Nanoparticles: the next step in endodontic medicaments

• Autores: Firas Elmsmari
• Directores de la Tesis: José Antonio González Sánchez (dir. tes.), Elena Sánchez López (codir. tes.)
• Lectura: En la Universitat Internacional de Catalunya ( España ) en 2021
• Idioma: español
• Programa de doctorado: Programa de Doctorado en Ciencias de la Salud por la Universitat Internacional de Catalunya
• Calificación: Sobresaliente Cum Laude
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• Resumen

  • Aim: The aim of the current study was to develop two formulations loading calcium hydroxide (Ca(OH)₂) and Clobetasol Propionate into poly lactic-co-glycolic acid (PLGA) biodegradable nanoparticles (NPs) to be used in the field of endodontics as an intracanal medications, including NPs optimization and characterization, plus drug release profile of the NPs compared to free drug counterpart. Additionally, comparison of the depth and area of penetration of the NPs inside the dentinal tubules against the free drug was carried out. Furthermore, the therapeutic efficacy for both formulations were examined using antibacterial and anti-inflammatory assays.

    Methodology: NPs were prepared using the solvent displacement method. Optimization of the NPs was carried out with a central composite design to obtain a final optimized formulation. The NPs morphology was examined under transmission electron microscopy (TEM), plus characterization was done by x-ray diffraction (XRD), fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). Moreover, the optimal conditions and cryoprotectants for freeze drying of the NPs were examined and tested. The drug release profile of the NPs and free drug was evaluated up to 48 hours. Additionally, the depth and area of penetration inside the dentinal tubules was examined for both the NPs and free drug. Antibacterial testing was performed on the antibacterial NPs to evaluate their efficacy in eliminating different bacterial strains. Agar disk-diffusion and broth dilution were used to determine the inhibition growth zones and the minimal inhibitory concentration (MIC). Likewise, anti-inflammatory testing was performed on the Clobetasol Propionate anti-inflammatory NPs, in which their response and reaction against inflammatory cells, in particular against macrophages was tested using the enzyme-linked immunosorbent assay (ELISA) to examine the cytokine release of IL-1β and TNF-α.

    Results: Using the solvent displacement method, both formulations of NPs were successfully optimized. The characteristics of the NPs utilizing the optimized formula showed a polidpsersity index (PI) value lower than 0.2, characteristic of monodisperse systems and an average size below 200 nm along with a highly negative surface charge (measured as zeta potential, ZP) and maximum entrapment efficiency (EE) percentage. For both the Ca(OH)₂/PLGA and the clobetasol propionate/PLGA NPs, the characterization of the NPs was performed, and the spherical morphology was confirmed using TEM. Moreover, interaction studies were carried out including XRD, FTIR and DSC to examine the profile of the NPs. These studies confirmed that no covalent bonds were formed during the preparation process and that the drug appeared to be encapsulated inside the NPs. Examining the drug release profile for the Ca(OH)₂/PLGA NPs, the Ca(OH)₂ NPs exhibited a prolonged and steady release with higher concentrations than those obtained with the free Ca(OH)₂, that remained stable up to 48 hours. Moreover, clobetasol propionate/PLGA NPs, exhibited a prolonged and steady release with only around 21 % of the encapsuled drug released after 48 hours, in comparison the free drug that was completely released after just 6 hours. For the freeze drying of the Ca(OH)₂/PLGA NPs, the combination of 5% of (2-Hydroxypropyl)-β-cyclodextrin and 15% D-mannitol gave rise to the most stable outcome and the best appearance after lyophilization being able to recover the initial physicochemical characteristics of the NPs after their resuspension. Unfortunately, for the clobetasol propionate/PLGA NPs, we were not able to achieve a suitable combination. Using the confocal laser scanning microscopy, it was shown that both the Ca(OH)₂/PLGA and the clobetasol propionate/PLGA NPs had a better depth and area of penetration inside the dentinal tubules when compared to the free drug. Plus, the NPs displayed higher values mean fluorescence intensity (MFI) and integrated density compared to the free drug. Assessing the antibacterial efficiency of the Ca(OH)₂/PLGA NPs using the agar diffusion test, it was noted that the NPs showed clear maximal zones of growth inhibition around the filter papers comparable only to the positive group of antibiotics. Additionally, the MIC of the NPs was measured in regard to inhibiting bacterial growth for 3 bacterial species. It was noted that after 24 hours of incubation the NPs were able to inhibit bacterial growth at all tested concentrations. The anti-inflammatory capacity of the clobetasol propionate/PLGA NPs was also examined, in which ELISA assays were used to evaluate the inflammatory cytokines release of the macrophages in response to the NPs. The releases of TNF-α was reduced considerably to almost undetectable amounts with NPs compared to the LPS negative control, displaying results similar and even less than the TCP positive control after 48 hours. However, resulted in the release of IL-1β in higher amounts which were similar to the TCP positive control and lesser than the LPS negative control group. Additionally, the inflammatory cytokines release of IL-1β and TNF-α from the LPS pretreated macrophages, was not reduced with the NPs at all tested concertation to limits below the amounts obtained by the LPS pretreated macrophages or the TCP positive control group after 48 hours.

    Conclusions: In the present study, Ca(OH)₂ loaded PLGA NPs and clobetasol propionate loaded PLGA NPs were successfully optimized and characterized, and their therapeutic efficacy was tested with aims to increase the antibacterial and anti-inflammatory effect through controlled drug release and reach areas of complicated root canal anatomy due to the smaller mean nanoscopic size. However, further future studies are still required in order for the developed NPs to take the next step to be utilized in a clinical condition and to achieve its goal as an intracanal medicament.