Covalent Organic Nanosheets Integrated Heterojunction with Two

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Covalent Organic Nanosheets Integrated Heterojunction with Two Strategies to Overcome Hypoxic-Tumor Photodynamic Therapy Kui Wang, Zhe Zhang, Lin Lin, Jie Chen, Kai Hao, Huayu Tian, and Xuesi Chen Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.9b00265 • Publication Date (Web): 16 Apr 2019 Downloaded from http://pubs.acs.org on April 17, 2019

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Chemistry of Materials

Covalent Organic Nanosheets Integrated Heterojunction with Two Strategies to Overcome Hypoxic-Tumor Photodynamic Therapy Kui Wang,†,‡,|| Zhe Zhang,†,|| Lin Lin,†,|| Jie Chen,†, ‡,|| Kai Hao,†,‡,|| Huayu Tian,*,†,‡,|| and Xuesi Chen†,‡,|| †Key

Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China ‡University of Science and Technology of China, Hefei 230026, China ||Jilin Biomedical Polymers Engineering Laboratory, Changchun 130022, China

ABSTRACT: Photodynamic therapy (PDT) still faces a key challenge associated with its oxygen-dependent property, which limits its therapeutic efficiency against hypoxic tumor. To address the problem, covalent organic nanosheets (CONs) are prepared with a donor-acceptor molecular heterostructure. CONs can address the hypoxic-tumor PDT by two strategies, that is, type I PDT and type I PDT combination with photothermal therapy (PTT). On the one hand, the molecular heterostructure of CONs can afford highly efficient charge carrier separation, a long lifetime of electrons and holes can be obtained, the electrons can reduce O2 to form O2•–, and at the same time, the holes can oxidize water to produce ·OH. Therefore, type I PDT is obtained, which can be used to diminish the limits of hypoxia in type II PDT. On the other hand, the recombination of photoexcited species result in an important nonradiative attenuation, and thus the energy is emitted as the heat. A combination of type I PDT and PTT serve as an effective way to get around the difficulties of hypoxia in PDT. Intravenous injection of CONs in nude mice followed by the combination of type I PDT and PTT under single wavelength irradiation achieve significant tumor ablation.

INTRODUCTION Photodynamic therapy (PDT) has received wide scientific attention because of its lack of initiating resistance, low systemic toxicity, and minimal invasive nature.1 PDT use a light-irradiated photosensitizer to produce reactive oxygen species (ROS), which can cause tumor cell apoptosis or necrosis. Many photosensitizers primarily generate singlet oxygens (1O2) through type II PDT, which are totally O2 dependent. Unfortunately, solid tumors have hypoxia tumor microenvironment.2‒3 The O2 deficiency in tumors give rise to an obvious reduction of tumor inhibiting efficiency for PDT. It is reported that type I PDT can diminish the oxygen limit of type II PDT, and PDT combination with other therapeutic methods also can overcome the therapeutic limit of type II PDT. For example, fluorinated covalent organic polymers (COPs) are prepared by Liu group, which can simultaneous tumor oxygenation and photodynamic treatment. Effective and sustained tumor oxygenation is significance in benefiting the treatment of solid tumors.4‒5 Covalent organic frameworks6‒7 (COFs) are an emerging class of porous crystalline materials that allow organic building blocks into long-range-ordered two-dimensional (2D) or three-dimensional (3D) networks via covalent bonds, and have attracted attention for a variety of potential applications, including separation,8‒9 environmental pollution,10‒11 proton conduction,12‒13 catalysis,14‒15 optoelectronics,16‒18 and drug delivery.19‒21 For example, in the optoelectronics field, COFs composed of two electroactive building blocks have been

reported by the Jiang17 and Bein18 group. If the energy levels of the building blocks are sufficiently aligned, an orderly interdigitated donor-acceptor molecular heterojunction can form,17‒18,22 which is beneficial in promoting charge carrier separation and transport after photoexcitation. Furthermore, a long lifetime of electrons and holes can be obtained. On the basis of the photocatalytic mechanism of a semiconductor,23‒24 the electrons on the conduction band can reduce the adsorbed O2 on the surface of the catalyst to form O2•–. At the same time, the holes on the valence band can oxidize the surrounding waters to produce ·OH. Both are high reactive oxygen species (ROS) causing cell damage.25 In addition, the Geminate and Non-Geminate recombination26‒28 of photoexcited species can result in an important nonradiative attenuation pathway, and thus their energy is emitted as the heat to achieve their photothermal conversion. Therefore, the COFs with a donor-acceptor molecular heterostructure may be exploited as a photosensitizer, which can diminish hypoxia limit of PDT by type I PDT and the combination of type I PDT with photothermal therapy (PTT).25,29 It is important to evaluate the PDT and PTT properties of COFs in vivo for antitumor application. However, the bulk COFs has low dispersibility in solution and bigger size, which cause in low bioavailability for the cells, and is also not fit for intravenous injection. Recently, CONs have appeared as a new member of 2D nanomaterials and have been extensively applied in antimicrobial,30-31 drug delivery,32 DNA detection,33 and battery materials.34‒36 CONs can be directly

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Figure 1. Schematic illustration of 2D CONs fabrication, in vivo tumor therapy, and the mechanism of PTT and type I PDT generation.

prepared from their bulk COFs via liquid ultrasonic exfoliation,37 because the strong interlayer π-π interactions of the COFs can be reduced.38 There are some advantages of CONs compared with COFs. On the one hand, the dispersibility and stability of CONs are improved in water. On the other hand, the CONs have smaller size, which is suitable for intravenous injection and possess enhanced permeability and retention (EPR) effect. The CONs also keep the ability that diminish hypoxia limit of PDT by type I PDT and type I PDT combination with PTT. Therefore, CONs of a heterostructure with excellent aqueous dispersibility can be an ideal alternative of COFs for in vivo tumor therapy. We chose porphyrin monomer as one of the building blocks for CONs. On the one hand, the hydrophobic property of porphyrin could be overcome by introduction of photosensitizer into frameworks, On the other hand, there was no need for encapsulate photosensitizer into CONs, which itself could act as a nano-photosensitizer. Herein, as a proof of concept, triphenylene-porphyrin (TP-Por) COF with donoracceptor molecular heterostructure were prepared.18 Impressively, COFs could be readily exfoliated into CONs via liquid ultrasonic delamination; thus, CONs could be obtained with good aqueous dispersibility in a period of time. In addition, the donor-acceptor CONs could be used as a novel theranostic platform for in vivo photoacoustic (PA) imaging and photodynamic/photothermal tumor therapy. CONs provided the following distinct advantages: (1) Twodimensional boronate ester-linked CONs had hydrolytic instability,39 which could provide potential biodegradability. (2) The CONs possessed excellent aqueous dispersibility

compared with hydrophobic porphyrin monomers. This would endow CONs with the potential for intravenous injection for in vivo application. Abundant hydroxyl groups on the margin endowed CONs with negative charges32,40 and a nano-sized morphology, which was beneficial in realizing the long circulation properties and EPR mediated tumor targeted delivery for in vivo application. (3) Compared to porphyrin monomer, CONs had strong absorption along with a high extinction coefficient at 635 nm, which were benefit to improving the efficiency of light conversion. (4) CONs contained a periodically ordered bicontinuous heterojunction network in intralayer skeletons and long-range ordered πcolumnar structures in the interlayer, which provided ambipolar pathways for exciton separation and charge carrier transport;18 thus, a long lifetime of electrons and holes could be obtained, which was beneficial to ROS generation through type I PDT mechanism. (5) CONs with mesoporous channels were suitably used as nano-photosensitizers. The mesoporous channels of CONs were beneficial to free diffusion of O2 and H2O molecules, which could significantly improve the contact possibility of O2 and H2O with the electrons and holes produced by CONs.41 Furthermore, the pores of CONs provided a pathway for facile diffusion of ROS out of the CONs interior, which could benefit their cytotoxic effects on tumor cells.42 (6) The Geminate and Non-Geminate recombination of photoexcited species could result in an important nonirradiative attenuation pathway, and thus their energy was emitted as the heat to achieve their photothermal conversion. (7) CONs had type I PDT and PTT capacities under a single wavelength irradiation, which could diminish


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oxygen-dependent property of PDT and guarantee powerful killing effects on tumor cells.43 (8) The PA imaging ability of CONs could make them as a multi-functional theranostic nano-platform. Detailed experiments were conducted in this work to determine CONs physical chemical properties, type I PDT and PTT capacities, and anti-tumor effects in vivo. This study conducted a meaningful exploration of the development of novel structured nanomaterials overcome hypoxia limit of type II PDT by type I PDT and type I PDT combination with PTT, which also extended the potential application of 2D polymers in the biomedical fields.44 RESULTS AND DISCUSSION Synthesis and Characterization of COFs and CONs. A 2D COF (TP-Por) was synthesized via the co-condensation reaction of 2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) and 5,15-bis(4-boronophenyl)-porphyrin (Por) (Figures S1-S5) according to the reported methods.18 The synthesis of the bulk TP-Por COF was conducted in a mixture of acetonitrile/mesitylene (7:3 v/v) under a solvothermal condition, which was isolated as a dark purple powder in approximately 70% yield. The as-prepared COF represented a porous hexagonal framework with HHTP units at the corners and porphyrin units at the edges (Figure 1). The successful formation of the bulk TP-Por COF material was confirmed using fourier-transform infrared spectroscopy (FT-IR) (Figure S6) and Solid-state NMR. FT-IR exhibited two characteristic peaks arising from the B-C bonds at 1333 and 1261 cm‒1. The result of Solid-state 13C cross-polarization/magic-anglespinning (CP/MAS) of boronic ester COF indicated broad peaks between 90 and 180 ppm consistent with the aromatic carbon atoms of the COF (Figure S7), which confirmed the formation of the expected structure, and its 11B NMR spectrum was made up of a single resonance consistent with the formation of a single type of boronate ester linkage (Figure S8). The boron content in the TP-Por COF was 28.5 mg/g, which was determined by inductively coupled plasma mass spectrometry, and the C, H, and N contents (Table S1) were evaluated using elemental analysis (EA). TP-Por COF composed of 68% porphyrin monomer and 32% HHTP monomer was calculated by nitrogen contents. The result showed that the C, H, N, and B contents were consistent with the theoretical values of infinite 2D polymer sheets. The thermal stability of the TP-Por COF investigated using thermogravimetric analysis (TGA) under argon indicated that the COF was stable up to ≈320 °C (Figure S9). Nitrogen sorption experiments of the TP-Por COF bulk material performed at 77 K yielded isotherms with a typical type IV shape (Figure S10), which was characteristic of mesoporous materials. The calculated Brunauer-Emmett-Teller (BET) surface area was 443 m2 g–1. Pore size distribution was calculated by quenched-solid density functional theory (QSDFT), which showed an average pore diameter of 2.6 nm (Figure S11). Powder X-ray diffraction (PXRD) confirmed the formation of a periodic structure (Figure S12). PXRD patterns of the COF exhibited an intense peak at 2θ = 4.7°, which could be attributed to the (100) crystal planes. The broad peak at 2θ = 28.0° corresponded to the (001) planes; therefore, the interlayer spacing between the (001) planes was found to be approximately 0.34 nm. Notably, transmission electron microscopy (TEM) images of COFs showed sheet-like

structures (Figure S13). Scanning electron microscopy (SEM) analysis of COFs also proved aggregation of sheet-like morphology (Figure S14). The bulk COF was a porous crystalline structure, resulting in low dispersibility in solution and further low bioavailability for the cells. However, because the bulk COF simultaneously had a sheet-like layered morphology and abundant hydroxyl groups on the periphery, it could be easily exfoliated into 2D CONs via one-step liquid sonication exfoliation. Therefore, the CONs were prepared by solvent-assisted liquid sonication. Nitrogen sorption experiments of the CONs performed at 77 K yielded isotherms (Figure S10). The calculated BET surface area was 30 m2 g–1. A sharp reduction of the BET surface area of CONs confirmed this exfoliation phenomenon. To verify the crystal property of the CONs, PXRD characterization was conducted (Figure S12). The results showed that the intensity of the first peak (100) planes at 2θ = 4.7° was reduced. And the peak at 2θ = 28.0° resulting from the (001) planes was broadened, which might have been caused by the partial loss of π-π stacking between the layers. The FT-IR spectra (Figure S15) and Solid-state NMR (Figure S16) of CONs remained the same as that of the bulk material, which confirmed the same molecular structures of the TP-Por COFs and 2D TP-Por CONs. TEM (Figure 2a) images clearly showed that the 2D CONs structures exhibited a sheet-like morphology. AFM analysis of the CONs was implemented by drop-casting the solution of CONs on a silicon wafer, where the average thickness was found to be approximately 25 nm (Figure 2b). Considering that the distance between the adjacent layers was approximately 3.4 Å, it was suggested that the CONs consisted of approximately 73 atomic layers. The CONs was dispersed in phosphate buffered saline (PBS) and the stability was observed at predetermined time intervals (0, 12 h). CONs maintained good aqueous stability for 12 h (Figure S17a). An obvious “Tyndall Effect” was observed when a red laser passed through the PBS solution of the CONs (Figure S17b), which confirmed their colloidal property. These results showed that the CONs could maintain good aqueous stability within a period of time. The enhanced water dispersibility could be attributed to the abundant hydroxyl groups on the surface of CONs. Dynamic light scattering (DLS) measurements of the CONs provided an average diameter of 345 nm with a polydispersity index of 0.11 (Figure S18a). The size of CONs kept stable in three days, which confirmed the degradation of CONs in PBS was a slower process (Figure S18b). The CONs showed a zeta potential of -37 mV (Figure S18c). The zeta potential value of the CONs was negative, as it contained surface hydroxyl (−OH) groups, which was beneficial for in vivo application of tumor therapy. Biodegradability of CONs. In general, biosafety is a critical concern when CONs are used in vivo. Therefore, it is of great importance to develop CONs that not only have theranostic ability but also possess sufficient biodegradability, which will be beneficial for CONs excretion from the body over a period of time. Generally, COFs are constructed by dynamic covalent bonds. The reversibility of the dynamic covalent bonds is indispensable for generation of the crystalline COFs. However, fundamental reversibility can not be neglected after preparation of COFs. The p-orbital for the boron in the ester linkage is shown to conjugate with the πelectrons on the phenyl rings and adjoining oxygen atoms,


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Figure 2. Characterization of CONs. (a) Transmission electron microscopy (TEM) image of CONs. (b) Atomic force microscopy (AFM) image of CONs with the thickness indicated. (c) UV-Vis absorption spectra of CONs and Por. (d) Normalized photoluminescence (PL) spectra of CONs and Por. (e) ROS generation from CONs using DPBF as a probe under 635 nm irradiation (1 W/cm2, 140 s). (f) Quantification of ROS generation ability of CONs in the dark, DPBF only, irradiation, and irradiation with vitamin C. (g) ESR spectra of CONs with DMPO in CH3CN under 635 nm laser irradiation for 0 or 2 min for demonstrated superoxide radical generation (a: No irradiation under hypoxia; b: Irradiation under hypoxia; c: No irradiation under normoxia; d: Irradiation under normoxia). (h) ESR spectra of CONs with DMPO in H2O under 635 nm laser irradiation for 0 or 2 min for demonstrated hydroxyl radical generation (a: hydroxyl radicals; b: alkane radical). (i) Temperature increase curves of CONs with time at various concentrations under irradiation (635 nm, 1 W/cm2, 5 min).

which can contribute to the relative stability of the CONs. This structure is also possibly attacked by nucleophiles,39 such as water. Therefore, CONs have potential biodegradability resulting from hydrolysis of the boronate ester. The biodegradability of CONs in PBS was evaluated in a horizontal shaker at 37 ℃ . Compared to the initial CONs absorption, there was hardly any absorption after 12 weeks because of the CONs hydrolysis (Figure S19). Furthermore, the dispersion of CONs turned transparent after 12 weeks, which indicated the degradation of CONs in this period. The morphological changes in the CONs were further assessed using SEM images. The original morphology of the CONs (Figure S20a) could be entirely disrupted, and a few residues of CONs could be observed after 12 weeks (Figure S20b). Furthermore, considering that the hydrogen peroxide (H2O2) level was approximately 0.1 mM in the tumor environment45 and the H2O2 sensitivity of boronate ester,46 the influence of the degradation of CONs with hydrogen peroxide (H2O2) was further evaluated. After adding 4 mM H2O2 9 times over 3 days, obvious morphological change and degradation could be observed via SEM, which indicated that H2O2 could significantly accelerate the biodegradable process of the CONs (Figure S20c). In Vitro ROS and Photothermal Property Evaluation of CONs. Compared to visible-near-infrared (Vis-NIR) absorption spectroscopy of Por monomer, an enhanced absorption of CONs could be obtained in the range of 450-

800 nm (Figure 2c). The extinction coefficient of CONs showed the light absorption ability at 635 nm; therefore, the optical absorption spectrum of CONs was acquired with variable concentrations (Figure S21). The normalized adsorption intensity for the length of the cell (A/L) at λ = 635 nm with alterable concentrations (C) was acquired. Following the Lambert-Beer law (A/L = εC, in which ε is the extinction coefficient), a linear relation of A/L to the concentration could be obtained, and the extinction coefficient at 635 nm was 14.8 L g–1 cm–1 (Figure S22), which was higher than some existing photoabsorbing agents such as gold nanorods (3.9 L g–1 cm–1) or graphene oxide (3.6 L g–1 cm–1) at 808 nm.47 The light energy of absorption by a photosensitive agent is usually released via four pathways: photochemistry, photon emission, molecular transfer, or heat production.48 The fluorescence spectra (Figure 2d) of CONs in water and Por monomer in DMF at the same molar concentration, the raw material (Por monomer) exhibited two fluorescence peaks. However, there was barely any fluorescence for the CONs compared to the Por monomer, indicating that a higher quenching yield was obtained for the CONs. Therefore, from the aspect of energy conservation, CONs had the potential to produce reactive oxygen species or photothermal effects under continuous 635 nm laser irradiation. The generation of various ROS for PDT of tumors normally involves two unique photochemical processes: type I and type II.49 Many photosensitizers50‒51 primarily generated 1O2


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through type II PDT process. Specific photosensitizers could produce superoxide anions (O2•–) and hydroxyl radicals (·OH) via type I PDT process.52‒53 The CONs were composed of an HHTP donor and porphyrin acceptor, in which the energy levels of the two subunits were adequately aligned. Each HHTP donor and porphyrin acceptor unit in the CONs interfaced; therefore, they endowed periodically ordered molecular heterojunctions, which were arranged into a 1D πstacked column. On the one hand, the CONs form an interdigitated donor-acceptor heterostructure. This nanoscopic segregation morphology forms a broad interface, which can be expected to promote charge separation upon photoexcitation. On the other hand, long-range ordered donor-on-donor and acceptor-on-acceptor π-columnar structures of CONs provide ambipolar pathways for charge carrier transport, which lead to a long lifetime for electrons and holes. The electrons on the conduction band can reduce the O2 to form O2•–. At the same time, the holes on the valence band can oxidize water to produce ·OH (Figure 1). Therefore, the CONs provided an ideal structure for the generation of ROS through type I PDT process, which could diminish the oxygen-dependent property of type II PDT. To examine the ROS generation capacity, CONs (100 μg/mL) were incubated with 1,3diphenylisobenzofuran (DPBF) under a 635 nm laser (1 W/cm2). There was a rapid reduced UV-Vis absorbance (Figure 2e). In contrast, negligible spectral changes were observed under control experiments, including the DPBF only or CONs in the dark experiments. A decrease in DPBF absorption at 415 nm was inhibited after the addition of vitamin C (Vc) as a ROS scavenger (Figure 2f). To distinguish different ROS production by CONs, we used dihydrorhodamine 123(DHR123) for superoxide radicals detection and hydroxyphenyl fluorescein (HPF) for hydroxyl radicals detection respectivly. CONs substantially increased the fluorescence intensity of DHR123 under normoxia (Figure S23a) upon laser irradiation. To test the performance of CONs under hypoxic conditions, we bubbled nitrogen in a suspension of CONs and DHR123 for 15 min, we found that the superoxide radicals production followed the same trend as in normoxic conditions, but the intensity was reduced (Figure S23b), which indicating that the CONs allowed the efficient utilization of oxygen even at hypoxic conditions. Fluorescence enhancement of HPF was observed (Figure S24a) under normoxic conditions. A few amount of ·OH was generated when CONs incubated with HPF after 18 min irradiation under hypoxic conditions, However, a significantly increasing of fluorescence after 21 min irradiation (Figure S24b), these results clearly indicated ·OH generation from CONs by hole oxidation and electron reduced other ROS. Electron spin resonance (ESR) was applied to identify the type of ROS from porphyrin monomer and CONs upon 635 nm irradiation. 5,5dimethyl-1-pyrroline-N-oxide (DMPO) and 2,2,6,6tetramethylpiperide (TEMP) were employed as the spintrapping agents of the superoxide radicals (O2•–) and hydroxyl radicals (·OH) and the singlet oxygen (1O2), respectively. Firstly, for the mixture of the porphyrin monomer and TEMP in tetrahydrofuran, there was an obvious characteristic 1:1:1 multiplicity for TEMP-1O2 adduct after 2 min of irradiation (Figure S25). This result indicated that porphyrin monomer generated singlet oxygen under light irradiation, which was a typical feature of type II PDT. In contrast, for the mixture of

CONs and TEMP in D2O, the signal of the TEMP-1O2 adduct was not detected after 2 min of irradiation (Figure S26). For the mixture of CONs and DMPO in acetonitrile, CONs induced the generation of a fourline spectrum with relative intensities of 1:1:1:1 upon irradiation under normoxia (Figure 2g), the signal of the DMPO-O2•– adduct could be detected. The signal of DMPO-O2•–adduct also could be observed under hypoxia, however, the intensity was reduced, these results indicated that CONs could produce O2•– even at hypoxia conditions. For the mixture of CONs and DMPO in water, an obvious ESR signal from an alkane radical and ·OH spintrapped adduct was observed after 2 min of irradiation (Figure 2h). These results indicated that CONs could produce ROS via the type I mechanism, that was, electron reduction or hole oxidation, which was different from that of the porphyrin monomer. This provided an opportunity for CONs to diminish the hypoxia challenge of PDT in a tumor area, which were the inherent features in the tumor microenvironment and could be deteriorated under type II PDT. This advantage could afford CONs more effective killing ability for tumor cells. To the best of our knowledge, this is the first time to report the type I PDT capacity of CONs with a donor-acceptor heterostructure. CONs as a donor-acceptor heterojunction could produce excitons upon light irradiation. The Geminate and NonGeminate recombination could result in an important nonradiative attenuation pathway for photoexcited species, and thus the energy emitted as heat to achieve photothermal conversion. Therefore, to explore whether CONs had photothermal properties, the temperature change of the PBS dispersion of CONs at different concentrations (0.05, 0.1, and 0.2 mg/mL) was evaluated under 635 nm laser irradiation (1 W/cm2), where deionized (DI) water was used as the control (Figure 2i). No obvious temperature increase was observed for pure water, while the temperature of the CONs (0.2 mg/mL) increased by 41 °C upon irradiation for 5 min upon 635 nm laser irradiation (1 W/cm2). To further investigate the photothermal stability of CONs, PBS dispersion of CONs (0.05 mg/mL, 0.2 mL) was illuminated using a 635 nm laser (1 W/cm2) until it reached a steady temperature, upon which the laser was shut off and the dispersion was cooled to room temperature. The temperature variation in the CONs was recorded for three cycles (Figure S27a). There was no obvious change in the maximal temperature during each cycle, indicating that the CONs were stable under conditions of 635 nm laser irradiation. The photothermal conversion efficiencies (η) of the CONs were measured following a reported method.54 Briefly, under conditions of 635 nm laser irradiation (1 W/cm2), the temperature of the aqueous dispersion of CONs (0.1 mg/mL) was allowed to increase to the point of steady state. After switching the laser off, the temperature decrease was recorded so as to estimate the heat transfer rate from the solution to the environment (Figure S27b). The efficiency (η) of the CONs was found to be approximately 19.1% (Figure S27c). These observations illustrated that the CONs could efficiently convert the 635 nm laser energy into thermal energy. This photothermal property indicated that CONs had potential in biomedical applications as a PTT agent for killing cancer cells. A combination of photodynamic/photothermal therapy served as an effective approach to diminish the oxygen-dependent therapeutic limit of type II PDT.


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Figure 3. In vitro cellular experiment. (a) Detection of CONs induced ROS for HeLa cells in normoxic and hypoxic culture media by fluorescence microscopy. Fluorescence images of HeLa cells treated with PBS, PBS+Laser, CONs+Laser under normoxia, and CONs+Laser under hypoxia. The 635 nm laser irradiation was at 1 W/cm2 for 5 min. (b) Viability of HeLa cells after incubation with CONs at various concentrations for 24 h in both normoxic and hypoxic conditions. (c) Cell viability of HeLa treated with CONs or Pplx at various concentrations in the presence of laser irradiation (635 nm, 1 W/cm2, and 5 min). (d) Fluorescence images of HeLa cells treated with PBS or CONs with or without laser irradiation (635 nm, 1 W/cm2, and 10 min), in which live and dead HeLa cells were co-stained with calcein-AM and propidium iodide (PI). (e) Annexin V-FITC/PI analysis of HeLa cells incubated with PBS or CONs with or without laser irradiation.

Photoacoustic Properties of the CONs. The absorption band of CONs could be observed in the region of 650-750 nm. Moreover, both excellent photostability and a high light-toheat conversion efficiency suggest that CONs were promising for photoacoustic (PA) imaging of mice. CONs at various mass concentrations were embedded in agar gel cylinders, which were investigated in a multi-spectral optoacoustic tomography (MSOT) imaging system. The quantitative result showed that the PA intensity was linearly correlated with the CON concentration (Figure S28). These findings demonstrated the potential of CONs as a theranostic nanoparticle. Cell Experiment of the CONs. The intracellular ROS of HeLa and 4T1 cells were further investigated using 2′,7′dichlorofluorescin diacetate (DCFH-DA) as an indicator under laser irradiation. DCFH-DA was deacetylated to a nonfluorescent compound once it spreads to cells, and the deacetylated compound was oxidized by ROS. Fluorescence microscopy indicated no obvious green fluorescence in PBS, PBS+Laser (Figure 3a for HeLa cells, Figure S29a for 4T1 cells), because there was no adequate ROS to oxidize DCFHDA into DCF. In contrast, intense intracellular green fluorescence was observed in cells cultured with CONs followed by 635 nm laser irradiation (1 W/cm2, 5 min) in normoxia, obvious green fluorescence was also observed for the cells incubated with CONs followed by 635 nm laser

irradiation (1 W/cm2, 5 min) under hypoxia, suggesting CONs could overcome the hypoxic environment and generate a high intracellular ROS level. Encouraged by the potential phototherapy result, we next evaluated the cytotoxicity and in vitro phototherapy efficacy of CONs on cancer cell lines (HeLa and 4T1). To assess the biocompatibility of CONs, the viability of HeLa and 4T1 cells that were treated with CONs at various concentrations (10100 μg/mL) for 24 h under both normaxic and hypoxic environments was evaluated using CCK-8. The CONs could not affect the cell viability at the tested concentrations (Figure 3b for HeLa cells, Figure S29b for 4T1 cells) in both normoxia and hypoxia. Then, the in vitro phototherapy efficacy of the CONs was evaluated in the HeLa and 4T1 cells, which were treated with CONs for 6 h, followed by 635 nm irradiation at 1 W/cm2 for 5 min. After another 24 h of incubation, the CCK-8 assay showed that the cell viability decreased with the increased concentration of CONs (Figure 3c), and nearly no cell viability was detected at 2 μg/mL for the HeLa cells. We also compared with benchmark PDT agents that are currently used in the clinic, as shown in Figure 3c, CONs had excellent therapy effect than protoporphyrin IX (Pplx) at various concentrations. Similar cytotoxicity evaluation results were also observed in the 4T1 cells (Figure S29c), further confirming the


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Figure 4. (a) In vivo PA images in the tumor area after the injection of CONs. (b) PA intensities of the tumor area at different time intervals (0, 8, 12, and 24 h) post-injection of CONs. (c) In vivo ROS generation detected using DCF fluorescence. DCF staining of the tumor regions were treated with PBS, CONs, CONs+Laser and CONs+Vc+Laser, respectively. The laser irradiation was at 1 W/cm2 for 5 min. (d) Infrared thermograph of the HeLa tumor-bearing mice treated with PBS and CONs during laser irradiation at 1 W/cm2 for 5 min. (e) Temperature elevation at the tumor region of the HeLa tumor-bearing mice treated with PBS and CONs during laser irradiation.

phototherapy effect of CONs. HeLa solid tumor had hypoxia tumor microenvironment. Therefore, in order to further examine photodynamic therapy effect of CONs under hypoxia, we irradiated HeLa cells under normoxia or hypoxia (oxygen concentration ∼5%) condition using LED light source. The cell viability also showed a decrease under hypoxic environment, which indicated that CONs could cause cell death even at ∼5% oxygen concentration (Figure S30). A live/dead cell staining assay was conducted. After laser irradiation, the live and dead cells were distinguished by calcein-AM and propidium iodide (PI) co-staining. The PBS, 635 nm laser irradiation, and CONs treatments exhibited negligible effects on cell viability. In contrast, the majority of cells were killed via the phototherapy ablation after treatment with CONs under laser irradiation (Figure 3d for HeLa cells, Figure S29d for 4T1 cells). An annexin V-FITC (AV)/PI staining assay was evaluated to analyze the apoptosis and necrosis of the HeLa and 4T1 cells using flow cytometry. Without laser irradiation, there were negligible apoptosis or necrosis for the HeLa and 4T1 cells after incubation with CONs (10 μg/mL). For the HeLa cells, laser irradiation could only induce 6.41% cell apoptosis and 2.66% cell necrosis; however, CONs with laser irradiation could induce 46.7% cell apoptosis and 18.8% cell necrosis (Figure 3e). CONs under laser irradiation could cause similar cell apoptosis and necrosis for the 4T1 cells (Figure S29e). These results clearly demonstrate that the CONs had an excellent antitumor effect for tumor ablation.

In Vivo Photoacoustic Imaging, ROS Evaluation, and Thermal Imaging. The in vivo PAI observation was conducted on xenografted HeLa tumor model. When the HeLa tumor volume reached approximately 100 mm3, the mice were administered an intravenous (i.v.) injection of CONs (5 mg/kg). The accumulation of CONs in the tumor area was monitored using the PA imaging system (Figure 4a). The PA images were recorded at four different time points (0, 8, 12, and 24 h). It was found that the PA signals gradually increased in the tumor position, and reached the maximal value at 12 h post-administration. Then, the PA signals in the tumor sites decreased at 24 h post-administration (Figure 4b). These results confirmed the effective tumor accumulation of CONs, which was attributed to the EPR effect of the nano-sized CONs. The PA imaging can provide accurate and timely spatio-temporal information for tumor location and photosensitizer accumulation, which can precisely guide the following in vivo tumor phototherapy. We further evaluated the generation of ROS in the tumor of the mice using DCFH-DA as an indicator.55 Explicit green fluorescence was observed in the tumor section from the mice treated with CONs upon 635 nm irradiation (Figure 4c). In contrast, the CONs caused less fluorescence in the absence of irradiation. After an intratumoral injection of Vc (2.5 mg/kg), the green fluorescence obviously decreased, which indicated that Vc can partially inhibit PDT56. Therefore, CONs could


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Figure 5. In vivo anti-tumor experiment. (a) Time-dependent tumor growth curves of HeLa-tumor-bearing mice after various treatments. Data are expressed as means ± s.d. (n = 5). P-values were calculated by t-test: (*P < 0.05, ***P < 0.001). (b) Photograph of tumors excised from mice after 22 d of treatment. (c) Body weight of the mice in different groups. (d) Photograph of mice at the end of treatment. (e) Hematoxylin and eosin (H&E)-stained histological section of tumor.

generate sufficient in vivo ROS in a tumor under irradiation, indicating a potential photodynamic therapy for tumors. To further demonstrate whether in vivo hyperthermia of CONs could be generated, CONs were injected into the mice bearing a HeLa tumor at a dose of 5 mg/kg, followed by an infrared thermograph after 12 h post-injection under the irradiation of a 635 nm laser (1 W/cm2) in the tumor region for 5 min. The PBS group resulted in a slight temperature increase upon irradiation, (Figure 4d and Figure 4e). However, the temperature in the CONs irradiation group rapidly increased above 50 °C within the first 2 min of irradiation and reached near 54 °C after another 3 min of irradiation. Therefore, CONs were able to afford a hyperthermia for tumor photothermal therapy. Reasonably, both efficient ROS generation and hyperthermia of CONs result from their strong absorption, preferable charge carrier separation and transportation, excitons annihilation, and enhanced tumor accumulation. The Pharmacokinetics of CONs and In Vivo Antitumor Therapy. The pharmacokinetics of CONs was evaluated to illuminate their in vivo circulation. According to blood circulation curve (Figure S31), the blood circulation of CONs was followed a two-compartment model and with a distribution half-life (t1/2α) of 0.11 h and a blood-elimination half-life (t1/2β) of 4.23 h. In most cases, the hypoxia tumor microenvironment and quick depletion of oxygen for the PDT would be severely confined therapeutic effects for PDT.57 Therefore, the CONs with a donor-acceptor molecular heterostructure could address hypoxia limit of PDT by type I PDT and the combination of type I PDT with PTT. In vivo antitumor efficacy of CONs was evaluated in the HeLa-tumorbearing mice. When the tumor volumes reached approximately 100 mm3, the mice were randomly divided into six groups: (1) PBS non-irradiation, (2) PBS irradiation, (3) CONs (5 mg/kg) non-irradiation, (4) CONs (5 mg/kg) plus slide plus irradiation, (5) CONs (5 mg/kg) plus Vc plus irradiation, (6) CONs (5 mg/kg) irradiation. The irradiation (635 nm, 1 W/cm2, and

5 min) was conducted at 12 h post-injection (iv) of CONs. Following irradiation, the tumor volumes of the six groups were measured every 2 days using a digital caliper (Figure 5a), and digital photos of the tumor regions were taken every 7 days during the observational period (Figure 5d, Figure S32). As shown in Figure 5a, PBS exhibited a rapid tumor growth with or without irradiation, suggesting that the light exposure nearly had no influence on the tumor growth. Compared to the PBS group, there were similar tumor growth curves when the mice were treated with CONs alone, suggesting that CONs were not able to inhibit the tumor growth because of their negligible dark cytotoxicity. However, the significant result was that total tumor ablation was achieved in the CONs+Laser treated group. Similar results were also obtained in the photographs of tumors (Figure 5b). There were two main factors for the tumor inhibition in the CONs+Laser group. First, efficient type I PDT could be generated by CONs, which could diminish hypoxia limit of PDT and effectively damage the tumors. Second, the local overheating induced by the PTT effect of the CONs could cause apoptosis or necrosis of the tumor cells, there was no need for oxygen participant. To further respectively illuminate the anticancer efficacy of type I PDT and PTT. On the one hand, an ice-cold slide was used to reduce the temperature of the tumor. Compared to CONs+Laser group, the tumor inhibition result of the CONs+Slide+Laser group was distinctly reduced, which were derived from the suppression of PTT. On the other hand, ROSscavenger Vc was injected into the tumors. Distinctly, compared to the CONs+Laser group, the therapeutic effect of the CONs+Vc+Laser group also decreased, indicating that ROS-mediated PDT played a key role in achieving tumor ablation. Therefore, these results potently demonstrated that the CONs could diminish hypoxia limit of PDT by type I PDT and the combination of type I PDT with PTT. To validate light-triggered anticancer efficacy, the tumor damage from CONs was further observed at 8 h postirradiation using hematoxylin & eosin (H&E) staining. PBS


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showed no distinct tumor necrosis with or without irradiation, while CONs with irradiation resulted in severe damage of the tumor cells (Figure 5e). Compared to that of the CONs+Laser group, the tumor necrosis decreased for the CONs+Slide+Laser group, which indicating that the tumor inhibition of CONs was affected by the ice-cold slide suppression of PTT. The morphological change in the tumor cells in the CONs+Vc+Laser group also decreased. Therefore, both type I PDT and PTT contribute to the tumor ablation. The body weight change of the mice was a critical indicator in the toxicity evaluation of CONs. Therefore, the body weights of the mice in each group were measured. They were not obviously affected in all the groups during the observational period (Figure 5c), indicating that the antitumor therapy treatment by CONs was biocompatible. Safety is a critical problem when involving in vivo application of CONs. Therefore, experiments, including histological analysis of major organs and blood hematology, were conducted to ensure the safety of CONs. H&E staining of major organs proved the good bio-safety of CONs (Figure S33). Blood hematology parameters including red blood cells (RBC), white blood cells (WBC), platelets (PLT), hemoglobin (HGB), hematocrit (HCT), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and mean corpuscular volume (MCV) were measured (Figure S34). Compared to the control group, no obvious changes were observed in these important parameters except for the platelet from of the CONs-treated groups. Although the platelet count showed a significant decrease at day 2 d,58 it recovered to a normal range after 22 d, indicating that CONs induced negligible toxicity to the treated mice over the longer term. These results demonstrated that CONs barely caused toxicity in the treated mice at least in our short-term observation of 22 d. CONCLUSIONS In summary, COFs were composed of an HHTP donor and porphyrin acceptor, in which the energy levels of the two subunits were aligned adequately. Each HHTP donor and porphyrin acceptor unit in the COF interfaced, thereby endowing periodically ordered molecular heterojunctions, which were arranged into a 1D π-stacked column. Subsequently, 2D CONs integrated heterojunction could be simply prepared via ultrasonic delamination of bulk COFs. The structure of CONs could afford highly efficient charge carrier separation and transportation. The electrons could reduce the adsorbed O2 to form O2•–, and at the same time, the holes could oxidize the surrounding waters to produce ·OH. Both were high reactive ROS causing cell damage. Therefore, type I PDT could be obtained for CONs, which is a new method to diminish the limits of hypoxia in PDT. The Geminate and Non-Geminate recombination resulted in an important non-radiative attenuation pathway of photoexcited species, and thus their energy was emitted as the heat to achieve the photothermal conversion, a combination of type I PDT and PTT served as a potent approach to address the oxygen-dependent limit of PDT. The size of CONs was good for achieving tumor accumulation by the EPR effect, which could ensure their favorable ROS generation and hyperthermia in the tumors. In vivo antitumor therapy could be performed by intravenous administration of CONs. Upon 635 nm light irradiation, ROS generation and photo-thermal capacity were observed during the in vitro and in vivo experiments. The PA imaging capacity of CONs could guide the antitumor therapy

in vivo. Thus, CONs promoted potently photoactive cell damage. Moreover, because CONs had potential biodegradability, no significant in vivo toxicity was found for CONs after 22 d. Therefore, hypoxia limitations of PDT was for the first time addressed by using type I PDT and the combination of type I PDT with PTT for 2D CONs integrated heterojunction. Encouraged by this work, more research progress in the application of CONs in biomedicine is expected. MATERIALS AND METHODS Materials. Paraformaldehyde, pyrrole, trifluorobonetherate (BF3·Et2O) were purchased from Aladdin (Shanghai, China). 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), 1,3diphenylisobenzofuran (DPBF), 2,3,6,7,10,11hexahydroxytriphenylene (HHTP), 4-formylphenylboronic acid, 1,3-propanediol, InCl3 were obtained from energy chemical (Shanghai, China). 2',7'-dichlorofluorescein-diacetate (DCFH-DA), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2,2,6,6-tetramethylpiperidine (TEMP), 4',6-diamidino-2phenyllindole dihydrochloride (DAPI), Calcein-AM, propidium iodide (PI), Dihydrorhodamine123 and 3’-(4hydroxyphenyl) fluorescein were purchased from SigmaAldrich (Missouri, USA). Cell culture products, including dulbecco's modified eagle's medium (DMEM) and fetal bovine serum (FBS), were purchased from Gibco (Grand Island, NY, USA). Reactive oxygen species (ROS) assay kit was obtained from Beyotime Biotechnology (Shanghai, China). The deionized water was prepared through a Milli-Q water purification equipment (Millipore Co., MA, USA). All of the starting materials were obtained commercially and were used without further purification. Characterization. Fourier transform infared (FT-IR) spectra were recorded on a Bio-Rad Win-IR instrument (BioRad Laboratories Inc., Cambridge, MA, USA) using potassium bromide (KBr) method. Dynamic light scattering (DLS) measurement was performed on a WyattQELS instrument with a vertically polarized He-Ne laser (DAWN EOS, Wyatt Technology Co., Santa Barbara, CA, USA). The scattering angle was fixed at 90o. The zeta potential of CONs was detected by Zeta PALS (Brookhaven instruments corporation, New York, USA). PXRD was performed by a Riguku D/MAX2550 diffractometer using Cu Kα radiation, 40 KV, 200 A with a scanning rate of 0.2o/min. The thermogravimetric analysis (TGA) was performed using a NetzchSta 449c thermal analyzer system at a rate of 10 ℃/min under an air atmosphere. 1H NMR spectra were recorded on an AV-300 NMR spectrometer (Bruker, Karlsruhe, Germany) in deuterated chloroform (CDCl3) or tetrahydrofuran (THF-d8). UV-Vis absorption spectra were monitored with a Shimadzu UV-2450 PC UV/vis spectrophotometer. The fluorescence intensity tests were obtained using a PerkinElmer LS-55 spectrofluorophotometer. The morphology of CONs was measured by transmission electron microscopy (TEM) using a JEOL JEM-1011 electron microscope operating at an acceleration voltage of 100KV. The degradability of CONs were characterized by scanning electron microscopy (SEM) using a Model XL 30 ESEM (Philips). Atomic force microscope (AFM) was performed on an MFP-3D-S AFM (Asylum Research, USA) using the tapping mode in air (NanoSensors SSS-NCH probe with the tip radius as small as 2 nm). Elemental analyses including C, H, N, were measured by VARIO EL-III Elemental Analyzer. The boron content of CONs was determined by inductively coupled plasma mass


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spectroscopy. The nitrogen adsorption isotherm was measured on a Micromeritics ASAP 2010 analyzer. Pore size distribution was estimated by the DFT method. A laser at λ = 635 nm was employed as the light source for experiments. The output power of the laser was controlled by a fiber coupled laser system.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental section, synthesis and characterization of COFs and CONs, size distribution and zeta potential of CONs, stability evaluation of CONs, degradation treatments of CONs, superoxide radical and hydroxyl radical measurements by fluorescence spectra and ESR; photothermal effect and in vitro PA imaging, in vitro cell experiment, H&E stained images and in vivo toxicity evaluation.

AUTHOR INFORMATION Corresponding Author *Email: [email protected]

ORCID Huayu Tian: 0000-0002-2482-3744

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The authors are thankful to the National Natural Science Foundation of China (51873208, 21474104, 51520105004 and 51390484, 51833010), National program for support of Top-notch young professionals, Jilin province science and technology development program (20160204032GX and 20180414027GH) for financial support to this work.

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Covalent Organic Nanosheets Integrated Heterojunction with Two

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