Nano Today 56 (2024) 102279

Contents lists available at ScienceDirect

Nano Today
journal homepage: www.elsevier.com/locate/nanotoday

Clinically used lipiodol as an effective radioenhancer

Shuang Zhu a, b, c, 1, You Liao b, c, 1, Chenglu Gu b, c, Dongmei Wang b, c, Haili Yan d, Long Gao e, f,
Duiping Feng e, f, Zhanjun Gu a, b, c, *
a
CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China
b
CAS Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Beijing 100049, China
c
College of Materials Science and Optoelectronic Technology, University of Chinese Academy of Sciences, Beijing 100049, China
d
Shanxi Key Laboratory of Intelligent Imaging and Nanomedicine, First Hospital of Shanxi Medical University, Taiyuan 030001, China
e
Department of Oncological and Vascular Intervention, First Hospital of Shanxi Medical University, Taiyuan, Shanxi Province 030001, China
f
Shanxi Provincial Clinical Research Center for Interventional Medicine, First Hospital of Shanxi Medical University, Taiyuan, Shanxi Province 030001, China

A R T I C L E I N F O A B S T R A C T

Keywords: High atomic number (Z) element-based radiosensitizers have been widely reported for enhancing radio­
Lipiodol therapeutic effect, while its limited tumor distribution and long-term safety are major limitations in clinics. In
Radiotherapy this study, we propose the clinically used lipiodol, a liquid radiological imaging agent with high iodine con­
High Z element
centration, as a potential radiosensitizer. Compared to clinically approved high Z nanoradiosensitizer HfO2,
Liquid radiosensitizer
Clinical formulation
lipiodol emulsion shows much better tumor spatial distribution and tumor inhibiting ability at in vivo level upon
radiation treatment. In addition to primary tumor, lipiodol emulsion also exhibited efficient targetability and
inhibition of lymph node metastasis with radiation treatment. Furthermore, the in vitro mechanism study in­
dicates that lipiodol enhances radiotherapeutic effect by generating extra reactive oxygen species to cause DNA
damage and cell death, irrespective of cancer type. Lastly, toxicity assessments demonstrate the safety of lipiodol
for practical medical applications. This report provides scientific evidence supporting the century-old lipiodol as
an effective liquid radiosensitizer ready for clinical implementation.

Introduction either directly damage cellular DNA or indirectly interact with water to
produce toxic reactive oxygen species (ROS) to promote cell death [6].
External ionizing radiation therapy play a critical role in the man­ Once located in tumor cells, high Z element enables to deposit more
agement of patients with solid tumor. Ample evidence shows that almost radiation energy, leading to increased tumor cell death compared to
half of cancer patients receive at least one course of radiotherapy during radiation alone [7,8]. Recently, an hafnium oxide nanoparticle-based
their treatment history [1,2]. Curability however, is limited by tolerance radiosensitizer NBTXR3 (Nanobiotix SA, France) has been approved in
of surrounding normal tissues under high-dose radiation. Over the years, clinics for the treatment of soft-tissue sarcoma using intratumoral
strategies have been developed to overcome this limitation. Despite the administration [9]. Similarly, the radiosensitizing ability of a Gd-based
technical improvement achieved in radiotherapy such as hypofractio­ nanoparticle (AGuIX, NH TherAguix, France) in brain tumor treatment
nated therapy or ultra-high dose rate radiotherapy, radiosensitizers that was also evaluated in phase 1 clinical trial [3]. However, although the
can sensitize tumor cells preferentially to radiation so as to realize dose clinical results demonstrated therapeutic improvement of these high Z
de-escalation and maintain normal tissue tolerance are widely studied nanoparticle with radiotherapy, unavoidable limitations of undegrad­
and applied as an effective method to reduce complication risk of ability and unknown long-term toxicity still limit their practical appli­
radiotherapy [3,4]. Nanoradiosensitizers based on high atomic Z cation [10]. A safe radiosensitizer with better understanding of its
element (such as hafnium, gold, iodine, etc.) are recognized and pharmacokinetic behavior and toxic profile is needed.
developed for boosting antitumor effect of radiotherapy [5]. During the Lipiodol, a World Health Organization listed essential medicine for
interaction with ionizing radiation, high Z element could release elec­ treating iodine deficiency [11], is a mixture of long-chain di-iodinated
trons (such as photoelectric, Compton and Auger electrons), which can ethyl esters of fatty acids of poppy seed, with a high iodine concentration

* Correspondence to: Institute of High Energy Physics, Beijing 100049, China.

E-mail address: [email protected] (Z. Gu).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.nantod.2024.102279
Received 8 January 2024; Received in revised form 25 March 2024; Accepted 16 April 2024
Available online 22 April 2024
1748-0132/© 2024 Elsevier Ltd. All rights reserved.
S. Zhu et al. Nano Today 56 (2024) 102279

up to 480 mg/mL. It was firstly synthesized by Macel Guerbet in 1901 conducted to determine cell viability.
and has vastly been used as in radiological imaging application since
1921 owing to its radiopaque properties, such as bronchography, hys­ Clonogenic assay
terosalpingography, lymphangiography and hepatography [12,13].
Therefore, lipiodol containing high-concentrated high Z element iodine Hepa 1–6 cells were planted in 6-well plates with 1 × 103 cell per
(Z=53) may also serve as an effective radiosensitizer. Some evidence well and incubated for 24 h. Next, cells were treated with 160 μg/mL
supports this hypothesis. For example, preclinical data show that iodine lipiodol-containing medium and exposed to Cu-filtered X-rays (160 kV,
could enhance radiotherapy by absorbing more X-ray and producing 15 mA) at 0, 1, 2 or 4 Gy after 6 h. Seven days later, the cells were fixed
more free radical at tumor site [14–17]. Furthermore, Monte Carlo with 4 % Polyformaldehyde (Biosharp Life Science) for 5 min and
stimulation of both physical and biological dose-enhancement analyses stained with Giemsa (Solarbio) working solution for 10 min. The clones
has revealed that the uptake of lipiodol showed an increased in-tumor were counted and the value of sensitization enhancement ratio (SER10)
dose by 10 % under X-ray irradiation, without increasing the dose to was calculated according to previous report [26]. Results were collected
normal tissue [18–20]. Additionally, as early as 1980 s, scientists found from three independent replicates.
that lipiodol could be selectively taken up and retained by neoplastic
tissue of the liver, while the deposits in hepatic parenchyma were nil on Free radical detection
the plain X-ray film [21–23]. This phenomenon of uptake and retention
(4 weeks or longer) was further utilized to targeted deliver cytotoxic DCFH-DA (10 mM) (Beyotime Biotechnology) in dimethyl sulfoxide
drugs by arterial injection of lipiodol in trans-arterial chemo­ (0.25 mL) and 0.01 M NaOH (1 mL) were mixed. After 30 min, the re­
embolization (TACE) of hepatocellular carcinoma (HCC) [24]. Lastly, action was terminated by PBS (5 mL) to obtain a working solution. Then,
unlike other heavy metal element-based radiosensitizers, iodine as a lipiodol (Hengrui Pharmaceutical Co., Ltd., 360 μg/mL) were added. X-
trace mineral is safe for human use. Over the century, its pharmacoki­ ray irradiation (6 Gy, 160 kV, 15 mA) were then performed by an irra­
netic and toxicity profile have been extensively characterized in both diator (MultiRad 160, Faxitron). At last, the fluorescence spectra were
pre-clinical and clinical application [25]. Such features can greatly detected by a fluorescence spectrophotometer (Fluorolog-3, Horiba).
reduce the escalating cost and timeline required for new drug develop­
ment in clinics, and thus accelerate the process of lipiodol repurposing Detection of intracellular ROS generation
from bench to bedside. Accordingly, evidence has indicated the suit­
ability of lipiodol as a safe radiosensitizer, but few systematic studies Hepa 1–6 cells were planted in confocal dishes at a density of 1 × 105
have assessed its radio-enhancing effect. cells per well. 24 h later, the culture medium was replaced with new
This report assessed the feasibility of lipiodol in radiotherapy complete medium with lipiodol (160 μg/mL, lipiodol was mixed in
enhancement using different tumor models. Firstly, we explored its high DMSO, Vlipiodol:VDMSO = 1:2). Control and X-ray group was treated with
Z-based therapeutic effect upon ionizing radiation at both in vitro and in same amount of DMSO only. After co-incubation for 6 h, the cells were
vivo levels. Comparing different clinically used formulation of lipiodol, carefully rinsed twice with PBS and incubated with 1 mL serum-free
we observed that lipiodol emulsion exhibits a strong impact on tumor medium containing DCFH-DA (Beyotime Biotechnology, dilution
inhibition, better than clinically used high Z radiosensitizer HfO2. This 1:1000) and Hoechst 33342 (Dojindo, dilution 1:100) at 37℃ for
may ascribe to the super-high effective high Z element concentration 20 min. Then the cells were rinsed three times with fresh serum-free
and evenly-distribution ability of lipiodol emulsion in solid tumors. It culture medium to remove DCFH-DA and then kept on ice without
also demonstrated efficacy in various types of tumors, indicating its light to get Cu-filtered X-ray irradiation (4 Gy, 160 kV, 15 mA). Finally,
universal radio-enhancing application. Furthermore, in addition to pri­ the fluorescence images of intracellular DCF were obtained from a
mary tumor, lipiodol emulsion also exhibited efficient targetability and confocal microscope (A1/LSM-Kit, Nikon, Japan/Germany).
inhibition of lymph node metastasis in animals with radiation treatment.
This discovery holds promise for controlling tumor metastasis via lymph DNA damage assay
nodes and potentially offers an alternative radiotherapy-based strategy
to lymphadenectomy. Lastly, comprehensive toxicity assessments also Hepa 1–6 cells were seeded in a 24-well plate with L-Polylysine (L-
indicate the safety of lipiodol emulsion for practical use. Therefore, this PLL) coated cover glass (2 × 104 cells per well). After 24 h, lipiodol with
report provides scientific evidence for the use of lipiodol as a readily concentration of 160 μg/mL was added and incubated for 6 h. Then, X-
available radiosensitizer. It may open a large field of clinical applica­ ray groups were exposed to Cu-filtered X-ray irradiation (4 Gy, 15 mA,
tions in solid cancers when radiotherapy is indicated. 160 kV). After incubation for another 1 h, the cells were fixed with 4 %
paraformaldehyde for 10 min, followed by 0.5 % Triton-X100 for
Methods 10 min, and then 5 % FBS in PBS for 1 h. Next, cells were incubated in
phospho-histone H2A.X (Ser139) rabbit mAb (1:800) at 4℃ for 12 h,
Cell culture followed by goat anti-rabbit IgG (H+L) (Alexa Fluor 488, 1:500) at 37◦ C
for 1 h. Finally, Hoechst 33342 (1:200) was added and incubated for
All melanoma cell lines were grown in dulbecco’s modified eagle 10 min. Finally, the cells were observed under a laser confocal micro­
medium (DMEM) (Gibco Life Technologies) supplemented with 10 % scope (A1/LSM-Kit, Nikon, Japan/Germany) after sealing.
FBS (Biological Industries Israel Beit Haemek Ltd.) and 1 % pen­
icillin–streptomycin 100X (Gibco Life Technologies). All cells were Apoptosis/necrosis assay
grown at 37 ◦ C in a humidified atmosphere with 5 % CO2. For all the
following cellular experiments, lipiodol was firstly mixed in DMSO at a Apoptosis level is studied using Annexin V-FITC apoptosis detection
volume ratio of 1:2 before use unless indicated otherwise. kit (Beyotime). The experiment was conducted according to manufac­
turer’s structure. Hepa 1–6 cells were seeded in a confocal well (5 × 105
In vitro biocompatibility assay cells per well). After 24 h, lipiodol with concentration of 120 μg/mL was
added and incubated for 6 h. Then, X-ray groups were exposed to Cu-
Hepa 1–6 cells were seeded in a 96-well plate (6 × 103 cell/well). filtered X-ray irradiation (4 Gy, 15 mA, 160 kV). After incubating for
After 24 h, the medium was replaced with different concentration of another 36 h, cells were mixed with Annexin V-FITC and PI dye. Finally,
lipiodol (0, 80, 160, 320, and 640 μg/mL, n = 6) containing medium. the cells were observed under a laser confocal microscope (A1/LSM-Kit,
Subsequently, the standard MTT (Beyotime Biotechnology) assay was Nikon, Japan/Germany).

2
S. Zhu et al. Nano Today 56 (2024) 102279

Radiation-induced cytotoxicity assay Tumor retention by inductive coupling plasma mass spectrometer

Human-derived melanoma cell lines (including Hela, PANC-1, A549, The quantitative analysis of the content of iodine element in tumor
HCT116, or U87) and mouse-derived B16 cells were seeded in a 96-well were tested by inductive coupling plasma mass spectrometer (ICP-MS,
plate (2–4 ×103 cells per well). After 24 h, the medium was replaced Thermo-X7, Thermo.Co., USA). The 4T1 tumor-bearing mice were
with a medium containing lipiodol (30 μg/mL) for 12 h. Then, the cells intratumorally injected with lipiodol and IG41 (10 μL). The mice were
were treated by Cu-filtered X-ray (2 or 4 Gy). After 72 h, cells were sacrificed, and tumor were collected on day 0 (2 h after injection) and
incubated in 10 % CCK-8 for 1.5–2 h, and the absorbance of the su­ day 16. Before being disposed with nitric acid, the collected samples
pernatant at 450 nm was measured with a microplate reader (n ≥ 5, were stored at − 20 ◦ C. For digestion and detection, the tumors were
Thermo Scientific, Multiskan MK3). dissolved in nitric acid overnight. Then hydrogen peroxide (30 %) was
added dropwise to the samples and heated up to 120 ◦ C until 0.5–1 mL
solution was left in each sample. After cooling down and filtration of the
Lipiodol emulsion preparation obtained solutions, the supernatants were diluted with 2 % HNO3 for
ICP-MS test to measure the content of the I element.
The emulsion was prepared using the 3-way stopcock method with
polycarbonate syringes. The aqueous phase (iopromide (Bayer Phar­ Lymph node metastasis model and therapy
maceutical Co., Ltd) or 5 % glucose) was first pushed towards the sy­
ringe containing Lipiodol in order to favor a water-in-oil emulsion. At 4T1/Luc cancer cell metastasis in lymph nodes was established by
least 30 vigorous pumping exchanges through the stopcock are per­ injecting 4T1/Luc cancer cells into the paw of BALB/c mice. On 6th day
formed before use. after tumor inoculation, lymph node metastasis was monitored through
an IVIS imaging system after intraperitoneal injection of potassium
luciferin. On 9th day, mice were randomly divided into five groups (n ≥
HfO2 synthesis and characterization 5): (i) 5 % Glucose, (ii) 5 % Glucose + X-ray, (iii) IO (lipiodol) + X-ray,
(iv) IW41 (lipiodol:iopromide = 4:1) + X-ray, (v) IG41 (lipiodol:5 %
HfO2 nanoparticle was synthesized according to the published pro­ Glucose = 4:1). Different solutions (25 μL) were injected intratumorally
tocol by NANOBIOTIX [27]. Briefly, A tetramethylammonium hydrox­ on paws. 2 h later, tumor and sentinel lymph node were treated with Al-
ide solution (Alfa) is added to HfCI4 solution (ThermoScientific), until filtered X-ray irradiation (6 Gy, 160 kV, 15 mA). Body weight, paw
the pH of the final suspension reaches a pH around 11.3. White pre­ tumor volumes and lymph node size were monitored at least twice a
cipitate is obtained after centrifuge and then transferred in an autoclave week. At the end of the experiment, lymph node was obtained for size
and heated at a temperature of 160◦ C to perform crystallization. After measurement and H&E analysis. The lymph node volume (V) was
cooling, the suspension is washed with de-ionized water and sodium measured with a vernier caliper and calculated as V = (4/3) ⋅ π ⋅ r3.
hexametaphosphate (Aladdin) was added to increase nanoparticle
biocompatibility. For characterization, the morphology and chemical CT and μCT imaging
composition were studied by transmission electron microscope analysis
(JEM-2100 plus, JEOL) and X-ray diffraction analysis (Rigaku D/MAX For ex vivo CT imaging, different solutions (including water, HfO2,
2500). lipiodol, IG41 or IG21) with the same volume were imaged in 1.5-mL
centrifuge tube by the CT instrument (Triumph X-SPECT/X-O CT,
USA). For in vivo CT imaging, 4T1 tumor bearing mice were intra­
In vivo tumor therapy tumorally injected with different solutions including HfO2 (10 μL), lip­
iodol (10 μL) or IG41 (10 μL) and CT imaging was performed
Hepa 1–6 tumor-xenografted mice were obtained by inoculating respectively one hour and seven days later. Lymph node CT imaging was
subcutaneously on the left abdomen of C57 mice. When tumor volume performed one hour and seven days after lipiodol/IG41/IW41 (25 μL)
reached 50–100 mm3, mice were randomly divided into seven groups (n treatment. For tumor distribution study based on μCT, 4T1 tumor were
≥ 8): (i) 5 % Glucose, (ii) 5 % Glucose + X-ray, (iii) IO (lipiodol) + X-ray, injected with different solutions including HfO2 (10 μL), lipiodol (10 μL)
(iv) HfO2 + X-ray, (v) IG21 (lipiodol:5 % Glucose = 2:1) + X-ray, (vi) or IG41 (10 μL). Afterwards, tumors were isolated and imaged using a
IG41 (lipiodol:5 % Glucose = 4:1) + X-ray, and (vii) IW41 (lipiodol: synchrotron-based μCT device (Beijing Synchrotron Radiation Facility).
iopromide = 4:1) + X-ray. Briefly, 50 μL of different solutions (5 %
Glucose or lipiodol or HfO2 or lipiodol emulsion) were administrated via Biosafety study
intratumoral injection. The mice (only tumor area) were then exposed to
Al-filtered X-ray irradiation (2 Gy * 3 times, 160 kV, 15 mA) for To study the in vivo biosafety of lipiodol emulsion, healthy BALB/c
continuous 3 times (once in two days). mice were used. The mice were injected with 100 μL of IG41 (lip­
For 4T1 model, BALB/c mice were used. When tumor volume iodol:5 % Glucose = 4:1) by subcutaneous injection (n = 4). The body
reached 40–60 mm3, mice were randomly divided into six groups (n = weight was recorded every two days, and the behaviors were also
7): (i) 5 % Glucose, (ii) 5 % Glucose + X-ray, (iii) IO (lipiodol) + X-ray, observed. Mice were sacrificed on day 1, 3, 7 and 15. Whole blood was
(iv) IG21 (lipiodol:5 % Glucose = 2:1) + X-ray, (v) IG41 (lipiodol:5 % collected for the subsequent analysis of blood routine and blood
Glucose = 4:1) + X-ray, and (vi) IW41 (lipiodol:iopromide = 4:1) + X- biochemical indexes. Major organs including heat, liver, spleen, lung
ray. Same to Hepa 1–6 model, 50 μL of different solutions were and kidney were obtained and fixed in 4 % paraformaldehyde for tissue
administrated via intratumoral injection and tumor area were exposed section analysis.
to Al-filtered X-ray irradiation (2 Gy × 3 times).
For tumor therapy experiment, body weight and tumor volumes Animals feeding
were monitored every other day. The tumor volume (V) was measured
with a vernier caliper and calculated as V = (Width2 × Length)/2. One The animal models are 6 ~ 8-week (18 ~ 22 g) C57 (Hepa 1–6
or two mice from each group were randomly collected and fixed in 4 % tumor) or BALB/c (4T1 tumor) female mice that were obtained from
paraformaldehyde for tissue section analysis two days after the X-ray Beijing HFK Biotechnology Co., Ltd. During the whole experiments, the
treatment. Tumor volume was tracked until euthanasia (tumor volume mice were fed into a polypropylene cage. The feeding conditions
reached 1500 mm3) or 80 days after tumor inoculation. included temperature at 25 ± 2 ◦ C 50 ± 5 % air humidity, a normal light

3
S. Zhu et al. Nano Today 56 (2024) 102279

cycle (12 h light/dark cycle), standard pelleted food, and tap water. due to the retrospective study design.

Ethical regulations Statistical analysis

The research performed in the present study complies with all ethical Data were expressed as means ± standard deviation. Comparison
regulations. All animal experimental procedures were approved by the between groups was analyzed by one-way analysis of variance (ANOVA,
animal ethics committee of the Institute of High Energy Physics Animal GraphPad Prism 9) with Turkey’s post hoc test. Student’s t-test was used
protocol number: IHEPLLSC-033. When required, tumor size did not when comparing between two groups. Significance is indicated by as­
exceed the maximum volume (1,500 mm3) allowed by the Ethical terisks: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Committee. The retrospective clinical study was conducted at First
Hospital of Shanxi Medical University between 2019 and 2022, which
complied with the ethical guidelines of the 1975 Declaration of Helsinki
and was approved by the ethics committee of First Hospital of Shanxi
Medical University. The requirement for informed consent was waived

Fig. 1. Radiosensitization of lipiodol at in vitro level. (a) X-Ray mass attenuation coefficients of iodine and human normal organ/tissues including bone, soft tissue
and adipose tissue (data received from NIST). Clonogenic formation (b) and survival curves (c) that were pretreated with or without lipiodol for 6 h followed by
exposure to radiation at doses from 0 to 4 Gy. The survival data were normalized to those of unirradiated control cells (n = 3). (d) DCF fluorescence images of Hepa
1–6 cells after different treatments. (e) Fluorescence images of γ-H2AX foci in Hepa 1–6 cells after different treatments. (f) Total radical oxygen species measurement
by DCFH-DA probes with different treatments. (g) Cell viability measurements in different tumor cell lines treated with or without lipiodol followed by exposure to
radiation. Error bars are means ± standard deviation (SD). Statistical significance between two groups were studied using two-tailed unpaired Student’s t-test (* p <
0.05, ** p < 0.01, *** p < 0.001).

4
S. Zhu et al. Nano Today 56 (2024) 102279

Results ROS, we determined whether lipiodol could enhance ROS production

under X-ray radiation. As expected, lipiodol increased global ROS [28]
Lipiodol sensitizes radiation effect in cancer cells owing to its enhanced radiolysis of water (Fig. 1f). Such ability was
further examined at intracellular level. Lipiodol + X-ray groups
The X-ray attenuation coefficient of iodine is presented in Fig. 1a, demonstrated a much stronger green fluorescence signal than X-ray
demonstrating a much higher attenuation ability compared to human alone group, where higher-concentrated lipiodol lead to more ROS
bone and normal tissue. This finding highlights the potential of lipiodol production than lower concentrated-group (Fig. 1d and S2). Moreover, a
for radiosensitization. To assess the radiosensitizing ability, we first major effect induced by radiotherapy is associated with DNA damage.
conducted in vitro clonogenic survival assays of lipiodol. Since lipiodol is We thus examined DNA damage using γ-H2AX staining, a marker of
clinically applied for treating HCC in TACE, an HCC-derived cell line double-stranded breaks. Consistent with ROS assay, the fluorescence
Hepa 1–6 was chosen as a primary cell model for our study (biocom­ intensity of γ-H2AX in the nuclei of Hepa 1–6 cells was significantly
patibility of lipiodol on Hepa 1–6 cell was verified in Figure S1). As increased in the lipiodol + X-ray treated groups compared with the X-ray
shown in Fig. 1b and c, lipiodol exhibited significantly enhanced radi­ alone group in a concentration-dependent manner (Fig. 1e and
osensitization on Hepa 1–6 cells in a radiation dose-dependent manner Figure S3). It is reported that apoptosis and necrosis of cancer cells
with a sensitizer enhancement rate (SER) of 1.23 (a SER of > 1 denotes induced by DNA damage are the major effects of radiation on tumor
synergistic radiosensitization). Subsequently, given that the radio­ tissue and are beneficial effects of radiation for cancer therapy [29]. We
sensitizing mechanism of high Z element is linked to the production of thus studied the cell death mechanism induced by lipiodol. As shown in

Fig. 2. Radiosensitization of lipiodol formulations at in vivo level. Average tumor volumes (a) and survival rate curve (b) of Hepa 1–6 xenograft tumors with the
indicated treatments at different time points (days). (c) Representative images of hematoxylin and eosin (H&E, scale bars 50 μm) and immunohistochemical staining
(Ki67, TUNEL, scale bars = 20 μm) of Hepa 1–6 xenograft tumors with the indicated treatments. (d) Average tumor volumes of 4T1 xenograft tumors with the
indicated treatments at different time points (days). Error bars are means ± SD. P values were determined using one-way ANOVA with post hoc Tukey test.

5
S. Zhu et al. Nano Today 56 (2024) 102279

Figure S4, both apoptotic and necrotic cells are observed in lipiodol plus behind the heightened radiosensitization of lipiodol emulsion. Firstly, X-
X-ray group, where apoptotic death plays a more dominant role in lip­ ray absorption ability of lipiodol, lipiodol emulsion and HfO2 solution
iodol radiosensitizer-mediated cell death. Lastly, we extended the was measured by CT. All lipiodol-based solutions exhibited stronger CT
radiosensitizing assessment of lipiodol to additional cancerous cell lines, signals than HfO2 solution (2.5-fold, Fig. 3a). This proved that lipiodol-
in which radiotherapy is commonly used, including A549 (lung carci­ based formulations possess much superior X-ray attenuation ability in
noma), Hela (cervical carcinoma), PANC1 (pancreatic cancer), HCT116 comparison to HfO2. Next, we examined the tumor distribution ability of
(colorectal carcinoma), U87 (glioblastoma) and B16 (melanoma) cells different radiosensitizers. Upon single intratumoral injection, HfO2 was
(Fig. 1g and Figure S5). The observations were consistent across all six predominantly observed in the margin area of tumor, leaving most of the
cell lines. The addition of lipiodol could decrease cell viability upon tumor volume unfilled (Fig. 3b). In comparison, both lipiodol and lip­
X-ray irradiation, especially at higher radiation doses. Our data strongly iodol emulsions were found to effectively cover nearly the entire tumor
suggest that iodine-based lipiodol can function as an effective radio­ area. To obtain more detailed information on spatial tumor distribution,
sensitizer and enhance the radiation effect in different cancer cells. we employed synchrotron radiation micro-CT. Consistent with the
findings in Fig. 3b, lipiodol emulsion exhibited a widespread distribu­
Lipiodol emulsion exhibits potent tumor inhibition effect in different tumor tion throughout the solid tumor (Fig. 3c), whereas only a small fraction
models of the tumor was exposed to HfO2 injection. Moreover, although lipiodol
exhibited better distribution than HfO2, it is not as homogenous and
Inspired by in vitro results, we sought to investigate the in vivo widespread as lipiodol emulsion. Lastly, tumor retention ability of
radiosensitization potential of lipiodol using different tumor models. lipiodol-based treatment was studied. As demonstrated in Fig. 3b, the CT
Initially, C57BL/6 mice harboring subcutaneous Hepa 1–6 tumors were performed one week after injection showed that lipiodol-based radio­
established. Clinically used lipiodol formulations including lipiodol and sensitizers were stably localized in the subcutaneous tumor on rodent
lipiodol emulsion were both studied [30]. Herein, to align with clinical animals. These findings were further confirmed using an ICP method.
guidelines of TACE for HCC treatement, it is suggested to use drug dil­ The quantitative analysis result of iodine in tumors 16 days after in­
uents such as 5 % dextrose and non-ionic contrast medium as aqueous jection verified that lipiodol and lipiodol emulsion have long-term
phase in lipiodol emulsion [31–33]. Therefore, we selected lipiodol-5 % retention at tumor site (Figure S13). Furthermore, in addition to intra­
glucose emulsion (IG) and lipiodol-iopromide emulsion (IW) in the ratio tumoral injection, data on lipiodol emulsion distribution and retention
of 2:1 and 4:1, respectively, as representative lipiodol emulsion in human HCC tumor via transarterial injection was also obtained. It is
(Figure S6) [34]. In addition, to better elucidate the efficacy of shown that lipiodol emulsion could be effectively taken up by tumor and
lipiodol-radiosensitizer, clinically approved high Z-based HfO2 radio­ exhibit long-retention ability (over one month) (Figure S14) Moreover,
sensitizer was also selected for comparison (Figure S7). The mice were lipiodol emulsion was found homogenously distributed throughout the
randomly assigned into seven groups: control, X-ray alone, lipiodol (IO) entire HCC tumor area, further confirming our previous μCT outcome in
+ X, HfO2 + X, IG21 + X, IG41+X and IW41 + X. As shown in Fig. 2a and the animal study.
S8, the tumor volume plot demonstrated a significant delay in tumor Lipiodol emulsion accumulated at lymph node and inhibited tumor
regrowth in all lipiodol emulsion + X-ray-treated groups compared to metastasis upon radiotherapy.
X-ray alone and HfO2 + X-ray (p ≤ 0.02 by Student’s t-test) group. An Lipiodol in clinics is widely used in lymphography. In addition, our
enhancement in tumor volume suppression was found in both IG41 and study demonstrated that lipiodol could be detected in lymph node
IW41 groups than IG21 group upon X-ray, indicating a following intratumoral injection (Figure S15). Therefore, apart from its
concentration-dependent therapeutic effect. These lipiodol emulsion local effect on tumors, we further assessed the radiotherapeutic effect of
treatments also resulted in a high survival rate among the mice (Fig. 2b, lipiodol emulsion on lymph node metastasis using a footpad lymphatic
over 50 %). Moreover, lipiodol itself also demonstrated limited thera­ metastasis model [35]. Tumor cells were injected into footpad and 6
peutic effect in terms of tumor growth inhibition and animal survival days later, noticeable tumor metastasis was observed in the popliteal
rate. Subsequently, we evaluated the therapeutic effects at tissue level (sentinel) lymph node (Figure S16). Treatment (including Ctrl, X-ray,
(Fig. 2c). As expected, the group receiving lipiodol emulsions + X-ray IO, IW41+X and IG41+X) was given after lymph node metastasis
irradiation caused the most severe cancer necrosis by Hematoxylin and occurred (Fig. 4a). As shown in Fig. 4b and S17, all lipiodol-based
Eosin (H&E) staining. By measuring Ki67 and terminal deoxynucleotidyl treatments resulted in significant lymph node size and weight
transferase dUTP nick end labeling (TUNEL) levels, tumor cell prolif­ decrease when compared with control (2.46-fold) and single X-ray
erative and apoptotic activity after different treatments were investi­ treatment (1.74-fold). This result indicated the enhanced radio­
gated, respectively. Although both HfO2 and lipiodol showed tumor therapeutic effect of lipiodol in repressing tumor growth at the sentinel
proliferation-inhibiting and apoptosis-inducing effect when compared lymph node. H&E results also confirmed the therapeutic effects of
to the X-ray group, these effects are less significant than those observed metastasis lymph node upon lipiodol-based treatments (Fig. 4c).
with lipiodol emulsions. (Figure S9). Furthermore, CT image demonstrated that all lipiodol-based solutions
In addition to the HCC model, we also assessed the radiotherapeutic could target and accumulate at lymph node for over a week upon a
effect of lipiodol formulations on a breast cancer model (4T1 cell) using single intratumoral injection (Fig. 4d). No noticeable changes in body
BALB/c mice. A similar result was obtained. All lipiodol emulsion-based weight were observed between the lipiodol-based treatment and
groups exhibited more significant tumor inhibition than X-ray group control/X-ray groups (Figure S18).
(Fig. 2d and S10a), along with a survival probability exceeding 40 % Lipiodol emulsion treatment is well tolerated with no appreciable
(Figure S10b). Moreover, higher-ratioed lipiodol emulsion demon­ toxicity.
strated better tumor suppression. H&E staining outcome were consistent Last, we evaluated the safety profile of lipiodol emulsion through
with Hepa 1–6 result (Figure S11). Mice body weight remains consistent blood examination and histopathological assessment of major organs.
among groups during the observation period in both Hepa 1–6 and 4T1 Healthy mice were received either the control (5 % glucose) or lipiodol
experiments (Figure S12). Taken together, the in vivo results verified the emulsion (IG41) treatment upon subcutaneous injection (for safety
efficient radiosensitizing ability of different lipiodol formulations, with study, injection dose is doubled than therapeutic purpose). Blood sam­
the clinically utilized lipiodol emulsion exhibiting a more potent tumor ples and weight data were obtained respectively at 1, 3, 7 and 15 days.
inhibition effect than HfO2 and lipiodol alone. As seen in Fig. 5a and b, there were no significant differences identified
Higher X-ray absorption and improved tumor distribution demon­ in blood biochemistry and blood routine tests that focused on liver,
strate enhanced therapeutic outcomes. kidney and hematological functions. Mice body weight also remain
The above results drove us to investigate the underlying mechanism stable during experimental period (Figure S19). In addition, no

6
S. Zhu et al. Nano Today 56 (2024) 102279

Fig. 3. CT imaging of different lipiodol formulations and HfO2. (a) The CT values in Hounsfield units (HU) for the indicated solutions. Statistical difference were
determined using one-way ANOVA with post hoc Tukey test. (b) In vivo CT images taken at different days after intratumoral injection of the indicated treatments
(arrow points at radiosensitizer’s location in tumor of each group). (c) Intratumoral distribution analysis by synchrotron radiation-based micro-CT of the indi­
cated treatments.

appreciable signs of inflammation, degeneration, or necrotizing lesions metastasis for radiotherapy; (iv) homogenous spatial distribution within
were observed in organs including heart, liver, spleen, lung and kidney solid tumors; and (v) prolonged tumor retention for fractionated
(Fig. 5c). These results collectively suggest that exposure to lipiodol radiotherapy. In addition to the therapeutic benefit, another major
emulsion did not induce any discernable tissue damage or abnormalities advantage of lipiodol-based radiosensitizer lies in its ease of clinical
following our treatment, suggesting its potential safety and tolerability translation. The lipiodol formulations assessed in this study are already
for future clinical use. employed in clinics, thereby the challenges during new drug develop­
ment such as scale-up manufacturing, safety assessment and regulatory
Discussion approval are greatly reduced. Instead of developing a completely new
medicine, which often faces a superlong journey from the laboratory to
This preclinical study aims to investigate the potential of lipiodol, a clinical implementation, our lipiodol-based radiosensitizer offers prac­
clinical radiological imaging agent containing rich high Z element tical and ready applicability for clinics.
iodine, as a liquid radioenhancer in tumor radiotherapy. We systemat­ The radiosensitizing ability of lipiodol was first examined on HCC
ically examined the key attributes necessary for an ideal sensitizer for tumor since lipiodol in cancer treatment is used in TACE of HCC.
clinical radiotherapy: (i) effective radiotherapeutic effect based on high Cellular results demonstrated that lipiodol significantly enhanced cell
Z element; (ii) universal radiosensitizer for a wide variety of solid tumor; death by producing extra ROS and causing DNA damage, while in vivo
(iii) ability to accumulate at both primary tumor and lymph node primary tumor outcomes also showed lipiodol emulsion effectively

7
S. Zhu et al. Nano Today 56 (2024) 102279

Fig. 4. Radiosensitization of lipiodol formulations on metastasis lymph node. (a) Schematic illustration of footpad lymphatic metastasis model and treatment
experiment (created with BioRender.com). (b) Metastatic lymph node size with the indicated treatments at the end of the experiment. (c) Representative histo­
pathological images of metastatic lymph nodes at the end of the experiment (Orange dashed lines represent the region of infiltrating tumor cells, T). Scale bar 100
μm. (d) In vivo CT images of metastasis lymph node taken at different days after intratumoral injection of the indicated treatments (arrow points at lymph node
metastasis imaged by lipiodol formulations).

suppressed tumor growth under X-ray radiation. This indicates that routes of administration have been verified to be safe, including oral
potential of lipiodol as a radiosensitizer for enhancing tumor radio­ administration, hypeodermic injection in muscle or subcutaneous tissue,
therapy. In addition to primary tumor, we assume that lipiodol also have lymphatic vessel injection, intra-arterial injection or direct injection into
radiotherapeutic potential in lymph node metastasis, given the innate certain tumours [45–49]. Our study further confirms the safety of sub­
ability of lipiodol in lymphography [13] and its selective accumulation cutaneous injection of lipiodol emulsion. However, direct intravenous
in metastatic lymph nodes [36]. Lymph node metastasis a significant injection or use in the presence of arteriovenous fistula should be
challenge in clinical cancer treatment, as many types of cancer spread to avoided. Thus, in practical applications of lipiodol in radiotherapy,
distant organs via regional lymphatics [37]. Thus locoregional lymph interventional techniques that allow safe administration under radio­
nodes are the commonest and earliest site of solid tumor metastasis [38]. logic guidance, preventing inadvertent venous administration or intra­
In treating lymph node metastasis, radiotherapy as a non-invasive vasation, are recommended.
treatment is attracting more attention over lymphadenectomy with As is widely accepted, the degree of high Z elements-mediated ra­
promising results [39–41]. However, the toxicity caused by high-dose diation sensitization largely depends on the quantity and spatial distri­
radiation to healthy tissue is still challenging in clinics [42]. Our study bution of these elements within the tumor [50]. From these points of
showed that lipiodol-based treatments could greatly sensitize radiation view, lipiodol-based formulations are practical radiosenstizer. In terms
therapy on lymph node metastasis than single X-ray treatment. Such of functioning high Z element amount, lipiodol solutions contain
radioenhancing ability of lipiodol-based formulations in both primary extremely high iodine concentration (at least 320 mg/mL in IG21),
and lymph node tumor could make it possible to maintain radiotherapy while the concentration of particulate-based radiosensitizer HfO2
efficacy while reducing dose per fraction, so as to limit the side effect product can only reach to 53.3 mg/mL [51]. It also exhibited a much
and improve patients’ quality of life. Moreover, the utilization of radi­ higher CT value than HfO2 nanoradiosensitizer. Such a high working
opaque lipiodol as a radiosensitizer enables target delineation and mo­ concentration in lipiodol emulsion is not possible to be realized by any
tion assessment during radiotherapy. These are crucial in the era of dose other clinically used high Z radiosensitizers (such as NBTXR3 or AGuIX)
escalation and stereotactic treatment of both primary tumor and lymph due to technical limitation. In addition, lipiodol emulsion also exhibited
node metastasis [43]. much more homogenous spatial distribution and tumor uptake than
Although lipiodol is only currently used in clinics for HCC, given that HfO2. This is due to the liquid nature of lipiodol solutions, which has
the high Z-based radiosensitizer is not custom-tailored to hepatic cancer, better tumor uptake and distribution ability than solid particles. Herein,
we envision lipiodol being a class solution for a wide range of tumors is lipiodol emulsion is also found to have better distribution than lipiodol
independent of histology or genetic makeup. Consequently, in addition itself in solid tumor. This may be the reason for the improved tumor
to HCC cell line, the lipiodol-activated radio-enhancement was also inhibition ability of lipiodol emulsion than single lipiodol in primary
demonstrated in other cancer cells originating different organ/tissues. solid tumor. This is consistent with previous report where lipiodol
This indicates that lipiodol may function as a universal radiosensitizer emulsion showed improved tumor uptake and retention ability than
for any type of tumors. They can be activated by ionising radiation to fit lipiodol itself [52]. As tumor environment is based on water, lipiodol as
the modern standards of radiotherapy technology, without requiring pure oil tend to accumulate once injected into tumor area, which may
changes in treatment protocol. Note that in treating different tumors greatly decrease its cellular uptake. While using three-way stopcock
located at different organs, the administration route of lipiodol and method to prepare lipiodol emulsion, lipiodol could be broken into
lipiodol emulsion may need take into consideration given its possible small-sized droplets by shear force, making it easier to diffuse in tumor.
pulmonary embolism [44]. Ever since the discovery of lipiodol, several Thus the distribution and tumor uptake ability of lipiodol is not as good

8
S. Zhu et al. Nano Today 56 (2024) 102279

Fig. 5. Toxicologic assessment of lipiodol emulsion in vivo. Hematological parameters (a) blood biochemistry (b) and representative histopathological images of
heart, liver, spleen, lung and kidney sections (c) at day 1, 3, 7 and 15 after administration of glucose or lipiodol emulsion (IG41) to mice (Scale bar 50 μm). C: control;
I: IG41 (lipiodol:5 % glucose=4:1). C1/I1 stands for day 1, C3/I3 stands for day 3, and so on.

as lipiodol emulsion. These results underscore the crucial role that examples highlighting the effective radiosensitizing effects of lipiodol-
tumor spatial distribution in achieving comprehensive tumor treatment based treatments in both primary tumor and lymph node metastasis
in clinical settings. following a single intratumoral application. Lipiodol has been used in
Last but not least, another merit of lipiodol-based radiosensitizers is clinics for over a century. It is now an opportune to capitalize on the
the long tumor retention ability upon single intratumoral injection. In ‘neglected’ knowledge of its intrinsic high X-ray absorption property
our study, all lipiodol-based formulations could remain within subcu­ beyond its conventional use as radiological contrast agents. The ability
taneous tumor for at least 15 days and popliteal lymph node over seven to efficiently absorb X-ray, coupled with homogenous and selective
days in rodent animals (animal experiment period is limited considering retention within tumors over an extended time, renders it highly suit­
animal ethics [53]) and one months in human. This prolonged retention able for practical radiotherapy. The imaging and interventional methods
is utmost importance in clinical applications, considering that frac­ developed for precise tumor treatment are the booster for the future
tionated radiotherapy usually last for weeks [54]. Long tumor retention development of this field. Admittedly that the extent to which the
could ensure the radioenhancing effect throughout the whole treatment enhanced nonclinical antitumor effect described herein with lipiodol
period. The current study also verified that lipiodol emulsion enhanced treatments will translate clinically to more effective radiotherapy regi­
the response of hepatic and breast tumors when radiation is delivered at mens or to decreased radiation dose thresholds that are sparing of
2 Gy per fraction (3 times) during 6 days of treatment period. In addi­ normal tissue will only be borne out by human clinical trials. However,
tion, single administration is also a clinically desirable approach over the phenomena and method of action described in this study are sup­
repeated procedures. It helps to reduce the risk of infection, variations in portive of such clinical trials. Starting from HCC, our report potentially
administration techniques and high healthcare burden [55]. Such ability paves the way for a broad range of clinical applications in the treatment
to enable long-term bioavailability in tumor upon one-time adminis­ of various cancer that necessitate radiotherapy. Leveraging its inherent
tration ease the clinical treatment procedure for sustained therapeutic drug delivery ability, lipiodol holds promise as a practical option in the
outcome. combination with other treatments, such as chemotherapy or immuno­
Taken together, our study provides compelling evidence and therapy, in the realm of clinical cancer treatment.

9
S. Zhu et al. Nano Today 56 (2024) 102279

CRediT authorship contribution statement [13] C.C. Pieper, S. Hur, C.M. Sommer, G. Nadolski, G. Maleux, J. Kim, M. Itkin, Invest.
Radiol. 54 (2019) 600.
[14] K.S. Iwamoto, S.T. Cochran, J. Winter, E. Holburt, R.T. Higashida, A. Norman,
Shuang Zhu: Writing – original draft, Methodology, Investigation, Radiother. Oncol. 8 (1987) 161.
Formal analysis, Conceptualization. You Liao: Writing – review & [15] J.F. Hainfeld, S.M. Ridwan, Y. Stanishevskiy, H.M. Smilowitz, Pharmaceutics 14
editing, Investigation, Formal analysis. Chenglu Gu: Writing – review & (2022) 508.
[16] M. Tamura, H. Ito, H. Matsui, Sci. Rep. 7 (2017) 43667.
editing, Investigation. Dongmei Wang: Writing – review & editing, [17] L. Mullenger, B.B. Singh, M.G. Ormerod, C.J. Dean, Nature 216 (1967) 372.
Investigation. Haili Yan: Writing – review & editing, Investigation. [18] D. Kawahara, S. Ozawa, H. Nakano, K. Kubo, T. Shiinoki, T. Kimura, Y. Nagata,
Long Gao: Writing – review & editing, Project administration. Duiping Rep. Pract. Oncol. Radiother. 24 (2019) 681.
[19] D. Kawahara, S. Ozawa, A. Saito, T. Nishio, T. Kimura, T. Suzuki, K. Hioki,
Feng: Writing – review & editing, Resources. Zhanjun Gu: Writing – T. Nakashima, Y. Ohno, Y. Murakami, Y. Nagata, Med. Phys. 44 (2017) 342.
review & editing, Supervision, Project administration, Funding acqui­ [20] A.V. Mesa, A. Norman, T.D. Solberg, J.J. Demarco, J.B. Smathers, Phys. Med. Biol.
sition, Conceptualization. 44 (1999) 1955.
[21] J.L. Raoul, P. Bourguet, J.F. Bretagne, R. Duvauferrier, S. Coornaert, P. Darnault,
A. Ramée, J.Y. Herry, J. Gastard, Radiology 168 (1988) 541.
Declaration of Competing Interest [22] R.A.M. Al-Mufti, R.B. Pedley, D. Marshall, R.H.J. Begent, A. Hilson, M.C. Winslet,
K.E.F. Hobbs, Br. J. Cancer 79 (1999) 1665.
[23] T. Kanematsu, K. Inokuchi, K. Sugimachi, T. Furuta, T. Sonoda, S. Tamura,
The authors declare that they have no known competing financial K. Hasuo, J. Surg. Oncol. 25 (1984) 218.
interests or personal relationships that could have appeared to influence [24] T. Konno, H. Maeda, I. Yokoyama, K. Iwai, K. Ogata, S. Tashiro, K. Uemura,
M. Mochinaga, E. Watanabe, K. Nakakuma, T. Morinaga, Y. Miyauchi, Gan Kagaku
the work reported in this paper. Ryoho 9 (1982) 2005.
[25] Lipiodol. 〈https://www.drugs.com/pro/lipiodol.html#:~:text=Use%20in%20Sp
Data availability ecific%20Populations〉 (accessed 16th, Jan).
[26] N.A.P. Franken, H.M. Rodermond, J. Stap, J. Haveman, C. van Bree, Nat. Protoc. 1
(2006) 2315.
Data will be made available on request. [27] L. Levy, A. Pottier, A. Rouet, J. Marill, C. Devaux, NANOBIOTIX, WIPO (2008).
[28] H. Kim, X. Xue, J. Vis. Exp. (2020) e60682.
[29] R. Yahyapour, P. Amini, S. Rezapour, M. Cheki, A. Rezaeyan, B. Farhood,
Acknowledgments
D. Shabeeb, A.E. Musa, H. Fallah, M. Najafi, Mil. Med. Res. 5 (2018) 9.
[30] J.M. Idee, B. Guiu, Crit. Rev. Oncol. Hematol. 88 (2013) 530.
This work was supported by National Basic Research Program of [31] C.G.Co.C. Interventionalists, Natl. Med. J. China 60 (2021) 599.
[32] Y. Aoyagi, T. Yoshida, S. Uchino, M. Takinami, S. Uezono, J. Intensive Care 8
China (2020YFA0710702 and 2021YFA1201200), National Natural
(2020) 69.
Science Foundation of China (22375205), Strategic Priority Research [33] T. de Baere, Y. Arai, R. Lencioni, J.F. Geschwind, W. Rilling, R. Salem, O. Matsui,
Program of Chinese Academy of Sciences (XDB36000000), Directional M.C. Soulen, Cardiovasc. Interv. Radiol. 39 (2016) 334.
Institutionalized Scientific Research Platform relies on Beijing Syn­ [34] E. Ahnfelt, O. Degerstedt, E. Lilienberg, E. Sjögren, P. Hansson, H. Lennernäs,
J. Drug Deliv. Sci. Technol. 53 (2019) 101143.
chrotron Radiation Facility of Chinese Academy of Sciences, Beijing [35] I. Carr, Cancer Metastasis Rev. 2 (1983) 307.
Natural Science Foundation (2222087), Shanxi Provincial Clinical [36] N. Ohtsuka, T. Konno, Y. Miyauchi, H. Maeda, Cancer 59 (1987) 1560.
Research Center for Interventional Medicine (202204010501004), Key [37] H. du Bois, T.A. Heim, A.W. Lund, Sci. Immunol. 6 (2021) eabg3551.
[38] K. Kawada, M.M. Taketo, Cancer Res 71 (2011) 1214.
Research and Development Projects of Shanxi Province [39] T. Thomas, Nat. Rev. Urol. 20 (2023) 197.
(202102130501014) and the Natural Science Foundation of Shanxi [40] B.A. Jereczek-Fossa, S. Ronchi, R. Orecchia, Rep. Pract. Oncol. Radiother. 20
Province (202203021211021). We sincerely thank for the professional (2015) 472.
[41] E. Van Cutsem, A. Cervantes, R. Adam, A. Sobrero, J.H. Van Krieken, D. Aderka,
medical guidance from Dr. Long Gao and Dr. Duiping Feng in First E. Aranda Aguilar, A. Bardelli, A. Benson, G. Bodoky, F. Ciardiello, A. D’Hoore,
Hospital of Shanxi Medical University. E. Diaz-Rubio, J.Y. Douillard, M. Ducreux, A. Falcone, A. Grothey, T. Gruenberger,
K. Haustermans, V. Heinemann, P. Hoff, C.H. Köhne, R. Labianca, P. Laurent-Puig,
B. Ma, T. Maughan, K. Muro, N. Normanno, P. Österlund, W.J.G. Oyen,
Appendix A. Supporting information D. Papamichael, G. Pentheroudakis, P. Pfeiffer, T.J. Price, C. Punt, J. Ricke,
A. Roth, R. Salazar, W. Scheithauer, H.J. Schmoll, J. Tabernero, J. Taïeb, S. Tejpar,
Supplementary data associated with this article can be found in the H. Wasan, T. Yoshino, A. Zaanan, D. Arnold, Ann. Oncol. 27 (2016) 1386.
[42] H. Matsushita, K. Jingu, R. Umezawa, T. Yamamoto, Y. Ishikawa, N. Takahashi,
online version at doi:10.1016/j.nantod.2024.102279. Y. Katagiri, N. Kadoya, Technol. Cancer Res. Treat. 17 (2018),
1533033818803597.
References [43] M.S. Kim, B.Y. Kim, H.Y. Choi, Y.J. Choi, S.H. Oh, J.H. Kang, S.R. Lee, J.H. Kang, G.
T. Kim, Y.S. Choi, E.H. Hwang, Imaging Sci. Dent. 45 (2015) 31.
[44] Lipiodol Prescribing Information. 〈https://www.drugs.com/pro/lipiodol.html#:~:
[1] R.A. Chandra, F.K. Keane, F.E.M. Voncken, C.R. Thomas Jr., Lancet 398 (2021)
text=Pulmonary%20embolism%20may%20occur%20immediately%20or%20after
171.
%20a,pulmonary%20infarction%2C%20acute%20respiratory%20distress%20syn
[2] M. Abdel-Wahab, S.S. Gondhowiardjo, A.A. Rosa, Y. Lievens, N. El-Haj, J.A.
drome%20and%20fatalities〉. (accessed 10 March).
P. Rubio, G.B. Prajogi, H. Helgadottir, E. Zubizarreta, A. Meghzifene, V. Ashraf,
[45] B. Bonnemain, M. Guerbet, Rev. Hist. Pharm. (Paris) 42 (1995) 159.
S. Hahn, T. Williams, M. Gospodarowicz, JCO Glob. Oncol. (2021) 827.
[46] J. Wu, Y. Ni, P. Huang, X. Li, Q. Wang, J. Clin. Hepatol. 17 (2001) 51.
[3] C. Verry, S. Dufort, B. Lemasson, S. Grand, J. Pietras, I. Troprès, Y. Crémillieux,
[47] J. Guan, X. He, Y. Chen, Q. Zeng, Q. Mei, Y. Li, J. Vasc. Interv. Radiol. 22 (2011)
F. Lux, S. Mériaux, B. Larrat, J. Balosso, G. Le Duc, E.L. Barbier, O. Tillement, Sci.
1216.
Adv. 6 (2020) eaay5279.
[48] M.A. Miszczuk, J. Chapiro, J.H. Geschwind, V. Thakur, N. Nezami, F. Laage-Gaupp,
[4] S.L. Liauw, P.P. Connell, R.R. Weichselbaum, Sci. Transl. Med. 5 (2013) 173sr2.
M. Kulon, J.M.M. van Breugel, A. Fereydooni, M. Lin, L.J. Savic, B. Tegel,
[5] K.T. Butterworth, S.J. McMahon, F.J. Currell, K.M. Prise, Nanoscale 4 (2012) 4830.
T. Wahlin, E. Funai, T. Schlachter, Transl. Oncol. 13 (2020) 100742.
[6] J. Xie, L. Gong, S. Zhu, Y. Yong, Z. Gu, Y. Zhao, Adv. Mater. 31 (2019) e1802244.
[49] A. Lintia-Gaultier, C. Perret, C. Ansquer, T. Eugène, F. Kraeber-Bodéré, E. Frampas,
[7] C. Hoffmann, V. Calugaru, E. Borcoman, V. Moreno, E. Calvo, X. Liem, S. Salas,
Nucl. Med. Commun. 34 (2013) 674.
B. Doger, T. Jouffroy, X. Mirabel, J. Rodriguez, A. Chilles, K. Bernois, M. Dimitriu,
[50] P.R. Rauta, Y. Mackeyev, K. Sanders, J.B.K. Kim, V.V. Gonzalez, Y. Zahra, M.
N. Fakhry, S.W. Hee Kam, C. Le Tourneau, Eur. J. Cancer 146 (2021) 135.
A. Shohayeb, B. Abousaida, G.V. Vijay, O. Tezcan, P. Derry, A.V. Liopo, E.
[8] Y. Chen, S. Liu, P. Gao, M. Shi, W. Pan, N. Li, B. Tang, Mater. Today Nano 20
R. Zubarev, R. Carter, P. Singh, S. Krishnan, Sci. Adv. 8 (2022) eabm9729.
(2022) 100253.
[51] S. Bonvalot, C. Le Pechoux, T. De Baere, G. Kantor, X. Buy, E. Stoeckle, P. Terrier,
[9] S. Bonvalot, P.L. Rutkowski, J. Thariat, S. Carrère, A. Ducassou, M.-P. Sunyach,
P. Sargos, J.M. Coindre, N. Lassau, R. Ait Sarkouh, M. Dimitriu, E. Borghi, L. Levy,
P. Agoston, A. Hong, A. Mervoyer, M. Rastrelli, V. Moreno, R.K. Li, B. Tiangco, A.
E. Deutsch, J.-C. Soria, Clin. Cancer Res. 23 (2017) 908.
C. Herraez, A. Gronchi, L. Mangel, T. Sy-Ortin, P. Hohenberger, T. de Baère, A. Le
[52] T. de Baere, X. Zhang, B. Aubert, G. Harry, C. Lagrange, J. Ropers, J. Dufaux,
Cesne, S. Helfre, E. Saada-Bouzid, A. Borkowska, R. Anghel, A. Co, M. Gebhart,
J. Lumbroso, P. Rougier, M. Ducreux, A. Roche, Radiology 201 (1996) 731.
G. Kantor, A. Montero, H.H. Loong, R. Vergés, L. Lapeire, S. Dema, G. Kacso,
L. Austen, L. Moureau-Zabotto, V. Servois, E. Wardelmann, P. Terrier, A.J. Lazar, J.
V.M.G. Bovée, C. Le Péchoux, Z. Papai, Lancet Oncol. 20 (2019) 1148.
[10] F. Vilotte, R. Jumeau, J. Bourhis, Lancet Oncol. 20 (2019) e557.
[11] Model List of Essential Medicines. 〈https://list.essentialmeds.org/〉 (accessed
2022.12).
[12] J. Sicard, J. Forestier, Rev. Neurol. 37 (1921) 1264.

10
S. Zhu et al. Nano Today 56 (2024) 102279

[53] Guide for the Care and Use of Laboratory Animals: Eighth Edition, Copyright 2011, %20external%20radiation%20therapy%20is,and%20the%20number%20of%20tre
NAS. 〈https://olaw.nih.gov/resources/publications/guide-care-2011.htm〉 atments%20is%20based%20on%3A (accessed March 10).
(accessed 15 March). [55] H.C. Liu, D. Davila Gonzalez, D.I. Viswanath, R.S. Vander Pol, S.Z. Saunders, N. Di
[54] Getting External Beam Radiation Therapy - American Cancer Society. https Trani, Y. Xu, J. Zheng, S.H. Chen, C.Y.X. Chua, A. Grattoni, Adv. Sci. 10 (2023)
://www.cancer.org/treatment/treatments-and-side-effects/treatment-types/radiat e2206873.
ion/external-beam-radiation-therapy.html#:~:text=The%20total%20dose%20of

11