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Hypoxia-Targeting, Tumor Microenvironment Responsive Nanocluster Bomb for RadicalEnhanced 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 210008, China 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 ‡
S Supporting Information *
ABSTRACT: Although ultrasmall 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 nanocluster 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 photothermal ablation of the tumor and 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 AktmTOR 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, photothermal therapy, tungsten
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microenvironment in solid tumors are crucial for achieving optimal therapeutic outcomes of RT. Although perfusion of oxygen gas into solid tumors can upregulate 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 ultrasmall metal NPs as radiosensitizers to potentiate the killing efficacy of RT has been tested.15−17 Upon irradiation, these NPs can strongly absorb the γ-ray 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 ultrasmall 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
adiotherapy (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 © 2017 American Chemical Society
Received: July 6, 2017 Accepted: October 9, 2017 Published: October 9, 2017 10159
DOI: 10.1021/acsnano.7b04737 ACS Nano 2017, 11, 10159−10174
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Cite This: ACS Nano 2017, 11, 10159-10174
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Figure 1. (a) Scheme illustration showing how MMP2-responsive WOACC NPs integrate active hypoxia-targeting properties with CT imaging, local PTT, and radical-enhanced radiotherapy. These WOACC NPs maintained their original size during blood circulation and destructed to release hypoxia microenvironment targeting WOAC NPs upon MMP2 simulation. (b) TEM image of WOACC NPs in low and high (inset) resolutions. (c) UV−vis-NIR profiles of the as-obtained WOACC NPs, WOAC NPs released from WOACC NPs, and the original W18O49 NPs. (d) Temperature variation profiles of the WOACC NP suspension (1 mg/mL) and WOAC NPs (1 mg/mL) receiving multilaser irradiation (1 and 0.2 W/cm2). PBS received laser irradiation (1 W/cm2) was taken as control group. (e) DLS analysis showing the size variation of WOACC NPs incubated with Cathepsin B (500 U/mL), Caspase-3 (200 U/mL), and MMP-2 (200 U/mL) enzymes for 2 h. Untreated WOACC NPs were taken for comparison.
matrix metalloproteinase-2 (MMP-2) cleavable peptide (ProLeu-Gly-Val-Arg-Gly).28 The half-life 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 photothermal conversion abilities of WO NPs,30−32 hypoxic conditions could be precisely investigated under the guidance of computed tomography (CT), followed by imaging-guided photothermal therapy (PTT). Meanwhile, highly reactive radicals were simultaneously produced in hypoxic regions during PTT, which could alleviate the hypoxic-induced resistance in Akt-mTOR-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 nanoplatform integrating the ability to active target hypoxia 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 hypoxiainduced resistance of tumors to therapy.
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, thus leading to reduced tumor accumulation and limited enhancement.20,21 In contrast, larger NPs exhibiting a long halflife in the circulatory system in vivo suffer from a short intratumor penetration depth, leading to compromised sensitization effect because of the limited accumulation in hypoxia region.22 Thus, platform featuring both enhanced accumulation in the 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 nanocluster bomb consisting of hypoxic microenvironment-targeting nonstoichiometric tungsten oxide NPs (WO NPs) being taken as radiosensitizers for RT. Since the CCL-28 chemokine is overexpressed in hypoxic tumor microenvironments and is validated to have a crucial role in driving tumor angiogenesis and tolerance,26,27 WO NPs were first modified with ligands targeting the CCL-28 chemokine (WOAC NPs). Then, clusters of WOAC NPs (WOACC NPs) were covalently bound using a
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 photothermal ablation of cancer cells.30 In this study, MMPresponsive, hypoxia-targeting cluster bomb-like NPs (WOACC 10160
DOI: 10.1021/acsnano.7b04737 ACS Nano 2017, 11, 10159−10174
Article
ACS Nano
ence 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 as-obtained 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 containing amino acid), and mouse serum as a more complicated case. We found that the incubation with cysteine induces 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 shifts of characteristic binding energies of tungsten, which 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
