Cytotoxicity and enzyme inhibition studies of polyoxometalates and their chitosan nanoassemblies

Abstract

Polyoxometalates (POMs) have become very significant in biomedical research for their structural diversity which renders them highly active against bacterial, viral and cancer diseases. In this study three different POMs were synthesized and nanoassemblies were made with chitosan (CTS), a natural biodegradable polymer with excellent drug carrier properties. The compounds were tested on two isoenzymes of alkaline phosphatases including tissue specific calf intestine alkaline phosphatase (CIAP) and tissue non-specific alkaline phosphatase (TNAP). Compound [TeW₆O₂₄]⁶⁻ (TeW₆) showed the highest activity (45.4 ± 11.3 nM) among tested compounds against TNAP. Similarly, chitosan-[TeW₆O₂₄]⁶⁻ (CTS-TeW₆) was proved to be a potent inhibitor of CIAP with K_i value of 22 ± 7 nM. A comparative study was made to evaluate their cytotoxic potential against HeLa cells. Among all tested compounds, Chitosan-[NaP₅W₃₀O₁₁₀]¹⁴⁻ (CTS-P₅W₃₀) has showed higher percent cytotoxicity (88 ± 10%) at 10 μM when compared with the standard anticancer drug vincristine (72 ± 7%). The study revealed that selected POMs proved excellent anticancer potential and were equally effective against alkaline phosphatase enzyme, an increased level of which may indicate cancer metastasis.

1. Introduction

Polyoxometalates (POMs) are discrete metal oxide clusters of early transition metal ions in high oxidation states (e.g. W⁶⁺, Mo⁶⁺, V⁵⁺), which exhibit exceptional diversity in shape, size, and structural properties [1], [2], [3], [4]. POMs are classified in two distinct families, namely, iso- and heteropolyanions [5], [6]. The number of heteropolyanions is larger than that of isopolyanions, thus making them highly interesting compounds for several applications and fields of study. Such attributes, in addition to their solubility and stability at physiological pH, render them very attractive in medicine [6], [7], [8], [9], [10]. Another promising feature of POMs is that they can easily interact and bind to the target macromolecule because of their diversity in size, shape, polarity, surface charge distribution, and redox potential which improves their bonding properties [11].

Bioinorganic metal complexes have been studied extensively for targeted anticancer drug therapy [12], [13], [14], [15]. The most promising metal-based anticancer drug is cisplatin, which is effective against lymphomas, head, neck, bladder and ovarian cancers [16], [17], [18]. POMs have excellent potential to treat various types of cancers including pancreatic cancer [19], leukemia [20], hepatocellular carcinoma [21], colon carcinoma [22], ovarian cancer [23], gastric cancer [24], among others. Moreover, POMs were reported to stimulate insulin secretion and regenerate pancreatic beta cell function in rodents [25], [26], [27], [28], [29], [30], [31]. POMs are also very effective in treating bacterial [32], [33], [34] and viral [35], [36], [37], [38] diseases. A very recent study shows the ability of POMs, specifically [TeW₆O₂₄]⁶⁻, to act as a co-crystallization agent in the isolation and crystallization of the latent isoform PPO4 mushroom (Agaricus bisporus) tyrosinase protein which helps in the catalytic activity of phenols oxidation to quinones which in turn are responsible for the generation of melanins by polymerization [39].

Encapsulation of POMs into a biodegradable polymer has been studied previously, which enhances its stability at physiological pH, hence improving antiviral, antibacterial and anticancer properties [33], [40], [41], [42], [43]. Chitosan (CTS) is a natural, biodegradable polymer that has become one of the most widely studied biomaterial so far. Chitosan is obtained by de-acetylation of chitin, an essential polysaccharide, which is found in the exoskeleton of shrimps and crab shells [44], [45]. Chitosan can be effectively used in extended release drug delivery system because it is degraded in the human body by ubiquitous enzymes chitosanase and lysozyme which aid in releasing the drug in a controlled and precise manner [46], [47]. A combination of chitosan with inorganic, bioactive compounds suggests high potential for the development of new drug molecules with low toxicity, high stability and effective bio-distribution [48], [49], [50], [51], [52].

Ectonucleotidases represent a membrane-bound enzymes family, which mainly includes ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and alkaline phosphatases that are involved in nucleotide metabolism. Inhibition of these enzymes may increase the immune response and helps in combating cancer, auto-immune diseases as well as bacterial and viral infections [53]. POMs have been identified as potent and selective inhibitors of NTPDases [54] and alkaline phosphatases [55], [56]. Alkaline phosphatase level usually rises in colon, breast, lung, thyroid and prostate cancer when it metastasizes to bone and liver [57], [58], [59], [60], [61], [62].

In the present study, POMs and their respective CTS associated nanoassemblies were synthesized, structurally characterized and evaluated for inhibition activity against alkaline phosphatase isoenzymes (TNAP and CIAP). In addition, a comparative in vitro cytotoxicity analysis of synthesized compounds with standard anticancer drug vincristine (VCN) was carried out on human cervical adenocarcinoma (HeLa) cells.

2. Materials and methods

All chemicals used for the synthesis of the POMs were purchased commercially and used as received without further purification. Chitosan (YC-100) (M ∼10,000 g/mol), tissue non-specific alkaline phosphatase enzyme (TNAP), L-phenylalanine, levamisole, Tris–HCl, Tris base, MgCl₂, ZnCl₂, RPMI-1640, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Sigma–Aldrich, Steinheim, Germany. Tissue specific alkaline phosphatase enzyme extracted from calf intestine (CIAP) was obtained from Calbiochem, Germany. Human cervical adenocarcinoma cell line (HeLa) was purchased from ATCC (ATCC CRL-5802) and vero cell line was acquired from RIKEN Bio Resource Center, Japan.

POMs were synthesized and encapsulated with CTS to form CTS-POMs nanoassemblies. The synthesis and characterization of these materials are given below.

2.1. Synthesis of (NH₄)₁₄[NaP₅W₃₀O₁₁₀]·31H₂O

(NH₄)₁₄[NaP₅W₃₀O₁₁₀]·31H₂O (NH₄-P₅W₃₀) was synthesized according to a previously described method [63], and characterized by FT-IR (Fig. 3b) and ³¹P NMR spectroscopy. ³¹P NMR spectrum (Fig. 2a) obtained shows the characteristic signal at −9.4 ppm The structure of the polyanion is shown in Fig. 1a.

Fig. 3.

The FT-IR spectra of (a) CTS-P₅W₃₀, (b) P₅W₃₀ (red line), and (c) CTS (blue line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 2.

A representative NMR spectra of POMs. (a) ³¹P NMR spectrum of (NH₄)₁₄[NaP₅W₃₀O₁₁₀]·31H₂O ((NH₄)₁₄-P₅W₃₀) in H₂O/D₂O (δ = −9.4 ppm); (b) ⁵¹V NMR spectrum of Na₆[V₁₀O₂₈]·18H₂O (Na₆-V₁₀) in H₂O/D₂O (δ = −423.3 V_10A, −498.8 V_10B, −514.4 V_10C and −576.5 V₁ ppm); (c) ¹⁸³W NMR spectrum of Na₆[TeW₆O₂₄]·22H₂O (Na₆-TeW₆) in H₂O/D₂O (δ = −115.6 ppm).

Fig. 1.

Structural representations of the POMs. (a) [NaP₅W₃₀O₁₁₀]¹⁴⁻ (P₅W₃₀), (b) [V₁₀O₂₈]⁶⁻ (V₁₀), (c) [TeW₆O₂₄]⁶⁻ (TeW₆).

2.2. Synthesis of chitosan-[NaP₅W₃₀O₁₁₀]¹⁴⁻ nanoassembly

Chitosan-[NaP₅W₃₀O₁₁₀]¹⁴⁻ (CTS-P₅W₃₀) nanoassembly was synthesized by dissolving 0.50 g (0.71 mM) of chitosan (CTS) in 70 mL of distilled water. The resultant solution was filtered (solution I). Similarly, 1.24 g (21.38 mM) of [NaP₅W₃₀O₁₁₀]¹⁴⁻ was dissolved in 30 mL of H₂O and the obtained solution was filtered (solution II). Solution II was added dropwise to solution I, and then gel-like precipitates were formed and separated from the solution by means of filtration, washed and air-dried. The obtained product was characterized by FT-IR spectroscopy (Fig. 3a), where the appearance of 936, 910, 795, 737 and 570 cm⁻¹ bands confirmed the formation of the nanoassembly.

2.3. Synthesis of Na₆[V₁₀O₂₈]·18H₂O

Compound Na₆V₁₀O₂₈·16H₂O (Na₆-V₁₀) (Fig. 1b) was prepared by adapting previously described methods [64], [65]. Typically, NaVO₃ (3 g, 24.6 mmol) is dissolved in 100 mL water and HCl (4 M) was used to reduce the pH to 4.8. The solution was filtered and HCl was added to maintain the pH at 4.5. The bulk sodium salt of the polyanion was obtained by the addition of 200 mL ethanol (95%, v/v), leading to an orange color precipitates, which were removed by filtration and air-dried. The product was characterized by FT-IR (Fig. 4b) and ⁵¹V NMR spectroscopy, where V₁ corresponds to the monomeric (VO₄³⁻) vanadate species whereas V_10A, V_10B and V_10C signals correspond to the V(3), V(1) and V(2) vanadium atoms in the decameric vanadate species (V₁₀O₂₈⁶⁻). ⁵¹V NMR spectrum obtained (Fig. 2b) shows the characteristic signal of decameric (V10A at −423.3 ppm; V_10B at −498.8 ppm; V_10C at −514.4 ppm) and monomeric species (V1 at −576.5 ppm). The structure of polyanion is shown in Fig. 1b.

Fig. 4.

The FT-IR spectra of (a) CTS-V₁₀, (b) V₁₀ (red line), and (c) CTS (blue line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

2.4. Synthesis of chitosan-[V₁₀O₂₈]⁶⁻ nanoassembly

Synthesis of the chitosan-[V₁₀O₂₈]⁶⁻ (CTS-V₁₀) nanoassembly was carried out by dissolving 0.50 g (0.71 mM) of chitosan (CTS) in 70 mL of distilled water and filtered (solution I). A 0.43 g (10.1 mM) of [V₁₀O₂₈]⁶⁻ was dissolved in 30 mL of distilled water and filtered (solution II). The solution (II) was added dropwise to solution (I). The gel-like precipitates were formed and separated from the solution by means of filtration, washed and air-dried. The obtained product was characterized by FT-IR spectroscopy (see Fig. 4a), where the appearance of 1074, 950, 821, and 744 cm⁻¹ bands confirmed the formation of the nanoassembly.

2.5. Synthesis of Na₆[TeW₆O₂₄]·22H₂O

Na₆[TeW₆O₂₄]·22H₂O (Na₆-TeW₆) (Fig. 1c) was synthesized by previously reported method with some modifications [66] Briefly, A mixture of Na₂WO₄·2H₂O (5.0 g, 15.2 mmol) and Te(OH)₆ (0.6 g, 2.6 mmol) were dissolved in 100 mL of water. The pH was adjusted to 5.0 with HCl (1 M) followed by heating at 110 °C until about one fourth of the solvent has been evaporated. The solution was allowed to cool and filtered. Slow evaporation of the filtrate at room temperature for one week led to the formation of colorless crystals, which were collected by filtration and air-dried. The product was characterized by FT-IR (Fig. 5b) and ¹⁸³W NMR spectroscopy. ¹⁸³W NMR spectrum obtained (Fig. 2c) shows the characteristic signal at 115.6 ppm. The structure of polyanion is shown in Fig. 1c.

Fig. 5.

The FT-IR spectra of (a) CTS-TeW₆, (b) TeW₆ (red line), and (c) CTS (blue line). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

2.6. Synthesis of chitosan-[TeW₆O₂₄]⁶⁻ nanoassembly

Chitosan-[TeW₆O₂₄]⁶⁻ (CTS-TeW₆) nanoassembly was synthesized by dissolving 0.50 g (0.71 mM) of CTS in 70 mL distilled water. The resultant solution was filtered (solution I). Similarly, 0.64 g (9.9 mM) of [TeW₆O₂₄]⁶⁻ was dissolved in 30 mL H₂O and the product was filtered (solution II). Solution (II) was added dropwise into solution (I) leading to the formation of gel-like precipitates which were separated by filtration and washed several times with water and air-dried. The obtained product was characterized by FT-IR spectroscopy (Fig. 5a), where the appearance of 948, 903, 679, 617 and 556 cm⁻¹ bands confirmed the formation of the nanoassembly.

2.7. Zeta potential and particle size measurement

The CTS-POMs nanoassemblies were characterized by zeta potential to check their stability in colloidal suspension. Furthermore the particle size of the selected compound CTS-P₅W₃₀ was also determined.

2.8. Enzyme inhibition studies against alkaline phosphatases

Each compound was tested on TNAP and CIAP enzymes. Each enzyme was diluted in a buffer having pH 9.5 prepared from 50 mM Tris–HCl, 5 mM MgCl₂, 0.1 mM ZnCl₂ and 50% glycerol. A 0.5 mM enzyme substrate p-nitrophenyl phosphate (p-NPP) solution was also prepared in assay buffer (pH 9.5) comprised of 50 mM Tris–HCl, 5 mM MgCl₂, 0.1 mM ZnCl₂ without glycerol.

The initial screening of test compounds was done at 0.1 mM concentration by using a previously described spectrophotometric method [67]. The standard inhibitors were employed for initial screening to compare and evaluate the inhibition capacity of test compounds. Levamisole was used as standard inhibitor of TNAP [68] while l-phenylalanine was used for CIAP [69]. Briefly, the compounds which showed ≥50% inhibition were selected and further tested to determine dose–response curve of each inhibitor. Generally, each compound was pre-incubated with 10 μL of enzyme and 70 μL of assay buffer at 37 °C for 10 min A 10 μL of p-NPP was added and the mixture was again incubated for 30 minutes The product (p-nitrophenolate) formed was measured on ELISA plate reader (Bio-TekELx 800™, Instruments, Inc. USA) at wavelength of 405 nm. The experiments were done in triplicate. The respective K_i value of each inhibitor was calculated by using Prism 5.0 (GraphPad Software, San Diego, CA, USA).

2.9. Cell lines and cell cultures

HeLa cells and vero cells were cultured in a medium containing RPMI-1640 supplemented with l-glutamine (2 mM), penicillin (100 U mL⁻¹) and streptomycin (100 μg mL⁻¹) accompanied with 10% FBS and stored in an incubator supplied with 5% CO₂ at a constant temperature of 37 °C. When both adherent cell lines were confluent the cells were cultivated in 96-well plates at a seeding density of 10⁴ cells in 100 μL to conduct cytotoxicity assays.

2.10. Cytotoxicity analysis by sulforhodamine B (SRB) assay

A previously described colorimetric cytotoxicity assay was adopted which measures cellular protein content of cells [70]. Concisely, Hela cells and vero cells were seeded in 96 well plates for 24 h. Different concentrations of test compounds (10, 1, 0.1 μM) were made and inoculated in test wells of 96 well plates and incubated for up to 48 h. Likewise, the control and blank wells were also prepared containing VCN and culture medium respectively. After incubation, the cells were fixed with 50 μL of 50% ice cold TCA solution for 1 h. The plates were washed 5 times with PBS and air dried. The fixed cells were further treated with 0.4% (w/v) sulforhodamine B dye prepared in 1% acetic acid solution and left at room temperature for 30 min The plates were rinsed with 1% acetic acid solution and allowed to dry. In order to solubilize the dye, a 10 mM Tris base solution was added and left for 10 min at room temperature. The absorbance was measured at 490 nm subtracting the background measurement at 630 nm.

3. Results and discussion

3.1. Synthesis of POMs and their chitosan based nanoassemblies

POMs and their chitosan linked nanoassemblies were synthesized and characterized by NMR, and FT-IR. The results are presented in Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5.

3.2. Characterization of chitosan-POMs nanoassemblies

The successful formations of chitosan-POM nanoassemblies were confirmed by the appearance of characteristic bands in the region of 1000–500 cm⁻¹ in the respective FT-IR spectra as compare to that of simple POM FT-IR spectra. The stability of chitosan-POM nanoassemblies was determined by zeta potential measurement which was found in the range of −23 to −45 mV (see Table 1). The maximum stability was given by CTS-P₅W₃₀ with zeta potential of −44.56 ± 2.48 mV (see Fig. 6b) while particle size of CTS-P₅W₃₀ was also determined with transmission electron microscopy (TEM) that was 100 nM (see Fig. 6a). A possible reason for negative zeta potential is the presence of a highly negative charge on the polyanions [71].

Table 1.

Zeta potential measurement of POMs and their chitosan nanoassemblies.

Code	Particle size ± SEMa (nm)	Zeta potential ± SEMa (mV)
CTS-P5W30	100	−44.56 ± 2.48
CTS-V10	–	−23.83 ± 1.09
CTS-TeW6	–	−37.06 ± 2.55

^aSEM of 3 experiments performed in triplicate.

Fig. 6.

A representation of particle size measurement and zeta potential of CTS-POMs, (a) TEM image of CTS-P₅W₃₀ (b) zeta potential measurement of CTS-POMs nanoassemblies.

3.3. Inhibition studies on alkaline phosphatases

An elevated level of the alkaline phosphatase enzyme has been studied as a non-specific tumor marker associated with certain abnormalities in physiological functions of human body [72], [73]. In recent years, the biological significance of POMs has become apparent and many studies have been reported in this context [21], [54], [74]. The present study was presented on the synthesis and evaluation of POMs and their CTS associated nanoassemblies, which were found potent inhibitor of alkaline phosphatases with K_i values in nano molar range

POMs and CTS-POMs nanoassemblies were tested against two different isoenzymes of alkaline phosphatase, i.e. CIAP and TNAP. The test compounds were initially screened at 0.1 mM end concentration, which showed more than 70% inhibition on both isoenzymes. In order to find K_i value, a 3-fold serial dilution of each compound was made and K_i value was determined in nano molar range. Among CTS-POMs nanoassemblies the highest inhibition against CIAP was given by CTS-TeW₆, with a K_i value of 22 ± 7 nM, while CTS-P₅W₃₀ and CTS-V₁₀ also showed a decent inhibition with 31.1 ± 7.8 nM and 509 ± 17 nM, respectively. The TeW₆ was the most potent TNAP inhibitor among all tested compounds with a K_i value of 45.4 ± 11.3 nM. A possible reason for higher selectivity and inhibition potential of CTS-TeW₆ and TeW₆ may be due to the presence of tellurium metal, which has been proved previously in reducing ALP levels in diabetic rats [75]. Dose–response curves of were made (Fig. 7) and presented in a comparative manner to evaluate the activity against ALP isoenzymes (see Table 2).

Fig. 7.

The potential activities of POMs and their chitosan based nanoassemblies on CIAP (blue line) and TNAP (black line) isoenzymes. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Table 2.

Activities of POMs and CTS based nanoassemblies against CIAP and TNAP.

No.	Code	Molecular formula/structure	Ki (nM) ± SEMa
			CIAPb	TNAPc
1	P5W30	(NH4)14[NaP5W30O110]·31H2O	921 ± 6	1112 ± 36
2	CTS-P5W30	Chitosan-[NaP5W30O110]14−	31.1 ± 7.8	3179 ± 84
3	V10	Na6[V10O28]·18H2O	79.1 ± 2.3	169 ± 21
4	CTS-V10	Chitosan-[V10O28]6−	509 ± 17	308 ± 63
5	TeW6	Na6[TeW6O24]·22H2O	520 ± 13	45.4 ± 11.3
6	CTS-TeW6	Chitosan-[TeW6O24]6−	22 ± 7	1921 ± 54
7	l-phenylalanine		75%	–
8	Levamisole		–	71%

^aSEM = standard error of mean of 3 experiments.

^bCIAP (calf intestinal alkaline phosphatase).

^cTNAP (tissue non-specific alkaline phosphatase).

3.4. Anticancer activity studies

3.4.1. Cytotoxicity assay

POMs and their chitosan-encapsulated nanoassemblies were tested as anticancer agents on HeLa cells. The test compounds were prepared in different concentration ranges of 10, 1 and 0.1 μM. All test compounds showed good cytotoxicity against HeLa cells, even at the lowest concentration of 0.1 μM with minimum toxicity against vero cells (see Table 3). The maximum cytotoxicity against HeLa cells was observed with the compound CTS-P₅W₃₀ having percent inhibition of 88 ± 10 (see Fig. 8), that may be due to the higher number of phosphorous (P) and tungsten (W) atoms [55].

Table 3.

The cytotoxicity comparison of vincristine, POMs and their chitosan based nanoassemblies on Hela and vero cells.

Codes	Percent inhibition ± SEMa at 10 μM on vero cells	Percent inhibition ± SEM at 10 μM on Hela cells	Percent inhibition ± SEM at 1 μM on Hela cells	Percent inhibition ± SEM at 0.1 μM on Hela cells
VCNb	10 ± 4	72 ± 7	–	–
P5W30	15 ± 6	74 ± 10	62 ± 13	54 ± 6
CTS-P5W30	12 ± 5	88 ± 10	78 ± 15	65 ± 9
V10	16 ± 3	66 ± 15	57 ± 13	49 ± 11
CTS-V10	11 ± 2	81 ± 9	73 ± 9	58 ± 11
TeW6	16 ± 8	69 ± 10	62 ± 10	53 ± 12
CTS-TeW6	11 ± 6	84 ± 10	71 ± 12	64 ± 7

^aSEM Standard error of mean of 3 experiments performed in triplicate.

^bVCN (vincristine).

Fig. 8.

A graphical representation of multiple comparisons of anticancer activities between POMs, their chitosan based nanoassemblies and VCN.

A difference of cytotoxicity behavior was observed between P₅W₃₀, TeW₆, CTS-P₅W₃₀ and CTS-TeW₆, which support the idea that the encapsulation of POMs into a biopolymer may render polyanion more target oriented, with minimal adverse effects [76]. The vanadium (V) containing compounds have previously been reported as good anticancer and antidiabetic agents [77], [78]. Here we have synthesized compounds V₁₀ and CTS-V₁₀ which also showed promising cytotoxicity behavior 66 ± 15% and 81 ± 9% respectively. The compound CTS-TeW₆ has anticancer activity of 84 ± 10% against HeLa cells which may be due to the presence of tellurium atom, which was previously reported as anticancer agent [79], [80].

4. Conclusions

We can state that much insight has already been gained on the bio-activity of POMs. Here we have adopted a previously used technique to synthesize and encapsulate three different POMs; P₅W₃₀, V₁₀ and TeW₆ with CTS to enhance drug carrier properties. The anticancer potential of these synthesized compounds were studied against alkaline phosphatases and Hela cells in a dose-dependent manner to explore their capacity in minimizing side effects through dose adjustment. The anticancer activity of these nanoassemblies, in particular, CTS-P₅W₃₀ allows for new strategies of treating cancer. Among the tested compounds, especially CTS-POMs nanoassemblies showed minimum toxicity against normal cells. Our results suggest that the CTS-POMs nanoassemblies have a high potential of treating cancer and its metastasis.