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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers

Nanoparticle Wettability Influences Nanoparticle-Phospholipid Interactions Nagarjun Konduru, Flavia Damiani, Svetla Stoilova-McPhie, Jason Tresback, Georgios Pyrgiotakis, Thomas C Donaghey, Philip Demokritou, Joseph Brain, and Ramon M. Molina Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03741 • Publication Date (Web): 12 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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Nanoparticle Wettability Influences Nanoparticle–Phospholipid Interactions

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Nagarjun V. Konduru a,b, Flavia Damiani a, Svetla Stoilova-McPhie c, Jason S. Tresback c,

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Georgios Pyrgiotakis a,b Thomas C. Donaghey a,b, Philip Demokritou a,b, Joseph D. Brain a,b,

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Ramon M. Molina a,b §

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a

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Health, Harvard T.H. Chan School of Public Health, 665 Huntington Avenue, Boston, MA

Molecular and Integrative Physiological Sciences Program, Department of Environmental

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02115, USA.

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b

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665 Huntington Avenue, Boston, MA 02115, USA.

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c

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Street, Cambridge, MA, 02138, USA.

Center for Nanotechnology and Nanotoxicology, Harvard T.H. Chan School of Public Health,

Center for Nanoscale Systems, Faculty of Art and Sciences, Harvard University, 11 Oxford

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Corresponding Author:

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§

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Department of Environmental Health, School of Public Health, 665 Huntington Avenue, Boston,

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MA 02115, USA. Tel: 617-432-2311

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Email: [email protected]

Ramon M. Molina, Molecular and Integrative Physiological Sciences Program, Harvard

ACS Paragon Plus Environment

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ABSTRACT

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We explored the influence of nanoparticle (NP) surface charge and hydrophobicity on

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nanoparticle-biomolecule interactions by measuring the composition of adsorbed phospholipids

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on four NPs, namely positively charged CeO2 and ZnO, and negatively charged BaSO4 and

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silica-coated CeO2 after exposure to bronchoalveolar lavage fluid (BALf) obtained from rats, and

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to a mixture of neutral dipalmitoyl phosphatidylcholine (DPPC) and negatively charged

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dipalmitoyl phosphatidic acid (DPPA). The resulting NP-lipid interactions were examined by

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cryogenic transmission electron microscopy (cryo-TEM) and by atomic force microscopy

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(AFM). Our data show that the amount of adsorbed lipids on NPs after incubation in BALf and

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the DPPC/DPPA mixture were highest in CeO2 than on the other NPs, qualitatively consistent

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with their relative hydrophobicity. The relative concentrations of specific adsorbed

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phospholipids on NP surfaces were different from their relative concentrations in the BALf.

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Sphingomyelin was not detected in the extracted lipids from the NPs despite its > 20%

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concentration in the BALf. AFM showed that the more hydrophobic CeO2 NPs tended to be

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located inside lipid vesicles while less hydrophobic BaSO4 NPs appeared to be outside. In

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addition, cryo-TEM analysis showed that CeO2 NPs were associated with the formation of

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multilamellar lipid bilayers, while BaSO4 NPs with unilamellar lipid bilayers. These data suggest

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that NP surface hydrophobicity predominantly controls the amounts and types of lipids adsorbed,

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as well as the nature of their interaction with phospholipids.


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INTRODUCTION

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When introduced into a biological milieu, nanoparticles (NPs) encounter a mixture of

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biomolecules (e.g., proteins, lipids, sugars, ions, and enzymes). Some may spontaneously adsorb

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onto the nanoparticle surface, forming the NP “corona” 1. These interactions are often dynamic,

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including sequential adsorption and desorption cycles 2. When inhaled, NPs encounter these

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biomolecules including pulmonary surfactant on lung surfaces. Events that occur even before the

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NPs deposit in lungs, such as adsorption of air contaminants or NP aggregation, can also affect

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corona formation. The lungs have two major components: the conducting airways and the gas

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exchange region, containing the alveoli. Depending on particle size, lung anatomy and breathing

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patterns, inhaled NPs, like larger particles may deposit in the airways or alveoli 3-4. When

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deposited in the parenchyma, particles first encounter the pulmonary surfactant layer, which

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resides on top of the alveolar lining fluid 5. The intrinsic physicochemical characteristics of NPs,

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such as size, hydrophobicity and surface charge, as well as the extrinsic properties of the

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suspending medium, such as protein and phospholipid (PL) concentrations influence

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nanoparticle-biomolecule interactions, corona formation, as well as subsequent NP biological

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effects and kinetics 6-8. Studying these interactions and their relative importance for different

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categories of NPs is a major challenge in the field of nanotoxicology 9.

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Pulmonary surfactant is composed of 85-90% w/w phospholipids; the remaining 10% w/w are

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proteins 10. The saturated lipid dipalmitoyl phosphatidylcholine (DPPC) is the most abundant,

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followed by monoenoic phosphatidylcholine, cholesterol, and anionic phospholipids 11.

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Structurally, DPPC contains a hydrophilic zwitterionic trimethyl ammonium and acidic


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phosphate head and two hydrophobic long chain fatty acid tails esterified to the glycerol. The

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DPPC-enriched lung lining fluid functions to prevent the collapse of alveoli as exhalation

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reduces alveolar diameter by producing a near-zero surface tension. Monolayers of DPPC endure

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a high surface pressure (or low surface tension) for a considerable time without collapsing 12-14.

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Predominantly, hydrogen bonding and electrostatic interactions control the interactions between

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DPPC and NP surfaces 15. While protein corona formation on NPs has been studied 16,

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phospholipid-NP interactions or formation of “corona” phospholipid is less explored. A recent

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study investigated the influence of hydrophobic and hydrophilic NPs on the mechanical

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properties of the DPPC monolayer. It was found that hydrophilic halloysite and bentonite NPs

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induced a concentration-dependent impairment of DPPC's ability to attain high surface pressures

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on interfacial compression, suggesting the possibility of reduced physiological function of lung

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surfactant 17.

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Few studies emphasize the role of surfactant phospholipids in lung macrophage-mediated

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particle uptake. Ruge et al. demonstrated that surfactant lipids that modulate protein-mediated

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particle agglomeration of NPs might play an important role in the clearance of NPs by alveolar

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macrophages 18. Kapralov et al. reported that coating single-wall carbon nanotubes (SWCNTs)

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with surfactant phospholipid significantly enhanced their uptake in a RAW 264.7 macrophage

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cell line 19. Furthermore, coating with phospholipids deficient in either phosphatidylglycerol

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(PG) or phosphatidylserine (PS) led to a significant reduction in macrophage phagocytosis of the

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coated SWCNTs. We also showed that coating of SWCNTs with PS and not PC promoted their

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uptake by professional phagocytes 20. Since alveolar macrophages play a critical role in NP-


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induced inflammation, oxidative stress, and innate immune function, it is possible that the

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formation of the NP lipid corona may influence NP fate and biological effects 21.

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Recently, Raesch et al. showed that magnetite NPs with different surface functionalizations

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(PEG-, PLGA-, and Lipid-NP) acquired different protein corona compositions but had similar

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classes of phospholipids 22. Formation of ‘corona-like’ supported bilayers around model silica

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nanoparticles after incubation in surfactant mixtures such as Curosurf® 23 and Survanta® 24 have

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been studied. Similarly, the interactions of inorganic nanomaterials with model PC bilayers has

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been studied 25. However, there is currently little information about the nature of PL-NP

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interactions in the lungs, particularly for industrially-relevant NPs. Obtaining such information

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will require both in vitro and in vivo experiments to quantify phospholipid ‘corona’ formation

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under a variety of conditions as well as mechanism-based models. We hypothesize that the NP

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surface characteristics influence the interactions of liposome-like structures present in BALf with

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NPs, their interactions with the NP surface, and the subsequent formation of ‘corona-like’

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bilayers around the NPs.

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In this study, we performed quantitative and qualitative analyses of the different phospholipid

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species adsorbing on to the surface of four different engineered NPs after exposure to rat

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bronchoalveolar lavage fluid (BALf). In addition, we also characterized the phospholipid-

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nanoparticle interactions in a more controlled formulation composed of a DPPC/DPPA mixture

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using cryogenic transmission electron microscopy (cryo-TEM) and atomic-force microscopy

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(AFM). To our knowledge, this is the first study examining the interactions of engineered

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nanoparticles with rat surfactant obtained by lung lavage using cryo-transmission electron


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microscopy. We believe that the data from this study using cryo-TEM and AFM techniques will

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provide important insights about the nature of interactions between engineered nanoparticles and

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phospholipids.

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MATERIALS AND METHODS

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Synthesis and characterization of nanoparticles. Uncoated CeO2, silica-coated CeO2, and ZnO

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NPs were synthesized by flame spray pyrolysis using the Versatile Engineered Nanomaterial

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Generation System (VENGES) at Harvard University 26. Detailed physicochemical and

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morphological characterization of these NPs was reported earlier 27-28. BaSO4 NPs (NM-220)

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were obtained from BASF SE (Ludwigshafen, Germany). This is a reference material for the

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Nanomaterial Testing Sponsorship Program of the Organization for Economic Cooperation and

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Development (OECD). Characterization of the NM-220 batch was previously published 29.

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Measurement of NP surface hydrophobicity. We used the Rose Bengal assay to determine the

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surface hydrophobicity of test NPs according to published protocols 30-31. The Partitioning

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Quotient (PQ) was determined as the ratio of RB bound to the NP surface to free RB in the liquid

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phase (PQ = RBbound/RBfree). The PQ values were plotted as a function of NP surface area and fit

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by a straight line using SAS statistical software (SAS Institute, Inc. Cary, NC). The slope was

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taken to represent the relative surface hydrophobicity, with increasing slope indicating increasing

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surface hydrophobicity 30.

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Hydrodynamic diameter and zeta potential. CeO2, silica-coated CeO2, BaSO4, and ZnO NPs

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were suspended in distilled water at specified concentrations and were sonicated in conical

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polyethylene tubes. A critical dispersion sonication energy (DSEcr) to achieve the smallest

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particle agglomerate size was used, as previously reported 28, 32. The suspensions were sonicated

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at 242 joules/ml (20 min/ml at 0.2-watt power output) in a cup sonicator fitted on a Sonifier S-

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450A (Branson Ultrasonics, Danbury, CT, USA). The sample tubes were immersed in running

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cold water to minimize heating of the particles during sonication. The hydrodynamic diameter

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(DH) and zeta potential (ζ) of each aggregate suspension were measured using a Zetasizer Nano-

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ZS (Malvern Instruments, Worcestershire, UK).

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Collection of bronchoalveolar lavage fluid. The protocols used in this study were approved by

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the Harvard Medical Area Animal Care and Use Committee. Twelve male Wistar Han rats (8

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weeks old) were obtained from Charles River Laboratories (Wilmington, MA), and were housed

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in standard microisolator cages under controlled conditions of temperature, humidity, and light at

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the Harvard Center for Comparative Medicine. Rats were euthanized and exsanguinated via the

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abdominal aorta under isoflurane anesthesia. The trachea was exposed and cannulated. The lungs

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were then lavaged with 5 mL of Ca++- and Mg++-free 0.9% sterile PBS by introducing the same

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solution in and out of the lungs three times to obtain representative samples of BALf. Cell-free

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BAL supernatant was obtained after centrifugation (350 x g at 4°C for 10 min). BALf samples

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from 3 rats were pooled for each NP incubation.

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Phospholipid extraction and analyses. Sonicated NP suspensions were added to BALf or

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DPPC/DPPA lipid mixture. In case of the DPPC/DPPA, the NP-lipid mixture were further


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sonicated for 30 minutes prior to incubation. After incubation in BALf and in DPPC/DPPA lipid

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mixture, the NP suspensions were centrifuged (14,500 x g for 10 min). The NP pellets containing

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the adsorbed phospholipids were suspended in 600 µL of DI water and transferred into a glass

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tube. Two milliliters of 1:2 chloroform:methanol solution (v/v) were added and vortexed. Then,

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600 µL of saline was added and vortexed. Finally, 600 µL of chloroform were added and the

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resulting suspension was spun at 3,000 x g for 10 min. Without disturbing the two phases, the

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lower phase containing the phospholipids was collected into separate glass vials and the samples

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were dried in nitrogen gas.

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The dried phospholipid samples were reconstituted with 0.5 mL of 1:1 chloroform:methanol

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(v/v). To extract phospholipids from BALf, 1ml of BALf was removed with 2 mL of 1:1 (v/v)

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chloroform:methanol. The lower layer was diluted to a final volume of 10 mL in 1:1 (v/v)

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chloroform:methanol. Analysis of extracted PL from BALf-incubated NPs was performed using

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an Acquity UPLC/AB Sciex 5500 tandem quadrupole mass spectrometer 19. An aliquot of each

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sample was diluted 1:10 into an internal standard solution (ISTD) containing 17:1 lyso and 17:0-

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30:1 phospholipid of phosphatidylethanolamine (PE), phosphatidylinositol (PI),

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phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidic acid (PA), and sphingomyelin

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(SM). Samples for PC were diluted 1:100 in the same ISTD mixture. The samples were injected

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under a reverse phrase gradient onto an Agilent EclipseTM XDB-C8 column of 50 X 4.6 (i.d)

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mm, 1.8 µM. The mass spectrometer was programmed for a multiple reaction monitoring

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(MRM) for the molecular species of each phospholipid class 19. Each molecular species

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identified above a 10:1 signal to noise was quantified against the response of the class relevant

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internal standard of known concentration.


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Nanoparticles for the DPPC/DPPA experiments were sonicated with 100 µg DPPC/50 µg DPPA

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at a NP:PL ratio of 6:1 (5 cycles 30 s) in a cold-water bath sonicator. Then, the NP-DPPC/DPPA

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suspension was incubated at 37°C for 30 min. The NPs were then centrifuged at 14,500 x g for

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30 min and the resulting pellet was washed 3 times with 25 mM HEPES, pH 7.4. After each

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washing, the samples were centrifuged at 14,500 x g for 30 min at 4°C. Phospholipids from

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coated NPs were extracted using the Folch procedure 33. Lipid phosphorus content was

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determined using a modification of the method for microdetection (1-10 nmol) 34. Aliquots of

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lipid extracts were transferred into test tubes and the solvent was evaporated to dryness under a

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stream of oxygen-free dry N2. Fifty µl of 70% (w/w) HClO4 was added to the dried samples,

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which were then incubated for 20 min at 170-180°C. After cooling, 0.4 ml of H2O was added to

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each tube followed in succession by 2 ml of sodium molybdate-malachite green reagent solution

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[1:3 (v/v) 4.2% (w/v) sodium molybdate in 5.0 M HCl-0.2% (w/v) aqueous malachite green] and

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80 µl of 1.5% (v/v) Tween 20. The tubes were shaken immediately to stabilize the developed

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color, which was measured at 660 nm in a Spectramax M5 spectrophotometer (Molecular

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Devices, Sunnyvale, CA).

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Cryogenic transmission electron microscopy. Cerium oxide and BaSO4 NPs were incubated in

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BALf or in a 2:1 mixture of DPPC and DPPA as described earlier. Holey carbon grids for

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electron microscopy (Quantifoil 2x1, Electron Microscopy Sciences, Hatfield, PA) were

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hydrophilized for 25 seconds. Five µl of the NP suspension was deposited on the grids and

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incubated for 1 minute. The grid was mounted on a semi-automatic Cryo-plunge – Gatan CP3

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(Gatan, Inc., Pleasanton, CA) and plunged in liquid ethane, cooled down by liquid nitrogen to


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preserve the fully hydrated structures in amorphous ice. The grids were then transferred under

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liquid nitrogen in a Tecnai Arctica operated at 200 kV equipped with a field emission gun (FEG)

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and autoloader (Thermofisher, Hillsboro, OR). Digital images were collected at low-dose

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conditions (~24 e-/Å 2 s) at 23,500 x and 39,000 x magnification with a 4096 x 4096 pixels CCD

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camera at 2.5 and 3.83 Å/pixels resolution, respectively.

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Atomic force microscopy. Atomic force microscopy (AFM) analyses of the same NP

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suspensions were performed using an MFP3d-BIO instrument (Asylum Research, Santa Barbara,

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CA) by drop casting and spin casting the NP-DPPC/DPPA aqueous suspension on freshly

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cleaved mica (V1 grade, Ted Pella, Redding, CA). Imaging was performed in a non-contact,

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AC/tapping mode in air using soft (2 N/m) cantilevers with a 7:1 aspect ratio silicon tip

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(AC240BSA, Asylum Research, Santa Barbara, CA). The height images were masked and

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flattened using instrument software.

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RESULTS AND DISCUSSION

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Nanoparticle characteristics. The physicochemical characteristics of the NPs used in this study

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are shown in Fig. 1 and Table 1. Values shown were obtained after the NPs were suspended in

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DI water and sonicated. Each NP suspension was then examined by TEM. The shapes and

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morphology of each NP are shown in Fig. 1. The zeta potential measurements for CeO2, Si-

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CeO2, BaSO4, and ZnO NPs in DI water were +34.5 ± 3.2 mV, -26.8 ± 0.3 mV, -18.5 ± 1.9 mV,

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and +23 ± 0.4 mV respectively. The hydrodynamic sizes of NPs measured in DI water using

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dynamic light scattering (DLS) showed that all four NP types formed agglomerates. Among the


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four NPs, ZnO showed a greater propensity to form larger agglomerates in DI water (221 nm)

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followed by Si-CeO2 (208 nm), BaSO4 (144 nm), and CeO2 (136 nm). The rod shape of ZnO

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NPs may have contributed to higher mean DLS agglomerate size compared to the other cubical

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and spherical NPs. The nanoparticles were incubated in cell-free BALf for 30 min at 37°C. After

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incubation, the zeta potentials changed to -23.2 ± 1.4 mV (CeO2), -16.7 ± 1.3 mV (Si-CeO2), -

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34.5 ± 2.7 mV (BaSO4), and -20.8 ± 1.7 mV (ZnO). After exposure to BALf, there were

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increases in agglomerate size in all NPs (Table 1). The relative increases were on the order ZnO

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> CeO2 > BaSO4 >Si-CeO2 NPs.

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Using the Rose Bengal partitioning assay on the four dry powders, we characterized the relative

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surface hydrophobicity of the four NPs. The linear relationships between NP surface areas and

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partitioning quotient (PQ) were plotted. The slopes of the linear regression lines are proportional

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to the relative hydrophobicities of NPs. The slopes of PQ for each NP were compared using SAS

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statistical software (SAS Institute, Inc. Cary, NC). We found that the relative surface

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hydrophobicity was on the order of CeO2 > ZnO = BaSO4 > Si-CeO2 NPs (Fig. 2).

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The surface chemistry of nanoparticles influences their agglomeration and the formation of

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protein coronas around them 16, 35. The interactions between lipids and surfaces of NPs are

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complex and is an active area of research. The NP-lipid interactions can be mediated by van der

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Waals, double layers, hydration, hydrophobic, thermal undulation and protrusion forces 36-37. In

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our experiments, we could not determine whether NPs aggregate first, and then interact with

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phospholipids, or whether phospholipids interact with individual particles which then aggregate.

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Perhaps both processes take place in parallel. Nevertheless, both possible processes can lower


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the surface energy of NPs 7, 35. It is important to note that although lung surfactant contains

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different phospholipids, it also contains proteins (10%). Other mechanism that might contribute

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to increased agglomerate size include lipid membrane fusion. It is a natural process of many

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biological events such as fertilization, viral membrane fusion with host cells, and exosome fusion

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to cell membranes 38. In some instances, proteins aid in such fusion process to form larger

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structures by acting as fusogenic agents. In other situations, the transition from lamellar bilayer

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phase to inverted hexagonal phase has been proposed as a step towards mixing of the outer

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bilayer of lipids to promote fusion 39-40.

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Adsorption of phospholipids from BAL fluid on nanoparticles. The composition of

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phospholipids extracted from NPs after incubation in BALf for 30 min at 37°C was analyzed by

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chloroform-methanol extraction followed by LC-MS analysis of the phospholipids. The amount

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of phospholipid bound per unit surface area (mg/m2) had the following order: CeO2 (1.76 ± 0.06)

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> Si-CeO2 (1.19 ± 0.06) = ZnO (1.14 ± 0.02) > BaSO4 (0.90 ± 0.10) NPs (Fig. 3a). Since these

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surface area calculations were based on BET analyses of dry powder, the influence of NP

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agglomeration in BALf on specific surface areas was not measured.

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The quantitative analyses of major phospholipids bound onto NPs are shown in Fig. 3b and

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Table 2. Phosphatidylcholine (PC) comprises more than 90% of the total phospholipid fraction

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bound to NPs (Fig. 3, Table S1, Online Supplement). At least 40 different species of PC were

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present (Table S1, Online Supplement). Of these, the seven most abundant were either saturated

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or monounsaturated fatty acid chains belonged to C32:0; C32:1; C34:2; C34:1; C34:0; C30:1;

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C16:0, where the first number is the total number of carbons in the two tails of the lipids and the


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second is the number of double bonds). Among the PC species, dipalmitoylphosphatidylcholine

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(DPPC, which is C32:0), the major surface-active component of lung surfactant, was the most

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abundant phospholipid species binding to all four NPs. Interestingly, the C18:2 PC species

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bound only to ZnO NPs. None were found in the other three NPs. Similarly, the phospholipid

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C30:0e species (with “e” standing for ether) were found on CeO2 and Si-CeO2 but was absent

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from both ZnO or BaSO4 NPs.

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Phosphatidic acid (PA; C38:5), an anionic phospholipid and a precursor phospholipid in the

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biosynthesis of lung surfactant, was the next most abundant phospholipid class in all four NPs. A

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larger mass percentage of adsorbed PA species was in CeO2 (5.7 %) than in BaSO4 (4.2 %), Si-

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CeO2 (4.4 %), and ZnO NPs (1.6 %). Interestingly, the highest percentage of another anionic

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phospholipid, phosphatidylserine (PS), was measured in ZnO NPs, followed by BaSO4 and then

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Si-CeO2. The levels of PS were lowest in the extracted phospholipids from CeO2 NPs despite

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their positively charged surfaces. The relative abundances of PA, phosphatidylinositol (PI), and

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phosphatidylglycerol (PG) species adsorbed onto NPs surfaces were found to be different from

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the concentrations in native rat BALf in which the particles had been incubated (Table 3),

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indicating preferential adsorption of some of the phospholipid species. Surprisingly, despite high

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sphingomyelin (SM) levels in rat lung BALf (22.4%), no detectable SM was found adsorbed on

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the NPs (Table S1, Online Supplement). While PA is a minor phospholipid in the normal lung

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lining fluid (only 0.15% of the total PL pool), it constituted 1.6 to 5.7% on the NP surfaces

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(Table 3). In contrast, 0.8 to 1% of the NP-bound lipid was comprised of PI, much lower than

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the 2.87 % of the total PL pool.

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Comparison of adsorption of phospholipids on nanoparticles: BAL fluid versus

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DPPC/DPPA mixture. We also studied adsorption onto NPs of a simpler lipid mixture than that

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found in BALf, namely a 2:1 mixture of DPPC and DPPA lipids. The lipid densities measured on

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the NP surfaces were 0.56 ±,0.01, 0.35 ± 0.02, 0.35 ±,0.02, and 0.34 ± 0.02 mg/m2 for CeO2, Si-

298

CeO2, BaSO4, and ZnO, respectively.

299 300

Our results from both the DPPC/DPPA mixture and from the BALf suggest that the total surface

301

densities of the phospholipids bound to the NP depend mainly on the NP hydrophobicity. The

302

lipid density on CeO2 NPs is significantly higher than the lipid densities of the other NPs, as seen

303

in Fig. 3 for the BALf extracts and for DPPC/DPPA mixtures. From the studies with BALf in

304

Fig. 3a, lipid adsorption onto BaSO4 is slightly lower than on ZnO and Si-CeO2. There were no

305

significant differences in extracted lipids among Si-CeO2, BaSO4, and ZnO NPs post-incubation

306

in the DPPC/DPPA mixture. The slope of the lines in the Rose-Bengal assay in Fig. 2 shows Si-

307

CeO2 to be slightly less hydrophobic than the other NPs, but this difference did not affect lipid

308

adsorption. Neither the sign nor the magnitude of the surface charge on the NPs correlates with

309

lipid adsorption. In regard to adsorption from BALf, similar adsorbed amounts were found on

310

ZnO as on Si-CeO2, despite having opposite surface charge. The least negatively charged NP,

311

BaSO4, (zeta potential -18.5 ±1.9 mV) adsorbs slightly less lipid than does a more negatively

312

charged NP, Si-CeO2 (zeta potential -26.8 ±0.3 mV), despite the greater repulsion of negatively

313

charged lipids that one might expect when the NP becomes more negatively charged. Thus,

314

although NP charge, size, shape, and other factors may influence phospholipid-nanoparticle

315

interactions, experimentally the only significant influence that is clearly demonstrated in our data

316

is NP hydrophobicity.


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317 318

Interaction of nanoparticles with phospholipids in BAL fluid. To examine the interactions

319

between phospholipids and NPs, using cryogenic TEM we examined samples of NPs incubated

320

in either BALf or a DPPC/DPPA mixture. We selected CeO2 and BaSO4 as model NPs since

321

these have different surface hydrophobicity and opposite surface charge. As BALf is a complex

322

mixture comprising both phospholipids and proteins, we also studied the PL-NP interactions

323

using model lipid vesicles prepared from a mixture of DPPC and DPPA phospholipids. DPPC is

324

a major phospholipid component of lung surfactant. These are also the predominant lipid species

325

interacting with NPs we observed after incubation in BALf. Although DPPA is not present in

326

lung surfactant, phosphatidic acids are present. We included DPPA because its tail groups are

327

identical to those of DPPC. Fig. S1 shows a cryo-TEM image of BALf showing large sheets of

328

lipid bilayers in micron sizes covering multiple holes of the grid. Cryo-TEM analysis showed

329

agglomerates of CeO2 (Fig. 4a) and BaSO4 (Fig. 4b) NPs interacting with these large liposome-

330

like structures present in the BALf. Based on cryo-TEM images, we could not determine exactly

331

how the lipid structures interact with individual or agglomerated NPs. There was no clear

332

evidence of “corona-like” lipid bilayers surrounding the NPs. We observed lipid bilayer vesicles

333

(~ 100-200 nm and > 500 nm) interacting with both BaSO4 and CeO2 NPs (Fig. 4). Importantly,

334

we observed that both NPs formed larger agglomerates after incubation in BALf than when

335

suspended in distilled water (Fig. 1). Furthermore, the particle agglomerates of CeO2 were larger

336

compared to BaSO4 NPs (Fig. 4). These observations are also supported by DLS measurements

337

of hydrodynamic diameters of NPs in distilled water and after incubation in BALf (Table 1).

338

Although cryo-TEM could not determine whether NPs were encapsulated or not in lipid vesicles,

339

it was apparent that agglomerates of both NPs were interacting with lipid vesicles.


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340 341

Interaction of nanoparticles with DPPC/DPPA phospholipids. We also examined the NP

342

interactions with a simpler phospholipid mixture of DPPC/DPPA. These two phospholipids

343

exhibit different charges (neutral and anionic, respectively). We hypothesized that their

344

interactions with NPs with opposite zeta potentials would be different. The cryo-TEM analysis of

345

CeO2 (Fig. 5a, 5b) and BaSO4 (Fig. 5c, 5d) NPs incubated in DPPC/DPPA lipid mixtures

346

showed agglomerates of NPs interacting with phospholipid vesicles with polyhedral shapes.

347

Interestingly, we observed CeO2 NPs interacting with concentrically enclosed multilamellar

348

phospholipid structures of variable sizes (Fig. 5a). These onion layer-like vesicular structures

349

were rarely present in the BaSO4 NPs. Instead, predominantly unilamellar phospholipid vesicles

350

were associated with the BaSO4 NPs (Fig. 5c). Unilamellar phospholipid vesicles consisting of a

351

single lipid bilayer synthesized in aqueous solutions such as ultrapure water contain a large

352

aqueous core, and therefore preferentially encapsulate hydrophilic molecules 41. Conversely,

353

multilamellar phospholipid vesicles with lipid bilayers arranged concentrically like in an onion-

354

skin, have high-lipid content which allows them for passively entrapping lipophilic molecules 41.

355

Our cryo-TEM findings show that the BaSO4 NPs were associated with the unilamellar

356

phospholipid vesicles and that the nanoparticle clusters displayed no direct interaction with the

357

lipid bilayers (Fig. 5c). Interestingly, BaSO4 NPs that were present in close proximity to the

358

vesicles were seen interacting with the bilayers (Fig. 5d). On the other hand, intact vesicles were

359

seen interacting with CeO2 NP agglomerates. There was no evidence of wrapping of lipid

360

bilayers around the CeO2 NPs. Nanoparticles decorating the surfaces of unilamellar and

361

multilamellar phospholipid vesicles were observed. In a similar study by Wang and Liu,

362

adsorption of 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes onto the surfaces of


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363

TiO2, ZnO and Fe3O4 NPs was observed with no evidence of bilayer formation around these NPs

364

25

365

only observed in SiO2 nanoparticles. The wrapping of lipid bilayers around SiO2 NPs was

366

attributed to the presence of a thin water layer between silica and the liposome surfaces. This

367

minimizes the steric hindrance and allows lipid membrane fusion and formation of supported

368

bilayers. Our results show that unilamellar and multilamellar vesicles exhibited a polyhedral

369

rather than a spherical shape. Similar findings by other groups have ascribed this behavior of

370

phospholipids to their existence in a gel phase under experimental temperatures used 42.

. In the same study, the formation of bilayers wrapping around the nanoparticles surface was

371 372

To verify whether the particle agglomerates were encapsulated within or interacting on vesicle

373

surfaces, we performed AFM analyses on the BaSO4 and CeO2 NPs incubated in DPPC/DPPA

374

lipids. The height images indicate that the single lipid bilayers are 5-6 nm above the mica (Fig.

375

6a). The CeO2 NPs agglomerated into larger clusters protruding up to 250 nm above the lipid

376

bilayers. These stack up around the NP clusters with 5 or more lipid bilayer sheets assembled

377

around and between the NPs. There is also evidence of the CeO2 NPs embedded in the lipid

378

bilayer as indicated by the line profile where the particles protrude 70-80 nm above the bilayers

379

(Fig. 6a).

380 381

Some of the BaSO4 NPs formed smaller agglomerates protruding 50-60 nm above the lipid

382

bilayers and their particle diameters appeared to range 10-30 nm (Fig. 6b). There was also

383

evidence of single BaSO4 NP embedded in the lipid bilayer shown at the end of the line profile in

384

Fig. 6b where the particle protrudes 15 nm above the 5 nm bilayer. Based on AFM topography,


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385

it was unclear whether the NPs were fully encapsulated within vesicles. However, there is

386

significant incorporation of BaSO4 NPs within the lipid bilayers.

Page 18 of 34

387 388

Furthermore, the phase contrast imaging for the CeO2 revealed small phase changes between the

389

lipid bilayer and the particle area (Fig. S2a, Online Supplement). This suggests that the materials

390

under the AFM tip did not change, indicating that the particles are either inside or on top of the

391

bilayer. If the particles were under the bilayer, we expected that these should also be present on

392

top, without any preferential partition. On the contrary, the BaSO4 had very strong phase-shifts

393

from the bilayers to the particle region indicating that the BaSO4 particles were sitting on top of

394

the bilayer (Fig. S2b, Online Supplement). Although these suggest that CeO2 NPs were inside

395

the bilayer, it is unclear whether the NPs are fully encapsulated within vesicles.

396 397

Another important phenomenon confirmed both by cryo-TEM and the AFM is that the CeO2 NPs

398

appeared to promote the creation of stacked bilayers (3-4) while the BaSO4 NPs tended to create

399

single lipid bilayers structures. The mechanisms for these observations are unknown. It can be

400

hypothesized that the particle size and wettability can influence the micelle formation and

401

surfactant interactions 43, as well as surface charge and the particle material characteristics 44.

402 403

CONCLUSIONS

404 405

We measured the compositions of extracted phospholipids from four NPs after incubation in

406

pooled rat bronchoalveolar lavage fluid, and in 2:1 mixtures of DPPC and DPPA. The

407

compositions of extracted phospholipids from positively charged CeO2 and ZnO, and negatively


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Langmuir

408

charged silica-coated CeO2 and BaSO4 NPs differed significantly. The hydrophobic CeO2 NPs

409

adsorbed the most lipids, while the other three NPs adsorbed lower amounts despite differences

410

in the surface charges. The relative amounts of phospholipids which bind to NPs were not

411

directly proportional to the amounts in the BAL fluid, indicating preferential adsorption of

412

certain phospholipid species. The AFM examination showed that the more hydrophobic CeO2

413

NPs tended to be partitioned inside lipid vesicles while less hydrophobic BaSO4 NPs appeared to

414

be outside lipid bilayers. Additionally, cryo-TEM analysis showed that CeO2 NPs were

415

associated with the formation of multilamellar stacked lipid bilayers, while BaSO4 NPs with

416

unilamellar lipid bilayers. These data suggest that NP surface hydrophobicity predominantly

417

controls the amounts and types of lipids adsorbed, as well as the nature of their interaction with

418

phospholipids. Further investigations are needed to improve our understanding of the

419

consequences of lung surfactant phospholipid-nanoparticle interactions in the lungs. Future

420

studies exploring the selectivity of NPs towards adsorption of specific phospholipid components

421

of the lung surfactant are also warranted. Especially, we need to explore how these interactions

422

affect in vivo biokinetics and biological responses.

423 424

Supplementary Material: Online supplement is available.

425 426

Acknowledgment: This work was supported by the National Institutes of Environmental Health

427

Sciences (NIH-P30ES000002, K99ES025813 and 1U24ES026946). Parts of this study were

428

performed at the Harvard University Center for Nanoscale Systems (CNS), a member of the

429

National Nanotechnology Coordinated Infrastructure Network (NNCI), which is supported by

430

the National Science Foundation under NSF award no. 1541959. The authors also gratefully


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Page 20 of 34

431

acknowledge the advice of Dr. Yi Zuo of University of Hawaii on hydrophobicity measurements

432

and Melissa Curran for editorial advice.

433

Table 1. Characterization of nanoparticles used in the study.

SSA (m /g) Dxrd (nm)

CeO2 28.0 32.9

DH (nm) ζ(mV)

136 34.5

DH (nm) ζ(mV)

1529 -23.2

2

Si-CeO2 BaSO4 27.8 33.0 32.6 36.0 In distilled water 208 144 -26.8 -18.50 Post-incubation in BALf 460 595 -16.7 -34.5

ZnO 41.0 29.0 221 23.00 2189 -20.8

SSA - Specific surface area Dxrd - diameter (based on X-ray diffraction) DH - hydrodynamic diameter ζ - zeta potential (ζ) BALf - bronchoalveolar lavage fluid 434


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435 Table 2. Quantitation of phospholipid components adsorbed on different nanoparticles after incubation in rat BALf. Phospholipid LPC

CeO2

Si-CeO2

BaSO4

ZnO

7.03 ± 0.25

7.15 ± 0.37

3.60 ± 0.38

5.69 ± 0.11

PC

1604.87 ± 57.19 1100.24 ± 56.47 834.48 ± 88.76

1079.10 ± 20.13

PE PI PG PS LPA PA Total

7.03 ± 0.25 3.58 ± 0.18 4.50 ± 0.48 21.09 ± 0.75 9.54 ± 0.49 8.99 ± 0.96 12.30 ± 0.44 14.30 ± 0.73 7.19 ± 0.77 3.52 ± 0.13 3.58 ± 0.18 2.70 ± 0.29 1.76 ± 0.06 1.19 ± 0.06 N.D. 100.19 ± 3.57 52.45 ± 2.69 37.77 ± 4.02 1757.80 ± 62.64 1192.03 ± 61.18 899.22 ± 95.65

5.69 ± 0.11 10.24 ± 0.19 14.80 ± 0.28 3.41 ± 0.06 N.D. 18.21 ± 0.34 1138.30 ± 21.23

Data are mean ± SE µg/m2, n=3 per NP. LPC - lysophosphatidylcholine; PC - phosphatidylcholine; PE - phosphatidyethanolamine; PI - phosphatidylinositol; PG - phosphatidylglycerol; PS - phosphatidylserine; LPA -lysophosphatidic acid; PA - phosphatidic acid; N.D. - not detectable


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Table 3. Relative abundance of phospholipid classes in rat bronchoalveolar lavage fluid and in adsorbed lipids on different nanoparticles.

Rat BALf LPC PC PE PI PG PS LPA PA SM

0.18 71.59 0.89 2.87 1.35 0.53 N.D. 0.15 22.41

NP-associated Phospholipids CeO2 Si-CeO2 BaSO4 ZnO 0.40 0.60 0.40 0.50 91.30 92.30 92.80 94.80 0.40 0.30 0.50 0.50 1.20 0.80 1.00 0.90 0.70 1.20 0.80 1.30 0.20 0.30 0.30 0.30 0.10 0.10 N.D. N.D. 5.70 4.40 4.20 1.60 N.D. N.D. N.D. N.D.

Data are relative amounts of each phospholipid class as % of total of all phospholipids measured. LPC - lysophosphatidylcholine; PC - phosphatidylcholine; PE - phosphatidyethanolamine; PI - phosphatidylinositol; PG - phosphatidylglycerol; PS - phosphatidylserine; LPA -lysophosphatidic acid; PA - phosphatidic acid; SM - sphingomyelin; BALf - bronchoalveolar lavage fluid; N.D. - not detectable 437 438


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439

Langmuir

Figure Legends

440 441

Fig. 1. Transmission electron micrographs of sonicated nanoparticle suspensions in DI water.

442

(a). CeO2. (b). Si-CeO2. Nanothin coatings of amorphous silica are shown (arrows). (c). BaSO4.

443

(d). ZnO nanorods.

444 445

Fig. 2. Surface hydrophobicity of CeO2, Si-CeO2, BaSO4 and ZnO NPs determined by Rose

446

Bengal partitioning. The relationships between NP surface areas and partitioning quotient are

447

plotted. The data were then analyzed by performing linear regression. Following model fitting,

448

the slopes of PQ for each NP were compared using SAS statistical software (SAS Institute, Inc.

449

Cary, NC). The slopes of the linear regression lines are proportional to the relative

450

hydrophobicity of NPs. The relative surface hydrophobicity was in the order of CeO2 > ZnO =

451

BaSO4 > Si-CeO2 NPs.

452 453

Fig. 3. Tandem quadruple mass spectroscopic analyses of lipids adsorbed on NPs after 30-

454

minute incubation in cell-free BAL fluid. (a). Total adsorbed phospholipids were calculated per

455

surface area as 1.76 ± 0.06 (CeO2), 1.19 ± 0.06 (Si-CeO2), 0.90 ± 0.10 (BaSO4) and 1.14 ± 0.02

456

(ZnO) mg/m2. (b). The amounts of different phospholipid classes are shown.

457

Phosphatidylcholine (PC) was the most abundant class bound to all NPs. (* CeO2 higher than the

458

other 3 NPs, # BaSO4 lower than the other 3 NPs, MANOVA, * # P Si-CeO2 NPs. 93x68mm (300 x 300 DPI)


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Fig. 3. Tandem quadruple mass spectroscopic analyses of lipids adsorbed on NPs after 30-minute incubation in cell-free BAL fluid. (a). Total adsorbed phospholipids were calculated per surface area as 1.76 ± 0.06 (CeO2), 1.19 ± 0.06 (Si-CeO2), 0.90 ± 0.10 (BaSO4) and 1.14 ± 0.02 (ZnO) mg/m2. (b). The amounts of different phospholipid classes are shown. Phosphatidylcholine (PC) was the most abundant class bound to all NPs. (* CeO2 higher than the other 3 NPs, # BaSO4 lower than the other 3 NPs, MANOVA, * # P

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