advances.sciencemag.org/cgi/content/full/5/12/eaay0044/DC1

Supplementary Materials for

FeSe quantum dots for in vivo multiphoton biomedical imaging

J. Kwon, S. W. Jun, S. I. Choi, X. Mao, J. Kim, E. K. Koh, Y.-H. Kim, S.-K. Kim, D. Y. Hwang, C.-S. Kim, J. Lee*

*Corresponding author. Email: nanoleelab@cnu.ac.kr

Published 6 December 2019, Sci. Adv. 5, eaay0044 (2019)

DOI: 10.1126/sciadv.aay0044

The PDF file includes:

Supplementary Texts Fig. S1. Physicochemical characterization of FeSe QDs. Fig. S2. Stability assessment of FeSe QDs dissolved in deionized water and PBS at 0.01 and 0.1 M. Fig. S3. Fluorescein diacetate assay. Fig. S4. Tumor cell targeting specificity of anti-HER2–QDs. Fig. S5. Tumor cell targeting specificity of anti-HER2–PEG-QDs. Fig. S6. Evaluation of physiological stability of anti-HER2–PEG-QDs in various conditions. Fig. S7. Hemocompatibility assay of FeSe QDs. Fig. S8. In vivo two-photon microscopic images before and after SHG signal removal. Fig. S9. Schematic diagrams presenting color replacement and removal process by using color threshold method. Legends for movies S1 to S3 Reference (43)

Other Supplementary Material for this manuscript includes the following: (available at advances.sciencemag.org/cgi/content/full/5/12/eaay0044/DC1)

Movie S1 (.avi format). In vitro two-photon microscopic images of HER2-overexpressed MCF7 cells (positive control) after treatment with anti-HER2–PEG-QDs (20 μg ml–1). Movie S2 (.avi format). In vitro two-photon microscopic images of MCF7 cells (negative control) after treatment with anti-HER2–PEG-QDs (20 μg ml–1). Movie S3 (.avi format). In vivo two-photon microscopic images (Z-scan) of cancer part were obtained 30 min after intravenous tail vein injection of anti-HER2–PEG-QDs by moving the excitation focal plane from 450 to 525 μm from the skin in 5-μm steps (λex = 800 nm, power = 100 mW).

Fig. S1. Physicochemical characterization of FeSe QDs. (A) Hydrodynamic diameter,

(B) Fe 2p and (C) Se 3d region of X-ray photoelectron spectra, (D) Kubelka-Munk plot

from UV-vis spectrum of FeSe QDs

Fig. S2. Stability assessment of FeSe QDs dissolved in deionized water and PBS at

0.01 and 0.1 M. (A) Digital photograph and (B) photoluminescence (PL) spectrum of

FeSe QDs dissolved in different solvents (water, 0.01 m PBS, and 0.1 m PBS). Neither

substantial aggregation nor notable difference in fluorescence was detected during

monitoring by a digital camera and fluorescence spectroscopy for five days.

Supplementary Texts

Measurement of quantum yield

The relative quantum yield of FeSe QDs was 40% when rhodamine B (RhB) was chosen

as the standard. The quantum yield was calculated using the following equation

QS = QR × IS/IR × AR/AS × (NS/NR)2

In the equation, R and S represent RhB and the sample, respectively. Q is the quantum

yield, I is the integral area of emission spectrum, A is the absorbance intensity at the

excitation wavelength and N is the refractive index of the solvent. Here, RhB was used as

the standard sample and dissolved in ethanol with a quantum yield of 95%, and samples

were dissolved in deionized water.

Cell Culture

The AGS human gastric adenocarcinoma, MG-63 human osteosarcoma, and NCI-H460

human lung carcinoma cell lines were purchased from the Korean Cell Line Bank (Seoul,

Korea). AGS and NCI-H460 cells were cultured in Roswell Park Memorial Institute

(RPMI) medium 1640 supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan,

UT), penicillin (100 IU mL-1

), and streptomycin (100 µg mL-1

). MG-63 cells were

cultured in Eagle's Minimum Essential Medium containing 10% FBS, penicillin (100 IU

mL-1

), and streptomycin (100 µg mL-1

). All cells were maintained in a humidified

incubator containing 5% CO2 at 37 °C. To evaluate the cell viability, an aqueous solution

of FeSe QDs was added to each cell line at different final concentrations (25, 50, and 70

μg mL-1

) dissolved in distilled water every other day (3, 5, and 7 days).

Flow Cytometry

To measure cell viability, the percentage of live cells was evaluated using a Muse™ Cell

Analyzer (PB4455ENEU; Millipore Co., Billerica, MA). The prepared cells were

suspended at a final cell concentrations 1 × 105 cells mL

-1. The suspended cells (100 μL)

were stained with Muse™ Annexin V & Dead Cell Reagent (MCH100105; Millipore Co.,

Darmstadt, Germany) for 20 min at 25°C, after which flow cytometry was carried out to

determine cell viability.

Fluorescein Diacetate Assay

To observe the morphological features of live cells, fluorescein diacetate (FDA, F7378;

Sigma-Aldrich, St. Louis, MO) staining was performed. FDA is a non-fluorescent,

hydrophobic substance that can penetrate the cell membrane and is metabolically

hydrolyzed by cytoplasmic lipase and emits green fluorescence.(43) The medium was

removed and the cells were washed with PBS and treated with an FDA stock solution (5

mg of FDA per mL of acetone) diluted in PBS (1:100). After 15 min of incubation at

37°C, cell morphology was observed using a fluorescence microscopy (Eclipse TS100,

Nikon, Japan) at an excitation wavelength 488 nm.

Fig. S3. Fluorescein diacetate assay. Fluorescence microscopic images of AGS, MG63,

and NCI-H460 cells after 3, 5, and 7 days of treatment with 0 (control) and 70 μg mL-1

FeSe QDs. Scale bar: 100 μm.

(Comments)

Three human cancer cell lines (AGS, MG63, NCI-H460) were chosen to evaluate the

biocompatibility of FeSe QDs based on the fluorescein diacetate assay because it is

possible for diverse cells of each organ to exhibit different responses to the QDs. The

fluorescence microscopic images of cells imply that the FeSe QDs possess superior

biocompatible nature regardless of its concentration. The QDs induced similar

proliferation as that of non-treated control cells for up to seven days. More details are

described in the main manuscript.

Fig. S4. Tumor cell targeting specificity of anti-HER2–QDs. Two-photon and three-

photon fluorescence images of HER2-non-expressed (MCF7) and HER2-overexpressed

(HER2) MCF7 cells stained with anti-HER2-QDs at 2, 20, and 100 μg mL-1

(λex: 800 nm

for two-photon and 1080 nm for three-photon fluorescence).

(Comment)

Figure S4 shows that without PEGylation, both the physically adsorbed and covalently

bound anti-HER2-QDs did not have distinct selectivity for the HER2-overexpressed cells

against HER2-non-expressed MCF7 cells, demonstrating the non-specific internalization

behavior of anti-HER2-QDs at concentrations of 20 and 100 μg/ mL-1

in the cell

cytoplasm. In other words, anti-HER2-QDs were internalized in the normal cells as well

as HER2-overexpressed cells depending on their concentration without selectivity.

Fig. S5. Tumor cell targeting specificity of anti-HER2–PEG-QDs. Two-photon

fluorescence images of HER2-non-expressed (MCF7) and HER2-overexpressed (HER2)

MCF7 cells stained with 2 μg mL-1

anti-HER2-PEG-QDs (λex: 800 nm).

(Comment)

Figure S5 shows the evaluation of the non-specific uptake and selectivity of anti-HER2-

PEG-QDs for human breast cancer cell targeting. FeSe QDs were encapsulated with PEG

before conjugation with HER2 antibodies to form anti-HER2-PEG-QDs to avoid non-

specific internalization. Both HER2-non-expressed and HER2-overexpressed MCF7 cells

were stained with anti-HER2-PEG-QDs and PEG-QDs that were not conjugated with

HER2 antibodies. The nuclei of the cells were stained with propidium iodide. Figure S5

shows that all samples exhibited red fluorescence signals from propidium iodide in the

nuclei and only HER2-overexpressed MCF7 cells stained with anti-HER2-PEG-QDs

emitted cyan fluorescence. Neither cellular uptake nor cell surface binding was observed

in the control samples, indicating that anti-HER2-PEG-QDs specifically targeted HER2

receptors.

Figure S6. The anti-HER2-PEG-QDs were physiologically stable and maintained optical

properties for seven days in serum and in various buffer solutions (fig. S6), with <1%

hemolysis rate at a QD concentration of 100 μg/ mL-1

(fig. S7).

Fig. S6. Evaluation of physiological stability of anti-HER2–PEG-QDs in various

conditions. (A) The hydrodynamic diameter (HD), (B) the zeta potential, and (C) relative

quantum yield of anti-HER2-PEG-QDs in deionized water (DW) (red square), 0.1 m PBS

(black diamond), 10% (blue triangle) and 20% (green circle) fetal bovine serum (FBS)

solution in 0.1 m PBS, and human blood serum (light pink square). QDs in PBS were used

as a standard control for the quantification of relative PL intensity.

Hemocompatibility

blood samples were gathered from the heart and caudal vena cava of mice. After

centrifugation at 1500 rpm for 5 min, the red blood cells were resuspended in PBS with a

final concentration of 2%. The cells were then incubated with FeSe QDs at different

concentrations at 37°C for 2 h. The absorption was determined at 560 nm using a

microplate reader (Tecan Group Ltd., Männedorf, Switzerland). Triton X-100 (1%) was

utilized as a positive control. The percentage of hemolysis was calculated as follows: the

hemolysis % = [(sample absorbance − negative control absorbance) / (positive control

absorbance – negative control absorbance)] × 100%.

Fig. S7. Hemocompatibility assay of FeSe QDs. Hemolysis rate of 2% red blood cells

incubated with different concentrations of FeSe QDs for 2 h.

Figures S8, S9.

The FeSe QDs, which were found around HER2-overexpressed MCF7 cells, were

identified in cyan. The color replacement method was applied, whereby cyan was

extracted using the color threshold method and separated from the original data. The

intensity of the extracted cyan FeSe QDs was replaced by magenta and combined with the

original data. This color replacement method was applied to the images from both HER2-

overexpressed and normal MCF7 cells, and the final images showed better contrast and

distinction (Fig. 3i and 3j). Moreover, the color threshold method was used to remove the

second harmonic generation (SHG) signal of collagen caused by a laser with a wavelength

of 800 nm.

Fig. S8. In vivo two-photon microscopic images before and after SHG signal removal.

Original in vivo two-photon microscopic images and images after removal of SHG signals

by color threshold technique before and after injecting anti-HER2-QDs. Scale bar: 20 μm.

Fig. S9. Schematic diagrams presenting color replacement and removal process by

using color threshold method. Process of (A) converting cyan to magenta and (B)

removal of SHG signal.

Other Supporting Materials

Movie S1. In vitro two-photon microscopic images of HER2-overexpressed MCF7

cells (positive control) after treatment with anti-HER2–PEG-QDs (20 μg ml–1

).

Cell nucleus: Propidium iodide (red fluorescence) (λex = 500 nm)

Cell membrane: anti-HER2-PEG-QDs (λex = 800 nm)

* Laser power: 40 mW at focal plane

Movie S2. In vitro two-photon microscopic images of MCF7 cells (negative control)

after treatment with anti-HER2–PEG-QDs (20 μg ml–1

). The acquisition parameters,

color channels, and scaling were the same as those of movie S1

Movie S3. In vivo two-photon microscopic images (Z-scan) of cancer part were

obtained 30 min after intravenous tail vein injection of anti-HER2–PEG-QDs by

moving the excitation focal plane from 450 to 525 μm from the skin in 5-μm steps (λex

= 800 nm, power = 100 mW). The SHG signal from collagen was high in the layer

between 450 μm and 470 μm (blue channel), and higher signal of QDs was detected in the

area between 470 μm and 500 μm from the surface (blue and green channel).

* Two-photon excitation wavelength: 800 nm.

* Laser power: 100 mW at focal plane.

* Imaging direction: from a depth of 450 μm to 525 μm (relative to skin surface) in the z-

direction, 5-μm steps.

* Scale bar: 20 μm.