A freeze-fracture transmission electron microscopy and small angle x-ray diffraction study of the effects of albumin, serum, and polymers on clinical lung surfactant microstructure

Andreas Braun, Patrick C. Stenger, Heidi E. Warriner, Joseph A. Zasadzinski, Karen W. Lu, H. William Taeusch

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Abstract

Freeze-fracture transmission electron microscopy shows significant differences in the bilayer organization and fraction of water within the bilayer aggregates of clinical lung surfactants, which increases from Survanta to Curosurf to Infasurf. Albumin and serum inactivate all three clinical surfactants in vitro; addition of the nonionic polymers polyethylene glycol, dextran, or hyaluronic acid also reduces inactivation in all three. Freeze-fracture transmission electron microscopy shows that polyethylene glycol, hyaluronic acid, and albumin do not adsorb to the surfactant aggregates, nor do these macromolecules penetrate the interior water compartments of the surfactant aggregates. This results in an osmotic pressure difference that dehydrates the bilayer aggregates, causing a decrease in the bilayer spacing as shown by small angle x-ray scattering and an increase in the ordering of the bilayers as shown by freeze-fracture electron microscopy. Small angle x-ray diffraction shows that the relationship between the bilayer spacing and the imposed osmotic pressure for Curosurf is a screened electrostatic interaction with a Debye length consistent with the ionic strength of the solution. The variation in surface tension due to surfactant adsorption measured by the pulsating bubble method shows that the extent of surfactant aggregate reorganization does not correlate with the maximum or minimum surface tension achieved with or without serum in the subphase. Albumin, polymers, and their mixtures alter the surfactant aggregate microstructure in the same manner; hence, neither inhibition reversal due to added polymer nor inactivation due to albumin is caused by alterations in surfactant microstructure.

Original languageEnglish (US)
Pages (from-to)123-139
Number of pages17
JournalBiophysical journal
Volume93
Issue number1
DOIs
StatePublished - Jul 2007

Bibliographical note

Funding Information:
FFTEM of the clinical lung surfactants Survanta, Curosurf, and Infasurf (FFTEM) shows significant differences in the bilayer organization of the as received materials. The major difference is the fraction of water within the bilayer aggregates, which increases from Survanta to Curosurf to Infasurf. Although each clinical surfactant has its own distinct bilayer organization, freeze-fracture images show that albumin and polymers do not adsorb to any of the clinical surfactant aggregates. Rather, both albumin and polymers act to dehydrate the surfactant aggregates by creating an osmotic pressure between the interior of the aggregate and the exterior. The aggregates expel any interior pockets of water and form well-ordered bilayers, with an interlayer spacing that depends on the osmotic pressure of the polymer or albumin solution. There is clear evidence of fusion of smaller aggregates to form larger aggregates, in addition to the flocculation observed via optical microscopy. Small angle x-ray scattering shows that the d-spacing s of Curosurf, Infasurf, and Survanta depend on the osmotic pressure, consistent with the polymer and albumin not being able to access the interior of the aggregates (67) . Albumin, polymers, and their mixtures alter the surfactant aggregate microstructure in the same manner; this implies that albumin does not inactivate surfactant by alterations in surfactant microstructure. In addition, the microstructure alterations of PEG and other polymers do not themselves reverse surfactant inactivation. This has been shown by comparing the effects of 5 wt % 10 kDa PEG with 0.25 wt % 250 kDa HA on surfactant microstructure and surfactant inactivation. These polymer concentrations are sufficient to reverse inactivation of the clinical surfactants; the lower HA concentration actually provides more complete inactivation reversal than does the higher PEG concentration in pulsating bubble experiments. However, the PEG solution has an osmotic pressure more than 100 times higher than the HA solution. FFTEM and small angle x-ray scattering show that high concentration of PEG necessary to reverse inhibition significantly alters the bilayer organization of the surfactant aggregates, whereas the lower concentrations of HA do not. In addition, the bilayer morphology of Survanta is minimally altered by PEG, whereas Infasurf and Curosurf aggregates undergo extensive reorganization, yet inactivation reversal occurs in all of the clinical surfactants. Hence, alternate mechanisms must be responsible for both inhibition of surfactant by surface active proteins and the polymer-induced inhibition reversal of clinical surfactants. We thank John Clements and Alan Waring for valuable discussion while conducting these experiments. Support for this work comes from National Institutes of Health grants HL-66410 (H.W.T.) and HL-51177 (J.A.Z.), the Tobacco Related Disease Research Program 14RT-0077 (J.A.Z.), and National Science Foundation grant CTS-0436124. P.S. was partially supported by a National Science Foundation graduate research fellowship.

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