Pulmonary Surfactant Monolayers Research Articles

Investigation of Drug for Pulmonary Administration-Model Pulmonary Surfactant Monolayer Interactions Using Langmuir-Blodgett Monolayer and Molecular Dynamics Simulation: A Case Study of Ketoprofen.

Pulmonary administration is widely used for the treatment of lung diseases. The interaction between drug molecules and pulmonary surfactants affects the efficacy of the drug directly. The location and distribution of drug molecules in a model pulmonary surfactant monolayer under different surface pressures can provide vivid information on the interaction between drug molecules and pulmonary surfactants during the pulmonary administration. Ketoprofen is a nonsteroidal anti-inflammatory drug for pulmonary administration. The effect of ketoprofen molecules on the lipid monolayer containing 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phospho-rac-glycerol (DPPG) is studied by surface pressure (π)-area (A) isotherms and compressibility modulus (Cs-1)-surface pressure (π) isotherms. The location and distribution of ketoprofen molecules in a lipid monolayer under different surface pressures are explored by surface tension, density profile, radial distribution function (RDF), and the potential of mean force (PMF) simulated by molecular dynamics (MD) simulation. The introduction of ketoprofen molecules affects the properties of DPPC/DPPG monolayers and the location and distribution of ketoprofen molecules in monolayers with various surface pressures. The existence of ketoprofen molecules hinders the formation of liquid-condensed (LC) films and decreases the compressibility of DPPC/DPPG monolayers. The location and distribution of ketoprofen molecules in the lipid monolayer are affected by cation-π interaction between the choline group of lipids and the benzene ring of ketoprofen, the steric hindrance of the lipid head groups, and the hydrophobicity of ketoprofen molecule itself, comprehensively. The contact state of lipid head group with water is determined by surface pressure, which affects the interaction between drug molecules and lipids and further dominates the location and distribution of ketoprofen in the lipid monolayer. This work confirms that ketoprofen molecules can affect the property and the inner structure of DPPC/DPPG monolayers during breathing. Furthermore, the results obtained using a mixed monolayer containing two major pulmonary surfactants DPPC/DPPG and ketoprofen molecules will be helpful for the in-depth understanding of the mechanism of inhaled administration therapy.

A Simulation Study on the Interaction Between Pollutant Nanoparticles and the Pulmonary Surfactant Monolayer.

A good understanding of the mechanism of interaction between inhaled pollutant nanoparticles (NPs) and the pulmonary surfactant monolayer is useful to study the impact of fine particulate matter on human health. In this work, we established coarse-grained models of four representative NPs with different hydrophilicity properties in the air (i.e., CaSO4, C, SiO2, and C6H14O2 NPs) and the pulmonary surfactant monolayer. Molecular dynamic simulations of the interaction during exhalation and inhalation breathing states were performed. The effects of NP hydrophilicity levels, NP structural properties, and cholesterol content in the monolayer on the behaviors of NP embedment or the transmembrane were analyzed by calculating the changes in potential energy, NP displacement, monolayer orderliness, and surface tension. Results showed that NPs can inhibit the ability of the monolayer to adjust surface tension. For all breathing states, the hydrophobic C NP cannot translocate across the monolayer and had the greatest influence on the structural properties of the monolayer, whereas the strongly hydrophilic SiO2 and C6H14O2 NPs can cross the monolayer with little impact. The semi-hydrophilic CaSO4 NP can penetrate the monolayer only during the inhalation breathing state. The hydrophilic flaky NP shows the best penetration ability, followed by the rod-shaped NP and spherical NP in turn. An increase in cholesterol content of the monolayer led to improved orderliness and decreased fluidity of the membrane system due to enhanced intermolecular forces. Consequently, difficulty in crossing the monolayer increased for the NPs.

Influence of volatile organic compounds (VOCs) on pulmonary surfactant monolayers at air-water interface: Implication for the pulmonary health

Understanding the interaction of pulmonary surfactant (PS) and volatile organic compounds (VOCs) at air-water interface is crucial for the assessment of pulmonary health. In this study, dipalmitoyl phosphatidylcholine (DPPC), the mainly effective components of PS, was used as a mimic of PS employed to explore its surface activity in the exposure of VOCs. The results showed that the surface tension of DPPC decreased significantly with the addition of VOCs. On a basis of structure-activity assessment, hydrogen-bond basicity and dispersion/cavity interaction of VOCs are responsible for the decreasing capacity of VOCs for DPPC’s surface tension. Surface pressure-molecular area (π-A) isotherms demonstrate that the phase conversion of DPPC monolayer from gas to liquid expanded phase was greatly impacted by VOCs. The collapse pressure of DPPC monolayer in VOCs exposure are lower compared to that of DPPC alone, which can be attributed to the lowest surface tension of DPPC solutions mixed with VOCs. Atomic force microscopy analysis elucidates that the presence of VOCs led to bigger apertures area of DPPC monolayer than that of DPPC alone, which is indicative of microstructure changes of DPPC monolayer in the exposure of VOCs. This study provides new evidences that the exposure of VOCs on DPPC may alter the air-water interface activity of PS.

An investigation into drug partitioning behaviour in simulated pulmonary surfactant monolayers with associated molecular modelling

Drug delivery to the body via the inhaled route is dependent upon patient status, device use, and respirable formulation characteristics. Further to inhalation, drug‐containing particles interact and dissolve within pulmonary fluid leading to the desired pharmacological response. Pulmonary surfactant stabilises the alveolar air‐liquid interface and permits optimal respiratory mechanics. This material represents the initial contacting surface for all inhaled matter. On dissolution, the fate of a drug substance can include receptor activation, membrane partitioning and cellular penetration. Here, we consider the partitioning behaviour of salbutamol when located in proximity to a simulated pulmonary surfactant monolayer at pH 7. The administration of salbutamol to the underside of the surfactant film resulted in an expanded character for the 2‐dimensional ensemble and a decrease in the compressibility term. The rate of drug partitioning was greater when the monolayer was in the expanded state (ie, inhalation end‐point), which was ascribed to more accessible areas for molecular insertion. Quantum mechanics protocols, executed via Gaussian 09, indicated that constructive interactions between salbutamol and integral components of the model surfactant film took the form of electrostatic and hydrophobic associations. The favourable interactions are thought to promote drug insertion into the monolayer structure leading to the observed expanded character. The data presented herein confirm that drug partitioning into pulmonary surfactant monolayers is a likely prospect further to the inhalation of respirable formulations. As such, this process holds potential to reduce drug‐receptor activation and/or increase the residence time of drug within the pulmonary space.

Physicochemical properties of nanoparticles affect translocation across pulmonary surfactant monolayer

ABSTRACTInteraction between nanoparticles (NPs) and pulmonary surfactant monolayer is one of the most important parts in NP-based pulmonary drug delivery system, which can affect the result of the inhaled nano-drugs and their potential efficacy. Here, we show how surface charge of NPs affects translocation across pulmonary surfactant monolayer with coarse-grained molecular dynamics simulations. The results reveal that the surface charge position of NPs can determine the fate of the inhaled NPs about whether they can have the ability of translocation across the pulmonary surfactant monolayer, which is that NPs with face surface charge can penetrate the pulmonary surfactant monolayer and NPs with edge surface charge cannot. Besides, dynamic process, phase state and the potential of mean force profiles further confirm this result. Moreover, compared to anionic NPs, there is a greater chance for cationic NPs to be adsorbed on the surface of the pulmonary surfactant monolayer, which can further decrease the thickness of the pulmonary surfactant monolayer and reduce the distance between charged NPs and the pulmonary surfactant monolayer. Our researches provide a novel simulation model of NPs on translocation across pulmonary surfactant monolayer and the study of NP-based pulmonary drug delivery system should consider the surface charge of NPs.

Effect of the surface charge density of nanoparticles on their translocation across pulmonary surfactant monolayer: a molecular dynamics simulation

Interaction between nanoparticles (NPs) and pulmonary surfactant monolayer plays a very significant role in nanoparticle-based pulmonary drug delivery system. Previous researches have indicated that different properties of nanoparticles can affect their translocation across pulmonary surfactant monolayer. Here we performed coarse-grained molecular dynamics simulation aimed at nanoparticles’ surface charge density effect on their penetration behaviours. Several hydrophilic nanoparticles with different surface charge densities were modelled in the simulations. The results show that NPs’ surface charge density affects their translocation capability: the higher the surface charge densities of NPs are, the worse their translocation capability is. It will cause the structural changes of pulmonary surfactant monolayer, and inhibit the normal phase transition of the monolayer during the compression process. Besides, charged NPs can be adsorbed on the surface of the monolayer after translocation as a stable state, and the adsorption capability of NPs increases generally with the increase of surface charge densities. Our simulation results suggest that the study of nanoparticle-based pulmonary drug delivery system should consider the nanoparticles’ surface charge density effect in order to avoid biological toxicity and improve efficacy.

The pH dependent interaction between nicotine and simulated pulmonary surfactant monolayers with associated molecular modelling

Pulmonary surfactant is an endogenous material that lines and stabilises the alveolar air–liquid interface. Respiratory mechanics can be compromised by exposure to environmental toxins such as cigarette smoke, which contains nicotine. This study aims to determine the influence of nicotine on the activity of simulated lung surfactant at pH 7 and pH 9. In all cases, the addition of nicotine to the test zone caused deviation in surfactant film performance. Importantly, the maximum surface pressure was reduced for each system. Computational modelling was applied to assess key interactions between each species, with the Gaussian 09 software platform used to calculate electrostatic potential surfaces. Modelling data confirmed either nicotine penetration into the two‐dimensional structure or interfacial/electrostatic interactions across the underside. The results obtained from this study suggest that nicotine can impair the ability of pulmonary surfactant to reduce the surface tension term, which can increase the work of breathing. When extrapolated to gross lung function, alveolar collapse and respiratory disease (e.g. chronic airway obstruction) may result. The delivery of nicotine to the (deep) lung can cause a deterioration in lung function and lead to reduced quality of life. Copyright © 2017 John Wiley & Sons, Ltd.

Crystallisation of aspirin via simulated pulmonary surfactant monolayers and lung‐specific additives

Pain is a prevalent condition that can have a serious impact upon the socioeconomic function of a population. Numerous methods exist to administer analgesic medication (e.g. aspirin) to the body however inherent drawbacks limit patient acceptability. The inhaled route offers promise to facilitate the administration of medication to the body. Here, we consider the crystallisation behaviour of aspirin, our model therapeutic agent, when in contact with material of relevance to the lung. Thus, our approach aims to better understand the interaction between drug substances and the respiratory tract. Langmuir monolayers composed of a mixed surfactant system were supported on an aqueous subphase containing aspirin (7.5 mg/ml). The surfactant film was compressed to either 5mN/m (i.e. inhalation end point) or 50 mN/m (i.e. exhalation end point), whilst located within a humid environment for 16 h. Standard cooling crystallisation procedures were employed to produce control samples. Antisolvent crystallisation in the presence or absence of lung‐specific additives was conducted. All samples were analysed via scanning electron microscopy and X‐ray diffraction. Drug‐surfactant interactions were confirmed via condensed Langmuir isotherms. Scanning electron microscopy analysis revealed plate‐like morphology. The crystallisation route dictated both the crystal habit and particle size distribution. Dominant reflections were the (100) and (200) aspects. The main modes of interaction were hydrogen bonding, hydrophobic associations, and van der Waals forces. Here, we have demonstrated the potential of antisolvent crystallisation with lung‐specific additives to achieve control over drug crystal morphology. The approach taken can be applied in respirable formulation engineering. Copyright © 2017 John Wiley & Sons, Ltd.

Ultrashort Single-Walled Carbon Nanotubes Insert into a Pulmonary Surfactant Monolayer via Self-Rotation: Poration and Mechanical Inhibition.

It has been widely accepted that longer single-walled carbon nanotubes (SWCNTs) exhibit higher toxicity by causing severe pneumonia once inhaled, yet relatively little is known regarding the potential toxicity of ultrashort SWCNTs, which are of central importance to the development of suitable vehicles for biomedical applications. Here, by combining coarse-grained molecular dynamics (CGMD), pulling simulations, and scaling analysis, we demonstrate that the inhalation toxicity of ultrashort SWCNTs (1.5 nm < l < 5.5 nm) can be derived from the unique behaviors on interaction with the pulmonary surfactant monolayer (PSM), which is located at the air-water interface of alveoli and forms the frontline of the lung host defense. Molecular dynamics (MD) simulations suggest that ultrashort SWCNTs spontaneously insert into the PSM via fast self-rotation. Further translocation toward the water or air phase involves overcoming a high free-energy barrier, indicating that removal of inhaled ultrashort SWCNTs from the PSM is difficult, possibly leading to the accumulation of SWCNTs in the PSM, with prolonged retention and increased inflammation potentials. Under certain conditions, the inserted SWCNTs are found to open hydrophilic pores in the PSM via a mechanism that mimics that of the antimicrobial peptide. Besides, the mechanical property of the PSM is inhibited by the deposited ultrashort SWCNTs through segregation of the inner lipid molecules from the outer phase. Our results bring to the forefront the concern of the inhalation toxicity of ultrashort SWCNTs and provide guidelines for future design of inhaled nanodrug carriers with minimized side effects.

The impact of cigarette/e‐cigarette vapour on simulated pulmonary surfactant monolayers under physiologically relevant conditions

Deviation in pulmonary surfactant structure–function activity can impair airway patency and lead to respiratory disorders. This novel study aims to evaluate the influence cigarette/e‐cigarette vapour has on model surfactant films located within a simulated pulmonary environment using a lung biosimulator. Chromatographic analysis confirmed that nicotine levels were consistent with the sampling regimen employed. On exposure to smoke vapour, Langmuir isotherms exhibited condensed character and a significant reduction in maximum surface pressure was noted in all cases. Langmuir isocycles, reflective of the human breathing cycle, demonstrated condensed character on smoke vapour delivery. A reduction in the maximum surface pressure was clear only in the case of cigarette vapour application. The components of cigarette vapour can cause oxidative damage to pulmonary surfactant and impair recycling. Neutral nicotine molecules can weaken the structure of the monolayer and cause destabilisation. A protective effect was evident in the case of repeated surfactant compression – relaxation cycles (i.e. the ability to reduce the surface tension term was impaired less), demonstrating a likely innate biological defensive mechanism of the lung. E‐cigarette vapour appeared to have a reduced impact on surfactant performance, which may hold value in harm reduction over the longer term. Copyright © 2017 John Wiley &amp; Sons, Ltd.

Transport of nanoparticles across pulmonary surfactant monolayer: a molecular dynamics study.

Pulmonary nanodrug delivery is an emerging concept, especially for targeted lung cancer therapy. Once inhaled, the nanoparticles (NPs) acting as drug carriers need to efficiently cross the pulmonary surfactant monolayer (PSM) of lung alveoli, which act as the first barrier for external particles entering the lung. Herein, by performing molecular dynamics simulations, we study how inhaled NPs interact with the PSM, particularly focusing on the transport of NPs with different properties across the PSM. While hydrophilic NPs translocate directly across the PSM, transport of hydrophobic NPs is achieved as the PSM wraps them. Intriguingly, when hydrophilic NPs are decorated with lipid molecules (LCNPs), they are wrapped by the PSM efficiently with mild PSM perturbation. Moreover, the structure formed is like a vesicle, which will likely fuse with cell membranes to accomplish the transport of hydrophilic NPs into secondary organs. This behavior makes the LCNP a prospective candidate for pulmonary nanodrug delivery. Herein, the effects of the physical properties of LCNPs on their transport are investigated. Increasing the LCNP size promotes its wrapping by reducing the PSM bending energy. The binding energy that drives transport can be strengthened by increasing the lipid coating density and the lipid tail length, both of which also reduce the risk of PSM rupture during transport. These results should help researchers understand how to better use surface decorations to achieve efficient pulmonary entry, which may provide useful guidance for the design of nano-based platforms for inhaled drug delivery.

Perturbation of the pulmonary surfactant monolayer by single-walled carbon nanotubes: a molecular dynamics study.

Single-walled carbon nanotubes (SWCNTs) are at present synthesized on a large scale with a variety of applications. The increasing likelihood of exposure to SWCNTs, however, puts human health at a high risk. As the front line of the innate host defense system, the pulmonary surfactant monolayer (PSM) at the air-water interface of the lungs interacts with the inhaled SWCNTs, which in turn inevitably perturb the ultrastructure of the PSM and affect its biophysical functions. Here, using molecular dynamics simulations, we demonstrate how the diameter and length of SWCNTs critically regulate their interactions with the PSM. Compared to their diameters, the inhalation toxicity of SWCNTs was found to be largely affected by their lengths. Short SWCNTs with lengths comparable to the monolayer thickness are found to vertically insert into the PSM with no indication of translocation, possibly leading to accumulation of SWCNTs in the PSM with prolonged retention and increased inflammation potentials. The perturbation also comes from the forming water pores across the PSM. Longer SWCNTs are found to horizontally insert into the PSM during inspiration, and they can be wrapped by the PSM during deep expiration via a tube diameter-dependent self-rotation. The potential toxicity of longer SWCNTs comes from severe lipid depletion and the PSM-rigidifying effect. Our findings could help reveal the inhalation toxicity of SWCNTs, and pave the way for the safe use of SWCNTs as vehicles for pulmonary drug delivery.

Surface patterning of single-walled carbon nanotubes enhances their perturbation on a pulmonary surfactant monolayer: frustrated translocation and bilayer vesiculation

In contrast to SWCNTs with unique surface properties, the surface patterning of SWCNTs is found to enhance their perturbation on the pulmonary surfactantsviafrustrated translocation and destructive bilayer vesiculation.

Nebulizing novel multifunctional nanovesicles: the impact of macrophage-targeted-pH-sensitive archaeosomes on a pulmonary surfactant.

In this study, a NE-U22 vibrating mesh Omron nebulizer was used to deliver the Lissamine™ rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine triethylammonium salt (Rh-PE) and 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS)/p-xylene-bis-pyridinium bromide (DPX) double-labelled macrophage-targeted pH-sensitive archaeosomes (ApH, 174 ± 48 nm, -30 ± 13 mV unilamellar nanovesicles made of dioleoyl-sn-glycero-3-phosphoethanolamine: [total polar archaeolipids from the hyperhalophile archaebacteria Halorubrum tebenquichense]: cholesteryl hemisuccinate 4.2 : 2.8 : 3 w : w : w) to J774A.1 cells covered by a Prosurf pulmonary surfactant (PS) monolayer at or below the equilibrium surface pressure πe. The uptake and cytoplasmic drug release from ApH were assessed by flow cytometry of Rh-PE and HPTS fluorescence, respectively. Despite being soft matter, nanovesicles are submitted to the dismantling interactions of shear stress of nebulization and contact with the surfactant barrier, and at least a fraction of nebulized ApH was found to be stable enough to execute higher cytoplasmic delivery than archaeolipid-lacking vesicles. Nebulized ApH increased the PS tensioactivity to just below πe, which was beyond the physiological range; this finding indicated that changes in lung surfactant function induced by nebulized nanovesicles were less likely to occur in vivo. The cytoplasmic delivery from ApH slightly decreased across monolayers at πe; this suggested that nanovesicles crossed the PS in a fashion inversely related to monolayer compression. Laurdan generalized polarization and fluorescence anisotropy were used to reveal that nanovesicles neither depleted B and C proteins of the PS nor increased the fluidity of the PS. Together with the feasibility of the cytoplasmic drug delivery upon nebulization, our results suggest that ApH are structurally unique nanovesicles that would not induce biophysical changes leading to PS inactivation and open the door to deeper future translational studies.

Lipid extraction mediates aggregation of carbon nanospheres in pulmonary surfactant monolayers.

Increasing evidence indicates that carbon nanoparticles (CNPs), which mainly originate from incomplete combustion of fossil fuels, have an adverse impact on the respiratory system. Recent in vivo experiments have shown that the pulmonary toxicity of CNPs is attributed to their aggregation in pulmonary surfactant monolayers (PSMs) while the underlying mechanism of aggregation remains unclear. Here, by performing coarse grained molecular dynamics simulations, we demonstrate for the first time that the aggregation of carbon nanospheres (CNSs) in PSMs is in fact size-dependent and mediated by lipid extractions. Upon CNS deposition, neighbouring lipid molecules are extracted from PSMs to cover CNSs from the top side. The extracted lipids induce clustering of CNSs to maximize the CNS-lipid interaction, by forming inverse micelles to wrap the aggregated CNSs cooperatively. The formed CNS clusters perturb the molecule structure of the PSM and thus affect its biofunction on respiration. Our simulations show that during the expiration process, CNSs form clusters that perturb the mechanical properties of the PSM in a manner depending on the CNS size. With deep inspiration, a high concentration of large CNSs may induce PSM rupture and thus have a potential impact on its biophysical properties.

Fluidizing and Solidifying Effects of Perfluorooctylated Fatty Alcohols on Pulmonary Surfactant Monolayers.

Pulmonary surfactant (PS) preparations based mainly on bovine or porcine extracts are commonly administered to patients with neonatal respiratory distress syndrome (NRDS) for therapy. The preparations are sufficiently effective to treat NRDS; however, they are associated with a risk of infection and involve costly purification procedures to achieve batch-to-batch reproducibility. Therefore, we investigated the mechanism and interfacial behavior of synthetic PS preparations containing a mimicking peptide (KLLKLLLKLWLKLLKLLL, Hel 13-5). In particular, a hybrid PS formulation with fluorinated amphiphiles is reported from the perspective of surface chemistry. Fluorinated amphiphiles are characterized by exceptional chemical and biological inertness, high oxygen-dissolving capacity, low surface tension, excellent spreading ability, and high fluidity. These properties are superior to those for the corresponding hydrocarbon analogs. Indeed, a small amount of fluorinated long-chain alcohols enhances the effectiveness of the model PS preparation for in vitro pulmonary functions. Moreover, the mode of the improved efficacy differs depending on the hydrophobic chain length in the alcohols. For alcohols with a short fluorocarbon (FC) chain, the monolayer phase of the model PS preparation remains disordered (fluidization). However, the addition of alcohols containing a long FC chain reduces the disordered/ordered phase transition pressure and the growth of ordered domains of the monolayer (condensation). Furthermore, repeated compression-expansion isotherms of the monolayers, which can simulate respiration in the lung, suggest irreversible elimination of the short-FC alcohol into the subphase and enhancement of the squeeze-out phenomenon of certain PS components by solid-like monolayer formation induced by the long-FC alcohol. We demonstrated that fluorinated amphiphiles may be used as additives for synthetic or commercial PS preparations for RDS treatment.

Molecular modeling of interaction between lipid monolayer and graphene nanosheets: implications for pulmonary nanotoxicity and pulmonary drug delivery

Understanding how nanoparticles interact with the pulmonary surfactant monolayer (PSM) is of great importance for safe applications in biomedicine and for evaluation of both health and environment impacts.

Understanding the Mutual Impact of Interaction between Hydrophobic Nanoparticles and Pulmonary Surfactant Monolayer

Interaction between hydrophobic nanoparticles (NPs) and a pulmonary surfactant (PS) film leads to a shift in molecular packing of surfactant molecules in the PS film around the interacting NPs. The resultant structural arrangement of surfactants around the NPs may be a potential structural factor responsible for their high retention ability within the film. Moreover, during this interaction, surfactant molecules coat the NPs and change their surface properties.

Physicochemical Properties of Nanoparticles Regulate Translocation across Pulmonary Surfactant Monolayer and Formation of Lipoprotein Corona

Interaction with the pulmonary surfactant film, being the first line of host defense, represents the initial bio-nano interaction in the lungs. Such interaction determines the fate of the inhaled nanoparticles and their potential therapeutic or toxicological effect. Despite considerable progress in optimizing physicochemical properties of nanoparticles for improved delivery and targeting, the mechanisms by which inhaled nanoparticles interact with the pulmonary surfactant film are still largely unknown. Here, using combined in vitro and in silico methods, we show how hydrophobicity and surface charge of nanoparticles differentially regulate the translocation and interaction with the pulmonary surfactant film. While hydrophilic nanoparticles generally translocate quickly across the pulmonary surfactant film, a significant portion of hydrophobic nanoparticles are trapped by the surfactant film and encapsulated in lipid protrusions upon film compression. Our results support a novel model of pulmonary surfactant lipoprotein corona associated with inhaled nanoparticles of different physicochemical properties. Our data suggest that the study of pulmonary nanotoxicology and nanoparticle-based pulmonary drug delivery should consider this lipoprotein corona.

Compositional and structural characterization of monolayers and bilayers composed of native pulmonary surfactant from wild type mice

This work comprises a structural and dynamical study of monolayers and bilayers composed of native pulmonary surfactant from mice. Spatially resolved information was obtained using fluorescence (confocal, wide field and two photon excitation) and atomic force microscopy methods. Lipid mass spectrometry experiments were also performed in order to obtain relevant information on the lipid composition of this material. Bilayers composed of mice pulmonary surfactant showed coexistence of distinct domains at room temperature, with morphologies and lateral packing resembling the coexistence of liquid ordered (lo)/liquid disordered (ld)-like phases reported previously in porcine lung surfactant. Interestingly, the molar ratio of saturated (mostly DPPC)/non-saturated phospholipid species and cholesterol measured in the innate material corresponds with that of a DOPC/DPPC/cholesterol mixture showing lo/ld phase coexistence at a similar temperature. This suggests that at quasi-equilibrium conditions, key lipid classes in this complex biological material are still able to produce the same scaffold observed in relevant but simpler model lipid mixtures. Also, robust structural and dynamical similarities between mono- and bi-layers composed of mice pulmonary surfactant were observed when the monolayers reach a surface pressure of 30mN/m. This value is in line with theoretically predicted and recently measured surface pressures, where the monolayer–bilayer equivalence occurs in samples composed of single phospholipids. Finally, squeezed out material attached to pulmonary surfactant monolayers was observed at surface pressures near the beginning of the monolayer reversible exclusion plateau (~40mN/m). Under these conditions this material adopts elongated tubular shapes and displays ordered lateral packing as indicated by spatially resolved LAURDAN GP measurements.