Synthesis of porphyrin-based 2D ytterbium metal organic frameworks for efficient photodynamic therapy

Photodynamic therapy (PDT), which relies on the photo-induced reactive oxygen species (ROS) to trigger tumor cells apoptosis, has attracted intense focus over the decades due to the minimum invasion, high-precision and controllable therapeutic processes. Tetra(4-carboxyphenyl) porphin (TCPP), as an effective PDT photosensitizer, can harness photons and generate singlet oxygen species (1O2) upon illumination; however, poor solubility and low loading rate greatly limit its further use. Although TCPP-based metal–organic-frameworks (MOFs) has been proposed to address these concerns, the relatively large size still limits their biomedical applications. Therefore, in this study, TCPP molecules are coordinated with Yb3+, growing into 2D Yb-TCPP MOFs by a wet chemical method; the as-prepared Yb-TCPP MOFs are around 200 nm in size and possess high 1O2 generation efficiency with low cytotoxicity. Due to TCPP is appeared as the organic frameworks of Yb-TCPP MOFs, the low loading rate problem is largely addressed; in addition, the absorbance of Yb-TCPP MOFs has been greatly expanded compared with free TCPP molecules due to the coordination with Yb3+, allowing the illumination at longer wavelength range, e.g. 655 nm, that possesses high penetration depth and low phototoxicity. Overall, we have prepared 2D Yb-TCPP MOFs suitable for the in vitro anticancer effect, revealing the potential of Yb-TCPP MOFs as the future anticancer agent.


Introduction
The local-precision, feasibility and excellent therapeutic effect offered by photodynamic therapy (PDT) have attracted lots of research attention in the past decades. 1,2 The reactive oxygen species (ROS) produced from therapeutic agents under illumination plays the key role in realizing the PDT. 3 Therefore, in most cases, the ROS production efficiency directly determines the PDT efficacy. In other words, in order to maximize PDT efficacy, the rst priority is to design a rational PDT therapeutic agent with high ROS generation efficiency.
Tetra(4-carboxyphenyl) porphin (TCPP), as an effective photosensitizer, can harness photons and generate singlet oxygen species ( 1 O 2 ) upon illumination, possessing great potential in PDT. 4 However, TCPP is poorly dissolved in water, making it hard to be directly used for clinical purposes. 5 Therefore, in practice, TCPP is usually packed within drug delivery systems (DDS) to improve the solubility and bioavailability. 6 In most TCPP DDS design, the carried TCPP will be released aer reaching to the lesion. Upon illumination, the released TCPP will produce 1 O 2 to initiate PDT. For example, Kan et al. prepared a UiO-66-type nMOF photosensitizer (UiO-66-TPP-SH) by post-surface modication. 7 Chen et al. constructed a functional nanoagent (MIL-101(Fe)@TCPP) by covalently binding a porphyrin photosensitizer on MIL-101(Fe) MOF nanoparticles. 8 Many efforts have been made, nevertheless, TCPP loading rate is still far from satisfactory, restricting it from practical application.
In recent decades, metal organic frameworks (MOFs) has drawn intensive focus due to the exible design strategies. 9,10 In MOFs, metal ions are distributed at the nodes, while organic ligands are coordinated with metal nodes and act as frameworks in 3D structure. 11 In MOFs, organic ligands and metal ions are highly replaceable, as long as the coordination and 3D periodic structure can be realized. As for TCPP, each molecule contains three carboxylic groups, which possess strong coordination ability with metal ions. Theoretically, TCPP can form MOFs with proper metal ions and synthetic strategy. Henceforth, TCPP MOFs with various metal ions as nodes have been synthesized and explored. For example, Wan et al. constructed a metal-organic framework (MOF) nanosystem based on the coordination of Mn(III) and porphyrin (TCPP), with controllable ROS generation and GSH depletion, which can signicantly improve the efficacy of photodynamic therapy. 12 Wang et al. synthesized 2D Cu-TCPP nanosheets by reacting with H 2 O 2 to generate 1 O 2 and cyclically consume GSH. 13 TCPP MOFs largely addressed the low loading rate of TCPP in DDS, as TCPP is now no longer a cargo but the framework of the nanostructure, therefore the low loading rate problem is largely alleviated. In addition, since TCPP is tted within MOFs 3D structures, the molecular movement freedom (vibration, rotation, stretching etc.) is largely restricted, it is also reported that the formation of TCPP MOFs can signicantly reduce the self-quenching problem of free TCPP molecules, thus improving the overall PDT efficiency. 14-16 Although many efforts have been made in the development of TCPP MOFs, some TCPP-based MOFs still lack systematic study. For example, ytterbium (Yb), which is an important lanthanide element with highly simplied isolated energy levels and high biocompatibility. 17 However, the application of lanthanide-TCPP MOFs, especially Yb-TCPP MOFs, has mostly been limited in the eld of catalysis, sensing and energy, while the biomedical application has been missing, with one of the major reasons being the relatively large size. 18,19 Therefore, in this study, we prepared nanosized Yb-TCPP MOFs and perform related characterization to evaluate the 1 O 2 production ability and PDT efficacy in vitro. To begin with, nanoscale Yb-TCPP MOFs are prepared by a relatively simple wet chemical methods with uniformly distributed size and 2D morphology, the as-prepared Yb-TCPP MOFs are around 200 nm in size, which are much smaller than previously reported ones. The small size is highly benecial to the following in vitro test of 1 O 2 production ability and PDT efficacy. To guide the following 1 O 2 generation test, the maximum absorbance of free TCPP and Yb-TCPP is obtained by using a UV-Vis spectrometer. Then, Yb-TCPP 1 O 2 production is characterized by using 1,3-diphenylisobenzofuran (DPBF). Under 655 nm illumination, the quick degradation of DPBF indicate the excellent 1 O 2 production ability of Yb-TCPP. Finally, Yb-TCPP MOFs are sent for in vitro test to examine the cell toxicity and PDT efficacy. With low cytotoxicity and high efficacy, this study highlights the superiority of Yb-TCPP MOFs in cancer treatment and provide a feasible Yb-TCPP MOFs preparation strategy. In addition, the Yb 3+ ions incorporated in the MOFs could be further developed to possess other biomedical features which could be coupled with TCPP to achieve combined therapy.

Detection of 1 O 2 generation in solution with DPBF probe
First, 10 mg of DPBF was dissolved in 10 mL of ethanol, and then 50 mL of this solution was added to an aqueous Yb-TCPP dispersion (100 mg mL −1 , 5 ml). Then, the solution was irradiated with 655 nm laser for 10 min, and the absorbance at 410 nm was measured with a UV-Vis absorption spectrophotometer every 2 min.
In vitro cytotoxicity assay using the CCK-8 protocol Cell viability was determined using the CCK-8 method. 4T1 cells were seeded into 96-well plates, the number of cells in each well was kept at 1 × 10 3 , and cultured in an incubator for 12 h. Aer the medium in the well plate was aspirated, DMEM containing PBS and Yb-TCPP were then added to each well, and incubated for another 8 h. Subsequently, the Yb-TCPP-containing medium was irradiated with a 655 nm laser for 10 min. The cells were further cultured for 16 h. Finally, the medium solution containing the nanomaterials was aspirated and washed three times with PBS. Then, each well was replaced with DMEM containing CCK-8 solution (100 mL, 10% CCK-8), and the cells were incubated for another 1 h, and measured its absorbance at 450 nm with a microplate reader.
Detection of intracellular 1 O 2 production Intracellular 1 O 2 generation was measured with CLSM. 4T1 cells were seeded into 6-well plates, the number of cells in each well was kept at 1 × 10 4 , and cultured in an incubator for 12 h. Aer the medium in the well plate was aspirated, DMEM containing PBS and Yb-TCPP were then added to each well, and incubated for another 8 h. Subsequently, the Yb-TCPP-containing medium was irradiated with a 655 nm laser for 10 min. The medium was aspirated, 0.2 mM DCFH-DA solution was added as a ROS detection probe and added to the culture dish, and incubated for another 20 min. Finally, the 4T1 cells were gently washed 3 times with PBS, and the intracellular 1 O 2 was detected with CLSM.

Observation of live and dead cell uorescence by CLSM
Live and dead cells were observed with CLSM, and 4T1 cells were seeded into 6-well plates, the cells in each well plate were maintained at 1 × 10 5 . The next procedure is roughly the same as that for measuring cytotoxicity. Aer addition of nanomaterials and illumination, 4T1 cells were further incubated for 16 h. Aer removing the medium and rinsing the dishes, 4T1 cells were stained with calcein and PI, live cells were stained green and dead cells were stained red. Aer 15 minutes of staining, the staining solution was aspirated, and the cells were rinsed twice with PBS, and then visualized with CLSM.

Synthesis and characterization of Yb-TCPP MOFs
The Yb-TCPP MOFs are synthesized by mixing and stirring ytterbium chloride hexahydrate (YbCl 3 $6H 2 O) and TCPP in N,Ndimethylformamide (DMF) and n-hexane solution containing triuoroacetic acid, polyvinyl pyrrolidone (PVP) and triethylamine (TEA) at elevated temperature. Under SEM and TEM images, the as-prepared Yb-TCPP MOFs exhibit a 2D morphology due the binding effect of PVP during the crystal growth, where the planar growth of MOFs crystal becomes dominated ( Fig. 1A and B). From XRD data and elemental mapping results, the as-prepared Yb-TCPP MOFs are well crystalized with Yb 3+ ions evenly distributed within Yb-TCPP MOFs, indicating the successful synthesis of Yb-TCPP MOFs (Fig. 1C-H). To study the absorbance proles and the correlation between free TCPP molecules and Yb-TCPP MOFs, UV-Vis spectrum is obtained for both. From the spectra, free TCPP molecules have sharp absorbance peaks located at 410 nm while Yb-TCPP MOFs have a much wider absorbance band, with good absorbance ability up to near-infrared region (Fig. 1I). This indicate that porphin centers of Yb-TCPP MOFs have been coordinated with Yb 3+ ions as a result of formation of MOFs. Due to the wide absorbance band of Yb-TCPP MOFs, longer wavelength illumination can be applied in practice, which is highly favorable in therapeutics as the phototoxicity could be greatly reduced and the penetration depth could be largely improved, thus, 655 nm illumination are used to study the 1 O 2 generation ability and PDT efficacy. Overall, materials characterization results demonstrate that the synthesis is successful, the as-prepared Yb-TCPP MOFs is thus used for the following experiments.

O 2 production test
Aer the successful synthesis of Yb-TCPP MOFs, the 1 O 2 generation ability is evaluated by using 1 O 2 detection probe (1,3diphenylisobenzofuran, DPBF). Briey, the presence of 1 O 2 in the environment will quickly degrade the DPBF, which can be reected on the decrement of UV-Vis absorbance spectra. Thus, the 1 O 2 generation can be deduced by comparing the UV-Vis spectra of the mixture. Therefore, DPBF is introduced into Yb-TCPP MOFs suspension. Without 655 nm illumination, from UV-Vis spectra, slight reduction of absorbance curve is observed for Yb-TCPP MOFs, due to the 1 O 2 produced under Russsel mechanism ( Fig. 2A and B). When Yb-TCPP MOFs are illuminated with 655 nm light, UV-Vis absorbance curves are reduced at a much faster speed, indicating the quick and efficient generation of 1 O 2 ( Fig. 2C and D). As the illumination continuous, the UV-Vis absorbance keeps decreasing, showing the stable 1 O 2 generation ability of Yb-TCPP MOFs which will not be bleached with up to 4 hours illumination (Fig. 2D). The stable 1 O 2 production behaviour also indicate the high photostability during the whole illumination period up to 4 h. On the contrary, due to the poor absorbing ability at longer wavelength region and self-quenching problem of free TCPP molecules, under 655 nm illumination, the decay absorbance curve is negligible in the 4 hour timescale (Fig. 2E and F), which further supports the superiority of Yb-TCPP MOFs as the PDT agent. The excellent 1 O 2 production of Yb-TCPP MOFs lays the foundation for following in vitro PDT experiments.
In addition, to guarantee the breakdown of Yb-TCPP MOFs will not happen upon illumination, Yb-TCPP MOFs are placed under 655 nm illumination with maximum power density for 1 h to evaluate the photostability under extreme condition. Results show that, Yb-TCPP MOFs still possess good 1 O 2 production ability as the absorbance of DPBF suspension quickly dropped once introduced to the Yb-TCPP MOFs suspension and illuminated with 655 nm light (Fig. S1 †); in addition, no observable degradation or damage of structure is captured on TEM image (Fig. S2 †).

In vitro PDT assessment
Aer establishing and conrming the excellent 1 O 2 generation ability of Yb-TCPP MOFs, we move on to explore the in vitro anticancer effects of Yb-TCPP MOFs. The experiments begin with the biocompatibility and cytotoxicity test of Yb-TCPP MOFs by using 3T3 cells as the indicators. 3T3 cells are incubated with different amount of Yb-TCPP MOFs. In the 24 h-incubation period, 3T3 cells can still maintain a high survival rate (Fig. 3A). Even with 100 mg per mL Yb-TCPP MOFs addition amount, more than 90% 3T3 cells are still alive, indicating that Yb-TCPP MOFs exhibit low cytotoxicity and high biocompatibility, suitable for the further therapeutic applications. Subsequently, 4T1 cancer cells are used to evaluate the anti-cancer effect of Yb-TCPP MOFs. 4T1 cells are cocultured with different amounts of Yb-TCPP MOFs. Without 655 nm illumination, Yb-TCPP MOFs do not exhibit noticeable kill effect on 4T1 cancer cells (Fig. 3B); however, due to the relatively high level of H 2 O 2 in cancel cells, Yb-TCPP MOFs can still present anti-cancer effect due to the Russell mechanism, still, only 30% 4T1 cells are dead at a concentration as high as 100 mg mL −1 (Fig. 3B). On the contrary, with 10 min 655 nm illumination, more than 80% 4T1 cells are quickly killed, due to the large amount of 1 O 2 produced by during PDT, inducing signicant oxidative damage to cells, causing 4T1 tumor cells apoptosis.
To further verify the anti-cancer effect of Yb-TCPP MOFs is due to the 1 O 2 produced under illumination, calcein AM/PI double stain kit is applied to reveal the live/death status of cells, simultaneously, 2,7-dichlorodihydrouorescein diacetate (DCFH-DA) is applied to evaluate the 1 O 2 within cells in a more intuitive way. From confocal laser scanning microscope (CLSM) image, in the control group, where the Yb-TCPP MOFs is missed, no green uorescence is received, indicating no 1 O 2 is generated (Fig. 4A). Correspondingly, in the live/dead staining test, 4T1 tumor cells are mostly alive (Fig. 4B). When Yb-TCPP MOFs are added to the system, without illumination, only traceable green uorescence is captured, indicating small amount of 1 O 2 is generated via the Fenton-like mechanism in the H 2 O 2 -rich environment within tumor cells (Fig. 4C). However, as the 1 O 2 level is low, only a few 4T1 cells lose the vitality, with traceable red uorescence appeared (Fig. 4D). Finally, when 655 nm illumination is applied to activate PDT, strong green uorescence can be detected under CLSM, indicating the high 1 O 2 production efficiency (Fig. 4E). Correspondingly, under live/death staining experiment, 4T1 cells are mostly killed in 1 O 2 rich environment, indicating the PDT is highly effective (Fig. 4F).
The apoptotic cells of the three groups are further measured by ow-cytometry using AnnexinV-FITC/PI-double-staining tool kit to statistically analyse the cells status in a more quantiable manner. It is obvious that 655 nm +Yb-TCPP MOFs treatment group has the highest ratio of apoptotic cells, much higher than Yb-TCPP group with 655 nm illumination, while the control group has no observable anticancer effect (Fig. 4g). Live/death staining also reveals the similar tread, with 655 nm +Yb-TCPP MOFs treatment group has the highest death rate (Fig. 4H), in addition, the highly correlated 1 O 2 production efficiency and anti-cancer effect indicate that the major anticancer mechanism is 1 O 2 -induced apoptosis (Fig. 4I).

Conclusions
In this study, Yb-TCPP MOFs are prepared with a relatively simple wet chemical method. The as-prepared Yb-TCPP MOFs exhibit a 2D morphology, with relatively small size at around 200 nm. XRD and elemental analysis reveals the well-crystalized structures with Yb 3+ ions evenly distributed within, indicating the successful synthesis of Yb-TCPP MOFs. UV-Vis absorbance of Yb-TCPP MOFs shows a wide absorbance band in visible range compared with free TCPP molecules, which only have a narrow absorption peak located at short wavelength range. The broadening of absorbance peak is largely due to the coordination between TCPP and Yb 3+ . Compared with free TCPP molecules, Yb-TCPP MOFs exhibit good absorbance ability in long wavelength range, e.g. 655 nm, which is highly favorable in therapeutic applications due to the deep penetration ability and low phototoxicity. Subsequently, the 1 O 2 production of TCPP and Yb-TCPP MOFs under 655 nm illumination is studied by using DPBF. The excellent 1 O 2 production efficiency of Yb-TCPP MOFs lays the foundation for the further in vitro PDT test; meanwhile, it also supports the superiority of Yb-TCPP MOFs in enhancing 1 O 2 production efficiency. Finally, in vitro biocompatibility and PDT efficacy studies are performed. Cytotoxicity experiments show the high biocompatibility of Yb-TCPP MOFs, with more than 90% 3T3 cells remain alive under 100 mg per mL Yb-TCPP MOFs concentration. When co-cultured with 4T1 tumor cells, with only 10 min 655 nm illumination, more than 80% of cancer cells are quickly killed. The highly correlated tumor cell death rate and 1 O 2 level directly supports the Yb-TCPP MOFs induced PDT is the primary anti-cancer mechanism. The successful synthesis of nanoscale 2D Yb-TCPP MOFs, high 1 O 2 production efficiency, low cytotoxicity and excellent PDT efficacy highlights the superiority of Yb-TCPP MOFs in biomedical, serving as a new powerful weapon in the ght against cancer and other disease.

Conflicts of interest
The authors declare no competing nancial interest.