The Mechanism of Chitosan Enhanced Lung Surfactant Adsorption at the Air-Liquid Interface in the Presence of Serum Proteins

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1 Mater. Res. Soc. Symp. Proc. Vol Materials Research Society 1061-MM10-03 The Mechanism of Chitosan Enhanced Lung Surfactant Adsorption at the Air-Liquid Interface in the Presence of Serum Proteins Patrick C Stenger, Omer M Palazoglu, and Joseph A Zasadzinski Department of Chemical Engineering, University of California, Santa Barbara, CA, ABSTRACT Pressure-area isotherms and fluorescence microscopy were used to investigate the impact of chitosan on the competitive adsorption between lung surfactant (LS) and serum proteins at the air-liquid interface. Isotherms demonstrate an optimum chitosan concentration to mediate LS adsorption; higher concentrations actually reduce the amount of LS which can adsorb. Fluorescence microscopy images show the transition from a serum protein to LS-covered interface for the optimum chitosan concentration; this transition goes through a sharply phase separated coexistence region. The results suggest that the cationic chitosan molecules mediate adsorption of the negatively charged LS aggregates by reducing the electrostatic barrier imposed by negatively charged interfacial serum proteins. INTRODUCTION Lung surfactant (LS) is a unique mixture of lipids and proteins that lines the alveolar air-liquid interface and lowers the surface tension in the lungs, thereby insuring negligible work of breathing and uniform lung inflation [1]. The absence of LS due to prematurity leads to Neonatal Respiratory Distress Syndrome (NRDS) which has been successfully treated in developed countries with animal-derived replacement LS [2]. In a related condition, the surface tension control imposed by LS is compromised during Acute Respiratory Distress Syndrome (ARDS) which afflicts 140,000 annually with a 40% mortality rate in the United States [3]. The complex pathogenesis of ARDS includes increased permeability of the alveolar-capillary barrier yielding an influx of blood serum proteins into the bronchial and alveolar fluid [4]. The animalderived replacement LS used to treat NRDS also loses its ability to reduce surface tension and is said to be inactivated when used to treat ARDS [5]. In vitro LS mixed with serum proteins shows an ARDS-like decrease in performance; surfactant inactivation caused by serum protein leakage into the alveoli is one reason why treatment of ARDS with replacement LS is unsuccessful [6]. One possible cause of LS inactivation is the competitive adsorption of surface-active serum proteins (such as albumin) that reduces or even eliminates the normal adsorption of LS to the interface [6]. Albumin is surface-active and has a saturation surface pressure, Π, (Π = γ w -γ; γ w is the surface tension of a clean air-water interface, 72 mn/m, and γ the measured surface tension) that is ~18 mn/m, much lower than the Π 70 (γ near zero) required for proper respiration [6]. This competitive adsorption of albumin to the alveolar air-liquid interface leads to a steric and electrostatic energy barrier to LS adsorption which can lower the rate of LS transport to the interface [7]. Several hydrophilic polymers, such as polyethylene glycol (PEG),

2 hyalaronic acid and chitosan have recently been shown to enhance the ability of replacement LS to resist serum inactivation both in vitro and in vivo [8-10]. Previous reports indicate that much lower chitosan concentrations are necessary to make LS resistant to albumin-induced inactivation than PEG or hyalaronic acid [9]. Chitosan is derived from deacetylated chitin, the skeletal material of invertebrates and has been shown to be biocompatible in numerous applications [11]. In our system, ~80% of the chitosan monomer units have an amine group which is 90% protonated at ph 5.5, making chitosan a highly charged cationic polyelectrolyte. The present study utilizes isotherms and fluorescence microscopy to investigate the effect of chitosan on competitive adsorption between LS and albumin. Our findings yield the first visualization of LS displacing albumin in the presence of chitosan during dynamic cycling. One possible mechanism for chitosan mediated LS adsorption is that it reduces the electrostatic repulsion between the negatively charged LS aggregates in the bulk and negatively charged albumin interfacial film, reversing albumin inactivation. EXPERIMENT Survanta (Abbott Laboratories, Columbus, Ohio), a clinical LS used for treating NRDS, was a generous gift of the Santa Barbara Cottage Hospital nursery. Survanta is an organic extract of minced bovine lungs that contains 80-90% wt. phosphatidylcholine, of which ~70% wt. is saturated dipalmitoylphosphatidylcholine (DPPC), about 10% wt. palmitic acid and < 2% wt. LS specific proteins [12]. There are approximately 5% wt. negatively charged phospholipids including phosphatidylglycerol and phosphatidylserine, making Survanta net anionic. Bovine serum albumin and 75-85% deacetylated chitosan (~ kda) were obtained from Sigma (St. Louis, MO) and used as received. Isotherms were recorded at 20 C in a Langmuir trough equipped with a Wilhelmy plate for surface pressure measurements and a continuous stainless steel ribbon barrier to change the trough area (Nima, Coventry, England). The trough had a surface area of 130 cm 2 and a typical compression/expansion cycle took 8 min (~0.42 cm 2 /sec). Albumin and chitosan were dissolved in the subphase buffer (NaCl 150 mm, CaCl 2 2 mm, NaHCO mm and ph = 7) at the stated concentrations for all experiments. To dissolve chitosan in the saline buffer, the solution ph was reduced to ~2.0 with 1M HCl and then raised to ~5.5 with 1M NaOH. The subphase ph for all experiments was ~5.5. Survanta was diluted in the same buffer to a lipid concentration of 2 mg/ml and was deposited dropwise onto the subphase of the Langmuir trough at the stated total surfactant amounts to initiate each adsorption experiment. A Nikon Optiphot optical microscope (Nikon, Tokyo, Japan) was positioned above the trough with a 10X extra long working distance objective designed for fluorescent light. Full-length movies and individual frames were recorded directly to the computer (Moviestar, Mountain View, CA). Contrast in the images was due to segregation of 1% mol. fluorescent lipid Texas Red-DHPE (Molecular Probes, Eugene, OR) which causes the Survanta monolayer to appear a light gray in images [10]. Larger aggregates of Survanta have significantly more dye and appear bright white, leading to an overall mottled texture for the surfactant film. The albumin was not labeled, does not fluoresce and appears black in the images.

3 RESULTS AND DISCUSSION Figure 1a shows a typical compression-expansion cyclic isotherm for 800 µg of Survanta adsorbing to a clean, saline buffered interface. The isotherm traces over itself on subsequent cycles and on compression exhibits a characteristic shoulder at Π ~42 mn/m and collapse plateau at Π max ~69 mn/m where the film begins to collapse and form cracks and folds [10, 13]. The hysteresis between compression and expansion cycles is typical of Survanta [1] and is due to the partial readsorption of the collapse structures into the monolayer [6]. On a clean interface, reexpanding the interface after monolayer collapse leads to a rapid drop in surface pressure until compression is resumed. There is no significant change in the Survanta isotherms from C [13]. Conversely, when the same amount of Survanta (800 µg) is deposited onto a subphase containing 2 mg/ml albumin (Figure 1b, black curve), the surface pressure does not increase above 40 mn/m even at the smallest trough area after the first cycle. Both the compression and expansion isotherms are not different than that of albumin alone (Figure 1b, red curve). Survanta inactivation under these conditions results from an inability of the LS to adsorb to the interface. Surface Pressure, mn/m Surface Pressure, mn/m a c Survanta Survanta, Albumin, Chitosan mg/ml Trough Area, % b Albumin Only d Survanta + Albumin Survanta, Albumin, Chitosan 0.5 mg/ml Trough Area, % Figure 1 Cyclic isotherms of Survanta on buffered saline subphases of varying composition. (a) 800 µg Survanta on a clean buffered saline subphase. (b) Black curve: 800 µg Survanta deposited onto a saline buffer containing 2 mg/ml albumin. Red curve: The isotherm for the albumin subphase, with no Survanta. (c) 800 µg Survanta on saline buffer containing 2 mg/ml albumin and mg/ml chitosan. (d) 800 µg Survanta on saline buffer containing 2 mg/ml albumin and 0.5 mg/ml chitosan.

4 Subphase concentrations of chitosan ranging from mg/ml were evaluated to determine their effect on reversing the albumin induced inactivation and restoring Survanta to the interface. Chitosan itself is not particularly surface active and only raises the surface pressure ~2 mn/m at a subphase concentration of 0.1 mg/ml. Addition of chitosan to the subphase does not change the albumin isotherm (Figure 1b, red curve), suggesting that chitosan does not alter the albumin interfacial film. Figure 1c shows Survanta (800 µg) deposited onto a subphase containing 2 mg/ml albumin and mg/ml chitosan; the isotherm resembles Survanta on a clean interface (Figure 1a) by the second compression-expansion cycle. At this concentration, chitosan reverses the albumin inactivation; the isotherm exhibits a characteristic shoulder at Π ~42 mn/m and a collapse plateau at Π max ~69 mn/m at trough areas similar to that of Figure 1a. Chitosan concentrations from mg/ml showed the greatest isotherm improvements and represent the optimum concentration for reversing albumin inactivation. In contrast, Figure 1d shows that Survanta (800 µg) deposited onto a subphase containing 2 mg/ml albumin and 0.5 mg/ml chitosan exhibits an isotherm which is intermediate between Survanta (Figure 1a) and Survanta-albumin (Figure 1b). Here the isotherm only reaches a maximum surface pressure of Π ~60 mn/m and the characteristic shoulder at Π ~42 mn/m occurs at a significantly lower trough area than Survanta (~35% vs. ~60%). Comparing Figures 1c and 1d, it is clear that increasing the chitosan concentration above the optimum values has an adverse effect on restoring the Survanta isotherm. Figure 2a shows a fluorescence image of the air-liquid interface after Survanta adsorption on a saline buffer subphase. Survanta (doped with 1 mol% Texas Red-DHPE) adsorbs to the interface as a mixture of monolayers (mottled light gray and dark gray) typical of a phase separated lipid/protein monolayer along with bright, three-dimensional aggregates that appear to be attached to the interface [13, 14]. This characteristic mottled texture is found at all surface pressures from 0 to collapse. In contrast, Figure 2b shows that Survanta films on a subphase containing 2 mg/ml albumin show isolated, out-of-focus bright regions with an overall dark homogeneous background. The Survanta does not form a monolayer at the interface and the surface pressure remains low throughout the compression/expansion cycle (Figure 1b). Figures 2c-h show Survanta deposited onto a subphase containing 2 mg/ml albumin and mg/ml chitosan during successive compression/expansion cycles; Survanta gradually displaces the albumin on the interface. Images from the first cycle at low surface pressure (Figure 2c, Π = 25) show an albumin-covered interface with limited out-of-focus bright spots, indicating that Survanta aggregates approach the albumin covered interface but cannot spread. However, images from the first cycle maximum surface pressure (Figure 2d, Π = 54) show that Survanta has broken through the albumin film; there is coexistence between the Survanta (mottled bright texture) and albumin (black) with a well-defined interface between the materials. The coexistence between the extended (>1000 µm) interfacial domains of Survanta and albumin continues through the second cycle compression (Figure 2e, Π = 43). However, Survanta can maintain a much higher dynamic surface pressure on compression than the albumin. Once sufficient Survanta adsorbs to the interface, it raises the surface pressure high enough (50-60 mn/m) to expel the albumin, resulting in a Survanta-covered interface. Images of the collapse plateau (Figure 2f, Π = 69) show only Survanta and are dominated by the cracks and folds (arrows) typical at monolayer collapse [10, 13]. On the third cycle compression, the mottled Survanta texture similar to Figure 2a is seen exclusively at all surface pressures (Figure 2g, Π = 43) and the system again forms a collapse plateau with the associated cracks and folds (Figure 2h, Π = 69).

5 b c d Cycle 1 Controls a Survanta f g h Cycle 3 Cycle 2 e Albumin Figure 2 Fluorescence images of 800 µg Survanta from the compression part of the isotherm spread at varying subphase compositions. Images are 1023 µm by 789 µm. Contrast in the images was due to segregation of 1% mol. fluorescent lipid Texas Red-DHPE in the Survanta monolayer. The albumin was not labeled and does not fluoresce. (a) Survanta on a clean, buffered subphase at Π = 43 mn/m. (b) Survanta on buffer containing 2 mg/ml albumin at Π = 25 mn/m. The remaining images show Survanta on a subphase containing 2 mg/ml albumin and mg/ml chitosan in successive expansion/compression cycles. First Cycle: (c) Π = 25 mn/m. (d) Π = 54 mn/m. Extended (>1000 µm) immiscible Survanta (mottled gray) and albumin (black) domains coexist on the interface. Second Cycle: (e) Π = 43 mn/m. (f) Π = 69 mn/m. The arrows indicate collapse cracks and folds. Third Cycle: (g) Π = 43 mn/m. (h) Π = 69 mn/m. The arrows indicate collapse cracks and folds. In contrast, when Survanta is deposited onto a subphase containing 2 mg/ml albumin and 0.5 mg/ml chitosan, the albumin is not totally displaced from the interface. The images from the first cycle compression (not shown) also illustrate an albumin-covered interface at low surface pressure and a coexistence of Survanta and albumin on the first expansion. However, the coexistence of Survanta and albumin persists throughout all four cycles, yielding an isotherm which requires significantly more compression to reach the requisite high surface pressures. The high chitosan concentration somehow prevents sufficient Survanta adsorption to raise the surface pressure high enough to completely expel the albumin from the interface. One possible mechanism for chitosan mediated LS adsorption is that it reduces the electrostatic repulsion between the negatively charged LS aggregates in the bulk and negatively charged albumin interfacial film, restoring LS to the interface. Polyelectrolytes frequently adsorb to oppositely charged surfaces, initially neutralizing and eventually reversing surface charge [15]. While the optimum chitosan concentration reduces the electrostatic repulsion due to charge neutralization, charge reversal at high chitosan concentration would slightly increase the electrostatic repulsion and reduce LS adsorption. CONCLUSIONS Isotherms and fluorescence microscopy were used to investigate the effect of chitosan on LS and serum protein competitive adsorption. At the optimum concentration (0.005 mg/ml),

6 chitosan fully reverses albumin inactivation and restores the healthy LS isotherm. Higher chitosan concentrations (0.5 mg/ml) result in an isotherm that is intermediate between Survanta and Survanta-albumin, indicating that increased chitosan adversely impacts LS adsorption. Fluorescence images at the optimum chitosan concentration show a transition from an albumin-covered interface to Survanta-covered interface during dynamic cycling. While Survanta and albumin coexist in distinct phases during the transition, enough Survanta eventually adsorbs expel the albumin from the interface. The isotherm and fluorescence microscopy results are consistent with chitosan reducing the electrostatic repulsion between the negatively charged LS aggregates and negatively charged albumin, mediating LS adsorption to the air-liquid interface. ACKNOWLEDGMENTS Support for this work comes from National Institute of Health Grants HL-66410, HL and the Tobacco Related Disease Research Program 14RT P.C.S. was partially supported by an NSF graduate research fellowship. REFERENCES 1. R. Notter, Lung surfactant: basic science and clinical applications vol New York: Marcel Dekker, G. K. Suresh and R. F. Soll, J Perinatol, 25, S40-S44, (2005). 3. G. D. Rubenfeld, E. Caldwell, E. Peabody, J. Weaver, D. P. Martin, M. Neff, E. J. Stern, and L. D. Hudson, New Engl J Med, 353, , (2005). 4. L. B. Ware and M. A. Matthay, New Engl J Med, 342, , (2000). 5. K. W. Lu, H. W. Taeusch, B. Robertson, J. Goerke, and J. A. Clements, Am J Resp Crit Care, 162, , (2000). 6. H. E. Warriner, J. Ding, A. J. Waring, and J. A. Zasadzinski, Biophys. J., 82, , (2002). 7. J. A. Zasadzinski, T. F. Alig, C. Alonso, J. B. de la Serna, J. Perez-Gil, and H. W. Taeusch, Biophys. J., 89, , (2005). 8. K. W. Lu, J. Goerke, J. A. Clements, and H. W. Taeusch, Pediatr Res, 58, , (2005). 9. Y. Y. Zuo, H. Alolabi, A. Shafiei, N. X. Kang, Z. Policova, P. N. Cox, E. Acosta, M. L. Hair, and A. W. Neumann, Pediatr Res, 60, , (2006). 10. P. C. Stenger and J. A. Zasadzinski, Biophys. J., 92, 3-9, (2007). 11. M. Kumar, R. A. A. Muzzarelli, C. Muzzarelli, H. Sashiwa, and A. J. Domb, Chem Rev, 104, , (2004). 12. A. Braun, P. C. Stenger, H. E. Warriner, J. A. Zasadzinski, K. W. Lu, and H. W. Taeusch, Biophys. J., 93, , (2007). 13. C. Alonso, T. Alig, J. Yoon, F. Bringezu, H. Warriner, and J. A. Zasadzinski, Biophys. J., 87, , (2004). 14. M. M. Lipp, K. Y. C. Lee, J. A. Zasadzinski, and A. J. Waring, Science, 273, , (1996). 15. P. M. Claesson, E. Poptoshev, E. Blomberg, and A. Dedinaite, Adv Colloid Interfac, 114, , (2005).