development of a deep-red fluorescent glucose …glu-1-o-dcsn stock solution (0.10 mg/ml, 0.19 mm)...
TRANSCRIPT
1
Development of a deep-red fluorescent glucose-conjugated bioprobe
for tumor targeting in vivo
Yinwei Cheng,†a Ghulam Shabir,†a,b Xiang Li,a Laiping Fang,b Liyan Xu,*a Hefeng Zhang,*b and Enmin Li*a
a. Department of Biochemistry and Molecular Biology, Comprehensive Building,
Shantou University Medical College, 22 Xinling Road, Shantou, 515041, China
b. Department of Chemistry, Shantou University, 243 Daxue Road, Shantou, 515063,
China
† These authors contributed equally.
*Corresponding author: [email protected], [email protected]; [email protected]
Contents
1. Experimental
1.1 Materials and Instruments
1.2 Synthesis of Glu-1-O-DCSN
1.3 Preparation of stock solutions of Glu-1-O-DCSN
1.4 Preparation of staining solution of Glu-1-O-DCSN
1.5 Cell culture and Glu-1-O-DCSN uptake assay
1.6 Glu-1-O-DCSN treatment and cell viability assay
1.7 Incubation and Staining of Living Cells
1.8 Animal experiments and in vivo image
1.9 Western blot analysis of GLUT1
2. Figures
S1. FTIR spectrum of compound 1 and isophorone
S2. 1H NMR spectrum of compound 1
S3. 13C NMR spectrum of compound 1
S4. FTIR spectrum of compound 2
S5. 1H NMR spectrum of compound 2
S6. 13C NMR spectrum of compound 2
Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2019
2
S7. FTIR spectrum of compound 3
S8. 1H NMR spectrum of compound 3
S9. FTIR spectrum of the Glu-1-O-DCSN probe and compound 3
S10. 1H NMR spectrum of the Glu-1-O-DCSN probe
S11. Cell imaging of HeLa, KYSE150 and NE1 cells labelled by probe Glu-1-O-DCSN
at concentration of 0.10 μM.
S12. Colocalization analysis of Glu-1-O-DCSN with NucBlue live cell stain in live-
cell imaging of HeLa, KYSE150 and NE1 cells.
S13. Glu-1-O-DCSN probe (0.10 μM) uptake was competitively inhibited by D-
glucose
1. Experimental
1.1 Materials and Instruments
All chemicals and reagents were purchased from Aladdin Chemical Company.
Solvents were purified and dried by standard methods before use. FTIR spectra were
run on a single beam Nicolet IR 100 (Fourier transform). UV-vis spectra were recorded
on a Shimadzu UV-2600 UV-Visible spectrophotometer. Fluorescence spectra were
measured on an F-7000 FL spectrophotometer. The absolute fluorescence quantum
yield was determined by using a Hamamatsu quantum yield spectrometer C11347
Quantaurus-QY. Proton and carbon nuclear magnetic resonance spectra (1H and 13C
NMR) were recorded on an AVANCE-400 MHz and 100 MHz NMR spectrometer,
respectively, with TMS as an internal reference. Compounds were routinely checked
by thin layer chromatography (TLC) on silica gel plates using petroleum ether
(PE)/ethyl acetate (EA) and chloroform: methanol. The crude products were purified
by flash column chromatography and re-crystallization techniques.
1.2 Synthesis of Glu-1-O-DCSN
The synthesis for the Glu-1-O-DCSN probe was profiled in Scheme 1 in the main
text, which involved the following steps:
a) malononitrile (3.96 g, 60.00 mmol) was dissolved in stirring ethanol (70 mL) at
room temperature followed by addition of 7.49 mL of isophorone (50.00 mmol). After
adding piperidine catalyst (10.00 mg), the reaction mixture was heated to reflux for
12 h. After cooled to room temperature, water (100.00 mL) was added into the
3
reaction mixture leading to precipitation. The precipitates were dissolved in a water
(60 mL)/ethanol (90 mL) for re-crystallization. Crystals of 2-(3,5,5- trimethylcyclohex-
2-en-1-ylidene) malononitrile (1) were collected and dried in an oven at 60oC (3.10 g,
75% yield, m.p. 71oC). Characteristic spectroscopic data of compound 1 was as follows:
FTIR (KBr, ῡ) = 2954, 2220, 1614, 1549, 1383, 1325, 893 cm-1. 1H NMR (400 MHz,
chloroform-d) δ 6.55 (q, J = 1.5 Hz, 1H), 2.44 (s, 2H), 2.10 (t, J = 1.3 Hz, 2H), 1.96 (t, J =
1.1 Hz, 3H), 0.94 (s, 6H). 13C NMR (100 MHz, CDCl3): δ=170.33, 159.69, 120.59, 113.16,
112.38, 99.98, 45.69, 42.64, 32.36, 27.81, 25.29;
b) To a stirred solution of acetobromo-α-D-glucose (3.92 g 9.60 mmol) in DCM
(60.00 mL), 4-diethyaminosalicylaldehyde (1.44 g, 8.00 mmol) and
tetrabutylammonium bromide (2.60 g, 8.00 mmol) were added at room temperature,
followed by the addition of 5% NaOH aqueous solution (40.00 mL). 2 h later, the
organic layer was collected, and the aqueous layer was extracted three times with
chloroform (90.00 mL). The organic layer was washed with brine solution (80.00 mL)
and dried with Na2SO4. After concentrated on a rotary evaporator, the resultant dark
violet residue was purified by silica gel column with PE : EA (4:1) as eluent to give
yellow oily liquid product of compound 2 (0.70 g, 25%). Characteristic spectroscopic
data of compound 2 is as follows: FTIR (KBr, ῡ) = 2975, 1752, 1664, 1596 cm-1. 1H NMR
(400 MHz, chloroform-d): δ= 7.76 (dd, J = 8.9, 1.6 Hz, 1H), 6.47 (d, 3JH,H = 9.1 Hz, 1H),
6.38 (s, 1H), 5.42-5.29 (m, 2H), 5.30-5.15 (m, 2H), 4.31 (dd, 3JH,H = 12.4, 4.5 Hz, 1H),
4.22 (dd, 3JH,H= 12.4, 2.4 Hz, 1H), 3.93-3.85 (m, 1H), 3.45 (qd, 3JH,H = 7.3, 2.7 Hz, 4H),
2.07 (dd,3JH,H = 6.8, 1.7 Hz, 12H), 1.25 (t, 3JH,H= 7.0 Hz, 6H).13C NMR (100 MHz, CDCl3):
δ= 186.66, 170.51, 170.21, 169.29, 169.18, 161.34, 145.96, 131.77, 130.05, 108.82,
107.22, 99.51, 72.53, 72.18, 70.94, 68.19, 61.91, 45.20, 20.65, 20.58, 12.46.
c) 2-(3,5,5- trimethylcyclohex-2-en-1-ylidene) malononitrile 1 (0.12 g, 0.66 mmol)
was added to compound 2 (0.35 g, 0.66 mmol) in 10 mL dry ethanol at room
temperature. After heated to 80oC, ammonium acetate (10.00 mg) was added and the
reaction solution was refluxed overnight. The reaction mixture was concentrated on a
rotary evaporator and the residue was purified on by silica gel column
chromatography (PE : EA = 1:1) to give compound 3 (0.20 g, 43%). Characteristic
spectroscopic data of compound 3 is as follows: FTIR (KBr, ῡ) = 2960, 2210, 1750, 1550,
1600, 1500, 1250, 1210, 1040 cm-1. 1H NMR (400 MHz, chloroform-d) δ= 7.51 (d, 3JH,H
4
= 7.3 Hz, 1H), 7.33 (d, 3JH,H= 16.0 Hz, 1H), 6.76 (d, 3JH,H = 16.0 Hz, 2H), 6.45 (s, 1H), 6.3-
6.07 (m, 1H), 5.47-5.31 (m, 2H), 5.26-5.10 (m, 2H), 4.32 (q, 1H), 3.88-3.57 (m, 1H), 3.41
(q, 3JH,H = 7.1, 4H), 2.54 (m, 3H), 2.21-1.87 (m, 12H), 1.85 (m, 1H), 1.22 (t, 3JH,H = 7.1 Hz,
7H), 1.09 (m, 5H).
d) To compound 3 (100.00 mg, 0.144 mmol) solution in methanol (5.00 mL), KOH
(4.00 mg, 0.72 mmol) was added at room temperature and the resultant solution was
stirred for 2 h. After the reaction solution was neutralized with acetic acid, methanol
was removed by using rotary evaporator followed by addition of water (15.00 mL).
The mixture was filtered and dried to give the final compound Glu-1-O-DCSN (39 mg,
55%). Characteristic spectroscopic data of compound Glu-1-O-DCSN is as follows:
FTIR (KBr, ῡ) = 2960, 2210, 1750, 1550, 1600, 1500, 1250, 1210, 1040 cm-1. 1H NMR
(400 MHz, methanol-d4): δ=7.73 (d, 3JH,H = 16.0 Hz, 1H), 7.57 (d, 3JH,H = 9.0 Hz, 1H), 6.91
(d, 3JH,H = 16.0 Hz, 1H), 6.69 (s, 1H), 6.58 (d, 3JH,H = 2.5 Hz, 1H), 6.47 (dd, 3JH,H = 9.0, 2.5
Hz, 1H), 4.85 (d, 3JH,H = 7.7 Hz, 1H), 4.56 (s, 2H), 3.88 (dd, 3JH,H = 11.9, 1.8 Hz, 1H), 3.79-
3.66 (m, 2H), 3.55 (t, J = 8.2 Hz, 1H), 3.49-3.46 (m, 1H), 3.42 (m, 9H), 3.20 (q, 3JH,H = 7.3
Hz, 2H), 2.61-2.54 (m, 2H), 1.31 (t, 3JH,H = 7.3 Hz, 2H), 1.19 (t, 3JH,H= 7.0 Hz, 6H), 1.07 (d, 3JH,H = 4.5 Hz, 6H).
1.3 Preparation of stock solutions of Glu-1-O-DCSN
To prepare a stock solution, Glu-1-O-DCSN was dissolved in absolute ethanol to a
final concentration of 0.10 mg/ml (0.19 mM) and the stock solution was stored in a
vial at room temperature and protected from light.
1.4 Preparation of staining solution of Glu-1-O-DCSN
Glu-1-O-DCSN stock solution (0.10 mg/ml, 0.19 mM) was diluted to the final working
concentration in live cell image solution (Cat. No. A14291DJ, Life Tech) and using
working concentration of 0.05 - 0.10 μM.
1.5 Cell culture and Glu-1-O-DCSN uptake assay
Human HeLa cervical carcinoma cells were cultured in Dulbecco’s modified Eagle’s
medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum,
human KYSE150 esophageal cancer squamous cells were cultured in RPMI 1640
medium (Thermo) containing 10% fetal bovine serum (GIBCO), and normal
immortalized NE1 esophageal epithelial cells were cultured in defined keratinocyte
serum-free medium (dKSFM, Invitrogen) at 37 oC, 5% CO2 (V/V).
5
1.6 Glu-1-O-DCSN treatment and cell viability assay
The CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega) was
used for determining cell proliferation after Glu-1-O-DCSN and vehicle treatment
(absolute ethanol). Briefly, cells were plated in triplicate in 96-well plates, at a density
of 10,000 cells per well (triplicate blank wells without cells were conducted for
normalization) and allowed to incubate overnight, and then serial dilutions of Glu-1-
O-DCSN (0.01 µM, 0.10 µM and 1.00 µM) were added, as well as vehicle control. After
treatment for 24 h, MTS reagent was added, and plates were read on a multi-well
scanning spectrophotometer at 492 nm, after incubating at 37 °C and 5% CO2 for 2 h,
and the cell viability ratio was calculated compared with vehicle treatment.
1.7 Incubation and staining of live cells and cell fluorescence intensity measurement
Cells were plated in fibronectin-coated 24-well NEST glass bottom cell culture plates
at 5,000 cells per well and incubated at 37 °C and 5% CO2 overnight to reach cells
confluency 60-70%. Remove cell culture medium from the well and cells were washed
twice with live cell imaging solution and add prewarmed (37 °C) staining solution
containing Glu-1-O-DCSN probe in the absence or presence of D-glucose at the
indicated concentrations. Live-cell imaging was immediately performed after Glu-1-
O-DCSN probe added using Zeiss confocal laser scanning microscope (LSM880) in
living image chamber (37 °C and 5% CO2) with time series mode (duration 240 cycles,
interval time 1 min), at excitation wavelength λex = 488 nm and emission wavelength
λem = 645 nm. In live cell imaging by using LSM880, both wavelength at 488 nm and
514 nm can excite Glu-1-O-DCSN efficiently with emission wavelength collected at
600-700nm. We choose 488 nm to excite Glu-1-O-DCSN as this excitation wavelength
is widely supplied in most fluorescent microscopes.
To investigate the subcellular localization of Glu-1-O-DSCN, a commercial
mitochondria probe Mito-Tracker Green (Cat. no. M7514, Invitrogen) (λex = 490 nm,
λem = 516 nm) and nuclear probe NucBlue Live Cell Stain (Cat. No. R37605, λex = 360
nm, λem = 460 nm) were used for colocalization analysis respectively. In cell imaging
using GLU-1-O-DCSN and MitoTracker Green, cells were incubated with 0.10 µM Glu-
1-O-DSCN and 0.10 µM Mito-Tracker green together for 30 minutes. Due to the
excitation wavelength of Mito-Tracker Green is 490 nm (λem = 516 nm) and at this
wavelength Glu-1-O-DCSN can also be excited, to avoid fluorescence crosstalk
6
between Glu-1-O-DCSN and Mito-Tracker Green, the excitation wavelength of Glu-1-
O-DCSN was chosen λex = 514 nm and collecting fluorescence in a range of 613 - 662
nm (at λex = 514 nm, λem, collected = 613 - 662 nm, only Glu-1-O-DSCN is visible), and Mito-
Tracker Green was chosen λex = 488 nm, collecting at 499-517 nm (at λex = 488 nm, λem,
collected = 499 - 517 nm, only Mito-Tracker Green is visible). In cell imaging using GLU-1-
O-DCSN and NucBlue Live Cell Stain, cells were incubated with 0.10 µM Glu-1-O-DCSN
and two drops of NucBlue Live Cell Stain together for 30 minutes according to the
manufacturer’s instructions. Glu-1-O-DCSN was imaged at excitation wavelength λex =
488 nm and emission wavelength λem = 645 nm, and NucBlue Live Cell Stain was
imaged at λex = 360 nm, λem = 460 nm. All image processing was done by ZEN (blue
edition) software, and co-localization analysis was done by Fuji software.
For measuring fluorescence intensity of each cell by ZEN software, we draw an area
as ROIs (regions of interest) which contains a cell in phase-contract image. ZEN
analyses images and let us know the digitalized mean of fluorescence intensity in the
ROIs that we determined. After subtracting the background intensity from the
fluorescence intensity, we get the fluorescence intensity value that a cell contains at
every time point. For each experiment, at least 3 images were performed and at least
10 cells are measured of each image.
1.8 Animal experiments and in vivo imaging
All experiments involving animals were performed in compliance with the policy on
animal use and ethics of Shantou University Medical College (SUMC), and
experimental procedures were performed in accordance with protocols approved by
SUMC Medical Animal Care & Welfare Committee. Five-week-old female nude mice
were purchased from Vital River Laboratories (Beijing, China) and maintained on a 12
h light/dark cycle under specific pathogen-free conditions, with free access to food
and water (Permit Number: 2017-0079). Mice (n=16) were randomized into two
groups, one is inoculated with KYSE150 cells (n=10) and the other is inoculated with
phosphate buffer saline (PBS) using as tumor-free control (n=6). Suspensions of 1×106
KYSE150 cells in 100 μL of serum-free 1640 medium were inoculated subcutaneously
into the right armpit of nude mice to establish the tumor model. Tumor growth was
monitored daily and tumor size were measured (length × width2). When tumor
reached about 100 mm3, mice were applied with Glu-1-O-DCSN probe as described
7
below.
For tumor labelling, the Glu-1-O-DCSN probe (20 nM, 100 μL) was intratumorally
injected into the tumor tissue of tumor-bearing mice (n=5). For control experiments
(n=3), the same dose of probe was injected subcutaneously into the right armpit of
tumor-free nude mice. All mice were anesthetized with 2% isoflurane throughout all
procedures. Real-time in vivo fluorescent images were performed using a Caliper IVIS
Kinetic small animal in vivo imaging system with an excitation filter of 400–650 nm at
different time intervals (0.5 h, 1.5 h, 18 h, 22 h) after injection. At 24 hours, mice were
sacrificed (operated on ice) and the main organs (including heart, liver, brain, lung,
spleen, stomach, kidney, colon and rectum) and tumor were removed, and
fluorescence imaging of the main organs was performed using the same above
imaging system.
For tumor targeting, the Glu-1-O-DCSN probe (20 nM, 100 μL) was intravenously
injected into tumor-bearing mice (n=5) and tumor-free control mice (n=3) via tail vein.
Mice and organ imaging were performed in the same way as the tumor labelling
experiments. Mice and organ imaging analyzed in a longitudinal series for each mouse
and were normalized using a look-up-table with common minimum and maximum
values. Relative fluorescence intensity was measured by LivingImage software by
background-corrected Regions of interest (ROI) measurement. ROI for both tumor and
background were derived from equivalent sized areas containing the same number of
pixels. Fluorescent intensity was calculated by mean ± SD, n=5.
1.9 Western blot analysis of GLUT1
NE1, KYSE150 and HeLa cells were lysed in 100 μL 1ⅹlaemmli sample buffer (bio-
rad,161-0747) for 10 minutes and sonicated for 10 seconds. Protein was loaded for
SDS-PAGE separation without additional boiling of lysate. Proteins were transferred
to PVDF membrane and blocking with 5% nonfat milk, then incubated with primary
antibody GLUT1 (1;1000, abcam, ab15309) and β-actin (1:1000, Santa Cruze, sc-
47778) at 4° overnight. Membrane is washed in TBST for 3 times and then incubated
with secondary antibody HRP-conjugated anti-rabbit (1;2000, CST, 7074) at room
temperature for 1 h. Chemiluminescence images were captured using bi0-rad chemi
imaging system after developing with ECL solution.
8
2. Figures
4000 3500 3000 2500 2000 1500 1000 500
020406080
100120140160180200
CN
CN
O
Tr
ansm
ittan
ce
Wavenumber/cm
2960
2930 22201610 1550
1430 1320
1660
14301380 1250
1150901
8912960
Isophorone
Compound 1
Figure S1. FTIR spectra of compound 1 and isophorone
CN
CNH
a
b
cd
a
b
c
d
e
e
9
Figure S2. 1H NMR spectrum of compound 1
CN
CN
b
a
c de
fg
h ij
k
a
b
cde
ij,kfg
h
Figure S3. 13C NMR spectrum of compound 1
4000 3500 3000 2500 2000 1500 1000 500
0
20
40
60
80
100
O
HO
N
OAcO
AcO
OAc
OAc
Tran
smitt
ance
Wavenumber/cm
3480
2970
17501590
16601520
136012201040
752Compound 2
Figure S4. FTIR spectrum of compound 2(图有改动)
10
OH
O
OO
O
O
O
O
O
O
O
Na
b
c
c
c
c
e
fg
hij
k
l m
a
b
c
nnk
l m
e, g, f
hi, j
Figure S5. 1H NMR spectrum of compound 2
OH
O
OO
O
O
O
O
O
O
O
Na
f
e
d
c
b
e
gh
kij
o
p r
n
n
m
m
ab,c,d,f
f
l
q
s
t
u
vs t
u, v
g
qo,p
r k,h,i,j,e
l
Figure S6. 13C NMR spectrum of compound 2
11
4000 3500 3000 2500 2000 1500 1000 500
0
20
40
60
80
100
CCN
CNO
N
OAcO
AcO
OAc
OAc
Tran
smitt
ance
Wavenumber/cm
2210
2960
1600
1750
1550 1500 1210
12701040
Compound 3
Figure S7. FTIR spectrum of compound 3(图有改动)
O
NC CN
N
O
O
O
O
O
O
O
OO
a
b
c
d
e
f
g
h
i
j
k
l
m n
o p
q
rs
tf,g,h,i
c
a
b
e d
l, n, m
k,u
u
o
p,q
rst
j
Figure S8. 1H NMR spectrum of compound 3
12
4000 3500 3000 2500 2000 1500 1000 500
020406080
100120140160180200
CCN
CNO
N
OAcO
AcO
OAc
OAc
CCN
CNHO
N
OHO
HO
OH
OH
Tran
smitt
ance
Wavenumber/cm
3390 2970 2210 1610
1540 1510 10701280
1390
2970
22101750
1610
1550 1510 12101270 10401150
Glu-1-O-DCSN
Compound 3
Figure S9. FTIR spectra of the Glu-1-O-DCSN probe and compound 3(图有改动)
OCN
CN
O
OH
HO
HOOH
Na
b
c
d e
rf
g
hi
j
k
l m
n
op
q
bi,l,hn
m j k
gf
o
p qr
a
ceb
Figure S10. 1H NMR spectrum of the Glu-1-O-DCSN probe
13
Figure S11. Cell imaging of HeLa, KYSE150 and NE1 cells labelled by probe Glu-1-O-DCSN. Three types of cells were incubated with 0.10 μM Glu-1-O-DCSN for 0.5 h and were visualized by using a confocal laser scanning microscope with excitation wavelength λex = 488 nm, λem, collected = 600-700 nm). Scale bar=20 μM
Figure S12. Colocalization analysis of Glu-1-O-DCSN (λex = 488 nm, λem, collected = 600-700 nm) and nuclei by probing with NucBlue live cell stain (λex = 305 nm, λem, collected = 460-490 nm) using live-cell imaging of HeLa, KYSE150 and NE1 cells. Scale bar = 20 μm (three cell lines were incubated in 0.10 μM aqueous Glu-1-O-DCSN solution for 0.5 h followed by NucBlue live cell stain for 0.5 h for visualization on a confocal laser scanning microscope).
14
Figure S13. Glu-1-O-DSCN probe (0.10 μM) (λex = 488 nm, λem, collected = 600-700 nm) in Cell imaging of HeLa, KYSE150 and NE1 cells under Glucose treatment (0 mM, 5.6 mM).
Table S1. Quantum yields of Glu-1-O-DCSN in different solvents Solvent Quantum yielda
Water 1.5%Water +20% ethanol 1.8%Water+50% ethanol 16.1%
Water + 20% DMSO
2.4%
Water + 50% DMSO
12.6%
DCM 7.8%THF 7.6%
Chloroform 5.8%Ethanol 17.2%
Methanol 15.2%DMF 24.3%
DMSO 35.1%PBS 7.4 0.1%
PBS 7.4+5%DMSO 2.4%
15
a. Determined at a concentration of 0.5 μM by using spectrophotometer with an integrating sphere detector.