Breathing Together: The Potential of Multidisciplinary Collaboration
Neutron reflectometry provides unique insights into pulmonary surfactant, advancing the development of novel therapeutic materials for surfactant-deficient lung disorders. These results mark the first of many to come from a close and sustained multidisciplinary collaboration between the ILL and Complutense University of Madrid.
Respiratory system schema illustrating the anatomical structure of the lungs, alveoli, and the process of oxygen and carbon dioxide exchange between alveoli and capillaries. (click to enlarge)
Alveoli are microscopic balloon-shaped structures in the lungs that expand and contract to enable the exchange of oxygen and carbon dioxide during breathing. Their surface is lined with pulmonary surfactant, a fluid that reduces surface tension and prevents their collapse. “In preterm infants, the surfactant of immature lungs is deficient making breathing impossible,” explains Jesús Pérez-Gil, Professor of Biochemistry and Molecular Biology at Complutense University of Madrid. “Treatment attempts with lipid-only surfactant mixtures failed, until the late 1980s when two highly unusual, hydrophobic proteins – SP-B and SP-C – were discovered.”
These two proteins – although making up less than 1% of surfactant mass – are indispensable: SP-B is absolutely essential for life, while SP-C, though not strictly required for survival, is linked to chronic respiratory disease when absent. Despite this breakthrough, many key details of the pulmonary surfactant system remain unknown, largely due to the difficulty of reproducing such a complex and inaccessible system in the laboratory, as well as the extreme hydrophobicity of its essential proteins, which makes them challenging to isolate and characterise. “Over the past 30 years, we have built substantial expertise in this field, purifying SP-B and SP-C from natural sources and reconstituting them into standardised lipid models that meaningfully represent physiological conditions,” explains Pérez-Gil. “Yet, even with these advances, we still do not fully understand how these proteins work or the specific contributions of each.”
Models suggest SP-B and SP-C play a critical role in forming multi-layered reservoirs at the alveolar surface during compression, but there was no direct experimental evidence. To investigate these 3D interfacial structures and clarify the specific role of each protein, the researchers turned to neutron reflectometry in collaboration with scientists at the ILL’s FIGARO instrument. “To mimic the air-liquid interface of the alveoli, we use a Langmuir trough – a small water-filled container where drops of the sample spread across the surface form a film, with surface pressure controlled by movable barriers,” explains Javier Carrascosa-Tejedor, research scientist at the ILL. “Installed within the FIGARO reflectometer, this setup allows a real-time investigation of compositional and structural changes at the interface during film compression and expansion, thanks to the ILL’s exceptionally high neutron flux, which enables data collection at an unmatched rate, resolution and quality.”
This unique capability was combined with a powerful contrast method that takes advantage of the significant difference in how neutrons interact with hydrogen and its isotope, deuterium. “The SP-B and SP-C proteins provided by Pérez-Gil’s group were combined with different hydrogenous or deuterated lipid substitutions, allowing specific parts of the system to be masked or highlighted,” explains Carrascosa-Tejedor. “This was essential for revealing the composition and structure of both the monolayer and the lipid reservoirs formed at high surface pressures.” In addition to neutron reflectometry experiments – performed under the most physiologically relevant conditions possible – complementary information was obtained using Langmuir trough and ellipsometry measurements at the Partnership for Soft Condensed Matter (PSCM), and epifluorescence microscopy at Complutense University. “While neutron reflectometry reveals the film’s structure perpendicular to the plane, microscopy corroborates and extends these nanoscale depth profiles by directly showing how the material is distributed within the plane,” explains Carrascosa-Tejedor.
This study provides mechanistic insights into how pulmonary surfactant maintains interfacial stability during respiration.
The results, recently published in the Journal of Colloid and Interface Science, provide experimental evidence of monolayer-to-multilayer transitions at the alveolar surface during compression, clarifying the specific role of each protein. “SP-B was shown to drive the formation and stabilisation of 3D multi-layered lipid reservoirs beneath the interface, confirming its essential role,” explains Ainhoa Collada, researcher at Complutense University of Madrid. “In contrast, no such structures were detected in systems containing only SP-C or lacking both proteins.” These findings align with previous knowledge: SP-B is critical for reservoir formation and, by extension, for breathing, while SP-C plays a more supportive role. “While this study focused on the role of each protein individually, our next article will explore the synergy between the two proteins, that always coexist, yet whose interaction remains little understood,” explains Collada.
Indeed, many more publications are expected to emerge from the close and sustained collaboration between Complutense University and the ILL. “This work – at the frontier of an extremely complex biological system and a cutting-edge experimental technique – illustrates the incredible potential of multidisciplinary collaboration to advance science,” explains Pérez-Gil. “It took years to learn to work together and create a structure in which we speak the same language through mutual effort, investment and adaptation,” continues Collada. The collaboration has already led to significant advances on both sides: improved materials, including the use of genetic engineering to produce human proteins, redesign of the Langmuir trough and its setup to reach higher surface pressures and temperatures, allowing experiments to progressively move even closer to physiological conditions. Looking ahead, the production of deuterated versions of SP-B and SP-C offers great potential for precise localisation, particularly in models containing both proteins simultaneously.
“Our guiding objective remains the development of novel therapeutic materials for patients with surfactant-deficient lung disorders, for which treatment options are currently limited,” explains Pérez-Gil. The structural insights gained from neutron reflectometry represent a significant step towards this goal and highlight the valuable contribution this technique can make to advancing pulmonary surfactant science.
Reference: Ainhoa Collada, Javier Carrascosa-Tejedor, Pablo Sanchez-Puga, Alessio Liguori, Philipp Gutfreund, Andreas Santamaría, José Carlos Castillo-Sanchez, Armando Maestro, Antonio Cruz, Jesús Pérez-Gil. "Escaping from Flatland: the role of proteins SP-B and SP-C in the formation of 3D structures in interfacial pulmonary surfactant films". Journal of Colloid and Interface Science 701 (2026) 138769
https://doi.org/10.1016/j.jcis.2025.138769
ILL instrument: FIGARO
ILL contact person: Javier Carrascosa Tejedor