Novel Phenobarbital-Loaded Nanostructured Lipid Carriers for Epilepsy Treatment: From QbD to In Vivo Evaluation

Abstract

Pharmacological treatments of central nervous system diseases are always challenging due to the restrictions imposed by the blood-brain barrier: while some drugs can effectively cross it, many others, some antiepileptic drugs among them, display permeability issues to reach the site of action and exert their pharmacological effects. The development of last-generation therapeutic nanosystems capable of enhancing drug biodistribution has gained ground in the past few years. Lipid-based nanoparticles are promising systems aimed to improve or facilitate the passage of drugs through biological barriers, which have demonstrated their effectiveness in various therapeutic fields, without signs of associated toxicity. In the present work, nanostructured lipid carriers (NLCs) containing the antiepileptic drug phenobarbital were designed and optimized by a quality by design approach (QbD). The optimized formulation was characterized by its entrapment efficiency, particle size, polydispersity index, and Z potential. Thermal properties were analyzed by DSC and TGA, and morphology and crystal properties were analyzed by AFM, TEM, and XRD. Drug localization and possible interactions between the drug and the formulation components were evaluated using FTIR. In vitro release kinetic, cytotoxicity on non-tumoral mouse fibroblasts L929, and in vivo anticonvulsant activity in an animal model of acute seizures were studied as well. The optimized formulation resulted in spherical particles with a mean size of ca. 178 nm and 98.2% of entrapment efficiency, physically stable for more than a month. Results obtained from the physicochemical and in vitro release characterization suggested that the drug was incorporated into the lipid matrix losing its crystalline structure after the synthesis process and was then released following a slower kinetic in comparison with the conventional immediate-release formulation. The NLC was non-toxic against the selected cell line and capable of delivering the drug to the site of action in an adequate amount and time for therapeutic effects, with no appreciable neurotoxicity. Therefore, the developed system represents a promising alternative for the treatment of one of the most prevalent neurological diseases, epilepsy.

Keywords: PTZ test; anticonvulsant; drug delivery; epilepsy; nanostructured lipid carrier (NLC); phenobarbital; release kinetic; solid lipid nanoparticles (SLNs).

FIGURE 1

Response surfaces for particle size (A), PDI (B), and Z-potential (C) as a function of the amount of lipid (mg) and surfactant (mg).

FIGURE 2

AFM images of NLC-PB. On the left, two independent 2D images are presented. The 3D image corresponds to the upper one. In the size curves, the X-axis has an arbitrary origin, and thus it serves to measure differences (diameters). Attractive forces with the support and weak forces of the probe generate flattening in the analyzed NPs resulting in smaller z particle size.

FIGURE 3

TEM images of: (A) 1:10 dilution, (B) 1:500 dilution of NLC-PB optimized formulation, and (C) 1:10 dilution of empty NLC (NLC-vehicle).

FIGURE 4

Stacked DSC thermograms corresponding to PB, myristyl myristate, Poloxamer 188, NLC-vehicle, and NLC-PB. Note: Y-axis scale is the same for all thermograms.

FIGURE 5

TGA thermograms of PB, myristyl myristate, Poloxamer 188, NLC-vehicle, and NLC-PB were obtained in the range of 30°C–600°C.

FIGURE 6

XRD patterns of raw materials and nanoparticles with and without phenobarbital. Note: Y-axis scale is the same for all x-ray diffraction patterns.

FIGURE 7

Overlaid FTIR spectra of PB, myristyl myristate, Poloxamer 188, NLC-vehicle, and NLC-PB.

FIGURE 8

Release profiles of PB from NLC with different drug loading. Two controls were included: immediate-release PB tablets (Bayer Luminal®, 100 mg) and a solution of PB inside the dialysis bag (discontinuous grey line). The inserted graph corresponds to the time range from 0 to 6 h. Initial point (t = 0) was included for graphical purposes.

FIGURE 9

Cell viability analysis by MTT assay. L929 cells were treated with different concentrations (1–4 mM) of NLC-PB, NLC-vehicle, and PB.

FIGURE 10

In vivo evaluation of the anticonvulsant activity expressed as proportion (%) of mice protected at each post-dose time. Green bars represent positive control (free PB), orange bars the optimized formulation (NLC-PB), and violet bars the vehicle without drug (NLC-vehicle). Both free PB and NLC-PB, were tested at a dose of 25 mg/kg.