Degradable Organically-Derivatized Polyoxometalate with Enhanced Activity against Glioblastoma Cell Line

High efficacy and low toxicity are critical for cancer treatment. Polyoxometalates (POMs) have been reported as potential candidates for cancer therapy. On accounts of the slow clearance of POMs, leading to long-term toxicity, the clinical application of POMs in cancer treatment is restricted. To address this problem, a degradable organoimido derivative of hexamolybdate is developed by modifying it with a cleavable organic group, leading to its degradation. Of note, this derivative exhibits favourable pharmacodynamics towards human malignant glioma cell (U251), the ability to penetrate across blood brain barrier and low toxicity towards rat pheochromocytoma cell (PC12). This line of research develops an effective POM-based agent for glioblastoma inhibition and will pave a new way to construct degradable anticancer agents for clinical cancer therapy.

Scientific RepoRts | 6:33529 | DOI: 10.1038/srep33529 2-amino-3-methylbenzoxyl group (AMB), which may benefit to its efficacy. As acy group could be cleaved in the environment of cell incubation 40 then destabilize the whole agent, POM-AMB-acy would be degraded, and eventually transformed into [MoO 4 ] 2− , which is the most common and easily-excreted form of the molybdenum element in human body 41 . In this way, a degradable POM-based compound may be developed as a promising candidate for glioblastoma inhibition with degradability. 6 O 19 ], 2-amino-3-methylbenzoic acid and N,N′-dicyclohexylcarbodiimide (DCC) in dry acetonitrile can afford POM-AMB-acy in 23% yield after 20 h, as monitored by ESI-MS ( Figure S1). The molecular structure of POM-AMB-acy has been clearly confirmed by single-crystal X-ray diffraction analysis (Fig. 2): its hexamolybdate cage is connected to the aromatic ring of 2-amino-3-methylbenzoxyl group via a Mo ≡ N triple bond with the Mo1-N1 bond length of 1.735 (4) Å and the linear C1-N1-Mo1 bond angle of 176.3 (8)°, which are in great agreement with the typical organoimido groups grafted at an octahedral d 0 metal center 33,38 . Thus, the proposed POM-AMB-acy has been successfully fabricated.

Synthesis and characterization. A refluxing reaction of [Bu 4 N] 2 [Mo
Inhibitory effect of POM against glioblastoma cell line. To understand whether POM-AMB-acy is indeed critical to glioblastoma's inhibition, the proliferation and morphology of malignant glioma cells The design of POM-AMB-acy and the overall process of blood brain barrier penetration, inhibition toward malignant glioma cell (U251) and degradation. treated with POM-AMB-acy was studied. For demonstration, U251 cells were chosen to be utilized in this model research. As shown in Table 1, IC 50 value for POM-AMB-acy is only 24.8 μ M, while that for TMZ, the clinically-used key therapeutic agent towards malignant gliomas, is around 500 μ M 29,42 . Moreover, examined by light microscopy (Fig. 3), U251 cells treated with 30.0 μ M POM-AMB-acy showed shrinkage, loss of neuritis, swelling of cell bodies, and a global disruption of the dendritic networks, in contrast to the control group. Therefore, this POM-based anticancer agent can stimulate apoptosis of malignant glioma cells indeed.
It needs to be figure out whether the synergistic effect originates from POM and organic functional moiety can indeed facilitate the inhibition performance of POM-AMB-acy towards U251 cells. Recently, it is reported that an organic functionalized POM derived from amantadine exhibited better anticancer performance against cancer cells than non-substituted hexamolybdates or amantadine independently 16 16 and POM-AMB-acy towards U251 cell were evaluated. As shown in Table 1, the IC 50 values of '6 equals of Na 2 MoO 4 ·2H 2 O' (53.4 μ M) was two-folds higher compared to POM-AMB-acy (24.8 μ M). This indicated that the functionalized POM, modified with N-acylureido group (acy) and 2-amino-3-methylbenzoxyl group (AMB), has better performance than metal ion itself. Furthermore, the difference between [Bu 4 N] 2 [Mo 6 O 19 ], POM-Ad, and POM-AMB-acy suggested that imidoylization can improve the inhibitory performance of POM towards malignant glioma cells on certain degree.
To investigate the location of POM-AMB-acy in U251 cells, scanning transmission electron microscopy (STEM) was employed to observe the location of molybdenum, and energy dispersive X-Ray spectroscopy (EDX) was utilized to track the ratio of molybdenum in each area. As shown in Fig. 4a, bright spots were clearly located inside the U251 cell and the ratio of molybdenum in this area reached 2.83% compared with the untreated cell (nearly 0%). Moreover, since the shading degree can indicate the concentration of molybdenum in STEM, there was an extremely bright oval area comparing with surroundings and the ratio of molybdenum in this area significantly climbed to 8.92% (Fig. 4b). Furthermore, the penetrating ability of POM-AMB-acy towards BBB was evaluated. The Balb/c mice were treated with POM-AMB-acy via tail vein injection. As shown in Table S1, after dosing with POM-AMB-acy (12.5 mg kg −1 ) for 10 min, the molybdenum level in brain rapidly reached 4.4 mg kg −1 , respectively. While in the control group, the molybdenum level was merely 0.2 mg kg −1 .
Degradability of POM-AMB-acy. Apart from the above characteristics, the degradability of POM-AMB-acy is of great importance. In order to prove this point, in vitro experiments were performed to investigate its degradability by utilizing the fresh cell incubation medium (MEM + 10% FBS) as demonstration. In order to investigate its stability in MEM, IR and ESI-MS was chosen to study its stability. According to Fig. 5a, POM-AMB-acy stayed intact in MEM within 40 min. While after 1 hour's treatment with MEM, new bands appeared at 1415 and 881 cm −1 compared with 40 min, and it can also be seen in the spectrum of Na 2 MoO 4 in MEM (Fig. 5b). Moreover, according to Figure S6, the time dependent IR spectra for another organoimido-derivatized POMs (POM-Ad) indicated that admantadine modified POM cannot be degraded into MoO 4 2− in MEM which furthermore proved that the optimized ligand was sufficient for POM's degradability.
Furthermore, the ESI-MS was also conducted to investigate the composition of degradation product. After maintaining the system (solute: POM-AMB-acy, solvent: MEM + 10% FBS) for 24 h, this inorganic degraded product was marked as complex T1. As shown in Figure S2, the ESI-MS spectrum for T1 was quite different from that for POM-AMB-acy itself. The characteristic peaks in POM-AMB-acy are located at 609.28, 1219.58 and 1460.86 ( Figure S1). However, the major peaks for T1 are located at 80. 95 4 2− in serum, which is corresponding to our result 43 . Therefore, in the cell incubation environment, POM-AMB-acy can be ultimately degraded into MoO 4 2− .
Cytotoxicity of POM-AMB-acy. To understand whether the degradable characteristic is critical to alleviating the toxicity of POM-AMB-acy, POM-AMB-acy itself and degraded POM-AMB-acy complex (T1) on cellular metabolism behavior towards PC12 cell was explored. As shown in Fig. 6a Fig. 6b, when the pretreatment was performed for 1 h before POM-AMB-acy being added into PC12 cells, the cell viability of it was higher than original POM-AMB-acy with 5% increase. Moreover, after 4 hours' pretreatment, the cell viability reached 91.9%, which was close to the degraded complex (T1). Therefore, POM-AMB-acy can be degraded and achieved low toxicity no more than 22 hours. Therefore, via degradation, the toxicity of POM-AMB-acy can be reduced and the long-term toxicity of POM can be alleviated on a certain degree.

Discussion
In conclusion, a degradable organically-derivatized POM was developed with high efficacy towards glioblastoma cancer cell. To prevent the side effects, this agent is endowed with degradability by introducing a cleavable functional group into its structure. This fundamental research can provide the guidance to fabricate other degradable agents based on POMs or nanoclusters for cancer therapy. All in all, this line of research represents a great demonstration as a POM-based compound for glioblastoma inhibition and provides an effective approach to solve the problems in the medicinal chemistry of POMs. This research will enrich the field of glioblastoma inhibition and medicinal chemistry of POMs with important advances.     absorption correction were performed with the software package of Rigaku RAPID AUTO (Rigaku, 1998, Ver2.30). Structures were solved by direct methods and refined against F 2 by full matrix least squares. All non-hydrogen atoms, except disordered atoms, were refined anisotropically. Hydrogen atoms were generated geometrically. All calculations were performed using the SHELXS-97 program package.
Cell culture. U251 (human malignant glioblastoma) and PC12 cells (rat pheochromocytoma) were obtained from the Cancer Institute of Chinese Academy of Medical Science (Beijing, China) and grown in MEM supplemented with 5% fetal bovine serum and 10% horse serum in a humidified 5% CO 2 environment at 37.0 °C. Cells were plated at a density of 1 × 10 6 cells per 100 mm culture dish and allowed to grow to approximately 70% confluence before experimentation.

Scanning transmission electron microscopy (STEM) and
Energy Dispersive X-ray Spectrum (EDX). U251 cells were seeded in 10 cm dishes at a density of 1 × 10 5 cells/mL. After 24 h incubation, cells were treated with 1 at the concentration of 60 μ M for 24 h. The cells were directly harvested with a cell scraper and centrifuged at 2500 rpm for 10 min. After fixation by a 2.5% (wt/vol) glutaraldehyde and 2% (wt/vol) paraformaldehyde, samples were then submitted to the Center of Biomedical Analysis (Tsinghua University) for subsequent treatment and sent to Analysis and Test Center (Tsinghua University) for STEM and EDX analysis. Of note, in order to clearly identify the distribution of molybdenum in cancer cells using STEM mode, it had to give up using osmium tetroxide for fixation, so the morphology of U251 cells lost on a certain degree.

Synthesis of POM-Ad.
According to the previous literature 16 , a mixture of (n-Bu 4 N) 4 (14)  Degrading process on cytotoxicity assays. To evaluate the cytotoxicity of degraded POM-AMB-acy complex, the PC12 cells were seeded at density of 5 × 10 4 cells per well in a 96-well microtiter plate in advance. To prepare the degraded POM-AMB-acy complex, POM-AMB-acy was pretreated with 100 μ L of mixture solution (10 μ L of DMSO solution and 90 μ L of cell medium) for corresponding period (0 h, 1 h, 4 h and 22 h) at the concentration of 80 μ M. After 24 h of incubation, the cells were treated with 100 μ L of cell medium and 100 μ L of mixture solution which contains the pretreated POM-AMB-acy, so the final concentration of POM-AMB-acy was 40 μ M and the cells were incubated for another 24 h. For each test, five replicates were employed. Twenty microliters of 5 mg/mL MTT solution were added to each well, and cells continued to be incubated for 4 h at 37.0 °C. After careful removal of the medium, dimethyl sulfoxide (DMSO) was added to each well, and the plate was then shaken for about 10 min. Absorbance was then measured at 490 nm in a microplate reader (Scientific Varioskan Flash, Thermo Fisher Scientific, U.S.A). The curves of viability were drawn by comparing the control group. The inhibitory rate was calculated using the following equation: inhibitory rate (%) = (OD control − OD treatment )/ OD control × 100%. The viability rate (%) = 100 − inhibitory rate (%).