18256 DOI: 10.1021/la103118d Langmuir 2010, 26(23), 18256–18265Published on Web 11/04/2010

pubs.acs.org/Langmuir

© 2010 American Chemical Society

Fluorocarbon-Hybrid Pulmonary Surfactants for Replacement

Therapy - A Langmuir Monolayer Study

Hiromichi Nakahara,† Sannamu Lee,† Marie Pierre Krafft,‡ and Osamu Shibata*,†

†Department of Biophysical Chemistry, Faculty of Pharmaceutical Sciences, Nagasaki International University,2825-7 Huis Ten Bosch, Sasebo, Nagasaki 859-3298, Japan, and ‡Syst�emes Organis�es Fluor�es �a Finalit�es

Th�erapeutiques (SOFFT), Universit�e de Strasbourg, Institut Charles Sadron (CNRS, UPR 22), 23 rue du Loess,BP 84047, 67034 Strasbourg Cedex 2, France

Received August 5, 2010. Revised Manuscript Received October 16, 2010

Effective additives to pulmonary surfactant (PS) preparations for therapy of respiratory distress syndrome (RDS) arebeing intensively sought.We report here the investigation of the effects of partially fluorinated amphiphiles (PFA) on thesurface behavior of a model PS formulation. When small amounts of a partially fluorinated alcohol C8F17CmH2mOH(F8HmOH,m=5and 11) are added to the PSmodel preparation (a dipalmitoylphosphatidylcholine (DPPC)/Hel 13-5peptide mixture) considered here, the effectiveness of the latter in in vitro pulmonary functions is enhanced. Themechanism for the improved efficacy depends on the hydrophobic chain length of the added PFAmolecules. The shorterPFA, F8H5OH, when incorporated in the monolayer of the PS model preparation, promotes a disordered liquid-expanded (LE) phase upon lateral compression (fluidization). In contrast, the addition of the longer PFA, F8H11OH,reduces the disorderedLE/ordered liquid-condensed (LC) phase transition pressure and promotes the growth of ordereddomains (solidification). Furthermore, compression-expansion cycles suggest that F8H5OH, when incorporated in thePS model preparation, undergoes an irreversible elimination into the subphase, whereas F8H11OH enhances thesqueeze-out phenomenon of the SP-B mimicking peptide, which is important in pulmonary functions and is related tothe formation of a solid-like monolayer at the surface and of a surface reservoir just below the surface. F8H11OHparticularly reinforces the effectiveness of DPPC in terms of minimum reachable surface tension, and of preservation ofthe integrated hysteresis area between compression and expansion isotherms, the two latter parameters being generallyaccepted indices for assessing PS efficacy. We suggest that PFA amphiphiles may be useful potential additives forsynthetic PS preparations destined for treatment of RDS in premature infants and in adults.

1. Introduction

Alveolus surfaces of mammalian lungs are covered with asurface-activematerial called pulmonary surfactant (PS). Specificroles of PS are roughly classified into two important categories:biophysical activity across the air-alveolar fluid interface andimmunological barrier against pathogens. These functions havebeen summarized in review articles.1-5 The interfacial and bio-physical PS functions include prevention of alveolar collapses atthe end of expiration and minimization of the work of breathingby reducing surface tension at the alveolar surface during arespiratory respiration.6 These functions are achieved by lipidmonolayers primarily dipalmitoylphosphatidylcholine (DPPC)and bilayer or multilayer structures (surface-associatedreservoirs) that are closely attached to the monolayers. PS defi-ciency causes neonatal respiratory distress syndrome (NRDS) inpremature infants, whereas lung injury often ends in acute RDS(ARDS) in children and adults. Surfactant preparations based onbovine or porcine lung extracts are commonly administered to

NRDS patients for therapy.7,8 Although these preparations areeffective, they carry a risk of infection and involve costly purifica-tion procedures in order to achieve batch-to-batch reproducibility.Therefore, preparations made of synthetic components andproteins or peptides are desirable for clinical surfactantreplacement therapy for NRDS and ARDS.9-13

Several additives to PS formulations have been considered forrestoration of pulmonary functions that have been inactivated byserum proteins such as albumin. Polymyxin B/PS mixtures haveincreased resistance to inactivation by meconium.14,15 Further-more, several hydrophilic polymers such as dextran, polyethyleneglycol, and hyaluronan enhance the ability of clinical PS prep-arations to resist inactivation by serum proteins.16-22 However,these polymers (especially the nonionic polymers) need to be

*Corresponding author. E-mail: wosamu@niu.ac.jp. URL: http://www.niu.ac.jp/∼pharm1/lab/physchem/indexenglish.html.(1) P�erez-Gil, J.; Keough,K.M.W.Biochim. Biophys. Acta 1998, 1408, 203–217.(2) Batenburg, J. J.; Haagsman, H. P. Prog. Lipid Res. 1998, 37, 235–276.(3) Cochrane, C. G. Am. J. Physiol. 2005, 288, L608–L609.(4) P�erez-Gil, J. Biochim. Biophys. Acta 2008, 1778, 1676–1695.(5) Notter, R. H. Lung surfactants: Basic science and clinical applications, 149th

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formulated at relatively high concentrations, which results inviscous solutions that hinder instillation to the patients.23

Recently, chitosan has been investigated as a PS additive.24-26

The resistance of chitosan/PS mixture to albumin-induced in-activationwas found to bemore effective than that induced by theabove-mentioned polymers.24

PS films show specific changes in fluidity and rigidity duringcompression and expansion of alveolar surfaces. Film rigidity isensured by dipalmitoylphosphatidylcholine (DPPC), which is amajor component of native PS extracts. However, DPPC alonecannot fulfill PS functions due to slow adsorption and poorrespreading on the surface. Partially fluorinated amphiphile(PFA) display unique features including high gas-dissolvingcapacity, low surface tension, and high fluidity and have triggerednumerous studies for applications in diagnosis and therapy.27-29

Although highly fluorinated compounds tend to accumulate in thehuman body and the environment, amphiphiles with short fluor-ocarbon chains (nFe 8) are acceptable in the clinical field. Indeed,perfluorooctyl bromide (PFOB, nF= 8) has been investigated foroxygen delivery and showed short organ retention.30,31 We havepreviously reported that certain partially fluorinated alcohols andderivatives of phosphocholineswith an eight-carbon fluorocarbonchain achieve a highly effective fluidizing effect on DPPC mono-layers and improve the stability of the monolayer at high surfacepressures.32-34 Such fluidization prevents the DPPC monolayerfrom crystallizing during compression and hence is expected toenhance the respreading of PSpreparations.Krafft and co-workershave recently examined the fluidization of DPPC monolayersutilizing fluorocarbon (FC) gases according to anovel technique inwhich the inspired air would be saturated by the FC.35-37 It wasfound that the fluorocarbon gases effectively inhibit the formationof the semicrystalline (liquid-condensed) DPPC domains andfacilitate the respreading of the DPPC monolayer. Moreover,gaseous PFOB promotes the respreading of a DPPC monolayerduring compression and expansion on an albumin-supplemented

subphase,38 suggesting potential usefulness of such fluorocarbonsin ARDS treatments.

In this paper we suggest, as a new approach to PS substituteadditives, the use of a hybrid-type PS formulation that incorpo-rates PFAmolecules to improve the efficiency of the PS prepara-tion for treatment of NRDS. The primary goal of the study is toevaluate the effect of partially fluorinated alcohols on modelPS preparations consisting of DPPC with and without a PSmimicking peptide (Hel 13-5)9,39 using the monolayer techni-que. Since PS forms monolayer films at the alveolar surface, itmake sense to study their surface properties using a Langmuirmonolayer model. (perfluorooctyl)pentanol (C8F17C5H10OH,F8H5OH) and (perfluorooctyl)undecanol (C8F17C11H22OH,F8H11OH), which both have a C8F17 chain, were selectedas the PFA molecules, because their molecular structure isrelatively simple and because they have thoroughly been inves-tigated previously.34,40,41 In order to understand the basic inter-facial behavior of the PS preparations used here, the surfacepressure-molecular area (π-A) and surface potential-area(ΔV-A) isotherm were systematically measured under physio-logical pH and ionic strength conditions.9 Phase and morphologyvariations of the monolayer were visualized using fluorescencemicroscopy (FM) and atomic force microscopy (AFM). Further-more,we investigated hysteresis of theπ-A andΔV-A isotherms,where the monolayer was alternatively compressed and expandedrepeatedly tomimic the biophysical situation found at the alveolarsurface during breathing, although the dynamics are somewhatdifferent.

2. Materials and Methods

2.1. Materials. The Hel 13-5 (NH2-KLLKLLLKLWLK-LLKLLL-COOH, seeFigureS1(A) in theSupporting Information)peptidewas synthesizedby theFmoc (9-fluorenylmethoxycarbonyl)technique and purified with reverse-phase HPLC as described inthe literature.42 More detailed procedures for the synthesis,purification, and analysis of Hel 13-5 were reported previ-ously.43,44 (Perfluorooctyl)pentanol (F8H5OH) and (perfluorooctyl)-undecanol (F8H11OH) were synthesized as reported previously (seeFigure S1(B)).45 L-R-Dipalmitoylphosphatidylcholine (DPPC;purity>99%) and the fluorescent probe, 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospho-choline (NBD-PC), were obtained from Avanti Polar Lipids, Inc.(Alabaster, AL) and were used without further purification.n-Hexane (>99.5%) and ethanol (>99.5%)were used as spread-ing solvents and were purchased from Cica-Merck (Uvasol,Tokyo, Japan) and nacalai tesque (Koto, Japan), respectively.Tris(hydroxymethyl) aminomethane (Tris) and acetic acid (HAc)of guaranteed reagent grade for the preparation of the subphasewere obtained from nacalai tesque. Sodium chloride (nacalaitesque)was roasted at 1023K for 24h to removeall surface-activeorganic impurities. The substrate solution was prepared usingthrice distilled water (the surface tension = 71.96 mN m-1 at298.2 K and the electrical resistivity = 18 MΩ cm).

2.2. Methods. 2.2.1. Surface Pressure-Area Isotherms.The surface pressure (π) of the monolayers was measured using

(18) William Taeusch, H.; Lu, K. W.; Goerke, J.; Clements, J. A. Am. J. Respir.Crit. Care Med. 1999, 159, 1391–1395.(19) Lu, K. W.; Taeusch, H. W.; Robertson, B.; Goerke, J.; Clements, J. A.Am.

J. Respir. Crit. Care Med. 2001, 164, 1531–1536.(20) Lu, K.W.;William Taeusch, H.; Robertson, B.; Goerke, J.; Clements, J. A.

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2009, 1788, 1033–1043.(27) Krafft, M. P. Adv. Drug Delivery Rev. 2001, 47, 209–228.(28) Riess, J. G. Chem. Rev. 2001, 101, 2797–2920.(29) Riess, J. G. Tetrahedron 2002, 58, 4113–4131.(30) Riess, J. G.Artif. Cells Blood Subst. Immobil. Biotechnol. 2006, 34, 567–580.(31) Riess, J. G. Artif. Cells Blood Subst. Immobil. Biotechnol. 2005, 33, 47–63.(32) Courrier, H. M.; Vandamme, T. F.; Krafft, M. P.; Nakamura, S.; Shibata,

O. Colloids Surf. A 2003, 215, 33–41.(33) Hiranita, T.; Nakamura, S.; Kawachi, M.; Courrier, H. M.; Vandamme,

T. F.; Krafft, M. P.; Shibata, O. J. Colloid Interface Sci. 2003, 265, 83–92.(34) Nakamura, S.; Nakahara, H.; Krafft,M. P.; Shibata, O.Langmuir 2007, 23,

12634–12644.(35) Gerber, F.; Krafft, M. P.; Vandamme, T. F.; Goldmann, M.; Fontaine, P.

Angew. Chem., Int. Ed. Engl. 2005, 44, 2749–2752.(36) Gerber, F.; Krafft Marie, P.; Vandamme Thierry, F.; Goldmann, M.;

Fontaine, P. Biophys. J. 2006, 90, 3184–3192.(37) Gerber, F.; Vandamme, T. F.; Krafft, M. P. Comptes Rendus Chim. 2009,

12, 180–187.(38) Gerber, F.; Krafft, M. P.; Vandamme, T. F. Biochim. Biophys. Acta 2007,

1768, 490–494.

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an automated homemade Wilhelmy balance. The surfacepressure balance (Mettler Toledo, AG-64) had a resolution of0.01mNm-1. The pressure-measuring systemwas equippedwith afilter paper (Whatman 541, periphery = 4 cm). The trough wasmade from Teflon-coated brass (area = 750 cm2), and Teflonbarriers (both hydrophobic and lipophobic) were used. The com-pression of a typical phospholipid such as DPPC can reach a sur-face pressure close to 70 mN m-1 without leakage of the materialby using hydrophilic barriers. The collapse value of DPPC mono-layers is a controversial subject discussed in the literature.46-50

Namely, an entirely Teflon-made system can prevent adsorptionof material onto the barriers, and allow accurate quantitativeanalyses of phospholipid monolayers up to collapse. In this study,the first kink point on theπ-A isotherms at higher surface pressures(>50 mN m-1) is defined as a monolayer collapse.47-50 Thesurface potential measurement supports this behavior. The π-Aisothermswere recorded at 298.2(0.1K.Stock solutionsofDPPC(1.0 mM), F8HmOH (1.0 mM), and Hel 13-5 (0.1 mM) wereprepared in n-hexane/ethanol (9/1, v/v) and (4.5/5.5, v/v forHel 13-5). The spreading solvents were allowed to evaporate for15 min prior to compression. The monolayer was compressed at aspeed of <0.11 nm2 molecule-1 min-1. Cyclic compression-expansion isotherms (hysteresis curves) were drawn by recordingfive compression and expansion cycles at a rate of ∼0.24 nm2

molecule-1 min-1 (or ∼90 cm2 min-1). The standard deviations(SD) formolecular surface area and surface pressurewere∼0.01 nm2

and ∼0.1 mN m-1, respectively. The pH of the subphase wascontrolled with 0.02 M Tris buffer and 0.13 M NaCl and wasadjusted to 7.4 with an adequate amount of HAc.

2.2.2. Surface Potential-Area Isotherms. The surfacepotential (ΔV) was recorded simultaneously with surface pressureusing Keithley 614 and 6517 electrometer, where the monolayerwas compressed and expanded at the air/water interface. Itwas monitored by using an ionizing 241Am electrode located at1-2 mm above the interface, while a reference electrode was

dipped in the subphase. The SD for the surface potential was5 mV.51,52

2.2.3. Fluorescence Microscopy (FM). The film balancesystem (KSV Minitrough) was mounted onto the stage of anOlympus microscope BX51WI (Tokyo, Japan) equipped with a100 W mercury lamp (USH-1030 L), an objective lens(SLMPlan50�, working distance=15mm), and a 3CCDcamerawith a camera control unit (IKTU51CU, Toshiba, Japan). Aspreading solution of the sample investigated was prepared anddoped with 1 mol% of the fluorescence probe (NBD-PC). Imageprocessing and analysis were carried out using the software,Adobe Photoshop Elements ver. 7.0 (Adobe Systems Incorpo-rated, CA). The total amount of ordered domains (dark contrastregions)was evaluated and expressed as a percentageper framebydividing the frame into dark and bright regions. For the percen-tage, the resolution was 0.1% and the maximum SD in this studywas 8.9%. More details concerning the FM measurements wereprovided in a previous paper.9

2.2.4. Atomic ForceMicroscopy (AFM).Langmuir-Blodgett(LB) film preparations were carried out with the KSVMinitrough. Freshly cleavedmica (Okenshoji Co., Tokyo, Japan)was used as a supporting solid substrate for film deposition (avertical dipping method). At selected surface pressures, a transfervelocityof 5mmmin-1was used for the film-formingmaterials ona 0.02 M Tris buffer and 0.13 M NaCl (pH 7.4) at 298.2 K. Thetransfer was achieved so that the hydrophilic part of the mono-layer was juxtaposed to the mica while the hydrophobic part wasexposed to air. LB films with deposition rate of ∼1 were used inthe experiments.TheAFMexperimentswereperformed in the air.The LB technique includes the risk that the original monolayerstructures may be modified by the electric charge of the samplesand the draining force during the deposition procedure. How-ever, AFM images of deposited LB films do provide usefulinformation for understanding pulmonary functions. AFMimages were obtained using an SPA 400 instrument (SeikoInstruments Co., Chiba, Japan) at room temperature in thetapping mode, which provided both topographical and phasecontrast images. Other details about AFM measurements werereported previously.9

Figure 1. π-A andΔV-A isotherms of monolayers containingDPPC, plus 0 to 20 wt% of F8H5OH (A) or 0 to 20 wt% of F8H11OH (B)spreadona 0.02MTris buffer solution (pH7.4) with 0.13MNaCl at 298.2K. (Inset) Enlargedπ-A isotherms at high surface pressures in thevicinity of the monolayer’s collapse.

(46) Warriner, H. E.; Ding, J.;Waring, A. J.; Zasadzinski, J. A.Biophys. J. 2002,82, 835–842.(47) Miano, F.; Zhao, X.; Lu, J. R.; Penfold, J. Biophys. J. 2007, 92, 1254–1262.(48) Miller, C. E.; Busath, D. D.; Strongin, B.;Majewski, J.Biophys. J. 2008, 95,

3278–3286.(49) Ma, G.; Allen, H. C. Langmuir 2007, 23, 589–597.(50) Sabatini, K.; Mattila, J.-P.; Megli, F. M.; Kinnuneny, P. K. J. Biophys. J.

2006, 90, 4488–4499.

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112, 6398–6403.

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3. Results

3.1. Effects of Added F8HmOH on DPPC Monolayers.

The π-A and ΔV-A isotherms of DPPCmonolayers containing0 to 20 wt % of F8H5OH and F8H11OH are shown in Figure 1,panles A and B, respectively. In the DPPC/F8H5OH system(Figure 1A), the π-A isotherm of DPPC monolayers is seen toshift toward larger molecular area as the percentage of addedF8H5OH increases. In the conditions used, DPPC monolayersundergo a first-order transition (at ∼11 mN m-1) from a liquid-expanded (LE) to a liquid-condensed (LC) phase upon compres-sion. Further compression leads tomonolayer collapse at∼55mNm-1. These phase transitions and properties of DPPCmonolayersincluding their ΔV behavior have been steadily discussed in theprevious papers.34,39 The LE/LC transition pressure increasesfrom ∼11 to ∼14 mN m-1 upon addition of 0 to 20 wt % ofF8H5OH (Figure 1A), which means that the DPPC monolayersundergo a fluidization process in the presence of this compound.34

Also, a slight increase of the monolayer’s collapse pressure by∼3mNm-1 is observed (see inset in Figure 1A). This rise providesevidence that DPPC and F8H5OH are miscible in the monolayerstate.34The general shape of theΔV-A isotherms does not changeupon addition of F8H5OH. However, the maximum ΔV value inthe close-packed state decreases from∼550 (curve 1) to∼370 mV(curve 5). This negative variation arises from the contribution of

the fluorocarbon moiety of F8H5OH, since negative ΔV valueshave previously been reported for pure F8H5OHmonolayers.34,41

Contrary to theDPPC/F8H5OH system, theDPPC/F8H11OHsystem (Figure 1B) exhibits a decrease of the LE/LC transitionpressure from∼11 to∼5 mNm-1 as the percentage of F8H11OHincreases. This reduction means that F8H11OH addition causesthe DPPC monolayers to become more rigid or ordered. In linewith this observation, the collapse pressure increases significantlyfrom55up to∼65mNm-1 (see inset in Figure 1B). TheΔV valuesnear the monolayer collapse decrease substantially from ∼550 to∼240 mV (curve 5). This larger ΔV reduction in the DPPC/F8H11OHsystemas compared toDPPC/F8H5OHsystem reflectsthe longer carbon chain ofF8H11OH(Figure S2 in theSupportingInformation). F8H11OH alone forms ordered monolayers and itsbehavior is scarcely affected by temperature rises. WhenF8H11OH monolayers are compressed up to the close-packedstate, the ΔV value reaches -700 mV at 298.2 K. F8H11OH isconsidered to be oriented more vertically in DPPC monolayersthan F8H5OH.

Figure 2 shows fluorescencemicrographs ofDPPCmonolayerswith 0, 5, and 10wt% F8HmOHadded in the LE/LC coexistenceregion. Fluorescent probes (here, NBD-PC) selectively dissolveinto disordered phases inmonolayers. Therefore, bright and darkregions in the FM images correspond to disordered (or LE) and

Figure 2. Fluorescentmicrographsofamonolayer ofDPPCalone (a),DPPCplus 5wt%ofF8H5OH(b), 10wt%ofF8H5OH(c), 5wt%ofF8H11OH (d), and 10wt%ofF8H11OH (e) spread on a 0.02MTris buffer solution (pH 7.4) with 0.13MNaCl at 298.2K.Numbers withinopen circles indicate the surface pressure (mNm-1). Themonolayers contain 1mol%of the fluorescent probe,NBD-PC. The scale bar in thelower right angle represents 100 μm.

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ordered (orLC) phases, respectively. Below the transition pressureof ∼11 mNm-1 and in the absence of F8HmOH, the FM imagesof DPPC monolayers are homogeneously bright. When thispressure is reached, nucleation of LC domains starts and furthermonolayer compression leads to growth of LC domains as seen inFigure 2a. The percentage of surface occupied byordered domainsin each FM image increases with increasing surface pressure andreaches ∼95% beyond 20 mN m-1 (Figure S3 in the SupportingInformation). The ordered domains with counterclockwise armsare characteristic of DPPC (L type) monolayers.53,54 Upon addi-tion of 5 wt % F8H5OH to DPPC (Figure 2b), the arms of thedomains become slimmer and start branching out. Further addi-tion of F8H5OH (Figure 2c) increases the morphological varia-tions. The shape of the ordereddomain is commonly controlled byline tension of phase boundaries and long-range dipole interac-tions. That is, the FM phase behavior in the DPPC/F8H5OHsystems is dominated by the long-range dipole interactionsinduced by the addition ofF8H5OH.This is readily understandablefrom the fact that monolayers of F8H5OH alone display anopposite variation ofΔV value as compared toDPPCmonolayers.As a result, the dipole interactions induce fluidization of theDPPCmonolayer.

Contrary to the DPPC/F8H5OH case, addition of 5 wt %F8H11OH causes an increase of the width of the arms of theordered domains (Figure 2d). For the DPPC monolayers with10 wt % F8H11OH (Figure 2e), the arms can no longer be distin-guished and the ordered domains are essentially round (at 13 and15 mN m-1) or elongated (at 20 mN m-1) in shape. This domainvariation means that addition of F8H11OH considerably changesthe line tension of the phase boundary between coexisting two-dimensional phases in monolayers and the long-range dipoleinteraction to the domain shape. In other words, a dominantfactor of the domain shape is expected to shift from the positiveline tension (πe 15mNm-1) to the dipole interaction (πg 20mNm-1). Considering that the ordered domains are larger in the

DPPC/F8H11OH system as compared to the DPPC/F8H5OHsystem, it appears that F8H11OHpreferably interacts with the LCphase of DPPC monolayers, which results in an improvement ofmonolayer order and rigidity.3.2. Effects of F8HmOH Addition on a Pulmonary Sur-

factant Model. 3.2.1. Compression Isotherms. The π-Aand ΔV-A isotherms of the binary DPPC/Hel 13-5 monolayer(XHel13-5 = 0.1), which is a simple model mixture of syntheticpulmonary surfactants,9,39,55 are shown inFigure 3 (curve 1). Thismonolayer has its disordered/ordered phase transition at∼14mNm-1, and the plateau at∼42mNm-1 (indicated by arrows), whenHel 13-5 begins to be squeezed out of DPPC monolayers.39

Indeed, the squeezed-out behavior of Hel 13-5 corresponds tothe vertical phase separations found for other phospholipidcontaining systems.56-58 Such squeezing-out behavior also takesplace for native pulmonary surfactants and has been discussedextensively.9,59-61 Upon addition of F8H11OH (Figure 3A), theπ-A isotherm shifts very slightly toward larger molecular areaswith increasing amount of added F8H5OH below the plateaupressure. Although the plateau pressure remains almost constantregardless of the amount of F8H5OH added, the plateau regionbecomes more pronounced. It is suggested that F8H5OHenhances the squeezed-out of Hel 13-5 upon compression. Thisview is also supported by the fact that the π-A isothermsmove tosmaller molecular areas upon F8H5OH addition when pressurebecomes larger than 42 mN m-1 (see inset in Figure 3A). ThemaximumΔV valuesmeasured formixtures containing 3 to 10wt%

Figure 3. π-A and ΔV-A isotherms of monolayers containing the model pulmonary surfactant preparation (the binary DPPC/Hel 13-5mixture with the fixed ratio ofXHel13-5= 0.1) plus 0 to 20 wt% of F8H5OH (A) and 0 to 20 wt% of F8H11OH (B) on a 0.02MTris buffersolution (pH 7.4) with 0.13MNaCl at 298.2 K. (Inset) Enlarged π-A isotherms at high surface pressures in the vicinity of the monolayer’scollapse.

(53) Cruz, A.; V�azquez, L.; V�elez, M.; P�erez-Gil, J. Langmuir 2005, 21, 5349–5355.(54) Weis, R. M.; McConnell, H. M. Nature 1984, 310, 47–49.

(55) Nakahara, H.; Lee, S.; Shibata, O. Biochim. Biophys. Acta 2010, 1798,1263–1271.

(56) Maaloum, M.; Muller, P.; Krafft, M. P. Langmuir 2004, 20, 2261–2264.(57) Krafft, M. P.; Giulieri, F.; Fontaine, P.; Goldmann,M. Langmuir 2001, 17,

6577–6584.(58) Wang, S.; Lunn, R.; Krafft, M. P.; Leblanc, R. M. Langmuir 2000, 16,

2882–2886.(59) Takamoto, D. Y.; Lipp, M. M.; Von Nahmen, A.; Lee, K. Y. C.; Waring,

A. J.; Zasadzinski, J. A. Biophys. J. 2001, 81, 153–169.(60) Diemel, R. V.; Snel, M. M. E.; Waring, A. J.; Walther, F. J.; van Golde,

L. M. G.; Putz, G.; Haagsman, H. P.; Batenburg, J. J. J. Biol. Chem. 2002, 277,21179–21188.

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of F8H5OH in the close-packed states are almost the same asthat for theDPPC/Hel 13-5mixture (curve 1), which indicates thecontribution of the F8H5OH addition to the ΔV value is small.Interestingly, it is found that F8H5OH is also squeezed out ofthe monolayer along with Hel 13-5. However, upon furtheraddition of F8H5OH (curve 5), the surplus amount of F8H5OHat the interface achieves a substantial negative contributions to theΔV value.

In the case of F8H11OH (Figure 3B), the π-A isothermsexhibit a different, more complex behavior. A reduction in thedisordered/ordered transition pressure by F8H11OH is seen atlower surface pressures. The π-A isotherms clearly move tolarger molecular areas in the vicinity of plateau pressures. Inaddition, the plateau region becomes wider as the amount ofadded F8H11OH increases. The plateau region in the F8H11OH-containing systems is larger compared with that of the F8H5OH-containing systems (Figure 3A). The decrease inΔV values in theclose-packed state with increasing F8H11OH amount is morepronounced than for the F8H5OH systems. This negative varia-tion indicates that F8H11OHmolecules remain in the monolayerabove the plateau regions. Therefore, it is suggested thatF8H11OH molecules improve the packing of DPPC monolayersby their presence at the interface and thus promote the squeeze-out motion of Hel 13-5 at the higher surface pressures.

3.2.2. Fluorescence Micrographs. FM images of the DPPC/Hel 13-5 monolayers (XHel13-5 = 0.1) with and withoutF8HmOH are shown in Figure 4. In Figure 4a, the ordered(dark) domains correspond to the LC phase of DPPC mono-layers, since Hel 13-5 itself is known to form a homogeneousdisordered (bright) phase.39 Although the LC domains grow inratio upon compression (see Figure S3), the domain boundariesbecome diffuse. This may be attributed to an increase in surfaceconcentration of Hel 13-5, which is relatively huge molecule ascompared to DPPC. In the case of the 5 wt% F8H5OH addition(Figure 4b), the number and size of the ordered domains diminishas a result of the fluidization effect of F8H5OH. However, thefurther addition of F8H5OH conversely brings about a growth insize of the ordered domains as seen in Figure 4c. The morphologyof the domains resembles that seen for pure DPPC monolayers(Figure 2a). Considering the phase variation observed inFigure 2a-c, this implies that F8H5OH hinders the interactionbetween DPPC and Hel 13-5, or that F8H5OH interacts pre-ferably with Hel 13-5 in the 10 wt % addition system. On theother hand, in theDPPC/Hel 13-5/F8H11OH system (Figure 4d,e),the phase behavior and its variation are similar to those observedin the absence of Hel 13-5 (Figure 2d,e). As opposed toF8H5OH, F8H11OH interacts favorably with the LC domainsof the DPPC monolayers, in spite of the presence of Hel 13-5.

Figure 4. Fluorescentmicrographs of the binaryDPPC/Hel 13-5mixture (XHel13-5=0.1) (a), with 5wt%ofF8H5OHadded (b), 10wt%ofF8H5OH added (c), 5 wt%of F8H11OHadded (d), and 10wt%ofF8H11OHadded (e) spread on a 0.02MTris buffer solution (pH 7.4)with 0.13MNaCl at 298.2 K. Numbers within open circles indicate the surface pressure (mNm-1). The monolayers contain 1 mol% of thefluorescent probe NBD-PC. The scale bar in the lower right angle represents 100 μm.

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3.2.3. AFM Topographic Images. AFM observations ofLangmuir-Blodgett (LB) films transferred onto mica were per-formed to elucidate the PFA addition effect at the nanometerlevel. The AFM topographies of the ordered domains (dark areasin the in situ FM observations) showed almost homogeneousimages (data not shown). Therefore, our attentionwas focused onthe disordered phase observed by FM. The AFM topographicimages collected for the PSmodel of DPPC/Hel 13-5 (XHel13-5=0.1) containing 0-10 wt % F8HmOH are shown in Figures 5and 6. For the DPPC/Hel 13-5 mixture alone (Figure 5A), thedark (lower in height) and bright (higher in height) domainsobserved at 35mNm-1 are composed ofHel 13-5 and ofDPPC,respectively.39 Further compression of the monolayers at45 mN m-1 (>the plateau pressure of ∼42 mN m-1 in pure Hel13-5) causes the emergence of protruding masses (indicated byarrows) that correspond to the squeezed-out Hel 13-5. Similarprotruding masses were also seen in monolayers of DOPG and apeptide dimer mimicking SP-B and called “nanosilos”.62 The

protruding masses grow in size and number at 55 mN m-1, andthe height difference (∼2.8 nm) between the masses and thesurrounding monolayers corresponds to 2-3 molecular layersofHel 13-5 peptides.39The formation of suchparticles is quite animportant process in pulmonary function.9,59

When F8H5OH is added (Figure 5B,C), the monolayer topo-graphy observed at 35 and 45 mNm-1 show little difference withthe AFM images of Figure 5A. However, at 55mNm-1 and for a10 wt% addition, the images show that the size of the protrudingmasses increases in diameter from 23( 7 nm (Figure 5A) to 52(13 nm (Figure 5C). The F8H5OH addition also enhances theirheight, which then reaches ∼6 nm (Figure 5D). According to theΔV-A isotherms (Figure 3A), the protrudingmasses are thoughtto comprise F8H5OH molecules excluded from the surfacemonolayers along with Hel 13-5.

For theF8H11OHaddition system (Figure 6), some changes ofthe monolayer’s morphology are observed as compared to theimages collected for the DPPC/Hel 13-5 mixture (Figure 5A).The Hel 13-5 network (dark) is preserved and continues to beinterlinked even at 55 mN m-1, which indicates that F8H11OHenhances phase separation between DPPC and Hel 13-5 in the

Figure 5. Typical topographic AFM images of the DPPC/Hel 13-5 preparation (A), the preparation with 5 wt % of F8H5OH added (B),andwith 10wt%ofF8H5OHadded (C) compressed at 35, 45, and 55mNm-1 on a 0.02MTris buffer solution (pH7.4) with 0.13MNaCl at298.2Kand transferred onmica. The scanned area is 1� 1μmand the scale bar in the lower-left corner represents 300 nm.The cross-sectionalprofiles along the white scanning lines a-c are given in panel D. The height difference between the arrowheads is indicated in the cross-sectional profiles.

(62) Ding, J.; Doudevski, I.; Warriner, H. E.; Alig, T.; Zasadzinski, J. A.;Waring, A. J.; Sherman, M. A. Langmuir 2003, 19, 1539–1550.

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monolayers by forming a closer-packed monolayer with DPPC.Huge protruding masses with a diameter of 137 ( 21 nm and aheight that reaches ∼13 nm are observed at 55 mN m-1

(Figure 6B). These protruding masses differ from those observedfor the F8H5OH systems in number, diameter, and height(Figure 6C). The formation of large protruding masses may beattributed to the stability of the rigid mixed DPPC/F8H11OHmonolayer at the interface, which induces the elimination of Hel13-5 from the monolayer. No surface micelles, such as thosereported for (F-alkyl)alkyldiblocks,63 were observed.3.2.4. Repeated Compression-Expansion Isotherms.

Repeated cycling π-A and ΔV-A isotherms of the preparationsinvestigated were measured systematically. The monolayers werecompressedup to their collapse pressure and then expanded to theinitial molecular areas. This process was repeated five times toascertain the repeatability of the respreading process. Typicalcompression-expansion isotherms are shown inFigure 7. For thePS model mixture (Figure 7A), both π-A and ΔV-A isothermsindicate a good repeatability throughout all five rounds, anddisplay a hysteresis loop between compression and expansionstages, which has been discussed previously in details.39 Oneshould notice that, in the absence of F8HmOH, beside thehysteresis phenomenon, the interfacial behavior, in terms ofmolecular packing, orientation, and surface activity of mono-layers depends simply on an extensive variable of molecular areasfor theDPPC/Hel 13-5 (XHel13-5= 0.1) mixture. In the 10 wt%F8H5OH addition system (Figure 7B), it is clear that the hyster-esis seen for the π-A isotherms during the first compression/expansion round is intensified as compared to that observed in theabsence of F8H5OH (Figure 7A). However, the hysteresis loopsare attenuated as the number of cycles increases. It is notable thattheΔV values in the close-packed state increase from∼460 at the

first round to ∼516 mV at the fifth round. Considering that theΔV value in the close-packed state is ∼532 mV in the absence ofadded alcohol (Figure 7A), it can be said that F8H5OH isgradually excluded from the interface by repeated compression/expansion cycles, and that the F8H5OH desorption is an irrever-sible process in the condition used. This interpretation is alsosupported by the hysteresis isotherms for the DPPC/F8HmOHsystemsmeasured in the absence ofHel 13-5 (see Figure S4 in theSupporting Information). On the other hand, in the 10 wt %F8H11OH addition system (Figure 7C), the hysteresis looprecorded during the first round is larger, and the magnitude ofthe size reduction of the hysteresis loops when the cycles arerepeated is smaller than with F8H5OH (Figure 7B). As for theΔV-A isotherms, the ΔV value in the close-packed state remainsconstant at ∼370 mV, regardless of the number of compression/expansion cycles. This means that F8H11OH molecules remainpresent at the interface during the successive cycling rounds, asopposed towhat is observed upon addition of F8H5OHmolecules.

4. Discussion

To our knowledge, this is the first investigation of a phospho-lipid/protein hybrid-type lung surfactant formulation incorporat-ing fluorinated molecules intended for the development of newsynthetic PS preparations. Besides the unique features mentionedin the Introduction, it should be noted that the PFA moleculesused are not macromolecules, contrary to Hel 13-5, and that thereis no need to worry about thermal stability and antigen recognitionrelated with biophylaxis. In particular, the relative thermal stabilityof PFA molecules is an advantage in terms of long-time storageand large-volume production. When a small amount of the PFAmolecules used here is added to the model PS preparation,the squeezing-out of Hel 13-5 during compression is enhanced.This squeezing-out phenomenon observed on the π-A isothermsat ∼42 mN m-1 is closely related with the reduction in work ofbreathing at the alveolar surface and thus is an important index to

Figure 6. Typical AFM topographic images of the DPPC/Hel 13-5 preparation with 5 wt % of F8H11OH added (A) and with 10 wt % ofF8H11OHadded (B) compressedat 35, 45, and55mNm-1ona0.02MTrisbuffer solution (pH7.4)with0.13MNaClat298.2Kandtransferredon mica. The scanned area is 1� 1 μm and the scale bar in the lower-left corner represents 300 nm. The cross-sectional profiles along the whitescanning line a and b are given in panel C. The height difference between the arrowheads is indicated in the cross-sectional profiles.

(63) Maaloum, M.; Muller, P.; Krafft, M. P. Angew. Chem., Int. Ed. Engl. 2002,41, 4331–4334.

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assess the efficacy of artificial PS preparations. From the point ofview of enhancing product effectiveness for NRDS patients,positive in vitro effects of PS additives include an increase ofthe maximum molecular collapse pressure, an increase ofthe integrated hysteresis area for the π-A isotherms, and animproved respreading ability at low additive concentrations.26,64

The respreading effect is complemented for NRDS treatment bythe fact that instillation of PS preparations reactivates alveolartype II cells so that native PS secretion is resumed. Our studyshows that incorporation of small amounts of both F8HmOHadditives has beneficial effects on the preparation.

The administration of PS formulations is a common treatmentfor NRDS infants, and many researchers investigate new syn-thetic preparations.9-13 In particular, use of air saturated withfluorocarbon gases has recently been considered for RDS treat-ment.35-38,65 This followed the observation that FC gasescan prevent the formation of an LC phase in Langmuir mono-layers of DPPC and can promote dissolution of existing LCdomain.35-38 A predominant symptom in RDS patients is adyspnea arising from dysfunction and inactivation of PS. Partialventilation with air and fluorocarbon gases has been considereduseful as RDS treatment.66,67 However, this application is stillunder investigation due to the difficulty of controlling the partialpressure of the gases, which strongly depends on the state of thelungs. In the present study, F8HmOHmolecules (m= 5 and 11)are incorporated in the PS preparation as potential additives.BothF8HmOHmolecules investigated improve the squeezing-outbehavior of Hel 13-5 during compression but appear to involvedifferent mechanisms; F8H5OH undergoes an irreversible ejection

from the surface monolayers along with Hel 13-5, whereasF8H11OH promotes Hel 13-5 squeeze-out by remaining presentwithin the DPPC monolayer at the surface even at high surfacepressures. It is noticeable that the squeeze-out enhancing mecha-nism drastically depends on the total length of the hydrophobicchain of F8HmOH, although both compounds have the sameperfluorooctyl moiety. Because it stays in the monolayer,F8H11OH is deemed to be more useful in terms of the improvedin vitro efficacy. Another additive to PS preparations, namelyhexadecanol (HD), which is present in the commercially availableformulation, Exosurf, has been investigated. A small amount ofHD induces a decrease of the LE/LC transition pressure ofDPPCmonolayers68 and in the collapse pressure of Infasurf,69 which isalso one of the commercial formulations. In terms of reduction ofthe transition pressure, HD exercises an effect similar to that ofF8H11OH rather than of F8H5OH.However, the morphologicalphase behavior induced by F8HmOH addition in this study issignificantly different from that observed with molecules withhydrocarbon chains.

A quantitative assessment of the variation of the integratedhysteresis areas during successive compression/expansion π-Acycles, which are related to the pressure-volume (P-V) curvesmea-sured in the lung,70-72 is indispensable to confirm such usefulness.In the case of F8H5OH incorporation (Figure 8A), the integratedhysteresis area decreases with increasing compression/expansioncycle number and eventually converges to almost the same valueas in the absence of additive. This result also indicates theirreversible elimination of F8H5OH from the surface monolayer.On the other hand, the integrated hysteresis area decreases

Figure 7. Cyclic compression and expansion (or hysteresis curves) for surface pressures and surface potentials ofmonolayers containing theDPPC/Hel 13-5 preparation (A), the preparation with 10 wt % of F8H5OH added (B), and with 10 wt % of F8H11OH added (C). Thesubphasewas a 0.02MTris buffer solution (pH7.4)with 0.13MNaCl. The temperaturewas 298.2K.The compression-expansion cycle wasperformed five times at the compression rate of ∼0.25 nm2 molecule-1min-1.

(64) Banerjee, R.; Bellare, J. R. J. Appl. Physiol. 2001, 90, 1447–1454.(65) Krafft, M. P.; Vandamme, T. F.; Gerber, F.; Shibata, O. Lung Surfactant

Supplements, US Patent 60/563,690 (April 19, 2004), PCT/IB 2005/001020,April 18, 2005.(66) Leach, C. L.; Greenspan, J. S.; Rubenstein, D.; Shaffer, T. H.; Wolfson,

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significantly more gradually against the number of cycles for theF8H11OHadditive (Figure 8B). At the fifth compression/expansionround, the integrated areas observed in the presence ofF8H11OHare still almost two times larger than that for the DPPC/Hel 13-5preparation. The reduction in integrated area between the firstand the fifth cycles is attributed to the formation of compact,solid-likeDPPC/F8H11OHmonolayers at high surface pressures,which may be difficult to respread on the surface during expan-sion. Nevertheless, a 5-10 wt % F8H11OH addition maycontribute to enhancing the in vitro effectiveness (or integratedhysteresis area) of the PS model preparation. Although theLangmuir monolayer behavior of the model PS preparationshas systematically been investigated in this study, a futureevaluation of the surface adsorption characteristics from aqueousPS suspensions could also provide important clues.

The major differences between the fluorinated amphiphiles usedhere and the fluorocarbon gases used in another approach to lungsurfactant replacement preparations35-38 concern their physicalstates (solid or gas) at room temperatures and the presence orabsence of hydrophilic headgroups in their structures. A priori,fluorocarbongases should interact onlywith thehydrophobic chainsof the monolayers, whereas F8HmOH can interact both with theirpolar head, which can be anchored at the water surface and withtheir hydrophobic chains in the air. Because F8HmOH compoundsare essentially restricted to the surface, mutual interactions (inparticular, van der Waals, electrostatic, and dipole-dipole inter-actions) between molecules of F8HmOH and PS components maybe more complex than those observed with fluorocarbon gases andthem.The results obtainedhere suggest that fluorinated amphiphilesmay be useful potential additive to PS formulations. Furthermore,

considering the application of gaseous fluorocarbons to albu-min-covered surfaces,38 the concept described here couldpossibly be extended to the use of such formulations in ARDSpatients. However, while a substantial amount of data is avail-able on the toxicity and pharmacodynamics of fluorocarbonsand fluorocarbon/hydrocarbon diblocks and generally indicateconsiderable biological inertness,73-75 the toxicity, pharma-codynamics and biodegradability of the fluorinated amphiphileswith polar headgroups are still poorly documented in the litera-ture, which could hinder the development of their possibleapplications.76,77 Therefore, the effect of highly fluorinated com-pounds on PS formulations warrants further research, in parti-cular in terms of adsorption kinetics and biocompatibility, as wellas of practical aspects, including feasibility, and acceptability.

In conclusion, the present in vitro surface chemistry studyprovides some fundamental information that supportsthe potential of fluorinated amphiphiles for NRDS surfactantreplacement therapy.TheFMimages illustrate themorphologicalvariations (shape and size) that occur in ordered domains of themodel PS formulation upon addition of F8HmOH (m = 5 and11). They suggest a fluidizing (for m = 5) or a condensing (form= 11) effect on the LC domains that depends on hydrophobicchain length. The topographicAFM image reveal that the formationof protruding masses (or surface-associated reservoirs) isimproved in size and number by F8HmOH addition above thesqueeze-out pressure of Hel 13-5. Finally, hysteresis measure-ments provide definite evidence that (i) F8H5OH undergoes anirreversible exclusion from the surface monolayer, (ii) F8H11OHenhances the squeeze-out behavior of Hel 13-5 by remaining atthe surface to form a solid-like monolayer with DPPC at highsurface pressures, and (iii) the 5-10 wt % F8H11OH additionenhances the effectiveness of the PSmodel preparation in terms ofboth integrated hysteresis area preservation and of maximumreachable surface pressure. These results suggest that a hybrid PSformulation incorporating fluorinated amphiphiles may providea useful basis for the design of novel synthetic PS preparationsand for effective formulations for NRDS therapy.

Acknowledgment. This work was supported by a Grant-in-Aid for Scientific Research 20500414 from the Japan Society forthe Promotion of Science (JSPS). This work was also supportedby a Grant-in-Aid for Young Scientists (B) 22710106 from JSPS(H.N.). The Centre National de la Recherche Scientifique(CNRS) and the Universit�e de Strasbourg (UdS), France, areacknowledged for support.

Supporting Information Available: Chemical structures ofHel 13-5 and of the fluorinated alcohols; the π-A andΔV-A isotherms of F8H11OH monolayers at differenttemperatures; the ratio of ordered domain areas in fluores-cence micrographs; the hysteresis curves for the DPPC/F8H5OH and DPPC/F8H11OH systems. This material isavailable free of charge via the Internet at http://pubs.acs.org.

Figure 8. Variation of the integrated hysteresis area betweencompression and expansion isotherms as a function of the numberof compression/expansioncycles; theDPPC/Hel 13-5preparationincorporating 0-20 wt % of F8H5OH (A) or 0-20 wt % ofF8H11OH (B).

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