Optimization of Diclofenac-Loaded Bicomponent Nanofibers: Effect of Gelatin on In Vitro and In Vivo Response

Abstract

The use of electrospun fibers as anti-inflammatory drug carriers is currently one of the most interesting approaches for the design of drug delivery systems. In recent years, biodegradable polymers blended with naturally derived ones have been extensively studied to fabricate bioinspired platforms capable of driving biological responses by releasing selected molecular/pharmaceutical signals. Here, sodium diclofenac (DicNa)-loaded electrospun fibers, consisting of polycaprolactone (PCL) or gelatin-functionalized PCL, were studied to evaluate fibroblasts' in vitro and in vivo response. In vitro studies demonstrated that cell adhesion of L929 cells (≈70%) was not affected by the presence of DicNa after 4 h. Moreover, the initial burst release of the drug from PD and PGD fibers, e.g., 80 and 48%, respectively, after 5 h-combined with its sustained release-did not produce any cytotoxic effect and did not negatively influence the biological activity of the cells. In particular, it was demonstrated that the addition of gelatin concurred to slow down the release mechanism, thus limiting the antiproliferative effect of DicNa, as confirmed by the significant increase in cell viability and collagen deposition after 7 days, with respect to PCL alone. In vivo studies in a rat subcutaneous model also confirmed the ability of DicNa-loaded fibers to moderate the inflammatory/foreign body response independently through the presence of gelatin that played a significant role in supporting the formation of small-caliber vessels after 10 days of implantation. All of these results suggest using bicomponent fibers loaded with DicNa as a valid therapeutic tool capable of supporting the wound healing process and limiting in vivo inflammation and rejection phenomena.

Keywords: animal model; anti-inflammatory drugs; drug delivery; electrospinning; nanofibers.

Figure 1

DicNa-loaded electrospun fibers: SEM images of PCL and PCL/gelatin nanofibers at different magnification (A) and fiber diameter distribution via image analysis (B).

Figure 2

DicNA-loaded electrospun fibers: detection of Na elements via EDS analysis. Detail of fiber morphology shown in square.

Figure 3

DicNa quantification via TGA analyses of DicNa-loaded (A) PCL (PD), and (B) PCL/gelatin nanofibers (PGD). DicNa is referred to as the thermogram of the drug, while CTR and CTRG, respectively, are reported as controls for unloaded PCL and PCL/gelatin nanofibers.

Figure 4

Comparison of cumulative release profiles of DicNa from electrospun fibers in PBS solution. Six independent experiments were performed, and results are expressed as mean values obtained (mean ± SD).

Figure 5

Biocompatibility assays. (A) Percentage of cell adhesion after 4 and 24 h. Results are presented as % of cell adhesion concerning control and cell culture plate (TCP), a statistically significant difference is represented as * p < 0.05. (B) Cell morphology captured by SEM (left, scale bar: 40 μm) and confocal (right, scale bar: 100 μm) microscopy images after 24 h.

Figure 6

In vitro response of DicNa-loaded nanofibers: (A) L929 viability after 2, 5, 7, 14, and 21 days (a statistically significant difference is represented as * p < 0.05; ** p < 0.01, and *** p < 0.001); (B) Sirius red assay for fibroblast collagen synthesis after 7, 14, and 21 days (a statistically significant difference is represented as ** p < 0.01, and *** p < 0.001).

Figure 7

H&E staining images at 10× magnification after four days of evaluation. CTR: the double arrow corresponds to the inflammatory infiltrate. PD: the formation of a pseudocapsule is observed around the material (arrow); the double arrow indicates the formation of immune cells. PGD: the double arrow indicates the presence of cellular remains. In all cases, the asterisk is located where the material was placed.

Figure 8

H&E staining images at 10× magnification after ten days of evaluation. CTR: the arrows correspond to foreign body giant cells. PD: immune response cells marked with the double arrow are observed, as well as the formation of blood vessels (arrow). PGD: the double arrow indicates the presence of inflammatory response cells, and the single arrow indicates the presence of blood vessels. In all cases, the asterisk is located where the material was placed.

Figure 9

H&E staining images at 10× magnification after 21 days of evaluation. CTR: the double arrow corresponds to granulomatous inflammation, and the arrows indicate the formation of blood vessels. PD: a decrease in immune cells is observed (double arrow), as well as the formation of blood vessels (arrows). PGD: the double arrow indicates a decrease in the inflammatory response, while the single arrow indicates the formation of blood vessels. In all cases, the asterisk is located where the material was placed.