Conjugates of Chitosan with β-Cyclodextrins as Promising Carriers for the Delivery of Levofloxacin: Spectral and Microbiological Studies

Abstract

In this work, we synthesized chitosan 5 kDa conjugates with β-cyclodextrins with various substituents as promising mucoadhesive carriers for the delivery of fluoroquinolones using the example of levofloxacin. The obtained conjugates were comprehensively characterized by spectral methods (UV-Vis, ATR-FTIR, 1H NMR, SEM). The physico-chemical properties of the complex formations were studied by IR, UV, and fluorescence spectroscopy. The dissociation constants of complexes with levofloxacin were determined. Complexation with conjugates provided four times slower drug release in comparison with plain CD and more than 20 times in comparison with the free drug. The antibacterial activity of the complexes was tested on model microorganisms Gram-negative bacteria Escherichia coli ATCC 25922 and Gram-positive Bacillus subtilis ATCC 6633. The complex with the conjugate demonstrated the same initial levofloxacin antibacterial activity but provided significant benefits, e.g., prolonged release.

Keywords: chitosan; cyclodextrin; drug delivery system; guest–host complexes; levofloxacin.

Figure 1

Synthesis of chitosan–β-cyclodextrin conjugates on sample NH₂-CD-Chit. (a) Synthesis of Ts-NH₂-CD. (b) ATR-FTIR spectrum of Ts-NH₂-CD (solid black line) and NH₂-CD (red dotted line) in water solution at 22 °C. (c) Synthesis of NH₂-CD-Chit. (d) ATR-FTIR spectrum of NH₂-CD-Chit (red solid line) and Ts-NH₂-CD (dotted black line) in water solution at 22 °C.

Figure 2

(a) Free amino group titration with TNBS. (b) Kinetic curves (420 nm) for chitosan solution (blue) and conjugate (red) NH₂-CD-Chit. Borate buffer solution. pH 9.0.

Figure 3

Scanning electron microscopy for NH₂-CD (a,c) and NH₂-CD-Chit (b,d). The acceleration voltage was set to 5 kV.

Figure 4

(a) UV spectrum of levofloxacin 3 × 10⁻⁵ M. (b) Dependence of absorbance 295 nm on the levofloxacin concentration. HCl solution, pH 4.0, T = 22 °C.

Figure 5

UV spectra reflecting the effects of complex formation of levofloxacin with β-cyclodextrin-containing ligands ((a)–Hp-CD and (b)–NH₂-CD-Chit) in the molar ratio of levofloxacin: ligand 10:1, 5:1, 2:1, 1: 1, 1:2, 1:5, 1:10. The concentration of levofloxacin in all samples was 3 × 10⁻⁵ M, HCl solution, pH 4.0, T = 22 °C.

Figure 6

Spectra of fluorescence emission of levofloxacin. (a) Levofloxacin solution 3 × 10⁻⁵ M, pH = 4.0, T = 22 °C. (b,c) UV spectra reflecting the effects of complex formation of levofloxacin with β-cyclodextrin-containing ligands ((b) NH₂-CD, (c) NH₂-CD-Chit) in the molar ratio of levofloxacin: ligand 10:1, 5:1, 2:1, 1: 1, 1:2, 1:5, 1:10. The concentration of levofloxacin in all samples 3 × 10⁻⁵ M, pH = 4.0, T = 22 °C, λ excitation 288 nm.

Figure 7

ATR-FTIR spectra of levofloxacin (a) and its complexes with NH₂-CD-Chit (b) in variable molar ratio. Levofloxacin concentration 3 × 10⁻⁵ M, HCl solution, pH = 4.0, T = 22 °C.

Figure 8

Levofloxacin release studies from complex with NH₂-CD (green dots) and Chit-NH₂-CD (red dots). Studies were carried out in 0.1 mM HCl solution (pH 4.0) at 37 °C 120 at rpm. Levofloxacin concentration in samples was 3 × 10⁻⁵ M, CD tori concentration in all samples was 1.5 × 10⁻⁵ M.

Figure 9

Inhibition zones appeared on Petri dishes, CLV = 0.1 μg/mL (a), 0.2 μg/mL (b), 0.5 μg/mL (c), and 1 μg/mL (d); C_conjugate = 0.5 μg/mL (a), 1 μg/mL (b), 2.4 μg/mL (c), 5 μg/mL (d), C_{CD’s torus in conjugate} = C_{Levofloxacin,} agar well diffusion method, B. subtilis ATCC 6633 pH 7.4 (0.02 M sodium phosphate buffer solution), 37 °C, 24 h of incubation. (e) The area of inhibition zones depending on CLV, B. subtilis ATCC 6633 and E. coli ATCC 25,922, agar well diffusion method, pH 7.4 (0.02 M sodium phosphate buffer solution), 37 °C, 24 h of incubation.