Cyclodextrins and Their Polymers Affect the Lipid Membrane Permeability and Increase Levofloxacin's Antibacterial Activity In Vitro

Abstract

Cyclodextrins (CDs) are promising drug carriers that are used in medicine. We chose CDs with different substituents (polar/apolar, charged/neutral) to obtain polymers (CDpols) with different properties. CDpols are urethanes with average Mw of ~120 kDa; they form nanoparticles 100-150 nm in diameter with variable ζ-potential. We studied the interaction of CD and CDpols with model (liposomal) and bacterial membranes. Both types of CD carriers cause an increase in the liposomal membrane permeability, and for polymers, this effect was almost two times stronger. The formation of CD/CDpols complexes with levofloxacin (LV) enhances LV's antibacterial action 2-fold in vitro on five bacterial strains. The most pronounced effect was determined for LV-CD complexes. LV-CDs and LV-CDpols adsorb on bacteria, and cell morphology influences this process dramatically. According to TEM studies, the rough surface and proteinaceous fimbria of Gram-negative E. coli facilitate the adsorption of CD particles, whereas the smooth surface of Gram-positive bacteria impedes it. In comparison with LV-CDs, LV-CDpols are adsorbed 15% more effectively by E. coli, 2.3-fold better by lactobacilli and 5-fold better in the case of B. subtilis. CDs and CDpols are not toxic for bacterial cells, but may cause mild defects that, in addition to LV-CD carrier adsorption, improve LV's antibacterial properties.

Keywords: antibacterial activity; cyclodextrin; levofloxacin; liposome; spectroscopy.

Figure 1

The structure of the β-cyclodextrin derivatives and their polymers, lipids used for liposome production and levofloxacin.

Figure 2

UV spectra of phenolphthalein (A), pH 10.7 (0.02 M Na-borate buffer solution), 22 °C. The release kinetics of PP from liposomes DPPC:CL = 80:20 (weight %) after adding CD (B) or CDpol (C), C_{CD torus} = 30 mg/mL, pH 7.4 (0.02 M Na-phosphate buffer solution), pH 10.7 (0.02 M Na-borate buffer solution), 37 °C.

Figure 3

Experimental PXRD patterns for LV (navy blue), MCD (red) and MCDpol (green); LV-MCD (brown) and LV-MCDpol complexes (baby blue). From 35° to 80°, no more signals were observed.

Figure 4

(A) The diameters of inhibition zones (mm) on Petri dishes with free LV and its complexes with SBECD and SBECDpol in the concentration range 50–100 μg/mL, agar well diffusion method, L. fermentum 90T-C4 pH 7.4 (0.02 M Na-phosphate buffer solution), 37 °C, 24 h of incubation. (B,C) The area of inhibition of E. coli ATCC 25922 bacterial growth depending on the concentration of the drug, agar well diffusion method, pH 7.4 (0.02 M Na-phosphate buffer solution), 37 °C, 24 h of incubation.

Figure 5

FTIR spectra of L. fermentum 90T-C4, B. subtilis ATCC6633 and E. coli ATCC 25922 in the range 2300–900 cm⁻¹ of pure cultures of bacteria, ~10¹¹ CFU/mL, H₂O, 22 °C.

Figure 6

The TEM micrographs of B. subtilis ATCC6633: overnight culture, cells incubated with LV, LV-SBECD or LV-SBECDpol for 24 h at 37 °C. C_LV = 2.5 μg/mL. Scale bar: 1 μm (A–D) and 5 μm (E–H).

Figure 7

The absorption curves of LV, LV–CD and LV-CDpol on bacterial strains, ~1 × 10⁹ cells/mL, pH 7.4 (0.02 M Na-phosphate buffer solution), 1 h of incubation, 37 °C. (A) E. coli JM109; (B) E. coli ATCC 25922; (C) E. coli MH1; (D) B. subtilis ATCC6633; (E) Lactobacillus fermentum 90T-C4; (F) Lactobacillus fermentum 90T-C4.

Figure 8

The morphology of bacterial surface: E. coli MH1 (A,B), E. coli JM109 (C,D), E. coli ATCC 25922 (E,F), L. fermentum 90T-C4 (G,H). Purple arrows indicate fimbria and red arrows indicate flagella. Scale bars: 1 μm (A–E) and 2 μm (F–H).

Figure 9

The TEM microphotographs of HPCDpol-Cu²⁺ particle adsorption on B. subtilis ATCC 6633 (A,B) and L. fermentum 90T-C4 (C,D), H₂O, 20 min of incubation, 37 °C, C(Cu²⁺) ~ 0.001 M, 80 mV. Scale bars: 1 μm (C) and 2 μm (A,B,D).