Theranostic Mn-Porphyrin MetalâOrganic Frameworks for Magnetic

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Biological and Medical Applications of Materials and Interfaces

Theranostic Mn-Porphyrin Metal-Organic Frameworks for Magnetic Resonance Imaging-Guided Nitric Oxide and Photothermal Synergistic Therapy Hui Zhang, Xue-Tao Tian, Yue Shang, Yu-Hao Li, and Xue-Bo Yin ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b09680 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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ACS Applied Materials & Interfaces

Theranostic Mn-Porphyrin Metal-Organic Frameworks for Magnetic Resonance ImagingGuided Nitric Oxide and Photothermal Synergistic Therapy Hui Zhang1, Xue-Tao Tian1, Yue Shang2, Yu-Hao Li2, Xue-Bo Yin*,1,3 1

State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of

Biosensing and Molecular Recognition, College of Chemistry, Nankai University, Tianjin, 300071, China 2

Tianjin Key Laboratory of Tumor Microenviroment and Neurovascular Regulation, School of

Medicine, Nankai University, Tianjin, 300071, China 3

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai

University, Tianjin, 300071, China * E-mail: [email protected]; Fax: (+86) 022-23503034 KEYWORDS: metal organic frameworks, Mn-porphyrin, magnetic resonance imaging, nitric oxide delivery, photothermal therapy

ACS Paragon Plus Environment

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Abstract

Chemotherapy remains restricted by its toxic adverse effects and resistance to drugs. The treatment of nitric oxide (NO) combined with imaging-guided physical therapy is a promising alternative for clinical application. Herein, we report nanoscale metal organic frameworks (NMOFs) system to integrate magnetic resonance (MR) imaging, spatiotemporally controllable NO delivery, and photothermal therapy (PTT) as a new means of cancer theranostic. As a proof of concept, the NMOFs are prepared with biocompatible Zr4+ ions and Mn-porphyrin as bridging ligand. By inserting paramagnetic Mn ions into porphyrin rings, Mn-porphyrin renders the NMOFs strong T1-weighted MR contrast capacity and high photothermal conversion for efficient PTT. S-nitrosothiol (SNO) is conjugated to the surfaces of the NMOFs for heat-sensitive NO generation. Moreover, single NIR light triggers the controllable NO release and PTT simultaneously for their efficient synergistic therapy with one-step operation. Upon intravenous injection, NMOF-SNO shows effective tumor accumulation as exposed by the MR images of tumor-bearing mice. When exposed to NIR laser, the tumors of mice injected with NMOF-SNO are completely inhibited, verifying the efficiency of NMOF-SNO. For the first time, Mnporphyrin NMOFs are developed to be an effective theranostic system for MR imaging-guided controllable NO-release and photothermal synergetic therapy under single NIR irradiation.


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1. Introduction Theranostic, which combines diagnostics and therapeutics to improve the management of healthcare, has driven cancer treatment to a new dimension.1-6 Imaging-guiding chemotherapy solves the toxicity and adverse effects of chemotherapeutic agents towards normal organs, but the continuous drug delivery may pose another devastating problem, drug resistance.6-11 The development of alternative strategy toward chemotherapy has been one of the critical challenges to provide effective therapy for cancer. Recently, various phototherapy, as “green” treatments with minimal toxicity to normal organs, have been developed, including photothermal therapy (PTT) and photodynamic therapy (PDT).12-16 The main obstacle of singlet oxygen (1O2)-based PDT is the local hypoxia of tumor, which is recognized as the characteristic hallmarks to lead to PDT resistance.17, 18 As an emerging treatment to tumor cells, free radical gas nitric oxide (NO) has drawn widespread attentions.19-24 The combination of NO therapy with other treatment protocols, such as chemotherapy,24, 25 radiotherapy,26 and PDT,27 has been confirmed to get excellent synergistic effects. Nanoscale metal organic frameworks (NMOFs), as efficient platform to combine imaging with cancer therapy together,5, 9, 16 are easily post-modified. MOFs have currently been used to the delivery of gas NO in terms of their excellent porosity. 28-30 However, NO gas is toxic and unstable, which makes the preparation of MOF-NO system difficult. Additionally, they can’t achieve the controlled and targeted NO release. In order to achieve precisely controlled NO release, real-time imaging monitoring and stimuli-responsive NO donors23,

31, 32

should be

integrated together.33-35 Consequently, the integration of stimuli-responsive NO donors, imaging guidance, and multi-modality therapeutic into NMOFs is expected for their optimal synergistic therapy.


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Porphyrin NMOFs, including DBP-UiO,13 PCN-224,36 or PMOF,9 have already been designed for PDT or fluorescence imaging because of the integration of the optical properties of free porphyrin. However, fluorescence imaging suffers from low penetration depth; PDT is resisted by the hypoxia environment of tumor. Fortunately, the incorporation of Mn into porphyrin rings, such as Mn-porphysome, is very promising to design and prepare magnetic resonance imaging (MRI) and PTT agent by taking the advantage of magnetic resonance response of Mn ions and photothermal conversion of Mn-porphyrin.37 Herein we for the first time demonstrate that the incorporation of Mn ions into porphyrin rings endows the capacity of MR contrast and photothermal conversion of the NMOFs without increasing their complexity. To this end, Mn-porphyrin NMOFs were prepared via selfassembling

of

biocompatible

Zr4+

ions

and

Mn-TCPP

(TCPP=tetrakis(4-

carboxyphenyl)porphyrin). The introduction of Mn ions into TCPP provided the photothermal and MR contrast capacity of the NMOFs, simultaneously.37-39 The NMOFs were then used to conjugate with S-nitrosothiol (SNO), a type of heat-unstable NO donor.40 Therefore, MR imaging-guided NO and photothermal synergistic therapy was realized with the NMOFs platform. Importantly, single NIR irradiation achieves photothermal conversion for PTT and controllable NO release for NO therapy simultaneously with simple operation and less photodamage. After intravenous injection into mice, NMOF-SNO showed efficient tumor accumulation as revealed with MR imaging. The tumors of the mice injected with NMOF-SNO were obviously inhibited upon the NIR irradiation, confirming the efficiency of in vivo NO and photothermal synergistic therapy. Low dose of DOX administration to tumor-bearing mice was designed to simulate the drug-resistance of tumor. Later on, normal dose of DOX and NMOFSNO were used to treat the drug-resistance tumor model. NMOF-SNO showed the complete


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inhibitation of the tumor growth compared to DOX chemotherapy. Therefore, NMOF-SNO composites developed here, as a new kind of biocompatible and effective agent, owned strong MR imaging capacity, efficient photothermal conversion, controllable NO release, and free from drug-resistance. The results may stimulate the development of non-drug therapeutics system as the alternative of chemotherapy. 2. Experimental Section 2.1 Materials. N, N-dimethylformamide (DMF) were obtained from Concord, Tianjin, China. Acetic acid, and zirconium chloride (ZrCl4) were purchased from Aladdin Chemistry Co. Ltd., Shanghai, China. Ammonia solution (28-30 wt % in water), tetraethoxysilane (TEOS, 99.9%), 3mercaptopropyl-trimethoxysilane (MPTES) and t-butyl nitrite were purchased from Meryer Co. Ltd. [5, 10, 15, 20-Tetrakis(4-methoxycarbonylphenyl) porphyrinato]-Mn (III) chloride (MnTCPPCl) was from Chemsoon Co Ltd., Shanghai, China. 3-Amino-4-aminomethyl-2’,7’difluorescein diacetate (DAFDA) was purchased from the Beyotime Institute of Biotechnology, Shanghai, China. Other reagents were obtained from Shanghai Aladdin Chemistry Co. Ltd. All solutions were prepared by using ultrapure water. All the BALB/c mice (18~22 g) used were purchased from the Institute of Hematology & Hospital of Blood Disease, Chinese Academy of Medical Sciences & Peking Union Medical College with the license No. SCXK-2014-0013, Tianjin, China. The mice were confined in a cage. They had open access to water and solid rodent chow, which was obtained from HFK, Beijing, China. The Institutional Animal Care Committee of Nankai University permitted all the experimental procedure. All the methods were consistent with the related provisions from the Institutional Animal Care Committee of Nankai University.


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2.2 Apparatus. Fourier transform infrared spectra (FT−IR) were obtained using a Bruker Tensor 27 Fourier transform infrared spectrometer (German). The transmission electron microscopy (TEM) images of NMOF and NMOF-SNO were obtained on a JEM-2010HR microscope (JEOL, Japan) with an operating voltage of 200 kV. The PTC-10ATG-DTA analyzer was used to represent the thermogravimetric analysis (TGA). Brunauer−Emmett−Teller (BET) surface area and pore size of NMOFs were investigated on a Tristar 3000 surface area and pore size analyzer (Micromeritics, Norcross, GA 30093-2901, U.S.A.). The X-ray diffraction (XRD) patterns were characterized on a D/max-2500 diffractometer (Rigaku, Japan) using Cu−K α radiation (λ=1.5418Å). UV-3900-visible spectrophotometer (HITACHI, Japan) was used to record the UV−vis absorption spectrum. The hydrodynamic sizes (DLS) and zeta potentials of NMOF and NMOF-SNO were tested by a Zetasizer Nano ZS, Malvern, England. The MR images were analysed by using a MRI system (1.2T, Huantong, Shanghai, China). 2.3 Synthesis of NMOFs and NMOF-SNO. ZrCl4(4.16 mg), MnTCPPCl (0.011 mmoL-1) and 0.42 mL acetic acid were dissolved in 16 mL of DMF using a 20 mL Pyrex vial. Then the mixture was heated at 120 °C in air oven for 12 h. After cooled down to room temperature, dark green fusiform shaped NMOFs were obtained and collected by centrifugation (9 mg, 63% yield). Anal. calcd. (%) for NMOFs: C, 56%; H, 2.7%; N, 5.3%. Found: C, 54%; H, 4.3%; N, 4.6%. The above obtained NMOFs was dissolved in 10 mL H2O and 96 µL (0.05 g/mL) of polyvinyl pyrrolidine (PVP) was added with agitation for 2 h at ambient temperature. Then, the mixture was centrifuged with ethanol and dispersed in 18 mL ethanol with 0.54 mL ammonium hydroxide. Subsequently, 34 µL TEOS was added to the mixture dropwise. Finally, we add 50 µL of 3-mercaptopropyl-trimethoxysilane and allow the reaction for 12 h. The resulted thiol-


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functionalized NMOFs was isolated through the centrifugal at 14000 rpm. The isolation product was washed with ethanol and distilled water, and then vacuum dried for further use. The introduction of NO donor on NMOFs was achieved by reacting the –SH groups with tbutyl nitrite. 10 mg of NMOF-SH nanocomposites and excess t-butyl nitrite were dispersed in 10 mL mixed solution of methanol/toluene (v/v=1:9) and agitated for 12 h at 25 °C. The synthetic nanocomposites were centrifuged at 14000 rpm, washed with anhydrous ethanol and ultrapure water for three times. The composites were then dried vacuum for 5 min and stored at -20 °C under dark conditions for further applications. 2.4 The cell internalization, cytotoxicity and photoxicity of NMOF-SNO. To compare the cell internalization of NMOF and NMOF-SNO, MCF-7 cells were cultured with fresh culture medium containing NMOF or NMOF-SNO (200 and 400 µg mL-1 based on NMOF) for 12 h. The cells were gathered, washing twice with cold PBS and counting. Finally, 500 µL PBS was added into the cells and digested with 500 µL aqua regia for inductively coupled plasma-atomic emission spectroscopy (ICP-AES) measurement. Repeat each experiment three times. The cytotoxicity and photoxicity of NMOF-SNO was evaluated with the viability of MCF-7 cells by a standard methyl thiazolyl tetrazolium (MTT) assay. Different methods were treated the cells and 96-well culture plates were used to incubate the cells with a density of 5 ×103 cells per well in culture medium. The cells were then treated with different concentration of NMOFs or NMOF-SNO (0, 50, 100, and 200 µg mL-1 based on NMOFs). The cells with only NIR laser (808 nm, 1.0 W cm-2) and the cells only incubated with NMOF-SNO were selected as the control groups. The PTT group of cells treated with NMOFs was exposed to 808 nm irradiation for 10 min at 1.0 W cm-2 level. The synergetic photothermal and gas therapy group of cells incubated with NMOF-SNO was also under NIR stimulation for 10 min (808 nm, 1.0 W cm-2). Then, the


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plates were maintained at 37 °C in 5% CO2 air incubator for another 24 h. Each well was added MTT solution (20 µL 5.0 mg mL-1, BBS). After 3 h, the residual MTT solution was removed. Then, each well was added 150 µL dimethyl sulfoxide (DMSO, Amresco, America) to dissolve the formazan crystals. The absorbance (OD value) of each well was measured on a microplate reader (Promega, China) at the wavelength of 550 nm. Cell viability was calculated by the following formula: Cell viability %=(ODdrug/ODcontrol) ×100% 2.5 Nitric oxide release in aqueous solution and intracellular nitric oxide staining. The Griess reagent kit was used to record NO release behavior of the nanocomposites, which could test the presence of nitrite ions in the solution effectively. The NO gas converted into nitrite in response to the Griess agent, which formed a pinkish diazo compound. UV-vis absorption spectroscopy was used to record the signal at 540 nm. The commercial NO indicator, DAFDA was used to stain nitric oxide in cells. MCF-7 cells were cultured in 6-well plates with 40000 cells per well. The cells were treated with DAFDA solution (5 µM, 1 mL) for 0.5 h when the cells were adherent. Subsequently, the cells were cultured with NMOFs or NMOF-SNO (400 µL, 1 mg mL-1 based on NMOFs) for 2 h and exposed to NIR laser for 10 min. Inverted fluorescence microscopy was used to analyze the stained cells. 2.6 In vitro and in vivo MR imaging with NMOF-SNO. The NMOFs and NMOF-SNO of MR imaging was tested at various Mn concentrations (0.076, 0.038, 0.019, 0.0096 and 0.0048 mM) with a 1.2 T MR imaging system. T1 value was recorded with the MR imaging system. Mn content of NMOF-SNO was tested with ICP-AES. The r1 value was the slope of the linear fitting equation between 1/T1 and Mn content. The r2 value was obtained as the same way. Images were


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acquired by a 50 mm animal coil and a fat-saturated 3D gradient echo imaging sequence. The MR imaging parameters were setted as shown: spin-echo T1 -weighted MRI sequence, FOV = 100 ×50 mm2, TR/TE = 100.0/8.8 ms, slice thickness = 1 mm, matrix = 512 × 512, 30.0 °C. In vivo MR imaging was carried out on the same nude mice anesthetized with 200 µL of 4 % chloral hydrate. The MR profiles were obtained by fixing the mice on an animal plate in the MR imaging system after intravenous injection of NMOF-SNO solution (400 µL, 1 mg mL-1 based on NMOFs) into the mice. The MR imaging was implemented on a 3.0 T MR imaging system (GE Signa Excite) using a small animal coil, before and after following injection, by a fatsaturated 3D gradient echo imaging sequence: FOV = 110 mm × 110 mm, TR/TE = 9.7/3.0 ms, FA = 13°, inversion time = 5.0 ms, slice thickness = 1 mm, and matrix = 256 × 256 without gap. 2.7 Biodistribution and clearance of NMOF-SNO nanocomposites. In order to confirm the biodistribution of NMOF-SNO, main organs from the nude mice injected with NMOF-SNO (400 µL, 1 mg mL-1 based on NMOFs) were sacrificed. After the organs were collected and weighted, they were solubilized in chloroazotic acid with boiling for 2 h. After each sample was diluted with ultrapure water to 5 mL, ICP-AES was used to test the content of Mn. For investigating the excretion of nanocomposites, mice were caged in metabolic case and their urine and feces were collected after injection with NMOF-SNO (400 µL, 1 mg mL-1 based on NMOFs). Chloroazotic acid was used to digest the urine and feces for the determination of Mn content with ICP-AES. 2.8 NO-PTT dual-therapy of tumor-bearing mice with NMOF-SNO as probe. The efficiency of NO-PTT synergetic therapy of NMOF-SNO was used with tumor-bearing mice (18-22 g, n=3 per group), which was anesthetized with 4% chloral hydrate (6 mL kg-1). The mice were divided into five groups. The negative control were treated with saline, while the mice treated with NMOF-SNO (400 µL, 1 mg mL-1 based on NMOFs) without any irradiation and the


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mice with only NIR light irradiation were considered as two kinds of positive controls. In PTT group, the mice after injection with NMOFs nanocomposites (400 µL, 1 mg mL-1 based on NMOFs) were exposed to NIR laser for 10 min (808 nm, 1.0 W cm-2). The synergetic NO and photothermal therapy group was injected with NMOF-SNO and irradiated under NIR laser (1.0 W cm-2, 10 min). The treatment for all the mice of different groups was repeated every 3 days. The tumor sizes and weights of the mice were measured every 2 day. The tumor volumes of mice were counted as (width2×length)/2 the same as the previous report.13, 16 The major organs of mice in different groups were collected. The biotoxicity was investigated by Hematoxylin and eosin (H&E) stained images. The mice were weighed in a balance within 14 days. The therapy efficiency of NMOF-SNO compared to free DOX was also evaluated with tumorbearing mice (18-22 g, n=3 per group) as models. In control group, the mice was first treated with low dose DOX (100 µL, 0.2 mg mL-1) every other day and then treated with high dose DOX (100 µL, 0.6 mg mL-1). The experiment group was first treated with low dose DOX (100 µL, 0.2 mg mL-1) and then with NMOF-SNO (400 µL, 1 mg mL-1 based on NMOFs) every other day. The tumor sizes and weights of the mice were measured and the biotoxicity was investigated by H&E stained images to evaluate the drug-resistance. 3. Results and Discussion 3.1 Preparation and characterization of NMOF-SNO. Figure 1A illustrated the fabrication of NMOF-SNO nanocomposites. Solvothermal reaction of ZrCl4, Mn-TCPP, and the modulating reagent acetic acid resulted in the formation of spindle-shaped NMOFs, similar to PCN-223.41 The size of NMOFs was 60×140 nm (Figure 1B). A relatively narrow size distribution was observed with the diameter of 224.3 nm from their dynamic light scattering (DLS) (Figure S1). High-resolution transmission electron microscope (TEM) images revealed the internal structure


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of the NMOFs with the triangular channel size of 1.25 nm, which was consistent with the results that calculated from single-crystal data (Figure 1C and 1D).41 Energy-dispersive X-ray (EDX) spectroscopy chemical mapping showed a uniform distribution of Zr and Mn dispersed in the NMOFs (Figure 1E). X-ray photoelectron spectroscopy (XPS) patterns disclosed that Zr, Mn, C, N, and O were successfully integrated in the NMOFs (Figure S2). The structure of the NMOFs was also confirmed by the comparison between powder X-ray diffraction (PXRD) patterns of the NMOFs and their simulated data (Figure S3). Concisely matched peaks in small angle sections validated that the crystal structure was maintained in the NMOFs. The composition of the NMOFs was investigated using inductively coupled plasma-atomic emission spectroscopy (ICPAES) and thermogravimetric-analysis (TGA) (Figure S4). The Zr/Mn ratio in the NMOFs was determined to be 2:1 and the calculated formula was Zr6C144H72N12O24Mn3 (Table S1). Moreover, the NMOFs dispersed well in water to form a transparent solution for their real application (Figure 1F).


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Figure 1. (A) Scheme for the synthesis of NMOF-SNO nanocomposite and the NIR light triggered nitric oxide release and PTT. (B) TEM images and (C) the high-resolution TEM images of NMOFs. The channel size of the NMOFs was highlighted in yellow. (D) View of NMOFs with triangular 1D channels in the structure. (E) EDX mapping of NMOFs on a TEM copper grid. Inset: the image of NMOFs captured with a CCD camera and the images of the signals of Zr and Mn in NMOFs. (F) Dispersity of NMOFs aqueous solution. N2 sorption measurements at 77 K were performed to assess the porosities of the NMOFs (Figure 2A and 2B). The experimental Brunauer-Emmett-Teller (BET) surface area of the NMOFs was calculated to be ~1900 m2/g and its pore volume was ~0.67 cm3/g. The pore with size of 1.26 nm was observed and consistent with the results from experimental result and theoretical prediction (Figure 1C and 1D). The UV-vis absorption spectra of TCPP ligand, MnTCPP ligand, and the NMOFs were recorded (Figure 2C). Mn-TCPP ligand had a strong peak at 466 nm and two peaks for Q band after chelating metal Mn ions,42 different to free porphyrin molecule with one peak at 414 nm for the Soret band and the four characteristic absorption peaks at 500-700 nm for Q band (Figure 2D). The NMOFs had a similar pattern to the Mn-TCPP, with some red shifting at 476 nm for the Soret band and two peaks at 573 nm and 603 nm for the Q band, which indicated that Mn-porphyrin was efficiently integrated into the frameworks. The NMOFs were thiol-functionalized to obtain NMOF-SH via a simple silanization procedure for the integration of NO precursor molecules through the reaction between thiol (-SH) groups and t-butyl nitrite. The morphology of NMOF-SNO was observed from their TEM images (Figure S5). The DLS diameter of the NMOF-SNO was 265.5 nm (Figure S6). The energy dispersive spectrometer (EDS) elemental mapping data clearly displayed that zirconium, manganese, silicon, nitrogen, and sulfur were successfully integrated into NMOF-SNO (Figure


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S7). The conjugation of the –SNO groups on the NMOF-SH was also confirmed with the characteristic peaks of -CH2-S-, –N=O, and–S-N= at 1298, 1511, and 794 cm-1 in FTIR spectra (Figure S8). To illustrate the structure and elemental distribution of the NMOF-SNO, XPS patterns were recorded (Figure S9). The peaks at 224 eV and 160 eV in wide scan XPS spectra undoubtedly disclose the presence of Zr and Mn from the NMOFs, while the peaks of Si2p, S2p, and N1s are also observed. TGA was utilized to study the component of different nanocomposites (Figure S10). Before 300 °C, the weight loss was ascribed to the removal of the solvent molecules. Based on the weight-losing ratio obtained from TGA result, the mass ratio of SNO shell in the nanocomposites was calculated to be 25.4%. All of the evidences illustrated that SNO was successfully conjugated onto the NMOFs.

Figure 2. (A) N2 adsorption isotherms of the NMOFs. (B) The pore size distribution for NMOFs using data measured with N2 at 77 K. (C) UV-vis spectra of TCPP, Mn-TCPP and the NMOFs. (D) The amplification of the UV-vis spectra of TCPP and Mn-TCPP solution at 500-700 nm 3.2 The photothermal property of NMOF-SNO. The Vis-NIR spectra of NMOF-SNO at different concentrations were recorded (Figure S11). Considering the deeper penetration into


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tissue and less damage to surrounding tissues, we selected 808 nm NIR laser to study the PTT property. The in vitro photothermal property of NMOF-SNO was studied by measuring the temperature of the solution under NIR laser irradiation (808 nm, 1 W cm-2, 600 s). The temperature enhanced with the increasement of the nanocomposite concentration, exposure time, and NIR laser power (Figure 3A and 3B). To validate the photo-thermal stability of the NMOFSNO nanocomposites to respond the photo-excitation, five cycles of laser ON/OFF with 808 nm laser were recorded (Figure 3C). The consistent temperature elevation was observed, indicating the high NIR photo-thermal stability of NMOF-SNO composites. The photothermal capacity was recorded with an infrared thermal imaging camera. As shown in Figure 3D, NMOF-SNO solution at the concentration of 400 µg mL-1 was quickly enhanced to 54 °C after light irradiation for 8 min. The elevated temperature was three times higher than that of saline solution, so NMOF-SNO was efficient to change NIR light into heat. The photothermal conversion efficiency of NMOF-SNO was 48.3%, which was calculated precisely (Figure S12). The efficiency was well than those of Bi2S3 (28.1%),43 Au nanorods (22.0%),44 and MoS2 (34.5%),45 possibly due to the high molar extinction coefficient (Figure S13), manifesting the potential of NMOF-SNO as a photothermal agent for NO generation and photothermal therapy.


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Figure 3. (A) The concentration-dependent temperature response curves of NMOF-SNO solution at different concentrations under 808 nm irradiation at 1 W cm-2. (B) The powerdependent temperature change of 400 µg mL-1 NMOF-SNO solution under the irradiation of 808 nm NIR laser. (C) Temperature variation of NMOF-SNO solution with 5 cycles of laser on/off under 808 nm irradiation at 1 W cm-2. (D) In vitro infrared thermal photographs of saline and NMOF-SNO under NIR laser irradiation taken at the specific time. 3.3 Magnetic resonance property of NMOF-SNO. T1-weighted MR imaging was performed to estimate the property of NMOF-SNO for theranostic application. The NMOFs exhibited the r1 value of 26.9 mM-1s-1, higher than that of previous Mn-porphyrin composites (Table S2).46-49 The high dispersibility of Mn in the frameworks attributed to the good T1-weighted imaging capacity of NMOFs core (Figure 4A). The diffusion of water molecules was promoted by the mesopores, improving the ability of Mn ions to approach water molecules for efficient T1-weighted MR imaging.46-49 The relaxation rate of NMOF-SNO had linear change with their increasing concentration. The longitudinal relaxivity value (r1) was 24.6 mM-1s-1, while the r2 value of NMOF-SNO was 12.1 mM-1s-1 (Figure S14). Thus, NMOF-SNO is very suitable for T1-weighted


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MR imaging. The T1-weighted MR efficiency of NMOF-SNO was confirmed by the apparent Mn concentration-reliant brightening property (Inset of Figure 4A). 3.4 The controlled in vitro release of NO. The release of NO, which was triggered by heat in aqueous solution, was tested by a typical Griess assay.50 NMOF-SNO and NMOFs solutions (400 µg mL-1 based on NMOFs) were irradiated under 808 nm laser for 10 min. No NO gas was observed from single NMOFs and only little NO gas was detected from NMOF-SNO without laser irradiation. However, NO was generated from NMOF-SNO quickly and strongly to respond the NIR laser irradiation at a power density of 1 W cm-2 (Figure 4B). Thus, the NO release was belonged to the breakage of S-NO triggered by the high temperature of the NMOF.24 The intracellular production of NO was verified by an NO probe, DAFDA, which reacts with NO to generate intense fluorescence.51 As observed in Figure 4C, the cells cultured with NMOF-SNO showed weak fluorescence compared to the NMOFs group. After irradiation with NIR laser, the cells treated with NMOF-SNO gave out brilliant green color, confirming the generation of plenty of NO.


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Figure 4. (A) The r1 relaxivity curves of NMOFs and NMOF-SNO. Inset: T1-weighted MR images of NMOF-SNO at different Mn concentrations. (B) Accumulated NO generation from the NMOF-SNO over 80 min after irradiation under NIR laser at 1W cm-2 for 10 min, where the NMOFs and NMOF-SNO without excitation were used as the controls. (C) Fluorescent images of MCF-7 cells dyed by DAPI and DAFDA cultured with NMOFs and NMOF-SNO with and without 808 nm laser irradiation at 1W cm-2 for 10 min. Scale bar: 50 µm. 3.5 Cytotoxicity of NMOF-SNO. To estimate the synergic treatment effects of NMOF-SNO, their cytotoxicity was evaluated using MCF-7 cells as a model by MTT assay (Figure 5A and B). More than 90% MCF-7 cells were alive when cultured with 200 µg mL-1 NMOF-SNO or NMOFs (based on NMOFs), which was due to the low toxicity of the nanocomposites. To further demonstrate the cell entry efficiency of NMOF and NMOF-SNO, we investigate the cellular internalization of NMOF and NMOF-SNO. As shown in Figure S15, the intracellular Mn content


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in the cells treated with NMOF was much the same with that of the cells treated with NMOFSNO, which indicates the same cell entry efficiency. By contrast, the cells cultured with single NMOFs then subjected with laser revealed obvious apoptosis to confirm the PTT efficiency. The lower cell viability at high concentration of NMOF-SNO demonstrated the significant effect from the NO and photothermal synergetic therapy. Because NO gas at low concentration has been reported as a signal molecule, we speculate that NO at low concentration does not have cytostatic effect to tumor cells 33 and the release of less NO hardly affects the cell viability. The CLSM images revealed that NMOF-SNO was endocytosed into the cells as observed from red fluorescence stemmed from Cy5-tagged NMOF-SNO after 2 h incubation (Figure 5C). Moreover, the intensity of fluorescence became intensive with the prolonged time of 4 h.

Figure 5. (A) Cell toxicity test of NMOFs and NMOF-SNO with recorded as cell viability. (B) Cell phototoxicity of NMOFs and NMOF-SNO nanoparticles at different concentrations with recorded as cell viability. (C) Cell internalization of NMOF-SNO towards MCF-7 cells for 2 h and 4 h revealed with DAPI and Cy5-labeled NMOF-SNO staining. Scale bar: 50 µm.


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Trypan blue experiment was used to further verify the synergetic treatment efficiency of NMOF-SNO. Dead cells were dyed by trypan blue and differentiated easily from the normal cells. Vast majority of cells were alive for the control group with single laser illumination (Figure S16), so the laser power is sufficiently safe to the cells. Besides, about 95% of the cells were alive observed from the NMOF-SNO group without NIR irradiation. As a comparison, the cell viability in NMOFs group became only 50% under laser irradiation to illustrate the efficiency of single PTT. The viability decreased to approximately 20% for the cells treated with the treatment of NMOF-SNO after NIR light irradiation. The efficient cell killing ability of NMOF-SNO+NIR confirmed the synergistic effect of NO and photothermal therapy. 3.6 In vivo T1-weighted MR imaging and biodistribution of NMOF-SNO. The T1-weighted MR imaging potential was investigated with mice as models after NMOF-SNO solution was intravenously injected. The kidney and liver evidently distinguished thanks to the good spatial resolution of MR imaging. As shown in Figure 6A, the dramatic bright signal was noticed in the liver and kidney part 1 h post injection. The signal of both the liver and kidney region decreased 24 h post injection (Figure 6B). Moreover, the MR imaging of liver region in the axial plane exhibited an obvious signal change. The consistent results were observed from the MCF-7 tumor-burdened mice for the accumulation in liver and kidney, while the tumor was obviously visualized after the intravenous injection of NMOF-SNO (Figure 6C). The strong signal enhancement in the tumor was revealed from the MR images, manifesting good tumor targeting of NMOF-SNO (Figure 6D). Therefore, NMOF-SNO clustered at the tumor site by enhanced permeability and retention (EPR) effect.52


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Figure 6. (A) T1-weighted MR imaging of mice in the coronal plane (upper) and in the axial plane (lower) at different time points. The white arrows referred to the liver and the red arrows referred to the kidney for their signal values of T1-weighted MR images. (B) The T1-weighted MR signal enhancement for the liver and kidney. (C) T1-weighted MR imaging of MCF-7 tumorbearing mice in the coronal plane (upper) and in the axial plane (lower) at different time points. The white arrows referred to the liver, the red arrows referred to the kidney, and the yellow arrows referred to the tumor for their signal values of T1-weighted MR images. (D) The T1weighted MR signal enhancement of the tumor-bearing mice from the liver, kidney, and tumor. (E) Biodistribution of Mn and (F) Excretion profiles of Mn in healthy mice after intravenous injection of NMOF-SNO.

Except for MR imaging, the in vivo behavior of NMOF-SNO was also quantitatively tested by measuring the content of Mn in main organs using ICP-AES. The tumor accumulation of NMOF-SNO was high at 24 h post injection, which was about 4.5% ID/g. The passive tumor


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targeting of NMOF-SNO should be belonged to the EPR effect of tumor tissues (Figure S17). For the sake of further studying the excretion kinetics, normal nude mice after intravenous injection of NMOF-SNO were sacrificed and the Mn content was analyzed by ICP-AES. The accumulation of Mn in liver and spleen was ~8.5 and ~8% ID/g after 24 h post injection and went down very low afterwards. In particular, kidneys brought out high Mn signals exactly as shown in MR imaging, implying that NMOF-SNO could be removed via kidney (Figure 6E). Later, Mn content in urinary and feces was detected by ICP-AES. It is calculated that 18 and 6% ID of Mn were excreted by urinary and feces at the beginning day. High content of Mn was found primarily in the urinary in the next days (Figure 6F). So the NMOF-SNO composites were excreted by renal excretion.15 3.7 In vivo NO and photothermal synergetic therapy. Inspired by its excellent tumor accumulation, NMOF-SNO was used for in vivo MR imaging-guided tumor treatment. The MCF-7 tumor bearing mice were chosen as model. To monitor the photothermal effect generated from NMOF-SNO, infrared camera was used to record the temperature change of the tumor site after intravenous injection and exposure to NIR laser (Figure 7A). In the case of NMOF-SNO injection, the temperature of the tumor site enhanced to 54 °C quickly in 8 min. The excellent temperature increasement were perfect for the ablation of tumor and NO release. The negligible temperature change of the group injected with the saline was observed. The photothermal capacity of NMOF-SNO also confirmed the potential for effective release of NO under 808 nm irradiation.


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Figure 7. (A) IR thermal images of tumor-bearing mice to reveal temperature changes after injected with NMOF-SNO or saline. The images were recorded after the tumor sites exposed to 808 nm laser. The arrows refer to the tumor site. (B) Tumor growth inhibition curve after different treatments. V0 and V refer to tumor volumes before and after each treatment (**p

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