Amphiphilic Cyclodextrin Nanoparticles as Delivery System for Idebenone: A Preformulation Study

Abstract

Idebenone (IDE), a synthetic short-chain analogue of coenzyme Q10, is a potent antioxidant able to prevent lipid peroxidation and stimulate nerve growth factor. Due to these properties, IDE could potentially be active towards cerebral disorders, but its poor water solubility limits its clinical application. Octanoyl-β-cyclodextrin is an amphiphilic cyclodextrin (ACyD8) bearing, on average, ten octanoyl substituents able to self-assemble in aqueous solutions, forming various typologies of supramolecular nanoassemblies. Here, we developed nanoparticles based on ACyD8 (ACyD8-NPs) for the potential intranasal administration of IDE to treat neurological disorders, such as Alzheimer's Disease. Nanoparticles were prepared using the nanoprecipitation method and were characterized for their size, zeta potential and morphology. STEM images showed spherical particles, with smooth surfaces and sizes of about 100 nm, suitable for the proposed therapeutical aim. The ACyD8-NPs effectively loaded IDE, showing a high encapsulation efficiency and drug loading percentage. To evaluate the host/guest interaction, UV-vis titration, mono- and two-dimensional NMR analyses, and molecular modeling studies were performed. IDE showed a high affinity for the ACyD8 cavity, forming a 1:1 inclusion complex with a high association constant. A biphasic and sustained release of IDE was observed from the ACyD8-NPs, and, after a burst effect of about 40%, the release was prolonged over 10 days. In vitro studies confirmed the lack of toxicity of the IDE/ACyD8-NPs on neuronal SH-SY5Y cells, and they demonstrated their antioxidant effect upon H₂O₂ exposure, as a general source of ROS.

Keywords: NMR studies; amphiphilic cyclodextrins; idebenone; in vitro antioxidant activity; molecular modeling; nanoparticles.

Figure 1

Chemical structure of IDE (a) and ACyD8 (b).

Figure 2

UV-vis spectra of IDE in the presence of increasing concentration of ACyD8. See Experimental Section for details.

Figure 3

The Benesi–Hildebrand plot of 1/A-A₀ vs. 1/[ACyD8] obtained from UV-vis data.

Figure 4

Stacked ¹H NMR spectra of free IDE (blue trace), ACyD8 (red trace) and IDE/ACyD8 (green trace). In the insets: chemical structures of IDE and ACyD8. Lowercase letters a–f were used for IDE proton assignment.

Figure 5

The 2D ROESY spectrum of IDE/ACyD8 complex (CDCl₃, at 25 °C). Inter- and intra-molecular interactions can be observed; only the cross-peaks of interest were circled in green. Dotted green circles identified cross-peaks slightly overlapped with the diagonal. Blue and red color represented negative- and positive-phase signals, respectively. Lowercase letters a–f were used for IDE proton assignment (see Figure 4).

Figure 6

(a) Representative structure of the most populated cluster from the MD simulation of complex1. (b) Representative structure of the most populated cluster from the MD simulation of complex2. (c) MD snapshot of complex1 at 348 ns of simulation time. (d) Representative structure of the second ranked (by population) cluster from the MD simulation of complex2. In every panel, IDE is represented by yellow thick sticks, and ACyD8 is represented by green thin sticks and molecular surfaces.

Figure 7

RMSD values of IDE atoms as a function of simulation time. The highlighted regions represent: IDE included in ACyD8 cavity (complex1—green solid line), IDE randomly interacting with the outer part of ACyD8 (complex1—green dashed line), cluster 1 of complex2 MD conformations (magenta solid lines) and cluster 2 of complex2 MD conformations (magenta dashed lines).

Figure 8

Plot of the correlation coefficients between IDE RMSD and force-field energy terms.

Figure 9

(a) RMSD of IDE atoms in complex1 as a function of MD simulation time; (b) RMSD of IDE atoms in complex2 as a function of MD simulation time. (c) Energetics of simulated complex1 as a function of simulation time. (d) Energetics of simulated complex2 as a function of simulation time.

Figure 10

STEM images of lyophilized IDE/ACyD8-NPs before (a) and after redispersion (b). The NPs observed at higher magnification showed both matrix structure (c) and nanoreservoir structure (d).

Figure 11

FT-IR spectra of IDE/ACyD8-NPs (sample C) compared to those of free components and the physical mixture. The inset shows magnification of the peaks in the range between 1750 cm⁻¹ and 1600 cm⁻¹, as highlighted by the dashed ellipse.

Figure 12

DSC traces of IDE/ACyD8-NPs (sample C), pure IDE and ACyD8, and the physical mixture.

Figure 13

In vitro release profiles of IDE from IDE/ACyD8-NPs in PBS (pH 7.4). Sample A (circle), sample B (triangle), sample C (diamond).

Figure 14

(a) Analysis of cell viability of neuronal SH-SY5Y cells treated with IDE/ACyD8-NPs, IDE and ACyD8 for 72 h. Data are presented as mean ± S.D. (b) Analysis of cytotoxicity on the same cells pretreated with IDE/ACyD8-NPs and exposed to 30 µM H₂O₂ for 72 h. Concentrations in IDE/ACyD8-NPs refers to the actual loading of IDE within NPs. Unloaded ACyD8-NPs (i) and (ii) were tested at the same ACyD8 concentration used for testing the drug-loaded NPs 20 µM and 40 µM, respectively. Data are presented as mean ± S.D. One-way ANOVA with Tukey’s multiple comparison test: in (a), all data are not statistically different; in (b), * p < 0.05 (H₂O₂ + IDE/ACyD8-NPs 2:1 40 µM versus H₂O₂).