipids are the driving force for reaching high surface pressure values, removal of unsaturated lipids should result in only minor effects in the ability of the surfactant to reach high surface pressure values; however, the surfactant inhibition caused by cigarette smoke in the current study is quite drastic. A second study on ETS effects on more complex, natural surfactants proposed a slightly different mechanism [24]. In this case, ETS exposure was shown to alter the lateral distribution of the porcine derived surfactant, Curosurf®, reducing the size of the ordered lipid domains and resulting in a surface that was enriched in unsaturated lipids and less effective in increasing the surface pressure [24]. The latter mechanism better aligns with the findings of the current study where cigarette smoke particles have resulted in an increase in the surface area of unsaturated multilayers.

Our tensiometric studies with the most abundant components in cigarette smoke clearly suggest that tar (i.e. the product of burning) is the main disruptive agent to surfactant interfacial properties (Fig. 5). These studies were performed with the components of highest concentration in cigarette smoke, based on the certificate of analysis of 1R6F cigarettes [47]. The smoke composition of 1R6F cigarettes generated by the University of Kentucky Center for Tobacco Reference Products closely mimics the smoke composition of other research cigarettes produced and analyzed by the same source and by others [48,49,50]. While we cannot rule out the presence of other surfactant inhibitory compounds in cigarette smoke, all other chemicals in cigarette smoke were at least one order of magnitude lower in concentration compared to those tested. The presence of a high amount of tar (as evidenced by Fig. 1b), suggests that perhaps modifications to the cigarette filters might be able to reduce some of these inhibitory compounds. These findings also explain why e-cigarette vapor was not detrimental to surfactant: e-cigarette is a result of e-liquid vaporization, but not burning. Little to no tar is expected from vaporization, which explains the lack of disruptive effects.

It should be noted that while the present study suggests that e-cigarette vapor does not directly affect the interfacial properties of lung surfactant, both e-cigarette vapor and cigarette smoke could impact lung surfactant function via indirect mechanisms. Such mechanisms could include protein/lipid oxidation and alterations in the expression or release of key surfactant components. E-cigarette vapor contains reactive free radicals [40] and reactive oxygen species [51, 52]. In addition, exposure to e-cigarette vapor has been shown to increase the expressions of genes involved in oxidative stress pathways of human bronchial epithelial cells [7]. While the downstream effects of such events on surfactant production and secretion are not yet known, it is quite plausible that exposure to reactive and oxidative species in e-cigarette vapor and increased oxidative stress might lead to oxidation of surfactant lipids and proteins and/or affect surfactant production or secretion. On the other hand, cigarette smoke has been shown to cause oxidative injury in type II alveolar cells [53, 54] and reduce the production and alter the secretion of surfactant phophospholipids by these cells [22, 55]. Thus, cigarette smoke is likely to inhibit surfactant function through both direct and indirect mechanisms while e-cigarette vapor might be capable of indirect surfactant disruption.

It is important to note some of the limitations of the current study and put the results in greater context. Here, exposure to smoke and vapor particulates was performed by bubbling the smoke and vapor in the subphase. This method has been previously used to study smoke cytotoxicity [55, 56] and was employed due to challenges in reproducible aerosol generation and quantification of the deposited particles, some of which have been addressed elsewhere [18, 57]. However, surfactant exposure to aerosols is a more physiologically-relevant exposure method and needs to be considered for future studies. Another limitation of the current study is that experiments were performed at room temperature; this is due to the fact that increased temperature reduces the size of surfactant domains, making them difficult to discern particularly at low surface pressure values. It should also be noted that the results presented in this study only focus on one aspect of potential e-cigarette toxicity. Thus, lack of surfactant disruption by e-cigarette vapor, does not suggest that e-cigarettes are safe. Increasing reports are emerging on the cytotoxicity, xenotoxicity, and inflammatory effects of e-cigarettes, which will help evaluate whether e-cigarette use will lead to other potential health effects. In addition, our study was focused on one brand and a limited number of e-liquid flavors and potential toxicity by other e-cigarettes cannot be ruled out. On the other hand, our results with conventional cigarettes further emphasize the association between cigarette use and respiratory toxicity. Changes in surfactant interfacial properties are associated with a number of respiratory diseases and can result in increased work of breathing and impaired gas exchange. While the effects of conventional cigarette smoke on surfactant production have been studied in the past [22, 44, 45], smoke effects on surfactant function and interfacial properties have received less attention and the results from this study help elucidate the disruptive effects of cigarette smoke on lung surfactant function and identify the component that is most harmful to surfactant interfacial properties.