Size Influences the Effect of Hydrophobic Nanoparticles on Lung Surfactant Model Systems

The alveolar lung surfactant (LS) is a complex lipid protein mixture that forms an interfacial monolayer reducing the surface tension to near zero values and thus preventing the lungs from collapse. Due to the expanding field of nanotechnology and the corresponding unavoidable exposure of human beings from the air, it is crucial to study the potential effects of nanoparticles (NPs) on the structural organization of the lung surfactant system. In the present study, we investigated both, the domain structure in pure DPPC monolayers as well as in lung surfactant model systems. In the pure lipid system we found that two different sized hydrophobic polymeric nanoparticles with diameter of ∼12 nm and ∼136 nm have contrasting effect on the functional and structural behavior. The small nanoparticles inserted into fluid domains at the LE-LC phase transition are not visibly disturbing the phase transition but disrupting the domain morphology of the LE phase. The large nanoparticles led to an expanded isotherm and to a significant decrease in the line tension and thus to a drastic disruption of the domain structures at a much lower number of nanoparticles with respect to the lipid. The surface activity of the model LS films again showed drastic variations due to presence of different sized NPs illustrated by the film balance isotherms and the atomic force microscopy. AFM revealed laterally profuse multilayer protrusion formation on compression but only in the presence of 136 nm sized nanoparticles. Moreover we investigated the vesicle insertion process into a preformed monolayer. A severe inhibition was observed only in the presence of ∼136 nm NPs compared to minor effects in the presence of ∼12 nm NPs. Our study clearly shows that the size of the nanoparticles made of the same material determines the interaction with biological membranes.     

Biophysical inhibition of synthetic lung surfactants

The biophysical activity and inhibition of a series of synthetic surfactant mixtures was studied and correlated with physiological effectiveness in restoring pressure-volume (P-V) mechanics of excised lungs. Results showed that several simple mixtures of dipalmitoyl phosphatidylcholine (DPPC) with fatty acids or diacylglycerols could be formulated to give good adsorption facility and dynamic surface tension lowering to less than 1 mN/m in pulsating bubble measurements at 37 degrees C. However, although biophysical activity approached that of natural lung surfactant (LS) and a related surfactant extract (CLSE) under normal conditions, surface properties were sharply inhibited by relatively small amounts of the plasma protein albumin (2 mg/ml) with minimum surface tensions greater than 30 nM/m even at high surfactant concentrations (5-20 mg lipids/ml). This sensitivity to biophysical inhibition was markedly increased compared to LS and CLSE, and had direct consequences for physiological efficacy: in spite of initially high activity, synthetic surfactants did not exert beneficial effects on P-V mechanics when instilled into surfactant-deficient excised rat lungs. Endogenous protein material was shown to be present upon surfactant recovery by lavage, and bubble measurements confirmed surface activity well below pre-instillation levels. Moreover, full biophysical activity was restored when lavage fluid was extracted to separate the synthetic surfactants from endogenous inhibitors. These results show that it is important to define relative sensitivity to biophysical inhibition in the development of effective lung surfactant substitutes. In addition, the existence of inhibition effects can generate an apparent lack of correspondence between initial biophysical activity and ultimate physiological actions of exogenous surfactant mixtures.     

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.     

Bacterial lipopolysaccharide promotes destabilization of lung surfactant-like films

The airspaces are lined with a dipalmitoylphosphatidylcholine (DPPC)-rich film called pulmonary surfactant, which is named for its ability to maintain normal respiratory mechanics by reducing surface tension at the air-liquid interface. Inhaled airborne particles containing bacterial lipopolysaccharide (LPS) may incorporate into the surfactant monolayer. In this study, we evaluated the effect of smooth LPS (S-LPS), containing the entire core oligosaccharide region and the O-antigen, on the biophysical properties of lung surfactant-like films composed of either DPPC or DPPC/palmitoyloleoylphosphatidylglycerol (POPG)/palmitic acid (PA) (28:9:5.6, w/w/w). Our results show that low amounts of S-LPS fluidized DPPC monolayers, as demonstrated by fluorescence microscopy and changes in the compressibility modulus. This promoted early collapse and prevented the attainment of high surface pressures. These destabilizing effects could not be relieved by repeated compression-expansion cycles. Similar effects were observed with surfactant-like films composed of DPPC/POPG/PA. On the other hand, the interaction of SP-A, a surfactant membrane-associated alveolar protein that also binds to LPS, with surfactant-like films containing S-LPS increased monolayer destabilization due to the extraction of lipid molecules from the monolayer, leading to the dissolution of monolayer material in the aqueous subphase. This suggests that SP-A may act as an LPS scavenger.     

Mechanics of edematous lungs.

Using the parenchymal marker technique, we measured pressure (P)-volume (P-V) curves of regions with volumes of approximately 1 cm3 in the dependent caudal lobes of oleic acid-injured dog lungs, during a very slow inflation from P = 0 to P = 30 cmH2O. The regional P-V curves are strongly sigmoidal. Regional volume, as a fraction of volume at total lung capacity, remains constant at 0.4-0.5 for airway P values from 0 to approximately 20 cmH2O and then increases rapidly, but continuously, to 1 at P = approximately 25 cmH2O. A model of parenchymal mechanics was modified to include the effects of elevated surface tension and fluid in the alveolar spaces. P-V curves calculated from the model are similar to the measured P-V curves. At lower lung volumes, P increases rapidly with lung volume as the air-fluid interface penetrates the mouth of the alveolus. At a value of P = approximately 20 cmH2O, the air-fluid interface is inside the alveolus and the lung is compliant, like an air-filled lung with constant surface tension. We conclude that the properties of the P-V curve of edematous lungs, particularly the knee in the P-V curve, are the result of the mechanics of parenchyma with constant surface tension and partially fluid-filled alveoli, not the result of abrupt opening of airways or atelectatic parenchyma.     

Pores of Kohn are filled in normal lungs: low-temperature scanning electron microscopy.

Interalveolar pores of Kohn, small uniform-sized epithelium-lined openings in alveolar walls of normal lung, have historically been demonstrated with electron-microscopic techniques that remove water. We show these pores to be present but almost invariably filled with material when water and surfactant are preserved in frozen hydrated lung examined with low-temperature scanning electron microscopy. In the normal mouse, 16 open empty pores per alveolus were found in instillation-fixed dried lung vs. less than 1 per alveolus in frozen hydrated lungs (P less than 0.001). In the normal rat, 13 pores were seen per alveolus in instillation-fixed dried lung vs. less than 1 per alveolus in frozen hydrated lungs (P less than 0.001). We suggest that pores of Kohn 1) function primarily as conduits for interalveolar movement of alveolar liquid, surfactant components, and macrophages, 2) provide distributed sites for tubular myelin storage without increasing gas diffusion pathway thickness in the alveolar subphase itself, and 3) do not function as pathways for collateral ventilation during normal breathing in the absence of atelectasis or obstruction.     

Nanoparticles modulate membrane interactions of human Islet amyloid polypeptide (hIAPP)

The dramatic expansion of nanotechnology applications, particularly the advent of nanomaterials and nanoparticles (NPs) into the consumer economy, have led to heightened awareness of their potential health risks. This study examines the impact of several NPs upon membrane-induced aggregation and bilayer interactions of the human Islet amyloid polypeptide (hIAPP). We report that several NPs – polymeric NPs, TiO₂ NPs, and Au NPs displaying coating layers exhibiting different electrostatic charges - did not significantly interfere with the fibrillation process and fibril morphology of hIAPP, both in buffer or in biomimetic DMPC:DMPG vesicle solutions. Spectroscopic and microscopic analyses suggest, in fact, that the NPs promoted membrane-induced fibrillation. Importantly, we find that all the NPs examined, regardless of composition or surface properties, gave rise to more pronounced, synergistic bilayer interactions when co-incubated with hIAPP. NP-enhanced bilayer interactions of hIAPP might point to possible toxicity and pathogenicity risks of amyloidogenic peptides in the presence of NPs.     

Influence of Plasmonic Nanoparticles on the Performance of Colorimetric Cell Viability Assays

The conventional colorimetric assays based on measurement of the metabolic activity are routinely used to evaluate the cytotoxicity of nanomaterials (NMs). However, due to the varying absorbance properties of plasmonic NMs in the visible region of the spectrum, obtained results can be misleading. In this study, MTT, MTS, and WST-1 colorimetric cell viability assays were evaluated in the presence of gold (AuNPs) or silver nanoparticles (AgNPs). Since a living cell a complex system containing many molecular and ionic species, the plasmonic AuNP and AgNPs may selectively interact with intracellular components possessing thiol, amino, and carboxyl group moieties change the aggregation behavior of the NMs and thus their absorbance. A series of UV/Vis and DLS experiments were conducted to understand the interference possibility of the tested plasmonic NMs. The results show that the AuNPs and AgNPs do not have absorption at the wavelength where MTT formazan is measured while the both NPs may interfere with absorbance of MTS and WST-1 formazan.The overall assessments show that MTT assay is more suitable for the cell viability evaluation of spherical AuNPs and AgNPs with an average diameter of 50 nm. This study also suggests that a preliminary ex situ evaluation of plasmonic nanoparticles can provide valuable information for the suitability of the assay.     

Interaction of densely polymer-coated gold nanoparticles with epithelial Caco-2 monolayers

We synthesized a library of polymer-coated gold nanoparticles (AuNPs) with well-defined sizes (5, 10, and 20 nm) and surface properties, and investigated their efficiency to cross the Caco-2 epithelial barrier and disrupt tight junctions connecting the cellular barrier. The positively charged and hydrophobic polymer-coated AuNPs showed little or no translocation across the model Caco-2 monolayer. Most of these positive and hydrophobic nanoparticles were either bound to the surface or internalized within the cell. The neutral and negatively charged polymer-coated AuNPs with a size of 5 nm showed a significantly higher translocation. All polymer-coated AuNPs induced the translocation of small molecules across the cellular monolayer, suggesting the loosening of the paracellular tight junction joining individual cells. The decrease in the TEER values of the monolayers supported the opening of the tight junctions. These tight junctions fully recovered for most polymer-coated AuNPs 12 h after removal of the nanoparticles. The exception was the cationic polymer-coated AuNPs in which the barrier function only recovered up to 62%. The library of polymer-coated AuNPs showed no apparent signs of hemolysis to erythrocytes at physiological pH. Our investigation has provided insight on the influence of polymer coatings on the epithelial barrier.     

Toxicity of gold nanoparticles (AuNPs): A review

Gold nanoparticles are a kind of nanomaterials that have received great interest in field of biomedicine due to their electrical, mechanical, thermal, chemical and optical properties. With these great potentials came the consequence of their interaction with biological tissues and molecules which presents the possibility of toxicity. This paper aims to consolidate and bring forward the studies performed that evaluate the toxicological aspect of AuNPs which were categorized into in vivo and in vitro studies. Both indicate to some extent oxidative damage to tissues and cell lines used in vivo and in vitro respectively with the liver, spleen and kidney most affected. The outcome of these review showed small controversy but however, the primary toxicity and its extent is collectively determined by the characteristics, preparations and physicochemical properties of the NPs. Some studies have shown that AuNPs are not toxic, though many other studies contradict this statement. In order to have a holistic inference, more studies are required that will focus on characterization of NPs and changes of physical properties before and after treatment with biological media. So also, they should incorporate controlled experiment which includes supernatant control Since most studies dwell on citrate or CTAB-capped AuNPs, there is the need to evaluate the toxicity and pharmacokinetics of functionalized AuNPs with their surface composition which in turn affects their toxicity. Functionalizing the NPs surface with more peculiar ligands would however help regulate and detoxify the uptake of these NPs.     

Three-dimensional reconstruction of alveoli in the rat lung for pressure-volume relationships

To determine alveolar pressure-volume relationships, alveolar three-dimensional reconstructions were prepared from lungs fixed by vascular perfusion at various points on the pressure-volume curve. Lungs from male Sprague-Dawley rats were fixed by perfusion through the pulmonary artery following a pressure-volume maneuver to the desired pressure point on either the inflation or deflation curve. Tissue samples from lungs were serially sectioned for determination of the volume fraction of alveoli and alveolar ducts and reconstruction of alveoli. Alveoli from lungs fixed at 5 cmH2O on the deflation curve (approximating functional residual volume) had a volume of 173 X 10(3) microns3, a surface area of 11,529 microns2, a mouth opening diameter of 72.7 microns, and a mean caliper diameter of 91.8 micron (SE). Alveolar shape changes during deflation from total lung capacity to residual volume was first (30 to 10 cmH2O) associated with little change in the diameter of the alveoli (102.7 +/- 2.4 to 100.3 +/- 3.3 microns). In the range overlapping normal breathing (10 to 0 cmH2O) there was a substantial decrease in diameter (100.3 +/- 3.3 to 43.3 +/- 2.3 microns). These measurements and others made on the relative changes in the dimensions of the alveolus suggest that the elastic network, particularly around the alveolar ducts, are predominant in determining lung behavior near the volume expansion limits of the lung while the elastic and surface tension properties of the alveoli are predominant in the volume range around functional residual capacity.     

Persistent adult zebrafish behavioral deficits results from acute embryonic exposure to gold nanoparticles

As the number of products containing nanomaterials increase, human exposure to nanoparticles (NPs) is unavoidable. Presently, few studies focus on the potential long-term consequences of developmental NP exposure. In this study, zebrafish embryos were acutely exposed to three gold NPs that possess functional groups with differing surface charge. Embryos were exposed to 50 μg/mL of 1.5 nm gold nanoparticles (AuNPs) possessing negatively charged 2-mercaptoethanesulfonic acid (MES) or neutral 2-(2-(2-mercaptoethoxy)ethoxy)ethanol (MEEE) ligands or 10 μg/mL of the AuNPs possessing positively charged trimethylammoniumethanethiol (TMAT). Both MES- and TMAT-AuNP exposed embryos exhibited hypo-locomotor activity, while those exposed to MEEE-AuNPs did not. A subset of embryos that were exposed to 1.5 nm MES- and TMAT-AuNPs during development from 6 to 120 h post fertilization was raised to adulthood. Behavioral abnormalities and the number of survivors into adulthood were evaluated at 122 days post fertilization. We found that both treatments induced abnormal startle behavior following a tap stimulus. However, the MES-AuNPs exposed group also exhibited abnormal adult behavior in the light and had a lower survivorship into adulthood. This study demonstrates that acute, developmental exposure to 1.5 nm MES- and TMAT-AuNPs, two NPs differing only in the functional group, affects larval behavior, with behavioral effects persisting into adulthood.     

Pulmonary and gastric surfactants. A comparison of the effect of surface requirements on function and phospholipid composition

Surfactant is present in the alveoli and conductive airways of mammalian lungs. The presence of surface active agents was, moreover, demonstrated for avian tubular lungs and for the stomach and intestine. As the surface characteristics of these organs differ from each other, their surfactants possess distinct biochemical and functional characteristics. In the stomach so-called 'gastric surfactant' forms a hydrophobic barrier to protect the mucosa against acid back-diffusion. For this purpose gastric mucosal cells secrete unsaturated phosphatidylcholines (PC), but no dipalmitoyl-PC (PC16:0/16:0). By contrast, surfactant from conductive airways, lung alveoli and tubular avian lungs contain PC16:0/16:0 as their main component in similar concentrations. Hence, there is no biochemical relation between gastric and pulmonary surfactant. Alveolar surfactant, being designed for preventing alveolar collapse under the highly dynamic conditions of an oscillating alveolus, easily reaches values of <5 mN/m upon cyclic compression. Surfactants from tubular air-exposed structures, however, like the conductive airways of mammalian lungs and the exclusively tubular avian lung, display inferior compressibility as they only reach minimal surface tension values of approximately 20 mN/m. Hence, the highly dynamic properties of alveolar surfactant do not apply for surfactants designed for air-liquid interfaces of tubular lung structures.     

In-vitro in-vivo correlation (IVIVC) in nanomedicine: Is protein corona the missing link?

One of the unmet challenges in nanotechnology is to understand and establish the relationship between physicochemical properties of nanoparticles (NPs) and its biological interactions (bio-nano interactions). However, we are still far from assessing the biofate of NPs in a clear and unquestionable manner. Recent developments in the area of bio-nano interface and the understanding of protein corona (PC) has brought new insight in predicting biological interactions of NPs. PC refers to the spontaneous formation of an adsorbed layer of biomolecules on the surface of NPs in a biological environment. PC formation involves the spatiotemporal interplay of an intricate network of biological, environmental and particle characteristics. NPs with its PC can be viewed as a biological entity, which interacts with cells and barriers in a biological system. Recent studies on the bio-nano interface have revealed biological signatures that participate in cellular and physiological bioprocesses and control the biofate and toxicity of NPs. The ability of in-vitro derived parameters to forecast in-vivo consequences by developing a mathematical model forms the basis of in-vitro in-vivo correlation (IVIVC). Understanding the effect of bio-nano interactions on the biological consequences of NPs at the cellular and physiological level can have a direct impact on the translation of future nanomedicines and can lead to the ultimate goal of developing a mathematical IVIVC model. The review summarizes the emerging paradigms in the field of bio-nano-interface which clearly suggests an urgent need to revisit existing protocols in nanotechnology for defining the physicochemical correlates of bio-nano interactions.     

Effects of hemoglobin and cell membrane lipids on pulmonary surfactant activity

These experiments characterize the effects of hemoglobin and erythrocyte membrane lipids on the dynamic surface activity and adsorption facility of whole lung surfactant (LS) and a calf lung surfactant extract (CLSE) used clinically in surfactant replacement therapy for the neonatal respiratory distress syndrome (RDS). The results show that, at concentrations from 25 to 200 mg/ml, hemoglobin (Hb) increased the minimum dynamic surface tension of LS or CLSE mixtures (0.5 and 1.0 mumol/ml) from less than 1 to 25 dyn/cm on an oscillating bubble apparatus at 37 degrees C. Similarly, erythrocyte membrane lipids (0.5-3 mumol/ml) also prevented LS and CLSE suspensions (0.5-2.0 mumol/ml) from lowering surface tension below 19 dyn/cm under dynamic compression on the bubble. Surface pressure-time adsorption isotherms for LS suspensions (0.084 and 0.168 mumol phospholipid/ml) were also adversely affected by Hb (0.3-2.5 mg/ml), having a slower adsorption rate and magnitude. Significantly, these inhibitory effects of Hb and membrane lipids could be abolished if LS and CLSE concentrations were raised to high levels. In complementary physiological experiments, instillation of Hb, membrane lipids, or albumin into excised rat lungs was shown to cause a decrease in pressure-volume compliance. This decreased compliance was most prominent in lungs made partially surfactant deficient before inhibitor delivery and could be reversed by supplementation with active exogenous surfactant. Taken together, these data show that molecular components in hemorrhagic pulmonary edema can biophysically inactivate endogenous LS and adversely affect lung mechanics. Moreover, exogenous surfactant replacement can reverse this process even in the continued presence of inhibitor molecules and thus has potential utility in therapy for adult as well as neonatal RDS.     

Alveolar sphingolipids generated in response to TNF-alpha modifies surfactant biophysical activity.

Sphingolipids represent a diverse group of bioactive lipid species that are generated intracellularly in response to tumor necrosis factor-alpha (TNF-alpha) and are implicated as potential mediators of acute lung injury. The purpose of these studies was to determine whether there was an extracellular, TNF-alpha-regulated pool of sphingolipids in the alveolus that modulates the surface tension lowering capacity of pulmonary surfactant. Intratracheal instillation of TNF-alpha in adult rats led to a twofold increase in the amount of surfactant-associated ceramide and tended to decrease levels of sphingomyelin without significantly altering sphingosine or sphinganine content. TNF-alpha induction of alveolar ceramide was associated with nearly an 80% increase in acid sphingomyelinase activity recovered in cell-free alveolar lavage. Ceramide administered in a dose-dependent manner potently antagonized the surface tension lowering effects of natural surfactant in vitro. Intratracheal TNF-alpha and ceramide treatment of rats significantly increased lung permeability, as was evidenced by extravasation of Evans blue dye into alveolar lavage and lung tissue. Thus these studies are the first to demonstrate the existence of a cytokine-regulated alveolar pool of sphingomyelin hydrolysis products that impairs the biophysical properties of the alveolar surfactant film. The results also suggest the presence of a secretory alveolar sphingomylinase that is TNF-alpha responsive and mediates effects of the cytokine on alveolar sphingolipid metabolism.     

Ultrasensitive electrical detection of protein using nanogap electrodes and nanoparticle-based DNA amplification

The present study describes an ultrasensitive protein biochip that employs nanogap electrodes and self-assembled nanoparticles to electrically detect protein. A bio-barcode DNA technique amplifies the concentration of target antigen at least 100-fold. This technique requires the establishment of conjugate magnetic nanoparticles (MNPs) and gold nanoparticles (AuNPs) through binding between monoclonal antibodies (2B2), the target antigen, and polyclonal antibodies (GP). Both GP and capture ssDNA (single-strand DNA) bonds to bio-barcode ssDNA are immobilized on the surface of AuNPs. A denature process releases the bio-barcode ssDNAs into the solution, and a hybridization process establishes multilayer AuNPs over the gap surface between electrodes. Electric current through double-layer self-assembled AuNPs is much greater than that through self-assembled monolayer AuNPs. This significant increase in electric current provides evidence that the solution contains the target antigen. Results show that the protein biochip attains a sensitivity of up to 1 pg/μL.     

Study on physisorption between phycocyanin and gold nanoparticles

Interactions between nanoparticles (NPs) and biomolecules, especially proteins, have attracted increasing attention. Photoresponsive proteins have shown high potential for optogenetic research. The combination between optogenetics and nanotechnology will bring a new biological era in which photoresponsive proteins will inevitably encounter NPs, therefore their interactions will be a key point to investigate. Here, we have systematically studied the interactions between a photoresponsive protein (called phycocyanin, PC) and a typical kind of amphiphilic polymer‐coated gold NPs (AP–AuNPs) using fluorescence quenching methods. The results showed that the binding constant between PCs and AP–AuNPs is 4.427 × 10⁶ M^–1 with a positive cooperativity, and the robust affinity was hydrophobic interaction driven mortise–tenon conjugation, which could even resist gel electrophoresis. These results could also shed light on potential designs for building up artificial protein–NP light‐harvesting systems.     

Role of lipid ordered/disordered phase coexistence in pulmonary surfactant function

The respiratory epithelium has evolved to produce a complicated network of extracellular membranes that are essential for breathing and, ultimately, survival. Surfactant membranes form a stable monolayer at the air-liquid interface with bilayer structures attached to it. By reducing the surface tension at the air-liquid interface, surfactant stabilizes the lung against collapse and facilitates inflation. The special composition of surfactant membranes results in the coexistence of two distinct micrometer-sized ordered/disordered phases maintained up to physiological temperatures. Phase coexistence might facilitate monolayer folding to form three-dimensional structures during exhalation and hence allow the film to attain minimal surface tension. These folded structures may act as a membrane reserve and attenuate the increase in membrane tension during inspiration. The present review summarizes what is known of ordered/disordered lipid phase coexistence in lung surfactant, paying attention to the possible role played by domain boundaries in the monolayer-to-multilayer transition, and the correlations of biophysical inactivation of pulmonary surfactant with alterations in phase coexistence.     

Surface forces in lungs. I. Alveolar surface tension-lung volume relationships

Alveolar surface tension (gamma)-lung volume relationships were obtained for quasi-static and dynamic lung pressure-volume (PV) histories from measurements of PV curves of liquid- and air-filled excised rabbit lungs. PV relationships were measured at room temperature in lungs filled with test liquids with constant liquid-liquid interfacial tensions with alveolar surface-active materials; and air-filled lungs before and after the normal alveolar surface film was covered with test liquids with constant values of liquid- and air-liquid interfacial tensions. Interfacial tensions of test liquids were measured in a surface balance on monolayers of dipalmitoyl phosphatidylcholine. Values of gamma for the normal air-filled lung were obtained either from points of intersection between PV curves with the normal and test liquid interface or from a general relationship between gamma and the component of recoil pressure due to surface tension derived from the data. In contrast to previous analyses that have used PV measurements, this approach does not depend on assumptions about lung microstructural geometry. Surface tension-volume relationships for the normal air-filled lung show a prominent hysteresis with surface tension ranging from near 0 at low volumes during lung deflation to transiently high values near 40 dyn/cm during inflation; value of equilibrium surface tension (gamma EQ) near 28 dyn/cm; and characteristic transitions in surface film compressibility and associated transitions in film kinetic behavior in nonequilibrium film states where gamma deviates from gamma EQ. These features are consistent with the behavior predicted from current models of alveolar surface film behavior.