Post on 28-Jul-2020
Nano Res
1
Single W18O49 nanowire: A multifunctional
nanoplatform for CT imaging and
photothermal/photodynamic/radiation synergistic
cancer therapy
Jianjian Qiu1, Qingfeng Xiao2, Xiangpeng Zheng1 (), Libo Zhang1, Huaiyong Xing2, Dalong Ni2, Yanyan
Liu2, Shengjian Zhang3, Qingguo Ren4, Yanqing Hua4, Kuaile Zhao5, and Wenbo Bu2 ()
Nano Res., Just Accepted Manuscript • DOI 10.1007/s12274-015-0858-z
http://www.thenanoresearch.com on July 10, 2015
© Tsinghua University Press 2015
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Nano Research
DOI 10.1007/s12274-015-0858-z
1
Graphical Table of Contents
Polyvinylpyrrolidone-decorated W18O49 nanowires have been synthesized by a simple
solvothermal approach as multifunctional nanotheranostic agents. These
nanotheranostics can be used as CT imaging probes, powerful photothermal agents,
photosensitizers, radiation dose intensifiers and more importantly, also generate
significant photothermal/photodynamic/radiation synergistic effects to ultimately
improve the in vivo anticancer efficacy.
2
Single W18O49 nanowire: A multifunctional nanoplatform for CT imaging and
photothermal/photodynamic/radiation synergistic cancer therapy
Jianjian Qiu, 1, † Qingfeng Xiao, 2, † Xiangpeng Zheng, 1, Libo Zhang, 1 Huaiyong
Xing, 2 Dalong Ni, 2 Yanyan Liu, 2 Shengjian Zhang,3 Qingguo Ren, 4 Yanqing Hua, 4
Kuaile Zhao,5 and Wenbo Bu2,
1Department of Radiation Oncology, Fudan University Huadong Hospital, Shanghai,
200040, China
2State Key Laboratory of High performance Ceramics and Superfine Microstructures,
Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai, 200050,
China
3Department of Radiology, Shanghai Cancer Hospital, Fudan University, Shanghai,
200032, China
4Department of Radiology, Fudan University Huadong Hospital, Shanghai, 200032,
China
5Department of Radiation Oncology, Shanghai Cancer Hospital, Fudan University,
Shanghai, 200032, China
† Jianjian Qiu and Qingfeng Xiao contributed equally to this work
Corresponding authors
Corresponding authors:
3
Xiangpeng Zheng, MD, PhD
Department of Radiation Oncology,
Fudan University Huadong Hospital
Shanghai, 200040 China
Tel: 86-21-62483180 ext 20211
Email: zhengxp@fudan.edu.cn
Wenbo Bu, PhD
State Key Laboratory of High performance Ceramics and Superfine Microstructures,
Shanghai Institute of Ceramics, Chinese Academy of Sciences,
Shanghai, 200050, China
Tel.: 86-21-52412712.
Fax: 86-21-52413122.
Email: wbbu@mail.sic.ac.cn
Short Running Title:
Single W18O49 nanowire-based multifunctional theranostic nanoplatform
4
Abstract
As a promising cancer treatment strategy, combined therapy is usually based on the
employment of complicated nanostructures with multi-components functioning as
photo-thermal energy transducer, photo-sensitizer or dose intensifier for photothermal
therapy (PTT), photodynamic therapy (PDT), or radiation therapy (RT). In this study,
ultrathin tungsten oxide nanowires (W18O49) were synthesized using solvothermal
approach and examined as a multifunctional theranostic nanoplatform. The in vitro
and in vivo experiments demonstrated that these nanowires could generate extensive
heat- and singlet oxygen-mediated damages to cancer cells under the 980 nm near
infrared-laser excitation, and also function as radiation dose intensifying agents to
enhance irradiative energy deposition locally and selectively during radiation therapy.
Compared to NIR-induced PTT/PDT and RT alone, W18O49-based synergistic
tri-modal therapy eradicated xenografted tumors and no recurrence was observed
within 9-month follow up. Moreover, strong X-ray attenuation ability of tungsten
element (Z = 74, 4.438 cm2/g, 100 KeV) qualified these nanowires as excellent
contrast agents in X-ray based imaging, for instance, diagnostic computed
tomography (CT) and cone-beam CT for image-guided radiation therapy. Toxicity
studies demonstrated minimal adverse effects on hematologic system and major
organs of mice within one month. In conclusion, these nanowires have shown
potentials in cancer therapy with inherent image guidance and synergistic effects from
phototherapy and radiation therapy, which warrants further investigation.
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Keywords
photodynamic therapy, photothermal therapy, radiation therapy, radiosensitization,
image guidance, synergistic therapy
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Despite of continuous financial and intellectual investments on cancer researches,
malignant tumors remain one of the major causes of human death. Various
approaches have been investigated to eradicate tumors, for instance surgery,
chemotherapy, radiation therapy, targeted therapy, phototherapy, and so on. Mounting
evidences have clearly shown that for most cancers, especially those in advanced
stages, multimodality or multidisciplinary treatments by combining different
approaches with different mechanisms work more effectively than single treatment
modality. In this regard, due to flexibility and tailorability on structures,
multicomponent nanomaterials with elaborately designed composite structures have
emerged as promising platforms for conducting multidisciplinary treatments,
including controlled drug delivery and release of chemotherapeutic agents, radiation
dose intensifier (as radiosensitizer), photosensitizers, photothermal transducers, etc.
Among them, near-infrared-based photothermal therapy (PTT) in combination with
radiation therapy has been receiving increasing interest due to the potential synergistic
interactions and limited toxicity profiles in comparison to conventional combination
of chemotherapy and radiation therapy [1-12]. The advantages of photothermal
therapy-radiotherapy combination could be attributed to underlying tumor biological
characteristics: (1) radioresistant cancer cells in S-phase and hypoxic
microenvironment are vulnerable to hyperthermia [13,14]; (2) local hyperthermia
from phototherapy improve blood circulation and hence oxygenation level in tumor,
which would be favorable for enhancing radiation damage to tumor cells [15]; (3)
7
local hyperthermia inhibits the repair process of DNA damages from ionizing
radiation [16].
Previously, our group synthesized a multifunctional core/satellite
nanotheranostic (CSNT) by attaching ultrasmall CuS nanoparticles onto the surface of
a silica-coated rare earth upconversion nanoparticle for PTT/RT combination therapy
[17]. Tumors could be completely eliminated in the presence of CSNT due to the
strong synergistic effects from PTT and CSNT-enhanced RT. However, the CSNT
system has some drawbacks that blockade its applications in further preclinical
experiments. For example, ultrasmall CuS nanoparticles on the surface of the CSNT
by electrostatic interaction may detach from the core, and the complicated structure
and time-consuming synthetic process of CSNT could be another disadvantage. In
light of the promising applications of nanotheranostics but their disadvantageous
complicated nanostructures, we proposed to construct a novel nanoplatform with
better stability, controllability and operability for multifunctional PTT/RT synergistic
treatment.
Recently, monoclinic PEGylated W18O49 nanowires as an emerging
photothermal agent have received extensive attention due to low cytoxicity and high
photothermal conversion efficacy [18]. The high atom number component (tungsten,
Z = 74) endows these nanowires with potentials for CT imaging and local radiation
dose enhancement. Coupled with the convenient one-step solvothermal approach,
these W-based nanomaterials are presumed to be a more efficient and cost-effective
platform than CSNT for PTT/RT synergistic therapy.
8
Additionally and more excitingly, these nanowires can serve as photosensitizers
to activate the formation of singlet oxygen under the excitation of 980 nm laser for
photodynamic therapy (PDT), which might generate additive therapeutic effects in
combination with either RT or PTT [19]. For example, PDT inhibits the repair of
DNA damage resulting from RT. The combination of PDT and PTT have multiple
benefits [6,7,20,21], including: (1) The appropriate heating by PTT can significantly
increase blood flow, improve oxygen supply and consequently enhance tumor cells’
sensitivity to PDT which is highly dependent on oxygen [22,23]; (2) PDT could
interfere with tumor physiology by disturbing microenvironmental conditions (e.g. a
decreased pH), resulting in increased heat sensitivity of cancer cells [24,25]; (3)
PDT-induced damages could be significantly inhibited enhanced by hyperthermia
[26,27]. Notably, PDT/PTT combination has been proved highly effective on
superficially located tumors, especially skin tumors (e.g. melanomas), in comparison
to deeply located tumors due to rapid attenuation of visible light/NIR intensity in
tissues [28]. In contrast, RT utilizes high-energy radiation (generally X-ray, -ray) to
deliver therapeutic dose to tumors within the body. Despite that RT is not subject to
tumor location and depth, normal tissues and organs surrounding tumors and within
the radiation path should be strictly protected by controlling their doses under certain
levels during radiation therapy, which limits the actual dose received by tumors. A
feasible strategy to increase tumor dose without scarifying normal tissue dose
limitation is to introducing targetable dose intensifying agents into tumors. Generally,
radiation dose intensifying agents are heavy element-contained and as radiation
9
bombards them, secondary electrons are produced, which may cause additional
irradiative damages to tumor cells [29-35]. Therefore, these W18O49 nanowires are
expected to be functional with both RT and PDT/PTT without the need of integrating
with additional components. To our best knowledge, the W18O49 nanowire platform
we proposed above has not been fully investigated yet.
In this study, we synthesized ultrathin polyvinylpyrrolidone (PVP)-decorated
tungsten oxide (W18O49) nanowires as a combinational therapeutic system by a
modified solvothermal appraoach. These nanowires possessed uniform size and
morphology with good dispersity in aqueous solution. Their effects on photothermal
conversion, production of singlet oxygen, and radiation dose enhancement in vitro
were investigated using aqueous solution heating, 1,3-diphenylisobenzofuran (DPBF)
quenching, and MRI-based polymer gel dosimetry experiments, respectively. In vivo
studies verified the feasibility and effectiveness of W18O49-PVP nanowires for
PTT/PDT/RT synergistic treatment. Impressively, tumor could be completely
eliminated with the synergistic effect without recurrence in at least 9 months. Last but
not least, the proposed nanoplatform showed favorable toxicity profile in
hematological and histological aspects.
RESULTS AND DISCUSSION
In this study, W18O49-PVP nanowires were prepared by a modified solvothermal
approach with PVP (Molecular weight = 40000 Da) as ligands and ethanol as the
solvent. Transmission electron microscopy (TEM) images showed that W18O49-PVP
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nanowires (Molecular weight = 40000 Da) possessed excellent microscopic dispersity
in aqueous solutions without significant visible aggregation, in contrast with the
serious agglomerations by using PEG-400 as ligands, indicating the role of PVP as an
excellent particle dispersant (Figure 1a, b and S1) [18,19]. And the molecular weights
of PVP had significant influences on the final morphology of W18O49-PVP nanowires.
Compared to the PVP-10000, the introduction of PVP-40000 remarkably improved
the dispersity of nanowires and generated single nanowires and/or double-nanowire
stacks (Figure S2). In addition, the high hydrophilicity of PVP molecules also
endowed these nanowires with superior solubility in water and other polar organic
solvents (e.g. ethanol, dimethylformamide, dimethyl sulfoxide) without any
flocculation at the concentration of 1.4 mg/mL for one week (Figure S3). Based on
these advantages above, PVP-40000 was finally chosen as surface ligands. To further
validate the decoration, Fourier transform infrared spectroscopy (FT-IR) was
performed to identify the characteristic functional groups (Figure S4). The
transmission bands located at 2948 and 2883 cm-1 could be assigned to asymmetric
and symmetric stretching vibrations of methylene groups (−CH2) and the peak at 1675
cm-1 corresponded to the stretching vibration of the carboxylic group (C=O) in the
PVP molecules in W18O49-PVP nanowires [36]. The peak at 809 cm-1 arised from the
vibrations mode of W-O [34]. The existence of these characteristic functional groups
indicated the PVP modification. The high-resolution TEM imaging showed good
crystallinity and the powder X-ray diffraction (XRD) pattern showed all reasonable
peaks corresponding to pure monoclinic W18O49 nanowires in the JCPDS card no.
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71-2450 (Figure S5a and b) [18,19]. Energy dispersive X-ray spectroscopic (EDS)
analysis proved the presence of W and O elements (Figure S6). The UV-Vis-NIR
spectroscopy was used to investigate the optical property of W18O49 nanowires. As
shown in Figure S7, the absorption in the NIR region became more and more
intensified with the increasing wavelength, indicating excellent thermotherapy
performance.
Subsequently, the effects on photothermal conversion, production of singlet
oxygen and radiation dose enhancement of W18O49 nanowires were investigated using
NIR irradiation of aqueous solution, DPBF quenching and MRI-based polymer gel
dosimetry experiments, respectively (Figure 2a) [38,39]. The heating curves showed
the clear temperature increased when the aqueous solution of W18O49 nanowires was
irradiated by 980 nm laser (1.2 W/cm2) for 5 min with comparison to no obvious
change in the deionized water as the control group, indicating that these nanowires
could induce heat under 980 nm NIR laser stimulation (Figure 2b). In addition, due to
the heavy metal element, tungsten in the nanowires, W18O49 nanowires have the
capability of intensifying radiation dose, which was demonstrated by the MRI-based
polymer gel dosimetry experiments, and hence the potentials in improving
radiotherapy efficacy [39]. As shown in Figure 2c, T2 signal of gels treated with RT
alone was slightly lower (grayer in imaging) than gels alone or gels containing
W18O49 without any treatment. In contrast, gels containing W18O49 and irradiated with
6 Gy (W18O49+RT group) showed lowest T2 signal, indicating that the existence of
W18O49 contributed to the radiation energy deposition in gels. More interestingly,
12
cytotoxic singlet oxygen (1O2), the major active agent in PDT can be produced as the
production of the interaction between nanowires and adjacent oxygen molecules
under the excitation of the 980 nm NIR laser [19]. As shown in Figure 2d and Figure
S8a and S8b, the absorption peak at 410 nm of DPBF solution containing W18O49
nanowires perceptibly decreased after the irradiation of 980 NIR laser for 20-min
intervals in contrast with an almost undetectable change observed in NIR laser
without nanowires group and heating alone group, which proved that W18O49
nanowires could be used as NIR photosensitizers to activate the formation of singlet
oxygen [19]. Combined together, experimental results showed the capabilities of
constructed nanowires as photothermal agents, photosensitizers and radiation dose
intensifying agents.
To validate the experimental findings, we conducted further studies using cells
and animal models. Cell counting kit-8 (CCK-8) assay was utilized to quantitatively
evaluate the therapeutic efficacy of these nanowires in combination with 980 nm NIR
laser and RT on cellular level. As showed in Figure 3, compared to control group, no
significant influence on cell viability was observed in either NIR alone group or
W18O49 nanowires alone group, even at a high concentration of 700 μg/mL. However,
the combination of the two treatments remarkably reduced the fraction of surviving
cells by 40.5%, indicating cellular damages caused by NIR-induced PDT/PTT. With
respect to RT, it was found that cell death rate was higher at the presence of W18O49
(29.8%) than at the absence (12.5%), suggesting that W18O49 nanowires enhanced the
RT efficacy with a mechanism of intensifying radiation dose deposition. Of note, the
13
highest cell killing was observed in the group receiving combination therapy of RT
and NIR along with administration of W18O49 nanowires. Only 30.5% of tumor cells
in this group were viable compared to projected additive value of 41.8% [1], implying
the synergistic effects from combination therapy.
Next, animal experiments were further conducted to examine the feasibility and
efficacy of PDT/PTT/RT combination therapy with W18O49. Herein, Balb/c mice
bearing subcutaneous 4T1 murine breast cancer tumors were randomly divided into
seven groups (n = 7) and subjected to treatment regimens as following: 1) PBS alone;
2) W18O49 alone; 3) NIR alone; 4) RT alone; 5) W18O49+RT; 6) W18O49+NIR; 7)
W18O49+NIR+RT. Daily behaviors of animals and tumor growth were recorded until
the end of experiment (Figure S9). The therapeutic effects were analyzed
quantitatively by monitoring the relative tumor volumes (V/V0) as a function of time
(Figure 4a and Table S1). Tumors in groups 1, 2 and 3 continuously grew and no
evident growth rate difference was observed (V/V0 = 6.7 ± 0.45, 6.5 ± 0.47, 6.6 ± 0.4,
respectively) at the 16th day, suggesting that either W18O49 nanowires alone or
NIR-laser (1.2 W/cm2) alone had no effect on tumor control. Comparatively, tumors
treated with intravenous W18O49 nanowires and NIR laser irradiation (group 6)
showed an inhibited growth pattern with a considerable tumor growth inhibition (TGI)
of 79.1% (V/V0 = 1.4 ± 0.45). The radiation dose enhancement effect of W18O49
nanowires was demonstrated by the significant difference between group 4 and 5 with
V/V0 values of 4.2 ± 0.48 and 2.7 ± 0.55, TGI of 37.3% and 59.7%, respectively.
Obviously, both W18O49+RT and W18O49+NIR treatment achieved partial response.
14
By contrast, the tumor receiving concurrent treatment of 980 nm NIR laser and RT
(group 7) completely shrinked in the fourth day and no evidence of recurrence was
observed within a followup of 9 months (Figure 4b). To put the complete response
seen in group 7 and partial responses in groups 6 and 5, it was evident that there
existed synergistic therapeutic effects between NIR-induced PDT/PTT and RT at the
presence of W18O49. Hematoxylin and eosin (H & E) staining showed that compared
to the NIR-laser alone and RT alone, both W18O49+NIR and W18O49+RT treatments
caused more severe tissue damages, such as irregular widening of intercellular spaces.
Undoubtedly, the most extensive tumor destruction was found in tumors receiving
combination treatments of W18O49 nanowires, NIR and RT, consistent with in vitro
observation and tumor growth curves (Figure 4c-i). Deoxynucleotidyl transferase
biotin-dUTP nick-end labeling (TUNEL) assay was used to assess tumor cell
apoptosis following specific treatments. In parallel with H&E staining findings,
W18O49+NIR+RT treatment induced most serious cell apoptosis among all groups
(Figure S10a). In addition, the body weight and survival status of mice were
monitored every two days. No remarkable declines of body weights and abnormal
behaviors were observed in all groups, indicating insignificant side effects of all
treatment regimens (Figure S11).
The high atom number (74) and hence high X-ray attenuation coefficient of the
tungsten element (4.438 cm2/g, 100 KeV) impart these nanowires with a favorable
ability of absorbing X-ray. [40-43]. CT imaging with W18O49 nanowires aqueous
solution as contrast medium showed an linear increase of HU measurements with
15
increasing concentrations, in consistence with findings in X-ray phantom imaging
(Figure S12a and b). CT imaging of a 4T1 tumor-bearing mouse intratumorally
injected with W18O49 nanowires displayed a significant enhancement compared to
imaging without nanowires with measurement of HU value increment from 52 HU to
271 HU (Figure S12c). These results showed that W18O49 nanowires could be a
promising CT contrast agent for cancer imaging.
Last, we investigated the toxicity profile of W18O49 nanowires in vivo.
Histological examination and hematological analysis were conducted on healthy mice
at day 3, 15, 30 after intravenous administration at a dosage of 12 mg/kg (150 μL for
each mouse). No remarkable tissue damage, toxicity and side effect to major organs
was found according to H & E staining (Figure 5). And nanowires had no
hematological toxicity in terms of blood chemistry and complete blood panel analysis
(Figure S13). Moreover, weight loss and behavioral changes (e.g. eating, drinking and
activity) were not found in mice receiving intravenous W18O49 nanowires (Figure
S14). These results demonstrated that W18O49 nanowires at the given dosage had a
favorable toxicity profile at least in a month.
CONCLUSION
Taken together, single ultrathin W18O49 nanowires have been synthesized using a
simple solvothermal approach for multifunctional CT imaging and
photodynamic/photothermal/radiation combination cancer therapy. Morphologically,
the constructed nanowires exhibit high water-solubility and uniformity. Functionally,
16
they perform as photosensitizers inducing cytotoxic heat and singlet oxygen under the
excitation of 980 nm NIR laser as well as radiation dose intensifying agents, proven
both in vitro and in vivo experiments. Breast tumor xenografts could be completely
eliminated due to the remarkable synergistic effects between W18O49
nanowire-mediated RT and PTT/PDT. The satisfactory performance of these
nanowires in CT imaging provides a potential application for seamless integrating
imaging guidance and radiation therapy with improved imaging quality and radiation
efficacy. Due to good biocompatibility, W18O49 nanowires have favorable toxicity
profile which could facilitate the preclinical and clinical trials.
Of note, W18O49 nanowires with multifunctions described above are structurally
simple, which offers them flexibility of conjugating or binding with other molecules
or structures to achieve more tuned functions. For example, it is very likely that
conjugation with drug carriers such as mesoporous silica or thermal-responsive
polymers may lead to further developments of various multifunctional platforms such
as novel photothermal-induced drug release [44,45]. Coupling with appropriate
surface ligands and targeting agents would empower them to be targetable for targeted
multimodal nanotheranostics [46,47]. And some other distinct intrinsic functional
characteristics of W18O49 nanowires, such as applications in photoacoustic and
ultrasonic imaging, remain to be further explored [48,49].
METHODS
17
Materials. Tungsten hexachloride (WCl6), Polyvinylpyrrolidone (40000/10000 Da)
were purchased from Sigma-Aldrich. Ethanol and polyethylene glycol (400 Da) were
purchased from Sinopharm Chemical Reagent Co., China. All reagents were of
analytical grade and used without any purification. Deionized water was used
throughout the experiments.
Synthesis of ultrathin PVP-decorated W18O49 nanowires. WCl6 (240 mg) was
dissolved in 40 mL ethanol by vigrous stirring until transparent yellow solution,
followed by the addition of another 40 mL ethanol containing 1 g PVP (40000/10000
Da) and subsequent stirring for 40 min. The resulted solution was transferred to an
autoclave for further heating at 200 oC for 48 h. Blue precipitates were obtained and
collected by ultracentrifugation, followed by washing with ethanol and deionized
water for several times, respectively. The product was finally dispersed in 15 mL
deionized water.
Assessment of W18O49 nanowires as radiosensitizers. The assessment of
radiosensitizing effect of W18O49 nanowires was conducted on the polymer gels
whose T2-weighted signal intensity changed with radiation dose. The radiosensitive
MAGIC (Methacrylic and Ascorbic acid in Gelatin Initiated by Copper) gel was
prepared as previously reported with minor modification. Typically, 8 g of gelatin was
mixed with 70 mL of water and stirred at 50 °C for 30 min before adding 200 mg of
hydroquinone in 4.8 mL of water. Then the solution was cooled down to 37 °C,
followed by adding ascorbic acid (35.2 mg in 5 mL of water), CuSO4•5H2O (2 mg in
3 mL of water) and 9 g of methacrylic acid, and stirred for 1 h. After that, 20 mg of
18
W18O49 nanowires were introduced and stirred for another 1 h. The MAGIC gels were
transferred in a refrigerator at 4 °C overnight. After irradiated under radiotherapy
instrument at the dose of 6 Gy, the Gels were scanned using a clinical MR
T2-weighted scanner.
In vitro X-ray irradiation treatment (W18O49-enhanced RT). DMEM solutions of
W18O49 nanowires with different concentrations of 0, 175, 350, and 700 μg/mL were
added to the wells and co-incubated for 4 h, followed by 6 Gy of X-ray radiations and
incubated another 20 h again. Cell viability was determined by CCK-8 assay.
In vitro NIR-laser irradiation treatment (photodynamic/photothermal). 4T1 cells
were incubated with different concentrations of 0, 175, 350, and 700 μg/mL of
W18O49 nanowires, followed by the irradiation of 980 NIR laser (1.2 W/cm2) for 5
min and then incubated for another 20 h. Cell viability was determined by CCK-8
assay.
In vitro PDT/PTT/W18O49-enhanced RT synergistic effect. 4T1 cells were seeded
into a 96-well cell-culture plate at 105/well and then incubated for 24 h at 37 °C under
5% CO2. Cells co-incubated with W18O49 nanowires (700 μg/mL) were exposed to the
980 laser (1.2 W/cm2) for 5 min and incubated for 30 min (PDT/PTT treatment),
followed by 6 Gy of X-ray radiations in 5 min (RT treatment) and incubated for 20 h
again. Cell viability was determined by CCK-8 assay. The projected additive value
was calculated by multiplying the cell viability of cells from W18O49+NIR treatment
by the cell viability of the W18O49+RT treatment.
19
In vivo investigation. Healthy Balb/c female mice (~ 20 g) were obtained and raised
at Laboratory animal center, shanghai medical college of Fudan University. Animal
procedures were in agreement with the guidelines of the Regional Ethics Committee
for Animal Experiments. All anesthetization was performed by intraperitoneal
injection of 10% chloral hydrate (50 μL). Hair on the flanks of the mice was removed
before they received any further treatments.
In vivo PDT/PTT/W18O49-enhanced RT synergistic effect. All animal experiments
were conducted under protocols approved by the Fudan University Laboratory animal
center. Tumors were inoculated by subcutaneously with 1.5 × 106 4T1 cells suspended
in 150 μL phosphate buffered saline (PBS) in the right side of each female Balb/c
mouse. When the tumor volume reached ≈ 75 mm3, the tumor therapy was performed
as follows: 1) PBS alone; 2) W18O49 alone; 3) laser alone; 4) RT alone; 5) W18O49+RT;
6) W18O49+laser; 7) W18O49+NIR+RT. A dosage of W18O49 nanowires (1.4 mg/mL,
150 μL) was intratumorally administrated into the mice in group 2, 5, 6, and 7. After 1
h, the mice received the treatment of RT for the group 5 and the irradiation of 980 nm
laser for 8 min for the group 6 in the tumor site, respectively. The W18O49-loaded
mice in group 7 received the RT treatment (5 min, 6 Gy) at 30 min following the
treatment with the exposure of 980 nm laser (1.2 W/cm2). Tumor sizes were measured
every 2 days after treatment using a vernier caliper and the whole process lasted 16
days. Tumor growth inhibition (TGI) was calculated with the formula: TGI (%) = 100
× (R1 - Rn) / R1, where R1 is the relative tumor volume of the group 1 and Rn is the
relative tumor volume of the nth group.
20
ACKNOWLEDGEMENTS
This work has been financially supported by the National Natural Science
Foundation of China (Grant No. 51372260, 51132009, 21172043), the Shanghai
Rising-Star Program (Grant No. 12QH1402500), the Science and Technology
Commission of Shanghai (Grant No.11nm0505000, 124119a0400), the Shanghai
Municipal Commission of Health (20134360), the Development Foundation for
Talents of Shanghai (Grant No.2012035).
21
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Figures
Figure 1. TEM images with different magnifications of the synthesized
W18O49-PVP40000 nanowires.
a b
31
Figure 2. (a) Schematic illustration of the W18O49 nanowires-based PDT/PTT/RT. (b)
Heating curves of W18O49 nanowires aqueous solution at different concentrations (980
nm, 1.2 W/cm2, 5 min). (c) Polymer gels experiment used to demonstrate the radiation
dose enhancement effect of W18O49 nanowires. Upper left: Gel without nanowires;
Lower left: Gel containing W18O49 nanowires; Upper right: RT on Gel without
nanowires; Lower right: RT on Gel containing W18O49 nanowires (Radiation dose = 6
Gy). (d) Time course absorption of DPBF solutions with (black line) and without (red
line) W18O49 nanowires under the photoirradiation of 980 nm NIR light (980 nm, 1.2
W/cm2).
32
Figure 3. (1) Relative viability of 4T1 cells incubated with W18O49 nanowires at varied
concentrations after receiving NIR, RT or NIR/RT combinational treatment. The
projected additive value was calculated by multiplying the cell viability of
W18O49+RT group by the cell viability of W18O49+NIR group. Statistical analysis was
performed using the Student’s two-tailed t test (*P<0.05). NIR: 980 nm, 1.2 W/cm2, 5
min; RT: 6 Gy (Radiation dose).
33
Figure 4. (a) Tumor response curves following the treatments in vivo with NIR, RT or
NIR/RT combined therapy as a function of time. (*P<0.05, **P<0.01 and ***P<0.001)
(b) The representative photographs of mice 1, 3, 5, and 9 months after
W18O49+NIR+RT treatment. NIR: 980 nm, 1.2 W/cm2, RT: 6 Gy (radiation dose). (c-i)
H&E-stained tumor tissue sections collected from different groups of mice receiving
various treatments: c) PBS; (d) W18O49 alone; (e) NIR alone; (f) RT alone; (g)
W18O49+RT; (h) W18O49+NIR; (i) W18O49+NIR+RT.
34
Figure 5. Histological examination in major organs (liver, spleen, heart, kidney, and
lung) of mice receiving single intravenous injection of PBS (control, 150 μL) or
W18O49 nanowires (12 mg/kg, 150μL for each mouse) followed by dissections in 3, 15,
and 30 days postinjection.