Evaluation of the antiproliferative effect of 18β -Glycyrrhetinic acid-loaded nanoparticles for treatment of drug-induced gingival overgrowth.

Introduction. Drug-induced gingival overgrowth (DIGO) is an oral disease that is detrimental to oral function and facial appearance; among them antiepileptics (i.e valproic acid) can increase the number of fibroblasts within gingival tissues (1). 18-β Glycyrrhetinic acid (GA) (a compound derived from liquorice) inhibits cell proliferation via cell cycle arrest and induction of apoptosis (2), and it has been demonstrated that could be used to treat Human Gingival Fibroblasts (HGFs) derived from DIGO patients (3). The aim of our study is to develop a delivery system able to provide a controlled release of GA in HGFs in vitro. For this purpose, we have selected polymeric nanoparticles (NPs) to enhance in situ release of GA, overcoming its poor solubility. Materials and Methods. Preparation of drug-loaded NPs: GA-loaded Poly-d,l-lactide-co-glycolide (PLGA) NPs were produced by using a one-step osmosis based methodology (patent Sapienza University n° RM2004A000555) and were successively coated with chitosane (CS) (4). Drug loading evaluation: The drug content of GA-loaded NPs was measured using a spectrophotometric method. NPs were dissolved in chloroform and the drug concentration was determined by measuring the absorbance of the solution at λ= 246 nm. Dynamic Light Scattering (DLS) Analysis: DLS experiments were carried out with a Zetasizer Nano S (Malvern Instruments, Malvern, U.K.) equipped with a 4 mW He-Ne laser (633 nm). Peak intensity analysis was used to determine the average hydrodynamic radius of the scattering particles. All samples were prepared at 0.1 mg/mL in filtered PBS. Isolation and culture of HGFs: Cells were obtained (with informed consent) from patients subjected to gingivectomy of the molar region. The specimens were plated in tissue culture flasks with complete DMEM, at 37°C, 5% CO2 atmosphere. The HGFs were used before the fifth passage. Cytotoxic Assay: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test was used to determine the GA concentration value able to provoke cytotoxicity. Statistical Analysis: Data were expressed as mean ± SD. Analysis was performed by ANOVA, p<0.05 was assumed significant. Results. The mean diameters of NPs was 200 nm with a GA content value of 26 μg/mg PLGA. No cytotoxic effect was observed in the presence of NPs. The toxic effect of GA was observed at concentration values higher than 100 μmol/L. Conclusions. Such formulations seem very promising for reducing cell proliferation in vitro, even if further studies will be needed to determine the concentrations of GA able to reduce the proliferation of HGFs derived from patients with DIGO pathology.