Evaluation of Drug Delivery and Efficacy of Ciprofloxacin-Loaded Povidone Foils and Nanofiber Mats in a Wound-Infection Model Based on Ex Vivo Human Skin

Topical treatment of wound infections is often a challenge due to limited drug availability at the site of infection. Topical drug delivery is an attractive option for reducing systemic side effects, provided that a more selective and sustained local drug delivery is achieved. In this study, a poorly water-soluble antibiotic, ciprofloxacin, was loaded on polyvinylpyrrolidone (PVP)-based foils and nanofiber mats using acetic acid as a solubilizer. Drug delivery kinetics, local toxicity, and antimicrobial activity were tested on an ex vivo wound model based on full-thickness human skin. Wounds of 5 mm in diameter were created on 1.5 × 1.5 cm skin blocks and treated with the investigated materials. While nanofiber mats reached the highest amount of delivered drug after 6 h, foils rapidly achieved a maximum drug concentration and maintained it over 24 h. The treatment had no effect on the overall skin metabolic activity but influenced the wound healing process, as observed using histological analysis. Both delivery systems were efficient in preventing the growth of Pseudomonas aeruginosa biofilms in ex vivo human skin. Interestingly, foils loaded with 500 µg of ciprofloxacin accomplished the complete eradication of biofilm infections with 1 × 109 bacteria/wound. We conclude that antimicrobial-loaded resorbable PVP foils and nanofiber mats are promising delivery systems for the prevention or topical treatment of infected wounds

Screening of Surfactants for Improved Delivery of Antimicrobials and Poly-Lactic-co-Glycolic Acid Particles in Wound Tissue

Topical wound management is often a challenge due to the poor penetration of antimicrobials in wound tissue and across the biofilm matrix where bacteria are embedded. Surfactants have been used for decades to improve the stability of formulations, increase drug solubility, and enhance penetration. In this study, we screened different detergents with respect to their cytotoxicity and their ability to improve the penetration of poly-lactic-co-glycolic acid (PLGA) particles in wound tissue. Among the tested surfactants, Kolliphor SLS and Tween 80 increased the penetration of PLGA particles and had a limited cytotoxicity. Then, these surfactants were used to formulate PLGA particles loaded with the poorly water-soluble antibiotic ciprofloxacin. The antimicrobial efficacy of the formulations was tested in a wound infection model based on human ex vivo skin. We found that even though PLGA particles had the same antimicrobial efficiency than the particle-free drug formulation, thanks to their solubilizing and anti-biofilm properties, the surfactants remarkably improved the antimicrobial activity of ciprofloxacin with respect to the drug formulation in water. We conclude that the use of Tween 80 in antimicrobial formulations might be a safe and efficient option to improve the topical antimicrobial management of chronic wound infections

Initial histologic examination of intact skin, induced wounds and infected wounds.

<p>(A and B) Richardson stained section of intact human skin (abdomen), shortly after the cosmetic surgery. (C and D) Induced superficial wound: the epidermis was removed with a ball shaped milling cutter. (E-G) Wound, intradermally infected with <i>P</i>. <i>aeruginosa</i>. The bacteria (visible as black dots—arrow 1) are found scattered in the upper woundlayers (F) as well as in deeper wound regions (G).</p

Quantification of selected interleukins in infected and uninfected wounds.

<p>(A) IL-1α, (B) IL-1β*, (C) IL-6, and (D) IL-8 concentrations of uninfected and infected wounds after 0 and 20 hours. (●,▴ and ■ represent triplicates of donors 1–3) (*Due to the limited volume of the testing material, triplicates had to be pooled). * p> 0.05, **p>0.01, *** p < 0.001.</p

Wound histology in the light microscope, twenty hours after the infection.

<p>(A-B). The Richardson stained sections show a dense layer of bacteria that takes up the upper part of the wound (circle 1). (C and D) Lower skin layers—close to the subcutis—reveal scattered bacteria.</p

Bacteria quantification and antimicrobial treatment.

<p>(A) Bacteria counts of uninfected and infected wounds after 0 and 20 hours (●,▴, ■ and ◆ represent triplicates of donors 1–4). (B) Bacteria counts of infected wounds with and without topical ciprofloxacin treatment (10 μL, 375 μg mL^-1, 20 hours,) (●,▴ and ■ represent triplicates of donors 5–7). *** p < 0.001.</p

Wound histology in the electron microscope, twenty hours after the infection.

<p>(A) Scanning electron microscopic image of the skin wound profile: the wound surface is entirely covered with layers of bacteria (circle 1), (B) whose hill-like structures consist of collagen bundles (arrow 1) colonized by bacteria (arrow 2). (C and D) (Transmission electron microscopic images) Ultrathin sectioning reveals the organization of the bacteria within the collagen bundles and physical contact between bacteria and collagen fibers (arrow 1).</p

Development, standardization and testing of a bacterial wound infection model based on ex vivo human skin

Current research on wound infections is primarily conducted on animal models, which limits direct transferability of these studies to humans. Some of these limitations can be overcome by using–otherwise discarded—skin from cosmetic surgeries. Superficial wounds are induced in fresh ex vivo skin, followed by intradermal injection of Pseudomonas aeruginosa under the wound. Subsequently, the infected skin is incubated for 20 hours at 37°C and the CFU/wound are determined. Within 20 hours, the bacteria count increased from 107 to 109 bacteria per wound, while microscopy revealed a dense bacterial community in the collagen network of the upper wound layers as well as numerous bacteria scattered in the dermis. At the same time, IL-1alpha and IL-1beta amounts increased in all infected wounds, while—due to bacteria-induced cell lysis—the IL-6 and IL-8 concentrations rose only in the uninfected samples. High-dosage ciprofloxacin treatment resulted in a decisive decrease in bacteria, but consistently failed to eradicate all bacteria. The main benefits of the ex vivo wound model are the use of healthy human skin, a quantifiable bacterial infection, a measureable donor-dependent immune response and a good repeatability of the results. These properties turn the ex vivo wound model into a valuable tool to examine the mechanisms of host-pathogen interactions and to test antimicrobial agents