Development of synthetic lung surfactants

We have previously reported the development of a reconstituted lung surfactant consisting of an organic solvent extract of natural bovine lung surfactant supplemented with synthetic lipids. This "artificial" surfactant was used successfully to treat surfactant deficiency states both in animals and humans. We now report on the successful testing of a synthetic lung surfactant consisting of a lipid-bound protein isolated from natural lung surfactant and the lipids present in the "artificial" lung surfactant and now used in the same concentration but in a synthetic, commercially available form. The synthetic lung surfactant possessed the in vitro and in vivo surface properties characterizing the "artificial" lung surfactant. In order to identify the components of the synthetic lung surfactant that are responsible for the required surface properties, a series of 25 simple mixtures was prepared. Of these, three possessed surface properties very similar to those of the "artificial" lung surfactant and the synthetic lung surfactant, in vitro as well as in vivo. These three mixtures had four components in common. Besides dipalmitoyl phosphatidylcholine and the lipid-bound protein, they each had a saturated fatty acid, palmitic or stearic, and they each had an acidic phospholipid, phosphatidylglycerol or phosphatidylserine.     

Rat eustachian tube synthesizes disaturated phosphatidylcholine

Otitis media results when the eustachian tube fails to adequately ventilate the middle ear. A surface tension-lowering substance may be required for normal tube opening, especially in young children with poorly developed naso-pharyngeal musculature. We report here that rat eustachian tube epithelium synthesizes disaturated phosphatidylcholine, which is recognized as the surface tension-lowering substance of pulmonary surfactant.     

Surfactant protein C: its unique properties and emerging immunomodulatory role in the lung

Surfactant protein C (SP-C) is a highly hydrophobic protein found in pulmonary surfactant. SP-C is synthesized exclusively in alveolar type II cells as a 21 kDa integral membrane precursor protein and subsequently proteolytically processed to a 3.7 kDa secretory protein. SP-C enhances the adsorption and spreading of phospholipids at the air-liquid interface thereby promoting the surface tension-lowering properties of surfactant. The importance of SP-C in normal lung function is underscored by the recent findings of inflammatory lung diseases associated both with absence of alveolar SP-C and with cellular expression of mutant SP-C isoforms. This review examines our current understanding of the role of SP-C in maintaining alveolar epithelial homeostasis and the potential role of abnormal SP-C expression in the development of lung diseases with particular emphasis on microbial pulmonary infection and inflammation.     

Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich alpha helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% alpha-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression pi-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.     

Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich α helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use.We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% α-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression π-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations.     

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.     

Surfactant protein A (SP-A): the alveolus and beyond.

Surfactant protein A (SP-A) is the major protein component of pulmonary surfactant, a material secreted by the alveolar type II cell that reduces surface tension at the alveolar air-liquid interface. The function of SP-A in the alveolus is to facilitate the surface tension-lowering properties of surfactant phospholipids, regulate surfactant phospholipid synthesis, secretion, and recycling, and counteract the inhibitory effects of plasma proteins released during lung injury on surfactant function. It has also been shown that SP-A modulates host response to microbes and particulates at the level of the alveolus. More recently, several investigators have reported that pulmonary surfactant phospholipids and SP-A are present in nonalveolar pulmonary sites as well as in other organs of the body. We describe the structure and possible functions of alveolar SP-A as well as the sites of extra-alveolar SP-A expression and the possible functions of SP-A in these sites.     

High-pressure liquid chromatography of cobalamins and cobalamin analogs

High-pressure liquid chromatography has been used to separate, identify, and quantitate 37 different cyanocobalamin analogs, including the most commonly occurring analogs that result from bacterial synthesis. This technique has also been used to simultaneously separate, identify, and quantitate five naturally occurring cobalamins that differ in their upper axial ligands: methylcobalamin, adenosylcobalamin, hydroxocobalamin, cyanocobalamin, and sulfitocobalamin. This method permits rapid quantitative detection and identification of cobalamins and naturally occurring and synthetic cobalamin analogs in complex mixtures.     

Comparative adsorption of natural lung surfactant, extracted phospholipids, and artificial phospholipid mixtures to the air-water interface

Adsorption to the air-water interface of natural lung surfactant obtained by bovine lung lavage is compared and contrasted with the adsorption of mixtures of synthetic phospholipids and of extracted mixed lung lipids containing minimal protein. Surface pressure-time (pi-t) adsorption isotherms are measured at 35 degrees C for the surfactant mixtures as a function of the presence or absence of divalent metal cations (Ca2+ and Mg2+) and of heating to 45 degrees C or 90 degrees C. The effect of aqueous dispersion technique (sonication or mechanical vortexing) on the adsorption process is also studied for the extracted or synthetic phospholipid mixtures. The results imply that the protein component is necessary for the optimal adsorption of natural lung surfactant. However, by taking advantage of different methods available for phospholipid dispersion in an aqueous phase in vitro, it is possible to formulate dispersions of extracted lung phospholipids containing of order 1% protein which adsorb as well as the complete surfactant system. These results suggest that protein concentrations in surfactant mixtures can be minimized for applications such as exogenous lung surfactant replacement for the neonatal Respiratory Distress Syndrome (RDS). However, for situations which may involve alterations in endogenous surfactant function such as in lung injury, effects involving pulmonary surfactant protein and protein-lipid interactions may be of functional significance.     

Content-dependent activity of lung surfactant protein B in mixtures with lipids

The content-dependent activity of surfactant protein (SP)-B was studied in mixtures with dipalmitoyl phosphatidylcholine (DPPC), synthetic lipids (SL), and purified phospholipids (PPL) from calf lung surfactant extract (CLSE). At fixed SP-B content, adsorption and dynamic surface tension lowering were ordered as PPL/SP-B approximately SL/SP-B > DPPC/SP-B. All mixtures were similar in having increased surface activity as SP-B content was incrementally raised from 0.05 to 0.75% by weight. SP-B had small but measurable effects on interfacial properties even at very low levels < or =0.1% by weight. PPL/SP-B (0.75%) had the highest adsorption and dynamic surface activity, approaching the behavior of CLSE. All mixtures containing 0.75% SP-B reached minimum surface tensions <1 mN/m in pulsating bubble studies at low phospholipid concentration (1 mg/ml). Mixtures of PPL or SL with SP-B (0.5%) also had minimum surface tensions <1 mN/m at 1 mg/ml, whereas DPPC/SP-B (0.5%) reached <1 mN/m at 2.5 mg/ml. Physiological activity also was strongly dependent on SP-B content. The ability of instilled SL/SP-B mixtures to improve surfactant-deficient pressure-volume mechanics in excised lavaged rat lungs increased as SP-B content was raised from 0.1 to 0.75% by weight. This study emphasizes the crucial functional activity of SP-B in lung surfactants. Significant differences in SP-B content between exogenous surfactants used to treat respiratory disease could be associated with substantial activity variations.     

Surfactant alterations in acute inflammatory lung injury from aspiration of acid and gastric particulates

This study examines surfactant dysfunction in rats with inflammatory lung injury from intratracheal instillation of hydrochloric acid (ACID, pH 1.25), small nonacidified gastric particles (SNAP), or combined acid and small gastric particles (CASP). Rats given CASP had the most severe lung injury at 6, 24, and 48 h based on decreases in arterial oxygenation and increases in erythrocytes, total leukocytes, neutrophils, total protein, and albumin in bronchoalveolar lavage (BAL). The content of large surfactant aggregates in BAL was reduced in all forms of aspiration injury, but decreases were greatest in rats given CASP. Large aggregates from aspiration-injured rats also had decreased levels of phosphatidylcholine (PC) and increased levels of lyso-PC and total protein compared with saline controls (abnormalities for CASP were greater than for SNAP or ACID alone). The surface tension-lowering ability of large surfactant aggregates on a bubble surfactometer was impaired in rats with aspiration injury at 6, 24, and 48 h, with the largest activity reductions found in animals given CASP. There were strong statistical correlations between surfactant dysfunction (increased minimum surface tension and reduced large aggregate content) and the severity of lung injury based on arterial oxygenation and levels of albumin, protein, and erythrocytes in BAL (P < 0.0001). Surfactant dysfunction also correlated strongly with reduced lung volumes during inflation and deflation (P = 0.0004-0.005). These results indicate that surfactant abnormalities are functionally important in gastric aspiration lung injury and contribute significantly to the increased severity of injury found in CASP compared with ACID or SNAP alone.     

Effects of α-tocopherol analogs on lysosome membranes and fatty acid monolayers

The surface pressures of α-tocopherol analogs, fatty acids, and their mixtures were measured in their spread monolayers at an air—water interface. The surface pressure—area isotherms for the mixed monolayers of α-tocopherol and either stearic acid, oleic acid or linoleic acid deviated positively from those calculated on the basis of the additivity rule, and the magnitude depended on the length of the phytyl side chain in α-tocopherol and on the degree of unsaturation of the fatty acid chains. Lysosome membranes of mouse liver were stabilized by addition of α-tocopherol. A decrease in the length of the phytyl side chain in α-tocopherol reduced its ability to stabilize lysosome membranes. A good correlation was obtained between the extent of stabilizing activity of α-tocopherol analogs on lysosome membranes and the degree of positive deviation of the surface pressure for their mixtures with fatty acids.     

Synthesis and activity of a novel diether phosphonoglycerol in phospholipase-resistant synthetic lipid:peptide lung surfactants()

This paper reports the chemical synthesis and purification of a novel phospholipase-resistant C16:0, C16:1 diether phosphonoglycerol with structural analogy to ester-linked anionic phosphatidylglycerol (PG) in endogenous pulmonary surfactant. This diether phosphonoglycerol (PG 1) is studied for phospholipase A(2) (PLA(2)) resistance and for surface activity in synthetic exogenous surfactants combined with Super Mini-B (S-MB) peptide and DEPN-8, a previously-reported diether phosphonolipid analog of dipalmitoyl phosphatidylcholine (DPPC, the major zwitterionic phospholipid in native lung surfactant). Activity experiments measured both adsorption and dynamic surface tension lowering due to the known importance of these surface behaviors in lung surfactant function in vivo. Synthetic surfactants containing 9 : 1 DEPN-8:PG 1 + 3% S-MB were resistant to degradation by PLA(2) in chromatographic studies, while calf lung surfactant extract (CLSE, the substance of the bovine clinical surfactant Infasurf?) was significantly degraded by PLA(2). The 9 : 1 DEPN-8:PG 1 + 3% S-MB mixture also had small but consistent increases in both adsorption and dynamic surface tension lowering ability compared to DEPN-8 + 3% S-MB. Consistent with these surface activity increases, molecular dynamics simulations using Protein Modeller, GROMACS force-field, and PyMOL showed that bilayers containing DPPC and palmitoyl-oleoyl-PC (POPC) as surrogates of DEPN-8 and PG 1 were penetrated to a greater extent by S-MB peptide than bilayers of DPPC alone. These results suggest that PG 1 or related anionic phosphono-PG analogs may have functional utility in phospholipase-resistant synthetic surfactants targeting forms of acute pulmonary injury where endogenous surfactant becomes dysfunctional due to phospholipase activity in the innate inflammatory response.     

Tautomeric equilibria of isoguanine and related purine analogs

Nucleobase pairs in DNA match hydrogen-bond donor and acceptor groups on the nucleobases. However, these can adopt more than one tautomeric form, and can consequently pair with nucleobases other than their canonical complements, possibly a source of natural mutation. These issues are now being re-visited by synthetic biologists increasing the number of replicable pairs in DNA by exploiting unnatural hydrogen bonding patterns, where tautomerism can also create mutation. Here, we combine spectroscopic measurements on methylated analogs of isoguanine tautomers and tautomeric mixtures with statistical analyses to a set of isoguanine analogs, the complement of isocytosine, the 5th and 6th “letters” in DNA.     

Surface properties of sulfur- and ether-linked phosphonolipids with and without purified hydrophobic lung surfactant proteins

Two novel C16:0 sulfur-linked phosphonolipids (S-lipid and SO(2)-lipid) and two ether-linked phosphonolipids (C16:0 DEPN-8 and C16:1 UnDEPN-8) were studied for surface behavior alone and in mixtures with purified bovine lung surfactant proteins (SP)-B and/or SP-C. Synthetic C16:0 phosphonolipids all had improved adsorption and film respreading compared to dipalmitoyl phosphatidylcholine, and SO(2)-lipid and DEPN-8 reached maximum surface pressures of 72mN/m (minimum surface tensions of <1mN/m) in compressed films on the Wilhelmy balance (23 degrees C). Dispersions of DEPN-8 (0.5mg/ml) and SO(2)-lipid (2.5mg/ml) also reached minimum surface tensions of <1mN/m on a pulsating bubble surfactometer (37 degrees C, 20cycles/min, 50% area compression). Synthetic lung surfactants containing DEPN-8 or SO(2)-lipid+0.75% SP-B+0.75% SP-C had dynamic surface activity on the bubble equal to that of calf lung surfactant extract (CLSE). Surfactants containing DEPN-8 or SO(2)-lipid plus 1.5% SP-B also had very high surface activity, but less than when both apoproteins were present together. Adding 10wt.% of UnDEPN-8 to synthetic lung surfactants did not improve dynamic surface activity. Surfactants containing DEPN-8 or SO(2)-lipid plus 0.75% SP-B/0.75% SP-C were chemically and biophysically resistant to phospholipase A(2) (PLA(2)), while CLSE was severely inhibited by PLA(2). The high activity and inhibition resistance of synthetic surfactants containing DEPN-8 or SO(2)-lipid plus SP-B/SP-C are promising for future applications in treating surfactant dysfunction in inflammatory lung injury.     

Monolayer and interfacial permeation

Transport across physical-chemical interfaces is considered in connection with three particular problems of biological interfaces: the structure and properties of cell membranes, the properties of the lung surfactant, and the effects of ionic currents across excitable membranes. With regard to cell membranes, studies of monolayer permeation suggest that permselectivity on the basis of size is a property of bilayer structure and probably gives rise to the observed dependence of the permeability on partition coefficients. The permeabilities of lipid and protein monolayers are consistent with the bimolecular leaflet (BML) model of the membrane and not with mosaic models. Experiments with the lung surfactant indicate that, in addition to its surface tension-lowering properties, it is unusual in its ability to form a strong two-dimensional network, which probably contributes to alveolar stability. Finally, the results of studies of interfacial ionic transference suggest a new way of accounting for the ionic fluxes in excitable membranes during an action potential without assuming ion-selective pores or carriers. In the suggested mechanism, it is possible to account for the change in ionic selectivity and the proper phasing of the fluxes, as well as other aspects of excitation in natural membranes.     

Enhancement of biophysical activity of lung surfactant extracts and phospholipid-apoprotein mixtures by surfactant protein A

The effects of surfactant protein (SP)-A on the dynamic surface tension lowering and resistance to inhibition of dispersions of calf lung surfactant extract (CLSE) and mixtures of synthetic phospholipids combined with SP-B,C hydrophobic apoproteins were studied at 37 degrees C and rapid cycling rate (20 cycles/min). Addition of SP-A to CLSE, which already contains SP-B and -C, gave a slight improvement in the time course of surface tension lowering on an oscillating bubble apparatus in the absence of inhibitory protein molecules such as albumin or hemoglobin. However, when these proteins were present at concentrations of 10-50 mg/ml, SP-A substantially improved the resistance of CLSE to their inhibitory effects. The beneficial effect of SP-A required the presence of Ca2+ ions, and disappeared when EDTA was substituted for this divalent cation in the subphase. The effect was also retained when SP-A was heated to 50 degrees C prior to addition to CLSE, but was abolished by heating SP-A to 99 degrees C. Additional studies showed that similar improvements in resistance to inhibition were found when SP-A was added to synthetic mixtures of dipalmitoyl phosphatidylcholine (DPPC):egg phosphatidylglycerol (PG) (80:20 by weight) reconstituted with 1% SP-B or SP-B and -C, but not to phospholipid mixtures containing only SP-C. The requirements for SP-B and calcium for the beneficial effects of SP-A on surface activity suggest that the formation of ordered, larger phospholipid-apoprotein aggregates may be involved in the process. The finding that SP-A enhances the ability of CLSE and other surfactant mixtures containing SP-B to resist inhibition is an advantage that will need to be weighed against other factors such as increased antigenicity and heat sensitivity in therapeutic applications in surfactant replacement therapy.     

Combined effects of polymers and KL4 peptide on surface activity of pulmonary surfactant lipids

Addition of ionic and nonionic polymers can improve the function of therapeutic surfactants in vitro and in vivo, especially under conditions that tend to inhibit surfactant activity. Since surfactant proteins also act to reduce surfactant inhibition, we studied the relative effects of a synthetic peptide (that mimics some of the properties of a surfactant protein), polymers, and their combination on function of surfactant phospholipid activity in vitro. We evaluated surface activity after adding polymers—polyethylene glycol or hyaluronan—to a lipid mixture with or without the synthetic peptide, sinapultide (KL₄). Using a pulsating bubble surfactometer, we measured peptide/polymer effects separately or combined at two peptide concentrations. Phospholipid mixtures, with or without KL₄ or polymers, all demonstrated good surface activity. With serum present as an inhibiting agent, adding either concentration of KL₄ reduced inhibition. Mixtures containing the higher concentration of KL₄ required higher concentrations of serum for inhibition to occur. Adding either polymer to mixtures with KL₄ further decreased susceptibility to inhibition (required higher serum concentrations). In the presence of serum, high molecular weight hyaluronan with KL₄ at 0.4 mg/ml improved surface activity to a greater degree than 0.8 mg/ml KL₄ without polymer. If the beneficial effects of adding polymer to KL₄-lipid mixtures are also borne out in the treatment of experimental lung injury, these peptide-polymer surfactant combinations may eventually prove useful in the treatment of some forms of acute lung injury in humans.     

Metabolic glycoengineering through the mammalian GalNAc salvage pathway

GalNAc is the initial sugar of mucin-type O-glycans, and is a component of several tumor antigens. The aim of this work was to determine whether synthetic GalNAc analogs could be taken up from the medium and incorporated into complex cellular O-glycans. The cell line employed was CHO ldlD, which can only use GalNAc and Gal present in the medium for the synthesis of its glycans. All GalNAc analogs with modified N-acyl groups (N-formyl, N-propionyl, N-glycolyl, N-azidoacetyl, N-bromoacetyl, and N-chloroacetyl) were incorporated into cellular O-glycans, although to different extents. The GalNAc analogs linked to Ser or Thr could be extended by the β3-galactosyltransferase glycoprotein-N-acetylgalactosamine 3β-galactosyl transferase 1 in vitro and in vivo and by α6-sialyltransferase α-N-acetylgalactosaminide-α-2,6-sialyltransferase 1. At the surface of CHO ldlD cells, all analogs were incorporated into sialylated O-glycan structures like those present on wild-type CHO cells, indicating that the GalNAc analogs do not change the overall structure of core-1 O-glycans. In addition, this study shows that the unnatural synthetic GalNAc analogs can be incorporated into human tumor cells, and that a tumor antigen modified by an analog can be readily detected by a specific antiserum. GalNAc analogs are therefore potential targets for tumor immunotherapy.