Structural determinants of collapse by a monomolecular mimic of pulmonary surfactant at the physiological temperature.

Pulmonary surfactant forms a thin film on the liquid that lines the alveolar air-sacks. When compressed by the decreasing alveolar surface area during exhalation, the films avoid collapse from the air/water interface and reduce surface tension to exceptionally low levels. To define better the structure of compressed films that determines their susceptibility to collapse, we measured how cholesterol affects the structure and collapse of dipalmitoyl phosphatidylcholine (DPPC) monolayers at physiological temperatures. Grazing incidence X-ray diffraction (GIXD) and grazing incidence X-ray off-specular scattering (GIXOS) established the lateral and transverse structures of films on a Langmuir trough at a surface pressure of 45 mN m-1, just below the equilibrium spreading pressure at which collapse begins. Experiments with captive bubbles at a surface pressure of 51 mN m-1 measured how the steroid affects isobaric collapse. Mol fractions of the steroid (Xchol) at 0.05 removed the tilt by the acyl chains of DPPC, shifted the unit cell from centered rectangular to hexagonal, and dramatically decreased the long-range order. Higher Xchol produced no further change in diffraction, suggesting that cholesterol partitions into a coexisting disordered phase. Cholesterol had minimal effect on rates of collapse until Xchol reached 0.20. Our results demonstrate that the decreased coherence length, indicating conversion of positional order to short-range, is insufficient to make a condensed monolayer susceptible to collapse. Our findings suggest a two-step process by which cholesterol induces disorder. The steroid would first convert the film with crystalline chains to a hexatic phase before generating a fully disordered structure that is susceptible to collapse. These results lead to far-reaching consequences for formulation of animal-derived therapeutic surfactants. Our results suggest that removal of cholesterol from these preparations should be unnecessary below Xchol = 0.20.

Validation of a high-performance liquid chromatography method for determining lysophosphatidylcholine content in bovine pulmonary surfactant medication.

Pulmonary surfactant replacement therapy is a promising improvement in neonatal care for infants with respiratory distress syndrome. Lysophosphatidylcholine (LPC) is an undesirable component that can hinder surfactant proteins from enhancing the adsorption of surfactant lipids to balance surface tensions by creating a saturated coating on the interior of the lungs. A novel normal-phase liquid chromatography method utilizing UV detection and non-toxic solvents was developed and validated for the first time to analyze LPC in the complex matrix of pulmonary surfactant medication. The analytical method validation included evaluation of system suitability, repeatability, intermediate precision, linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ), stability and robustness. The method yielded detection and quantification limits of 4.4 and 14.5 µg/ml, respectively. The calibration curve was modified linearly within the LOQ to 1.44 mg/ml range, with a determination coefficient of 0.9999 for standards and 0.9997 for sample solutions. Given the lack of reliable published data on LPC analysis in pulmonary surfactant medications, this newly developed method demonstrates promising results and offers advantages of HPLC methodology, including simplicity, accuracy, specificity, sensitivity and an exceptionally low LOD and LOQ. These attributes contribute to considering this achievement as an innovative method.

Impact of Nebulization on the Physicochemical Properties of Polymer-Lipid Hybrid Nanoparticles for Pulmonary Drug Delivery.

Nanoparticles (NPs) have shown significant potential for pulmonary administration of therapeutics for the treatment of chronic lung diseases in a localized and sustained manner. Nebulization is a suitable method of NP delivery, particularly in patients whose ability to breathe is impaired due to lung diseases. However, there are limited studies evaluating the physicochemical properties of NPs after they are passed through a nebulizer. High shear stress generated during nebulization could potentially affect the surface properties of NPs, resulting in the loss of encapsulated drugs and alteration in the release kinetics. Herein, we thoroughly examined the physicochemical properties as well as the therapeutic effectiveness of Infasurf lung surfactant (IFS)-coated PLGA NPs previously developed by us after passing through a commercial Aeroneb® vibrating-mesh nebulizer. Nebulization did not alter the size, surface charge, IFS coating and bi-phasic release pattern exhibited by the NPs. However, there was a temporary reduction in the initial release of encapsulated therapeutics in the nebulized compared to non-nebulized NPs. This underscores the importance of evaluating the drug release kinetics of NPs using the inhalation method of choice to ensure suitability for the intended medical application. The cellular uptake studies demonstrated that both nebulized and non-nebulized NPs were less readily taken up by alveolar macrophages compared to lung cancer cells, confirming the IFS coating retention. Overall, nebulization did not significantly compromise the physicochemical properties as well as therapeutic efficacy of the prepared nanotherapeutics.

A Review on Investigating the Interactions between Nanoparticles and the Pulmonary Surfactant Monolayer with Coarse-Grained Molecular Dynamics Method.

Pulmonary drug delivery has garnered significant attention due to its targeted local lung action, minimal toxic side effects, and high drug utilization. However, the physicochemical properties of inhaled nanoparticles (NPs) used as drug carriers can influence their interactions with the pulmonary surfactant (PS) monolayer, potentially altering the fate of the NPs and impairing the biophysical function of the PS monolayer. Thus, the objective of this review is to summarize how the physicochemical properties of NPs affect their interactions with the PS monolayer. Initially, the definition and properties of NPs, as well as the composition and characteristics of the PS monolayer, are introduced. Subsequently, the coarse-grained molecular dynamics (CGMD) simulation method for studying the interactions between NPs and the PS monolayer is presented. Finally, the implications of the hydrophobicity, size, shape, surface charge, surface modification, and aggregation of NPs on their interactions with the PS monolayer and on the composition of biomolecular corona are discussed. In conclusion, gaining a deeper understanding of the effects of the physicochemical properties of NPs on their interactions with the PS monolayer will contribute to the development of safer and more effective nanomedicines for pulmonary drug delivery.

Towards quantifying biomarkers for respiratory distress in preterm infants: Machine learning on mid infrared spectroscopy of lipid mixtures.

Neonatal respiratory distress syndrome (nRDS) is a challenging condition to diagnose which can lead to delays in receiving appropriate treatment. Mid infrared (IR) spectroscopy is capable of measuring the concentrations of two diagnostic nRDS biomarkers, lecithin (L) and sphingomyelin (S) with the potential for point of care (POC) diagnosis and monitoring. The effects of varying other lipid species present in lung surfactant on the mid IR spectra used to train machine learning models are explored. This study presents a lung lipid model of five lipids present in lung surfactant and varies each in a systematic approach to evaluate the ability of machine learning models to predict the lipid concentrations, the L/S ratio and to quantify the uncertainty in the predictions using the jackknife + -after-bootstrap and variant bootstrap methods. We establish the L/S ratio can be determined with an uncertainty of approximately ±0.3 mol/mol and we further identify the 5 most prominent wavenumbers associated with each machine learning model.

The biophysical function of pulmonary surfactant.

The type II pneumocytes of the lungs secrete a mixture of lipids and proteins that together acts as a surfactant. The material forms a thin film on the surface of the liquid layer that lines the alveolar air sacks. When compressed by the decreasing alveolar surface area during exhalation, the films reduce surface tension to exceptionally low levels. Pulmonary surfactant is essential for preserving the integrity of the barrier between alveolar air and capillary blood during normal breathing. This review focuses on the major biophysical processes by which endogenous pulmonary surfactant achieves its function and the mechanisms involved in those processes. Vesicles of pulmonary surfactant adsorb rapidly from the alveolar liquid to form the interfacial film. Interfacial insertion, which requires the hydrophobic surfactant protein SP-B, proceeds by a process analogous to the fusion of two vesicles. When compressed, the adsorbed film desorbs slowly. Constituents remain at the surface at high interfacial concentrations that reduce surface tensions well below equilibrium levels. We review the models proposed to explain how pulmonary surfactant achieves both the rapid adsorption and slow desorption characteristic of a functional film.

Understanding the Retention of Vaping Additives in the Lungs: Model Lung Surfactant Membrane Perturbation by Vitamin E and Vitamin E Acetate.

Deviations from the normal physicochemical and functional properties of pulmonary surfactants are associated with the incidence of lung injury and other respiratory disorders. This study aims to evaluate the alteration of the 2D molecular organization and morphology of pulmonary surfactant model membranes by the electronic cigarette additives α-tocopherol (vitamin E) and α-tocopherol acetate (vitamin E acetate), which have been associated with lung injury, termed e-cigarette or vaping-use-associated lung injury (EVALI). The model membranes used contained a 7:3 molar ratio of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol) to which α-tocopherol and α-tocopherol acetate were added to form mixtures of up to 20 mol % additive. The properties of the neat tocopherol additives and DPPC/POPG (7:3) mixtures with increasing molar proportions of additive were evaluated by surface pressure-area isotherms, excess area calculations, Brewster angle microscopy, grazing incidence X-ray diffraction, X-ray reflectivity, and atomic force microscopy. The addition of either additive alters the essential phase balance of the model pulmonary surfactant membrane by generating a greater proportion of the fluid phase. Despite this net fluidization, both tocopherol additives have space-filling effects on the liquid-expanded and condensed phases, yielding negative excess areas in the liquid-expanded phase and reduced tilt angles in the condensed phase. Both tocopherol additives alter the stability of the fluid phase, pushing the eventual collapse of this phase to higher surface pressures than the model membrane in the absence of an additive.

Evaluating ß2-agonists as siRNA delivery adjuvants for pulmonary surfactant-coated nanogel inhalation therapy.

The lung is an attractive target organ for inhalation of RNA therapeutics, such as small interfering RNA (siRNA). However, clinical translation of siRNA drugs for application in the lung is hampered by many extra- and intracellular barriers. We previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel (nanogel) core coated with Curosurf®, a clinically used pulmonary surfactant. The surfactant shell was shown to markedly improve particle stability and promote intracellular siRNA delivery, both in vitro and in vivo. However, the full potential of siRNA nanocarriers is typically not reached as they are rapidly trafficked towards lysosomes for degradation and only a fraction of the internalized siRNA cargo is able to escape into the cytosol. We recently reported on the repurposing of widely applied cationic amphiphilic drugs (CADs) as siRNA delivery enhancers. Due to their physicochemical properties, CADs passively accumulate in the (endo)lysosomal compartment causing a transient permeabilization of the lysosomal membrane, which facilitates cytosolic drug delivery. In this work, we assessed a selection of cationic amphiphilic ß2-agonists (i.e., salbutamol, formoterol, salmeterol and indacaterol) for their ability to enhance siRNA delivery in a lung epithelial and macrophage cell line. These drugs are widely used in the clinic for their bronchodilating effect in obstructive lung disease. As opposed to the least hydrophobic drugs salbutamol and formoterol, the more hydrophobic long-acting ß2-agonist (LABA) salmeterol promoted siRNA delivery in both cell types for both uncoated and surfactant-coated nanogels, whereas indacaterol showed this effect solely in lung epithelial cells. Our results demonstrate the potential of both salmeterol and indacaterol to be repurposed as adjuvants for nanocarrier-mediated siRNA delivery to the lung, which could provide opportunities for drug combination therapy.

Exploring protein-protein interactions and oligomerization state of pulmonary surfactant protein C (SP-C) through FRET and fluorescence self-quenching.

Pulmonary surfactant (PS) is a lipid-protein complex that forms films reducing surface tension at the alveolar air-liquid interface. Surfactant protein C (SP-C) plays a key role in rearranging the lipids at the PS surface layers during breathing. The N-terminal segment of SP-C, a lipopeptide of 35 amino acids, contains two palmitoylated cysteines, which affect the stability and structure of the molecule. The C-terminal region comprises a transmembrane α-helix that contains a ALLMG motif, supposedly analogous to a well-studied dimerization motif in glycophorin A. Previous studies have demonstrated the potential interaction between SP-C molecules using approaches such as Bimolecular Complementation assays or computational simulations. In this work, the oligomerization state of SP-C in membrane systems has been studied using fluorescence spectroscopy techniques. We have performed self-quenching and FRET assays to analyze dimerization of native palmitoylated SP-C and a non-palmitoylated recombinant version of SP-C (rSP-C) using fluorescently labeled versions of either protein reconstituted in different lipid systems mimicking pulmonary surfactant environments. Our results reveal that doubly palmitoylated native SP-C remains primarily monomeric. In contrast, non-palmitoylated recombinant SP-C exhibits dimerization, potentiated at high concentrations, especially in membranes with lipid phase separation. Therefore, palmitoylation could play a crucial role in stabilizing the monomeric α-helical conformation of SP-C. Depalmitoylation, high protein densities as a consequence of membrane compartmentalization, and other factors may all lead to the formation of protein dimers and higher-order oligomers, which could have functional implications under certain pathological conditions and contribute to membrane transformations associated with surfactant metabolism and alveolar homeostasis.

Sensitive Detection of Biomolecular Adsorption by a Low-Density Surfactant Layer Using Sum-Frequency Vibrational Spectroscopy.

Small molecules or proteins interact with a biomembrane in various ways for molecular recognition, structure stabilization, and transmembrane signaling. In this study, 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP), having a choline group, was used to investigate this interaction by using sum-frequency vibrational spectroscopy. The sum-frequency spectrum characteristic of a neat monolayer changed to that of a bare air/water interface at a larger molecular area of the DPTAP molecules due to local laser heating. Upon introduction of the aromatic molecules in the subphase at around 120 Å2 per molecule, the sum-frequency signal suddenly reappeared due to molecular adhesion, and this was utilized to probe the adsorption of the aromatic ring molecules in the water subphase to the choline headgroup of the DPTAP by cation-π interaction. The onset concentrations of this sum-frequency signal change allowed a comparison of the relative interaction strengths between different aromatic molecules. A zwitterionic surfactant molecule (DPPC) was found to interact weakly compared to the cationic DPTAP molecule.

Pulmonary Surfactant: A Mighty Thin Film.

Pulmonary surfactant is a critical component of lung function in healthy individuals. It functions in part by lowering surface tension in the alveoli, thereby allowing for breathing with minimal effort. The prevailing thinking is that low surface tension is attained by a compression-driven squeeze-out of unsaturated phospholipids during exhalation, forming a film enriched in saturated phospholipids that achieves surface tensions close to zero. A thorough review of past and recent literature suggests that the compression-driven squeeze-out mechanism may be erroneous. Here, we posit that a surfactant film enriched in saturated lipids is formed shortly after birth by an adsorption-driven sorting process and that its composition does not change during normal breathing. We provide biophysical evidence for the rapid formation of an enriched film at high surfactant concentrations, facilitated by adsorption structures containing hydrophobic surfactant proteins. We examine biophysical evidence for and against the compression-driven squeeze-out mechanism and propose a new model for surfactant function. The proposed model is tested against existing physiological and pathophysiological evidence in neonatal and adult lungs, leading to ideas for biophysical research, that should be addressed to establish the physiological relevance of this new perspective on the function of the mighty thin film that surfactant provides.

Synthetic surfactant with a combined SP-B and SP-C analogue is efficient in rabbit models of adult and neonatal respiratory distress syndrome.

Respiratory distress syndrome (RDS) in premature infants is caused by insufficient amounts of endogenous lung surfactant and is efficiently treated with replacement therapy using animal-derived surfactant preparations. On the other hand, adult/acute RDS (ARDS) occurs secondary to for example, sepsis, aspiration of gastric contents, and multitrauma and is caused by alveolar endothelial damage, leakage of plasma components into the airspaces and inhibition of surfactant activity. Instillation of surfactant preparations in ARDS has so far resulted in very limited treatment effects, partly due to inactivation of the delivered surfactants in the airspace. Here, we develop a combined surfactant protein B (SP-B) and SP-C peptide analogue (Combo) that can be efficiently expressed and purified from Escherichia coli without any solubility or purification tag. NMR spectroscopy shows that Combo peptide forms α-helices both in organic solvents and in lipid micelles, which coincide with the helical regions described for the isolated SP-B and SP-C parts. Artificial Combo surfactant composed of synthetic dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol, 1:1, mixed with 3 weights % relative to total phospholipids of Combo peptide efficiently improves tidal volumes and lung gas volumes at end-expiration in a premature rabbit fetus model of RDS. Combo surfactant also improves oxygenation and respiratory parameters and lowers cytokine release in an acid instillation-induced ARDS adult rabbit model. Combo surfactant is markedly more resistant to inhibition by albumin and fibrinogen than a natural-derived surfactant in clinical use for the treatment of RDS. These features of Combo surfactant make it attractive for the development of novel therapies against human ARDS.

Biophysical function of pulmonary surfactant in liquid ventilation.

Liquid ventilation is a mechanical ventilation technique in which the entire or part of the lung is filled with oxygenated perfluorocarbon (PFC) liquids rather than air in conventional mechanical ventilation. Despite its many ideal biophysicochemical properties for assisting liquid breathing, a general misconception about PFC is to use it as a replacement for pulmonary surfactant. Because of the high PFC-water interfacial tension (59 mN/m), pulmonary surfactant is indispensable in liquid ventilation to increase lung compliance. However, the biophysical function of pulmonary surfactant in liquid ventilation is still unknown. Here, we have studied the adsorption and dynamic surface activity of a natural surfactant preparation, Infasurf, at the PFC-water interface using constrained drop surfactometry. The constrained drop surfactometry is capable of simulating the intra-alveolar microenvironment of liquid ventilation under physiologically relevant conditions. It was found that Infasurf adsorbed to the PFC-water interface reduces the PFC-water interfacial tension from 59 mN/m to an equilibrium value of 9 mN/m within seconds. Atomic force microscopy revealed that after de novo adsorption, Infasurf forms multilayered structures at the PFC-water interface with an average thickness of 10-20 nm, depending on the adsorbing surfactant concentration. It was found that the adsorbed Infasurf film is capable of regulating the interfacial tension of the PFC-water interface within a narrow range, between ∼12 and ∼1 mN/m, during dynamic compression-expansion cycles that mimic liquid ventilation. These findings have novel implications in understanding the physiological and biophysical functions of the pulmonary surfactant film at the PFC-water interface, and may offer new translational insights into the development of liquid ventilation and liquid breathing techniques.

The anti-inflammatory and antiviral properties of anionic pulmonary surfactant phospholipids.

The pulmonary surfactant system of the lung is a lipid and protein complex, which regulates the biophysical properties of the alveoli to prevent lung collapse and the innate immune system in the lung. Pulmonary surfactant is a lipoprotein complex consisting of 90% phospholipids and 10% protein, by weight. Two minor components of pulmonary surfactant phospholipids, phosphatidylglycerol (PG) and phosphatidylinositol (PI), exist at very high concentrations in the extracellular alveolar compartments. We have reported that one of the most dominant molecular species of PG, palmitoyl-oleoyl-phosphatidylglycerol (POPG) and PI inhibit inflammatory responses induced by multiple toll-like receptors (TLR2/1, TLR3, TLR4, and TLR2/6) by interacting with subsets of multiprotein receptor components. These lipids also exert potent antiviral effects against RSV and influenza A, in vitro, by inhibiting virus binding to host cells. POPG and PI inhibit these viral infections in vivo, in multiple animal models. Especially noteworthy, these lipids markedly attenuate SARS-CoV-2 infection including its variants. These lipids are natural compounds that already exist in the lung and, thus, are less likely to cause adverse immune responses by hosts. Collectively, these data demonstrate that POPG and PI have strong potential as novel therapeutics for applications as anti-inflammatory compounds and preventatives, as treatments for broad ranges of RNA respiratory viruses.

Surfactant Proteins SP-B and SP-C in Pulmonary Surfactant Monolayers: Physical Properties Controlled by Specific Protein-Lipid Interactions.

The lining of the alveoli is covered by pulmonary surfactant, a complex mixture of surface-active lipids and proteins that enables efficient gas exchange between inhaled air and the circulation. Despite decades of advancements in the study of the pulmonary surfactant, the molecular scale behavior of the surfactant and the inherent role of the number of different lipids and proteins in surfactant behavior are not fully understood. The most important proteins in this complex system are the surfactant proteins SP-B and SP-C. Given this, in this work we performed nonequilibrium all-atom molecular dynamics simulations to study the interplay of SP-B and SP-C with multicomponent lipid monolayers mimicking the pulmonary surfactant in composition. The simulations were complemented by z-scan fluorescence correlation spectroscopy and atomic force microscopy measurements. Our state-of-the-art simulation model reproduces experimental pressure-area isotherms and lateral diffusion coefficients. In agreement with previous research, the inclusion of either SP-B and SP-C increases surface pressure, and our simulations provide a molecular scale explanation for this effect: The proteins display preferential lipid interactions with phosphatidylglycerol, they reside predominantly in the lipid acyl chain region, and they partition into the liquid expanded phase or even induce it in an otherwise packed monolayer. The latter effect is also visible in our atomic force microscopy images. The research done contributes to a better understanding of the roles of specific lipids and proteins in surfactant function, thus helping to develop better synthetic products for surfactant replacement therapy used in the treatment of many fatal lung-related injuries and diseases.

Synchrotron radiation circular dichroism spectroscopy reveals that gold and silver nanoparticles modify the secondary structure of a lung surfactant protein B analogue.

Inhaled nanoparticles (NPs) depositing in the alveolar region of the lung interact initially with a surfactant layer and in vitro studies have demonstrated that NPs can adversely affect the biophysical function of model pulmonary surfactants (PS), of which surfactant protein B (SP-B) is a key component. Other studies have demonstrated the potential for NPs to modify the structure and function of proteins. It was therefore hypothesised that NPs may affect the biophysical function of PS by modifying the structure of SP-B. Synchrotron radiation circular dichroism (SRCD) spectroscopy was used to explore the effect of various concentrations of gold nanoparticles (AuNPs) (5, 10, 20 nm), silver nanoparticles (AgNPs) (10 nm) and silver citrate on the secondary structure of surfactant protein B analogue, SP-B1-25, in a TFE/PB dispersion. For Au and Ag NPs the SRCD spectra indicated a concentration dependent reduction in the α-helical structure of SP-B1-25 (5 nm AuNP ≈ 10 nm AgNP ≫ 10 nm AuNP > 20 nm AuNP). For AuNPs the effect was greater for the 5 nm size, which was not fully explained by consideration of surface area. The impact of the 10 nm AgNPs was greater than that of the 10 nm AuNPs and the effect of AgNPs was greater than that of silver citrate at equivalent Ag mass concentrations. For 10 nm AuNPs, SRCD spectra for dispersions in, the more physiologically relevant, DPPC showed a similar concentration dependent pattern. The results demonstrate the potential for inhaled NPs to modify SP-B1-25 structure and thus potentially adversely impact the physiological function of the lung, however, further studies are necessary to confirm this.

Fisiología respiratoria: fisiología del surfactante pulmonar / Physiology of pulmonary surfactant

El proceso de respiración y el intercambio gaseoso requiere la interacción de variadas fuerzas en los distintos tejidos y órganos involucrados. La tensión superficial a nivel alveolar provocaría colapso de dichas estructuras de no ser por las características del surfactante que lo recubre. Revisaremos en este articulo la fisiología involucrada en su estructura física, producción y efectos pulmonares.

The process of breathing and gas exchange requires the interaction of various forces in the different tissues and organs involved. The surface tension at the alveolus would cause collapse of these structures without of the surfactant that covers it. We will review in this article the physiology involved in its physical structure, production, and pulmonary effects.

Reply to the 'Comment on "Bilayer aggregate microstructure determines viscoelasticity of lung surfactant suspensions"' by J.-F. Berret, DOI: 10.1039/d2sm00653g.

In their comment, Berret suggests that Curosurf, one of three clinical lung surfactant aqueous suspensions examined in the Soft Matter, 2021, 17, 5170-51820 is a Newtonian liquid rather than a shear-thinning soft solid with a small, but measurable yield stress. We postulate that these discrepancies may be due to the size of the magnetic wire measurement probe used in their paper (Thai et al., Colloids Surf., B, 2019, 178, 337-345) the diameter of which is similar in size to the Curosurf bilayer agregates (1-10 µm). The cone and plate rheometer used by Ciutara and Zasadzinski measures averaged effects over the entire macroscopic sample. Our combined results point out that the local viscoelastic properties of a moderately dense suspension may be different than its bulk properties.

Comment on "Bilayer aggregate microstructure determines viscoelasticity of lung surfactant suspensions" by C. O. Ciutara and J. A. Zasadzinski, Soft Matter, 2021, 17, 5170-5182.

For applications of pulmonary surfactant delivery to the lungs, the question of rheology of the existing clinical formulations is of upmost importance. Recently, Ciutara and Zasadsinky (C. O. Ciutara and J. A. Zasadzinski, Soft Matter, 2021, 17, 5170-5182.) measured the rheological properties of Infasurf®, Survanta® and Curosurf®, three of the most used pulmonary surfactant substitutes. This study revealed that these fluids are shear-thinning and characterized by a yield stress. The results obtained by Ciutara et al. on Curosurf® differ from our results published in L.-P.-A. Thai, F. Mousseau, E. Oikonomou, M. Radiom and J.-F. Berret, Colloids Surf., B, 2019, 178, 337-345. and in L.-P.-A. Thai, F. Mousseau, E. Oikonomou, M. Radiom and J.-F. Berret, ACS Nano, 2020, 14, 466-475. In contrast, we found that Curosurf® suspensions are viscous Newtonian or slightly shear-thinning fluids, with no evidence of yield stress. The purpose of this Comment is to discuss possible causes for the discrepancy between the two studies, and to suggest that for biological fluids such as surfactant substitutes, the microrheology technique of rotational magnetic spectroscopy (MRS) can provide valuable results.