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from Fisher Scientific (Pittsburgh, PA), Sigma-Aldrich (St. Louis, MO),
and Acros Organics (Pittsburgh, PA), unless noted otherwise. LysoTracker
was obtained from Molecular Probes (Eugene, OR), and IR786 was
purchased from Sigma-Aldrich. For synthesis of CTNF126 and CTNF103,
heptamethine fluorophores were synthesized first, and they were assorted
with side groups. Chloro-substituted dyes were synthesized as previously
fluorescent signal retention during histological analysis, a standard
method using cell pellets was preceded. PC3 pellets were seeded onto
100 mm × 15 mm petri dishes and incubated at 37 °C in humidified 5%
CO incubator in the media solution until the cell confluency reached
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7
90%. 1 × 10 cells (≈5 petri dishes) were stained with CTNF126 or IR786
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6
at a concentration of 2 × 10 m and incubated for 30 min at 37 °C. After
washing with media solution, the cells were collected using cell scraper
and centrifuged at 1200 rpm for 10 min. After the same histological
preparation was conducted, the sectioned slides were imaged using the
microscope using 20× objective.
reported, through salt condensation with Vilsmeier–Haack reagent to
build the heptamethine cyanine core.[
16,17,27,28]
Then, substitution of
different ionizable docking functional groups (phenoxide ions) was made
on the mesochlorine atom of the heptamethine cyanine core to compare
selective cellular retention of each fluorophore and further docking in the
subcellular component (Scheme S1, Supporting Information). The final
Quantitation and Statistical Analysis: Fluorescent intensity (FI) of a
region of interest (ROI) over various tissues was quantified using Image
J software (NIH, Bethesda, MD). Signal-to-background ratio (SBR) is
FI of ROI/background (BG) intensity. Results were presented as mean
(n = 5) ± standard deviation (s.d.). Statistical analysis was performed
using a one-way analysis of variance (ANOVA) between multiple groups.
A P-value of less than 0.05 was considered significant (*P < 0.05;
**P < 0.01; ***P < 0.001).
1
13
compounds were analyzed by H NMR, C NMR, and high resolution
mass spectrometry (HRMS) (see the Supporting Information for detailed
chemical syntheses and analysis).
Measurement of Optical Properties: Absorbance and fluorescence
were measured using fiber optic HR2000 (200–1100 nm) spectrometers
(Ocean Optics, Dunedin, FL). Fluorescence excitation was provided
by a 5 mW, 655 nm laser diode (Opcom Inc., Xiamen, China) coupled
through a 300 mm core diameter, NA 0.22 fiber (Fiberguide Industries,
Stirling, NJ). All optical measurements were made in serum-containing
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6
media. 5 × 10 m of each fluorophore solution was prepared in 10% Supporting Information
fetal bovine serum (FBS) solutions and incubated at 37 °C for 4 h to
Supporting Information is available from the Wiley Online Library or
from the author.
evaluate their photostability and physicochemical stability. In silico
calculations of physicochemical properties such as molecular weight,
charge, distribution coefficient (logD at pH 7.4), pKa, refractivity,
topological polar surface area (TPSA), H-bond donors/acceptors,
and rotatable bonds were calculated using JChem calculator plugins
Acknowledgements
(ChemAxon, Budapest, Hungary). Data plotting was performed using
Prism version 4.0a software (GraphPad, San Diego, CA) and Microsoft
Excel (Redmond, WA).
G.K.P., J.H.L., and A.L. contributed equally to this work. The authors
thank Dr. John V. Frangioni for many helpful discussions and
experimental support and Ivey Choi for manuscript editing. This
study was supported by the NIH (Grant Nos. R01-EB022230 and
R21CA223270), and the Marine Biotechnology Program (Grant No.
20170263) funded by the Ministry of Oceans and Fisheries, South
Korea. M.H. also thanks the Atlanta Clinical & Translational Science
Institute for the Healthcare Innovation Program Grant, the Georgia
Research Alliance for the Ventures Phase 1 Grant. G.K.P., J.H.L., A.L.,
and M.H. performed the experiments. G.K.P., J.H.L., N.S.H., G.E.F.,
and H.S.C. reviewed, analyzed, and interpreted the data. G.K.P., J.H.L.,
M.H., and H.S.C. wrote the paper. All authors discussed the results and
commented on the manuscript.
Live Cell Labeling and In Vitro Imaging: Human prostate cancer cells
4
(
PC3 cells) were seeded into 24-well plates (5 × 10 cells per well) and
incubated at 37 °C in humidified 5% CO incubator in DMEM containing
0% FBS and 1% penicillin streptomycin for 2 days. After washing twice
2
1
with media solution, CTNF126, IR786, and CTNF103 were added to each
−6
well at a concentration of 2 × 10 m and incubated for 30 min at 37 °C.
To improve image contrast, cells were washed three times with media
solution prior to imaging. The images were acquired either right after the
triple wash, or right after additional washing with 1% Tween 20 to compare
fluorescent signal reduction among the contrast agents. The live cell
imaging was performed using Nikon TE2000 epifluorescence microscope
equipped with a 75 W Xenon light source and an Orca-ER (Hamamatsu,
Bridgewater, NJ) camera. The filter set (Chroma Technology, Brattleboro,
VT) composed of 710 ± 25 nm excitation filter, 785 dichroic mirror, and
8
10 ± 20 nm emission filter was used to detect all NIR fluorophores. These Conflict of Interest
cells were observed again after 2 days to compare long-term stability.
In Vivo Cell Tracking and Histological Analysis: Animals were housed
in an AAALAC-certified facility and were studied under the supervision
of BIDMC IACUC in accordance with the approved institutional
protocol (#057-2014). Six-week-old C57BL/6 mice (male; 20–25 g) were
The authors declare no conflict of interest.
purchased from Charles River Laboratories (Wilmington, MA). Animals Keywords
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1
−1
were anesthetized with 100 mg kg ketamine and 10 mg kg xylazine
intraperitoneally (Webster Veterinary, Fort Devens, MA). PC3 cells
fixable bioprobe, histological analysis, in vivo cell tracking, near-infrared
fluorescence, optical imaging
−
6
were stained with 2 × 10 m of CTNF126 or IR786 for 30 min at 37 °C,
and detached with cell scraper. 1 × 10 cells per mouse were injected
6
intravenously into the mice. Animals were sacrificed 5 min and 24 h
postinjection of the cells, respectively.
Received: September 25, 2018
Revised: January 22, 2019
Published online:
The lung cavity was opened to observe injected cells in the lung using
the custom-built intraoperative NIR imaging system, which equipped
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7
60 nm excitation light (3.6 mW cm ) and white light (400–650 nm)
at 5500 lux. Color and NIR fluorescence images were acquired
simultaneously with custom software at rates of up to 15 Hz over a field of
view with a diameter of 15 cm. The lungs were resected and embedded in
Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA).
The tissues were then cryosectioned at 10 µm interval and
stained with hematoxylin and eosin for further visualization with the
customized TE2000 NIR fluorescence microscope. For validation of
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Adv. Mater. 2019, 1806216
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