**2.1. Delivery of antibiotics**

Wound healing is a complex process that often requires treatment with antibiotics. To optimize and improve the usage of currently available antibiotics, DDS of antibiotics have attracted much attention. Antibiotic drugs used in delivery systems for wound healing are cefazolin [17], gentamicin sulfate [6], ceftazidime pentahydrate [18], ciprofloxacin [19], gentamicin [20], doxycycline hyclate [21], and the anti‐inflammatory drug diclofenac [20]. Various biodegrad‐ able polymeric scaffolds (electrospun nanofibers, microspheres, composites, and films) were

investigated for antibiotic delivery systems, including electrospun nanofibers of poly(lactide‐ co‐glycolide) (PLAGA) [17], composites of a polyglyconate core and a porous poly(dl‐lactic‐ co‐glycolic acid) shell [18], chitosan (CS)‐gelatin composite films [19], a three‐dimensional (3D) polycaprolactone‐tricalcium phosphate (PCL‐TCP) mesh [6], bacterial cellulose (BC) mem‐ branes grafted with RGDC peptides (R for arginine, G for glycine, D for aspartic acid, C for cysteine) [20], poly(vinyl alcohol) (PVA) microspheres sandwiched poly(3‐hydroxybutyric acid) (PHB) electrospun fibers [21], and β‐cyclodextrin‐conjugated hyaluronan hydrogels [22].

Antibiotic agents used in wound healing typically incur adverse effects (e.g., nephrotoxicity for vancomycin, cytotoxicity for ciprofloxacin, and hemolysis for antimicrobial polymers). Loading of antibiotics within polymeric vesicles could attenuate side effects, which has been demonstrated recently [23]. Li et al. reported a general strategy to construct a bacterial strain‐ selective delivery system for antibiotics based on responsive polymeric vesicles. That was in response to enzymes, including penicillin G amidase (PGA) and β‐lactamase (Bla) that are closely associated with drug‐resistant bacterial strains. A sustained release of antibiotics enhanced stability and reduced side effects. The results demonstrated that methicillin‐resistant *Staphylococcus aureus* (*S. aureus*) (MRSA)‐triggered release of antibiotics from Bla‐degradable polymeric vesicles *in vitro* inhibited MRSA growth, and enhanced wound healing in an *in vivo* murine model.

### **2.2. Delivery of silver**

To solve the problem of the increased prevalence and growth of multidrug‐resistant bacteria, silver is used to reduce and eliminate wound infections using methodologies that limit the ability of bacteria to evolve into further antibiotic‐resistant strains. In recent decades, the developments of silver (colloidal silver solution, silver proteins, silver salts, silver sulfadiazine (SSD) and nanosilver)‐containing wound dressings for healing promotion and infection reduction have provided promising approaches [24]. The main synthesis approaches of silver monocrystalline silver (nanosilver or silver nanoparticle) include chemical reduction, micro‐ organism reduction, microwave‐assisted photochemical reduction, and laser ablation. Antibacterial wound dressings in the formats of AgNP‐embedded poly(vinyl pyrrolidone) (PVP) hydrogels were prepared by γ‐irradiation at various doses: 25, 35, and 45 kGy [25]. Antibacterial tests showed that the 1 and 5 mM AgNP‐embedded PVP hydrogels were effective, with 99.99% bactericidal activity at 12 and 6 h, respectively. A gamma‐irradiated PVA/ nanosilver hydrogel was also developed for potential use in burn dressing applications [26]. Interestingly, the wound healing activity of 0.1% w/w AgNPs in Pluronic F127 gels was enhanced to a considerable extent [27]. A new type of high surface area metallic silver in the form of highly porous silver microparticles (AgMPs) was studied [28]. Polylactic acid (PLA) nanofibers were successfully loaded with either highly porous AgMPs or AgNPs. A simulated three‐dimensional (3D) coculture system was designed to evaluate human epidermal kerati‐ nocytes and *S. aureus* bacteria on the wound dressings. PLA nanofibers containing highly porous AgMPs exhibited steady silver ion release at a greater rate of release than nanofibers containing AgNPs.

Due to its antimicrobial activity, good coagulation and immunostimulating activities, chitosan is one of the native polymers chosen to control infection and enhance wound healing. Chitosan‐ based wound dressings can be gels, microparticles or nanoparticles, sponges and films [29]. Sacco et al. combined the two antimicrobial agents, silver and chitosan, to develop a silver‐ containing antimicrobial membrane based on chitosan‐tripolyphosphate (TPP) hydrogel for wound treatments. Based on the slow diffusion of TPP, the macroscopic chitosan hydrogels were obtained that included AgNPs stabilized by a lactose‐modified chitosan. Besides the good bactericidal properties of the material, the biocompatibility assays on keratinocytes (HaCaT) and fibroblasts (NIH‐3T3) cell lines did not prove to have any harmful effects on the viability of cells using the MTT [1‐(4,5‐dimethylthiazol‐2‐yl)‐3,5‐diphenylformazan] method [8]. Chitin was also used to form the composite scaffolds with nanosilver. These chitin/nanosilver composites were found to be bactericidal against *S. aureus* and *Escherichia coli* (*E. coli*) with good blood‐clotting ability [30].

Bioelectric wound dressing can also deliver silver to wound beds. *Pseudomonas aeruginosa (P. aeruginosa)* is a common bacterium associated with chronic wound infection. An US Food and Drug Administration (FDA)‐approved wireless electroceutical dressing (WED), which in the presence of conductive wound exudate is activated to generate an electric field (0.3–0.9 V), was investigated to test its anti‐biofilm properties using a pathogenic *P. aeruginosa* strain PAO1. WED markedly disrupted biofilm integrity in a setting where normal silver dressing was ineffective. Biofilm thickness and number of live bacterial cells were decreased in the presence of WED because WED served a spontaneous source of reactive oxygen species [31].

### **2.3. Delivery of other drugs**

investigated for antibiotic delivery systems, including electrospun nanofibers of poly(lactide‐ co‐glycolide) (PLAGA) [17], composites of a polyglyconate core and a porous poly(dl‐lactic‐ co‐glycolic acid) shell [18], chitosan (CS)‐gelatin composite films [19], a three‐dimensional (3D) polycaprolactone‐tricalcium phosphate (PCL‐TCP) mesh [6], bacterial cellulose (BC) mem‐ branes grafted with RGDC peptides (R for arginine, G for glycine, D for aspartic acid, C for cysteine) [20], poly(vinyl alcohol) (PVA) microspheres sandwiched poly(3‐hydroxybutyric acid) (PHB) electrospun fibers [21], and β‐cyclodextrin‐conjugated hyaluronan hydrogels [22].

Antibiotic agents used in wound healing typically incur adverse effects (e.g., nephrotoxicity for vancomycin, cytotoxicity for ciprofloxacin, and hemolysis for antimicrobial polymers). Loading of antibiotics within polymeric vesicles could attenuate side effects, which has been demonstrated recently [23]. Li et al. reported a general strategy to construct a bacterial strain‐ selective delivery system for antibiotics based on responsive polymeric vesicles. That was in response to enzymes, including penicillin G amidase (PGA) and β‐lactamase (Bla) that are closely associated with drug‐resistant bacterial strains. A sustained release of antibiotics enhanced stability and reduced side effects. The results demonstrated that methicillin‐resistant *Staphylococcus aureus* (*S. aureus*) (MRSA)‐triggered release of antibiotics from Bla‐degradable polymeric vesicles *in vitro* inhibited MRSA growth, and enhanced wound healing in an *in vivo*

To solve the problem of the increased prevalence and growth of multidrug‐resistant bacteria, silver is used to reduce and eliminate wound infections using methodologies that limit the ability of bacteria to evolve into further antibiotic‐resistant strains. In recent decades, the developments of silver (colloidal silver solution, silver proteins, silver salts, silver sulfadiazine (SSD) and nanosilver)‐containing wound dressings for healing promotion and infection reduction have provided promising approaches [24]. The main synthesis approaches of silver monocrystalline silver (nanosilver or silver nanoparticle) include chemical reduction, micro‐ organism reduction, microwave‐assisted photochemical reduction, and laser ablation. Antibacterial wound dressings in the formats of AgNP‐embedded poly(vinyl pyrrolidone) (PVP) hydrogels were prepared by γ‐irradiation at various doses: 25, 35, and 45 kGy [25]. Antibacterial tests showed that the 1 and 5 mM AgNP‐embedded PVP hydrogels were effective, with 99.99% bactericidal activity at 12 and 6 h, respectively. A gamma‐irradiated PVA/ nanosilver hydrogel was also developed for potential use in burn dressing applications [26]. Interestingly, the wound healing activity of 0.1% w/w AgNPs in Pluronic F127 gels was enhanced to a considerable extent [27]. A new type of high surface area metallic silver in the form of highly porous silver microparticles (AgMPs) was studied [28]. Polylactic acid (PLA) nanofibers were successfully loaded with either highly porous AgMPs or AgNPs. A simulated three‐dimensional (3D) coculture system was designed to evaluate human epidermal kerati‐ nocytes and *S. aureus* bacteria on the wound dressings. PLA nanofibers containing highly porous AgMPs exhibited steady silver ion release at a greater rate of release than nanofibers

murine model.

**2.2. Delivery of silver**

76 Wound Healing - New insights into Ancient Challenges

containing AgNPs.

Besides silver, other drugs can be used to improve wound healing, for example, the anti‐scar drug astragaloside IV [32]. In a rat full‐skin excision model, the\*\*\*\* *in vivo* regulation of 9% astragaloside IV‐based solid lipid nanoparticles‐gel enhanced the migration and proliferation of keratinocytes, increased drug uptake on fibroblasts *in vitro* (*P* < 0.01) through the caveolae endocytosis pathway, and inhibited scar formation *in vivo* by increasing wound closure rate (*P* < 0.05) and by contributing to angiogenesis and collagen regular organization.

Different from most antibiotics that select for resistant bacteria, curcumin acts using multiple mechanisms. Curcumin (diferuloylmethane) is a bioactive and major phenolic component of turmeric derived from the rhizomes of *Curcuma longa linn*. Owing to its antioxidant and anti‐ inflammatory properties, curcumin plays a significant beneficial and pleiotropic regulatory role not only in cancers, cardiovascular disease, Alzheimer's disease, inflammatory disorders, and neurological disorders but also in wound healing because of its innate antimicrobial properties. However, the clinical implication of native curcumin is hindered due to low solubility, physicochemical instability, poor bioavailability, rapid metabolism, and poor pharmacokinetics, but these issues can be overcome by efficient delivery systems [33]. A biodegradable sponge, made from chitosan (CS) and sodium alginate (SA) with water uptake ability ranging between 1000 and 4300%, was developed to deliver curcumin as a wound dressing material up to 20 days. The *in vivo* animal test using SD rats showed that this CS/SA sponge had a better effect than cotton gauze, and adding curcumin into the sponge enhanced the therapeutic healing effect and improved collagen arrangement [34]. Curcumin nanoparti‐ cles (Curc‐np) with an average diameter of 222 ± 14 nm were synthesized [35]. Curc‐np represent a significant advance for reducing bacterial load. They can inhibit *in vitro* growth of methicillin‐resistant *S. aureus* (MRSA) and *P. aeruginosa* in dose‐dependent fashion, and so may represent a novel topical antimicrobial and wound healing adjuvant for infected burn wounds and other cutaneous injuries. Bacterial cellulose (BC) can be used for drug loading and controlled release [36]. The topical or transdermal drug delivery systems of two model drugs (lidocaine hydrochloride and ibuprofen) were developed. Diffusion studies with Franz cells showed that the incorporation of lidocaine hydrochloride in BC membranes provided lower permeation rates than those obtained with the conventional formulations [37].

There is a high mortality in patients with diabetes and severe pressure ulcers, resulting from the reduced neovascularization caused by the impaired activity of the transcription factor hypoxia‐inducible factor‐1 alpha (HIF‐1α). To improve HIF‐1α activity, Duscher et al. devel‐ oped the drug delivery system of an FDA‐approved small molecule deferoxamine (DFO), which is an iron chelator that increases HIF‐1α transactivation in diabetes by preventing iron‐ catalyzed reactive oxygen stress [38]. The animal study on a pressure‐induced ulcer model in diabetic mice showed a significantly improved wound healing using the transdermal delivery of DFO. DFO‐treated wounds demonstrated increased collagen density, improved neovascu‐ larization, and reduction of free radical formation, leading to decreased cell death.
