Nanostructure Changes in Lung Surfactant Monolayers Induced by

Nanostructure Changes in Lung Surfactant Monolayers Induced by Interactions between Palmitoyloleoylphosphatidylglycerol and Surfactant Protein B Junqi Ding, Ivo Doudevski, Heidi E. Warriner, Timothy Alig, and Joseph A. Zasadzinski

What does a lung surfactant do? • • • Forms a monolayer Lowers surface tension upon compression Respreads quickly on expansion Reduces the work of breathing Prevents illnesses of the lungs

Surface Tension • • Tendency of molecules to be pulled toward the center Surface tension of pure water is about 70 m. N/m Surface tension of lung surfactant is near 0 The higher the surface tension, the more the monolayer would like to maintain small area • Low surface tension in monolayer is critical http: //www. quest. arc. nasa. gov

What are the components in LS? • A complex mixture of lipids and proteins – Consists primarily of saturated dipalmitoylphosphatidylcholine (DPPC) – Smaller fractions of unsaturated phosphatidylcholines (PCs) – Anionic phospholipids, such as phospatidylglycerols (PGs) – Anionic lipids, such as palmitic acid (PA) – Neutral components, such as cholesterol – 4 lung surfactant-specific proteins

Lung surfactant proteins • SP-A and SP-D – Larger proteins – Responsible for host defense mechanisms – Aid in transport and recycling of lung surfactant • SP-B and SP-C – Smaller proteins – Intensely hydrophobic – Important to surface activity

Why is this complex mixture so complex? • Conflicting requirements of surfactant • Multiple lipids and proteins are necessary • DPPC alone forms rigid monolayers but can not adsorb or respread quickly • Unsaturated PGs are fluid enough and fast spreading, but can not lower surface tension sufficiently • Monolayer collapse is also a factor in this complexity

Monolayer Collapse • The maximum pressure sustained is set by the instability known as monolayer collapse • Monolayer collapse is the point at which the surfactant breaks • The surfactant must respread quickly in order to continue breathing • Proteins and lipids work together in order to continue with normal breathing

Squeeze-out Theory • Most of the unsaturated anionic lipids in the LS monolayer have a low collapse pressure relative to DPPC • The selective removal of lipids with low collapse pressures occurs as pressure increases – Low collapse pressure components are squeezed out of the monolayer – DPPC continues to lower the surface tension near zero – Squeezed out lipids are reincorporated into the monolayer upon expansion – Surfactant proteins aid in this reincorporation Ka Yee C. Lee, et al. Biophysical Journal 2001, 81, 572 -585

Compression-Expansion. Compression Isotherms of DPPG Monolayer Takamoto, et al. Biophysical Journal 2001, 81, 153 -169

Monolayer Collapse www. nsr. bioeng. washington. edu

Why is it important? • Respiratory distress syndrome (RDS) occurs in premature babies carried less than 34 weeks • The immature lung lacks the necessary surfactant in order to keep their lungs from collapsing • It is a serious lung condition that affects 40, 000 infants in the US each year, resulting in thousands of deaths http: //www. lungusa. org

Not Convinced Yet? ? • Cystic fibrosis (CF) – CF patients have a decrease of SP-A • Since SP-A is responsible for bacterial defense, its loss may increase the possibility of lung infection in patients – Alterations in surfactant lipid composition in patients impairs the surface tension lowering function • Pneumonia – Patients with pneumonia have a reduced PC and PG content, leading to the impairment of the surface tension lowering function – The amount of SP-A is decreased, causing patient host defense properties to lower http: //respiratory-research. com/

Smoke • Smoking alters surfactant compostion and function • Levels of SP-A and SP-D are decreased, which can contribute to the increased incidence of respiratory infections • Cigarette smoke’s nitrites and oxidants can inactivate alpha-1 -proteinase inhibitor, which is responsible for preventing the breakdown of tissue in the lungs, causing negative effects on surfactant function http: //respiratory-research. com/

2 Surfactant Replacement Therapies • Exosurf is a synthetic formulation that contains 80% DPPC with hexadecanol and tyloxapol – DPPC is found in natural LS but the other 2 components are not • Survanta is an extract of bovine lung surfactant that contains SP-B and SP-C and is supplemented with PA, triglycerides, and DPPC – The properties exhibited by Survanta can provide a benchmark for new synthetic replacement surfactants http: //www. sciencedaily. com http: //www. survanta. com

Need for New Surfactant Replacement Therapy • Surfactant replacement therapy has reduced mortality rates by 30%-50% for infants with RDS • Animal sources are difficult – expensive to purify – risk of containing contaminants – lack consistency between batches • Currently, surfactant replacement therapies can not used to treat diseases of the mature lung in adults http: //www. survanta. com Frank Bringezu, et al. Langmuir 2001, 17, 4641 -4648

Research Limitations • Lack of fundamental understanding of: – The roles of the individual components of LS – The way components interact in the monolayer and – Their affect on monolayer collapse and respreading • A better understanding is necessary in order to design new synthetic replacement therapies that can be tailored for treatment of different diseases and illnesses

Nanostructure Changes in Lung Surfactant Monolayers Induced by Interactions between Palmitoyloleoylphosphatidylglycerol and Surfactant Protein B Junqi Ding, Ivo Doudevski, Heidi E. Warriner, Timothy Alig, and Joseph A. Zasadzinski

Abstract • Langmuir isotherms, Brewster angle microscopy (BAM), and Atomic force microscopy AFM will be used to try and replicate some of the properties Survanta exhibits • They will use a synthetic dimeric peptide based on the SP-B protein to determine its possible use in LS replacement therapies • DPPC/POPG/PA monolayers will be studied both before and after the addition of the d. SP-B and compared to the results found with Survanta monolayers

Langmuir Trough for measuring Pressure-Area Isotherms Ising. phys. cwru. edu/surfactants/measurement. html

Pressure-Area Isotherm www. abo. fi/fak/mnf/fysik/mole/LB. html

AFM • Contact mode AFM is used to view samples that are too small to see under the optical microscope • Provides details of the morphology • Tip scans the surface in order to get height profiles

Brewster Angle Microscopy • Uses an argon laser as a light source • Mirror and polarizer are placed between the laser and the trough • Provides light at the Brewster angle (53. 1 o) • Additional polarizer and analyzer improve contrast and determine changes in molecular tilt angle • Contrast is due to local differences in monolayer refractive index caused by molecular density or packing • Useful to follow morphology in absence of any fluorescence dye Henson, S; Meunier, J. Rev. Sci. Instrum. 1991, 62, 936 -939

The Importance of SP-B in LS • SP-B is the only protein necessary for postnatal lung function and survival • Possibly responsible for the prevention of squeeze-out • 78 -residue, lipid associating protein • Can the synthetic peptide prove useful as a replacement?

SP-B A: hypothetical structure of native SP-B homodimer B: d. SP-B 1 -25 mimetic peptide C: molecular surface representation N-terminal domain is shown in purple, midsequences are shown in blue, C-termial sequence in green, and the disulfide connectivity in yellow. Ding, et al. Langmuir 2003, 19, 1539 -1550

Survanta Isotherm at o 25 C A plateau occurs around a surface pressure of 40 m. N/m. Ding, et al. Langmuir 2003, 19, 1539 -1550

AFM Images of Survanta Monolayers Before and After Plateau C, E: Surface pressure of 45 m. N/m B, D: Surface pressure of 30 m. N/m Nanosilos are seen in the fluid phase Ding, et al. Langmuir 2003, 19, 1539 -1550

What are These Nanosilos? • Nanosilos are lipid-protein structures from 40 -300 nm in diameter and 5 -8 nm in height • Nanosilos may be a mechanism of stabilizing the unsaturated lipids and SP-B protein in the vicinity of the monolayer for subsequent reincorporation as the monolayer is expanded • Are they only an intrinsic feature of Survanta? • Do they only occur above the plateau? • Is it possible to replicate these nanosilos and determine what causes their formation?

Isotherms of DPPC/POPG/PA Ding, et al. Langmuir 2003, 19, 1539 -1550

Isotherms of DPPG: POPG Monolayers Takamoto, et al. Biophysical Journal 2001, 81, 153 -169

Isotherms of DPPC/POPG/PA with d. SP-B 1 -25 Ding, et al. Langmuir 2003, 19, 1539 -1550

BAM Images of MA Increasing POPG content Increasing DPPC content Ding, et al. Langmuir 2003, 19, 1539 -1550

BAM Images of MB Increasing POPG content Increasing DPPC content Ding, et al. Langmuir 2003, 19, 1539 -1550

AFM Images Of MA 2 and MB 2 MA 2 at 30 m. N/m at 25 o. C MA 2: (DPPC/POPG/PA=50/40/8) MA 2 at 40 m. N/m at 25 o. C MB 2 at 30 m. N/m at 25 o. C MB 2: (DPPC/POPG/PA/d. SP-B 1 -25=50/40/8/10) Ding, et al. Langmuir 2003, 19, 1539 -1550

AFM of DPPC/POPG/PA/d. SP-B 1 -25 Mixtures Deposited at 40 m. N/m at 25 o. C 50/40/8/10 60/30/8/10 70/20/8/10 Ding, et al. Langmuir 2003, 19, 1539 -1550 80/10/8/10

AFM of Various Lipid Mixtures with d. SP-B 1 -25 DPPC/PA/d. SP-B 1 -25 75/8/10 DPPC/POPG/d. SP-B 1 -25 75/15/10 PA/d. SP-B 1 -25 Ding, et al. Langmuir 2003, 19, 1539 -1550 DPPC/d. SP-B 1 -25 POPG/d. SP-B 1 -25

AFM of DPPC/POPG/PA (75/15/8) with varying d. SP-B 1 -25 0% d-SP-B 1 -25 2. 5% d-SP-B 1 -25 7. 5% d-SP-B 1 -25 10% d-SP-B 1 -25 Ding, et al. Langmuir 2003, 19, 1539 -1550

AFM Images of DPPC/POPG/PA/d. SPB 1 -25 (75/15/8/10) on Freshly Cleaved Mica and Oxidized Silica Wafers Ding, et al. Langmuir 2003, 19, 1539 -1550

Discussion • Individual components of LS are either good at lowering surface tension or fluidizing the monolayer, no single lipid or protein exhibits both properties • Current thought is that lipids and proteins that are squeezed out from the monolayer occupy a “surface-associated reservoir” near the interface – From AFM images , it appears that nanosilos are part of this surface-associated reservoir

Conclusions • Plateaus in the isotherms occur only for SPB containing monolayers and the extent of the plateaus depend on the concentration of POPG • Nanosilos are present above the plateau pressure in monolayers containing SP-B and POPG • Nanosilos are present in both Survanta and the model synthetic surfactant with the SP-B peptide

Conclusions Cont’d • If POPG is squeezed out from the monolayer without being held in a nanosilo, it is likely that it does not reincorporate on expansion • Nanosilos retain both protein and fluid lipid in the immediate vicinity of the monolayer at high surface pressure and allows both molecules to reincorporate in the monolayer at a lower pressure • Model lung surfactant mixture and synthetic peptide capture the morphologies present in the bovine extract Survanta that contains the native SP -B