Sustainable whey proteins-nanostructured zinc oxide-based films for the treatment of chronic wounds: New insights from biopharmaceutical studies.

Chronic wounds represent silent epidemic affecting a large portion of the world population, especially the elders; in this context, the development of advanced bioactive dressings is imperative to accelerate wound healing process, while contrasting or preventing infections. The aim of the present work was to provide a deep characterization of the functional and biopharmaceutical properties of a sustainable thin and flexible films, composed of whey proteins alone (WPI) and added with nanostructured zinc oxide (WPZ) and intended for the management of chronic wounds. The potential of whey proteins-based films as wound dressings has been confirmed by their wettability, hydration properties, elastic behavior upon hydration, biodegradation propensity and, when added with nanostructured zinc oxide, antibacterial efficacy against both Gram-positive and Gram-negative pathogens, i.e. Staphylococcus aureus and Escherichia coli. In-vitro experiments, performed on normal human dermal fibroblasts, confirmed film cytocompatibility, also revealing the possible role of Zn2+ ions in promoting fibroblast proliferation. Finally, in-vivo studies on rat model confirmed film suitability to act as wound dressing, since able to ensure a regular healing process while providing effective protection from infections. In particular, both films WPI and WPZ are responsible for the formation in the wound bed of a continuous collagen layer similar to that of healthy skin.

Influence of Electrospun Fibre Secondary Morphology on Antibiotic Release Kinetic and Its Impact on Antimicrobic Efficacy.

Vascular graft infections are a severe complication in vascular surgery, with a high morbidity and mortality. Prevention and treatment involve the use of antibiotic- or antiseptic-impregnated artificial vascular grafts, but currently, there are no commercially available infection-proof small-diameter vascular grafts (SDVGs). In this work we investigated the antimicrobic activity of two SDVGs prototypes loaded with tobramycin and produced via the electrospinning of drug-doped PLGA (polylactide-co-glycolide) solutions. Differences in rheological and conductivity properties of the polymer solutions resulted in non-identical fibre morphology that deeply influenced the hydration profile and consequently the in vitro cumulative drug release, which was investigated by using a spectrofluorimetric technique. Using DDSolver Excel add-in, modelling of the drug release kinetic was performed to evaluate the release mechanism involved: Prototype 1 showed a sustained and diffusive driven drug release, which allowed for the complete elution of tobramycin within 2 weeks, whereas Prototype 2 resulted in a more extended drug release controlled by both diffusion and matrix relaxation. Time-kill assays performed on S. aureus and E. coli highlighted the influence of burst drug release on the decay rate of bacterial populations, with Prototype 1 being more efficient on both microorganisms. Nevertheless, both prototypes showed good antimicrobic activity over the 5 days of in vitro testing.

Polysaccharide-protein microparticles based-scaffolds to recover soft tissue loss in mild periodontitis.

Periodontal regeneration is extremely limited and unpredictable due to structural complications, as it requires the simultaneous restoration of different tissues, including cementum, gingiva, bone, and periodontal ligament. In this work, spray-dried microparticles based on green materials (polysaccharides - gums - and a protein - silk fibroin) are proposed to be implanted in the periodontal pocket as 3D scaffolds during non-surgical treatments, to prevent the progression of periodontal disease and to promote the healing in mild periodontitis. Arabic or xanthan gum have been associated to silk fibroin, extracted from Bombyx mori cocoons, and loaded with lysozyme due to its antibacterial properties. The microparticles were prepared by spray-drying and cross-linked by water vapor annealing, inducing the amorphous to semi-crystalline transition of the protein component. The microparticles were characterized in terms of their chemico-physical features (SEM, size distribution, structural characterization - FTIR and SAXS, hydration and degradation properties) and preclinical properties (lysozyme release, antibacterial properties, mucoadhesion, in vitro cells adhesion and proliferation and in vivo safety on a murine incisional wound model). The encouraging preclinical results highlighted that these three-dimensional (3D) microparticles could provide a biocompatible platform able to prevent periodontitis progression and to promote the healing of soft tissues in mild periodontitis.

Characterization and Molecular Modelling of Non-Antibiotic Nanohybrids for Wound Healing Purposes.

The healing process of chronic wounds continues to be a current clinical challenge, worsened by the risk of microbial infections and bacterial resistance to the most frequent antibiotics. In this work, non-antibiotic nanohybrids based on chlorhexidine dihydrochloride and clay minerals have been developed in order to design advanced therapeutic systems aimed to enhance wound healing in chronic lesions. To prepare the nanohybrids, two methodologies have been compared: the intercalation solution procedure and the spray-drying technique, the latter as a one-step process able to reduce preparation times. Nanohybrids were then fully studied by solid state characterization techniques. Computational calculations were also performed to assess the interactions between the drug and the clays at the molecular level. In vitro human fibroblast biocompatibility and antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa were assessed to check biocompatibility and potential microbicidal effects of the obtained nanomaterials. The results demonstrated the effective organic/inorganic character of the nanohybrids with homogeneous drug distribution into the clayey structures, which had been confirmed by classical mechanics calculations. Good biocompatibility and microbicidal effects were also observed, especially for the spray-dried nanohybrids. It was suggested that it could be due to a greater contact area with target cells and bacterial suspensions.

Electrospun Scaffolds Based on Poly(butyl cyanoacrylate) for Tendon Tissue Engineering.

Tendon disorders are common medical conditions that could lead to significant disability, pain, healthcare costs, and a loss of productivity. Traditional approaches require long periods of treatment, and they largely fail due to the tissues weakening and the postoperative alterations of the normal joint mechanics. To overcome these limitations, innovative strategies for the treatment of these injuries need to be explored. The aim of the present work was the design of nano-fibrous scaffolds based on poly(butyl cyanoacrylate) (PBCA), a well-known biodegradable and biocompatible synthetic polymer, doped with copper oxide nanoparticles and caseinphosphopeptides (CPP), able to mimic the hierarchical structure of the tendon and to improve the tissue healing potential. These were developed as implants to be sutured to reconstruct the tendons and the ligaments during surgery. PBCA was synthetized, and then electrospun to produce aligned nanofibers. The obtained scaffolds were characterized for their structure and physico-chemical and mechanical properties, highlighting that CuO and CPP loading, and the aligned conformation determined an increase in the scaffold mechanical performance. Furthermore, the scaffolds loaded with CuO showed antioxidant and anti-inflammatory properties. Moreover, human tenocytes adhesion and proliferation to the scaffolds were assessed in vitro. Finally, the antibacterial activity of the scaffolds was evaluated using Escherichia coli and Staphylococcus aureus as representative of Gram-negative and Gram-positive bacteria, respectively, demonstrating that the CuO-doped scaffolds possessed a significant antimicrobial effect against E. coli. In conclusion, scaffolds based on PBCA and doped with CuO and CPP deserve particular attention as enhancers of the tendon tissue regeneration and able to avoid bacterial adhesion. Further investigation on the scaffold efficacy in vivo will assess their capability for enhancing the tendon ECM restoration in view of accelerating their translation to the clinic.

Dual mode antibacterial surfaces based on Prussian blue and silver nanoparticles.

Prussian Blue (PB) is an inexpensive, biocompatible, photothermally active material. In this paper, self-assembled monolayers of PB nanoparticles were grafted on a glass surface, protected with a thin layer of silica and decorated with spherical silver nanoparticles. This combination of a photothermally active nanomaterial, PB, and an intrinsically antibacterial one, silver, leads to a versatile coating that can be used for medical devices and implants. The intrinsic antibacterial action of nanosilver, always active over time, can be enhanced on demand by switching on the photothermal effect of PB using near infrared (NIR) radiation, which has a good penetration depth through tissues and low side effects. Glass surfaces functionalized by this layer-by-layer approach have been characterized for their morphology and composition, and their intrinsic and photothermal antibacterial effect was studied against Gram+ and Gram- planktonic bacteria.

A Supramolecular Approach to Antimicrobial Surfaces.

In this paper, we report on the preparation of Imidazole-functionalized glass surfaces, demonstrating the ability of a dinuclear Cu(II) complex of a macrocyclic ligand to give a "cascade" interaction with the deprotonated forms of grafted imidazole moieties. In this way, we realized a prototypal example of an antimicrobial surface based on a supramolecular approach, obtaining a neat microbicidal effect using low amounts of the described copper complex.

A chemometric assessment and profiling of the essential oils from Hibiscus sabdariffa L. from Kurdistan, Iraq.

Hibiscus sabdariffa L. is a tropical plant belonging to the Malvaceae family. In Kurdistan, the Autonomous Region of Iraq, water infusion of H. sabdariffa calyces is recommended for the treatment of hypotension and the common cold. Three distillation techniques: hydrodistillation (HD), steam distillation (SD), and solvent-free microwave-assisted extraction (SFME) have been compared to obtain the essential oils from calyces. The composition of the extracts was investigated by GC-FID and GC-MS. A total of 62 compounds have been identified, from which 55 components were found in HD distillates (95.75%), 37 components in SFME (96.06%), and 29 in SD (99.63%). Chemometric tools were applied to optimise and evidence the relation between distillation techniques and composition of the obtained essential oils as an investigation for the essential oils commercialisation approach of Hibiscus sabdariffa L. that have been done from a long time using conventional hydrodistillation in the local Herbal and Tea markets in Kurdistan.

Gold Nanostars Embedded in PDMS Films: A Photothermal Material for Antibacterial Applications.

Bacteria infections and related biofilms growth on surfaces of medical devices are a serious threat to human health. Controlled hyperthermia caused by photothermal effects can be used to kill bacteria and counteract biofilms formation. Embedding of plasmonic nano-objects like gold nanostars (GNS), able to give an intense photothermal effect when irradiated in the NIR, can be a smart way to functionalize a transparent and biocompatible material like polydimethylsiloxane (PDMS). This process enables bacteria destruction on surfaces of PDMS-made medical surfaces, an action which, in principle, can also be exploited in subcutaneous devices. We prepared stable and reproducible thin PDMS films containing controllable quantities of GNS, enabling a temperature increase that can reach more than 40 degrees. The hyperthermia exerted by this hybrid material generates an effective thermal microbicidal effect, killing bacteria with a near infrared (NIR) laser source with irradiance values that are safe for skin.

Tobramycin Supplemented Small-Diameter Vascular Grafts for Local Antibiotic Delivery: A Preliminary Formulation Study.

Peripheral artery occlusive disease is an emerging cardiovascular disease characterized by the blockage of blood vessels in the limbs and is associated with dysfunction, gangrene, amputation, and a high mortality risk. Possible treatments involve by-pass surgery using autologous vessel grafts, because of the lack of suitable synthetic small-diameter vascular prosthesis. One to five percent of patients experience vascular graft infection, with a high risk of haemorrhage, spreading of the infection, amputation and even death. In this work, an infection-proof vascular graft prototype was designed and manufactured by electrospinning 12.5% w/v poly-L-lactic-co-glycolic acid solution in 75% v/v dichloromethane, 23.8% v/v dimethylformamide and 1.2% v/v water, loaded with 0.2% w/wPLGA. Polymer and tobramycin concentrations were selected after viscosity and surface tension and after HPLC-UV encapsulation efficiency (EE%) evaluation, respectively. The final drug-loaded prototype had an EE% of 95.58% ± 3.14%, with smooth fibres in the nanometer range and good porosity; graft wall thickness was 291 ± 20.82 µm and its internal diameter was 2.61 ± 0.05 mm. The graft's antimicrobic activity evaluation through time-kill assays demonstrated a significant and strong antibacterial activity over 5 days against Staphylococcus aureus and Escherichia coli. An indirect cell viability assay on Normal Human Dermal Fibroblasts (NHDF) confirmed the cytocompatibility of the grafts.

PVA Films with Mixed Silver Nanoparticles and Gold Nanostars for Intrinsic and Photothermal Antibacterial Action.

PVA films with embedded either silver nanoparticles (AgNP), NIR-absorbing photothermal gold nanostars (GNS), or mixed AgNP+GNS were prepared in this research. The optimal conditions to obtain stable AgNP+GNS films with intact, long lasting photothermal GNS were obtained. These require coating of GNS with a thiolated polyethylene glycol (PEG) terminated with a carboxylic acid function, acting as reticulant in the film formation. In the mixed AgNP+GNS films, the total noble metal content is <0.15% w/w and in the Ag films < 0.025% w/w. The slow but prolonged Ag+ release from film-embedded AgNP (8-11% of total Ag released after 24 h, in the mixed films) results in a very strong microbicidal effect against planktonic Escherichia coli and Staphylococcus aureus bacterial strains (the release of Au from films is instead negligible). Beside this intrinsic effect, the mixed films also exert an on-demand, fast hyperthermal bactericidal action, switched on by NIR laser irradiation (800 nm, i.e., inside the biotransparent window) of the localized surface plasmon resonance (LSPR) absorption bands of GNS. Temperature increases of 30 °C are obtained using irradiances as low as 0.27 W/cm2. Moreover, 80-90% death on both strains was observed in bacteria in contact with the GNS-containing films, after 30 min of irradiation. Finally, the biocompatibility of all films was verified on human fibroblasts, finding negligible viability decrease in all cases.

Microstructured Lipid Carriers (MLC) Based on N-Acetylcysteine and Chitosan Preventing Pseudomonas aeruginosa Biofilm.

The aim of this work was the development of microstructured lipid carriers (MLC) based on chitosan (CH) and containing N-acetylcysteine (NAC), a mucolytic and antioxidant agent, to inhibit the formation of Pseudomonas aeruginosa biofilm. MLC were prepared using the high shear homogenization technique. The MLC were characterized for morphology, particle size, Z potential, encapsulation efficiency and drug release. The antioxidant properties of NAC-loaded microstructured carriers were evaluated through an in vitro spectrophotometer assay. Finally, the activity of NAC-CH-MLC on biofilm production by Pseudomonas aeruginosa was also evaluated. Results obtained from this study highlighted that the use of chitosan into the inner aqueous phase permitted to obtain microstructured particles with a narrow size range and with good encapsulation efficiency. NAC-loaded MLC showed higher antioxidant activity than the free molecule, demonstrating how encapsulation increases the antioxidant effect of the molecule. Furthermore, the reduction of biofilm growth resulted extremely high with MLC being 64.74% ± 6.2% and 83.74% ± 9.95%, respectively, at 0.5 mg/mL and 2 mg/mL. In conclusion, this work represents a favorable technological strategy against diseases in which bacterial biofilm is relevant, such as cystic fibrosis.

GMP-compliant sponge-like dressing containing MSC lyo-secretome: Proteomic network of healing in a murine wound model.

Chronic wounds account for 3% of total healthcare expenditure of developed countries; thus, innovative therapies, including Mesenchymal Stem Cells (MSCs) end their exosomes are increasingly considered, even if the activity depends on the whole secretome, made of both soluble proteins and extracellular vesicles. In this work, we prove for the first time the in vivo activity of the whole secretome formulated in a sponge-like alginate wound dressing to obtain the controlled release of bioactive substances. The product has been prepared in a public GMP-compliant facility by a scalable process; based on the murine model, treated wounds healed faster than controls without complications or infections. The treatment induced a higher acute inflammatory process in a short time and sustained the proliferative phase by accelerating fibroblast migration, granulation tissue formation, neovascularization and collagen deposition. The efficacy was substantially supported by the agreement between histological and proteomic findings. In addition to functional modules related to proteolysis, complement and coagulation cascades, protein folding and ECM remodeling, in treated skin, emerged the role of specific wound healing related proteins, including Tenascin (Tnc), Decorin (Dcn) and Epidermal growth factor receptor (EGFR). Of note, Decorin and Tenascin were also components of secretome, and network analysis suggests a potential role in regulating EGFR. Although further experiments will be necessary to characterize better the molecular keys induced by treatment, overall, our results confirm the whole secretome efficacy as novel "cell-free therapy". Also, sponge-like topical dressing containing the whole secretome, GMP- compliant and "ready-off-the-shelf", may represent a relevant point to facilitate its translation into the clinic.

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

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.

Self-Assembled Monolayers of Copper Sulfide Nanoparticles on Glass as Antibacterial Coatings.

We developed an easy and reproducible synthetic method to graft a monolayer of copper sulfide nanoparticles (CuS NP) on glass and exploited their particular antibacterial features. Samples were fully characterized showing a good stability, a neat photo-thermal effect when irradiated in the Near InfraRed (NIR) region (in the so called "biological window"), and the ability to release controlled quantities of copper in water. The desired antibacterial activity is thus based on two different mechanisms: (i) slow and sustained copper release from CuS NP-glass samples, (ii) local temperature increase caused by a photo-thermal effect under NIR laser irradiation of CuS NP-glass samples. This behavior allows promising in vivo applications to be foreseen, ensuring a "static" antibacterial protection tailored to fight bacterial adhesion in the critical timescale of possible infection and biofilm formation. This can be reinforced, when needed, by a photo-thermal action switchable on demand by an NIR light.

Development of a Mucoadhesive in Situ Gelling Formulation for the Delivery of Lactobacillus gasseri into Vaginal Cavity.

Local administration of vaginal probiotics, especially lactobacilli, has been recently proposed as an effective prevention strategy against candidosis recurrences, which affect 40-50% of women. In this context, the aim of the present work was the development of a mucoadhesive in situ gelling formulation for the vaginal administration of Lactobacillus gasseri. Mixtures of poloxamer 407 (P407) and methylcellulose (MC), two thermosensitive polymers, were prepared and subjected to rheological analyses for the assessment of their sol/gel transition temperature. The association of P407 (15% w/w) with MC (1.5% w/w) produced an increase in gelation extent at 37 °C even after dilution in simulated vaginal fluid (SVF). The presence of 0.5% w/w pectin (PEC) produced a reduction of vehicle pH and viscosity at 25 °C that is the vehicle resistance to flow during administration. The presence of a low concentration of xyloglucan (XYL) (0.25% w/w) increases the mucoadhesive properties and the capability to gelify at 37 °C of the formulation after dilution with SVF. A three-component (P407/MC/PEC; 3cM) and a four-component (P407/MC/PEC/XYL; 4cM) mixture were selected as promising candidates for the delivery of L. gasseri to the vaginal cavity. They were able to preserve L. gasseri viability and were cytocompatible towards the HeLa cell line.

High Stability Thiol-Coated Gold Nanostars Monolayers with Photo-Thermal Antibacterial Activity and Wettability Control.

The adhesion and proliferation of bacteria on abiotic surfaces pose challenges in both health care and industrial applications. Gold nanostars (GNSs) monolayers grafted on glass have demonstrated to exert antibacterial action due to their photo-thermal features. Here, these GNS layers were further functionalized using thiols monolayers, in order to impart different wettability to the surfaces and thus adding a feature that could help to fight bacterial proliferation. Thiol that has different functional groups was used and the thiol-protected surfaces were characterized by means of UV-vis spectroscopy, contact angles, SEM and surface enhanced Raman spectroscopy (SERS). We verified that (i) coating with the proper thiol allows us to impart high hydrophilicity or hydrophobicity to the surfaces (with contact angle values ranging from 10 to 120°); (ii) GNS monolayers are strongly stabilized by functionalization with thiols, with shelf stability increasing from a few weeks to more than three months and (iii) photo-thermal features and subsequent antibacterial effects caused by hyperthermia are not changed by thiols layers, allowing us to kill at least 99.99% of representative bacterial strains.

High Bactericidal Self-Assembled Nano-Monolayer of Silver Sulfadiazine on Hydroxylated Material Surfaces.

Anti-infective surfaces are a modern strategy to address the issue of infection related to the clinical use of materials for implants and medical devices. Nanocoatings, with their high surface/mass ratio, lend themselves to being mono-layered on the material surfaces to release antibacterial molecules and prevent bacterial adhesion. Here, a "layer-by-layer" (LbL) approach to achieve a self-assembled monolayer (SAM) with high microbicidal effect on hydroxylated surfaces is presented, exploiting the reaction between a monolayer of thiolic functions on glass/quartz surfaces and a newly synthesized derivative of the well-known antibacterial compound silver sulfadiazine. Using several different techniques, it is demonstrated that a nano-monolayer of silver sulfadiazine is formed on the surfaces. The surface-functionalized materials showed efficient bactericidal effect against both Gram-positive and Gram-negative bacteria. Interestingly, bactericidal self-assembled nano-monolayers of silver sulfadiazine could be achieved on a large variety of materials by simply pre-depositing glass-like SiO2 films on their surfaces.

Adipose Mesenchymal Extracellular Vesicles as Alpha-1-Antitrypsin Physiological Delivery Systems for Lung Regeneration.

Accumulating evidence shows that Mesenchymal Stem/Stromal Cells (MSCs) exert their therapeutic effects by the release of secretome, made of both soluble proteins and nano/microstructured extracellular vesicles (EVs). In this work, for the first time, we proved by a proteomic investigation that adipose-derived (AD)-MSC-secretome contains alpha-1-antitrypsin (AAT), the main elastase inhibitor in the lung, 72 other proteins involved in protease/antiprotease balance, and 46 proteins involved in the response to bacteria. By secretome fractionation, we proved that AAT is present both in the soluble fraction of secretome and aggregated and/or adsorbed on the surface of EVs, that can act as natural carriers promoting AAT in vivo stability and activity. To modulate secretome composition, AD-MSCs were cultured in different stimulating conditions, such as serum starvation or chemicals (IL-1ß and/or dexamethasone) and the expression of the gene encoding for AAT was increased. By testing in vitro the anti-elastase activity of MSC-secretome, a dose-dependent effect was observed; chemical stimulation of AD-MSCs did not increase their secretome anti-elastase activity. Finally, MSC-secretome showed anti-bacterial activity on Gram-negative bacteria, especially for Klebsiellapneumoniae. These preliminary results, in addition to the already demonstrated immunomodulation, pave the way for the use of MSC-secretome in the treatment of AAT-deficiency lung diseases.

Montmorillonite-norfloxacin nanocomposite intended for healing of infected wounds.

Background: Chronic cutaneous wounds represent a major issue in medical care and are often prone to infections. Purpose: The aim of this study was the design of a clay mineral-drug nanocomposite based on montmorillonite and norfloxacin (NF, antimicrobial drug) as a powder for cutaneous application, to enhance wound healing in infected skin lesions. Methods: The nanocomposite has been prepared by means of an intercalation solution procedure. Adsorption isotherm, solid-state characterization of the nanocomposite, drug loading capacity and its release have been performed. Moreover, cytocompatibility, in vitro fibroblast proliferation and antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus were assessed. Results: The clay drug adsorption isotherm demonstrates that the mechanism of NF intercalation into montmorillonite galleries is the adsorption as one single process, due to the charge-charge interaction between protonated NF and negatively charged montmorillonite edges in the interlayer space. Nanocomposite is biocompatible and it is characterized by antimicrobial activity greater than the free drug: this is due to its nanostructure and controlled drug release properties. Conclusion: Considering the results obtained, NF-montmorillonite nanocomposite seems a promising tool to treat infected skin lesions or skin wounds prone to infection, as chronic ulcers (diabetic foot, venous leg ulcers) and burns.