ACS Nano Volume Issue 2017 (Doi 10.1021/acsnano.7b04737) Huo, Da Liu, Sen Zhang, Chao He, Jian Zhou, Zhengyang Zhang - Hypoxia-Targeting, Tumor Microenvironment Responsive Nano-Cluster Bomb Fo PDF

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Article
Hypoxia-Targeting, Tumor Microenvironment Responsive
Nano-Cluster Bomb for Radical-Enhanced Radiotherapy
Da Huo, sen liu, Chao Zhang, Jian He, Zhengyang Zhou, Hao Zhang, and Yong Hu
ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b04737 • Publication Date (Web): 09 Oct 2017
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Hypoxia-Targeting, Tumor Microenvironment Responsive Nano-
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10 Cluster Bomb for Radical-Enhanced Radiotherapy
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15 Da Huo,†,‡ Sen Liu,‡ Chao Zhang,‡ Jian He,† Zhengyang Zhou,†* Hao Zhang,§* and Yong Hu†,‡*
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Department of Radiology, Drum Tower Hospital, School of Medicine, Nanjing University,
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27 Nanjing, Jiangsu, China, 210008, China.
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29 Collaborative Innovation Center of Chemistry for Life Sciences, College of Engineering and
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31 Applied Sciences, Nanjing University, Nanjing, Jiangsu, 210093, China.
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Department of Oncology First Affiliated Hospital of Nanjing Medical University, Nanjing,
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ABSTRACT:
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5 Although ultra-small metal nanoparticles (NPs) have been used as radiosensitizers to
6 enhance the local damage to tumor tissues while reducing injury to the surrounding organs, their
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8 rapid clearance from the circulatory system and the presence of hypoxia within the tumor
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10 continue to hamper their further application in radiotherapy (RT). In this study, we report a size
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12 tunable nano-cluster bomb with a initial size of approximately 33 nm featuring a long half-life
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during blood circulation, and destructed to release small hypoxia microenvironment-targeting
15 NPs (~5 nm) to achieve deep tumor penetration. Hypoxic profiles of solid tumors were precisely
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17 imaged using NP-enhanced computed tomography (CT) with higher spatial resolution. Once
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19 irradiated with a 1064-nm laser, CT-guided, local photo-thermal ablation of the tumor and the
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21 production of radical species could be achieved simultaneously. The induced-radical species
22 alleviated the hypoxia-induced resistance and sensitized the tumor to the killing efficacy of
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24 radiation in Akt-mTOR pathway dependent manner. The therapeutic outcome was assessed in
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26 animal models of orthotopical breast cancer and pancreatic cancer, supporting the feasibility of
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28 our combinational treatment in hypoxic tumor management.
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33 KEYWORDS: nanoparticles, hypoxia, radiotherapy, radiosensitization, photo-thermal therapy,
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35 tungsten
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Radiotherapy (RT) has long been regarded as the one of the most feasible and routinely used
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5 therapeutic modalities in the battle against cancer.1,2 However, the clinical application of RT in
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7 cancer therapy is seriously hampered by the hypoxia-induced resistance of the tumor and the
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9 unexpected injury to normal tissue by the high dosage of irradiation.3-5 Hypoxia, described as
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insufficient oxygen supply, has proven to be one of the primary driving forces for tumor
12 angiogenesis and metastasis.6-8 Unfortunately, traditional low dosage RT might exacerbate the
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14 hypoxic conditions, rendering tumor cells less vulnerable to radiation-induced killing.9
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16 Furthermore, the tumor cells that survive RT become more apoptosis-resistant and contribute
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18 greatly to tumor relapse.10,11 Moreover, the enrichment of tumor-promoting cells (e.g., alternative
19 activated macrophages and cancer stem cells) in hypoxic regions have also been confirmed,12
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21 which also integrate abilities to protect themselves from RT-induced cytolysis and thus
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23 contribute to rapid local recurrence.13 Therefore, clinical methods that increase the sensitivity of
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25 hypoxic tumor cells to RT and reverse or alleviate the hypoxic microenvironment in solid tumors
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are crucial for achieving optimal therapeutic outcomes of RT.
28 Although perfusion of oxygen gas into solid tumors can up-regulate the oxygen pressure
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30 inside the tumor, the high interstitial pressure in the hypoxic region greatly hinders the
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32 penetration of oxygen gas, making this method less valid for the sensitization of whole tumor
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34 mass.14 Recently, the potential of ultra-small metal NPs as radiosensitizers to potentiate the
35 killing efficacy of RT has been tested.15-17 Upon irradiation, these NPs can strongly absorb the γ-
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37 ray or X-ray irradiation to produce secondary electrons, thus enhancing the localized irradiation
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39 dosage and boosting the susceptibility of cancer cells to the radiation while protecting the
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41 surrounding healthy tissue from RT-induced injury.18 In addition, these ultra-small NPs can
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penetrate deeply and homogeneously inside the tumor, where an even killing induced by
44 radiation could be achieved thereafter.19 The major drawback of this method is the lack of ability
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46 to alleviate the hypoxic condition within the tumor, which drives the local relapse post RT.
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48 Moreover, considering their size, these NPs are subjected to limited blood availability, and thus
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50 leading to reduced tumor accumulation and limited enhancement.20,21 In contrast, larger NPs
51 exhibiting a long half-life in the circulatory system in vivo suffer from a short intra-tumor
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53 penetration depth, leading to compromised sensitization effect because of the limited
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55 accumulation in hypoxia region.22 Thus, platform featuring both enhanced accumulation in the
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tumor tissue and deep penetration into the dense collagen matrix in the tumor are essential to
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5 reach optimal radiosensitization effect for RT.
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7 Recently, size tunable NPs were successfully fabricated as drug delivery systems and
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9 provided additional tenability in the spatial control of delivery to solid tumors.23-25 Most of these
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NPs maintain their initially larger size during circulation to achieve the longer half-life and
12 passive targeting ability to the tumor via the enhanced permeability and retention (EPR) effect.
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14 Upon reaching the tumor site, NPs are transformed into small NPs in response to stimuli (e.g.
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16 enzyme, pH) from the tumor microenvironment to facilitate deep tumor penetration. Although
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18 these sophisticated nanomedicines have successfully resolved the contradictory observations of a
19 long blood half-life and deep tumor penetration, they are not suitable for RT because most of
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21 them are composed of polymers, which are less sufficient for radiosensitization.
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23 To this end, we developed a type of nano-cluster bomb consisting of hypoxic
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25 microenvironment-targeting non-stoichiometric tungsten oxide NPs (WO NPs) being taken as
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radiosensitizers for RT. Since the CCL-28 chemokine is over-expressed in hypoxic tumor
28 microenvironments and is validated to have a crucial role in driving tumor angiogenesis and
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30 tolerance,26,27 WO NPs were firstly modified with ligands targeting the CCL-28 chemokine
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32 (WOAC NPs). Then, clusters of WOAC NPs (WOACC NPs) were covalently bound using a
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34 matrix metalloproteinase-2 (MMP-2) cleavable peptide (Pro-Leu-Gly-Val-Arg-Gly).28 The half-
35 life of WOACC NPs in blood was increased compared to that of WOAC NPs because of their
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37 enlarged size. Once these WOACC NPs accumulated inside the tumor due to the EPR effect,29
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39 up-regulated expression of the MMP-2 enzyme in the tumor microenvironment triggered the
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41 destruction of these WOACC NPs to release small WOAC NPs (Figure 1a), which deeply
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penetrated into the tumor attributed to their small sizes. Considering the robust CT-enhancing
44 and photo-thermal conversion abilities of WO NPs,30-32 hypoxic conditions could be precisely
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46 investigated under the guidance of computed tomography (CT), followed by imaging-guided
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48 photo-thermal therapy (PTT). Meanwhile, highly reactive radicals were simultaneously produced
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50 in hypoxic regions during PTT, which could alleviate the hypoxic induced resistance in Akt-
51 mTOR dependent manner,33 subsequently enhancing the susceptibility of hypoxic cancer cells to
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53 radiation.34 Outstanding therapeutic outcomes of the synergistic treatments were confirmed in
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55 both orthotopic breast cancer and pancreatic cancer tumor models. Compared to several previous
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57 studies,35,36 our study reports a nano-platform integrating the ability to active target hypoxia
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region, penetrate deeply into hypoxia region, together with real-time imaging of the hypoxic
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5 microenvironment and radical-assisted RT. We believe that such radical-assisted radiotherapy
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7 will play an active role in future clinical practice by overcoming the hypoxia-induced resistance
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9 of tumors to therapy.
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12 RESULTS AND DISCUSSION
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14 Characterization. Ultrafine W18O49 NPs have high light absorption efficacy in the near
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16 infrared region (NIR)-II (1000-1500 nm). Due to their small size, these W18O49 NPs rapidly
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18 diffuse in the lymphatic system and may be applied for the photo-thermal ablation of cancer
19 cells.30 In this study, MMP-responsive, hypoxia-targeting cluster bomb-like NPs (WOACC NPs)
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21 were fabricated by covalently bonding PAA-functionalized W18O49 NPs (WO NPs) with a
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23 metalloproteinase-2 (MMP-2) cleavable peptide (Pro-Leu-Gly-Val-Arg-Gly). Firstly, polyacrylic
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25 acid (PAA) modified W18O49 NPs (Zeta potential: -23.2 mV) were fabricated. Then, the carboxyl
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groups on the surface of these PAA-modified W18O49 NPs were transformed to thiol groups by
28 grafting cysteamine (T-W18O49 NPs, Zeta potential: -14.3 mV). Next, these T-W18O49 NPs were
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30 modified with the MMP-2 enzyme cleavable peptide by performing a coupling reaction between
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32 the maleimide and thiol groups. Then, amino group-modified W18O49 NPs (A-W18O49 NPs, Zeta
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34 potential: +3.7 mV) were prepared by conjugating APTES to the surface of the W18O49 NPs. The
35 obtained A-W18O49 NPs were then added to the MMP-2-modified T-W18O49 NP suspension
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37 (ratio of A-W18O49 NPs: T-W18O49 NPs equaling 6:1) to obtain clusters of W18O49 NPs (WOC
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39 NPs). Finally, the hypoxia-targeting group of anti-CCL28 was conjugated to the surface of WOC
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41 NPs to form the WOACC NPs (Zeta potential: -4.5 mV). The individual anti-CCL28-modified
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A-W18O49 NPs were denoted by WOAC NPs.
44 These WOACC NPs exhibited a strawberry-like structure, with a size about 33 nm as
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46 observed in Figure 1. In the XRD spectra shown in supporting information Figure S1, all the
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48 peaks of the as-obtained WOACC NPs can be well indexed into monoclinic phase of W18O49,
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50 confirming that the crystal structure was not affected during the formation of nanocluster. Upon
51 closer observation, these WOACC NPs consisted of ultrafine W18O49 NPs (with a mean diameter
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53 less than 5 nm), confirming the cluster like structure. NIR light absorption profiles of original
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55 W18O49 NPs and WOAC NPs released from WOACC NPs are shown in Figure 1c. No obvious
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57 differences between the two groups were observed, demonstrating the optical stability of these
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W18O49 NPs. The enhanced light absorption of the WOACC NPs might be attributed to the
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5 assembly of the WOAC NPs. Taken together, the covalent bonding procedure did not affect the
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7 NIR light absorption property of W18O49 NPs, and the WOAC NPs released from the WOACC
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9 NPs maintained their local surface plasmon resonance properties. Light absorption of the WOAC
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NP in NIR II (900 nm-1200 nm) is essential for the success of our study because NIR II light can
12 penetrate the deep regions of the tumor and transform the light to heat. Both the WOACC NPs
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14 and WOAC NPs released from the WOACC NPs had excellent photo-thermal conversion
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16 abilities upon irradiation with a 1064 nm laser (1 W/cm2). Even when irradiated with a laser
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18 power density as low as 0.2 W/cm2, the suspension could still achieve a temperature elevation
19 over 40 oC in the presence of WOACC NPs, indicating that superb light to heat conversion
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21 ability of WOACC NPs (Figure 1d). MMP-2 is over-expressed in almost all types of human
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23 tumor microenvironments, whose level correlates positively with tumor progression and
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25 metastasis.38 On this basis, the selective response to MMP-2 enzyme is crucial for WOACC NPs
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to distinguish cancerous lesions from normal tissue. To verify this, we incubated WOACC NPs
28 with different enzymes such as MMP-2, Cathepsin B, and Caspase-3. Changes in the sizes of the
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30 WOACC NPs were analyzed by dynamic light scattering (DLS) (Figure 1e). Pronounced size
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32 variations in the WOACC NPs were observed only in the presence of MMP-2 (from 81 nm to 11
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34 nm), suggesting that the obtained WOACC NPs were disrupted to release WOAC NPs only upon
35 MMP-2 treatment. The TEM images of these WOAC NPs released from WOACC NPs after
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37 MMP-2 treatment showed nearly negligible difference compared to the original WOAC NPs
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39 (Figure S2). The MMP-2 dependent destruction is important to avoid the premature release of
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41 WOAC NPs before they reached the tumor site, which may result in unnecessary loss of cargo
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during circulation in the blood. Based on these results, we concluded that the MMP-2-responsive,
44 cluster bomb-like WOACC NPs were successfully fabricated.
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46 In Vitro Stability and Cytotoxicity. The stability of nanomedicine has profound effect in
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48 regulating its fate both in vitro and in vivo. As such, we tested the optical properties of the as-
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50 obtained WOACC NPs after incubation with freshly harvested mouse serum, in an attempt to
51 mimic the harsh in vivo microenvironment. As can be seen in Figure S3, an incubation with
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53 mouse serum for 48 h only slightly mitigated the featuring adsorption in NIR, suggesting that
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55 WOACC is fairly stable. By using X-ray photoelectron spectrometer (Figure S4), we analyzed
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57 the oxidation status of tungsten in WOACC before and after interaction with cysteine (sulfur
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containing amino acid), and mouse serum as a more complicated case. We found that the
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5 incubation with cysteine induce little changes of the bonding energy of W6+, indicating that the
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7 interaction between sulfur containing amino acid and WOACC is regulated by electrostatic
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9 forces. Meanwhile, from the spectra of WOACC incubated with mouse serum, one can see that
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the bonding energy of W6+ greatly changed from 38.28 and 36.08 ev (before incubation) to 38.58
12 and 36.58 ev (post incubation) of 4f5/2 and 4f7/2, respectively, suggesting that there exists
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14 potential covalent bonding between the serum proteins and WOACC. Furthermore, we analyzed
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16 the profiles of binding energy of tungsten by, clearly, nearly negligible shift of characteristic
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18 binding energies of tungsten indicates the less tendency of forming covalent binding between
19 sulfur (in cysteine) and WOACC.
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21 Specifically, the cytotoxicity of WOACC was tested in both human epithelial HUVEC cell
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23 line and cancerous HeLa cell line (Figure S5). It can be seen that WOACC NPs with a
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25 concentration of less than 100 µg/mL exert near negligible cytotoxic effect regardless of cell
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types. Even when challenged with a 5 mg/mL of WOACC NPs, HUVEC and HeLa cells still can
28 exhibit 82.5% and 84.2% viability post 48 h incubation, respectively. We think this finding can
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30 be taken as a solid evidence to support the compatibility of WOACC for practical application.
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32 Furthermore, we have analyzed the potential of cytotoxicity of WOACC upon stimulation (laser
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34 irradiation and radiation). In particular, 200 µL of DMEM culture medium containing 1 mg/mL
35 WOACC was irradiated with 1064-nm laser (1 W/cm2) for 10 min. Then, the suspension was
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37 allowed to cool down naturally, and the WOACC NPs were collected via centrifugation every 24
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39 hours. The concentration of tungsten in the supernatant was analyzed by inductively-coupled
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41 plasma mass spectrometer (ICP-MS) as shown in Figure S6. It can be seen that the laser
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irradiation-induced degradation of WOACC is nearly negligible with an up to 1.4% of tungsten
44 released into conditioned medium during the observation period. Furthermore, when switched
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46 the laser irradiation to radiation (3 Gy), we observed a higher content of tungsten release profiles
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48 (at 72 h, 4.1%). It can be envisioned that the release of heavy metal ions might induce
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50 cytotoxicity in normal tissue or partially being responsible for the cytolysis of cancer cells
51 induced by metal-enhanced radiation. To test this hypothesis, HUVEC and HeLa cells were co-
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53 cultured with the supernatant containing the released tungsten ions for 48 h, with the viability of
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55 each well analyzed via MTT assay as mentioned above. It can be seen that the release of tungsten
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57 ions caused by either laser irradiation or radiation induce negligible cytotoxicity regardless of
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cell types. It is reasonable to see this result as the cytotoxicity induced by heavy metal should be
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5 dosage-dependent, and the concentration of non-specific released tungsten ions is believed to be
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7 within tolerable range.
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9 In Vivo Distribution Profiles. The penetration of NPs into the tumor is a key factor for
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achieving cytolysis in hypoxia region, which are located deeply inside the solid tumor. Therefore,
12 the penetrating potency of these WOACC NPs was studied in a 3D spheroid tumor model in
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14 vitro, so as to mimic the relevant obstacles faced by the WOACC NPs in solid tumors33. All
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16 these samples were labeled with FITC and showed a green color in the confocal laser scanning
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18 microscope (CLSM) images (Figure. 2a). For FITC-labeled WO NPs (a1) and the cluster of
19 W18O49 NPs covalently bound by the non-cleavable peptide (WOCC NPs) (a2), green
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21 fluorescence was observed only in the vicinity of the peripheral tumor, indicating that the rigid
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23 cluster with an unchangeable diameter had a limited capability to penetrate deep into tumors.
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25 However, abundant green fluorescence was observed from the periphery to the center of the
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multicellular spheroids for both WOACC NPs (a4) and WOAC NPs (a3), clearly supporting their
28 penetration abilities with respect to non-targeting counterparts like WOCC. Once the activity of
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30 MMP-2 was blocked prior to the incubation with WOACC (a5), the green fluorescence was
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32 observed only around the periphery of the tumor model (same as the results a2), clearly
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34 illustrating that the penetration depth of WOACC NPs was suppressed. Thus, the deep
35 penetration of WOAAC NPs can be attributed to their disassembly by MMP-2. Notably, it can be
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37 seen that the penetration depth of WO NPs is limited. These observations were further corfirmed
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39 by ICP-MS analysis of the tungsten content (Figure S7), collectively supporting the essential role
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41 of proper surface functionalization in active accumulation in hypoxia lesion.
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Although the MMP-2-triggered release of WOAC NPs is essential for the drug delivery to
44 the tumor center, the blood circulation time of WOACC NPs is also important because it affects
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46 the tumor accumulation of WOACC NPs via the EPR effect. Blood retention profiles of both
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48 WOACC NPs and WOAC NPs were analyzed post-i.v. injection, as shown in Figure 2b. The
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50 assembly of WOAC NPs into larger clusters clearly increased their half-life in blood (274 min
51 versus 124 min for WOACC NPs and WOAC NPs, respectively), which greatly benefits the
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53 tumor accumulation of WOACC NPs via the EPR effect. The distribution of these two samples
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55 inside the tumors was also measured and shown in Figure 2c. For WOAC NPs, only an
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57 accumulation amount of 4% ID/g tumor tissue could be achieved at 24 h post injection, and the
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amount of the WOAC NPs did not change, even after 48 h. However, 17.5% of the injected dose
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5 of WOACC NPs per gram of tumor was achieved in the tumor at 24 h post injection and further
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7 increased to 27.5% ID/g tumor tissue after 48 h. We attribute the enhanced tumor accumulation
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9 of MMP-2-responsive WOACC NPs to their extended blood availability in vivo, especially in
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comparison with that of WOAC NPs. Due to the high level of MMP-2 enzyme in the tumor
12 tissue, these WOACC NPs were then degraded to release WOAC NPs, which penetrated deeply
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14 inside the tumor because of their small size. Based on these results, our objective to design a
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16 nano-platform with a prolonged blood retention time and deep tumor penetration was
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18 successfully achieved.
19 In Vitro Radiosensitization. The radiosensitization effect of WOACC NPs on 4T1 cells
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21 that had received different treatments and grew under both normal and hypoxic conditions was
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23 first verified using colony formation assays, as shown in Figure S8. The lowest cell survival
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25 could be observed in cells received synergistic treatment (WOACC NPs assisted, PTT-enhanced
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RT, denoted by PTT+RT). Considering that radiation in identical dosage achieve much less
28 cytolytic effect under hypoxia condition, it is suggested that the simultaneous treatment with
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30 WOACCNPs and PTT greatly enhanced the susceptibility of tumor cells to radiation under
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32 hypoxic conditions. Although PTT had been proven to be an effective cancer therapy due to its
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34 ability to induce cytolysis, neither PTT plus WOACC NPs nor RT plus WOACC NPs alone
35 exerted a comparable therapeutic effect as that of the synergistic treatment, indicating that
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37 sequential therapy with PTT and WOACC NP-assisted RT was of great importance. The in vitro
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39 viabilities of 4T1 tumor cells that had received the different treatments under normoxic and
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41 hypoxic conditions were also quantitatively investigated using Annexin-V/PI staining and flow
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cytometry (Figure 3a and Figure S9). Again, it can be seen that hypoxic condition makes 4T1
44 cells less vulnerable to RT, which reduced the killing efficacy of the RT treatment from 30.7%
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46 (normoxia) to 12.5% (hypoxia), indicating that radiation exerted a cytolytic effect in an oxygen-
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48 dependent manner, consistent with the clinical observations.39 In contrast, a comparable killing
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50 efficacy was observed following the combined treatment (PTT+RT group), regardless of the
51 oxygen conditions (57.9% and 63.4% for normoxia and hypoxia, respectively).
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53 Since the therapeutic effect of PTT was not long-lasting, we envisioned that the thermo-
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55 induced radical production might be responsible for the enhanced effects.33 To test this
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57 hypothesis, we incubated cells with N-acetylcysteine (NAC), a broad spectrum quencher of
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reactive oxygen species (ROS), prior to the radiation. The killing efficacy of the combination
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5 treatment (right axis) was reduced thereafter (from 63.4% to 38.6%) (Figure 3a). Meanwhile,
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7 NAC had much less influence on the killing efficacy of PTT alone (30.4% and 28.3%; in the
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9 absence or presence of NAC). Thus, it is safe to conclude that the ROS induced by PTT, rather
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than thermolysis itself, is responsible for the radiosensitization. The production of radicals by
12 WOACC NPs upon laser irradiation was confirmed by EPR spectroscopy using DEPMPO as a
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14 spin trapping molecule. As shown in Figure 3b, multiple radical signals were obtained in the
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16 EPR spectrum and identified as hydroxyl- (·OH) and superoxide radicals (·O2-), while cells
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18 incubated with WOACC NPs without laser irradiation failed to induce such radicals.40 This
19 phenomenon was further confirmed by using flow-cytometry to detect the level of intracellular
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21 ROS in cells received different treatments including synergistic treatments, PTT or RT alone,
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23 and WOACC incubation in the presence of H2DCFDA, a ROS responsive probe, as shown in
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25 Figure 3c. Cells receiving PTT+RT treatment produce the highest amount of ROS.
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Furthermore, the enhancement of killing efficacy was also observed in normoxic cells
28 incubated with WOACC NPs followed by RT (41.9%, compared to 30.7% without WOACC
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30 NPs). It is not surprising to see such radiosensitization effect as heavy metal elements like
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32 tungsten, have been tested to be valid as radiosensitizers to strengthen the RT therapeutic
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34 index.41 The enhancement also positively correlated with the oxygen content (killing efficacy in
35 hypoxic cells, 16.3% and 12.5%, in the presence and absence of WOACC, respectively). We
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37 speculated that the abnormal enhanced DNA repair process in cancer cells received the radiation-
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39 induced ionization might be responsible for the resistance to metal-enhanced RT.34 To verify this
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41 hypothesis, the phosphorylation of several key proteins underlying the stress-induced DNA
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damage repair mechanism such as mTOR,41 Akt,42 and MAPK,43 was investigated using Western
44 blotting as shown in Figure 3d. It can be seen that WOACC NPs-assisted PTT leaded to obvious
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46 reduction of phosphorylation of mTOR and Akt, and that of AMPK were less affected,
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48 suggesting that this treatment enhanced the susceptibility of hypoxic cells to radiation in mTOR-
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50 Akt dependent manner. Furthermore, when the cells were treated with either mTOR or Akt
51 agonist prior to WOACC NPs assisted PTT, the phosphorylation of mTOR largely recovered,
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53 clearly suggesting that the Akt located in the upstream of this pathway, in consistent with Bodine
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55 et al. reported.44 In addition, as shown in Figure 3e, when we tested the expression of several
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57 genes underlying cell proliferation (MT 3), hypoxia-driven radiation resistance (HIF-1α and
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ANGPTL 4), cell growth (IGFBP-1), angiogenesis (VEGFA), and apoptosis (CASP-1 and BAX)
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5 by using high-throughput qRT-PCR array, it seems that the radiosensitization effect is more
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7 relevant to the reverse of hypoxia-driven apoptosis resistance that angiogenesis inhibition as
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9 indicated by the significant reduced expression of related genes. Taken together, these findings
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suggest that the WOACC assisted PTT enhances the susceptibility of hypoxic cancer cells by
12 reversing their apoptosis resistance driven by hypoxia in Akt-mTOR dependent manner.
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14 In Vivo Imaging. In vivo non-invasive imaging of tumor hypoxia with high spatial
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16 resolution is of great therapeutic significance. Unfortunately, limited progress has been achieved
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18 till now. W18O49 NPs are good candidates for PTT and can also act as CT contrast agents due to
19 their high X-ray attenuation.30 Here, the potential of WOACC NPs as contrast agents to locally
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21 image hypoxic regions under CT guidance was tested. As shown in Figure 4a, a heterogeneous
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23 enhancement of the tumor lesion, particularly in the center of solid tumors (Figure 4a, b1 inner
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25 area) in the breast tumor-bearing mouse, was observed post-i.v. injection of WOACC NPs. This
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result is reasonable because hypoxia primarily occurs in regions lacking oxygen and nutrient
28 supplements (normally in the center of the tumor). To better understand the intra-tumoral
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30 diffusion of NPs, an artificial diffusion pathway was defined by a line start from the tumor
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32 boundary (highlighted by red star) and end in the center of the tumor (highlighted by yellow star)
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34 in Figure 4a for simplicity. Distribution profiles (as indicated by their HU values) from three
35 randomly selected regions of interest along the diffusion path in the tumors were analyzed
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37 (Figure 4d). WO NPs and WOAC NPs showed the lowest values, indicating that least NPs
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39 penetrated into the tumor tissues. The low tumor accumulation of NPs was mainly ascribed to the
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41 small size of WO NPs and WOAC NPs, which fail to accumulate in tumor to certain amount via
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the EPR effect. These NPs would leak out from the blood vessels and be eliminated by renal
44 clearance long before they get interact with the tumor,45 as confirmed by our previous finding of
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46 the in vivo circulation and related tumor distribution. For the WOCC NPs, the HU value at the
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48 peripheral zone of the tumor was as high as the value of the WOACC NPs and then rapidly
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50 decreased toward the center of the tumor. The larger size of the WOCC NPs extended their half-
51 life in the circulatory system, thus leading to a passively accumulation of WOCC NPs at the
52
53 tumor site, which consequently resulted in the highest HU value at the boundary. In contrast,
54
55 larger counterpart WOCC NPs exhibited a poor tumor penetration ability, with fewer WOCC
56
57 NPs capable of accessing the tumor center, as demonstrated by the low HU values. In the mice
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3
received an i.v. injection of WOACC NPs, we observed the highest accumulation in tumor tissue,
4
5 consistent with the results of the in vivo tumor distribution experiment. The HU value was higher
6
7 around the tumor boundary. Considering the high level of MMP-2 in tumor microenvironment,
8
9 WOACC NPs will be degraded to release small WOAC NPs once they enter the tumor through
10
11
leaky dysfunctional blood vessels. These released small WOAC NPs are likely to penetrate
12 deeply into the tumor tissue and distributed heterogeneous depending on the hypoxia condition
13
14 (more specifically, the expression of CCL-28 chemokine), as indicated by the distinct HU values
15
16 along the diffusion path. The presence of WOAC NPs (released from the WOACC NPs) in the
17
18 center of the tumor was further verified by the Bio-TEM (Figure 4b). The small WOAC NPs,
19 with a size less than 5 nm, were clearly observed in enlarged image (Figure 4b), demonstrating
20
21 the successful deep penetration of WOAC NPs into the tumor center. In particular, the tissue
22
23 located in the middle layer of the tumor showed much higher HU values (273 HU, 6.5 mm from
24
25 boundary) than that of the tumor center (110 HU, 10 mm boundary), indicating that more
26
27
WOAC NPs were located between the boundary and center of the tumor, in consistent with the
28 observation of fluorescence imaging (Figure S10). We anticipated that despite the surface
29
30 conjugation of anti-CCL-28 Abs, who promote the active accumulation in hypoxia regions, the
31
32 vasculature available in hypoxia region is also attributable to the enhanced accumulation. Given
33
34 the fact that WOCC NPs in sizes similar to that of WOACC NPs can barely access such regions
35 as indicated by HU values, we argue that the contribution of blood vessels-induced accumulation
36
37 is limited, and collectively support the dominant role of CCL-28 targeting ligand in active
38
39 accumulation at hypoxic region. Thus, it seems that the middle section of the solid tumor suffers
40
41 from a more severe hypoxic condition than that of the central part. We speculated that the
42
43
prolonged shortage of nutrients and oxygen, rather than short-period stress, in the central tumor
44 section, induce permanent cell necrosis, thus reducing the levels of hypoxia-related cytokines
45
46 and chemokines,46,47 while the limited oxygen supplement to the middle section of tumor tissue
47
48 still support the growth of cancer cell under hypoxia condition. Future studies are required to get
49
50 a better understanding of this phenomenon.
51 The centers of tumor tissues were collected and dissected post-i.v. injection of FITC-labeled
52
53 samples and subjected to immunofluorescence analysis to confirm this hypothesis. Hypoxic
54
55 conditions were labeled with HIF-1 (red), as shown in Figure 5a. The co-localization of green
56
57 (WOACC NPs) and red fluorescence (HIF-1) was observed in tissues surrounding the tumor
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3
center, confirming the accuracy of the hypoxia-specific WOACC NPs-enhanced CT imaging
4
5 (a1). However, fewer hypoxic cells were observed in the tumor center (red), together with lower
6
7 concentrations of NPs (green). These results further indicated that the lower HU values observed
8
9 in the tumor center were caused by necrosis-induced reduction of CCL-28 expression in the
10
11
center of the solid tumor. In the mice treated with WOCC NPs, WOAC NPs, or WO NPs, almost
12 no NPs were observed in the center of the tumor, consistent with our previous observation of the
13
14 in vivo penetration (Figure 4d).
15
16 The hypoxia-targeting ability of the CCL-28 chemokine and the MMP-2-responsiveness of
17
18 WOACC NPs were further verified by using flow cytometry, as shown in Figure 5b and c.
19 WOACC NPs preferred to accumulate in HIF-1-positive regions (29.3% NPs+ HIF+). When the
20
21 activity of MMP-2 was blocked, the amount of WOACC NPs accumulated in hypoxia region
22
23 decreased to 8.7% (NPs+ HIF+), indicating the reduced ability to penetrate into the solid tumor.
24
25 However, when the expression of CCL-28 chemokine was blocked via intratumoral injection of
26
27
antibody prior to the administration of WOACC NPs, we still observed a small amount of
28 WOACC NPs accumulated in hypoxic region (17.5% NPs+ HIF+). These results were attributed
29
30 to the superb penetration ability of small WOAC NPs released from the WOACC NPs. Based on
31
32 these results of the enzyme and chemokine blocking tests, we concluded that the MMP-2-
33
34 sensitive peptide played a vital role during the sequential delivery process as it is essential for the
35 release of ultrafine WOAC NPs from the WOACC cluster bomb. When the interaction between
36
37 the CCL-28 chemokine and WOACC NPs was inhibited, a small fraction of WOAC NPs still
38
39 could access the peripheral hypoxic regions via passive diffusion. However, this accumulation
40
41 was non-specific and likely to suffer from limited therapeutic index as the majority of hypoxic
42
43
regions located deeply within the solid tumors. Furthermore, the expression of a DNA damage
44 marker (phosphorylated histone H2AX, pH2AX)48 and late stage apoptosis activation indicator
45
46 (Poly [ADP-Ribose] Polymerase, PARP)49 after RT treatment were investigated using
47
48 immunofluorescence staining (Figure 5d) to confirm the cytolysis effect. The WOACC NPs
49
50 assisted PTT plus RT with WOACC NPs induce more DNA damage (indicated by spot-like
51 pH2AX fluorescence, red) than PTT or WOACC-enhanced RT alone, which could be safely
52
53 ascribed to the alleviated RT resistance. Increased cleaved PARP protein, which was positively
54
55 correlated with the number of apoptotic hypoxic cancer cells, was observed after the synergistic
56
57
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3
treatment (green color) compared with that in the PTT or NP-enhanced RT groups, supporting
4
5 the practical significance of the synergistic treatment.
6
7 PET Imaging. Long-term response of tissue post treatment stands as another important
8
9 concern because different nanomedicines may exert distinct effects on the tumor
10
11
microenvironment. Hypoxia, among various inflammatory reactions, has been taken as the
12 primary concern when considering its critical role in driving angiogenesis and local relapse. On
13
14 this basis, we evaluated the hypoxic conditions in tumor tissue of mice received different
15
16 treatments. PET imaging with 18F fluoromisonidazole (FMISO) has long been taken as the gold
17
18 standard for tracking hypoxia due to its high sensitivity, despite its low spatial resolution.
19 Residual hypoxic zones inside the tumor after the different treatments were verified using PET.
20
21 Almost no positive hypoxic zones were observed inside the tumor after the synergistic treatment
22
23 (Figure 6a, II, white circle) with respect to that of control group (I, white circle). This region had
24
25 the lowest PET signal and the smallest amount of 18F-MISO accumulation (Standardized uptake
26
27
values (SUV): mean: 0.0186±0.0024; Max: 0.0374±0.0103, p<0.001; uptake: 0.059% ID/g
28 tumor, Figure 6b and c). In contrast, intra-tumoral hypoxia has been confirmed in mice received
29
30 both the WOACC PTT treatment (SUV: mean: 0.3122 ± 0.1032; uptake: 2.43% ID/g tumor) and
31
32 WOACC NP-sensitized RT (SUV: mean: 0.1862 ± 0.0298; uptake: 2.11% ID/g tumor), again
33
34 highlighting the anti-tumor effect of the synergistic therapy. Notably, WOACC NP-sensitized
35 RT led to a pronounced reduction in 18
FMISO uptake in the central tumor, as indicated in the
36
37 transverse images (Figure 6a, IV), suggesting the potential presence of necrosis induced by
38
39 WOACC NP-assisted RT. Collectively, these findings from the PET analysis and in vitro assays
40
41 suggested that the WOACC NP-assisted synergistic treatment reduced the RT resistance of
42
43
hypoxic cells together with a long-term protection to inhibit the recurrence of hypoxia.
44 In Vivo Therapy. The long-term survival and anti-tumor profiles are shown in Figure 7.
45
46 The WOACC NP-assisted synergistic therapy markedly reduced the tumor volume (Figure 7A),
47
48 consistent with results of PET imaging. Mice received the WOACC NP-assisted synergistic
49
50 therapy showed the smallest residual tumor volume (approximately 1.7% of the initial burden)
51 among all the tested animals at the end of the first week after two cycles of treatments,
52
53 confirming the excellent effectiveness of the synergistic treatment. No local relapse was found
54
55 after prolonged observations without additional therapy, further indicating the long-term
56
57 validness of our synergistic treatment in inhibiting the hypoxia-driven local tumor recurrence
58
59
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1
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3
compared with that of other treatments. Although PTT or WOACC NP enhanced RT alone could
4
5 also exert moderate tumoral lysis effects (50.13% and 41.74% of the initial tumor volume for
6
7 PTT and NP-enhanced RT, respectively, compared to the saline group after 5 weeks) with
8
9 extended survival rates (57.14% both for PTT and NP-enhanced RT after 4 weeks), their
10
11
therapeutic efficacies were lower than the synergistic treatment (survival rates: 100% and 85.74%
12 after 5 and 8 weeks, respectively, Figure 7b).
13
14 Another process governing the survival of tumor-bearing mice is the distant metastasis from
15
16 the primary tumor. Because hypoxia tightly correlated with metastasis,50 we were interested in
17
18 the anti-metastasis potential of our synergistic treatment targeting hypoxia. Firstly, the status of
19 the typical epithelial mesenchymal transition (EMT), which is activated prior to tumor invasion
20
21 and metastasis occurs, was investigated. As shown in Figure 8, the WOACC NP-assisted
22
23 synergistic treatment greatly reduces the expression of N-cadherin, which is positively correlated
24
25 with metastasis. Thus, tumor invasion and metastasis were suppressed by the WOACC NP-
26
27
assisted combined treatment. The expression of E-cadherin, which plays an opposing role to N-
28 cadherin, is up-regulated when comparing with that of the control group, collectively confirming
29
30 the role of synergistic therapy in inhibiting tumor metastasis. A similar trend was observed in the
31
32 PTT group, indicating that the ability of synergistic therapy to block metastasis might originate
33
34 from the WOACC-assisted PTT with less relevance to radiation. The RT treatment without the
35 WOACC NPs surprisingly promoted the EMT, consistent with the report by Dewhirst et al.34
36
37 Furthermore, the metastatic profiles in the liver and lungs were recorded using necropsy and
38
39 pathological studies. Small nodules were clearly observed in the images of the lungs and liver
40
41 (Figure 9a). In addition, as shown in Figure 9b, only one mouse showed metastasis in the lung 4
42
43
weeks after the synergistic therapy, which was obviously less than the other treatments (PTT:
44 liver 1/3, lung 2/3; NP-enhanced RT: liver 2/3, lung 3/3) and control group (liver 3/3, lung 3/3).
45
46 These findings collectively confirmed that the WOACC NP assisted synergistic treatment
47
48 effectively inhibited distant metastasis, which was beneficial for the long-term survival.
49
50 Pancreatic Cancer Model. Encouraged by the results of the WOACC NP assisted
51 synergistic therapy on breast cancer, we further challenged these nano-cluster bomb-like NPs
52
53 with another representative yet hard-to-handle hypoxic tumor: pancreatic carcinoma (PC).
54
55 Among the various types of malignancies, pancreatic carcinoma (PC) exhibits the poorest
56
57 prognosis (less than 2% five-year survival).51 Pathological study suggests that the tumor
58
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1
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3
protective dense stroma drives the establishment of a hypoxic microenvironment and hinders the
4
5 effective delivery of drugs into tumor mass.52 Anti-stroma therapy, identified by utilizing several
6
7 inhibitors targeting the tumor stroma, has been taken as one of the appealing methods in the
8
9 battle against PC. Although promising, the complicated or even contradicted roles of stroma
10
11
played during the progress of PC make it hard to achieve optimal outcome by solely inhibiting
12 the stroma. Thus, finding potential robust treatment integrating abilities to target stroma-
13
14 surrounded tumor mass and alleviate its hypoxia condition alternative to anti-stroma
15
16 chemotherapy is of great research and clinical significance, and remains challenging. To this end,
17
18 we are interested to test the therapeutic potential of the WOACC NP-assisted synergistic
19 treatment in an orthotopic human PC model. By using bioluminescence imaging (BLI), the
20
21 proliferation of firefly luciferase-expressing pancreatic cancer cells could be tracked in vivo non-
22
23 invasively over time after receiving different treatments. As shown in Figure 10a, the synergistic
24
25 therapy effectively slowed the progression of PC, and the tumor burden was only slightly
26
27
increased at week 4 post-treatment, according to the bioluminescence photon intensity (2.34-fold,
28 compared with the control) compared with the groups of RT (12.07-fold) or gemcitabine (4.02-
29
30 fold). Necropsy images (Figure 10b) provided further evidence supporting the success of the
31
32 WOACC NP-assisted treatment in conquering hypoxia tumor.
33
34 Inflammation, particularly chronic inflammation, has been proven to be one of the major
35 driving forces for tumor angiogenesis and growth.47 Based on accumulating evidence, non-
36
37 specific PTT might exert its cytolytic effect by inducing necrotic cell death, risking a local
38
39 chronic inflammatory response, which promotes local malignancy.53 To avoid this, next-
40
41 generation of PTT should act in inflammation-free manner if considered for clinical translation.
42
43
Therefore, we verified the influence of the combined treatment with WOACC NPs on the tumor
44 microenvironment by analyzing the expression of 39 inflammation-related cytokines (Th1, Th2,
45
46 and Th17) and chemokines using an inflammation antibody array. As shown in Figure 10c,
47
48 expression of the majority of inflammatory factors did not experience obvious increase post
49
50 synergistic treatment, indicating that the absence of inflammatory response, further excluding the
51 possibility of local recurrence driven by chronic inflammation. All these findings strongly
52
53 support the feasibility of using synergistic therapy as a valuable treatment for PC.
54
55 Long-Term Cytotoxicity. The long-term safety of nanomedicines is another important
56
57 concern before their clinical translation. Thus, hematological, biochemical, and pathological
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3
assays were applied to study the potential cytotoxicity. As displayed in Figure 11b, liver function
4
5 was determined by measuring the alanine amino transferase (ALT), total protein (TP), and total
6
7 bilirubin (TBIL) levels; kidney function was determined by measuring the blood urea nitrogen
8
9 (BUN) and creatinine (CRE) levels; and spleen function was determined by measuring the
10
11
platelet (PLT) counts. The levels of these metrics exhibited only slight changes within 7 days
12 after the injection of WOACC NPs, and their levels recovered rapidly over time. In addition, red
13
14 blood cell (RBC) and white blood cell (WBC) counts, indicators of hemopoietic and aerobic
15
16 capacity, also remained nearly unchanged 3 days after administration. In addition, no observable
17
18 variation or accumulation of inflammatory immune cells was observed in the heart, lung, liver,
19 spleen, and kidney (Figure 11a), further confirming the biocompatibility of WOACC for
20
21 practical in vivo applications.
22
23
24
25 CONCLUSION
26
27
In summary, we synthesized a type of nano-cluster bomb-like NPs integrating both an active
28 hypoxia-targeting ability and passive tumor accumulation potency via the EPR effect. Based on
29
30 the in vitro observations, these NPs stimulated the production of radical species upon laser
31
32 irradiation, which potentiated the killing efficacy of radiation by inhibiting the phosphorylation
33
34 of mTOR. Hypoxic profiles inside the tumor were precisely analyzed using contrast-enhanced
35 CT, which was beneficial for imaging-guided PTT. Furthermore, the therapeutic outcomes was
36
37 assessed in animals orthotopically bearing either breast cancer or pancreatic cancer using the
38
39 WOACC NP-assisted combined treatments, supporting the feasibility of our radical-assisted
40
41 strategy in future clinical trials.
42
43
44 MATERIALS and METHODs
45
46 Chemicals and Materials. Tungsten hexachloride (WCl6, ≥99.9%), cobalt (II) chloride
47
48 hexahydrate (CoCl2·6H2O, Bioreagent) (3-aminopropyl) triethoxysilane (APTES, ≥99.0%),
49
50 poly(acrylic acid) (PAA, MW~2 kDa), diethylene glycol (DEG, ≥99.0%), cysteamine (≥95.0%),
51 Trypan blue solution (0.4%), tris(hydroxymethyl)aminomethane (Tris, ≥99.8%), N-acetyl-L-
52
53 cysteine (NAC, ≥99.0%), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC,
54
55 Bioxtra), N-hydroxy-succinamide (NHS, 98.0%), DL-dithiothreitol (DTT, ≥99.5%),
56
57 (diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO), and N-succinimidyl-4-
58
59
60 17

1
2
3
(maleimidomethyl) cyclohexane-1-carboxylate ester (SMCC, ≥98.0%) were all obtained from
4
5 Sigma-Aldrich (St. Louis, MO). Gemcitabine (99.97%) was purchased from Selleck (Shanghai,
6
7 P. R. China). The MMP-2 enzyme cleavable peptide (NHS-Pro-Leu-Gly-Val-Arg-Gly-
8
9 maleimide, ≥97.0%) was purchased from Yarebio, Shanghai, China. The recombinant human
10
11
MMP-2 protein, recombinant human Cathepsin B protein, and recombinant human Caspase-3
12 protein were all obtained from R&D Systems (Minneapolis, MN). The MMP-2 inhibitor (N-
13
14 [(1,1'-Biphenyl)-4-ylsulfonyl]-D-phenylalanine, Rofecoxib) was obtained from Abcam
15
16 (Shanghai, P. R. China). Dulbecco’s Modified Eagle’s Medium (DMEM), RPMI-1640,
17
18 phosphate-buffered saline (PBS), and penicillin/streptomycin were obtained from Invitrogen
19 (Thermo Fisher Scientific, Carlsbad, CA). D-Luciferin potassium salt (≥95.0%) was purchased
20
21 from Sciencelight (Shanghai, P. R. China). All reagents were used as received without further
22
23 purifications. Deionized (DI) water with a resistivity of 18.2 MΩ·cm was used in all experiments
24
25 and was prepared using a Millipore ultrapure water system (Billerica, MA).
26
27 Antibodies. The monoclonal rat anti-mouse CCL-28 antibody (IgG2B, Clone#134306) was
28 obtained from R&D Systems. The monoclonal mouse anti-mouse HIF-1α (IgG1, Clone#1A3),
29
30 monoclonal mouse anti-human α-tubulin (IgG1, Clone#DM1A), monoclonal mouse anti-mouse
31
32 E-cadherin (IgG1, Clone#M168), monoclonal rabbit anti-mouse N-cadherin (IgG,
33
34 Clone#EPR1791-4), secondary Alexa Fluor 488-conjugated goat anti-mouse IgG (H+L), and
35
36
Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L) antibodies were all obtained from
37 Abcam (Shanghai China). The monoclonal rabbit anti-mouse mTOR antibody (IgG, Clone#7C10)
38
39 and phospho-mTOR antibody (Ser2481) were obtained from the mTOR pathway antibody
40
41 sampler kit (Cell Signaling Technology, Danvers, MA). The FITC-labeled mouse anti-mouse
42
43 cleaved PARP antibody (IgG1, Clone#F21-852) and Alexa Fluor 647-labeled mouse anti-mouse
44 phosphorylated H2AX antibody (IgG1, Clone#N1-431) were purchased from BD Pharmingen
45
46 (San Jose, CA).
47
48 Cell Lines. The 4T1 mouse breast cancer cell line was purchased from the Type Culture
49
50 Collection of the Chinese Academy of Sciences (Shanghai, P. R. China). 4T1 cells were cultured
51
and maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS, Hyclone,
52
53 Thermo Fisher Scientific), 1% penicillin/streptomycin, 1.5 mg/mL NaHCO3, 2.5 mg/mL glucose,
54
55 and 0.11 mg/mL sodium pyruvate. The SH-SY5Y human neuroblastoma cancer cell line was
56
57 purchased from the Shanghai Institute of Cell Biology (Shanghai, P. R. China). SH-SY5Y cells
58
59
60 18

1
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3
were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The
4
5 BxPC3 human pancreatic cancer cell line was purchased from the American Type Culture
6
7 Collection (ATCC, Virginia, USA). BxPC3 cells were transfected with the firefly luciferase
8
9 vector to generate the BxPC3/Luc+ cell line. The obtained BxPC3/Luc+ cells were cultured in
10
11
DMEM supplemented with 20% FBS and 1% penicillin/streptomycin. Cell cultures were
12 maintained in an incubator at 37°C in a humidified atmosphere with 5% CO2. The medium was
13
14 replaced every other day until the cells were approximately 85% confluent.
15
16 Animal Models. For the breast tumor model, female BALB/c mice (4-6 weeks old) were
17
18 purchased from the Comparative Medicine Centre of Yangzhou University and raised in a
19 specific pathogen-free (SPF) facility. The orthotopic breast cancer tumor model was established
20
21 in mice by subcutaneously injecting the 4T1 mouse breast cancer cell line (2 × 105 cells per
22
23 mouse) seeded in a 25% (vol/vol) Matrigel basement membrane matrix (Corning) into the second
24
25 mammary fat pad on the right side.
26
27
For the pancreatic tumor model, female BALB/c nu/nu nude mice (4-6 weeks old) were
28 purchased from the Comparative Medicine Centre of Yangzhou University and raised in a
29
30 specific pathogen-free (SPF) facility. Firstly, 3 × 106 BxPC3/Luc+ pancreatic cancer cells were
31
32 subcutaneously injected into the flank of a donor nude mouse. After 4 weeks, the donor nude
33
34 mouse was sacrificed, the tumor tissue was harvested, and the necrotic tissue was removed from
35 the section. Then, the tumor was minced into approximately 1 mm3 pieces. Nude mice were
36
37 anesthetized with an intraperitoneal injection of ketamine (20 mg/kg body weight) and xylazine
38
39 (10 mg/kg body weight). After careful sterilization with 75% alcohol, the mouse abdomen was
40
41 opened using a left longitudinal laparotomy. The pancreatic tail was gently exteriorized, and a
42
43
tissue pocket was created. A freshly prepared piece of tumor tissue was placed into the tissue
44 pocket and immediately covered with a gelatin sponge. After 5 min, the pancreas was carefully
45
46 relocated into the abdominal cavity, and the abdominal wall was closed in two layers. Mice were
47
48 imaged regularly with a bioluminescence imaging modality during the investigation.
49
50 All the protocols for the animal tests were reviewed and approved by the Committee on
51 Animals at Nanjing University and performed in accordance with the guidelines provided by the
52
53 National Institute of Animal Care.
54
55 Synthesis. PAA-modified W18O49 NPs were synthesized according to our previously
56
57 reported method.30 Briefly, 500 mg of WCl6 and 200 mg of PAA were dissolved in 100 mL of
58
59
60 19

1
2
3
DEG in an N2 atmosphere at 60 °C for 30 min. Then, the temperature was increased to 180 °C
4
5 and maintained for an additional 30 min to complete the reaction. After cooling to room
6
7 temperature, DI water was added to the suspension to induce the precipitation of the PAA-
8
9 modified W18O49 NPs. The blue product was re-suspended in DI water to a final concentration of
10
11
1018 NPs/mL.
12 For the sulfhydrylation of W18O49 NPs, 50 mg of EDC and 36 mg of sulfo-NHS were added
13
14 to 5 mL of the PAA-modified W18O49 NP suspension and allowed to react for 2 hours at room
15
16 temperature. Then, 19 mg of cysteamine was added to the solution and allowed to react for an
17
18 additional 8 hours at room temperature with gentle magnetic stirring. Thiol group-modified
19 W18O49 (T- W18O49) NPs were collected via centrifugation (16,000 rpm for 30 min) and washed
20
21 with DI water three times to remove the excess cysteamine. The T-W18O49 NPs were re-
22
23 suspended in DI water to a final concentration of 1016 NPs/mL and stored at 4 °C.
24
25 The parameters used to synthesize the amino group-modified W18O49 NPs (A-W18O49, also
26
27
denoted by WO NPs) NPs were identical to the parameters used to synthesize PAA-modified
28 W18O49 NPs, although PAA was omitted. APTES (9.85 mL) was added to 10 mL of a solution
29
30 (Ethanol: DI water, 3:1; vol/vol) containing 10 mg/mL bare W18O49 NPs. The reaction was
31
32 allowed to proceed for 24 hours at room temperature. Then, the A-W18O49 NPs were collected
33
34 via centrifugation and washed with DI water three times to remove unreacted APTES. The
35 product was suspended in DI water to a final concentration of 1017 NPs/mL.
36
37 The WOC and WOACC NPs were synthesized by adding 18.5 mg of the MMP-2 enzyme
38
39 cleavable peptide to 5 mL of the T-W18O49 NP suspension, and the coupling reaction between the
40
41 maleimide group and thiol group was allowed to proceed for 6 hours at room temperature.
42
43
Afterwards, products were collected via centrifugation and washed with PBS three times to
44 remove non-specific absorbed peptides. The precipitation was re-suspended in DI water. The A-
45
46 W18O49 NP suspension was added to obtain a final ratio of 6:1 (A-W18O49 NPs: T-W18O49 NPs),
47
48 and the coupling reaction between the NHS group and amino group was allowed to proceed for
49
50 24 hours. The final product (WOC NPs) was collected via centrifugation, washed with PBS three
51 times, and re-suspended in PBS to a final concentration of 1018 NPs/mL. Twenty micrograms of
52
53 anti-CCL28 antibody (in a 20 µL of PBS containing 0.5% NaN3) was added to 2 mL of 50 mM
54
55 Tris (pH~8.0) containing 10 mM DTT and reacted for 4 hours at 4 °C to cleave the disulfide
56
57 bond in the antibody. The SMCC-conjugated half antibody was purified using size exclusion
58
59
60 20

1
2
3
chromatography (Shimadzu, Japan) and added to 5 mL of the WOC suspension. The conjugation
4
5 process was allowed to proceed for 24 hours at 4 °C. The final product (WOACC NPs) was
6
7 collected via centrifugation and washed with PBS three times to remove the non-reacted half
8
9 antibodies. The obtained WOACC NPs were stored as powders at -20 °C until further use.
10
11
For the synthesis of WOAC NPs, the SMCC-conjugated anti-CCL-28 antibody was
12 obtained as described above, and 30 µL of PBS containing ca. 25 µg of modified half antibodies
13
14 was added to 5 mL of the A-W18O49 suspension. The reaction was allowed to proceed at 4 °C for
15
16 24 hours. The synthesized WOAC NPs were collected via centrifugation and washed three times
17
18 with PBS. The obtained WOAC NPs were stored as powders at -20 °C until further use.
19 Fluorescent labeling of various samples with DyLight 488-NHS (Thermo Fisher) was
20
21 performed according to the manufacturer’s instructions. All the fluorescently labeled platforms
22
23 were purified via centrifugation and washed with PBS five times to remove non-specific
24
25 absorbed dyes.
26
27
Electron Paramagnetic Resonance (EPR) Analysis. Freshly trypsinized 4T1 cells were
28 cultured in 24-well plates for approximately 24 hours under normal conditions. Then, cells were
29
30 co-cultured with 1 mL of medium containing WOACC NPs (1 mg of tungsten/well) for 6 hours
31
32 followed by the PTT treatment (1064-nm laser with a power density of 0.75 W/cm2 for 15 min).
33
34 Next, DEPMPO was added to this mixture, and the EPR spectra of DEPMPO/radicals adducts
35 were obtained with a Bruker EMX ESR Spectrometer (Billerica, MA, USA).
36
37 Culture of 3D Spheroid. The production of 3D tumor spheroids in vitro was performed
38
39 using a previously reported method.37 Briefly, a thin layer of poly(2-hydroxyethyl methacrylate)
40
41 (PHEMA, Sigma-Aldrich) was coated on the bottom of cell culture dishes and sterilized under
42
43
ultraviolet light for 2 hours. SH-SY5Y cells were freshly trypsinized and counted with a
44 hemocytometer (Eppendorf). Approximately 5 × 105 cells in 5 mL of fresh DMEM medium were
45
46 added to the PHEMA-coated culture dish. Cells were incubated as described above, and the
47
48 culture medium was replaced every other day. SH-SY5Y MCs (ca. 300 µm in diameter) formed
49
50 spontaneously within 7 days. All images were acquired on a scanning confocal microscope
51 (LSM 700, Zeiss) and processed on the work station.
52
53 CT Imaging. BALB/c mice bearing orthotopic breast tumors were randomly divided into
54
55 four groups and anesthetized with ketamine (20 mg/kg body weight) and xylazine (10 mg/kg
56
57 body weight) via an intraperitoneal injection. Afterwards, the mice were administered a tail vein
58
59
60 21

1
2
3
injection of 120 µL of a PBS suspension containing different NPs (WOACC NPs, WOC NPs,
4
5 WOAC NPs, and WO NPs, 30 mg tungsten/kg body weight). Images were collected before and
6
7 after the injections using an animal micro-CT (Sky scan, Bruker). Images were acquired with the
8
9 following parameters: camera pixel size 12.59 mm; source voltage 50 kV; source current 500 µA;
10
11
and number of rows and columns 1336 and 2000, respectively. Volume-rendered 3D-
12 reconstructed images were processed with Sky scan 3Dvox software using a breast cancer-
13
14 specific window.
15
16 Micro-PET Imaging. Mice were intravenously injected with 100 µL of a saline suspension
17
18 containing 18F-MISO (75 µCi/mouse) to evaluate the hypoxic conditions in breast tumor-bearing
19 mice following the different treatments. Micro-positron emission tomography (PET) scans were
20
21 performed on an Inveon PET/CT system (Siemens, Malvern, PA) 2 hours after the injection. All
22
23 the PET scanners were cross-calibrated periodically. All the data were processed and
24
25 reconstructed on the workstation.
26
27
Bioluminescence. In vivo imaging of mice bearing orthotopic pancreatic tumors was
28 performed using a bioluminescence imaging modality. Mice received an intraperitoneal injection
29
30 of a PBS solution containing D-luciferin (200 mg/kg body weight) 20 min prior to being
31
32 anesthetized with isoflurane and imaged using an IVIS 200 system (Caliper Life Sciences). Data
33
34 were processed and reconstructed on the workstation.
35 Protein Array Assay. Mice were sacrificed after receiving treatments, and tumor tissues
36
37 were collected under sterilized conditions and stored in liquid nitrogen. Antibody array assays
38
39 (Inflammation cytokine array, Ray Biotech Inc.) were conducted according to the manufacturer’s
40
41 instructions. Briefly, supernatants containing the corresponding cell lysates were incubated with
42
43
the blocked microarray glass slide at room temperature with gentle rotation, and unreacted media
44 were removed via gentle shaking with washing buffer. Next, the detection antibody cocktail and
45
46 Cy3 equivalent dye-conjugated Streptavidin were added in a stepwise manner, followed by at
47
48 least five washes with PBS. The signals were visualized using a laser scanner equipped with a
49
50 Cy3 wavelength (Axon GenePix). Data extraction was performed using ScanArray Express
51 (PerkinElmer, USA). Hierarchical clustering of differentially expressed cytokines was performed
52
53 using Cluster (version 3.0, Stanford University) and TreeView (version 1.60, Stanford University)
54
55 software.
56
57
58
59
60 22

1
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qRT-PCR Array. The analysis of miRNA using qRT-PCR array were conducted by
4
5 wcgene biotechnology corporation (Shanghai, P. R. China). Briefly, total RNAs were isolated
6
7 from 100 µL of cells samples received different treatments using single-step acidified
8
9 phenol/chloroform purification method. The viability of RNA extraction during purification was
10
11
preserved by the introduction of synthesized-exogenous RNAs, followed by polyadenylation
12 through polymerase reaction and reverse-transcription into cDNA. The content of individual
13
14 miRNA in these samples were quantified using real-time SYBR Green qRT-PCR reaction with
15
16 specific MystiCq miRNA qPCR primers (Sigma-Aldrich). All the protocols in details can be
17
18 found at the official website of wcgene (http://www.wcgene.com) free of charge.
19 Flow Cytometry Analysis. Mice were sacrificed after receiving different treatments, and
20
21 tumor tissues were removed and minced with eye scissors into 5 mm3 pieces. The tumor
22
23 fragments were incubated in DMEM supplemented with 20% FBS, 30 µg of type IV collagenase
24
25 (Sigma-Aldrich), and type I DNase (Roche) for 6 hours at 37°C. Single cancer cells were
26
27
collected via centrifugation (1,200 rpm for 5 min) and re-suspended in PBS. The cells were fixed,
28 permeabilized using the FoxP3 staining buffer set (Miltenyi Biotec), and re-suspended in 1 mL
29
30 of PBS after centrifugation. After intracellular staining with a HIF-1α primary antibody (dilution
31
32 ratio of 1: 500) and Alexa Fluor 647-labeled secondary antibody (dilution ratio of 1: 250), the
33
34 cells were characterized by flow cytometry (FACS verse, BD Bioscience), and the raw FACS
35 data were processed with FlowJo software (version 7.6.1).
36
37 Immunofluorescent Staining. Freshly dissected solid tumors were first embedded in
38
39 optimal cutting temperature compound and frozen on ice. Frozen blocks were sectioned to a
40
41 thickness of 10 µm and mounted onto glass slides. Blocking and hybridization were performed in
42
43
3% (wt/vol) BSA in PBS. The HIF-1α, E-cadherin, and N-cadherin primary antibodies were
44 diluted to 1: 500, 1: 2,000, and 1: 2,000, respectively. The secondary antibodies were diluted
45
46 1:500. The FITC-labeled cleaved PARP antibody and Alexa Fluor 647-labeled phosphorylated
47
48 H2AX antibody were diluted 1: 250. All images were acquired on a fluorescence microscope
49
50 (BX51, Olympus, Japan) and processed using Imaris software (version 7.2.3, Bitplane).
51 Colony Formation Assay. Freshly trypsinized-4T1 cells were cultured in 6-well plates for
52
53 approximately (800 cells/well) 24 hours under normal conditions. The cells were co-cultured
54
55 with 2 mL of medium containing WOACC NPs (3 mg tungsten/well) for 6 hours. For the NAC
56
57 treatment, NAC was added to the culture medium (5 mg/mL) 4 hours prior to the addition of
58
59
60 23

1
2
3
WOACC NPs. Hypoxic conditions were induced with CoCl2. X-ray irradiation was performed in
4
5 a micro X-ray irradiator (RS 2000, Radsource) with a total of 3 Gy of irradiation. The PTT
6
7 treatment (1 prior to the radiation) was conducted using a 1064-nm laser with a power density of
8
9 0.75 W/cm2 for 15 min. After a 14 day of incubation, the colonies were fixed with methanol,
10
11
stained with 0.5% crystal violet (Beyotime Biotechnology, Shanghai, P. R. China) in ethanol,
12 and colonies with ≥40 cells were counted under a bright field microscope.
13
14 Bio-Distribution. Mice bearing breast tumors were randomly divided into two groups
15
16 followed by i.v. injection of different NPs at a dose of 30 mg tungsten/kg body weight. Mice
17
18 were sacrificed, tumor tissues and blood were harvested, and tumors were weighed at different
19 time intervals. The tissue and blood samples were digested with freshly prepared aqua regia by
20
21 repeatedly heating the samples to boiling until the solution became pellucid. Nitric acid was
22
23 added to reach a final volume of 1 mL. The concentration of tungsten in each sample was
24
25 determined with inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7500ce). The
26
27
bio-distribution profiles were presented as percentage of the injected dose per gram organ weight
28 (% ID/g tissue) as a function of time.
29
30 Bio-TEM. The tissue was harvested from the central solid tumor and fixed in modified
31
32 Karnovsky’s fixative (1.5% glutaraldehyde, 3% paraformaldehyde, and 5% sucrose in 0.1 M
33
34 cacodylate buffer at pH ca. 7.4), followed by incubation with 2% osmium tetroxide fixation
35 buffer (Sigma-Aldrich) for an additional 2 hours to determine the tumor ultrastructure using bio-
36
37 TEM. Then, the sections were stained with 1% uranyl acetate and dehydrated in ethanol.
38
39 Samples were embedded in epoxy resin, sectioned into pieces in 60-70 µm thickness, and placed
40
41 on copper grids. The sample was further stained with lead nitrate and uranyl acetate prior to
42
43
characterization.
44 Long-Term Cytotoxicity. Nude mice that had received an i.v. injection of WOACC NPs
45
46 were sacrificed 1, 7, 15, and 30 days after treatment. Blood was collected in a sodium EDTA
47
48 anticoagulant tube for the hematology study. Potential cytotoxic and allergic effects were
49
50 verified by determining the variations in the red blood cell (RBC), eosinophil (EOS), and
51 basophil (BAS) counts. The immune response was determined by investigating the numbers of
52
53 lymphocytes (LYM), monocytes (MON), and neutrophils (NEU). Spleen function was verified
54
55 by examining platelet (PLT) production. Biochemical analyses were performed by collecting
56
57 serum from the blood in a coagulation tube containing a separation tube. Liver function was
58
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60 24

1
2
3
evaluated by determining the serum levels of alanine aminotransferase (ALT), total protein (TP),
4
5 and total bilirubin level (TBIL). Kidney function was determined by measuring the serum blood
6
7 urea nitrogen (BUN) and creatinine (CRE) levels.
8
9 Statistics. Triplicate data were analyzed with Student’s t test using GraphPad Prism
10
11
software (version 7.0); the significance level was p<0.05. Significant differences are indicated by
12 asterisks in corresponding figures.
13
14
15
16 ASSOCIATED CONTENT
17
18 Supporting Information
19 The supporting Information is available free of charge on the ACS Publication website at DOI:
20
21 10.1021/acsnano.xx.
22
23
24
25 The authors have no competing financial interests to disclose.
26
27
28 AUTHOR INFORMATION
29
30 Corresponding Authors
31
32 *E-mail: zyzhou@nju.edu.cn;
33 *E-mail: dndxzh@njmu.edu.cn;
34 *E-mail: hvyong@nju.edu.cn;
35
36
37 Author contributions
38
39
The manuscript was drafted with contributions from all authors. All authors have approved the
40 final version of manuscript.
41
42
43
44 ACKNOWLEDGMENTS
45
46 This work was supported by the National Key R&D Program of China: 2017YFA0205400,
47 National Natural Science Foundation of China (No. 21474047, 81671751, 81371516, and
48
49 81502608), the Technique Development Foundation of Nanjing (Outstanding Youth Foundation,
50
51 JQX15004) and the Industry-University-Research Collaboration project of Jiangsu Province
52
53 (BY2015041-01). D.H. would like to give special thanks to wcgene biotechnology corporation
54
for their help of the qRT-PCR analysis.
55
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REFERENCES
4
5 (1) Travis, L. B.; Ng, A. K.; Allan, J. M.; Pui, C. H.; Kennedy, A. R.; Xu, X. G. Second
6
7 Malignant Neoplasms and Cardiovascular Disease Following Radiotherapy. J. Natl. Cancer Inst.
8
9 2012, 106, 229-46.
10
11
(2) Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. Global Cancer
12 Statistics. CA Cancer J. Clin. 2011, 61, 69-90.
13
14 (3) Baumann, R.; Depping, R.; Delaperriere, M. Targeting Hypoxia to Overcome Radiation
15
16 Resistance in Head & Neck Cancers: Real Challenge or Clinical Fairytale. Expert Rev.
17
18 Anticancer Ther. 2016, 16, 751-758.
19 (4) Schaue, D.; McBride, W. H. Opportunities and Challenges of Radiotherapy for Treating
20
21 Cancer. Nat. Rev. Clin. Oncol. 2015, 12, 527-540.
22
18
23 (5) Hendrickson, K.; Phillips, M.; Smith, W. Hypoxia Imaging with FMISO-PET in Head
24
25 and Neck cancer: Potential for Guiding Intensity Modulated Radiation Therapy in Overcoming
26
27
Hypoxia-Induced Treatment Resistance. Radiother. Oncol. 2011, 101, 369-375.
28 (6) Masuda, S.; Belmonte, J. C. I. The Microenvironment and Resistance to Personalized
29
30 Cancer Therapy. Nat. Rev. Clin. Oncol. 2012, 10, 79.
31
32 (7) Mo, R.; Gu, Z.; Tumor Microenvironment and Intracellular Signal-activated
33
34 Nanomaterials for Anticancer Drug Delivery. Mater. Today 2016, 19, 274-283.
35 (8) Vaupel, P.; Mayer, A. Hypoxia in Cancer: Significance and Impact on Clinical Outcome.
36
37 Cancer Metastasis Rev. 2007, 26, 225-239.
38
39 (9) Barker, H. E.; Paget, J. T. E.; Khan, A. A.; Harrington, K.J.; The Tumour
40
41 Microenvironment after Radiotherapy: Mechanisms of Resistance and Recurrence. Nat. Rev.
42
43
Cancer 2015, 15. 409-25.
44 (10) Barnett, G. C.; West, C. M. L.; Dunning, A. M.; Elliott, R. M.; Coles, C. E.; Pharoah, P.
45
46 D. P.; Burnet, N. G. Normal Tissue Reactions to Radiotherapy: Towards Tailoring Treatment
47
48 Dose by Genotype. Nat. Rev. Cancer 2009, 9, 134-142.
49
50 (11) Kim, J. J.; Tannock, I. F. Repopulation of Cancer Cells During Therapy: An Important
51 Cause of Treatment Failure. Nat. Rev. Cancer 2005, 5, 516-25.
52
53 (12) Song, M.; Liu, T.; Shi, C.; Zhang, X.; Chen, X. Bioconjugated Manganese Dioxide
54
55 Nanoparticles Enhance Chemotherapy Response by Priming Tumor-Associated Macrophages
56
57 Toward M1-Like Phenotype and Attenuating Tumor Hypoxia. ACS Nano 2016, 10, 633-647.
58
59
60 26

1
2
3
(13) Teicher, A. Hypoxia and Drug Resistance, Cancer Metastasis Rev. 1994, 13,139-168.
4
5 (14) Baumann, M.; Appold, S.; Zimmer, J.; Scharf, M.; Beuthien-Baumann, B.; Dubben, H.
6
7 H.; Enghardt, W.; Schreiber, A.; Eicheler, W.; Petersen, C. Radiobiological Hypoxia, Oxygen
8
9 Tension, Interstitial Fluid Pressure and Relative Viable Tumour Area in Two Human Squamous
10
11
Cell Carcinomas in Nude Mice During Fractionated Radiotherapy. Acta Oncol. 2001, 40, 519-
12 528.
13
14 (15) Kunz-Schughart, L. A.; Dubrovska, A.; Peitzsch, C.; Ewe, A.; Aigner, A.; Schellenburg.,
15
16 S.; Muders, M. H.; Hampel, S.; Cirillo, G.; Lemma, F.; Tietze, R.; Alexiou, C.; Stephan, H.;
17
18 Zarschler, K.; Vittorio O.; Kavallaris, M.; Parak, W. J.; Mädler, L.; Pokhrel, S. Nanoparticles for
19 Radiooncology: Mission, Vision, Challenges. Biomaterials 2016, 120, 155-184.
20
21 (16) Zhang, X. D.; Wu, D.; Shen, X.; Chen, J.; Sun Y. M.; Liu, P. X.; Liang, X. J. Size-
22
23 Dependent Radiosensitization of PEG-Coated Gold Nanoparticles for Cancer Radiation Therapy.
24
25 Biomaterials 2012, 33, 6408-6419.
26
27
(17) Wang, Y.; Wu, Y. Y.; Liu, Y. J.; Shen, J.; Lv, L.; Li, L. B. BSA-mediated Synthesis of
28 Bismuth Sulfide Nanotheranostic Agents for Tumor Multimodal Imaging and
29
30 Thermoradiotherapy. Adv. Funct. Mater. 2016, 26, 5335-5344.
31
32 (18) Kwatra, D., Venugopal1, A.; Anant, S. Nanoparticles in Radiation Therapy: A Summary
33
34 of Various Approaches to Enhance Radiosensitization in Cancer. Transl. Cancer Res. 2013, 2,
35 330-342.
36
37 (19) Zhang, X. D.; Chen, J.; Luo, Z. T.; Wu, D.; Shen, X.; Song, S. S.; Sun, Y. M.; Liu, P. X.;
38
39 Zhao, J.; Huo, S. D.; Fan, S. J.; Fan, F. Y.; Liang, X. J.; Xie, J. P. Enhanced Tumor
40
41 Accumulation of Sub-2 nm Gold Nanoclusters for Cancer Radiation Therapy. Adv. Healthcare
42
43
Mater. 2014, 3, 133-141.
44 (20) Yu, M.; Liu, J.; Ning, X.; Zheng, J. High-Contrast Noninvasive Imaging of Kidney
45
46 Clearance Kinetics Enabled by Renal Clearable Nanofluorophores. Angew. Chem. Int. Ed. 2015,
47
48 54, 15434-15438.
49
50 (21) Huo, S.; Ma, H.; Huang, K.; Liu, J.; Wei, T.; Jin, S.; Zhang, J.; He, S.; Liang, X.-J.
51 Superior Penetration and Retention Behavior of 50 nm Gold Nanoparticles in Tumors. Cancer
52
53 Res. 2012, 73, 319-330.
54
55 (22) Black, K. C. L.; Wang, Y.; Luehmann, H. P.; Cai, X.; Xing, W.; Pang, B.; Zhao, Y.;
56 198
57 Cutler, C. S.; Wang, L. V.; Liu, Y.; Xia, Y. Radioactive Au-Doped Nanostructures with
58
59
60 27

1
2
3
Different Shapes for In Vivo Analyses of Their Biodistribution, Tumor Uptake, and Intratumoral
4
5 Distribution. ACS Nano 2014, 8, 4385-4394.
6
7 (23) Sun, Q.; Zhou, Z.; Qiu, N.; Shen, Y. Rational Design of Cancer Nanomedicine:
8
9 Nanoproperty Integration and Synchronization. Adv. Mater. 2017, 29, 1606628.
10
11
(24) Sun, W.; Sun, X.; Ma, X.; Zhou, Z.; Jin, E.; Zhang, B.; Shen, Y.; Van Kirk, E. A.;
12 Murdoch, W. J.; Lott, J. R.; Lodge, T. P.; Radosz. M.; Zhao, Y. Integration of Nanoassembly
13
14 Functions for An Effective Delivery Cascade for Cancer Drugs. Adv. Mater. 2014, 26, 7615-
15
16 7621.
17
18 (25) Lu, Y.; Aimetti, A.; Langer, R.; Gu, Z. Bioresponsive Materials. Nat. Rev. Mater. 2016,
19 2, 16075.
20
21 (26) Facciabene, A.; Peng, X.; Hagemann, I. S.; Balint, K.; Barchetti, A.; Wang, L. P.;
22
23 Gimotty, P. A.; Gilks, C. B.; Lal, P.; Zhang, L.; Coukos, G. Tumor Hypoxia Promotes Tolerance
24
25 and Angiogenesis via CCL28 and Treg Cells. Nature 2011, 475, 226-230.
26
27
(27) Balkwill, F. R. The Chemokine System and Cancer. J. Pathol. 2012, 226, 148-157.
28 (28) Turk, B. E.; Huang, L. L.; Piro, E. T.; Cantley, L. C. Determination of Protease Cleavage
29
30 Site Motifs Using Mixture-Based Oriented Peptide Libraries. Nat. Biotechnol. 2001, 19, 661-667.
31
32 (29) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Horia, K. Anaplastic Lymphoma Kinase
33
34 Inhibition in Non-Small-Cell Lung Cancer. J. Control. Rel. 2000, 65, 271-284.
35 (30) Huo, D.; He, J.; Li, H.; Huang, A. J.; Zhao, H. Y.; Ding, Y.; Zhou, Z. Y.; Hu, Y. X-ray
36
37 CT Guided Fault-Free Photothermal Ablation of Metastatic Lymph Nodes with Ultrafine HER-2
38
39 Targeting W18O49 Nanoparticles. Biomaterials 2014, 35, 9155-9166.
40
41 (31) Kalluru, P.; Vankayala, R.; Chiang, C. S.; Hwang, K. C. Photosensitization of Singlet
42
43
Oxygen and In Vivo Photodynamic Therapeutic Effects Mediated by PEGylated W18O49
44 Nanowires. Angew. Chem. Int. Ed. 2013, 52, 12332-12336.
45
46 (32) Chen, Z.; Wang, Q.; Wang, H.; Zhang, L.; Song, G.; Song, L.; Hu, J.; Wang, H.; Liu, J.;
47
48 Zhu, M.; Zhao, D. Ultrathin PEGylated W18O49 Nanowires as A New 980 nm-Laser-Driven
49
50 Photothermal Agent for Efficient Ablation of Cancer Cells In Vivo. Adv. Mater. 2013, 25, 2095-
51 2100.
52
53 (33) Wang, L.; Sun, Q.; Wang, X.; Wen, T.; Yin, J. J.; Wang, P.; Bai, R.; Zhang, X. Q.; Zhang,
54
55 L. H.; Lu, A. H.; Chen, C. Using Hollow Carbon Nanospheres as a Light-Induced Free Radical
56
57 Generator to Overcome Chemotherapy Resistance. J. Am. Chem. Soc. 2015, 137, 1947-1955.
58
59
60 28

1
2
3
(34) Dewhirst, M. W.; Cao, Y.; Moeller, B. Cycling Hypoxia and Free Radicals Regulate
4
5 Angiogenesis and Radiotherapy Response. Nat. Rev. Cancer 2008, 8, 425-437.
6
7 (35) Wong, C.; Stylianopoulos, T.; Cui, J.; Martin, J.; Chauhan, V. P.; Jiang, W.; Popovíc, Z.;
8
9 Jain, R. K.; Bawendi, M. G.; Fukumura, D. Multistage Nanoparticle Delivery System for Deep
10
11
Penetration into Tumor Tissue. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 2426-2431
12 (36) Lia, H. J.; Du, J. Z.; Du, X. J.; Xu, C. F.; Sun, C. Y.; Wang, H. X.; Zhi-Ting Cao, Z. T.;
13
14 Yang, X. Z.; Zhu, Y. H.; Nie, S. M.; Wang, J. Stimuli-Responsive Clustered Nanoparticles for
15
16 Improved Tumor Penetration and Therapeutic Efficacy, Proc. Natl. Acad. Sci. 2016, 113, 4164-
17
18 4169.
19 (37) Wang, X.; Zhen, X.; Wang, J.; Zhang, J.; Wu, W.; Jiang, X. Doxorubicin Delivery to 3D
20
21 Multicellular Spheroids and Tumors Based on Boronic Acid-Rich Chitosan Nanoparticles
22
23 Biomaterials 2013, 34, 4667-4679.
24
25 (38) Quail, D. F.; Joyce, J.A. Microenvironmental Regulation of Tumor Progression and
26
27
Metastasis. Nat. Med. 2013, 19, 1423-1437.
28 (39) Nair, C. K. K.; Parida, D. K.; Nomura, T. Radioprotectors in Radiotherapy. J. Radiat. Res.
29
30 2001, 42, 21-37.
31
32 (40) Liu, J. Q.; Sham, J. S. K.; Shimoda, L. A.; Kuppusamy, P.; Sylvester, J. T. Hypoxic
33
34 Constriction and Reactive Oxygen Species in Porcine Distal Pulmonary Arteries. Am. J. Physiol.:
35 Lung Cell. Mol. Physiol. 2003, 285, 322-333.
36
37 (41) Beuvink, I.; Boulay, A.; Fumagalli, S.; Zilbermann, F.; Ruetz, S.; O'Reilly, T.; Natt, F.;
38
39 Hall, J.; Lane, H. A.; Thomas, G. The mTOR Inhibitor RAD001 Sensitizes Tumor Cells to
40
41 DNA-Damaged Induced Apoptosis Through Inhibition of p21 Translation. Cell 2005, 120, 747-
42
43
759.
44 (42) Kao, G. D.; Jiang, Z.; Fernandes, A. M.; Gupta, A. K.; Maity, A. Inhibition of
45
46 Phosphatidylinositol-3-OH Kinase/Akt Signaling Impairs DNA Repair in Glioblastoma Cells
47
48 Following Ionizing Radiation. J. Biol. Chem. 2007, 282, 21206-21212.
49
50 (43) Lavin, M. F.; Kozlov, S.; ATM Activation and DNA Damage Response. Cell cycle 2007,
51 6, 931-942.
52
53 (44) Bodine, S. C.; Stitt, T. N.; Gonzalez, M.; Kline, W. O.; Stover, G. L.; Bauerlein, R.;
54
55 Zlotchenko, E.; Scrimgeour, A.; Lawrence, J. C. Glass, D. J.; Yancopoulos, G. D. Akt/mTOR
56
57
58
59
60 29

1
2
3
Pathway is A Crucial Regulator of Skeletal Muscle Hypertrophy and Can Prevent Muscle
4
5 Atrophy In Vivo. Nat. Cell Biol. 2001, 3, 1014-1019.
6
7 (45) Zhang, Y.; Poon, W.; Tavares, A. J.; McGilvray, I. D.; Chan, W. C. Nanoparticle-Liver
8
9 Interactions: Cellular Uptake and Hepatobiliary Elimination. J. Control. Rel. 2016, 240, 332-348.
10
11
(46) O'Rourke, R. W.; White, A. E.; Metcalf, M. D.; Olivas, A. S.; Mitra, P.; Larison, W. G.;
12 Cheang, E. C.; Varlamov, O.; Corless, C. L.; Roberts, C. T. Jr.; Marks, D. L. Hypoxia-Induced
13
14 Inflammatory Cytokine Secretion in Human Adipose Tissue Stromovascular Cells. Diabetologia
15
16 2011, 54, 1480-1490.
17
18 (47) Balkwill, F.; Mantovani, A. Inflammation and Cancer: Back to Virchow? The Lancet.
19 2001, 357, 539-545.
20
21 (48) Stucki, M.; Clapperton, J. A.; Mohammad, D.; Yaffe, M. B.; Smerdon, S. J.; Jackson, S.
22
23 P. MDC1 Directly Binds Phosphorylated Histone H2AX to Regulate Cellular Responses to DNA
24
25 Double-Strand Breaks. Cell 2005, 123, 1213-1226.
26
27
(49) Tewari, M.; Quan, L. T.; O'Rourke, K.; Desnoyers, S.; Zeng, Z.; Beidler, D. R.; Poirier,
28 G. G.; Salvesen, G. S.; Dixit, V. M. Yama/CPP32β, A Mammalian Homolog of CED-3, is A
29
30 CrmA-Inhibitable Protease That Cleaves the Death Substrate Poly(ADP-ribose) Polymerase. Cell
31
32 1995, 81, 801-809.
33
34 (50) Vaupel, P.; Mayer, A. Hypoxia in Cancer: Significance and Impact on Clinical Outcome.
35 Cancer Metastasis Rev. 2007, 26, 225-239.
36
37 (51) Von Hoff, D. D.; Ervin, T.; Arena, F. P.; Chiorean, E. G.; Infante, J.; Moore, M.; Seay, T.
38
39 Randomized Phase III Study of Weekly Nab-Paclitaxel Plus Gemcitabine Versus Gemcitabine
40
41 Alone in Patients with Metastatic Adenocarcinoma of the Pancreas (MPACT). N. Engl. J. Med.
42
43
2013, 369, 1691-1703.
44 (52) Sherman, M. H.; Yu, R. T.; Engle, D. D.; Ding, N.; Atkins, A. R.; Tiriac, H. Vitamin D
45
46 Receptor-Mediated Stromal Reprogramming Suppresses Pancreatitis and Enhances Pancreatic
47
48 Cancer Therapy. Cell 2014, 159, 80-93.
49
50 (53) Melamed, J. R.; Edelstein, R. S.; Day, E. S. Elucidating the Fundamental Mechanisms of
51 Cell Death Triggered by Photothermal Therapy. ACS Nano 2015, 9, 6-11.
52
53
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21 Figure 1. (a) Scheme illustration showing how MMP2-responsive WOACC NPs integrate active
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23 hypoxia-targeting properties with CT imaging, local PTT, and radical-enhanced radiotherapy.
24
25 These WOACC NPs maintain their original size during blood circulation, and destructed to
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release hypoxia microenvironment targeting WOAC NPs upon MMP2 simulation. (b) TEM
28 image of WOACC NPs in low and high (inset) resolutions. (c) UV-vis-NIR profiles of the as-
29
30 obtained WOACC NPs, WOAC NPs released from WOACC NPs, and the original W18O49 NPs.
31
32 (d) Temperature variation profiles of the WOACC NP suspension (1 mg/mL) and WOAC NPs (1
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34 mg/mL) receiving multi-laser irradiation (1 and 0.2 W/cm2). PBS received laser irradiation (1
35 W/cm2) was taken as control group. (e) DLS analysis showing the size variation of WOACC NPs
36
37 incubated with Cathepsin B (500 U/mL), Caspase-3 (200 U/mL), and MMP-2 (200 U/mL)
38
39 enzymes for 2 hours. Untreated WOACC NPs were taken for comparison.
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29 Figure 2. (a) CLSM images of SH-SY5Y 3D multicellular spheroids treated with (a1) FITC-
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31 labeled WO NPs, (a2) FITC-labeled WOCC NPs, (a3) FITC-labeled WOAC NPs, (a4) FITC-
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33 labeled WOACC NPs, and (a5) FITC-labeled WOACC NPs that had been pretreated with an
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35 MMP-2 inhibitor. Scale bars: 50 µm. In vitro distribution profiles of WOACC NPs and WOAC
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NPs (injection dose: 30 mg tungsten/kg body weight) in (b) blood and (c) tumor tissue. (n = 3
38 each time point) *p<0.05, **p<0.01, ***p<0.001.
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34 Figure 3. (a) Flow cytometry data for Annexin V-FITC/PI-stained normoxic and hypoxic cells
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36 received different treatments. PTT+RT: Synergistic treatment with WOACC; PTT: Photothermal
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38 treatment with WOACC; RT+WOACC: Radiosensitization with WOACC; RT: Radiation alone;
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40 WOACC: Incubation with WOACC without treatment. (b) EPR spectra showing the presence of
41 radicals produced by WOACC NPs upon laser irradiation. (c) Flow-cytometry showing the
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43 expression of reactive oxygen species in 4T1 cells received different treatments. The cells were
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45 stained with H2DCFDA probe within 30 min post treatment. (d) Western blotting results
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47 showing the expression of phosphorylation of the mTOR, Akt, and AMPK proteins in cells
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received different treatments. I: Control; II: Radiation; III: WOACC; IV: PTT with WOACC; V:
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50 PTT with WOACC plus mTOR agonist; VI: PTT with WOACC plus Akt agonist; VII: PTT with
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52 WOACC; VIII: Normal non-cancerous cell (HUVEC). Tubulin was taken as gate-keeper protein.
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54 (e) qRT-PCR results showing the expression of several miRNA in cells received WOACC PTT
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in the presence or absence of mTOR agonist. #p<0.001
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25 Figure 4. (a) Contrast-enhanced CT images of tumor lesions post-i.v. injection of (b1) WOACC
26 NPs, (b2) WOCC NPs, (b3) WOAC NPs, and (b4) WO NPs (30 mg tungsten/kg body weight).
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28 The orange arrow highlights the diffusion path of samples from the tumor boundary (red star) to
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30 the deep tumor region (yellow star). (b) Bio-TEM images of deep tumor tissues showing the
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32 presence of WOAC NPs. Right panel: WOAC NPs (pointed by red arrow) released from
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WOACC NPs were observed in high resolution TEM images of randomly depicted sections
35 (boxed in b). (c) HU values of regions of interest at different depths along the diffusion pathway
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37 (red star to yellow star in (a)).
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28 Figure 5. (a) Immunofluorescence images showing the intra-tumor distribution of fluorescently
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labeled NPs (a1: WOACC NPs; a2: WOCC NPs; a3: WOAC NPs; a4: WO NPs). Scale bar: 5
31 µm. (b) The accumulation profiles of different NPs in hypoxic regions (HIF-1 positive) pots-i.v.
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33 injection as verified by flow cytometry. (c) Co-localization of NPs with HIF-1-positive cancer
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35 cells (indicated by the red frame in (b)). The activities of the MMP-2 enzyme (+MMP-2 inhibitor)
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37 and CCL-28 chemokine (+CCL-28 antibody) were blocked prior to the administration of NPs. (d)
38 Immunofluorescent staining of tumor tissue sections from mice that received (I) synergistic
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40 treatment (WOACC NPs plus PTT and RT), (II) PTT with WOACC NPs, (III) radiation alone,
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42 and (IV) WOACC NPs-sensitized radiotherapy. Scale bar: 5 µm.
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Figure 6. (a) PET images of mice received different treatments (at day 25, I: Saline II:
29 Combined treatment; III: PTT post-i.v. injection of WOACC NPs; IV: RT post-i.v. injection of
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31 WOACC NPs). Tumor lesions are highlighted by a white circle. Right panel in each group shows
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33 the images of the tumor in transverse view. (b) SUV and (c) normalized values (to the weight) in
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35 solid tumor regions were obtained from PET images captured at 1 h post-i.v. of F-MISO.
36 #p<0.001.
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18 Figure 7. (a) Tumor volumes of orthotopic breast tumors of mice received different treatments.
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20 #p<0.001. (b) Survival profiles of tumor-bearing mice received different treatments. I:
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22 synergistic treatment; II: PTT post-i.v. injection of WOACC NPs (1064 nm laser, 1 W/cm2 for 15
23 min); III: Radiation (3 Gy) post-i.v. injection of WOACC NPs; IV: i.v. injection of WOACC (20
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25 mg/Kg body weight); V: i.v. injection of saline. (n = 7 in each group)
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28 Figure 8. Immunofluorescent images showing the expression of E-cadherin and N-cadherin in
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30 tumor tissues of mice received different treatments. PTT+RT: synergistic treatment with
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32 WOACC NPs; RT: radiation; PTT: Photothermal treatment with WOACC; Control: i.v. injection
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34 of saline. Scale bar: 5 µm.
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38 Figure 9. (a) Representative pathological features of spontaneous metastasis to the lung and liver,
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40 as indicated by both necropsy and H&E staining. The boundary between normal tissue and
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42 neoplasia was highlighted by black line to guide the eyes. (b) Liver and lung metastasis profiles
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of mice received different treatments. (each “+” indicates that one mouse exhibited positive
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45 metastasis in a specific tissue. I: synergistic treatment; II: RT post-i.v. injection of WOACC NPs;
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47 III: PTT post-i.v. injection of WOACC NPs; IV: i.v. injection of saline.
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28 Figure 10. (a) BLI images of orthotopic pancreatic tumor-bearing nude mice following
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30 treatments with saline (Control), gemcitabine, radiotherapy (RT), and synergistic treatment with
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WOACC NPs (WOACC). (b) Necropsy results of tumor lesions in mice received different
33 treatments (St: stomach; T: tumor (black line); P: pancreas (red line); Sp: spleen). The pancreas
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35 is highlighted (inset in each graph). (c) The hierarchical clustering analysis showing the
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37 inflammatory response in tumor tissue of mice received different treatments (I: Control; II:
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39 synergistic treatment; III: chemotherapy with gemcitabine; IV: Radiation).
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34 Figure 11. (a) Studies of the long-term toxicity of WOACC NPs in the brain, lung, heart,
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36 stomach, pancreas, and colon at days 7, 15, and 30 post-i.v. injection of WOACC NPs using
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38 H&E staining. (b) The levels of immune cells in the blood circulation and biomarkers in serum
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TABLE of CONTENTS
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7 Cluster bomb-like nanoparticles with enhanced blood availability were constructed from
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9 covalently bound ultrafine tungsten nanoparticles. These cluster bomb-like nanoparticles were
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degraded into small nanospheres once in the tumor microenvironment and actively targeted the
12 deep hypoxic tumor lesion. Photo-thermal therapy and radical production can be realized
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14 simultaneously upon laser irradiation, in an order to enhance the radiation-susceptibility of
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16 hypoxic cancer cells.
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ACS Nano Volume Issue 2017 (Doi 10.1021/acsnano.7b04737) Huo, Da Liu, Sen Zhang, Chao He, Jian Zhou, Zhengyang Zhang - Hypoxia-Targeting, Tumor Microenvironment Responsive Nano-Cluster Bomb Fo PDF

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ACS Nano Volume Issue 2017 (Doi 10.1021/acsnano.7b04737) Huo, Da Liu, Sen Zhang, Chao He, Jian Zhou, Zhengyang Zhang - Hypoxia-Targeting, Tumor Microenvironment Responsive Nano-Cluster Bomb Fo PDF

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