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University of Toronto L L University of Toronto C C F F E E Laboratory for Applied Surface Thermodynamics Laboratory of Colloid and Formulation Engineering predicting lung mechanics from dynamic surface tension evaluations of lung surfactants Edgar Acosta, Z. Policova, S. Saad , A. W. Neumann. February 22, 2012 Workshop on Surfactant Driven Thin Film Flows 1 to be held at the Fields Institute

Lung Surfactants and Lung Physiology Upon compression (exhalation) the lung surfactants produce a near zero surface tension that reduce the pressure difference between the smaller alveoli and the airways Laplace Pressure: ΔP ~ γ /R (R, radius of the alveolus) 2

The Engineering Approach Surfactant chemistry and In vitro In vivo additives 35 (c) 2 25 15 Humid air 5 m m S n o n a u e e s c J / , t r f i 0 0.8 1.0 Relative Area A r Surfactant and lung mechanics 3

Composition of lung surfactants •Phospholipids ~ 85-90% •Mainly phostphatidyl cholines (zwitterionic), and particularly dipalmitoyl phosphatidyl cholines (DPPC) to give solid-like properties. •Phosphatidyl glycerols (anionic) that impart appropriate dynamic folding/unfolding properties to the surfactant film •Neutral Lipids ~ 1-5% (cholesterol) •Proteins ~ 5-10% •Surfactant Proteins A and D => anionic, hydrophilic •Surfactant Proteins B and C => cationic, hydrophobic •Surfactant Protein B is essential 4

Surfactant Evaluation => Compression isotherms “film collapse” “solid” Elasticity ε=dγ/dln(A) = -dπ/dln(A) “condensed” “Ideal gas” Molecular area = 1/surface Wilhelmy Balance concentration=1/ Γ Surface pressure = surface tension of the pure liquid ( γ 0 )- surface tension ( γ )

Evaluation of Surfactant Dynamics Constrain Sessile Drop Captive Bubble Pendant Drop

Dynamic Evaluation => adsorption and relaxation effects 1 Relative area A/Ao 0.9 Adsorption and relaxation 0.8 effects depend on: 0.7 Compression dynamics 0 0.5 1 1.5 2 normalized time, t/cycle period 35 Environment Adsorption Adsorption Surface tension, mJ/m^2 10 s/cycle 30 25 Surfactant composition 20 15 10 3 s/cycle 5 0 0 0.5 1 1.5 2 7 Normalized time, t/cycle period Relaxation

Compression Relaxation Model γ eq Equilibrium surface tension γ min,c Minimum surface tension at collapse k a , k r First order adsorption and relaxation constants ε c , ε e Elasticity during compression and expansion 8

Compression Relaxation Model Typical fit of CRM model Parameters for specific scenarios Formulation ε c , ε e , k a , s - k r , γ min , γ eq , mJ/m 2 mJ/m 2 s -1 mJ/m 2 mJ/m 2 1 BLES 120 130 2.5 0.0 2 22 BLES-albumin 72 78 1.5 2.5 20 25 Formulation ε c , ε e , k a , s - k r , γ min , γ eq , mJ/m 2 mJ/m 2 s -1 mJ/m 2 mJ/m 2 1 9

CRM - Pressure-Volume Model Ventilator waveform: V=f(t) CRM parameters: γ eq , γ min,c , k a , k r , ε c , ε e γ =f(A, t) Prokop et al. (1999) A =f(V, γ) Smith et al. (1986) P =P tissue (V) +P capillary (γ) 10

Tissue contribution to lung pressure 100 75 % Total lung capacity (TLC) 50 Dixon et al, 2009 (rats) 25 Smith and Stamenovic, 1987 (rabbits) Simon et al, 2010 (mice) 0 0 5 10 15 20 Recoil pressure - tissue, cm H2O 11

CRM-PV algorithm (P) t = 0.027γ t + a/(b-V t ) A t = 0.0324V t + 1.734 - 0.707γ t 0.366 Save γt ,Vt, (P)t , At V t+δt from ventilation function γ a = γ t A t +δt = 0.0324V t+δt + 1.734 - 0.707γ a γ a = γ t+δt 0.366 δ −   dA A A =   + t t t no δ  dt  t t γ t+δt = γ a ? If (dA/dt) t <0, ε= ε c , else ε= ε e yes If γ a < γ eq , k=k r , else k=k a t= t+δt γ ε ( )  d   dA  no = + γ − γ     k eq a  dt  A  dt  t t t > t max ? γ n = γ t +(dγ/dt) t δt If γ n < γ min , γ t+δt = γ min , else γ t+δt = γ n yes 12 End

CRM – PV –rabbit model CRM-PV prediction –BLES Bachofen et al. (1987) 100 Compression 100 Compression % Total lung capacity (TLC) % Total lung capacity 80 75 (TLC) Expansion Expansion 60 50 25 40 0 10 20 30 40 0 10 20 30 Surface tension, mJ/m2 Pressure, cm H2O 3 Compression Lung area, m2 2 Expansion 1 13 40 60 80 100 % Total lung capacity (TLC)

CRM – PV –mice model 100 80 60 Experimental % C T T CRM-PV c a p a c g n L u a o y ) ( t t i l l 40 0 15 30 Pressure, cm H 2 O Experiments of Allen and Bates 100 0.75 % Total lung capacity (TLC) CRM-PV CONTROL BLES Volume, ml 80 0.5 60 0.25 CRM-PV HCl injury BLES - model of ALBUMIN ARDS 40 0 14 0 10 20 30 -5 5 15 25 Pressure, cm H2O Pressure, cm H2O

CRM – PV, dynamic properties 70 60 50 40 30 20 m m O H 10 n e 2 a a E c / c s t l , l 0 0 1 2 3 4 Lung volumes of air ventilated CRM-PV prediction of lung elastance (ΔP/ ΔV) – left – and experimental values –right - using variable ventilation *** low minimum surface tension is not always important *** 15 Fast surfactant adsorption is essential

Conclusions 1 – In vitro – in vivo correlations are closer to reality => integrated approach to design surfactant therapies 2 – Much to be learned of the physics of surfactant membranes at the molecular scale 3 – A combination of strategies: surfactant additives, method of ventilation may be used in alternative therapies 4 – Need to introduce flow-driven pressure drop 5 – Need to incorporate surfactant spreading 16

Acknowledgements • Canada Institute for Health Research (CIHR) • BLES Biochemicals (London, Ontario) 17

Surfactant membrane conformations air water 18

Compression Relaxation Model % Area reduction (compression) Elasticity slightly improves with 19 surfactant concentration

Compression Relaxation Model % Area reduction (compression) Relaxation constant is not a function of 20 surfactant concentration

Compression Relaxation Model BLES 2 mg/ml BLES 27 mg/ml % Area reduction (compression) Adsorption constant tends to increase 21 with surfactant concentration

Cationic Surfactant Additives Reasoning: Air Cationic additives can be use to induce flocculation and + + + larger, more active, surfactant + + + aggregates Liquid + + Anchors SP-B, a cationic protein, is essential to life The anionic headgroup of phosphatidyl glycerols seems NH 3 + to easily hydrate, weakening Chitosan the surfactant film 22

Effect of Chitosan on BLES Optimal molar ratio of number of cationic groups in polymer to anionic groups in lipids Addition of chitosan, up to a certain ratio, induce larger aggregates to form, also improving the surface activity 23

Effect of Chitosan on BLES 200 Film collapse Elasticity, mJ/m2 150 100 50 0 0 0 0.5 0.5 0.5 1 1 1 1.5 1.5 1.5 2 2 2 2.5 2.5 2.5 n+/n- binding ratio Cationic surfactant additives can improve the elasticity of exogenous surfactant and reduce the relaxation constant

Cationic additives may be the answer to ARDS Serum threshold dosage, μl/ml 160 140 120 100 80 60 40 Optimized BLES + Chitosan 20 BLES only 0 0 2 4 6 8 10 Lung surfactant (BLES) concentration, mg/L 550 μl/ml serum simulates the high protein content in the lungs of ARDS patients. Even a high exogenous surfactant 25 concentration ~ 27 mg/ml BLES would not work

Effect of cationic peptides Polymyxin B 24 2mg/ml BLES +additive NH 3+ NH 3+ Minimum surface tension , mJ/m2 BLES only 18 0.20 mg/ml Polylysine 50kDa 0.10 mg/ml Polymxyin B Complete lung surfactant 12 NH 3+ Physiologically active formulations NH 3+ 6 NH 3+ 0 0 250 500 750 Serum content, μl/ml 26