Disassembly of Hydrophobic Photosensitizer by Bio-degradable

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Biological and Medical Applications of Materials and Interfaces

Disassembly of Hydrophobic Photosensitizer by Bio-degradable Zeolitic Imidazolate Framework-8 for Photodynamic Cancer Therapy Dandan Xu, Yongqiang You, Fanyu Zeng, Yong Wang, Chunyan Liang, Huanhuan Feng, and Xing Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b03831 • Publication Date (Web): 20 Apr 2018 Downloaded from http://pubs.acs.org on April 20, 2018

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ACS Applied Materials & Interfaces

Disassembly of Hydrophobic Photosensitizer by Bio-degradable Zeolitic Imidazolate Framework-8 for Photodynamic Cancer Therapy Dandan Xu,†,#,‡ Yongqiang You,†,#,‡ Fanyu Zeng,†,# Yong Wang, †,# Chunyan Liang, †,# Huanhuan Feng*,†,# and Xing Ma*,†,#,§ †

State Key Laboratory of Advanced Welding and Joining, Harbin Institute of Technology

(Shenzhen), Shenzhen 518055, China #

Research Centre of Printed Flexible Electronics, School of Materials Science and Engineering,

Harbin Institute of Technology (Shenzhen), Shenzhen 518055, China §

Key Laboratory of Micro-systems and Micro-structures Manufacturing of Ministry of

Education, Harbin Institute of Technology, Harbin 150001, China KEYWORDS: Metal Organic Framework, Photodynamic Therapy, Zeolitic Imidazolate Framework-8, Zinc(II) Phthalocyanine, Hydrophobic Photosensitizer

ABSTRACT: Photodynamic therapy (PDT), an alternative to conventional cancer therapeutics, has gained increasing attentions for its non-invasive advantage and simultaneous fluorescence imaging property. PDT is a tripartite process that functions in 1 Environment ACS Paragon Plus

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the simultaneous presence of a photosensitizer (PS), light and available oxygen molecules. However, many highly efficient PSs are hydrophobic and highly tend to selfaggregate in aqueous solution, leading to quick quenching of PDT effect. Here we construct

zeolitic

imidazolate

framework-8

(ZIF-8)

containing

water

insoluble

photosensitizer zinc(II) phthalocyanine (ZnPc), a typical hydrophobic PS, by one-step coprecipitation process, named as ZnPc@ZIF-8. The micropores of ZIF-8 act as molecular cages to separate and maintain hydrophobic ZnPc in monomeric state and protect it against self-aggregation, which enables the encapsulated ZnPc to generate cytotoxic singlet oxygen (1O2) under light irradiation (650 nm) in aqueous condition. The formed nano-system of ZnPc@ZIF-8 can be endocytosed by cancer cells and exhibits red fluorescent emission with excellent photodynamic activity for cancer treatment in vitro. Besides, ZnPc@ZIF-8 are acid sensitive and would completely degrade after PDT, which can be monitored by the self-quenching of fluorescence emission of ZnPc. This work paves a facile way for resolving the problem of solubility and bioavailability of hydrophobic PS by utilizing metal–organic frameworks as nano-carriers.

INTRODUCTION Photodynamic therapy (PDT) with non-invasive clinical effectiveness and intrinsic fluorescence imaging nature has been a promising approach in cancer treatment since the early 21st century.1-3 Compared with conventional therapeutics, such as radio-therapy, surgery and chemotherapy, PDT has the advantages of repeatable administration, controllable light dose, quick curative effect, as well as site-specific treatment by localized irradiation.4-6 PDT functions under the condition that the photosensitizer (PS) excited by light irradiation transfers energy to molecular oxygen (O 2) and generate

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cytotoxic singlet oxygen (1O2) to cause tumor cell apoptosis and/or necrosis. 7-8 However, the recently developed highly efficient PSs are generally hydrophobic, leading to selfaggregation in aqueous solution, which not only quenches their PDT effect but also causes ineffective delivery of PS molecules to tumor site. In addition, the aggregation of PS molecules greatly reduces the opportunity of contact between PS molecules and O 2, which is also detrimental to PDT. 9-10 Nanoscale metal organic framework (NMOF) as an emerging nanomaterial has been extensively exploited in a variety of applications due to their merits of high porosity, large specific surface area, and controllable composition.11-16 In particular, recent researches have utilized NMOFs as effective nanoparticle-based delivery platforms for biomedical applications.17-27 Until now, a few examples show robust and crystallized NMOFs with well-defined pore structures can serve as nano-carriers to incorporate various guests of small molecules.28-32 Researchers have used NMOF as a nanoscale scaffold for the disassembly of hydrophobic PS to maintain their monomeric state in aqueous solution, thus realizing effective PS delivery for enhanced PDT efficacy.22 For example, porphyrinbased MOFs were prepared by using porphyrin derivatives as bridging ligands to coordinate with metal ions in periodic arrays, which greatly reduced the self-quenching of the porphyrin and remained their PDT efficacy.23, 27, 33-34 Although directly incorporating PS molecules into the MOF scaffold can realize well-ordered disassembly of hydrophobic PS molecules for efficient PDT, the design and synthesis of the PS-based MOFs are usually quite complicated and only feasible for several specific PS molecules. Thus, there is a huge demand for a facile and universal strategy to solve the solubility and bioavailability of hydrophobic PSs.



Herein, we incorporated zinc(II) phthalocyanine (ZnPc, a typical hydrophobic PS) molecules into zeolitic organic framework (ZIF-8), named ZnPc@ZIF-8, by a facile and universal one-step coprecipitation strategy. As shown in Figure 1a, the process of encapsulation of ZnPc molecules took place in the formation process of the micropores of ZIF-8 in methanol solution. The synthesized ZnPc@ZIF-8 is excepted to mechanically block the encapsulated ZnPc molecules from free diffusion, thus physically realizing the separation of ZnPc molecules. Moreover, O2 molecules can easily diffuse into the micropores of ZIF-8, which facilitates the contact between PS molecules and O2 molecules to ensure high photodynamic efficacy. After nano-sized ZnPc@ZIF-8 was endocytosed by tumor cells, cytotoxic

1

O2 was efficiently generated under light

irradiation (650 nm), exhibiting excellent photodynamic activity for cancer treatment in vitro, as shown in Figure 1c.


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Figure 1. Schematic illustrating of the one-step fabrication of ZnPc@ZIF-8 (a), TEM image of ZnPc@ZIF-8 (b) and the PDT process of ZnPc@ZIF-8 for HepG-2 cells in vitro (c). RESULTS AND DISCUSSIONS The prepared nanoparticles of ZIF-8 and ZnPc@ZIF-8 were characterized by both SEM and TEM as shown in Figure S2 in the SI. The morphology of the MOF-based nanoparticle was further verified by high resolution TEM image (Figure 1b). No obvious difference between ZIF-8 and ZnPc@ZIF-8 was observed in terms of morphology, and no apparent aggregation or crystal of ZnPc was observed by microscopy, which suggests that ZnPc molecules are encapsulated into the micropores inside ZIF-8. We further characterized the as-prepared ZnPc@ZIF-8 by powder X-ray diffraction (XRD). The powder of pure ZnPc and ZIF-8 were measured as comparison. As Figure 2a shows, the peaks of ZnPc@ZIF-8 and ZIF-8 are in good agreement. We found characteristic peaks of MOF structure of ZIF-8 at 2θ=7.28, 12.64 and 17.92 degree, which can be attributed to coherent diffraction from (011), (112) and (222) planes.35 In addition, the corresponding diffraction peaks of ZnPc@ZIF-8 exhibit slight deviation to lower values at 2θ=7.22, 12.60 and 17.88 degree compared to those of ZIF-8, indicating an increase in the crystal space according to the Bragg’s Law. The increase of interplane distance could be explained by the encapsulation of ZnPc guest molecules inside the micropores of ZIF-8. However, we do not find any obvious peaks from crystal of pure ZnPc, which further confirms that ZnPc molecules were dispersed inside the micropores of ZIF-8, rather than self-aggregated induced crystallization. Fourier transform infrared (FTIR) spectra of the



as-prepared products show the representative signal of ZIF-8, also evidence the formation of ZIF-8 (Figure S3 in the SI).30

Figure 2. XRD patterns of ZnPc@ZIF-8, ZIF-8 and ZnPc (a), UV-vis spectra (b) and corresponding picture (inset) of ZnPc dissolved in DMF (①) and ZnPc (②), ZIF-8 (③) and ZnPc@ZIF-8 (④) suspended in DI water, luminescence spectra of ZIF-8, ZnPc and ZnPc@ZIF-8 dissolved in DI water (c) with the inset of fluorescence image of ZnPc@ZIF-8 in DI water under red light irradiation (Ex=610±20 nm, Em=680±20 nm) and variation of the luminescence intensity of ZnPc@ZIF-8 with different addition of ZnPc (d).


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Subsequently, we investigated the optical properties of ZnPc@ZIF-8. As shown in Figure 2b, ZIF-8 suspended in deionized (DI) water does not show any absorbance peaks between 550 nm and 750 nm. Since the absorbance spectrum of ZnPc (DMF) shows two characteristic peaks at 605 nm and 670 nm (dash line in Figure 2b), indicating the monomeric state of ZnPc in DMF. Hydrophobic ZnPc molecules easily aggregate in aqueous solution, leading to the disappearance of the absorbance peaks of ZnPc (DI). However, the absorbance spectrum of ZnPc@ZIF-8 suspended in DI water displays two strong peaks at 605 nm and 670 nm, which are consistent with those of ZnPc (DMF). It is proved that the micropores of ZIF-8 successfully separate and keep ZnPc molecules in monomeric state, and protect it against aggregating in aqueous solution.36 What’s more, it is worth mentioning that ZnPc@ZIF-8 has excellent stability, whose nano-structure was not destroyed in aqueous condition at neutral pH, and the fluorescent intensity was well maintained after more than one-month storage (Figure S4 in the SI). The inset picture of Figure 2b shows that ZnPc aggregates in aqueous solution, while ZnPc@ZIF-8 is well dispersed in DI water and exhibits blue color. In addition, the luminescence spectrum of ZnPc@ZIF-8 in aqueous solution presents a strong peak at 680 nm which is not found in those of ZIF-8 and ZnPc solution (Figure 2c), further confirming the monomeric state of ZnPc molecules inside the micropores of ZIF-8. The luminescence property of ZnPc@ZIF-8 nanoparticles was validated by fluorescence microscopy image with red fluorescence emission, as shown in the inset picture of Figure 2c. Notably, the aqueous suspension of ZnPc would not exhibit any emitted light observed by fluorescent microscopy. To prove the universality of our strategy, we chose another typical hydrophobic PS, Coumarin 6, and prepared Coumarin 6@ZIF-8 by the same strategy.



While the luminescence peak of Coumarin 6 in aqueous solution completely quenches due to the self-aggregation of Coumarin 6 molecules, the luminescence spectrum of Coumarin 6@ZIF-8 suspended in aqueous solution shows a strong peak at around 506 nm (Figure S5 in the SI). It is also proved that Coumarin 6 molecules was successfully encapsulated inside ZIF-8 as monomeric state. We optimized the addition of ZnPc during the preparation process. With the increasing addition of ZnPc, the luminescence intensity of ZnPc@ZIF-8 keeps increasing at the beginning, suggesting that ZnPc molecules are gradually filling the micropores of ZIF-8. Then, the luminescence intensity reaches the peak value when 0.25 mg ZnPc was added, which is regarded as the optimal addition of ZnPc (Figure 3d). After that, ZnPc molecules monodispersed inside the micropores of ZIF-8 would aggregate due to excessive presence of ZnPc molecules, leading to a decrease in the luminescence intensity of ZnPc@ZIF-8. The typical luminescence spectra of ZnPc@ZIF-8 with different addition of ZnPc amount are shown in Figure S6 in the SI. In order to quantify the loaded ZnPc inside ZIF-8, we suspended the optimized ZnPc@ZIF-8 sample in DMF and extracted all the loaded ZnPc by vigorous sonication. By comparing the UV-vis spectrum of the extracted ZnPc (DMF) with the standard curve of ZnPc (DMF), the optimal mass ratio of ZnPc to ZnPc@ZIF-8 is found to be 5.9 μg ZnPc/mg ZnPc@ZIF-8. Detailed quantification results can be found in Figure S7 in the SI. Next, the capability of photo-induced 1O2 generation of ZnPc@ZIF-8 was detected using 9,10-anthracenediyl-bis-(methylene)dimalonic acid (ABDA) as the 1O2 trapped agent. ABDA reacts with 1O2 to produce corresponding endoperoxide, which induces the quenching of absorbance peaks at 350−415 nm to monitor the generation of 1O2. We


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suspended ZnPc@ZIF-8 in DI water saturated with ABDA, and the solution was irradiated by red light with the power density of about 200 mW·cm-2. UV-vis spectra of ABDA with ZnPc@ZIF-8 and ABDA only in dark have been provided as control in Figure S8 in the SI. The absorbance of only ABDA in dark remains unchanged. However, the absorbance of ABDA with ZnPc@ZIF-8 decreases continuously under 650 nm light irradiation within 50 min, and that in dark declines slightly (Figure 3a). The photobleaching of ABDA clearly reveals the 1O2 generation capability of ZnPc inside ZIF-8.

Figure 3. UV−vis spectra of ABDA with ZnPc@ZIF-8 under irradiation (650 nm) and the inset showing the variation trend of the peak values at 380 nm of ABDA with ZnPc@ZIF-8 under irradiation and without irradiation within 50 min (a), cell viability of HepG-2 cancer cells after incubated with ZIF-8 and ZnPc@ZIF-8 for 24 h in dark (b), photodynamic cytotoxicity of ZnPc@ZIF-8 under irradiation (650 nm) for 0 min, 10 min and 30 min, respectively and further incubated for 24 h (c), and the bright field image, the fluorescent images of ZnPc emission (red) and DAPI emission (blue) respectively, and



the merged image of HepG-2 cells treated with ZnPc@ZIF-8 after PDT. The cell nucleus was stained with DAPI (d). Scale bar = 50 μm. Meanwhile, the PDT efficacy of ZnPc@ZIF-8 was demonstrated in HepG-2 cells in vitro. The experimental details about the PDT experiment in vitro is given in the SI. Obviously, control groups, cells incubated with ZnPc@ZIF-8 and ZIF-8 at a series of concentrations up to 50 µg·mL-1 for 24 h in dark, show that the ZIF-8 based nano-carriers have no cytotoxicity to HepG-2 cells (Figure 3b). The cell viability of ZnPc@ZIF-8 is slightly lower than that of ZIF-8, probably because of the generated photodynamic cytotoxicity of ZnPc@ZIF-8 in the process of MTT analysis under visible light. Figure 3c shows notable photodynamic cytotoxicity of ZnPc@ZIF-8 under 650 nm light irradiation with the power density of about 3.3 mW·cm-2 on the surface of the cells, and the photodynamic cytotoxicity demonstrates increasing dependence on the concentration of ZnPc@ZIF-8. When the irradiation time is increased from 10 min to 30 min, the cell viability does not significantly decrease. It is probably due to the limitation of available oxygen molecules surrounding the ZnPc@ZIF-8 nanoparticles. On the one hand, the result proves the highly efficient 1O2 generation capability of ZnPc@ZIF-8. On the other hand, it suggests that future endeavors should be contributed to acquire more oxygen molecules in order to make full use of the photodynamic activity of ZnPc@ZIF-8. The biocompatibility of ZnPc@ZIF-8 was examined by MTT assay using MC3T3-E1 cell line. As shown in Figure S9a, the cell viability of MC3T3-E1 cells treated by ZnPc@ZIF-8 at the concentration of 50 µg·mL-1 maintained up to 80%, indicating that ZnPc@ZIF-8 has little cytotoxicity to normal cells and is safe for biomedical applications within proper concentration range. Moreover, the photodynamic cytotoxicity induced only by ZIF-8 has been


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performed as control. ZIF-8 only did not show photodynamic cytotoxicity (Figure S9b in the SI) under the same condition with cells treatment with ZnPc@ZIF-8, which further proves that the photodynamic cytotoxicity of ZnPc@ZIF-8 is only induced by ZnPc disassembled within ZIF-8. In addition, fluorescence images of HepG-2 cells treated with ZnPc@ZIF-8 incubated for 24 h after PDT are shown in Figure 3d. The cell nucleus was labelled by 4',6-diamidino-2-phenylindole (DAPI) with blue fluorescence emission. The localization and accumulation of ZnPc@ZIF-8 within cytoplasm are visualized by red fluorescence color. The morphologies of HepG-2 cells significantly change with apparent shrinkage after PDT, which demonstrates that ZnPc@ZIF-8 could bring serious photodynamic damage to tumor cells and eventually lead to apoptosis and/or necrosis of tumor cells.36 The biodegradability of nano-carriers has been a bottleneck hindering the biomedical applications of nanoparticles-based drug delivery systems (DDS).37 Due to the sensitivity of ZIF-8 towards acidic pH, the ZnPc@ZIF-8 based nano-carriers here would degrade under intracellular acidic condition, which could also result in the releasing and selfquenching of ZnPc molecules in tumor sites.38 Taking advantage of the dynamics of the fluorescence emission from ZnPc@ZIF-8 nanoparticles, we can monitor the degradation process of ZnPc@ZIF-8 inside cells, which can be used for fluorescence image guided PDT in future. First, we investigated the change of luminescence intensity of ZnPc@ZIF8 in phosphate buffered saline (PBS) buffer at pH values of 7.4 and 5.0, chosen as the physiological and intracellular conditions. It is anticipated that the acid condition induced degradation of ZIF-8 would lead to the escape of the encapsulated ZnPc molecules which would quickly aggregate and result in the fluorescence quenching of ZnPc@ZIF-8. As shown in Figure 4a and b, the luminescence intensity of ZnPc@ZIF-8 in neutral PBS



keeps steady up to 72 hours, while that in acidic PBS drops remarkably within 72 h, which indicates that the ZIF-8 molecular cages are stable at physiological pH and would be destroyed under acidic condition. The acidic sensitive degradation of ZIF-8 was further confirmed by dynamic light scattering measurement. The average size of ZnPc@ZIF-8 suspended in neutral PBS buffer (pH=7.4) is stabilized around 255 nm after 72 h, while that suspended in acidic PBS buffer (pH=5.0) gradually reduces to 78 nm after 24 h immersion and completely degrades without any peak of size distribution after 48 h immersion (Figure S10 and Table S1 in the SI). We also monitored the intracellular degradation process of the ZnPc@ZIF-8 by capturing the fluorescence images of HepG-2 cells incubated with ZnPc@ZIF-8 for 24 h, 48 h and 72 h (Figure 4c). With the incubation time increasing, the fluorescence intensity inside the cells gradually decreases, suggesting the degradation of ZnPc@ZIF-8 inside cells. But there are still some ZnPc@ZIF-8 nanoparticles (marked with green circles) outside cells exhibit strong red fluorescence emission as shown in Figure 4b. It is because that these nanoparticles stay in the cell culture medium (pH=7.4) and do not degrade. The results in return evidence the intracellular acidic condition induced degradation and consequent fluorescence quenching of ZnPc@ZIF-8 nano-carriers.


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Figure 4. Luminescence spectra (a) and intensity variation (b) of 0.1 mg·mL-1 ZnPc@ZIF-8 PBS (pH=7.4 and pH=5.0) solutions, and the merged, bright field and fluorescent images of HepG-2 cells treated with ZnPc@ZIF-8 for 24 h, 48 h and 72 h, respectively. The fluorescence intensity profiles of the arrow across cells are shown at bottom row (c). Scale bar = 25 μm.

CONCLUSION In summary, we present a one-step co-precipitation approach for the synthesis of ZnPc@ZIF-8 nanoparticles. ZIF-8 with well-defined porous structure offers nanoscale molecular cages to encapsulate and separate hydrophobic ZnPc molecules, keeping them monomeric in aqueous solution for effective PS delivery. The as-prepared ZnPc@ZIF-8 nanoparticles possess excellent luminescence intensity, highly efficient 1O2 generation



capability, as well as photodynamic activity towards HepG-2 cancer cell line. Meanwhile, the MOF-based nano-carriers show low cytotoxicity and are fully biodegradable after PDT, which can be monitored in-situ by the self-quenching of the fluorescent emission of ZnPc. Overall, this work presents not only a solution for solubility and bioavailability of hydrophobic PS by utilizing MOFs as nano-carriers but also a potential biodegradable therapeutic platform for imaging-guided PDT.

ASSOCIATED CONTENT Supporting Information The following file is available free of charge. SEM and TEM images of ZIF-8 and ZnPc@ZIF-8; FTIR spectra of ZIF-8, ZnPc and ZnPc@ZIF-8; luminescence spectra of ZnPc@ZIF-8 suspended in aqueous condition at different time, Coumarin 6@ZIF-8 and Coumarin 6 suspended in DI water, Coumarin-6 dissolved in DMF, and ZnPc@ZIF-8 with different addition of ZnPc suspended in DI water; UV-vis spectra of ZnPc dissolved in DMF with different concentrations and the extracted ZnPc in DMF from the optimized ZnPC@ZIF-8 sample; the standard curve of ZnPc dissolved in DMF; DLS measurements and the average size of ZnPc@ZIF-8 suspended in PBS at different pH conditions (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], fenghuanhuan@ hit.edu.cn.


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Author Contributions ‡These authors contributed equally. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was financially supported by the Key Laboratory of Micro-systems and Microstructures Manufacturing of Ministry of Education, Harbin Institute of Technology (Project No. 2016KM007), Shenzhen Peacock Plan (KQTD2015071616442225), Shenzhen Peacock Innovation Project (KQJSCX20170726104623185).

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Figure 1. Schematic illustrating of the one-step fabrication of ZnPc@ZIF-8 (a), TEM image of ZnPc@ZIF-8 (b) and the PDT process of ZnPc@ZIF-8 for HepG-2 cells in vitro (c). 563x412mm (72 x 72 DPI)

ACS Paragon Plus Environment


Figure 2. XRD patterns of ZnPc@ZIF-8, ZIF-8 and ZnPc (a), UV-vis spectra and corresponding picture (inset) of ZnPc dissolved in DMF (①) and ZnPc (②), ZIF-8 (③) and ZnPc@ZIF-8 (④) suspended in DI water, luminescence spectra of ZIF-8, ZnPc and ZnPc@ZIF-8 dissolved in DI water (c) with the inset of fluorescence image of ZnPc@ZIF-8 in DI water under red light irradiation (Ex=610±20 nm, Em=680±20 nm) and variation of the luminescence intensity of ZnPc@ZIF-8 with different addition of ZnPc (d). 493x376mm (72 x 72 DPI)


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Figure 3. UV−vis spectra of ABDA with ZnPc@ZIF-8 under irradiation (650 nm) and the inset showing the variation trend of the peak values at 380 nm of ABDA with ZnPc@ZIF-8 under irradiation and without irradiation within 50 min (a), cell viability of HepG-2 cancer cells after incubated with ZIF-8 and ZnPc@ZIF-8 for 24 h in dark (b), photodynamic cytotoxicity of ZnPc@ZIF-8 under irradiation (650 nm) for 0 min, 10 min and 30 min, respectively and further incubated for 24 h (c), and the bright field image, the fluorescent images of ZnPc emission (red) and DAPI emission (blue) respectively, and the merged image of HepG-2 cells treated with ZnPc@ZIF-8 after PDT. The cell nucleus was stained with DAPI (d). Scale bar = 50 µm. 632x306mm (72 x 72 DPI)



820x435mm (72 x 72 DPI)


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Disassembly of Hydrophobic Photosensitizer by Bio-degradable

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