**5. Gene delivery systems in wound healing**

Gene delivery is an emerging technology in the field of tissue repair and is being used to promote wound healing. Gene delivery is targeted to develop sustained release, to reduce side effects, and to enable both spatial and temporal control of gene silencing afterward. For example, chemical modifications were used to stabilize and reduce nonspecific effects of siRNA molecules using effective delivery [77]. The controlled delivery of nucleic acids (DNA and RNA) to selected tissues remains an inefficient process are affected by low transfection efficacy, poor scalability because of varying efficiency with cell type and location, and ques‐ tionable safety as a result of toxicity issues arising from the typical materials (e.g., viral vectors) and procedures employed. Biocompatible materials, in the formats of micro/nanoparticles, scaffolds, hydrogels and electrospun fibers, made from cationic polymers and lipids, have been used as nonviral vectors, which has attracted much attention recently.

### **5.1. Viral vectors in gene delivery**

biocompatible biomaterials have been applied for stem cell delivery. A silk fibroin‐chitosan (SFCS) scaffold serving as a delivery vehicle for human adipose‐derived stem cells (ASCs) was evaluated in a murine soft tissue injury model [71]. Microvessel density at wound bed biopsy sites at 2 weeks postoperative was significantly higher in the ASC‐SFCS group vs. SFCS alone (7.5 ± 1.1 vs. 5.1 ± 1.0 blood vessels per high‐power field). A newly developed thermorespon‐ sive poly(ethylene) glycol (PEG)‐hyaluronic acid (HA) hybrid hydrogel with multiple acrylate functional groups provides an efficient delivery dressing system for human adipose‐derived stem cells (hADSCs) [72]. Although cellular proliferation was inhibited, cellular secretion of growth factors, such as VEGF and PDGF production, increased over 7 days, whereas IL‐2 and IFNγ release were unaffected. Injectable gelatin microcryogels (GMs) were used to load human ASCs [73]. The results demonstrated the priming effects of GMs on the upregulation of stemness genes and improved secretion of growth factors of hASCs for potential augmented wound healing. In a full‐thickness skin wound model in nude mice, multisite injections and dressings of hASC‐laden GMs significantly accelerated the healing compared to free stem cell

Endothelial cells (ECs), keratinocytes, and fibroblasts are the most studied cells in terms of accelerated wound healing and improved skin tissue regeneration. A growing number of studies indicate that endothelial cells (ECs) and endothelial progenitor cells (EPCs) may regulate vascular repair in wound healing via paracrine mechanisms [61]. Using dried reagent patches that incorporate dextran (DEX) and a bulk aqueous phase comprising a cell culture medium containing poly(ethylene) glycol (PEG), Bathany et al. made a micro‐patterned localized delivery of fluorescent molecules and enzymes for cell detachment [74]. Keratino‐ cytes were delivered to dermal wounds in mice via cell‐adhesive peptides attached to chitosan membranes [75]. Two peptides of 12 or 13 amino acids each that bind to cell surface heparin‐ like receptors (A5G27 and A5G33) were found to promote strong keratinocyte attachment, whereas the one that binds to integrin (A99) was inactive. Recombinant human collagen III (rhCol‐III) gel was used as a delivery vehicle for cultured autologous skin cells (keratinocytes only or keratinocyte‐fibroblast mixtures) [76]. Its effect on the healing of full‐thickness wounds in a porcine wound‐healing model was examined. Two Landrace pigs were used for the study. Fourteen deep dermal wounds were created on the back of each pig with an 8‐mm biopsy punch. The scaffold enhanced early granulation tissue formation. Interestingly, fibroblast‐ containing gel was effectively removed from the wound, whereas gels without cells or with

Gene delivery is an emerging technology in the field of tissue repair and is being used to promote wound healing. Gene delivery is targeted to develop sustained release, to reduce side effects, and to enable both spatial and temporal control of gene silencing afterward. For example, chemical modifications were used to stabilize and reduce nonspecific effects of

injection.

**4.2. Other cells**

82 Wound Healing - New insights into Ancient Challenges

keratinocytes only remained intact.

**5. Gene delivery systems in wound healing**

The TGFβ family plays a critical regulatory role in repair and coordination of remodeling after cutaneous wounding. TGFβ3 has been implicated in an antagonistic role regulating overt wound closure and promoting ordered dermal remodeling. A mutant form of TGFβ3

**Figure 2.** Transgenic overexpression of TGFβ3 decreases fibroblast to myofibroblast differentiation at the site of cutane‐ ous wounding *in vivo*. (A) and (B) wound sections were stained immunohistochemically for fibroblast (a: vimentin) and myofibroblast (b: SMA) markers after treatment with [a and b(i)] PBS, [a and b(ii)] Lnt‐TGFβ3, or [a and b(iii)] Lnt‐ mutTGFβ3. (C) Real‐time reverse transcription‐PCR showed that both TGFβ3 application groups and the PBS control (n = 4) as well as a significant decrease between the Lnt‐mutTGFβ3 and Lnt‐TGFβ3 treatment groups. PBS, phosphate‐ buffered saline; SMA, smooth muscle actin; TGF, transforming growth factor.

(mutTGFβ3) was generated by ablating its binding site for the latency‐associated TGFβ‐ binding protein (LTBP‐1) [78]. A localized intradermal transduction using a lentiviral vector expressing the mutTGFβ3 in a mouse skin wounding model was demonstrated to reduce reepithelialization density and fibroblast/myofibroblast trans‐differentiation within the wound area. Both of which reduced scar tissue formation (**Figure 2**). Using a noninvasive imaging system, the kinetics of luciferase gene expression was studied when delivered in an adenoviral vector (replication‐deficient adenovirus, Ad5). A peak of gene expression occurred at 7 days after delivery [79]. The esophageal cancer‐related gene‐4 (Ecrg4) delivering a viral‐ mediated gene was evaluated in a cutaneous wound healing model [80]. Both Ecrg4 mRNA and its protein product were localized to the epidermis, dermis, and hair follicles of healthy mouse skin.

### **5.2. Nonviral vectors in gene delivery**

Gene delivery using adenoviral vectors in tissue regeneration is hindered by a short duration of transgene expression. A fibrin scaffold was used to enhance delivery of the adenovirus to a wound site, precluding the need for high repeated doses [81]. An anti‐fibrotic interfering RNA (RNAi) delivery system using exogenous microRNA (miR)‐29B was proposed to modulate ECM remodeling following cutaneous injury. A collagen scaffold was used as the carrier of (miR)‐29B. The mRNA expressions of collagen type I and collagen type III were reduced up to 2 weeks after fibroblasts culture. *In vivo* evaluation in full‐thickness wounds treated with miR‐29B delivery revealed that collagen type III/I ratio and matrix metalloproteinase (MMP)‐ 8 to TIMP‐1 ratio were improved [82]. Porous (100 and 60 μm) and nonporous (n‐pore) hyaluronic acid‐MMP hydrogels with encapsulated reporter (pGFPluc) or proangiogenic (pVEGF) plasmids are used as a scaffold‐mediated gene delivery [83]. Alginate‐DNA gels were used to treat diabetic wounds, which provided sustained release of bioactive factors, such as neuropeptides and VEGF [13]. Silver nanoparticles (AgNPs) can be further augmented for gene delivery applications. The biofunctionalized stable AgNPs with good DNA‐binding ability for efficient transfection and minimal toxicity were developed [84]. Polyethylene glycol (PEG)‐ stabilized chitosan‐g‐polyacrylamide was used to modify AgNPs. To enhance the efficiency of gene transfection, the Arg‐Gly‐Asp‐Ser (RGDS) peptide was immobilized on the surface of AgNPs. The transfection efficiency of AgNPs increased significantly after immobilization of the RGDS peptide reaching up to 42 ± 4% and 30 ± 3% in HeLa and A549 cells, respectively. The transfection efficiency was significantly higher than 34 ± 3% and 23 ± 2%, respectively, with the use of polyethylenimine (PEI, 25 kDa).

For treating diabetic patients with a threat of limb amputations, genes of various growth factors have been proposed in delivery systems. A simple nonviral gene delivery using minicircle plasmid DNA encoding VEGF was combined with an arginine‐grafted cationic dendrimer PAM‐RG4 [85]. Mouse ASCs were transfected with DNA plasmid encoding VEGF or green fluorescent protein (GFP) using biodegradable poly (β‐amino) esters (PBAE). Cells transfected with Lipofectamine™ 2000, a commercially available transfection reagent, were included as controls. ASCs transfected using PBAEs showed an enhanced transfection efficiency and 12– 15‐folds higher VEGF production compared with the controls (\**P* < 0.05) [86]. Keratinocyte growth factor‐1 (KGF‐1) DNA was delivered using NTC8385‐VA1 plasmid, a novel minimal‐ ized, antibiotic‐free DNA expression vector [87].

**Figure 3.** (A) Time course of nanoneedles incubated in cell‐culture medium at 37°C. Scale bar = 2 μm. (B) Nanoneedles mediate neovascularization in wound healing. (C) The number of nodes in the vasculature per millimeter square. (D) Within each field of view acquired for untreated control, intramuscular injection (IM), and nanoinjection. P < 0.05, P <  0.01, P < 0.001.

DNA‐incorporated electrospun nanofibrous matrix was fabricated to control the release of DNA in response to high concentration of MMPs (matrix metalloproteinases) such as diabetic ulcers [88]. High efficiency and minimal toxicity *in vitro* have been demonstrated that can be used for an intracellular delivery of nucleic acids by using nanoneedles [89]. Biodegradable nanoneedles were fabricated by metal‐assisted chemical etching of silicon. These nanoneedles mediated the in situ delivery of an angiogenic gene, VEGF165, and triggered the patterned formation of new blood vessels. The nanoneedles were designed for extremely localized delivery to a few superficial layers of cells (two‐dimensional patterning). This gene delivery can access the cytosol to co‐deliver DNA and siRNA with an efficiency greater than 90%. *In vivo* studies show that the nanoneedles transfected the VEGF165 gene, improved wound healing and scar‐tissue remodeling, and induced sustained neovascularization and a localized sixfold increase in blood perfusion in the target region of the muscle (**Figure 3**). This confined intracellular delivery has the potential to target specific exposed areas within a tissue, further reduce the invasiveness of the injection, and limit the impact on the overall structure of the tissue.
