1

Supplementary Data

Not Nanocarbon but Dispersant Induced Abnormality in Lysosome in

Macrophages In Vivo

Masako Yudasaka*1, Minfang Zhang

1, Sachiko Matsumura*

2, Ryota Yuge

3, Toshinari Ichihashi

3, Hiroshi

Irie4, Kiyotaka Shiba

2, Sumio Iijima

1, 3, 5

1 National Institute of Advanced Industrial Science and Technology, 1-1-1 Higashi, Tsukuba, 305-8565,

Japan

2 Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550, Japan

3 NEC Corporations, Smart Energy Research Laboratories, 34 Miyukigaoka, Tsukuba, 305-8501, Japan

4 Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi, Tokyo, Japan

5 Meijo University, Shiogamaguchi, Tempaku, Nagoya, 468-8502, Japan

Equally contributed: M. Yudasaka, M. Zhang, S. Matsumura

Corresponding authors: M. Yudasaka (m-yudasaksa@aist.go.jp), S. Matsumura

(sachiko.matsumura@jfcr.or.jp)

Supplementary Data 1. Optical microscopy observation of liver and spleen tissues of

glucose-CNH administered mouse

Black CNH agglomerates were observed in mouse liver tissues (Figures SD 1a, 1b) at 14

days post-injection of glucose-CNH into a tail vein. The tissue was stained with nuclear fast

red and berlin blue in Figure SD 1a. The berlin blue gives blue color to hemosiderin. The blue

color was observed in the tissue as indicated with red circle in Figure SD 1a, where the black

spots of CNHs were also localized. There were not so many places stained with berlin blue in

liver, which was the same with the control mouse (no CNH administration). In the

STEM/TEM observation, CNHs were often found in lysosomes of macrophages and

coexisted with Fe-containing substances. Therefore, we infer that most of the CNHs did not

exist with hemosiderin in macrophages or the black color of CNHs hindered the observation

of the blue color.

The macrophages cells exhibit brown color by anti Iba-1 antibody staining. When the

tissues were stained with the anti Iba-1 antibody (Figures SD 1b), CNHs were found to be

resident within macrophages (stained brown). The observation indicated that CNHs were

resident within macrophage, which coincided with the STEM/EDX observation (see text).

2

We also stained spleen tissues with nuclear fast red and berlin blue (Figures SD 1c to 1f).

These staining showed that the CNHs (black spots) were present with hemosiderin (blue

spots) as seen in Figures SD 1c-1d and the CNHs were localized in macrophages at marginal

zones and red pulps (Figures SD 1e-1f). These results suggest that the CNHs were engulfed

by splenic macrophages and, coexisted with hemosiderin within the macrophages.

Figure SD-1. Optical microscopy images of liver (a, b) and spleen (c-f) tissue samples

taken from mice after 14 days post-intravenous injection of glucose-CNHs. Tissues were

stained with nuclear fast red and berlin blue (a, c, d) or stained with an anti Iba-1 antibody (b,

e, f).

a b

c

f

10 mm10 mm

100 mm

10 mm

Liver Liver

Spleen

Spleen

10 mm

Spleen

100 mm

Spleene

d

Nuclear fast red, Berlin blue

anti Iba-1

anti Iba-1

Nuclear fast red, Berlin blue Nuclear fast red, Berlin blue

anti Iba-1

3

Supplementary data 2. Optical microscopy observation of liver and spleen tissues of

CPEG-CNH administered mouse

The histological observation with optical microscopy showed the same tendency as seen for

the glucose-CNH cases (Figure SD 2).

Figure SD-2. Optical microscopy images of liver (a, b) and spleen (c-f) tissue samples

taken from mice after 7 days post-intravenous injection of CPEG-CNHs. Tissues were stained

with nuclear fast red and berlin blue (a, c, d) or stained with an anti Iba-1 antibody (b, e, f).

f

20 mm100 mm

e

50 mm

c

20 mm

d

10 mm10 mm

a bLiver Liver

Spleen Spleen

Spleen Spleen

Hemaotoxylin, anti Iba-1Nuclear fast red, Berlin blue

Nuclear fast red, Berlin blue Nuclear fast red, Berlin blue

anti Iba-1 anti Iba-1

4

Supplementary data 3. High resolution transmission electron microscopy (HRTEM)

observation of hemosiderin clusters.

Structure of hemosiderin attached to carbon nanohorns surface localized in macrophages in

spleen tissue of SIC:ICR mouse after 7 days from the injection was observed with HRTEM

(Topcon 002B, 120 kV). The tissue was fixed with osmium but not stained with uranium nor

lead. The HRTEM micrographs of hemosiderin clusters showed that hemosiderin had

amorphous-like structure and did not show any fringes characteristic of any ordered

structures (Figure SD 3). If the particles attached to CNHs were not hemosiderin but ferritin,

the lattice fringe of ferrihydite which is characteristic of ferritin1 should be observed by

HRTEM as reported by Chasteen et al.1

Thus we consider that the iron-rich particles attaching

to the CNH surfaces were hemosiderin but not ferritin.

5

Figure SD 3. HRTEM micrographs of a spleen tissue of SIC:ICR mouse after 7 days from

the injection of CNHs observed with Topcon 002B (Lab6 electron gun, 120 kV acceleration

voltage). Here the tissues were not stained. A macrophage (a) had a lysosome (b) that does

not have the limiting membrane. In the lysosome, CNHs (yellow asterisks) and hemosiderin

clusters (dark particles such as those indicated with a yellow arrow) existed. Magnified

images of the hemosiderin clusters did not show any regular lattice fringes (d, e; yellow

arrows) characteristic of ferritin1, suggesting that they were hemosiderin clusters but not

a b

c

d e

**

*

6

ferritin.

Supplementary Data 4. Electron microscopy observation of liver of a mouse in which

glucose-CNHs were intravenously injected.

Figure SD 4.1 indicates the liver tissue of a mouse, in which glucose-CNH was

intravenously injected at a dose quantity of 1 mg/kg. This dose was 1/10 of that introduced in

the main text. The other experimental conditions were the same as written in the main text.

Abnormality in the liver tissues was not found in the electron microscopy observation.

Figure SD 4.1 shows the liver tissue and the magnified images of phagosomes of

macrophages. A lot of CNHs were localized in the phagosomes (Figure SD 4.1 b-c),

The images of control mouse, in which glucose solution was injected, is shown in Figure

SD 4.2. Indeed, no CNHs were found in the liver tissues.

Figure SD 4.1. STEM images of liver tissues. The Magnified images (b, c) show

phagosomes enclosing CNHs.

1 mm1 mm

1 mm

a

b c

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Figure SD 4.2. STEM images of liver tissues of a control mouse. A macrophage is visible in

the image (a) and a part of it is shown with higher magnification (b).

Supplementary Data 5. Electron microscopy observations of liver and spleen of a

tumor-bearing mouse in which phospholipid-CNHs were injected.

The experimental details are reportedly shown previously.2 Briefly, CNHs in which Gd2O3

nanoparticles were embedded were prepared and coated with phospholipid PEG in 5%

glucose solution (PLPEG-CNH). PLPEG used in this study was

N-(carbonyl-methoxypolyethyleneglycol5000)-1,2-distearoyl-sn-glycero-3-phospho

ethanolamine, sodium salt (SUNBRIGHT DSPE-050CN; NOF Corp., Tokyo). The

concentration of CNH was 2 mg/mL.

Female 6-week-old BALB/cAnNCrlCrlj mice (Japan Charles River Co.) were housed in

Japanese Foundation for Cancer Research laboratory. Vascular endothelial growth factor

(VEGF)-secreting SBC-3 cells (human small cell lung cancer cell line, SBC-3/VEGF)

provided by Dr. Y. Matsumura (National Cancer Center Hospital East, Chiba) was

subcutaneously injected into the right backs of mice. Fourteen days after, PLPEG-CNH

solution was injected via the tail vein at a dose of 0.2 mL. The mice were sacrificed 21 hours

later, and their organs were removed and fixed in 2% glutaraldehyde in 0.1 M phosphate

buffer (pH7.4). The fixed tissues were treated with osmium tetroxide solution (1%, 70%, and

100% each for 30 min), propylene oxide (1 h, twice), and Epon embedding medium (3 days,

60 C). The Epon-embedded blocks were sectioned and stained with uranyl acetate for 10

min and Reynolds lead for 3 min. The stained sections were observed with STEM and TEM

(Hitachi HD-2300) at an acceleration voltage of 120 kV and 61 mA current. All animal care

and experimentation were performed in compliance with the Guidelines for Animal

Experiments at the Japanese Foundation for Cancer Research.

Typical STEM images of liver are shown in Figure SD 5.1. CNHs were localized in

phagosomes in macrophages. Except the engulfment of CNHs by macrophages, unusual

6 mm 1.5 mm

8

phenomena were not found.

The tissues of spleen were similarly observed with STEM and TEM. The CNHs were

localized in the vascular endothelial cells and in follicles/cells. The unusual membrane

degradation was not observed (Figure SD 5.2).

Figure SD 5.1. Electron microscopy images of CNHs in liver. (a) A large agglomerate of

CNHs globular-aggregates are localized inside a macrophage. (b-e) Magnified images of

parts of the agglomerate in (a). (H: Hepatocyte, N: Nucleus). Z-contrast images observed in

scanning transmission electron microscopy (a-d) and transmission electron microscopy image

(e).

500 nm

100 nm 100 nm

3 mm

500 nm

CNHs

CNH aggregates

CNHsCNHs

Macrophage

N

H

a b

c

d e

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Figure SD 5.2. Electron microscopy images of CNHs in spleen. (a) CNHs existed in vascular

endothelial cells (*) and follicles/cells (*). (b) Magnified image of an endothelial cell in (a).

(c, d) Magnified images of CNHs in the endothelial cell. Particles indicated with white

arrows are Gd2O3 nanoparticles that are embedded within the CNH as labels for electron

microscopy observation.

Supplementary Data 6. Electron microscopy observation of liver and spleen tissues of a

mouse in which CPEG-CNH was injected. (This mouse was another one in a group (n=5) in

which the mouse shown in the main text was included.)

Accumulation of CNHs and hemosiderin in lysosome and the degradation of the lysosome

membrane in liver and spleen were also observed in a different mouse as shown below.

Experimental conditions were the same as described in the main text. CPEG-CNHs were

injected in tail veins, and the mice were sacrificed and dissected after 7 days.

SD 6.1. Liver

The image of Figure SD 6.1a shows macrophage in the hepatic sinusoid. The magnified

image of the area in a yellow box (Figure SD 6.1 c-f) shows the existence of CNHs (Figure

SD 6.1 f, yellow asterisks). The dark linear lines, corresponding to the side view of the

nanohorn tubule walls, are visible in Figure 6.1 f. The iron of hemosiderin located around

CNHs was detected by EELS measurement at the three bright particles indicated with yellow

arrows in Figure SD 6.1 c. A typical EELS is presented in Figure 6.1 b which was measured

at a bright particle designated with “A” in Figure SD 6.1 c. The hemosiderin particles seem to

1 mm 200 nm

60 nm200 nm

600 nm6 mm

60 nm TEM 60 nm

TEM

CNH aggregates

CNH aggregates

A CNH-aggregate A CNH-aggregate

One CNH-aggregate

A CNH-aggregate A CNH-aggregate

*

*

*

** *

*

*

a b

c d

e f

g h

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be agglomerated with CNHs (Figures SD 6.1 d-f). At a blue arrow point, iron was not

detected by EELS measurements. Lysosome membranes were not obviously seen in STEM

and TEM images, indicating that the membranes could be degraded. These results coincided

with those presented in the main text, suggesting that these phenomena were not for the

special mice.

Figure SD 6.1. STEM and TEM images of liver tissues (a, c-f). An EELS (b) was measured

at a position indicated with the yellow arrow “A” in (c). The other yellow arrows in (c)

indicate the places where EELS showed iron peaks, while a blue arrow indicates the place

where EELS did not show iron peaks. Yellow asterisks denote CNHs in “f”. Images of “c-f”

were taken from the same places.

A

Measured at A in “b”.

EELS

STEM

STEM

STEM TEM

TEM

Co

un

ts

660 700 740 780Energy(eV)

*

*

*

*

*

*

*

Fe L3Fe L2

6 mm

500 nm

500 nm 100 nm

500 nm

a

c

d

b

e

f

11

SD 6.2. Spleen

CNHs were also found in lysosomes of spleen macrophages (Figures SD 6.2 a-d). The

images showed the same conclusion written in the main text: The lysosome membranes were

not clearly observed (Figures SD 6.2 c, d) and TEM (Figures SD 6.2 b) images. Iron of

hemosiderin was found around CNHs (Figure SD 6.2 d). A typical EELS with iron peaks are

shown in Figure SD 6.2 e. The iron was not detected at blue arrow point.

Figure SD 6.2. STEM-TEM images of spleen tissues (a-d). An EELS measured at a position

indicated with “A” in (d) is shown in (e). The red arrows indicate the places where EELS

showed iron peaks, while a blue arrow indicates the place where EELS did not show iron

peak. Yellow asterisks in “d” denote CNHs. Images of “b” and “d” were taken from the same

place.

SD 6.3. STEM images of liver and spleen tissues of control mouse.

The mouse experimental condition was the same as written in the main text except the

injected materials. PBS solution of CPEG (dose of CPEG: 4.6 mg/kg) was intravenously

injected from the tail vein of mice and the mice were sacrificed and dissected after 7 days.

The liver and spleen tissues did not show any abnormality in the STEM observation. Typical

images are shown in Figure SD 6.3.

A

AFe L3

Co

un

ts

EELS spectrum

660 700 740 780Energy(eV)

Fe L2

STEM STEM

TEM STEM150 nm 100 nm

600 nm6 mm

Measured at A in “b”.

a

b

c

d

e

**

12

Figure SD 6.3. STEM images of liver (left) and spleen (right) tissues of control mouse.

References

1. Chasteen ND, Harrison PM. Mineralization in ferritin: An efficient means of iron storage. J. Struct. Biol. 1999; 126: 182-94.

2. Matsumura S, Yuge R, Sato S, Tomida A, Ichihashi T, Irie H, Iijima S, Shiba K, Yudasaka M. Ultrastructural localization of intravenously injected carbon nanohorns in tumor. Int.

Nnational J. Nanomedicine 2014; 9: 3499-508

6 mm 3 mm

Liver Spleena b