Design and synthesis of a novel mitochondria-targeted osteosarcoma theranostic agent based on a PIM1 kinase inhibitor

Article abstract of DOI:10.1016/j.jconrel.2021.02.028

Osteosarcoma (OS) is the most common malignancy of the skeletal system, with a poor prognosis and high rate of recurrence. Adequate surgical margin and adjuvant chemotherapy improve the overall survival and limb salvage rate of osteosarcoma patients. Previous studies have showed that OS exhibits an increase in the expression of proviral integration site for Moloney murine leukemia virus 1 (PIM1) kinase, and high levels of PIM1 are also associated with poor OS prognosis and metastasis. We exploited the overexpression of proto-oncogenic PIM1 in OS towards the development of a novel near-infrared imaging and targeted therapeutic agent, namely QCAi-Cy7d by conjugating a PIM1 small molecule inhibitor and heptamethine cyanine dye, for simultaneous guiding surgery and chemotherapy. QCAi-Cy7d showed targeted imaging and anticancer activities against OS in vitro and vivo without any obvious toxicity, and its antitumoral activity was much greater than the parent PIMI inhibitor. These results demonstrated the potential of new conjugate of PIM1 inhibitor and near-infrared imaging, supporting structure-based design and development of theranostic agents for precise tumor imaging and targeted treatment.

Full text of DOI:10.1016/j.jconrel.2021.02.028

Journal of Controlled Release 332 (2021) 434–447
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Design and synthesis of a novel mitochondria-targeted osteosarcoma
theranostic agent based on a PIM1 kinase inhibitor
,
,
,
,*
Xinzeyu Yia 1, Zhi Caob 1, Ying Yuana, Wen Lib, Xinyue Cuib, Zilin Chenb *, Xiang Hua
,
,
*
Aixi Yua
a Department of Orthopedics Trauma and Microsurgery, Zhongnan Hospital of Wuhan University, Wuhan 430071, China
b Key Laboratory of Combinatorial Biosynthesis and Drug Discovery Ministry of Education Hubei Province Engineering and Technology Research Center for Fluorinated
Pharmaceuticals, Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Osteosarcoma (OS) is the most common malignancy of the skeletal system, with a poor prognosis and high rate of
recurrence. Adequate surgical margin and adjuvant chemotherapy improve the overall survival and limb salvage
rate of osteosarcoma patients. Previous studies have showed that OS exhibits an increase in the expression of
proviral integration site for Moloney murine leukemia virus 1 (PIM1) kinase, and high levels of PIM1 are also
associated with poor OS prognosis and metastasis. We exploited the overexpression of proto-oncogenic PIM1 in
OS towards the development of a novel near-infrared imaging and targeted therapeutic agent, namely QCAi-
Cy7d by conjugating a PIM1 small molecule inhibitor and heptamethine cyanine dye, for simultaneous guid-
ing surgery and chemotherapy. QCAi-Cy7d showed targeted imaging and anticancer activities against OS in vitro
and vivo without any obvious toxicity, and its antitumoral activity was much greater than the parent PIMI in-
hibitor. These results demonstrated the potential of new conjugate of PIM1 inhibitor and near-infrared imaging,
supporting structure-based design and development of theranostic agents for precise tumor imaging and targeted
treatment.
PIM1
Kinase inhibitor
Osteosarcoma
Near-infrared imaging
Anti-tumor
1. Introduction
and inhibit cell apoptosis by phosphorylating targeted proteins such as
MAP3K5 and BCL-2 [10,11]. Since PIM1 kinase is overexpressed in
Osteosarcoma (OS), the most common primary malignant bone
tumor, occurs in the metaphysis of long bones. Overall survival rates of
OS patient remain low for many years [1,2] while being improved to
some degrees with the introduction of adjuvant chemotherapy and ra-
diation therapy [3,4]. Indeed, an adequate surgical margin is an
important factor to determine the survival rate of patients and reduce
the risk of amputation [5,6]. Therefore, a multifunctional agent with
targeted imaging and stronger antitumor ability will be helpful to ach-
ieve simultaneous treatment and guided surgical resection of OS.
However, classic imaging agents based on antibodies or peptides have
various limitations for applications, due to their very large steric hin-
drance and potential immunogenicity. Therefore, small molecule im-
aging agents such as fluorescent agents based on small molecule
inhibitors are under active investigation [7–9].
many solid and hematopoietic tumors, including OS [12,13], some PIM1
kinase inhibitors have been synthesized towards tumor treatment
[14,15]. The overexpression of PIM1 in OS is associated with metastasis
and poor prognosis, whereas there are no reports on the effects of PIM1
inhibitors on OS in vivo [16,17]. In addition, a positron emission to-
mography (PET) imaging agent based on a PIM1 inhibitor was devel-
oped for kinase-targeted imaging [18]; however, the radioactivity and
short half-life of radionuclides limit their application to the guided
surgical resection of tumors. Subsequently, Guo et al. [19] constructed
and used a red-emitting fluorescent probe and to image tissue and
subcutaneous xenograft tumor models. Nevertheless, the shorter emis-
sion wavelength may limit its applications to whole-body in vivo im-
aging, especially for deep tissues. Hence, it should be valuable to
develop a near-infrared (NIR) imaging and therapeutic agent based on a
PIM1 inhibitor for OS imaging and therapy.
PIM1, a serine/threonine-specific proviral integration site for
Moloney murine leukemia virus (PIM) kinase, can regulate the cell cycle
Compared to imaging in the visible region, near-infrared imaging has
* Corresponding authors at: Department of Orthopedics Trauma and Microsurgery, Zhongnan Hospital of Wuhan University, Wuhan, 430071, China
E-mail addresses: chenzl@whu.edu.cn (Z. Chen), shawnhu2002@whu.edu.cn (X. Hu), yuaixi@whu.edu.cn (A. Yu).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.jconrel.2021.02.028
Received 22 December 2020; Received in revised form 17 February 2021; Accepted 24 February 2021
Available online 1 March 2021
0168-3659/© 2021 Elsevier B.V. All rights reserved.

X. Yi et al.
Journal of Controlled Release 332 (2021) 434–447
lower photon attenuation, tissue scattering and autofluorescence
because its imaging wavelength is located in the tissue-optical-
transparent window [20–22]. Many studies have suggested the bene-
fits of near-infrared imaging in sarcoma resection through the reduction
of the recurrence rate [6,23]. Heptamethine cyanine dyes (HMCDs),
members of the organic near-infrared cyanine dye family, have been
reported to accumulate in the mitochondria of tumor cells, involving the
z: 310.2 [M + H]+.
2.2.3. Synthesis of compound QCAi
A solution of compound 3 (2.00 g, 6.47 mmol, 1.00 eq) in MeOH (20
mL) was added to a solution of K2CO3 (2.68 g, 19.40 mmol, 3.00 eq) in
H2O (1 mL), and the mixture was stirred at 70 ◦C for 4 h. The reaction
mixture was diluted with H2O (50 mL), and 2 N HCl was added to the
mixture to reach pH = 3–4. Then, the mixture was filtered and
concentrated under reduced pressure to obtain QCAi (1.50 g, 5.31
mmol, 82.1% yield) as a yellow solid, whose structure was confirmed by
1H NMR and MS. The 1H NMR (400 MHz, DMSO) results are as follows:
10.69 (s, 1H), 9.48 (m, 1H), 7.97 (d, J = 8.1 Hz, 1H), 7.87–7.71 (m, 2H),
7.62–7.59 (m, 1H), 7.56 (ddd, J = 2.0, 6.2, 8.2 Hz, 1H), 7.24–7.22 (m,
1H), 7.19–7.15(m, 1H), 6.50 (d, J = 7.9 Hz, 1H). MS (ESI) m/z: 282.0
[M + H]+.
tumor hypoxia and the HIF1α/organic anion transporting polypeptide
(OATP) signaling axis [24,25]. PIM1 kinase is mainly distributed in the
mitochondria [19], and the mitochondrial subcellular targeting of PIM1
kinase plays a key role in the mitochondrial fission and apoptosis
pathway by interacting with the Drp1 and BCL-2 family of proteins
[11,26,27]. QCAi, a selective PIM1 kinase inhibitor based on the
structure of quinoxaline-2-carboxylic acids, was first reported by Oyal-
lon et al. in 2018. Being a strong inhibitor of HsPim-1 kinase (IC50 =
0.074
μ
M), QCAi showed a rather weak antitumor activity against
KU812 cells (IC50 = 38.9 ± 3.4
μ
M) [28]. This discrepancy may prob-
2.2.4. Synthesis of compound QCAi-Cy7d
ably attribute to its poor permeability or accumulation within tumor
cells. Therefore, we hypothesized that, by conjugating the PIM1 inhib-
itor QCAi with the HMCD Cy7 derivative (Cy7d), the resulting QCAi-
Cy7d conjugate may retain the characteristics of both compounds. In
particular, QCAi would strengthen the tumor retention of HMCD to
optimize the imaging effects, and Cy7d may help deliver QCAi into the
carcinoma cells to improve antitumor effects. Herein we report the
synthesis of a novel compound that combines HMCD Cy7d and the PIM1
inhibitor QCAi for OS localization and targeted therapy. We further
demonstrated the tumor-targeted imaging and therapeutic potential of
the QCAi-Cy7d conjugate against OS in vitro and in vivo.
A solution of QCAi (100 mg, 0.355 mmol, 1.00 eq), hydroxybenzo-
triazole (HOBt) (48 mg, 0.355 mmol, 1.00 eq) and N, N-diisopro-
pylcarbodiimide (DIC) (45 mg, 0.355 mmol, 1.00 eq) in dimethyl
formamide (DMF) (4 mL) was added to a solution of tert-butoxy
carbonyl (BOC)-hexanediamine (120 mg, 0.554 mmol, 1.56 eq) in
DMF (4 mL) and the mixture was stirred at room temperature for 3 h.
Then, the reaction mixture was diluted with H2O (50 mL), and 95%
trifluoroacetic acid (TFA) (8 mL) was added to remove the BOC pro-
tecting group and incubated for 2 h after lyophilization. Afterwards, the
mixture was lyophilized again and purified using HPLC to obtain com-
pound 4 (70 mg, 0.185 mmol, 52.1% yield). A solution of Cy7d-NHS (50
mg, 0.079 mmol, 1.00 eq) and N, N-diisopropylethylamine (DIEA) (30.6
mg, 0.237 mmol, 3.00 eq) in DMF (2 mL) was added to a solution of
compound 4 (50 mg, 0.132 mmol, 1.67 eq) in DMF (2 mL), and the
mixture was stirred at room temperature overnight. The reaction
mixture was diluted with H2O (50 mL) and purified using HPLC after
lyophilization to yield QCAi-Cy7d (15 mg, 0.017 mmol, 21.5% yield) as
a green solid, whose structure was confirmed by 1H NMR and MS. The
particle size distributions of QCAi-Cy7d in DMSO and PBS containing
2% DMSO were measured using dynamic light scattering (DLS) (Zeta-
sizerNano; Malvern).The 1H NMR (400 MHz, CDCl3) results are as fol-
lows: 1H NMR (400 MHz, Acetone) δ 11.69 (s, 1H), 9.08 (s, 1H), 7.97 (m,
2H), 7.86 (d, J = 8.1 Hz, 1H), 7.77 (m, 4H), 7.57–7.46 (m, 3H), 7.37 (dd,
J = 13.8, 5.5 Hz, 5H), 7.24 (d, J = 7.4 Hz, 2H), 7.16 (t, J = 8.0 Hz, 1H),
6.68–6.47 (m, 3H), 6.41 (t, J = 11.6 Hz, 2H), 4.26–4.00 (m, 4H), 3.51 (s,
4H), 2.15 (m, 2H), 1.69 (d, J = 9.2 Hz, 12H), 1.39–1.16 (m, 16H), 1.02
(t, J = 7.3 Hz, 3H). MS (ESI) m/z: 899.30 [M + H] + .
2. Materials and methods
2.1. Materials
Human OS cell lines 143B and U2OS were purchased from the Cell
Storage Center of Wuhan University (Wuhan, China). Sulfobromoph-
thalein sodium hydrate (BSP) was purchased from Acros Organic (Geel,
Belgium). Ethyl 3-oxo-3, 4-dihydro-2-quinoxalinecarboxylate (1) was
obtained from Wuxi AppTec Company (Shanghai, China). Heptamethine
cyanine dye derivative Cy7 (Cy7d) was obtained from Nanjing Goyoo
Biotech Co. Ltd. (Nanjing, China). MitoTracker Green and Hoechst
33342 were purchased from Invitrogen (Groningen, The Netherlands)
and Beyotime (Shanghai, China), respectively. All other chemicals and
solvents were of reagent grade or HPLC grade.
2.2. Synthesis and structural characterization of QCAi-Cy7d
2.3. Molecular docking analysis of QCAi-Cy7d with the PIM1 protein
2.2.1. Synthesis of compound 2
The human PIM1 structure (PDB ID 4I41) was prepared using the
Protein Preparation Wizard, and the active site was defined by the
cocrystal ligand, with the 3D coordinates of 17.769469, ꢀ 37.141063,
and 2.569375, and a radius of 20.0 Å. Then, QCAi was docked using the
A solution of compound 1 (4.50 g, 20.6 mmol, 1.00 eq) in POCl3
(74.2 g, 484 mmol, 45 mL, 23.48 eq) was stirred at 110 ◦C for 1 h. The
◦
mixture was cooled to 15 C and diluted with ethyl acetate (EA) (500
mL). The organic phase was quenched with ice water (500 mL), adjusted
to pH = 7 with solid NaHCO3, and extracted with EA (300 mL × 2). The
combined organic layers were washed with brine (400 mL), dried over
Na2SO4, filtered and concentrated under vacuum to yield compound 2
(4.50 g, 18.9 mmol, 91.7% yield, 99.4% pure) as a red oil, whose
structure was confirmed by mass spectrometry (MS). MS (ESI) m/z:
237.1 [M + H]+.
¨
extra precision (XP) modes of Glide in the Schrodinger Suite, and the
pharmacophoric conformation of QCAi was considered the reference for
QCAi-Cy7d to generate conformations within the PIM1 pocket. Finally,
the QCAi-Cy7d complex was used for molecular dynamics simulations
(Desmond program, 20 ns) and binding free energy calculations (MM-
GBSA).
2.2.2. Synthesis of compound 3
2.4. Determination of the optical properties
A solution of compound 2 (5.00 g, 21.1 mmol, 1.00 eq), 3-aminophe-
nol (2.77 g, 25.3 mmol, 1.20 eq) and p-toluene sulfonic acid (TsOH)
(727 mg, 4.23 mmol, 0.2 eq) in ethanol (EtOH) (100 mL) was stirred at
80 ◦C for 16 h. Then, the reaction mixture was concentrated under
reduced pressure to obtain a residue, that was triturated with EA (30
mL) at 20 ◦C for 30 min to yield compound 3 (6.10 g, 19.7 mmol, 93.3%
yield) as a red solid, whose structure was confirmed by MS. MS (ESI) m/
The absorption and emission spectra of QCAi-Cy7d (1 μM) in tris
(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer at
different pH values and in different solvents were determined with an
FL-4600 fluorescence spectrometer (Hitachi Ltd., Tokyo, Japan). The
relative fluorescence quantum yields in different solutions were deter-
mined using the equation provided by Guo et al. [19] with rhodamine B
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Journal of Controlled Release 332 (2021) 434–447
Fig. 1. Synthesis of the theranostic QCAi-Cy7d conjugate consisting of NIRF dye and PIM1 inhibitor.
Fig. 2. Binding of Cy7d-conjugated QCAi to the ATP pocket of PIM1 (PDB ID 4I41), as determined by the docking program Glide. The carbon atoms of QCAi-Cy7d are
in green, and the amino acid residues of the PIM1 protein are in blue. (For interpretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
as the standard. The absorbance data of rhodamine B in ethanol and
QCAi-Cy7d (1 M) in different solvents were obtained using a UV–vi-
sible spectrophotometer (UV-1240, SHIMADZU, Japan). The pH value of
2.6. Cytotoxicity evaluation
μ
The cytotoxicities of QCAi, Cy7d, and QCAi-Cy7d were evaluated
using cell-counting kit-8 (CCK-8) assay kits (Biosharp Co., Ltd., China).
the Tris-HCl buffer was measured with a precision pH meter. A 1 μM
solution of QCAi-Cy7d in ethanol was continuously irradiated for 1800 s
with a 150 W xenon lamp to evaluate the photostability of QCAi-Cy7d.
Briefly, 100 μL of U2OS and 143B cells were placed into 96-well plates
(5 × 103 cells/well) and cultured for 12 h. Then, the cells were incubated
The fluorescence intensity of 1
μ
M QCAi-Cy7d (excitation wavelength =
with serial dilutions of QCAi, Cy7d, and QCAi-Cy7d for another 24 h.
750 nm, scan range = 780 nm) in an ethanol/water solution (ethanol:
water = 1:1) containing different ions was also determined. Finally, we
After the incubation, the medium was discarded, and 100 μL of fresh
medium containing 10 μL of CCK-8 reagent were added and incubated
examined the emission spectra of different concentrations (1–15
μ
M) of
for 4 h. Finally, a Multiskan FC plate reader (Thermo Fisher Scientific,
USA) was used to measure the absorbance of each well, and the cell
viability was calculated as follows: Cell viability = (OD value of the
sample - OD value of the blank)/ (OD value of the control - OD value of
the blank) × 100%.
QCAi-Cy7d in ethanol to assess the effects of the concentration and
aggregation.
2.5. Cell culture
The human OS cell line U2OS was cultured in McCoy 5A medium
(Gibco, USA), and 143B cells were cultured in MEM/EBSS (HyClone,
USA). All media were supplemented with 10% fetal bovine serum (FBS)
2.7. Apoptosis assay
A CytoFLEX flow cytometer (Beckman Coulter) and an annexin V/
propidium iodide (AV/PI) cell apoptosis assay kit (KeyGen Biotech,
China) were used to evaluate cell apoptosis. Briefly, after 24 h of incu-
and penicillin (100IU/mL, HyClone)/streptomycin (100μg/mL,
HyClone). The cells were grown in a humidified 5% CO2 incubator at
37 ◦C.
bation with 5 μ
M QCAi, Cy7d, or QCAi-Cy7d at 37 ◦C for 24 h, cells were
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Journal of Controlled Release 332 (2021) 434–447
Fig. 3. The optical properties of QCAi-Cy7d. (A) Emission spectra of QCAi-Cy7d (1
μ
M) in different solvents. (B) Absorption spectra of QCAi-Cy7d (1
μM) in different
solvents. (C) Emission of QCAi-Cy7d at different concentrations (1–15
μ
M) in ethanol. (D) Fluorescence intensities of QCAi-Cy7d (1
μ
M) in Tris/HCl buffer at various
pH conditions. (E) Fluorescence intensity of QCAi-Cy7d (1
μ
M) in the presence of 50 μM different ions in ethanol/water solution (ethanol: water = 1:1). (F) Pho-
tostability of QCAi-Cy7d upon continuous excitation with a xenon lamp. (E-F: excitation wavelength = 750 nm, scan range = 780 nm. DMSO: dimethyl sulfoxide;
DMF: N, N-dimethylformamide; THF: tetrahydrofuran; ACN: acetonitrile; MEM: minimum essential medium; FBS: fetal bovine serum).
and washed 3 times with PBS (HyClone, USA). Hoechst 33342 was used
Table 1
to stain the nuclei. Finally, the cells were observed with a confocal laser-
Photophysical properties of QCAi-Cy7d in various solvents. λabs: maximal ab-
scanning microscope (Leica, Germany). To further verify the cellular
sorption wavelength; λem: maximum emission wavelength; Φ: fluorescence
uptake of QCAi-Cy7d, the cells were preincubated with a 100-fold excess
quantum yield. (N/A: not measured).
of the nonfluorescent ligand QCAi (1 mM), 250
μM BSP (an OATP
Solvent
λabs (nm)
λem (nm)
Δλ (nm)
Φ
competitive inhibitor) or both for 30 min. The mean fluorescence in-
tensities were calculated using ImageJ software.
Ethanol
DMSO
DMF
THF
748.6
752.6
750.6
752
778.4
787.4
784.2
784.6
777.6
779.2
29.8
34.8
33.6
32.6
31.4
10.2
0.09902
0.10294
0.06964
0.04052
0.16663
N/A
The subcellular localization of QCAi-Cy7d on the mitochondria in OS
cells was assessed using the commercial probe MitoTracker Green. After
Hoechst 33342 staining, the cells were sequentially incubated with 200
ACN
746.2
769
FBS
nM MitoTracker Green for 30 min and 10 μM QCAi-Cy7d for 30 min.
Images were obtained using a confocal laser microscope after rinses with
PBS. ImageJ software was used to analyze the colocalization of QCAi-
Cy7d and MitoTracker Green. All cells used for imaging were live
cells, and the cells were washed 3 times for 3 min per wash before every
reagent change.
collected and washed with cold PBS. Then, the cells were diluted to a
density of 1 × 106 cells/mL with 500
μL of 1× binding buffer per assay
and 5 μL of AV-EGFP and PI, stained at room temperature in the dark and
finally analyzed using flow cytometry.
2.9. Animal tumor xenograft model
2.8. Uptake and subcellular localization of QCAi-Cy7d in OS cells
The protocol used to establish the xenograft tumor model was
approved by the ethics committee of the Animal Care and Use Com-
mittee at Wuhan University. Male BALB/c nude mice of 18–22g were
purchased from Sibeifu Biotechnology Co., Ltd. (Beijing, China). They
The cells (143B and U2OS) were seeded into 20-mm confocal laser
dishes (NEST, China) at a density of 1 × 105 cells/well and cultured for
12 h. Then, the cells were incubated with 10 μM QCAi-Cy7d for 30 min
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Journal of Controlled Release 332 (2021) 434–447
Fig. 4. The inhibition of 143B and U2OS cell viability by QCAi-Cy7d is associated with the induction of apoptosis. The dose-dependent, anti-viability activities of
QCAi, Cy7d and QCAi-Cy7d against 143B (A) and U2OS (B) cells were determined by using CCK-8 assay. (C) Effects of QCAi, Cy7d and QCAi-Cy7d on apoptosis of
143B (C) and U2OS (D) cells were determined by using annexin V/propidium iodide apoptosis assay.
were housed under specific pathogen-free conditions with free access to
were prepared for the histological analysis. Finally, frozen sections
stained with DAPI and paraffin-embedded tissue sections stained with
hematoxylin and eosin (H&E) were observed under a confocal laser
scanning microscope for observation. Normal nude mice were allocated
into five groups (n = 3), injected with 5 nmol of QCAi-Cy7d and sacri-
ficed at different time points (0.5 h 6, 12, 24 and 48 h) after the injection
to explore the metabolism and clearance of QCAi-Cy7d in vivo. Major
organs were collected and imaging signals were detected.
food and water. Human 143B OS cells (100
μ
L) (5 × 106) in 50%
Matrigel (BD Biosciences, USA) were subcutaneously injected to estab-
lish a tumor xenograft model.
2.10. QCAi-Cy7d for near-infrared fluorescence (NIRF) imaging of OS
In the in vivo NIRF imaging studies, mice bearing 143B tumor xe-
nografts (n = 3) were injected with 5 nmol of QCAi-Cy7d (control group)
and Cy7d (Cy7d group) via the tail vein. A 100-fold dose of nonfluo-
rescent ligand QCAi (QCAi group) and a 25-fold dose of OATPs inhibitor
BSP (BSP group) were injected together with QCAi-Cy7d (n = 3) to
determine whether QCAi-Cy7d specifically targets PIM1 kinase in vivo.
Moreover, a 4-fold dose of QCAi-Cy7d (20 nmol) was injected to eval-
uate the dose-dependent effects of QCAi-Cy7d (high-dose group; n = 3).
At 48 h after the injection, NIRF images of the entire body were captured
using a Bruker Xtreme BI imaging system (Billerica, USA). The mice
were sacrificed after whole-body imaging, and imaging signals of the
major organs and tumors tissue were detected. Then, frozen sections and
paraffin-embedded tissue sections of the subcutaneous xenograft tumors
2.11. In vivo tumor inhibitory effects and toxicity assay
Twenty BALB/c nude mice bearing 143B tumors were randomly
classified into 4 groups (n = 5) when the tumor sizes reached 75–125
mm3 to evaluate the therapeutic effects of QCAi-Cy7d: (1) a control
group (treated with 2% DMSO in PBS), (2) QCAi group (3.75
μ
mol/kg),
mol/
L each) were injected via the tail vein
(3) Cy7d group (3.75
μmol/kg), and (4) QCAi-Cy7d group (3.75 μ
kg). These preparations (100
μ
once every 2 days for a total of 7 injections (14 days). The body weights
and tumor sizes were measured, and the tumor volumes were calculated
as follows: length × width × width ×0.5. Two days after the last
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Journal of Controlled Release 332 (2021) 434–447
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Journal of Controlled Release 332 (2021) 434–447
Fig. 5. Uptake study results in 143B and
U2OS cells. (A) Images of QCAi-Cy7d in the
two cell lines costained with MitoTracker
Green (200 nM). (B) Intensity profile of the
costained region in the image of 143B cells.
(C) Colocalization analysis in 143B cells. (D)
Images of the QCAi-Cy7d target blocking test
in 143B and U2OS cells after treatment with
250
μ
M BSP, 1 mM QCAi and both com-
QCAi-Cy7d). (E)
pounds (control: 10
μ
M
Average fluorescence intensities of 143B cells
in the four groups. (n = 10). (For interpre-
tation of the references to colour in this figure
legend, the reader is referred to the web
version of this article.)
injection, the mice were sacrificed and serum was collected. Serum
biochemical indices, including ALT, AST, BUN, Cr, CK and total bilirubin
(T-Bil), were detected with an automatic biochemical analyzer (Rayto
Life and Analytical Sciences Co., Ltd., Shenzhen, China) to assess the
potential toxicity of QCAi-Cy7d.
there were a water bridge between Asp245 and the agent and hydro-
phobic interactions between QCAi-Cy7d and Tyr 207. The total binding
free energy of QCAi-Cy7d was ꢀ 15.9345 kcal/mol, slightly higher than
that of QCAi was ꢀ 21.8543 kcal/mol. These results show that Cy7d-
conjuagted QCAi-Cy7d could directly bind to PIM1, despite that the
binding strength was slightly weaker than that of QCAi.
The tumors and major organs were collected for further pathological
assessments. The tumor burden (weight of tumors/average body weight)
and tumor inhibition rate (TIR; (1-Wsample/Wcontrol) × 100) were
calculated. A TUNEL assay (Roche, Basel, Switzerland) was performed to
assess tumor cell apoptosis in the tissue. The major organs were fixed
with formalin, embedded in paraffin and stained with H&E. Images of all
3.3. Optical properties of QCAi-Cy7d
QCAi-Cy7d in acetonitrile (ACN) exhibited the most intense fluo-
rescence emission, while the agent in FBS and MEM + 10% FBS showed
weak fluorescence intensity (Fig. 3A-B). This is also manifested by
fluorescence quantum yield values (Table 1). The absorption spectra
were similar among all solvents. Except for FBS, the Stokes shifts of the
solvents were approximately 30 nm (29.8–34.8 nm), and the quantum
yields were in the range of 4.052% (tetrahydrofuran, THF)-16.663%
(ACN). In the excitation test with different concentrations of QCAi-Cy7d,
the excitation peak was enhanced, and a slight redshift was observed
when the concentration increased (Fig. 3C). This redshift was possibly
caused by the intermolecular conjugation of the agent [21]. Interest-
ingly, the performance of the agent at different pH values was symmetric
at pH = 7, and the fluorescence intensities at pH = 5 and 9 were stronger
than those at pH = 6 and 8, whereas the intensity at pH = 7 was in
between (Fig. 3D). We measured the intensities of QCAi-Cy7d in the
stained samples were captured with
(Olympus IX73, Japan).
a fluorescence microscope
2.12. Statistical analysis
The data are presented as the means ± standard deviations. A one-
way ANOVA was used to determine the differences. The differences
were considered statistically significant at 0.01 < *P ≤ 0.05, highly
significant at 0.001 < **P ≤ 0.01, and extremely significant at ***P ≤
0.001.
3. Results
presence of 15 interfering ions at 50
μ
M (1–15: K+, SO24ꢀ , Mn2+, NO3ꢀ ,
3.1. Synthesis and structural characterization of QCAi-Cy7d
Co2+, Ni2+, Brꢀ , Fe2+, Cu2+, HPO24ꢀ , CO23ꢀ , Cr3+, S2ꢀ , SO32ꢀ , Fe3+), and
no significant quenching was observed except for the last 3 ions, S2ꢀ
,
QCAi and QCAi-Cy7d were synthesized by following a sequence of
procedures shown in Fig. 1. Ethyl 3-hydroxyquinoxaline-2-carboxylate
(1) was chlorinated in the presence of POCl3 to obtain ethyl 3-chloroqui-
noxaline-2-carboxylic acid ethyl ester (2). Then, 3-aminophenol was
coupled to compound 2 via a Buchwald-Hartwig coupling reaction in the
presence of EtOH and TsOH to offer compound 3. QCAi was obtained
after the hydrolysis of compound 3, and the structure of QCAi was
confirmed by 1H NMR and MS (Fig. S1, S2).
SO23ꢀ and Fe3+ (Fig. 3E). Finally, 1
μM QCAi-Cy7d was continuously
excited by a 150 W xenon lamp for 1800 s to verify its photostability,
and the data indicate that there was almost no loss of intensity under
continuous excitation within short periods of time (<30 min) (Fig. 3F).
3.4. In vitro anticancer activity and apoptosis
The target molecule QCAi-Cy7d was prepared by following two
amidation reactions between QCAi, hexanediamine (as the linker) and
Cy7d. The final product was purified using HPLC, and the chemical
structure was confirmed by 1H NMR and MS (Fig. S3, S4). The chemical
structures of QCAi and QCAi-Cy7d are shown in Fig. S5 and the particle
size distribution of QCAi-Cy7d is shown in Fig. S6.
The anticancer activities of QCAi, Cy7d, and QCAi-Cy7d against
143B and U2OS cells were determined with CCK-8 assay. As shown in
Fig. 4, QCAi exhibited rather a weak cytotoxicity against 143B cells
(Fig. 4A) and U2OS cells (Fig. 4B), showing an IC50 of 277.6 ± 83.5 μM
and 118.7 ± 20.9
viabilities against 143B (IC50, 57.3 ± 6.4
M) cells, the anticancer activities of QCAi-Cy7d were 8.0-fold (7.2 ±
1.0 M) and 3.2-fold (11.8 ± 1.6 M) higher, respectively, than Cy7d.
Cell apoptotic profiles were further examined through flow cytom-
μ
M, respectively. While Cy7d exhibited some anti-
μ
M) and U2OS (38.3 ± 5.0
μ
3.2. Molecular docking analysis of QCAi-Cy7d with the PIM1 protein
μ
μ
QCAi-Cy7d was able to bind to the same binding pocket of PIM1 as
QCAi, while the larger molecular volume of QCAi-Cy7d caused some
differences in the surrounding amino acid residues (Fig. 2). Residual
Asp167 and Val206 were revealed to form hydrogen bonds with the
amide nitrogen atom and carbonyl oxygen of QCAi-Cy7d, respectively.
Meanwhile, Asn172 was able to form a hydrogen bond with the amide
oxygen atom of QCAi-Cy7d mediated by water molecules. In addition,
etry analyses of annexin V/propidium iodide-stained cells (Fig. 4C and
D). The apoptosis rates revealed that the incubation with QCAi-Cy7d
(37.81% in 143B and 20.19% in U2OS) caused a significant increase
in apoptotic cells, compared to the rates after incubation with only QCAi
(9.68% in 143B and 5.22% in U2OS) and only Cy7d (11.56% in 143B
and 5.30% in U2OS), while the latter two were not significantly different
from the control. These results demonstrated that QCAi-Cy7d is more
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Journal of Controlled Release 332 (2021) 434–447
Fig. 6. NIRF imaging and biodistribution of
QCAi-Cy7d in vivo. (A) Whole-body NIR
imaging results at 48 h after the injection of
the five different preparations (red arrow:
tumors). (B) NIR fluorescence images of or-
gans and tumors at 48 h post-injection. (C)
Fluorescence intensity of organs and tumors
treated with different preparations (expo-
sure time: 2 s). (D) NIRF signal from frozen
sections and H&E staining of subcutaneous
tumor tissues from mice in the control (5
nmol QCAi-Cy7d) group. Scale bar = 50 μm.
(For interpretation of the references to
colour in this figure legend, the reader is
referred to the web version of this article.)
potent than the unconjugated parent compounds in the inhibition of OS
cell viability in vitro, associated with the induction of apoptosis to
greater degrees.
3.7. In vivo antitumor effects
We thus investigated the antitumor effects of QCAi-Cy7d in vivo
following the establishment of a 143B xenograft mouse model. PBS
(with 2% DMSO), QCAi, Cy7d and QCAi-Cy7d were injected into tumor-
bearing mice via the tail vein every 3 days for a total of 5 injections. As
shown in Fig. 7A and B, while both QCAi and Cy7d exhibited moderate
antitumor activities (P < 0.05), QCAi-Cy7d treatment led to a more
pronounced antitumor effect than the Cy7d (P < 0.01) and PBS (P <
0.001) groups. As mouse body weights did not differ among the 4 groups
(Fig. 7C), mice subjected to QCAi-Cy7d therapy showed a lower tumor
burden than the PBS and Cy7d groups (P < 0.001 and P < 0.01,
respectively). The QCAi-Cy7d group had a TIR of 72.42%, higher than
those of QCAi (38.42%) and Cy7d (44.91%) groups (Fig. 7D). The
TUNEL staining study indicated a remarkable increase in cell apoptosis
in the tumor samples from mice treated with QCAi-Cy7d, as compared to
the other 3 groups (Fig. 7E). Finally, the blood chemistry profiles were
determined and H&E staining of the major organ sections were con-
ducted to evaluate possible toxicity of these preparations. Neither the
hematological parameters nor the histological staining indicated
noticeable toxicity (Figs. 8 and 9).
3.5. In vitro cellular uptake and subcellular localization
The 143B and U2OS cells were costained with QCAi-Cy7d and
MitoTracker Green to explore the subcellular colocalization of QCAi-
Cy7d in OS cells. The merged confocal microscopy images showed
that QCAi-Cy7d accumulated in OS cells and was located in the mito-
chondria (Fig. 5A), and a region of interest (ROI) of the 143B cell overlay
image was selected to conduct a 2-channel curve-fitting analysis. As
shown in Fig. 5B, the fluorescence intensity distributions in the ROIs
were similar. The scatter plots confirmed these same results (Fig. 5C).
Moreover, Pearson’s correlation factor was 0.776, while Manders’
colocalization coefficients were 0.756 (green) and 0.788 (red).
The 143B and U2OS cells were pretreated with 250
fold concentration of QCAi (1 mM, unconjugated parent construct) or
both for 30 min before an incubation with 10 M QCAi-Cy7d to further
μM BSP, a 100-
μ
examine the cellular uptake mechanism and ability of QCAi-Cy7d to
target the PIM1 kinase. The confocal images (Fig. 5D) showed a signif-
icant decrease in uptake of the agent in the other 3 groups compared to
the control group. The mean intensities of the 143B cells in the 4 groups
were calculated (n = 10 cells per group). These data revealed the same
results (P < 0.001) and the cotreatment group showed a stronger
blocking effect than the QCAi pretreated group (P < 0.01).
4. Discussions
An adequate surgical margin guided and confirmed by imaging is the
critical factor for surgical treatment for cancer patients. In addition, the
introduction of adjuvant chemotherapy improves overall survival and a
higher limb salvage rate. Hence, multifunctional agents that can
simultaneously achieve early diagnosis, guide the surgery and pursue
targeted therapy are under active development. PIM1 kinase, an onco-
genic kinase, plays a key role in intercellular signaling cascades to
promote tumor growth [29], and its overexpression is associated with
OS metastasis and poor prognosis. Therefore, we considered PIM1 ki-
nase as a potential target for OS imaging and therapy. A few selective
and high-affinity PIM1 inhibitors have been identified in recent years
[30,31], and they are excellent candidates as imaging probes [8]. Rather
only a PET probe and a classic fluorescent probe targeting PIM1 kinase
have been developed for imaging and it may not be applicable to guide
surgery and therapy because of the radiation or a short emission
wavelength.
3.6. In vivo imaging of OS and biodistribution of QCAi-Cy7d
Five different preparations were injected into mice bearing 143B
xenograft tumors to further evaluate the fate of QCAi-Cy7d in vivo. In
the whole-body images, appreciable fluorescence was observed in ROIs
from the QCAi-Cy7d-injected groups and BSP group but not in ROIs from
the other two groups (Fig. 6A). Equivalent results were obtained from ex
vivo images of the major organs and tumors (Fig. 6B). Compared to the
tumors from the control group, the tumors from the BSP group showed
no significant differences in fluorescence intensity (P > 0.05), whereas
the QCAi and Cy7d groups displayed weaker fluorescence intensity (P <
0.05). The mean fluorescence intensity of tumors was stronger than that
in other organs, except the liver and kidney, at 48 h (Fig. 6C), and the
imaging results from major organs at different time points showed that
the fluorescence intensity was decreased in the liver and did not change
Two elements determining the efficacy of an inhibitor are its access
and binding to the target in vivo. Our molecular docking study
demonstrated that there was minimal difference in binding to PIM1
between QCAi and QCAi-Cy7d. By contrast, the cytotoxicity and
apoptosis assays revealed significantly greater activities for QCAi-Cy7d
in the induction of apoptosis and inhibition of OS cell viability than the
parent compounds. The subcellular localization study indicated the
preferential accumulation of QCAi-Cy7d in the mitochondria, where
PIM1 kinase mainly distributes and interacts with apoptosis-related
proteins, which could be blocked by the pretreatment with overdose
–
considerably in the kidney over time (Fig. S7A B). These data indicate
that QCAi-Cy7d was generally metabolized by the liver and excreted via
the kidney. A strong NIRF signal was also detected in the frozen sections
of the subcutaneous tumor tissues from the control group, and H&E
staining of the subcutaneous tumor suggested the presence of OS in these
xenografts (Fig. 6D).
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Journal of Controlled Release 332 (2021) 434–447
(caption on next page)
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Journal of Controlled Release 332 (2021) 434–447
Fig. 7. In vivo antitumor effects of QCAi-Cy7d in 143B xenograft mouse model. (A) Tumor growth in mice subjected to four different treatments (n = 5). (B) Image of
the tumors harvested from 143B xenograft mice at the end of study (n = 5). (C) Mouse body weights over time following drug administration. (D) Tumor burden
(weight of tumor/average body weight) and TIR ((1-Wsample/Wcontrol) × 100) of mice with different treatments. (E) TUNEL assay images of the four groups. Scale bar
= 50 μm.
Fig. 8. Levels of blood biomarkers for hepatic, renal or cardiac functions in 143B xenograft mice following different treatments.
QCAi (100-fold) and BSP (250
μ
M). These results indicated sufficient
that tumoral QCAi-Cy7d fluorescent signal could be blocked with an
overdose of QCAi, suggesting the importance of QCAi moiety in tumor
targeting. The in vivo therapy study demonstrated the anti-OS effec-
tiveness and safety of the QCAi-Cy7d in vivo, showing much greater
intracellular accumulation of QCAi-Cy7d and binding to PIM1. To
further evaluate the fate of QCAi-Cy7d in vivo, tissue distribution and
antitumor studies were conducted. The body distribution study revealed
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Journal of Controlled Release 332 (2021) 434–447
Fig. 9. H&E staining of some major organs isolated from mice subjected to different treatments. There are no obvious hydropic damages or necrotic lesions after the
treatment with QCAi, Cy7d and QCAi-Cy7d. Scale bar = 50 m.
μ
efficacy than the parent compounds. Our findings indicate the potential
of QCAi-Cy7d as a theranostic agent for the treatment of OS.
confirmed by our results, consistent with previous studies [9,37].
Compared to the control groups, significant decreases in tumor fluo-
rescent were observed in the QCAi groups both in vitro and vivo. Our
vivo images captured 48 h after the injection showed that the control
and BSP competition groups retained measurable fluorescence in the
ROI, but not the QCAi competition and Cy7d groups. These results
indicate that OATPs may contribute to early entry, but not prolonged
retention. In addition, HMCDs without a meso-chloride moiety, such as
Cy7d, do not remain in tumor tissues for a long period because they are
unable to form covalent adducts with biomolecules possessing free thiols
[34,38,39]. Therefore, the PIM1 inhibitor QCAi plays a crucial role in
the targeting and retention abilities of the agent. The targeting ability of
QCAi-Cy7d and its longer tumor retention time reinforce the HMCD
imaging results and antitumor effects of the PIM1 inhibitor, which
provides a basis for its multifunctional applications to guide surgery and
molecular targeted therapy. Moreover, based on the results of our study,
fluorescent probes based on small-molecule inhibitors with excellent
selectivity and specificity provide new insights into the development of
novel multifunctional theranostic agents.
As OATP substrates, the heptamethine cyanine dye Cy7d is inter-
nalized by tumor cells that overexpress OATPs on their surface [32]. It
shows modest cytotoxicity towards OS cells because it accumulates in
tumor cell mitochondria and disrupts mitochondrial function [33]. The
amidation of the peripheral carboxylic acids of Cy7d prolongs its
retention time in tumors [34]. In addition, QCAi exerts a weak antitumor
effect probably due to its poor passive diffusion into OS cells. Thus,
QCAi-Cy7d displayed significantly stronger antitumor activity than
QCAi in vitro and vivo, although the inhibitory effects of QCAi on PIM1
kinase reach the nanomolar level. Moreover, the higher mitochondrial
transmembrane potential of tumor cells determines the preferential
accumulation of QCAi-Cy7d in the mitochondria due to the properties of
HMCDs [35], while PIM1 kinase has also been reported to regulate
hypoxia-induced
chemoresistance
and
mitochondria-mediated
apoptosis [27,36]. Because of the same subcellular localization, Cy7d
can help to accurately deliver QCAi to the mitochondria of tumor cells to
inhibit the PIM1 kinase activity and achieve better antitumor effects.
Our study further confirms the therapeutic potential of a PIM1 inhibitor
combined with other agents and the potential of HMCDs for tumor im-
aging and treatment by specifically conjugating antitumor drugs with
small-molecule inhibitors.
In conclusion, a tumor-targeting multifunctional agent QCAi-Cy7d
was designed and synthesized, and its utility in tumor imaging and
therapy was demonstrated in OS xenograft mouse models. With the
OATP-mediated delivery supported by HMCDs and the targeting ability
of a PIM1 inhibitor, QCAi-Cy7d can accumulate in the mitochondria of
OS to enhance the antitumor activity from the PIM1 inhibition, and
In addition to the mitochondrial targeting ability of Cy7d, the similar
function provided by the PIM1 inhibitor QCAi in the agent was
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[14] R. De Smedt, S. Peirs, J. Morscio, F. Matthijssens, J. Roels, L. Reunes,
QCAi-Cy7d can be applied as a theranostic agent for simultaneous NIRF
imaging and OS treatment. By combining NIRF with conventional im-
aging modalities, anatomical details can be completed so that QCAi-
Cy7d serves as an intraoperative noninvasive cancer imaging agent.
Showing effective antitumor activity, attributable to PIM1 inhibition
and mitochondrial targeting, QCAi-Cy7d shall be a valuable candidate
for targeted imaging and treatment of OS.
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Competing Interests
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This work was supported by Hubei Province Scientific and Technical
Innovation Key Project (No. 2019ACA136), the Interdisciplinary Project
of Wuhan University (No. 2042018kf0216), the National Natural Sci-
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Appendix A. Supplementary data
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