Dimethyl Fumarate-Loaded Transethosomes: A Formulative Study and Preliminary Ex Vivo and In Vivo Evaluation

Abstract

In this study, transethosomes were investigated as potential delivery systems for dimethyl fumarate. A formulative study was performed investigating the effect of the composition of transethosomes on the morphology and size of vesicles, as well as drug entrapment capacity, using cryogenic transmission electron microscopy, photon correlation spectroscopy, and HPLC. The stability of vesicles was evaluated, both for size increase and capability to control the drug degradation. Drug release kinetics and permeability profiles were evaluated in vitro using Franz cells, associated with different synthetic membranes. The in vitro viability, as well as the capacity to improve wound healing, were evaluated in human keratinocytes. Transmission electron microscopy enabled the evaluation of transethosome uptake and intracellular fate. Based on the obtained results, a transethosome gel was further formulated for the cutaneous application of dimethyl fumarate, the safety of which was evaluated in vivo with a patch test. It was found that the phosphatidylcholine concentration affected vesicle size and lamellarity, influencing the capacity to control dimethyl fumarate's chemical stability and release kinetics. Indeed, phosphatidylcholine 2.7% w/w led to multivesicular vesicles with 344 nm mean size, controlling the drug's chemical stability for at least 90 days. Conversely, phosphatidylcholine 0.9% w/w resulted in 130 nm sized unilamellar vesicles, which maintained 55% of the drug over 3 months. These latest kinds of transethosomes were able to improve wound healing in vitro and were easily internalised by keratinocytes. The selected transethosome gel, loading 25 mg/mL dimethyl fumarate, was not irritant after cutaneous application under occlusion, suggesting its possible suitability in the treatment of wounds caused by diabetes mellitus or peripheral vascular diseases.

Keywords: cryogenic transmission electron microscopy; dimethyl fumarate; transdermal delivery; transethosomes; wound healing.

Figure 1

Cryo-TEM images of TET_0.9-DMF_0.5 (a) and TET_2.7-DMF_0.5 (b). The bar corresponds to 200 nm in both panels.

Figure 2

Effect of storage on ethosomes and transethosomes kept at 22 °C for 3 months: (a) Z average mean diameters measured via PCS on TET_0.9 (o), TET_0.9-DMF (•), TET_2.7 (□), and TET_2.7-DMF (▪), p values < 0.05; (b) DMF residual content in T-ETO_0.9-DMF (•), T-ETO_0.9-DMF (▪) and SOL-DMF (▼). The percentage refers to the total DMF content evaluated after sample preparation; p values < 0.05.

Figure 3

DMF release kinetics from TET_0.9-DMF (•), TET_2.7-DMF (▪), and SOL-DMF (▼), as determined by Franz cells associated with PTFE. Data are the mean of 6 independent experiments ± s.d.

Figure 4

DMF permeability kinetics from TET_0.9-DMF (•), TET_2.7-DMF (▪), and SOL-DMF (▼), as determined by Franz cells associated with Strat-M^®. (a) linear part of the kinetic profile (0–8 h); (b) 0–24 h kinetic. Data are the mean of 6 independent experiments ± s.d.

Figure 5

HaCaT cell viability evaluated using an MTT test after 24 h of treatment with SOL-DMF differently diluted (from 0.625 µg/mL to 80 µg/mL) (a) and with differently diluted DMF-loaded or unloaded TET_0.9 and TET_2.7 (b). Data are the results of three independent experiments performed in triplicate. * p ≤ 0.01; ** p ≤ 0.005; *** p ≤ 0.0001 by two-way ANOVA followed by Tukey’s multiple comparison test; treatment vs. ctrl.

Figure 6

Effect of DMF-loaded or unloaded formulations on the wound closure in HaCaT cells: (a) a scratch test was performed on a confluent monolayer of HaCaT cells; different images were taken to measure the wound area right after the scratch (left panel T₀) and 24 h after (right panel T24) (scale bar 200 μm); (b) quantification of the wound area (within the red edges) 24 h after the scratch analysed by using ImageJ software, compared with T₀. Data are the results of three independent experiments performed in triplicate. **** p ≤ 0.0001 using one-way ANOVA followed by Tukey’s multiple comparison test.

Figure 7

Transmission electron micrographs of keratinocytes incubated with TET_0.9-DMF (a–c) or TET_2.7-DMF (d–f) for 2 h (a,d), 6 h (b,e), and 24 h (c,f). Bars: 1000 nm (a), 2000 nm (b,c), 3000 nm (d), and 1000 nm (e,f).

Figure 8

DMF permeability kinetics from TET_0.9-DMF50 (o), TET_0.9-DMF25-gel (□), and TET_0.9-DMF10-gel (x), as determined by Franz cells associated with Strat-M^®. Data are the mean of 6 independent experiments ± s.d.