Norfloxacin-Loaded Electrospun Scaffolds: Montmorillonite Nanocomposite vs. Free Drug

Abstract

Infections in nonhealing wounds remain one of the major challenges. Recently, nanomedicine approach seems a valid option to overcome the antibiotic resistance mechanisms. The aim of this study was the development of three types of polysaccharide-based scaffolds (chitosan-based (CH), chitosan/chondroitin sulfate-based (CH/CS), chitosan/hyaluronic acid-based (CH/HA)), as dermal substitutes, to be loaded with norfloxacin, intended for the treatment of infected wounds. The scaffolds have been loaded with norfloxacin as a free drug (N scaffolds) or in montmorillonite nanocomposite (H-hybrid-scaffolds). Chitosan/glycosaminoglycan (chondroitin sulfate or hyaluronic acid) scaffolds were prepared by means of electrospinning with a simple, one-step process. The scaffolds were characterized by 500 nm diameter fibers with homogeneous structures when norfloxacin was loaded as a free drug. On the contrary, the presence of nanocomposite caused a certain degree of surface roughness, with fibers having 1000 nm diameters. The presence of norfloxacin-montmorillonite nanocomposite (1%) caused higher deformability (90%-120%) and lower elasticity (5-10 mN/cm²), decreasing the mechanical resistance of the systems. All the scaffolds were proven to be degraded via lysozyme (this should ensure scaffold resorption) and this sustained the drug release (from 50% to 100% in 3 days, depending on system composition), especially when the drug was loaded in the scaffolds as a nanocomposite. Moreover, the scaffolds were able to decrease the bioburden at least 100-fold, proving that drug loading in the scaffolds did not impair the antimicrobial activity of norfloxacin. Chondroitin sulfate and montmorillonite in the scaffolds are proven to possess a synergic performance, enhancing the fibroblast proliferation without impairing norfloxacin's antimicrobial properties. The scaffold based on chondroitin sulfate, containing 1% norfloxacin in the nanocomposite, demonstrated adequate stiffness to sustain fibroblast proliferation and the capability to sustain antimicrobial properties to prevent/treat nonhealing wound infection during the healing process.

Keywords: antimicrobial properties; chitosan; electrospinning; fibroblasts proliferation; glycosaminoglycans; scaffolds.

Figure 1

UV spectra of N (red line) and N subjected to heat treatment (1 h at 150 °C) (black line) obtained from the HPLC analysis at N peak maximum.

Figure 2

SEM microphotographs of chitosan-based (CH), chitosan/chondroitin sulfate-based (CH/CS) and chitosan/hyaluronic acid-based (CH/HA) scaffolds loaded with 1% or 2% norfloxacin, as a free drug (1% N or 2% N), or in VHS-N nanocomposite. In each image, the nanofiber diameters (nm, mean values ± SD; n = 30) and Si content for hybrid scaffolds are reported. Statistics: Mann–Whitney (Wilcoxon) W test p < 0.05: CH2H vs. CH/CS2H; CH/CS2H vs. CH/HA2H; CH/HA1H vs. CH/HA2H; CH/HA2H vs. CH/HA2N (scale bar 5 μm).

Figure 3

Transmission electron microscope (TEM) microphotographs and EDX spectra for CH, CH/CS and CH/HA scaffolds loaded with 2% norfloxacin in nanocomposite CH (A–C), CH/CS (G–I) and CH/HA (D–F).

Figure 4

Fourier transform infrared spectroscopy (FTIR) spectra evaluated for norfloxacin-loaded scaffolds (CH-N2, CH/CS-N2, CH/HA-N2) and VHS-N loaded scaffolds (CH-H2, CH/CS-H2, CH/HA-H2), both types containing 2% w/w norfloxacin in norfloxacin–montmorillonite nanocomposite (VHS-N).

Figure 5

X-ray powder diffraction (XRPD) diffractograms of the scaffolds loaded with VHS-N at 2% norfloxacin (CH-H2, CH/CS-H2, CH/HA-H2) compared to unloaded CH scaffolds (CH) and VHS-N.

Figure 6

Mechanical properties (force at break mN, a-b; elongation %, c-d; Young’s modulus mN.cm², e-f) for dry (a,c,e) and wet (b,d,f) scaffolds loaded with 1% (a) or 2% (b) of norfloxacin as a free drug (N) or as nanocomposite (H) (mean values ± SD; n = 3). Statistics: * = Mann–Whitney (Wilcoxon) W test p < 0.05.

Figure 7

Release profiles (%) of norfloxacin from the N or H scaffolds loaded with 1% (a) or 2% (b) as a free drug (N) or as nanocomposite (H), in saline solution (mean values ± SD; n = 3).

Figure 8

Norfloxacin released (%) in lysozyme from the scaffolds loaded with 1% (a) or 2% (b) of norfloxacin as a free drug (N) or as nanocomposite (H) and glucosamine released (%) from the scaffolds loaded with 1% (c) or 2% (d) of norfloxacin as a free drug (N) or as nanocomposite (H) subjected to lysozyme activity (mean values ± SD; n = 3)

Figure 9

SEM microphotographs of the scaffolds loaded with 1% or 2% of norfloxacin as a free drug (N) or as nanocomposite (H) subjected to lysozyme activity for 10 days (scale bar: 5 µm).

Figure 10

Cytocompatibility (optical density (OD)) of fibroblasts grown for 3 days (a) and 6 days (b) onto CH (blue), CH/CS (red), CH/HA (green) loaded with norfloxacin at 1% (plain color) and 2% (oblique lines), and with norfloxacin–montmorillonite nanocomposite (N-VHS) at 1% (horizontal lines) and 2% (dots) in norfloxacin. N norfloxacin as a free drug and H (N-VHS nanocomposite) at the same concentrations of the scaffolds are evaluated (mean values ± SD; n = 8). Statistics: * = Mann–Whitney (Wilcoxon) W test p < 0.05.

Figure 11

Confocal laser scanning microscopy (CLSM) microphotographs of fibroblasts grown for 6 days onto CH, CH/CS, CH/HA loaded with norfloxacin as a free drug (N) or norfloxacin–montmorillonite nanocomposite (H, N-VHS) at 1% or 2% (in blue: nuclei; in green: cytoskeleton) (scale bar: 50 μm).

Figure 12

SEM microphotographs of fibroblasts grown for 6 days onto CH, CH/CS, CH/HA loaded with norfloxacin as a free drug (N) or norfloxacin–montmorillonite nanocomposite (H, N-VHS) at 1% or 2% (scale bar: 50 μm).

Figure 13

Microbicidal effect evaluated for 1% norfloxacin as a free drug (a,c) and (b,d) as nanocomposite loaded into CH, CH/CS and CH/HA scaffolds against Pseudomonas aeruginosa (a and b) and Staphylococcus aureus (c and d), in comparison to norfloxacin as a free drug and as nanocomposite, with the same concentration as in the scaffolds (mean values ± SD; n = 3).