Cinnamaldehyde-cucurbituril complex: investigation of loading efficiency and its role in enhancing cinnamaldehyde in vitro anti-tumor activity

Abstract

This study aimed to clarify the physico-chemical properties of cucurbit[7]uril (CB[7]) and cinnamaldehyde (Cinn) inclusion complexes (CB[7]-Cinn) and their resulting antitumor activity. CB[7]-Cinn inclusion complexes were prepared by a simple experimental approach and fully characterized for their stoichiometry, formation constant, particle size and morphology. Quantum chemical calculations were performed to elucidate the stable molecular structures of the inclusion complexes and their precursors and to investigate the probable stoichiometry and direction of interaction using three different DFT functionals at the 6-31G(d,p) basis set. The UV-vis spectrophotometric titrations as well as the Job plot, based on ¹H NMR spectroscopy, suggested 1 : 1 and 1 : 2 stoichiometries of CB[7] : Cinn. The formation constants of the complexes were calculated using Benesi-Hildebrand equations and non-linear fittings. Moreover, the theoretical calculations confirmed the potential formation of 1 : 1 and 1 : 2 stoichiometries and clarify the orientation of binding from the Cinn phenyl moiety. The nanoparticles' TEM images showed a crystal-like spherical shape, smooth surface, with a small tendency to agglomerate. CB[7]-Cinn inclusion complexes were analyzed for their antitumor activity against MDA-MB-231 breast cancer and U-87 glioblastoma cell lines. The IC₅₀ values were calculated after 72 hours of incubation with different concentrations of CB[7]-Cinn inclusion complexes and compared to free Cinn and free CB[7]. The IC₅₀ values for free Cinn and CB[7]-Cinn inclusion complexes were 240.17 ± 32.46 μM and 260.47 ± 20.83 μM against U-87 cells and 85.93 ± 3.35 μM and 176.3 ± 7.79 μM against MDA-MB-231 cells, respectively, despite the enhanced aqueous solubility. No significant cytotoxicity was noticed for the free CB[7].

Fig. 1. Chemical structure of (a) cinnamaldehyde and (b) cucurbit[7]uril.

Fig. 2. ¹H-NMR spectrum of (a) CB[7] and (b) Cinn, in D₂O at room temperature, with the protons assigned.

Fig. 3. ¹H NMR spectra of 1 : 1 CB[7]–Cinn inclusion complex in D₂O compared to the free Cinn in D₂O.

Fig. 4. Benesi–Hildebrand plots of (a) 1/ΔA versus 1/[CB[7]] and (b) 1/ΔA versus 1/[CB[7]]² for CB[7]–Cinn inclusion complex using UV-vis spectrophotometric titration.

Fig. 5. Job's plot of CB[7]–Cinn inclusion complex with respect to (a) Cinn and (b) CB[7], where χ is the mole fraction.

Fig. 6. ¹H NMR stacked spectra in D₂O solvent of Cinn with CB[7] at different mole fractions according to Job's plot method.

Fig. 7. The different possible complexes present at equilibrium.

Fig. 8. Average size distribution by intensity with different time point for 1 : 1 inclusion complex nanoparticles of CB[7]–Cinn.

Fig. 9. Transmission electron microscope (TEM) images of CB[7]–Cinn.

Fig. 10. A proposed structure for inclusion of Cinn into CB[7] with different orientations (a) aldehyde side and (b) phenyl side (by Gaussian view 09).

Fig. 11. Stability energy diagrams of the inclusion complexation of the Cinn from the aldehyde side into CB[7] at different DFT level of theory using (a) PBE0, (b) M06-2X and (c) B3LYP functional with 6-31G(d,p) basis set and CPMC model with solvation.

Fig. 12. Stability energy diagrams of the inclusion complexation of the Cinn from the phenyl side into CB[7] at different DFT level of theory using (a) PBE0, (b) M06-2X and (c) B3LYP functional with 6-31G(d,p) basis set and CPMC model with solvation.

Fig. 13. (a) A proposed molecular structure for inclusion of two molecules of Cinn into CB[7] from the phenyl side (by Gaussian view 09). (b) Stability energy diagrams of the inclusion complexation of the two Cinn from the phenyl side into CB[7] at DFT level of theory using B3LYP functional with 6-31G(d,p) basis set and CPMC model with solvation.

Fig. 14. The dose–response curve for (a) U-87 and (b) MDA-MB-231 cells treated with free Cinn, free CB[7] and CB[7]–Cinn. All cytotoxicity values represent the average ± SD of three independent experiments.