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Supporting information for: Endosome-triggered ion-releasing
nanoparticles as therapeutics to enhance the angiogenic efficacy of human
mesenchymal stem cells
Gwang-Bum Im 1,†, Euiyoung Jung 2,3,†, Yeong Hwan Kim 1, Yu-Jin Kim 1, Sung-Won Kim 1,
Gun-Jae Jeong 4, Tae-Jin Lee 1, Dong-Ik Kim 4, Jinheung Kim 3, Taeghwan Hyeon 5, 6,
Taekyung Yu 2,*, Suk Ho Bhang 1,*
1School of Chemical Engineering, Sungkyunkwan University, Suwon 16419, Republic of
Korea
2Department of Chemical Engineering, Kyung Hee University, Youngin 17104, Republic of
Korea
3Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750,
Republic of Korea
4Division of Vascular Surgery, Samsung Medical Center, Sungkyunkwan University School of
Medicine, Seoul 06351, Republic of Korea
5Center for Nanoparticle Research, Institute for Basic Science (IBS), Seoul 08826, Republic
of Korea
6School of Chemical and Biological Engineering, and Institute of Chemical Process, Seoul
National University, Seoul 08826, Republic of Korea
†These authors contributed equally to this work.
*Author to whom correspondence should be addressed: 1
Suk Ho Bhang, Ph.D., E-mail: [email protected], Tel.: +82-31-290-7242, Fax: +82-31-
290-7272
Taekyung Yu, Ph.D., E-mail: [email protected], Tel.: +82-31-201-2064, Fax: +82-31-204-
8114
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Content list
Materials and methods 4-12
Supporting Figures 13
Fig. S1 13
Fig. S2 14
Fig. S3 15
Fig. S4 16
Fig. S5 17
Fig. S6 18
Fig. S7 19
Fig. S8 20
Fig. S9 21
References 22
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Additional Methods Section
Characterization. TEM images and EDS profiles were captured using a field-emission
electron microscope (JEM-2100F, JEOL, Tokyo, Japan) operating at 200 kV. XRD patterns
were obtained using an X-ray diffractometer (D-MAX/A, Rigaku, Tokyo, Japan) at 35 kV
and 35 mA. The UV–Vis spectra were recorded using a Jasco UV–Vis spectrophotometer
(Cary 60 UV–vis, Agilent Technologies, Santa Clara, CA, USA) within the range of 250–850
nm. The elemental ratio of ETIN was measured using a direct reading Echelle inductively
coupled plasma (ICP) spectrometer (Direct Reading Echelle IPC, Leeman, Hudson, USA).
Cell culture. hMSCs were purchased from Lonza (Basel, Switzerland). The hMSCs were
cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL), supplemented with
10% (v/v) fetal bovine serum (Gibco BRL), and 1% (v/v) penicillin/streptomycin (Gibco
BRL). The cells were incubated at 37 °C with 5% CO2 saturation. The medium was changed
every 2 days. Cells within 8 passages were used for the experiments. For hypoxic cell culture,
hMSCs were incubated in a serum-free medium with 2% O2 for 48 h. For proliferation and
cell viability test in hypoxic cell culture, HDFs and hMSCs were incubated in hMSC CM or
ETIN CM with 2% O2 for 72 h. For CM extraction, hMSCs were treated with or without
ETIN for 1 hour. hMSC CM or ETIN CM was extracted from hMSCs culture dish on 1 day
after treatment. To confirm effect of Fe ion to hMSCs, FeCl3 (Sigma) solution was treated to
hMSCs for 1 and 3 h.
Measurement of cytotoxicity of ETIN. Cell viability was evaluated using a CCK-8 assay
(Dojindo Molecular Technologies, Inc., Kumamoto, Japan). The CCK-8 assay measures the
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amount of formazan dye that is reduced by intracellular dehydrogenase activities. The
number of living cells is proportional to the amount of formazan dye. Briefly, hMSCs (1 ×
104 cells/well in 400 μL serum-free medium) were cultured on 24-well plates with various
concentrations of ETIN for 24 h and rinsed with PBS three times. After replenishing the wells
with fresh medium, CCK-8 solution was added into each well, and the cells were incubated
for 2 h. Then, the absorbance was measured at 450 nm using a plate reader (Infinite F50,
Tecan, Männedorf, Switzerland). The cell viability was calculated as the percentage of viable
cells relative to the ETIN-untreated cells (n = 4 per group). A TUNEL assay was performed
using ApopTag® Fluorescent In Situ Apoptosis Detection Kit (Millipore, Bedford, USA)
according to the manufacturer’s instruction to examine the apoptotic activity of hMSCs,
which were cultured with ETIN for 24 h. Cellular membrane and cell adhesion was evaluated
by DiI (Sigma-Aldrich) staining. After cells were treated with various concentration of ETIN
for 24 h, the cells were treated with the DiI solution (6.25 µM) and incubated for 30 min at 37
°C. The cells were then washed twice in PBS. Cells were fixed with 4% paraformaldehyde
solution for 10 min and washed in PBS. After 4',6-diamidino-2-phenylindole (DAPI, Vector
Laboratories, Burlingame, USA) staining, DiI fluorescence was measured using a
fluorescence microscope (IX71, Olympus, Tokyo, Japan). Live/dead assays were performed
with FDA (sigma) and EB (sigma). FDA (green) stains the cytoplasm of viable cells, whereas
EB (red) stains the nuclei of nonviable cells. The staining solution was freshly prepared by
mixing 10 mL of FDA stock solution (1.5 mg/mL of FDA in dimethyl sulfoxide), 5 mL of
EB stock solution (1 mg/mL of EB in PBS), and 3 mL of PBS. Then, the staining solution
was applied to the cells, and the cells were incubated for 3–5 min at 37 °C. After staining, the
samples were washed twice with PBS and examined using a fluorescence microscope (DFC
3000 G, Leica, Wetzlar, Germany).
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Quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR was used to
quantify the relative gene expression levels of VEGF, FGF2, CXCL12, CXCR4, CD31, and
SM-α. Total ribonucleic acid (RNA) was extracted from samples (105 cells per each sample)
using 1 mL TRIzol reagent (Life Technologies, Inc., Carlsbad, CA, USA) and 200 μL
chloroform. The lysed samples were centrifuged at 12,000 rpm for 10 min at 4 °C. The RNA
pellets were washed with 75% (v/v) ethanol in water and dried. After drying, samples were
dissolved in RNase-free water. For qRT-PCR, the SsoAdvanced™ Universal SYBR Green
Supermix kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instruction and
the CFX Connect™ real-time PCR detection system (Bio-Rad) according to the
manufacturer’s instruction were used. Table 1 shows the primers used for qRT-PCR.
Table 1. Primer sequences of qRT-PCR
Primer Sequence
Human
GAPDH
F: 5’-GTC GGA GTC AAC GGA TTT GG-3’
R: 5’-GGG TGG AAT CA TTG GAA CAT-3’
Human
VEGF
F: 5’-GAG GGC AGA ATC ATC ACG AAG T-3’
R: 5’-CAC CAG GGT CTC GAT TGG AT-3’
Human
FGF2
F: 5’-GAC GGC AGA CTT GAC GG-3’
R: 5’-CTC TCT CTT CTG CTT GAA GTT-3’
Human F: 5’-TGC ATC AGT GAC GGT AAG CCA-3’
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CXCL12 R: 5’-ATC CAC TTT AAT TTC GGG TCA A-3’
Human
CXCR4
F: 5’-TGC TTG CTG AAT TGG AAG TG-3’
R: 5’-AGT CAT AGT CCC CTG AGC CC-3’
Human
HIF-1α
F: 5’-CAG TTA CGT TCC TTC GAT CAG TTG-3’
F: 5’-TTT GAG GAC TTG CGC TTT CA-3’
Human
BCL-2
F: 5’-CTT GAC AGA GGA TCA TGC TGT AC-3’
R: 5’-GGA TGC TTT ATT TCA TGA GGC-3’
Human
BAX
F: 5’-GCA ACT TCA ACT GGG GCC GGG-3’
R: 5’-GAT CCA GCC CAA CAG CCG CTC-3’
Human
CASPASE-3
F: 5’-CCT GGT TAT TAT TCT TGG CGA AA-3’
R: 5’-GCA CAA AGC GAC TGG ATG AA-3’
Human
KI67
F: 5’-TGACCCTGATGAGAAAGCTCAA-3’
R: 5’-CCCTGAGCAACACTGTCTTTT-3’
Mouse
β-actin
F: 5’-GGC TGT ATT CCC CTC CAT CG-3’
R: 5’-CCA GTT GGT AAC AAT GCC TG T-3’
Mouse
CD31
F: 5’-CAA ACA GAA ACC CGT GGA GAT G-3’
R: 5’-ACC GTA ATG GCT GTT GGC TTC-3’
Mouse
SM-α
F: 5’-CAG GCA TGG ATG GCA TCA ATC AC-3’
R: 5’-ACT CTA GCT GTG AAG TCA GTG TCG-3’
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Mouse VEGF F: 5’-AGA TGT CCA CCA GGG TCT CA-3’
R: 5’-CTC ACA AAT CTG GGT GGC GA-3’
Mouse TNF-
α
F: 5’-CCA TTC CTG AGT TCT GCA AAG G-3’
R: 5’-AGG TAG GAA GGC CTG AGA TCT TAT C-3’
Mouse
IL-12
F: 5’-AAG CCT TCC TCC TAT CAG CC-3’
R: 5’-TTC AGG TCT CTC CCA ACC CAA-3’
Mouse
Vimentin
F: 5’-TCC AGA GAG AGG AAG CCG AA-3’
R: 5’-AAG GTC AAG ACG TGC CAG AG-3’
Reverse transcription-polymerase chain reaction (RT-PCR). The 105 hMSCs with or
without ETIN treatment for 1 h were lysed in TRIzol reagent. Total RNA was extracted and
precipitated with isopropanol, and the RNA pellets were washed with 75% (v/v) solution of
ethanol in water, air-dried, and dissolved in 0.1% (v/v) diethyl pyrocarbonate-treated water.
Reverse transcription was performed using 10 μL of 2×Easy Taq SuperMix (TransGen
Biotechnology, Beijing, China), 0.5 μL of cDNA, 0.5 μL of each primer, and 8.5 μL of sterile
pure H2O, followed by PCR amplification of the synthesized complementary
deoxyribonucleic acid. PCR consisted of 35 cycles of denaturing (94 °C, 30 s), annealing (58
°C, 45 s), and extension (72 °C, 45 s), with a final extension at 72 °C for 10 min. PCR was
followed by electrophoresis on a 2% (w/v) agarose gel, and visualization was performed by
ethidium bromide staining. PCR products were analyzed using a gel documentation system
(WGD-30, Daihan Scientific, Korea). β-actin served as an internal control. Table 2 shows the
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primers used for RT-PCR.
Table 2. Primer sequences for RT-PCR.
Primer Sequence
Human
β-actin
F: 5’-GCA CTC TTC CAG CCT TCC TTC C-3’
R: 5’-TCA CCt TCA CCG TTC CAG TTT TT-3’
Human
VEGF
F: 5’-GCA GAA GGA GGA GGG CAG AAT-3’
R: 5’-ACA CTC CAG GCC CTC GTC ATT-3’
Human
FGF2
F: 5’-TCC ACC TAT AAT TGG TCA AAG TGG T-3’
R: 5’-TCA GTA GAT GTT TCC CTC CAA TGT-3’
Human
HIF-1α
F: 5’-TAT GAC CTG CTT GGT GCT GA-3’
R: 5’-GGG AGA AAA TCA AGT CGT GC-3’
Human
GAPDH
F: 5’-CCC TCC AAA ATC AAG TGG GG-3’
R: 5’-CGC CAC AGT TTC CCG GAG GG-3’
Human & Mouse
β-actin
F: 5’-GCT CCG GCA TGT GCA A-3’
R: 5’-AGG ATC TTC ATG AGG TAG T-3’
Intracellular distribution of ETIN. The hMSCs were cultured on a 150 mm dish (1 × 106
cells/well) and incubated with 15 μg/mL ETIN for 1 h. The cells were then fixed using 9
Karnovsky’s fixative for 4 h at 4 °C and rinsed three times with cold 0.05 M cacodylate
buffer. The cells were fixed with 1% osmium tetraoxide for 2 h at 4 °C and washed twice
with cold distilled water. The samples were treated with 0.5% uranyl acetate overnight at 4
°C, dehydrated using graded concentrations of ethanol of 30, 50, 70, 80, 90, 95, and 100%,
rinsed with propylene oxide, and finally, embedded in Spurr’s resin, which was then
polymerized at 70 °C for 24 h. Thin sections of 100 nm were obtained using an
ultramicrotome (Leica, Wetzlar, German), collected on 200-mesh copper grids, and observed
by TEM (JEM-1010, JEOL, Tokyo, Japan). Quantitative concentration of intracellular Au
and Fe was measured by ICP-MS.
Wound treatment. Four-week-old female athymic mice ((20–25) g body weight, Orient,
Seoul, Korea) were anesthetized with 200 µL xylazine (20 mg/kg) and ketamine (100 mg/kg).
A 2.0 cm × 2.0 cm sized skin defect was made on the back of each mouse with surgical
scissors. Epidermis, dermis, and stratum corneum were removed, and the muscle fascia was
exposed. To prevent the wound by contracture, 8 sutures were placed at the border of the
wound with 6–0 sutures (AILEE Co., Ltd., Busan, Korea), and the wound margins were
anchored to the underlying muscle fascia. The wound-induced mice were injected with (n =
6) or without (n = 6) ETIN-treated hMSCs (1 × 106 cells/mouse). After cell injection, the
wounds were covered with polyurethane film (Tegaderm, 3 M Healthcare, St. Paul, MN).
Tegaderm is commercialized wound dressing and has been used as a positive control in our
experiment following the previous studies [1,2]. Wound-induced mice with only Tegaderm
treatment served as a control (n = 6). All animals received care according to the guidelines
for the care and use of laboratory animals of Sungkyunkwan university (Approved number:
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SKKUIACUC2017-05-03-3, September 2018).
In vivo imaging. The macroscopic wound area was quantified by processing photographs
taken at various time points by tracing the wound margin and calculating the pixel area
related to it with a ruler using a fine-resolution computer mouse. The location of the
advancing margin of wound closure was defined as the grossly visible margin of epithelial
migration toward the center of the wound and over the granulation tissue bed. The wound
area was calculated as the percentage of the initial wound area ([wound area at time]/[initial
wound area] × 100%). Morphometric analysis was performed on digital images using the
imaging software (Photoshop CC, Adobe Systems).
Histology. Microscopic tissue regeneration was observed by H&E-and Masson’s trichrome-
stained tissue sections using a light microscope (CKX53, Olympus, Tokyo, Japan). Skin
tissue samples were fixed in formaldehyde, dehydrated with a concentration of 20% sucrose,
and embedded in optimum cutting temperature (OCT) compound (SciGen Scientific,
Gardenas, CA, USA). Specimens were sliced into 10 µm-thick sections and stained with
H&E and Masson’s trichrome to examine tissue regeneration.
Immunohistochemistry. For immunohistochemical staining, samples embedded in OCT
compound were cut into 10 μm-thick sections at -22 °C. To stain microvessels,
immunohistochemistry was performed on sections with CD31 (Abcam, Cambridge, UK),
SM-α (Abcam), involucrin (Abcam), laminin (Abcam), and HNA (Abcam) antibodies.
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CD31+, SM-α+, involucrin+, and laminin+ signals were visualized with fluorescein-
isothiocyanate-conjugated secondary antibodies (Jackson Immuno Research Laboratories,
West Grove, PA). HNA+ signals were visualized with rhodamine (TRITC)-conjugated
secondary antibodies (Jackson Immuno Research Laboratories) The sections were
counterstained with DAPI and examined by fluorescence microscopy (IX71, Olympus,
Tokyo, Japan).
Statistical Analysis. All quantitative data were expressed as the mean ± standard deviation.
Statistical analysis was performed by analysis of one-way ANOVA using a Bonferroni test.
However, two-way repeated measures ANOVA followed by Bonferroni t-test was used to
analyze the time-course for the wound-healing process. P values of less than 0.05 were
considered statistically significant.
Acknowledgments. Kazunori Kataoka at the University of Tokyo and Kawasaki Institute
of Industrial Promotion in Japan is acknowledged for his helpful advices on the manuscript
development. This research was supported by the National Research Foundation of Korea
(NRF), funded by the Ministry of Science and ICT (NRF-2018M3A9E2023255, NRF-
2017R1A5A1070259, NRF-2017R1A5A1015365, and NRF-2019R1C1C1007384); by the
Bio & Medical Technology Development Program of the NRF funded by the Ministry of
Science, ICT and Future Planning (NRF-2016M3A9B4919711) and by a grant of the Korea
Health Technology R&D Project through the Korea Health Industry Development Institute
(KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant
HI17C1728). This work was supported by the NRF grant funded by the Korean government
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(MSIP) (NRF-2014R1A5A1009799, NRF- 2019M3E6A1103866, and NRF-
2016M3D1A1021140).
Supporting Figures
Fig. S1. (A) TEM and (B) EDS mapping images of ETIN stored for 6 months in an aqueous solution. (C) EDS mapping images of ETIN after synthesizing. Abbreviation: TEM, transmission electron microscopy; EDS, energy dispersive spectrometry; ETIN, endosome triggered iron-ion-releasing nanoparticle.
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Fig. S2. Half maximal inhibitory concentration (IC50) value of ETIN on hMSCs.
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Fig. S3. (A) Duration of HIF-1α expression in hMSCs treated with ETIN for 1 h compared with the no treatment (NT) group (n = 4, *P < 0.05 versus NT group). (B) HIF-1α expression from hMSCs at 12 h after treating ETIN for 1 h. Abbreviation: HIF-1α, hypoxia inducible factor-1 alpha.
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Fig. S4. Relative mRNA expression of (A) VEGF and (B) HIF-1α in hMSCs after treating 15 µg/mL of Fe ion solution (n = 6). Abbreviation: VEGF, vascular endothelial growth factor; HIF-1α, hypoxia inducible factor-1 alpha.
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Fig. S5. Representative images of FDA/EB staining in figure 4K with high resolution and magnification. Abbreviation: FDA/EB, fluorescein diacetate/ethidium bromide.
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Fig. S6. Representative images of FDA/EB staining in figure 4O with high resolution and magnification.
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Fig. S7. Angiogenesis related protein expressions from hMSCs affected by ETIN treatment (15 µg/mL). Abbreviation: FGF, fibroblast growth factor; uPA, urokinase-type plasminogen activator.
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Fig. S8. Cellular uptake test with ETIN concentration and time, as determined by quantifying the amounts of Au in the hMSCs using ICP-MS (n = 4). Abbreviation: ICP-MS, inductively coupled plasma-mass spectroscopy.
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1. F. Yergoz, N. Hastar, C. E. Cimenci, A. D. Ozkan, T. Tekinay, M. O. Guler, A. B.
Tekinay, Heparin mimetic peptide nanofiber gel promotes regeneration of full
thickness burn injury. Biomaterials. 134 (2017) 117-127.
2. S. S. Hosseini Salekdeh, H. Daemi, M. Zare-Gachi, S. Rajabi, F. Bazgir, N. Aghdami,
M. S. Nourbakhsh, H. Baharvand, Assessment of the Efficacy of Tributylammonium
Alginate Surface-Modified Polyurethane as an Antibacterial Elastomeric Wound
Dressing for both Noninfected and Infected Full-Thickness Wounds. ACS Appl Mater
Interfaces 12 (2020) 3393-3406.
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