Nanoparticle Wettability Influences Nanoparticle-Phospholipid

Subscriber access provided by University of Pennsylvania Libraries

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 34 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

Langmuir

1

Nanoparticle Wettability Influences Nanoparticle–Phospholipid Interactions

2

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

3

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

4

Ramon M. Molina a,b §

5 6 7 8

a

9

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

Molecular and Integrative Physiological Sciences Program, Department of Environmental

10

02115, USA.

11

b

12

665 Huntington Avenue, Boston, MA 02115, USA.

13

c

14

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

15 16 17 18

Corresponding Author:

19

§

20

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

21

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

22

Email: [email protected]

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

ACS Paragon Plus Environment

1

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

23

Page 2 of 34

ABSTRACT

24 25

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

26

nanoparticle-biomolecule interactions by measuring the composition of adsorbed phospholipids

27

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

28

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

29

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

30

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

31

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

32

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

33

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

34

with their relative hydrophobicity. The relative concentrations of specific adsorbed

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

as well as the nature of their interaction with phospholipids.


2

Page 3 of 34 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

43

Langmuir

INTRODUCTION

44 45

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

46

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

47

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

48

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

49

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

50

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

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61 62

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

63

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

64

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

65

Structurally, DPPC contains a hydrophilic zwitterionic trimethyl ammonium and acidic


3

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

Page 4 of 34

66

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

67

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

68

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

69

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

70

Predominantly, hydrogen bonding and electrostatic interactions control the interactions between

71

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

72

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

73

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

74

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

75

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

76

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

77

surfactant 17.

78 79

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

80

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

81

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

82

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

83

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

84

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

85

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

86

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

87

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


4

Page 5 of 34 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

Langmuir

88

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

89

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

90 91

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

92

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

93

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

94

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

95

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

96

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

97

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

98

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

99

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

100

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

101

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

102

bilayers around the NPs.

103 104

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

105

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

106

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

107

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

108

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

109

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

110

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


5

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

Page 6 of 34

111

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

112

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

113

phospholipids.

114 115

MATERIALS AND METHODS

116 117

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

118

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

119

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

120

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

121

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

122

Nanomaterial Testing Sponsorship Program of the Organization for Economic Cooperation and

123

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

124 125

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

126

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

127

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

128

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

129

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

130

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

131

surface hydrophobicity 30.

132


6

Page 7 of 34 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

Langmuir

133

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

134

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

135

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

136

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

137

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

138

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

139

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

140

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

141

ZS (Malvern Instruments, Worcestershire, UK).

142 143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

from 3 rats were pooled for each NP incubation.

153 154

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

155

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


7

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

Page 8 of 34

156

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

157

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

158

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

159

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

160

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

161

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

162

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

163

were dried in nitrogen gas.

164 165

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

166

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

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

internal standard of known concentration.


8

Page 9 of 34 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

Langmuir

179 180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

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

189

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

190

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

191

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

192

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

193

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

194

Devices, Sunnyvale, CA).

195 196

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

197

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

198

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

199

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

200

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

201

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


9

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

Page 10 of 34

202

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

203

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

204

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

205

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

206

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

207 208

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

209

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

210

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

211

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

212

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

213

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

214

flattened using instrument software.

215 216

RESULTS AND DISCUSSION

217 218

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

219

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

220

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

221

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

222

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

223

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

224

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


10

Page 11 of 34 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

Langmuir

225

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

226

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

227

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

228

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

229

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

230

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

231

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

232

> CeO2 > BaSO4 >Si-CeO2 NPs.

233 234

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

235

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

236

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

237

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

238

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

239

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

240 241

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

242

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

243

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

244

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

245

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

246

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

247

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


11

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

Page 12 of 34

248

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

249

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

250

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

251

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

252

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

253

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

254

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

255

bilayer of lipids to promote fusion 39-40.

256 257

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

258

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

259

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

260

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

261

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

262

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

263

agglomeration in BALf on specific surface areas was not measured.

264 265

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

266

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

267

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

268

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

269

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

270

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


12

Page 13 of 34 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

Langmuir

271

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

272

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

273

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

274

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

275

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

276

from both ZnO or BaSO4 NPs.

277 278

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

279

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

280

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

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

the 2.87 % of the total PL pool.

293


13

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

Page 14 of 34

294

Comparison of adsorption of phospholipids on nanoparticles: BAL fluid versus

295

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

296

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

297

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.


14

Page 15 of 34 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

Langmuir

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.


15

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

Page 16 of 34

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


16

Page 17 of 34 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

Langmuir

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,


17

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

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


18

Page 19 of 34 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

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


19

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

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


20

Page 21 of 34 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

Langmuir

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


21

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

Page 22 of 34

436

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


22

Page 23 of 34 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

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

Nanoparticle Wettability Influences Nanoparticle-Phospholipid

Recommend Documents