Novel Tocopherol Succinate-Polyoxomolybdate Bioconjugate as Potential Anti-Cancer Agent

Despite the promising anti-cancer properties of the polyoxometalates (POMs) compound, they have not yet been reported for clinical use due to their general toxicity. This study reports the synthesis of tocopherol succinate (TS)-polyoxomolybdate (POMo) conjugate (T2POMo) as a novel organic–inorganic hybrid conjugate of POMo and evaluating its anti-cancer properties in vitro. The aim was to introduce a more potent derivative with less general toxicity than initial POMo to cancer treatment studies. The T2POMo conjugate was synthesized using amide bond formation between POMo and TS based on the carbodiimide strategy. The chemical structure of T2POMo conjugate was fully investigated and confirmed using spectroscopy and elemental analysis techniques. The anti-cancer properties of T2POMo conjugate were evaluated on Brest (MCF-7) and prostate cancer (LNCAP) cell lines carefully by the MTT protocol, and the general toxicity was studied on human umbilical vein endothelial cells (HUVEC) similarly. Finally, the quantity of induced apoptosis was carefully evaluated using the flow cytometry technique for the T2POMo conjugate compared to POMo. The cytotoxicity studies showed that tocopherol succinate conjugation altered and regulated the activity and seems to induce great synergistic cytotoxic effects on cancerous cell lines. The half-maximal inhibitory concentration (IC50) on the MCF-7 cell line was about 167.3 μg/mL, and on the LNCAP cell line was about 234.1 μg/mL. The cytotoxicity of T2POMo was significantly greater than that of POMo, and the toxic effects on normal cells were significantly reduced. Flow cytometry results showed that the hybrid conjugate could produce about 61% of apoptosis in the MCF-7 cell line than POMo (36%) alone. Therefore, tocopherol succinate hybrid conjugate (T2POMo) can be introduced as a promising potent anti-cancer agent to further pre-clinical studies.


Introduction
Based to the World Health Organization (WHO), cancer is a group of diseases associated with abnormal cell growth and the potential to attack other parts of the body and one of the leading causes of million deaths worldwide. It was responsible for more than 9 million mortalities in 2018, especially in low to middle-income countries [1].
All of the chemotherapy drugs suffer from some drawbacks, such as high price, numerous side effects, and low bioavailability [2]. Thus, it has always been fascinating to find new cytotoxic agents to overcome these limitations by replacing previous drugs in the clinic.
Polyoxometalates (POMs), macroanionic clusters, are chemical structures with early transition metals in which the metal ions in their highest oxidation states are linked together through an oxygen bridge [3]. They have been studied in various fields, such as catalysis, material science, pharmaceutical science, medicine, and biosensors, because of their unique properties and reactivity [4]. During the last decades, POMs have attracted much attention in pharmaceutical research as therapeutic agents like anti-cancers, antibiotics, antivirals, etc. It seems that due to their low price of preparation, simple synthesis, easy modification, and other eminent characteristics, they have a unique chance of being considered as drugs in the future.
Many reports on the anti-cancer activity of POMs and their organic hybrids regardless of the structure, identity, and chemical composition of POMs are available. Even because of their unique potency in this regard, they were introduced as the next generation of metallodrugs by Bijelic et al. [5].
Yamase published the first report on the anti-cancer activity of POMs in 1988 around anti-tumor activity (NH3Pri) (Mo7O24) polyoxometalate animal transplantable tumors and human cancer xenograft [6]. Still, in the late twentieth century, the anti-cancer studies reported by the Sabarinathan team [7] on silicotungstate cluster coordinated organic-inorganic hybrid material [Cu(dmbpy)]2 [SiW12O40]·8H2O, and Li et al. [8] on Keggin-type rare earth-containing (POMs) specifically in 2019 and 2020 showed the appeal of this research area.
Despite these excellent studies, two significant problems have remained in this regard that impede the clinical application of POMs in cancer therapy; first, compared to other anti-cancer drugs, with an inhibitory concentration (IC50) in the nanomolar range, relatively large quantities of POMs are needed to initiate the anti-cancer effect in-vitro and in-vivo. Second, the cytotoxicity in healthy cells and side effects of POMs as a drug should be considered by either increasing the cell-selectivity through targeting strategy or conjugation to bioactive molecules [3,9]. Molecular hybridization is expected to open up new interdisciplinary perspectives in medicinal chemistry [10]; in most cases, the hybrid compounds benefit from integrating their biological effects. In this regard, combining different pharmacophores, organic or inorganic substructures, is possible with an appropriate rationale to get the desired outcomes.
Hybridization strategy, specifically the covalent modification, seems to be a valuable strategy to control the inherent cytotoxicity of POMs. This strategy offers additional advantages, including better biological stability and, in some cases, better selectivity in POMs [11][12][13]. In this regard, hybridization with some specific bio-molecules, such as peptides, vitamins, proteins, and other bioactive ligands, has created more research appeal in this kind of cytotoxic agent.
Some of the recent papers on the subject of anti-cancer activity of organic or biological hybrids of POMs are as follows; Boulmier et al. in 2017, reported the anti-cancer activity of a series of polyoxometalate-bisphosphonate complexes containing Mo(VI)O 6 octahedra, zoledronate, or an N-alkyl zoledronate analog, in their structures Mn was heterometal. They found promising activities against human non-small cell lung cancer (NCI-H460) cells with IC50 values for growth inhibition of ∼5 μM per bisphosphonate ligand [14].
Hosseini et al. in 2020 reported the cytotoxicity of biotinconjugated manganese polyoxomolybdate on MCF-7 cell line (IC50; 0.082 mM) and HepG2 cell line (IC50; 0.091 mM). Meanwhile, they approved the lower cytotoxicity on the HUVEC cell line [15]. Ventura et al. in 2018 reported functionalization of the Anderson-Evans polyoxomolybdate [(MnMo6O24) 3-] with a Bombesin antagonist peptide. They studied the anti-cancer activity of this conjugate against MCF-7 and Hela cell lines; in both cytotoxicity assays, they found IC50 about 75 nM [12].
Vitamin E is well-known as a lipophilic vitamin with anti-oxidant activity, which has also been demonstrated to lower the cancer risk [16]. Vitamin E has eight varieties, four tocopherols (α-, β-, γ-and δ-tocopherols) and four tocotrienols (α-, β-, γ-, and δ-tocotrienols), that are up-and-coming candidates for the synthesis of anti-cancer hybrid conjugates [17]. α-tocopherol is a significant variant of vitamin E with unique features [18], some of the previous studies revealed that high-dose (100 µM) α-tocopherol inhibited cell proliferation in ER+ breast cancer, including MCF-7 and T47D cells, in a dose-dependent manner [16,19].
Among all vitamin E derivatives with unique characteristics, it has been demonstrated that α-tocopherol succinate (TS) can inhibit the proliferation rate of various cancers in vitro and in vivo [17]. Some recent studies have also revealed that α-TS has anti-cancer activities in various hormone-dependent breast cancers, and even such as MCF-7, MDA-MB-435, 4T1, and MDA-MB-453 cells [16]. Furthermore, TS has proven its synergistic effects when it has been used with other therapeutic agents in clinic, in vitro, and in vivo studies [20,21].
Herein, for the first time following our recent studies and interests, we aimed to evaluate the synergistic effect of α-TS on the cytotoxicity of an Anderson-type polyoxomolybdate (POMo). So, T 2 POMo conjugation was synthesized using amide covalent bonds, and the cytotoxicity of this novel conjugation was studied on two types of cancerous cell lines besides the normal cells by MTT assay. Furthermore, the apoptosis value was studied quantitatively using Annexin V/propidiumiodide (PI) kit.

Synthesis of Compounds
Synthesis of POMo, the synthesis of POMo was achieved in two steps from sodium molybdate and manganese acetate precursors based on our previous study [22].

Step 1: Synthesis of [TBA] 4 [α-Mo 8 O 26 ], (POM-1)
Briefly, a solution of sodium molybdate dihydrate (NaMoO 4 ⋅2H 2 O) (2.50 g, 10.35 mmol) in 6 mL of water was acidified with 6.0 N HCl while stirred vigorously for about 2 min at an ambient temperature. An aqueous solution of TBAB (1.67 g, 5.20 mmol) was then added to the above solution, white precipitates that were immediately formed were collected and washed respectively with water, ethanol, acetone, and diethyl ether. The product was dissolved in the minimum amount of acetonitrile and stored at − 10 °C around 30 h. The bright, colorless, block-shaped crystals were collected and washed with deionized water, ethanol, acetone, and diethyl ether, respectively. The obtained crystals were dried in the vacuum oven overnight; the yield was about 75% based on NaMoO4 [23].

Step 2: Synthesis of ([TBA] 3 [MnMo 6 O 18 ((HOCH 2 ) 3 CNH 2 ) 2 ]); POMo
A mixture of POM-1, Mn(OAc) 3 ⋅ 2H 2 O, and (HOCH 2 ) 3 CNH 2 [24] was refluxed for 16 h in acetonitrile. The orange solution was cooled to room temperature and filtered to remove impurities, and the orange filtrate was exposed to the diethyl ether for several days (5 days). The POMo was obtained as large orange crystals, filtered and washed with a small amount of cold acetonitrile and diethyl ether, and dried in the vacuum [25].

Synthesis of Tocopherol succinate POMo conjugate (T 2 POMo)
α-Tocopherylsuccinate (TS) was synthesized based on the procedure reported by Mai et al. [26] by the reaction of α-Tocopherol and succinic anhydride. TS (0.25 g, 1 mmol) was first activated in the presence of NHS (0.12 g, 1.04 mmol) and EDC (0.19 g, 1.20 mmol) in anhydrous DMF/CH3CN, the conjugation between TS and the amine moieties of POMo (0.94 g, 0.5 mmol) was carried out in the presence of the catalytic amount of triethylamine for 48 h in a room temperature. The T 2 POMO bio-conjugation was precipitated by adding diethyl ether, filtered and further purified by acetone and water to remove residual impurities and finally, the pure product was obtained from acetonitrile [27]. FTIR, 1 H NMR, and UV-Visible spectroscopy and CHNS analysis were used for characterization of the final conjugate.

T 2 POMo Stability Study
For this purpose, a solution of T 2 POMo (40 µg/mL) was prepared at pH = 7.4 in PBS-0.3% DMSO (as close as possible to cell culture media) as reported by Geisberger et al. [28]. Upon mixing with PBS, the clear solution was retained. The solution was scanned immediately, after 24 h, after 48 h, and after 72 h by UV-Vis spectrophotometer.

In-Vitro Cell Viability Assay
The cells were cultured in a standard condition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in an atmosphere of 5% CO 2 , then were retained in RPMI-1640 (GIBCO) medium for following cytotoxicity evaluation using MTT protocol [29]. Typically, stock solutions were prepared in the concentration of 500 μg/mL in phosphate-buffered saline (PBS) (pH 7.4). Cells in a density of 5 ×10 3 per well were seeded, then incubated in the same condition as culturing step for 24 h. Different concentrations of POMo, TS, and T 2 POMo (ranging from 50, 100, 200, 300, and 400 μg/mL) were treated regularly on MCF-7, LNCAP, and HUVEC Cells plates. After incubating for 24 h, the medium was removed, and 20 µL of MTT solution (5 mg/mL) was added to each well, and incubation was continued for 4 h. After that, the medium was replaced with 150 µL DMSO to solubilize the purple formazan precipitates, and the absorbance was read using a microplate reader at 570 nm. The cell viability was calculated using the following equation; A t , A b and A c represent mean absorbance of the treatment, blank and negative control, respectively [30]. For each treatment, the average of 9 runs was considered, and results were given as Mean ± SD.

Hemolysis Assay
The hemocompatibility of POMo and T 2 POMo was evaluated using a procedure reported by Shi et al. [31]. Briefly, 1 mL of fresh rat blood was centrifuged at 3000 rpm for about 15 min and precipitated RBCs were isolated and rinsed thoroughly with PBS solution for purification and stored properly. The hemolysis test solutions of POMo and T 2 POMo were prepared by adding 40 μL of RBCs to 960 μL of PBS solutions of test groups in different concentrations. The test concentrations were in the range of 50-400 μg/mL; the samples were incubated at 37 ºC for 6 h. After that, the dispersions were centrifuged (3000 rpm, 20 min), and the supernatant was evaluated by UV-visible spectroscopy at 540 nm. The ionized water and PBS were respectively used as positive and negative controls, and the hemolysis ratio was calculated using the following equation: A t , A n and A p refer to absorption of the test group, absorption of negative control, and positive control absorption.

Flow Cytometry Analysis of Cell Apoptosis
To approve the apoptosis pathway, the binding proportion of annexin V and propidium iodide uptake was checked using a phosphatidylserine detection kit (IQ product, Netherlands). In this regard, MCF-7 cells were seeded in a 12-well plate with a density of 10 4 cells/well and incubated for 24 h at 37 °C. POMo and T 2 POMo solutions with a 200 mg/mL concentration were treated on cells, and incubation was followed for the next 24 h in the same condition. After that, the cells were washed three times with cold PBS and then harvested using Trypsin. To evaluate the apoptosis ratio, the cells were doubled stained with Annexin-V-FITC and PI respectively according to the manufacturer's proposed procedure and incubated in the dark for 15 min a room temperature and analyzed using FACS Calibur flow cytometer. Only single cells were gated for fluorescence analysis [32].

Statistical Analysis
The data were analyzed using one-way ANOVA using SPSS software (the version of 21) followed by student t-test to evaluate the difference between groups or by Post Hoc LSD test for more than two groups. The p value was lower than 0.05, considered as a significant difference between averages.  [25]. The reaction of tocopherol succinate with both ends of POMo led to the final conjugation T 2 POMo as a pale orange powder (Fig. 1).

Synthesis of compounds
Since the synthesis and the characterization of Anderson type polyoxomolybdates (POM-1 & POMo) were previously reported, so in this study, the spectral data ( 1 H NMR, FTIR), elemental analysis (CHNS), and XRD pattern were compared with those were reported earlier [25] to ensure about the accurate synthesis of required polyoxometalate subunit. The FTIR, 1 H NMR, CHNS data for POM-1 and POMo are as follows, and the XRD pattern for POMo in comparison to that report by Marcoux et al. has been provided in Fig. 2. As it can be seen, the similarity between two patterns of prepared POMo and that retrieved as standard XRD is definite. [

3
The structure of (T 2 POMo) was characterized by 1  Based on the available reports, six edge-sharing MoO 6 octahedral are arranged around a core of the MnO 6 unit, making the Anderson structure. The TRIS are bound to the Mn(III) ion in the core via its alkoxy groups, so two amine groups of TRIS are oriented outside of POM and are available for further modification. The organic groups cover both sides of the planar hexagon through the chemical bonding to TRIS amine groups [25].
As shown in Fig. 1, we used both amine groups of POMo for the functionalization with TS. The amidation reaction between TS carboxylic acid and POMo was carried out through the carbodiimide strategy using EDC/NHS [33]. Purification through precipitation afforded the final product with a relatively high yield. The chemical structure of the T 2 POMo conjugation was confirmed by elemental analysis, FTIR spectroscopy, and 1 H NMR spectroscopy. With an indepth look at the FTIR spectrum (Fig. 3) of final conjugation compared to the POMo, we find some changes after the conjugation; for example, N-H stretching frequency has increased to some extent from 3448 cm −1 to 3489 cm −1 , the carbonyl group stretching frequency moves from 1719 cm −1 in TS to 1688 cm −1 in T 2 POMo due to the conjugation, we also see the primary ester band of TS in the related place around 1741 cm −1 . Finally, some spectral details related to TS have appeared in the corresponding area in the final spectra. Furthermore, we can see the characteristic bands of Anderson-type POMo in proper regions around 939.2, 917.7, and 662.8 cm −1 , respectively, after conjugation with TS. These IR proofs undoubtedly supported the correct amide formation between TS and POMo.
The data of 1 H NMR of T 2 POMo is the best complementary one, all fundamental signals of TBA and TRIS in the POMo scaffold are relocated intact in T 2 POMo. Marcoux et al. [25] showed that because of the strong electron-withdrawing identity of POM, its methylene protons (belong to TRIS) appear around 60-62 ppm in 1 H NMR spectra with the right signal ratio to other related peaks. Along with characteristic signals of TS and POMo, the signal of NH amide has correctly appeared around 8.6 ppm with the exact signal ratio to the POMo CH2 moieties around 61 ppm (as shown in Fig. 4). Based on these spectral proofs, the conjugation of two TS molecules to the POMo scaffold was approved initially. The best complementary evidence was obtained from elemental analysis; according to these results and comparing with theoretical values, the chemical structure and formula were finally approved [25]. Furthermore, UV-Visible spectroscopy (Fig. 5) showed the characteristic bands for both TS and POMo accordingly; it seems that upon the conjugation, the shape and details of the spectrum have changed completely in comparison to its sub-groups (TS & POMo). The general shape of the bioconjugate (T 2 POMo) spectrum confirms the combination of the two components. Some small changes in the maximum absorption wavelength are in line with those reported by others for hybrid organic-inorganic conjugations [34].

Stability of T 2 POMo Conjugate
Before in vitro cytotoxicity evaluation, the stability of the T 2 POMo conjugation should be checked in the same condition as the MTT assay protocol. In this regard, the stability of T 2 POMo conjugation was analyzed using the UV-Visible spectrum of the dissolved sample after specified times (instantly, 24, 48, and 72 h after) [35,36].
The UV/vis spectrum of T 2 POMo in PBS (Fig. 6) clearly indicates its stability around neutral pH conditions through monitoring of the characteristic of POM absorption bands, i.e. The characteristic of T 2 POMo absorption bands did not undergo significant changes at any wavelength over a period of 3 days. These results agree well with the previously observed stability, which was reported by Geisberger et al. [28].

In Vitro Cytotoxicity Assessments (MTT Assay)
To study the effect of TS conjugation on the cytotoxicity profile of POMo in the final product, two cancer cell lines comprising MCF-7 and LNCAP were selected due to their relatively high level of tocopherol receptor on them based on previous reports [37,38]. The cells were treated with different concentrations ranging from 50 to 400 μg/mL of TS, POMo and T 2 POMo. Furthermore, the normal cell cytotoxicity was evaluated on human umbilical vein endothelial cells (HUVEC) in the same way using a concentration of 400 μg/mL.  Figure 7 represents the cytotoxicity profile of T 2 POMo in two different incubation times and different concentrations on the MCF-7 cell line. As it can be seen, the cytotoxicity profile is completely time and dose-responsive (p < 0.05 for each comparison). Based on these initial results, we selected the 24 h for incubation time, and the comparative cytotoxicity of POMo and T2 2 POMo have been evaluated on both MCF-7 and LNCaP cell lines (Fig. 8). Eventually, Fig. 9 represents the comparative cytotoxicity of POMo and T 2 POMo on the HUVEC normal cells.
Previous reports have been repeatedly referred to the anti-cancer properties of TS and the synergistic effects of TS on the cytotoxic properties of some anti-cancer drugs and agents [17]. So it seems that TS is a good candidate for enhancing the anti-cancer properties, and based on these studies, TS was selected to bind to polyoxomolybdate.
As can be deduced from Fig. 8 (up), T 2 POMo exhibited a better growth inhibition effect considerably on MCF-7 cells compared to POMo and TS. The IC50 of the T 2 POMo and POMo on MCF-7 was 167.3 and 321.7 μg/ mL, respectively. On the other hand, both POMo and T 2 POMo showed somewhat less cytotoxic effects on LNCAP (Fig. 8-down), the IC50 of T 2 POMO and POMo on normal cell line (LNCAP) were, respectively 234.1 and 382.2 μg/mL estimated. The better-detected activity on MCF-7 could be attributed to the higher value of tocopherol responsivity in this cell type, as human protein atlas implied [39].
However, the complementary and adjuvant effects of TS on the cytotoxicity effects in both cell lines are well evident, and the main hypothesis of this study seems to be confirmed.
The second hypothesis of this study was to reduce the cytotoxicity effects on HUVEC normal cells, which is confirmed by the normal cell line results (Fig. 9).
The protective effects of tocopherol, mentioned earlier [40], appear to reduce toxicity on the normal cell line. The cytotoxicity of T 2 POMo and POMo were evaluated on the HUVEC cells at the concentration of 400 μg/mL, which was high enough to see the cytotoxic effects. Interestingly, we did not get any considerable cytotoxicity on HUVEC compared to the positive control (cis-platin) at the same concentration for T 2 POMO. As seen in Figs. 8 and 9, both POMo and T 2 POMO have higher cell viability compared to the cis-platin at the same concentration (* and $ mean the significant difference between each group and positive control), and this effect is recognized much profoundly in the case of T 2 POMo comparing Cis-Platin (p value < 0.05). Furthermore, there is a significant difference between all treating groups and the control group with 100% viability (@ means significant difference with control).
The more cytotoxicity of T 2 POMo than the POMo can result from the inherent toxicity of the POMo, besides its facilitated cell entry through tocopherol receptors. In other words, the cytotoxicity of the T 2 POMo was improved by higher cell endocytosis of the conjugation through the tocopherol receptors. Although the cellular behavior of T 2 POMo is not precisely apparent, we find the better activity of the conjugation against the MCF-7 cancerous cells than the LNCAP ones. This lower effect on LNCAP cell lines can be explained by the lower expression level of tocopherol-binding proteins on LNCAP cells, or probably, the lower sensitivity of the LNCAP cells compared to the MCF-7 cells in the culturing process or other intracellular

Hemolysis Assay
The evaluation of possible toxicity in red blood cells (RBC), with measuring the rate of hemolysis, is the best initial biological assay among the different cytotoxicity assays. This essay is based on red cell membrane rupturing in the presence of any xenobiotic. RBC are the main cells in blood circulation, which xenobiotics encounter initially following intravenous injection. Thus, any interruption in the membrane of RBC would certainly disrupt their vital function and could be lethal [42]. The hemolytic activity of the POMo and its bioconjugation T 2 POMo (Fig. 10) was evaluated in erythrocytes from rats employing the standard methodology. The subsequent release of hemoglobin was used to assess hemolytic activity as the concentration function, with concentrations ranging from 50 to 400 μg/mL. The T 2 POMo conjugate is significantly safer (p value < 0.05) than the POMo, even at 400 μg/mL. This safety is profoundly apparent in higher concentrations, and as it can be seen, even at a concentration of 400 μg/ml, the total percent of hemolysis is still below five percent in the case of T 2 POMo, which is the promising outcome [31]. It seems that for both POMo and T 2 POMo, the best concentration for being safe to RBC is 200 μg/mL.

Apoptosis Quantification Using the Flow Cytometry Protocol
To quantify the cell apoptosis, MCF-7 cells (the better cytotoxic effects were obtained on it) were treated with the same concentration of both POMo and T 2 POMo (200 μg/mL), incubated for 24 h, and finally were stained by Annexin V/propidiumiodide (PI). The Annexin V binds to cells in the early apoptosis stage, which can be used as a very specific apoptotic marker and PI stains cells in late apoptosis and dead cells [43]. The results have been shown in Fig. 11, the upper left quadrant shows the percent of necrosis in cell death, the upper right shows late apoptotic cells, the lower left shows normal alive cells, and the lower right quadrant shows the cells in the early apoptosis stage. The results showed that the proportion of apoptotic cells (in the early and late phase) increased by adding POMo and T 2 POMo to 36.56 and 60.88%, respectively. The proportion of late apoptotic cells induced by the T 2 POMo is significantly higher than the POMo (18.36% vs. 5%) and Fig. 9 The comparative cytotoxicity of T 2 POMo, a-TS, and POM on the HUVEC cell line compare to the Cis-platin at the concentration of 400 μg/mL and control untreated cells (* refers to the significant difference between POMo and T 2 POMo results, & refers to the significant difference between cytotoxicity of T 2 POMo and Cis-Platin; p value < 0.05) Fig. 10 Hemolysis activity of POMo & T 2 POMo more profoundly increased regarding the control group (2.88%). So, it can be concluded that the conjugation of TS to the POMo improved the cytotoxicity of POMo through the valuable mechanism of programmed cell death. Peers have reported the same results in this area [32].
Finally, as Zamolo et al. have stated, this approach provides an efficient cytotoxic bioactive inorganic agent and paves the way to bio-functionalize the POMs for biorecognition, cell internalization and biomimetic catalysis [44].
This result is always promising for a new cytotoxic compound, reducing the side effects or improving the biocompatibility accompanying the better cytotoxicity profile.

Conclusion
Achieving new cytotoxic agents with improved effects compared to previous generations has always been the focus of chemists in medicine. Polyoxometalates are considered the next generation of inorganic anti-cancer compounds, so designing and synthesizing hybrid conjugates of these compounds using bioactive molecules can be a promising path for further development. In this study, a new generation of polyoxomolybdate hybrid conjugation was evaluated. Preliminary in vitro cytotoxicity results showed that polyoxomolybate conjugation with tocopherol succinate could increase cytotoxicity on cancer cells and reduce cell toxicity on normal healthy cells. Also, it seems that tocopherol can facilitate the entry of polyoxomolybdate into the cell and, besides, create synergistic anti-cancer effects.
This synergistic effect can be related to the targeting ability of the tocopherol and the intrinsic cytotoxicity of the POMo. As a complementary fact, the designed hybrid conjugation of polyoxomolybdate and tocopherol succinate showed a significantly improved apoptosis compared to peer polyoxomolybdate, which is the valuable outcome of this study.
Our preliminary findings in this study convinced us to continue to synthesis bioactive POMs with enhanced antitumor properties.