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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 Downloaded from http://pubs.acs.org on October 10, 2017
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Revised MS# nn-2017-047375.R1
Hypoxia-Targeting, Tumor Microenvironment Responsive NanoCluster Bomb for Radical-Enhanced Radiotherapy
Da Huo,†,‡ Sen Liu,‡ Chao Zhang,‡ Jian He,† Zhengyang Zhou,†* Hao Zhang,§* and Yong Hu†,‡*
†
Department of Radiology, Drum Tower Hospital, School of Medicine, Nanjing University, Nanjing, Jiangsu, China, 210008, China.
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Collaborative Innovation Center of Chemistry for Life Sciences, College of Engineering and Applied Sciences, Nanjing University, Nanjing, Jiangsu, 210093, China.
§
Department of Oncology First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, 210029, China.
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ABSTRACT: Although ultra-small metal nanoparticles (NPs) have been used as radiosensitizers to enhance the local damage to tumor tissues while reducing injury to the surrounding organs, their rapid clearance from the circulatory system and the presence of hypoxia within the tumor continue to hamper their further application in radiotherapy (RT). In this study, we report a size tunable nano-cluster bomb with a initial size of approximately 33 nm featuring a long half-life during blood circulation, and destructed to release small hypoxia microenvironment-targeting NPs (~5 nm) to achieve deep tumor penetration. Hypoxic profiles of solid tumors were precisely imaged using NP-enhanced computed tomography (CT) with higher spatial resolution. Once irradiated with a 1064-nm laser, CT-guided, local photo-thermal ablation of the tumor and the production of radical species could be achieved simultaneously. The induced-radical species alleviated the hypoxia-induced resistance and sensitized the tumor to the killing efficacy of radiation in Akt-mTOR pathway dependent manner. The therapeutic outcome was assessed in animal models of orthotopical breast cancer and pancreatic cancer, supporting the feasibility of our combinational treatment in hypoxic tumor management.
KEYWORDS: nanoparticles, hypoxia, radiotherapy, radiosensitization, photo-thermal therapy, tungsten
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Radiotherapy (RT) has long been regarded as the one of the most feasible and routinely used therapeutic modalities in the battle against cancer.1,2 However, the clinical application of RT in cancer therapy is seriously hampered by the hypoxia-induced resistance of the tumor and the unexpected injury to normal tissue by the high dosage of irradiation.3-5 Hypoxia, described as insufficient oxygen supply, has proven to be one of the primary driving forces for tumor angiogenesis and metastasis.6-8 Unfortunately, traditional low dosage RT might exacerbate the hypoxic conditions, rendering tumor cells less vulnerable to radiation-induced killing.9 Furthermore, the tumor cells that survive RT become more apoptosis-resistant and contribute greatly to tumor relapse.10,11 Moreover, the enrichment of tumor-promoting cells (e.g., alternative activated macrophages and cancer stem cells) in hypoxic regions have also been confirmed,12 which also integrate abilities to protect themselves from RT-induced cytolysis and thus contribute to rapid local recurrence.13 Therefore, clinical methods that increase the sensitivity of hypoxic tumor cells to RT and reverse or alleviate the hypoxic microenvironment in solid tumors are crucial for achieving optimal therapeutic outcomes of RT. Although perfusion of oxygen gas into solid tumors can up-regulate the oxygen pressure inside the tumor, the high interstitial pressure in the hypoxic region greatly hinders the penetration of oxygen gas, making this method less valid for the sensitization of whole tumor mass.14 Recently, the potential of ultra-small metal NPs as radiosensitizers to potentiate the killing efficacy of RT has been tested.15-17 Upon irradiation, these NPs can strongly absorb the γray or X-ray irradiation to produce secondary electrons, thus enhancing the localized irradiation dosage and boosting the susceptibility of cancer cells to the radiation while protecting the surrounding healthy tissue from RT-induced injury.18 In addition, these ultra-small NPs can penetrate deeply and homogeneously inside the tumor, where an even killing induced by radiation could be achieved thereafter.19 The major drawback of this method is the lack of ability to alleviate the hypoxic condition within the tumor, which drives the local relapse post RT. Moreover, considering their size, these NPs are subjected to limited blood availability, and thus leading to reduced tumor accumulation and limited enhancement.20,21 In contrast, larger NPs exhibiting a long half-life in the circulatory system in vivo suffer from a short intra-tumor penetration depth, leading to compromised sensitization effect because of the limited 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 reach optimal radiosensitization effect for RT. Recently, size tunable NPs were successfully fabricated as drug delivery systems and provided additional tenability in the spatial control of delivery to solid tumors.23-25 Most of these NPs maintain their initially larger size during circulation to achieve the longer half-life and passive targeting ability to the tumor via the enhanced permeability and retention (EPR) effect. Upon reaching the tumor site, NPs are transformed into small NPs in response to stimuli (e.g. enzyme, pH) from the tumor microenvironment to facilitate deep tumor penetration. Although these sophisticated nanomedicines have successfully resolved the contradictory observations of a long blood half-life and deep tumor penetration, they are not suitable for RT because most of them are composed of polymers, which are less sufficient for radiosensitization. To this end, we developed a type of nano-cluster bomb consisting of hypoxic microenvironment-targeting non-stoichiometric tungsten oxide NPs (WO NPs) being taken as radiosensitizers for RT. Since the CCL-28 chemokine is over-expressed in hypoxic tumor microenvironments and is validated to have a crucial role in driving tumor angiogenesis and tolerance,26,27 WO NPs were firstly modified with ligands targeting the CCL-28 chemokine (WOAC NPs). Then, clusters of WOAC NPs (WOACC NPs) were covalently bound using a matrix metalloproteinase-2 (MMP-2) cleavable peptide (Pro-Leu-Gly-Val-Arg-Gly).28 The halflife of WOACC NPs in blood was increased compared to that of WOAC NPs because of their enlarged size. Once these WOACC NPs accumulated inside the tumor due to the EPR effect,29 up-regulated expression of the MMP-2 enzyme in the tumor microenvironment triggered the destruction of these WOACC NPs to release small WOAC NPs (Figure 1a), which deeply penetrated into the tumor attributed to their small sizes. Considering the robust CT-enhancing and photo-thermal conversion abilities of WO NPs,30-32 hypoxic conditions could be precisely investigated under the guidance of computed tomography (CT), followed by imaging-guided photo-thermal therapy (PTT). Meanwhile, highly reactive radicals were simultaneously produced in hypoxic regions during PTT, which could alleviate the hypoxic induced resistance in AktmTOR dependent manner,33 subsequently enhancing the susceptibility of hypoxic cancer cells to radiation.34 Outstanding therapeutic outcomes of the synergistic treatments were confirmed in both orthotopic breast cancer and pancreatic cancer tumor models. Compared to several previous studies,35,36 our study reports a nano-platform integrating the ability to active target hypoxia 4
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region, penetrate deeply into hypoxia region, together with real-time imaging of the hypoxic microenvironment and radical-assisted RT. We believe that such radical-assisted radiotherapy will play an active role in future clinical practice by overcoming the hypoxia-induced resistance of tumors to therapy.
RESULTS AND DISCUSSION Characterization. Ultrafine W18O49 NPs have high light absorption efficacy in the near infrared region (NIR)-II (1000-1500 nm). Due to their small size, these W18O49 NPs rapidly diffuse in the lymphatic system and may be applied for the photo-thermal ablation of cancer cells.30 In this study, MMP-responsive, hypoxia-targeting cluster bomb-like NPs (WOACC NPs) were fabricated by covalently bonding PAA-functionalized W18O49 NPs (WO NPs) with a metalloproteinase-2 (MMP-2) cleavable peptide (Pro-Leu-Gly-Val-Arg-Gly). Firstly, polyacrylic acid (PAA) modified W18O49 NPs (Zeta potential: -23.2 mV) were fabricated. Then, the carboxyl groups on the surface of these PAA-modified W18O49 NPs were transformed to thiol groups by grafting cysteamine (T-W18O49 NPs, Zeta potential: -14.3 mV). Next, these T-W18O49 NPs were modified with the MMP-2 enzyme cleavable peptide by performing a coupling reaction between the maleimide and thiol groups. Then, amino group-modified W18O49 NPs (A-W18O49 NPs, Zeta potential: +3.7 mV) were prepared by conjugating APTES to the surface of the W18O49 NPs. The obtained A-W18O49 NPs were then added to the MMP-2-modified T-W18O49 NP suspension (ratio of A-W18O49 NPs: T-W18O49 NPs equaling 6:1) to obtain clusters of W18O49 NPs (WOC NPs). Finally, the hypoxia-targeting group of anti-CCL28 was conjugated to the surface of WOC NPs to form the WOACC NPs (Zeta potential: -4.5 mV). The individual anti-CCL28-modified A-W18O49 NPs were denoted by WOAC NPs. These WOACC NPs exhibited a strawberry-like structure, with a size about 33 nm as observed in Figure 1. In the XRD spectra shown in supporting information Figure S1, all the peaks of the as-obtained WOACC NPs can be well indexed into monoclinic phase of W18O49, confirming that the crystal structure was not affected during the formation of nanocluster. Upon closer observation, these WOACC NPs consisted of ultrafine W18O49 NPs (with a mean diameter less than 5 nm), confirming the cluster like structure. NIR light absorption profiles of original W18O49 NPs and WOAC NPs released from WOACC NPs are shown in Figure 1c. No obvious differences between the two groups were observed, demonstrating the optical stability of these 5
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W18O49 NPs. The enhanced light absorption of the WOACC NPs might be attributed to the assembly of the WOAC NPs. Taken together, the covalent bonding procedure did not affect the NIR light absorption property of W18O49 NPs, and the WOAC NPs released from the WOACC NPs maintained their local surface plasmon resonance properties. Light absorption of the WOAC NP in NIR II (900 nm-1200 nm) is essential for the success of our study because NIR II light can penetrate the deep regions of the tumor and transform the light to heat. Both the WOACC NPs and WOAC NPs released from the WOACC NPs had excellent photo-thermal conversion abilities upon irradiation with a 1064 nm laser (1 W/cm2). Even when irradiated with a laser power density as low as 0.2 W/cm2, the suspension could still achieve a temperature elevation over 40 oC in the presence of WOACC NPs, indicating that superb light to heat conversion ability of WOACC NPs (Figure 1d). MMP-2 is over-expressed in almost all types of human tumor microenvironments, whose level correlates positively with tumor progression and metastasis.38 On this basis, the selective response to MMP-2 enzyme is crucial for WOACC NPs to distinguish cancerous lesions from normal tissue. To verify this, we incubated WOACC NPs with different enzymes such as MMP-2, Cathepsin B, and Caspase-3. Changes in the sizes of the WOACC NPs were analyzed by dynamic light scattering (DLS) (Figure 1e). Pronounced size variations in the WOACC NPs were observed only in the presence of MMP-2 (from 81 nm to 11 nm), suggesting that the obtained WOACC NPs were disrupted to release WOAC NPs only upon MMP-2 treatment. The TEM images of these WOAC NPs released from WOACC NPs after MMP-2 treatment showed nearly negligible difference compared to the original WOAC NPs (Figure S2). The MMP-2 dependent destruction is important to avoid the premature release of WOAC NPs before they reached the tumor site, which may result in unnecessary loss of cargo during circulation in the blood. Based on these results, we concluded that the MMP-2-responsive, cluster bomb-like WOACC NPs were successfully fabricated. In Vitro Stability and Cytotoxicity. The stability of nanomedicine has profound effect in regulating its fate both in vitro and in vivo. As such, we tested the optical properties of the asobtained WOACC NPs after incubation with freshly harvested mouse serum, in an attempt to mimic the harsh in vivo microenvironment. As can be seen in Figure S3, an incubation with mouse serum for 48 h only slightly mitigated the featuring adsorption in NIR, suggesting that WOACC is fairly stable. By using X-ray photoelectron spectrometer (Figure S4), we analyzed the oxidation status of tungsten in WOACC before and after interaction with cysteine (sulfur 6
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containing amino acid), and mouse serum as a more complicated case. We found that the incubation with cysteine induce little changes of the bonding energy of W6+, indicating that the interaction between sulfur containing amino acid and WOACC is regulated by electrostatic forces. Meanwhile, from the spectra of WOACC incubated with mouse serum, one can see that the bonding energy of W6+ greatly changed from 38.28 and 36.08 ev (before incubation) to 38.58 and 36.58 ev (post incubation) of 4f5/2 and 4f7/2, respectively, suggesting that there exists potential covalent bonding between the serum proteins and WOACC. Furthermore, we analyzed the profiles of binding energy of tungsten by, clearly, nearly negligible shift of characteristic binding energies of tungsten indicates the less tendency of forming covalent binding between sulfur (in cysteine) and WOACC. Specifically, the cytotoxicity of WOACC was tested in both human epithelial HUVEC cell line and cancerous HeLa cell line (Figure S5). It can be seen that WOACC NPs with a concentration of less than 100 µg/mL exert near negligible cytotoxic effect regardless of cell types. Even when challenged with a 5 mg/mL of WOACC NPs, HUVEC and HeLa cells still can exhibit 82.5% and 84.2% viability post 48 h incubation, respectively. We think this finding can be taken as a solid evidence to support the compatibility of WOACC for practical application. Furthermore, we have analyzed the potential of cytotoxicity of WOACC upon stimulation (laser irradiation and radiation). In particular, 200 µL of DMEM culture medium containing 1 mg/mL WOACC was irradiated with 1064-nm laser (1 W/cm2) for 10 min. Then, the suspension was allowed to cool down naturally, and the WOACC NPs were collected via centrifugation every 24 hours. The concentration of tungsten in the supernatant was analyzed by inductively-coupled plasma mass spectrometer (ICP-MS) as shown in Figure S6. It can be seen that the laser irradiation-induced degradation of WOACC is nearly negligible with an up to 1.4% of tungsten released into conditioned medium during the observation period. Furthermore, when switched the laser irradiation to radiation (3 Gy), we observed a higher content of tungsten release profiles (at 72 h, 4.1%). It can be envisioned that the release of heavy metal ions might induce cytotoxicity in normal tissue or partially being responsible for the cytolysis of cancer cells induced by metal-enhanced radiation. To test this hypothesis, HUVEC and HeLa cells were cocultured with the supernatant containing the released tungsten ions for 48 h, with the viability of each well analyzed via MTT assay as mentioned above. It can be seen that the release of tungsten ions caused by either laser irradiation or radiation induce negligible cytotoxicity regardless of 7
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cell types. It is reasonable to see this result as the cytotoxicity induced by heavy metal should be dosage-dependent, and the concentration of non-specific released tungsten ions is believed to be within tolerable range. In Vivo Distribution Profiles. The penetration of NPs into the tumor is a key factor for achieving cytolysis in hypoxia region, which are located deeply inside the solid tumor. Therefore, the penetrating potency of these WOACC NPs was studied in a 3D spheroid tumor model in vitro, so as to mimic the relevant obstacles faced by the WOACC NPs in solid tumors33. All these samples were labeled with FITC and showed a green color in the confocal laser scanning microscope (CLSM) images (Figure. 2a). For FITC-labeled WO NPs (a1) and the cluster of W18O49 NPs covalently bound by the non-cleavable peptide (WOCC NPs) (a2), green fluorescence was observed only in the vicinity of the peripheral tumor, indicating that the rigid cluster with an unchangeable diameter had a limited capability to penetrate deep into tumors. However, abundant green fluorescence was observed from the periphery to the center of the multicellular spheroids for both WOACC NPs (a4) and WOAC NPs (a3), clearly supporting their penetration abilities with respect to non-targeting counterparts like WOCC. Once the activity of MMP-2 was blocked prior to the incubation with WOACC (a5), the green fluorescence was observed only around the periphery of the tumor model (same as the results a2), clearly illustrating that the penetration depth of WOACC NPs was suppressed. Thus, the deep penetration of WOAAC NPs can be attributed to their disassembly by MMP-2. Notably, it can be seen that the penetration depth of WO NPs is limited. These observations were further corfirmed by ICP-MS analysis of the tungsten content (Figure S7), collectively supporting the essential role of proper surface functionalization in active accumulation in hypoxia lesion. Although the MMP-2-triggered release of WOAC NPs is essential for the drug delivery to the tumor center, the blood circulation time of WOACC NPs is also important because it affects the tumor accumulation of WOACC NPs via the EPR effect. Blood retention profiles of both WOACC NPs and WOAC NPs were analyzed post-i.v. injection, as shown in Figure 2b. The assembly of WOAC NPs into larger clusters clearly increased their half-life in blood (274 min versus 124 min for WOACC NPs and WOAC NPs, respectively), which greatly benefits the tumor accumulation of WOACC NPs via the EPR effect. The distribution of these two samples inside the tumors was also measured and shown in Figure 2c. For WOAC NPs, only an accumulation amount of 4% ID/g tumor tissue could be achieved at 24 h post injection, and the 8
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amount of the WOAC NPs did not change, even after 48 h. However, 17.5% of the injected dose of WOACC NPs per gram of tumor was achieved in the tumor at 24 h post injection and further increased to 27.5% ID/g tumor tissue after 48 h. We attribute the enhanced tumor accumulation of MMP-2-responsive WOACC NPs to their extended blood availability in vivo, especially in comparison with that of WOAC NPs. Due to the high level of MMP-2 enzyme in the tumor tissue, these WOACC NPs were then degraded to release WOAC NPs, which penetrated deeply inside the tumor because of their small size. Based on these results, our objective to design a nano-platform with a prolonged blood retention time and deep tumor penetration was successfully achieved. In Vitro Radiosensitization. The radiosensitization effect of WOACC NPs on 4T1 cells that had received different treatments and grew under both normal and hypoxic conditions was first verified using colony formation assays, as shown in Figure S8. The lowest cell survival could be observed in cells received synergistic treatment (WOACC NPs assisted, PTT-enhanced RT, denoted by PTT+RT). Considering that radiation in identical dosage achieve much less cytolytic effect under hypoxia condition, it is suggested that the simultaneous treatment with WOACCNPs and PTT greatly enhanced the susceptibility of tumor cells to radiation under hypoxic conditions. Although PTT had been proven to be an effective cancer therapy due to its ability to induce cytolysis, neither PTT plus WOACC NPs nor RT plus WOACC NPs alone exerted a comparable therapeutic effect as that of the synergistic treatment, indicating that sequential therapy with PTT and WOACC NP-assisted RT was of great importance. The in vitro viabilities of 4T1 tumor cells that had received the different treatments under normoxic and hypoxic conditions were also quantitatively investigated using Annexin-V/PI staining and flow cytometry (Figure 3a and Figure S9). Again, it can be seen that hypoxic condition makes 4T1 cells less vulnerable to RT, which reduced the killing efficacy of the RT treatment from 30.7% (normoxia) to 12.5% (hypoxia), indicating that radiation exerted a cytolytic effect in an oxygendependent manner, consistent with the clinical observations.39 In contrast, a comparable killing efficacy was observed following the combined treatment (PTT+RT group), regardless of the oxygen conditions (57.9% and 63.4% for normoxia and hypoxia, respectively). Since the therapeutic effect of PTT was not long-lasting, we envisioned that the thermoinduced radical production might be responsible for the enhanced effects.33 To test this hypothesis, we incubated cells with N-acetylcysteine (NAC), a broad spectrum quencher of 9
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reactive oxygen species (ROS), prior to the radiation. The killing efficacy of the combination treatment (right axis) was reduced thereafter (from 63.4% to 38.6%) (Figure 3a). Meanwhile, NAC had much less influence on the killing efficacy of PTT alone (30.4% and 28.3%; in the absence or presence of NAC). Thus, it is safe to conclude that the ROS induced by PTT, rather than thermolysis itself, is responsible for the radiosensitization. The production of radicals by WOACC NPs upon laser irradiation was confirmed by EPR spectroscopy using DEPMPO as a spin trapping molecule. As shown in Figure 3b, multiple radical signals were obtained in the EPR spectrum and identified as hydroxyl- (·OH) and superoxide radicals (·O2-), while cells incubated with WOACC NPs without laser irradiation failed to induce such radicals.40 This phenomenon was further confirmed by using flow-cytometry to detect the level of intracellular ROS in cells received different treatments including synergistic treatments, PTT or RT alone, and WOACC incubation in the presence of H2DCFDA, a ROS responsive probe, as shown in Figure 3c. Cells receiving PTT+RT treatment produce the highest amount of ROS. Furthermore, the enhancement of killing efficacy was also observed in normoxic cells incubated with WOACC NPs followed by RT (41.9%, compared to 30.7% without WOACC NPs). It is not surprising to see such radiosensitization effect as heavy metal elements like tungsten, have been tested to be valid as radiosensitizers to strengthen the RT therapeutic index.41 The enhancement also positively correlated with the oxygen content (killing efficacy in hypoxic cells, 16.3% and 12.5%, in the presence and absence of WOACC, respectively). We speculated that the abnormal enhanced DNA repair process in cancer cells received the radiationinduced ionization might be responsible for the resistance to metal-enhanced RT.34 To verify this hypothesis, the phosphorylation of several key proteins underlying the stress-induced DNA damage repair mechanism such as mTOR,41 Akt,42 and MAPK,43 was investigated using Western blotting as shown in Figure 3d. It can be seen that WOACC NPs-assisted PTT leaded to obvious reduction of phosphorylation of mTOR and Akt, and that of AMPK were less affected, suggesting that this treatment enhanced the susceptibility of hypoxic cells to radiation in mTORAkt dependent manner. Furthermore, when the cells were treated with either mTOR or Akt agonist prior to WOACC NPs assisted PTT, the phosphorylation of mTOR largely recovered, clearly suggesting that the Akt located in the upstream of this pathway, in consistent with Bodine et al. reported.44 In addition, as shown in Figure 3e, when we tested the expression of several genes underlying cell proliferation (MT 3), hypoxia-driven radiation resistance (HIF-1α and 10
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ANGPTL 4), cell growth (IGFBP-1), angiogenesis (VEGFA), and apoptosis (CASP-1 and BAX) by using high-throughput qRT-PCR array, it seems that the radiosensitization effect is more relevant to the reverse of hypoxia-driven apoptosis resistance that angiogenesis inhibition as indicated by the significant reduced expression of related genes. Taken together, these findings suggest that the WOACC assisted PTT enhances the susceptibility of hypoxic cancer cells by reversing their apoptosis resistance driven by hypoxia in Akt-mTOR dependent manner. In Vivo Imaging. In vivo non-invasive imaging of tumor hypoxia with high spatial resolution is of great therapeutic significance. Unfortunately, limited progress has been achieved till now. W18O49 NPs are good candidates for PTT and can also act as CT contrast agents due to their high X-ray attenuation.30 Here, the potential of WOACC NPs as contrast agents to locally image hypoxic regions under CT guidance was tested. As shown in Figure 4a, a heterogeneous enhancement of the tumor lesion, particularly in the center of solid tumors (Figure 4a, b1 inner area) in the breast tumor-bearing mouse, was observed post-i.v. injection of WOACC NPs. This result is reasonable because hypoxia primarily occurs in regions lacking oxygen and nutrient supplements (normally in the center of the tumor). To better understand the intra-tumoral diffusion of NPs, an artificial diffusion pathway was defined by a line start from the tumor boundary (highlighted by red star) and end in the center of the tumor (highlighted by yellow star) in Figure 4a for simplicity. Distribution profiles (as indicated by their HU values) from three randomly selected regions of interest along the diffusion path in the tumors were analyzed (Figure 4d). WO NPs and WOAC NPs showed the lowest values, indicating that least NPs penetrated into the tumor tissues. The low tumor accumulation of NPs was mainly ascribed to the small size of WO NPs and WOAC NPs, which fail to accumulate in tumor to certain amount via the EPR effect. These NPs would leak out from the blood vessels and be eliminated by renal clearance long before they get interact with the tumor,45 as confirmed by our previous finding of the in vivo circulation and related tumor distribution. For the WOCC NPs, the HU value at the peripheral zone of the tumor was as high as the value of the WOACC NPs and then rapidly decreased toward the center of the tumor. The larger size of the WOCC NPs extended their halflife in the circulatory system, thus leading to a passively accumulation of WOCC NPs at the tumor site, which consequently resulted in the highest HU value at the boundary. In contrast, larger counterpart WOCC NPs exhibited a poor tumor penetration ability, with fewer WOCC NPs capable of accessing the tumor center, as demonstrated by the low HU values. In the mice 11
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received an i.v. injection of WOACC NPs, we observed the highest accumulation in tumor tissue, consistent with the results of the in vivo tumor distribution experiment. The HU value was higher around the tumor boundary. Considering the high level of MMP-2 in tumor microenvironment, WOACC NPs will be degraded to release small WOAC NPs once they enter the tumor through leaky dysfunctional blood vessels. These released small WOAC NPs are likely to penetrate deeply into the tumor tissue and distributed heterogeneous depending on the hypoxia condition (more specifically, the expression of CCL-28 chemokine), as indicated by the distinct HU values along the diffusion path. The presence of WOAC NPs (released from the WOACC NPs) in the center of the tumor was further verified by the Bio-TEM (Figure 4b). The small WOAC NPs, with a size less than 5 nm, were clearly observed in enlarged image (Figure 4b), demonstrating the successful deep penetration of WOAC NPs into the tumor center. In particular, the tissue located in the middle layer of the tumor showed much higher HU values (273 HU, 6.5 mm from boundary) than that of the tumor center (110 HU, 10 mm boundary), indicating that more WOAC NPs were located between the boundary and center of the tumor, in consistent with the observation of fluorescence imaging (Figure S10). We anticipated that despite the surface conjugation of anti-CCL-28 Abs, who promote the active accumulation in hypoxia regions, the vasculature available in hypoxia region is also attributable to the enhanced accumulation. Given the fact that WOCC NPs in sizes similar to that of WOACC NPs can barely access such regions as indicated by HU values, we argue that the contribution of blood vessels-induced accumulation is limited, and collectively support the dominant role of CCL-28 targeting ligand in active accumulation at hypoxic region. Thus, it seems that the middle section of the solid tumor suffers from a more severe hypoxic condition than that of the central part. We speculated that the prolonged shortage of nutrients and oxygen, rather than short-period stress, in the central tumor section, induce permanent cell necrosis, thus reducing the levels of hypoxia-related cytokines and chemokines,46,47 while the limited oxygen supplement to the middle section of tumor tissue still support the growth of cancer cell under hypoxia condition. Future studies are required to get a better understanding of this phenomenon. The centers of tumor tissues were collected and dissected post-i.v. injection of FITC-labeled samples and subjected to immunofluorescence analysis to confirm this hypothesis. Hypoxic conditions were labeled with HIF-1 (red), as shown in Figure 5a. The co-localization of green (WOACC NPs) and red fluorescence (HIF-1) was observed in tissues surrounding the tumor 12
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center, confirming the accuracy of the hypoxia-specific WOACC NPs-enhanced CT imaging (a1). However, fewer hypoxic cells were observed in the tumor center (red), together with lower concentrations of NPs (green). These results further indicated that the lower HU values observed in the tumor center were caused by necrosis-induced reduction of CCL-28 expression in the center of the solid tumor. In the mice treated with WOCC NPs, WOAC NPs, or WO NPs, almost no NPs were observed in the center of the tumor, consistent with our previous observation of the in vivo penetration (Figure 4d). The hypoxia-targeting ability of the CCL-28 chemokine and the MMP-2-responsiveness of WOACC NPs were further verified by using flow cytometry, as shown in Figure 5b and c. WOACC NPs preferred to accumulate in HIF-1-positive regions (29.3% NPs+ HIF+). When the activity of MMP-2 was blocked, the amount of WOACC NPs accumulated in hypoxia region decreased to 8.7% (NPs+ HIF+), indicating the reduced ability to penetrate into the solid tumor. However, when the expression of CCL-28 chemokine was blocked via intratumoral injection of antibody prior to the administration of WOACC NPs, we still observed a small amount of WOACC NPs accumulated in hypoxic region (17.5% NPs+ HIF+). These results were attributed to the superb penetration ability of small WOAC NPs released from the WOACC NPs. Based on these results of the enzyme and chemokine blocking tests, we concluded that the MMP-2sensitive peptide played a vital role during the sequential delivery process as it is essential for the release of ultrafine WOAC NPs from the WOACC cluster bomb. When the interaction between the CCL-28 chemokine and WOACC NPs was inhibited, a small fraction of WOAC NPs still could access the peripheral hypoxic regions via passive diffusion. However, this accumulation was non-specific and likely to suffer from limited therapeutic index as the majority of hypoxic regions located deeply within the solid tumors. Furthermore, the expression of a DNA damage marker (phosphorylated histone H2AX, pH2AX)48 and late stage apoptosis activation indicator (Poly [ADP-Ribose] Polymerase, PARP)49 after RT treatment were investigated using immunofluorescence staining (Figure 5d) to confirm the cytolysis effect. The WOACC NPs assisted PTT plus RT with WOACC NPs induce more DNA damage (indicated by spot-like pH2AX fluorescence, red) than PTT or WOACC-enhanced RT alone, which could be safely ascribed to the alleviated RT resistance. Increased cleaved PARP protein, which was positively correlated with the number of apoptotic hypoxic cancer cells, was observed after the synergistic
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treatment (green color) compared with that in the PTT or NP-enhanced RT groups, supporting the practical significance of the synergistic treatment. PET Imaging. Long-term response of tissue post treatment stands as another important concern because different nanomedicines may exert distinct effects on the tumor microenvironment. Hypoxia, among various inflammatory reactions, has been taken as the primary concern when considering its critical role in driving angiogenesis and local relapse. On this basis, we evaluated the hypoxic conditions in tumor tissue of mice received different treatments. PET imaging with 18F fluoromisonidazole (FMISO) has long been taken as the gold standard for tracking hypoxia due to its high sensitivity, despite its low spatial resolution. Residual hypoxic zones inside the tumor after the different treatments were verified using PET. Almost no positive hypoxic zones were observed inside the tumor after the synergistic treatment (Figure 6a, II, white circle) with respect to that of control group (I, white circle). This region had the lowest PET signal and the smallest amount of 18F-MISO accumulation (Standardized uptake values (SUV): mean: 0.0186±0.0024; Max: 0.0374±0.0103, p
