Hydroxypropyl-β-cyclodextrin Enhance Apoptosis Induced by Articaine on Squamous Carcinoma Cells

Objectives: To perform the physicochemical characterization of articaine-hydroxypropyl-β-cyclodextrin inclusion complex (ATC_HPβCD) and to assess the cytotoxic effect on human oral squamous cell carcinoma (SCC) in vitro.
Methods: ATC_HPβCD was prepared by mixing equimolar quantities of ATC and HPβCD. ATC_HPβCD was characterized by UV-Vis absorption, fluorimetry, differential scanning calorimetry (DSC), high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR) and scanning electronic microscopy (SEM). Three different SCC cell lines and HaCaT cells (control) were exposed for 24-48h to different concentrations of ATC and ATC_HPβCD. Cell viability was assessed by using formazan dye (MTT) and Live/Dead® staining assay. Apoptosis were assessed by flow cytometry using Annexin V-FITC and 7-Amino-Acimomycin (7-AAD) staining. Anti-proliferative effect was assessed by using sulforhodamine-B dye.
Results: Maximum fluorescence peaks of ATC and ATC_HPβCD were at 348nm. Complexation kinetics between the ATC and HPβCD showed an equilibration time of 400min. There was a loss of endothermic peak of ATC (169°C) when complexed in HPβCD, as shown by DSC. NMR analysis showed an association constant of 117.8M^-1 between ATC and HPβCD and a complexed fraction of 41%. SEM showed the physical differences between these substances. ATC induced cell death in a concentration-dependent manner, especially when complexed in HPβCD. ATC_HPβCD LD₅₀ significantly lower than ATC LD₅₀ in all cell lines (P < 0.05). ATC_HPβCD induced earlier than later apoptosis in all cell lines when compared to ATC at 3.12mM. ATC_HPβCD also showed a significantly greater anti-proliferative effect than ATC in all cell lines (P < 0.05) in concentrations above 3.12mM.
Conclusions: Inclusion complex of ATC_HPβCD showed molecular stability at molar ratio 1:1. At equivalent concentrations, the cytotoxicity of the ATC and ATC_HPβCD is related to late and early apoptosis, respectively. ATC_HPβCD induced greater cytotoxic and anti-proliferative effects than the ATC, especially in concentrations above 3.12mM.