Supplementary Information
An Innovative Nanoformulation Utilizing Tumor Microenvironment
-
Responsive PEG
-
Polyglutamic Coating and Dynamic Charge Adjustment for Specific Targeting of ER Stress
Inducer, MicroRNA, and
Immunoadjuvant in Pancreatic Cancer: In Vitro Investigations
Ching
-
Yao Li
1
,
Tsui
-
Fen Chou
2,3
,
and
Yu
-
Li Lo
1,4
*
1
Department and Institute of Pharmacology, National Yang Ming Chiao Tung University, Taipei 112304,
Taiwan
2
Division of Biology and
Biological Engineering, California Institute of Technology, Pasadena, CA
91125, United States
3
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena,
CA 91125, United States
4
Faculty of Pharmacy, National
Yang Ming Chiao Tung University, Taipei 112304, Taiwan
*
Corresponding author. Tel: +886
-
2
-
2826
-
7095
E
-
mail address:
yulilo@nycu.edu.tw
(
Yu
-
Li Lo
)
Table S1.
Combination index (CI) and synergistic levels
a
CI
Description
<0.1
Very strong
synergism
0.1
-
0.3
Strong synergism
0.3
-
0.7
Synergism
0.7
-
0.85
Moderate synergism
0.85
-
0.90
Slight synergism
0.90
-
1.10
Nearly additive
1.10
-
1.20
Slight antagonism
1.20
-
1.45
Moderate antagonism
1.45
-
3.3
Antagonism
3.3
-
10
Strong antagonism
>10
Very antagonism
a
According to
Chou
–
Talalay method
[1]
Figure
S1.
(A) Measurement of intracellular intensity of
DiI
-
CB, miR
-
, and/or R
-
loaded
formulations
in NIT
-
1 cells for 24 h by flow cytometry. (B)
Evaluation of fluorescence intensity of CB, FAM
-
miR
-
,
and/or R
-
loaded formulations in
NIT
-
1 cells
for 24 h by flow cytometry. (AB)
at the equivalent
concentrations of CB: 210 nM, miR: 100 nM,
and R: 1.5
μ
M. The
mean
±
SD
of
results
from
three
independent experiments (n = 3; statistical significance at **
p
< 0.01; ***
p
< 0.001).
Figure
S2.
(A) Evaluation of the expression of EGFR, VCP, and PD
-
L1 in Panc
-
02 and NIT
-
1 cells
by western blot.
(B) PD
-
L1 targeting and subcellular localization of
FAM
-
miR+CB+R/SLN
-
CSW
in
Panc
-
02 cells for 1, 3, and 24 h as overserved by CLSM. Blue: Hoechst (a nuclear dye); green: ER;
red: DiI
-
CB; gray:
Alexa 680
-
PD
-
L1. Scale Bar: 20 μm. (C) EGFR
-
targeting and intracellular
distribution of
FAM
-
miR+CB+R/SLN
-
CSW
in Panc
-
02 cells for 1, 3,
and 24 h as imaged by CLSM.
Blue: Hoechst (a nuclear dye); green: ER; red: DiI
-
CB; gray: Cy5.5
-
EGFR. Scale Bar: 20 μm. (A
-
C)
Representative images
of
three repeated experiments
(n = 3)
.
Figure
S3.
(A)
Relative cell viability (%) of
Panc
-
02
cells treated with various CB concentrations
for
24 h
by
SRB assay.
(B)
Relative cell viability (%) for
the co
-
treatment of miR (50, 100, and 200 nM)
and CB (100, 200, and 400 nM).
The combination index (CI) was calculated to examine the degree
of drug in
teractions, whereas CI < 1, = 1, and > 1 specify synergism, additive effect, and antagonism,
respectively, based on Chou’s definition for drug combination therapies
[1]
.
Figure
S4.
Visualization of reactive oxygen species (ROS) stained with a fluorescent probe of 2',7'
-
Dichlorodihydrofluorescein diacetate (DCFH
-
DA)
using an OLYMPUS FV10i CLSM
after treatment
of Panc
-
02 cells with miR (100 nM)
-
, CB (210 nM)
-
, and/or R (1.5
μ
M)
-
loaded
formulations for 24
h. Blue: DAPI (a nuclear dye); green: ROS. Scale Bar: 20 μm.
Figure
S5.
(A) Visualization of HMGB1 expression by CLSM after treatment of Panc
-
02 cells by
miR (100 nM)
-
, CB (210 nM)
-
, and/or R (1.5
μ
M)
-
loaded formulations for 24 h. Blue: Hoechst (a
nuclear dye); green: ER;
red
:
Alexa 680
-
HMGB1. Scale Bar: 20 μm. (B) Detection o
f intracellular
ATP level using ATP Detection Assay Kits after the above treatment
.
The
mean
±
SD
of
results
from
three independent experiments (n = 3; statistical significance at **
p
< 0.01; ***
p
< 0.001
.
References
[1] T.C. Chou, Drug c
ombination studies and their synergy quantification using the Chou
-
Talalay
method, Cancer Res 70 (2010) 440
-
6.