**1. Introduction**

The skin is the major protective barrier against the environment and the loss its integrity, as a result of injury or illness, may lead to morbidity or even death.

Wound healing is a complex event, based on overlapping but well-orchestrated cellular and molecular processes, to repair damaged tissue and restore skin function [1,2]. The process of healing proceeds through different phases (hemostasis, inflammatory, proliferative and remodeling) and involves extracellular matrix (ECM) molecules, soluble mediators, as cytokines and growth factors, various resident cells, and infiltrating leucocytes. In nonhealing wounds, the healing process stops at the inflammatory state, and chronic wounds, such as venous leg ulcers, arterial ulcers, diabetic ulcers, and pressure ulcers, i.e., bed sores, fail to proceed through an orderly and timely process to restore skin anatomical and functional integrity [1,2]. Moreover, all of these wounds are contaminated by proliferating bacteria from the surrounding skin, the local environment, and the endogenous patient sources, resulting in wound colonization [3,4]. This could enhance or impair wound healing, depending on the bacterial load. In the absence of an effective immune response, impeded by underlying morbidity, as venous and arterial insufficiency, diabetes, or ageing, bacterial colonization becomes critical and an unavoidable transition towards infection occurs [3,4]. In fact, the exposed subcutaneous tissue provides a favorable substrate for the microbial growth of a wide variety of microorganisms. Moreover, a longer healing time could dramatically increase the possible occurrence of infection and biofilm formation [4,5].

Infections in nonhealing wounds remain one of the major challenges. Although appropriate systemic antibiotics are considered essential for the treatment of clinically infected wounds, topical antibiotics are not recommended since they could promote bacterial resistance. Recently, a nanomedicine approach, creating antimicrobial nanotherapeutics, has appeared to be a valid option to eliminate bacterial infections, since nanomaterials can overcome antibiotic resistance mechanisms, owing to their unique and advantageous physico-chemical properties [6,7]. In fact, several studies report that nanosystems interact with microorganisms upon multiple mechanisms, including electrostatic attraction, hydrophobic and Van der Waals forces through surface interactions, and this makes them promising candidates to achieve enhanced therapeutic efficacy against multidrug resistant (MDR) infections [6,7]. Considering this evidence, in this work, a norfloxacin–montmorillonite nanocomposite (VHS-N), previously prepared by an intercalation solution procedure, was encapsulated in nanofibrous scaffolds, since it proved to increase drug potency against both *Pseudomonas aeruginosa* and *Staphylococcus aureus* (probably due to the high surface area to volume ratio, which increases the contact area with target organisms), maintaining cytocompatibility towards fibroblasts in vitro [8].

Given this premise, the aim of this study was the loading of montmorillonite norfloxacin nanocomposite (VHS-N) in three types of biopolymer–polysaccharide-based scaffolds (chitosan-based (CH), chitosan/chondroitin sulfate-based (CH/CS), chitosan/hyaluronic acid-based (CH/HA) (H hybrid scaffolds) to obtain dermal substitutes, intended for the treatment of wounds prone to infection, such as chronic ulcers (diabetic foot, venous leg ulcers) and burns.

The hybrid scaffolds were compared with scaffolds with the same compositions in polysaccharides, but loaded with norfloxacin as a free drug (N scaffolds).

The unloaded scaffolds were previously designed and developed [9,10]. Briefly, chitosan and chitosan/glycosaminoglycan electrospun scaffolds were manufactured using electrospinning by means of a simple/single-step process. Polymeric blends in water/acetic acid mixture were electrospun and the resulting random scaffolds were crosslinked by heating to obtain water resistant systems. The scaffolds proved their effectiveness in enhancing cell growth in vitro (fibroblasts and endothelial cells) and wound healing in vivo in a murine, burn/excisional model [9]. Moreover, lysozyme, normally secreted by macrophages and polymorphonuclear neutrophilis during the inflammatory phase of the healing process, proved to degrade the scaffolds in vitro [10].

Chitosan, glycosaminoglycans and pullulan were selected since they are polysaccharide biopolymers (organic molecules synthesized by the living organisms [11]), and biopolymers are recognized as the most promising materials in wound healing since they are characterized by having many advantages over synthetic materials because of their biocompatibility, biodegradability, lower antigenicity and renewability [11]. Therefore, although there are some examples in the literature focused on the enhancement of wound healing using antimicrobial loaded electrospun scaffolds/ dressings [12–15], those were in large part based on synthetic polymers, as polycaprolactone [12–14] or polyethylene glycol [15], and produced using critical solvents such as formic acid [12], or chloroform [15].

Furthermore, biomaterial-based complex nanostructures developed by electrospinning could lead to great advancements in the drug delivery and bioengineering/biomedical panorama [16]. In fact, electrospinning is a robust and on-demand process with high-throughput capable of making available broadly used drugs, such as antibiotics/chemotherapeutics, and enhancing their activities thanks to the nanostructure. Moreover, the electrospun materials are characterized by high mimicry and mechanical properties capable of modulating biological processes and determining cell fate, as the case of biochemical signals [17].
