- Synthesis of compounds
[TBA]4[α-Mo8O26] (POM-1) was synthesized as the basis for POMo according to the previous reports without any difficulty, after addition of Mn(OAc)3 and TRIS subunits, [TBA]3[MnMo6O18{(OCH2)3CNH2}2](POMo) was isolated as orange crystals in 80% yield from POM-1 according to the previous reports (25) . The reaction of tocopherol succinate with both ends of POMo led to the final conjugation T2POMo as a pale orange powder (figure 1).
Since the synthesis and the characterization of Anderson type polyoxomolybdates (POM-1 & POMo) were previously reported, so in this study, the spectral data (1HNMR, 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, 1HNMR, CHNS data for POM-1 and POMo are as follow, and the XRD pattern for POMo in compared to that report by Marcoux et al. has been provided in figure 2. As it can be seen, the similarity between two patterns of prepared POMo and that retrieved as standard XRD is definite.
[TBA]4[α-Mo8O26] (POM-1); FTIR (KBr): n (cm−1) 3445 (w, br), 2968(s), 2938 (s), 2875 (s), 1615 (w), 1473 (s), 1371(m), 1339 (w), 1149 (w), 957 (s), 928 (s), 910 (s), 862 (s), 810 (s), 663 (s), 562 (w), 505 (w), 413 (w).
[TBA]3[MnMo6O18(24)2] (POMo); FTIR (KBr): n (cm−1), 3448 (w, br), 2960 (s), 1479 (s), 1040 (s), 939 (s), 917 (s), 900 (s), 663 (s); 1H-NMR (400 MHz, DMSO-d6): 0.94 (t, 36 H), 1.32 (m, 24 H), 1.57 (m, 24 H), 3.12 (m, 24 H), 61.8 (s, 12 H); Elemental analysis: calculated for C56H124MnMo6N5O24: Elemental Analysis: C 35.73, H6.64, Mn 2.92, Mo 30.59, N 3.72; found experimentally C 35.70 %, H 6.75%, N 3.61, Mn 2.86%, Mo 30.28%; UV-Vis. (CH3CN): emax, 220, 254, 356 nm.
The structure of (T2POMo) were characterized by 1HNMR spectroscopy, FTIR spectroscopy, and CHNS elemental analysis as well as UV-vis. spectroscopy, the results are as follow:
T2POMo bio-conjugate;
FTIR (KBr): n (cm−1) 3489 (w, br), some medium weight bands below 3000 cm-1, 1741.8 (m, ester C=O of TS), 1688.7 (m, newly formed amide C=O), 1479 (m), 1041 (s), 939 (s), 917 (s), 662 (s).
1H-NMR (400 MHz, DMSO-d6): 0.96 (t, 36 H, POMo), 1.32 (m, 24 H, POMo), 1.56 (m, 8 H, TS), 1.69 (m, 4H, TS), 1.72 (m, 24 H, POMo), 3.16 (m, 24 H, POMo), 3.34-342 (m, 6 H, TS), 4.15 (s, 2H, TS), 4.30 (s, 2H, TS), 6.38 (m, 4H, TS), 9.38 (bs, 2H, newly formed amide NH), 61.60 (s, 12 H, POMo);
Elemental analysis:
calculated for C122H230MnMo6N5O32: C, 50.36 %; H, 7.97 %; N, 2.41 %; Mn, 1.89 %; Mo, 19.79 %
found experimental C, 50.10 %; H, 8.05 %; N, 2.49 %, Mn, 1.81 %; Mo, 19.65 %.
UV-Vis. (CH3CN): 207, 224, 256, 293, 388 nm.
Based on the available reports, six edge-sharing MoO6 octahedral are arranged around a core of the MnO6 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 to outside of POM and are available for further modification The organic groups cover both sides of the planar hexagon through the chemical bonding to the amine groups of TRIS (25).
As can be seen in Figure 1, we used both amine groups of POMo for the functionalization with TS. The amidation reaction between the carboxylic acid of TS with POMos 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 T2POMo conjugation was confirmed by elemental analysis, FT-IR spectroscopy, and 1H NMR spectroscopy. With an in-depth look at FTIR spectrun (figure 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 T2POMo due to the conjugation, we also see the primarily ester band of TS in the related place around 1741 cm-1. Finally, some spectral details related to TS have been appeared in corresponding area in final conjugation 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 1HNMR of T2POMo is the best complementary one, the all fundamental signals of TBA and TRIS in the POMo scaffold are relocated intact in T2POMo. As shown by Marcoux et al. (25), because of strong electron-withdrawing identity of POM, its methylene protons (belong to TRIS) are appeared around 60-62 ppm in 1HNMR spectra with the right signal ratio to other related peaks. Along characteristic signals of TS and POMo, the signal of NH amide was correctly appeared around 8.6 ppm with the exact signal ratio to the POMo CH2 moieties around 61 ppm (as shown in figure 4). Based on these spectral proofs, the conjugation of two nolecules of TS to 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 approved finally (25). Furthermore, UV-vis. spectroscopy (figure 5), showed the characteristic bands for both of 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 bio-conjugate (T2POMo) spectrum confirms the combination of the two components as well. There are some small changes in maximum absorption wave length of components which are in line with those reported by others for hybrid organic-inorganic conjugations (34).
- Stability of T2POMo conjugate
Before in vitro cytotoxicity evaluation, the stability of the T2POMo conjugation should be checked in the same condition as MTT assay protocol. In this regard, the stability of T2POMo conjugation was analyzed using the UV-Vis. spectrum of the dissolved sample after specified times (instantly, 24h, 48h, and 72h after) (35, 36).
The UV/vis spectrum of T2POMo in PBS (Figure 6) clearly indicates its stability around neutral pH conditions through monitoring of the characteristic of POM absorption bands, i.e.
The characteristic of T2POMo 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 Cytotoxicit Assessments (MTT assay)
To study the effect of TS conjugation on the cytotoxicity profile of POMo in 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 – 400 mg/mL of TS, POMo and T2POMo. Furthermore, the normal cell cytotoxicity was evaluated on human umbilical vein endothelial cells (HUVEC) in the same way using a concentration of 400 mg/mL.
The results of in vitro cytotoxicity for the final conjugation (T2POMo) in comparison to the POMo and TS, on the MCF-7, LNCAP, and HUVEC cells are presented in figures (7, 8 and 9). Figure 7 represents the cytotoxicity profile of T2POMo in two different incubation times and different concentrations on MCF-7 cell line. As it can be seen, the cytotoxicity profile is fully time and dose responsive (p<0.05 for each comparing). Based on these initial results, we selected the 24h for incubation time and the comparative cytotoxicity of POMo and T2POMo have been evaluated on both of MCF-7 and LNCaP cell lines (figure 8). Eventually, Figure 9 represents the comparative cytotoxicity of POMo and T2POMo 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 figure 8 (up), T2POMo exhibited considerably a better growth inhibition effect on MCF-7 cells compared to POMo and TS. The IC50 of the T2POMo and POMo on MCF-7 were 167.3 mg/mL and 321.7 mg/mL respectively. On the other hand, both of POMo and T2POMo showed somewhat less cytotoxic effects on LNCAP (figure 8 down), the IC50 of T2POMO and POM on the LNCAP were respectively 234.1 mg/mL and 382.2 mg/mL estimated. The better detected activity on MCF-7 could be attributed to the higher value of tocopherol responsivity in this type of cells 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 results of normal cell line (figure 7).
The protective effects of tocopherol, mentioned earlier (40), appear to help reduce toxicity on the normal cell line. The cytotoxicity of T2POMo and POMo were evaluated on the HUVEC cells at the concentration of 400 mg/mL, which was high enough to see the cytotoxic effects. Interestingly, we did not get any considerable cytotoxicity on HUVEC in comparison to the positive control (cis-platin) at the same concentration for T2POMO. As seen in Figure 8, both POMo and T2POMO have higher cell viability compared to the cis-platin at the same concentration (* and $ mean significant difference between each groups and positive control), and this effect is recognized much profoundly in the case of T2POMo comparing Cis-Platin (pvalue < 0.05). Furthermore, there are significant difference between all treating groups and control group with 100% of viability (@ means significant difference with control).
The more cytotoxicity of T2POMo compared to the POMo, can be a result of the inherent toxicity of the POMo, besides its facilitated cell entry through tocopherol receptors. In other words, the cytotoxicity of the T2POMo was improved by higher cell endocytosis of the conjugation through the tocopherol receptors. Although the cellular behavior of T2POMo 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 mechanisms that are predominant in MCF-7 cells relative to the LNCAP ones. Based on the evidence obtained from the Human Protein Atlas database, the MCF-7 tumor cells have more expression of the tocopherol-binding protein (HLCS gene) than the prostate tumor cells; then, we can attribute the observed results to this fact (41).
-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 haemolytic activity of the POMo and its bioconjugation T2POMo (figure 9) was evaluated in erythrocytes from rat employing standard methodology. The subsequent release of haemoglobin was used to assess haemolytic activity as the function of concentration, with concentrations ranging from 50 to 400 μg/mL. Based on obtained results, in all concentrations, the T2POMo conjugation is significantly safer (Pvalue <0.05) than the POMo even at 400 mg/mL. This safety is profoundly apparent in higher concentrations, and as it can be seen even at a concentration of 400 mg/mL the total percent of hemolysis is still below 5 percent in the case of T2POMo which is the promising outcome (31). It seems that for both POMo and T2POMo the best concentration for being safe to RBC is 200 mg/mL.
-Apoptosis quantification using flowcytometry 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 T2POMo (200 mg/mL), incubated for 24h, and finally were stained by Annexin V/propidiumiodide (PI). The Annexin V binds to cells in 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 figure 10, 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 early apoptosis stage. The results showed that the proportion of apoptotic cells (in early and late phase) increased by the addition of POMo and T2POMo to 36.56%, and 60.88 % respectively. The proportion of late apoptotic cells induced by the T2POMo is significantly higher than the POMo (18.36 % vs. 5%), and 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. The same results have been reported by peers 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-functionalization of the POMs for bio-recognition, cell internalization and biomimetic catalysis (44).
This result always is promising for a new cytotoxic compound, reducing the side effects or improving the biocompatibility accompanying by the better cytotoxicity profile.