Near-Infrared Fluorescent Probe Activated by Nitroreductase for in Vitro and in Vivo Hypoxic Tumor Detection

Vitro and In Vivo Hypoxic Tumor Detection

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Near-Infrared Fluorescent Probe Activated by Nitroreductase for In

Sanu Karan, Mi Young Cho, Hyunseung Lee, Hwunjae Lee, Hye Sun Park, Mahesh Sundararajan,

Jonathan L. Sessler,* and Kwan Soo Hong *

Cite This: J. Med. Chem. 2021, 64, 2971 −2981

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sı Supporting Information

ABSTRACT: Tumor hypoxia is correlated with increased

resistance to chemotherapy and poor overall prognoses across a

number of cancer types. We present here a cancer cell-selective and

hypoxia-responsive probe (fol-BODIPY) designed on the basis of

density functional theory (DFT)-optimized quantum chemical

calculations. The fol-BODIPY probe was found to provide a rapid

ﬂuorescence “oﬀ−on” response to hypoxia relative to controls,

which lack the folate or nitro-benzyl moieties. In vitro confocal

microscopy and ﬂow cytometry analyses, as well as in vivo near-

infrared optical imaging of CT26 solid tumor-bearing mice,

provided support for the contention that fol-BODIPY is more

readily accepted by folate receptor-positive CT26 cancer cells and

provides a superior ﬂuorescence “oﬀ−on” signal under hypoxic

conditions than the controls. Based on the ﬁndings of this study, we propose that fol-BODIPY may serve as a tumor-targeting,

hypoxia-activatable probe that allows for direct cancer monitoring both in vitro and in vivo.

INTRODUCTION

biologically convenient spectral region (λ /λ = 685/730

nm).

ex em

■

Hypoxia is a common characteristic of solid tumor tissues,

resulting from limited blood supply and inadequate oxygen

Tumor hypoxia is correlated with increased levels of various

enzymes, such as nitroreductase (NTR), azo-reductase, and

delivery. Various disease states are associated with chronic

8,19

−5

quinonereductase.

Not surprisingly, therefore, many

hypoxia, including cancer,

intracellular infectious diseases,

hypoxia-responsive probes have been conjugated to nitro,

coronary and peripheral artery diseases, alcohol-induced liver

8,9

azo, selenium, and quinone moieties, which are sensitive to

injury, and gastrointestinal inﬂammatory conditions. Tumor

20−23

hypoxic microenvironments.

Nevertheless, probes that

hypoxia is correlated with treatment resistance, aggressive

may be used to image both intracellular hypoxia and hypoxia in

vivo remain all but unknown. Alternative approaches to

hypoxia sensing, including positron emission tomography and

phenotypes, a large metastatic potential, and decreased

11,12

overall survival rates.

Therefore, it would be potentially

beneﬁcial to be able to detect and monitor hypoxia since doing

so might allow therapies to be tailored to a particular status. It

might also allow for tumor monitoring under conditions of

clinical use, including surgical resection. Hitherto, several

methods have been developed to detect hypoxia, such as blood

ﬂow velocity monitoring, oxygen electrodes, and methods

based on hypoxia markers. However, given their ease of

potential use, speciﬁc probes for hypoxic tumors could prove

complementary to these existing methods. More generally,

various F-labeled radiotracer-based strategies, have been

reported; however, they too are plagued by major challenges,

such as high background noise and limited spatial reso-

24,25

lution.

In an eﬀort to overcome some of the drawbacks associated

with optical probes for in vivo cancer detection, increasing

attention is being devoted to exploring the use of NIR

ﬂuorophores (i.e., agents with emission wavelengths above 700

14−16

nm). To date, several hypoxia-sensitive NIR ﬂuorophores

there is a need for cancer detection probes.

Unfortunately,

in many instances, the probes suﬀer from poor sensitivity. In

the speciﬁc case of hypoxia probes, drawbacks include poor

penetration, high scattering of the excitation and emission light

through the tissues, and limits in the increase of ﬂuorescence

intensity seen in animal or clinical studies. Here, we report a

conjugate (fol-BODIPY) that acts as a near-infrared (NIR)

ﬂuorescent hypoxia-sensitive probe and which operates in a

Received: December 15, 2020

Published: March 12, 2021

2021 American Chemical Society

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Journal of Medicinal Chemistry

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Scheme 1. Chemical Structures of fol-BODIPY and Controls 1 and 2 and Schematic Representation of Cancer Targeting and

the Active Switching on of Fluorescence Expected under Hypoxic Conditions

Figure 1. DFT-optimized structures, computed HOMO, and LUMO gaps for fol-BODIPY (A) and control 1 (B). The calculated energy gap for

fol-BODIPY is ΔE_f_o_l_‑_B_O_D_I_P_Y= (HOMO−LUMO) = −0.933 eV, while that for the control 1 is −1.166 eV.

have been reported; ⁶⁻³¹however, few of these ﬂuorophores

cancer cells. Therefore, there remains a need for a tumor-

targeting, hypoxia-responsive NIR probe that emits in the >700

nm spectral region.

32,33

possess a cancer-targeting moiety.

these ﬂuorophores can diﬀerentiate between normal and

Furthermore, few of

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Figure 2. (A) Normalized ﬂuorescence spectra of fol-BODIPY, control 1, and control 2 in the presence and absence of NTR (1 μg/mL) and (B)

corresponding quantitative comparison of the ﬂuorescence intensity. All experiments were carried out in the presence of NADH hydrate (200 μM).

The data shown are the average of four independent experiments; error bars are the standard deviation (nonpaired Student’s t-test). ***p < 0.001.

BF -chelated azadipyrromethane class 1 (λ = 685 nm, λ

730 nm) probes (Scheme S1 ) are modiﬁed versions of

With such design attributes in mind, a BF -chelated

azadipyrromethane NIR probe was conjugated to an NTR-

responsive p-nitrobenzyl moiety via a phenolic ether linker

giving the fol-BODIPY probe (Scheme 1). This probe was

synthesized in three successive steps as shown in Scheme S1.

BODIPY (λ = 493 nm, λ = 503 nm), where class 1 refers to

the nature and position of the pyrrole ring substitution (e.g.,

Ph at positions 3 and 5). These derivatives typically display

good chemical stability in aqueous media, possess high

ﬂuorescence quantum yields, and are characterized by excellent

Precursors and mono-O- Bu-BODIPY were synthesized in

8,49

accordance with reported methods.

Control 1 (Scheme

photo-stability at an absorption/emission λ

of 696/727

S1 ) was formed by reacting mono-O- Bu-BODIPY with 4-

nitrobenzyl bromide with an yield of 78.3%. Compound 6a,

produced by deprotection of the BOC group in control 1 using

triﬂuoroacetic acid, was reacted with N-hydroxysuccinimide in

the presence of diisopropylethylamine to form a reactive

intermediate (N-hydroxysuccinimide ester). This intermediate

max

4,35

nm.

Thus, to create a highly sensitive ﬂuorescent oﬀ/on

probe for hypoxic regions with cancer-targeting speciﬁcity, we

aimed to build oﬀ a BF -chelated azadipyrromethane core, a

class 1 probe known for its excellent photo-physical properties,

high ﬂuorescence quantum yield (0.3−0.4), and good photo-

6,37

stability.

As detailed below, this core was conjugated to a

was subsequently reacted with folate ethylenediamine to

produce fol-BODIPY (Scheme S1 ). Control 2 was prepared

using the same method but starting with compound 4 and

benzyl bromide. The fol-BODIPY conjugate and controls 1

folate subunit to produce a conjugate (fol-BODIPY) that acts

as an NIR ﬂuorescent hypoxia-sensitive probe (λ /λ = 685/

ex em

30 nm). The alpha isoform of the folate receptor (FR-α) is

common in about 40% of human cancers and is especially

prevalent in malignant tissues such as ovarian and colorectal

cancers. This makes folate a useful cancer-targeting group. In

contrast, folic acid or its conjugates do not accumulate

and 2 were characterized by H and C NMR spectroscopy, as

well as by HRMS (Figures S1 −S18) and high-performance

liquid chromatography (HPLC) analysis (Figure S19).

Figure 1 shows the density functional theory (DFT)-

optimized structures and the frontier molecular orbitals

expected to deﬁne the optical properties of fol-BODIPY (A)

as well as those of control 1 (B). In both species, a presumed

38−41

appreciably in normal tissues.

Not surprisingly, therefore,

a range of folate-conjugated drugs and imaging agents have

42−46

been developed recently.

The fol-BODIPY probe also contains a p-nitrobenzyl ether

hydrogen bonding interaction between the -BF group and the

linkage. This is a known NTR cleavable trigger. In the case of

fol-BODIPY, it permits the selective targeting of hypoxic

cancer cells overexpressing NTR in vitro, as well as hypoxic

tumors in vivo as demonstrated using a xenograft mouse model.

phenyl ring is inferred based on their proximity (ca. 1.99 Å).

The computed energy gaps between the highest occupied

molecular orbital (HOMO) and the lowest unoccupied

molecular orbital (LUMO) between fol-BODIPY and control

are 0.93 and 1.17 eV, respectively. This leads us to suggest

RESULTS AND DISCUSSION

■

that fol-BODIPY will be more reactive and sensitive than its

congener control 1. Based on this DFT-optimized structure,

fol-BODIPY was selected for further study as a potential

hypoxia probe. We then optimized the structure of the reduced

aniline (but uncleaved) form of fol-BODIPY using a protocol

Design and Synthesis of fol-BODIPY. Our goal was to

generate an eﬃcient ﬂuorescent probe that would allow for in

vivo hypoxic tumor detection. We felt that such a putative

probe would need to fulﬁll several speciﬁc requirements,

including having an NIR optical window while displaying

enhanced ﬂuorescence with excellent sensitivity and selectivity.

51−53

analogous to that used in our previous studies

S20A ). We also computed the absorption spectrum of control

(Figure

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Figure 3. (A) UV absorption and (B) ﬂuorescence emission (λ_e_x= 642 nm) spectra of fol-BODIPY (5 μM) recorded in the presence of various

concentrations of NTR (0 −1 μ g/mL). All data points were acquired 30 min after the addition of NTR into the medium at 37 °C.

Figure 4. Fluorescence response of fol-BODIPY (5 μM) in the presence of NADH (50 μM) to various species (A): (1) fol-BODIPY + NADH,

(

2) 150 mM KCl, (3) 2.5 mM CaCl , (4) 2.5 mM MgCl , (5) 10 mM glucose, (6) 1 mM vitamin C, (7) 1 mM vitamin B , (8) 100 μM HSA, (9)

•

0 μM H O , (10) 10 μM OH, (11) 1 mM glutamic acid, (12) 1 mM arginine, (13) 1 mM serine, (14) 5 mM glutathione, (15) 1 mM cysteine,

2 2

(16) 1 mM homocysteine, (17) 1 mM dithiothreitol (DTT), and (18) 0.5 μg/mL NTR. (B) Fluorescence response at λ_e_m= 730 nm of fol-

BODIPY (5 μM) with diﬀerent species present in blood serum (graphically). (C) Time-dependent ﬂuorescence intensity change of the probe (5

μM) in the presence of NTR (0.5 μg/mL). (D) Maximum ﬂuorescence intensity increase seen when the fol-BODIPY probe (5 μM) is treated with

NTR at diﬀerent pHs per the conditions of (A). All experiments were carried out in the presence of NADH (200 μM) and an excitation wavelength

(λ_e_x) of 642 nm was used in all cases.

using a range-separated DFT (CAM-B3LYP) in conjunction

(NADH) under physiological conditions (pH 7.4). The optical

changes, if any, were then monitored using UV−vis and

ﬂuorescence spectroscopies (Figures S21 −S22). Although no

signiﬁcant changes were observed for control 2 or in the

absence of NTR, a remarkable enhancement in the

ﬂuorescence intensity at 730 nm was observed for fol-

BODIPY in the presence of NTR (Figure 2). Such ﬁndings

are consistent with the design expectation that NTR promotes

with the def2-SV(P) basis set as implemented in the ORCA

package. We used 10 roots to predict the spectrum. The

computed spectrum qualitatively matches the experimental

data, as shown in Figure S20B. To gain further insights into the

guiding structure−function relationships, we likewise carried

out DFT calculations to understand the nature of Frontier

orbitals that dictate the origin of absorption/emission

spectrum of fol-BODIPY and control 1. The HOMO−

LUMO gap of fol-BODIPY (0.933 eV) is smaller by 0.23 eV

compared to those of control 1 (1.167 eV), leading us to infer

that the former is more reactive than control 1.

reduction of the electron-withdrawing nitro group (−NO ) to

the corresponding electron-donating amine group (−NH ),

which then leads to self-immolative ether cleavage. The net

result is release of the free ﬂuorophore and a readily discernible

enhancement in the ﬂuorescence intensity. Since control 2

lacks a 4-nitro group, it and its associated optical features

remain unchanged in the presence of NTR.

Optical Properties of Fol-BODIPY. Based on consid-

erable literature precedent, it was expected that NTR would

promote cleavage of the phenolic ether (−O−) bond in fol-

BODIPY so as to release the active ﬂuorophore. To test this

hypothesis, fol-BODIPY and control 2 were treated with 1 μg/

mL NTR in the presence of nicotinamide adenine dinucleotide

Figure 3 shows that the absorption maxima at 685 nm and

the emission maxima at 730 nm increase steadily upon

addition of NTR. The results presented in Figure 3A reveal

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Figure 5. In vitro ﬂuorescence imaging of CT26 cells. Representative confocal laser scanning microscopy (CLSM) images of CT26 cells incubated

with fol-BODIPY or controls 1 and 2 (5 μM each) for 15 min under hypoxic (A) and normoxic (B) conditions. Scale bar, 20 μm. Images were

obtained using an excitation wavelength of 633 nm and emission wavelengths in the 638−737 nm spectral region. (C) Corresponding quantitative

hypoxia−normoxia ratio for control 1, control 2, and fol-BODIPY. Relative ﬂuorescent intensity was measured from seven individual cells using

ImageJ software (nonpaired Student’s t-test). (D) FACS analyses of CT26 cells treated with fol-BODIPY and controls 1 and 2. Cells, incubated

with PBS under hypoxic or normoxic conditions for 24 h, were treated with fol-BODIPY or controls 1 and 2 (5 μM each) for 15 min before ﬂow

cytometry analysis. (E) Comparison of the MFI measured using ﬂow cytometry. Plotted data are the average of three independent experiments;

error bars are the standard deviations (nonpaired Student’s t-test). ***p < 0.001.

that the intensity of the UV−vis absorption spectral feature

centered at 685 nm in the case of fol-BODIPY gradually

increases as a function of NTR concentration (0−1 μg/mL) up

an apparent 8-fold maximum in the presence of 1 μg/mL

NTR. Similarly, the ﬂuorescence intensity of fol-BODIPY was

seen to increase ∼20-fold upon the addition of 1 μg/ml NTR

cleavage process. Species tested in this regard included metal

ions (Na , K , Ca , and Mg ), amino acids (cysteine, serine,

homocysteine, glutamic acid, arginine, and glutathione),

NADH, DTT glucose, vitamin C, vitamin B , and H O

(Figure 4A). We also monitored the evolution of the NTR-

mediated ﬂuorescence emission intensity increase in the case

of fol-BODIPY (cf. Figures 4C and S26). Saturation was

reached within 25 min following initial exposure to NTR (1

μg/mL) in the presence of NADH (200 μM) at 37 °C. We

also examined the reactivity of fol-BODIPY toward NTR as a

function of pH. As shown in Figure 4D, the ﬂuorescence

emission intensity at λ_e_m= 730 nm recorded in the presence of

NTR (1 μg/mL) was near maximal at pH 7. Good plasma

stability was also seen for fol-BODIPY over the course of 48 h

(Figure S27). Taken in aggregate, these predicative studies

provide support for the notion that fol-BODIPY could serve as

a useful probe for hypoxic cancer cells and hypoxic tumor

microenvironments.

(Figure 3B). Based on a regression analysis, the calculated

detection limit of the fol-BODIPY probe toward NTR was

found to be 1.52 ng/mL (Figure S23). This represents a lower

detection limit than that seen for previously reported

2−29

probes

and is equivalent to an ultrasensitive probe.

To gain further insights into the NTR-mediated release of an

active ﬂuorophore from fol-BODIPY, it was treated with 1 μg/

mL NTR in phosphate-buﬀered saline (PBS) for 1 h in the

presence of NADPH. An aliquot was subjected to HR-MS

analysis, which revealed inter alia two major peaks, one

ascribable to the p-nitrobenzyl ether-free ﬂuorophore ([M +

H] = 1053.37) and one corresponding to 4-aminobenzyl

alcohol ([M + H] = 124.076) (Figure S24). Moreover, new

In Vitro Probe Activation and Imaging of Hypoxic CT-

26 Cells. Cytotoxicity is a major issue to consider in the

context of biological probe development. Cell proliferation

studies [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium

bromide (MTT) assays] were thus carried out in the CT26

bands, consistent with the NTR-promoted self-immolative

ether bond cleavage, were observed in the HPLC chromato-

gram (Figure S25). It was also observed that the presence of

potential biological interferants had little adverse eﬀects on the

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Figure 6. In vitro ﬂuorescence imaging of cell spheroids with fol-BODIPY. Representative ﬂuorescence (E /E = 685/730 nm) and bright-ﬁeld

images of tumor CT26 (A) and normal WI38 (B) cell spheroids incubated under culture conditions with fol-BODIPY or controls 1 and 2 (10 μM,

each) and corresponding quantitative comparison of the MFI measured in ROIs (regions of interest) of the spheroid center (C) and periphery (D).

Two independent experiments were performed, and the error bars represent the standard deviation (nonpaired Student’s t-test). Scale bar: 100 μm.

*p < 0.05.

54,55

and WI38 cell lines using fol-BODIPY and control 1.

cells.

We thus believe that fol-BODIPY may be used to

Gratifyingly, both probes proved essentially nontoxic with

distinguish eﬀectively hypoxic cells from normoxic cells.

CT26 cells were also incubated for diﬀerent times (2, 4, 6, 8,

and 24 h) under the hypoxic conditions (1% oxygen) before

being treated with either fol-BODIPY or control 1 (1 μM in

each) for 15 min prior to analysis Figure S31). In contrast to

what was seen for control 1, the ﬂuorescence intensity of fol-

BODIPY was found to increase as a function of pre-incubation

time (Figure S32). These ﬁndings are rationalized in terms of

the eﬀectiveness of hypoxia increasing as a function of time,

which translates into a more reducing environment leading to

greater activation of fol-BODIPY.

≥

90% cell viability being seen after incubation with 0−50 μM

for 24 h (Figures S28 ).

As noted above, the fol-BODIPY probe comprises a folate

tumor-targeting element. Therefore, we expected it to target

selectively folate receptor-enriched CT26 cells relative to folate

receptor-negative cell lines, such as WI38. After conﬁrming

that hypoxia was maintained in the 1% oxygen incubator used

for these studies by Western blot analysis of hypoxia-inducible

factor-1α protein (HIF-1α) as a hypoxia marker (Figure S29 ),

we incubated CT-26 cells with fol-BODIPY and, separately,

control probes 1 and 2 (5 μM in all cases) under both

normoxic and hypoxic (1% oxygen) conditions. The cellular

uptake of fol-BODIPY and controls was conﬁrmed using

confocal laser scanning microscpy (CLSM) to aﬀect live cell

imaging. As shown in Figure 5A, a strong ﬂuorescence was

seen in CT26 cells 15 min post-incubation with fol-BODIPY

To test the eﬀectiveness of fol-BODIPY in hypoxic tumor

microenvironments, ﬂuorescence microscopic images of folate

6,57

receptor-positive

tumor CT26 (Figure 6A) and folate

normal WI38 cells (Figure 6B) in 3D

8,59

receptor-negative

spheroid culture condition were recorded after incubating with

fol-BODIPY or control 1 or 2 (10 μM). In both the spheroid

center and periphery, higher ﬂuorescence signals were

observed only in the fol-BODIPY group, while only a minimal

ﬂuorescence signature was seen in the case of control 1 and 2

groups. There was a nonsigniﬁcant diﬀerence in the spheroid

center between CT26 and WI38 cells (Figure 6C; 52 ± 1 vs 41

± 4, p = 0.25), indicating that fol-BODIPY can travel

intercellularly to the spheroid center, where it is then

presumably reduced via hypoxia to produce a ﬂuorescent

signal. Enhanced (2.5-fold higher) mean ﬂuorescence intensity

(MFI) in the fol-BODIPY group was observed in the folate

receptor-positive CT26 tumor spheroid periphery compared to

the normal WI-38 cells (Figure 6D; 91 ± 2 vs 35 ± 6; p =

0.031). This diﬀerence is ascribed to folate receptor-mediated

intracellular uptake and accumulation of fol-BODIPY as it

encounters cells in the spheroid periphery, an eﬀect that is

expected to be enhanced in the case of CT26-derived

spheroids. In the CT26 tumor spheroids incubated with fol-

BODIPY, higher ﬂuorescent MFI was observed in the tumor

periphery than in the tumor core region (center) (91 ± 2 vs 52

± 1; p = 0.043). This ﬁnding is consistent with fol-BODIPY

accumulating relatively well in the peripheral region notwith-

standing the fact that the cores of these tumor models are more

(

10 μM) under hypoxic conditions; the eﬀect was greater than

under normoxic conditions (Figure 5B). The relative

normoxia−hypoxia ﬂuorescent intensity was determined

using ImageJ software (Java, Maryland, USA) in CT26 cells

treated with either control 1, control 2, or fol-BODIPY

(

Figure 5C). These studies revealed very high normoxia−

hypoxia ﬂuorescence intensity ratios in the case of fol-

BODIPY. Further evidence of selective hypoxic cell imaging

in the case of CT26 cells came from live cell ﬂuorescence-

activated cell sorting (FACS) studies, which revealed that fol-

BODIPY gives rise to a high level of ﬂuorescence under these

simulated hypoxic conditions (Figure 5C,D). In contrast, only

a subtle ﬂuorescence diﬀerence was seen under otherwise

identical experiment conditions in the case of WI38 cells

Figure S30). Little or no ﬂuorescence intensity increase or

hypoxic versus normoxic diﬀerentiation was seen in the case of

controls 1 and 2. Adventitious reduction of fol-BODIPY in the

case of normoxic cells could serve as a potential limitation.

However, in nitroaromatic compounds with suitable reduction

potentials (approximately −330 to −450 mV), the ﬁrst radical

anions formed by one-electron addition can be eﬃciently

scavenged by molecular oxygen. As a result, the fully cleaved

daughter products are only observed in appreciably hypoxic

60,61

hypoxic than the peripheral regions.

In other words for

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Figure 7. Tumor detection in vivo carried out by means of ﬂuorescence imaging with fol-BODIPY. (A) Time-dependent in vivo optical imaging of

xenograft CT26 tumor-bearing mice following intravenous injection of fol-BODIPY or control 1 (1 mM each, 100 μL) and (B) corresponding

quantitative comparison of the MFI obtained in the tumor region of CT26 tumor-bearing mice (n = 5) at 22 h post-injection (nonpaired Student’s

t-test). (C) Representative ex vivo ﬂuorescence images obtained from several organs including the liver, lungs, spleen, heart, and tumor, extracted

after ﬁnal in vivo imaging at 22 h, for the fol-BODIPY group compared with the control 1 group. (D) Corresponding comparison of the relative

mean ﬂuorescence intensty (MFI) measured in tumor tissues (n = 3), and error bars are the standard deviations (nonpaired Student’s t-test). *p <

0.05 and ***p < 0.001.

spheroids, it is predicted that the nature of the cells plays a

dominant role in determining signal intensity. Support for this

conclusion comes from the ﬁnding that a relatively weak signal

is seen in the case of the folate receptor-negative WI38

spheroids.

of fol-BODIPY was seen based on the observation of an

enhanced tumor-speciﬁc ﬂuorescence emission (Figure 7A).

Quantitative analyses revealed that the ﬂuorescence intensity at

the tumor site was approximately 2-fold higher in the case of

fol-BODIPY as compared to control 1 (Figure 7C). After

sacriﬁce, organs and tumor tissues were harvested from the

mice and their respective ﬂuorescence intensities were

compared. Appreciable ﬂuorescence was observed only in the

solid tumors obtained from the animals treated with fol-

BODIPY; no ex vivo ﬂuorescence was detected in the heart,

body, liver, lung, and spleen (Figure 7B, left side), unlike the

results observed in the corresponding studies carried out with

control 1 (Figure 7B). Quantiﬁcation of the data revealed a

In Vivo and Ex Vivo Fluorescence Imaging of the

Hypoxic CT26 Tumor-Bearing Mouse Model Using fol-

BODIPY. The potential use of fol-BODIPY as a speciﬁc

tumor-targeting ﬂuorescent probe for bio-imaging was further

investigated by monitoring the ﬂuorescence emission in

xenograft tumor models and in diﬀerent organs in vivo.

Toward this end, CT26 hypoxic tumor-bearing nude mice

were injected with fol-BODIPY, control 1, or PBS at the

tumor site. In vivo images were obtained at various times. A

readily discernible NIR ﬂuorescence was seen immediately

after injection of the probe (Figure S33A). The NIR

ﬂuorescence initially increased with time before eventually

fading. At 25 h, the ﬂuorescence intensity seen with fol-

BODIPY in the tumor region was ∼2.4-fold higher than that

produced by control 1 (Figure S33B). The folate subunit

provides a degree of tumor targeting, leading to preferential

uptake in tumor cells. This, in turn, allows a readily

distinguishable ﬂuorescent signal to be observed for fol-

BODIPY. Importantly, these data serve to underscore the fact

that fol-BODIPY is more eﬀective in detecting hypoxia than

controls. fol-BODIPY and control 1 in PBS buﬀer were also

administered to CT26 cell-inoculated xenograft mice via

intravenous injection. After 22 h, evidence of accumulation

.5-fold higher ﬂuorescence intensity for fol-BODIPY at the

tumor region than in the case of control 1 (Figure 7D). On the

basis of these combined ﬁndings, we propose that fol-BODIPY

may be used to target hypoxic tumors eﬀectively without

inducing acute side eﬀects in other major organs, such as the

liver, lungs, spleen, and heart. No adverse reactions were

observed during or after imaging with our probes, nor was seen

appreciable animal weight loss. We thus believe that fol-

BODIPY constitutes an attractive hypoxia-responsive ﬂuo-

rochrome that permits eﬀective NIR tumor imaging.

CONCLUSIONS

■

The cancer cell-targeting hypoxia-activatable fol-BODIPY

probe was designed based on DFT-optimized quantum

chemical calculations to permit cancer cell labeling and

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tumor imaging. The ﬂuorescence emission of fol-BODIPY

proved responsive to hypoxia; it was roughly 20-fold more

intense under hypoxic conditions when activated by exposure

to NTR than two control probes 1 and 2. Moreover, a fast

response time (<5 min) and ultra-sensitivity (detection limit of

ca. 1.5 ng/mL) were seen. In vitro CLSM and FACS studies

revealed that compared with the control probes, fol-BODIPY

is more easily taken up by folate receptor-positive CT26 cancer

cells and produces a greater NIR signal under hypoxic

conditions. These imaging beneﬁts were recapitulated in 3D-

cultured hypoxia-mimicking spheroid cells. In vivo studies,

involving CT26 cell-derived mouse xenografts, served to

conﬁrm that relative to control 1, which lacks a folate-based

tumor-targeting moiety, fol-BODIPY selectively accumulates

in the solid tumor region after tail vein injection, giving rise to

maximally enhanced ﬂuorescence intensity within 1 day. This

tumor-targeting ability and the observation of speciﬁc

activation in hypoxic tumor regions leads us to suggest that

fol-BODIPY is a promising ﬂuorescence “oﬀ−on” probe that

could be used in potential applications, such as initial diagnosis

of surgery, where the ﬂuorescence-guided image detection of

tumor tissue would be beneﬁcial.

with brine and dried over anhydrous Na

to dryness under reduced pressure. The residue was washed with 20%

acetone in ether, acetone, and ether. The product fol-BODIPY was

obtained as a dark green solid (175 mg, 73.5%). H nuclear magnetic

, ﬁltered, and evaporated

resonance (NMR) (400 MHz, DMSO-d ): δ 11.43 (s, 1H); 8.65 (d, J

8.68, 1H); 8.30−8.21 (m, 2H); 8.19−8.1 (m, 5H); 8.08−8.2 (m,

3H); 7.83 (d, J = 5.76, 2H); 7.78−7.73 (m, 3H); 7.69−7.63 (m, 4H);

.55−7.46 (m, 5H); 7.47 (d, J = 6.44, 1H); 7.26−7.22 (m, 2H); 7.17

(d, J = 8.92, 2H); 6.94 (d, J = 6.56, 2H), 6.63 (s, 2H); 5.44 (s, 2H);

4.63 (t, J = 5.6, 2H); 4.49 (d, J = 6.2, 2H); 4.30 (s, 1H); 3.34 (m,

2H); 3.08 (d, J = 11.76, 2H); 2.40 (d, J = 7.0, 2H); 2.28 (d, J = 6.64,

H). C NMR (100 MHz, DMSO-d ): 180.72, 172.62, 172.50,

72.30, 172.23, 166.90, 154.21, 151.19, 149.07, 148.95, 144.93,

44.80, 133.68, 132.30, 132.05, 129.51, 129.02, 128.78, 128.72,

28.68, 128.59, 128.48, 128.22, 124.08, 123.98, 121.72, 120.13,

16.26, 115.47, 115.65, 68.72, 67.29, 65.35, 53.62, 46.26, 38.79, 34.56,

2.43 ppm. Electrospray ionization (ESI) high-resolution mass

spectrometry (HR-MS) [M + Na] : calcd 1210.40; found,

1210.405. The purity of fol-BODIPY was conﬁrmed as 98.7% by

HPLC analysis.

Synthesis of Control 2. Control 2 (yield: 78.25%) was prepared

using a procedure analogous to that used to prepare fol-BODIPY

except for starting with compound 6b (Scheme S1 ). H NMR (400

MHz, DMSO-d ): δ 8.07 (d, J = 7.28, 4H); 8.00 (m, 5H); 7.56 (m,

H); 7.50 (m, 8H); 7.38 (m, 4H); 7.28 (d, J = 8.4, 2H); 7.12 (d, J =

.16, 2H); 5.24 (s, 2H); 4.78 (m, 1H); 4.48 (s, 2H); 3.17 (s, 2H);

EXPERIMENTAL SECTION

.89 (s, 4H); 2.73 (s, 4H). C NMR (100 MHz, DMSO-d ): 162.34,

■

60.28, 158.15, 157.84, 149.38, 147.91, 140.44, 136.71, 133.44,

33.33, 128.63, 128.58, 128.52, 128.28, 128.19, 128.02, 127.85,

24.12, 123.52, 118.81, 115.83, 115.63, 114.82, 69.56, 60.22, 48.58,

5.32, 35.80, 30.78, 20.79 ppm. ESI HR-MS m/z [M + H] : calcd

142.42; found, 1142.4102.

Materials and Instrumentation. Commercial analytical grade

chemicals were purchased and used without further puriﬁcation.

Further details including the chemical synthesis of compounds are

provided in the Supporting Information .

Synthesis of Control 1. A dimethylformamide (DMF) solution

5 mL) of compound 4 (Scheme S1 , 643.5 mg, 1.0 mmol) and

Absorption and Fluorescence Studies. Fluorescence and UV−

(

K CO (276.42 mg, 2 mmol) were stirred at 0 °C under a nitrogen

vis spectra were recorded using a spectrophotometer (S-3100; Scinco,

Seoul, Korea) in a 1 cm standard quartz cell and a UV-1800

spectrophotometer (Scinco), respectively. NTR from Escherichia coli

was used in our experiments. Stock solutions of various analytes

atmosphere for 10 min. Then, 4-nitrobenzylbromide (324 mg, 1.5

mmol) in 3 mL of DMF was added slowly to the reaction mixture,

which was stirred at room temperature (RT) overnight. The reaction

mixture was diluted with water and extracted with EtOAc. After

collecting the organic layer and removing the bulk of the solvent

under reduced pressure, the crude compound was puriﬁed by silica gel

(NADH, KCl, CaCl

, MgCl , glucose, vitamin C, 1 mM vitamin B ,

HSA, H , ·OH, glutamic acid, arginine, serine, glutathione,

cysteine, homocysteine, and DTT) were prepared using double-

distilled water. A stock solution of fol-BODIPY (20 μM) was

prepared using Dulbecco’s modiﬁed Eagle’s medium (DMEM)

containing 10% fetal bovine serum (FBS) (pH 7.4) and 0.2%

DMSO. The ﬂuorescence spectra were obtained at excitation

wavelengths under 685 nm using a 5 nm slit width. The ﬂuorescence

changes of fol-BODIPY (5.0 μM) were recorded in the presence of

increasing concentrations of NTR (0−1 μg/mL) in DMEM with 10%

FBS (pH 7.4) and 0.2% DMSO. For these studies, fol-BODIPY was

incubated with NTR for 30 min at 37 °C. The ﬂuorescence quantum

yield of fol-BODIPY was measured in the presence and absence of

NTR in the cell medium (pH 7.4).

column chromatography using ethyl acetate/hexanes (EtOAc/Hex)

(

1:2) as the eluent; this aﬀorded 710 mg (91.19%) of control 1. H

NMR (400 MHz, DMSO-d ): δ 8.29 (d, J = 6.4, 2H); 8.17 (m, 8H);

.77 (d, J = 5.28, 2H); 7.60 (m, 2H); 7.53 (d, J = 4.44, 2H); 7.48 (m,

H); 7.24 (d, J = 4.8, 2H); 7.12 (d, J = 8.36, 2H); 5.42 (s, 1H); 4.83

(s, 1H); 1.46 (s, 9H). C NMR (100 MHz, DMSO-d ): 170.30,

161.03, 160.89, 157.89, 157.77, 154.40, 154.26, 147.56, 145.01,

144.89, 142.61, 133.39, 132.25, 132.16, 130.02, 129.57, 129.36,

129.13, 128.81, 128.76, 124.18, 124.13, 124.12, 120.28, 116.29,

116.00, 115.67, 115.35 ppm. ESI HR-MS m/z (M + Na): calcd

801.26; found, 801.2667.

Synthesis of fol-BODIPY. Compound 6a (Scheme S1 ) was

Quantum Chemical Calculations. Quantum chemical calcu-

lations were carried out by using a quantum chemical calculation

synthesized from 4-hydroxychalconone (purchased commercially) in

six consecutive steps as detailed in the Supporting Information .

Brieﬂy, a mixture of compound 6a (145 mg, 0.2 mmol) and N-(3-

dimethyl-aminopropyl)-N′-ethylcarbodiimide hydrochloride (77 mg,

program (ORCA 4.0.1, Max-Planck Institute, Berlin, Germany).

The structures were optimized using the BP86 exchange correlation

3,64

DFT

in conjunction with a Karlsruhe split valence polarization

65,66

.4 mmol) was dissolved in anhydrous dimethyl sulfoxide (DMSO, 2

def2-SVP basis set

for all atoms. Due to the large system size, a

mL) and stirred at RT for 3 h under a N atmosphere. The solution

was extracted with dichloromethane (DCM) (50 mL) and 0.5 M HCl

medium-sized split valence basis set was used; it proved adequate to

support our experimental predictions. This method and basis set

combination has previously been used by us and found to give rise to

(

50 mL). The organic phases were washed with 0.5 M HCl brine and

51−53

dried over anhydrous Na SO , ﬁltered, and evaporated to dryness on a

acceptable predictions of geometric and electronic structure.

rotary evaporator, while the temperature of the water bath was kept

below 40 °C. The solid material obtained in this way and folate

ethylenediamine (compound 2) (87 mg, 0.18 mmol) were then

dissolved in 2 mL of anhydrous DMSO and stirred at RT for 18 h

NTR Detection Assay Using HPLC. HPLC was performed using

an HPLC system (YL9100, Youngin, Seoul, South Korea) with a

Sunﬁre C18 column (4.6 mm × 150 mm, 5 μm, Waters,

Massachusetts, USA). The conditions were as follows: volume ratio

under a N atmosphere. The volatiles were removed by means of a

of acetonitrile/H O = 100:0 (0 min) to 60:40 (20 min); ﬂow rate 1

short path distillation column under reduced pressure at RT. The

crude product obtained in this way was partitioned between DCM

mL/min; and UV detection at ≤254 nm.

Linear Range and Detection Limit. The detection limit of the

fol-BODIPY probe toward NTR was calculated based on

ﬂuorescence spectroscopic titrations. The ﬂuorescence emission

(

50 mL) and 1 M Na CO (50 mL). The aqueous phase was

2 3

extracted using DCM (3 × 30 mL). The organic layers were washed

978

J. Med. Chem. 2021, 64, 2971−2981

Journal of Medicinal Chemistry

The Supporting Information is available free of charge at

Featured Article

spectrum of fol-BODIPY (2 μM) was measured 10 times. The

standard deviation of the blank solution was also measured. The

ﬂuorescence intensity (λ_e_m= 730 nm) of the probe was plotted versus

the concentrations of NTR. The detection limit was calculated using

the following equation: detection limit = 3σ/k, where σ is the standard

deviation of the blank measurement and k is the slope between the

ﬂuorescence intensity versus the NTR concentration.

the left ﬂank region of 6 week-old male mice (Balb/c-nude), and the

animals were split into two groups (n = 3 each group). Ten days after

tumor inoculation, tumors reached an average diameter of 7 mm

before injection. At this point, the fol-BODIPY or control 1 probe (2

mg/kg) dissolved in 2.5% DMSO (100 μL) was intravenously

administrated via the tail vein, and in vivo ﬂuorescence images were

acquired at 3, 6, and 22 h post-intravenous (IV) injection of the

probes using an optical imaging system (IVIS Spectrum, PerkinElm-

er). The ﬂuorescence images were collected using an optical imaging

Determination of the Fluorescence Quantum Yield. The

ﬂuorescence quantum yield of fol-BODIPY in the presence and

absence of NTR was determined in PBS buﬀer (10 mM, pH 7.4)

using BF -chelated tetra-arylazadipyrromethene (Φ = 0.36) as a

system (IVIS Spectrum; E /E = 675//740 nm) equipped with a 30

x m

nm bandpass ﬁlter. The animals were sacriﬁced 24 h post-IV injection,

and several organs including the liver, lungs, spleen, heart, and tumor

were sampled. Subsequently, ex vivo imaging was performed using the

same imaging system.

standard. The ﬂuorescence quantum yield was calculated using the

following equation: Φ = Φ (A F /A F ), where Φ is the standard

ﬂuorescence quantum yield, Φ is the sample ﬂuorescence quantum

yield, A and A are the absorbance of the sample and the reference,

respectively, at the same excitation wavelength, and F and F are the

corresponding relative integrated ﬂuorescence intensities.

ASSOCIATED CONTENT

sı Supporting Information

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02162.

Materials and methods; 1H NMR, 13_C_N_M_R_,_H_R_-_M_S

■

Cells. The CT26 (mouse colon cancer; folate receptor-positive)

cell line was kindly provided by Y. S. Gho (POSTECH, Korea) and

cultured in minimum essential media (MEM, Gibco, Grand Island,

NY, USA) containing 10% FBS (Gibco) and 1% antibiotic-

antimycotic (Gibco). The WI-38 (human lung ﬁbroblast; folate

receptor-negative) cell line was obtained from the American Type

Culture Collection (ATCC, Rockville, MD, USA). The WI-38 cells

were maintained in Eagle’s minimum essential medium (EMEM,

ATCC) supplemented with 10% FBS and 1% antibiotic−antimycotic

ESI, HPLC chromatogram, optimized structure and

absorption spectrum, UV absorption spectrum, ﬂuo-

rescence emission spectra, detection limit, HPLC

proﬁles, stability test, cell cytotoxicity, western blot

analysis, ﬂow cytometric analysis, and CLSM images;

and spectroscopic data of compounds (PDF)

at 37 °C in a humidiﬁed atmosphere containing 5% CO .

In Vitro Cell Imaging. Cells were seeded at a density of 2 × 10

cells/well in eight-well plates (Ibidi, Martinsried, Germany) at 37 °C

in a humidiﬁed incubator and kept overnight. The next day, the cells

Molecular formula strings (CSV)

were transferred to a hypoxia (1% O ) incubator chamber

(STEMCELL Technologies, Inc., Vancouver, BC, Canada) or kept

AUTHOR INFORMATION

Corresponding Authors

■

under normoxic conditions for 8 h and then incubated either with 5

μM fol-BODIPY or a control probe for 15 min. Fluorescence images

were acquired using a confocal laser scanning ﬂuorescence micro-

scope (LSM710, Carl Zeiss, Oberkochen, Germany) equipped with a

BODIPY ﬁlter set. For ﬂuorescence imaging of spheroids, cells were

Jonathan L. Sessler − Department of Chemistry, The

University of Texas at Austin, Austin, Texas 78712-1224,

United States; orcid.org/0000-0002-9576-1325;

Email: sessler@cm.utexas.edu

seeded at a density of 2 × 10 cells/well in ultra-low attachment 96-

Kwan Soo Hong − Research Center for Bioconvergence

Analysis, Korea Basic Science Institute, Cheongju 28119,

Republic of Korea; Graduate School of Analytical Science and

Technology, Chungnam National University, Daejeon 34134,

Republic of Korea; orcid.org/0000-0003-4342-5183;

Email: kshong@kbsi.re.kr

BODIPY and the control probes. Cells were seeded at a density of

Authors

0,000 cells/well in 96-well plates and grown overnight at 37 °C in an

Sanu Karan − Research Center for Bioconvergence Analysis,

Korea Basic Science Institute, Cheongju 28119, Republic of

Korea; Graduate School of Analytical Science and

Technology, Chungnam National University, Daejeon 34134,

Republic of Korea

Mi Young Cho − Research Center for Bioconvergence Analysis,

Korea Basic Science Institute, Cheongju 28119, Republic of

Korea

Hyunseung Lee − Research Center for Bioconvergence

Analysis, Korea Basic Science Institute, Cheongju 28119,

Republic of Korea

Hwunjae Lee − Research Center for Bioconvergence Analysis,

Korea Basic Science Institute, Cheongju 28119, Republic of

Korea

Hye Sun Park − Research Center for Bioconvergence Analysis,

Korea Basic Science Institute, Cheongju 28119, Republic of

Korea

Mahesh Sundararajan − Theoretical Chemistry Section,

Bhabha Atomic Research Centre, Mumbai 400 085, India;

orcid.org/0000-0002-1522-124X

incubator with 5% CO . The next day, the cells were incubated with

diﬀerent concentrations of fol-BODIPY and control 1 for 24 h before

being subject to exchange with a fresh culture medium containing

10% MTT assay (5 mg/ml in PBS) solution and incubating for an

additional 2 h at 37 °C. Subsequently, the medium was removed and

the cells were dissolved in 100 μL of DMSO. The absorbance was

measured using a microplate reader (XMark, Bio-Rad, Berkeley, CA,

USA) at 570 nm.

Flow Cytometric Analyses. CT26 and WI-38 cells were seeded

at a density of 1.5 × 10 cells/well in six-well plates, grown for 20 h,

and then pre-incubated for 8 h under hypoxic or normoxic conditions

prior to incubation with fol-BODIPY or controls 1 or 2. After

incubation for 15 min, the cells were gently scraped, suspended in

PBS (Gibco), and transferred to ﬂow cytometry tubes. Subsequently,

the cells were analyzed using a ﬂow cytometer (Attune NxT, Thermo

Fisher) for red RL2 ﬂuorescence (638 nm excitation, 720/30 nm

emission). All analyses were carried out in triplicate using at least

0,000 cells.

In Vivo and Ex Vivo Animal Imaging. All animal experiments

were performed according to protocols approved by the Institutional

Animal Care and Use Committee of the Korean Basic Sciences

Institute (KBSI-AEC-1803). A mouse tumor model was established

by injecting CT26 cells (5 × 10 cells per mouse) subcutaneously into

Complete contact information is available at:

979

J. Med. Chem. 2021, 64, 2971−2981

Journal of Medicinal Chemistry

https://pubs.acs.org/10.1021/acs.jmedchem.0c02162

Featured Article

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manuscript was written through the contribution of all authors.

All authors have given approval to the ﬁnal version of the

manuscript.

Notes

(

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

Hoecher, B.; Lambert, I. B. Oxygen-Insensitive Nitroreductases:

This work was supported by the National Research Council of

Science and Technology (NST) grant from the Korea

government (MSIT) (K.S.H., CAP-18-02-KRIBB) and the

National Research Foundation of Korea (NRF) grant funded

by the Korea government (MSIT) (K. S. H. ,

(

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ABBREVIATIONS

■

BOC, tert-butoxycarbonyl; DCC, N,N′-dicyclohexylcarbodii-

mide; DCM, dichloromethane; DFT, density functional

theory; DIEA, diisopropylethylamine; DMAP, 4-(N,N-

dimethylamino)pyridine; DMF, dimethylformamide; DMSO,

dimethyl sulfoxide; E , excitation; E , emission; fol, folate; FR-

(19) Hodgkiss, R. J.; Jones, G. W.; Long, A.; Middleton, R. W.;

Parrick, J.; Stratford, M. R. L.; Wardman, P.; Wilson, G. D.

Fluorescent markers for hypoxic cells: a study of nitroaromatic

compounds, with fluorescent heterocyclic side chains, that undergo

bioreductive binding. J. Med. Chem. 1991, 34, 2268−2274.

(20) Liu, Y.; Xu, Y.; Qian, X.; Liu, J.; Shen, L.; Li, J.; Zhang, Y. Novel

α, folate receptor; HIF-1α/β, hypoxia-inducible factors;

HOMO, highest occupied molecular orbital; HPLC, high-

performance liquid chromatography; HRMS, high-resolution

mass spectrometry; J, coupling constant (in NMR spectrom-

etry); LUMO, lowest unoccupied molecular orbital; m/z,

mass-to-charge ratio; MFI, mean ﬂuorescence intensity; MS,

mass spectrometry; NADH, reduced nicotinamide adenine

dinucleotide; NHS, N-hydroxysuccinimide; NIR, near infra-

red; NMR, nuclear magnetic resonance; NTR, nitroreductase;

OD, optical density; PBS, phosphate-buﬀered saline; PET,

positron emission tomography; Ph, phenyl; t-Bu, tert-butyl;

TFA, triﬂuroacetic acid; TLC, thin-layer chromatography; UV,

ultraviolet

fluorescent markers for hypoxic cells of naphthalimides with two

heterocyclic side chains for bioreductive binding. Bioorg. Med. Chem.

21) Tanabe, K.; Hirata, N.; Harada, H.; Hiraoka, M.; Nishimoto, S.-

006, 14, 2935−2941.

i. Emission under hypoxia: one-electron reduction and fluorescence

characteristics of an indolequinone-coumarin conjugate. Chembiochem

2008, 9, 426−432.

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Techniques for Hypoxia Imaging. J. Nanomed. Res. 2017 , 6 , 00160.

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