Biomarker Displacement Activation: A General HostâGuest Strategy

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Biomarker Displacement Activation: a General HostGuest Strategy for Targeted Phototheranostics in Vivo Jie Gao, Jun Li, Wen-Chao Geng, Fang-Yuan Chen, Xingchen Duan, Zhe Zheng, Dan Ding, and Dong-Sheng Guo J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b02331 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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Journal of the American Chemical Society

Biomarker Displacement Activation: a General Host-Guest Strategy for Targeted Phototheranostics in Vivo Jie Gao†,#, Jun Li‡,#, Wen-Chao Geng†, Fang-Yuan Chen†, Xingchen Duan‡, Zhe Zheng†, Dan Ding*,‡, and Dong-Sheng Guo*,†,§ †

College of Chemistry, State Key Laboratory of Elemento-Organic Chemistry, Key Laboratory of Functional Polymer Materials, Ministry of Education, Nankai University Tianjin 300071, P. R. China. ‡ State Key Laboratory of Medicinal Chemical Biology, Key Laboratory of Bioactive Materials, Ministry of Education, and College of Life Sciences, Nankai University, Tianjin 300071, P. R. China. § Collaborative Innovation Center of Chemical Science and Engineering, Nankai University Tianjin 300071, P. R. China. KEYWORDS supramolecular chemistry; molecular recognition; self-assembly; fluorescence imaging; photodynamic therapy.

ABSTRACT: Activatable phototheranostics is highly appealing to meet the demand of precision medicine. However, although displaying the efficacy in construction of activatable photosensitizers (PSs), direct covalent decoration still shows some inevitable issues, such as complex molecular design, tedious synthesis, possible photoactivity changes and potential toxicity. Herein, we propose a novel concept of biomarker displacement activation (BDA) using host-guest strategy. To exemplify BDA, we engineered a PS-loaded nanocarrier by utilizing a macrocyclic amphiphile, where the fluorescence and photoactivity of PS were completely annihilated by the complexation of macrocyclic receptor (OFF state). When nanocarriers were accumulated into tumor tissues via the enhanced permeability and retention effect, the overexpressed biomarker adenosine triphosphates displaced PSs out, accompanied with their fluorescence and photoactivity recovered (ON state). These reinstallations are unattainable in normal tissues, allowing us to concurrently achieve selective tumor imaging and targeted therapy in vivo. Compared with widely used covalent approach, the present BDA strategy provides the following advantages: (1) employment of approved PSs without custom covalent decoration; (2) traceless release of PSs with high fidelity by biomarker displacement; (3) adaptability to different PSs for establishing a universal platform and promised facile combination of diverse PSs to enhance photon utility in light window. Such a host-guest BDA strategy is easily amenable to other ensembles and targets, so that versatile biomedical applications can be envisaged.

of suppressing fluorescence emission and blocking the energy transfer pathways between PSs and oxygen after light irradiation.8,20-22 To achieve this goal, one frequently used strategy is covalently anchoring quenchers or energy acceptors to the vicinity of PSs, or conjugating a high number of PSs at the polymeric backbone that giving rise to self-quenching at the correct conjugation density, either of which can be re-activated in response to various stimuli (Scheme 1a).12-15,23-25 However, direct covalent decoration, although demonstrating efficacy in architecting activatable PSs, always suffers from problems such as complex molecular design, time- and cost-consuming synthesis/purification, possible photoactivity changes, and potential toxicity arising from the introduction of exogenous moieties on PSs.21 Supramolecular chemistry has broad potential applications for dye chemistry by leveraging reversible noncovalent molecular recognition motifs.26 Host-guest motifs, for example, typically comprise a discrete macrocyclic host with a cavity that is selective for complementary binding to certain guest ligands.27-28 The photophysical modulation of chromophores via macrocyclic encapsulation has always been an active re-

INTRODUCTION Manipulating the photophysical properties of optical agents has served a critical role in modern medicine through phototheranostics, the application of light for diagnosis and treatment of diseases.1-4 Among numerous optical agents, photosensitizers (PSs) are quite fascinating by displaying the dual functions of fluorescence imaging and photodynamic therapy (PDT).5-7 Although PDT has emerged as a noninvasive and reliable cancer-therapy modality, its clinical translation is hindered by the unexpected dark toxicity due to the “always-on” model and low tumor specificity of currently approved PSs.8 Thus, the currently available PSs are obviously not smart enough to meet the demand of precision medicine.9-11 An ideal PS for PDT application is to be selectively switched on in the tumor microenvironment or inside the cancer cells by responding to the biological stimulus while staying inert during its systemic circulation. In this regard, activatable PSs that can be activated by tumor-associated stimuli have been developed to minimize nonspecific activation of phototoxicity and to increase the signal-to-background ratio for bioimaging.12-19 In general, the design of activatable PSs is based on the principle 1

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search area.29 One particular macrocycle, calix[n]arene (CnA, n = 4, 5, 6, 8 generally),30 is distinguished by its par excellence complexation-induced fluorescence quenching of chromophores,29,31-32 which has been abundantly engaged in detecting biological analytes,33-34 monitoring enzymatic activity and biomembrane transport in real time,35-38 and screening drugs via indicator displacement assay or tandem assay (realizing the desired “turn-on” sensing for competitive analytes by regeneration of the intrinsic fluorescence of chromophores).39-40 Moreover, most of the CnA derivatives showed low or no toxicity in animal models,41 promising their rich biomedical applications including drug delivery and gene transfection.42-44 Collectively, these intrinsic features of CnA naturally open up the opportunity for activatable phototheranostics, realizing both tumor-selective imaging and targeted therapy in vivo. Herein, we propose a new and general host-guest strategy for activatable phototheranostics, termed as biomarker displacement activation (BDA). In BDA (Scheme 1b and 1c), a PS is pre-loaded into the cavity of the receptor with the fluorescence and photoactivity annihilated (OFF state); when encountering a biomarker overexpressed in tumor tissues, the competitive inclusion of biomarker gives rise to the release of PS, accompanied with the recovery of fluorescence and photoactivity (ON state). Consequently, by using the commercial PSs without the need of any covalent modification, the tumor-selective imaging and targeted photodynamic cancer ablation are achieved. As a proof-of-concept example of BDA, we succeed in the adenosine triphosphate (ATP)-activated imaging-guided therapy in vivo. ATP represents an ideal tumor biomarker that severely overexpressed in tumor tissues ([ATP] > 100 µM) than in normal tissues ([ATP] = 1−10 nM).45-46 A new macrocyclic amphiphile, guanidinium-modified calix[5]arene pentadodecyl ether (GC5A-12C), is prepared, affording extraordinarily strong binding (nanomolar range) to commercial PSs as well as ATP (Figure 1). Strong binding to PSs is prerequisite to avoid unwarranted off-target leaking during blood circulation, and strong binding to ATP is then necessary to trigger the PS release. In order to apply this strat-

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egy in vivo, we engineered a pegylated GC5A-12C nanocarrier (115 nm), securing the efficiently enhanced permeability and retention (EPR) effect. Collectively, these design principles led us to develop a general phototheranostics nanoplatform of dual selectivity for targeted imaging and therapy that result from the EPR effect of the nanocarrier and tumor-ATP response. This study not only demonstrates that BDA is a promising approach to develop advanced activatable optical agents with generality and simplicity not achievable by other existing strategies, but also manifests that calixarene amphiphiles can serve as an ideal nanoplatform to achieve activatable phototheranostics in vivo.

RESULTS AND DISCUSSION Receptor Synthesis and Nanocarrier Construction. An ideal receptor for BDA should be subtly designed to possess the following features: (1) strong binding to commercial PSs (four anionic PSs were employed herein, Figure 1b); (2) drastic annihilating the fluorescence and photoactivity of PSs; (3) strong binding to tumor-targeted biomarkers (ATP in this work); (4) robust self-assembly into 20−200 nm nanoparticles promising EPR.47 Accordingly, a new macrocyclic receptor, GC5A-12C, was designed. First, calixarenes were employed as the macrocyclic scaffold benefiting from their facile modification as well as their capability of quenching fluorescence.29,31 Second, among the CnA family, we screened C5A on account of its larger cavity size than C4A and its easier conformational immobilization than C6A and C8A.48 Third, guanidinium groups were decorated at the upper rim to donate charge-assisted hydrogen bonds (salt bridge) with the anionic groups of PSs and ATP,49-50 which provide additionally anchoring points that supplement the binding interactions intrinsic to the calixarene cavities. Finally, dodecyl chains were attached at the lower rim to impart the amphiphilic aggregation besides immobilizing the cone conformation.51-53 The well-tailored GC5A-12C was prepared in 5 steps (Figure 1a), mainly following the reported syntheses of C4A analogues.54

Scheme 1. Schematic illustration of activatable phototheranostics. (a) The typically covalent approach for designing activatable PSs. (b) The proposed host-guest strategy in this work, biomarker displacement activation (BDA). (c) Simplified energy level diagram showing the working principle of BDA: If the PS bonds with the receptor, the excited singlet state of PS returns to ground state through the photoinduced electron transfer (PET) pathway, leading to fluorescence and photoactivity completely annihilated (OFF state). Once the PS is displaced by the targeted biomarker, its photophysical properties will be restored with high fidelity (ON state).

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With respect to in vivo application, the pegylated nanocarrier was constructed by co-assembling GC5A-12C and 4-(dodecyloxy)benzamido-terminated methoxy poly(ethylene glycol) (PEG-12C) with a 1:1 molar ratio. PEG has been well demonstrated to give nanomedicines a stealth property. It can hinder adsorption of opsonic proteins and thus prevent scavenging by the reticuloendothelial system (RES) and mononuclear phagocytic system screening, enabling nanomedicines to remain in blood circulation for long times.55 The obtained GC5A-12C nanocarrier shows an average hydrated diameter of 115±5 nm, identified by dynamic light scattering (DLS) measurement (Figure S5).Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images show the spherical-like morphology with consistent size with the DLS result (Figure S5). Moreover, upon complexation with AlPcS4, no appreciable change was observed in the size of the GC5A-12C nanocarrier, while the zeta-potential shifts to less positive as a result of charge offset (Figure S5). Such a size-distributed and pegylated nanocarrier is of capability to

preferentially accumulate in tumor tissues by exploiting tumor’s EPR effect as result of their pathologically leaky vasculature and poor lymphatic drainage.55 Binding with PSs and Modulating their Photophysical Properties. To verify the generality of the GC5A-12C nanocarrier as the BDA platform, we employed four commercial PSs (AlPcS4, TPPS, EY, and RB in Figure 1b), which are widely used as phototheranostics agents in cancer research, and even in clinical practice.56 Moreover, to dissect the host-guest complexation between calixarene receptor and PSs clearly, we engaged guanidinium-modified calix[5]arene pentaisohexyl ether (GC5A-6C in Figure 1a) in binding titrations. GC5A-6C with a critical aggregation concentration of 0.4 mM is molecularly dispersed during the course of fluorescence titrations.57 Investigating the binding of GC5A-12C with PSs at the molecular level could not be implemented with respect to its extremely tight amphiphilic aggregation. The employment of GC5A-6C is therefore to avoid any complication of amphiphilic aggregation on host-guest complexation.

Figure 1. Receptor synthesis and nanocarrier construction. (a) Synthetic routes of GC5A-12C and GC5A-6C: (i) K2CO3, RBr, CH3CN, reflux; (ii) HNO3, AcOH, dry CH2Cl2, r.t.; (iii) SnCl2⋅2H2O, C2H5OH/AcOEt (1:1, v:v), reflux; (iv) 1,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea, Et3N, AgNO3, dry CH2Cl2, r.t.; (v) SnCl4, AcOEt, r.t. (b) Four commercial PSs employed in this work, including sulfonated aluminum phthalocyanine (AlPcS4), 5, 10, 15, 20-tetrakis-(4-sulfonatophenyl)-porphyrin (TPPS), rose bengal (RB) and eosin Y (EY). (c) Schematic illustration of the pegylated GC5A-12C nanocarrier for BDA, where the fluorescence and photoactivity of PS were annihilated by the complexation of GC5A-12C, while reactivated by ATP displacement.

As expected, each PS can be strongly encapsulated in GC5A-6C, accompanied with a drastic fluorescence quenching

as shown in Figure S10 (Binding affinities: AlPcS4, (1.7±0.8)×108 M−1; TPPS, (1.1±0.3)×108 M−1; RB, 3



(9.6±0.2)×107 M−1; EY, (5.7±0.2)×108 M−1, Figure S6-9. Fluorescence suppression upon complexation: AlPcS4, (99.5±0.1)%; TPPS, (96.1±0.1)%; RB, (94.9±0.4)%; EY, (99.4±0.1)%.). The exceptionally large fluorescence response upon complexation of PSs warrants detailed photophysical attention. Taking AlPcS4 as example, we studied the rationale behind the complexation-induced fluorescence quenching in detail, which is crucial for understanding the BDA strategy. As implied by the steady-state spectra (Figure 2a), upon 1:1 complexation with GC5A-6C, the fluorescence of AlPcS4 is quantitatively quenched without any shift of emission maximum, indicating a PET quenching mechanism.58 Rigorous confirmation for static quenching was derived from time-resolved fluorescence measurements (Figure S13). The addition of GC5A-6C greatly reduced the overall fluorescence intensity (indicative of a very short-lived species, the statically quenched complex), but left the fluorescence lifetime of the detectable component unaltered, within error (indicative of the absence of dynamic quenching of the residual uncomplexed AlPcS4). The formation of a ground-state complex was established by UV-Vis titration (Figure 2b), which revealed significant changes in the band shapes and intensities, as well as characteristic isosbestic points. The latter provides a corroborative evidence for a two-state complexation model (uncomplexed form and 1:1 inclusion complex). Calixarenes are well-demonstrated fluorescence quenchers via the PET mechanism.29 It is reasonably acceptable that the PET quenching mechanism is transferable to the other three PSs. Based on the aforementioned fundamental insights, we transferred our system from GC5A-6C to the GC5A-12C nanocarrier, which is preferable for the following in vivo application. Adding the GC5A-12C nanocarrier to PSs gives rise to the reasonably consistent fluorescence quenching (Figure 2c and Figure S11). Moreover, the corresponding photoactivity (1O2 generation) is almost completely annihilated too (Figure 2d and Figure S12) (photoactivity annihilation: AlPcS4, (94.5±0.1)%; TPPS, (86.7±0.3)%; RB, (92.2±0.1)%; EY, (87.5±0.2)%). Both fluorescence quenching and photoactivity annihilation are paralleled to each other with a 0.99 Pearson’s correlation coefficient. It is reasonably acceptable by comparing the rate constant of PET (kPET = 2.0×1010 s−1) with those of fluorescence and intersystem crossing (ISC) (kFluo = 1.0×108 s−1 and kISC = 4.9×107 s−1) (see ESI). PET is a much more favorable process to deactivate the singlet excited state than fluorescence emission and ISC (Scheme 1c). This efficient quenching mechanism rationalizes the working principle of BDA. Consequently, the complexation of calixarene leads to both fluorescence “super”-quenching and photoactivity “super”-annihilation of PSs. Both processes can be reversed by addition of competitive biomarkers. Reactivation of Fluorescence and Photoactivity by ATP Displacement in Inanimate Milieu and Cancer Cells. ATP represents one of promising tumor biomarkers, as the concentration of extracellular ATP in tumor tissues ([ATP] > 100 µM) is more than 4 orders of magnitude higher than that in normal tissues ([ATP] = 1−10 nM).45-46 Although a few examples of ATP-triggered imaging and drug delivery systems have been reported,59-65 these systems were designed to distinguish extracellular and intracellular matrices (ATP is highly present within the cells at a concentration range of 1−10 mM,66

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whereas low concentrations can be found in the extracellular milieu). More recently, Aida and coworkers reported molecular glues for ATP-responsive activity modulation of trypsin in vitro and in cellular systems, showing promising therapeutic application of tumor targeting.67 However, in vivo tumor-selective imaging and targeted therapy directed by the overexpression of ATP in tumor tissues has never been attempted before. One challenge is designing artificial receptors capable of strong binding to ATP with exquisite specificity. In nature, ATP carrier protein has a funnel-like structure (∼20 Å in length and 8 Å in diameter) capped by arginine lids.68 Inspired by this, we designed GC5A as the artificial receptor for ATP, possessing both intrinsic cavity and guanidinium anchoring points. GC5A-6C affords extremely strong binding affinity ((4.7±1.4) × 108 M−1) to ATP.57 In BDA, such a strong binding to biomarker is also prerequisite to trigger the substantial release of PSs. Two concentrations (10 nM and 100 µM) of ATP were employed to evaluate the reactivation of fluorescence and photoactivity of PSs by ATP displacement, where 10 nM is to mimic the normal tissues and 100 µM is to mimic the tumor tissues. The fluorescence, as well as photoactivity, is almost completely recovered at 100 µM ATP, while a negligible change at 10 nM ATP is seen (Figure 2c-d). That means the reactivation highly depends on the ATP production. As a result, the PS-loaded GC5A-12C nanoplatform is capable of imaging and treating tumor specifically. It is also necessary to confirm the specificity of ATP-triggered release since drug delivery needs to bear a long period of blood circulation to reach the tumor site. In this regard, we tested the displacement of PSs by other biologically important species found in blood. No significant fluorescence regeneration of four PSs was observed upon addition of these biological species (Figure 2e and S14). Furthermore, we examined the fluorescence intensity of the AlPcS4-loaded GC5A-12C nanoparticle in mouse serum. Comparing with free AlPcS4, almost no appreciable emission was detected, which validates the robust complexation stability of the AlPcS4/GC5A-12C nanoparticle. Such high binding affinities at nanomolar level would avoid unwarranted off-target leaking of PSs from the nanocarrier during blood circulation. According to the aforementioned results, all four PSs are appropriate for the BDA strategy. Furthermore, taking the tissues penetrability of light in in vivo application into account, AlPcS4 was screened as the optimal PS in the following cell and in vivo experiments owing to its near-infrared light absorption. We next investigated whether PS/GC5A-12C nanoparticles are capable of releasing PSs in response to the biological ATP level in cancer cells. After incubation with the AlPcS4/GC5A-12C nanoparticle at 37 oC for 6 h, 4T1 breast cancer cells were imaged by confocal laser scanning microscopy (CLSM). Obvious intracellular fluorescence signal from AlPcS4 is clearly observed (Figure S15), indicating that AlPcS4 with restored fluorescence are significantly released from the AlPcS4/GC5A-12C nanoparticle within the cancer cells. To further confirm that such PS release with activated emission stems from the action of ATP, sodium azide (NaN3) which is known to efficiently decrease intracellular ATP generation, was employed to pre-treat 4T1 cancer cells.69

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Figure 2. Fluorescence and photoactivity annihilation by the complexation of GC5A and reactivation by ATP displacement. (a) Fluorescence spectra of AlPcS4 (2.0 µM) upon increasing concentration of GC5A-6C (0−2.2 µM), λex = 606 nm. (b) Direct UV-Vis titration of AlPcS4 (2.0 µM) with GC5A-6C (0−2.6 µM). (c) Fluorescence spectra of AlPcS4 (2.0 µM) in the absence and presence of the GC5A-12C nanocarrier (2.0 µM), and upon addition of ATP (10 nM and 100 µM, respectively). (d) 1O2 generation from AlPcS4 (2.0 µM) in the absence and presence of the GC5A-12C nanocarrier (2.0 µM), and upon addition of ATP (10 nM and 100 µM, respectively) by using p-nitroso-N,N-dimethylaniline as sensor (recording absorption changes at 440 nm). A0: Initial absorption intensity; A: Absorption intensity after irradiation. (e) Competitive release of AlPcS4 from the AlPcS4/GC5A-12C (2.0/2.0 µM) nanoparticle in response to various representative components of blood, AlPcS4 alone as control. (f) Fluorescence images of free AlPcS4 (10 µM) and the AlPcS4/GC5A-12C (10/10 µM) nanoparticle in mouse serum.

mM of Ca2+ leads to significant enhancement of intracellular AlPcS4 fluorescence (Figure S15). Taken together, these results not only verify that the PS release from our nanoparticles is indeed triggered by ATP, but also demonstrate that the amount of PS released is directly proportional to the intracellular ATP level. High 1O2 generation efficiency is the most important prerequisite for effective PDT. Hence, we assessed the capacity of released PS from the AlPcS4/GC5A-12C nanoparticle to produce effective 1O2 in cancer cells. 2, 7-Dichlorofluorescein

As shown in Figure S15, pre-treatment of 4T1 cancer cells with 10 mM of NaN3 results in tremendous reduction of the AlPcS4 fluorescence compared with that in the cells without inhibitor treatment. On the other hand, it has been well established that Ca2+ is able to activate dehydrogenases in mitochondria and thus increase NADH and ATP production.70 Accordingly, Ca2+ is also used in cellular experiments to increase the intracellular ATP concentration. As expected, in contrast to AlPcS4/GC5A-12C nanoparticle-incubated 4T1 cancer cells in the absence of Ca2+, pre-treatment of the cancer cells with 5 5



diacetate (DCFH-DA) was used as the intracellular 1O2 indicator, since non-fluorescent DCFH-DA could permeate into live cells and be oxidized to yield green emissive dichlorofluorescein (DCF) in the presence of 1O2.11 After the AlPcS4/GC5A-12C nanoparticle-treated 4T1 cancer cells were exposed to 660 nm laser irradiation for 3 min, rather intense green fluorescence from DCF is clearly observed throughout the cells (Figure S16a). It is noteworthy that such green fluorescence enhancement of the indicator can be greatly restrained via pre-treatment of cancer cells with N-acetylcysteine

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to scavenge the produced 1O2 (Figure S16c). As controls, there is extremely low DCF fluorescence detected in the AlPcS4/GC5A-12C nanoparticle-treated 4T1 cancer cells without 660 nm laser irradiation or untreated cancer cells in the presence and absence of laser irradiation (Figure S16b, d-e). These data reasonably illustrate that the released AlPcS4 from the AlPcS4/GC5A-12C nanoparticle within the cancer cells retrieves its ability to generate 1O2 accompanied with its fluorescence recovery.

Figure 3. In vivo selective fluorescence visualization of tumors. (a) Schematic illustration of the tumor-selective imaging and targeted PDT performed on tumor-bearing mice with dual selectivity arising from the EPR effect of the nanocarrier and tumor-ATP response. (b) In vivo fluorescence imaging of the 4T1 tumor-bearing nude mice at 1, 4, 6, 8 and 24 h after intravenous injection of AlPcS4/GC5A-12C and free AlPcS4. Arrows indicate the tumor sites. Ex vivo imaging of tumor and major organs harvested from the euthanized 4T1 tumor-bearing nude mice at 6 h post-injection. 6


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After validating the restored 1O2 production capability, the PDT efficacy of the released AlPcS4 against cancer cells was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. As shown in Figure S17, bare GC5A-12C nanocarrier without loading of PS exhibits negligible cytotoxicity, as indicative of the > 90% cell viability even at the highest concentration of 20 µM based on the GC5A-12C unit. Moreover, Figure S18 shows the AlPcS4/GC5A-12C nanoparticle without 660 nm laser irradiation is similarly favorable biocompatibility to bare GC5A-12C nanocarrier, suggesting that AlPcS4 itself is biocompatible. In sharp comparison, when the AlPcS4/GC5A-12C nanoparticle-treated cancer cells were exposed to 660 nm laser for 3 min, a dose-dependent cytotoxicity is observed owing to PDT. For example, 63.5% of total cells are dead when 5 µM of the AlPcS4/GC5A-12C nanoparticle was used, whereas only 7.2% cancer cells survive when the nanoparticle concentration elevates to 20 µM. We have achieved activatable phototheranostics by BDA, which was validated not only in inanimate milieu but also in cancer cells. The complexation of calixarene receptor “super”-quenches both the fluorescence and photoactivity of commercial PSs (OFF state), which minimizes the imaging background and off-target phototoxicity during its systemic delivery. When encountering the tumor-overexpressed biomarker ATP, the competitive inclusion of ATP gives rise to the traceless release of PSs, accompanied with the recovery of fluorescence and photoactivity (ON state). These fundamental results form the chemical basis for applying the BDA strategy in vivo to realize tumor-targeted phototheranostics. In Vivo BDA for Selective Tumor Imaging and Targeted PDT. The in vivo application of the AlPcS4/GC5A-12C nanoparticle was studied using xenograft 4T1 tumor-bearing nude mouse model. It is hypothesized that the non-emissive AlPcS4/GC5A-12C nanoparticle is capable of significantly accumulating in tumor tissues via the EPR effect and then releasing active AlPcS4 due to the BDA by ATP overexpressed in tumors, which allows for selective fluorescence imaging of tumor tissues and precision PDT (Figure 3a). To validate this hypothesis, we first tested whether BDA can selectively occur in tumor tissues. Accordingly, the AlPcS4/GC5A-12C nanoparticle was directly injected into the tumor, whereas the same amount of nanoparticle was subcutaneously injected into the normal skin as a control. As displayed in Figure S19, within tumor tissues, AlPcS4 is rapidly released from the nanoparticle and becomes emissive at 5 min post-injection, and the released AlPcS4 signal gradually increases over time. In sharp contrast, no detectable AlPcS4 signal is observed in normal skin even at 3 h post-injection. This result reveals that tumor-selective BDA works in vivo, and the difference in ATP concentration between tumor and normal tissues is beneficial to selectively light-up the tumor. We next investigated whether systemically administrating the AlPcS4/GC5A-12C nanoparticle can specifically visualize tumor tissues by virtue of both EPR effect and BDA. As such, 4T1 tumor-bearing mice were injected with the AlPcS4/GC5A-12C nanoparticle via the tail vein, followed by in vivo fluorescence imaging of the released AlPcS4 using a Maestro system. Figure 3b shows the time-dependent in vivo biodistribution of the released AlPcS4 resulting in high fluorescence. The tumors can be clearly delineated by AlPcS4 fluorescence since 1 h post-injection of the nanoparticle, and the fluorescent signal from released AlPcS4 increases gradually and reaches a peak at 6 h post-injection. The ex vivo tissue

imaging reveals that there is weak fluorescence detected in normal tissues such as liver, kidney and intestine at 6 h post-injection. These results reveal that the AlPcS4/GC5A-12C nanoparticle possesses prominent EPR effect, permitting effective tumor uptake, and that AlPcS4 is able to be significantly released from the nanoparticle and recover its fluorescence triggered by the overexpressed ATP in tumor tissues. It has been well-established that nanomaterials are prone to uptake by RES organs such as liver and spleen.71 The low fluorescence signals in RES organs suggest that active AlPcS4 molecules are hardly released from the nanoparticle owing to the low ATP levels in normal organs. As a result, rather high fluorescence intensity ratio of tumor to normal tissues is achieved thanks to the large difference in ATP level between tumor and normal tissues. As a control, free AlPcS4 with the same AlPcS4 concentration as that for the AlPcS4/GC5A-12C nanoparticle was intravenously injected into 4T1 tumor-bearing mice. Although free AlPcS4 is also enriched in tumor tissues to some extent, far stronger fluorescent signal is seen in the abdomen of the mice and especially in the intestines (Figure 3b). Noteworthy is that the fluorescence signal in tumor from nanoparticle-treated mice is significantly higher than that from free AlPcS4-treated mice at each tested time point, and the fluorescence intensity ratio of tumor to normal tissues in nanoparticle-treated mice is also significantly larger than that in free AlPcS4-treated mice. These highlight the advantages of our probe design in terms of nanoformulation and BDA. Encouraged by the high enrichment and ATP-triggered release of AlPcS4 from nanoparticle in tumor tissues, we next studied whether the released PS can realize effective PDT by virtue of BDA. In this experiment, 4T1 tumor-bearing mice were randomly divided into 6 groups with 5 mice in each group, named "Saline", "Saline 660 nm", "AlPcS4", "AlPcS4 660 nm", "AlPcS4/GC5A-12C", and "AlPcS4/GC5A-12C 660 nm". For "Saline", "AlPcS4", and "AlPcS4/GC5A-12C" groups, 4T1 tumor-bearing mice were intravenously administrated with saline, free AlPcS4 (1.34 mg⋅kg−1), and the AlPcS4/GC5A-12C nanoparticle (at dose of AlPcS4 1.34 mg⋅kg−1), respectively, on day 0. For "Saline 660 nm", "AlPcS4 660 nm", and "AlPcS4/GC5A-12C 660 nm" cohorts, after injecting with saline, free AlPcS4 (1.34 mg⋅kg−1), and the AlPcS4/GC5A-12C nanoparticle (at dose of AlPcS4 1.34 mg⋅kg−1), respectively, via the tail vein on day 0, the mice were irradiated with 660 nm laser for 5 min each at 6 h and 24 h post-injection, generating 1O2 for PDT by PS. The in vivo antitumor activities of the aforementioned treatments were subsequently investigated by monitoring the tumor volumes during 16 days. As displayed in Figure 4a, the treatments of "AlPcS4" and "AlPcS4/GC5A-12C" have negligible inhibition effect on tumor growth as compared to "Saline". Moreover, the treatment of "Saline 660 nm" also has similar tumor growth profile to "Saline", indicating that 660 nm laser irradiation alone possesses no antitumor effect. It is found that the treatment of "AlPcS4 660 nm" can suppress the tumor growth to some extent due to the PDT of free AlPcS4, although the average tumor volume on day 16 is around 12 times larger than that on day 0. Dramatically, the treatment of "AlPcS4/GC5A-12C 660 nm" shows the most efficacious antitumor efficacy, as evidenced by the tumor growth stagnation and even smaller average tumor volume on day 16 than that on day 0. The best antitumor activity of "AlPcS4/GC5A-12C 660 nm" is further confirmed by the average weights of excised tumors and the corresponding photographs providing intuitive 7



evidence (Figure 4b). Moreover, negligible body weight losses were observed in the mice of 6 groups, suggesting low in vivo toxicities of various treatments (Figure 4c), which are further confirmed by the hematoxylin and eosin (H&E) staining of liver and spleen tissues from the mice in each group, indicating no significant lesions to normal organs (Figure S20). On day 16, all the mice in the 6 groups were sacrificed and the tumor tissues were collected, sliced and stained with H&E, in situ

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terminal deoxynucleotidyl transferased dUTP nick end labeling (TUNEL) and fluorescent proliferating cell nuclear antigen (PCNA), respectively. The images in Figure 4d demonstrate that among different treatments, "AlPcS4/GC5A-12C 660 nm" is the most effective in causing necrotic regions within tumors, inducing cancer cell apoptosis, and impeding cancer cell proliferation. These histological results validate the superb anticancer efficacy of "AlPcS4/GC5A-12C 660 nm".

Figure 4. In vivo targeted photodynamic tumor ablation. (a) Relative tumor volumes in different groups. (b) Tumor weights obtained on day 16 and representative photographs of tumor tissues. (c) The body weight changes in different groups. (d) Histological observation of the tumor tissues after treatment. The tumor sections were stained with H&E, PCNA and TUNEL, respectively. All the scale bars are 100 µm. Green fluorescence indicates the signal from PCNA and TUNEL staining, while the blue fluorescence is from the nucleus staining. Group 1: Saline. Group 2: Saline 660 nm. Group 3: AlPcS4. Group 4: AlPcS4 660 nm. Group 5: AlPcS4/GC5A-12C. Group 6: AlPcS4/GC5A-12C 660 nm. Error bars are based on standard error of mean. (***p< 0.001)

fidelity, easy adaptability to different PSs and promised facile combination of them to enhance photon utility in light window. The key factor for BDA is the subtle design of biocompatible receptors that (1) affording high affinities to PSs to avoid undesired off-target leaking during its systemic delivery; (2) giving rise to “super”-quenching of fluorescence and photoactivity to minimize the imaging background and phototoxicity to normal tissues; (3) strong binding to biomarkers to

CONCLUSIONS In summary, we developed a host-guest BDA strategy achieving in vivo selective tumor diagnosis and targeted photodynamic cancer ablation, which paves a new avenue for precision medicine. As comparison to the most frequently used covalent approach for activatable phototheranostics, the non-covalent BDA strategy exhibits intrinsic superiorities of employment of currently approved PSs, traceless release of PSs with high 8


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Journal of the American Chemical Society (8) Li, X. S.; Kolemen, S.; Yoon, J.; Akkaya, E. U. Adv. Funct. Mater. 2017, 27, 1604053. (9) Taniguchi, A.; Shimizu, Y.; Oisaki, K.; Sohma, Y.; Kanai, M. Nat. Chem. 2016, 8, 974-982. (10) He, J.; Wang, Y.; Missinato, M. A.; Onuoha, E.; Perkins, L. A.; Watkins, S. C.; St Croix, C. M.; Tsang, M.; Bruchez, M. P. Nat. Methods 2016, 13, 263-268. (11) Zhu, H.; Fang, Y.; Miao, Q.; Qi, X.; Ding, D.; Chen, P.; Pu, K. ACS Nano 2017, 11, 8998-9009. (12) Feng, B.; Zhou, F. Y.; Xu, Z. A.; Wang, T. T.; Wang, D. G.; Liu, J. P.; Fu, Y. L.; Yin, Q.; Zhang, Z. W.; Yu, H. J.; Li, Y. P. Adv. Funct. Mater. 2016, 26, 7431-7442. (13) Park, S. Y.; Baik, H. J.; Oh, Y. T.; Oh, K. T.; Youn, Y. S.; Lee, E. S. Angew. Chem., Int. Ed. 2011, 50, 1644-1647. (14) Yuan, Y. Y.; Zhang, C.-J.; Gao, M.; Zhang, R. Y.; Tang, B. Z.; Liu, B. Angew. Chem., Int. Ed. 2015, 54, 1780-1786. (15) Zhou, Z. J.; Song, J. B.; Tian, R.; Yang, Z.; Yu, G. C.; Lin, L. S.; Zhang, G. F.; Fan, W. P.; Zhang, F. W.; Niu, G.; Nie, L. M.; Chen, X. Y. Angew. Chem., Int. Ed. 2017, 56, 6492-6496. (16) Li, X.-S.; Ke, M.-R.; Huang, W.; Ye, C.-H.; Huang, J.-D. Chem. - Eur. J. 2015, 21, 3310-3317. (17) Wang, X.-Q.; Lei, Q.; Zhu, J.-Y.; Wang, W.-J.; Cheng, Q.; Gao, F.; Sun, Y.-X.; Zhang, X.-Z. ACS Appl. Mater. Interfaces 2016, 8, 22892-22899. (18) Song, Z.; Mao, D.; Sung, S. H. P.; Kwok, R. T. K.; Lam, J. W. Y.; Kong, D.; Ding, D.; Tang, B. Z. Adv. Mater. 2016, 28, 7249-7256. (19) Zhu, Z.; Tang, Z.; Phillips, J. A.; Yang, R.; Wang, H.; Tan, W. J. Am. Chem. Soc. 2008, 130, 10856-10857. (20) Majumdar, P.; Nomula, R.; Zhao, J. Z. J. Mater. Chem. C 2014, 2, 5982-5997. (21) Lovell, J. F.; Liu, T. W.; Chen, J.; Zheng, G. Chem. Rev. 2010, 110, 2839-2857. (22) Ng, K. K.; Zheng, G. Chem. Rev. 2015, 115, 11012-11042. (23) Tian, J.; Ding, L.; Xu, H. J.; Shen, Z.; Ju, H.; Jia, L.; Bao, L.; Yu, J. S. J. Am. Chem. Soc. 2013, 135, 18850-18858. (24) Piao, W.; Hanaoka, K.; Fujisawa, T.; Takeuchi, S.; Komatsu, T.; Ueno, T.; Terai, T.; Tahara, T.; Nagano, T.; Urano, Y. J. Am. Chem. Soc. 2017, 139, 13713-13719. (25) Kim, H.; Mun, S.; Choi, Y. J. Mater. Chem. B 2013, 1, 429-431. (26) Ramamurthy, V.; Inoue, Y. Supramolecular Photochemistry: Controlling Photochemical Processes; John Wiley & Sons: Hoboken, USA, 2011. (27) Houk, K. N.; Leach, A. G.; Kim, S. P.; Zhang, X. Angew. Chem., Int. Ed. 2003, 42, 4872-4897. (28) Ma, X.; Zhao, Y. Chem. Rev. 2015, 115, 7794-7839. (29) Dsouza, R. N.; Pischel, U.; Nau, W. M. Chem. Rev. 2011, 111, 7941-7980. (30) Böhmer, V. Angew. Chem., Int. Ed. 1995, 34, 713-745. (31) Guo, D.-S.; Wang, K.; Liu, Y. J. Inclusion Phenom. Macrocyclic Chem. 2008, 62, 1-21. (32) Guo, D.-S.; Liu, Y. Acc. Chem. Res. 2014, 47, 1925-1934. (33) Guo, D.-S.; Uzunova, V. D.; Su, X.; Liu, Y.; Nau, W. M. Chem. Sci. 2011, 2, 1722-1734. (34) Minaker, S. A.; Daze, K. D.; Ma, M. C.; Hof, F. J. Am. Chem. Soc. 2012, 134, 11674-11680. (35) Hennig, A.; Bakirci, H.; Nau, W. M. Nat. Methods 2007, 4, 629-632. (36) Ghale, G.; Lanctot, A. G.; Kreissl, H. T.; Jacob, M. H.; Weingart, H.; Winterhalter, M.; Nau, W. M. Angew. Chem., Int. Ed. 2014, 53, 2762-2765. (37) Ghale, G.; Nau, W. M. Acc. Chem. Res. 2014, 47, 2150-2159. (38) Norouzy, A.; Azizi, Z.; Nau, W. M. A Angew. Chem., Int. Ed. 2015, 54, 792-795. (39) You, L.; Zha, D.; Anslyn, E. V. Chem. Rev. 2015, 115, 7840-7892. (40) Dsouza, R. N.; Hennig, A.; Nau, W. M. Chem. - Eur. J. 2012, 18, 3444-3459. (41) Nimse, S. B.; Kim, T. Chem. Soc. Rev. 2013, 42, 366-386. (42) Zhou, Y.; Li, H.; Yang, Y.-W. Chin. Chem. Lett. 2015, 26, 825-828.

trigger the targeted release of PSs in tumor tissues, permitting maximized tumor-to-normal tissue ratio. Taking ATP as the biomarker, a BDA platform was engineered using the pegylated GC5A-12C nanocarrier. The general phototheranostics nanoplatform is adaptive to various anionic PSs commercially available. More importantly, the self-assembled nanosystem is of dual selectivity for tumor-targeted phototheranostics in vivo that result from the EPR effect and tumor-ATP response. Comparing with free PS, the PS/GC5A-12C nanoparticle not only is able to selectively visualize tumor in real time, but also gives rise to much more efficient cancer ablation. To be envisaged, this concept of BDA is easily amenable to other ensembles and targets, so that versatile biomedical applications can be envisaged.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Experimental conditions, supplementary methods for chemical synthesis and characterization of compounds, supplementary experiments and data.

AUTHOR INFORMATION Corresponding Author *[email protected] *[email protected]

ORCID Dan Ding: 0000-0003-1873-6510 Dong-Sheng Guo: 0000-0002-0765-5427

Author Contributions #

J.G. and J.L. contributed equally to this work.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was supported by the NSFC (21672112 and 51622305), the PCSIRT (IRT13023), the Science & Technology Project of Tianjin of China (15JCYBJC29800), the National Basic Research Program of China (2015CB856503), and the Fundamental Research Funds for the Central Universities and Program of Tianjin Young Talents, which are gratefully acknowledged.

REFERENCES (1) Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P. L.; Urano, Y. Chem. Rev. 2010, 110, 2620-2640. (2) Garland, M.; Yim, J. J.; Bogyo, M. Cell. Chem. Biol. 2016, 23, 122-136. (3) Gao, M.; Yu, F.; Lv, C.; Choo, J.; Chen, L. Chem. Soc. Rev. 2017, 46, 2237-2271. (4) Wang, L.; Yang, P. P.; Zhao, X. X.; Wang, H. Nanoscale 2016, 8, 2488-2509. (5) Dolmans, D. E.; Fukumura, D.; Jain, R. K. Nat. Rev. Cancer 2003, 3, 380-387. (6) Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.; Verma, S.; Pogue, B. W.; Hasan, T. Chem. Rev. 2010, 110, 2795-2838. (7) 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. J. Am. Chem. Soc. 2017, 139, 7595-7602. 9



(43) Rodik, R. V.; Klymchenko, A. S.; Mely, Y.; Kalchenko, V. I. J. Inclusion Phenom. Macrocyclic Chem. 2014, 80, 189-200. (44) Bagnacani, V.; Franceschi, V.; Bassi, M.; Lomazzi, M.; Donofrio, G.; Sansone, F.; Casnati, A.; Ungaro, R. Nat. Commun. 2013, 4, 1721. (45) Pellegatti, P.; Raffaghello, L.; Bianchi, G.; Piccardi, F.; Pistoia, V.; Di Virgilio, F. PLoS ONE 2008, 3, e2599. (46) Trabanelli, S.; Ocadlikova, D.; Gulinelli, S.; Curti, A.; Salvestrini, V.; Vieira, R. P.; Idzko, M.; Di Virgilio, F.; Ferrari, D.; Lemoli, R. M. J. Immunol. 2012, 189, 1303-1310. (47) Dai, Y.; Xu, C.; Sun, X.; Chen, X. Chem. Soc. Rev. 2017, 46, 3830-3852. (48) Guo, D.-S.; Liu, Y. Chem. Soc. Rev. 2012, 41, 5907-5921. (49) Mogaki, R.; Hashim, P. K.; Okuro, K.; Aida, T. Chem. Soc. Rev. 2017, 46, 6480-6491. (50) Geng, W.-C.; Liu, Y.-C.; Wang, Y.-Y.; Xu, Z.; Zheng, Z.; Yang, C.-B.; Guo, D. S. Chem. Commun. 2017, 53, 392-395. (51) Helttunen, K.; Shahgaldian, P. New J. Chem. 2010, 34, 2704-2714. (52) Xu, Z.; Peng, S.; Wang, Y.-Y.; Zhang, J.-K.; Lazar, A. I.; Guo, D.-S. Adv. Mater. 2016, 28, 7666-7671. (53) Shulov, I.; Rodik, R. V.; Arntz, Y.; Reisch, A.; Kalchenko, V. I.; Klymchenko, A. S. Angew. Chem. Int. Ed. 2016, 55, 15884-15888. (54) Sansone, F.; Dudic, M.; Donofrio, G.; Rivetti, C.; Baldini, L.; Casnati, A.; Cellai, S.; Ungaro, R. J. Am. Chem. Soc. 2006, 128, 14528-14536. (55) Sun, Q.; Zhou, Z.; Qiu, N.; Shen, Y. Adv. Mater. 2017, 29, 1606628. (56) Ormond, A. B.; Freeman, H. S. Materials 2013, 6, 817-840. (57) Zheng, Z.; Geng, W.-C.; Gao, J.; Wang, Y.-Y.; Sun, H.; Guo, D.-S. Chem. Sci. 2018, 9, 2087-2091. (58) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Springer US: New York, USA, 2006. (59) Lu, Y.; Aimetti, A. A.; Langer, R.; Gu, Z. Nat. Rev. Mater. 2016, 2, 16075. (60) Mo, R.; Jiang, T.; DiSanto, R.; Tai, W.; Gu, Z. Nat. Commun. 2014, 5, 3364. (61) Mo, R.; Jiang, T.; Gu, Z. Angew. Chem., Int. Ed. 2014, 53, 5815-5820. (62) Biswas, S.; Kinbara, K.; Niwa, T.; Taguchi, H.; Ishii, N.; Watanabe, S.; Miyata, K.; Kataoka, K.; Aida, T. Nat. Chem. 2013, 5, 613-620. (63) Naito, M.; Ishii, T.; Matsumoto, A.; Miyata, K.; Miyahara, Y.; Kataoka, K. Angew. Chem., Int. Ed. 2012, 51, 10751-10755. (64) Deng, J.; Wang, K.; Wang, M.; Yu, P.; Mao, L. J. Am. Chem. Soc. 2017, 139, 5877-5882. (65) Yu, G.; Zhou, J.; Shen, J.; Tang, G.; Huang, F. Chem. Sci. 2016, 7, 4073-4078. (66) Traut, T. W. Mol. Cell. Biochem. 1994, 140, 1-22. (67) Okuro, K.; Sasaki, M.; Aida, T. J. Am. Chem. Soc. 2016, 138, 5527-5530. (68) Pebay-Peyroula, E.; Dahout-Gonzalez, C.; Kahn, R.; Trezeguet, V.; Lauquin, G. J.; Brandolin, G. Nature 2003, 426, 39-44. (69) Bowler, M. W.; Montgomery, M. G.; Leslie, A. G.; Walker, J. E. Proc. Natl. Acad. Sci. USA 2006, 103, 8646-8649. (70) Griffiths, E. J.; Rutter, G. A. Biochim. Biophys. Acta 2009, 1787, 1324-1333. (71) Wang, B.; He, X.; Zhang, Z.; Zhao, Y.; Feng, W. Acc. Chem. Res. 2013, 46, 761-769.

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