Optimized HSP90 mediated fluorescent probes for cancer-specific bioimaging

Article abstract of DOI:10.1039/c9tb02505g

Cancer-specific bioimaging has been correlated with fluorescence-guided tumor therapy, garnering extensive interest from researchers. Herein, a highly efficient tumor-targeting fluorescent probe (NP-001), which is integrated with 4-hydroxy-1,8-naphthalimide and NVP-AUY922, for tumor imaging has been established. 4-Hydroxy-1,8-naphthalimide is a fluorescent molecule with remarkable imaging compatibility. NVP-AUY922 is a heat shock protein 90 (HSP90) inhibitor with preferential tumor selectivity that is conjugated to 4-hydroxy-1,8-naphthalimide as a tumor-targeting ligand. NP-002, a resorcinol-blocked probe which prevented binding with an amino acid residue of the HSP90 ATP binding pocket, was also synthesized as a control. In vitro and ex vivo assays showed that NP-001 could arrest cell proliferation, induce apoptosis and accumulate to inhibit HSP90. Confocal laser scanning microscopy (CLSM) also confirmed that NP-001 could be selectively internalized by tumor cells for cancer-specific bioimaging. Moreover, pharmacokinetic studies and histological analysis also indicated that NP-001 had a relatively longer retention time and showed no major organ-related toxicities. Overall, these encouraging data suggest that NP-001 is a promising new candidate for the early diagnosis of metastatic disease as well as targeted tumor imaging.

Full text of DOI:10.1039/c9tb02505g

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Materials for biology and medicine
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21 January 2018
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Wei Wei, Guanghui Ma et al.
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Optimized HSP90 mediated fluorescent probes for cancer-specific
bioimaging
3
Shulei Zhu,a Yalei Li,b Yushu Huang,a Minmin Zhang,b Xiaofan Gu,a Yang He,a Hongchun Liu,b
Mingliang Ma,*, a, c Wei Lu*, a
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a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Molecular Engineering, East China Normal University, 3663 North
Zhongshan Road, Shanghai, 200062, PR China.
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Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
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Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, PR China.
c Key Laboratory of Brain Functional Genomics-Ministry of Education, School of Life Science, East
China Normal University, 3663 North Zhongshan Road, Shanghai, 200062, PR China.
Keywords: fluorescent probe, heat shock protein 90, cancer-specific, bioimaging
Abstract
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Cancer-specific bioimaging has been correlated with fluorescence-guided tumor therapy,
garnering extensive interest from researchers. Herein, a highly efficient tumor-targeted
fluorescent probe (NP-001), which integrates with 4-hydroxy-1, 8-naphthalimide and
NVP-AUY922 for tumor imaging has been established. 4-Hydroxy-1, 8-naphthalimide is a
fluorescent molecule with remarkable imaging compatibility. NVP-AUY922 is a heat shock
protein 90 (HSP90) inhibitor with preferential tumor selectivity that is conjugated to 4-hydroxy-1,
8-naphthalimide as a tumor-targeted ligand. NP-002, a resorcinol-blocked probe which
prevented binding with an amino acid residue of the HSP90 ATP binding pocket was also

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synthesized as a control. In vitro and ex vivo assays showed that NP-001 could aDrrOeI:s1t0.1c0e3l9l/C9TB02505G
proliferation, induce apoptosis and accumulate to inhibit HSP90. Confocal laser scanning
microscopy (CLSM) also confirmed that NP-001 could be selectively internalized by tumor cells
for cancer-specific bioimaging. Moreover, pharmacokinetic studies and histological analysis also
indicated NP-001 had a relatively longer retention time and showed no major organ-related
toxicities. Overall, these encouraging data suggest that NP-001 is a promising new candidate for
the early diagnosis of metastatic disease as well as targeted tumor imaging.
Introduction
Cancer remains a major public health problem worldwide, which has been expected to rank as the leading cause
of death.1 Generally, surgical removal is the best treatment method for cancer if the tumor can be entirely
removed, depending on precise tumor delineation and surgical skill.2-6 Monitoring the presence of tumors to
avoid incomplete removal is critical and has attracted extensive interest from researchers.7 Recently,
fluorescence imaging technology8 has played a dominant role in the localization and dynamic monitoring of
disease-related biomarkers due to its low cost and high sensitivity and spatial resolution, as well as its
damage-free real-time imaging capabilities.9,10 Conjugation of small fluorophores and biomarker-specific ligands
has become one of the main strategies for fluorescence imaging.11 Various types of ligands are used, such as
small molecules, aptamers, peptides and antibodies, which have high affinities for various tumor receptors.7,12-14
What we are interested in this study was HSP90-related ligand, which is one of a family of molecular
chaperones.15
Molecular chaperones have been the subject of biological and translational research for many years.15 HSP90
plays a critical role in folding, stabilization, activation, and degradation of essential protein substrates.16-19 It is

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also known to simultaneously target many oncogenic proteins20 such as FAK (integrin pathway), IL6R (IAK/STAT
pathway), CDK4, CDK6, CDK9 (cell cycling), APAF-1 (apoptosis), and IRB kinases (NF-kB pathway).21,22 HSP90
interacts with more than 200 different “client” proteins and many of these proteins are involved in signal
transduction, protein trafficking, receptor maturation, and innate and adaptive immunity.23,24 As a result,
inhibition of HSP90 has attracted much interest as a viable molecular target for cancer treatment. HSP90
functions as a homodimer, and dimerization is essential for its function in vivo.25 An HSP90 dimer consists of
three highly conserved domains: the N-terminal domain (NTD), the middle domain (MD), and the C-terminal
domain (CTD).26 The NTD of HSP90 contains a nucleotide binding region that mediates both ATP and ADP,27-29
and it is also the target of many clinical compounds. Although, it comprises 1%-3% of the cellular proteins,27
HSP90 derived from tumor cells with high ATPase activity has special selectivity over the latent form expressed in
normal cells regarding its binding affinity to HSP90 inhibitors.30 Many typical ATP competitive Hsp90 inhibitors
have undergone clinical trials.31-33 As shown in Figure 1, such HSP90 inhibitors include geldanamycin,34,35
radicicol,35,36 NVP-AUY922,37 SNX-5422,38 STA9090,39 and AT13387,40 which have been evaluated in clinical trials
for various indications. Furthermore, HSP90 is very susceptible to inhibition and is sensitive to cell-permeable
HSP90 inhibitors.41 However, clinical HSP90 inhibitors have shown problematic off-target effects and/ or
HSP90-related toxicities28,42-44 such as excess hepatotoxicity,45 neurological toxicities,46 visual disturbances,47
ocular toxicities38 and so on. Therefore, new methods of delivering selective HSP90 inhibitors are urgently
required.

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O
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HO
OH
Cl
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OH
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OH
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Geldanamycin
Radicicol
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NH2
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OH
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N
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NH2
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OH
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HO
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F3C
NH
OH
O
SNX-5422
STA9090
AT13387
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Figure 1. Some typical HSP90 inhibitors.
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While HSP90 is mostly located in the intracellular space, it has also been found extracellularly, particularly in
tumor models.48-50 Over the past few years, extracellular expression HSP90 (eHSP90) by tumor cells has emerged
as an effective target associated with tumor invasion and metastasis,51-53 which may be the result of
oncogene-driven factors that alter its normal cellular behavior.54 The first evidence for eHSP90 involvement in
tumor cell invasion was reported in 2004.55 eHSP90 may be found as a secreted form and expressed on the cell
surface.54 Previous work has demonstrated that eHSP90 as a chemotherapeutic target could be actively
internalized from the extracellular space, confirming trafficking of HSP90 to the cell surface but also showing that
the protein is reinternalized.56,57 Above all, eHSP90 has a specific function as a carrier transporting diagnostic
reagents or drugs into tumor cells.
To explore the potential role of a HSP90 inhibitor (NVP-AUY922) and fluorophore conjugate for cancer-specific
bioimaging, we developed two conjugates, active type NP-001 and its inactive resorcinol-blocked analog NP-002
(Scheme 1), to confirm the tumor selectivity. The conjugate was made up of NVP-AUY922 and
4-hydroxy-1,8-naphthalimide58. 1, 8-Naphthalimide has been widely used over the past several decades to
monitor the critical enzymes or factors in human biological processes due to its suitability for confocal

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microscopic screening.58-60 4-Hydroxy-1,8-naphthalimide, with an emission wavelength of 560 nm, is favored for
its minimum photo-damage to samples and its deep tissue penetration.61,62 Microscopic analysis showed a
significant distinct fluorescence window between NP-001 and NP-002. NP-001 with an active HSP90 inhibitor
scaffold has strong fluorescence while the inactive resorcinol-blocked analog displays almost no visible
fluorescence in A549 and HCT116 cells and on ex vivo HCT116 xenografts tumor slides, suggesting NP-001
accumulation in tumor cells and cancer-specific bioimaging.
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Scheme 1. Chemical structures and schematic illustration of NP-001 and NP-002 for cancer specific imaging.
NP-001 was selectively internalized into tumor cells for imaging via binding to extracellular HSP90, while NP-002
with low HSP90 affinity had almost no effect.
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Results and discussion
Synthesis of key intermediate 6
As illustrated in Scheme 2, the synthesis of 6 was started from commercially available 4-bromo-1,8-naphthalic
anhydride (1). Firstly, compound 1 was reacted with 4-aminobutyric acid in EtOH to generate compound 2 with

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90% yield. Then, compound 2 was reacted with methanol in the presence of K2CO3 to result in compound 3.
Subsequently, compound 4 was obtained by demethylation of methyl ether in hydriodic acid at reflux. Finally,
compound 4 was treated with tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate followed by addition of
HATU and Hunig’s base to obtain compound 5 and the t-butyloxy carbonyl (Boc) group of 5 was removed in 10%
TFA/DCM to produce the PEG-NH2 key intermediate 6.
O
O
O
O
O
O
NHBoc
O
O
NH2
TFA
OH
OH
N
O
N
O
OH
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H
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OH
OH
OH
Br
Br
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Scheme 2. Reagents and conditions: (a) 4-Aminobutyric acid (1.1 equiv), EtOH, 70-80 °C, 8 h, 90%; (b) K2CO3 (8.0
equiv), CH3OH, 70-80 °C, 12 h; HCl, H2O, 95%; (c) 55% w/w HI, H2O, 140 °C, 6 h, 60%; (d) tert-butyl
(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (1.1 equiv), HATU, DIEA, anhydrous DMF, rt, 3 h, 70%; (e) 10%
TFA/DCM, rt, 3 h, 80%.
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Synthesis of NP-001
NP-001 was synthesized from the key intermediate 7 (Scheme 3). Compound 7 was synthesized according to the
literature37 and was first hydrolyzed by KOH in the mixed solvents THF/MeOH/H2O to give compound 8 with an
excellent yield. The benzyl-protecting groups were removed with 10% Pd/C under H2 in MeOH to produce
compound 8. Compound 8 was reacted with compound 6 by condensation reaction in the presence of HATU and
Hunig’s base to produce compound NP-001 with an acceptable yield.
O
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BnO
c
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OH
NP-001
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Scheme 3. Reagents and conditions: (a) KOH (3.0 equiv), THF/MeOH/H2O (V:V:V/ 1:1:1), 60 °C, 2 h, 85%; (b) 10%
Pd/C, MeOH, RT, 6 h, 90%; (c) compound 6 (1.2 equiv), HATU, DIEA, anhydrous DMF, RT, 4 h, 75%.
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Synthesis of NP-002
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The route used for the synthesis of NP-002 is shown in Scheme 437, starting from commercially available
1-(2,4-dihydroxy-5-isopropylphenyl)ethenone 10. Both phenolic hydroxyls were reacted with iodomethane to
generate 11, which was subsequently reacted with diethyl oxalate to generate keto-enol ester 12. Construction
of the pyrazole heterocycle of 13 was achieved by reaction of 12 with hydrazine hydrochloride in anhydrous
ethanol with a relatively high yield. Iodination at the 4-position of the pyrazole was achieved with
N-iodosuccinimide and a catalytic amount of ceric ammonium nitrate to improve the reaction rate and yield.
Compound 14 then underwent
a
palladium catalyzed Suzuki cross-coupling reaction with
4-(morpholinomethyl)phenylboronic acid to generate 15, which was further converted to 16 by ester
saponification. Finally, NP-002 was achieved by condensation of 16 and 6 in moderate condition.
O
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OH
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OH
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NP-002
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Scheme 4. Reagents and conditions: (a) CH3I (2.0 equiv), K2CO3, DMF, RT, 3 h, 40%; (b) Diethyl oxalate (1,2 equiv),
60% NaH, anhydrous THF, 60 °C, 4 h, 80%; (c) Hydroxylamine hydrochloride (1.2 equiv), absolute EtOH, 80 °C, 2 h,
85%; (d) N-iodosuccinimide (3.0 equiv), CAN, MeCN, 85 °C, 18 h, 77%; (e) 4-4-(Morpholinomethyl)phenylboronic
acid (1.2 equiv), Pd(PPh3)2Cl2, NaHCO3, DMF/H2O (2:1), 90°C, 8 h, 76%; (f) KOH (1.5 equiv), MeOH/THF/H2O
(V:V:V/ 1:1:1), 60 °C, 4 h, 85%; (g) compound 6 (1.2 equiv), HATU, DIEA, anhydrous DMF, RT, 5 h, 58%.
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HSP90 binding affinity assay

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First, both synthesized compounds were tested for their potential HSP90 inhibitory properties. MeasuDreOmI:e1n0.t1s0o39f/C9TB02505G
binding were conducted by a fluorescence polarization (FP) assay63 using NVP-AUY922 and unconjugated probe 4
as positive control at the concentration of 1000 nM, 333.3 nM, 111.1 nM, 37.0 nM, 12.3 nM, and 4.1 nM in
duplicate, and the results of the mean values were expressed as IC50 values. As presented in Table 1, NP-001
showed significant differences compared with NP-002. It was apparent that NP-001 showed appreciable binding
to recombinant human HSP90α protein with an inhibition potency that was slightly less than that of NVP-AUY922,
while NP-002 and unconjugated probe 4 were shown to have a negligible impact on HSP90α that was much
weaker than NVP-AUY922. These results showed that the conjugation of NVP-AUY922 and fluorescent probe did
not affect the binding affinity of the hybrids to HSP90, which indicated that NP-001 with active HSP90 scaffold
was suitable for constructing HSP90 inhibitor conjugates by this method, suggesting the possibility of higher
selectivity in tumor cells. Thus, these results inspired us conducting a further evaluation of compound NP-001.
Table 1. Data for compound NP-001and NP-002: Binding on HSP90 (FP Assay).
Comp.
NP-001
IC50a (nM ± SD)
158.90 ± 34.73
> 1000
NP-002
NVP-AUY922b
4c
11.20 ± 0.20
> 1000
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a Data presented is the mean ± SD value of three independent determinations.
b Used as positive control.
c Unconjugated probe used as a control.
In vitro cytotoxicity assay of NP-001 and NP-002

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Next, the in vitro anti-proliferative activities of NP-001 and NP-002 were evaluated by MTT assay inDAO5I:4190.c1e0l3ls9/C9TB02505G
and HCT116 cells. First, the cytotoxicity of both compounds (0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM,
32 μM and 64 μM) in A549 and HCT116 were evaluated for totally 24 h, 48 h and 72 h. As illustrated in Figure 2,
cell viability was decreased with increasing drug concentrations and exposure, which demonstrated the
cytotoxicity of NP-001 was dose-dependent and time-dependent, and may be attributed to the inhibition of
HSP90 in both cell lines. As for NP-002 with reduced HSP90 binding affinity, there was almost negligible toxicity
at all concentrations and treatment durations during the assay, demonstrating that HSP90 affinity had a
non-negligible role in cell viability and may lead to cumulative probe accumulation.
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Figure 2. In vitro cytotoxicity evaluation of NP-001 and NP-002 on A549 cells and HCT116 cells for 24 h (A), (D),
48 h (B), (E) and 72 h (C), (F). Data are expressed as mean ± standard deviation (SD). (n = 6).
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To confirm that the cytotoxicity of NP-001 (0.25 μM, 0.5 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM and 64 μM)
differed between tumor cells and normal cells, in vitro cytotoxicity assays were performed in HL-7702 cells and
HepG2 cells for 24 h (Figure 3). The aggressive HepG2 cells were more affected compared with the
non-oncogenic HL-7702 cells, due to the high affinity of activated HSP90. The in vitro cytotoxicity results
suggested that covalent conjugation of NVP-AUY922 to 4-hydroxy-1,8-naphthalimide interestingly had high

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selectivity and high cytotoxicity against tumor cells while sparing normal cells, indicating the possibility of NP-001
as a cancer-specific bioimaing probe.
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Figure 3. In vitro cytotoxicity evaluation of NP-001 on HL-7702 (A) and HepG2 (B) cells for 24 h. Data are
expressed as mean + SD. (n = 6).
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Photophysical properties of NP-001 and NP-002
To unequivocally establish that our newly developed probes were indeed good fluorescent systems capable of
emitting strong fluorescence during in vitro cellular uptake in tumor cells, the UV-vis absorption of all compounds
was investigated (Figure 4). Both compounds were dissolved in DMSO and mixed with PBS buffer (1: 100/ V: V).
Compared with the absorption peak of the two test compounds at pH 6.0 and pH 7.4, no obvious shift of the
absorption peak was observed in the absorption of NP-001 and NP-002 (Figure 4A). Maximum fluorescence
emission peaks of all compounds (50 μM) were also evaluated at pH 6.0 and 7.4, and the results showed that the
emission peaks were around 575 nm. The normalized intensity of NP-001 was less than that of NP-002 at the
equivalent concentrations at both pH values (Figure 4B), which meant that the fluorescence of NP-001 should be
even weaker than NP-002 under equal cellular uptake of the drugs. Overall, NP-001 and NP-002 exhibited almost
the same absorption and fluorescence emission properties, which suggested that the covalent conjugation and
pH values did not affect the normalized absorbance and fluorescence intensity of the hybrids. Together, these

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results showed that the emission of both probes could be directly used to visualized cellular uptakDeOinI: 1t0u.m10o39r/C9TB02505G
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lesions.
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Figure 4. (A) UV−vis absorbance spectra (PBS as solvent), and (B) fluorescence emission spectra (PBS as solvent)
of NP-001 and NP-002 at concentration of 50 μM.
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Cellular uptake of NP-001 and NP-002 in A549 cells, HCT116 cells and HL-7702 cells
We next employed both probes for the imaging of a tumor microenvironment to demonstrate whether NP-001
could be efficiently internalized into tumor cells. The cellular uptake behavior of NP-001 and NP-002 in HCT116
and A549 cells was measured by confocal laser scanning microscopy (CLSM).64 Cells were treated with 25 μM
compound at 37 °C for 6 h and quantitation of cellular uptake was conducted by flow cytometry. As shown in
Figure 5A and 5C, when A549 cells and HCT116 cells were treated with NP-001 and NP-002 for 6 h, respectively,
the NP-001 groups were observed to have significantly stronger green fluorescence in the cytoplasm than the
NP-002 groups, in which it was almost undetectable. The quantified results showed that mean fluorescence
intensities of A549 and HCT116 cells treated with NP-001 were approximately 3.5- and 4.5-fold stronger than
those of cells treated with NP-002, respectively (Figure 5B and 5D). Both compounds showed almost negligible
fluorescence in HL-7702 cells (Figure 5E), which is a normal cell line with low HSP90 expression. Flow cytometry
analysis data also confirmed the results (Figure 5F). These differences indicate that NP-001 has a higher tumor

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cell internalization ability than NP-002 in both tumor cell lines while sparing the normal cell line, and confirm that
resorcinol groups are critical for inhibitor binding in the active site of HSP90, thus leading to effective cellular
uptake and imaging.
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Figure 5. The cellular uptake behavior of NP-001 and NP-002 (25 μM) in A549 and HCT116 cells. (A)
Representative confocal laser scanning microscopic (CLSM) images of A549 cells (Scale bar = 20 μm). (B) Mean

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fluorescence intensity (MFI) of A549 cells incubated with both compounds for 6 h conducted by flowDcOyIt:o1m0.1e0tr3y9/C9TB02505G
analysis. (C) Representative confocal laser scanning microscopic (CLSM) images of HCT116 cells (Scale bar = 50
μm). (D) Mean fluorescence intensity (MFI) of HCT116 cells incubated with both compounds for 6 h conducted by
flow cytometry analysis. (E) Representative confocal laser scanning microscopic (CLSM) images of HL-7702 cells
(Scale bar = 50 μm). (F) Mean fluorescence intensity (MFI) of HL7702 cells incubated with both compounds for 6
h conducted by flow cytometry analysis. The experiments were performed three times, and the results of the
representative experiments are shown. Data were expressed as mean + SD, ***P < 0.001, n = 3, Student’s t-test.
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In vitro HSP90 expression
To further evaluate the relation between surface HSP90 expression and the cellular uptake, the expression levels
of HSP90 in A549, and HCT116 tumor cell lines and HL7702 normal cell line were determined by
immunohistochemical fluorescence analysis. Cells with proper densities were treated with anti-HSP90 rabbit
polyclonal antibody, Cy3 conjugated goat anti-rabbit IgG, and DAPI. As shown in Figure 6A,6B,6C, anti-HSP90
rabbit polyclonal antibody was found to stain the surface of HSP90 and then co-labeled with Cy3 conjugated goat
anti-rabbit IgG to show the presence of HSP90 with red fluorescence. The tumor cells groups were observed to
have significantly stronger red fluorescence than that of the normal cell line groups, demonstrating high surface
expression of HSP90 in tumorigenic cells than that in non-tumorigenic cells. Flow cytometry analysis of in vitro
expression profiles of membrane-bound HSP90 on the three cell lines using Cy3 conjugated Goat Anti-rabbit IgG
also confirmed fluorescence results (Figure 6D). Western blotting analysis was also performed to show HSP90
expression in all cell lines. Figure 6E showed that HSP90 in A549 and HCT116 cells was relatively higher than
HL7702 cells. These results are consistent with previous cellular uptake behaviors. Collectively, our data highlight

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that Hsp90 is not only trafficked to the membrane but is actively internalized and the cellular upDtaOkIe: 10o.r1d0e39r/C9TB02505G
correlates with the relative HSP90 expression of the cells.
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Figure 6. In vitro HSP90 expression in A549, HCT116, and HL7702 cells. Representative immunofluorescence
images of the cells (A) A549 cells, (B) HCT116 cells, and (C) HL7702 cells, following HSP90 staining, with
anti-HSP90 rabbit polyclonal antibody, Cy3 conjugated Goat Anti-rabbit IgG, and DAPI. Red, Cy3 conjugated Goat
Anti-rabbit IgG and blue, DAPI. (Scale bar = 100 μm). (D) Flow cytometry analysis of in vitro expression profiles of
membrane-bound Hsp90 on A549, HCT116 tumor cell lines, and normal HL7702 cell line using Cy3 conjugated
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Goat Anti-rabbit IgG. (E) Western blotting analysis of HSP90 expression in A549, and HCT116 tumorDOceI:ll10li.n10e3s9./C9TB02505G
Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. These experiments were
performed three times, and the results of the representative experiments are shown. Data were expressed as
mean + SD, ***P < 0.001, n = 3, Student’s t-test.
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Subcellular distribution of NP-001 and NP-002
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To further investigate the subcellular distribution of the probes after uptake into tumor cells, blue Hoechst 33258
was used, which is known to selectively and strongly stain intact nuclei. The subcellular distribution behavior of
NP-001 and NP-002 in A549 and HCT116 cells was measured by CLSM. Cells were treated with 25 μM compounds
at 37 °C for 6 h and then stained with Hoechst 33258 (1 μg/ mL) for 5 min. As shown in Figure 7A and 7B, when
A549 and HCT116 cells were treated with Hoechst 33258 for 5 min, strong blue fluorescence was observed in the
nuclei. It is obvious that NP-001 was mostly located in the cytoplasm and with very little in the nuclei, also
demonstrating effective cellular uptake of the probe.

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Figure 7. Subcellular distribution of NP-001 and NP-002 in A549 and HCT116 cells. Cells were treated with 25 μM
NP-001 and NP-002 for 6 h, respectively, then stained with Hoechst 33258 (1 μg/ mL) for 5 min. (A)
Representative CLSM images of A549 cells (Scale bar = 75 μm). (B) Representative CLSM images of HCT116 cells
(Scale bar = 75 μm). The experiments were performed three times, and the results of the representative CLSM
images are shown.
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Competitive experiments of NP-001

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To further confirm the mechanism of HSP90-mediated NP-001 cellular uptake, A549 and HCT116DcOeIl:ls10w.1e0r3e9/C9TB02505G
incubated with 100-fold molar free NVP-AUY922 mixed with NP-001 (25 μM, 100:1 n/n) for 6 h, which can block
the binding between NP-001 and HSP90. Vehicle mixed with NP-001 in culture medium (25 μM) was also
incubated in the other groups for 6 h. The cellular uptake behavior was observed by CLSM. Compared with the
cells incubated with NVP-AUY922 and NP-001 (Figure 8A and 8B), brighter green fluorescence was observed in
both cell lines incubated with vehicle and NP-001 (Figure 8A and 8B) indicating a higher NP-001 cellular uptake.
In the normal HL7702 cell groups, both test groups showed no obvious fluorescence (Figure 8C), which was in
consistent with the previous in vitro cellular uptake results. As shown in Figure 8D, quantitative analysis was
further performed. Notably, the mean fluorescence intensity (MFI) values of NP-001 uptake in the NVP-AUY922
and NP-001 groups were approximately 2.6- and 3.6-fold stronger than those of the groups incubated with
vehicle and NP-001.
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Figure 8. The cellular uptake of tumor cells treated with NVP-AUY922 mixed with NP-001 (25 μM, 100:1, n/n) or
NP-001 mixed with vehicle (25 μM). (A) A549 cells, (B) HCT116 cells and (C) HL7702 cells were treated with 25
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μM NVP-AUY922 (100 folds molar) or vehicle mixture with NP-001 for 6 h. (A), (B) and (C) RepresentaDtOivI:e10C.L1S0M39/C9TB02505G
images of A549 cells, HCT116 cells and HL-7702 cells, respectively (Scale bar = 50 μm). (D) Mean fluorescence
intensity (MFI) of A549 cells, HCT116 cells and HL7702 cells conducted by flow cytometry analysis. The
experiments were performed three times, and the results of the representative experiments are shown. Data
were expressed as mean + SD, ***P < 0.001, n = 3, Student’s t-test.
An HSP90-mediated NP-001 cellular uptake mechanism was also confirmed by competition assays with
recombinant HSP90 alpha protein. A549 cells and HCT116 cells were incubated with NP-001/ HSP90 protein (25
μM, 100:1 n/n) for 6 h. The other groups were also incubated with vehicle and NP-001 in culture medium (25 μM)
for 6 h. Cellular uptake behavior was observed by CLSM. Compared with the cells incubated with recombinant
HSP90 alpha and NP-001 (Figure 9A and 9B), brighter green fluorescence was observed in both cell lines
incubated with vehicle and NP-001 (Figure 9A and 9B) indicating higher NP-001 cellular uptake. In the normal
HL7702 cells groups, both test groups showed no obvious fluorescence (Figure 9C), which was in consistent with
the previous HSP90 inhibitor competitive cellular uptake results. All these results from HSP90 inhibitor and
recombinant HSP90 protein competitive experiments revealed that occupation of HSP90 could significantly
decrease the NP-001 uptake, confirming the HSP90-mediated targeting mechanism, which can simultaneously
penetrate the cell membranes and accumulate in tumor cells to improve NP-001 delivery. As shown in Figure 9D,
quantitative analysis was also further performed. Notably, the MFI values of NP-001 uptake in the vehicle and
NP-001 groups were approximately 2.8- and 3.3-fold stronger than those of the groups incubated with
recombinant HSP90 protein and NP-001.

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Figure 9. The cellular uptake of tumor cells treated NP-001/ HSP90 protein (1/100, n/n) and NP-001 solution (25
μM). (A) A549 cells, (B) HCT116 cells and (C) HL7702 cells were treated with NP-001/ HSP90 protein (1/100, n/n)
or vehicle mixed with NP-001 for 6 h. (A), (B) and (C) Representative CLSM images of A549 cells, HCT116 cells
(Scale bar = 75 μm) and HL-7702 cells (Scale bar = 50 μm), respectively. (D) MFI of A549 cells, HCT116 cells and
HL7702 cells conducted by flow cytometry analysis. The experiments were performed three times, and the
results of the representative experiments are shown. Data were expressed as mean + SD, ***P < 0.001, n = 3,
Student’s t-test.
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Time-dependent cellular uptake and cellular accumulation of NP-001 in A549 cells
After validating the mechanism, a fluorescence experiment was also performed to demonstrate whether NP-001
could increase intracellular drug accumulation in a time-dependent manner. The cellular uptake behavior was
investigated in A549 cells by CSLM. A549 cells were incubated with 25 μM NP-001 for different incubation time
periods (1 h, 2 h, 4 h, and 6 h). As shown in Figure 10A, when A549 cells were incubated with NP-001 for 1 h,

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green fluorescence in the cytoplasm could be observed, indicating the successful internalization of DNOPI-:01001.1.03In9/C9TB02505G
addition, with the increase in incubation time from 1 h to 6 h, stronger fluorescence appeared in A549 cells. A
further quantitative measurement of fluorescence intensity was undertaken in A549 cells by cell lysis analysis
and demonstrated that the fluorescence intensity of NP-001 within A549 cells was greatly enhanced (Figure 10B).
The MFI of the group treated with NP-001 for 6 h exhibited a 3-fold increase compared with the 1 h group. Taken
together, the results indicated that HSP90 binding may result in a time-dependent increase in the accumulation
of higher concentrations of probes or drugs in tumor cells for imaging.
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Figure 10. Time-dependent cellular uptake of both compounds by A549 cells. (A) Representative CLSM images
(Scale bar = 50 μm). (B) MFI of A549 cells incubated with 25 μM NP-001 for different incubation time periods (1 h,
2 h, 4 h, 6 h) conducted by microplate spectrophotometer analysis. The experiments were performed three times,
and the results of the representative experiments are shown. Data were expressed as mean + SD, ***P < 0.001, n
= 3, Student’s t-test.
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In vivo drug accumulation and biodistribution of NP-001 and NP-002
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Previous in vitro studies suggested that aggressive tumor cell lines, almost exclusively, internalized eHSP90,
leading to the accumulation of NP-001. To further investigate the biological properties of NP-001 and NP-002 in
vivo, drug accumulation and biodistribution profiles in major organs were evaluated in HCT116 xenografts. After

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a single intravenous administration of 15 mg kg-1 of both compounds, the mice were sacrificed andDtOhIe: 1m0.1a0jo39r/C9TB02505G
organs were excised and homogenized in 0.5 mL DMSO. The concentration of NP-001 and NP-002 was examined
by HPLC. As shown in Figure 11A, the level of NP-002 accumulation was much lower than that of NP-001. In
addition, with the increase in single intravenous administration time from 1 h to 12 h, the tumor tissues
concentration of NP-001 in 12 h group was greatly enhanced exhibiting a 4.7-fold increase compared with the 1 h
group. Furthermore, biodistribution analysis (Figure 11B) showed that NP-002 distributed without significant
tumor selectivity. In contrast, NP-001 clearly showed more enhanced accumulation at the tumor site than
NP-002 compared with other organs, suggesting favorable tumor selectivity. Additionally, the concentration of
NP-001 in excised tumor tissue was 5.0-fold higher than NP-002 at 12 h post-injection. Overall, these results
indicate the therapeutic superiority of NP-001, demonstrating preferential accumulation in tumor tissue.
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Figure 11. Drug accumulation and biodistribution on HCT116 xenografts. (A) Drugs concentration in tumor tissues,
1, 4, 6, and 12 h after i.v. injection of NP-001 and NP-002 solution at a dose of 15 mg kg-1. (B) Drugs
concentration in tumor tissues and major organs 12 h after i.v. injection of NP-001 and NP-002 solution at a dose
of 15 mg kg-1. The concentration was analyzed by HPLC. Results were expressed as mean + SD, ***P < 0.001, n
= 3, Student’s t-test.
Confocal laser scanning microscopy (CLSM) assay of ex vivo mice tumor sections

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Encouraged by these results, sections from mice tumor tissues (12 h-treatment at 15 mg kg-1) embedded in
paraffin were also cut into a section of 8 μm-thick sections for CLSM assay. As shown in Figure 12A, the
fluorescence of tumor sections treated with NP-002 was much weaker than that in those treated with NP-001,
suggesting that tumor accumulation was significantly decreased when both phenolic hydroxyls were blocked.
Quantitative analysis of these images by the Image J software also showed that the MFI of the NP-001 groups
was 6-fold stronger than those of the NP-002 groups (Figure 12B). These results are in consistent with those of
the in vitro and ex vivo biodistribution assays, further elucidating the tumor cumulative effect of NP-001.
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Figure 12. CLSM assay of ex vivo mice tumor sections and pharmacokinetic assessment for different compounds.
(A) Representative confocal laser scanning microscopic images of tumor sections after 12 h-treated of NP-001
and NP-002 at 15 mg/kg and 0.9% NaCl. (B) Mean fluorescence intensity (MFI) of tumor issues. (C)
Representative plasma concentration-time profiles of NP-001, NP-002 and NVP-AUY922 after i.v. injection into
mice (a dose of 15 mg kg-1). The experiments were performed three times, and the results of the representative
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experiments are shown. (Scale bar = 25 μm). Data were expressed as mean + SD, ***P < 0.001, n = 3, DSOtuI:d1e0n.1t0’s39t/C9TB02505G
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test.
Pharmacokinetics studies
Previous studies have demonstrated that NP-001 has enhanced tumor selectivity, which promotes its
accumulation at tumor sites. To further determine that NP-001 has this property in vivo, a pharmacokinetic study
was undertaken by intravenously injection of 15 mg kg-1 of NP-001, NP-002, and free NVP-AUY922 to BALB/c
mice of approximately 20 g in weight. As shown in Figure 12C, NP-001 was retained at a relatively higher
concentrations in the blood for up to 12 h, whereas NP-002 and free NVP-AUY922 were rapidly cleared from the
blood after i.v. administration. A comparison of all tested groups showed that the NP-001 group had a relatively
longer retention time. NP-001 with active HSP90 inhibitor scaffold could bind with HSP90 in vivo and prolong the
retention time, further confirming the possibility of enhanced probe accumulation in the tumor tissues for
imaging.
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Histological analysis
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Finally, to validate the therapeutic efficacy, heart, liver, spleen, lung, kidney and tumor issues were excised from
HCT116-bearing mice at day 14 after the first injection for further histological analysis. As illustrated in Figure
13A, typical features of apoptosis, such as nuclear damage, were observed from haematoxylin and eosin (H&E)-
stained sections, and the results suggest almost no noticeable major organ-related toxicities. In situ
TdT-mediated dUTP nick end labeling (TUNEL) assay was further employed to observe tumor cell apoptosis, with
apoptotic cells represented in green (Figure 13B). The NP-001 group showed a relatively higher positive rate of
tumor cell apoptosis than the NP-002 group and the control group (Figure 13B, C), exhibiting the same tendency

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as in the HSP90 binding assay. All of these results are in good agreement with the biodistribution assay, further
validating the superior efficacy of the NVP-AUY922-based scaffold NP-001.
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Figure 13. Representative images of ex vivo histological analyses of tumor sections at 14 days after the first
injection with NP-001, NP-001 (a dose of 15 mg/kg) and 0.9% NaCl. (A) Representative H&E staining (100 μm). (B)
TUNEL assay (100 μm). (C) Statistical analysis of the apoptotic ratios in the tumor tissues from the TUNEL assay.
Data were expressed as mean + SD, ***P < 0.0001, n = 3, Student’s t-test.
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Conclusions
In summary, we have successfully developed a HSP90-mediated tumor targeting probe, NP-001, for
cancer-specific bioimaging. Binding properties and in vitro anti-proliferation activities confirmed that NP-001

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retains HSP90 affinity. Furthermore, biological imaging evaluation revealed distinctly different results DfoOrI:N1P0.-1000319/C9TB02505G
and resorcinol hydroxyl-blocked NP-002. NP-001 with active HSP90 inhibitor scaffold was demonstrated to highly
accumulate in tumor cells compared with normal cells in a time-dependent manner mediated by eHSP90.
Moreover, biodistribution, pharmacokinetics studies, and histological analysis further revealed that NP-001
showed enhanced tumor tissue selectivity and no obvious major organ-related toxicities. Collectively, we have
shown that NP-001 is a promising imaging probe for the detection and visualization of tumor lesions, offering a
highly effective tumor response, low side effects and good biocompatibility, meriting further evaluation.
Materials and instrumentation
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All reagents were obtained from commercial sources and dried prior to use unless noted otherwise. Anhydrous
solutions of reaction mixtures were transferred via an oven-dried syringe or cannula.
3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma) was used as received without
further purification. Recombinant HSP90α protein and FITC-labeled geldanamycin were obtained from Abcam
(Cambridge, UK). Reactions were monitored by thin-layer chromatography on 0.25 mm silicon gel plates (GF254)
1
and visualized under UV light. H NMR and 13C NMR spectra were recorded at 23 ℃ in CDCl3 and DMSO-d6 on a
Bruker DRX-400 (400 MHz) using TMS as internal standard. Chemical shifts were reported as δ (ppm) and signal
splitting patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint), or multiplet (m),
with coupling constants (J) in hertz. The mass spectra (MS) were recorded on a Finnigan MAT-95 mass
spectrometer. High-resolution mass spectra (HRMS) were performed on an electron spray injection (ESI)
ThermoFisher Scientific LTQ FTICR mass spectrometer. High performance liquid chromatography (HPLC) analysis
was performed at room temperature using a Diamonsil C18 (250 mm*4.6 mm). Purity of all tested compounds
was ≥95 %, a mobile phase gradient from 5% CH3CN/buffer (0.1% TFA/H2O) for 2 min, 5% CH3CN/buffer (0.1%

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TFA/H2O) to 95% CH3CN/buffer (0.1% TFA/H2O) for 20 min, 95% CH3CN/buffer (0.1% TFA/H2O) to 5%
CH3CN/buffer (0.1% TFA/H2O) for 3 min, a flow rate of 1.0 mL min-1, and plotted at 373 nm. All images were
mounted and observed with a LEICA TCS SP8 Confocal Microscope System and Nikon Eclipse Ti2 fluorescence
microscope. Flow cytometry analysis measured by a FACSCalibur flow cytometry (BD Biosciences, USA). Ultrapure
water was used to prepare all aqueous solutions.
In vitro HSP90 assay
To assess the effects on HSP90 activity in vitro, fluorescence polarization (FP) assay was performed. Briefly,
various concentration of compounds and FITC-labeled geldanamycin were added to each reaction well.
Recombinant HSP90 protein was diluted with the reaction buffer (20 mM HEPES pH 7.3, 50 mM KCl, 5 mM MgCl2,
20 mM Na2MoO4, 0.01% Triton X-100, 100 μg/mL BSA, and 2 mM DTT) and added to each well to get the final
concentration of 22.2 nM. The enzyme reaction was initiated as soon as the purified HSP90 was added. After
incubation for 60 min at 4°C, the fluorescent polarization of the samples was determined by microtiter-plate
reader (Envision, Perkin Elmer).
Cell culture
Human adenocarcinoma A549 cells，human colorectal carcinoma HCT116 cells, human liver carcinoma HepG2
cells and normal human liver HL-7702 cells were purchased from the cell bank of the Chinese Academy of
Sciences (Shanghai, China). A549 cells were cultured in F12 media. HCT116 cells were cultured in McCoy's 5A
medium. HepG2 cells were cultured in DMEM media. HL-7702 cells were cultured in RPMI 1640 medium. All
media were supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/ streptomycin. Cells
were incubated in a humid atmosphere of 5% CO2 at 37 °C.
In vitro cytotoxicity assay

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A549 cells and HCT116 cells were seeded in a 96-well plate at proper density in growth media and incubated for
24 h. Afterwards, a range of concentrations of NP-001 and NP-002 were added to the above cells and incubated
at 37 °C for 24 h, 48 h and 72 h, respectively. The cell viability was then evaluated with an MTT assay. 20 μL of
MTT (5 mg mL-1) solution was added to each well of the plate and incubated at 37 °C for 4 h. 200 μL of cell lysis
buffer ( 10g SDS, 5 mL isobutanol and 0.1 mL 10 M HCl diluted to 100 mL with H2O) was added to dissolve the
formazan crystals after removing the media. Thereafter, the absorbance at 462 nm of each well was measured by
using a microplate reader (BioTek, American).
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HL-7702 cells and HepG2 cells were seeded in a 96-well plate at proper density in growth media and incubated
for 24 h. Afterwards, a range of concentrations of NP-001 and NP-002 were added to the above cells and
incubated at 37 °C for 24 h, respectively. The cell viability was then evaluated with an MTT assay. 20 μL of MTT (5
mg mL-1) solution was added to each well of the plate and incubated at 37 °C for 4 h. 200 μL of cell lysis buffer
was added to dissolve the formazan crystals after removing the media. Thereafter, the absorbance at 462 nm of
each well was measured by using a microplate reader (BioTek, American).
The cytotoxicity of all tested compounds is expressed as a percentage of the control using the following
equation:
Cell viability (%) = (AS – AB)/ (AControl – AB) × 100
(1)
Where AS, AB and AControl are the absorbance of the sample, blank and control wells.
Photophysical properties of NP-001 and NP-002
NP-001 and NP-002 were dissolved in DMSO to form a solution of concentration 5 mM, respectively. And then
the solutions were diluted with PBS buffer for 100 times in different pH value (6.0 and 7.4). The resulting

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solutions were measured by using an ultraviolet spectrophotometer (UV-1900PCS, China) and a fluorescence
spectrophotometer (Agilent Cary Eclipse, American).
In vitro cellular uptake.
For the purpose of observing the cellular uptake behavior of NP-001 and NP-002, A549 cells, HCT116 cells and
HL-7702 cells were plated in 20 mm glass bottomed culture dishes at proper density and incubated for 24 h.
Afterwards, 25 μmol L-1 NP-001 or NP-002 solution were equally added to the above cells and the mixture was
incubated for 6 h. The cells were washed with PBS and stained with 4% paraformaldehyde and subsequently
imaged using a Leica TCS SP8 confocal fluorescence microscope by excitation at λex 488 nm (Germany).
For the purpose of determining competitive property of NP-001 and NVP-AUY922, A549 cells, HCT116 cells and
HL-7702 cells were plated in 20 mm glass bottomed culture dishes at proper density and incubated for 24 h.
Afterwards, NP-001/NVP-AUY922 (1/100, n/n) and NP-001 solution at a concentration of 25 μmol L-1 was added
to the above cells and the mixture was incubated for 6 h. The cells were washed with PBS and stained with 4%
paraformaldehyde and subsequently imaged using a Leica TCS SP8 confocal fluorescence microscope by
excitation at λex 488 nm (Germany).
For the purpose of determining competitive property of NP-001 and recombinant HSP90 protein, A549 cells,
HCT116 cells and HL-7702 cells were plated in 20 mm glass bottomed culture dishes at proper density and
incubated for 24 h. Afterwards, NP-001/ HSP90 protein (1/100, n/n) and NP-001 solution at a concentration of 25
μmol L-1 was added to the above cells and the mixture was incubated for 6 h. The cells were washed with PBS and
stained with 4% paraformaldehyde and subsequently imaged using a Leica TCS SP8 confocal fluorescence
microscope by excitation at λex 488 nm (Germany).
For the purpose of determining time-dependent cellular uptake behavior of NP-001, A549 cells were plated in 20

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mm glass bottomed culture dishes at proper density and incubated for 24 h. Afterwards, 25 μmolDLO-1I:N1P0.-1000319/C9TB02505G
solution was added to above cells and the mixture was incubated for 1 h, 2 h, 4 h and 6 h. The cells were washed
with PBS and stained with 4% paraformaldehyde and subsequently imaged using a Leica TCS SP8 confocal
fluorescence microscope by excitation at λex 488 nm (Germany).
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In vitro surface staining assay
For the purpose of assessing the surface staining of HSP90 in vitro, immunohistochemistry was studied in A549,
HCT116, and HL7702 cells. Cells were seeded in a 6-well plate at proper density in growth media and incubated
for 24 h. Afterwards, the cells were washed with PBS, stained with 4% paraformaldehyde for 10 min, and Triton
X-100 (50 μL) for 20 min. Subsequently, cells were washed with PBS for 3 times and incubated with 3% BSA for 30
min. Next, cells were treated with 1% anti-HSP90 rabbit polyclonal antibody (200 μL) and incubated for 12 h at
4 °C. Next, cells were treated with 1% Cy3 conjugated Goat Anti-rabbit IgG (200 μL) for 50 min and treated with
DAPI for 10 min. Finally, the cells were washed with PBS and subsequently imaged using a Nikon ECLIPSE Ti2
epifluorescence microscope by excitation at λex 405 nm and 550 nm (Japan).
Western bolt assay
Cells at logarithmic phase were seeded in 6-well plates and cultured overnight. Compounds were added to the
wells and cells were incubated at 37ꢀ°C for 24ꢀh. The cells were harvested and washed with cold PBS for three
times. The proteins of whole cell lysates were prepared in RIPA buffer, and quantified by the bicinchoninic acid
(BCA) method. GAPDH was used as internal control. Proteins were separated on SDS-PAGE and transferred to
PVDF membrane by using the semi-dry method. The membranes were blocked with 5% nonfat dry milk and
incubated with the respective primary antibodies and HRP-conjugated secondary antibodies. Western blot was
visualized using ChemiDoc MP detection system (Bio-Rad).