**3. Bioactive protein delivery systems in wound healing**

Wound healing in skin is an evolutionarily conserved, complex, multicellular process, which is executed and regulated by an equally complex signaling network involving numerous growth factors, cytokines, and chemokines [39]. Growth factors are soluble secreted proteins capable of affecting a variety of cellular processes important for tissue regeneration. However, the application of growth factors in clinics remains limited due to lack of good delivery systems and carriers. Recently, biomaterial carriers and sophisticated delivery systems such as nano‐ particles and nanofibers for delivery of growth factors and peptides related in wound healing are a main focus in this research area [40].

### **3.1. Delivery of growth factors**

EGF, PDGF, FGF2, keratinocyte growth factor (KGF) [41], transforming growth factor‐β (TGF‐ β), insulin‐like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte macrophage colony stimulating factor (GM‐CSF), and connective tissue growth factor (CTGF) are the main growth factors correlated with the wound healing process of skin [16]. Growth factors usually have short half‐life time leading to a rapid deactivation at local wound beds in the body and resulting in a low efficacy. In order to enhance the efficacy of growth factor delivery systems, some bioactive and biodegradable matrixes including extracellular matrixes, have been used as carriers [42].

EGF is one of the most common growth factors used for treating skin wounds. Succinoylated dextrin (~85,000 g/mol; ~19 mol% succinoylation), a clinically well‐tolerated polymer, was used to deliver EGF and led to sustained release of free recombinant human EGF over time (52.7% release after 168 h) [43]. Using a layer‐by‐layer assembly technique, EGF was successfully encapsulated using poly(acrylic acid) (PAA)‐modified polyurethane (PU) films [44] or chitosan and alginate films [45]. Johnson and Wang treated the full‐thickness wounded mice with a heparin‐binding epidermal growth factor coacervate delivery system, and the results exhibited the enhanced migration of keratinocytes with retained proliferative potential, forming a confluent layer for regained barrier function within 7 days [46]. Chitosan‐based gel formula‐ tions containing egg yolk oil and EGF are better alternatives compared to Silverdin® (1% silver sulfadiazine), given their significant difference (*P* < 0.05) treating wounds in Wistar rats [47]. Since the healing rate of wound is an important factor influencing the outcome of clinical treatments, as well as a crucial step in burn wound treatment, and the quality of wound healing has a direct bearing on the life quality of patients, FGF2 clearly has clinical efficacy in a variety of wound managements [48]. Skin flap survival is a major challenge in reconstructive plastic surgery. A sustained delivery system of FGF2 using heparin‐conjugated fibrin was used to improve skin flap survival significantly in a rat animal model [49]. A delivery system composed of fibrin hydrogels doped with bFGF‐loaded double emulsion increased the proliferation of endothelial cells compared to sham controls, indicating that the released bFGF was bioactive [50]. An injectable delivery system of PDGF using two‐component polyurethane scaffolds was reported to achieve a sustained release for up to 21 days. The *in vitro* bioactivity of the released PDGF was largely preserved by a lyophilized powder. The presence of PDGF attracted both fibroblasts and mononuclear cells, significantly accelerating the degradation of the polymer and enhancing the formation of new granulation tissue as early as day 3 [51]. Hyaluronan‐ based porous nanoparticles were also investigated for the delivery of PDGF [52]. Recombinant human stromal cell‐derived factor‐1 (SDF‐1), a naturally occurring chemokine that is rapidly overexpressed in response to tissue injury, was delivered in an alginate gel to accelerate wound closure and reduce scar formation [53]. SDF‐1 delivery systems were evaluated using an acute surgical Yorkshire pig model. Wounds treated with SDF‐1 protein (*n* = 10) and plasmid (*n* = 6)‐ loaded alginate patches healed faster than the sham (*n* = 4) or control (*n* = 4). At day 9, SDF‐1‐ treated wounds significantly accelerated wound closure (55.0 ± 14.3% healed) compared to nontreated controls (8.2 ± 6.0%, *p* < 0.05).

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

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‐

Wound healing in skin is an evolutionarily conserved, complex, multicellular process, which is executed and regulated by an equally complex signaling network involving numerous growth factors, cytokines, and chemokines [39]. Growth factors are soluble secreted proteins capable of affecting a variety of cellular processes important for tissue regeneration. However, the application of growth factors in clinics remains limited due to lack of good delivery systems and carriers. Recently, biomaterial carriers and sophisticated delivery systems such as nano‐ particles and nanofibers for delivery of growth factors and peptides related in wound healing

EGF, PDGF, FGF2, keratinocyte growth factor (KGF) [41], transforming growth factor‐β (TGF‐ β), insulin‐like growth factor (IGF), vascular endothelial growth factor (VEGF), granulocyte macrophage colony stimulating factor (GM‐CSF), and connective tissue growth factor (CTGF) are the main growth factors correlated with the wound healing process of skin [16]. Growth factors usually have short half‐life time leading to a rapid deactivation at local wound beds in the body and resulting in a low efficacy. In order to enhance the efficacy of growth factor delivery systems, some bioactive and biodegradable matrixes including extracellular matrixes,

permeation rates than those obtained with the conventional formulations [37].

larization, and reduction of free radical formation, leading to decreased cell death.

**3. Bioactive protein delivery systems in wound healing**

are a main focus in this research area [40].

78 Wound Healing - New insights into Ancient Challenges

**3.1. Delivery of growth factors**

have been used as carriers [42].

Recently, it has been increasingly recognized that biodegradable and biocompatible scaffolds incorporated with multiple growth factors might serve as the most promising medical devices for skin tissue regeneration. Beyond drug delivery, BC hydrogel is used to deliver bFGF, EGF, and KGF with modifications of different extracellular matrices (ECMs; collagen, elastin, and hyaluronan) [54]. *In vitro* and *in vivo* evaluation of the applicability of a dextran hydrogel loaded with chitosan microparticles (255 ± 0.9 μm) containing EGF and VEGF were performed, and they accelerated wound healing [55]. Moreover, the histological analysis revealed the absence of reactive or granulomatous inflammatory reaction in skin lesions. Multiple epidermal induction factors (EIF), such as EGF, insulin, hydrocortisone, and retinoic acid (RA), were prepared for blended and core‐shell electrospinnings with gelatin (gel) and poly(l‐lactic acid)‐co‐poly‐(e‐caprolactone) (PLLCL) solutions [56]. An initial 44.9% burst release from EIF blended electrospun nanofibers was observed over a period of 15 days. The epidermal differentiation potential of adipose‐derived stem cells (ADSCs) was used to evaluate the scaffolds prepared either by core‐shell spinning or by blend spinning. After 15 days of cell culture, the proliferation of ADSCs on EIF‐encapsulated core‐shell nanofibers was the highest. Moreover, a higher percentage of ADSCs were differentiated to epidermal lineages on EIF‐ encapsulated core‐shell nanofibers compared to the cell differentiation of EIF‐blended nanofibers, and this can be attributed to the sustained release of EIF from the core‐shell nanofibers. A method for coating commercially available nylon wound dressings using the layer‐by‐layer process was utilized to control the release of VEGF165 and PDGF‐BB [57]. Animal evaluation was performed using a db/db mouse model of chronic wound healing. This combination delivery system promotes significant increases in the formation of granulation tissue and/or cellular proliferation when compared to dressings utilizing single growth factor therapeutics.

### **3.2. Delivery of peptides**

Current therapeutic regiments of wounded patients are static and mostly rely on matrices, gels, and engineered skin tissue. Accordingly, there is a need to design next‐generation grafting materials to enable biotherapeutic spatiotemporal targeting from clinically approved matrices. Peptides are good candidates for controlling wound infections. A drug carrier system was designed for delivering an insect metalloproteinase inhibitor (IMPI) drug to enable treatment of chronic wound infections [58]. Poly(lactic‐co‐glycolic acid) (PLGA) supplies lactate that accelerates neovascularization and promotes wound healing. Delivery systems of LL37 peptide encapsulated in PLGA nanoparticles (PLGA‐LL37 NP) were evaluated in full‐ thickness excisional wounds. A significantly higher collagen deposition, re‐epithelialized and neovascularized composition were found in PLGA‐LL37 NP‐treated group. *In vitro*, PLGA‐ LL37 NP induced enhanced cell migration but had no effect on the metabolism and prolifer‐ ation of keratinocytes. Interestingly, it displayed antimicrobial activity on *E. coli* [59]. CM11 peptide (WKLFKKILKVL‐NH2) (128 mg/L), a short cecropin‐melittin hybrid peptide, was delivered by an alginate sulfate‐based hydrogel as the antimicrobial wound dressing, and its healing effects were tested on skin infections caused by MRSA (200 μL, 3 × 108 CFU/mL) in a mouse model [60]. During 8‐day period, the 2% mupirocin treatment group and hydrogel containing peptide treatment groups showed similar levels of wound healing.
