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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Near-Infrared Light-Initiated Molecular Superoxide Radical Generator: Rejuvenating Photodynamic Therapy against Hypoxic Tumors Mingle Li,† Jing Xia,‡ Ruisong Tian,† Jingyun Wang,‡ Jiangli Fan,† Jianjun Du,† Saran Long,† Xiangzhi Song,∥ James W. Foley,§ and Xiaojun Peng*,† †
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China Department School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China ∥ College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China § Rowland Institute at Harvard, Harvard University, Cambridge, Massachusetts 02142, United States
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‡
S Supporting Information *
ABSTRACT: Hypoxia, a quite universal feature in most solid tumors, has been considered as the “Achilles’ heel” of traditional photodynamic therapy (PDT) and substantially impairs the overall therapeutic efficacy. Herein, we develop a near-infrared (NIR) light-triggered molecular superoxide radical (O2−•) generator (ENBS-B) to surmount this intractable issue, also reveal its detailed O2−• action mechanism underlying the antihypoxia effects, and confirm its application for in vivo targeted hypoxic solid tumor ablation. Photomediated radical generation mechanism study shows that, even under severe hypoxic environment (2% O2), ENBS-B can generate considerable O2−• through type I photoreactions, and partial O2−• is transformed to high toxic OH· through SODmediated cascade reactions. These radicals synergistically damage the intracellular lysosomes, which subsequently trigger cancer cell apoptosis, presenting a robust hypoxic PDT potency. In vitro coculture model shows that, benefiting from biotin ligand, ENBS-B achieves 87-fold higher cellular uptake in cancer cells than normal cells, offering opportunities for personalized medicine. Following intravenous administration, ENBS-B is able to specifically target to neoplastic tissues and completely suppresses the tumor growth at a low light-dose irradiation. As such, we postulated this work will extend the options of excellent agents for clinical cancer therapy.
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INTRODUCTION
causes untoward side effects, for instance, the hyperoxic seizures and barotrauma problems in HBO treatments.20,21 In sharply different from O2 supply strategies, directly reducing the O2 requirement in essence by creating low oxygendependent agents may be a more promising way for clinical application, but rare efforts have been dedicated in this field. Superoxide radical (O2−•), one of the primary and most toxic reactive oxygen species (ROS), has been identified as the main oxidant for cancer therapy and as adjuvant to synergize the chemotherapy.22−24 Excessive O2−• in body reacts with proteins, DNA, and lipids, irreversibly damaging cellular components and resulting in dysfunction in cell metabolism.25,26 Notably, the intracellular superoxide dismutase (SOD) can initiate disproportionation reactions to catalyze O2−• to form hydrogen peroxide (H2O2) and O2.27,28 Then the accumulation H2O2 in cancer cells further converts down-
Tumor hypoxia caused by aberrant neoplastic cell proliferation and apoptosis as well as distorted tumor vasculatures has been considered as one of the major factors of the limited treatment outcome and poor prognosis for many cancer therapeutic proposals,1−4 especially photodynamic therapy (PDT) in which the tumoricidal effect relies highly on the oxygen level.5−10 Moreover, during the PDT process, the vascular damage further worsens the O2 shortage. As a result, tumor hypoxia is known as the “Achilles’ heel” of traditional PDT.5,9,11 Recently, to fight the negative consequences of hypoxia, many innovative approaches have been introduced, such as hyperbaric oxygen (HBO) therapy to increase intratumor oxygen perfusion,12,13 “oxygen shuttles” to deliver oxygen into tumors (e.g., perfluorocarbon and artificial red blood cells),14−16 and oxygen self-supplement systems to promote in situ oxygen generation (e.g., MnO2, CaO2, and catalase).17−19 However, employing these auxiliary strategies in the PDT procedure still cannot obtain ideal results, and often © XXXX American Chemical Society
Received: August 12, 2018 Published: October 17, 2018 A
DOI: 10.1021/jacs.8b08658 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society stream into more chemically reactive and highly toxic hydroxyl radical (OH·), which may dramatically aggravate the oxidative injury and improve the anticancer efficacy.24,29 A further benefit is that, at least in part, O2 is recyclable in these cascade bioreactions, thereby facilitating to ameliorate antihypoxia performance.27 Thus, exogenous O2−• generators would be an appealing candidate, which can open up a new avenue for hypoxic solid tumor treatment. To date, engineering functional inorganic and metallic (e.g., TiO2, ZnO, etc.) nanocomposites to produce O2−• are among the most studied strategies, in which such nanomaterials are able to be activated by UV light.30−32 However, several disadvantages exist; one is that the therapeutic efficiency depends largely on the highly energetic power density of light sources, which inevitably exerts superficial tissue injury. In addition, the poor biodegradability and increased pharmacokinetic complexity of nanocomposites represent potential limitations to widely clinical translation efforts.33 What is worse, the low penetration depth of UV light compared to NIR, which lies in the “therapeutic window (600−900 nm)”, also severely restricts its utility in deep-seated tumor PDT.5,8 Contrarily, owing to the obvious advantages (e.g., optimal biosafety, flexible preparation/modification, and excellent reproducibility) of organic small molecules relative to nanomaterials, it is reasonable to envision that NIR-triggered molecular O2−• generators are highly promising for rejuvenating PDT against tumor hypoxia. Unfortunately, such robust agents have not been reported yet. Here, we developed a NIR light-activated molecular O2−• generator, ENBS-B, for selective hypoxic tumor ablation in vivo, and revealed the detailed O2−• action mechanism underlying the antihypoxia effects for the first time. Previously, we and other researchers have shown that Nile blue analogs,34−36 after structural modification with “heavy atom”, can be applied in PDT against bacteria and tumors,37−47 however, their ROS functioning mechanism remains poorly understood, and prior to our work, rare efforts have noted that benzophenothiazine compounds are actually powerful O2−• generators, and in vivo hypoxic solid tumors treatment by using such compounds via O2−• mechanism has not been reported. Encouragingly, in this study, we found that, even under severe hypoxic environment (2% O2), ENBS-B enabled to generate sufficient O2−• under light irradiation via Type I photoreactions, thereby ideal PDT activities were still remained in hypoxic tumors. In addition, the formed O2−• could not only serve as oxidant to kill cancer cells, but also participated in SOD-mediated proportionation reaction to form H2O2 and its downstream highly toxic OH· (Scheme 1), which subsequently promoted the anticancer performance. Meanwhile, as evidenced by our constructed in vitro coculture model, ENBS-B could selectively target and internalize into biotin receptor positive cancer cells; as a result, strikingly selective PDT effect was achieved on cancers both in vitro and in vivo, whereas negligible toxicity toward normal cells, realizing the principle of clinical precision medicine.
Scheme 1. Schematic Illustration of Photo-Induced Radical Generation Mechanism of ENBS-B
was prepared as control (Figure 1a). The synthetic routes are detailed in Figure S1, and all chemical structures were fully
Figure 1. (a) Molecular structures (b) UV−vis absorption and (c) fluorescence spectra of ENBS-B and ENBS-C6-NH2.
confirmed by 1H NMR, 13C NMR, and ESI-MS analytical data (Figures S2−S7). As shown in Figure 1b, both ENBS-C6-NH2 and ENBS-B displayed an intense absorption profile that localizes in the therapeutic window, promising deeper permeability and less phototoxicity to normal organisms.5,7,8 After 660 nm excitation, considerably high NIR fluorescence (Φf = 0.21) was exhibited (Figure 1c). Such superb emission property was unique and rare over common PSs (e.g., Φf = 0.1 for BODIPY PS),49−51 qualifying ENBS-B as an ideal agent for noninvasively guiding the in vivo PDT in real time. Light Triggered O2−• Generation. To determine the O2−• generation of ENBS-B, electron paramagnetic resonance (EPR) spectroscopy was initially carried out. For this method, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied as the spin-trap agent for O2−•. As shown in Figure 2a, neither ENBS-B in dark nor light irradiation alone had any impact on the EPR signals. In contrast, for ENBS-B under NIR light irradiation, a characteristic paramagnetic adduct was observed and matched with that of referential KO2 (a well-known O2−• generator),22 indicating the production of O2−•. However, upon the addition of SOD, corresponding EPR amplitude was dramatically reduced to background level, which was ascribed to that SOD can effectively catalyze the dismutation of O2−•.27,28
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RESULTS AND DISCUSSION Synthesis and Photophysical Properties. Because most malignant cells, such as liver, lung, breast, and ovarian tumor cells, overexpress high-affinity biotin transporter on the cell surface,48 biotin unit was used as targeting ligand, serving to achieve preferential tumor homing. To better understand the targeting potency, the biotin-lacking analog, ENBS-C6-NH2 B
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to O2−• was observed in HepG2 cells by confocal laser scanning microscopy (CLSM), and the average fluorescence intensity increased from 82 to 2146 as the light dose processed from 0 to 14.4 J/cm2. Notably, the relative intracellular O2−• was sharply blocked by 3.4-fold after pretreatment with Vc owing to the O2−• scavenging effect (Figure 3b). We next proceeded to examine the O2−• generation under severe hypoxia (2% O2). To better mimic the tumor hypoxic environment, HepG2 cells were incubated in an incubator chamber (MIC-101, Billups-rothenberg) under a humidified, 2% O2, and 5% CO2 atmosphere for 8 h. Intracellular hypoxia was evidenced by anaerobic indicator ROS-ID, the fluorescence of which can recover dramatically due to the nitro group reduces to amino group under hypoxic conditions.57 CLSM images demonstrated that hypoxia leaded to 9.3-fold increase of ROS-ID fluorescence, confirming the in vitro hypoxic model was successfully constructed (Figure 3c). Encouragingly, after hypoxia treatment, despite of some influence, remarkable DHE fluorescence for O2−• was still detected in HepG2 cells (Figure 3d), implying ENBS-B might have satisfactory PDT activity under hypoxia. On the contrary, negligible 1O2 signals were obtained. These findings were matched well with the results of ROS studies in Figure 2. But, we did find remarkable green fluorescence of HPF for OH· in HepG2 cells no matter under 2% or 21% O2 environments after 660 nm light irradiation, significantly conflicting with the results in aqueous solution. That was to say OH· was formed in a manner that was not directly dependent on the photosensitization of ENBS-B. Because O2−• is the primary precursor of the most powerful oxidant,58 we hypothesized that one possible mechanism attributable to the above remarkable difference might be the O2−• induced by ENBS-B first underwent superoxide dismutase (SOD) disproportionation under acidic condition and, subsequently, a Haber−Weiss reaction or Fenton reaction to form the most toxic OH·.59,60 To validate this, the potent SOD inhibitor, 2-methoxyestradiol was used to weaken the activity of intracellular SOD,61 which could in turn prevent O2−• from transforming to OH·. As illustrated in Figure 3e, after the HepG2 cells were incubated with SOD inhibitor alone for 1 h, the fluorescence intensity of DHE in cells was not altered in comparison with control group no matter in the presence of irradiation or not, thereby excluding the interference of endogenous O2−•. Interestingly, with the addition of 2-methoxyestradiol, upon 5 min irradiation for ENBS-B, the O2−• level was dramatically boosted as compared with those cells not pretreated with SOD inhibitor. In the meantime, the OH· generation diminished significantly under the identical conditions (Figure 3f). These results revealed that the deactivation of intracellular SOD strikingly suppressed the dismutation of O2−• to H2O2 and O2, which subsequently hindered the OH· formation. Notably, in these cascade reactions, molecular oxygen was a recyclable unit in some degree, this might be the reason why O2−• and OH· could maintain at a favorable level under hypoxia. In Vitro Cytotoxic Study under Normoxia and Hypoxia. Afterward, the in vitro anticancer potency of ENBS-B was tested by methyl thiazolyltetrazolium (MTT) assay. It was obvious that ENBS-B had negligible dark toxicity (Figure S11), suggesting its good biocompatibility in vitro. After exposure to light, ENBS-B effectively inhibited the cell proliferation in a light and PS dose-dependent manner (Figure 4a), and the PDT efficiency of our ENBS-B even was much superior to clinical photosensitizer Ce6, with the IC50 (half
Figure 2. (a) EPR signals of DMPO for O2−• characterization, KO2 as the reference. (b) Fluorescence spectra for O2−• using DHR123 as fluorescence probe. (c) Fluorescence intensity of DHR123 at 526 nm after 660 nm irradiation for 6 min. (d) Fluorescence intensity of SOSG at 525 nm and HPF at 516 nm as a function of irradiation time in the presence of ENBS-B. (e) Photodegradation curves of ABDA with ENBS-B under 660 nm light irradiation. (f) Schematic illustration of O2−• generation through electron transfer process.
Then we further confirmed the O2−• production by O2−• probe dihydrorhodamine 123 (DHR123),52 which is nonfluorescent but can react with O2−• to emit strong green fluorescence centered at 526 nm. Similar to EPR analysis, ENBS-B distinctly increased the fluorescence intensity of DHR123 by 8 times within 6 min irradiation, while nearly no fluorescence was detected in the presence of SOD (Figure 2b and Figure S8). Moreover, vitamin C (Vc), a radical scavenger, was added into ENBS-B solutions to further validate that the enhanced DHR123 signal was indeed caused by generated O2−•, as expected, the fluorescence showed an 3.9-fold decrease (Figure 2c). On the other hand, no other ROS were observed when we employed singlet oxygen sensor green (SOSG) and hydroxyphenyl fluorescein (HPF) as the specific indicators for 1O2 and OH·, respectively (Figure 2d and Figure S9).53,54 Also, the 1O2 measurement result using 9,10anthracenedipropanoic acid (ABDA) was consistent with that of SOSG (Figure 2e). Considering that PDT process can take place via two different photochemical reaction mechanisms: energy transfer pathway to generate 1O2 and electron transfer pathway to produce O2−•,6,9 we reasonably speculated that ENBS-B predominately underwent electron transfer in the PDT process (Figure 2f). In Vitro ROS Generation Evaluation. Given the strong O2−• generation potency of ENBS-B, we then studied its photoactivity in vitro under both hypoxic and normoxic conditions by standard droethidium (DHE) staining assay. The detection mechanism is illustrated in Figure 3a.55,56 As shown in Figure S10, in the normoxic environment (21% O2), after exposure to 660 nm light, bright red fluorescence assigned C
DOI: 10.1021/jacs.8b08658 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Figure 3. (a) Schematic illustration of DHE for O2−• detection. (b) ROS generation of ENBS-B in HepG2 cells upon 660 nm light irradiation and CLSM images of cellular O2−• after exposure to 14.4 J/cm2 light dose in the absence or presence of Vc. (c) Intracellular hypoxia imaging using ROS-ID as anaerobic indicator. (d) ROS detection in HepG2 cells under normoxia (21% O2) amd hypoxia (2% O2) conditions using DHE, SOSG, and HPF as the O2−•, 1O2, and OH· fluorescence indicator, respectively. SOD inhibitor-mediated cellular (e) O2−• and (f) OH· generation. *P < 0.05, ***P < 0.001, and ****P < 0.0001 determined by Student’s t test.
Figure 4. (a) Cell viability of HepG2 cells subjected to a range of ENBS-B concentration and light doses. (b) PDT effect comparison of ENBS-B and Ce6 in HepG2 cells. (c) Cell viability of HepG2 cells in the (c) absence and (b) presence of light irradiation under normoxia (21% O2) or hypoxia (2% O2) conditions. (e) Dark toxicity and (f) phototoxicity effect of ENBS-B on HepG2 and COS-7 cells. (g) Cellular fluorescence images of ENBS-B or ENBS-C6-NH2 in in vitro coculture model. Data were expressed as mean ± SD *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 determined by Student’s t test.
maximal inhibitory concentration) value of merely 0.026 μM, 114-fold lower than that of Ce6 (Figure 4b). Benefiting from the good performance of ENBS-B in O2−• generation, we
proceeded to examine its PDT effect in 2% O2 hypoxic environment. As shown in Figure 4c, oxygen tension had no impact on the dark toxicity of ENBS-B. Upon 12 J/cm2 D
DOI: 10.1021/jacs.8b08658 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Figure 5. (a) In vivo NIR tumor-targeting images. (b) Relative fluorescence change of ENBS-B in the tumor and adjacent muscle tissues. (c) Fluorescence intensity profile of ENBS-B and ENBS-C6-NH2 in the tumor sites at different time point. (d) Immunofluorescence imaging of tumor slices. The tumor blood vessels (red) are stained with the anti-CD31 antibody, hypoxia-related protein HIF-α is stained with anti-HIF-1α antibody (green). (e) Relative tumor volume of mice after different treatments. (f) Average weight of tumors harvested at 14 d post-treatment (g) H&E staining of tumor sections from different treatment groups after 14 d of treatment, scale bar = 100 μm. Data were shown as mean ± SD (n = 6), **P < 0.01, ***P < 0.001, and ****P < 0.0001 determined by Student’s t test.
endowed ENBS-B with outstanding cellular selectivity, which was highly important for tumor-triggered targeting PDT. In Vivo Tumor NIR Imaging and Therapy. Because of NIR fluorescence facilitates bioimaging and the less susceptible interference from tissue autofluorescence, a small animal fluorescence imaging system was applied to study the tumor preferential accumulation of ENBS-B in tumor-bearing BALB/ c mice (Figure 5a). After intravenous injection of ENBS-B, the tumor sites could be clearly visualized and distinguished from surrounding tissues, showing a NIR signal-to-background ratio value (contrast ratio between tumor and adjacent muscle) as high as 7 ± 0.54 (Figure 5b), whereas according to previous studies that if such value exceeds 2.5, it means substantially preferential accumulation.62 In sharply contrast, nearly no ENBS-C6-NH2 signals at tumors were observed and, at 4h postinjection, the tumor accumulation of ENBS-B was 53.5fold higher than that of ENBS-C6-NH2 (Figure 5c), thus revealing that appreciable tumor targeting was achieved in ENBS-B. Since the tumor hypoxia is associated with tumor size,63 until the tumor volume reached about 200 mm3, these mice were used to explore the in vivo PDT potency of ENBSB. Besides, immunofluorescence staining also confirmed that the tumors were at severe hypoxic state as indicated by the high level of hypoxia inducible factor 1-α (HIF-1α), a hypoxiaassociated proteins which is overexpressed in hypoxic solid tumors.1 Moreover, the small amount of tumor blood vasculatures labeled by anti-CD31 antibody validated the tumor hypoxia again (Figure 5d).1 Then we conducted the hypoxic tumor treatment. As presented in Figure 5e, the tumors in the PBS treated group grew remarkably over the course of therapy regardless of 660 nm irradiation, exhibiting a 14-fold increment, which suggested that 660 nm light irradiation had little influence on the tumor inhibition. Also, ENBS-B alone treated mice showed no tumor suppression behavior, confirming the biosecurity of ENBS-B. However, when tumors were subjected to 660 nm light
irradiation, although 2% O2 slightly overwhelmed the PDT outcome, ENBS-B successfully leaded to severe cell disruption (Figure 4d). For example, at a low ENBS-B concentration of 0.63 μM, up to 94% cancer cells were killed. The LIVE/DEAD cell costaining assay further intuitively confirmed the ideal PDT performance of ENBS-B in hypoxia, as evidenced by the intense homogeneous red PI fluorescence which was comparable to that in normoxia (Figure S12). It is thus validated that ENBS-B was not seriously susceptible to oxygen, and could work well in hypoxic solid tumors in vivo. Another item worth noting is that selectively killing cancer cells while no cytotoxicity on normal cells is the fundamental principle in precision medicine; otherwise, patients will endure immense suffering. To study whether ENBS-B owned selective PDT ability, in this work, biotin receptor positive HepG2 cells and biotin receptor negative COS-7 cells, which have huge morphological differences from each other, were used as representative. Obviously, ENBS-B had no cytotoxicity toward these two cell lines in the absence of light (Figure 4e); however, after exposure to 12 J/cm2 irradiation, ENBS-B significantly killed the HepG2 cells, but little photodamage on COS-7 cells was observed (IC50 = 0.10 μM in HepG2 cells vs 0.56 μM in COS-7 cells). Meanwhile, the cell viability of COS7 cells remained up to 89% at the ENBS-B concentration of 0.16 μM (Figure 4f). To further intuitively observe this exciting result, a more challengeable experiment was performed. As depicted in Figure 4g, we constructed an in vitro cocultured model (mixed cultivation of HepG2 and COS7 cells) to simulate the actual tumor environment. After staining with ENBS-B for 0.5 h, the cellular uptake in HepG2 cells was 87-fold more than that in COS-7 cells. As a result, upon irradiation, HepG2 cells exhibited a 16-fold higher O2−• generation than COS-7 cells (Figure S13). In marked contrast, there was no such difference for ENBS-C6-NH2. Consequently, the conjugation of biotin moieties successfully E
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Journal of the American Chemical Society irradiation for 15 min, ENBS-B significantly suppressed the tumor growth, and the tumors began to shrink and scab. The average tumor weight and representative images also confirmed the excellent anticancer performance (Figure 5f and Figure S14). To further probe the antitumor efficacy of ENBS-B, tumors from four groups were sliced and stained with hematoxylin and eosin (H&E) for histological analysis (Figure 5g). Obviously, prominent cell necrosis was observed in tumors treated by ENBS-B with light. But in other groups, no remarkable damage was caused. On the other hand, the biodistribution data (Figure S15) as well as ex vivo imaging of main organs at 36 h postinjection (Figure S16) suggested that ENBS-B could be easily metabolized through kidney. Moreover, we did not observed noticeable cell necrosis and inflammation lesions in all major organs including heart, liver, spleen, lung, and kidneys (Figure S17), and all mice did show no noticeable abnormal body weight changes during the course of treatment (Figure S18), thereby suggesting that ENBS-B was biocompatible and applicable to in vivo systems. Therapeutic Mechanism of ENBS-B-Mediated PDT. To understand the detailed therapeutic mechanism underlying the excellent anticancer effect of ENBS-B, we then measured the intracellular distribution using commercial organelle-selective trackers. As manifested in Figure 6a, red fluorescence of ENBS-B overlapped well with the green fluorescence of lysosome dye LysoTracker Green DND 26 (correlation coefficient of 0.89), suggesting the internalization of ENBS-B and specific localization in lysosomes. In contrast, poor correlation coefficients were found between ENBS-B and mitochondrion or nuclear tracker (Figure S19). Recently, several different groups, including ours, have reported that PDT can induce intracellular organelles disruption by generated ROS to trigger cancer cell apoptosis.54,64−67 To elucidate the ENBS-B-mediated lysosome destruction, acridine orange (AO) was used as the lysosomal integrity indicator. For untreated-, ENBS-B-, and light-treated cells, a massive red fluorescence (white arrow) of AO was observed, indicating the lysosomal compartments were still intact. However, upon 660 nm light irradiation, the integrity of lysosomes was severely disrupted as identified by the disappeared AO red fluorescence (Figure 6b). Moreover, nuclear morphology becomes smaller and shrinks, and the cells were collapsed (yellow arrow). This was probably attributed to the formed OH·, because OH· could cause a larger wide range of oxidative damage to cancer cells, including lipids, DNA, and functional proteins.25,68 Then, Annexin V-FITC and propidium iodide (PI) were used as the indicators to investigate the cell apoptosis and death pathway (Figure 6c). After HepG2 cells were incubated with 1 μM ENBS-B for 30 min followed by stained with Annexin V-FITC/PI, invisible apoptosis signals were detected, similar to those treated with irradiation alone. Contrarily, intense green and red fluorescence were observed in the PDT group, indicating that most cancer cells underwent late-stage apoptosis. Besides, Vc significantly prevented the cell damage, which further implied that the radicals induced by ENBS-B were indeed responsible for the cell death. Overall, the possible mechanism of cell death induced by ENBS-B under irradiation could be reasonably speculated as the generated radicals damaged the lysosomes and nuclei DNA of cancer cells, leading to serious apoptotic cell death.
Figure 6. (a) Fluorescence imaging of HepG2 cells labeled with ENBS-B and its colocalization with Lyso-Tracker. P1, P2, and P3 are the correlation coefficient of ENBS-B with Lyso-, Mito-, and Nucleartracker, respectively. (b) CLSM images of AO staining for lysosomal integrity. (c) Annexin V-FITC/PI costaining on HepG2 cells after different treatments.
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CONCLUSION In summary, we developed a target-specific NIR light activated molecular O2−• generator, and confirmed its in vivo application for overcoming solid tumor hypoxia. Comprehensive characterizations have been performed to understand the O2−• action mechanism of ENBS-B. Our findings revealed that, even under severe O2 shortage environment, ENBS-B was able to sensitize abundant O2−• generation, and partial O2−• could be transformed to high toxic OH· through SOD-mediated cascade reactions. As a result, these radicals synergistically disrupted the integrities of cellular lysosomes and nuclei, subsequently triggering cancer cell apoptosis to enhance the therapeutic efficiency. In vitro PDT results confirmed the ideal antihypoxia activities of ENBS-B and also ENBS-B showed superior targeted phototoxicity toward biotin receptor positive cancer cells, with negligible side effects on normal cells. Benefiting from the superb NIR fluorescence emitting property, ENBS-B was intrinsically suited for precise tumor imaging with high sensitivity. The distinct in vivo hypoxic solid tumor ablation F
DOI: 10.1021/jacs.8b08658 J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Journal of the American Chemical Society further highlighted the potential of ENBS-B as a potent O2−• generator for overcoming anaerobic defects of traditional PDT. We expect this work will lay the foundation for future development to exploit more safe, effective, and valuable agents for clinical solid tumor treatment.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.8b08658. Detailed experimental conditions and methods (PDF)
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AUTHOR INFORMATION
Corresponding Author
*
[email protected] ORCID
Jingyun Wang: 0000-0003-3248-9747 Jiangli Fan: 0000-0003-4962-5186 Jianjun Du: 0000-0001-7777-079X Xiangzhi Song: 0000-0002-4034-6386 Xiaojun Peng: 0000-0002-8806-322X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by National Science Foundation of China (project 21421005, 21576037, and U1608222). REFERENCES
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