Superoxide Radical Photogenerator with Amplification Effect

Subscriber access provided by TULANE UNIVERSITY

Article

Superoxide Radical Photogenerator with Amplification Effect: Surmounting the Achilles’ heels of Photodynamic Oncotherapy Mingle Li, Tao Xiong, Jianjun Du, Ruisong Tian, Ming Xiao, Lianying Guo, Saran Long, Jiangli Fan, Wen Sun, Kun Shao, Xiangzhi Song, James W. Foley, and Xiaojun Peng J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13141 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society

Superoxide Radical Photogenerator with Amplification Effect: Surmounting the Achilles’ heels of Photodynamic Oncotherapy , , , Mingle Li,† Tao Xiong,† Jianjun Du,† ⊥ Ruisong Tian,† Ming Xiao,† Lianying Guo,‡ Saran Long,† ⊥ Jiangli Fan,† ⊥ , , , , Wen Sun,† ⊥ Kun Shao,† ⊥ Xiangzhi Song,ǀǀ James W. Foley,§ and Xiaojun Peng* † ⊥

†State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China ‡Department of Pathophysiology, Dalian Medical University, Dalian 116044, China ǀǀCollege of Chemistry and Chemical Engineering, Central South University, Changsha 410083, China §Rowland Institute at Harvard, Harvard University, Cambridge, Massachusetts 02142, United States ⊥Research Institute of Dalian University of Technology in Shenzhen, Shenzhen 518057, China ABSTRACT: Strong oxygen dependence, poor tumor targeting, and limited treatment depth have been considered as the “Achilles’ heels” facing the clinical usage of photodynamic therapy (PDT). Different from common approaches, here, we propose an innovative tactic by using photon-initiated dyad cationic superoxide radical (O2−•) generator (ENBOS) featuring “0 + 1 > 1” amplification effect to simultaneously overcome these drawbacks. In particular, by taking advantage of the Förster resonance energy transfer theory, the energy donor successfully endows ENBOS with significantly enhanced NIR absorbance and photon utility, which in turn lead to ENBOS more easily activated and generating more O2−• in deep tissues, thus dramatically intensifies the type I PDT against hypoxic deep tumors. Moreover, benefiting from the dyad cationic feature, ENBOS achieves superior “structure-inherent targeting” abilities with the signal-to-background ratio as high as 25.2 at 48 h post intravenous injection, offering opportunities for accurate imaging-guided tumor treatment. Meanwhile, the intratumoral accumulation and retention performance are also markedly improved (> 120 h). Based on these unique merits, ENBOS selectively inhibits the deep-seated hypoxic tumor proliferation at a low light-dose irradiation. Therefore, this delicate design may open up new horizons and cause a paradigm change for PDT in future cancer therapy.

INTRODUCTION Photodynamic therapy (PDT), a noninvasive treatment modality, has garnered tremendous popularity in personalized medicine, and in some cases it also has proven to be efficient where clinical protocols, such as surgery, chemotherapy, and radiotherapy, have failed.1-3 However, PDT as a first-line therapy still suffers from several “Achilles’ heels”.4,5 For instance, its effectiveness is hindered by the poor tumor selectivity of most current available photosensitizers (PSs), inevitably causing off target toxicity and prolonged cutaneous photosensitivity.6-8 In addition, due to the rapid tumor progression and insufficient O2 supply,9,10 the inherent tumor hypoxia drastically decreases the anticancer outcome, because the tumoricidal effect of traditional PDT is strongly oxygen-tension dependent.11-14 Moreover, the limited therapeutic depth further makes the treatment of deep-seated or large tumors fundamentally difficult.5,15-17 In order to overcome these drawbacks, various methods are being intensively explored. For example, the therapeutic accuracy of PSs can be improved by cancer specific ligands

(e.g., antibodies, metabolic substrates, and small interfering RNA),18-21 and nanotechnologies based on enhanced permeability and retention (EPR) effects.22,23 To fight against hypoxia, recently, Yoon and Liu have reviewed the facile approaches for ameliorating anti-hypoxia efficiencies,24 such as packaging O2 self-supplement materials including MnO2,10 catalase,25-27 or perfluorocarbon,28 with PSs in one “package”, hypoxia responsive prodrugs,29,30 etc.. While for deep PDT, utilizing smart excitation tools (e.g., X-ray,31 two photon,32,33 upconversion,34 and internal self-luminescence35,36) are the most common patterns. Indeed, these approaches have achieved significant advance, however, they can hardly overcome all three challenges at the same time. Besides, owing to above-mentioned strategies depend largely on the inorganic nanocomposites, the substantial risk of unknown immunotoxicities, high cost/benefits ratio, and limited reproducibility further hamper their FDA approval and clinical translation.37-39 Therefore, it is reasonable to envision that developing organic small-molecule PSs capable of simultaneously surmounting these Achilles’ heels may be of more clinical benefits. Unfortunately, no such molecules have

ACS Paragon Plus Environment

Journal of the American Chemical Society 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

been reported to date. Recently, we and others have shown that benzophenothiazine compounds are appealing PSs for anticancer and antibacterial.40-48 In particular, they are greatly ideal for hypoxic tumor treatment, because such molecules predominantly undergo type I mechanism to generate superoxide radicals (O2−•), which can subsequently take part in superoxide dismutase mediated disproportionation reactions,49 as well as Haber-Weiss/Fenton reactions50 to synergistically augment the antihypoxia outcome in a low O2-dependent manner.40 Unfortunately, owing to their limited intratumoral accumulation/retention capacities (even if conjugated by targeting ligands, e.g., biotin40), especially for intravenous (i.v.) injection, the overall in vivo therapeutic indexes are not satisfactory. Moreover, since the strong light absorption, reflection and scattering profiles of body tissues, the light fluence attenuates exponentially as the light penetrating more deeply (Scheme 1).5,51 Thus, although benzophenothiazine behaves NIR absorption within the “therapeutic window”, the suboptimal absorbance significantly limits or even terminates its application in deep tumors, because of PSs with weak optical absorbance in deep tissues usually failing to be activated (Scheme 1).

is perfectly suitable for elevating the molecular absorbance,59,60 we thus speculated that dyad cationic O2−• generator constructed by FRET might be an optimal choice to facilitate the selective hypoxic deep PDT response. Along this line, a typical NIR chromophore (Nile blue derivative, here denoted as ENBO) was applied to pair with a benzophenothiazine analog (energy acceptor, ENBS) to induce the FRET process (Figure 1a). As expected, the energy donor ENBO, as an absorbance “adjuvant”, successfully provided the prepared dyad O2−• generator (ENBOS) with significantly boosted NIR absorbance, photon utility, and O2−• generation (> 2.2-fold). As a result, once exposure to NIR light, ENBOS was more easily triggered in hypoxic deep tissues through type I mechanism, achieving an amplified effect of “0 + 1 > 1” both in vitro and in vivo. Besides, due to the powerful SIT function, ENBOS acquired markedly improved intratumoral accumulation/retention abilities (> 120 h), and could specifically “light up” the tumor site with a signal-to-background ratio (SBR) as high as 25.2, which thus highlighted the advantages of our strategy in terms of precision medicine. To our knowledge, this is the first report of molecular SIT O2−• generator for simultaneous cancer diagnosis/therapeutics and resolution of the “Achilles’ heels” of traditional PDT.

Scheme 1. Concept Illustration That PS with Low ε Only Enables Superficial Tumor-Confined Phototoxicity, Whereas PS with High ε can Result in Deep-Seated Tumor Eradication.

Motivated by above concerns, herein, for the first time, we described a fundamentally different approach by using Förster resonance energy transfer (FRET) theory to fabricate photon-initiated “structure-inherent targeting (SIT)” O2−• generator with encouraging “0 + 1 > 1” effect to realize substantially intensified selective ablation of deep-seated hypoxic tumors. It is well documented that compounds featuring lipophilic cationic chemical structures possess apparent self-recognition function toward neoplasms because malignant tumors usually develop higher degree of membrane potential than normal tissues,52,53 also termed as SIT,53-55 which has shown immense clinic values in biological imaging and diagnosis in recent years.56-58 Additionally, FRET mechanism

Figure 1. Chemical structures of (a) ENBOS, (b) ENBS, and (c) ENBO. (d) Absorption and (e) emission spectra of ENBO, ENBS, and ENBOS. (f) Photophysical data. a ε value at 660 nm.

RESULTS AND DISCUSSION Synthesis and Spectroscopic Characterization. The SIT O2−• generator (ENBOS) and its analogue without FRET effect (ENBS, reference compound) were designed and synthesized (Figure S1). The proposed structures were confirmed by HRMS, 1H NMR and 13C NMR (Figures S2-S10). As shown in Figure 1d, compared with bare ENBS, ENBOS presented a obviously broader and stronger NIR absorption band ranging from 600-750 nm due to the spectra overlap of other two units, and meanwhile, the extinction coefficient (ε) of ENBOS at 660 nm was increased from 45100 to 71000 M-1cm-1(Figure 1f),


Page 2 of 9

Page 3 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society which indicated that the NIR absorbance and concomitant photon utility of ENBOS have been significantly improved by the FRET effect. Then, the efficient energy transfer was ascertained by fluorescence emission spectra (Figure 1e).

Specifically, the energy donor ENBO displayed an intense emission at 684 nm; however, upon coupling, this donor signal was completely quenched in ENBOS, suggesting the reasonable construction of intramolecular FRET action.

Figure 2. (a) Fluorescence spectra of DHR123 for O2−• detection. (b) The fluorescence intensity of DHR123 at 526 nm after different treatments (c) Comparison of O2−• generation as a function of irradiation time. (d) The fluorescence intensity of Singlet Oxygen Sensor Green (SOSG) for 1O2 detection. (e) Illustration of enhanced O2−• generation due to FRET effect for ENBOS. (f) Relative intracellular O2−• level generated by ENBOS upon light irradiation. (g) Intracellular hypoxia confirmation using ROS-ID as the anaerobic probe and O2−• imaging under normoxic (21% O2) and hypoxic (2% O2) environments. *P < 0.05, ***P < 0.001, and ****P < 0.0001.

FRET-Amplified O2−• Generation and the Photosensitization Mechanism Confirmation for ENBOS. Since strong optical absorption of PS can facilitate its photon utilization such that generates more ROS, we then assessed the FRET-amplified PDT performance of ENBOS by using the O2−• probe, dihydrorhodamine 123 (DHR123).61 Monochromatic light of 660 nm was used as the irradiation source in this study. Because of the absence of intersystem crossing (ISC) process, ENBO did not produce measurable O2−•, which was similar to the results of DHR123 alone (Figure 2a). Surprisingly, under light irradiation, ENBOS substantially increased the fluorescence intensity of DHR123 by 4.8 times, but ENBS without energy transfer only showed limited O2−• signals (Figures 2a, b), indicating better O2−• generation ability of ENBOS than ENBS. Remarkably, ENBOS also led to a faster O2−• generation rate in comparison with ENBS (Figure 2c). These results fully validated that the intramolecular FRET effect indeed promoted the NIR photon utilization, thereby greatly boosting the O2−• production of ENBOS, which was critically important for robust PDT actions. On the other hand, under the same conditions, we did not found obvious singlet oxygen (1O2) (Figures 2d and S11) and hydroxyl radical (OH•) generation (Figure S12), revealing that ENBOS predominantly underwent the type I mechanism, also known as the electron transfer photochemical process (Figure 2e). The excellent photostability of ENBOS also allowed us to further use it for various biomedical applications (Figure S13). Light-Induced Intracellular O2−• Generation under Normoxia (21% O2) and Hypoxia (2% O2). Next, we evaluated the O2−• formation in living cells under normoxic

and hypoxic environments using the in vitro O2−• fluorescent indicator dihydroethidium (DHE),62 and the corresponding detection mechanism was displayed in Figure S14. For 4T1 cells treated with ENBOS followed by NIR light irradiation in normoxia (21% O2), brightly red signals attributing to O2−• were noted (Figure S15), and the average fluorescence intensity increased from 94 to 2429 as the rise of light-dose from 1.2 to 48 J/cm2 (Figure 2f), suggesting ENBOS could highly efficiently induce the O2−• formation in vitro. And also, the intracellular O2−• was almost completely scavenged by the radical scavenger Vitamin C (Vc), further confirming the robust O2−• induction capacity of ENBOS. To better mimic the hypoxic state of solid tumors, 4T1 cells were firstly cultured under a severe hypoxic atmosphere (2% O2) for longer than 8 h within an incubator chamber (MIC-101, Billups-rothenberg). Afterwards, the intracellular hypoxia was determined by using the anaerobic probe ROS-ID.63 As illustrated in Figure 2g, the ROS-ID fluorescence intensity in hypoxia-treated cells indeed showed 31-fold increment relative to normoxia (21% O2), thus suggesting the successful construction of in vitro hypoxic model. Importantly, even under severe hypoxic condition (2% O2), ENBOS still had satisfactory O2−• level as indicated by the bright DHE signals, meaning that ENBOS was capable of surmounting the tumor hypoxia, because widely perceived mechanism holds that O2−• can act as ROS initiator to convert into other highly cytotoxic oxidants, such as OH•, through intracellular cascade reactions (e.g. Haber-Weiss reaction and Fenton reaction)50 to synergistically augment the antihypoxia efficiency in an O2 reuse manner, just like evidenced in our



previous work.40 Amplification Effect Enables ENBOS to Be More Easily Activated in Simulated Hypoxic Deep-Tissue Settings. To investigate whether the enhanced NIR absorbance could lead to ENBOS more easily activated in deep-seated hypoxic tumor cells, we compared ENBOS- and ENBS-mediated O2−• generation in a simulated deep-tissue setting (Figure S16). As control experiments, when 4T1 cells were directly exposed to NIR light, no appreciable difference in DHE signals was noticed for ENBOS and ENBS (Figure 3a). In sharp contrast, once a piece of 5 mm pork tissue was placed between cells and light source, the cellular O2−• generation induced by ENBS dramatically decreased by 92% and was barely detectable, whereas the DHE signals still remained at an ideal level in ENBOS-loaded cells (Figure 3b). These data unambiguously proved that, with the help of non-PS ENBO as the absorbance enhancer, ENBOS enabled to be more easily triggered in internal tissues, yielding the effect of “0 + 1 >1” to amplify the deep PDT efficiency in essence.

light dose- and ENBOS concentration-dependent manner (Figure 4a). In particular, the in vitro anticancer efficiency of our ENBOS even was superior to the clinical PS, Ce6 (Figure S22), behaving a half maximal inhibitory concentration (IC50) of merely 0.033 M, 32-fold lower than that of Ce6 (IC50 = 1.064 M). To our gratification, because of the ideal O2−• generation potency in hypoxic environments, ENBOS successfully led to potent photodamage toward hypoxic cancer cells (Figure S23). Corresponding IC50 values have been summarized in Table S1. It was noteworthy that, even the light source was blocked by 5 mm pork slice, ENBOS still promised an excellent PDT response, however, the anticancer efficacy of ENBS was sharply impaired due to its inability to produce O2−• in deep-tissues (Figure 4b). In detail, the inhibition rate was only reduced by 23%, 8%, and 2% at ENBOS concentration of 0.08, 0.16, and 0.32 M, respectively; whereas the phototherapy outcome was drastically decreased with 47%, 53%, and 39% for the same three ENBS dose, respectively. Similar results were also observed in Live/Dead cell co-staining assay (Figure 4c). These findings were consistent with the data from intracellular O2−• detection in a simulated deep-tissue, clearly verifying the superior “0 + 1 >1” effect of our ENBOS in treating large or deep-seated hypoxic tumors.

Figure 3. Relative O2−• generation in 4T1 cells after ENBOS- or ENBS-mediated PDT treatment (48 J/cm2), with excitation light (a) unblocked or (b) blocked with a piece of 5 mm pork tissue. ***P < 0.001.

In Vitro O2−•-Mediated PDT Evaluation under Hypoxia and Normoxia. The ENBOS-induced photodynamic cancer cell killing was first studied by fluorescence imaging. As shown in Figure S17, owing to the dyad cationic structural property, ENBOS efficiently penetrated into 4T1 cells, and meanwhile, the NIR fluorescence staining was matched well with the commercial lysosome dyes (LysoTracker green), indicating the specific lysosome anchoring of ENBOS (Figure S18). Upon light irradiation, as evidenced by the AO staining,64,65 the formed O2−• severely disrupted the lysosome integrities (Figure S19) and subsequently resulted in serious cancer cell apoptosis and death (Figure S20). Then the in vitro PDT efficiency was further explored in details by standard methyl thiazolyltetrazolium (MTT) and live/dead cell co-staining assays. Obviously, negligible cytotoxicity of ENBOS was observed in the absence of light (Figure S21), however, under NIR light irradiation, ENBOS induced severe damage to tumor cells, and the cell viability exhibited both

Figure 4. (a) Light- and ENBOS-dose dependent viabilities in 4T1 cells. (b) In vitro anticancer efficiency of ENBS and ENBOS with light source blocked by a piece of 5 mm pork tissue under hypoxia (2% O2) (48 J/cm2). (g) Live/Dead cell co-staining assays using Calcein AM and Propidium Iodide as fluorescence probes. **P < 0.01 and ***P < 0.001.

In Vivo Xenograft Tumor-Targeting Accumulation and Imaging. Since specific tumor targeting capability is of paramount importance for precise PDT, we then investigated the SIT potency of ENBOS by i.v. injection (Figure 5a). Evidently, the 4T1 tumors could be clearly visualized from neighboring tissues with a SBR value as high as 25.2 at 48 h post-injection (Figure 5b). Of note, such high SBR was greatly encouraging and relative rare because a SBR value more than 2.5 is generally regarded as preferential accumulation in essence.66 24 h after administration, the maximal tumor signal


Page 4 of 9

Page 5 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society was attained, and even 120 h later, the tumor site still could be clearly distinguished. In contrast, nearly no ENBS signals were found during the observation course (Figures 5c and S24), revealing the more durable intratumoral accumulation/retention ability of ENBOS than ENBS. And

also, the targeting potency of ENBOS was obviously better than the previously reported biotin-conjugated O2−• generator which could merely maintained at tumor for 24 h,40 thereby highlighting the advantages of our SIT strategy in terms of tumor-targeting and imaging-guided precision therapy.

Figure 5. (a) In vivo real time fluorescence imaging of 4T1 tumor-bearing mice after i.v. injection of ENBOS. (b) Fluorescence change of ENBOS in the tumor and adjacent muscle tissues. (c) Relative fluorescence intensity of ENBOS and ENBS in 4T1 tumors at different time points. (d) Immunofluorescence imaging of tumor slice. (e) Schematic illustration of ENBOS-mediated PDT in simulated hypoxic deep tumors. (f) Relative tumor volume, (g) average tumor weights, and (h) representative photographs from different groups. Data were expressed as mean ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001 determined by Student’s t test.

Photodynamic Anticancer efficiencies of ENBOS on Deep-Seated Hypoxic Tumors. In order to explore the advantages of ENBOS in overcoming the deficiencies of PDT, we examined the anticancer efficacy of ENBOS in 4T1 tumor-bearing Balb/c mice by i.v. injection. The immunofluorescence imaging of hypoxia inducible factor (HIF-1) and tumor blood vasculatures (CD-31) indicated that the tumors were in a severely hypoxic state (Figure 5d).11 Then, as a proof of principle experiment, the subcutaneous tumor was covered by 5 mm pork tissue so as to simulate the hypoxic tumors which locate deeply inside body, and followed by NIR light exposure (0.1 W/cm2, 15 min) to conduct PDT treatment (Figure 5e). Similar to control group (PBS alone), neither ENBOS injection by itself or irradiation alone could delay the tumor growth during the study period, showing  15-fold increment (Figure 5f). For tumors exposed directly to NIR light, ENBOS exhibited prominent tumor suppression with the tumor growth inhibition (TGI) rate as high as 84%. Surprisingly, even in the case of pork tissue blocking, the TGI rate remained up to 68%, which highlighted the predominant targeted PDT effect of ENBOS on hypoxic deep tumors. The average tumor weights (Figure 5g) and corresponding tumor photographs (Figure 5h) validated the accurate antitumor results of ENBOS again. Subsequently, to more intuitively evidence the superb potency of ENBOS for deep-seated hypoxic tumors, we evaluated the in vivo therapeutic efficacy through intratumoral (i.t.) administration of ENBOS and ENBS so as to ensure the same PS dosage within the tumors (Figure 6a). Under blocked

NIR light irradiation, despite ENBS showing some extent tumor inhibition, the TGI rate was lower than 30%. However, nearly complete tumor regression was presented in mice injected with ENBOS (Figure 6b), and the tumor weigh was only 1/3 of ENBS-treated tumors (Figure 6c). This optimal therapeutic efficacy was mainly attributed to the more robust O2−• production of ENBOS in deep-tissues, thanks to its significantly enhanced NIR absorbance resulting from FRET construction. Furthermore, as shown in the T2-weighted MRI in vivo on day 15 after PDT treatment, much better tumor suppression was found for ENBOS (Figure 6d). The histological analysis also revealed that the destruction depth of tumor by ENBOS was greater than ENBS, as indicated by the shallower color of staining region for apoptotic and necrotic cancer cells (Figure 6e). During the course of therapy, all mice did not have noticeable abnormal body weight changes (Figure S25), and no obvious destructive cell necrosis or inflammation lesions in all major organs including heart, liver, spleen, lung and kidneys were observed (Figure S26), thereby implying the high biocompatibility and safety of ENBOS for biomedical applications.

CONCLUSION In summary, by using FRET mechanism, we have developed an appealing dyad SIT O2−• generator to amplify the selective anticancer performance against deep-seated hypoxic tumors, as a result, successfully addressing the Achilles’ heels of photodynamic therapy with an encouraging amplification effect of “0 + 1 > 1”. Owing to the outstanding SIT feature and



low oxygen sensitivity, ENBOS could spontaneously target to and work well within the hypoxic compartment of malignant tissues through type I photoreactions. Meanwhile, the enhanced NIR absorbance and photon utility enabled ENBOS to be more easily triggered in deep tissues, thereby ensuring the PDT efficiency against hypoxic deep tumors. Better still, other unique merits were also achieved, such as significantly enhanced intratumoral accumulation/retention ability (> 120 h) as well as excellent SBR value (> 25.2), which are critically important for precise anticancer actions. More importantly, this facile molecule design method was also suitable for improving the photosensitization activities of other photoactive agents, such as BODIPYs, phthalocyanines, and porphyrins. As such, we postulated that this work would cause a paradigm change of hypoxic deep PDT, and might be of great clinical benefits for future cancer precision therapy.

Figure 6. (a) Schematic diagram of PDT strategy after i.t. injection. (b) Relative tumor volume and (c) average tumor weights from different groups. (d) T2-weighted MRI in vivo on day 15. (e) H&E staining of tumors from different treatment groups. **P < 0.01, ****P < 0.0001. #P < 0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 determined by Student’s t test, #reference to ENBS + light group.

ASSOCIATED CONTENT Supporting Information. Detailed experimental conditions and methods, Figures S1-S26 and Table S1.

AUTHOR INFORMATION

Corresponding Author

*[email protected] Notes The authors declare no competing financial interests.

ACKNOWLEDGEMENTS We thank the National Natural Science Foundation of China (Project Nos. 21421005, 21576037) and the NSFC-Liaoning United Fund (Project No. U1608222) for financial support.

REFERENCES (1) Dolmans, D. E. J. G. J.; Fukumura, D.; Jain, R. K. Photodynamic therapy for cancer. Nat. Rev. Cancer. 2003, 3, 380-387. (2) Li, X.; Lee, S.; Yoon, J. Supramolecular photosensitizers rejuvenate photodynamic therapy. Chem. Soc. Rev. 2018, 47, 1174-1188. (3) Luby, B. M.; Walsh, C. D.; Zheng, G. Advanced photosensitizer activation strategies for smarter photodynamic therapy beacons. Angew. Chem., Int. Ed. 2018, 57, 2-14. (4) Li, X.; Yu, S.; Lee, D.; Kim, G.; Lee, B.; Cho, Y.; Zheng, B.-Y.; Ke, M.-R.; Huang, J.-D.; Nam, K. T.; Chen, X.; Yoon, J. Facile Supramolecular Approach to Nucleic-Acid-Driven Activatable Nanotheranostics That Overcome Drawbacks of Photodynamic Therapy. ACS Nano 2018, 12, 681-688. (5) Fan, W.; Huang, P.; Chen, X. Overcoming the Achilles' heel of photodynamic therapy. Chem. Soc. Rev. 2016, 45, 6488-6519. (6) Li, X.; Kolemen, S.; Yoon, J.; Akkaya, E. U. Activatable Photosensitizers: Agents for Selective Photodynamic Therapy. Adv. Funct. Mater. 2016, 27, 1604053. (7) Zhao, X.; Li, M.; Sun, W.; Fan, J.; Du, J.; Peng, X. An estrogen receptor targeted ruthenium complex as a two-photon photodynamic therapy agent for breast cancer cells. Chem. Commun. 2018, 54, 7038-7041. (8) Jung, H. S.; Lee, J.-H.; Kim, K.; Koo, S.; Verwilst, P.; Sessler, J. L.; Kang, C.; Kim, J. S. Mitochondria-Targeted Cryptocyanine-Based Photothermogenic Photosensitizer. J. Am. Chem. Soc. 2017, 139, 9972-9978. (9) Vaupel, P.; Höckel, M.; Mayer, A. Detection and Characterization of Tumor Hypoxia Using pO2 Histography. Antioxid. Redox Signaling 2007, 9, 1221-1236. (10) Chen, Q.; Feng, L.; Liu, J.; Zhu, W.; Dong, Z.; Wu, Y.; Liu, Z. Intelligent Albumin-MnO2 Nanoparticles as pH-/H2O2-Responsive Dissociable Nanocarriers to Modulate Tumor Hypoxia for Effective Combination Therapy. Adv. Mater. 2016, 28, 7129-7136. (11) Gong, H.; Chao, Y.; Xiang, J.; Han, X.; Song, G.; Feng, L.; Liu, J.; Yang, G.; Chen, Q.; Liu, Z. Hyaluronidase To Enhance Nanoparticle-Based Photodynamic Tumor Therapy. Nano Lett. 2016, 16, 2512-2521. (12) Jung, H. S.; Han, J.; Shi, H.; Koo, S.; Singh, H.; Kim, H.-J.; Sessler, J. L.; Lee, J. Y.; Kim, J.-H.; Kim, J. S. Overcoming the Limits of Hypoxia in Photodynamic Therapy: A Carbonic Anhydrase IX-Targeted Approach. J. Am. Chem. Soc. 2017, 139, 7595-7602. (13) Liu, J.-n.; Bu, W.; Shi, J. Chemical Design and Synthesis of Functionalized Probes for Imaging and Treating Tumor Hypoxia. Chem. Rev. 2017, 117, 6160-6224. (14) Kolemen, S.; Ozdemir, T.; Lee, D.; Kim, G. M.; Karatas, T.; Yoon, J.; Akkaya, E. U. Remote-Controlled Release of Singlet


Page 6 of 9

Page 7 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society Oxygen by the Plasmonic Heating of Endoperoxide-Modified Gold Nanorods: Towards a Paradigm Change in Photodynamic Therapy. Angew. Chem., Int. Ed. 2016, 55, 3606-3610. (15) Cao, H.; Wang, L.; Yang, Y.; Li, J.; Qi, Y.; Li, Y.; Li, Y.; Wang, H.; Li, J. An Assembled Nano-complex for Improving both Therapeutic Efficiency and Treatment Depth in Photodynamic Therapy. Angew. Chem., Int. Ed. 2018, 57, 7759-7763. (16) Ji, C.; Gao, Q.; Dong, X.; Yin, W.; Gu, Z.; Gan, Z.; Zhao, Y.; Yin, M. A Size-Reducible Nanodrug with an Aggregation-Enhanced Photodynamic Effect for Deep Chemo-Photodynamic Therapy. Angew. Chem., Int. Ed. 2018, 57, 11384-11388. (17) Huang, L.; Li, Z.; Zhao, Y.; Zhang, Y.; Wu, S.; Zhao, J.; Han, G. Ultralow-Power Near Infrared Lamp Light Operable Targeted Organic Nanoparticle Photodynamic Therapy. J. Am. Chem. Soc. 2016, 138, 14586-14591. (18) Yuan, Y.; Zhang, C.-J.; Gao, M.; Zhang, R.; Tang, B. Z.; Liu, B. Specific Light-Up Bioprobe with Aggregation-Induced Emission and Activatable Photoactivity for the Targeted and Image-Guided Photodynamic Ablation of Cancer Cells. Angew. Chem., Int. Ed. 2014, 54, 1780-1786. (19) Yu, Z.; Pan, W.; Li, N.; Tang, B. A nuclear targeted dual-photosensitizer for drug-resistant cancer therapy with NIR activated multiple ROS. Chem. Sci. 2017, 7, 4237-4244. (20) Mitsunaga, M.; Ogawa, M.; Kosaka, N.; Rosenblum, L. T.; Choyke, P. L.; Kobayashi, H. Cancer cell-selective in vivo near infrared photoimmunotherapy targeting specific membrane molecules. Nat. Med. 2011, 17, 1685. (21) Li, X.; Kim, C. y.; Lee, S.; Lee, D.; Chung, H.-M.; Kim, G.; Heo, S.-H.; Kim, C.; Hong, K.-S.; Yoon, J. Nanostructured Phthalocyanine Assemblies with Protein-Driven Switchable Photoactivities for Biophotonic Imaging and Therapy. J. Am. Chem. Soc. 2017, 139, 10880-10886. (22) Blanco, E.; Shen, H.; Ferrari, M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 2015, 33, 941. (23) Lucky, S. S.; Soo, K. C.; Zhang, Y. Nanoparticles in Photodynamic Therapy. Chem. Rev. 2015, 115, 1990-2042. (24) Li, X.; Kwon, N.; Guo, T.; Liu, Z.; Yoon, J. Innovative Strategies for Hypoxic-Tumor Photodynamic Therapy. Angew. Chem., Int. Ed. 2018, 57, 11522-11531. (25) Chen, H.; Tian, J.; He, W.; Guo, Z. H2O2-Activatable and O2-Evolving Nanoparticles for Highly Efficient and Selective Photodynamic Therapy against Hypoxic Tumor Cells. J. Am. Chem. Soc. 2015, 137, 1539-1547. (26) Wang, H.; Chao, Y.; Liu, J.; Zhu, W.; Wang, G.; Xu, L.; Liu, Z. Photosensitizer-crosslinked in-situ polymerization on catalase for tumor hypoxia modulation & enhanced photodynamic therapy. Biomaterials 2018, 181, 310-317. (27) Cheng, H.; Zhu, J.-Y.; Li, S.-Y.; Zeng, J.-Y.; Lei, Q.; Chen, K.-W.; Zhang, C.; Zhang, X.-Z. An O2 Self-Sufficient Biomimetic Nanoplatform for Highly Specific and Efficient Photodynamic Therapy. Adv. Funct. Mater. 2016, 26, 7847-7860. (28) Song, G.; Liang, C.; Yi, X.; Zhao, Q.; Cheng, L.; Yang, K.; Liu, Z. Perfluorocarbon-Loaded Hollow Bi2Se3 Nanoparticles for Timely Supply of Oxygen under Near-Infrared Light to Enhance the Radiotherapy of Cancer. Adv. Mater. 2016, 28, 2716-2723. (29) Zhou, Y.; Maiti, M.; Sharma, A.; Won, M.; Yu, L.; Miao, L. X.; Shin, J.; Podder, A.; Bobba, K. N.; Han, J.; Bhuniya, S.; Kim, J. S. Azo-based small molecular hypoxia responsive theranostic for tumor-specific imaging and therapy. J. Control. Release 2018, 288, 14-22. (30) Verwilst, P.; Han, J.; Lee, J.; Mun, S.; Kang, H.-G.; Kim, J.

S. Reconsidering azobenzene as a component of small-molecule hypoxia-mediated cancer drugs: A theranostic case study. Biomaterials 2017, 115, 104-114. (31) Kaščáková, S.; Giuliani, A.; Lacerda, S.; Pallier, A.; Mercère, P.; Tóth, É.; Réfrégiers, M. X-ray-induced radiophotodynamic therapy (RPDT) using lanthanide micelles: Beyond depth limitations. Nano Res. 2015, 8, 2373-2379. (32) Huang, H.; Yu, B.; Zhang, P.; Huang, J.; Chen, Y.; Gasser, G.; Ji, L.; Chao, H. Highly Charged Ruthenium(II) Polypyridyl Complexes as Lysosome-Localized Photosensitizers for Two-Photon Photodynamic Therapy. Angew. Chem., Int. Ed. 2015, 54, 14049-14052. (33) Shen, Y.; Shuhendler, A. J.; Ye, D.; Xu, J.-J.; Chen, H.-Y. Two-photon excitation nanoparticles for photodynamic therapy. Chem. Soc. Rev. 2016, 45, 6725-6741. (34) Sun, Y.; Feng, W.; Yang, P.; Huang, C.; Li, F. The biosafety of lanthanide upconversion nanomaterials. Chem. Soc. Rev. 2015, 44, 1509-1525. (35) Yuan, H.; Chong, H.; Wang, B.; Zhu, C.; Liu, L.; Yang, Q.; Lv, F.; Wang, S. Chemical Molecule-Induced Light-Activated System for Anticancer and Antifungal Activities. J. Am. Chem. Soc. 2012. 134, 13184-13187. (36) Liu, S.; Yuan, H.; Bai, H.; Zhang, P.; Lv, F.; Liu, L.; Dai, Z.; Bao, J.; Wang, S. Electrochemiluminescence for Electric-Driven Antibacterial Therapeutics. J. Am. Chem. Soc. 2018, 140, 2284-2291. (37) Cheng, Z.; Al Zaki, A.; Hui, J. Z.; Muzykantov, V. R.; Tsourkas, A. Multifunctional Nanoparticles: Cost Versus Benefit of Adding Targeting and Imaging Capabilities. Science 2012, 338, 903. (38) Yu, G.; Zhao, X.; Zhou, J.; Mao, Z.; Huang, X.; Wang, Z.; Hua, B.; Liu, Y.; Zhang, F.; He, Z.; Jacobson, O.; Gao, C.; Wang, W.; Yu, C.; Zhu, X.; Huang, F.; Chen, X. Supramolecular Polymer-Based Nanomedicine: High Therapeutic Performance and Negligible Long-Term Immunotoxicity. J. Am. Chem. Soc. 2018, 140, 8005-8019. (39) Kang, Y.; Sun, W.; Fan, J.; Wei, Z.; Wang, S.; Li, M.; Zhang, Z.; Xie, Y.; Du, J.; Peng, X. Ratiometric real-time monitoring of hydroxyapatite-doxorubicin nanotheranostic agents for on-demand tumor targeted chemotherapy. Mater. Chem. Front. 2018, 2, 1791-1798. (40) Li, M.; Xia, J.; Tian, R.; Wang, J.; Fan, J.; Du, J.; Long, S.; Song, X.; Foley, J. W.; Peng, X. Near-Infrared Light-Initiated Molecular Superoxide Radical Generator: Rejuvenating Photodynamic Therapy against Hypoxic Tumors. J. Am. Chem. Soc. 2018, 140, 14851-14859. (41) Akilov, O. E.; Kosaka, S.; O'Riordan, K.; Song, X.; Sherwood, M.; Flotte, T. J.; Foley, J. W.; Hasan, T. The Role of Photosensitizer Molecular Charge and Structure on the Efficacy of Photodynamic Therapy against Leishmania Parasites. Chem. Biol. 2006, 13, 839-847. (42) Zheng, X.; Sallum, U. W.; Verma, S.; Athar, H.; Evans, C. L.; Hasan, T. Exploiting a Bacterial Drug-Resistance Mechanism: A Light-Activated Construct for the Destruction of MRSA. Angew. Chem., Int. Ed. 2009, 48, 2148-2151. (43) O'Riordan, K.; Akilov, O. E.; Chang, S. K.; Foley, J. W.; Hasan, T. Real-time fluorescence monitoring of phenothiazinium photosensitizers and their anti-mycobacterial photodynamic activity against Mycobacterium bovis BCG in in vitro and in vivo models of localized infection. Photoch. Photobio. Sci. 2007, 6, 1117-1123. (44) Abrahamse, H.; Hamblin, Michael R. New photosensitizers for photodynamic therapy. Biochem. J. 2016, 473, 347-364.



(45) Hung, H.-I.; Klein, O. J.; Peterson, S. W.; Rokosh, S. R.; Osseiran, S.; Nowell, N. H.; Evans, C. L. PLGA nanoparticle encapsulation reduces toxicity while retaining the therapeutic efficacy of EtNBS-PDT in vitro. Sci. Rep. 2016, 6, 33234. (46) Klein, O. J.; Bhayana, B.; Park, Y. J.; Evans, C. L. In Vitro Optimization of EtNBS-PDT against Hypoxic Tumor Environments with a Tiered, High-Content, 3D Model Optical Screening Platform. Mol. Pharmaceutics 2012, 9, 3171-3182. (47) Vecchio, D.; Bhayana, B.; Huang, L.; Carrasco, E.; Evans, C. L.; Hamblin, M. R. Structure–function relationships of Nile blue (EtNBS) derivatives as antimicrobial photosensitizers. Eur. J. Med. Chem. 2014, 75, 479-491. (48) Klein, O. J.; Yuan, H.; Nowell, N. H.; Kaittanis, C.; Josephson, L.; Evans, C. L. An Integrin-Targeted, Highly Diffusive Construct for Photodynamic Therapy. Sci. Rep. 2017, 7, 13375. (49) Yao, J.; Cheng, Y.; Zhou, M.; Zhao, S.; Lin, S.; Wang, X.; Wu, J.; Li, S.; Wei, H. ROS scavenging Mn3O4 nanozymes for in vivo anti-inflammation. Chem. Sci. 2018, 9, 2927-2933. (50) Benov, L. How superoxide radical damages the cell. Protoplasma 2001, 217, 33-36. (51) Li, M.; Peng, X. Research Progress on the Phthalocyanine Based Targeting Photosensitizers in Photodynamic Therapy. Huaxue Xuebao 2016, 74, 959-968. (52) Luo, S.; Tan, X.; Fang, S.; Wang, Y.; Liu, T.; Wang, X.; Yuan, Y.; Sun, H.; Qi, Q.; Shi, C. Mitochondria-Targeted Small-Molecule Fluorophores for Dual Modal Cancer Phototherapy. Adv. Funct. Mater. 2016, 26, 2826-2835. (53) Li, M.; Long, S.; Kang, Y.; Guo, L.; Wang, J.; Fan, J.; Du, J.; Peng, X. De Novo Design of Phototheranostic Sensitizers Based on Structure-Inherent Targeting for Enhanced Cancer Ablation. J. Am. Chem. Soc. 2018, 140, 15820-15826. (54) Hyun, H.; Park, M. H.; Owens, E. A.; Wada, H.; Henary, M.; Handgraaf, H. J. M.; Vahrmeijer, A. L.; Frangioni, J. V.; Choi, H. S. Structure-inherent targeting of near-infrared fluorophores for parathyroid and thyroid gland imaging. Nat. Med. 2015, 21, 192. (55) Owens, E. A.; Hyun, H.; Dost, T. L.; Lee, J. H.; Park, G.; Pham, D. H.; Park, M. H.; Choi, H. S.; Henary, M. Near-Infrared Illumination of Native Tissues for Image-Guided Surgery. J. Med. Chem. 2016, 59, 5311-5323. (56) Hyun, H.; Owens, E. A.; Wada, H.; Levitz, A.; Park, G.; Park, M. H.; Frangioni, J. V.; Henary, M.; Choi, H. S. Cartilage-Specific Near-Infrared Fluorophores for Biomedical

Imaging. Angew. Chem., Int. Ed. 2015, 54, 8648-8652. (57) Hyun, H.; Wada, H.; Bao, K.; Gravier, J.; Yadav, Y.; Laramie, M.; Henary, M.; Frangioni, J. V.; Choi, H. S. Phosphonated Near-Infrared Fluorophores for Biomedical Imaging of Bone. Angew. Chem., Int. Ed. 2014, 53, 10668-10672. (58) Choi, H. S.; Nasr, K.; Alyabyev, S.; Feith, D.; Lee, J. H.; Kim, S. H.; Ashitate, Y.; Hyun, H.; Patonay, G.; Strekowski, L.; Henary, M.; Frangioni, J. V. Synthesis and In Vivo Fate of Zwitterionic Near-Infrared Fluorophores. Angew. Chem., Int. Ed. 2011, 50, 6258-6263. (59) Huang, L.; Li, Z.; Zhao, Y.; Yang, J.; Yang, Y.; Pendharkar, A. I.; Zhang, Y.; Kelmar, S.; Chen, L.; Wu, W.; Zhao, J.; Han, G. Enhancing Photodynamic Therapy through Resonance Energy Transfer Constructed Near-Infrared Photosensitized Nanoparticles. Adv. Mater. 2017, 29, 1604789. (60) Zhang, C.; Zhao, J.; Wu, S.; Wang, Z.; Wu, W.; Ma, J.; Guo, S.; Huang, L. Intramolecular RET Enhanced Visible Light-Absorbing Bodipy Organic Triplet Photosensitizers and Application in Photooxidation and Triplet-Triplet Annihilation Upconversion. J. Am. Chem. Soc. 2013, 135, 10566-10578. (61) Nam, J. S.; Kang, M.-G.; Kang, J.; Park, S.-Y.; Lee, S. J. C.; Kim, H.-T.; Seo, J. K.; Kwon, O.-H.; Lim, M. H.; Rhee, H.-W.; Kwon, T.-H. Endoplasmic Reticulum-Localized Iridium(III) Complexes as Efficient Photodynamic Therapy Agents via Protein Modifications. J. Am. Chem. Soc. 2016, 138, 10968-10977. (62) Park, W.; Bae, B.-c.; Na, K. A highly tumor-specific light-triggerable drug carrier responds to hypoxic tumor conditions for effective tumor treatment. Biomaterials 2016, 77, 227-234. (63) Zheng, D.-W.; Li, B.; Li, C.-X.; Fan, J.-X.; Lei, Q.; Li, C.; Xu, Z.; Zhang, X.-Z. Carbon-Dot-Decorated Carbon Nitride Nanoparticles for Enhanced Photodynamic Therapy against Hypoxic Tumor via Water Splitting. ACS Nano 2016, 10, 8715-8722. (64) Li, M.; Tian, R.; Fan, J.; Du, J.; Long, S.; Peng, X. A lysosome-targeted BODIPY as potential NIR photosensitizer for photodynamic therapy. Dyes Pigm. 2017, 147, 99-105. (65) Bian, Y.; Li, M.; Fan, J.; Du, J.; Long, S.; Peng, X. A proton-activatable aminated-chrysophanol sensitizer for photodynamic therapy. Dyes Pigm. 2017, 147, 476-483. (66) Choi, H. S.; Gibbs, S. L.; Lee, J. H.; Kim, S. H.; Ashitate, Y.; Liu, F.; Hyun, H.; Park, G.; Xie, Y.; Bae, S.; Henary, M.; Frangioni, J. V. Targeted zwitterionic near-infrared fluorophores for improved optical imaging. Nat. Biotechnol. 2013, 31, 148.


Page 8 of 9

Page 9 of 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Journal of the American Chemical Society


Superoxide Radical Photogenerator with Amplification Effect

Recommend Documents