Co-delivery of Cu(I) chelator and chemotherapeutics as a new strategy for tumor theranostic

Article abstract of DOI:10.1016/j.jconrel.2020.02.023

Chelating Cu from tumors has been verified as an effective and promising strategy for cancer therapy through antiangiogenesis. However, systematic removal Cu by injecting with Cu chelators will result unavoidable side effects, since Cu is indispensable to the body. In this work, a micelle targeting to tumors' newborn vessels based on a polypeptide was developed to co-load DOX and Probe X, which can go through an “OFF-to-ON” procedure to report the Cu⁺-capture events in vivo in a real-time way by giving near infrared (NIR) fluorescence and photoacoustic signal. By co-delivering antiangiogenesis and chemotherapeutic reagents, the tumor can be significantly suppressed, meanwhile with a low systematic toxicity. Hopefully, this work can offer new insights in designing sophisticated antitumor strategy.

Full text of DOI:10.1016/j.jconrel.2020.02.023

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Co-delivery of Cu(I) chelator and chemotherapeutics as a new
strategy for tumor theranostic
Tao Sun, Guangping Zhang, Zhongyuan Guo, Qinjun Chen, Yujie
Zhang, Yongchao Chu, Qin Guo, Chao Li, Wenxi Zhou, Yiwen
Zhang, Peixin Liu, Hongyi Chen, Haijun Yu, Liping Jiang, Chen
Jiang
PII:
S0168-3659(20)30113-9
DOI:
https://doi.org/10.1016/j.jconrel.2020.02.023
COREL 10176
Reference:
To appear in:
Journal of Controlled Release
Received date:
Revised date:
Accepted date:
23 December 2019
6 February 2020
12 February 2020
Please cite this article as: T. Sun, G. Zhang, Z. Guo, et al., Co-delivery of Cu(I) chelator
and chemotherapeutics as a new strategy for tumor theranostic, Journal of Controlled
Release (2020), https://doi.org/10.1016/j.jconrel.2020.02.023
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2020 Published by Elsevier.

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Co-delivery of Cu(I) Chelator and Chemotherapeutics As
A New Strategy for Tumor Theranostic
a
b
a
a
Tao Sun , Guangping Zhang , Zhongyuan Guo , Qinjun Chen , Yujie
a
a
a
a
a
Zhang , Yongchao Chu , Qin Guo , Chao Li , Wenxi Zhou , Yiwen
a
a
a
c
d
Zhang , Peixin Liu , Hongyi Chen , Haijun Yu , Liping Jiang , and Chen
a,
Jiang *
a
Key Laboratory of Smart Drug Delivery (Ministry of Education), Minhang Hospital,
State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain
Science, Institutes of Brain Science, Department of Pharmaceutics, School of
Pharmacy, Research Center on Aging and Medicine, Fudan University, Shanghai
2
01203, PR China
b
Shandong Province Key Laboratory of Medical Physics and Image Processing
Technology, School of Physics and Electronics & Institute of Materials and Clean
Energy, Shandong Normal University, 1 University Road, Jinan, 250358, PR China
c
State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, PR
China
d
State key Laboratory of Molecular Engineering of Polymers, Department of
Macromolecular Science, Fudan University, Shanghai 200438, PR China
Corresponding author: Prof. Chen Jiang, E-mail: jiangchen@shmu.edu.cn

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Graphical abstract

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Highlights
+


A theranostic specific Cu chelator was proposed.
+
Targeting micelle co-loaded with Cu -chelator and chemotherapeutic reagents
was fabricated.


Antiangiogenesis and tumor’s oxygen saturation reduction were reduced.
Fine antitumor efficacy was achieved with low side effect.

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ABSTRACT: Chelating Cu from tumors has been verified as an eff ective and
promising strategy for cancer therapy through antiangiogenesis. However, systematic
removal Cu by injecting with Cu chelators will result unavoidable side eff ects, since
Cu is indispensable to the body. In this work, a micelle targeting to tumors’ newborn
vessels based on a polypeptide was developed to co-load DOX and Probe X, which
+
can go through an “OFF-to-ON” procedure to report the Cu -capture events in vivo in
a real-time way by giving near infrared (NIR) fluorescence and photoacoustic signal.
By co-delivering antiangiogenesis and chemotherapeutic reagents, the tumor can be
significantly suppressed, meanwhile with a low systematic toxicity. Hopefully, this
work can offer new insights in designing sophisticated antitumor strategy.
Keywords
Cu chelator; Antiangiogenesis; Theranostic; Real-time reporting; Co-delivery

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1
. Introduction
Cu concentration, which is found extraordinarily elevated in many types of tumor,
often correlates with tumor burden and recurrence [1–2]. Plentiful research suggests
that Cu can promote the proliferation and migration of endothelial cells among the
tumor sites [3], and Cu is also indispensable for tumor cells to secrete angiogenic
factors [4]. There are already research on animal models focusing on suppressing
tumor angiogenesis direct by inhibiting in vivo copper trafficking [5]. A famous case
under clinical trial is tetrathiomolybdate, a strong copper chelator, which has been
proved able to suppress angiogenesis and tumor growth via copper deficiency [6].
Chelating Cu can remarkably lower Cu concentration systematically by promoting
the excretion after chelating [7]. However, Cu is a required element for life and an
essential element functionalizing in most aerobic organisms, mainly playing as a
structural and catalytic cofactor, and consequently involved in many biological
pathways. Notably, lowering the Cu concentration systematically by simply injecting
with Cu chelators was already found to cause severe side effects, including erythra,
optic neuritis, emesis and leucopenia [8]. From another perspective, without enough
reliable information on “when, where, how and how much” of the chelators’
fictionalization, clinical medication could be implicit [9]. As to our knowledge, there
are very few reported systems concerning chelating Cu in vivo meanwhile with a
reporting system [10]. It remains challenging to integrate a specific drug carrier to
deliver Cu chelators to tumors to avoid systematic toxicity and meanwhile provide the
real-time information on pharmacodynamics.
+
Cu, especially its monovalent form Cu , serves as a critical signaling species in
+
cells and can function in multiple organelles of tumor cells [11–12]. Cu plays a more
2+
important role than Cu in tumor development and metastasis. On the other hand, in
+
tumors’ reducing microenvironment, Cu can stay in the Cu state [13] and be
+
stabilized with GSH or Vitamin C within the cytosol [14]. Removal of Cu is more
direct and Cu concentration will be thus synchronously lowered through the redox
+
2+
balance of Cu -Cu [15].
From both clinic and theory, the synergistic therapy of chemotherapy and

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antiangiogenesis is reasonable, where chemotherapy directly kills tumor cells and
antiangiogenesis modulates the microenvironment by reducing new vessels that
support tumors [16]. In clinic, there are already attempts trying to combine
chemotherapy and antiangiogenesis together to suppress the cancer progress [17],
while the clinical therapeutic efficacy by using combinations (such as
bevacizumab/5-fluorouracil and trastazumab/paclitaxel for treating metastatic
colorectal/breast cancers) has been well approved [18]. However, it is still challenging
in specifically delivering chemotherapeutic agents and Cu chelators to the tumor sites
for better synergy, since they often perform greatly different physical properties. The
strategy can be further pushed forward by the targeting nanomedicine technology with
finer efficacy and improved biocompatibility.
+
In this work, we co-loaded DOX and a Cu Probe X (both as the chelator and tracer)
into a micellar system built from a body-friendly polypeptide amphiphile (Scheme 1
and Figure 1). The micelle was integrated with cRGD to target tumors’
neoangiogenesis, based on which, the chemotherapeutics and chelator can be
+
delivered to tumor simultaneously. The Cu chelating events can be monitored by NIR
fluorescence and photoacoustic imaging in a non-invasive and real-time way. After
treatment, Cu level and oxygen saturation were significantly down-regulated in
tumors, and the antineoangiogenesis mechanism was studied. The improved
combined anti-tumor efficacy from antiangiogenesis and chemotherapy was achieved.
Finally, the in vivo toxicity was also investigated. These findings, hopefully, can
provide new insights to design, fabricate and tailor artificial antitumor nanosystems.

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+
Scheme 1. Illustration of the co-delivery of Cu chelator and chemotherapeutics as a
new strategy for tumor theranostic. (A) “OFF-to-ON” procedure of Probe X induced
+
by the specific molecular recognition with Cu ; (B) Targeting micelle co-assembled
from CPLP/PLP/Probe X/DOX; (C) the tumor-targeting delivery (EPR effect and
cRGD receptor) and tumor theranostics mechanism.
2
2
. Materials and methods
.1 Materials
N -PEG-NH (MW 5000) and PEG-NH (MW 5000) were purchased from JenKem
3
2
2
Technology Co. Ltd. (Beijing, China). Lys (cbz), phe, triphosgene and cyclohexanone
were from Aladdin (Shanghai, China). HBr, AcOH, NaOH, NaOAc and CuI were
from Energy Chemical (Shanghai, China). Thiourea, POCl , ethyl 2-hydroxyethyl
3
sulfide, PMDETA and VcNa were commercially available from Sigma-Aldrich
(Shanghai, China). Deuterium reagents for nuclear magnetic resonance (NMR)
spectroscopy including CDCl and DMSO-δ , CD OD and D O were from J&K
3
6
3
2
Scientific Ltd. (Beijing, China). All other chemicals and mentioned solvents were
from Sinopharm Chemical Reagent (Shanghai, China). Thin layer chromatography

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(TLC) boards and silica gel for column (SilicaFlash F60, 230~400 mesh) were from
Haiyang Chemical (Qingdao, China), while Ar was from Lyumin Gas Co. Ltd.
Shanghai, China). cRGD-alkyne was purchased from China Peptide (Hangzhou,
(
China). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS)
were from Gibco BRL (Carlsbad, CA, USA). Nonessential amino acids,
trypsin-EDTA (0.25%), streptomycin, and penicillin were from Invitrogen Co.
(Waltham, Massachusetts, USA). Dialysis bags and filter membrane were from
Amicon Ultra (Millipore, MA, USA).
2
.2 Equipment
1
H NMR spectra were undertaken on Varian Oxford NMR spectrometer (400 MHz,
1
3
Palo Alto, CA, USA) at room temperature. C NMR spectra were recorded on Bruker
NMR Ascend spectrometer (600 MHz, Billerica, MA, USA) at room temperature.
FT-IR spectra were obtained employing ATR geometry on a Perkin-Elmer infrared
spectrophotometer (Waltham, MA, USA) at room temperature. Electron spray
ionization mass spectrometry (ESI-MS) was from Agilent LCMSD (Santa Clara, CA,
USA). Transmission electron microscopy (TEM) images were carried on a
JEM-100CX electron microscope from JEOL Ltd. (Tokyo, Japan). DLS and
ζ-potential results were obtained from a Wyatt QELS Technology DAWN HELEOS
instrument (Santa Barbara, CA, USA) a 12-angle replaced detector in a scintillation
o
vial and a 50 mW solid-state laser operated at 25 C. Chemical structures were drawn
on ChemBio 3D Ultra (14.0 version, Cambridge Soft-ware, MA, USA). Chemical
quantification was carried out on an analytical HPLC (Agilent Technologies Inc., CA,
USA), equipped with gradient flow control pump (1260 Quat Pump-G1311B),
auto-sampler (1260 ALS-G1329B), diode array detector (1260DAD-G4212B),
fluorescence detector (FLD-G1321A), column oven (1290 TCC-G1316C) and a
2
50-4.6 mm column (reversed phase-C18 (5 mm), Agilent Technologies Inc., CA,
USA). Cells were observed on a fluorescence microscope (Leica, Wetzlar, Germany).
Small animal in vivo fluorescence imaging was photographed on a Molecular Biology
Workstations (Image Visualization and Infrared Spectroscopy, IVIS, Caliper, Newton,

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MA, USA). Cu concentration in tumors was determined by Inductively Coupled
Plasma-Mass Spectrometry (ICP-Ms, Varian, Palo Alto, CA, USA) authorized to
National Center of testing Technology (Shanghai, China).
2
2
.3 Synthesis
.3.1 Synthesis and characterizations of the amphiphilic polypeptide
Peptide monomers: Lys (cbz)-NCA and phe-NCA were synthesized via the classic
Fuchs-Farthing method using triphosgene [19]. Briefly, lys (cbz) (3 g, 10.7 mmol) and
triphosgene (1.27 g, 4.28 mmol) in anhydrous THF (30 mL) were stirred for 3 h at 50
o
C under Ar. The mixture was cooled down to room temperature and slowly dripped
into cold anhydrous cyclohexane under rigorous stirring. The suspension was allowed
o
to crystallize at -20 C overnight, and the appeared white solid was filtered and dried
1
to give Lys (cbz)-NCA. Phe-NCA was obtained via a similar way. The H NMR and
1
3
C NMR spectra were in accordance with the reported literature [19].
XPEG-pLys-pPhe (X=MeO or N ): The triblock polymer XPEG-pLys-pPhe (X=MeO
3
or N ) contains the units of ethylene glycol (112, MW 5000), lysine (12) and
3
phenylalanine (18), and was prepared from a ring-opening polymerization reaction
(
Figure 1) with XPEG-NH as the initiator. To a stirred solution of XPEG (1 g, 0.2
2
mmol) in anhydrous DMF (10 mL), dried Lys (cbz)-NCA (736 mg, 2.4 mmol) from
o
the last step was added and maintained under stirring at 50 C under Ar for 48 h. Then
Phe-NCA (1.15 g, 6 mmol) in anhydrous DMF (20 mL) was injected into the
o
suspension cooled to room temperature, and the mixture was maintained at 50 C
under Ar for another 48 h. Then the suspension was cooled to room temperature and
slowly dripped into cold diethyl ether to allow the precipitation (×3). The white solid
was filtered and isolated by repeated precipitation from DMF into XPEG-pLys
(cbz)-pPhe, which was then treated with a mixed solvent of TFA (10 mL) and
HBr/HOAc (0.5 mL, 5wt HBr%) for 3 h at room temperature to deprotect the cbz
groups on lysine. The acidic solution was precipitated thrice in diethyl ether,
redissolved into pure water (5 mL) and dialyzed against water with a membrane

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(MWCO 1000) for 24 h (It should be noted that if the acidic solution was directly
treated with a dialysis against water, possible hydrolysis of the polypeptide could
occur catalyzed by residual acid in the dialysis bag.). Then the solution in the bag was
freeze-dried to give XPEG-pLys-pPhe.
cRGD-PEG-pLys-pPhe: cRGD was coupled onto the terminus of N -PEG-pLys-pPhe
3
via a Click reaction catalyzed by CuI. cRGD-alkyne (5 eq) and N -PEG-pLys-pPhe (1
3
eq) were dissolved in anhydrous DMF under Ar. A solution of CuI (0.05 eq), sodium
ascorbate (10 eq), and PMDETA (1 eq) in anhydrous DMF was injected into the
mixture, which was stirred at room temperature at dark under Ar for 12 h. The
solution was finally dialyzed against EDTA aqueous solution (10 mM, 48 h, to
+
2+
remove Cu and Cu ) and then deionized water (48 h) using a membrane (MW 1000),
followed by a freeze-drying to give the final product cRGD-PEG-pLys-pPhe.
+
2
.3.2 Synthesis and characterizations of Cu chelator
3
,6,12,15-Tetrathia-9-monoazaheptadecane (Compound 2): Compound 2 was
synthesized following a reported literature [20], and the characterizations were all in
accordance with reported literature (Figure S1 to S8). A mixture of ethyl
2
-hydroxyethyl sulfide (4.25 g, 40 mmol) and thiourea (3.05 g, 40 mmol) in
hydrobromic acid (48wt%, 8.5 mL, 75 mmol) was refluxed overnight under Ar. The
mixture was cooled to room temperature, and NaOH (3.2 g, 80 mmol) aqueous
solution (10 mL) was added slowly. The mixture was further refluxed overnight under
Ar, and then cooled to room temperature, neutralized with HCl aqueous solution (1
M), followed by an extraction with DCM (50 mL×3). The organics were combined,
washed with water (100 mL) and brine (100 mL), dried over Na SO , and evaporated
2
4
to dryness to harvest compound 1 as a colorless oil, which was used without
purification.
The oxidizing layer of sodium lumps was carefully removed with a knife. Sodium
(1.29 g, 56 mmol) and 3-thiapentan-1-thiol (4.1 g, 33.6 mmol) were suspended in

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absolute ethanol (60 mL) and the resulting suspension was heated to reflux. A solution
of bis(2-chloroethyl) amine hydrochloride (2.0 g, 11.2 mmol) in absolute ethanol (35
mL) was then injected into the above solution, which was then refluxed for 4 h. The
mixture was cooled to room temperature and EtOH was removed under vacuum. The
residual solid was carefully quenched with water in an ice-bath. DCM (150 mL) was
used to extract the suspension. The organics were washed with water, dried over dried
over Na SO , and evaporated to dryness to give a crude product, which was further
2
4
purified with a flash column chromatography (DCM:MeOH, 50:1 to 10:1, v:v) to give
compound 2 as a chocolate oil (2.3 g, 66.1%).
IR780 (Compound 5): IR780 was synthesized following a reported literature [21], and
the characterizations were all in accordance. To a solution of DMF (20 mL, 273 mmol)
in ice-bathed anhydrous DCM (20 mL) under Ar, POCl (17.5 mL, 115 mmol) in
3
anhydrous DCM (5 mL) was added dropwise in 0.5 h. Then, cyclohexanone (5 g, 50
mmol) was injected slowly into the above solution. The resulting mixture was stirred
o
vigorously at 80 C for 3 h, and poured into ice-cold water under stirring to obtain a
yellowish precipitation. The solid was filtered off, washed with water, and dried under
vacuum to give compound 3 (8.1 g, 93.2%) as a yellowish solid with a fine purity
(
Figure S9).
To a solution containing 2,3,3-trimethyl-3H-indole (2 g, 12.5 mmol) in ACN (20 mL),
-iodopropane (10.6 mL, 62 mmol) was added, and the mixture was refluxed under
1
stirring overnight. ACN was then removed under vacuum. The residual solid was
washed with diethyl ether, air-dried and recrystallized in acetone to obtain compound
4
as a white solid (3.3 g, 80.4%).
Compound 3 (0.5 g, 2.9 mmol) and compound 4 (1.91 g, 5.81 mmol) were dissolved
in BuOH-toluene (25 mL, 7:3, v:v) under Ar, and the solution was refluxed overnight
in a 100-mL flask with three necks installed with a Dean-Stark condenser to remove
generated water. Then, all solvents were removed under vacuum and the residual solid
was purified with a flash chromatography (DCM:MeOH, 50:1 to 20:1, v:v) to give

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compound 4 (IR780, 1.6 g, 85.3%) as a dark green solid.
Probe X: Probe X was synthesized following a reported literature [22], and the
characterizations were all in accordance. Compound 5 (0.14 g, 0.20 mmol) and
compound 2 (0.20 g, 0.80 mmol) were dissolved in anhydrous DMF (20 mL), and the
o
mixture was maintained at dark/80 C for 4 h under Ar. DMF was then removed under
vacuum, and the resulting residual solid was purified by flash columnchromatography
(DCM:EtOH, 200:1 to 20:1, v:v) to give Probe X as a dark blue solid (0.081 g, 0.18
mmol, 38.8%) with a fine purity (Figure S10).
2
.4 UV-vis spectrometer for Job’s plot
+
The complex stoichiometry of Probe X with Cu in solution (water:ACN=1:1, v:v)
was determined by Job’s continuous variation method using a UV-vis spectrometer. A
set of working solutions were obtained by mixing Vg mL of the stock Probe X
-4
-4
solution (10 mol/L) with (Vt-Vg) mL of the stock Probe X solution (10 mol/L),
where Vt is a fixed total volume and Vg is a variable volume for guest (from 0 to 10
mL, 0≤Vg≤Vt). To obtain the complex constant, the concentration of
-5
[
Cu(MeCN) ][PF ] (2×10 mol/L) was kept constant with the concentration of Probe
4 6
-4
-3
X ranging from 4×10 to 3.6×10 mol/L.
2
.5 FT-IR spectrometer
+
The sample of Probe X/Cu complex for FT-IR was obtained by freeze-drying the
Probe X/[Cu(MeCN) ][PF ] (1:1, molar ratio) aqueous solution, while the physical
4
6
mixture was prepared by just freshly mixing solid Probe X and [Cu(MeCN) ][PF ]
4
6
(1:1, molar ratio) together. Notably, during grinding with KBr for the FT-IR cake, the
+
molecular recognition between Probe X/Cu could occur, so solid Probe X and
[
Cu(MeCN) ][PF ] should be grinded with dry KBr separately, mixed together rapidly
4 6
and tableted into a single cake before measurement to minimize the unexpected
complexion.

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2
.6 Geometric optimization
In order to verify the possibility of the molecular recognizing mechanism model, a
geometric optimization was performed and the result was shown in the supporting
video. Atomic-based simulation can play a useful tool in indicating the properties at
an amicroscopic level and is regarded as necessary complements in explaining the
capturing process shown in Figure 1. In order to verify the possibility of the molecular
recognizing mechanism model, a molecular geometric optimization for the host-guest
system by using Gaussian 16 package is performed, where the B3LYP
exchange-correlation functional and a basis set of 6-31g(d) for H, C, N, S and
Lanl2dz for Cu are used. In this calculation, the surrounding water solvent is
considered by the widely used polarizable continuum model (PCM). It would be
relatively time-consuming for the host-guest system to spontaneously form a complex
from a completely disordered initial state that has been used in many simulation
+
calculation [23], a pre-setup where Cu is close to the middle N atom of Probe X was
+
applied. However, to our surprise, instead of continuing to complex with N atom, Cu
spontaneously approached to the S atoms and form stable coordinate bonds as shown
in the video. The final result of the configuration was shown in Figure 2H.
2
.7 Drug formulation and characterizations
The classic emulsion-solvent evaporation method was used to prepare the micelles
used in this project. Briefly, the mixed solid with all compositions (Probe X, DOX
and polymers, 10 mg) was dissolved in methanol (5 mL) to form a transparent
solution, then methanol was totally removed under vacuum to yield a membrane on
the inside wall. Afterwards, deionized water (10 mL) was slowly injected into the
flask and sonicated for 0.5 h at room temperature to obtain the micellar solution with
obvious opalescence. All micelles were freshly prepared for DLS/TEM
characterizations and antitumor efficacy. The TEM samples were obtained by
dripping the micellar solution to a copper TEM net, followed by complete drying
under an infrared lamp.

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HPLC was used to evaluate the loading content (LC) and encapsulation efficiency (EE)
of micelles. ACN (1 mL) was added to collapse the assembly of drug-loading micelle
(0.1 mL), 20 μL of which was submitted to HPLC for determination. The HPLC
condition for Probe X: InertSustain® C18 column (5um, 4.6×250 mm); Column
o
temperature: 25 C; flowing rate: 1 mL/min; detector: VWD (UV-vis): 214 nm;
gradient flowing phase: 0~18 min, 60% to 30% ACN in water, 18~18.5 min, 30% to
6
0% ACN in water, 18.5~20 min, 60% back to 30% ACN in water. The HPLC
condition for DOX is: Agilent ODS C18 column (4.6×250 mm, 5 μm particle size);
flowing phase: KH PO (10 mM, aqueous):ACN:acetic acid=70:30:0.3 (v:v:v);
2
4
o
flowing rate: 1.0 mL/min; column temperature: 25 C; detector: fluorescence detector
λex/em = 480/560 nm.
LC and EE of Probe X or DOX were calculated according to the following
formulas.
LC (%) = (the mass of loaded drug/the total mass of whole micellar
materials)×100%.
EE (%) = (the mass of loaded drug/the total mass of fed drug)×100%.
2
.8 Confocal imaging of cellular uptake
MDA-MB-231 cells were incubated in DMEM supplemented with 10% FBS plus 1%
4
(
v:v) antibiotic. MDA-MB-231 cells were seeded with a density of 2 × 10 per well in
o
2
4-well plates (Corning, Shanghai, China), and cultured at 37 C and 4% CO for 48 h
2
until an 80–90% confluency. The cells were incubated with glucose-free DMEM
medium for 12 h to achieve a balance. The cells were then cultured with targeting
CPLP-X micelles (constructed with 20% CPLP and 80% PLP, based on our previous
research) for 15~60 min. Then, the cells were washed thrice with glucose-free Hank’s
solution, monitored by fluorescence microscopy (Leica, Wetzlar, Germany).
2
.9 In vivo distribution observed by IVIS imaging
The formulations of free X, free IR780, PLP-X, CPLP-X and CPLP-IR780 were

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firstly prepared and injected into tumor-bearing mice (X: 5mg/kg body weight) via the
tail vein, where IR780 plays as the “Always-ON” reagent. The in vivo imaging of
tumor-bearing mice post i.v. injection over time with different formulations were
observed by IVIS. The ex vivo images by IVIS (PerkinElmer, Waltham, USA) of
excised main organs isolated from tumor-bearing mice observed 12 h post injection
with different formulations
2
.10 Cell viability and in vitro antitumor efficacy
The in vitro cytotoxicity of Probe X and DOX on HEK-293 and MD-MBA-293 were
evaluated using the MTT assay. Briefly, the cells were seeded into 96-well plates and
then incubated separately with Probe X or DOX with various concentrations for 48 h.
The MTT reagent was added to each well and further incubated for 0.5 h. Then, the
medium was removed and 200 μL of DMSO was then added to each well. After
shaking for 0.5 h at room temperature at dark, the absorbance of each well at 490 nm
was measured with a microplate reader (BioTek Epoch, Winooski, VT, USA).
2
.11 In vivo antitumor efficacy
All animal experiments and benefits were strictly complied with Institutional Animal
Care and Use Committee of School of Pharmacy Fudan University. Female balb/c
nude mice of 6 weeks with about 20 g body weight were from Department of
Experimental Animals of School of Pharmacy Fudan University, and cultured under
6
requested aseptic conditions. 1.5×10 MDA-MB-231 cells in 120 μL of Matrigel (5
mg/mL, BD Biosciences, Bedford, MA, USA) in PBS 7.4 were injected into the
second right mammary fat-pad of nude mice to establish the tumor-bearing mice. Two
weeks post the injection, the mice were randomly divided into 6 groups (n=6). The
tumor-bearing mice were intravenously administrated with the formulations of saline,
free X, free DOX, PLP-DOX/X, CPLP-X, CPLP-DOX, and CPLP-DOX/X at an
equivalent DOX dosage of 5 mg/kg and X dosage of 5 mg/kg body weight (7 days×3).
The tumor size and body weight of the mice were recorded every 3 days. The tumor
volume was measured with a vernier caliper and calculated with the formula: tumor

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2
th
volume=(lump length)×(lump width) /2. All mice were sacrificed on the 30 day. The
tumors and main organs were carefully excised and rinsed with PBS 7.4 for further
photographs. TUNEL assay was carried on 5 μm frozen tumor slices using a DNA
fragmentation detection kit according to the regular protocols provided by the
producer and observed by fluorescent microscope (Leica, Wetzlar, Germany).
2
.12 Photoacoustic imaging
The animal and phantom tests were performed by a multispectral Photoacoustics
Imaging System (Visusal Sonics, Vevo LAZR, Toronto, Canada). For the in vivo
photoacoustic imaging of the tumors, the mice were i.v. injected with different
formulations 24 h before the imaging (λex=730 nm). To monitor the tumor’s
modulated oxygen saturation, the mice experienced a 3-round treatment with different
formulations.
2
.13 In vitro tube-formation and wound-healing
Probe X (25 μg/mL) was mixed with Matrigel matrix (BD Biosciences, MA, USA)
o
and added to the 48-well plates, which were placed at 37 C for 1 h to solidify. Then,
human umbilical vein endothelial cells (HUVECs) in 200 μL basic DMEM containing
5
2
×10 cells were seeded onto these matrices and further incubated for 12 h. Then,
random fields in each well were observed at different intervals under a microscope
Olympus, Tokyo, Japan).
HUVECs were seeded into the 6-well plates at a density of 10 cells/well and
(
5
incubated for 48 h to form a monolayer. Then, a single linear wound to the layer was
caused with a pipette tip carefully, further incubated with Probe X (25 μg/mL) in basic
DMEM for 24 h. Defined wound line at fixed part in each well was observed at
different intervals under a microscope (Olympus, Tokyo, Japan).
2
.14 Cu determination by ICP-Ms
The tumors randomly selected from four mice each group were homogenized and then
digested in 70% nitric acid at 80 °C for 24 h until no solid tissues were left. The

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suspension was dispersed in ultrapure water and the copper contents in all samples
were detected using ICP-Ms.
2
.15 Histochemistry analysis
The main organs were excised, fixed with 4% paraf-ormaldehyde solution (v/v in
water) for 12 h, followed by being frozen in the OCT embedding medium (Sakura,
o
Torrance, CA, USA) at -80 C. Pictures of the stained slices were photographed under
the inverted fluorescent microscope (Leica, Wetzlar, Germany) for histochemistry
analysis.
2
.16 Data analysis and statistics
All the data were presented as means ± SD, and comparison between groups was
performed by GraphPad Prism. Statistical significance was defined as p < 0.05.
3
3
. Results and discussion
.1 Design, theoretical calculation and synthesis
We first started the project by selecting a specific chelating ligand, as well as a tracker,
+
for Cu both in vitro and in vivo. With the aim of facially capturing metal ions, various
types of ligands have been developed and widely applied in MRI and biomedical
sciences, to name a few, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid
(DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) [24] and
tetrathiomolybdate (TM) [25]. Most of the developed chelating ligands, however,
have limited selectivity to metal ions, which will easily result an unexpected loss of
entogenous essential metals once applied. It is with great realistic meaning in finding
+
2+
+
a specific chelating ligand for Cu . Different from Cu chelators, Cu strongly prefers
ligands with softer donor atoms such as thioether S and aromatic N [26].
On the other hand, human body is a “black box” to scientists, and upon the
substrates delivered into the body, the information of “where/when/how/how much”
of the distribution and spatio-temporal accumulation is difficult to be monitored in a
real-time way. Tissue extraction method can definitely provide useful messages,

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which, however, is time-consuming, hysteretic and traumatogenic. Since Cu is an
essential element for life, while alterations in its cellular homeostasis other than
tumors are connected to serious diseases or side effects, including leukoderma,
Menkes and Wilson diseases by undiscriminatingly capturing Cu from the Cu-relying
enzymes [27–28]. In this regard, real-time reporting the Cu-caging phenomenon in a
theranostic strategy, especially upon in vivo situation, can provide rich information on
drug tracking, efficacy prediction and clinical guidance with lowered side-effect.
+
However, developing reliable optical imaging ligands with Cu -selective fluorescent
sensors is still facing tremendous challenge, since Cu is normally used as a
fluorescent-quenching reagent. Indeed, instead of the “OFF-to-ON” type, we initially
did plan to create an “ON-to-OFF” type fluorescent sensor upon the recognition of the
+
ligand to Cu . However, the fluorescence quenching can be attributed to various
unpredictable reasons (photobleaching, solvent effect, aggregation caused quenching,
+
etc), besides Cu capture. After attempts, we finally successfully obtained an
“OFF-to-ON” type NIR fluorescent sensor (Probe X) derived from IR780. Probe X
+
+
can be used as a Cu tracker, as well as a Cu chelator. The synthesis steps of the
chelators (compound 2 and Probe X) were shown in Figure 1.
In order to realize the targeting delivery of the ligands and chemotherapeutic,
through a ring-opening reaction initiated by PEG-NH , we also developed a
2
bio-friendly polypeptide-based amphiphile (PLP, Figure 1), which can load the
hydrophobic substrates and self-assemble into micelles in the aqueous solution. The
cyclic peptide cRGD, that is proved capable to target the tumors’ newborn vessels,
was anchored to the polymer to obtain CPLP. Through the active (cRGD) and passive
(EPR effect) targeting, the micelles are hopefully able to reach the aimed sites
effectively.

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Figure 1. Synthesis of (A) the amphiphilic polypeptides (CPLP and PLP, blue:
+
hydrophobic; green: hydrophilic; purple: cRGD) and (B) Cu chelator (Probe X, gray:
OFF state; red: ON state).
+
3
.2 Molecular recognition between Probe X and Cu
+
We then investigated the specific Cu capturing capability of Probe X in aqueous
+
solutions by multiple means. In presence of increasing amount of Cu , the intensity of
excitation spectrum of Probe X tended to be stronger (Figure 2A), which suggests the

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+
addition of Cu can help Probe X gain more energy from the irradiating photons. On
the other hand, the emission is found simultaneously increasing (Figure 2B). In
+
+
absence of Cu , the fluorescence is relatively weak, while the Probe X/Cu complex
+
can give much stronger fluorescence. The UV-vis spectra of sole X and X+Cu were
+
also undertaken, where a clear red-shift was noticed for X+Cu (Figure S11). This
indicated the formation of a new species, which performed a stronger absorbance at
the excitation wavelength of fluorescence. The combined results suggest an
+
“
OFF-to-ON” ability of Probe X to detect/complex Cu , where the mechanism could
be an increased energy-gaining capability for the fluorescence, since fluorescence is
essentially an energy-transfer phenomenon.
Then we employed the classic Job’s continuous variation method to determine the
+
complex stoichiometry of Probe X/Cu , where the maximum value was found at a
+
+
molar ratio (Probe X/(Probe X+Cu )) of 0.5. This signified that Probe X/Cu tended
to stay in a 1:1 stoichiometry mode. We then explored the selectivity of Probe X to
+
different cations that are common in vivo. It was noticed that only Cu can induce
+
strong fluorescence. When facing cations other than Cu , Probe X is still in an “OFF”
+
state, while upon addition with Cu to the above solutions, strong fluorescence can be
recovered. FT-IR can be used in studying the supramolecular interactions between
+
ligands and metal ions. The FT-IR spectra of Probe X, Cu , the corresponding
+
physical mixture and complex of X/Cu in KBr were undertaken. The physical
+
mixture and complex of X/Cu have the exactly same chemical composition, however,
they gave greatly distinctive results. The spectrum of physical mixture is clearly the
+
overlying of the spectra of starting materials (X/Cu ), for example, the peaks at the
-1
wavelength at 2937 and 831 cm . Meanwhile, new peaks can be recognized at the
spectrum of the complex, revealing the complex is a new species and new interactions
+
were formed between X/Cu .
We then employed geometric optimization to mimic the recognition procedure
+
(
Figure 2G and supporting video). Cu was released to the system in a free state, and
+
after a relatively long balancing, Cu automatically approached the ligand area to
form a stable complex. Meanwhile, the original configuration of Probe X was slightly

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perturbated, especially observed from the lateral view. Both ligands were attached to
the molecule via rotatable covalent bonds, allowing Probe X with appropriate
+
configuration to hunt Cu in the solvent. From the vedio of the detailed molecular
+
recognition process, Cu firstly attached with the middle N atom of Probe X
+
temporarily. After short interactions with N atom, Cu more tended to be attracted by
+
the S-containing ligands, and both ligands became curved and curled to cage Cu in
the middle to form a stable complex. Upon completion the molecular recognition, the
systematic energy slightly rises 0.07 eV based on the calculation, which is easily
overcome from the environment disturb. The Off-to-ON procedure mechanism could
+
be: the electron rich S atoms (also Cu receptor) are able to quench the fluorescence of
+
the tricarbocyanine backbone, and upon capturing with Cu , the quenching influence
is inhibited to further recover the fluorescence.
+
Then the Cu recognizing capability of Probe X was evaluated on MD-MBA-231
(human triple negative breast cancer cells) and HEK-293 (human embryonic kidney)
+
cells. Since endogenous Cu is much richer in tumor cells than somatic cells, the
fluorescence intensity is much stronger on MD-MBA-231 than HEK-293 (Figure 2I)
+
in accordance with the cell types. Introduction with exogenous Cu can increase, and
+
extra Cu chelator (compound 2) can decrease the fluorescence intensity of both cells.
The fluorescence intensity of tumor cells was also found obviously increased upon
introduction with Vitamin C (VC) that can trigger the release of labile endogenous
+
Cu in living cells [29]. The results were further verified using flow cytometry, where
a large number of cells with “Turned-ON” fluorescence emission was found upon
treated with sole Probe X (1 mM). At this concentration of Probe X, introduction with
+
exogenous Cu cannot increase the number of “Turned-ON” cells, suggesting the
+
+
buffered Cu -capturing ability of 1 mM Probe X and endogenous Cu can already
satisfy the chelating of Probe X. We further carried on the same experiments on
+
HEK-293 cells, and found exogenous Cu can give an apparent rise to the number of
+
“
Turned-ON” cells, since exogenous Cu in HEK-293 cells is initially at a low level.
+
Taken together, the combined results suggest tumor cells contain much more Cu than
body cells. Probe X can be employed in tracking/capturing endogenously produced

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+
Cu , no matter in buffers or physiological medium.
Figure 2. (A) The excitation and (B) the emission spectra of Probe X in presence with
+
different Cu concentration in mixed methanol/water (2.5 mM, v:v, 1:1),
λex/em=750/792 nm; (C) the red-shift of the UV-vis absorbance of Probe X in
+
presence of Cu in mixed methanol/water (v:v, 1:1); (D) The complex stoichiometry
+
of Probe X/Cu determined by Job’s continuous variation method using a UV-vis
spectrometer; (E) fluorescence responses of the Probe X (2.5 mM) to various cations
in the aqueous buff er (pH 7.0, 25 mM PBS buff er with 10% ethanol, 2.5 mM), where
the black bars represent the addition of an excess of cations (2.5 mM) to a solution of
+
Probe X, and red bars represent the subsequent addition of 2.5 mM Cu to the above
solutions (λex/em=750/792 nm); (F) the comparision of the FT-IR spectra of Probe X,
+
Cu+, physical mixture and complex of X/Cu in KBr; (G) the Probe X’s molecular

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+
configuration variation before and after the Cu capture; (H) fluorescence images of
MD-MBA-231 and HEK-293 cells treated with different formulations (control: no
+
2+
treatment; Probe X (5 μM); Probe X+Cu : pretreated with CuCl (200 mM, Cu can
2
+
be rapidly transformed into Cu in the cells in presence of GSH) for 7 h and further
o
+
+
incubated with Probe X (5 mM) for 10 min at 37 C; Probe X+Cu then Cu
chelators: pretreated with CuCl (200 mM) for 7 h, further incubated with Probe X (5
2
o
+
mM) for 10 min at 37 C, and subsequently treated with a competing Cu chelator
o
compound 2 (50 μM) for an additional 10 min at 37 C; Probe X+VC: pretreated with
o
VC (1 mM) for 4 h and then incubated with Probe X (5 mM) for 10 min at 37 C;
+
Probe X+VC then Cu chelators: pretreated with VC (1 mM) for 4 h, further
o
incubated with Probe X (5 mM) for 10 min at 37 C, and subsequently treated with a
+
o
competing Cu chelator compound 2 (50 μM) for an additional 10 min at 37 C); (I)
the quantitative determination of MDA-MB-231 cells with NIR fluorescence using
+
flow cytometry (Probe X (5 μM, 10 min); Probe X+Cu : pretreated with CuCl (200
2
o
mM) for 7 h and further incubated with Probe X (5 mM) for 10 min at 37 C) with
scale bars=25 μm; the quantitative determination of (J) MD-MBA-231 and (K)
HEK-293 cells with recovered fluorescence using flow cytometry (Probe X (5 μM,
+
1
0 min); Probe X+Cu : pretreated with CuCl (200 mM) for 7 h and further
2
o
incubated with Probe X (5 mM) for 10 min at 37 C).
3
.3 Drug formulation
Both the selected cytotoxic drug (DOX) and Probe X possess conjugated systems, we
decided to employ PEG-pLys-pPhe (PLP) as the building block to construct the
nanomicelle, where poly-phenylalanine plays as the hydrophobic part of the
amphiphile. The pPhe part will self-assemble into hydrophobic core to encapsulate
DOX and Probe X via hydrophobic interactions, and more important, π-π stacking
effect, with the aim to form a micelle with high drug loading rate and stability.
The preparation of the micelles loading with DOX/X was optimized, and the
PLP-DOX/X and CPLP-DOX/X micelles (the formulation with active-targeting
ability, doped with 20% cRGD-PLP composition, based on our previous research [30])

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in PBS 7.4 were obtained. The micellar solutions were dried under vacuum and
observed under TEM, where spherical micelles with uniform morphology ranging
from 20 to 60 nm were found for both micelles (Figure 3A and B). The PLP-DOX/X
and CPLP-DOX/X micellar aqueous solution is homogeneous with opalescence, and
typical Tyndall effect can be found, while obvious solid can be found if DOX/X in
DMSO was directly dispersed in PBS 7.4. The size distribution was further verified
using dynamic light scattering (DLS, 56.1±5.2 nm for PLP-DOX/X and 62.7±3.4 nm
for CPLP-DOX/X) and nanoparticle tracking analysis (NTA). No aggregates smaller
than 10 nm were tested, meaning a fine dispersion and encapsulation. It is
understandable the diameters measured by DLS were slightly larger than TEM, since
DLS data are from the hydrodynamic diameters, while TEM is measuring the solid
spheres [31]. We also found an increase of the Z potential before and after the
encapsulation (Figure 3F), which could be ascribed to the loaded DOX and X with
potential positive charges. After optimization, the drug loading rate and encapsulation
efficiency are measured to be 19.3% and 89.7% by HPLC, respectively (DOX:X=1:1,
w:w).
Figure 3. TEM images of (A) PLP-DOX/X and (B) CPLP-DOX/X micelles; (C)
Tyndall effect of PLP-DOX/X and CPLP-DOX/X micelles in PBS 7.4; size
distribution of PLP-DOX/X and CPLP-DOX/X micelles in PBS 7.4 measured by (D)
DLS and (E) NTA; (F) Z potentials of PLP-DOX/X and CPLP-DOX/X micelles in

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PBS 7.4.
3
.3 Drug distribution
CPLP-X micelle (“OFF” state) was used to treat MD-MBA-231 cells and monitored
under confocal imaging system to study the cellular internalization pathway and
reveal the detailed “OFF-to-ON” process (Figure 4A). When incubated for 15 min
with CPLP-X micelle, clear nanoparticles with fluorescence (“ON” state) were
observed and co-localized with lysosomes. The nanoparticles still stayed in an
orbicular state, meaning the fluorescence of Probe X molecules were still
+
encapsulated in the micellar core but already triggered-ON. On the other hand, Cu is
relatively rich in tumor cells and can be rapidly absorbed into the micellar core,
though normally regarded hydrophobic. Upon 60 min, a lysosome-escape and
extensive fluorescence in cytoplasm were found. In this procedure, Probe X that is
+
still available was dissociated into the cytoplasm to capture residual Cu in the cell. In
combination, an apparent internalization transfer via lysosomes into cytoplasm,
accompanied with the “OFF-to-ON” procedure was revealed.
Photoacoustic imaging is a noninvasive technique with high imaging depth (ca. 10 cm)
that can provide higher optical resolution at depths inaccessible by other optical
approaches [32]. It was reported that Cu can strengthen the photoacoustic imaging
capability of heptamethine cyanine dye [33]. We further used the Probe X-loading
micelles as photoacoustic imaging reagents. The targeting CPLP-X micelle can give
the strongest photoacoustic signal at the tumor sites in all tested four groups (Figure
4
B and C), which should be due to the highest tumor accumulation of CPLP-X from
both passive (EPR effect) and active targeting (cRGD) effect.
Encouraged by these results, we further investigated the drug distribution and
“OFF-to-ON” procedures in vivo by IVIS (Figure 4E). Formulations of free Probe X
(OFF), free IR780 (already-ON), PLP-X, CPLP-X and CPLP-IR780 with the same
molar amount of the dyes were injected into the tumor-bearing mice via tail vein. No
obvious signal can be detected in Probe X group at 2 h, which could be explained by
+
the uncontrollable distribution and the relatively low concentration of Cu in vivo. It is

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possible that Probe X mostly distributed in a specific organ, but it showed low
+
florescence due to the low concentration of Cu . Surprisingly, even without specific
formulation, clear NIR fluorescence signal could be monitored only at the tumor sites,
which tended to be even stronger as time extended until 24 h. This further verified the
+
higher concentration of Cu in tumors that is essential in expanding new vessels. As to
the IR780 group, since fluorescence signal is always-ON, we noticed strong
fluorescence signal systemically that vanished over time possibly due to the
metabolism and excretion. At 24 h, only residual signal can be found at the tumor
sites. It is understandable since the dyes of indocyanine green family have been
approved by FDA in clinical angiography [34]. Noteworthy, evident liver metabolism
of PLP-X was observed at 2 h. However, due to the EFR effect, the tumor
accumulation predominated. On the other hand, tumor accumulation of CPLP-X is
stronger, suggesting the important role of the active targeting inherited from cRGD.
No severe liver accumulation but a kidney excretion was noticed. The NIR signal
elevation in the tumors is from the increasing accumulation, as well as the
+
“
OFF-to-ON” procedure of Probe X capturing the in situ Cu . As the control, the NIR
signal at the tumor site is always strong for the CPLP-IR780 group that is already ON.
The results were further verified by the ex vivo images of excised main organs
isolated from tumor-bearing mice observed 12 h post injection with different
formulations. Limited signal was monitored in the liver. We deduce the NIR light has
limited penetration capability, which makes IVIS more suitable in monitoring drug
distribution in relatively superficial tumor [35], such as breast cancer. On the other
hand, liver is located deep in cavum abdominis, which is sometimes difficulty and
direct to be observed by IVIS, though useful information can be obtained in a
tempo-way. The spatio-information is more reliable if we try to investigate the
distribution ex vivo on the isolated organs (Figure 4D), where the liver accumulation
is obviously unneglectable. In combination, CPLP micelle can target and accumulate
into the tumor to result an “OFF-to-ON” NIR signal. The real-time activatable
theranostic phenomenon is of potential application in clinic [36].

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Figure 4. (A) Confocal imaging of cellular uptake of the targeting CPLP-X micelles
after incubation for 15 and 60 min on MD-MBA-231 cells with scale bar=5 μm; (B)
in vivo photoacoustic imaging of the tumors in white circle 24 h post i.v. injection
with different formulations; (C) semi-quantitation of the in vivo photoacoustic
imaging (λex=730 nm) of the tumors in white circle 24 h post i.v. injection with
different formulations (n=4); (D) ex vivo images by IVIS of excised main organs
isolated from tumor-bearing mice observed 12 h post injection with different
formulations; (E) in vivo imaging of tumor-bearing mice post i.v. injection over time
with different formulations observed by IVIS, where the white circles represent tumor
positions.
3
.4 Antitumor efficacy
With above data in hand, the antitumor efficacy of the proposed strategy was further
+
evaluated. Cu is not just impotent in building new vessels in the tumor, but also in
serving as an essential catalytic cofactor for enzymes that function in antioxidant
defense, iron homeostasis and cellular respiration [37]. Down-regulating the
+
+
intracellular Cu concentration and trafficking of Cu in the cells provides a
promising route to enhance the cytotoxicity of chemotherapeutic agents by disrupting

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these above processes. It was found the IC concentration of DOX can decrease 52
5
0
times (from 8.649 to 0.1623 μg/mL) when co-treated with Probe X (Figure 5A and
S12). The applied concentration of Probe X is 6 pmol/L, at which concentration, the
cell viability (just Probe X) can still reach over 80%. At nearly all concentrations,
Probe X can enhance the antitumor efficacy of DOX. This suggests a fine synergistic
effect of DOX and Probe X in the anti-tumor applications. Motivated by this, we then
carried out the in vivo antitumor efficacy test. The tumor-bearing mice were
intravenously administrated with the formulations of saline, free X, free DOX,
PLP-DOX/X, CPLP-X, CPLP-DOX, and CPLP-DOX/X at an equivalent DOX
dosage of 5 mg/kg body weight every (7 days×3). The tumor size and body weight of
the mice were recorded every 3 days. Among all groups, CPLP-DOX/X shows the
most evident suppression effect on tumor growth (Figure 5B~D). In addition, the
body-weight of all groups was rather stable with regular fluctuations, suggesting
minimized systemic toxicity (Figure 5E). During the treatment, the body weight
decreased apparently upon treated with free Probe X and DOX, suggesting the
unneglectable systematic toxicity.
After 3-round therapy, the tumors were excised from the sacrificed mice in all
groups to investigate the neoangiogenesis. Triple negative breast cancer (TNBC), used
as the model in this study, is a typical type of tumor marked with adequate blood
perfusion, and neoangiogenesis is crucial in the cancer progression [38]. Trafficking
+
Cu could be of great beneficence in attenuating the proliferation and motility of
endothelial cells in tumors [39]. CPLP-DOX/X demonstrated the most significant
effect in decreasing neoangiogenesis (Figure 5F). On the other hand, formulations
only containing DOX show little efficacy in decreasing neoangiogenesis. The
combined results verified a favorable synergistic effect of DOX and Probe X in
anti-tumor applications by means of cytotoxic effect and decreasing neoangiogenesis
simultaneously.

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Figure 5. (A) In vitro cell viability to MD-MBA-231 cells of DOX in absence and
presence (Probe X is set constant at 6 pmol/L.) of X evaluated by MTT assay; in vivo
antitumor efficacy study of tumor volume (B), excised tumor weight (C), tumor