prevention of vascular inflammation by prevention of ... · coated nanoparticles were characterized...

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Prevention of Vascular Inflammation by Nanoparticle Targeting

of Adherent Neutrophils

Zhenjia Wang, Jing Li , Jaehyung Cho, and Asrar B. Malik

Mice: Adult (6-8 weeks) male wild-type (C57BL/6), FcγRIII-/- (B6.129-Fcgr3<tm1Sjv>/j),

Mac-1-/- (CD11b/CD18), and LFA1-/- (CD11a/CD18) mice were purchased from Jackson

Laboratory (Bar Harbor, Maine). CD1 mice (6-8 weeks) were purchased from Charles

River Laboratories (Wilmington, MA). The University of Illinois Institutional Animal Care

and Use Committee approved all animal care and experimental protocols used in these

studies. All experiments were made under anesthesia using intraperitoneal injection of

the mixture of ketamine (80 mg/kg), xylazine (2 mg/kg), and acepromazine (2 mg/kg) in

saline. CD1 male mice were used in the LPS-induced acute lung injury studies.

Reagents and Antibodies: Lipopolysaccharide (LPS) and bovine serum albumin (BSA)

were purchased from Sigma (St. Louis, MO). Glutaraldehyde was obtained from

Electron Microscopy Sciences (Hatfield, PA). Piceatannol, the water-insoluble

Syk/Zap70 kinase inhibitor1,2, was purchased from Tokyo Chemical Industry (TCI)

(Pittsburgh, PA). Cy5 (HIDCI, 2-[5-(1,3-dihydro-1, 3, 3-trimethyl-2H-indol-2-ylidene)-1,

3-pentadienyl]-1, 3, 3-trimethyl-3H-indolium iodide, 635nm (excitation)/660nm

(emission)) was purchased from Exciton (Dayton, OH). Alexa Fluor-488 and Alex Fluor-

647-labeled anti-mouse Gr-1(Ly-6G/Ly-C6) and anti-mouse F4/80 antibodies, Alexa

Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2014.17

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

© 2014 Macmillan Publishers Limited. All rights reserved.

http://www.nature.com/doifinder/10.1038/nnano.2014.17

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Fluor-488-labeled IgG 2b κ as isotype control for anti-mouse Gr-1 and F4/80, and

recombinant mouse TNF-α were purchased from Biolegend (San Diego, CA).

Carboxylated polystyrene fluorescent yellow-green nanoparticles (100 nm in a diameter,

excitation/emission: 505nm/515nm) and Alex-Fluor 647-NHS for bio-conjugation were

purchased from Invitrogen (Grand Island, NY). EDC (1-Ethyl-3-(3-

dimethylaminopropyl)carbodiimide) and Sulfo-NHS (N-hydroxysulfo succinimide) were

purchased from Pierce (Rockford, IL). Diff-Quick staining kit was purchased from Fisher

Scientific. N-formyl-methionyl-leucyl-phenylalanine (fMLF) and other chemicals were

from Sigma (St. Louis, MO). Rabbit anti-phospho Syk (Tyr525/526) and anti-mouse β-

actin antibodies were purchased from Cell Signaling (Danvers, MA). CellTiter 96®

AQueous one solution cell proliferation assay was purchased from Promega (Madison,

WI). Isolated mouse lung endothelial cells (MLECs) were cultured as described3.

Purified human fibrinogen was kindly provided by Deane F. Mosher (University of

Wisconsin, Madison, WI).

Measuring size of albumin nanoparticles: The size of albumin nanoparticles was

measured using dynamics light scattering (Dyatt Inc.) at 633 nm. Diluted albumin

nanoparticle solution was spread on a copper grid (300 mesh) and the size of

nanoparticles was determined by transmission electron microscope (Jeol JEM-1220).

Synthesis of BSA-conjugated polystyrene nanoparticles: BSA molecules were

covalently conjugated with yellow-green polystyrene nanoparticles (excitation/emission:

505nm/515nm) with a diameter of 100 nm according to manufacturer’s instruction. To


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activate COOH groups on fluorescent polystyrene nanoparticles with EDC and Sulfo-

NHS for reaction of 15 min. The mixture was centrifuged using Nanosep-50K

(OD50C33) (Pall Inc.) and was dissolved in PBS buffer (pH 7.4). After activation of

nanoparticles, a defined amount of BSA solution was added to the activated particle

solution, and incubated for 2 hours at RT. Free BSA molecules were separated from

BSA-coated nanoparticles using Nanosep-300K centrifuge device (OD300C33). BSA-

coated nanoparticles were characterized by absorption spectra using Carey 300 Bio4. In

the study of uptake of BSA-coated nanoparticles in neutrophils (Figure 2e), we

intravenously infused 5 µl of 6.5 nM nanoparticles in each mouse.

In vitro cytotoxicity assay. Mouse neutrophils were isolated as described5.

Neutrophils (1 x 105 cells) were stimulated with or without 10 µM fMLF for 10 minutes at

37ºC. After washing, cells were resuspended in RPMI1640 medium and treated with

albumin nanoparticles or piceatannol-loaded nanoparticles, 800 µg/ml (200 µM as

piceatannol), at 37ºC for 1 hour. Cells were then incubated with MTT solution at 37ºC

for 4 hours in the dark. Absorbance was recorded using a plate reader (PHERAstar,

BMG biotech) at a wavelength of 490 nm. Incubation of MTT solution with each reagent

without cells was counted as the blank for each sample. Cell viability after treatment

with 150 µM H2O2 was used as a positive control.

Syk phosphorylation. Isolated mouse neutrophils (2 x 106 cells) were plated onto a

fibrinogen-coated wells of a 6 well plate and incubated with albumin nanoparticles or

piceatannol-loaded nanoparticles, 800 µg/ml (200 µM as piceatannol), at 37ºC in the

presence of 50 ng/ml TNF-α for 30 minutes. Non-adherent neutrophils were collected


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and lysed with RIPA buffer (Tris-HCl, pH 7.4 containing 1% Triton-X100, 0.05% SDS,

proteinase inhibitor cocktail, 1 mM PMSF, 1 mM Na3VO4, and 1 mM NaF). Also,

adherent neutrophils were lysed and combined with the lysate of non-adherent cells.

The final lysate was electrophoresed under reduced conditions and immunoblotted with

an anti-phosphoSyk-Tyr525/526 antibody. The band density was measured by

densitometry using Scion Image (v4.0).

Flow cytometric analysis. Mouse neutrophils treated with or without 10 µM fMLF were

incubated with 100 µg/ml of Cy5-labeled albumin nanoparticles, followed by incubation

with Alexa Fluor 488-conjugated anti-mouse Gr-1 antibodies. Cells were fixed and

analyzed by flow cytometry (Cyan ADP, Beckman Coulter). Mouse neutrophils were

identified by forward and side scatter as well as Gr-1 expression.

Flow chamber assay. A flow chamber assay was performed as described with

modifications5. Confluent mouse lung endothelial cells (MLECs) on fibrinogen-coated

glass coverslips were stimulated by murine TNF-α (20 ng/ml) for 6 hours and placed

into a parallel plate flow chamber (Bioptech). Mouse neutrophils, 2 x 106, were treated

with albumin nanoparticles or piceatannol-loaded nanoparticles, 800 µg/ml (200 µM as

piceatannol), for 20 minutes at 37ºC and then incubated with fMLF for 10 minutes. After

being washed and resuspended in RPMI1640 medium containing 0.1% BSA,

neutrophils were perfused for 10 minutes over activated MLECs under venous shear (1

dyne/cm2). The flow chamber and microscope system were kept at 37ºC in a plexiglass

chamber. Real-time images were acquired using a Nikon microscope (ECLIPSE Ti,

Melville, NY) equipped with 10 x/0.25 NA objective lens and recorded with a digital

camera (CoolSNAP ES2). The data were analyzed through NIS Elements (AR 3.2).


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Adherent (determined either as round and spread) and migrating neutrophils were

counted in a field of 0.15 mm2. The number of firmly adherent neutrophils was counted

in 5 separate fields.

Lung tissue myeloperoxidase (MPO) activity: Acute lung injury was induced by

intraperitoneal injection of LPS (10 mg/kg body weight) in male CD1 mice (6-8 weeks

old). At 2 hour after LPS injection, piceatannol-loaded albumin nanoparticles in saline

were injected into mice via tail vein. At 8 hours post-LPS injection, lungs were isolated,

and the half were used for MPO measurement and the others were fixed with 4%

paraformaldehyde for histological analysis. MPO activity in the lung tissues was

measured as described 6. Briefly, lungs were isolated and homogenized in 1 ml of PBS

(pH 6.0, 50 mM) containing 0.5% hexadecyltrmethylammonium bromide for 20 seconds.

The homogenates were centrifuged at 14,000 rpm for 20 minutes at 4°C, and the pellet

was collected and resuspended with the same buffer (0.5 ml). After homogenization and

centrifugation, the supernatant was collected and mixed with PBS containing 0.2 mg/ml

o-dianisidine hydrochloride and 0.05% H2O2 at a final concentration of 1/30 (v/v).

Absorbance change was measured at 460 nm for 3 minutes using DU-530/UV-Vis

spectrometer (Beckman Coulter). MPO activity was calculated as change in absorbance

over time and normalized with dry lung weight. To microscopically image infiltration of

neutrophils in lungs, chemo-staining was done as described7. Briefly, formalin-fixed,

paraffin-embedded tissue samples were sectioned at 5µm thickness and mounted on

Starfrost/Plus slides. Slides were incubated in Naphthol AS-D chloroacetate (Sigma)

according to manufacturer’s instructions. Slides were rinsed in distilled water, stained


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with hematoxylin, and mounted with aqueous solution. The slides were imaged using a

microscope and stained neutrophils were quantified per field (0.6 mm2).

Neutrophil and monocyte counts in mouse bronchoalveolar lavage (BAL). At 2

hour after intraperitoneal injection of LPS (10 mg/kg mouse weight), PBS, BSA

nanoparticles, or piceatannol-loaded BSA nanoparticles (piceatannol concentration at

4.3 mg/kg mouse weight) were intravenously infused into CD1 mice. At 6 hours later,

mouse BAL fluid was collected by inserting a needle into the upper trachea. Lavage was

performed by introducing 3 sequential 1 ml of PBS into the lungs and carefully

withdrawing BAL fluid. The BAL fluid was centrifuged at 1600 rpm for 5 min. Cells were

resuspended in 0.5 ml PBS and counted in a hemocytometer. Slides were also

prepared using an aliquot of each sample and stained with the mixture of xanthene dye

and thiazine dye using Diff-Quick staining protocol8. Neutrophils and monocytes were

quantified under a microscope.


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Movie 1. The merged intravital microscopic images show that Cy5-loaded albumin

nanoparticles (red) are internalized by leukocytes adherent to venular endothelial cells

following TNF-α-induced cremaster venular inflammation in WT mice.

Movie 2. Cy5-loaded albumin nanoparticles are internalized by adherent and the very

slowly moving Gr-1 positive neutrophils post-TNF-α-induced venular inflammation in WT

mice. Neutrophils were visualized by infusion of Alexa Fluor 488-conjugated anti-Gr-1

antibodies.

Movie 3. Cy5-loaded albumin nanoparticles are not internalized by circulating

neutrophils in non-TNF-α-treated cremaster muscle venules in WT mice. Neutrophils

were visualized by infusion of Alexa Fluor 488-conjugated anti-Gr-1 antibodies.

Movie 4. Cy5-loaded albumin nanoparticles are not internalized by monocytes on TNF-

α-stimulated venular endothelial cells in WT mice. Monocytes were visualized by

infusion of Alexa Fluor-488-labeled anti-mouse F4/80 antibodies.

Movie 5. Alexa Fluor-647-conjugated albumin nanoparticles (red) show puncta after

internalization by adherent neutrophils (green) on the TNF-α-stimulated venular

endothelial cells in WT mice. Puncta (red) in neutrophils represent single albumin


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nanoparticles and their aggregates. Neutrophils were visualized by infusion of Alexa

Fluor-488-labeled anti-mouse Gr-1 antibodies.

Movie 6. BSA-conjugated polystyrene nanoparticles (100 nm in diameter) bind to the

surface of adherent neutrophils without internalization post-TNF-α-induced venular

inflammation in WT mice. Neutrophils were visualized by infusion of Alexa Fluor-647-

labeled anti-mouse Gr-1 antibodies.

Movie 7-8. Neutrophil adhesion to TNF-α-stimulated venular endothelial cells in WT

mice before (movie 7) and 1 hour after piceatannol-loaded albumin nanoparticles

(movie 8). Neutrophils were visualized by infusion of Alexa Fluor-488-labeled anti-

mouse Gr-1 antibodies.


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Supplementary Figures

Figure S1. Scheme for preparing albumin nanoparticles using BSA (bovine serum

albumin) incorporated with fluorescent molecules or a drug.

BSAEthanol

Crosslinking

Nanoparticles

Drug or imaging agents


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Figure S2. Transmission electron microscopy (TEM) shows size of albumin

nanoparticles averaging 100 nm in a diameter. Scale bar: 500 nm.


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Figure S3. Distribution of size of albumin nanoparticles measured using dynamic light

scattering.

100 200 300 4000

10

20

30

Inte

nsity

(a.

u)

Particle Diameter (nm)


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Figure S4. Specificity of anti-Gr-1 antibody in mouse neutrophils. Alexa-Fluor-488

isotype matched IgG 2b, κ (1.5 µg/mouse) is the control. Alexa-Fluor-488 anti-mouse

Gr-1 (1.5 µg/mouse) is infused i.v. in 3 hr TNF-α-treated cremaster muscle venulee.

Cremaster muscle venules are imaged using intravtial microscope.

Control

Anti-Gr-1

Tissue IsotypeIgG

Merge

Tissue Anti-Gr-1 Merge


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Figure S5. Loading of piceatannol in albumin nanoparticles is dependent of the amount

of initially added piceatannol. Amount of picetannol loading in albumin nanoparticles

was determined using the absorption spectrum of piceatannol. Results represent mean

± SEM.

0.0 0.5 1.00.0

0.3

0.6

0.9

Pic

ea

tan

no

l-lo

ad

ed in

P

art

icle

s (m

g)

Added piceatannol (mg)


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Figure S6. Binding of albumin nanoparticles to fMLF-activated neutrophils. Mouse

neutrophils, 3 x 105, were stimulated with 10 µM fMLF for 10 minutes. Cells were

washed and resuspended in RPMI1640 medium, followed by incubation with Cy5-

loaded albumin nanoparticles (NP), 100 µg/ml, and 488-conjugated anti-Gr-1

antibodies. Cells were analyzed by flow cytometry. (A) Gating of isolated mouse

neutrophil is shown in R1. (B) Graph shows staining of Gr-1 and NP in R1 gating. (C-D)

Surface expression of Gr-1 and signal of NP on unstimulated and fMLF-stimulated

neutrophils. (E) Confocal image of a neutrophil internalizing Cy5-loaded albumin

nanoparticles

NP

even

t

- fMLF+ fMLF

Gr-1

even

t

- fMLF+ fMLF

R1

FS

SS

NP

Gr-1

R2R3

R5R499.62%

A B

C D E


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Figure S7. Lung tissue section stained with neutrophil-specific esterase for evaluating

effects of piecatannol-loaded albumin nanoparticles. Pink objects are neutrophils and

blue objects for cell nuclei. We quantified the numbers of neutrophils per field and

averaged over 20 fields (0.6 mm2) in 3 mice per group (results shown in Figure 4I)

PBS Pic-loaded Alb Nanoat 0.86 mg (pic)/kg

Pic-loaded Alb Nanoat 4.3 mg (pic)/kg


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5 Hahm, E. et al. Extracellular protein disulfide isomerase regulates ligand-binding activity of alphaMbeta2 integrin and neutrophil recruitment during vascular inflammation. Blood 121, 3789-3800 (2013).

6 Garrean, S. et al. Caveolin-1 regulates NF-kappaB activation and lung inflammatory response to sepsis induced by lipopolysaccharide. J. Immunol.177, 4853-4860 (2006).

7 Lomas-Neira, J. L., Chung, C. S., Wesche, D. E., Perl, M. & Ayala, A. In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury. J. Leukoc. Biol. 77, 846-853 (2005).

8 Huang, X., Wu, J., Spong, S. & Sheppard, D. The integrin alphavbeta6 is critical for keratinocyte migration on both its known ligand, fibronectin, and on vitronectin. J. Cell Sci. 111, 2189-2195 (1998).


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