Anticancer Activity of a Hydrogel Containing Folic Acid Towards MCF-7 and MDA-MB-231 Cells

Materials and Methods

Reagents. All solvents of analytical grade were purchased from Carlo Erba Reagents (Milan, Italy): acetone, chloroform, dichloromethane, ethanol, ethyl ether, methanol, n-hexane, acetonitrile and triethylamine. L-Glycine (MW=75.07), 4,4’-dimetoxytrityl chloride (DMTrCl), trimethyl chlorosilane (Me₃SiCl), ellagic acid (MW=302.19), dicyclohexyl carbodiimide (DCC), dimethylaminoyiridine (DMAP), acryloyl chloride, ammonium persulfate (NH4)₂S₂O₈, N,N-dimethylacrylamide (DMAA), FA, tert-butyl hydroperoxide (t-BOOH) trichloroacetic acid (TCA) acid, 2-thiobarbituric (TBA), butylated hydroxytoluene (BHT), 4,5-dimethylthiazol-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulphoxide (DMSO) were purchased from Sigma-Aldrich (Sigma Chemical Co., St. Louis, MO, USA).

Cell culture. MCF-7 and MDA-MB-231 cells, obtained from the American Type Culture Collection (Manassas, VA, USA), were maintained in Dulbecco's modified Eagle's medium/Ham's F-12 containing 5% fetal bovine serum and supplemented with 1% L-glutamine and 1% penicillin/streptomycin (Sigma, Milan, Italy). The cells were cultured in serum-free medium (SFM) for at least 24 h before treatments.

Instruments. Infrared spectra were performed on KBr pellets using an FT-IR Perkin-Elmer 1720 spectometer (Norwalk, Connecticut, USA), in the range 4000-400 cm⁻¹ (16 scans). The ¹H-MNR were performed by a Bruker VM30 spectometer (Bruker, Zürich, Switzerland), the chemical shifts are expressed in δ and are related to the solvent. The structures of synthesized compounds was confirmed by (GC-MS) Hewlett Packard 5972 (HAZLET, New Jersey, USA). The UV-VIS spectra were carried out using JASCO-530 UV spectrophotometer (JASCO Europe s.r.l., Milan, Italy). Samples were freeze-dried using a freeze-drying Micro Modulyo Edwards apparatus (Hastings, UK).

Synthesis of N-trytilglycine (1). The reaction was conducted according to the procedure reported in literature (22). In a three-neck flask fitted with a reflux condenser, funnel dripper, magnetic stirrer, thoroughly flamed and maintained under nitrogen bubbling, L-glycine (0.27 g, 13 mmoles) was dissolved in 1.22 ml mixture of chloroform (0.98 ml) and acetonitrile (0.24 ml). The reaction mixture was maintained at 30°C under magnetic stirring. After dissolution, 0.42 ml (3.3 mmol) of trimethyl chlorosylane were added. The reaction was conducted for 2 h at 30°C. Thereafter, 0.97 ml (6.9 mmol) of dry triethylamine and dropwise 4,4’-dimethoxytrytil chloride (1.20 g, 13 mmol), dissolved in chloroform (15.77 ml) were added. The addition of 4,4’-dimethoxytrytil chloride caused a chromatic change from white to red. This mixture was maintained for one hour under magnetic stirring; then 0,71 ml of methanol were added causing chromatic change from red to grey-green. The mixture was left stirring for 2 h. Finally the colour was pale yellow. The reaction was monitored through (TLC) on aluminum oxide (eluent mixture: chloroform). The solvents were then evaporated under reduced pressure and the obtained solid was washed with diethyl ether and then with an aqueous solution of citric acid 5% w/v. The organic phase was treated with a solution of NaOH 1 N (7.18 ml) and water (3.59 ml). The phase was treated with cold diethyl ether (17.18 ml) and neutralized with glacial acetic acid and the obtained precipitate was extracted with diethyl ether, while the organic phase was washed with distilled water and dried for 2 h with magnesium sulphate. The solution was filtered and dried under reduced pressure. The obtained product (1), orange in colour, was characterized through FT-IR.

Esterification of N-trytilglycine with ellagic acid (2). The reaction was conducted in agreement with the procedure reported in literature (23). In a two-neck flask fitted with a reflux condenser, magnetic stirrer, thoroughly flamed and maintained under nitrogen bubbling, 0.50 g (1.32 mmol) of (1) and 0.39 g (1.32 mmol) of ellagic acid were dissolved in 2.64 ml of dry dichloromethane. After dissolution, DCC (0.35 g, 1.7 mmol) and DMAP (0.08 g, 0.65 mmol) were added. The reaction mixture was maintained at room temperature under magnetic stirring for 12 hours. The reaction was monitored through TLC on silica gel (eluent mixture chloroform/n-exane 5:5). Finally, the product was filtered to be purified from dicyclohexyl urea formed during the reaction. After that it was purified through a chromatographic column on silica gel (eluent mixture dichloromethane/petroleum ether 4:6). The obtained product (2) was characterized through FT-IR.

Detritylation reaction (3). The reaction was conducted in agreement with the procedure reported in literature (24). In a three-neck flask fitted with a reflux condenser, magnetic stirrer, thoroughly flamed and maintained under nitrogen bubbling, 0.15 g of (2) (0.22 mmol) were added to a solution of trifluoroacetic acid in dichloromethane 60% giving a red-colored solution. After that, 0.45 ml of methanol (0.59 mmol) were added and the mixture was stirred for one hour at room temperature until the observation of a chromatic shift from red to yellow-orange. The solvent was evaporated at reduced pressure and the obtained salt was dried. This latter was subsequently washed with 2.26 ml of a 1 N HCl solution in MeOH. The solvent was evaporised and the product was dried and washed with diethyl ether in order to eliminate the trytil moieties. The obtained product (3) was dried under reduced pressure and characterized through FT-IR and ¹H-NMR.

Acrylation of 3. In a three-neck flask fitted with a reflux condenser, magnetic stirrer, thoroughly flamed and maintained under nitrogen bubbling, product 3 (0.30 g, 0.83 mmol) was dissolved in 6 ml of dichloromethane. After that, 0.08 ml (0.99 mmol) of acryloil chloride and 0.14 ml (0.1 mmol) of triethylamine were added and the solution appeared yellow coloured. The reaction was maintained for 12 hours at room temperature and was monitored through TLC on silica gel (eluent mixture chloroform/methanol 9:1). Finally, the solvent was evaporated under reduced pressure and the obtained product (4) was characterized by FT-IR e ¹H-NMR.

Hydrogel preparation. In a two-neck flask fitted with a reflux condenser, magnetic stirring thoroughly flamed and maintained under nitrogen bubbling, product 4 (0.65 g, 1.57 mmol) was solubilized in an aqueous solution of NH₃/urea. Subsequently, DMAA (0.16 ml, 0.1 mmol) and ammonium persulphate (80.35 g, 0.35 mol) were added. Finally, the reaction was carried out at 60°C until the formation of the hydrogel. The obtained hydrogel was washed with diethyl ether in a porous filter, dried under vacuum and characterized through FT-IR (9).

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Figure 1.

Ellagic acid and glicine-based hydrogel containing folic acid.

Antioxidant activity evaluation. The ability of the obtained hydrogel to protect against lipid peroxidation induced by tert-BOOH, was examined in rat liver microsomal membranes during 120 min of incubation. Aliquots of both ellagic acid and ellagic acid-based hydrogel (EAH) were added to the microsomal suspension (9, 25-27). Then the suspensions were incubated at 37°C in a shaking bath under air in the dark. After incubation, thiobarbituric acid-malondialdehyde complex (TBA-MDA) formation was monitored by the use of UV-VIS spectrophotometry at 535 nm (25). The experiment was repeated in triplicate (n=3).

Swelling studies. The swelling characteristics of the hydrogel were determined in order to check for its hydrophilic affinity (9). Typically, aliquots (40-50 mg) of hydrogel dried to constant weight were placed on a tared 5-ml sintered glass filter (Ø10 mm; porosity, G3), weighed, and left to swell by immersing the filter plus support in a beaker containing the swelling media, namely phosphate buffers at pH 6.2 to mimic the conditions typical of tumor pathology, and pH 7.4 to simulate the physiological environment. At a predetermined time, the excess of water was removed by percolation at atmospheric pressure. Then the filter was placed in a centrifuge test tube, fixed with the help of a bored silicone stopper, then centrifuged at 1372 rcf for 15 min and weighed. This operation was repeated at the different times (1, 6, 12 and 24 h). The filter weight was determined after centrifugation with water alone. The weights recorded at the different times were averaged and used to give the water content (α, %) by the following equation: where Ws and Wd are weights of swollen and dried hydrogel, respectively.

Incorporation of FA into preformed hydrogel. The folic acid incorporation into microspheres was performed (9) as follows: 500 mg of preformed empty hydrogel (prepared as described above) were wetted with 6 ml in a concentrated solution of folic acid (15 mg/ml). After three days, under slow stirring at 37°C, the microspheres were filtered and dried at reduced pressure in the presence of P₂O₅ to a constant weight. The loading efficiency (LE, %) of all samples were determined by UV-Vis spectrophotometric analysis of filtered solvent according to the following equation: where C_i was the concentration of FA in solution before the loading study, C₀ the concentration of FA in solution after the loading study. LE was measured spectrophotometrically (λ=365 nm, ϵ=7595 mol⁻¹dm³cm⁻¹).

Drug release studies. Dried hydrogel (10 mg) was dispersed in 6 ml of swelling media (phosphate buffers at pH 6.2 and pH 7.4 (9). The test tubes were maintained at 37°C in an horizontal shaking bath and shaked at a rate of 100 rpm. At predetermined intervals, the samples were centrifuged, 5 ml of supernatant were removed and the medium was replaced with fresh solution to maintain the same total volume throughout the study. The concentration of FA was determined spectrophotometrically at 365 nm. The experiment was repeated in triplicate (n=3). The data were calculated in terms of the drug release percentage.

(MTT) assay. The cytotoxic effects of EAH and of EAH containing folic acid on human breast cancer cells were evaluated by MTT dye test (cell viability) (27, 28). The cells were seeded at a density of 1×10⁴ cells in 96-well tissue culture plates for 24 h at 37°C and 5% CO₂ to allow the adhesion of the cells. After 24 hours of incubation, the culture medium was replaced with SFM and EAH or EAH+FA were added at 50 μM. The toxicity experiments were carried out at 24 and 48 h of incubation. Untreated cells were used as control.

At the end of the incubation time, 50 μl of MTT tetrazolium salt (5 mg/ml dissolved in SFM) were added to each well. The plates were incubated for an additional four hours and then the medium was discarded. A volume of 150 μl of DMSO was added to each well, and the solution was vigorously mixed to dissolve the reacted dye. The absorbance of each well was read on a microplate reader (Multiskan EX, Thermo Scientific, USA) at a test wavelength of 570 nm with a reference wavelength of 690 nm. The sample was tested in triplicate and the percentage of viable cells, directly proportional to the amount of formazan crystals formed, was calculated using the following equation: % Cell viability=AbsT/AbsU ×100, where AbsT is the absorbance of treated cells and AbsU is the absorbance of untreated cells.

Statistical analysis. Data are expressed as the mean±S.D. of at least three independent experiments. Statistical analysis was performed using Student's t-test. p-Values ≤0.05 were considered statistically significant.