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: sukhobhang@skku.edu, Tel.: +82-31-290-7242, Fax: +82-31-

290-7272

Taekyung Yu, Ph.D., E-mail: tkyu@khu.ac.kr, Tel.: +82-31-201-2064, Fax: +82-31-204-

8114

2

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

3

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).

5

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’

6

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’

7

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

8

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:

10

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.

11

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.

13

Fig. S2. Half maximal inhibitory concentration (IC50) value of ETIN on hMSCs.

14

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.

15

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.

16

Fig. S5. Representative images of FDA/EB staining in figure 4K with high resolution and magnification. Abbreviation: FDA/EB, fluorescein diacetate/ethidium bromide.

17

Fig. S6. Representative images of FDA/EB staining in figure 4O with high resolution and magnification.

18

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.

19

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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