S1

Supplementary material for

Tailored Black Phosphorus for Erythrocyte Membrane Nanocloaking

with Interleukin-1α siRNA and Paclitaxel for Targeted, Durable, and

Mild Combination Cancer Therapy

Wenquan Ou1, Jeong Hoon Byeon

2*, Zar Chi Soe

1, Bo Kyun Kim

1, Raj Kumar Thapa

1, Biki Gupta

1,

Bijay Kumar Poudel1, Sae Kwang Ku

3, Chul Soon Yong

1, Jong Oh Kim

1*

Mr. Wenquan Ou, Mr. Zar Chi Soe, Mr. Bo Kyun Kim, Mr. Raj Kumar Thapa, Dr. Biki Gupta, Dr.

Bijay Kumar Poudel, Dr. Chul Soon Yong, and Dr. Jong Oh Kim

1. College of Pharmacy, Yeungnam University, Gyeongsan 38541, Republic of Korea.

Dr. Jeong Hoon Byeon

2. School of Mechanical Engineering, Yeungnam University, Gyeongsan 38541, Republic of Korea.

Dr. Sae Kwang Ku

3. College of Korean Medicine, Daegu Haany University, Gyeongsan 38610, Republic of Korea.

*Corresponding authors: J. H. Byeon. Email: postjb@yu.ac.kr and J. O. Kim. Email:

jongohkim@yu.ac.kr

S2

Figure S1

In-flight tailoring of coarse BP flakes and basic material characterizations. (A) A schematic of in-

flight tailoring for the continuous production of size-classified (tailored) BP particles. Coarse BP flakes

were first pulverized using a probe sonicator after being dispersed in deoxygenated water (1 mg/mL).

The dispersion was mechanically sprayed as droplets with nitrogen gas, and the droplet-laden nitrogen

gas flow entered a diffusion dryer to form BP aerosol particles by absorbing water molecules. The

aerosols were then injected into a NDMA after passing through a soft X-ray charger (creating

Boltzmann charge distribution) for a size classification of 60 nm. (B) Size distributions of untailored

(nonclassified; GMD = 61.3 nm, GSD = 1.55) and tailored (classified; GMD = 60.0 nm, GSD = 1.08)

BP particles. (C) TEM images of the BP particles. The classified configuration exhibited a more

uniform distribution than that of the nonclassified configuration. The high-magnification images show

the wrinkled shape and characteristic microstructure of BP (021 plane). (D, E) Concentration-dependent

cytotoxicities (MC-38 cells) and NIR-activated (808 nm, 0.5 W/cm2, 5 min) temperature elevations of

the tailored (naked) and EM-cloaked BP specimens. The tailored BP exhibited better biocompatibility

than that of the untailored BP, and this was further enhanced by EM cloaking. Cloaking restrained the

temperature elevations of naked BP particles (ΔT ≤ 9oC) to enhance the mild hyperthermic

microenvironment.

0.0E+00

5.0E+05

1.0E+06

1.5E+06

2.0E+06

2.5E+06

3.0E+06

3.5E+06

4.0E+06

4.5E+06

5.0E+06

0.0E+00

5.0E+06

1.0E+07

1.5E+07

2.0E+07

2.5E+07

3.0E+07

3.5E+07

4.0E+07

4.5E+07

5.0E+07

1 10 100 1000

dN

/dlo

gD

p (

pa

rtic

les/

cm3)

Equivalent mobility diameter (nm)

Nonclassified

Classified

HV

ELECTROSTATIC CLASSIFIERBOLTZMANN CHARGE

DISTRIBUTION

50 nm 50 nm

10 nm

0.34 nm

Wrinkled

Region

COARSE BP

ULTRASONIC

BUBBLER

NONCLASSIFIED CLASSIFIED-60 nm

MICROSTRUCTURE

GMD = 61.3 nm

GSD = 1.55

= 60.0 nm

= 1.08

A B

C

D E

0

2

4

6

8

10

12

14

16

18

20

0 2 4 6

Tem

per

atu

re e

lev

ati

on

(oC

)

Irradiation time (min)

Naked BP (200 μg/mL)

Naked BP (100 μg/mL)Naked BP (50 μg/mL)

BP-H@EM-YSA (200 μg/mL)

BP-H@EM-YSA (100 μg/mL)BP-H@EM-YSA (50 μg/mL)

PBS

0

20

40

60

80

100

0.1 1 5 10 50

Cel

l v

iab

ilit

y (

%)

Concentration (μg/mL)

Nonclassified BP Classified BP BP (Classified)-H@EM-YSA

S3

Figure S2

Zeta potential changes of BP-H (H-grafted BP) particles after ILsi loading (N = 3). The decrease in the

positive potential of BP-H could be the result of ILsi’s negative charge, representing successful loading

of ILsi on BP-H.

Figure S3

Changes in EE and LC of the nanosystem as a function of siRNA content (N = 3).

si content(%)

S4

Figure S4

FTIR spectrum of anchorable YSA (YSA-PEG2000-DSPE) (A) and XRD profile of BP-H-ILsi-X@EM-

YSA (B) with spectra of individual components to verify the loading of functional molecules on core

BP particles.

Figure S5

Western blot analyses of CD47 and CD235a proteins in EMs before and after the assembly of BP-H-

ILsi-X@EM-YSA.

N-H C=O

-OH

C-N

C=O

A B

DSPE-PEG2000-COOH

Pristine YSA

YSA-PEG2000-DSPE

H

EM-YSA

X

BP

BP-H-ILsi-X@EM-YSA

4000

Tra

nsm

itta

nce

(%

)

4000 3500 3000 2500 2000 1500 1000

Wavenumber (cm-1)

6000

2000

0

2θ (o)

5 10 15 20 25 30 35 40 45 50 55 60 65

Inte

nsi

ty (

a.u

.)

GAPDH

CD47

CD235a

gapdh

S5

Figure S6

(A) Absorbance monitoring of DPBF at 410 nm for 5 min NIR irradiation (808 nm, 0.5 W/cm2) after

treatment with naked BP particles and BP-H-ILsi-X@EM-YSA nanosystems to examine the generation

of singlet oxygen (as potent ROS generation). The concentration of BP for this monitoring was 100

µg/mL (N = 6). (B) Monitoring of the DLS size and X content of the nanosystem for 10 h after being

dispersed in distilled water, PBS, and mouse serum to examine dispersion stability (N = 6; **

p < 0.01).

0 60 120 180 240 300

Time (s)

1.5A

bsorban

ce

1.0

0.5

0

Naked BP

BP-H-ILsi-X@EM-YSA

in distilled waterin PBSin serum

Size (nm) X (% )in distilled waterin PBSin serum

200

Parti

cle

siz

e (n

m)

150

100

50

0

X c

on

ten

t (%

)

100

80

60

40

20

00 2 4 6 8 10

Time (h)

S6

Figure S7

(A) In vitro measurement of IL secretion in MC-38 cells after treatment with BP-H-ILsi-X@EM-YSA

nanosystem as a function of ILsi concentration in the nanosystem using flow cytometry. Relative levels

of ILm in MC-38 cells determined by qRT-PCR after treatment with (B) the nanosystem as a function

of ILsi concentration in the nanosystem or (C) the different configurations (1–6) (N = 6). CCL22

expressions and corresponding m levels in RAW264.7 macrophages (D, E) or BMDCs (F, G) after

being co-cultured with MC-38 cells pretreated with the different configurations (1–6) (N = 6; **

p <

0.01).

100

IL

600

Cou

nt

400

200

0101 102 103 104

PBSControlBP-H-ILsi-X

@EM-Scr5102550

**

****

(1)(2)(3)(4)(5)(6)

Rela

tive

IL

m

ex

pre

ssio

n

0.8

0.6

0.4

0.2

0 Rela

tive

IL

m

ex

pre

ssio

n

2.0

1.5

1.0

0.5

0

(1): Control

(2): ILsi-X

(3): BP-H-X@EM

(4): BP-H-ILsi-X@EM

(5): BP-H-ILsi-X@EM-YSA

(6): BP-H-ILsi-X@EM-Scr

CC

L22 (

pg

/mL

)

20

40

60

80

0

**

****

RAW264.7

**

****

**

**

**

(1)(2)(3)(4)(5)(6) (1)(2)(3)(4)(5)(6)

RAW264.7

Rela

tive

CC

L22m

expre

ssio

n

2.0

1.5

1.0

0.5

0

CC

L22 (

pg/m

L)

1000

1500

500

0

DC

(1) (2) (3) (4) (5) (6)

Re

lati

ve C

CL

22

m

ex

pre

ssio

n

2.5

2.0

1.5

1.0

0.5

0

DC

**

****

(1) (2) (3) (4) (5) (6)

5 10 25 50

nM

nM

S7

Figure S8

Viabilities of MC-38 (A) and 293T (B) cells incubated with BP, free X, BP@EM-YSA, BP-H-X@EM-

YSA, and BP-H-ILsi-X@EM-YSA for 24 h in the absence and presence of NIR irradiation (808 nm, 0.5

W/cm2, 5 min) using MTT assay (N = 6).

Figure S9

(A) Digital images and (B) percentages of EM hemolysis at different concentrations of nonclassified

BP, classified BP, and BP (classified)-H-ILsi-X@EM-YSA (N = 3).

A B

A B

(μg/mL)

S8

Figure S10

Photothermal contours of the treated (PBS, BP-H-ILsi-X@EM, BP-H-ILsi-X@EM-YSA) mice (A) and

corresponding temperature elevation profiles in the tumor regions (B) during NIR irradiation (808 nm,

0.5 W/cm2, 5 min, N = 6).

Figure S11

Mean serum levels of IFN-γ (A) and TNF-α (B) in mice treated with the different configurations (1–6)

(N = 6; **

p < 0.01).

A B

80

IFN

-γ(p

g/m

L)

60

40

20

0(1) (2) (3) (4) (5) (6) (1) (2) (3) (4) (5) (6)

**

**

**

**

**

**100

TN

F-α

(pg

/mL

)

80

60

40

20

0

(1): PBS

(2): ILsi-X

(3): BP-H-ILsi-X@EM

(4): BP-H-ILsi-X@EM-YSA

(5): BP-H-X@EM-YSA (NIR)

(6): BP-H-ILsi-X@EM-YSA (NIR)

S9

Figure S12

Representative digital images of tumor masses isolated from MC-38 tumor-bearing mice treated with

the different configurations.

Figure S13

Immunohistochemical analysis of Ki-67 and CD31 in tumor tissues after treatment with the different

configurations.

PBS

ILsi-X

BP-H-ILsi-X@EM

BP-H-ILsi-X@EM-YSA

BP-H-X@EM-YSA (NIR)

BP-H-ILsi-X@EM-YSA (NIR)

PBS ILsi-X BP-H-ILsi-X@EM

BP-H-ILsi-X@

EM-YSA

BP-H-X@

EM-YSA (NIR)

BP-H-ILsi-X@

EM-YSA (NIR)

Ki-

67

CD

31

120 μm

S10

Figure S14

Representative H&E staining of the heart, liver, spleen, lung, and kidney in MC-38 tumor-bearing mice

treated with the different configurations.

Heart Liver Spleen Lung Kidney

PB

SIL

si-X

BP

-H-I

Lsi

-X@

EM

BP

-H-I

Lsi

-X@

EM

-YS

A

BP

-H-X

@

EM

-YS

A (

NIR

)

BP

-H-I

Lsi

-X@

EM

-YS

A (

NIR

)

120 μm

S11

TABLE S1

Histomorphometrical analysis of tumor masses taken from MC-38 tumor-bearing C57BL/6 mice

Values are expressed as the mean ± SD of six tumor mass histological fields.

Treatment groups: G1 = PBS, G2 = ILsi-X, G3 = BP-H-ILsi@EM, G4 = BP-H-ILsi-X@EM-YSA, G5 = BP-H-

X@EM-YSA (NIR), and G6 = BP-H-ILsi-X@EM-YSA (NIR).

PECAM-1: platelet/endothelial cell adhesion molecule-1.

ap < 0.01, as compared with G1 by Mann–Whitney (MW) test. bp < 0.01, as compared with G2 by MW test. cp < 0.01 and dp < 0.05, as compared with G3 by MW test. ep < 0.01, as compared with G4 by MW test. fp < 0.01, as compared with G5 by MW test.

Item

Group

Tumor cell volume

(%/mm2)

Immunoreactive cell percentage

(%/mm2 of tumor mass)

Immunoreactive cell number

(cells/mm2 of tumor mass)

Ki-67 CD31 (PECAM-1) CD8+ Foxp3

Control (G1) 77.56 10.95 62.99 12.48 50.27 13.16 65.67 14.39 375.00 60.49

Treatment

G2 76.95 11.34 58.06 13.54 47.96 11.65 69.33 13.19 363.67 48.90

G3 54.60 7.47ab 38.29 5.22ab 28.62 4.24ab 167.33 24.74ab 150.33 18.26ab

G4 39.37 6.69abd 29.96 2.47abc 20.40 2.32abc 237.33 35.55abc 88.50 15.90abc

G5 28.18 4.64abce 17.46 2.01abce 12.91 2.85abce 248.00 180.60abce 221.00 48.67abce

G6 15.38 3.85abcef 8.54 3.95abcef 5.13 1.62abcef 3052.33 944.75abcef 43.83 19.17abcef