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Poly(ethylene glycol) enhances the surface activity of a pulmonary surfactant
Colloids and Surfaces B: Biointerfaces 36 (2004) 167–176
Poly(ethylene glycol) enhances the surface activity of a pulmonary surfactant Laura M.Y. Yu a , James J. Lu a , Idy W.Y. Chiu b , Kin Shun Leung c , Yawen W. Chan a , Ling Zhang a , Zdenka Policova a , Michael L. Hair a , A. Wilhelm Neumann a,∗ a b
Department of Mechanical and Industrial Engineering, University of Toronto, 5 King’s College Road, Toronto, Ont., Canada M5S 3G8 University of Toronto, Division of Molecular Genetics and Molecular Biology, 100 St. George Street, Toronto, Ont., Canada M5S 3G3 c Department of Systems Design Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ont., Canada N2L 3G1 Received 22 August 2003; accepted 8 June 2004
Abstract The primary role of lung surfactant is to reduce surface tension at the air–liquid interface of alveoli during respiration. Axisymmetric drop shape analysis (ADSA) was used to study the effect of poly(ethylene glycol) (PEG) on the rate of surface film formation of a bovine lipid extract surfactant (BLES), a therapeutic lung surfactant preparation. PEG of molecular weights 3350; 8000; 10,000; 35,000; and 300,000 in combination with a BLES mixture of 0.5 mg/mL was studied. The adsorption rate of BLES alone at 0.5 mg/mL was much slower than that of a natural lung surfactant at the same concentration; more than 200 s are required to reach the equilibrium surface tension of 25 mJ/m2 . PEG, while not surface active itself, enhances the adsorption of BLES to an extent depending on its concentration and molecular weight. These findings suggest that depletion attraction induced by higher molecular weight PEG (in the range of 8000 to 35,000) may be responsible for increasing the adsorption rate of BLES at low concentration. The results provide a basis for using PEG as an additive to BLES to reduce its required concentration in clinical treatment, thus reducing the cost for surfactant replacement therapy. © 2004 Elsevier B.V. All rights reserved. Keywords: Poly(ethylene glycol); Pulmonary surfactant; BLES; Adsorption; Surface tension
1. Introduction Lung surfactant plays a crucial role in respiration. Its primary function is to reduce the surface tension at the air–liquid interface in alveoli during respiration. The reduction in surface tension reduces the energy required to inflate the lungs and the change in surface tension with lung volumes stabilizes the lungs against collapse during breathing [1]. A deficiency of lung surfactant in the alveolar cavity may lead to lung collapse, decreased pulmonary compliance, oxygen starvation, and pulmonary edema [1,2]. Lung surfactant is a surface active material that lines the inner surface of the alveolus. The lamellar bodies of lung surfactant are synthesized, stored, and secreted by Type II alveolar epithelial cells [3–6]. Lung surfactant lining the alveo-
∗
Corresponding author. Tel.: +1 416 978 1270; fax: +1 416 978 7753. E-mail address: [email protected] (A.W. Neumann).
0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2004.06.005
lus is a complex mixture of lipids and proteins. It is comprised of about 90% lipids and 10% proteins by weight. Of the lipid fraction, phospholipids account for approximately 85–90%, the remaining fraction being neutral lipids, including cholesterol. The phospholipid fraction is in turn made up of 30% dipalmitoyl phosphatidylcholine (DPPC), a saturated phospholipid, and 70% of other monosaturated and unsaturated phospholipids [7–9]. DPPC is the main contributor in attaining near zero surface tension at the air–liquid interface in alveoli during respiration [10,11]. There are four surfactant-associated proteins (SP), denoted as SP-A, SP-B, SP-C, and SP-D. SP-A and SP-D are high molecular weight hydrophilic proteins, whereas SP-B and SP-C are low molecular weight hydrophobic and surface active proteins. The function of SP-A is to facilitate the rate of surfactant adsorption onto the air–liquid interface by promoting the formation of phospholipids into tubular myelin structures together with SP-B and Ca2+ [8,10]. SP-D is believed to be involved in the defense mechanism of the lung and may have a regula-
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tory role in surfactant production and metabolism [11–13]. Nevertheless, it has not been demonstrated to participate in any surface activity of the lung [11–13]. Both SP-B and SP-C have been shown to accelerate surface film formation, enhance adsorption of phospholipid mixtures, and improve film re-spreading during dynamic compression and expansion of surface films, respectively [3,6,7,9,13]. Deficiency of lung surfactant in the alveolar space causes various lung diseases due to high surface tension in the alveoli. Consequently, higher energy is required to inflate the lungs during respiration. Neonatal respiratory distress syndrome (nRDS) is the disease associated with lung surfactant deficiency in the alveolar space in premature babies. An acute form of RDS (aRDS) also exists and is usually caused by chronic lung injury. Other complications of prematurity and injury induced by mechanical ventilator or hyperoxia during intensive care of infants may also lead to acute RDS (aRDS) [1]. Surfactant replacement therapy using natural and synthetic formulations have been successful in treating nRDS patients. Bovine lipid extract surfactant (BLES) is an example of a natural formulation extracted from bovines with SP-A and SP-D removed during processing. It is widely used in many Canadian hospitals. Exosurf® on the other hand is one of the FDA approved synthetic formulations that contains the main surface tension reducing component DPPC supplemented with synthetic non-biological emulsifying agents that have no relation to the natural lung surfactant. While surfactant replacement therapy has been successful in reducing the number of premature infant deaths, its production from animal sources is expensive and purification is difficult. The use of animal sources also poses a risk of transferring other viral diseases or contaminants to the patient [14]. There are similar concerns with respect to synthetic formulations, including variability in composition from batch to batch. Surfactant replacement therapy is not as effective in treating patients with acute RDS as the treatment of the neonatal form of RDS. The cause is because leakage of blood proteins, as observed in patients with aRDS, into the alveolar cavity, leading to surfactant dysfunction [15–18]. Hence, understanding the mechanism of surfactant dysfunction and designing more effective treatment for RDS patients by avoiding this surfactant inhibition continues to be the main impetus for current research in lung surfactant. The efficacy of surfactant replacement therapy using the natural formulation of BLES depends on the repeated administration of a large dosage of the concentrated surfactant, i.e. 27 mg/mL. The surface activity of BLES at low concentration is impaired by the removal of SP-A during the extraction process. Previous studies focusing on SP-A have demonstrated that this protein is able to resist surfactant inactivation by plasma proteins [15,17,19] and thus to improve the pulmonary mechanics of animals deficient in surfactant in the presence of plasma proteins [19]. Another study showed that animals with malfunctioning SP-A gene could breathe normally [20]. However, alveolar lavages of
these animals showed deteriorated adsorption behaviour at low concentrations [20]. These studies suggest that SP-A may play an important role under certain limiting conditions [15,19–21]. Concentration of pulmonary surfactant in the body and in clinical treatment is high. At such high concentrations BLES adsorbs too quickly and the surfactant film performs well enough dynamically, so that experimental methods in present use can not detect any subtle differences in the surface activity. However, at low concentration, adsorption of lung surfactant is slow so that changes in surface tension can be monitored closely with current methodologies. If lung surfactant function can be improved at low concentrations without compromising its surface activity, it may be possible to reduce the concentration of surfactant required in surfactant replacement therapy. The cost of the treatment can also be decreased. As alluded to earlier, the adsorption rate of lung surfactant deficient in SP-A is low (see Fig. 2). A possible remedy to overcome the slow rate of film formation of a low concentration of lung surfactant deficient in SP-A is by the addition of water soluble, nonionic polymers, such as poly(ethylene glycol) (PEG). The use of nonionic polymers to enhance the performance of lung surfactant seemed possible since PEG is a well-known fusogen for cells. It has also been shown to bind phospholipid vesicles [22,23]. Research findings by Tashiro, et al. [16], and Taeusch, et al. [17] suggest that nonionic polymers including PEG and dextran can reverse the inactivation of lung surfactant by meconium and plasma proteins. Dextran was also demonstrated to restore the surface activity of BLES inhibited by albumin [24]. Taeusch, et al. [17] introduced the use of simple nonionic polymers such as PEG and dextran on the rationale that their simple molecular structure may have similar properties to the carbohydrate-binding region of SP-A. They believe that depletion attraction may be the underlying mechanism responsible for the reversal of inhibition. Hence, the objective of this study is to enhance the surface tension performance of BLES (a therapeutic preparation without SP-A and SP-D) at a low concentration (0.5 mg/mL) by using PEG as an additive. The rate of surface film formation at this concentration is low enough that adsorption kinetics could be captured using a suitable surface tension technique.
2. Materials and methods Bovine lipid extract surfactant (BLES) and bovine natural lung surfactant were used in this study. BLES and natural lung surfactant were generously donated by BLES Biochemicals Inc. (London, Ont., Canada). The BLES and natural lung surfactant samples were supplied at 27 and 2.99 mg/mL, respectively. BLES samples were subdivided into vials while natural lung surfactant samples were suspended in salt solution before subdividing into vials. Both subdivided samples were sealed in nitrogen gas and stored at −20 ◦ C until the day of the experiment and were used without further treat-
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ment. BLES consists of approximately 98% phospholipids and 2% proteins by weight. The phospholipid components are 45% dipalmitoyl phosphatidylcholine, 35% unsaturated phosphatidylcholine (PC), 12% phosphatidylglycerol (PG), 2% phosphatidylethanolamine (PE), 1% phosphatidylinositol (PI), 1% lysophosphatidylcholine (LPC), and 2% sphingomyelin (SM) (Composition of BLES was provided by BLES Biochemicals Inc., London, Ont.). The low molecular weight, hydrophobic, surfactant-associated proteins (SP) B and C are present, while the high molecular weight, surfactant proteins SP-A and SP-D are removed by processing. There are 0.55 mg/mL of SP-B and C in BLES according to the specifications given by the manufacturer. The BLES samples were prepared from the bovine natural lung surfactant obtained from the lungs of cattle which had been slaughtered for human consumption. The bronchopulmonary lavage was washed with saline/magnesium chloride/calcium chloride solution. Bovine natural lung surfactant was then extracted with chloroform/methanol and precipitated with acetone. SP-A and SP-D were removed during this precipitation procedure. The acetone precipitate was re-suspended in saline solution composed of 0.10 N NaCl and 1.5 mM calcium chloride. The term, natural lung surfactant in this paper, refers to the extracted bovine natural lung surfactant before the removal of SP-A and D. BLES and natural lung surfactant were diluted to the desired concentration using a salt solution. PEG samples with average molecular weights 3350; 8000; 10,000, and 35,000 were purchased from Sigma Chemicals Co., USA and PEG sample of MW 20,000 and PEO sample of MW 300,000 were purchased from Polysciences Inc., USA. It is noted that the polymer at very high molecular weight is made by a different process and so it is no longer a polyglycol but a polyoxide. All PEG and PEO solutions were prepared by dissolving the desired amount in salt solution in a 10 mL volumetric flask. The salt solution was composed of 0.6 wt.% NaCl and 1.5 mM CaCl2 solution. Water used in the experiments was demineralized and distilled.
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A pendant drop arrangement was used in this study to measure the rate of surface film formation of pulmonary surfactant mixtures. A quartz capillary tube with an outer diameter of 3 mm and inner diameter of 1 mm was used to form a drop of the pulmonary surfactant. The drop was formed inside a quartz glass cuvette (Hellma Ltd.) and a Teflon stopper was used to seal the cuvette to prevent evaporation and contamination from the outside environment. The glass cuvette was placed inside an aluminum temperature cell (Ramé Hart Inc.), where the temperature was maintained at 37 ± 0.3 ◦ C by a water bath (Neslab Instruments Inc., USA). The quartz capillary was connected to a motor-driven syringe through Teflon tubing and connectors. The syringe was mounted onto a stepping motor that controls the volume and speed of drop formation. Images of the drop were acquired using a CCD monochrome camera (Cohu Inc., Canada) and a microscope (Leitz Wetzlar, Germany). A light source and diffuser were used to ensure a uniformly lit background during image acquisition. Fig. 1 shows a schematic diagram of the experimental set-up. All images were stored and processed on a Sun workstation, Ultra Sparc 1 (Sun Microsystems Inc., USA). They were acquired at a rate of 20–30 frames per second and later analyzed. SOBEL operator edge detection with a sub-pixel resolution procedure was used to extract profiles of the drop. The image analysis techniques, which include optical distortion and vertical alignment corrections were performed after edge detection. Axisymmetric drop shape analysis (ADSA) was used to analyze the digitized drop images. ADSA is a technique for determining liquid–fluid interfacial tension based on the numerical integration of the Laplace equation of capillarity which describes the shape of liquid–fluid menisci [25]. The strategy used in the methodology is to minimize the deviation between the experimental and the theoretical Laplacian curve. The best match yields the interfacial tension of a pendant or sessile drop with an accuracy of ±0.01 mJ/m2 . Detailed development of ADSA has been discussed elsewhere [25–27]. The output of ADSA
Fig. 1. A schematic diagram of the experimental set-up.
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Fig. 2. Surface tension comparison of BLES and natural lung surfactant at 37 ◦ C.
includes surface tension, volume, surface area, and curvature at the apex of the drop.
3. Results The rate of surface film formation of surfactant in mixtures containing BLES and PEG as a function of concentration and molecular weight were analyzed over an adsorption period of 300 s. The adsorption rate during the initial period is of importance because it indicates how effectively surfactant molecules adsorb at the air–liquid interface and form surfactant films. Fast adsorption is physiologically important since the surfactant film must be formed rapidly during the initial opening of the lungs [8].
SP-A, which facilitates the adsorption of lung surfactant by forming tubular myelin structures together with SP-B and calcium ions [10], is absent from BLES and this reduces the rate of adsorption at the air–liquid interface at low concentrations [21]. This is shown in Fig. 2 where the adsorption rates of BLES and natural lung surfactant (both at 0.5 mg/mL and 37 ◦ C) are compared. The results shown are the average of at least four individual runs, except for the adsorption isotherm of BLES (0.5 mg/mL) due to the random occurrence of adsorption clicks as explained in detailed in [28,29]. From Fig. 2, it can be seen that more than 200 s elapse before BLES reaches its equilibrium surface tension of 25 mJ/m2 , whereas natural lung surfactant attained its equilibrium within 10 s.
Fig. 3. Surface tension of BLES, natural lung surfactant, PEG only (MW 10,000) and BLES + PEG at 37 ◦ C.
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Fig. 4. Surface tension of BLES + PEG as a function of PEG (MW 10,000) concentration at time 0 and after 10 s of adsorption.
The slow rate of surface film formation of BLES at 0.5 mg/mL can be enhanced by the addition of PEG (Fig. 3). It should be noted that the number of data points on each curve in Fig. 3 was reduced for graphical clarity. Fig. 3 shows that PEG at MW 10,000 enhances the adsorption rate of BLES (0.5 mg/mL) and that this enhancement is a function of polymer concentration. It is clear from Fig. 3 that the improved surface activity is not due to adsorption of PEG since its surface tension remained at about 58 mJ/m2 during the entire 300 s. As seen in Fig. 3 the adsorption isotherm of BLES + PEG (28 mg/mL) is very close to the adsorption isotherm of natural lung surfactant at 0.5 mg/mL. The experimental results also show that increasing the PEG
concentration beyond 28 mg/mL gave no further improvement in the adsorption rate of BLES. This is more clearly shown in Fig. 4, which shows the surface tension recorded immediately after drop formation (t = 0 s) and after 10 s of adsorption (t = 10 s). The ability of PEG to enhance the adsorption of a low concentration BLES was also found to depend on its molecular weight. Fig. 5 shows the adsorption isotherms of BLES in mixture with different molecular weight of PEG at 10 mg/mL. There appears to be an optimal range of molecular weight at which PEG is able to enhance the performance of BLES at the chosen PEG concentration of 10 mg/mL. It should be noted that with PEO of MW
Fig. 5. Effect of PEG molecular weight (10 mg/mL) on the surface tension of BLES.
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Table 1 Surface tension of BLES + PEG (30 mg/mL) attained as a function of molecular weight at time 0 and after 10 s of adsorption Molecular weight of PEG
Surface tension (mJ/m2 ) t =0
3350 8000 10000 20000 35000
33.7 33.4 32.7 33.4 33.0
± ± ± ± ±
t = 10 s 0.7 2.6 0.6 0.1 1.0
27.8 28.7 28.4 28.6 28.9
± ± ± ± ±
0.7 1.2 0.7 0.1 0.1
300,000 the surface tension remained high and almost unchanged at about 57 mJ/m2 . This PEG concentration of 10 mg/mL was chosen since for each molecular weight, there is a minimum concentration of PEG that optimizes the performance of BLES (see Fig. 3). At higher concentration of each molecular weight, the effect of PEG concentration on the performance of BLES would mask the effect of molecular weight. This relationship is shown in Table 1, which reports the results when using a constant PEG concentration of 30 mg/mL for each molecular weight studied in Fig. 5. PEO (MW 300,000) is omitted from Table 1 since at 30 mg/mL, the polymer becomes so difficult to dissolve in salt solution that the experiment could not be readily performed. It is concluded that PEG enhances the adsorption of BLES at low concentration. The effect is simultaneously dependent on both the concentration and molecular weight of PEG. Thus, the optimum range of concentration and/or molecular weight of PEG for film formation can not be determined independent of each other. The key findings of this study are visualized in Fig. 6a–c which show pendant drop images of BLES only at 0.5 mg/mL, PEG only (MW 10,000) at 28 mg/mL, and BLES + PEG (28 mg/mL, MW 10,000) after 10 s of adsorption. The round drop shapes of BLES and PEG shown in Fig. 6a and b indicate high surface tension and the values obtained from ADSA are 68.7 ± 0.8 and 58.2 ± 0.2 mJ/m2 , respectively. Fig. 6c shows a drop image of BLES + PEG (28 mg/mL, MW 10,000). The drop shape has an elongated profile that contrasts with the high surface tension profiles of BLES and PEG in Fig. 6a and b. The measured surface tension corresponding to Fig. 6c is 27.9 ± 0.1 mJ/m2 .
4. Discussions BLES is a complex mixture of phospholipids, SP-B and SP-C. The phospholipids interact with the surfactant proteins in order to produce the rapid adsorption rates which are observed in the bulk adsorption studies of BLES [28]. The performance of BLES is compromised significantly at low concentrations compared to the natural extract at the same
Fig. 6. Pendant drop images of (a) BLES (0.5 mg/mL), (b) PEG (28 mg/mL, MW 10,000), and (c) BLES (0.5 mg/mL) + PEG (28 mg/mL, MW 10,000) after 10 s of adsorption.
concentration, as was shown in Fig. 2 and in agreement with [20]. Previous studies have shown that larger aggregates of lung surfactant have greater ability to lower surface tension than their smaller counterparts [15]. Hence, it is postulated that the surface activity of BLES at low concentration may be improved if formation of larger aggregates can be induced. PEG is by far the most commonly used agent for cell fusion. Theories regarding its efficacy have been described in the literature [22,23,30]. Studies on the PEG-induced aggregation of phospholipid vesicles have shown that the underly-
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ing mechanism involves the restructuring of water molecules around the hydration sphere of the vesicles due to the high affinity of the polymer for water. Water molecules interact with PEG through hydrogen bonding due to the presence of polyether oxygen along the backbone of the polymer. Consequently a depletion attraction force can be induced as was first described by Asakura and Oosawa [31] for colloidal systems in the presence of polymers. These authors explained that in any colloidal system, addition of water-soluble polymer causes disruption of the osmotic balance in the system. Consequently, an attractive force, referred to as depletion attraction, is induced and this causes colloid particles to aggregate. The magnitude of these forces have been directly measured by Kuhl et al. [23] in phospholipids in which vesicle aggregation was induced by the addition of PEG. Although depletion attraction was studied on pure phospholipid vesicle systems [22,23], the authors provide evidence in their study suggesting that depletion attraction may be responsible for the increased adsorption rate in our BLES–PEG systems. The work of Meyuhas et al. [22] showed that aggregation of phospholipid vesicles (made from sonicated egg phosphatidylcholine) is induced when dextran or PEG is added at their respective overlap concentration. These authors studied the aggregation of phosphatidylcholine (PC) vesicles by measuring turbidity of both large and small PC unilamellar vesicles. They found that at a PC concentration of 1 mM or higher the threshold concentration required to induce aggregation always occurred at the overlap concentration of the added polymers. At this overlap concentration, the polymer chains begin to interact and form entanglements, which would allow them to trap more water molecules than in their extended conformation [22,23]. As the molecular weight of polymer increased, the threshold concentration required for aggregation decreased accordingly. The authors confirmed the findings from their turbidity measurements in their dialysis experiment. By measuring the osmotically driven shrinkage of dialysis bags, both dextran and PEG were able to induce size growth in the vesicles. At much higher polymer concentrations, the size growth of the PC vesicles was irreversible. Kuhl et al. [23] performed similar studies to Meyuhas et al. [22] between phospholipid bilayers and PEG. The authors studied the nature of bilayer–PEG interaction using a surface force apparatus, 31 P NMR, and quasi-elastic light scattering techniques. The molecular volume of the polymer increases as it binds more water and becomes excluded from the spaces between the lipid bilayers. As a result of this concentration difference inside and outside the gap between the lipid bilayers, an osmotic force is induced. The surface force profiles obtained by the authors showed that such an osmotic attractive force is much stronger than the van der Waals force of interaction. From the various techniques used in their study, they confirmed that PEG of molecular weight 8000 to 10,000 was effective in causing vesicle aggregation. In the surface force measurement, these authors found
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that low molecular weight PEG (MW < 1000) is ineffective in generating a depletion layer due to the small size of the polymer molecules. Surface force profiles of bilayers measured in aqueous solution of higher PEG molecular weight (MW 20,000) showed only a repulsive force. The authors suggest that the high molecular weight PEG may adsorb on the surfaces of the bilayer, or that the slow mobility of the polymer chains may cause them to be trapped between the surfaces of the bilayer. Thus the repulsive force measured in their experiment may be due to entropic confinement and compression of the trapped polymer chains [23]. Evans and Needham have performed adhesion experiment on giant phospholipid vesicles [32]. These authors used synthetic phospholipids of 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and dioleolyl phosphatidylglycerol (DOPG), and digalactosyl diacylglycerol (DGDG), a natural lipid. They have found that PEG and dextran both enhance the adhesion of the vesicles to each other in their micropipette aspiration measurement. The attraction between phospholipid vesicles was three times greater in PEG solution than in dextran solution at the same volume fraction of the polymers [32]. Their findings are in close agreement with those of Meyuhas et al. [22] and Kuhl et al. [23] using different techniques as discussed above. Accordingly, Evans and Needham [32] developed a depletion force theory, the self-consistent mean field theory (SCMF), to explain their findings in the presence of non-adsorbing polymers. The SCMF theory also predicted the absence of polymer in the gap between vesicles of neutral lipids at equilibrium separation [32]. The presence of pure water in the gap induces an attractive osmotic force between the vesicle surfaces that causes them to aggregate [23,32]. Although depletion attraction had only been studied on pure phospholipid vesicle systems in the literature [22,23,32], the studies strongly suggest that depletion attraction may be responsible for the increased adsorption rate in our BLES–PEG systems. However, it is not known if PEG has an effect on the phospholipid vesicles in our BLES–PEG system. It is suggested that in our experiments, aggregation of lung surfactant may have resulted because of a depletion attraction force induced by osmotic imbalance as a result of the presence of PEG. The large surfactant aggregates which are formed would presumably aid in the adsorption of BLES in agreement with previous observations [15,22,23]. Larger particles were indeed observed in our pendant drops when PEG was added to BLES at increasing concentrations (>20 mg/mL). The size of these particles was not measured in our study. Similar observations were seen in captive bubble experiments of BLES + PEG mixture when PEG (MW 10,000) was added at 28 mg/mL and 50 mg/mL [36]. Fig. 7a and b are captive bubble images of BLES (0.5 mg/mL) and BLES (0.5 mg/mL) + PEG (28 mg/mL, MW 10,000), respectively. A comparison of these two images clearly show large par-
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Fig. 7. A comparison of captive bubble images of (a) BLES (0.5 mg/mL), and (b) BLES (0.5 mg/mL) + PEG (28 mg/mL, MW 10,000) clearly show the formation of aggregates when PEG is added to BLES.
ticles in the subphase of Fig. 7b, i.e. in the case of addition of PEG. Fig. 3 shows that when PEG of MW 10,000 was added to BLES at 28 mg/mL, the adsorption rate of BLES was increased significantly and the resulting isotherm becomes comparable to that of natural lung surfactant. The overlap concentration of PEG (MW 10,000) was measured by Kuhl et al. using dynamic light scattering and is reported to be 28 mg/mL [23]. According to Meyuhas et al. [22], aggregation of PC vesicles always occurred at the overlap concentration. As shown in Fig. 4, the minimum concentration of PEG (MW 10,000) required to observe improvement in the adsorption rate of BLES is about 15 mg/mL. Although the adsorption of BLES enhanced by PEG (MW 10,000) does not appear to maximize at the overlap concentration of 28 mg/mL measured by Kuhl et al. [23], it is noted that in our more complex natural system, the addition of PEG at concentrations lower than the overlap concentration may be sufficient to promote or increase
the interaction between phospholipids and surfactant proteins (SP-B and SP-C) in BLES to improve its adsorption rate. The effect of molecular weight of PEG on the performance of BLES is also reported in this study. It is found that moderately high molecular weight (i.e. MW > 8000) best enhances adsorption of BLES to the air–liquid interface. According to the literature [23,36], PEG does not induce aggregation in phospholipid vesicles at MW 20,000. Rupert et al. [36] suggests that at MW of 20,000 the interaction between polymer chains is presumably favoured over interactions between phospholipids or water. Consequently the surfaces of phospholipid vesicles become covered with PEG. As discussed above, Kuhl et al. [23] also suggested that PEG adsorbs onto surfaces of phospholipid vesicles. The vesicles become sterically stabilized and thus impede the close approach of phospholipid vesicles [36]. However, improvement in the adsorption of BLES persisted for PEG at MW 35,000 (Fig. 5). A recent publication by Platikanov et al. [37] showed that phosphatidylcholine vesicles take on different bilayer arrangement and are packed differently when prepared under different conditions and subject to different environment. Hence, the discrepancy between our results and the literature is not unexpected since BLES is a more complex system than those previously studied in the literature [22,23,31,36]. The arrangement of the lung surfactant aggregates at the interface and in the bulk would be very different from those vesicle systems studied by Meyuhas et al. [22], Kuhl et al. [23], and Evans and Needham [32]. The adsorption of BLES was hindered significantly at very high molecular weight of 300,000. At such a high molecular weight, the polymer itself has a very large molecular volume and the bulky polymer chains may well be trapped between lung surfactant aggregates due to its slow diffusion and mobility [23]. This agrees with the near constant surface tension for BLES with PEO of MW 300,000 (Fig. 5). An experiment with extended adsorption period of 500 s was performed for the BLES + PEG (MW 300,000). During the first 300–400 s surface tension remained constant near 57–59 mJ/m2 . The surface tension then decreased steadily and required a further 100 s to reach equilibrium. At low molecular weight (i.e. MW < 3350), a much higher concentration of PEG would be required to achieve overlap concentration and induce a strong enough depletion attraction force to cause appreciable aggregation of surfactant [23]. However, at these higher concentrations, viscosity of the BLES–PEG mixture is increased and this would hinder in vitro experiment. (Viscosity effects on the rate of drop formation, however, were not the focus of this study and their significance on the adsorption of lung surfactant film is unknown). High viscosity also hinders any in vivo application (Our previous studies in rabbit were unsuccessful using BLES + dextran mixture, the main problem being that dextran was used at such a high concentration that the mixture became too viscous to be instilled into the lungs of the rabbit.). Our experimental results
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suggest an optimum range of molecular weight from 8000 to 35,000 at which the performance of BLES is enhanced. Within this optimum molecular range, the improvement in the adsorption of BLES is dependent on the concentration used (Fig. 5). As the molecular weight of PEG increased, the concentration required to enhance the adsorption of BLES (0.5 mg/mL) decreased correspondingly, which is in agreement with findings of Meyuhas et al. [22]. This result is not unexpected if the overlap concentration plays a significant role [33]. Hence, an optimum concentration depends on molecular weight. This inter-dependence will provide flexibility when other parameters need to be considered, such as the effect of PEG on the gas transfer properties across the surfactant film [34] and the use of PEG to reverse protein inactivation of surfactant (i.e. albumin) [16,17,35]. Similar results were also observed in preliminary pendant drop studies using poly(vinyl pyrrolidone) (PVP) and dextran in mixture with BLES suspension. We believe that addition of PVP and dextran also induces depletion attraction in BLES suspensions. However, the patterns in such systems are more complicated than those observed in BLES–PEG systems.
5. Conclusions From this study, it has been shown that PEG is able to enhance the rate of surface film formation of a low concentration BLES. The effect of PEG on the performance of BLES is simultaneously dependent on both its concentration and molecular weight. We have proposed that depletion attraction may likely be the underlying mechanism for the observed effect. It is hoped that this work will shed light on the possibility of reducing the clinical concentration in surfactant replacement therapy and thus reduce the cost of treatment. This work will also help understand the reversibility of inactivated lung surfactant systems by plasma proteins, such as albumin and fibrinogen by nonionic polymers.
Acknowledgements This work was supported by a grant from the Canadian Institute of Health Research (grant MOP38037) and Ontario Graduate Scholarship for Science and Technology (L.M.Y. Yu). We wish to thank BLES Biochemicals Inc. for their generous donation of the BLES and natural lung surfactant samples.
References [1] R.H. Notter, in: C. Lenfant (Ed.), Lung Surfactants Basic Science and Clinical Applications, Marcel Dekker, New York, NY, 2000 (Chapter 10).
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[2] A.C. Guyton, Basic Human Physiology: Normal Function and Mechanis of Disease, first ed., W.B. Saunders Company, Philadelphia, 1971 (Chapter 28). [3] H. Hamm, H. Fabel, W. Bartsch, The Clinical Investigator 70 (1992) 637. [4] J.A. Clements, Am. Rev. Respir. Dis. 115 (Suppl) (1997) 67. [5] R.H. Notter, in: C. Lenfant (Ed.), Lung Surfactants: Basic Science and Clinical Applications, Marcel Dekker Inc., New York, NY, 2000 (Chapter 1). [6] R.J. King, in: B. Robertson, L.M.G. van Golde, J.J. Batenburg (Eds.), Pulmonary Surfactant, Elsevier Science, New York, NY, 1984, Chapter 1. [7] S. Hawgood, in: R.G. Crystal (Ed.), The Lung: Scientific Foundation, Lippincott-Raven Publishers, Philadelphia, 1997 (Chapter 38). [8] R.H. Notter, in: C. Lenfant (Ed.), Lung Surfactants: Basic Science and Clinical Applications, Marcel Dekker, New York, NY, 2000 (Chapter 6). [9] S. Yu, P.G.R. Harding, N. Smith, F. Possmayer, Lipids 18 (1983) 522. [10] R.H. Notter, Lung Surfactants: Basic Science and Clinical Applications, first ed., Marcel Dekker, New York, NY, 2000 (Chapter 3). [11] F. Possmayer, Am. Re. Rev. Respir. Dis. Editorial 142 (1990) 749. [12] K.M.W. Keough, Lung Surfactant: Cellular and Molecular Processing, in: S.A. Rooney (Ed.), R.G. Landes Company, TX, 1998 (Chapter 1). [13] R.H. Notter, in: C. Lenfant, Lung Surfactants: Basic Science and Clinical Applications, Marcel Dekker, New York, NY, 2000 (Chapter 8). [14] J.A. Zasadzinski, J. Ding, H.E. Warriner, F. Bringezu, A.J. Waring, Current Opinion Col. Interf. Sci. 6 (2001) 506. [15] R.H. Notter, in: C. Lenfant (Ed.), Lung Surfactants: Basic Science and Clinical Applications, Marcel Dekker, New York, NY, 2000 (Chapter 9). [16] K. Tashiro, T. Kobayashi, B. Robertson, Acta Paediatr. 89 (2000) 1439. [17] H.W. Taeusch, K.W. Lu, J. Goerke, J.A. Clements, Am. J. Respir. Crit. Care Med. 159 (1999) 1391. [18] B.A. Holm, Z. Wang, R.H. Notter, Pediatric Res. 46 (1999) 85. [19] K. Yukitake, C.L. Brown, M.A. Schlueter, J.A. Clements, S. Hawgood, Pediatric Res. 37 (1995) 21. [20] T.R. Korfhagen, M.D. Bruno, G.F. Ross, K.M. Huelsman, M. Ikegami, A.H. Jobe, S.E. Wert, B.R. Stripp, R.E. Morris, S.W. Glasser, C.J. Brahurski, H.S. Iwamoto, J.A. Whitsett, Proc. Nat. Acad. Sci. U.S.A. 93 (1996) 9594. [21] J. Pérez-Gil, K.M.W. Keough, Biochim. Biophy. Acta 1408 (1998) 203. [22] D. Meyuhas, S. Nir, D. Lichtenberg, Biophys. J. 71 (1996) 2602. [23] T. Kuhl, Y. Guo, J.L. Alderfer, A.D. Berman, D. Leckband, J. Israelachvili, S.W. Hui, Langmuir 12 (1996) 3003. [24] J.J. Lu, W.W.Y. Cheung, L.M.Y. Yu, Z. Policova, D. Li, M.L. Hair, A.W. Neumann, Respir. Physiol. Neurobiol. 130 (2002) 169. [25] D.Y. Kwok, D. Vollhardt, R. Miller, D. Li, A.W. Neumann, Colloids Surf. A 88 (1994) 51. [26] R. Rotenberg, L. Boruvka, A.W. Neumann, J. Colloid Interf. Sci. 93 (1983) 169. [27] P. Cheng, D. Li, L. Boruvka, Y. Rotenberg, A.W. Neumann, Colloids Surf. 43 (1990) 151. [28] J.J. Lu, L.M.Y. Yu, W.W.Y. Cheung, Z. Policova, D. Li, M.L. Hair, A.W. Neumann, Colloids Surf. B 29 (2002) 119. [29] S. Schürch, H. Bachofen, J. Goerke, F. Possmayer, J. Appl. Physiol. 67 (6) (1989) 2389. [30] K. Arnold, L. Pratsch, K. Gawrisch, Biochim. Biophys. Acta 728 (1983) 121. [31] S. Asakura, F. Oosawa, J. Poly. Sci. 33 (1985) 183.
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Poly(ethylene glycol) enhances the surface activity of a pulmonary surfactant Laura M.Y. Yu a , James J. Lu a , Idy W.Y. Chiu b , Kin Shun Leung c , Yawen W. Chan a , Ling Zhang a , Zdenka Policova a , Michael L. Hair a , A. Wilhelm Neumann a,∗ a b
Department of Mechanical and Industrial Engineering, University of Toronto, 5 King’s College Road, Toronto, Ont., Canada M5S 3G8 University of Toronto, Division of Molecular Genetics and Molecular Biology, 100 St. George Street, Toronto, Ont., Canada M5S 3G3 c Department of Systems Design Engineering, University of Waterloo, 200 University Avenue West, Waterloo, Ont., Canada N2L 3G1 Received 22 August 2003; accepted 8 June 2004
Abstract The primary role of lung surfactant is to reduce surface tension at the air–liquid interface of alveoli during respiration. Axisymmetric drop shape analysis (ADSA) was used to study the effect of poly(ethylene glycol) (PEG) on the rate of surface film formation of a bovine lipid extract surfactant (BLES), a therapeutic lung surfactant preparation. PEG of molecular weights 3350; 8000; 10,000; 35,000; and 300,000 in combination with a BLES mixture of 0.5 mg/mL was studied. The adsorption rate of BLES alone at 0.5 mg/mL was much slower than that of a natural lung surfactant at the same concentration; more than 200 s are required to reach the equilibrium surface tension of 25 mJ/m2 . PEG, while not surface active itself, enhances the adsorption of BLES to an extent depending on its concentration and molecular weight. These findings suggest that depletion attraction induced by higher molecular weight PEG (in the range of 8000 to 35,000) may be responsible for increasing the adsorption rate of BLES at low concentration. The results provide a basis for using PEG as an additive to BLES to reduce its required concentration in clinical treatment, thus reducing the cost for surfactant replacement therapy. © 2004 Elsevier B.V. All rights reserved. Keywords: Poly(ethylene glycol); Pulmonary surfactant; BLES; Adsorption; Surface tension
1. Introduction Lung surfactant plays a crucial role in respiration. Its primary function is to reduce the surface tension at the air–liquid interface in alveoli during respiration. The reduction in surface tension reduces the energy required to inflate the lungs and the change in surface tension with lung volumes stabilizes the lungs against collapse during breathing [1]. A deficiency of lung surfactant in the alveolar cavity may lead to lung collapse, decreased pulmonary compliance, oxygen starvation, and pulmonary edema [1,2]. Lung surfactant is a surface active material that lines the inner surface of the alveolus. The lamellar bodies of lung surfactant are synthesized, stored, and secreted by Type II alveolar epithelial cells [3–6]. Lung surfactant lining the alveo-
∗
Corresponding author. Tel.: +1 416 978 1270; fax: +1 416 978 7753. E-mail address: [email protected] (A.W. Neumann).
0927-7765/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2004.06.005
lus is a complex mixture of lipids and proteins. It is comprised of about 90% lipids and 10% proteins by weight. Of the lipid fraction, phospholipids account for approximately 85–90%, the remaining fraction being neutral lipids, including cholesterol. The phospholipid fraction is in turn made up of 30% dipalmitoyl phosphatidylcholine (DPPC), a saturated phospholipid, and 70% of other monosaturated and unsaturated phospholipids [7–9]. DPPC is the main contributor in attaining near zero surface tension at the air–liquid interface in alveoli during respiration [10,11]. There are four surfactant-associated proteins (SP), denoted as SP-A, SP-B, SP-C, and SP-D. SP-A and SP-D are high molecular weight hydrophilic proteins, whereas SP-B and SP-C are low molecular weight hydrophobic and surface active proteins. The function of SP-A is to facilitate the rate of surfactant adsorption onto the air–liquid interface by promoting the formation of phospholipids into tubular myelin structures together with SP-B and Ca2+ [8,10]. SP-D is believed to be involved in the defense mechanism of the lung and may have a regula-
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tory role in surfactant production and metabolism [11–13]. Nevertheless, it has not been demonstrated to participate in any surface activity of the lung [11–13]. Both SP-B and SP-C have been shown to accelerate surface film formation, enhance adsorption of phospholipid mixtures, and improve film re-spreading during dynamic compression and expansion of surface films, respectively [3,6,7,9,13]. Deficiency of lung surfactant in the alveolar space causes various lung diseases due to high surface tension in the alveoli. Consequently, higher energy is required to inflate the lungs during respiration. Neonatal respiratory distress syndrome (nRDS) is the disease associated with lung surfactant deficiency in the alveolar space in premature babies. An acute form of RDS (aRDS) also exists and is usually caused by chronic lung injury. Other complications of prematurity and injury induced by mechanical ventilator or hyperoxia during intensive care of infants may also lead to acute RDS (aRDS) [1]. Surfactant replacement therapy using natural and synthetic formulations have been successful in treating nRDS patients. Bovine lipid extract surfactant (BLES) is an example of a natural formulation extracted from bovines with SP-A and SP-D removed during processing. It is widely used in many Canadian hospitals. Exosurf® on the other hand is one of the FDA approved synthetic formulations that contains the main surface tension reducing component DPPC supplemented with synthetic non-biological emulsifying agents that have no relation to the natural lung surfactant. While surfactant replacement therapy has been successful in reducing the number of premature infant deaths, its production from animal sources is expensive and purification is difficult. The use of animal sources also poses a risk of transferring other viral diseases or contaminants to the patient [14]. There are similar concerns with respect to synthetic formulations, including variability in composition from batch to batch. Surfactant replacement therapy is not as effective in treating patients with acute RDS as the treatment of the neonatal form of RDS. The cause is because leakage of blood proteins, as observed in patients with aRDS, into the alveolar cavity, leading to surfactant dysfunction [15–18]. Hence, understanding the mechanism of surfactant dysfunction and designing more effective treatment for RDS patients by avoiding this surfactant inhibition continues to be the main impetus for current research in lung surfactant. The efficacy of surfactant replacement therapy using the natural formulation of BLES depends on the repeated administration of a large dosage of the concentrated surfactant, i.e. 27 mg/mL. The surface activity of BLES at low concentration is impaired by the removal of SP-A during the extraction process. Previous studies focusing on SP-A have demonstrated that this protein is able to resist surfactant inactivation by plasma proteins [15,17,19] and thus to improve the pulmonary mechanics of animals deficient in surfactant in the presence of plasma proteins [19]. Another study showed that animals with malfunctioning SP-A gene could breathe normally [20]. However, alveolar lavages of
these animals showed deteriorated adsorption behaviour at low concentrations [20]. These studies suggest that SP-A may play an important role under certain limiting conditions [15,19–21]. Concentration of pulmonary surfactant in the body and in clinical treatment is high. At such high concentrations BLES adsorbs too quickly and the surfactant film performs well enough dynamically, so that experimental methods in present use can not detect any subtle differences in the surface activity. However, at low concentration, adsorption of lung surfactant is slow so that changes in surface tension can be monitored closely with current methodologies. If lung surfactant function can be improved at low concentrations without compromising its surface activity, it may be possible to reduce the concentration of surfactant required in surfactant replacement therapy. The cost of the treatment can also be decreased. As alluded to earlier, the adsorption rate of lung surfactant deficient in SP-A is low (see Fig. 2). A possible remedy to overcome the slow rate of film formation of a low concentration of lung surfactant deficient in SP-A is by the addition of water soluble, nonionic polymers, such as poly(ethylene glycol) (PEG). The use of nonionic polymers to enhance the performance of lung surfactant seemed possible since PEG is a well-known fusogen for cells. It has also been shown to bind phospholipid vesicles [22,23]. Research findings by Tashiro, et al. [16], and Taeusch, et al. [17] suggest that nonionic polymers including PEG and dextran can reverse the inactivation of lung surfactant by meconium and plasma proteins. Dextran was also demonstrated to restore the surface activity of BLES inhibited by albumin [24]. Taeusch, et al. [17] introduced the use of simple nonionic polymers such as PEG and dextran on the rationale that their simple molecular structure may have similar properties to the carbohydrate-binding region of SP-A. They believe that depletion attraction may be the underlying mechanism responsible for the reversal of inhibition. Hence, the objective of this study is to enhance the surface tension performance of BLES (a therapeutic preparation without SP-A and SP-D) at a low concentration (0.5 mg/mL) by using PEG as an additive. The rate of surface film formation at this concentration is low enough that adsorption kinetics could be captured using a suitable surface tension technique.
2. Materials and methods Bovine lipid extract surfactant (BLES) and bovine natural lung surfactant were used in this study. BLES and natural lung surfactant were generously donated by BLES Biochemicals Inc. (London, Ont., Canada). The BLES and natural lung surfactant samples were supplied at 27 and 2.99 mg/mL, respectively. BLES samples were subdivided into vials while natural lung surfactant samples were suspended in salt solution before subdividing into vials. Both subdivided samples were sealed in nitrogen gas and stored at −20 ◦ C until the day of the experiment and were used without further treat-
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ment. BLES consists of approximately 98% phospholipids and 2% proteins by weight. The phospholipid components are 45% dipalmitoyl phosphatidylcholine, 35% unsaturated phosphatidylcholine (PC), 12% phosphatidylglycerol (PG), 2% phosphatidylethanolamine (PE), 1% phosphatidylinositol (PI), 1% lysophosphatidylcholine (LPC), and 2% sphingomyelin (SM) (Composition of BLES was provided by BLES Biochemicals Inc., London, Ont.). The low molecular weight, hydrophobic, surfactant-associated proteins (SP) B and C are present, while the high molecular weight, surfactant proteins SP-A and SP-D are removed by processing. There are 0.55 mg/mL of SP-B and C in BLES according to the specifications given by the manufacturer. The BLES samples were prepared from the bovine natural lung surfactant obtained from the lungs of cattle which had been slaughtered for human consumption. The bronchopulmonary lavage was washed with saline/magnesium chloride/calcium chloride solution. Bovine natural lung surfactant was then extracted with chloroform/methanol and precipitated with acetone. SP-A and SP-D were removed during this precipitation procedure. The acetone precipitate was re-suspended in saline solution composed of 0.10 N NaCl and 1.5 mM calcium chloride. The term, natural lung surfactant in this paper, refers to the extracted bovine natural lung surfactant before the removal of SP-A and D. BLES and natural lung surfactant were diluted to the desired concentration using a salt solution. PEG samples with average molecular weights 3350; 8000; 10,000, and 35,000 were purchased from Sigma Chemicals Co., USA and PEG sample of MW 20,000 and PEO sample of MW 300,000 were purchased from Polysciences Inc., USA. It is noted that the polymer at very high molecular weight is made by a different process and so it is no longer a polyglycol but a polyoxide. All PEG and PEO solutions were prepared by dissolving the desired amount in salt solution in a 10 mL volumetric flask. The salt solution was composed of 0.6 wt.% NaCl and 1.5 mM CaCl2 solution. Water used in the experiments was demineralized and distilled.
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A pendant drop arrangement was used in this study to measure the rate of surface film formation of pulmonary surfactant mixtures. A quartz capillary tube with an outer diameter of 3 mm and inner diameter of 1 mm was used to form a drop of the pulmonary surfactant. The drop was formed inside a quartz glass cuvette (Hellma Ltd.) and a Teflon stopper was used to seal the cuvette to prevent evaporation and contamination from the outside environment. The glass cuvette was placed inside an aluminum temperature cell (Ramé Hart Inc.), where the temperature was maintained at 37 ± 0.3 ◦ C by a water bath (Neslab Instruments Inc., USA). The quartz capillary was connected to a motor-driven syringe through Teflon tubing and connectors. The syringe was mounted onto a stepping motor that controls the volume and speed of drop formation. Images of the drop were acquired using a CCD monochrome camera (Cohu Inc., Canada) and a microscope (Leitz Wetzlar, Germany). A light source and diffuser were used to ensure a uniformly lit background during image acquisition. Fig. 1 shows a schematic diagram of the experimental set-up. All images were stored and processed on a Sun workstation, Ultra Sparc 1 (Sun Microsystems Inc., USA). They were acquired at a rate of 20–30 frames per second and later analyzed. SOBEL operator edge detection with a sub-pixel resolution procedure was used to extract profiles of the drop. The image analysis techniques, which include optical distortion and vertical alignment corrections were performed after edge detection. Axisymmetric drop shape analysis (ADSA) was used to analyze the digitized drop images. ADSA is a technique for determining liquid–fluid interfacial tension based on the numerical integration of the Laplace equation of capillarity which describes the shape of liquid–fluid menisci [25]. The strategy used in the methodology is to minimize the deviation between the experimental and the theoretical Laplacian curve. The best match yields the interfacial tension of a pendant or sessile drop with an accuracy of ±0.01 mJ/m2 . Detailed development of ADSA has been discussed elsewhere [25–27]. The output of ADSA
Fig. 1. A schematic diagram of the experimental set-up.
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Fig. 2. Surface tension comparison of BLES and natural lung surfactant at 37 ◦ C.
includes surface tension, volume, surface area, and curvature at the apex of the drop.
3. Results The rate of surface film formation of surfactant in mixtures containing BLES and PEG as a function of concentration and molecular weight were analyzed over an adsorption period of 300 s. The adsorption rate during the initial period is of importance because it indicates how effectively surfactant molecules adsorb at the air–liquid interface and form surfactant films. Fast adsorption is physiologically important since the surfactant film must be formed rapidly during the initial opening of the lungs [8].
SP-A, which facilitates the adsorption of lung surfactant by forming tubular myelin structures together with SP-B and calcium ions [10], is absent from BLES and this reduces the rate of adsorption at the air–liquid interface at low concentrations [21]. This is shown in Fig. 2 where the adsorption rates of BLES and natural lung surfactant (both at 0.5 mg/mL and 37 ◦ C) are compared. The results shown are the average of at least four individual runs, except for the adsorption isotherm of BLES (0.5 mg/mL) due to the random occurrence of adsorption clicks as explained in detailed in [28,29]. From Fig. 2, it can be seen that more than 200 s elapse before BLES reaches its equilibrium surface tension of 25 mJ/m2 , whereas natural lung surfactant attained its equilibrium within 10 s.
Fig. 3. Surface tension of BLES, natural lung surfactant, PEG only (MW 10,000) and BLES + PEG at 37 ◦ C.
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Fig. 4. Surface tension of BLES + PEG as a function of PEG (MW 10,000) concentration at time 0 and after 10 s of adsorption.
The slow rate of surface film formation of BLES at 0.5 mg/mL can be enhanced by the addition of PEG (Fig. 3). It should be noted that the number of data points on each curve in Fig. 3 was reduced for graphical clarity. Fig. 3 shows that PEG at MW 10,000 enhances the adsorption rate of BLES (0.5 mg/mL) and that this enhancement is a function of polymer concentration. It is clear from Fig. 3 that the improved surface activity is not due to adsorption of PEG since its surface tension remained at about 58 mJ/m2 during the entire 300 s. As seen in Fig. 3 the adsorption isotherm of BLES + PEG (28 mg/mL) is very close to the adsorption isotherm of natural lung surfactant at 0.5 mg/mL. The experimental results also show that increasing the PEG
concentration beyond 28 mg/mL gave no further improvement in the adsorption rate of BLES. This is more clearly shown in Fig. 4, which shows the surface tension recorded immediately after drop formation (t = 0 s) and after 10 s of adsorption (t = 10 s). The ability of PEG to enhance the adsorption of a low concentration BLES was also found to depend on its molecular weight. Fig. 5 shows the adsorption isotherms of BLES in mixture with different molecular weight of PEG at 10 mg/mL. There appears to be an optimal range of molecular weight at which PEG is able to enhance the performance of BLES at the chosen PEG concentration of 10 mg/mL. It should be noted that with PEO of MW
Fig. 5. Effect of PEG molecular weight (10 mg/mL) on the surface tension of BLES.
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Table 1 Surface tension of BLES + PEG (30 mg/mL) attained as a function of molecular weight at time 0 and after 10 s of adsorption Molecular weight of PEG
Surface tension (mJ/m2 ) t =0
3350 8000 10000 20000 35000
33.7 33.4 32.7 33.4 33.0
± ± ± ± ±
t = 10 s 0.7 2.6 0.6 0.1 1.0
27.8 28.7 28.4 28.6 28.9
± ± ± ± ±
0.7 1.2 0.7 0.1 0.1
300,000 the surface tension remained high and almost unchanged at about 57 mJ/m2 . This PEG concentration of 10 mg/mL was chosen since for each molecular weight, there is a minimum concentration of PEG that optimizes the performance of BLES (see Fig. 3). At higher concentration of each molecular weight, the effect of PEG concentration on the performance of BLES would mask the effect of molecular weight. This relationship is shown in Table 1, which reports the results when using a constant PEG concentration of 30 mg/mL for each molecular weight studied in Fig. 5. PEO (MW 300,000) is omitted from Table 1 since at 30 mg/mL, the polymer becomes so difficult to dissolve in salt solution that the experiment could not be readily performed. It is concluded that PEG enhances the adsorption of BLES at low concentration. The effect is simultaneously dependent on both the concentration and molecular weight of PEG. Thus, the optimum range of concentration and/or molecular weight of PEG for film formation can not be determined independent of each other. The key findings of this study are visualized in Fig. 6a–c which show pendant drop images of BLES only at 0.5 mg/mL, PEG only (MW 10,000) at 28 mg/mL, and BLES + PEG (28 mg/mL, MW 10,000) after 10 s of adsorption. The round drop shapes of BLES and PEG shown in Fig. 6a and b indicate high surface tension and the values obtained from ADSA are 68.7 ± 0.8 and 58.2 ± 0.2 mJ/m2 , respectively. Fig. 6c shows a drop image of BLES + PEG (28 mg/mL, MW 10,000). The drop shape has an elongated profile that contrasts with the high surface tension profiles of BLES and PEG in Fig. 6a and b. The measured surface tension corresponding to Fig. 6c is 27.9 ± 0.1 mJ/m2 .
4. Discussions BLES is a complex mixture of phospholipids, SP-B and SP-C. The phospholipids interact with the surfactant proteins in order to produce the rapid adsorption rates which are observed in the bulk adsorption studies of BLES [28]. The performance of BLES is compromised significantly at low concentrations compared to the natural extract at the same
Fig. 6. Pendant drop images of (a) BLES (0.5 mg/mL), (b) PEG (28 mg/mL, MW 10,000), and (c) BLES (0.5 mg/mL) + PEG (28 mg/mL, MW 10,000) after 10 s of adsorption.
concentration, as was shown in Fig. 2 and in agreement with [20]. Previous studies have shown that larger aggregates of lung surfactant have greater ability to lower surface tension than their smaller counterparts [15]. Hence, it is postulated that the surface activity of BLES at low concentration may be improved if formation of larger aggregates can be induced. PEG is by far the most commonly used agent for cell fusion. Theories regarding its efficacy have been described in the literature [22,23,30]. Studies on the PEG-induced aggregation of phospholipid vesicles have shown that the underly-
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ing mechanism involves the restructuring of water molecules around the hydration sphere of the vesicles due to the high affinity of the polymer for water. Water molecules interact with PEG through hydrogen bonding due to the presence of polyether oxygen along the backbone of the polymer. Consequently a depletion attraction force can be induced as was first described by Asakura and Oosawa [31] for colloidal systems in the presence of polymers. These authors explained that in any colloidal system, addition of water-soluble polymer causes disruption of the osmotic balance in the system. Consequently, an attractive force, referred to as depletion attraction, is induced and this causes colloid particles to aggregate. The magnitude of these forces have been directly measured by Kuhl et al. [23] in phospholipids in which vesicle aggregation was induced by the addition of PEG. Although depletion attraction was studied on pure phospholipid vesicle systems [22,23], the authors provide evidence in their study suggesting that depletion attraction may be responsible for the increased adsorption rate in our BLES–PEG systems. The work of Meyuhas et al. [22] showed that aggregation of phospholipid vesicles (made from sonicated egg phosphatidylcholine) is induced when dextran or PEG is added at their respective overlap concentration. These authors studied the aggregation of phosphatidylcholine (PC) vesicles by measuring turbidity of both large and small PC unilamellar vesicles. They found that at a PC concentration of 1 mM or higher the threshold concentration required to induce aggregation always occurred at the overlap concentration of the added polymers. At this overlap concentration, the polymer chains begin to interact and form entanglements, which would allow them to trap more water molecules than in their extended conformation [22,23]. As the molecular weight of polymer increased, the threshold concentration required for aggregation decreased accordingly. The authors confirmed the findings from their turbidity measurements in their dialysis experiment. By measuring the osmotically driven shrinkage of dialysis bags, both dextran and PEG were able to induce size growth in the vesicles. At much higher polymer concentrations, the size growth of the PC vesicles was irreversible. Kuhl et al. [23] performed similar studies to Meyuhas et al. [22] between phospholipid bilayers and PEG. The authors studied the nature of bilayer–PEG interaction using a surface force apparatus, 31 P NMR, and quasi-elastic light scattering techniques. The molecular volume of the polymer increases as it binds more water and becomes excluded from the spaces between the lipid bilayers. As a result of this concentration difference inside and outside the gap between the lipid bilayers, an osmotic force is induced. The surface force profiles obtained by the authors showed that such an osmotic attractive force is much stronger than the van der Waals force of interaction. From the various techniques used in their study, they confirmed that PEG of molecular weight 8000 to 10,000 was effective in causing vesicle aggregation. In the surface force measurement, these authors found
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that low molecular weight PEG (MW < 1000) is ineffective in generating a depletion layer due to the small size of the polymer molecules. Surface force profiles of bilayers measured in aqueous solution of higher PEG molecular weight (MW 20,000) showed only a repulsive force. The authors suggest that the high molecular weight PEG may adsorb on the surfaces of the bilayer, or that the slow mobility of the polymer chains may cause them to be trapped between the surfaces of the bilayer. Thus the repulsive force measured in their experiment may be due to entropic confinement and compression of the trapped polymer chains [23]. Evans and Needham have performed adhesion experiment on giant phospholipid vesicles [32]. These authors used synthetic phospholipids of 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and dioleolyl phosphatidylglycerol (DOPG), and digalactosyl diacylglycerol (DGDG), a natural lipid. They have found that PEG and dextran both enhance the adhesion of the vesicles to each other in their micropipette aspiration measurement. The attraction between phospholipid vesicles was three times greater in PEG solution than in dextran solution at the same volume fraction of the polymers [32]. Their findings are in close agreement with those of Meyuhas et al. [22] and Kuhl et al. [23] using different techniques as discussed above. Accordingly, Evans and Needham [32] developed a depletion force theory, the self-consistent mean field theory (SCMF), to explain their findings in the presence of non-adsorbing polymers. The SCMF theory also predicted the absence of polymer in the gap between vesicles of neutral lipids at equilibrium separation [32]. The presence of pure water in the gap induces an attractive osmotic force between the vesicle surfaces that causes them to aggregate [23,32]. Although depletion attraction had only been studied on pure phospholipid vesicle systems in the literature [22,23,32], the studies strongly suggest that depletion attraction may be responsible for the increased adsorption rate in our BLES–PEG systems. However, it is not known if PEG has an effect on the phospholipid vesicles in our BLES–PEG system. It is suggested that in our experiments, aggregation of lung surfactant may have resulted because of a depletion attraction force induced by osmotic imbalance as a result of the presence of PEG. The large surfactant aggregates which are formed would presumably aid in the adsorption of BLES in agreement with previous observations [15,22,23]. Larger particles were indeed observed in our pendant drops when PEG was added to BLES at increasing concentrations (>20 mg/mL). The size of these particles was not measured in our study. Similar observations were seen in captive bubble experiments of BLES + PEG mixture when PEG (MW 10,000) was added at 28 mg/mL and 50 mg/mL [36]. Fig. 7a and b are captive bubble images of BLES (0.5 mg/mL) and BLES (0.5 mg/mL) + PEG (28 mg/mL, MW 10,000), respectively. A comparison of these two images clearly show large par-
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Fig. 7. A comparison of captive bubble images of (a) BLES (0.5 mg/mL), and (b) BLES (0.5 mg/mL) + PEG (28 mg/mL, MW 10,000) clearly show the formation of aggregates when PEG is added to BLES.
ticles in the subphase of Fig. 7b, i.e. in the case of addition of PEG. Fig. 3 shows that when PEG of MW 10,000 was added to BLES at 28 mg/mL, the adsorption rate of BLES was increased significantly and the resulting isotherm becomes comparable to that of natural lung surfactant. The overlap concentration of PEG (MW 10,000) was measured by Kuhl et al. using dynamic light scattering and is reported to be 28 mg/mL [23]. According to Meyuhas et al. [22], aggregation of PC vesicles always occurred at the overlap concentration. As shown in Fig. 4, the minimum concentration of PEG (MW 10,000) required to observe improvement in the adsorption rate of BLES is about 15 mg/mL. Although the adsorption of BLES enhanced by PEG (MW 10,000) does not appear to maximize at the overlap concentration of 28 mg/mL measured by Kuhl et al. [23], it is noted that in our more complex natural system, the addition of PEG at concentrations lower than the overlap concentration may be sufficient to promote or increase
the interaction between phospholipids and surfactant proteins (SP-B and SP-C) in BLES to improve its adsorption rate. The effect of molecular weight of PEG on the performance of BLES is also reported in this study. It is found that moderately high molecular weight (i.e. MW > 8000) best enhances adsorption of BLES to the air–liquid interface. According to the literature [23,36], PEG does not induce aggregation in phospholipid vesicles at MW 20,000. Rupert et al. [36] suggests that at MW of 20,000 the interaction between polymer chains is presumably favoured over interactions between phospholipids or water. Consequently the surfaces of phospholipid vesicles become covered with PEG. As discussed above, Kuhl et al. [23] also suggested that PEG adsorbs onto surfaces of phospholipid vesicles. The vesicles become sterically stabilized and thus impede the close approach of phospholipid vesicles [36]. However, improvement in the adsorption of BLES persisted for PEG at MW 35,000 (Fig. 5). A recent publication by Platikanov et al. [37] showed that phosphatidylcholine vesicles take on different bilayer arrangement and are packed differently when prepared under different conditions and subject to different environment. Hence, the discrepancy between our results and the literature is not unexpected since BLES is a more complex system than those previously studied in the literature [22,23,31,36]. The arrangement of the lung surfactant aggregates at the interface and in the bulk would be very different from those vesicle systems studied by Meyuhas et al. [22], Kuhl et al. [23], and Evans and Needham [32]. The adsorption of BLES was hindered significantly at very high molecular weight of 300,000. At such a high molecular weight, the polymer itself has a very large molecular volume and the bulky polymer chains may well be trapped between lung surfactant aggregates due to its slow diffusion and mobility [23]. This agrees with the near constant surface tension for BLES with PEO of MW 300,000 (Fig. 5). An experiment with extended adsorption period of 500 s was performed for the BLES + PEG (MW 300,000). During the first 300–400 s surface tension remained constant near 57–59 mJ/m2 . The surface tension then decreased steadily and required a further 100 s to reach equilibrium. At low molecular weight (i.e. MW < 3350), a much higher concentration of PEG would be required to achieve overlap concentration and induce a strong enough depletion attraction force to cause appreciable aggregation of surfactant [23]. However, at these higher concentrations, viscosity of the BLES–PEG mixture is increased and this would hinder in vitro experiment. (Viscosity effects on the rate of drop formation, however, were not the focus of this study and their significance on the adsorption of lung surfactant film is unknown). High viscosity also hinders any in vivo application (Our previous studies in rabbit were unsuccessful using BLES + dextran mixture, the main problem being that dextran was used at such a high concentration that the mixture became too viscous to be instilled into the lungs of the rabbit.). Our experimental results
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suggest an optimum range of molecular weight from 8000 to 35,000 at which the performance of BLES is enhanced. Within this optimum molecular range, the improvement in the adsorption of BLES is dependent on the concentration used (Fig. 5). As the molecular weight of PEG increased, the concentration required to enhance the adsorption of BLES (0.5 mg/mL) decreased correspondingly, which is in agreement with findings of Meyuhas et al. [22]. This result is not unexpected if the overlap concentration plays a significant role [33]. Hence, an optimum concentration depends on molecular weight. This inter-dependence will provide flexibility when other parameters need to be considered, such as the effect of PEG on the gas transfer properties across the surfactant film [34] and the use of PEG to reverse protein inactivation of surfactant (i.e. albumin) [16,17,35]. Similar results were also observed in preliminary pendant drop studies using poly(vinyl pyrrolidone) (PVP) and dextran in mixture with BLES suspension. We believe that addition of PVP and dextran also induces depletion attraction in BLES suspensions. However, the patterns in such systems are more complicated than those observed in BLES–PEG systems.
5. Conclusions From this study, it has been shown that PEG is able to enhance the rate of surface film formation of a low concentration BLES. The effect of PEG on the performance of BLES is simultaneously dependent on both its concentration and molecular weight. We have proposed that depletion attraction may likely be the underlying mechanism for the observed effect. It is hoped that this work will shed light on the possibility of reducing the clinical concentration in surfactant replacement therapy and thus reduce the cost of treatment. This work will also help understand the reversibility of inactivated lung surfactant systems by plasma proteins, such as albumin and fibrinogen by nonionic polymers.
Acknowledgements This work was supported by a grant from the Canadian Institute of Health Research (grant MOP38037) and Ontario Graduate Scholarship for Science and Technology (L.M.Y. Yu). We wish to thank BLES Biochemicals Inc. for their generous donation of the BLES and natural lung surfactant samples.
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