Rutin/Sulfobutylether-β-Cyclodextrin as a Promising Therapeutic Formulation for Ocular Infection

Abstract

Ocular pathologies present significant challenges to achieving effective therapeutic results due to various anatomical and physiological barriers. Natural products such as flavonoids, alone or in association with allopathic drugs, present many therapeutic actions including anticancer, anti-inflammatory, and antibacterial action. However, their clinical employment is challenging for scientists due to their low water solubility. In this study, we designed a liquid formulation based on rutin/sulfobutylether-β-cyclodextrin (RTN/SBE-β-CD) inclusion complex for treating ocular infections. The correct stoichiometry and the accurate binding constant were determined by employing SupraFit software (2.5.120) in the UV-vis titration experiment. A deep physical-chemical characterization of the RTN/SBE-β-CD inclusion complex was also performed; it confirmed the predominant formation of a stable complex (K_c, 9660 M^-1) in a 1:1 molar ratio, with high water solubility that was 20 times (2.5 mg/mL) higher than the free molecule (0.125 mg/mL), permitting the dissolution of the solid complex within 30 min. NMR studies revealed the involvement of the bicyclic flavonoid moiety in the complexation, which was also confirmed by molecular modeling studies. In vitro, the antibacterial and antibiofilm activity of the formulation was assayed against Staphylococcus aureus and Pseudomonas aeruginosa strains. The results demonstrated a significant activity of the formulation than that of the free molecules.

Keywords: antibacterial activity; biofilm; resistant strains; rutin; sulfobutylether-β-cyclodextrin.

Figure 12

The results are expressed as mean ± SD. RTN/SBE-β-CD inclusion complex significantly reduced S. aureus ATCC 43300 (MRSA) biofilm formation at 0.5 MIC (0.61 μg/mL in RTN). (a) Biofilm biomass is expressed as crystal violet optical density (O.D. 497 nm) (** p < 0.01); (b) cell viability is expressed as Log₁₀ CFU/mL (**** p < 0.0001).

Figure 1

Molecular structure of RTN (left) and schematic structure of SBE-β-CD (right).

Figure 2

UV-vis spectra of free RTN and in the presence of an increasing amount of SBE-β-CD in water:MeOH 80:20 mixture (%, v/v). The experiments were carried out in triplicate.

Figure 3

Phase solubility diagram of RTN/SBE-β-CD inclusion complex. The experiments were carried out in triplicate.

Figure 4

Stacked portions of the ¹H NMR spectra relative to the free RTN (blue line) and RTN/SBE-β-CD (black line) inclusion complex. Only those diagnostic signals relative to RTN are reported.

Figure 5

The 3D full minimized structure of RTN/SBE-β-CD inclusion complex. Top view (left) and side view (right). Hydrogen bonds were represented as a yellow dotted line.

Figure 6

WAXD patterns of free RTN (green line), SBE-β-CD (black line), physical mixture (blue line), and inclusion complex (orange line).

Figure 7

TGA curves of free RTN (green line), SBE-β-CD (black line), physical mixture (blue line), and inclusion complex (orange line).

Figure 8

FT-IR spectra recorded at 25 °C of free RTN (green line), SBE-β-CD (black line) physical mixture (blue line), and inclusion complex (orange line). Characteristic peaks were labeled; the absorptions of RTN and SBE-β-CD functional groups are in red and black, respectively.

Figure 9

Scanning electron microscope (SEM) images of free RTN (a), SBE-β-CD (b), physical mixture (c), and inclusion complex (d) at different magnifications.

Figure 10

Dissolution profiles of free RTN (green line) and RTN/SBE-β-CD inclusion complex (orange line) in water at 25.0 ± 0.5 °C. All values of the inclusion complex are statistically significant compared to free RTN data (*** p < 0.001). The experiments were carried out in triplicate. The results are presented as the mean of three experiments ± standard deviation (SD). The error bar, if not shown, is inside the symbol.

Figure 11

The results are expressed as mean ± SD. RTN/SBE-β-CD inclusion complex significantly reduced S. aureus ATCC 6538 biofilm formation at 0.5 MIC (0.61 μg/mL in RTN). (a) Biofilm biomass is expressed as crystal violet optical density (O.D. 497 nm) (* p < 0.05); (b) cell viability is expressed as Log₁₀ CFU/mL (**** p < 0.0001).