Post on 28-Sep-2020
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