*2.8. Data Analysis*

Data are reported as arithmetic means and standard deviations of at least three experiments. Calculations, data processing, and graphics were prepared with Excel 2018 (Microsoft, Redmond, WA, USA).

### **3. Results and Discussion**

#### *3.1. Preparation and Characterization of Drug-Loaded Foils and Nanofiber Mats*

Starting from aqueous solutions of PVP, acetic acid, and ciprofloxacin at di fferent concentrations, transparent films and nanofiber mats were fabricated using the solvent casting and electrospinning methods, respectively (Figure 1).

**Figure 1.** Schematic representation of the methods used for the film and nanofiber mat fabrication, and pictures of the discs used in the experimental set up. Cipro—ciprofloxacin; PVP—polyvinylpyrrolidone.

The final dry transparent foils had a thickness of 150–180 μm and the absence of crystal formation was verified using SEM (Figure A1, Appendix A). As recently demonstrated, the obtained PVP-based foils represent suitable wound dressings due to their flexibility, adhesion, and resorption properties [19,28]. The nanofiber had an average diameter of 360 ± 80 nm and did not show any defects or beads (Figure A2, Appendix A).

#### *3.2. Foils and Nanofiber Mats Had Di*ff*erent Drug Delivery Profiles*

Foils and nanofiber mats (discs of 8 mm in diameter) were applied on the top of the 5 mm in diameter wounds created on the 1.5 × 1.5 cm ex vivo skin tissue to provide an applied drug dosage of approximately 250 μg/wound. The skin explants were incubated in humidified chambers in an incubator with 100% humidity. This maintained the wetness of the skin blocks, which favored the dissolution of foils and nanofibers. After different incubation times, the non-penetrated material was removed, 8 mm biopsies were taken, and extracts were prepared to measure the amount of penetrated drug (Figure 2).

**Figure 2.** Time-dependent concentration of ciprofloxacin into wound tissue after the topical application of ciprofloxacin-loaded foils (**a**) and nanofibers (**b**). Ciprofloxacin in wound tissue extracts was measured by means of a fluorescence microplate reader and drug concentration was calculated from standard curves. Means and standard deviations from three independent experiments using skin from three different donors are reported.

Both penetration kinetics could be well-fitted to a second-degree polynomial trend line. Amounts ranging between 12 and 18 μg of ciprofloxacin/wound were detected for foils, whereas amounts ranging between 5 and 10 μg/wound were measured for nanofiber discs. Foils delivered high amounts of ciprofloxacin very quickly (approximately 12 μg after only 2 h), whereas nanofiber mats reached similar concentrations later, after 6 h. Interestingly, foils maintained the reached drug concentration in the wound over 24 h. On the contrary, in samples treated with nanofiber mats, the maximal concentration was maintained for a shorter time range (6–16 h). The decreased amounts of drug in the wound tissue observed for nanofiber mats (Figure 2b) were due to drug di ffusion to the neighboring tissue (Figure A3, Appendix B). We suppose that for nanofiber mats, a lower amount of drug penetrated the wound tissue and that, due to di ffusion to the nearby skin tissue, a reduction of local drug concentration in the wound was measured after 24 h. On the contrary, foils delivered a higher amount of drug to the wound and could saturate both the wound and the nearby tissue.

In a previous study, the dissolution rate and drug release for both materials was investigated in a phosphate bu ffer and on a mice full-thickness wound model [19]. Results showed that nanofibers dissolved and released the loaded drug very quickly, within the first two hours, whereas foils took longer. The fast dissolution of nanofibers might result in a high local concentration causing drug precipitation or crystallization, which in turn can hinder drug penetration. Another important aspect to be considered is that the investigated PVP-foils contained higher amounts (1.4%) of residual acetic acid than the nanofiber mats (0.3%) [19], which might also have had an influence on drug solubility and penetration.

In summary, drug penetration experiments using the ex vivo wound model showed that both drug delivery systems could deliver the loaded antibiotic to the wound tissue in a controlled manner. The ability of PVP-foils to maintain a high drug concentration in the wound over 24 h is of significance, especially for the treatment of biofilm infections, which need high antibiotic concentrations to be resolved. On the other side, the more controlled delivery profile of the nanofiber mats might be more useful for the prevention of wound infections.

#### *3.3. Local Toxicity of Ciprofloxacin in Full-Thickness Ex Vivo Skin*

The wound model was also used to test the potential cytotoxicity of the drug and investigated materials after topical application. For these experiments, tissue blocks were cultured in supplemented RPMI 1640 medium at the air–liquid interface. After 20 h of treatment, tissue viability was tested by means of an XTT test (Figure 3a).

Many cells in skin explants were still metabolically active and an evident formation of the red formazan product was observed. PVP foils did not show any toxicity. This was somehow expected with PVP being a well-tolerated, FDA-approved polymer with many uses, such as a food additive, binder in tablets, and plasma volume expander [29]. Also, foils prepared from a PVP solution in 30% acetic acid resulted in having no e ffect on skin viability due to the fact that only residual amounts of acetic acid molecules remained within the PVP polymers after solvent evaporation during the preparation process [19]. Finally, the PVP foils loaded with the three concentrations of ciprofloxacin also resulted in having no influence on the overall viability of skin cells after 20 h of incubation. Wounds treated with silver nanoparticles, which release toxic silver ions, served as positive controls. The evident reduction of formazan formation was indicative of silver toxicity toward wound cells. Thus, even if this test gave no information about the type of cells being a ffected by the tested substances, it was a useful method to detect overall acute toxic e ffects.

Previous studies have shown that ciprofloxacin has toxic e ffects on fibroblasts [30] and keratinocytes [31]. Toxicity was shown to be time- and concentration-dependent. For this reason, we tested the e ffect of ciprofloxacin delivered by the investigated foils with regard to re-epithelialization. After 9 days of incubation in supplemented RPMI-1640 medium, a partial re-epithelialization had occurred in controls, with the re-growing of a keratinocyte layer on the edges and in the wound bed (Figure 3b,d,e). Cell nuclei and the collagen network presented a normal morphology in samples treated with empty PVP foils with residual acetic acid. Only a delayed re-epithelialization was observed, probably due to oxygen deprivation (data not shown). On the contrary, in wounds treated with ciprofloxacin, only a thin epithelial layer was observed. Cells' nuclei appeared small with an irregular morphology typical of necrotic cells and the collagen matrix was less organized than in the

controls (Figure 3c,f,g). This effect was visible in all samples treated with ciprofloxacin at different concentration and was independent of the formulation (data not shown).

In recent years, it has been recognized that fluoroquinolones, despite being well tolerated by a broad portion of patients, can have rare and very disabling side effects like a tendon rupture and irreversible nerve damage [13]. There are several hypotheses on the mechanism of toxicity and on the reason why some persons develop these side effects more than others do. One theory is that there might be a gene variant responsible for a disrupted quinolone metabolism. Our results show that, despite ciprofloxacin inducing no changes in whole skin metabolic activity, it negatively influenced the re-epithelialization of ex vivo wounds. Even if this side effect is tolerable in the case of severe wound infections, these results stress the importance of a more local and controlled release of drugs like ciprofloxacin that have narrow therapeutic windows.

#### *3.4. Ciprofloxacin-Loaded Foils and Nanofiber Mats E*ffi*ciently Reduced P. aeruginosa Infections*

Next, we investigated the antimicrobial efficacy of the investigated drug delivery systems using the ex vivo skin infection model (Figure 4). The PAO1 strain was used, which possesses several proteolytic enzymes, among those collagenases, and was shown to build biofilm-like infections on ex vivo skin [21]. Bacteria inoculated in the wounds grew from 1 × 10<sup>7</sup> to 1 × 10<sup>9</sup> bacteria per wound after 20 h of incubation. Macroscopically, the surface of the PAO1-infected wound appeared shiny and yellowish (Figure 4c). Scanning electron microscopy images of the wound surface showed bacteria conglomerates typical of a biofilm. These three-dimensional structures are made of bacteria, extracellular materials, and probably of degraded collagen material (Figure 4d,e). The microscopic pictures of H&E-stained skin sections (Figure 4f) revealed an approximately 10-μm-thick bacteria

film on the surface of the wound, but also groups of bacteria deep in the wound tissue (circles). The scanning electron microscopic analysis of the wound profile (Figure 4g,h) confirmed the formation of a bacterial biofilm within the superficial wound tissue, as well as the presence of small agglomerates or scattered bacteria deep in the wound.

**Figure 4.** Wound infection model with the *P. aeruginosa* strain PAO1. (**a**) A superficial wound of approximately 5 mm in diameter was produced on skin explants. (**b**) Bacteria (1 × 10<sup>7</sup> per wound) were inoculated and skin explants, which were incubated for 20 h in the previous analysis. (**c**) Microscopic appearance of a representative wound 20 h after the PAO1 injection. (**d**,**<sup>e</sup>**) SEM images of the wound surface at two different magnifications showing the typical morphology of bacterial communities in the biofilm. (**f**) Wound section stained with H&E showing a bacteria layer on the surface of the wound and bacteria agglomerates deep into the tissue (circles). (**g**,**h**) SEM images of wound sections at different magnifications confirming bacterial growth on the surface (**g**) and deep in the wound tissue (**h**).

In the ex vivo model, most of bacteria grew on the wound's surface, despite the fact that they were inoculated deep in the connective tissue. *P. aeruginosa* can grow in both aerobic and anaerobic conditions. However, the less favorable conditions and shortage of oxygen in the deeper wound layers resulted in a slower proliferation of bacteria. Nevertheless, these small colonies can be the reason for recurrent infections if not eradicated during a treatment. For this reason, the penetration of adequate drug concentrations deep in the wound tissue and the eradication of all bacterial colonies is a crucial factor for a successful therapy.

*Pseudomonas aeruginosa* has already been cultured on ex vivo skin [24,32–35]. In this study, we used the ex vivo wound infection model as a three dimensional set up to test the capacity of ciprofloxacin-loaded PVP drug delivery systems to eradicate bacteria located not only on the surface, but also deep in the wound's tissue. The investigated foils and nanofiber mats were loaded with three different concentrations of ciprofloxacin, corresponding to final doses of 11, 250, and 500 μg per wound. The treatments were applied on the top of the wounds 1 h or 20 h after bacteria inoculation and kept for a further 20 h. After 1 h of inoculation, bacteria were still planktonic and thus less resistant to antibiotics. After 20 h of inoculation, the number of bacteria had increased and bacteria were organized in a biofilm, which was more difficult to treat. Samples treated 1 h after bacteria inoculation served to test the drug efficacy toward a moderate infection with approximately 1 × 10<sup>7</sup> planktonic bacteria, whereas samples treated 20 h after PAO1 inoculation served to test the efficacy of foils and nanofiber mats on a severe infection with approximately 1 × 10<sup>9</sup> bacteria primarily organized into a biofilm. The experiment terminated after a total of 40 h.

Macroscopically, a thick, yellow biofilm was visible on the surface of untreated wounds, whereas no bacterial film was visible on the treated samples (Figure 5a).

**Figure 5.** Antimicrobial efficacy of PVP foils and nanofiber mats with different ciprofloxacin payloads applied on wounds infected with planktonic bacteria in a biofilm. (**a**) Representative pictures of untreated and treated wounds after 40 h of incubation. (**b**,**<sup>c</sup>**) Bacteria count in tissue extracts of wounds treated with foils (**b**) or nanofiber mats (**c**). (**d**) Bacteria counts in control wounds treated with plain PVP foils prepared from solutions in water or 30% acetic acid. Means and standard deviations of three independent experiments (three donors) run in triplicate are reported. Cipro—ciprofloxacin; AcCOOH—acetic acid.

Drug crystals were visible on the wound edges, where higher concentrations of the drug were reached after the foils or nanofibers had dissolved. When applied 1 h after bacterial inoculation, both foils and nanofiber mats resulted in a complete eradication of the bacterial infection independent from the amount of drug that was applied. This result shows the potential of these drug delivery materials for the prevention of wound infections. In particular, nanofibers should be tailored to obtain a more controlled drug delivery in order to have effective antimicrobial concentrations and limit adverse side effects.

In contrast, the 20-h-biofilm infection was more difficult to treat. PVP foils had the best performance (Figure 5b). Foils loaded with 500 μg ciprofloxacin achieved a complete eradication of the bacteria, and the two lower concentrations resulted in a 3-log reduction of bacteria concentration with respect to the untreated controls. Nanofiber mats had a lower antimicrobial activity with a maximal 3-log reduction achieved by the highest concentration. No effect on bacterial growth was measured for plain PVP foils or foils with residual acetic acid (Figure 5d).

The most striking result was the complete eradication of the biofilm infection achieved with the 500-μg PVP foils. Foils have a higher percentage of residual acetic acid (1.4%) than nanofiber mats (0.1%) [19]. Because acetic acid also has antimicrobial activity, it might have acted synergistically with ciprofloxacin. Nevertheless, the superior efficacy of the foils compared to the nanofiber mats correlated well with the delivery kinetics. Foils delivered higher amounts of the drug and over a longer time than nanofibers did. Phillips et al. assessed the efficacy of several commercially available treatments in a porcine ex vivo wound model and found that time-release silver gel and cadexomer iodine dressings were the most effective in reducing a mature biofilm with a reduction between 5 and 7 logs out of a 7-log total [24]. Using an in vivo mouse model, Roy et al. found that ciprofloxacin-loaded keratin-based hydrogels with a sustained drug release profile could reduce the amount of *P. aeruginosa* in the wound bed by 99.9% without interfering with the key processes of wound healing [36]. Thus, these and our results underline the importance of drug delivery systems with a sustained release profile for the efficient treatment of *P. aeruginosa* wound infections.
