A Sigma1 Receptor Agonist Alters Fluidity and Stability of Lipid Monolayers.

Interactions between the sigma1 receptor agonist PRE-084 and various lipid monolayers, including dipalmitoylphosphatidylcholine (DPPC), DPP-ethanolamine (DPPE), DPP-glycerol (DPPG), DPP-serine (DPPS), palmitoylsphingomyelin (PSM), and cholesterol (Ch), were investigated to elucidate the effects of PRE-084 on membrane fluidity and stability. Their interactions with sigma1 receptor agonists have potential implications for neuroprotection, antidepressant, analgesic, and cognitive enhancement effects. In this study, we observed that the presence of PRE-084 in the subphase led to increased fluidity in DPPC and DPPE monolayers, whereas decreasing fluidity was observed in DPPG, DPPS, and PSM monolayers. The interaction of PRE-084 with Ch monolayers was found to be distinct from its interaction with other lipids. Fluorescence microscopy images revealed changes in the size and shape of liquid-condensed domains in the presence of PRE-084, supporting the notion of altered membrane fluidity. Our findings provide new insights into the interaction of PRE-084 with lipid monolayers and its potential implications for biological and membrane science.

How Self-Assembled Nanodomains Can Impact the Organization of a Phospholipid Monolayer-Flower-Like Arrays.

We found that monolayers of dipalmitoylphosphatidylcholine (DPPC) and semi-fluorinated tetrablock di(F10H16) self-assemble to form a new type of large, complex flower-like patterns on the surface of water and on solid substrates. The hierarchical organization of these unusual self-assemblies was investigated using compression and surface potential isotherms, in situ fluorescence and Brewster angle microscopies, and atomic force microscopy after transfer.

Inverse Electron Demand Diels-Alder Reactions in the Liposomal Membrane Accelerates Release of the Encapsulated Drugs.

Bio-orthogonal inverse electron demand Diels-Alder (IEDDA) reactions between liposomes containing a tetrazine-based (Tz) compound and 2-norbornene (2-NB) could be a novel trigger for accelerating drug release from the liposomes via temporary membrane destabilization, as shown in our previous report. Herein, we evaluated the in vitro drug release using NB derivatives with carboxyl groups [5-norbornene-2-carboxylic acid (NBCOOH) and 5-norbornene-2,3-dicarboxylic acid (NB(COOH)2)] to investigate the effects of substituents at the NB backbone on the drug release rate. First, POTz-liposome composed of a Tz compound (2-hexadecyl-N-(6-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)pyridin-3-yl)octadecanamide) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) were prepared. The mass spectrometry analysis revealed the binding of NB derivatives to the Tz compound via the IEDDA reaction after the POTz-liposome reacted with the NB derivatives. Indium-111-labeled diethylenetriaminepentaacetic acid (111In-DTPA) was encapsulated inside the liposomes, and the drug release rate was quantified by measuring radioactivity. At 24 h after incubation with 2-NB, NBCOOH, and NB(COOH)2, the release rates of 111In-DTPA from POTz-liposome were 21.0, 80.8, and 23.3%, respectively, which were significantly higher than those of POTz-liposome that was not treated with NB derivatives (4.2%), indicating the involvement of the IEDDA reaction for prompting drug release. Additionally, a thermodynamic evaluation using Langmuir monolayers was conducted to explore the mechanism of the accelerated drug release. An increase in membrane fluidity and a reduction in intermolecular repulsion between POPC and the Tz compound were observed after the reaction with NB derivatives, especially for NBCOOH. Thus, the IEDDA reaction in the liposomal membrane could be a potent trigger for accelerating the release of encapsulated drugs by regulating membrane fluidity and intermolecular repulsion in the liposomal membrane.

Interactions of a Tetrazine Derivative with Biomembrane Constituents: A Langmuir Monolayer Study.

Tetrazine (Tz) is expected to be used for bioimaging and as an analytical reagent. It is known to react very fast with trans-cyclooctene under water in organic chemistry. Here, to understand the interaction between Tz and biomembrane constituents, we first investigated the interfacial behavior of a newly synthesized Tz derivative comprising a C18-saturated hydrocarbon chain (rTz-C18) using a Langmuir monolayer spread at the air-water interface. Surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms were measured for monolayers of rTz-C18 and biomembrane constituents such as dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyl sphingomyelin (PSM), and cholesterol (Ch). The lateral interaction between rTz-C18 and the lipids was thermodynamically elucidated from the excess Gibbs free energy of mixing and two-dimensional phase diagram. The binary monolayers except for the Ch system indicated high miscibility or affinity. In particular, rTz-C18 was found to interact more strongly with DPPE, which is a major constituent of the inner surface of cell membranes. The phase behavior and morphology upon monolayer compression were investigated by using Brewster angle microscopy (BAM), fluorescence microscopy (FM), and atomic force microscopy (AFM). The BAM and FM images of the DPPC/rTz-C18, DPPG/rTz-C18, and PSM/rTz-C18 systems exhibited a coexistence state of two different liquid-condensed domains derived mainly from monolayers of phospholipids and phospholipids-rTz-C18. From these morphological observations, it is worthy to note that rTz-C18 is possible to interact with a limited amount of the lipids except for DPPE.

Ionic Nature of a Gemini Surfactant at the Air/Water Interface.

The ionic state of an adsorbed gemini surfactant at the air/water interface was investigated using a combination of surface potential and surface tension data. The combined model was developed and successfully described the experimental data. The results verified the existence of three ionic states of the gemini surfactant in the interfacial zone. Furthermore, the model can quantify the adsorbed concentrations of these species. At low concentrations, the fully dissociated state dominates the adsorption. At high concentrations, the fully associated state dominates, accounting for up to 80% of the total adsorption. In the middle range, the adsorption is dominated by the partially associated state, which has a maximum percentage of 80% at a critical micelle concentration of 0.5. The variation in the ionic state is a unique characteristic of gemini surfactants, which can be the underlying mechanism for their advantages over conventional surfactants.

Improvement of pulmonary surfactant activity by introducing D-amino acids into highly hydrophobic amphiphilic α-peptide Hel 13-5.

The high costs of artificial pulmonary surfactants, ranging in hundreds per kilogram of body weight, used for treating the respiratory distress syndrome (RDS) premature babies have limited their applications. We have extensively studied soy lecithins and higher alcohols as lipid alternatives to expensive phospholipids such as DPPC and PG. As a substitute for the proteins, we have synthesized the peptide Hel 13-5D3 by introducing D-amino acids into a highly lipid-soluble, basic amphiphilic peptide, Hel 13-5, composed of 18 amino acid residues. Analysis of the surfactant activities of lipid-amphiphilic artificial peptide mixtures using lung-irrigated rat models revealed that a mixture (Murosurf SLPD3) of dehydrogenated soy lecithin, fractionated soy lecithin, palmitic acid (PA), and peptide Hel 13-5D3 (40:40:17.5:2.5, by weight) superior pulmonary surfactant activity than a commercially available pulmonary surfactant (beractant, Surfacten®). Experiments using ovalbumin-sensitized model animals revealed that the lipid-amphiphilic artificial peptide mixtures provided significant control over an increase in the pulmonary resistance induced by premature allergy reaction and reduced the number of acidocytes and neutrophils in lung-irrigated solution. The newly developed low-cost pulmonary surfactant system may be used for treatment of a wide variety of respiratory diseases.

Molecular dynamics investigation on adsorption layer of alcohols at the air/brine interface.

Alcohols are a significant group of surfactants which have been employed extensively in industry to improve the interfacial effects. Recently, the change in surface potential (ΔV) of two isomeric hexanols, methyl isobutyl carbinol (MIBC) and 1-hexanol, was investigated by using an ionizing (241)Am electrode. It clearly showed the opposite effects between MIBC and 1-hexanol in the interfacial zone: one enhanced the presence of cations, whereas the other enhanced the presence of anions. This study employs molecular dynamics simulation to provide new insights into the interactions between alcohol molecules and ions as well as water at the molecular level. The results qualitatively agreed with the experimental data and verified the significance of MIBC branching structure on the molecular arrangement within the interfacial zone. The results also highlighted the role of the second water layer on the interfacial properties.

Surface pressure induced structural transitions of an amphiphilic peptide in pulmonary surfactant systems by an in situ PM-IRRAS study.

Pulmonary surfactant model peptide, Hel 13-5, in binary and ternary lipid mixtures has been characterized employing the polarization-modulation infrared reflection-absorption spectroscopy (PM-IRRAS) in situ at the air-water interface for a monolayer state and the polarized ATR-FTIR for a bilayer film. In the bilayer form, Hel 13-5 predominantly adopts an α-helical secondary structure in the lipid mixtures. It had been made clear from CD measurements that the Hel 13-5 structure is mainly in the α-helical form in aqueous solutions. In the monolayer state, however, the secondary structure of Hel 13-5 exhibits an interconversion of the α-helix into ß-sheet with increasing surface pressures. The difference in the secondary structure is attributed to formation of a surface-associated reservoir just below the surface monolayer. The reservoir formation is a key function of pulmonary surfactants and is induced by a squeeze-out of the fluid components in their monolayers. Compression and expansion cycles of the monolayers generate a hysteresis in molecular orientation of the lipid monolayer as well as in peptide structure. The formation and deformation of reservoirs are, in common, deeply related to the hysteresis behavior. Thus, the transition of peptide structures across the interface is a quite important matter to clarify the role and its mechanism of the reservoirs in pulmonary functions. The present study primarily reveals roles of the anionic lipids in control of the peptide secondary structure. Accordingly, it is demonstrated that they prevent the protein structure transition from α-helix into ß-sheet by incorporating the peptide during the squeeze-out event.

Investigation of interfacial behavior of glycyrrhizin with a lipid raft model via a Langmuir monolayer study.

An interaction of glycyrrhizin (GC) with a lipid raft biomembrane model that consisted of N-palmitoyl-d-erythro-sphingosylphosphorylcholine (PSM), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol (CHOL) was systematically studied using the Langmuir monolayer technique. To construct the lipid raft model, the surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms for three-component (PSM/DOPC/CHOL) systems on 0.02M Tris buffer with 0.13M NaCl (pH7.4) were primarily measured by changing their compositions. Thermodynamic and interaction parameters for binary PSM/DOPC and PSM/CHOL systems revealed that PSM interacts more strongly with CHOL than with DOPC. In addition, a morphological analysis performed with Brewster angle microscopy (BAM) and fluorescence microscopy (FM) revealed an optimal ratio of PSM/DOPC/CHOL (1/1/1, by mole) as a model of lipid rafts. Second, the interaction of GC with the ternary PSM/DOPC/CHOL monolayers was investigated on Tris buffer solutions containing different GC concentrations (1, 5, 10, 25, and 50µM). In BAM and FM images, microdomains were found to become smaller by increasing the GC concentration in the subphase, suggesting that GC regulates the size of raft domains, which provide dynamic scaffolding for numerous cellular processes. More interestingly, the distinctive GC striped regions were formed at the interface at 50µM, which shows that GC divides the ternary monolayer into pieces. This phenomenon was observed only in the presence of CHOL in the monolayer. These results suggest that CHOL plays an essential role in the interaction with GC, which results in one of the major activities associated with saponins' membrane disruption.

Solubilization of n-alkylbenzenes into gemini surfactant micelles in aqueous medium.

Solubilization of benzene, toluene, ethylbenzene, n-propylbenzene, n-butylbenzene, and n-pentylbenzene into micelles of decanediyl-1-10-bis(dimethyltetradecylammonium bromide) (14-10-14,2Br(-)) has been investigated in the temperature range from 288.2 to 308.2 K. The equilibrium concentrations of all the solubilizates are determined spectrophotometrically. The concentration of the solubilizates remains constant below the critical micelle concentration (cmc) and increases linearly with an increase in 14-10-14,2Br(-) concentration above the cmc. Compared to the mother micelle, the solubilized micelles indicate much larger hydrodynamic diameters, which are determined by dynamic light scattering. Therefore, the Gibbs energy change for the solubilization of n-alkylbenzenes has been evaluated by the partitioning of the solubilizates between the aqueous and micellar phases. Furthermore, the enthalpy and entropy changes for the solubilization could be calculated from temperature dependence of the Gibbs energy change. From the thermodynamic parameters, it is found that the solubilization for the present system is entropy-driven and that the location of the solubilizates moves into the inner core of the micelle with an elongation of their alkyl chains. The movement on the location is also supported by the results of absorption spectra, Fourier transform infrared (FTIR) spectra, and two-dimensional nuclear Overhauser effect spectroscopy (2-D NOESY).

Ionic Distribution of an Unequal Electrolyte Near an Air/Water Surface.

The distribution of electrolytes near the air/water surface plays an essential role in many processes. While the general distribution is governed by classic Poisson-Boltzmann statistics, the analytical solution is only available for symmetric electrolytes. From the recent studies in the literature, it is evident that surface adsorption is dependent on specific ions as well as the H-bond structure at the surface. Experimental data can capture the macro properties of the surface, such as surface tension and surface potential. Yet, the underpinning mechanisms behind this experimental macro-observation remain unclear. To address the challenge, we developed a framework combining experimental studies and numerical calculations. The model was developed for electrolytes with unequal cationic and anionic charges. The asymmetric model was successfully applied to describe the surface charge of MgCl 2 aqueous solution. The results can be explained by the role of cationic size and charge on the surface layer.

Interplay of long-chain tetrazine derivatives and biomembrane components at the air-water interface.

Tetrazine (Tz) is an emerging bioorthogonal ligand that is expected to have applications (e.g., bioimaging) in chemistry and chemical biology. In this review, we highlight the interactions of reduced tetrazine (rTz) derivatives insoluble in aqueous media with biological membrane constituents or their related lipids, such as dipalmitoyl-phosphatidylcholine, dipalmitoyl-phosphatidylethanolamine, dipalmitoyl-phosphatidylglycerol, palmitoyl-sphingomyelin, and cholesterol in the Langmuir monolayer state at the air-water interface. The two-component interaction was thermodynamically elucidated by measuring the surface pressure (π) and molecular area (A) isotherms. The monolayer miscibility between the two components was analyzed using the excess Gibbs energy of mixing and two-dimensional phase diagram. The phase behavior of the binary monolayers was studied using the Brewster angle, fluorescence, and atomic force microscopy. This study discusses the affinities of the rTz moieties for the hydrophilic groups of the lipids used.

Inverse electron-demand diels-alder reactions of tetrazine and norbornene at the air-water interface.

The surface chemistry of the inverse electron-demand Diels-Alder (IEDDA) reaction at the air-water interface is elucidated. Tetrazine (C18-Tz) and norbornene derivatives (C16-NCA) were used as the reactants. Langmuir monolayers of C18-Tz, C16-NCA, and their binary mixtures were prepared on aqueous substrates. The surface properties were analyzed using the surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms, as well as fluorescence microscopy to monitor the progress of the reaction. First, to provide comparison data to evaluate the reaction on the surface, the two components were mixed in stock solutions of organic solvents for the IEDDA reaction. The Langmuir monolayer spread from the reaction solution was characterized as a function of the reaction time. In the subsequent experiments, the Langmuir monolayers were deposited onto the surface of the substrate solutions by spreading from separate stock solutions of C18-Tz and C16-NCA. The variation of the surface behavior of the monolayers with the molecular area, surface composition of the two components, compression speed of the monolayers, and the temperature was studied. We discuss the effects of the air phase in the reaction field on the reaction efficiency by comparing the results obtained from the two methods.

Specific interaction restrains structural transitions of an amphiphilic peptide in pulmonary surfactant model systems: an in situ PM-IRRAS investigation.

In situ polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) at the air-water interface has been used to determine secondary structure of the pulmonary surfactant model peptide, Hel 13-5, in the absence and the presence of phospholipid monolayers. Herein, fully saturated phospholipids of DPPC and DPPG are utilized to understand the effect of specific interaction between anionic DPPG and cationic Hel 13-5 on the peptide secondary structure. The spectrum frequency in the amide region (1500-1700cm(-1)) obtained from PM-IRRAS has been confirmed by comparing with that from ATR-FTIR for the corresponding bulk films. The PM-IRRAS spectra of single Hel 13-5 monolayers indicate the alpha-helical contour in the amide region, which coincides with the result from CD measurements in aqueous solutions. In the presence of phospholipid monolayers, however, Hel 13-5 changes its conformation from the alpha-helix to the extended beta-sheet as surface pressure increases upon compression at the interface, and this interconversion is found to be irreversible even during expansion process of monolayers. Furthermore, it is notable that the electrostatic interaction between DPPG and Hel 13-5 inhibits to some extent the interconversion to the beta-sheet during compression. These features are completely different from the bulk behavior, which demonstrates different roles of native proteins in the bulk phase and at the interface for pulmonary functions. In addition, the conformational variation of Hel 13-5 does not indicate close correlation with surface activity, which is common characteristic even for reversible hysteresis curves in pulmonary surfactant systems. This suggests that the secondary structure of native proteins is not strongly related to the surface activity during respiration. This work contributes to secondary structure determination of Hel 13-5 in the phospholipid domains in situ at the air-water interface and will provide insight into the molecular and physiological mechanism for SP-B and SP-C actions across the interface.

Fluorocarbon-hybrid pulmonary surfactants for replacement therapy--a Langmuir monolayer study.

Effective additives to pulmonary surfactant (PS) preparations for therapy of respiratory distress syndrome (RDS) are being intensively sought. We report here the investigation of the effects of partially fluorinated amphiphiles (PFA) on the surface behavior of a model PS formulation. When small amounts of a partially fluorinated alcohol C(8)F(17)C(m)H(2m)OH (F8HmOH, m = 5 and 11) are added to the PS model preparation (a dipalmitoylphosphatidylcholine (DPPC)/Hel 13-5 peptide mixture) considered here, the effectiveness of the latter in in vitro pulmonary functions is enhanced. The mechanism for the improved efficacy depends on the hydrophobic chain length of the added PFA molecules. The shorter PFA, F8H5OH, when incorporated in the monolayer of the PS model preparation, promotes a disordered liquid-expanded (LE) phase upon lateral compression (fluidization). In contrast, the addition of the longer PFA, F8H11OH, reduces the disordered LE/ordered liquid-condensed (LC) phase transition pressure and promotes the growth of ordered domains (solidification). Furthermore, compression-expansion cycles suggest that F8H5OH, when incorporated in the PS model preparation, undergoes an irreversible elimination into the subphase, whereas F8H11OH enhances the squeeze-out phenomenon of the SP-B mimicking peptide, which is important in pulmonary functions and is related to the formation of a solid-like monolayer at the surface and of a surface reservoir just below the surface. F8H11OH particularly reinforces the effectiveness of DPPC in terms of minimum reachable surface tension, and of preservation of the integrated hysteresis area between compression and expansion isotherms, the two latter parameters being generally accepted indices for assessing PS efficacy. We suggest that PFA amphiphiles may be useful potential additives for synthetic PS preparations destined for treatment of RDS in premature infants and in adults.

Surface Potential of the Air/Water Interface.

The surface charge/surface potential of the air/water interface plays a key role in many natural and industrial processes. Since the first decade of the 20th century, there are many theoretical proposals to describe the surface charge in the presence of different moieties. However, a complete and consistent description of the interfacial layer remains elusive. More recently, the theoretical frameworks and experimental data get complementary support from the simulation at a molecular level. This paper reviews the recent developments from the theoretical, experimental and simulation aspects. The combined results indicated that the interaction between hydration shells of adsorbed ions and the H-bonds network of surface water plays a critical role in the ionic adsorption. The factor should be incorporated into the conventional theories to correctly predict the ion distribution near the air/water surface.

Solubilization of Progesterone and its Derivatives into Gemini Surfactant Solutions.

The solubilization of poorly water-soluble progesterone derivatives into micelles of a gemini surfactant was investigated in an aqueous medium. The aqueous solubility at different temperatures was determined spectroscopically using an ultraviolet visible light spectrophotometer. Thermodynamic parameters for the solubilization were calculated under the basis of the solubility change against temperature. The solubility of progesterone was quite low and remained constant below the critical micelle concentration (cmc) of the surfactant. On the other hand, the solubility increased considerably with increasing surfactant concentration above the cmc. It was suggested that the solubilizates were located in the vicinity of polar regions of micelles.

Pulmonary surfactant model systems catch the specific interaction of an amphiphilic peptide with anionic phospholipid.

Interfacial behavior was studied in pulmonary surfactant model systems containing an amphiphilic alpha-helical peptide (Hel 13-5), which consists of 13 hydrophobic and five hydrophilic amino acid residues. Fully saturated phospholipids of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) were utilized to understand specific interactions between anionic DPPG and cationic Hel 13-5 for pulmonary functions. Surface pressure (pi)-molecular area (A) and surface potential (DeltaV)-A isotherms of DPPG/Hel 13-5 and DPPC/DPPG (4:1, mol/mol)/Hel 13-5 preparations were measured to obtain basic information on the phase behavior under compression and expansion processes. The interaction leads to a variation in squeeze-out surface pressures against a mole fraction of Hel 13-5, where Hel 13-5 is eliminated from the surface on compression. The phase behavior was visualized by means of Brewster angle microscopy, fluorescence microscopy, and atomic force microscopy. At low surface pressures, the formation of differently ordered domains in size and shape is induced by electrostatic interactions. The domains independently grow upon compression to high surface pressures, especially in the DPPG/Hel 13-5 system. Under the further compression process, protrusion masses are formed in AFM images in the vicinity of squeeze-out pressures. The protrusion masses, which are attributed to the squeezed-out Hel 13-5, grow larger in lateral size with increasing DPPG content in phospholipid compositions. During subsequent expansion up to 35 mN m(-1), the protrusions retain their height and lateral diameter for the DPPG/Hel 13-5 system, whereas the protrusions become smaller for the DPPC/Hel 13-5 and DPPC/DPPG/Hel 13-5 systems due to a reentrance of the ejected Hel 13-5 into the surface. In this work we detected for the first time, to our knowledge, a remarkably large hysteresis loop for cyclic DeltaV-A isotherms of the binary DPPG/Hel 13-5 preparation. This exciting phenomenon suggests that the specific interaction triggers two completely independent processes for Hel 13-5 during repeated compression and expansion: 1), squeezing-out into the subsolution; and 2), and close packing as a monolayer with DPPG at the interface. These characteristic processes are also strongly supported by atomic force microscopy observations. The data presented here provide complementary information on the mechanism and importance of the specific interaction between the phosphatidylglycerol headgroup and the polarized moiety of native surfactant protein B for biophysical functions of pulmonary surfactants.

Lateral interaction of cholesterol with a semifluorinated amphiphile at the air-water interface.

Lipid rafts consisting mainly of sphingomyelin and cholesterol (Ch) on biomembrane surfaces are deeply related to cellular processes such as protein trafficking and signal transduction. During the processes, the raft microdomains affect the fluidty of biological membranes, which is controlled to large extents by Ch. In this paper, we have investigated the interaction between Ch and a semiflurinated alcohol (F6H9OH) from the aspect of a fluidty control using surface chemistry. The two-component Langmuir monolayer at the air-water interface was characterized by the surface pressure (π)-molecular area (A) and surface potential (ΔV)-A isotherms. The compressibility modulus and excess Gibbs free energy of mixing were calculated from the π-A isotherms. And also the two-dimensional phase diagram was constructed on the basis of phase transition pressures and monolayer collapse pressures. Furthermore, the phase behavior of binary monolayers was visualized with fluorescence microscopy (in situ) and atomic force microscopy (ex situ). The result here indicates a possibility of fluidity control of Ch-related membranes by arranging the fluorination degree of the constituent lipids.

Development of low cost pulmonary surfactants composed of a mixture of lipids or lipids-peptides using higher aliphatic alcohol or soy lecithin.

The artificial pulmonary surfactant composition in the present study is characterized by a lipid mixture system composed of higher aliphatic alcohol, egg yolk phosphatidylcholine (egg PC), soy lecithin and higher aliphatic acid as the major components or a peptide-lipid mixture system composed of a combination of the lipid mixture system to which a peptide is added. Three peptides with amphiphilic surface-staying, membrane spanning, and both properties were designed and synthesized. The evaluation of pulmonary surfactant assay was performed by a hysteresis curve drawn upon the measurement for the surface tension-area curve with the Wilhelmy surface tensometer in vitro and the recovery of lung compliance for the pulmonary surfactant-deficient rat models in vivo. Lipid-mixture systems composed of octadecanol or soy lecithins containing no peptide were favorable hysteresis curves as compared with commercially available Surfacten, but were not prominent. The peptide-lipid mixture systems composed of a combination of the lipid mixture of alkyl alcohol or soy lecithin to which peptides designed were added were desirable hysteresis curves similar to Surfacten and amphiphilic Hel 13-5 peptide-lipids mixture systems were much more effective than the lipid mixture system. Particularly, the recovery of lung compliance treated with hydrogenated soy lecithin-fractionated soy lecithin PC70-palmitic acid-peptide Hel 13-5 (40:40:17.5:2.5, w/w) was comparable to that with Surfacten. Because the artificial pulmonary surfactant compositions of this study can be prepared at lower costs, they are useful for the treatment of respiratory distress syndrome and acute respiratory distress syndrome as well as for inflammatory pulmonary diseases, dyspnea caused by asthma, etc.