Hel 13-5

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.

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.

Pulmonary functions such as rapid adsorption, respreading, and hysteresis behavior of pulmonary surfactants are very important for respiratory movement. The interfacial behavior of pulmonary preparations containing an amphiphilic peptide (Hel 13-5) has recently investigated. An orientation of hydrophobic chains in a dipalmitoylphosphatidylcholine (DPPC) with or without palmitic acid (PA) is associated with a collapse of alveoli during respiration process. Therefore, the present study focused on the acyl chain orientation in model pulmonary surfactants (DPPC/Hel 13-5 and DPPC/PA/Hel 13-5). A successive change in the orientation during cyclic compression and expansion of films at the air-water interface can be probed directly by an infrared reflection-absorption spectrometry (IRRAS) technique. The hysteresis behavior, one of very important pulmonary functions, was previously observed in surface pressure (pi)-molecular area (A) isotherms for the both model pulmonary surfactant systems (Langmuir 22(2006)1182-1192 and Langmuir 22(2006)5792-5803). In addition, it was reported that Hel 13-5 was squeezed-out of the surface on compression like native pulmonary surfactant proteins. The data obtained for the binary and ternary systems were compared with those of the equivalent pure DPPC and DPPC/PA mixtures, respectively. For an asymmetric methylene stretching vibration (nu(a)-CH(2)) RA intensity, the absolute RA values increased with shifting to small surface area, monotonously. For the corresponding wavenumber, on the other hand, the values gradually decreased into approximately 2920cm(-1). However, they were kept constant in the squeeze-out region in spite of a further decrease of surface area. These results suggested that the orientation of hydrophobic chains in DPPC and DPPC/PA mixtures became in the most packed state soon after emergence of the squeeze-out process of Hel 13-5 and then the packed orientation was retained up to the collapse state. This indicated that the squeezed-out Hel 13-5 stabilized monolayers left at the interface. For the DPPC/PA/Hel 13-5 system, in particular, dissociated PA molecules were excluded together with Hel 13-5 and the surface monolayers were refined to DPPC and undissociated PA components during the compression process. And the similar behavior in the second and third cycles supported the good respreading ability of the monolayers containing Hel 13-5.