**4. Metal nanoantimicrobials for wound dressing applications**

Wound healing still represents a clinical challenge, which requires efficient wound management strategies [69]. Indeed, a crucial component of wound care is the choice of dressing. Many modern wound dressings have been developed to promote wound healing, such as dressings designed to absorb exudate, to provide an ideal moisture balance at the wound surface, to prevent maceration of surrounding tissue and infections and to reduce the bacterial load [70, 71]. Biomaterials, such as chitosan, alginate and collagen, play an important role as wound dressing materials by accelerating the healing of wounds and also because they can embed many nanoparticles for the development of metal nanoparticles-based wound dressings [69, 72]. Hydrogel-based wound dressings provide a cooling sensation and a moisture environment [73]. Several systems based on the combination of hydrogel and metal nanoparticles, such as zinc, copper and silver, have been recently proposed by many authors, aiming to develop wound dressings with antibacterial and enhanced wound-healing properties. For example, an alginate hydrogel/zinc oxide nanoparticles composite bandage was developed by Mohandas *et al*. using a freeze-drying method. The results obtained demonstrated controlled degradation profile and faster blood clotting ability, along with excellent antimicrobial activity against different microorganisms such as *Escherichia coli*, *Staphylococcus aureus*, *Candida albicans* and methicillin resistant *S. aureus* (MRSA) [74]. β-chitin hydrogel/nZnO composite bandages with interconnected micro-porous structure were also obtained by freeze-drying technique and proposed for infected wounds with large volume of exudate. Indeed, the wounds treated with the composite bandages promoted the healing and the re-epithelialization, enhanced collagen deposition and showed reduced number of bacterial colonies [75]. Other formulations involving the use of chitin hydrogel/nano ZnO composite bandages were proposed by Kumar *et al*. for burn, diabetic and chronic wound defects, because of the enhanced swelling, blood clotting and antibacterial properties achieved [76]. Kumar *et al*. have also developed a flexible and microporous chitosan hydrogel/nano zinc oxide composite bandages by incorporating the zinc oxide nanoparticles into chitosan hydrogel. *In vivo* woundhealing evaluations proved the enhanced healing ability of the materials without causing toxicity to cells [73]. Chitosan and copper nanoparticles co-introduced into an ointment preparation were investigated by Rakhmetova *et al*. and their combination at certain ratio of components, concentrations and physicochemical characteristics enhanced the antibacterial and wound-healing properties of the individual components [77]. Babushkina *et al*. demonstrated the efficacy of local application of a suspension of copper and zinc nanoparticles and of a drug based on chitosan and copper/zinc on bacterial contaminated purulent wound in rats [78]. Copper (II) cross-linked alginate hydrogels with body fluid absorption ability and haemostatic properties were developed and suggested by Klinkajon and Supaphol for the treatment of exudation/bleeding wounds and burns [79].

In combination with silver, copper nanoparticles may give rise to more complete bactericidal effect against a mixed bacterial population [56]. The broad-spectrum antimicrobial activity of silver has been demonstrated against a wide range of microorganisms, including methicillin resistant bacteria, fungi and viruses [58]. Although the exact antimicrobial mechanism still represents a debated topic, many theories on the action of silver nanoparticles on microbes have been proposed. One of them involves the anchorage and penetration of the nanoparticles into the bacterial cell wall, which cause structural changes in the cell membrane such as permeability and respiration [59–62]. *E. coli* cells treated with silver nanoparticles appear damaged and show the formation of 'pits' in the cell wall of the bacteria, where the silver nanoparticles accumulate [59, 63]. Another antibacterial mechanism involves the release of silver ions and their interaction with the enzymes of the respiratory chain, the cell membrane and the DNA. The binding of silver to the membrane can inhibit the passage of nutrients through the membrane, interfering with normal concentration gradients between the cell and the surrounding environment, so leading to cell death [64, 65]. The formation of free radicals has also the ability to damage the cell membrane and makes it porous, thus causing the death

Nanosilver products safety data available in EPA's formal incident reporting database indicates that nanosilver products are safe. Silver nanoparticles can be easily incorporated into matrix materials and have demonstrated a great potential in applications of huge interest in nanotechnology [66]. When incorporated into wound treatment systems, silver nanoparticles can provide clinically relevance in the development of ideal environment for rapid and effective healing. These systems may significantly reduce the time required for the homeostatic equilibrium, while reducing the risk of complications and improving the physical appearance of the scar [67]. Silver nanoparticles induce rapid healing and improved cosmetic appearance in a dose-dependent manner and exert positive effects through their antimicrobial properties,

Wound healing still represents a clinical challenge, which requires efficient wound management strategies [69]. Indeed, a crucial component of wound care is the choice of dressing. Many modern wound dressings have been developed to promote wound healing, such as dressings designed to absorb exudate, to provide an ideal moisture balance at the wound surface, to prevent maceration of surrounding tissue and infections and to reduce the bacterial load [70, 71]. Biomaterials, such as chitosan, alginate and collagen, play an important role as wound dressing materials by accelerating the healing of wounds and also because they can embed many nanoparticles for the development of metal nanoparticles-based wound dressings [69, 72]. Hydrogel-based wound dressings provide a cooling sensation and a moisture environment [73]. Several systems based on the combination of hydrogel and metal nanoparticles, such as zinc, copper and silver, have been recently proposed by many authors, aiming to develop wound dressings with antibacterial and enhanced wound-healing properties. For example, an alginate hydrogel/zinc oxide nanoparticles composite bandage was developed

reduction in wound inflammation and modulation of fibrogenic cytokines [68].

**4. Metal nanoantimicrobials for wound dressing applications**

of bacteria [66].

440 Wound Healing - New insights into Ancient Challenges

Among the recent trends against burn infections involving the use of noble metal antimicrobials, the most prevalent is represented by silver [80]. For nearly 50 years, silver-containing compounds have been the mainstay of burn wound care and silver sulfadiazine (SSD) has been the standard topical antimicrobial for burn wounds for decades [64, 81].

Silver has been used as an antimicrobial agent for a long time in the form of metal silver and silver sulfadiazine ointments [41], and today, there is scientific evidence supporting the use of silver-based wound dressings highlighting antimicrobial efficacy on biofilms within the *in vitro* and *in vivo* environments [40]. A number of wound dressings developed using silver have been approved by the US Food and Drug Administration (FDA) [82]. In addition to antimicrobial activity, silver dressings may modulate or reduce wound pain and limit the frequency of changes [83]. While topical silver creams and solutions require frequent application, the dressings can control the release of silver to the wound and require to be changed with less frequency [84]. Nanocrystalline silver dressings are considered as the gold standard in the conservative treatment of wounds and burns. It has been demonstrated that nanosilver has both anti-inflammatory effects and improves wound healing [85]. The healing response studied by Chowdhury *et al*. in laparotomy wounds after application of silver nanoparticles determined increased collagen expression from dermal fibroblasts, improved wound healing and reduced microbial load [86]. Rigo *et al*. have observed that the application of Ag NP-based dressing for prolonged time does not affect the proliferation of fibroblasts and keratinocytes, leading to the restoration of the organized skin structure in previously unhealed parts of the wound [87]. Polyvinyl alcohol (PVA) hydrogels loaded with a controlled concentration of silver could combine the hydrogel property of keeping a moisturized environment, thus stimulating healing, with the effect of silver of inhibiting or killing the bacteria [88]. PVA-Ag NPs mats, fabricated by Nguyen *et al*. from a suspension of PVA and Ag NPs after microwave irradiation, possess high tensile stress and anti-bacterial activities at the same time and were proposed as a promoter of wound healing [89].

Hydrogels with polyvinyl pyrrolidone (PVP) and alginate were synthesized by Singh *et al*., and silver nanoparticles were incorporated in hydrogel network using gamma radiation. The hydrogel-containing nanosilver demonstrated strong antimicrobial effect and complete inhibition of microbial growth, absorption capacity, moisture permeability and the ability to prevent fluid accumulation in exudating wound [90]. Chitosan-PVP-nanosilver oxide wound dressings showed excellent results such as good swelling capability, good antibacterial activity and also transparency of the film, which helps to regularly monitor the condition of wound without removing it from the wound site [81]. The silver nanocrystalline chitosan dressing described by Lu *et al*. significantly increased the rate of wound healing and was associated with silver levels in blood and tissues well below those associated with the silver sulfadiazine dressing (p < 0.01) [91]. Silver released in a moist wound surface environment significantly increases the rate of re-epithelialization compared to a standard antibiotic solution, as demonstrated by Demling *et al*. [92].

The application of both silver dressings and antibiotic therapy can have a synergistic effect in improving wound healing, since the interaction of silver released from the dressings significantly increases the susceptibility of bacterial cells within biofilms to antibiotics. Moreover, the reduction of the silver particle size to nanoscale level provides better penetration and accumulation of silver within biofilms, thus contributing to the effectiveness of the silver based product [93]. As silver is the most widely used substance to obtain antimicrobial effects, different formulations involving the use of silver-containing solution or silver nanoparticles have been developed. Among the most widespread antimicrobial dressings, silver foam dressings and silver alginate dressings are applied to exuding wounds and demonstrate improved performances than the traditional gauze dressings [94]. Silver alginate wound dressings have demonstrated beneficial effects on wound healing, in terms of wound exudates levels and prevention from wound infections [95–97]. Silver alginate dressings are particularly known for the prolonged antimicrobial efficacy, which indicates sustained availability of ionic silver and suggests the necessity of reduced dressings changes [98]. Excellent and sustainable controllability of Ag+ release were obtained by the AgNP-bacterial cellulose hybrid nanostructure developed by Wu *et al*., which offered promising results for antimicrobial wound dressing through the addition of silver nanoparticles. Indeed, bacterial cellulose has attracted great attention as novel wound dressing material, but it has no antimicrobial activity [99]. The silver nanoparticle/bacterial cellulose gel membranes developed by Wu *et al*. demonstrated *in vivo* excellent healing effects in a second-degree rat wound model and were proposed as promising antimicrobial wound dressing with good biocompatibility to promote scald wound healing [100].

The use of cellulose/nanosilver sponge materials was strongly encouraged in case of serious wound infection and *in vivo* tests confirmed accelerate infected wound healing and absorbing capacity for wound exudate [101]. Other examples of composite scaffolds are biocomposite films containing alginate and sago starch impregnated with silver nanoparticles [102], chitin/ nanosilver composite scaffolds and electrospun mats doped with nanosilver, zinc oxide, etc., as degradable and non-degradable polymers [103, 104]. For example, polymeric nanofilmcontaining silver nanoparticles exhibit antimicrobial activity at loadings and release rates of silver lower than conventional dressings. When placed on a moist wound, the PVA dissolves and the silver-loaded nanofilm results immobilized on the wound bed, thus allowing the normal and complete wound closure by re-epithelialization [104]. A general overview of some relevant techniques adopted to incorporate nanometals into hydrogel network for wound dressing production is reported in **Table 1**.

leading to the restoration of the organized skin structure in previously unhealed parts of the wound [87]. Polyvinyl alcohol (PVA) hydrogels loaded with a controlled concentration of silver could combine the hydrogel property of keeping a moisturized environment, thus stimulating healing, with the effect of silver of inhibiting or killing the bacteria [88]. PVA-Ag NPs mats, fabricated by Nguyen *et al*. from a suspension of PVA and Ag NPs after microwave irradiation, possess high tensile stress and anti-bacterial activities at the same time and were proposed as

Hydrogels with polyvinyl pyrrolidone (PVP) and alginate were synthesized by Singh *et al*., and silver nanoparticles were incorporated in hydrogel network using gamma radiation. The hydrogel-containing nanosilver demonstrated strong antimicrobial effect and complete inhibition of microbial growth, absorption capacity, moisture permeability and the ability to prevent fluid accumulation in exudating wound [90]. Chitosan-PVP-nanosilver oxide wound dressings showed excellent results such as good swelling capability, good antibacterial activity and also transparency of the film, which helps to regularly monitor the condition of wound without removing it from the wound site [81]. The silver nanocrystalline chitosan dressing described by Lu *et al*. significantly increased the rate of wound healing and was associated with silver levels in blood and tissues well below those associated with the silver sulfadiazine dressing (p < 0.01) [91]. Silver released in a moist wound surface environment significantly increases the rate of re-epithelialization compared to a standard antibiotic solution, as

The application of both silver dressings and antibiotic therapy can have a synergistic effect in improving wound healing, since the interaction of silver released from the dressings significantly increases the susceptibility of bacterial cells within biofilms to antibiotics. Moreover, the reduction of the silver particle size to nanoscale level provides better penetration and accumulation of silver within biofilms, thus contributing to the effectiveness of the silver based product [93]. As silver is the most widely used substance to obtain antimicrobial effects, different formulations involving the use of silver-containing solution or silver nanoparticles have been developed. Among the most widespread antimicrobial dressings, silver foam dressings and silver alginate dressings are applied to exuding wounds and demonstrate improved performances than the traditional gauze dressings [94]. Silver alginate wound dressings have demonstrated beneficial effects on wound healing, in terms of wound exudates levels and prevention from wound infections [95–97]. Silver alginate dressings are particularly known for the prolonged antimicrobial efficacy, which indicates sustained availability of ionic silver and suggests the necessity of reduced dressings changes [98]. Excellent and sustainable

ture developed by Wu *et al*., which offered promising results for antimicrobial wound dressing through the addition of silver nanoparticles. Indeed, bacterial cellulose has attracted great attention as novel wound dressing material, but it has no antimicrobial activity [99]. The silver nanoparticle/bacterial cellulose gel membranes developed by Wu *et al*. demonstrated *in vivo* excellent healing effects in a second-degree rat wound model and were proposed as promising antimicrobial wound dressing with good biocompatibility to promote scald wound healing

release were obtained by the AgNP-bacterial cellulose hybrid nanostruc-

a promoter of wound healing [89].

442 Wound Healing - New insights into Ancient Challenges

demonstrated by Demling *et al*. [92].

controllability of Ag+

[100].


**Table 1.** Overview of some relevant techniques for production of nanometal-based antimicrobial wound dressings.

The widespread use of silver-based dressings in surgery is promising, inexpensive and well tolerated. The placement of silver-nylon dressings over incision sites in colorectal, neurological, spinal, cardiovascular and orthopaedic procedures at the time of primary closure has been described by Abboud *et al*. as effective in reducing surgical site infection rates [105]. Commercial dressings impregnated by immersion in solutions of AgNPs using different concentrations of silver from 125 to 1000 ppm demonstrated anti-biofilm efficacy against *Pseudomonas aeruginosa* [70]. Conventional cotton gauzes were modified by Sannino *et al*. through the deposition of silver-based nanocoatings obtained by a patented photo-assisted deposition process, which allows the silver treatment of natural and synthetic materials for different applications [106–108]. Particularly, the technology adopted involves the preparation of a silver-based solution, and then the deposition of the silver solution onto the surface of the material through spray coating or dip coating and the following exposure of the wet material to ultraviolet light, in order to induce the photo-chemical deposition of silver nanoparticles on the surface of the product. Indeed, the synthesis and deposition of the silver nanoparticles occur simultaneously onto the surface of the material because the photo-reduction reaction induced by UV irradiation determines the conversion from the silver precursor to metal silver nanoparticles. The silver coatings deposited are characterized by a strong adhesion to the substrate, good antimicrobial capability and biocompatibility and low silver release [109]. Cotton gauzes treated with low amounts of silver have demonstrated good antimicrobial activity against different bacterial strains and fungi, and the good antibacterial properties were further confirmed in simulated working conditions such as after incubation in artificial exudate inoculated with bacteria [110]. **Figure 1** reports the agar diffusion test performed on untreated gauze and gauze treated with silver by adopting the technology described using *Staphylococcus aureus* as tester microorganism.

**Figure 1.** Agar diffusion tests on untreated gauze and cotton gauze treated by photo-reduction technology.


**Table 2.** Examples of commercial silver-containing dressings.

Although the impregnating silver solution was prepared by using a percentage of silver lower than 0.5 wt/v%, the antibacterial test clearly demonstrated that the presence of the silver coating successfully inhibited the bacterial growth beneath and around the sample, thus indicating a good potential of product as antibacterial wound dressing. Also flax substrates have been treated with silver by adopting the same technology and the microbiological activity was still confirmed after industrial washing, thus suggesting the excellent stability of the coating on the surface of the textile material [111, 112]. In order to provide flax substrates with a moist environment and antibacterial capability at same time, Paladini *et al*. has developed a wound dressing biomaterial based on silver-doped self-assembling di-phenylalanine hydrogels. These peptide-based hydrogels have some similarities to the extracellular matrix due to their high hydration and nanofibrous architecture, which make them suitable for wound dressing applications where the wound environment needs to be controlled to prevent microbial invasion and to favour tissue regeneration [113]. Along with research efforts, in recent years, many silver-based wound dressings have been marketed for medical problems such as wide-body burns, sepsis in traumatic wounds and chronic diabetic ulcers [114, 115]. Some examples are collected in **Table 2**.
