Postsynthetic Ligand Exchange of Metal–Organic Framework for

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Post-Synthetic Ligand Exchange of Metal Organic Framework for Photodynamic Therapy Xueyan Zhao, Zhixiang Zhang, Xuechao Cai, Binbin Ding, Chunqiang Sun, Guofeng Liu, Chunling Hu, Shuai Shao, and Maolin Pang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00740 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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Post-Synthetic Ligand Exchange of Metal Organic Framework for Photodynamic Therapy Xueyan Zhao,†,‡ Zhixiang Zhang,† Xuechao Cai,†,§ Binbin Ding,†,§ Chunqiang Sun,† Guofeng Liu,† Chunling Hu,†,§ Shuai Shao,†,‡ and Maolin Pang*,† †State

Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, PR China. ‡Changchun §University

University of Science and Technology, Changchun 130022, PR China.

of Science and Technology of China, Hefei 230026, PR China.

ABSTRACT: Attributed to the large pore size and excellent stability, the metal organic framework (MOF) NU-1000, which is formed by the coordination of Zr cluster and 1,3,6,8-tetrakis(p-benzoicacid)pyrene (H4TBAPy) ligand, has been widely studied in the catalysis research field, however, only a few reports about the biomedical application of NU-1000 could be found in the open literature. In this study, a functional ligand, tetrakis(4-carboxyphenyl)porphyrin (TCPP) was introduced into NU-1000 via post-synthetic ligand exchange method, and the resultant mixed ligand MOF possesses excellent photodynamic effect. Finally, in vitro and in vivo assessment about the antitumor efficacy was investigated for the first time. It demonstrates the feasibility of TCPP substituted NU-1000 to be used for photodynamic therapy, and also provides an alternative approach to enrich the function of MOF for various applications via post-synthetic method.

KEYWORDS: Metal Organic Frameworks; NU-1000; post-synthetic modification; ligand exchange; photodynamic therapy.

1. INTRODUCTION Recently, photodynamic therapy (PDT) received considerable interests.1-5 Under light irradiation, photosensitizers could generate highly active reactive oxygen species (ROS), which would oxidize neighboring biological macromolecules and produce cytotoxic effects, eventually leading to cell injury and necrosis.6 Besides the widely investigated inorganic materials, porphyrin or similar macrocyclic related derivatives with tetrapyrrole structure was usually selected as photosensitizers.7,8 However, due to the instability and hydrophobic nature of porphyrin, their biomedical applications are largely limited, therefore, an effective approach to encapsulate or introduce porphyrin into the crystal structure and maximum their photodynamic effect to kill cancer cells is greatly needed.9-11 Metal-organic frameworks (MOFs) constructed from organic ligands and metal ions or clusters, refer to a kind of periodical crystalline materials with permanent internal pores.12 Controlling the morphology of MOFs and reducing the size to nanoscale are vitally important.13-22 Owing to the excellent stability and low cytotoxicity, Zrbased MOFs have been widely investigated in the past decades, especially in the biomedical field.23-30 However, another Zr-based MOF named NU-1000,31-37 with much bigger pore size, has been largely overlooked, and a few

reports about the biomedical application could be found in the literature.38,39 NU-1000 is composed of Zr6 cluster and 1,3,6,8-tetrakis(p-benzoicacid)pyrene (H4TBAPy) ligand, which is mainly used for catalysis applications.31-37 NU-1000 also shows great potential in the biomedical application, e.g., the large pore size permits high drug uptake capacity, and the instability in phosphate buffer saline (PBS) solution allows controlled drug release.38,39 Ligand exchange method has been widely investigated in MOF community, and the organic ligands could be substituted with functional groups via solvothermal or other post-synthetic approaches.40 During the ligand exchange process, analogous ligands could be partially or completely introduced into the MOF structure through the competition between the different ligands in solution, and new functions could be achieved by such a postsynthetic modification method.41-45 [5,10,15,20-tetrakis(4carboxyphenyl)porphyrin] (TCPP) is a pyriocyclic compound with four carboxylic acid groups, which is similar to the structure of H4TBAPy. In purpose of improving the stability of NU-1000 in PBS and render it for biomedical application,38,39 postsynthetic modification method was used to stabilize NU-1000 by introducing TCPP ligand in this study. Herein, nanoscale NU-1000 was prepared first, and then a mixed ligand MOF based on

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Scheme 1. Post-synthetic modification of NU-1000 with TCPP, and application for photodynamic therapy.

NU-1000 (abbreviated as NT) was formed by substitution of NU-1000 with TCPP. Subsequently, polyethylene glycol (PEG) and folic acid (FA) were introduced to establish a NT-based targeted therapy nanoplatform. Finally, in vitro and in vivo assessment about the antitumor efficacy was investigated for the first time (Scheme 1). 2. EXPERIMENTAL SECTION 2.1. Synthesis of NU-1000 nanorods. 97 mg of ZrOCl2 and 1.6 g of BA were dissolved in 8 mL of DMF, and 20 mg of H4TBAPy was also dissolved in 8 mL of DMF. The two solutions were mixed and heated to 80 oC for 1 h, and then 40 μL of Trifluoroacetic acid was added, finally, the mixture was heated to 100 oC and held for another 30 min. The products were washed with ethanol for two times. 2.2. Synthesis of NT nanorods. 2 mg of NU-1000 and 3 mg of TCPP were mixed in 3.0 mL of DMF, and the mixture was stirred at 40 oC for 12 h in a water bath. The products were washed with ethanol for three times. 2.3. Synthesis of NT@PEG@FA. In a typical procedure, NT (10 mg) and NH2-PEG-2000-NH2 (4 mg) were mixed in 3 mL of DI water under magnetic stirring. After 6 h, the precipitates were washed with ethanol for several times. And then, 1 mL of FA (3 mg, in DMSO) solution was introduced into 2 mL of NT@PEG (10 mg) aqueous solution under magnetic stirring for 2 h. The precipitates were washed with ethanol for three times. All of the experiments were carried out at room temperature. The other detailed experimental procedures could be found in supporting information. 3. RESULT AND DISCUSSION In this study, nanoscale NU-1000 was synthesized first, and then TCPP ligand in DMF was added to start the ligand exchange reaction. Figure 1, S1 and S2 show the scanning electron microscope (SEM) images of the assynthesized NU-1000 and TCPP substituted NU-1000 (NT). As shown in Figure 1a, 1b and S1, rice-like NU-1000 particles with an average size of 100 nm were produced, and the resultant NT preserved the original size and morphology of NU-1000 after ligand exchange process (Figure 1c, 1d and S2). The ligand exchange process was also conducted in different solvents, such as acetonitrile, and ethanol etc. However, the morphology can’t be maintained and the particles were aggregated seriously in

Figure 1. SEM images of (a, b) NU-1000 and (c,d) NT.

those solvents (Figure S3). Therefore, in order to preserve the morphology and dispersibility of NT, DMF was used as the solvent in this study. The color of the product was changed from yellow to purple after the ligand exchange reaction, suggesting the successful ligand exchange process (Figure S4a). According to the dynamic light scattering (DLS) measurement result (Figure S5a), the mean diameters were about 100 nm for both NU-1000 and NT, which are coincident with the result observed from SEM images. Moreover, after functionalized with PEG and FA, the size of the resultant NT@PEG@FA was increased to about 160 nm (Figure S5a). Furthermore, NU-1000 showed a high positive zeta potential of +34.7 mV in water, and this value decreased to about -17.1 mV after the ligand exchange process. The zeta potential was further decreased to around -20.9 mV after coating with PEG and FA (Figure S5b). The DLS and zeta potentials of NT in DMEM at different times were also measured. No obvious sediment was observed in the bottom of the centrifuge tubes (Figure S6), and the DLS as well as zeta potential results didn’t change significantly within 24 h, indicating the excellent dispersibility of NT in DMEM (Figure S7). The powder X-ray diffraction (PXRD) patterns for the resultant products are shown in Figure 2a. After the ligand exchange process, the diffraction peaks of the assynthesized samples exactly match the standard simulated PXRD patterns for NU-1000. Attributed to the identical symmetry and connectivity of H4TBAPy and TCPP, the two ligands could coordinate with Zr6 cluster to form two isostructural MOFs (csq network topology), i.e., NU-1000,31 and PCN-222,23 respectively. Therefore, after the H4TBAPy ligands were partially replaced with TCPP, the structure of NU-1000 was probably not changed, and maybe only hybrid materials of NU-1000 and PCN-222 were obtained. 1H NMR spectra of NU-1000 and NT are shown in Figure 2b, which was used to determine the ratio of the replacement. It could be found from Figure 2b that around 20% of H4TBAPy ligand was replaced by TCPP, and several new peaks in the range of 8.4-8.8 ppm belonging to porphyrin molecule could be observed. 1H NMR spectra further confirmed the successful substitution of H4TBAPy with TCPP. Moreover,

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Figure 3. UV-vis spectra of DPBF solution in the (a) absence and (b) presence of NT under laser irradiation (12 min, 650 nm, 100 mW cm-2).

Figure 2. (a) PXRD patterns, (b) 1H NMR spectra, (c) UV-vis spectra, and (d) N2 sorption isotherms at 77 K for NU-1000 and NT, respectively.

Figure 2c show the Fourier transformed infrared (FT-IR) spectra of NU-1000, TCPP and NT, respectively. For NU1000, the peaks at 1670 cm-1 and 1200 cm-1 correspond to the C=O and C-C bonds, respectively. The two bonds disappeared in the FT-IR spectrum of NT, and a new peak at 1700 cm-1 belonged to C-N bond appeared, which is quite similar to the FT-IR spectra of TCPP.17-19 Therefore, it is indicated that TCPP was successfully introduced into NU-1000. In addition, we also obtained the UV-vis spectra of NU-1000 and NT. As shown in Figure S8, a broad band centered at around 410 nm could be found for NT, which lies between the two broad bands of NU-1000 and TCPP, implying the successful partially substitution of H4TBAPy with TCPP. Figure 2d shows the N2 sorption isotherms for NU-1000 and NT, respectively. Both NU-1000 and NT exhibit typical type-I isotherms, and the BrunauerEmmett-Teller (BET) surface area for NU-1000 and NT are 1921 m2 g−1 and 1853 m2 g−1, respectively. The pore volume for NT is 0.98 cm3 g−1, and this value is about 1.0 cm3 g−1 for NU-1000.31,46 Since the BET surface area of NT didn’t decrease significantly, it is also proved that the TCPP ligand was successfully introduced into the framework structure of NU-1000, rather than being capsulated in the mesopores of NU-1000. Furthermore, the thermogravimetric analysis (TGA) result implied that the resultant NT was stable up to around 400 oC, and totally decomposed at about 500 oC (Figure S9a). The total weight loss for pure NT, NT@PEG and NT@PEG@FA was around 71.1%, 73.8% and 82.2% (Figure S9b), respectively. Therefore, about 2.7% of PEG and 8.4% of FA were covered on the surface of NT.47-50 In order to further confirm that the TCPP ligand is indeed introduced into the framework structure of NU1000, the following experiments were conducted. First, instead of TCPP, tetraphenylporphyrin (TPP) was introduced to react with NU-1000.51 However, under the identical experimental conditions, yellow powder rather than purple product was obtained after the reaction (Figure S4b), which indicates that although TPP is similar to TCPP, it can’t coordinate with Zr6 cluster. In another

word, TCPP is indeed coordinated with Zr6 cluster to form NT through the indispensable carboxyl group, rather than simply being adsorbed in the pores of NU-1000. Moreover, the as-synthesized NT particles were immersed in 8 M HCl for 12 h under stirring condition, and then washed with DMF and acetone, respectively. It could be found from Figure S4c that the supernatant is still colorless under such a harsh washing condition, implying that no porphyrin molecule escaped from the products. Therefore, all of the above results demonstrated the successful replacement of H4TBAPy with TCPP by such a post-synthetic solution-based ligand exchange process. It should be emphasized that the stability of NU-1000 in PBS was largely increased after the ligand exchange process (Figure S10-S12). As shown in Figure S12, after soaking in PBS for 24 h, the crystallinity of NU-1000 decreased seriously, while NT can still maintain its crystallinity, demonstrating the excellent stability of NT and increased stability of NU-1000 in PBS. In an effort to evaluate the photodynamic effect of NT and determine the capability of generation of singlet oxygen under laser irradiation (650 nm), 1,3-diphenylisobenzofuran (DPBF) was used as an indicator.52,53 In the absence of NT, the absorption band of DPBF at 420 nm didn’t change too much under laser irradiation (Figure 3a). However, as shown in Figure 3b, this band decreased rapidly in the presence of NT (650 nm, 12 min, 100 mW cm-2), implying the generation of singlet oxygen and excellent photodynamic effect of NT. The intracellular ROS generation was detected by DCFH-DA.17 Figure 4 shows the fluorescent photographs of HeLa cells incubated with NT. There were almost no green emissions, indicating no ROS generated in the absence of NT or in PBS solutions (650 nm, 100 mW cm-2). However, strong green emissions were observed for the cells in the presence of NT under laser irradiation, suggesting the laser induced generation of 1O2. It should be emphasized that pure NU-1000 didn’t produce ROS under a 650 nm laser irradiation, and the photodynamic effect only could be observed after the introduction of porphyrin-based TCPP ligand by the ligand exchange reaction. To evaluate the photodynamic effect of TCPP and determine the capability of generation of singlet oxygen under laser irradiation, Indocyanine green (ICG) was used as an indicator. The absorption band of ICG at 780 nm decreased rapidly with increasing times (0-200 s, 650 nm, 100 mW cm-2) in the presence of

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ACS Applied Materials & Interfaces Table 1. Electron transition configurations, excitation energies, and oscillator strengths (f) for main absorption band of porphyrin, and pyrene. H4TBAPy

State S1 S2

TCPP

S1 S2

a

Figure 4. Fluorescence photographs of HeLa cells after incubation with NT under a 650 nm laser irradiation (10 min, 100 mW cm-2).

TCPP (Figure S13), implying the generation of singlet oxygen and excellent photodynamic effect of TCPP. As to the mechanism of producing ROS for NT, a possible explanation was briefly given below. After substituted with functional TCPP, NT contains a large amount of TCPP. Under laser irradiation, TCPP will be pumped from the ground singlet state to the excited triplet state, and then relaxed back to the ground state to generate ROS via various complex photochemical processes.1,7,8 Furthermore, the influence of the substitution of H4TBAPy with TCPP on the photodynamic effect of NT was calculated by the DFT/TD-DFT methods. Both the optimization and absorption spectra simulation were performed using PBE0 as functional and def2-tzvp as basis set under Gaussian09 program package.54,55 Table 1 listed the simulated absorption spectra data, and Figure 5 shows the corresponding molecular orbitals. The optimized structures of H4TBAPy and TCPP are similar. The central pyrene and porphyrin molecules

Energy(nm/eV) 410/3.02 358/3.46

f 0.8673 0.0568

571/2.17

0.0177

536/2.31

0.0397

possess planar structure and a large dihedral angle with peripheral carboxyphenyl can be detected (55.9°and 68.6°, respectively). For H4TBAPy, the maximum absorption band is located at 410 nm, which is attributed to the transition from the highest occupied molecular orbital (HOMO) to the lowest unoccupied molecular orbital (LUMO). The HOMO in H4TBAPy is mainly located on pyrene, which can be assigned to its π bonding orbital, and the LUMO is assigned as its π* anti-bonding orbital with its distribution extends to carboxyphenyl. In a word, the excitation of H4TBAPy is a typical π→π* transition involving a weak charge transfer from central to peripheral, indicating a poor Energy Transfer (EnT) process and a negligible photodynamic effect of NU-1000. However, attributed to the transitions from HOMO1→LUMO and HOMO→LUMO +1, the maximum absorption band shifted to 571 nm for TCPP, which originates from the decrease of energy gap between HOMO and LUMO. Likewise, the occupied and unoccupied ones can also be assigned to π and π* orbital, respectively. However, the difference is that they are all localized on porphyrin. According to the results above, after the replacement of H4TBAPy with TCPP, the energy gap of NT becomes narrower and the absorption peak red shifted, which permits NT nanoparticles to be excited easily with the light with lower energy (650 nm laser in this study), finally an excellent photodynamic effect NU

120

NU+NIR

NT

NT+NIR

100 80 60 40 20 0

Figure 5. Frontier molecular orbitals and energy diagram of pyrene and porphyrin.

Major contrib.a H→L (97.1%) H→L+1 (58.5%) H-1→L (25.6%) H-1→L (62.9%) H→L+1 (36.3%) H→L (61.8%) H-1→L+1 (37.7%)

‘H’ means HOMO and ‘L’ means LUMO.

Cell Viability (%)

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

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0

2.5

5

10 25 50 100 200 500

Concentration (g/mL)

Figure 6. In vitro cell viability data of cultured HeLa cells after incubation with NU-1000, NT, NU-1000 + NIR, and NT + NIR for 24 h.

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Figure 9. Flow cytometry data for apoptosis in the HeLa cells treated with PBS, 650 nm laser, NU, NT, NU + NIR, NT + NIR, respectively. The four quadrants (Q1-Q4) represent necrotic, late-stage apoptotic, early apoptotic, and live, respectively.

Figure 7. Fluorescence images of HeLa cells treated with NT (100 μg mL-1) for different times.

is achieved via an efficient EnT process in NT.51 Since NT can generate ROS upon laser irradiation, the in vitro cell toxicity and phototoxicity were then evaluated by cultivating HeLa cells with MTT assay. In the absence of light irradiation, the cell viability can still maintain over 80% after incubating with NU or NT even with a high concentration of 200 μg mL-1 (Figure 6), implying the low toxicity and good biocompatibility of NU and NT. However, the cell viability of the group incubated with NT decreased significantly under a 650 nm laser irradiation (5 min, 50 mW cm-2), indicating the generation of ROS to kill cancer cells under laser irradiation. Moreover, there were no obvious cell apoptosis or death after laser irradiation in the absence of NT, which further proved that NT could generate ROS to induce cancer cells death under laser irradiation.17-19 Hence, NT is an effective photodynamic agent for PDT treatment.

Figure 8. Fluorescence images of calcein-AM (green, live cells) and propidiumiodide (red, dead cells) cocultured with HeLa cells after the different treatments.

To investigate the cellular uptake process of NT, the cell membrane was stained with DiI and monitored with a fluorescence microscope. As shown in Figure 7, after incubation with NT for different times, bright blue fluorescence was observed, and the intensity increased with increasing times, demonstrating that more and more NT nanoparticles were endocytosed into HeLa cells.17-19 In order to further visualize the photodynamic effect of NT on cancer cells, the fluorescence microscopy was utilized to distinguish the live or dead cells by co-staining the cells with Calcein-AM and propidium iodide (PI), and then the stained cells were treated with PBS, 650 nm laser, NU, NT, NU + NIR, NT + NIR, respectively. As shown in Figure 8, almost all of the HeLa cells were green for the PBS and NIR groups, and a few red cells were observed for the NU, NU+NIR and NT groups, suggesting the poor killing effecton HeLa cells of these treatment methods and the negligible photodynamic effect of NU. However, the cells were all red for the NT+NIR group, which also indicated that NT could effectively kill HeLa cells under a 650 nm laser irradiation.

Figure 10. (a) The body weight, (b) relative tumor volumes, and (c) images of the excised tumors of mice after treatment with (I) PBS, (II) NIR, (III) NU-1000, (IV) NT, (V) NU-1000 + NIR, and (VI) NT + NIR, respectively.

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Figure 11. Hematoxylin and eosin (H&E) stained images of major organs (Heart, Liver, Spleen, Lung, and Kidney) for all groups.

Furthermore, the flow cytometry measurements were performed to study the cell apoptosis process.17 As shown in Figure 9, only a few cells were dead for the NU (9.95%), NU + NIR (4.91%), or NT group (11.3%). However, after irradiated by a 650 nm laser, a remarkably cell apoptosis was observed for NT + NIR group (about 49.3%, 37.2% for apoptosis and 12.1% for necrosis), which demonstrated that NT could induce cell apoptosis efficiently by generating ROS under a 650 nm laser irradiation. Finally, the in vivo anti-tumor efficacy was evaluated.1719 As shown in Figure 10a, the mean body weight was slightly increased for all of the mice, further indicating good biocompatibility and low toxicity of NT in vivo. The best antitumor efficacy was achieved for the NT + NIR group, and the average tumor volume was the smallest (Figure 10b and 10c), demonstrating the excellent in vivo antitumor efficacy of NT under a 650 nm laser irradiation. Moreover, the main organs of mice for each group were dissected for pathological morphological analysis. No obvious lesions were found for all of the mice (Figure 11),

1h 6h 12 h 1 Day 3 Day 7 Day

50 40 30 20

which further confirmed the good in vivo biocompatibility of NT. The biodistribution of NT@PEG@FA in main organs and tumor at different times was also investigated.18,49,50 As shown in Figure 12, the concentration of NT@PEG@FA gradually increased and reached the highest accumulation level in tumor at the time of 12 h after intravenous injection, and then decreased thereafter.18 In order to confirm the targeting effect of FA, the biodistribution of NT@PEG and NT@PEG@FA were studied.18 As shown in Figure S14, a much higher concentration of Zr was observed for the NT@PEG@FA group, and a clear contrast between NT@PEG and NT@PEG@FA group could be found in tumors. Therefore, the injected NT@PEG@FA nanocomposites is targeting at tumors after modification with FA. Additionally, to further prove tumor targeting effect of FA, the in vivo antitumor efficacy of NT@PEG and NT@PEG@FA were also investigated, and the best antitumor efficacy was achieved for the NT@PEG@FA group, demonstrating the good tumor targeting effect of FA (Figure S15). 4. CONCLUSION NU-1000 Nanoparticles with sizes around 100 nm were prepared first, and then the H4TBAPy ligand in NU-1000 was successfully partially replaced with TCPP via the post-synthetic modification method. Under such a ligand exchange process, the crystal structure and morphology of NU-1000 didn’t change obviously. Attributed to the presence of TCPP, the resultant NT nanoparticles could generate reactive oxygen species under a 650 nm laser irradiation efficiently. Because of low toxicity and good photodynamic effect, NT illustrated excellent anti-tumor efficacy in vitro and in vivo for the first time. This is the first report about PDT application of NU-1000 based materials in the biomedical research field, which demonstrates the great potential of treatment of tumors. This work not only expands the application of NU-1000 based materials in the biomedical research field, but also provides an alternative approach to functionalize and permit the existing MOFs for various applications via the post-synthetic modification method.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org. Experimental procedure, SEM images, XRD, DLS, zeta potential, TGA, UV-vis spectra, and images of samples etc.

AUTHOR INFORMATION

10

Tu m or

K id ne y

Lu ng

er Li v

H ea

rt

0

en

Corresponding Author

Sp le

Concentration of Zr (g/g)

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

Figure 12. The in vivo biodistribution of Zr after tail vein injection with NT@PEG@FA nanoparticles at different times.

* E-mail address: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT

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ACS Applied Materials & Interfaces This project was financially supported by the National Natural Science Foundation of China (NSFC 21471145), the Science and Technology Development Planning Project of Jilin Province (20170101179JC), and the ‘‘Hundred Talents Program” of Chinese Academy of Science (Y620021001). The supercomputing center of University of Science and Technology of China (USTC) is greatly acknowledged for the computational support.

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