Oxygen-Self-Produced Nanoplatform for Relieving Hypoxia and

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Oxygen Self-Produced Nanoplatform for Relieving Hypoxia and Breaking Resistance to Sonodynamic Treatment of Pancreatic Cancer Jie Chen, Honglin Luo, Yan Liu, Wei Zhang, Hongxue Li, Tao Luo, Kun Zhang, Yongxiang Zhao, and Junjie Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.7b08225 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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Oxygen Self-Produced Nanoplatform for Relieving Hypoxia and Breaking Resistance to Sonodynamic Treatment of Pancreatic Cancer Jie Chen,#,† Honglin Luo,#,† Yan Liu,† Wei Zhang,† Hongxue Li,† Tao Luo,† Kun Zhang,†,‡,* Yongxiang Zhao†,§,* and Junjie Liu†,* † Affiliated Tumor Hospital of Guangxi Medical University, 71 He-di Road, Nanning 530021, P.R. China; ‡ Department of Medical Ultrasound, Shanghai Tenth people’s Hospital, Tongji University School of Medicine, 301 Yan-chang-zhong Road, Shanghai, 200072, P. R. China; § National Center for International Research of Biological Targeting Diagnosis and Therapy, Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy Research, Collaborative Innovation Center for Targeting Tumor Diagnosis and Therapy, Guangxi Medical University, 6 Shuang-yong Road, Nanning, Guangxi 530021, China. * Address correspondence to [email protected], [email protected] and [email protected]

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ABSTRACT: Hypoxia as one characteristic hallmark of solid tumors has been demonstrated to involve in cancer metastasis and progression, induce severe resistance to oxygen-dependent therapies and hamper the transportation of theranostic agents. To address these issues, an oxygen self-produced sonodynamic therapy (SDT) nanoplatform involving modified fluorocarbon (FC) chains-mediated oxygen delivery protocol has been established to realize highly-efficient SDT against hypoxic pancreatic cancer. In this nanoplatform, mesopores and FC chains of FC chainsfunctionalized hollow mesoporous organosilica nanoparticles (FHMONs) carriers can provide sufficient storage capacity and binding sites for sonosensitizers (IR780) and oxygen, respectively. In vitro and in vivo experiments demonstrate the nanoplatform involving this distinctive oxygen delivery protocol indeed breaks the hypoxia-specific transportation barriers, supplies sufficient oxygen to hypoxic PANC-1 cells especially upon exposure to ultrasound irradiation and relieves hypoxia. Consequently, hypoxia-induced resistance to SDT is inhibited and sufficient highly reactive oxygen species (ROS) are produced to kill PANC-1 cells and shrinkage hypoxic PANC1 pancreatic cancer. This distinctive FC chains-mediated oxygen delivery method provides an avenue to hypoxia oxygenation, and holds great potential in mitigating hypoxia-induced resistance to those oxygen-depleted therapies, e.g., photodynamic therapy (PDT), radiotherapy, chemotherapy, etc.. KEYWORDS： Hypoxia reversion, Oxygen delivery, Sonodynamic treatment, Reactive active species, Hypoxia-induced resistance


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Insufficient oxygen supply termed as hypoxia ubiquitously arises in various solid tumors, and can be recognized as one characteristic hallmark of advanced solid tumors.1 It has been extensively accepted that hypoxia can promote tumor angiogenesis and cancer metastasis.2-6 Moreover, the hostile hypoxia also leads to some inherent resistances to medical therapies (e.g., radiotherapy, chemotherapy and PDT) wherein oxygen is essential in the process of cancer cell destruction, consequently resulting in the failures of many anti-tumor technologies.1,5 Therefore, protocols capable of relieving or even reversing hypoxia is desirable but challenging. In an attempt to treat hypoxic tumor, two pathways are highlighted, one of which is to develop less oxygen-dependent therapy or hypoxia-activated prodrugs.7-10 In practice, the two methods fail to truthfully alter the pre-existing hypoxia.11 As a consequence, the intrinsic characteristics associated with hypoxia, e.g., inadequate blood supply, reduced susceptibility, drug-resistant genes expression and deficiency in targeting sites, remain unresolved in these two pathways. As a comparison, tumor oxygenation has aroused considerable attention, since it can effectively relieve hypoxia microenvironment via delivering oxygen to hypoxic regions of solid tumor and further attenuate resistance to various oxygen-dependent therapies.12-15 Currently, various oxygen-delivery strategies have been designed to modulate tumor hypoxia to reinforce radiotherapy and PDT, e.g., hyperbaric oxygen (HBO) injection,16 oxygencarried microbubbles,12,17,18 in-situ oxygen production by reaction between H2O2 and metals or catalase,19-22 photo-activated H2O splitting-mediated oxygen production,23 and dissolved oxygen delivery in perfluorocarbon (PFC) compounds or hemoglobin.15,24 These strategies have been demonstrated to realize hypoxia oxygenation via high-efficient oxygen delivery, significantly relieve hypoxia and intensify the oxygen-dependent therapy efficiency against malignancies, providing candidate pathways to inhibiting tumor metastasis and progression. However, these


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oxygen delivery strategies suffer from some potential issues, e.g., H2O2 dependence in H2O2mediated oxygen production, side effects in HBO injection, poor light penetration in photoactivated oxygen production, large size and poor stability in microbubbles-mediated oxygen delivery, etc..16-26 More significantly, some hypoxia-specific transport barriers consisting of inadequate blood supply, increased interstitial fluid pressure, thick storma barrier and disabled targeting due to lack of specific receptors hampered these oxygen delivery carriers from entering hypoxic regions of tumor.2,10,27 Therefore, more advanced and effective oxygen delivery strategies capable of breaking hypoxia-specific transport obstacles are urgently desirable. In this report, an oxygen self-produced nanoplatform with a diameter of less than 200 nm has been prepared to modulate hypoxia, attenuate hypoxia-induced resistance to sonodynamic therapy (SDT) and improve SDT efficiency against highly-aggressive and hypoxic PANC-1 pancreatic cancer. FC chains-functionalized HMON (FHMON) that combines the structural and functional advantages of HMONs was employed as the oxygen reservoir and IR780 sonosensitizer carrier, respectively. It is highly expected that FHMON carriers can absorb oxygen via the functionalized FC chain, since the modified FC chains similar to PFC compounds exhibited a strong affinity towards oxygen via the hydrophobic interaction and H bonding.11, 28,29 In particular, the oxygen delivery strategy using chelated FC chains instead of free PFC compound can avoid troublesome loading, leach and short half-life of super-hydrophobic PFC oxygen reservoir and stabilize O2 delivery. It has been extensively accepted that SDT is more preferable than PDT in treating deep tumors due to the deeper penetration of ultrasound (US) trigger in SDT than light in PDT.26 Nevertheless, SDT also suffers from the hypoxia-induced resistance because of its oxygendepletion treatment principle.17,18 Herein, this oxygen self-produced SDT nanoplatform can


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effectively relieve hypoxic regions of PANC-1 solid tumor via the local US-responsive oxygen release manner, diminish hypoxia-induced resistance to SDT, produce sufficient reactive oxygen species (ROS) and enhance SDT efficiency against PANC-1 solid tumor. Besides triggering SDT process and oxygen release, US could promote more nanoparticles or drugs enter hypoxic solid tumor via the enhanced permeability mediated by bubbles-mediated cavitation affect.30-34 Inspired by it, the marriage strategy of oxygen bubbles from this nanoplatform and US irradiation is expected to break hypoxia-specific transport barriers and enable more oxygen self-produced SDT nanoplatforms to enter hypoxic regions of PANC-1 solid tumor, which further contribute to the intensified SDT. More significantly, the local irradiation and deep penetration of US trigger, the small particle size (< 200 nm) and exogenous oxygen delivery independent of H2O2 in this oxygen self-produced SDT system determine this modified FC chains-mediated oxygen delivery protocol can overcome their respective limitations of aforementioned oxygen delivery strategies. Moreover, this distinctive oxygen delivery strategy holds great potentials in inhibiting metastasis of hypoxic tumor and breaking hypoxia-induced resistance to other oxygen-dependent therapies, e.g., PDT, radiotherapy, chemotherapy. RESULTS AND DISCUSSION Synthesis of oxygen self-produced SDT nanoplatform (IR780@O2-FHMON). As one type of mesoporous silica nanoparticles (MSNs), hollow mesoporous organosilica nanoparticles (HMONs) have been regarded as an ideal carrier due to their excellent biocompatibility and large surface area.35-37 Very recently, they have been well documented in loading organic sonosensitizers.38 In this work, FHMON that combines structural and functional advantages of HMONs in loading sonosensitizers with the ability of FC chains to carry oxygen was employed as carriers of sonosensitizers and oxygen. FHMON can be obtained via a well-established


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method, wherein in-situ modification of FC chains can be simultaneously accomplished when fabricating FHMON.36 This method could also avoid the potential flaws of post-modification, e.g., uneven modifications, tedious synthetic procedures and potential mesopore occlusion.36 Besides acting as sonosensitizer,39 IR780 was also endowed with abilities of NIR fluorescence imaging and photothermal ablation,40,41 which will provide an opportunity for the future multifunctional theranostic nanomedicine. The synthetic procedure of IR780@O2-FHMON is depicted in Figure 1a, wherein FHMON carrier is firstly fabricated. In detail, solid silica cores were firstly obtained via the hydrolysis and condensation of tetraethoxysilane (TEOS), followed by coating of hybrid FC chains-contained organosilica shell by co-condensation of TEOS and FC chains-contained 1H,1H,2H,2Hperfluorodecyltriethoxysilane (PDES). Immediately afterwards, the etching process was carried out to remove the inner silica cores and keep the FC chains-contained hybrid mesoporous shell intact, obtaining FHMON. The emerging C-F vibration peaks in FTIR spectrum of FHMON at 1308 cm-1 and 1344 cm-1 indicate the presence of FC chains (Figure S1). The uniformlydistributed FHMON carriers with an average particle size of 180 nm in diameter (Figures 1c-j and S1-S4) feature large surface area (214 m2/g) that supplies sufficient capacity for loading IR780. The apparent characteristic peak of IR780 in IR780@FHMON in comparison to FHMON suggests the successful encapsulation of hydrophobic IR780 (Figure 2a), and its loading percentage (i.e., 9.3%) is obtained via the linear correlation of characteristic peak intensity with concentration. Electrostatic interaction between FHMON and IR780 is responsible for IR780 loading, as demonstrated by the increased Zeta potential (Figure 2b). As well, IR780 encapsulation also leads to the increase of particle size (Figure S4).


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Moreover, the uniform distribution of an enormous quantity of F atoms in hybrid shell (Figure 1j) can provide sufficient binding sites for oxygen loading. Oxygen bubbling was employed to facilitate the binding of FC chains in IR780@FHMON with oxygen and yield the ultimate product, i.e., oxygen self-produced SDT nanoplatform (abbreviated into IR780@O2FHMON). The presence of O2 characteristic peak in IR780@O2-FHMON via gas chromatograph (GC) (Figure 2c) demonstrates the successful binding of oxygen to FC chains. Oxygen adsorption in FC chains further results in the increase of Zeta potential (Figure 2b), but fails to alter particle size (Figure S4). In particular, O2 bubbles are clearly observed via the optical microscopy and ultrasound imaging technology when heating IR780@O2-FHMON dispersion in degassed water (Figures 2d and S5), which also suggests the presence of oxygen in IR780@O2FHMON. Noticeably, depending on its hydrophobicity, IR780 shows a neglectable release in PBS solution at pH=7.4, promising a robust bio-stability in in vivo blood circulation (Figure S6). Nevertheless, an evidently intensified IR780 release in the presence of acidic buffer (pH=6.0) or US irradiation is found. This US or acidic-responsive IR780 release will benefit the specific SDT of PANC-1 solid tumor and guarantee therapeutic safety, since acidity is a hallmark of tumor microenvironment and US irradiation is local.34, 42 In vitro hypoxia modulation via oxygen supply from IR780@O2-FHMON. Sufficient oxygen supply is the pre-requisite of relieving hypoxia and diminishing resistance to various therapies. IR780@O2-FHMON as an oxygen reservoir is expected to realize the oxygen supply especially upon exposure to US irradiation, because US irradiation as the trigger of SDT can provide energy to cleave the affinity between oxygen and F atoms and promote oxygen release.15 To demonstrate it, extracellular oxygen content was detected (Figure 2e). Results show that the oxygen content in the group of IR780@O2-FHMON is much higher than that in


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IR780@FHMON, suggesting oxygen release from IR780@O2-FHMON. Especially upon exposure to US irradiation, more oxygen release from IR780@O2-FHMON is triggered, resulting in a more effective hypoxia modulation. This sufficient oxygen supply will be beneficial for mitigating resistance to SDT and improving SDT efficiency of PANC-1 pancreatic cancer. Moreover, time-dependent oxygen concentration curve was monitored. The highest oxygen concentration in US+IR780@O2-FHMON within 24 h (Figure 2f) demonstrates the occurrence of continuous oxygen supply from IR780@O2-FHMON. This result also suggests that US irradiation indeed activated oxygen adsorbed in FC chains of FHMON to become free oxygen and significantly relieve hypoxia. Furthermore, intracellular oxygenation for reversing hypoxia was evaluated, and the intracellular O2 level indicator, i.e., [Ru(dpp)3]Cl2(dpp, 4,7-diphenyl-1,10-phenanthroline), was used to monitor the intracellular oxygen level.43 An efficient intracellular oxygen supply from IR780@O2-FHMON via the spontaneous oxygen release is determined in comparison to IR780@FHMON alone or US+IR780@FHMON, suggesting the successful modulation of intracellular hypoxic milieu (Figure 2g). Once applying US irradiation, lower fluorescence intensity in US+IR780@O2-FHMON than that in IR780@O2-FHMON alone is observed, denoting more intracellular oxygen supply. This phenomenon can be primarily attributed to the US-accelerated oxygen release from IR780@O2-FHMON. In vitro and intracellular ROS generation. ROS-mediated apoptosis can be regarded as the principle of many therapeutic methods, but it is severely suppressed in hypoxic regions of tumor due to the absence of sufficient oxygen content.22 After demonstrating extracellular and intracellular tumor hypoxia modulation, oxygen-dependent ROS generation that was involved in sonocatalytic process of SDT was explored to investigate the influence of oxygen supply from


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IR780@O2-FHMON on the resistance of hypoxic PANC -1 cells to SDT. The principle is depicted in Figure 1b, wherein IR780 can absorb US energy and change oxygen into ROS, i.e., singlet oxygen (1O2). To demonstrate it, 1,3-diphenylisobenzofuran (DPBF) agent was firstly adopted to quantitatively evaluate the production of 1O2 associated with SDT, since it can be oxidized by 1O2 and result in a decreased absorbance intensity at 410 nm in UV-vis spectrum.38 The rapidly decreased absorbance intensity of DPBF in the presence of US irradiation and IR780@O2-FHMON indicates mass production of 1O2 via sonocatalysis-mediated oxygen conversion due to sufficient oxygen supply from IR780@O2-FHMON (Figure 3a). Electron spin resonance (ESR) spectroscopy was employed to further evaluate

1

O2 and 2,2,6,6–

tetramethylpiperidine (TEMP) as a spin trap was used to realize the signal visualization of 1O2 produced from [email protected] Typical 1O2-induced characteristic signals can be observed in US+IR780@FHMON (Figure 3b), while no evident signal emerges in US+FHMON. This result demonstrates the sonocatalytic conversion process from oxygen to 1O2 indeed occurred to the encapsulated IR780 in IR780@FHMON in the presence of US. In particular, more oxygen supply in the group of US+IR780@O2-FHMON results in stronger 1O2 signal and more 1O2 production, which will enable the highly-efficient SDT against hypoxic PANC-1 cells. Intracellular ROS production of hypoxic PANC-1 cells was also examined by using 2’,7’dichlorofluorescindiacetate (DCFH-DA) that is a typical ROS probe.44 Similar to in vitro experiments, sufficient oxygen supply in US+IR780@O2-FHMON result in the highest ROS content, as evidenced by the strongest DCF fluorescence signal detected by flow cytometry in (Figure 3c). This result also indicates sufficient oxygen is indispensible for the sonocatalytic process of 1O2 production in IR780-mediated SDT. Moreover, larger power density of US can promote IR780@O2-FHMON to generate more ROS (Figure S7). In this regard, this oxygen


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supply-enhanced SDT using IR780@O2-FHMON can significantly inhibit cell proliferation of hypoxic PANC-1 cells via the ROS-mediated apoptosis pathway, as evidenced by the comparison between US+IR780@FHMON and US+IR780@O2-FHMON (Figure 3d). As well, the SDT outcome against hypoxic PANC-1 cells is dependent on the mass concentration of IR780@O2-FHMON and the power density of US (Figure S8). In vitro inhibition of hypoxia-induced resistance to SDT and enhanced SDT efficacy. More detailed SDT evaluation has been carried out to demonstrate whether the successful hypoxia modulation in aforementioned experiments can diminish hypoxia-induced resistance to SDT and improve SDT efficiency for hypoxic PANC-1 cells. Hypoxic PANC-1 cells treated with different groups were stained by propidium iodide (PI) & calcein AM to differentiate dead and viable cells via laser confocal scanning microscopy (LCSM) (Figure 4a). It is found that most hypoxic PANC-1

cells

remain

alive

(represented

by

green

color)

after

treatment

with

US+IR780@FHMON, suggesting the hypoxic PANC-1 cells displays a robust hypoxia-induced resistance to SDT. In contrast, the treatment of US+IR780@O2-FHMON harvests a large quantity of dead cells. This result sufficiently demonstrates IR780@O2-FHMON in the presence of US indeed attenuates the hypoxia-induced resistance to SDT and realizes the highly-efficient SDT of hypoxic PANC-1 cells. This intriguing result is resulted from the relieved extracellular and intracellular hypoxic milieu via the continuously US-accelerated oxygen release from IR780@O2-FHMON, as demonstrated in above experiments. Similar results can be obtained using flow cytometry (Figure 4b), wherein all of hypoxic PANC-1 cells after different treatments were stained by PI and annexin V-FITC. The percentage of late apoptotic cells that truthfully reflect the ultimate cell death is the largest (58.3%) in US+IR780@O2-FHMON, reflecting a highly-effective SDT outcome. This intriguing result was


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resulted from the considerably inhibited resistance to SDT that hypoxia reversion originating from the sufficient oxygen supply in US+IR780@O2-FHMON causes. To determine the intensified SDT principle of IR780@O2-FHMON in the presence of US irradiation, DCFH-DA was qualitatively used to detect the ROS during SDT. The strongest fluorescence intensity of DCF in US+IR780@O2-FHMON indicates the most ROS due to the most sufficient oxygen supply. This result also demonstrates the occurrence of oxygen transformation into ROS via the sonocatalysis process (Figure 4c), and explains why US+IR780@O2-FHMON causes the largest apoptosis in above LCSM observation or flow cytometry. It is worth noting that the treatment with IR780@O2-FHMON alone also causes cell apoptosis (Figures 3d and 4a,b), which can be probably ascribed to the local HBO therapy originating from the spontaneous oxygen release in IR780@O2-FHMON alone (Figure 2e-g). In particular, the lower ROS level but more cell death in IR780@O2-FHMON alone is found when comparing to US+IR780@FHMON. This intriguing phenomenon can be attributed to the fact that the continuous oxygen release in IR780@O2FHMON alone can sustain HBO beneficial for continuously generating ROS,45 while in US+IR780@FHMON, ROS will rapidly vanish due to short half-life once US irradiation is completed, as demonstrated in Figure S9. Bio-safety of FHMON carriers and IR780@O2-FHMON were evaluated on two types of normal cell lines, i.e., normoxic mouse fibroblast cells (L929) and alpha mouse liver (AML-12) cells. Neglectable cytotoxicity of FHMON carriers on L929 and AML-12 cells within the range of 800 µg/mL is observed in Figure S10, suggesting an excellent biosafety of FHMON carriers. It has been extensively documented that HBO can kill cancer cells via generating ROS,45 but it is safe for normal cells or tissues and can induce antioxidant gene expression,46-48 and play a stimulus for proliferation, differentiation, cytoprotective and angiogenic response of normal cells,


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e.g., endothelial cells, osteoblasts and fibroblasts.49-51 Therefore, the local HBO originating from the spontaneous oxygen release in IR780@O2-FHMON alone can kill hypoxic PANC-1 cells, but it promotes proliferation of L929 and AML-12 cells (Figure S11). Similar to SDT outcomes of PANC-1 cells, the normal sonocatalytic process of SDT in both US+IR780@FHMON and US+IR780@O2-FHMON plays a pro-apoptotic role for L929 and AML-12 cells due to the normoxic milieu and sufficient ROS (Figure S11). Fortunately, in vivo experiments, US irradiation is local and confined to the site of tumor, thus the cytotoxicity of US+IR780@FHMON and US+IR780@O2-FHMON on normal cells or tissues can be avoided. In vivo highly-efficient SDT principle against hypoxic solid tumor. After in vitro, extracellular and intracellular explorations, in vivo evaluations using this oxygen self-produced nanoplatform to relieve hypoxic regions, diminish resistance to SDT and improve SDT against hypoxic PANC-1 pancreatic solid tumor were also implemented. The schematic of in vivo SDT against hypoxic solid tumor using this specific oxygen self-produced SDT nanoplatform (IR780@O2-FHMON) is shown in Figure 5a. Herein, IR780@O2-FHMON is expected to favorably realize three functions named as 3F (F1-F3), i.e., F1: breaking barriers of nanoplatform delivery, F2: releasing oxygen and relieving hypoxia, F3: diminishing hypoxia-induced resistance to SDT and improving SDT efficiency against hypoxic PANC-1 solid tumor. Besides accelerating oxygen release and activating IR780 to generate sonocatalytic conversion of oxygen into ROS, US could also enhance the permeability of tumor tissue and break extracellular stroma barrier.32-34 This provides an opportunity to break the hypoxic-specific transportation barriers and realize a considerable accumulation of IR780@O2-FHMON in hypoxic PANC-1 solid tumor. As expected, more accumulation of IR780@O2-FHMON in hypoxic PANC-1 cells after US irradiation in comparison to IR780@O2-FHMON alone validates


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US-triggered O2 bubbles-enhanced cavitation effect indeed enhance permeability of cell membrane (Figures S12). More significantly, more retention of IR780@O2-FHMON in hypoxic PANC-1 solid tumor after experiencing US irradiation further demonstrates that US-triggered O2 bubbles-enhanced cavitation effect indeed breaks hypoxic-specific stroma barriers, as shown in in vivo distribution of Si atoms (Figure 5b). These results sufficiently demonstrate the feasibility of F1 and concurrently lay a solid foundation for F2 and F3. Moreover, the large blood half-life (128 min) will further promote F1 (Figure 5c). In vivo tumor oxygenation using IR780@O2-FHMON. The nude mice bearing PANC-1 solid tumor in each group received three repeated SDT treatments at day =0, 5, 13 (Figure 6a). During in vivo SDT, oxygen partial pressure (pO2) was monitored in a real-time manner to demonstrate F2. Before and after the 1st SDT treatment, time-dependent normalized pO2 curve within 24 h is obtained, as exhibited in Figure 6b. No obvious variation of pO2 in PANC-1 solid tumor in three groups (i.e., control, US+FHMON and IR780@FHMON) is observed. In contrast, pO2 in PANC1 tumor after treatment with IR780@O2-FHMON alone gradually increases due to the spontaneous oxygen release, suggesting IR780@O2-FHMON can relieve hypoxic regions of malignant solid tumor. More intriguingly, once IR780@O2-FHMON unites to US irradiation, US-mediated enhanced accumulation and accelerated oxygen release of IR780@O2-FHMON result in a drastic elevation of pO2 in hypoxic PANC-1 solid tumor, which further relieves in vivo hypoxia microenvironment. In particular, pO2 in hypoxic PANC-1 solid tumor after three repeated treatments was also monitored at day=0, 5, 13 (Figure 6c). After each SDT treatment, an identical variation trend of pO2, i.e., rapid elevation and slow recession, is observed in PANC1 solid tumor treated with IR780@O2-FHMON alone and US+IR780@O2-FHMON. Inspiringly,


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the lowest pO2 remain much higher than the critical pO2 value of hypoxia, suggesting the complete hypoxia reversion by this oxygen self-produced SDT nanoplatform. To visualize the hypoxia variation, the residual tumor at the end of SDT experiments (day 28) was collected after resection, and then immunochemically stained by CD31-Cy3, DAPI and hypoxyprobe (Hypoxyprobe-1 plus kit) to discern blood vessels, cell nucleus and hypoxic regions, respectively.15 In comparison to control, the LCSM image of PANC-1 xenograft tumor slices in US+IR780@O2-FHMON displays a considerably decreased hypoxia distribution (Figure 6d), further demonstrating the permanent hypoxia reversion (F2). The hypoxia reversion means the termination of hypoxia-induced resistance to oxygen-dependent therapies,52 which will benefit the following in vivo SDT against hypoxic PANC-1 solid tumor (F3). In vivo SDT of PANC-1 solid tumor using IR780@O2-FHMON. Encouraged by F2 and F1, highly-efficient SDT against highly-aggressive PANC-1 pancreatic cancer can be expected. Nude mice bearing hypoxic PANC-1 solid tumor were randomly divided into five groups, and each

group

received

different

treatments,

e.g.,

control

(PBS),

US+FHMON,

US+IR780@FHMON, IR780@O2-FHMON and US+IR780@O2-FHMON. It is found that the group of US+IR780@O2-FHMON performs best in inhibiting tumor growth among all five groups, eventually resulting in the shrinkage of tumor volume (Figure 7a,b). This result sufficiently demonstrates O2 supply in IR780@O2-FHMON in presence of US irradiation can significantly reinforce in vivo SDT (F3) against hypoxic PANC-1 solid tumor after mitigating hypoxia-induced resistance (F2) and enhancing IR780@O2-FHMON accumulation (F1). Depending on the excellent SDT outcome, the survival rate of nude mice in the group of US+IR780@O2-FHMON is considerably prolonged (Figure 7c), and results in a slow increase of weight (Figure S13). As explained in cellular-level experiment, IR780@O2-FHMON alone in


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the absence of US irradiation can spontaneously release oxygen to trigger the HBO therapy against PANC-1 tumor, similar to in vitro SDT. To comprehensively understand the molecular mechanism of SDT against hypoxic PANC-1 solid tumor, various pathological examinations including hematoxylin and eosin (H&E), proliferation cell nuclear antigen (PCNA) immunohistochemical staining and TUNEL immunofluorescent assay were carried out. In comparison to control, some characteristics associated with necrosis, e.g., cell lysis, collapsed cytoskeleton, chromatin condensation and nucleus disintegration, are obviously observed in the group of US+IR780@O2-FHMON (Figure 7d). US+IR780@O2-FHMON also induces the most apoptotic cells, and simultaneously acquire the largest inhibitory effect against the proliferation of PANC-1 cells, as evidenced in LCSM images of TUNEL (Figure 7e) and optical images of PCNA (Figure 7f), respectively. In particular, the ranking order of apoptotic cells and inhibitory proliferation performance are consistent with that of therapeutic effect in all groups. On this ground, these pathology information confirmatively suggests the oxygen self-produced SDT realized the most excellent treatment outcome through inducing PANC-1 apoptosis and inhibiting their proliferation. No evident variations in blood biochemical indices (Figure S14) and H&E pathological examination of normal organ slices (Figure S15) determine the local US irradiation guarantees the excellent bio-safety of this oxygen self-produced SDT nanoplatform. The excellent bio-safety is favorable for future clinical translation. CONCLUSIONS

In summary, we have successfully developed an oxygen self-produced SDT nanoplatform and established a FC chains-mediated oxygen delivery strategy for highly-efficient SDT against hypoxic malignant tumor. This approach takes the advantages of FHMONs-based nanosystems


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for constructing the oxygen self-produced SDT nanoplatform, e.g., the well-defined mesoporous structure facilitates the high loading of IR780, and the in-situ & uniform FC chains modification during FHMON synthesis supplies sufficient binding sites for oxygen delivery. In vitro, extracellular, intracellular and in vivo experiments demonstrate US can promote more accumulation of this biocompatible nanoplatform in hypoxic tumor, accelerate oxygen release and permanently relieve hypoxia. More significantly, the hypoxia reversion has been demonstrated to diminish the hypoxia-induced resistance to SDT, and eventually realize the highly-efficient SDT against of hypoxic PANC-1 pancreatic cancer via producing more ROS benefiting from sufficient oxygen supply. It is highly expected that this special oxygen selfproduced SDT nanoplatform involving the FC chains-mediated oxygen delivery protocol will realize hypoxia modulation, highly-efficient treatment and metastasis inhibition of highlyaggressive hypoxic malignant cancers. MATERIALS AND METHODS Synthesis of FHMONs and FITC-labeled FHMONs carriers FHMONs were obtained basing on a modified ‘in-situ hydrophobic layer-protected selective etching strategy’ that has been well established in a previous report.36 Typically, 5 mL of DI water was mixed with EtOH with a certain volume ratio of 1:7, followed by adding 1.57 mL of ammonia solution. The mixture was continuously stirred at 30 °C for 30 min. Afterwards, 3 mL of TEOS was added quickly and stirred for another 45 min so as to completely hydrolysis and condensation of TEOS, yielding the solid silica cores (s-SiO2). Subsequently, a mixture consisting of TEOS (2.5 mL) and PDES (1 mL) was dropwise injected into the milky-white dispersion within 3 min. Another 80 min-incubation was carried out at 30 °C, and the organosilica hybrid shell was coated onto the solid silica cores, obtaining core-shell structured


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colloidal nanoparticles (abbreviated into s-SiO2@h-SiO2). Afterwards, the white colloidal nanoparticles were harvested and rinsed with DI water, and then divided into duplicate in average. Each one was re-dispersed in 50 mL Na2CO3 aqueous solution (0.6 M) for 30 min at 80 °C during which solid inner core (i.e., s-SiO2) was etched out and obtain the ultimate product, i.e., fluorocarbon

chains-functionalized hollow mesoporous organosilica nanoparticles

(FHMONs). The harvested FHMONs via the high-speed centrifugation at 12000 rpm/min for 10 min were washed with DI water for three times. As for FITC-labeled FHMONs, FITC-APTES (100 µL) should be added in the mixture of TEOS and PETES when coating the organosilica hybrid shell onto s-SiO2, and other synthetic procedures were approximately identical to those of FHMONs. Synthesis of IR780@FHMON and IR780@O2-FHMON 100 mg of IR780 was dissolved in methanol (50 mL) via the ultrasonication-assisted dispersion method. After completely dissolution, FHMONs (100 mg) were added and evenly dispersed in the IR780 solution in methanol via sonication. Afterwards, continuous stirring was carried out for 24 h so as to realize the highly-efficient loading of IR780 by FHMONs via electrostatic interaction. After that, the IR780@FHMON product was collected via high-speed centrifugation and washed with methanol for three times to remove the residual IR780. To check the successful loading of IR780, UV-Vis spectrometer detection was carried out on a Nicolet Avatar 370 FT-IR spectrophotometer using KBr pellets as reference. In terms of IR780@O2-FHMON, aforementioned IR780@FHMON with a certain amount was re-dispersed in DI water or PBS via ultrasonication according to the actual demand. Afterwards, O2 bubbling was enforced to make the chelation of O2 onto fluorocarbon chains of FHMON, and the chelation process lasted 4 h for the sufficient chelation of O2, obtaining


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IR780@O2-FHMON dispersion in DI water or PBS. As well, the zeta potentials and particle size distributions of FHMON, IR780@FHMON and IR780@O2-FHMON were inspected on Malvern Nano-ZS90. In vitro O2 release via ultrasound molecular imaging, GC detection, optical microscopic observation 2 mL of IR780@O2-FHMON solution in PBS (5 mg/mL) was added in the little finger chamber of nitrile powder-free gloves, and then exhausted the gas via twisting the little finger chamber and sealed the chamber. Ultrasound molecular imaging was conducted on so as to validate the presence of O2 in IR780@O2-FHMON, wherein the B-mode fundamental imaging was employed on LOGIQ E9 (GE Co. LTD) with a transducer of 6 - 13 MHz. The ultrasonic images were captured before and after therapeutic US irradiation. Furthermore, 200 µL of IR780@O2-FHMON solution in degassed PBS (5 mg/mL) via boiling was dropped into the groove of the LCSM-specific culture dishes (Diameter/Dimension: 35 mm/15 mm, Cat. No.: 801002, NEST Biotechnology Co.LTD., Wuxi, China), and then covered by the square glass coverslip (1.5 cm ×1.5 cm). Subsequently, they were sealed along the periphery of coverslip using transparent nail polish. After drying, 1 mL of PBS was added. Immediately afterwards, US irradiation was applied on the bottom of dish, between which agar gel with 1 cm in thickness was inserted in case of hyperthermia. Optical microscopy observation was carried out to validate the emergence of O2 bubbles. Noticeably, the power density of US was 1 W/cm2, and duty cycle was 100%. The irradiation duration was 30 s per cycle with two cycles in total and 30 s intervals between two cycles. As well, gas chromatography (GC) was also used to detect O2 production from IR780@O2-FHMON solution with mixed gas (H2/N2 =0.03 MPa : 0.1 MPa) as carrier gas. In detail, 50 mL of IR780@O2-FHMON solution (5 mg/mL) was added in three-necked, round-


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bottomed flask that was placed in heating mantle with a set temperature of 40 °C lower than 42 °C that was the accepted-universally apoptotic limit temperature. The middle inlet was sealed by ground glass stopper, while other two-side inlets inserted into the admission line of GC 2060 apparatus at a total flow rate of 50 mL/min. In vitro quantitative analysis of 1O2 DPBF was used as an indicator to investigate the generation of 1O2, because it could react irreversibly with 1O2 to result in decrease in of DPBF absorption intensity at about 410 nm. All media were deoxygenated via N2 bubbling before use. The IR780@O2-FHMON (5 mg) was suspended into acetonitrile (3 mL), followed by adding DPBF solution in acetonitrile (20 µL, 8 mM). The mixture was treated by US irradiation with 9 cycles at some given time points (0.5 h, 1 h, 2 h, 4 h), and each cycle lasted 20 s and the interval time between two cycles was 30 s, and the ultrasound parameters were given: frequency=1.0 MHz, power density: 1.0 W/cm2, duty cycle=100%. At given time points (0 h, 0.5 h, 0.55 h, 1 h, 1.05 h, 2 h, 2.05 h, 4 h, 4.05 h, 8 h), the absorption intensity of the mixture at 410 nm was monitored on a UV-vis spectrophotometer (UV-3600). For control, no sample and no US irradiation were carried out and other procedures were the same with above. As for US+FHMON and US+IR780@FHMON, IR780@O2-FHMON was replaced by FHMON and IR780@FHMON, respectively, and other operations followed the procedures of US+IR780@O2-FHMON. Experiments were performed in triplicate with results presentation as mean value ± standard deviation (SD). Differences between US+IR780@O2FHMON and control and between US+IR780@FHMON and control were analyzed using the Student’s two-tailed t test (*P < 0.05, **P < 0.01 and ***P < 0.001). 1

O2 detection using ESR


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Electron spin resonance (ESR) spectroscopy was used to further confirm 1O2 production after adding the capturing agent of 1O2 (TEMP). Typically, 20 µL of TEMP buffer solution (1 mM) was firstly added into 80 µL of IR780@O2-FHMON buffer solution (500 µg/mL) in a dark eppendorf tube. Subsequently, US irradiation (1.0 MHz, 1.0 W/cm2, 100% duty cycle) was carried out for 120 s. Immediately afterwards, the 1O2 signal was immediately detected by the electron spin resonance (ESR) spectrometer (Bruker EMX-8/2.7 spectrometer). Moreover, US+FHMON and US+HIR780@FHMON groups were also tested for comparison. Quantitative detection of extracellular O2 Five groups were set, and named as control, US, IR780@FHMON, IR780@O2-FHMON, US+IR780@O2-FHMON, respectively. PANC-1 cells at a density of 2 × 105 cells per mL DMEM were firstly seeded in the LCSM-specific culture disk (Diameter/Dimension: 35 mm/15 mm, Cat. No.: 801002, NEST Biotechnology Co.LTD., Wuxi, China), and allowed to adhere overnight at 37 °C in hypoxic incubator (MCO-5M CO2 incubator, Sanyo, Moriguchi, Japan) that was full of hypoxic gas stream (0.1% O2: 5% CO2: 94.9% N2 in volume). Afterwards, these cells in each groups experienced the corresponding treatments, and the treatments and subsequent incubation were still carried out in the hypoxic incubator infused with hypoxic gas stream (0.1% O2: 5% CO2: 94.9% N2 in volume) with the minimum gap that merely allowed the ultrasound transducer to enter, which is beneficial for minimizing potential oxygenation of hypoxic cells from normal gas atmosphere. In detail, US group represented only US irradiation without any other samples, while IR780@FHMON group and IR780@O2-FHMON group represented co-incubation of PANC-1 cells with IR780@FHMON and IR780@O2-FHMON solution (0.5 mg/mL) in fresh DMEM after removing the previous culture medium. In particular, US+IR780@O2-FHMON group indicated that 30 min later after replacing the previous culture


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medium with IR780@O2-FHMON solution in fresh DMEM, US irradiation was carried out. The power density and duty cycle of US were 1.0 W/cm2 and 100 %, respectively. The irradiation duration was 20 s per cycle with 9 cycles in total and 30 s intervals between two cycles, and during each interval, the gate of hypoxic incubator was closely shut, which further lowered the influence of potential oxygenation on the hypoxic cells when implementing the in vitro hypoxic experiments. The employed mass concentration was 500 µg/mL based on IR780@O2-FHMON and 46.5 µg/mL based on I780. After 12 h, the dissolved O2 concentration in culture media was measured by using a Unisense oxygen microelectrode. As well, time-dependent O2 release profile in US+IR780@O2-FHMON group was also obtained at the same conditions via monitoring the O2 concentration at some given time points (0 h, 0.5 h, 1.0 h, 2 h, 4 h, 8 h, 12 h, 18 h, 24 h). Three independent culture disks per group were carried out for measuring the dissolved-oxygen level. Noticeably, PBS used for cell washing as well as DMEM cell culture media was deoxygenated via bubbling with nitrogen gas before use Intracellular O2 monitoring O2 indicator, i.e., [Ru(dpp)3]Cl2, was used to detect the O2 supply of IR780@O2-FHMON. In detail, four groups were set, i.e., IR780@FHMON, US+IR780@FHMON, IR780@O2FHMON and US+IR780@O2-FHMON. PANC-1 cells (2 × 105 cells per mL DMEM, pH = 7.4) were firstly seeded in the LCSM-specific culture disk (Diameter/Dimension: 35 mm/15 mm, Cat. No.: 801002, NEST Biotechnology Co.LTD., Wuxi, China), and then allowed to adhere overnight at 37 °C under hypoxic incubator (MCO-5M CO2 incubator, Sanyo, Moriguchi, Japan). Afterwards, these cells in each group experienced their corresponding treatments that were still carried out in hypoxic incubator full of hypoxic gas stream (0.1% O2: 5% CO2: 94.9% N2 in volume) with the minimum gap that merely allowed the ultrasound transducer to enter. The


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employed mass concentration was 500 µg/mL based on IR780@O2-FHMON and 46.5 µg/mL based on I780. After 12 h, these cells were then incubated with [Ru(dpp)3]Cl2 (Sigma-Aldrich, Co. LTD.) at a concentration of 10 µg/ml for another 12 h in hypoxic incubator so as to evaluate the outcomes of continuous hypoxia modulation within 24 h, followed by rinsing with PBS for three times to remove the free [Ru(dpp)3]Cl2 and residual particles. Ultimately, 1 mL of fresh DMEM culture media was added and the PANC-1 cells were further hermetically cultured in a confocal dish for 2 h at 37 °C in hypoxic incubator (MCO-5M CO2 incubator, Sanyo, Moriguchi, Japan). The level of intracellular O2 was evaluated by detecting the fluorescence of [Ru(dpp)3]2+ (λex = 450 nm, λem = 610 nm) when placed in live cell culture system of LCSM (FV 1000, Olympus) that maintained hypoxic condition via infusing hypoxic gas stream. This live cell culture system is also called as cell growth environment control system equipped in Olympus FV1000. Noticeably, PBS used for cell washing as well as DMEM cell culture media was deoxygenated via bubbling with nitrogen gas before use. Cell apoptosis assay via flow cytometry Panc-1 cells seeded in a 6-well cell-culture plate at a density of 2×105 cells per well were cultured in hypoxic atmosphere at 37 °C for 24 h. Five groups were set, and they were control, FHMON combing US irradiation (US+FHMON), IR780@FHMON combining US irradiation (US+IR780@FHMON),

IR780@O2-FHMON

and

IR780@O2-FHMON

combining

US

irradiation (US+IR780@O2-FHMON). After corresponding treatment of each group in hypoxic incubator, another 24 h hypoxic incubation was carried out. The treatment in each group was still carried out in hypoxic incubator full of hypoxic gas stream (0.1% O2: 5% CO2: 94.9% N2 in volume) with the minimum gap that merely allowed the ultrasound transducer to enter. The parameters of US irradiation were indicated as follows: power density=1 W/cm2, transducer


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frequency=1 MHz, duration time= 20 s per cycle, total cycle=9, interval between two cycles=30 s during which the hypoxic incubator gate was shut. Agar gel was embedded into the middle site between LCSM-specific disk and US transducer. Subsequently, isolated PANC-1 cells were firstly collected and adherent cells were also harvested via the trypsin digestion method. Afterwards, all of collected cells were re-suspended in 1.0 mL of binding buffer, and stained by propidium iodide (PI) and annexin V-FITC (2 µL of annexin V-FITC and 2 µL of PI per ml binding buffer). To acquire convincing outcomes, the incubation duration before analysis by flow cytometry was 30 min. Ultimately, the cells were re-collected and re-suspended in 1 mL of PBS for flow cytometry. The employed mass concentration was 500 µg/mL based on IR780@O2-FHMON and 46.5 µg/mL based on I780. Noticeably, PBS used for cell washing as well as DMEM cell culture media was deoxygenated via bubbling with nitrogen gas before use. ROS detection using flow cytometry PANC-1 cells (2 × 105 cells per well in 6-well plates) were incubated in hypoxic incubator. Four groups were set, i.e., PBS, FHMON (0.5 mg/mL), IR780@FHMON (0.5 mg/ml) and IR780@O2-FHMON (0.5 mg/mL), and each group experienced the same US irradiation in hypoxic incubator, meaning 46.5 µg/mL based on I780. All cells were firstly incubated with 1 mL of FBS-free DMEM containing DCFH-DA (1:1000) for 20 min in the dark prior to incubation with different samples and US irradiation in hypoxic incubator. After another 4 h incubation in hypoxic incubator, isolated and adherent cells in each group were collected. The collected cells were then washed with FBS-free culture medium and PBS in sequence, and their fluorescence intensity was assessed by flow cytometry. In particular, the relative ROS under different power densities (0, 0.6, 1.0, 1.2, 1.5 W/cm2) were also detected. Three independent


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dishes in each group were adopted at one given power density. Agar gel was embedded into the middle site between LCSM-specific disk and US transducer. As well, time-dependent ROS variations in two groups (i.e., IR780@O2-FHMON and US+IR780@O2-FHMON) were also monitored, and 7 time points were set in each group. In detail, PANC-1 cells seeded in 6-well culture disk at a density of 2 × 105 cells per well were cultured in hypoxic atmosphere at 37 °C for 24 h, and allowed the cells to adhere. For the group of IR780@O2-FHMON, at time point = 0, the media in 3 independent culture dishes were firstly replaced by 1 mL of FBS-free DMEM containing DCFH-DA (1:1000), followed by adding IR780@O2-FHMON, respectively. On the contrary, at time point = 0, PANC-1 cell in other 18 independent culture dishes were firstly treated with IR780@O2-FHMON. Afterwards, the media in three dishes were replaced by 1 mL of FBS-free DMEM containing DCFH-DA (1:1000) at each given time point (0.5 h, 1 h, 2 h, 4 h, 8 h and12 h) for staining. The staining was carried out for 20 min in the dark, and another 4 h incubation in hypoxic incubator was enforced prior to collecting the isolated and adherent cells in each dish. The subsequent procedures were identical to those in aforementioned experiment. For the group of US+IR780@ FHMON, the treatment with IR780@O2-FHMON alone were replaced by the treatment with US+IR780@ FHMON, and other procedures were identical. Noticeably, the initial ROS level represented by fluorescence intensity via LCSM (time point = 0) was normalized to 1, and performed as the reference. Ultrasound irradiation was still carried out in hypoxic incubator full of hypoxic gas stream (0.1% O2: 5% CO2: 94.9% N2 in volume) with the minimum gap that merely allowed the ultrasound transducer to enter. The employed parameters of US were indicated as follows: power density=1 W/cm2, transducer frequency=1 MHz, duration time = 20 s per cycle, total cycle=9, interval between two cycles=30 s during which the hypoxic incubator gate was shut. Agar gel


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was embedded into the middle site between LCSM-specific disk and US transducer. Noticeably, PBS used for cell washing as well as DMEM cell culture media was deoxygenated via bubbling with nitrogen gas before use. Qualitative cellular-level ROS monitoring via LCSM observation PANC-1 cells seeded in the LCSM-specific culture disks at a density of 2 × 105 cells per well were cultured in hypoxic atmosphere at 37 °C for 24 h. Subsequently, five groups were set, i.e., control, FHMON combing US irradiation (US+FHMON), IR780@FHMON (0.5 mg/mL) combining US irradiation (US+IR780@FHMON), IR780@O2-FHMON and IR780@O2FHMON combining US irradiation (US+IR780@O2-FHMON). It is worth noting that all cells in the five groups firstly incubated with 1 mL of FBS-free DMEM containing DCFH-DA (10 µM) for 20 min, followed by their perspective corresponding treatment in each group. After another 4 h incubation in hypoxic incubator, the level of intracellular ROS was evaluated by detecting the fluorescence of DCF (λex = 488 nm, λem = 525 nm) with confocal laser scanning microscopy (FV 1000, Olympus, Japan). Ultrasound irradiation was still carried out in hypoxic incubator, and the employed parameters of US were indicated as follows: power density=1 W/cm2, transducer frequency=1 MHz, duration time = 20 s per cycle, total cycle=9, interval between two cycles=30 s during which the hypoxic incubator gate was shut. Agar gel was embedded into the middle site between LCSM-specific disk and US transducer. Noticeably, PBS used for cell washing as well as DMEM cell culture media was deoxygenated via bubbling with nitrogen gas before use. In vitro O2-supplied SDT via LCSM observation Panc-1cells seeded in the LCSM-specific dish at a density of 2 × 105 cells per well were cultured in hypoxic incubator with mixed atmosphere of N2/O2/CO2 (94.9:0.1:5) at 37 °C for 24


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h until they were adhered to the bottom of the dish. Five groups identical to those in cell apoptosis assay via flow cytometry were set, i.e., control, US+FHMON, US+IR780@FHMON, IR780@O2-FHMON

and

US+IR780@O2-FHMON.

The

corresponding

treatment

and

subsequent incubation in each group was still carried out in the hypoxic incubator. The mass concentration of any sample is 0.5 mg/mL (that is, 46.5 µg/mL based on I780) and the employed parameters of US were indicated as follows: power density=1 W/cm2, transducer frequency=1 MHz, duration time= 20 s per cycle, total cycle=9, interval between two cycles=30 s during which the hypoxic incubator gate was shut. Agar gel was embedded into the middle between LCSM-specific disk and US transducer. After 24-incubation post-treatment in hypoxic incubator, both isolated cells and adherent cells were harvested, and then were stained by PI (100 µL, 20 µM) and Calcein-AM (100 µL, 20 µM) for determining dead and viable cells via LCSM observation. Noticeably, PBS used for cell washing as well as DMEM cell culture media was deoxygenated via bubbling with nitrogen gas before use. Intracellular endocytosis by LCSM observation Typically, PANC-1 cells at a density of 2 × 105 were seeded into the LCSM-specific dish and incubated for 12 h in hypoxic incubator at 37 °C, and two groups, i.e., IR780@O2-FHMON and US+IR780@O2-FHMON, were set. The culture media in each group were replaced with IR780@O2-FHMON (1 mL, 100 µg/mL in DMEM), but only in the group of US+IR780@O2FHMON, additional US irradiation was carried out. Ultrasound irradiation was still carried out in hypoxic incubator, and the employed parameters of US were indicated as follows: power density=1 W/cm2, transducer frequency=1 MHz, duration time = 20 s per cycle, total cycle=9, interval between two cycles=30 s during which the hypoxic incubator gate was shut. Afterwards, the cells were cultured for 4 h in hypoxic incubator. 4',6-diamidino-2-phenylindole (DAPI, 100


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µL) in methanol was added into the dish to stain the cell nuclei. The cells were then observed in cell growth environment control system by LCSM after staining for 15 min. Noticeably, PBS used for cell washing as well as DMEM cell culture media was deoxygenated via bubbling with nitrogen gas before use. In vivo O2-supplied SDT against Panc-1 xenografted solid tumor on nude mice model Panc-1 pancreatic solid tumor-bearing nude mice (36 in sum) were randomly allocated into 5 groups (n=6), i.e., Control (PBS), US+FHMON, US+IR780@FHMON, IR780@O2-FHMON, and US+IR780@O2-FHMON, and each group experienced their perspective corresponding treatments. The experiment was approved ethically and scientifically by Guangxi Medical University, and complied with Practice for Laboratory Animals in China. The 1st treatment occurred on day 0. The detailed procedures were depicted, e.g., in the groups of control and IR780@O2-FHMON, only i.v. injections of PBS and R780@O2-FHMON were carried out; while in three groups of US+FHMON, US+IR780@FHMON and US+IR780@O2-FHMON, after receiving i.v. injections of FHMON, IR780@FHMON and IR780@O2-FHMON, the PANC-1 tumor on nude mice was further treated with US irradiation after 6 h post-injection. The employed power density of US was 1.0 W/cm2 with a 100% duty cycle, and irradiation duration was 20 s per cycle with 9 cycles in total and 1 min interval between two cycles. Between tumors and therapeutic US transducer, the sound-absorbing panel with a hole whose diameter was consistent with tumor size was embedded. The administering method is intravenous injection via tail vein. As well, on day 5 and day 13, other two same treatments in each group were carried out. The injection dose was 100 mg FHMON /Kg in total, thus the injections dose per injection based on FHMON carriers and IR780 were 33.33 mg FHMON carrier /Kg and 3.4 mg IR780/Kg per injection, respectively. During the experimental period, tumor volume, body weight and


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intratumoral O2 partial pressure (pO2) were measured at some given days. The tumor volume was calculated according to the following formula: Tumor volume (V) = L × W2/2 wherein L and W were the measured length and width of tumors, respectively using a digital caliper. The relative tumor volume (Vt/V0) was normalized to the initial volume (V0). In particular, the intratumoral pO2 within 24 h after the 1st treatment was measured in detail by a Unisense oxygen microelectrode equipped with a hand-operated micropropeller. At the end of experimental period (day 28), the nude mice were euthanized via injecting excessive anesthetics (2.5% pentobarbital), and all tumors in each group were dissected and stained by H&E, PCNA and TUNEL immunofluorescence or immumohistochemistry for histological analysis via optical microscopic or LCSM observation. Statistical analysis All the experiments were performed in triplicate. The obtained data were expressed as the mean value ± standard deviation (SD) and any statistical comparison between two groups was analyzed using the Student’s two-tailed t test. *P < 0.05 (significant), **P < 0.01 (moderately significant) and ***P < 0.001 (highly significant).

ASSOCIATED CONTENT Supporting Information Available: Materials and characterization, additional experimental details and supplementary figures from Figure S1 to Figure S15. This material is available free of charge via the Internet at http://pubs.acs.org. The authors declare no competing ﬁnancial interest.

AUTHOR INFORMATION


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Author Contributions #

The authors contributed equally to this work.

K. Chen, K. Zhang and J. Liu supervised the project. K. Zhang conceived and designed the experiments. J. Chen and H. Luo performed the synthesis of different samples and cell experiments, J. Chen, H. Luo, Y Liu, W. Zhang and H. Li performed the animal experiments and related analysis. J. Chen, K. Zhang, Y. Zhao and J. Liu wrote the manuscript. All authors discussed the results and commented on the manuscript. ACKNOWLEDGMENT This work was supported by the Opening Project of Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy Research (Grant No. GXSWBX201507), National Natural Science Foundation of China (Grant No.81701721, 81771836, 81501473), and Fostering and Action Planning of Tongji University for Young Excellences (Grant No. 2015KJ061). REFERENCES (1)

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Figures

Figure 1. Synthetic process & action principle of IR780@O2-FHMON and characterization of FHMON carriers. (a) Schematic of IR780@O2-FHMON; (b) Principle of intensified SDT using IR780@O2-FHMON; (c-e) TEM, dark-field, bright-field images of FHMON carriers; (f-j) Merged atom mapping image (f), Si (g), C (h), O (i), F (j) atom mapping images of FHMON carriers.


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Figure 2. Characterization and extracellular & intracellular hypoxia modulation of IR780@O2-FHMON. (a) Uv-vis spectra of FHMON carrier and IR780@FHMON with a characteristic peak of IR780 at around 780 nm; (b) Zeta potentials of FHMON, IR780@FHMON and IR780@O2-FHMON; (c,d) GC characteristic peak (c) and optical microscopic image (d) of O2 released from IR780@O2-FHMON after heating and US irradiation, respectively, and scale bar: 200 µm. (e) Extracellular O2 concentration of hypoxic PANC-1 cells after treatments with different groups, i.e., control, IR780@FHMON, US+IR780@FHMON, IR780@O2-FHMON and US+IR780@O2-FHMON, and data are presented as the mean value ± SD (n=3). (f) Time-sweep O2 concentration curves of hypoxic PANC-1 cells after treatments with different groups, i.e., control, IR780@FHMON, US+IR780@FHMON, IR780@O2-FHMON and US+IR780@O2FHMON; Data are presented as the mean value ± SD (n=3) and significances are obtained via comparing to control. (g) LCSM images of hypoxic PANC-1 cells stained by O2 indicator, i.e., (Ru(dpp)3)Cl2 after treatments with different groups (i.e., control, IR780@FHMON, US+IR780@FHMON, IR780@O2-FHMON and US+IR780@O2-FHMON) and scale bar: 40 µm. Note ‘*’, ‘**’ and ‘***’ represent P

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