Renal-clearable ultrasmall covalent organic framework nanodots as photodynamic agents for effective cancer therapy

Journal Pre-proof Renal-clearable ultrasmall covalent organic framework nanodots as photodynamic agents for effective cancer therapy Yan Zhang, Lu Zhang, Zhenzhen Wang, Faming Wang, Lihua Kang, Fangfang Cao, Kai Dong, Jinsong Ren, Xiaogang Qu PII:

S0142-9612(19)30561-7

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119462

Reference:

JBMT 119462

To appear in:

Biomaterials

Received Date: 25 April 2019 Revised Date:

13 August 2019

Accepted Date: 29 August 2019

Please cite this article as: Zhang Y, Zhang L, Wang Z, Wang F, Kang L, Cao F, Dong K, Ren J, Qu X, Renal-clearable ultrasmall covalent organic framework nanodots as photodynamic agents for effective cancer therapy, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119462. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Renal-Clearable Ultrasmall Covalent Organic Framework Nanodots as Photodynamic Agents for Effective Cancer Therapy Yan Zhang,a,c Lu Zhang,a,c Zhenzhen Wang,a,c Faming Wang,a,c Lihua Kang,b* Fangfang Cao,a,c Kai Dong,a Jinsong Rena and Xiaogang Qua,* a. Laboratory of Chemical Biology, State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China b. Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061,

PR China c. University of Chinese Academy of Sciences, Beijing 100039, China

Corresponding Author

E-mail addresses: [email protected]; [email protected], [email protected].

Abstract Covalent organic frameworks (COFs) and their derivatives represent an emerging class of crystalline porous materials with broad potential applications. However, the biomedical applications of them were limited by the large size, low dispersivity, poor bioavailability within cells and metabolic problems. Herein, renal-clearable ultrasmall COF nanodots have been synthesized and utilized as efficient cancer therapy agents. A simple liquid exfoliation strategy was used to prepare COF nanodots. After polyethylene glycol (PEG) conjugation, the PEG coated COF nanodots (COF nanodots-PEG) showed improved physiological stability and biocompatibility. In addition, the well isolated porphyrin molecules endowed COF nanodots-PEG good light-triggered reactive oxygen species production ability, which showed excellent photodynamic therapy efficiency with good tumor accumulation ability. In particular, due to the ultrasmall size, COF nanodots-PEG could be cleared from the body through the renal filtration with no appreciable in vivo toxicity. Our study highlights the potential of COFs-based nanoparticles for biomedical applications. Keywords: Covalent organic framework, nanodots, reactive oxygen species, photodynamic therapy, renal clearance

1. Introduction Covalent organic frameworks (COFs) represent an emerging class of porous crystalline materials, which are built from organic building blocks linked by dynamic covalent bonds that crystallize into highly ordered two-dimensional (2D) layered stacking structures or three-dimensional (3D) polymeric networks [1-3]. The tunable nature and porous characteristic make COFs promising platforms for the applications in sensor, catalysis, gas storage and separation, energy storage, etc. [4-9]. Further research on them has resulted in the discovery of covalent organic nanosheets (CONs) via exfoliation of the COFs [10]. The exfoliated CONs with adjustable atomic structures and periodical channels have emerged as a new member in the family of 2D nanomaterials and received increasing research interest with plenty of applications [11,12]. Very recently, due to their diverse functionality and chemical stability, there has been significant interest in applying COFs and CONs in biological field, for example, enzyme immobilization, photothermal conversion and drug delivery [13-19]. However, their further in vivo biomedical applications were limited by the large size, low dispersivity and poor bioavailability within cells. Compared to 2D nanosheets and their bulk counterparts, zero-dimensional nanodots usually possess new physical and optical properties [20-23]. Of particularly note, the nanoscale size and good biocompatibility of nanodots would make it suitable for biomedical applications. Thus, the construction of COF nanodots as nanoscale therapy agents would expand the applications of COFs in biological field. Cancer nanomedicine, which was based on using nanomaterials to realize tumor

therapy, has received tremendous attention in the past few decades [24]. Because of the outstanding physicochemical properties, various types of nanoparticles, such as metallic/metallic oxide nanoparticles, mesoporous silica and liposomes, have been synthesized and utilized to prepare nanomedicines [25-28]. However, the biocompatibility of nanomaterials is the first consideration to ensure the safety use. Especially, the particle sizes influence their toxicity and clearance characteristics [29-31]. For example, to avoid the potential long-time toxicity, the therapy agents should be cleared from the body within a rational period in clinical applications, which typically require the agents to be smaller than 10 nm for efficient renal clearance. Various nanoparticles with ultrasmall size have been proved to possess renal clearance characteristic, such as gold nanoparticles, black phosphorus quantum dots, porphyrin-PEG polymers, etc. [32-34]. Towards this end, we speculated that COF nanodots with ultrasmall size would be good candidates for cancer nanomedicines. Photodynamic therapy (PDT), as an emerging therapeutic modality, has attracted considerable research interest and plays a key role in current cancer therapy [35-37]. PDT is based on the concept that light-excited photosensitizers (PSs) interact with oxygen to generate reactive oxygen species (ROS), especially singlet oxygen (1O2), for destructing cancer cells. The minimal invasive nature, high efficacy and fast healing process make PDT particularly attractive than other conventional therapies [38,39]. Molecular PSs, such as porphyrin and phthalocyanine, are classic PDT agents with high PDT efficiency and have been widely used for cancer therapy [40].

Especially, the localization of PSs is critical for the efficiency of PDT [41-44]. However, most current existing molecular PSs are free molecules with the characteristics of low water solubility and aggregation tendency, which would cause low PDT efficacy and low selectivity toward target tissues. They often need to be transformed into pharmaceutical formulations or be used for the building blocks to fabricate nanoparticles, which thus can be selectively delivered to tumors based on the enhanced permeation and retention (EPR) effect or recognizing tumor-specific receptors [45-48]. Although promising, the concerns about the toxicity, stability, PDT efficiency and metabolic pathway of these materials restrict their applications [49,50]. Therefore, it is still highly desirable to develop stable PSs-based nanoagents with good biocompatibility as well as high efficiency for PDT. Herein, ultrasmall porphyrin-based COF nanodots have been synthesized and utilized as highly effective PDT agents for cancer therapy by us, which displayed good biocompatibility, efficient tumor accumulation and renal clearance property. A simple liquid exfoliation strategy was developed for the synthesis of highly dispersed ultrasmall porphyrin-based COF nanodots (Scheme 1) and PEG was further modified to improve the physiological stability and biocompatibility of them (COF nanodots-PEG). In addition, the well isolated porphyrin molecules endowed the COF nanodots-PEG good light-triggered ROS production ability, which showed excellent PDT efficiency both in vitro and in vivo. Specifically, due to the ultrasmall size, COF nanodots-PEG could be cleared from the body through the renal filtration without causing long-term toxicity. Our study presents a simplified approach to fabricate

biocompatible ultrasmall COF nanodots with good ROS generation ability, which highlights the clinical application potential of COF-based nanoparticles for cancer therapy. 2. Experiment section 2.1. Materials Dichlorobenzene and butyl alcohol were purchased from Aladdin Reagent. 5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphine

(Tph),

2,5-dihydroxyterephthalaldehyde (Dha), 4,4’,4”-(1,3,5-triazine-2,4,6-triyl)trianiline (TAPT)

and

2,5-dihydroxyterephthalaldehyde

(DHTA)

were

obtained

from

Changchun Third Party Pharmaceutical Technology Co. Ltd. 2’,7’-Dichlorofuorescein diacetate (DCFH-DA), 3-[4,5-dimethylthiazolyl-2-]-2,5-diphenyltetraolium bromide (MTT), propidium iodide (PI), Calcein AM and 1,3-diphenylisobenzofuran (DPBF) were

purchased

from

Sigma-Aldrich.

DSPE-PEG

(Mw=5k),

acetic

acid,

tetrahydrofuran (THF) and all other reagents were of analytical reagent grade and used as received. Ultrapure water (18.2 MΩ; Millpore Co., USA) was used in all experiments and to prepare all buffers. 2.2. Characterization Transmission electron microscope images were recorded using an FEI TECNAI G2 20 high-resolution transmission electron microscope operating at 200 kV. The morphology and composition of the as-prepared samples were tested using a field emission scanning electron microscope (FESEM, S4800, Hitachi) equipped with an energy-dispersive X-ray spectrum. A JASCO V-550

UV/vis spectrometer was used for determining the UV–vis spectroscopy. An Olympus BX-51 optical equipped with a CCD camera was used for capturing fluorescence images. X-ray photoelectron Spectroscopy (XPS) spectra were analyzed by Thermo Fisher Scientific ESCALAB 250Xi Spectrometer Electron Spectroscopy (America). Dynamic light scattering (DLS) measurements were performed using a WyattQELS instrument with a vertically polarized He-Ne laser (DAWN EOS, Wyatt Technology) at 90° collecting optics. ICP-MS measurements were performed on a Thermo Scientific X series

inductively

coupled plasma mass spectrometer. The flow cytometry data was obtained by BD LSRFortessaTM Cell Analyzer. The Laser emission wavelength used in this article were 638 nm±10 nm. The power was 90 mW and the beam size was 0.1 cm×0.9 cm. 2.3. Preparation of bulk porphyrin-based COFs The bulk porphyrin-based COFs were firstly prepared according to the literature [51] by mixing Dha (19.9 mg, 0.12 mmol) and Tph (40.5 mg, 0.06 mmol) in dichlorobenzene/butyl alcohol/6 M acetic acid (5/5/1, v/v/v, 3.3 mL). After sonication for 10 minutes, the mixture was degassed in a Pyrex tube (20 mL) through freeze-pump-thaw cycles for three times and then sealed off. The tube was heated 3 days at 120 °C. After that, the product was collected and washed with anhydrous THF, acetone. The powder was dried under vacuum overnight in 78.7% isolated yield. 2.4 Preparation of ultrasmall COF nanodots and COF nanodots-PEG In brief, COFs (1 mg mL-1) in DMF/water (9/1, v/v) was sonicated in an

ultrasonic bath for 5 h at the power of 300 W. The dispersion was then sonicated with a sonic probe for 5 h at the power of 1200 W, 30%. The sonic probe worked every 3 s with the interval of 4 s. Next, the dispersion was again sonicated with ultrasonic bath for 5 h. After that, the resulting solution was centrifuged at 12,000 rpm for 15 min. Finally, the supernatant containing COF nanodots was collected for future use. Then, the dispersion containing COF nanodots was mixed with DSPE-PEG (1 mg mL-1) under sonication in an ultrasonic bath for 30 min. After that, DMF in the mixture was removed by vacuum rotary evaporation and the resulted sample was dissolved in water and dialyzed against water to remove unreacted DSPE-PEG. In addition, for removing the micelles formed by DSPE-PEG, the sample was further filtered by 100 nm membrane. The amount of DSPE-PEG on COF nanodots was about 50.9 % (w/w) of the COF nanodots-PEG. In addition, as the composition molar ratio of 2,5-dihydroxyterephthalaldehyde

(Dha)

and

5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphine (Tph) in COF nanodots was 2:1, the content of Tph in COF nanodots-PEG could be calculated as:

Ccof was the content of COF nanodots in COF nanodots-PEG, which was 100 %-50.9 %=49.1 %; MrTph was the relative molecular weight of Tph, which was 647.81 g mol-1; MrDha was the relative molecular weight of Dha, which was 166.13 g mol-1; So, the content of Tph in COF nanodots-PEG was calculated as 32.9 %.

2.5 Preparation of another imine-based COF (TAPT-DHTA-COF) and the derived nanodots The TAPT-DHTA-COF were prepared according to the literature [7,8], which was similar to the preparation method of porphyrin-based COFs except TAPT (113.4 mg, 0.32 mmol) and DHTA (79.7 mg, 0.48 mmol) were used. The obtained materials were in 82.7% isolated yield. After that, the nanodots were obtained with the same preparation technique of COF nanodots. 2.6 Preparation of Co-COF nanodots-PEG COF nanodots-PEG (4 mg) was dispersed in sodium acetate buffer (4 mL, pH 5.5), firstly. Subsequently, 20 µL of CoCl2•6H2O (10 mg) was drop wisely added into the above solution. The solution was stirred for 1 h at 37°C. Finally, product was dialyzed against water to remove the unbounded CoCl2. 2.7 The ROS generation ability of COF nanodots-PEG For detecting the generated ROS, DCFH (10 µM) was mixed with COF nanodots-PEG (20 µg mL-1, 1 mL) and irradiated with 638 nm±10 nm light at the power of 90 mW for 3 min. The beam size of laser was 0.1 cm×0.9 cm. After the irradiation, the fluorescence of the solution was measured. For detecting the generated singlet oxygen, 1 mL ethanol solution of DPBF (20 µg mL-1) was mixed with COF nanodots-PEG (20 µg mL-1) or not. After irradiated with 638 nm±10 nm light for a certain time, the samples were taken for UV-vis measurements. For comparing the singlet oxygen generation ability of COF nanodots-PEG,

TPPS4, PPIX and Tph in water, indocyanine green (ICG) was used as the trapping agent. 1 mL ICG solution (9 µg mL-1) was mixed with COF nanodots-PEG (30 µg mL-1, the amount of Tph in COF nanoodots-PEG was abous 15 µM), TPPS4 (15 µM), PPIX (15 µM) or Tph (15 µM). PPIX and Tph were dissolved in 50 µL DMSO firstly. After irradiated with 638 nm±10 nm light for a certain time, the samples were taken for UV-vis measurements. The generation rate of ROS was evaluated by the absorption decrease of ICG at 779 nm. 2.8 Preparation of Cy7 labeled COF nanodots-PEG Firstly, DSPE-PEG-NH2 was used to modify COF nanodots instead of DSPE-PEG. After that, the purified COF nanodots-PEG (1 mg ml-1) was reacted with Sulfo-Cy7 NHS at 0.1 mg ml-1 in pH 7.4 phosphate buffer. The reaction was reacted overnight by avoiding light at room temperature. Excess dye molecules were removed by dialysis against water. The purified nanoparticles were used for NIR fluorescence imaging in mice. Before the experiments, mouse hair was removed. 2.9 In vitro photodynamic therapy After adding COF nanodots-PEG to cells and incubated for 4 h, the plates were irradiated with 638 nm±10 nm laser at a power density of 90 mW for 5 min. After that, cells were incubated for 24 h and then the cell activity was evaluated by MTT assay. For validating the tissue penetration depth of the laser, chicken breast tissues with different thickness (0 mm, 1 mm, 2 mm, 3 mm and 4 mm) were used as the tissue models plated on the top of the cells and the respective cell viability was tested by MTT assay.

To prove the generation of ROS in cells, HeLa cells were seeded in 6-well plate (flow cytometry analysis) or 12 mm sterile cover slips in a 24-well plate (fluorescence microscopy) and DCFH-DA was added. After incubating with certain materials (the concentration of COF nanodots-PEG was 40 µg mL-1), cells were irradiated with 638 nm±10 nm light was 3 minutes. Then, cells were imaged by fluorescence microscopy or analyzed by flow cytometry. Fluorescence microscopy and flow cytometry analysis were performed to show the effect of COF nanodots-PEG on cells directly. After incubated with certain materials (COF nanodots-PEG 40 µg mL-1) and received certain treatments, HeLa cells were stained with Calcein AM and propidium iodide for 15 min. Then cells were washed with PBS and imaged. The apoptosis of the cells was detected by Annexin V-FITC Apoptosis detection Kit. Briefly, after various treatments, HeLa cells were incubation with Annexin V and PI, and then used for flow cytometry analysis. 2.10 Co-Labeling Stability Study Due to the ultrastrong binding force with metal ions, EDTA was used for testing the Co-labeling stability. A solution containing EDTA (1 mM) and Co-COF nanodots-PEG (1mg mL-1) was prepared and incubated at 37°C under stirring. At different time points, portions of the mixture were sampled. Owing to the ultrasmall size, Co-COF nanodots-PEG was hard to be separated by centrifuge. So, the samples were dialyzed against water. The water was collected after dialysis and tested with ICP-MS. 2.11 Animal Experiments

Female Kunming mice weighted about 25 g were obtained from the Laboratory Animal Center of Jilin University (Changchun, China) and handing procedures were according to the guidelines of the Regional Ethics Committee for Animal Experiments. Hepatoma 22 (H22) of 2 × 105 cells were subcutaneously injected into mice to establishing the tumor bearing mice model. 2.12 In vivo biodistribution and pharmacokinetic profile of COF nanodots-PEG Co-COF nanodots-PEG were administered intravenously into healthy mice (n=3). Blood of mice was collected at different periods. The content of Co-COF nanodots-PEG in blood was quantified by the amount of Co2+. For studying the clearance properties, Co-COF nanodots-PEG were administered intravenously in healthy mice (n=3, each time point). The major organs, feces and urine of mice were collected at different periods. The content of Co-COF nanodots-PEG was quantified by the amount of Co2+. H22 tumor bearing mice were injected with Co-COF nanodots-PEG intravenously (n=3, each time point). The organs and tumors were collected at different periods and content of Co-COF nanodots-PEG was quantified by the amount of Co2+. In vivo photodynamic therapy: H22 tumor bearing mice were randomly divided into four groups (n=6, each group): (1) saline alone; (2) saline+light; (3) COF nanodots-PEG and (4) COF nanodots-PEG+light. The solution of COF nanodots-PEG (40 mg kg-1) was injected into mice though i.v.. At 4 h post injection, mice in group 2 and 4 were irradiated by the 638 nm±10 nm laser (90 mW, every 3 min with 1 min

interval for total 15 min). After that, every two days the tumor size and body weight were measured. After 2 weeks, the mice were sacrificed, and the tumors were collected. 2.13 Histology At 24 h post treatment, the tumor tissues (n=3) in each group were harvested and were dissected to make paraffin section. Then, H&E staining and TUNEL staining assay were conducted. The tumor bearing mice were sacrificed at two weeks post treatment. The major organs of mice in COF nanodots-PEG+light group were dissected to make paraffin section for further H&E staining. The one of healthy mice were used for control. After the injection of COF nanodots-PEG, the major organs (heart, liver, spleen, lung, and kidneys) of mice were collected at 30 days post injection for a histology analysis. 2.14 Blood Biochemical Assay After i.v. injection of COF nanodots-PEG, mice (n=6, each time point) were sacrificed at the first, 7th, 30th day post injection. Blood samples were collected from the mice for blood biochemical assay. Six healthy mice were used for control. 2.15 Statistical Analysis All the experiments shown were performed at least three independent times. The data were expressed as means ± standard deviation (SD). The statistical analysis was performed by using Origin 8.0 software. The significance level was set as *P < 0.05 and **P < 0.01, which was performed by Student’s t-test.

3. Results and Discussion The porphyrin-based COFs were synthesized firstly by the Schiff base chemistry between

5,10,15,20-tetrakis(4-aminophenyl)-21H,23H-porphine

(Tph)

and

2,5-dihydroxyterephthalaldehyde (Dha) in approximately 78.7% yield [51]. The successful preparation confirmed by the powder X-ray diffraction (PXRD) pattern, which showed peaks at 2θ=~3.4°, 6.9° and 20-23°, corresponding to the 100, 200 and 001 facets (Fig. S1A), which was consistent with selected area electron diffraction (SAED) pattern of COFs (Fig. S1B). The Fourier transform infrared (FTIR) spectra of COFs and their raw materials were also obtained (Fig. S1C). Comparing with the one of the raw materials, the N-H stretching bands (3350cm-1) and the C=O stretching bands (1660 cm-1) disappeared and the C=N stretching bands (1610 cm-1) existed in the FTIR spectrum of the obtained COFs. In addition, the morphology and composition elements of the obtained COFs was further characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), and the energy dispersive X-ray (EDX) spectroscopy (Fig. S1D-F). After the successful preparation of porphyrin-based COFs, a simple liquid exfoliation technique was employed to prepare COF nanodots. As both water bath sonication and probe sonication are commonly used in obtaining sheet like exfoliated materials and preparing quantum dots [30,52-54], the combination of them with appropriate sonication time and solvent was exploited to obtain COF nanodots. As illustrated in Scheme 1A, the strategy consisted of water bath sonication and ultrasound probe sonication was utilized to exfoliate bulk COFs in 90% N, N-dimethylformamide aqueous solution. The COF

nanodots was obtained with a yield of about 28.6%. Afterwards, to enhance the solubility, 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)] (DSPE-PEG) was employed to decorate the surface of COF nanodots based on the hydrophobic interactions [30,55]. The PEG modified COF nanodots were obtained by rotary evaporator and followed by dialysis with water. The COF nanodots-PEG showed average sizes of 3.46 nm as observed under TEM image (Fig. 1A, B), while the hydrodynamic size was about 9.45 nm as measured by dynamic light scattering (Fig. S2A). In addition, the atomic force microscopy (AFM) was further utilized to characterize the nanodots, which were determined to be around 2.0 nm for COF nanodots (Fig. S2B, C) and 2.9 nm for COF nanodots-PEG (Fig. 1C). The slight increase in thickness and hydrodynamic size were resulted from the PEG coating. Furthermore, the PXRD patterns of COF nanodots and COF nanodots-PEG were obtained. Comparing with COFs, the peak at 2θ=~3.4° of COF nanodots reduced significantly (Fig. S3A). This could be attributed to the loss of π–π stacking through layer-by-layer exfoliation within the COFs layers owing to the ultrasonication. In addition, the broadening of the peak at 2θ=20-23°arising from the (001) planes signified the exfoliation of COF into nanodots with a smaller number of layers [12]. However, the peaks at 6.9°, corresponding to the (200) facet, still existed, which meant that COFs nanodots still showed the crystallinity. For the pattern of COF nanodots-PEG (Fig. S4A), it not only showed the peaks at 6.9° and 20-23° belong to COFs but also the peaks at 19.1°, 21.9°, 23.2° and 26.2° belong to DSPE-PEG [56].

The results further proved the successful preparation of COF nanodots-PEG. In addition, the high-resolution TEM image of COF nanodots (Fig. S3B) and COF nanodots-PEG (Fig. 1A) all showed the lattice around 0.22 nm. The result was consistent with the (002) diffraction planes of COFs, implying that COF nanodots and COF nanodots-PEG kept the similar crystallinity with bulk COFs. The SAED pattern of COF nanodots also proved this (Fig. S3C). The EDX patterns of COF nanodots and COF nanodots-PEG were further obtained (Fig. S3D and S4B). The presence of phosphorus element in COF nanodots-PEG but not COF nanodots proved the successful modification of DSPE-PEG on COF nanodots-PEG. Moreover, the COF nanodots-PEG showed high stability in different physiological solutions (water, phosphate buffered saline and fetal bovine serum), while COF nanodots without PEG modification aggregated in the above solutions after 24 h (Fig. S5). The improved stability also proved the successful PEG modification. To further understand the mechanism for the structural transformation, X-ray photoelectron spectroscopy (XPS) N 1s data of COF nanodots and its bulk COFs were obtained (Fig. 1D and Fig. S6). The spectrum of COFs showed four types of nitrogen in the binding energy range 393.0-410.0 eV: the components at 397.0 eV and 400.0 eV were corresponding to the two electronically inequivalent N species in the core of Tph molecule [57], the component at 398.2 eV represented the imine nitrogen and the component at 401.5 eV corresponded to the nitrogen in a hemiaminal structure [58], which were the intermediate states in the Schiff-base condensation reaction. However, the nitrogen in a hemiaminal structure disappeared and the nitrogen in proton NH3+ (402.5 eV) [59]

appeared in the spectrum of COF nanodots, in which the proton may obtained from phenolic hydroxyl or water. Based on these results, we speculated that most of the relatively unstable hemiaminal structures and part of the imine bonds in COFs fractured into NH3+ in the preparation process. Furthermore, the noncovalent van der Waals force and π-π interaction force between the layers were broken by high-energy jets, which were generated by ultrasonic waves, so the number of COFs layers also decreased. The broken of bonds between and within the layers of COFs induced the formation of COF nanodots. In addition, the FITR spectra of COF nanodots and COF nanodots-PEG were further obtained (Fig. 1E). Comparing with the spectrum of bulk COFs, the one of COF nanodots showed the N-H stretching bands (3350 cm-1), the C=O stretching bands (1660 cm-1) and the C-N stretching bands (1090 cm-1). In addition, the C=N stretching bands (1610 cm-1) still existed in the spectrum of COF nanodots. The results further proved the process of the imine bonds in COFs partly fractured into NH3+. Meanwhile, the existence of C-H stretching bands (2880 cm-1) in the spectrum of COF nanodots-PEG also proved the successful modification of DSPE-PEG on COF nanodots. The UV-Vis spectrum of COF nanodots-PEG was further characterized (Fig. S7). The presence of Soret peak and Q-bands absorption revealed the electron-hole separation and photon absorption ability of COF nanodots. In addition, to demonstrate the generality of our method, the liquid exfoliation technique was also applied to another imine-based 2D COFs. A new imine-based COF,

which

consisted

of

4,4’,4”-(1,3,5-triazine-2,4,6-triyl)trianiline

and

2,5-dihydroxyterephthalaldehyde, with ribbons-like morphology was prepared (Fig.

S8A-C) [7,8]. After liquid exfoliation, the nanodots were formed (Fig. S8D, E), which verified that our approach could be broadly used for the preparation of zero-dimensional COF nanodots from imine-based 2D COFs. Then, the ROS generation ability of COF nanodots-PEG was further examined with light irradiation. Firstly, the production of ROS was evaluated by 2’,7’-dichlorofluorescein (DCFH), which could emit fluorescence after being oxidized by ROS. As shown in Fig. 2A, a large amount of ROS was detected for the light-irradiated COF nanodots-PEG system. Then, to clarify the types of ROS, 1,3-diphenylisobenzofuran (DPBF), which could show a decrease of the absorption intensity at about 410 nm after reacting with 1O2, was employed to prove the generation of

1

O2. The results proved the

1

O2 generation ability of COF

nanodots-PEG under light irradiation (Fig. 2B, C). Moreover, the 1O2 generation ability of COF nanodots-PEG was compared with that of some free photosensitizers (5,10,15,20-Tetrakis(4-sulfonatophenyl)-porphyrin (TPPS4, common hydrophilic photosensitizer), protoporphyrin IX (PPIX, common hydrophobic photosensitizer) and Tph) by using indocyanine green (ICG) as the trapping agent [60]. Although the similar efficiency with TPPS4 was shown, COF nanodots-PEG showed higher 1O2 production efficiency compared with free Tph and PPIX (Fig. S9A, B), which was attributed to the well isolated porphyrin molecules and better solubility after modification. In addition, hydroxyl radicals (·OH) was also proved being produced by COF nanodots-PEG under light irradiation (Fig. S10). All the experiments indicated the good ROS generation ability of COF nanodots-PEG. Furthermore, the

photothermal effect of COF nanodots-PEG was also studied, which indicated that no efficient heating effect existed even under laser irradiation with a high power of 3.0 W cm-2 (Fig. S9C). After that, the fluorescence property of COF nanodots-PEG was further tested, which exhibited a fluorescence peak at ∼665 nm with excitation wavelength of 430 nm (Fig. S9D). However, the fluorescence of COF nanodots-PEG was much weaker than that of Tph, we speculated that the configuration transformation of Dha would exist in COF nanodots and result in enhanced intersystem crossing, which led to the decrease of fluorescence intensity. Moreover, the stacking interaction between layers in COF nanodots further decreased the fluorescence. However, the weakening of fluorescence did not affect the ability of COF nanodots to produce singlet oxygen as evidenced by our experiment results and supported by some references [42,61]. Encouraged by the outstanding ROS generation ability in vitro, we then tested the PDT efficiency of COF nanodots-PEG on cancer cells. Before that, the cytotoxicity of COF nanodots-PEG was measured by MTT cell viability assay on tumor cells (HeLa and MDA-MB-231) and normal cells (RAW 264.7 and L929). No significant cell cytotoxicity of COF nanodots-PEG was observed even the concentration reached 200 µg mL-1 (Fig. 3A). Then, the ROS generation ability of COF nanodots-PEG in cells was studied with 2’,7’-dichlorofluorescein diacetate (DCFH-DA) and detected by fluorescence microscopy. Negligible fluorescence was observed when the cells were in PBS, PBS+light or COF nanodots-PEG group. In contrast, cells treated with COF nanodots-PEG (40 µg mL-1) under light irradiation

for 3 min showed obvious green fluorescence, suggesting that a large amount of ROS was generated (Fig. 3C). The results were also confirmed by flow cytometry (Fig. S11). Then, the PDT efficiency of COF nanodots-PEG on HeLa cells were identified with Calcein AM/prodium iodide cell-survival assay. As shown in Fig. 3D, COF nanodots-PEG (40 µg mL-1) showed significant cytotoxicity in cells under light irradiation, while PBS, light irradiation or COF nanodots-PEG alone showed no obvious cytotoxicity. The result also confirmed by the MTT assay (Fig. 3B). Under light irradiation, COF nanodots-PEG showed concentration related cytotoxicity on HeLa cells. These results indicated the high PDT efficiency of COF nanodots-PEG. Comparing with ultraviolet and visible light, near infrared light showed improved tissue penetration ability. To test the tissue penetration depth of the laser we used, chicken breast tissues with different thickness were used as the tissue models plated on the top of the cells and the respective PDT efficiency of COF nanodots on HeLa cells under laser irradiation were evaluated. As shown in Fig. S12, with the increase of tissue thickness, the cell viability increased. However, ~25% inactivation of cells still existed with 4 mm tissue. The results proved the possibility of our nanodots to be used as agents for PDT. Furthermore, the possible death mechanism was explored with annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) staining assay by flow cytometry experiments. The results in Fig. 3E demonstrated that the cell toxicity was associated with apoptosis and necrosis [62]. All these results confirmed the high efficiency of COF nanodots-PEG as a PDT agent.

Before investigating the tumoricidal efficacy of COF nanodots-PEG in vivo, the biodistribution and pharmacokinetic profile were studied. For benefitting the study, COF nanodots-PEG were labeled with Co (Co-COF nanodots-PEG) due to the strong binding effect of Co with porphyrin core of Tph. After confirming the stability of Co in Co-COF nanodots-PEG (Fig. S13A), the inductively coupled plasma mass spectrometry (ICP-MS) was utilized to measure the levels of Co in blood and different organs. Firstly, time-dependent blood circulation studies of COF nanodots-PEG were carried out on healthy mice with intravenous (i.v.) injection. The blood circulation curve showed that the pharmacokinetic profiles of Co-COF nanodots-PEG fitted by a two-compartment model, which indicated the blood circulation half-lives of distribution and clearance phases was 0.27 h and 4.36 h, respectively (Fig. 4A), indicating that COF nanodots-PEG exhibited a fast blood circulation profile. In addition, the accurate distributions of Co-COF nanodots-PEG in the main organs and excretions (feces and urine) were also measured. It was found that only a small amount of Co was detained in the major organs and the contents were markedly decreased over time due to the stepwise clearance of nanoparticles (Fig. 4B). In addition, most contents of Co were detected in the urine (Fig. 4C), which suggested that the clearance of Co-COF nanodots-PEG was through the renal filtration. Considering the presence of COF nanodots-PEG in liver and the truth that majority of Co was existed in the urine but not feces, we speculated that the clearance of Co-COF nanodots-PEG may occur via two ways: (1) The Co-COF nanodots-PEG in kidney directly passed through glomerular filtration and were excreted out, which

was proved by the TEM image of collected urine (Fig. S13B). (2) For Co-COF nanodots-PEG retained in the liver, the PEG attached by hydrophobic interaction would gradually fall off the COF nanodots-PEG and the resulted COF nanodots could be eliminated from the body through the renal pathway due to the smaller size, similar to some other nanoparticles reported previously [29,63]. After that, the accurate biodistribution of porphyrin-based COF nanodots-PEG on H22 tumor bearing mice with i.v. injection was further studied. As illustrated in Fig. 4D, due to the capture of reticuloendothelial system, a portion of Co-COF nanodots-PEG were accumulated in liver and spleen. However, there was also a part of Co-COF nanodots-PEG accumulated in tumors by EPR effect and reached the peak level at 4 h post injection. In addition, the contents in the organs also decreased over time, indicating the fast metabolism of Co-COF nanodots-PEG. Furthermore, Cy7 modified COF nanodots-PEG were prepared and the biodistribution of COF nanodots-PEG in tumor-bearing mouse was monitored by fluorescence imaging. The accumulation in bladder and tumor was clearly shown in Fig. S13C, D. The results further proved the enrichment of COF nanodots-PEG in tumor site and renal clearable characteristic. Then, the therapy efficiency of COF nanodots-PEG was evaluated on tumor-bearing mice. Once the average tumor volume reached ~100 mm3, the mice with tumor were randomly divided into four groups with six mice in each group: (1) saline

alone;

(2)

saline+light;

(3)

COF

nanodots-PEG

and

(4)

COF

nanodots-PEG+light. The saline or COF nanodots-PEG (40 mg kg-1) was injected into

mice though i.v.. Then the mice in group 2 and 4 were irradiated by laser at 4 h post injection. The tumor size of mice was recorded for 14 days after the irradiation. As presented in Fig. 5A-C, Tumors of mice in saline, saline+light and COF nanodots-PEG groups all grew quickly, suggesting that light irradiation or COF nanodots-PEG alone had no effect on inhibiting tumor growth, while tumors of mice in COF nanodots-PEG+light group showed negligible growth. Furthermore, to gain insight into the cytotoxic effect of COF nanodots-PEG under light, the tumor tissues were collected from the mice 24 h post treatment and processed for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and hematoxylin and eosin (H&E) staining analysis. The results indicated that COF nanodots-PEG caused more serious damages to tumor cells under light irradiation than those control groups (Fig. 5D, E). Furthermore, the intratumor ROS generation ability of COF nanodots-PEG was proved by using DCFH-DA as the ROS probe (Fig. S14). All the experiment results proved the good PDT efficiency of COF nanodots-PEG with light irradiation. In addition, after the PDT with COF nanodots-PEG under light irradiation, no notable side effects on mice emerged, which was illustrated by body weight analysis and histology analysis of major organs collected after treatments (Fig. S13E, F). To ensure the safety for further bioapplications, in vivo long-term toxicology of COF nanodots-PEG on healthy mice was investigated. Liver function markers (ALB, ALP, ALT, AST, and TP), kidney function markers (BUN and CREA), as well as blood biochemistry (WBC, RBC, HGB, HCT, MCV, MCH, MCHC, and PLT) were

tests after i.v. injection of COF nanodots-PEG at different time points. As shown in Fig. S15A, all the parameters were within their normal ranges following 1 day, 7 days and 30 days, relative to healthy mice. Besides, negligible influence toward the mice body weight was noted after the injection of COF nanodots-PEG (Fig. S15B). In addition, the major organs (heart, liver, spleen, lung, and kidneys) of the COF nanodots-PEG treated mice were collected at 30 days after injection for a histology analysis. The H&E results indicated that no significant damage was shown in COF nanodots-PEG group (Fig. S15C). All these results indicated that COF nanodots-PEG could be used as safe PDT agents, which was partly due to the fast renal clearance characteristic of COF nanodots-PEG. 4. Conclusion In summary, we succeed in fabricating the ultrasmall COF nanodots with excellent PDT efficiency and renal-clearable characteristic. The ultrasmall COF nanodots were prepared by a simple liquid exfoliation assay and the fabricated COF nanodots-PEG showed good physiological stability and biocompatibility. In addition, the well isolated porphyrin molecules endowed COF nanodots-PEG good light-triggered ROS production ability, which showed excellent PDT efficiency in treating cancer cells and inhibiting tumor growth as a result of efficient tumor accumulation. Notably, owing to their ultrasmall size, COF nanodots-PEG could be excreted via the renal system from treated mice after i.v. injection and showed no obvious long-term side effects. With the high stability, superior biocompatibility, efficient tumor accumulation, excellent PDT efficiency and effective metabolism, COF nanodots-PEG were promising

candidates for clinical cancer therapy. Furthermore, due to the characteristics of high affinity with metal ions and easy to be modified, COF nanodots-PEG would bring more opportunities for biological and medical applications with attaching additional imaging reagents and therapy molecules. We expect that this study will open up an exciting research direction for COFs-based materials in biomedical field. Acknowledgements Financial support was provided by the National Natural Science Foundation of China (Grants21431007, 21533008, 21871249, 81502277, 91856205 and 21820102009), Key Research Pro-gram of Frontier Sciences of CAS (QYZDY-SSW-SLH052).

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Scheme 1. Schematic illustration showing COF nanodots-PEG as photodynamic agents for cancer therapy. (A) The fabrication process of COF nanodots-PEG. (B) The utilization of COF nanodots-PEG as photodynamic agents for effective cancer therapy with renal clearance behaviour.

Fig. 1. Characterization of prepared COF nanodots and COF nanodots-PEG. (A) A TEM image of as-synthesized COF nanodots-PEG. Scale bar: 20 nm. Inserted: Representative image of individual COF nanodots-PEG. (B) Diameter distribution measured from TEM image of prepared COF nanodots-PEG. (C) An AFM image of COF nanodots-PEG and height profiles along the corresponding lines. Scale bar: 500 nm. (D) The XPS N 1s analysis of COF nanodots. (E) The FTIR spectra of bulk COFs, COF nanodots, COF nanodots-PEG and DSPE-PEG.

Fig. 2. ROS generation ability of COF nanodots-PEG. (A) ROS generation evaluated by the measurement of DCFH fluorescence. Time dependent absorption spectra of DPBF after incubating with (B) or without (C) COF nanodots-PEG under light irradiation.

Fig. 3. In vitro photodynamic therapy effect of COF nanodots-PEG. (A) Cell viability after incubation with COF nanodots-PEG at different concentrations. (B) Cell viability of HeLa cells treated with COF nanodots-PEG under irradiation of 638 nm±10 nm laser lamp for 5 min at different concentrations. (C) Fluorescence microscopy analysis of ROS generation with DCFH-DA in HeLa cells after different treatments. Scale bar, 10 µm. (D) Fluorescence images of Calcein AM- and PI-costained cells with different treatments. Scale bar, 20 µm. (E) Flow cytometry analysis of apoptosis cells following treatment with different formulations.

Fig. 4. In vivo pharmacokinetic and biodistribution profile of COF nanodots-PEG. (A) Blood circulation of Co-COF nanodots-PEG in healthy mice after i.v. injection measured by ICP-MS. Time-dependent biodistribution (B) and the excretion profiles (C) of Co-COF nanodots-PEG after i.v. injection in healthy mice. (D) The biodistribution of Co-COF nanodots-PEG in H22 tumor bearing mice at various time points after i.v. injection based on ICP-MS analysis.

Fig. 5. In vivo photodynamic therapy effect of COF nanodots-PEG. (A) Representative photos of the excised tumors on day 14 after the various treatments. (B) Change of tumor volumes after various treatments indicated. **P < 0.01. (C) Photographs of the H22 tumor-bearing mice before treatment and on day 14 after the various treatments. The representative micrographs of TUNEL (green) and nucleus (blue) co-stained (D) and H&E stained (E) tumor slices 24 h after the corresponding treatment. Scale bar, 50 µm. 1. saline; 2. saline+light; 3. COF nanodots-PEG; 4. COF nanodots-PEG+light.