Experimental Lung Research, 31:563–579, 2005 Copyright # Taylor & Francis Inc. ISSN: 0190-2148 print/1521-0499 online DOI: 10.1080/019021490951531
COMPARISON OF THREE LIPID FORMULATIONS FOR SYNTHETIC SURFACTANT WITH A SURFACTANT PROTEIN B ANALOG
Frans J. Walther & Los Angeles Biomedical Research Institute at Harbor–UCLA Medical Center, Torrance, California, USA; and Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands !ndez-Juviel and Larry M. Gordon & Los Angeles Biomedical Jose´ M. Herna Research Institute at Harbor–UCLA Medical Center, Torrance, California, USA Alan J. Waring & Los Angeles Biomedical Research Institute at Harbor–UCLA Medical Center, Torrance, California, USA; and Department of Medicine, UCLA School of Medicine, Los Angeles, California, USA Patrick Stenger and Joseph A. Zasadzinski & Department of Chemical Engineering, UCSB, Santa Barbara, California, USA
& Surfactant protein B (SP-B) is an essential component of pulmonary surfactant. Synthetic dimeric SP-B1–25 (SP-B1–25), a peptide based on the N-terminal domain of human SP-B, efficiently mimics the functional properties of SP-B. The authors investigated the optimum lipid composition for SP-B1–25 by comparing the effects of natural lung lavage lipids (NLL), a synthetic equivalent of NLL (synthetic lavage lipids SLL), and a standard lipid mixture (TL) on the activities of SP-B1–25. Surfactant preparations were formulated by mixing 2 mol% SP-B1–25 in NNL, SLL, and TL. Calfactant, a calf lung lavage extract with SP-B and SP-C, was a positive control and lipids without peptide were negative controls. Minimum surface tension measured on a captive bubble surfactometer was similar for the three SP-B1–25 surfactant preparations and calfactant. The effects on lung function were compared in ventilated, lavaged, surfactant-deficient rats. Oxygenation and lung volumes were consistently higher in rats treated with calfactant and SP-B1–25 in NLL or SLL than in rats treated with SP-B1–25 in TL. Fourier transform infrared spectra observed abnormal secondary conformations for SP-B1–25 in TL as a possible cause for the reduced lung function. Lipid composition plays a crucial role in the in vitro and in vivo functions of SP-B1–25 in surfactant preparations.
Received 7 October 2004; accepted 18 February 2005. This work was supported by grant HL55534 from the National Institutes of Health. The REI Bruker Vector 22 FTIR spectrometer was funded by a grant from the Harbor–UCLA REI Common Use and Replacement Equipment Program. Calfactant (Infasurf) was a generous gift of Ony Inc., Amherst, New York. Address correspondence to Frans J. Walther, MD, PhD, Los Angeles Biomedical Research Institute at Harbor–UCLA Medical Center, 1124 West Carson Street, Building F-5 South, Torrance, CA 90502, USA. E-mail:
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F. J. Walther et al. Keywords captive bubble surfactometry, dimeric SP-B1–25 peptide, Fourier transform infrared spectroscopy, lung function, rats
Lung surfactant is a mixture of lipid and surfactant proteins that is synthesized and secreted into the alveolar fluid by alveolar type II cells to reduce surface tension at the air-liquid interface in the alveolus. Surfactant obtained by lung lavage is composed of approximately 80% phospholipids, 10% neutral lipids, and 10% protein [1]. The most abundant phospholipid constituent of lung surfactant is phosphatidylcholine (PC), in particular dipalmitoylphosphatidylcholine (DPPC). DPPC contributes to the formation of a rigid interfacial film that reduces surface tension to very low values during dynamic compression, whereas fluid phospholipids and neutral lipids significantly improve film spreading [2, 3]. The highly hydrophobic surfactant proteins B and C (SP-B and SP-C) enhance the absorption of phospholipids into the air-water interface and optimize surface tension reduction. SP-B is critical for normal lung function, as hereditary SP-B deficiency is lethal in newborn infants [4] and SP-B knockout mice [5]. Current therapy for respiratory distress syndrome (RDS) in preterm infants includes intratracheal administration of pulmonary surfactant containing naturally occurring SP-B and SP-C [6]. Formulations of lipids with synthetic peptide analogs, based on the known SP-B primary sequence [7] or a structural equivalent [8], are being developed as an alternative to animal surfactants extracted from cow or pig lungs. Dimeric SP-B1–25, a homodimer of a 25–amino acid residue peptide based on the N-terminal of human SP-B, produces a range of physical changes in lung surfactant monolayers similar to those observed for native SP-B, including an enhanced coexistence of buckled and flat monolayers [9], modified phase behavior of lipid mixtures [10], and increased formation of ‘‘nanosilo’’ structures associated with the lipid monolayers [11]. Synthetic surfactant preparations containing dimeric SP-B1–25 improve lung function in animal models of neonatal RDS and acute RDS (ARDS) [12]. In previous in vivo studies, we have formulated synthetic surfactant SP-B peptides with a standard lipid mixture (TL) consisting of DPPC, palmitoyloleoylphosphatidylglycerol (POPG), and palmitic acid (PA) (69:22:9 w=w=w), originally developed by Tanaka and colleagues [13]. However, this lipid mixture is quite different from natural surfactants, such as the commercially available calf lung lavage extract calfactant (Infasurf), which contains approximately 55% of disaturated PC species and the remaining phospholipids are unsaturated [2, 14]. Here, we investigated the optimum lipid composition for dimeric SP-B1–25 by performing in vitro and in vivo experiments with 2 mol% dimeric SP-B1–25 in 3 different lipid mixtures: (i) a lipid extract of calfactant (natural lavage lipids, NLL), (ii) a synthetic
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lipid equivalent of NLL (synthetic lavage lipids, SLL), and (iii) the standard Tanaka lipid mixture (TL). We then investigated dimeric SP-B1–25 function and structure in vitro with captive bubble surfactometry and Fourier transform infrared (FTIR) spectral experiments and in vivo by measuring lung function in surfactant-deficient rats. METHODS Materials Peptide synthesis reagents were purchased from Applied Biosystems (Foster City, CA), high-performance liquid chromatography (HPLC) solvents from Fisher (Pittsburgh, PA), and all other chemicals from Sigma (St. Louis, MO) and Aldrich (Milwaukee, WI). Palmitic acid, DPPC, dioleoylphosphatidylcholine (DOPC), POPG, palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylserine (POPS), and cholesterol were from Avanti Polar Lipids (Alabaster, AL). Calfactant (Infasurf) was a generous gift of Ony (Amherst, NY). Adult male Sprague-Dawley rats, weighing 200 to 225 g, were obtained from Harlan (San Diego, CA). Synthesis and Characterization of Dimeric SP-B1–25 (SP-B1–25) Dimeric SP-B1–25 was prepared by first synthesizing monomeric SP-B1–25 on a 0.25-mmol scale with an Applied Biosystems Model 431A peptide synthesizer using a FastMoc strategy [15]. The sequence was based on the N-terminus of human SP-B (Figure 1) with one modification, cysteine in position 11 was replaced by alanine. The peptide was synthesized with prederivatized Fmoc-Gly resin (Calbiochem-Nova, La Jolla) or PEG-PA resin (Perceptive Biosystems, Old Connecticut Path, MA) and single coupled for all residues. After purification by reverse-phase HPLC, its molecular mass was confirmed by fast atom bombardment mass spectroscopy or electrospray mass spectroscopy. The disulfide linked homodimer SP-B1–25 (Figure 1) was formed by oxidizing the monomeric SP-B1–25 [16]. The molecular mass of dimeric SP-B1–25 was confirmed by electrospray mass spectroscopy and indicated the yield of dimeric product to be essentially 100% [16]. The dimeric peptide was then freeze-dried to remove the organic solvent prior to formulation with lipid. Dimeric SP-B1–25 will be referred to as SP-B1–25. Surfactant Preparations Calfactant, a calf lung lavage extract consisting of 0.9% SP-B, SP-C, phospholipids, and cholesterol, contains 35 mg phospholipids=mL of 150 mM phosphate-buffered saline (pH 7.0) [3] and was used as a positive
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FIGURE 1 Amino acid sequences of the native, human N-terminal SP-B1–25 peptide [24], variant SP-B1–25 (Cys-11 ! Ala-11) monomer peptide, and dimeric SP-B1–25 peptide. Amino acids are indicated by single-letter codes. Dimeric SP-B1–25 consists of two variant monomeric SP-B1–25 (Cys-11 ! Ala-11), covalently linked by a disulfide bond (see "S"S" at Cys-8) [16].
control. NLL was obtained by extraction of calfactant and removal of native SP-B and SP-C by Folch [17] partitioning of the lipid-protein suspension, followed by C8 reverse-phase chromatography and verified by nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) [18]. NLL lipids were suspended at 35 mg phospholipids=mL of 150 mM phosphate buffer saline (pH 7.0). SSL, a synthetic mimic of NLL, was based on the known composition of calf lung surfactant lipids [2] and contained 16 mg=mL of DPPC (fatty acids C16:0-C16:0), 10 mg=mL of DOPC (C18:1C18:1), 3 mg=mL of POPG (C16:0-C18:1), 1 mg=mL of POPE (C16:0C18:1), 3 mg=mL of POPS (C16:0-C18:1), and 2 mg=mL of cholesterol, or a total of 35 mg lipids=mL of 150 mM phosphate-buffered saline solution (pH 7.0). The third lipid mixture was a standard lipid mixture (TL ¼ DPPC:POPG:PA 69:22:9 w=w=w) developed by Tanaka and colleagues [13] at a concentration of 35 mg=mL. SP-B1–25 at 2 mol% was mixed into the NLL, SLL, or TL preparations. Purified lipid and protein fractions were quantitated by FTIR analysis [19]. Viscosity Bulk viscosities of the lipid and peptide mixtures were measured with a Cannon U-tube viscometer and an Advanced Rheometric Expansion System (ARES) viscometer utilizing both cone and plate and conicylinder
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geometries. Sample temperature was kept constant at 37 # 1$C by a circulating water bath, while the viscosity was sampled over a large range of shear rates (ARES viscometer only). Captive Bubble Surfactometry Surface activity of the surfactant preparations was checked with a captive bubble surfactometer, which has been described in detail elsewhere [16, 20, 21]. In short, the instrument consists of a sample chamber cut from cylindrical glass tubing of high quality with an inner diameter of 1 cm. A Teflon piston with a tight O-ring seal is fitted into the glass tubing from the top end. A plug of buffered agarose gel is inserted between the piston and solution. The other end is fitted into a plate of stainless steel, which is provided with an inlet port in the center for adding solutions and the air bubble to the chamber. Chamber and piston are vertically mounted within a sturdy rack of steel whose height is regulated by a micrometer gear with minimal redundancy. For usual measurements, the chamber is filled with 10% sucrose Goerke’s buffer, to which 1 mL surfactant is added. The chamber content is stirred with a small magnetic bar, and its temperature is maintained at 37$C. A small air bubble is introduced from beneath, whose volume and hence surface area can be changed by compression and decompression brought about by changing the height of the rack. The agarose plug’s ionic composition inhibits bubble adhesion to its surface, thus creating an uninterrupted surface area and a perfect bubble. During the compression and re-expansion cycles, bubble images are continuously recorded to the hard drive and on a video recorder. Selected single frames are stored in RAM for later image processing and analysis [22]. Bubble areas and volumes are calculated by an original algorithm relating bubble height and diameter to areas of revolution, and the bubble surface tension is determined by using the method of Malcolm and Elliot [23]. Secondary Structural Analysis of SP-B1–25 in Surfactant Preparations Using FTIR Spectroscopy Infrared spectra were recorded at 25$C using a Bruker Vector 22 FTIR spectrometer, fitted with a Pike Technologies horizontal attenuated-totalreflectance (ATR) accessory (Madison, WI) and a deuterated triglycine sulfate (DTS) detector [12, 24]. Aliquots (100 mL) of the NLL, SLL, or TL surfactant lipid dispersions, prepared with or without 2 mol% SP-B1–25, were spread on the 50 % 20 % 2-mm germanium ATR crystal, and allowed to air dry. The sample film was then hydrated with deuterated water vapor
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for 1 hour prior to measurement. The sample was scan averaged for 256 spectra, at a resolutions of 2 cm"1 and a gain of 4. The peptide amide I band of the FTIR spectra of SP-B1–25 in surfactant lipids was analyzed for the various secondary conformations. For measurements of 2 mol% SP-B1–25 mixed with surfactant lipids (see above), the FTIR spectrum of the lipid film without peptide (i.e., band centered at 1730 cm"1) was subtracted from the corresponding spectrum of peptide with lipid. The proportions of a-helix, b-turn, b-sheet, and disordered conformations were determined by Fourier self-deconvolutions for band narrowing and area calculations of component peaks determined with curve-fitting software (GRAMS/AI; ThermoGalactic, Salem, NH). The frequency limits for the different conformations were as follows: a-helix (1662 to 1645 cm"1), b-sheet (1637 to 1613 cm"1 and 1710 to 1682 cm"1), b-turns (1682 to 1662 cm"1), and disordered structures (1650 to 1637 cm"1) [12, 24, 25–28]. Ventilated Rats The animal experiments were performed with the approval of the Harbor–UCLA Research and Education Institute Animal Care and Use Committee. Anesthesia, surgery, lavage, ventilation, and monitoring used in this study are the same as previously described [12]. Briefly, adult male Sprague-Dawley rats, weighing 200 to 225 g, were anesthetized with 35 mg=kg pentobarbital sodium and 80 mg=kg ketamine by intraperitoneal injection, intubated, and ventilated with a rodent ventilator (Harvard Apparatus, South Natick, MA) with 100% oxygen, a tidal volume of 7.5 mL=kg, and a rate of 60 per minute. An arterial line was placed in the abdominal aorta for measurements of arterial blood pressure and blood gases. The rats were paralyzed with 1 mg=kg pancuronium bromide intravenously. Only animals with PaO2 values > 400 mm Hg while ventilated with 100% oxygen and with normal blood pressure values were included in the experiments. Airway pressures were measured with a pressure transducer (Gould, Cleveland, OH) and tidal volume with a pneumotachometer (Validyne, Northridge, CA) connected to a multichannel recorder (Gould). The lungs were lavaged 8 to 12 times with 8 mL of prewarmed 0.9% NaCl. After the PaO2 in 100% oxygen had reached stable values of
