Near-infrared heptamethine cyanine dye-based nanoscale coordination polymers with intrinsic nucleus-targeting for low temperature photothermal therapy

Article abstract of DOI:10.1016/j.nantod.2020.100910

Effective photothermal therapy (PTT) under low temperature (< 50 °C) and power density (< 0.4 W/cm²) avoiding the damage of skin and normal organs. Here, we report a novel heptamethine cyanine dye-based nanoscale coordination polymer (NCP), Hf-HI-4COOH, as an exceptionally effective photosensitizer for nucleus-targeting low temperature PTT. Hf-HI-4COOH shows strong near-infrared absorption and high photothermal conversion efficiency (39.51 %). Further, it exhibits intrinsic nucleus-targeting property and cell inhibition under low power density in vitro. Moreover, the long blood circulation, high tumor accumulation, and nucleus-targeting contribute to significant inhibition of breast tumors in tumor-bearing mice under low temperature (48 °C) and power density (0.3 W/cm²) without obvious toxicity. In general, we establish a nucleus-targeting Hf-HI-4COOH for enhanced PTT with low temperature and power density.

Full text of DOI:10.1016/j.nantod.2020.100910

Nano Today 34 (2020) 100910
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Near-infrared heptamethine cyanine dye-based nanoscale
coordination polymers with intrinsic nucleus-targeting for low
temperature photothermal therapy
Zhenqi Jianga,b, Bo Yuana, Yinjie Wanga,b, Zhenni Weia, Shan Suna,
Ozioma Udochukwu Akakurua,b, Yong Lia, Juan Lia,∗, Aiguo Wua,∗
a Cixi Institute of Biomedical Engineering, CAS Key Laboratory of Magnetic Materials and Devices, Zhejiang Engineering Research Center for Biomedical
Materials, Ningbo Institute of Materials Technology and Engineering, Chinese Academy of Sciences, Ningbo, 315201, PR China
b University of Chinese Academy of Sciences, Beijing, 100049, PR China
a r t i c l e i n f o
a b s t r a c t
Effective photothermal therapy (PTT) under low temperature (< 50 ◦C) and power density (< 0.4 W/cm2)
avoiding the damage of skin and normal organs. Here, we report a novel heptamethine cyanine dye-based
nanoscale coordination polymer (NCP), Hf-HI-4COOH, as an exceptionally effective photosensitizer for
nucleus-targeting low temperature PTT. Hf-HI-4COOH shows strong near-infrared absorption and high
photothermal conversion efficiency (39.51 %). Further, it exhibits intrinsic nucleus-targeting property and
cell inhibition under low power density in vitro. Moreover, the long blood circulation, high tumor accu-
mulation, and nucleus-targeting contribute to significant inhibition of breast tumors in tumor-bearing
mice under low temperature (48 ◦C) and power density (0.3 W/cm2) without obvious toxicity. In gen-
eral, we establish a nucleus-targeting Hf-HI-4COOH for enhanced PTT with low temperature and power
density.
Article history:
Received 25 May 2020
Accepted 7 June 2020
Keywords:
Nanoscale coordination polymer
Photothermal therapy
Nucleus targeting
Near-infrared
Heptamethine cyanine
© 2020 Elsevier Ltd. All rights reserved.
Introduction
sensitive to heat [13]. Some encouraging attempts to increase
PTT efficiency have been explored by increasing accumulation in
Photothermal therapy (PTT) under near-infrared (NIR) light is
a promising cancer treatment approach [1–4]. It converts light
energy to kill cancer cells with high efficiency and selectivity [4,5].
However, the power density of current available photothermal
agents is often higher than the human skin-sustainable maximum
value (∼0.4 W/cm2) permitted by the American National Standards
Institute [6]. Furthermore, the required temperature to induce
complete cell necrosis is over 50 ◦C that will not only destroy the
tumor but also do harm to nearby normal organs, for the non-
specificity of lasers [7]. Therefore, development of strategies to
effectively destruct tumor under low power density and tempera-
ture would be efficient way to improve tumor therapeutic efficacy
[8].
nuclei [13], including cell-penetrating peptides (CPPs) [14] and
amino-rich polymers [15]. Conjugating or assembling CPPs and
amino-rich polymers with photosensitizers (PS) could improve
nucleus accumulation [6,12,14], but the nucleus-entry capacity
of CPPs was limited after conjugation to PS [16]. Additionally,
hemagglutination, severe serum inhibition and subsequently rapid
clearance from the blood circulation of amino-rich polymers [17]
have obstructed the further development of nucleus-targeted PTT.
Furthermore, many reported inorganic nanoparticles based-PS are
lack of biodegradability and with potential long-term toxicity
[18], whilst organic dyes based-PS are easily to be photobleached
[19–21], which hinder the progress of these PS in future clinical
translation. To settle the above issues, it is important to develop
a biocompatible, high photothermal conversion effective PS with
strong NIR absorbance, excellent photostability and instinctive
nucleus-targeting [22,23].
Luckily, nuclei, as the central governor and one of the most
thermolabile intracellular structure, was expected to improve
PTT efficiency under low power density and temperature [9–12],
because the DNA and proteins in nucleus appear to be hyper-
Nanoscale coordination polymers (NCPs), self-assembling of
metal ions and organic ligands, are thought to be a bright
nanocarrier platform for tumor imaging and therapy for its
intrinsic biodegradability, structural/chemical diversities, and
highly enriched functionalities [24–26]. Previous reports reported
∗
Corresponding authors.
E-mail addresses: lij@nimte.ac.cn (J. Li), aiguo@nimte.ac.cn (A. Wu).
https://doi.org/10.1016/j.nantod.2020.100910
1748-0132/© 2020 Elsevier Ltd. All rights reserved.

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Z. Jiang, B. Yuan, Y. Wang et al. / Nano Today 34 (2020) 100910
Scheme 1. Synthesis of Hf-HI-4COOH NCPs and the description of nucleus targeted low temperature PTT.
Scheme 2. Synthesis path of HI-4COOH.
nanoZIF-90, a subclass of NCPs, improved tumor therapeutic
efficacy by mitochondria-targeting without modification [27,28].
Thence, subcellular-targeting NCPs with strong near-infrared
absorption and high photothermal conversion efficiency, such as
nucleus, might improve therapeutic efficacy of PTT. So far, there
were less report about NCPs showed efficient PTT therapy under
NIR light, especially with the intrinsic nucleus-targeting ability. We
maybe need to develop a new NCPs based on a new designed NIR
organic probe.
Herein, we report the synthesis of a Hf-heptamethine indocya-
nine dye (HI-4COOH)-based NCPs as intrinsic nucleus-targeting PS
with highly photothermal conversion efficiency for PTT under low
power density and temperature (Scheme 1). For the strong coordi-
nation between Hf and carboxyl group and its wide application of
Hf in the synthesis of CP [29,30], we choose Hf as a metal element
to coordinate with HI-4COOH to form NCPs. Although heptame-
thine indocyanine dye-based NCPs for PTT have been realized [31],
the nucleus-targeting PTT by heptamethine indocyanine dye-based
NCPs has not been reported until now. We hypothesized that the
incorporation of a heptamethine indocyanine dye-derived bridg-
ing ligand into robust NCPs structure of appropriate size would
show several advantages compared to the existing PTT agents:
first, the dye molecules could be well separated in the polymer,
avoiding aggregation and self-quenching under NIR light; second,
coordination of heptamethine indocyanine dye ligands to heavy Hf
centers via the carboxylate of heptamethine indocyanine dye could
enhance photothermal conversion efficiency; third, the nucleus-
targeting could exert thermal therapeutic effects at low power
density and temperature. In this Hf-HI-4COOH design, a high load-
ing of HI-4COOH dye and nucleus targeting can be achieved to
allow efficient low power density and temperature PTT. To our best
known, this is the first NCPs that shows both intrinsic nucleus-
targeting and strong NIR absorption without further modification
to increase the tumor inhibition under low power density and tem-
perature.
Results and discussion
Synthesis and characterization of Hf-HI-4COOH
The newly designed heptamethine indocyanine dye derivative,
4-((5-carboxy-2-((E)-2-((E)-3-(2-((E)-5-carboxy-1-(4-carboxyb-

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Fig. 1. Characterization and photothermal ability of Hf-HI-4COOH. TEM images of (a) Hf-HI-4COOH and (b) incubating in complete RPMI 1640 cell culture medium after 24
h. (c) UV–vis spectra of HI-4COOH in methanol and Hf-HI-4COOH in water. (d) Temperature change of different concentrations of Hf-HI-4COOH solution under an 808 nm
laser (0.6 W/cm2). (e) Temperature change of Hf-HI-4COOH (100 ␮g/mL) solution under different power densities of 808 nm laser. (f) Temperature curves of Hf-HI-4COOH
(100 ␮g/mL) solution for five cycles under an 808 nm laser (0.6 W/cm2).
enzyl)-3,3-dimethylindolin-2-ylidene)
ethylidene)-2-
highest cellular uptake of Hf (Fig. S11) and was used in subsequent
studies.
chlorocyclohex-1-en-1-yl)vinyl)-3,3-dimethyl-3H-indol-1-ium-1-
yl) methyl)benzoate (HI-4COOH), was synthesized by a third-step
reaction (Scheme 2), further the structure and chemical com-
ponents were determined by 1H and 13C NMR and ESI-TOF-MS
(Fig. S1-S8). For the strong coordination between Hf and car-
boxyl groups, the tetra-carboxylate groups of HI-4COOH ligand
allow the construction of CPs with Hf. Hf-HI-4COOH NCPs was
synthesized through a solvothermal reaction by dissolving HfCl4
and HI-4COOH in N, N-dimethylformamide (DMF) and methanol
(MeOH) at 90 ◦C for 24 h. The resulting product was purified
by DMF (two times) and ethanol (four times) successively. By
changing the volume of glacial acetic acid (Table S1), we could
regulate and control the size of products. The sample 2 showed the
The obtained Hf-HI-4COOH NCPs showed good dispersibility
in water. Powder X-ray diffraction (XRD) measurements and Fast
Fourier Transform of the High Resolution TEM image indicated the
amorphous nature of Hf-HI-4COOH NCPs (Fig. S12-S13). The TEM
image showed the diameter of Hf-HI-4COOH was 30 nm (Fig. 1a).
Dynamic light scattering (DLS) measurements showed the average
diameter was 36.7 nm for Hf-HI-4COOH (Fig. S14). Notably, this size
is smaller than the size that could cross the nucleus pore complex
to enter the cell nucleus [32]. Meanwhile, the ␨-potential of Hf-
HI-4COOH was −18.4 2.3 mV. The composition of Hf-HI-4COOH
was confirmed by plasma optical emission spectrometer (ICP-OES)
and organic element analyzer, the ratio of Hf to HI-4COOH was 2.72,

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Fig. 2. Cellular uptake and PTT efficiency of Hf-HI-4COOH in vitro. (a) XFM images of S (cell), Zn (nucleus) and Hf (Hf-HI-4COOH) in 4T1 cells incubated with Hf-HI-4COOH
for 8 h. (b) LSCM images of 4T1 cells incubated with Hf-HI-4COOH for 8 h and DAPI was used as a nucleus tracker. (Scale bar = 10 ␮m) (c) Time-dependent enrichment of
Hf-HI-4COOH in the nucleus. (d, e) Cell viability of 4T1 cells incubated with Hf-HI-4COOH for 8 h, then under an 808 nm laser of 0.3 or 0.6 W/cm2 (5 min) and for another 24
and 48 h incubation. (f) Cell viability of 4T1 cells under different power densities of 808 nm laser (5 min) for another 24 and 48 h incubation. The cells were incubated with
Hf-HI-4COOH for 8 h before irradiation. Mean SD (n = 3).
which was slightly lower than calculated value (the ratio = 3) due to
the nano size and defect of Hf-HI-4COOH (Table S2). This result also
indicated much carboxylate groups in HI-4COOH were free, which
may be the major reason for good water dispersibility and negative
␨-potential of Hf-HI-4COOH. The stability of Hf-HI-4COOH in phys-
iologically relevant media was determined by incubating particles
in complete RPMI 1640 medium with 10 % FBS for 24 and 48 h at 37
◦C. TEM images and DLS results showed an unaltered morphology
and obvious size change after incubation (Fig. 1b, S16a and S17).
high photo-stability (Fig. 1f). The TEM images, DLS results and dig-
ital photos of Hf-HI-4COOH showed there were no obvious change
after NIR laser exposure (Fig. S16b, S17 and S19).
Cellular location and in vitro photo-therapy efficacy of
Hf-HI-4COOH
To confirm the location of Hf-HI-4COOH after incubation with
cells, X-ray fluorescence imaging (XFM) and laser scanning con-
focal microscopy (LSCM) were used [35]. Once cell nucleus was
labeled with Zn as previous report [36], the Hf-HI-4COOH were
found in nuclei when either Hf or HI-4COOH was used as label
while Hf and HI-4COOH were dispersed in the cytoplasm when
incubated with only HfCl4 or HI-4COOH (Fig. 2a and b). Further-
more, the Hf contents of cells and nuclei were determined by
ICP-OES. The Hf-HI-4COOH could be efficiently internalized into
the cells with an uptake percent of about 34 % and 37 % at 4 h
and 12 h, respectively. The Hf-HI-4COOH were quickly enriched in
nuclei and attained saturation after 4 h incubation. After 4 h, over
90 % of Hf-HI-4COOH internalized by 4T1 cells were found in the
nuclei (Fig. 2c). Taken together, Hf-HI-4COOH could be taken up by
tumor cells and internalized into their nuclei. To further explore
Photothermal ability of Hf-HI-4COOH
Similar to HI-4COOH in methanol, Hf-HI-4COOH revealed strong
absorption at 797 nm (Fig. 1c). The mass extinction coefficient at
808 nm of NCPs was calculated to be 67.11 L/ (g*cm), which was
higher than that of many previously reported PSs [18,33]. In addi-
tion, the fluorescence of Hf-HI-4COOH was close to the solution
under 808 nm laser (Fig. S18), which could improve the effect of
PTT [34]. Furthermore, Hf-HI-4COOH showed efficient photother-
mal conversion efficiency (39.51 %) and could be fastly heated under
808 nm NIR laser (Fig. 1d and e, Table S3). Exposed to NIR laser
for five cycles (5 min for one, 0.6 W/cm2), Hf-HI-4COOH showed

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Fig. 3. (a) Blood circulation of Hf-HI-4COOH after i.v. injection. The pharmacokinetics followed the two-compartment model. (b) Biodistribution of Hf-HI-4COOH in 4T1-
tumor-bearinng mice at 24 h after i.v. injection. (c) Time-dependent distribution of Hf in main organs of healthy mice after i.v. injection of Hf-HI-4COOH. (d) Serum levels
of aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin and alkaline phosphatase (ALP) of health mice after 1, 7 and 14 d post injection (i.v.) of the
Hf-HI-4COOH. (e) H&E-stained images of major organs from healthy control mice after i.v. injection of PBS and Hf-HI-4COOH after 60 days (Scale Bar = 100 ␮m) (The injection
dosage of Hf-HI-4COOH was 20 mg/kg).
the mechanism of nucleus-targeting ability, nucleus accumulation
of Hf-HI-4COOH with different particle size and zeta potential were
investigated (Fig. S11 and S21). These results showed a decrease
of nucleus accumulation in 4T1 cancer cells with the particle size
increasing, and the zeta potential change from negative to positive
with the pH value decreasing. Therefore, proper particle size and
positive charge of Hf-HI-4COOH in cancer cells might contribute
to the nucleus-targeting. To further compare the nucleus-targeting
ability difference of Hf-HI-4COOH between cancer cells and normal
cells, the cellular and nucleus accumulation of Hf-HI-4COOH in nor-
mal cell line MCF-10A were also tested. Fig. S22 showed the cellular
uptake of Hf-HI-4COOH in MCF-10A cells was only one-sixth of 4T1
cancer cells, and less than 30 % of Hf was found in the nucleus of
normal cells. Above results showed that both particle size and pos-
itive charge contributed to the excellent nucleus-targeting ability
of Hf-HI-4COOH in cancer cells [13].
To further evaluate biocompatibility of Hf-HI-4COOH on normal
cells, MCF-10A were incubated with Hf-HI-4COOH after different
treatment or under NIR laser various concentration. The results
showed the Hf-HI-4COOH were biocompatible with normal cells
after NIR laser irradiation or high temperature (Fig. S24). In
addition, the MCF-10A kept 80 % cell viability after irradiation, indi-
cating normal cells were minimally affected by this treatment (Fig.
S25).
To evaluate therapeutic efficacy of Hf-HI-4COOH upon expo-
sure to NIR laser various concentration of Hf-HI-4COOH and
laser power were used. The CCK-8 results showed that 4T1 can-
cer cells can be inhibited within 24 h at a high laser power
(0.6 W/cm2) in a concentration-dependent manner for the high
temperature. Furthermore, the tumor cells were also inhibited
on exposure to a lower laser power (0.3 W/cm2) after 48 h
(Fig. 2d, e and f), because cell death caused by heat induced
nucleus damage is time-dependent [37]. Afterwards, the apopto-
sis analysis of 4T1 cells revealed that the major types of induced
cell death were different for the 0.3 and 0.6 W/cm2 laser pow-
ers, which was early apoptosis and necrosis respectively (Fig.
S26).
The biocompatibility of the Hf-HI-4COOH was explored by cell
counting kit-8 (CCK-8) assay. The results demonstrated that Hf-HI-
4COOH showed no obvious toxicity to both cancer and normal cells
(ECA-109, 4T1, U87-MG and MCF-10A cells) after being incubated
at a high concentration (100 ␮g/mL) for 24 or 48 h (Fig. S23).

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In vivo pharmacokinetics and biocompatibility of Hf-HI-4COOH
ture increasement and cytotoxicity assays in vitro. PTT efficacy in
in vivo studies demonstrated tumor decrease under low power den-
sity and temperature and complete tumor eradication in mice that
were treated with Hf-HI-4COOH without obvious toxicity during
the therapy. Further, the Hf-HI-4COOH could be clean-up within
14 days and showed no obvious change of blood analyses. There-
fore, the nucleus-targeting Hf-HI-4COOH afford a new generation
of highly effective PS for safe PTT under low power density and
temperature.
Encouraged by the excellent in vitro low laser power PTT effi-
ciency, the behavior of Hf-HI-4COOH in vivo was also expected.
At first, the concentration of Hf4+ in blood and major organs were
measured by ICP-OES. The concentration of Hf-HI-4COOH in blood
showed a gradual decay within 24 h and the half-life was 5.13 0.35
h by a two-compartment model (Fig. 3a). The long blood half-life of
Hf-HI-4COOH contributed to passive tumor accumulation via the
enhanced permeability and retention effect. It showed a relatively
high tumor accumulation of 7.29 0.47 % ID g−1 at 24 h post injec-
tion of Hf-HI-4COOH for tumor-bearing mice (Fig. 3b). Moreover,
the Hf concentration of major organs were tested by ICP-OES. The
concentration of Hf decreased to a rather low concentration within
14 days, which indicated that most of Hf-HI-4COOH were cleaned-
up from the mice body (Fig. 3c). The side effects of the Hf-HI-4COOH
were also important to evaluate safety of Hf-HI-4COOH. The H&E
stained major organs of PBS and Hf-HI-4COOH groups also showed
that there were no significant signs of inflammatory lesion or organ
damage after 60 days (Fig. 3e). Furthermore, blood panel, biochem-
ical and serum analyses were conducted for hematology assay.
Within 14 days, the mice treated with Hf-HI-4COOH remained con-
stant compared to the control (Fig. 3d and S27-S29)[38]. All results
showed the Hf-HI-4COOH have good biocompatibility and will not
result in obvious toxicity during therapy.
Experimental section
Materials and cell lines
All of the starting materials were purchased from Sigma-Aldrich
and Fisher (USA), unless otherwise noted, and used without further
purification.
The human esophageal cancer cells ECA-109, mouse breast can-
cer cells 4T1, human brain glioma cancer cells U87-MG, human
epithelial cell line MCF-10A were purchased from Shanghai Cell
Bank. The ECA-109 and 4T1 cells were cultured in 1640 medium
(Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum
(Hyclone, Utah, USA). U87-MG cells were cultured in MEM medium
(Gibco, Grand Island, NY, USA) containing 10 % fetal bovine serum.
MCF-10A cells were cultured in Complete culture medium of mam-
mary epithelial cells (Zhongqiaoxinzhou Co.Ltd, Shanghai, China).
Antitumor efficacy of Hf-HI-4COOH in vivo
Synthesis of HI-4COOH
To evaluate the antitumor efficacy in vivo, the 4T1 tumor-
bearing mice were intravenously administrated with PBS and
Hf-HI-4COOH (20 mg/kg). After 24 h the photothermal heating pro-
files in vivo were recorded. There was a rapidly rise ∼15 ◦C within 5
min under 808 nm laser at 0.3 W/cm2 for Hf-HI-4COOH, while the
PBS showed only ∼1.5 ◦C increase at the same condition (Fig. 4a and
b). Notably, the photothermal heating profile of Hf-HI-4COOH after
intratumorally injection were stably within 5 min at 0.6 W/cm2,
whilst the HI-4COOH showed an obvious decrease after 2 min (Fig.
S30-S33), which indicated that Hf-HI-4COOH showed higher pho-
tothermal stability than HI-4COOH in vivo. Then the tumors of
group which treated by Hf-HI-4COOH with light decreased during
the treatment and were eliminated and without recurrence within
14 days post-treatment (Fig. 4c). However, tumors of other three
groups showed a rapid growth. Further these mice survived for
more than 60 days, whilst the other three groups survived no more
than 26 days (Fig. 4d). The body weight of mice after PTT showed
no significant decrease, indicating no acute toxicity to mice major
organs (Fig. S34). The H&E staining of tumors after treatment also
showed that there were much vacuolar chromatin and cell necrosis
(Fig. 4e). Comparing to the PBS group, there was no severe dam-
age in other main organs for the mice treated by Hf-HI-4COOH
with light, indicating the Hf-HI-4COOH was with good biosafety
(Fig. 5 and S35). Besides that, the H&E staining of skins after treat-
ment also showed no obvious damage comparing to the skin of
healthy mice (Fig. S36). In general, the Hf-HI-4COOH showed the
effective inhibition of tumor growth without obvious toxicity. The
nucleus-targeting with high photothermal conversion efficiency
of Hf-HI-4COOH contributed to the significantly improved tumor
inhibition under low temperature and power density.
Synthesis of 2,3,3-trimethyl-3H-indole-5-carboxylic acid
4-hydrazinobenzoic acid (3.0 g, 19.8 mmol), methyl isopropyl
ketone (3.3 mL, 29.7 mmol) and sodium acetate (3.3 g, 39.6 mmol)
was mixed in a round-bottomed fitted with a condenser. Another
glacial acetic acid (45 mL) was added. The brown suspension was
refluxed for 8 h, and the solvent was removed under reduced pres-
sure with a rotavapor. The residue was re-dissolved into a clear
solution using 100 mL water and methanol mixture solution (9/1,
v/v). Undissolved material was filtered off, the filtrate was allowed
to stand at room temperature, and the 2,3,3-trimethyl-3H-indole-
5-carboxylic acid (2.82 g, 70 %) was collected by filtration. 1H-NMR
(400 MHz, MeOD, ppm): ␦ = 8.04 (m, 2 H), 7.51 (t, 1 H), 2.32 (s, 3
H), 1.37 (s, 6 H). m/z: calcd., 204.1; found 204.0895. (Fig. S1-S2)
Synthesis of
5-carboxy-1-(4-carboxybenzyl)-2,3,3-trimethyl-3H-indol-1-ium
bromide
2,3,3-trimethyl-3H-indole-5-carboxylic acid (9.8 g, 48 mmol)
and alpha-bromo-p-toluic acid (12.3 g, 57 mmol) were dissolved
in 40 mL o-dichlorobenzene and stirring under 120 ◦C for 30 min.
The solid crude product was then filtrated and recrystallized in
methanol. The purified product 5-carboxy-1-(4-carboxybenzyl)-
2,3,3-trimethyl-3H-indol-1-ium bromide (10.06 g, 62 %) was dried
under vacuum. 1H-NMR (400 MHz, DMSO-D6, ppm): ␦ = 8.10 (d, 1
H), 8.00 (d, 3 H), 7.76 (s,2 H), 7.34 (s, 1 H), 3.18 (s, 2 H), 2.40 (s, 3 H),
1.36 (s, 6 H). m/z: calcd., 338.1; found 338.1347. (Fig. S3-S4)
Synthesis of 2-chloro-3-(hydroxymethylidene)
cyclohexene-1-carbaldehyde
Conclusion
80 mL DMF/dichloromethane mixture (1/1, v/v) was added
dropwisely into a solution of phosphoryl chloride (37 mL) and
anhydrous dichloromethane (35 mL) under stirring in ice/water
bath; afterwards, 10 g cyclohexanone was added dropwisely. The
ice/water bath was removed, and the solution was then heated and
refluxed for 3 h. The mixture was poured into ice, yielding a solid
In summary, a newly designed Hf-HI-4COOH for highly effective
nucleus-targeted low temperature PTT was developed. The synthe-
sized Hf-HI-4COOH showed great nucleus-targeting and worked
as an efficient PS for PTT, as demonstrated by both fast tempera-

Z. Jiang, B. Yuan, Y. Wang et al. / Nano Today 34 (2020) 100910
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Fig. 4. PTT efficiency of Hf-HI-4COOH in vivo. (a) IR thermal images and (b) temperature change of 4T1 tumor-bearing mice with intravenous injection PBS or Hf-HI-4COOH
under 808 nm (0.3 W/cm2) laser irradiation taken at different time intervals. (c) Tumor volume change and (d) survival rate of tumor-bearing mice after different treatments.
Mean SD (n = 5). (e) Photography of tumor-bearing mice at 0, 3rd and 7th days treatment and H&E staining of tumors for one treatment after 24 h. (Scale bar = 100 ␮m).
product which was collected by filtration and washed with iced
diethyl ether. The resulting yellow product was used directly.
methanol, and stirred for 1 h at 60 ◦C under N2. The solvent was
removed by rotatory evaporation, and the crude product was puri-
fied by dissolving in DMF and precipitating in diethyl ether. After
filtration, the HI-4COOH (0.984 g, 64 %) was dried under vacuum.
1H NMR (400 MHz, MeOD, ppm): ␦ = 8.43(d, 1 H), 8.34(d, 1 H),
8.25(t, 1 H), 8.18(t, 1 H), 8.13(d,2H), 8.05(d, 2 H), 7.97(d,3 H), 7.31(t,
3 H), 6.28 (s, 1 H), 5.48(s, 3 H), 3.24(s, 2 H), 2.51(s, 2 H), 1.80(t, 12 H),
1.31(m, 6 H). 13C-NMR (101 MHz, DMSO-D6, ppm): ␦ = 170.90(a),
Synthesis of HI-4COOH
5-carboxy-1-(4-carboxybenzyl)-2,3,3-trimethyl-3H-indol-
1-ium bromide (1.32 g, 3.9 mmol), 2-chloro-3-(hydroxy-
methylidene)cyclohexene-1-carbaldehyde (0.34 g, 1.9 mmol)
and sodium acetate (1.65 g, 19.8 mmol) were mixed in 260 mL

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Z. Jiang, B. Yuan, Y. Wang et al. / Nano Today 34 (2020) 100910
Fig. 5. H&E staining of vital organs after tail vein injection of PBS, only light, Hf-HI-4COOH and Hf-HI-4COOH with light for 14 days. (Scale Bar = 100 ␮m).
168.30(k, ag, ap), 156.31(p), 152.34(aa), 149.79(aj), 148.69(g),
145.77(m), 144.54(ad), 141.65(j), 141.32(af), 138.58(b), 137.94(ao),
132.23(s), 131.05(e), 130.77(al), 130.35(i, l), 130.10(c), 130.00(d),
129.37(w), 128.88(h), 127.17(an), 127.10(am), 126.95(ae, ah),
126.53(aj), 123.32(r), 120.59(y), 106.69(q), 106.36(z), 100.36(q,
z), 95.88(x), 60.25(f), 55.54(n), 49.38(ab), 48.67(ak), 28.18(ac),
28.02(o), 21.28(t), 20.46(v), 14.54(u). m/z: for [C48H42ClN2O8]-,
calcd. 811.2625; found 811.2276. The purity of HI-4COOH was
tested by HPLC (Agilent 1260) (MeOH: water containing 0.1 %
H3PO4 = 75 : 25, flow rate =1 mL/min, temperature: 30 ◦C, C18
UV absorption of Hf-HI-4COOH NCPs was determined by UV–vis
detection (Lambda 950, Perkin Elmer, U.S.).
Intracellular localization and cellular uptake of NCPs in the cells
For the investigation of elements uptake in 4T1 cells, X-ray flu-
orescence microscopy at Shanghai Synchrotron Radiation Facility
(SSRF, Shanghai, China) was used as our group pervious reported
[28]. In general, the cells were grown on sterile Malay films for 24 h,
and then were cultured with Hf-HI-4COOH or HfCl4 (Hf: 5 ␮g/mL)
for another 4 h. After fixing by tissue fixative and washed with pure
water, the cells were tested. The hard X-rays BL15U beamline at SSR
was also used to get distribution mapping of elements (Cl, Zn and
Hf) at the condition of energy of the X-ray was 10 keV with the
beam spot was 0.5 × 0.5 ␮m/(step*s).
column) (97.0562 %). ␧(808 m, in MeOH) = 1.342 × 105 M−1 cm−1
.
(Fig. S5-S8)
Synthesis and characterization of the Hf-HI-4COOH NCPs
30 mg HI-4COOH and 10 mg HfCl4 was added to a 50 mL reactor.
Another 10 mL DMF/MeOH (9/1, v/v) and different volume glacial
acetic acid (Table S1) was added to get a solution. The reaction mix-
ture was kept in 90 ◦C for 24 h. The resulting powder was washed
with copious amounts of DMF, 1 % triethylamine in ethanol (v/v),
and ethanol successively. For sample 2 as an example, 11.7 mg
product was obtained with a yield of 68.2 % calculated with the
concentration of Hf. Sample 2 was used for the rest experiment
and named as Hf-HI-4COOH.
Particle size and zeta potential of Hf-HI-4COOH NCPs was mea-
sured by a particle size-zeta potential analyzer (Nano-ZS, Malvern,
England). High resolution transmission electron microscopy
(HRTEM) images were recorded by a Talos (Thermofisher, USA).
The structural properties of samples were investigated by X-ray
diffraction (XRD) using an X-ray powder diffractometer (XRD, D8
Discover, Bruker AXS). The weight of element was tested by ICP-OES
(Optima 2100, Perkin-Elmer, USA) for Hf, organic element analyzer
(Elementar, Germany) for N, C and H. In order to determine ther-
mal stability, thermogravimetric and derivative thermogravimetric
analysis (TG-DTG) (Pyris Diamond, PerkinElmer, U.S.) were used.
TG analysis was performed with an increase in temperature from
room temperature to 800 ◦C, and the heating rate was 10 ◦C/min.
For LSCM analysis, confocal culture dishes (NETS Co. U.S) were
used for the experiment. 4T1 cells on complete DMEM medium
were seeded into each dish at at 1 × 105 cells and allowed to adhere
for 24 h under the condition of 5% CO2 at 37 ◦C. The medium was
then removed and treated with fresh medium containing Hf-HI-
4COOH or HI-4COOH solution (HI-4COOH: 10 ␮g/mL). After further
4 h incubation, the cells were washed three times with PBS to
remove any absorbed free nanoparticles. Afterwards, the cells were
fixed with 4 % formaldehyde for 30 min, treated with 0.1 % triton
for 5 min, and then treated with 1.0 % BSA for 30 min and treated
with Phalloidin with Fluorescein Isothiocyanate Labeled (5 ␮g/mL)
and DAPI (10 ␮g/mL) for 30 min at room temperature.
The 4T1 cells or MCF-10A cells were allowed to adhere for 24 h
and then treated by Hf-HI-4COOH (Hf: 5 ␮g/mL) with for 1, 2, 4, 12 h.
After removing the culture medium and washing with PBS for three
times, the cells were trypsinized and collected. In order to quantify
the nuclear uptake, cell nucleus was separated from cell cytosol by
nucleus extraction. The extraction solution consisted 1 mM EDTA,
1 % Triton X-100, 100 mM NaCl, and 10 mM Tris buffer (pH 7.4). The
collected cells were treated with the nuclei solution at 4 ◦C. After 10
min, cell nuclei were collected by centrifugation. The nuclei were
treated by aqua regia overnight. The nuclear uptake was tested by

Z. Jiang, B. Yuan, Y. Wang et al. / Nano Today 34 (2020) 100910
9
ICP-OES. The cellular uptakes of were detected following the above-
mentioned procedure without nuclei extraction.
Y.W. and Z.W carried out the experiments and data acquisition. Z.J.,
S. S. and Y.L. lead in data curation and analysis. Z.J. and J.L. took the
lead in writing of original draft and designing the figures. O. U. A.
took the lead in revising the manuscript. J.L. and A.W. lead in fund-
ing acquisition and project administration. All authors provided
critical feedback and worked on the manuscript.
Establishment of 4T1 breast tumor xenograft
Under the approvement of the Regional Ethics Committee
for Animal Experiments at Ningbo University (Permit No. SYXK
(Zhe) 2019–0005). All Balb/C mice used in this study all bought
from Kawensi Biological products sales center (Nanjing, China). To
develop the tumor model, 4T1 cells (1 × 106) suspended in 100 ␮l
of serum-free 1640 medium were subcutaneously injected into the
back of each Balb/c mouse. The tumor size was calculated by the
long size for a, the short size for b, V = a*b2/2. When the tumor grew
to 40∼60 mm3, the tumor-bearing mice could be used for further
experiment.
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgements
This work was financially supported by National Key R&D Pro-
gram of China (2018YFC0910601), Natural Science Foundation of
China (Grant No. 81871411, 32011530115), Youth Innovation Pro-
motion Association Foundation of CAS (2017340), and Key R&D
Program of Zhejiang Province (2020C03110). We would also like to
thank Mr. Yong Yang at Ningbo University Hospital for hematolog-
ical analysis. Furthermore, the authors also thank the use of X-ray
fluorescence imaging in Shanghai Synchrotron Radiation Facility at
Line BL15U.
Pharmacokinetics and bio-distribution
To determine the pharmacokinetics of Hf-HI-4COOH, the mice
were intravenously (i.v.) injection with 100 ␮l Hf-HI-4COOH (HI-
4COOH: 20 mg/kg) through the tail vein. The blood was collected
from each mouse at indicated time points (0.17, 0.5, 1, 2, 4, 8, 12
and 24 h), weighed and then dissolved with digestive aqua regia to
analyze the total amount of Hf in the blood by using ICP-OES.
To evaluate the in vivo biodistribution of Hf, three tumor mice
were i.v. injection with 100 ␮l Hf-HI-4COOH (HI-4COOH: 20 mg/kg)
through the tail vein. After one day, all the mice were sacrificed,
and the major organs and tumors were collected, and then the dis-
tribution of Hf was determined by ICP-OES analysis. For ICP-OES
analysis, these organs were freeze dried and weighted. All organs
were treated in aqua regia for 4 h at 95 ◦C for dissolution of the
tissues.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at doi:https://doi.org/10.1016/j.nantod.2020.
100910.
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