Formulation and Characterisation of Lignocaine-Adrenaline Nanosuspension Gingival Retraction Cord Preliminary In Vitro and In Vivo Evaluation for Painless and Haemostatic Effects

Abstract

Dental anxiety (DA) refers to fear, anxiety, or stress associated with dental procedures, and is often triggered by discomfort during treatment. The placement of gingival retraction cords, which are necessary for complex restorations and crown work, can cause gingival bleeding when removed. Current retraction cords do not offer painless placement, and while local anaesthesia (lignocaine) is used to numb tissues, it can exacerbate DA. Ferric sulphate, a haemostatic agent, may cause discolouration and does not prevent recurrent bleeding after cord removal, affecting the aesthetic outcomes. Thus, the lignocaine-adrenaline combination is valuable for both haemostasis and pain relief. This study aimed to characterise and evaluate the stability of an optimised lignocaine-adrenaline nanogel using a central composite design (CCD), conducting an in vitro cytotoxicity test, and in vivo for efficacy testing of the formulation. Compatibility was assessed using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) and ultraviolet-visible (UV-Vis) spectroscopy. 18 lignocaine-adrenaline nanoemulsions (LANEs) derived via CCD were characterised by their zeta potential, polydispersity index (PdI), pH, and particle size. All LANEs were transformed into nanoemulsion-based gels (NBG) by incorporating 0.1% Carbopol 940. Stability studies of LANE and NBG were conducted for over 12 months at 25 �C. The stability data for LANE and NBG were integrated with the CCD predictions to produce the optimised NBG. The optimised NBG was validated in triplicate and centrifuged at 5000 rpm for 30 min, freeze-thaw cycles at -5 �C and 25 �C, and repeated thermocycling at 4 �C and 40 �C. The in vitro drug release profile was gathered using Franz diffusion cells with cellulose acetate membrane 0.45 �m pore size. The samples were taken at predetermined times of 0, 5, 10, 15, 17, 20, 25, 30, 40, 60, 90, and 120 min, and the absorbance was read with UV-Vis at the respective lignocaine and adrenaline absorbance wavelengths. The in vitro cytotoxicity test was performed by MTT assay using the Ea.hy926 cell line with two-fold serial dilutions in 96-well plate, and the reading was performed using an ELISA microplate reader. In the in vivo study, the efficacy of a gingival retraction cord impregnated with lignocaine-adrenaline nanogel was evaluated in 18 Dunkin Hartley guinea pigs, divided into experimental, positive, and negative control groups. The investigation included clinical assessment of bleeding time and pain management, along with histopathological examination (HPE) analysis of tissue samples to assess inflammation and toxicity, with comparisons within the three control groups. ATR-FTIR and UV-Vis confirmed compatibility between lignocaine-adrenaline and excipients. The nanogel exhibited viscosity comparable to that of ferric sulphate (24 � 1 mPa.s) at 20 �C. Post-stability assessment, LANEs and NBGs showed no phase separation. The optimised LANE exhibited a zeta potential of -26.47 � 0.02mV, PdI of 0.36 � 0.01, pH of 6.28 � 0.02, and particle size of 61.76 � 0.25 nm. The optimised NBG was stable under stress conditions. The in vitro drug release studies showed that lignocaine followed the Korsmeyer-Peppas model (R2 = 0.95, n = 1.01), indicating non-Fickian diffusion, whereas adrenaline displayed zero-order release (R2 = 0.9862), providing consistent and prolonged drug release. Cytotoxicity studies revealed an IC50 to be 7.98 mM, which was lower than the 2% (85.29 mM) lignocaine concentration in the nanogel. However, the IC50 concentration was ineffective in controlling bleeding, in contrast to the 2% lignocaine-adrenaline nanogel, for which the concentration was emulated from the market LA anaesthesia concentration. Therefore, the assessment of 2% toxicity was performed in in vivo studies in which necrosis was absent in HPE findings. The lignocaine-adrenaline control group had a significantly shorter bleeding time (p-value < 0.001) with 41.17 � 1.47 seconds and a pain score of 4, indication no pain. The group also shows significant reduction in haemorrhage and increasing of cell count from 96.17 � 0.75 cells/mm2 at 10 minutes to 97.50 � 0.85 cells/mm2 at 15 minutes, with statistically significant p-value < 0.01, suggesting the nanogel may enhance cellular proliferation or activity in the tissue. Overall, the lignocaine-adrenaline nanogel demonstrated good haemostatic control, reduced inflammation, and minimised pain, making it a promising addition to the clinical dental practice for gingival retraction procedures.