Formulation and Characterization of β-Cyclodextrins-Nitazoxanide Inclusion Complexes: Enhanced Solubility, In Vitro Drug Release, and Antiviral Activity in Vero Cells

Abstract

Background/Objectives: Nitazoxanide (NTX) exhibits promising therapeutic potential; its effectiveness is constrained by its low oral bioavailability due to its poor water solubility and limited permeability. Methods: This study focused on developing a complex of NTX with β-cyclodextrins (β-CDs), specifically β-CD and hydroxypropyl-β-cyclodextrin (Hβ-CD), to enhance the solubility and antiviral activity of NTX. Results: The formation of the β-CD:NTX in an aqueous solution was verified using UV-visible spectroscopy, confirming a 1:1 inclusion complex. Characterization of the solid β-CD:NTX complexes was confirmed via FTIR, X-ray diffraction (XRD), scanning electron microscopy (SEM), and DSC-TGA analyses. Molecular docking studies revealed that the NTX thiazole ring with the nitro group was positioned within the β-CDs cavity, while the benzene ring remained outside. Phase solubility tests showed that β-CD:NTX complexes were formed with high stability constants, demonstrating a linear increase in NTX solubility as the β-CD concentration increased. Dissolution tests revealed rapid and nearly complete NTX release within 90 min for β-CD:NTX and Hβ-CD:NTX complexes. The β-CD:NTX complexes were tested for their antiviral activity against Herpes simplex virus (HSV-1) cultures. Results showed that the Hβ-CD:NTX complex had significantly higher antiviral efficacy than β-CD:NTX and free NTX alone. Moreover, cytotoxicity and cellular uptake studies on Vero cells indicated that the Hβ-CD:NTX complex demonstrated lower cytotoxicity and had the highest IC₅₀ value, followed by β-CD:NTX and free NTX. Conclusions: These findings suggest that Hβ-CD:NTX inclusion complexes may serve as effective carriers for delivering NTX in HSV-1 treatments using Vero cell models.

Keywords: antiviral activity; in vitro drug release; inclusion complexes; nitazoxanide; solubility; β-cyclodextrins.

Figure 1

UV–visible absorption spectra of nitazoxanide (NTX) in the presence of (a) β-cyclodextrin (β-CD) and (b) hydroxypropyl-β-cyclodextrin (Hβ-CD) at pH 7.4 in phosphate-buffered saline solutions. Benesi–Hildebrand plots of 1/[A-A₀] vs. (c) 1/[β-CD] and (d) 1/[Hβ-CD]. β-CD and Hβ-CD concentrations ranged from 0 to 0.012 M for measurements 1 to 7 (Black line to Purple line).

Figure 2

Fourier transform infrared spectroscopy (FTIR) spectra of β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (Hβ-CD), nitazoxanide (NTX), and their β-CD:NTX and Hβ-CD:NTX inclusion complexes.

Figure 3

X-ray diffraction (XRD) patterns of β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (Hβ-CD), nitazoxanide (NTX), and their β-CD:NTX and Hβ-CD:NTX inclusion complexes.

Figure 4

Scanning electron microscopy (SEM) analysis of β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (Hβ-CD), nitazoxanide (NTX), and their β-CD:NTX and Hβ-CD:NTX inclusion complexes.

Figure 5

(a) Thermogravimetric analysis (TGA) and (b) differential scanning calorimetry (DSC) analysis of β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (Hβ-CD), nitazoxanide (NTX), and their β-CD:NTX and Hβ-CD:NTX inclusion complexes.

Figure 6

Molecular docking of β-cyclodextrin (β-CD), hydroxypropyl-β-cyclodextrin (Hβ-CD), nitazoxanide (NTX), and their β-CD:NTX and Hβ-CD:NTX inclusion complexes. (NTX; Tan, red, blue, yellow, and white colors corresponding to the carbon, oxygen, nitrogen, sulfur, and hydrogen atoms).

Figure 7

Phase solubility study of nitazoxanide (NTX) in the presence of (a) β-cyclodextrin (β-CD) and (b) hydroxypropyl-β-cyclodextrin (Hβ-CD).

Figure 8

In vitro drug release studies of pure NTX and its β-CD:NTX and Hβ-CD:NTX inclusion complexes.

Figure 9

Antiviral activity test at (a) 24 h and (b) 48 h. Controlled exposure antiviral assay for (c) 6 h and (d) 12 h (data are the mean ± SD of experiments * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001), below the limit of detection (using the TCID₅₀ method).

Figure 10

(a) Effect of different nitazoxanide (NTX) drug formulations on the viability of Vero cells as a function of free NTX and its β-cyclodextrin (β-CD):NTX and hydroxypropyl-β-cyclodextrin (Hβ-CD):NTX inclusion complexes at 48 h and (b) fluorescence microscopy images using phase-contrast, 4′,6-diamidino-2-phenylindole (DAPI), and fluorescein isothiocyanate (FITC) staining of control, NTX, and their β-CD:NTX and Hβ-CD:NTX inclusion complexes at 150 μg/mL for 48 h in Vero cells. Scale bar = 50 μm.