Therapeutic Strategies for Enhancing Angiogenesis in Wound Healing

Advanced Drug Delivery Reviews 146 (2019) 97–125

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Advanced Drug Delivery Reviews

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Therapeutic strategies for enhancing angiogenesis in wound healing

Austin P. Veith a,1, Kayla Henderson a,1, Adrianne Spencer a, Andrew D. Sligar a, Aaron B. Baker a,b,c,d,⁎ a Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA b Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA c The Institute for Computational Engineering and Sciences, University of Texas at Austin, Austin, TX, USA d Institute for Biomaterials, Drug Delivery and Regenerative Medicine, University of Texas at Austin, Austin, TX, USA article info abstract

Article history: The enhancement of wound healing has been a goal of medical practitioners for thousands of years. The develop- Received 17 March 2018 ment of chronic, non-healing wounds is a persistent medical problem that drives patient morbidity and increases Received in revised form 15 September 2018 healthcare costs. A key aspect of many non-healing wounds is the reduced presence of vessel growth through the Accepted 24 September 2018 process of angiogenesis. This review surveys the creation of new treatments for healing cutaneous wounds Available online 26 September 2018 through therapeutic angiogenesis. In particular, we discuss the challenges and advancement that have been made in delivering biologic, pharmaceutical and cell-based therapies as enhancers of wound vascularity and Keywords: Wound healing healing. Angiogenesis © 2018 Elsevier B.V. All rights reserved. Growth factor therapy Cell-based therapies Stem cell therapy Neovascularization Diabetic ulcers Chronic wounds Non-healing wounds

Contents

1. Introduction...... 98 2. Proteintherapeuticsforwoundangiogenesis...... 99 2.1. Growthfactors...... 99 2.1.1. Plateletderivedgrowthfactor...... 99 2.1.2. Epidermalgrowthfactor...... 100 2.1.3. Fibroblastgrowthfactors...... 100 2.1.4. Vascularendothelialgrowthfactor...... 101 2.2. Non-growthfactorproteindelivery...... 101 2.3. Peptides...... 103 2.4. Bloodderivedfactors...... 103 3. Geneandnucleicacid-basedtherapiesforwoundangiogenesis...... 104 3.1. MicroRNAsinwoundangiogenesis...... 104 3.1.1. MicroRNAsthattargetangiogenesisinwoundhealing...... 104 3.2. Deliverymethods...... 107 3.2.1. Freeviralvectordelivery...... 107 3.2.2. Scaffoldsandhydrogelsemployedingenedelivery...... 107 3.2.3. Electroporationandsonoporationenhanceddelivery...... 108 4. Druganddrug-likecompoundsforenhancingwoundangiogenesis...... 108 4.1. Strategiesusingdeliveryofsmallmoleculestopromoteangiogenesisandrescueimpairedwoundhealinginanimalmodels...... 108 4.2. Adenosinetriphosphate...... 108 4.3. Statins...... 109

⁎ Corresponding author at: Department of Biomedical Engineering, University of Texas at Austin, University Station BME 5.202D, Austin, TX 78712, USA. E-mail address: [email protected] (A.B. Baker). 1 Authors contributed equally to this work.

4.4. Deferoxamine...... 110 4.5. Naturalcompounds...... 111 4.6. Hyaluronanoligosaccharides...... 113 5. Stemcellbasedtherapiesforwoundangiogenesis...... 114 5.1. Challengesandstrategiesfortherapeuticcelldelivery...... 114 5.2. Adiposederivedstemcells...... 114 5.3. Bonemarrowderivedmesenchymalstemcells...... 116 5.4. Inducedpluripotentstemcells...... 118 5.5. Otherstemcellpopulations...... 118 6. Conclusion...... 118 Acknowledgements...... 119 References...... 119

1. Introduction factors that regulate new blood vessel growth [21–23]. The M2 phenotype is thought to be more strongly associated with angiogenic activity In modern times, non-healing, cutaneous wounds have become [24,25]. The presence of therapeutics and/or biomaterials for delivery of major medical and social burden in the United States and worldwide. compounds can also induce a response from macrophages. The foreign Diabetes is the leading cause of chronic, non-healing ulcers that leave body response to some biomaterials involves the formation of foreign patients at increased risk for limb amputation [1,2]. It is estimated that body giant cells and fibrotic encapsulation of the material [26]. The for- chronic wounds cost the U.S. over $25 billion annually and significantly eign body reaction to implanted biomaterials can be separated into contribute to the rising cost of healthcare [3–6]. Astonishingly, diabetic stages including protein adsorption on blood or biofluid contact, initial ulcers are responsible for 25-50% of the total cost of diabetes treatment cells response/recruitment from cells of the innate immune system in- [7] and are the most common cause for limb amputations in the United cluding macrophages, production of cytokines and soluble factors that States [8]. Debridement of necrotic tissues, offloading, infection control, can control inflammation and the recruitment of cells involved in fi- surgical revascularization, and limb elevation/compression are often re- brous tissue formation, and finally formation of fibrous capsule around quired concurrently and lead to the immense cost and complexity the foreign body [27]. The polarization of macrophages can be altered [9,10]. Additionally, a host of techniques and dressings have been ap- by interactions with biomaterials and this is a potential strategy for al- plied with the goal of improving chronic wound healing, including the tering the course of wound healing and modulating the result of the for- delivery of growth factors, gene therapy, stem cell therapies and eign body reaction [28,29]. mechanical/pressure-based stimulation [11–15]. Currently, only The injured tissue at this stage undergoes rapid proliferation in a platelet-derived growth factor-BB (PDGF-BB; Becaplermin/Regranex), loose provisional extracellular matrix (ECM). In a well healing wound, is approved in the U.S. for clinical treatment of chronic wounds. How- this granulation tissue is characterized by intense angiogenesis giving ever, the mixed efficacy of this treatment has prevented it from achiev- it its characteristic pink swollen appearance [30]. Angiogenesis in the ing widespread clinical adoption [16,17]. A major aspect of non-healing provisional matrix is guided by several factors including the develop- wounds in diabetes and peripheral vascular disease is a reduced ability ment of gradients of hypoxia caused by local microvascular disruption to regrow microvasculature through the process of angiogenesis [18]. and cytokines produced by macrophages. Among other processes, hyp- This disruption of the normal healing process contributes to the dys- oxia activates the transcription factor hypoxia-inducible factor-1-alpha functional healing response and is major therapeutic target for creating (HIF-1α), which promotes angiogenesis through multiple mechanisms new treatments for non-healing and ischemic wounds. In this review, including the regulation of a large number of angiogenesis related we will examine recent advances in therapies targeting the enhance- genes [31] and the production of angiogenic growth factors including ment of angiogenesis in wound healing. for Ang-2, stromal cell-derived factor-1 (SDF-1) and vascular endothe- The healing of wounds is a complex process that greatly depends on lial growth factor (VEGF-A) [32]. Heparan sulfate proteoglycans also the extent and mechanism of injury. Broadly speaking, the wound play a key role in regulating the angiogenic activity of many of these healing process can be separated into a several stages that include im- growth factors including VEGF-A and fibroblast growth factor-2 (FGF- mediate hemostasis, acute inflammation, proliferation and maturation 2) [33,34]. [19]. A key accompanying activity in the proliferation stage is the forma- In dermal wounds with robust healing, the angiogenic activity dur- tion of new blood vessels, a process known as angiogenesis. In intact tis- ing the proliferative phase initially creates a disorganized vascular net- sues, the microvasculature remains in a state of homeostasis where work with vessels of high tortuosity and many dead-end flow adequate nutrients and oxygen are delivered to the tissues in balance pathways, often reaching higher vessel numbers than the skin prior to with removal of waste products and carbon dioxide. Upon injury, the injury [35]. Following this peak in vessel density, there is increased ex- microvasculature is disrupted leading to fluid accumulation, inflamma- pression of anti-angiogenic growth factors, such as Sprouty2 and pig- tion and the development of hypoxia. Hypoxia and inflammatory cyto- ment epithelium-derived factor (PEDF), leading to subsequent kines activate endothelial cells, leading to recruitment of immune cells. regression of the vascular network [36,37]. Maturation of the vascular The injured tissue recruits an early influx of neutrophils followed later network to include larger vessels and prevent regression requires the by macrophages. Macrophages have been recently recognized to polar- stabilization of the capillaries by pericytes and vascular smooth muscle ize into a continuum of phenotypes and the nature of this polarization cells (vSMCs) [38]. A key growth factor in promoting stabilization of the has a critical effect on wound healing, scar formation and angiogenesis vascular network is PDGF-BB, which aids in recruitment and differenti- [20]. Classically, the polarization of macrophages ranges in a continuum ation of pericytes [39]. In addition, angiopoietin-1 (Ang-1) and from a pro-inflammatory M1 phenotype to alternatively activated M2a- angiopoietin-2 (Ang-2), in combination with the receptor Tie2, play a d phenotypes that have been associated with anti-inflammatory and complex role in regulating angiogenesis and maturity of vascular net- wound healing activity [20]. Macrophages are essential to the coordi- works [40–42]. Specifically, Tie2 is expressed on pericytes and signaling nate the angiogenic response in wound healing and produce many through Ang-2/Tie2 in pericytes can regulate angiogenesis [43,44]. A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 99

Reduced angiogenesis plays a prominent role in the non-healing na- 2. Protein therapeutics for wound angiogenesis ture of diabetic foot ulcers and other chronic wounds [45]. Multiple mechanisms are supported for the reduced angiogenesis in these 2.1. Growth factors chronic wounds. Diabetic patients with foot ulcers have higher circulatory levels of PEDF in their plasma in comparison to non-diabetic pa- There have been many years of intensive study into the use of tients and diabetic patients without foot ulcers [46]. In diabetic skin, growth factors for both inducing angiogenesis and enhancing wound there is also a marked reduction in syndecan-4 and glypican-1, two closure in chronic wounds. Endogenous growth factors play a critical key cell surface heparan sulfate proteoglycans that enable effective role in orchestrating the wound healing process and are essential for ef- binding of FGF-2 and other growth factors to their cognate receptors fective wound healing [53]. While the delivery of exogenous growth [47,48]. Similarly, wound exudates isolated from venous leg ulcers factors has shown benefits to wound closure and angiogenesis in animal have anti-angiogenic properties, even in spite of increased expression studies, almost all growth factor-based agents for wound healing have of angiogenic factors [49]. These analyses are consistent with a proteo- not shown clear benefits for enhancing wound healing in patients in mic analysis of venous leg ulcer exudates that found increases in anti- clinical trials. One hypothesis regarding the failure of these trials is angiogenic proteins in non-healing ulcers [50]. In chronic venous ulcers, that human patients develop resistance to angiogenic growth factors there was also increased of proteolysis of VEGF-A [51]aswellasin- during the development of vascular disease and the aging process creased levels of soluble VEGFR-1, which may serve to neutralize [54]. It is also uncertain whether a single growth factor therapy could VEGF-A activity [52]. be effective for wound healing [55]. The mixed results from clinical tri- In this review, we summarize the recent progress in the delivery of als, combined with the prohibitive cost of the therapies, have slowed compounds to wounds with the goal of inducing therapeutic angiogen- the clinical adoption of these treatments, however research is still un- esis. In particular, we will focus on new systems and approaches for de- derway to develop more effective protein therapies for wound healing. livering compounds and cells for enhancing angiogenesis in cutaneous wounds. As non-healing wounds are major clinical problem, we will 2.1.1. Platelet derived growth factor emphasize studies with results relevant to chronic wounds. The review Platelet-derived growth factors (PDGFs) are important is arranged by the overall therapeutic strategy of using proteins, gene/ chemoattractants in the wound healing process. Currently, becaplermin nucleic acid delivery, small molecules/drug-like compounds or cell ther- (100 μg/g PDGF-BB) is the only FDA approved growth factor treatments apies (Fig. 1). for wound healing [16,56]. In some clinical trials, becaplermin has been shown expedite wound healing in venous leg ulcers [57,58]. In a clinical trial on becaplermin for treating diabetic foot ulcers, 48% of patients (29/ 61) had healed ulcers versus 25% (14/57) in the placebo group with a dosage of 2.2 μg/cm2 of ulcer area [59]. Clinical trials in 1998 and 1999 showed statistically significant improvements in this type of chronic wound with becaplermin treatment [60,61]. In the groups treated once a day with becaplermin, diabetic foot ulcers exhibited decreased wound areas and faster healing rates over the placebo controls. These positive results can be attributed to the ability of PDGF-BB to enhance recruitment of cells to the wound site and the stimulation of an angiogenic response. However, other clinical trials have not found improvements in wound healing with becaplermin treatment [62]. Therapeutic resistance may also play a significant role in preventing the effectiveness of PDGF-BB therapies, thus necessitating higher concentrations and increasing the risk of side effects of the growth factor therapy. In addition, the FDA gave becaplermin a black box warning due to a five-fold increase in death rate among patients that received three tubes of becaplermin and who had pre-existing malignancy [63]. While the risk of getting a new cancer was not increased in patients without cancer, this finding highlights the risk of using pro-growth and angiogenic therapies in patients with an existing cancer. Novel delivery strategies are needed to increase the efficacy of PDGF-BB without increasing the dose and to increase its cost effectiveness [64]. The simultaneous application of multiple growth factors and proteins may provide the extra benefit needed to see therapies applied in a clinical environment. The simultaneous application of PDGF-BB and epidermal growth factor (EGF) has been shown to direct pericyte recruitment to help stabilize blood vessels [65,66]. Other multiple growth factor studies have shown that the simultaneous application of PDGF-BB and FGF-2 has improved vascular stability in hind limb ischemia models in rats and rabbits [67]. The dual delivery of PDGF-BB and VEGF-A from electrospun nanofiber scaffolds accelerated wound healing in rats and promoted angiogenesis at the wound site [68]. Similar results were achieved in the simultaneous delivery of VEGF-A and PDGF-BB bound by laminin heparin binding domains from fibrin matrices [69]. In another study, researchers created an electrospun nanofiber scaffold of Fig. 1. Summary diagram of the cellular and molecular components of wound healing and collagen and hyaluronic acid (HA) that released FGF-2, EGF, VEGF-A wound angiogenesis (created using BioRender and Adobe Illustrator). and PDGF-BB with varying release kinetics. This construct accelerated 100 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125

Table 1 Multi-growth factor approaches for enhancing wound healing and angiogenesis.

Growth factors Results Exp. model Ref.

PDGF-BB and VEGF-A Promotes angiogenesis at the wound site, with signiﬁcantly increased numbers of capillaries in the wound bed Rats [68] PDGF-BB and FGF-2 Increased the levels of neovascularization signiﬁcantly Rats [67] PDGF-BB, FGF-2, VEGF-A and EGF Accelerates wound closure and enhances the maturation of blood vessels in the wound bed in diabetic rats Diabetic Rats [70] PlGF domains and VEGF-A Chimeric protein promoted angiogenesis with reduced vascular permeability Rats [135] PDGF-BB and IGF-1 Increased numbers of blood vessels at the wound site, Increased VEGF expression Rats [324]

wound closure and the maturation of blood vessels in the wound beds comparison to the non-EGF dressings and control wounds. Finally, to of rats with streptozotocin-induced diabetes [70]. Finally, combining address another major issue in protein therapeutics, dextrin conjuga- PDGF-BB with a syndecan-4 proteoliposomes significantly increased tion has been used to reduce the proteolytic degradation of EGF in circu- its activity in wound closure, angiogenesis and increased the proportion lation, thereby increasing its wound healing capacity [90]. By day 11 in a of M2 macrophages in a diabetic mouse model of wound healing [71]. study of full thickness excisional wounds in diabetic mice, neo-dermal Table 1 summarizes the recent studies using multiple growth factors tissue generation was greatest in the group that received both 1 μg/mL to enhance angiogenesis in wound healing. and 10 μg/mL of dextrin-EGF over the control that received 10 μg/mL of EGF without dextrin conjugation. The dextrin-EGF construct also ac- 2.1.2. Epidermal growth factor celerated wound closure and the formation of granulated tissue at the Epidermal growth factor (EGF) is another promising growth factor 10 μg/mL dose over the control. Further research into novel materials for wound healing therapies and a known mediator of angiogenesis. and protective strategies will help develop more effective EGF therapies. Early clinical trials showed reduced healing times and increase re- epithelialization in wounds treated with recombinant EGF [72,73], how- 2.1.3. Fibroblast growth factors ever, concerns over the role of EGF in cancer have reduced enthusiasm Fibroblast growth factors (FGFs) are a family of growth factors that for these therapies in the U.S. [74]. Despite this fact, there has been in- show great promise for applications in dermal wound healing as they creased interest in EGF treatment to enhance wound healing in chronic affect a large number of physical processes [91]. There are several differ- wounds in other parts of the world. There are several commercially ent FGF family members expressed during the healing process including available forms of recombinant human EGF outside of the U.S. including FGF-1, FGF-2, FGF-7, FGF-10 and FGF-22 [92,93]. In general, FGF ligands Easyef (Nepidermin), Regen-D 150, and Heberprot-P. Easyef is available bind to their receptors in the presence of heparin or heparan sulfate and as a spray or ointment and has shown showed benefits for diabetic foot these signals can promote angiogenesis and proliferation/migration of ulcers in a prospective, open-label trial, crossover study with 89 patients endothelial cells [94]. The most studied member of the FGF family [75]. Regen-D 150 is a gel formulation containing EGF that has shown used in wound healing applications is FGF-2 [95], partly due to its strong improvements in wound healing time and reduces the risk of amputa- association with wound angiogenesis. The other FGFs are more often as- tion in patients with chronic diabetic foot ulcers [76]. Heberprot-P is a sociated with the enhancement of re-epithelization [93,96,97], the most lyophilized formulation of EGF that has been tested in over 100,000 pa- notable being FGF-7. FGF-7 has been shown to be integral in the migra- tients in Cuba and has also been shown to increase healing of chronic ul- tion of keratinocytes to the wound area [98] and a lack of FGF-7 delays cers and reduce diabetes-related amputations [77]. Finally, clinical trials cutaneous wound healing in mice [99]. Further, the dual delivery of in Vietnam have supported that recombinant human EGF has positive FGF-7 and FGF-2 has been shown to enhance skin wound healing, healing effects on moderate to severe diabetic foot ulcers [78,79]. showing more developed vascular networks than the scaffold control Mechanistically, EGF has been shown to enhance angiogenesis [100]. through phosphatidylinositide 3-kinase/protein kinase B (PI3K/Akt) Multiple studies have reported the benefits of delivery of FGF-2 for and extracellular signaling-regulated kinase-1/2 (ERK1/2) through wound healing. In freeze injured skin grafts, the delivery of FGF-2 stim- VEGF-mediated pathways [80]. EGF can induce production of VEGF-A ulated angiogenesis and accelerated wound healing [101]. More recent to enhance tumor angiogenesis in cancer cells [81] and is a powerful studies have corroborated this evidence in a wide range of wound chemoattractant for keratinocyte migration to encourage wound re- models from skin flaps to incisional hernias [102,103]. In one study, epithelialization [82] with heparin-bound EGF being the one of the FGF-2 released from heparin-fibrin composite matrices increased fibro- most potent forms [83]. EGF also regulates nuclear factor-κB (NF-κB) blast proliferation and collagen density, decreased the necrotic area in controlled activation of CCCTC-binding factor (CTCF), a gene regulator the skin flap and the vascular density in comparison to control wounds that plays a significant role in epithelial cell migration [84]. In mice [102]. Research on incisional hernias has shown that tissues treated with a knockout of the NF-κB p50 subunit, the wound closure percent- with FGF-2 after the incision had increased breaking strength and age was significantly reduced in comparison to wild type (WT) control lower hernia development over untreated incisions [103]. In clinical mice upon EGF treatment [85]. studies, FGF-2 increased wound healing in patients with burns and Recently, new delivery methods have been explored to help localize chronic ulcers [104,105]. A second clinical study also demonstrated in- EGF and increase its circulation time [86]. Silk biomaterials have been creased wound closure in patients with burns and enhanced strength developed to deliver EGF to promote angiogenesis and wound closure in wounds treated with FGF-2 [106]. in cutaneous excisional wounds in mice [87,88]. In a study by Gil et al. Novel FGF-2 delivery methods have been explored to enhance its three different material structures and two different drug incorporation wound healing activity. Delivery of FGF-2 from gelatin sheets methods were examined [87]. The silk biomaterial was formulated as a crosslinked with glutaraldehyde accelerated re-epithelialization, forma- solid film, a porous film, or electrospun nanofibers. Silk biomaterials tion of granulation tissue and angiogenesis in comparison to control show wound closure percentages significantly greater than control wounds in a mouse model [107]. For injectable applications, chitosan- wounds in a full thickness dermal wound model. The delivery of EGF heparin hydrogels have been developed to deliver FGF-2 via injection from silk biomaterials decreased scar tissue formation and increased ep- to subcutaneous wound sites [108]. When the gel was injected into ithelialization over a Tegaderm control. Another group developed a the ischemic hind limb of a diabetic mice, there was increased blood hyaluronic acid (HA)/collagen sponge that could be used to deliver vessel formation after four days. Furthermore, there was a significant in- EGF as a wound dressing [89]. In a wound model in rats, the EGF- crease in blood flow and oxygen saturation in the calf and ankle until at containing dressing increased wound closure and angiogenesis in least day 28. Diabetic patients have a reduction in cell surface A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 101 proteoglycans that are essential for robust FGF-2 signaling, including infiltration at a greater rate than the control treatments. VEGF-A encap- syndecan-4 and glypican-1 [48,54]. Several studies support that co- sulated PLGA microspheres and nanoparticles released from chitosan/ delivering syndecan-4 or glypican-1 in a proteoliposome with FGF-2 hyaluronic acid hydrogels and fibrin scaffolds respectively have also can markedly enhance its activity for angiogenesis and wound healing been used to promote angiogenesis in non-healing infected wounds in diabetic animal models [47,48,54,109,110]. and in diabetic mice [126,127]. The release of VEGF-A from calcium algi- The delivery of growth factors and specifically FGF-2 from nano- nate microspheres has been shown to increase capillary network forma- particles and nanocrystals has also been investigated [111–113]. tion and neovascularization as well in subcutaneous implantation Mesoporous silica nanoparticles (MSNs) are a means to create con- models in rats [128,129]. When histological analysis was performed, trolled drug release profiles by fine tuning the surface area to volume all sites displayed a low level of fibrotic tissue and the microspheres ratio, pore sizes, and surface properties. Zhang et al. delivered FGF-2 loaded with VEGF-A showed considerable capillary growth and newly from MSNs with a formulation that allowed release over 20 days formed blood vessels. [111]. In addition, Li et al. combined tunability of gelatin micro- Placenta growth factor (PlGF) has also been shown to enhance an- spheres and the structural stability of a collagen/cellulose nanocrystal giogenesis and can have angiogenic synergy with VEGF-A [130–132]. scaffold shown by Li et al. provided a suitable environment for en- Mice overexpressing PlGF exhibit increased vascularization in both hancing angiogenic growth [112]. Microspheres, impregnated with fetal and adult life [133]. These mice display hypervascularized skin FGF-2, were loaded into a scaffold composed of collagen and cellulose and enlarged blood vessels. Gene transfer of PlGF increases wound an- nanocrystals (CNCs), which increased the mechanical stability of the giogenesis in health and diabetic mice [131]. PlGF also promotes chemo- collagen network. In a subcutaneous implantation model, the FGF-2 taxis of mesenchymal stem cells (MSCs), which may express angiogenic releasing gelatin microspheres significantly increased the number of factors or induce increased angiogenesis/angiogenic factor secretion in new blood vessels compared to FGF-2 from the scaffold alone or con- other cell types. When MSCs were cultured with fibroblast-like trol group. synoviocytes, the synoviocytes were found to secrete higher levels of PlGF and induce greater angiogenesis compared to fibroblast-like 2.1.4. Vascular endothelial growth factor synoviocyte injection controls [134]. The ECM binding domains from The vascular endothelial growth factor (VEGF) family is com- PlGF have also been used to engineer variants of other growth factors prised of five separate growth factors, VEGF-A, -B, -C, -D and pla- (VEGF-A, PDGF-BB and BMP-2) with super-affinity to the ECM, leading centa growth factor (PlGF) [114]. These growth factors are to increased repair in chronic wounds in rodents, over their wild-type primarily responsible for regulating angiogenesis and counterparts [135]. The overall concept is that the localization of the lymphangiogenesis in both embryonic and adult development growth factor may be better controlled with super-affinity to the ECM, [114]. The VEGF family also plays a significant role in the perme- reducing the side effects of angiogenic therapy. The super-affinity ability of blood vessels, vasodilation, and have been shown to stabi- VEGF-A variant promoted angiogenesis with reduced induction of per- lize new blood vessel growth during wound healing. However, the meability of the vasculature for equivalent wild type VEGF-A treatment application of VEGF therapies in clinical settings has been limited, [135]. due in part to the vasopermeability induced by the treatment [115]. Several clinical trials have been performed using recombi- 2.2. Non-growth factor protein delivery nant VEGF-A to enhance angiogenesis in critical limb or myocardial ischemia [116–119], but the clinical benefits of VEGF therapy were Many other proteins have displayed therapeutic effects on wound minor and further studies were not pursued. angiogenesis in preclinical wound healing models. Insulin is a key ex- During the course of wound healing, the levels of VEGF-A and FGF-2 ample of a protein hormone shown to be a regulator of angiogenesis in the serum have a negative correlation with one another, first high and wound healing [136,137]. The delivery of insulin from PLGA micro- levels of FGF-2 involved in the initial recruitment of endothelial cells spheres encapsulated in an alginate sponge dressing enhanced healing followed by high levels of VEGF-A used to stabilize the new vessel and reduced scarring from burn injuries in rats by stimulating angiogen- growth and provide a prolonged angiogenic signaling period [120]. esis [137]. In addition, injection of insulin into the skin of mice induced Losi et al. showed that concurrent delivery of FGF-2 and VEGF-A in- angiogenesis suggesting it is a suitable candidate for ischemic wounds creased the number of endothelial cells around the immediate vicinity [138]. Furthermore, in a double-blind placebo-controlled clinical trial, of their scaffold in a hind limb ischemia model, as well as increased per- the topical delivery of insulin to diabetic skin ulcers resulted in im- fusion of blood in the hind limbs of rats after ischemic injury [121]. Fur- proved wound healing [139]. This result presents insulin as a low-cost ther, the delivery of granulocyte-macrophage colony stimulating factor alternative to growth factor therapies. (GM-CSF) to diabetic patients with leg ulcers increased the level of The hormone erythropoietin (EPO) is another non-growth factor VEGF-A produced by macrophages and monocytes [95,122]. Application protein that has been shown to increase angiogenesis [140–143]. In of VEGF-A to wounds in db/db mice increased wound closure and angio- second-degree burn injury model where a substantial portion of genesis [123]. In wounds treated with VEGF-A there was a greater num- the original vasculature is damaged or lost completely, mice and ber of endothelial progenitor cells that lead to new blood vessel rats treated with topical application or infusion of EPO displayed formation, as well as increased amounts of pro-angiogenic soluble rates of angiogenesis that were significantly increased over the con- factors. trol that did not receive EPO [140]. Further, wound closure rates in To further enhance the effect of VEGF-A at the wound site, many the rats treated with EPO were accelerated over the control, with therapies also involve the delivery of the growth factor from biomate- the wounds on average 98.8% closed by day 7. In addition, EPO has rials. During degradation, poly(lactic-co-glycolic acid) (PLGA) scaffolds been used to treat ischemic injuries, where significant revasculariza- hydrolyze into lactic and glycolic acids [124]. This lactate helps promote tion is required to restore adequate blood flow [143,144], and it is angiogenesis and helps recruit endothelial progenitor cells at wound suggested that EPO works synergistically with VEGF to promote an- sites and, when combined with VEGF-A, promotes healing [125]. giogenesis at the wound site [145]. On the other hand, EPO has VEGF-A released from PLGA scaffolds lead to significantly greater been implicated in tumor angiogenesis in hepatic tumors and Lewis wound closure in full thickness skin wounds in diabetic mice (N80% of lung carcinoma tumors [146,147], so similar precautions should be the initial wound area) 10 days post injury compared to controls that re- taken as in many growth factor therapies. ceived only VEGF-A or the scaffold alone. The PLGA scaffolds promoted Stromal-cell derived factor-1 (SDF-1) is another protein that angiogenesis at the wound site and the tissue showed greater collagen has been shown to regulate angiogenesis by recruiting endothe- deposition, dermal remodeling, granulation, and blood capillary lial progenitor cells (EPC) to the wound site [148,149]and 102 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 promoting sustained cell proliferation [150]. SDF-1 is also a scaffold provided a proliferative environment for the cells and upon strong chemotactic agent for monocytes, such as macrophages introduction to a skin wound model in mice, the 1F9 scaffold helped [151], has been shown to promote the local recruitment of to promote wound healing and angiogenesis. Moreover, 1F9 treated monocytes with anti-inflammatory activity enhances the expan- wounds experienced marked increases in wound closure over the sion of local vasculature [152]. SDF-1 has also been shown to controls. Thymosin Beta 4 (Tβ4) is another protein has been shown promote bone mesenchymal stem cells (bMSC) and EPC chemo- to increase wound healing and angiogenesis in diabetic rats [157] taxis to wound sites [153,154]. The migration of bMSCs and and improve tissue repair. Tβ4 accelerated wound closure, with signif- EPCs promotes vascularization and in turn helps accelerate the icantly greater wound closing rates over control groups. Further, Tβ4 wound healing process. SDF-1 has also been shown to promote was shown to promote angiogenesis at the wound site as seen by angiogenesis in a diabetic mouse model, with a three-fold in- marked increased in endothelial cells and greater neovascularization. crease in blood vessel formation over a control group that did Relative capillary density was also increased in rats that were treated not receive SDF-1 [149]. The delivery of SDF-1 significantly accel- with Tβ4. erated wound closure as well as vascularization in full thickness The development of therapeutic resistance is one hypothesis for the skin wounds. Finally, in a novel approach, Vågesjö et al. trans- reason for the failure of clinical trials for growth factor therapies formed lactobacilli with a plasmid encoding SDF-1 [155]. These [48,110,158]. In diabetic patient skin, there is a marked reduction in bacteria were then applied topically to wound sites in mouse syndecan-4 and glypican-1 [47,48] proteins that are key in stabilizing models of hyperglycemia and peripheral ischemia, leading to ac- the interactions of growth factors with their receptors [109]. celerated wound closure and efficacy of bioavailable SDF-1. Syndecan-4 has been shown to modulate the migration of fibroblasts Other non-growth factor-based protein therapeutics have also and keratinocytes in dermal wounds [159]. Delivery of syndecan-4 in been explored for enhancing angiogenesis during wound healing. a liposomal formulation led to enhanced revascularization in a hind Spidroin, the protein found in spider’s silk, has been explored for its limb ischemia model in mice, diabetic ob/ob mice, and rats advanced scaffolding capacity [156]. Recombinant spidroin (1F9) was [54,109,110]. This treatment also enhanced wound closure and angio- used as a cell scaffold to promote the proliferation of cells while un- genesis in response to treatment with FGF-2 or PDGF-BB (Fig. 2A-E) dergoing bioresorption. 1F9 could potentially be used in a wide vari- [47,71]. Formulating recombinant syndecan-4 in a proteoliposome en- ety of applications. When cultured with 3T3 fibroblasts, the 1F9 hanced the activation of FGF-2 mediated signaling, increased nuclear

Fig. 2. Wound angiogenesis enhancement by syndecan-4 proteoliposomes. (A) von-Willebrand Factor (vWF) staining in the wound bed. Bar = 250 um. (B) Quantification of the small and (C) large vessels in the wound bed. (D) PECAM-1 fluorescence immunostaining for (red), αSMA (green) and nuclei (blue). Bar = 50 um. (E) Quantification of PECAM-1+/α-SMA+ vessels ⁎ † in the wound beds. p b 0.05 versus control group. p b 0.05 versus control and S4PL groups (p b 0.05). Used with permission from [71]. A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 103 trafficking of FGF-2 and causes increased recycling of syndecan-4/FGF-2 cells, levels of VEGF-A and proliferating keratinocytes. Other natu- during uptake [47,110]. Glypican-1 proteoliposomes also enhanced re- ral AMPs from non-human sources have been shown to increase vascularization in a hind limb ischemia model in WT and ob/ob mice angiogenesis and wound healing. The topical delivery of 200 and increased FGF-2 activity on endothelial cells [48]. Monteforte et al. μg/mL of CW49, a peptide derived from frog’sskin,hasshownto isolated exosomes by immunoselection from glioblastoma cells (one increase angiogenesis in vitro and in vivo, through HUVEC tube for- of the most angiogenic tumors), applied them in a mouse hind limb is- mation assays and a diabetic mouse model respectively [182]. Sim- chemia model and found marked improvement in the recovery of per- ilarly, tylotoin, a peptide present in salamander skin, showed fusion and formation of blood vessels [160]. Other examples of growth comparable wound healing capability with EGF in a full thickness factor co-receptors include the neuropilins (NRP1, NRP2), which help dermal wound mouse model [183]. The topical application of 20 modulate the angiogenic potential of VEGF-A [161–163]. Thus, novel ef- μLof20μg/mL tylotoin to the wound site twice a day resulted in fective protein therapies may involve the delivery of co-receptors to en- almost complete closure of the wounds by day 10, similar to a hance growth factor signaling. treatment of 20 μL of 100 μg/mL EGF. Combined with tylotoin’sin- duction of endothelial tube formation in vitro,suggeststhatitmay 2.3. Peptides play an angiogenic role in wound healing. Artificial AMPs engineered in lab have also shown promise for The delivery of short, anti-microbial peptides (AMPs) for therapeu- wound healing applications. SR-0379 is a key example of the new tic angiogenesis and wound healing is a promising development in the wave of artificial AMPs [184]. In full thickness skin ulcers in rats, field [164,165]. These peptides can be cost effective [166], and may treatment with 200 μg/mL of SR-0379 significantly reduced the offer an efficacious alternative to traditional growth factor therapies. wound area over a 60 μg/mL dose of FGF2 after just two days and Cathelicidins are a notable class of natural AMPs, LL-37 being the decreased the wound healing time overall. These results are similar only human cathelicidin [167–169]. Chereddy et al. showed improved to another artificial AMP, WRL3 [185]. In a MRSA infected burn wound closure in a full thickness excisional wound model in mice wound model in mice, the delivery of 4 μg/mL of WRL3, increased with up to a 7.5 mg/wound dose of LL-37 delivered from a tunable, angiogenesis, as shown by an increased number of blood vessels nanoparticle carrier. The wounds that were treated with LL-37 nano- upon histological analysis, at the burn site as well as helped miti- particles experienced an average wound closure percentage of 90% gate the infection. by day 10, which was significantly higher than the negative control groups, who received either only LL-37 or PLGA scaffold with nano- 2.4. Blood derived factors particles. Further, LL-37 delivery upregulated IL-6 and VEGF-A production and showed higher re-epithelialization than the control tissues. Blood derived factors present a promising new area of research in Other groups have also seen the upregulation of VEGF-A by LL-37 de- the field. Platelets contain growth factors and cytokines that can en- livery [170]. Concerns remain over LL-37 treatment, as elevated levels hance normal wound healing process and the delivery of a high num- of the peptide in circulation can result in the hemolytic activity ber of these factors to a wound site could provide a relatively [171,172], and, in specific cases, the delivery of LL-37 may promote straightforward way to enhance wound healing and angiogenesis. the growth of other pathogens [173] or inhibit the innate immune re- Platelet-rich plasma (PRP) is derived from gradient centrifugation sponse to bacteria [174]. However, engineered hybrid versions of the of a patient’s whole blood [186], and some components of PRP in- LL-37 peptide have been shown to mitigate this danger [175]. AP-57 clude PDGF-BB, TGF-β1, FGF-2, VEGF-A, EGF, and platelet factor-4 is another natural AMP that is commonly found in the mucosa of to name a few [187]. The potential strengths of PRP therapy include the stomach and colon [176]. A dose of 40 μg per treatment of AP- the fact that autologous PRP is easily obtainable and that it may 57 from nanoparticles delivered in an amphiphilic polylactic acid mimic the healing process with a combination of growth factors, fi- (PLA)-Pluronic F127-PLA block copolymer hydrogel was sufficient to brin and other components, as to a single growth factor therapy. accelerate wound closure and promote angiogenesis [177]. The deliv- Some studies have supported that PRP can improve neovasculariza- ery of AP-57 to the wound site increased the number of endothelial tion and wound repair [188], however, the clinical evidence for cells in the area and increased the vascular density significantly after using PRP in chronic wounds comes from trials with relatively 14 days in vivo. small patient populations, making the current support for use in LL-37 derivatives have also shown promise in enhancing angiogen- humans inconclusive [189]. esis in wounds [178]. LLKKK18 is an 18-mer peptide derivative of LL- In animal studies, PRP therapy can induce neovascularization and 37 that displays the most powerful lipopolysaccharide neutralizing ac- has also been shown to promote graft survival in chondrocutaneous tivity of all the peptide derivatives of LL-37 [179]. For burn wound composite grafts in rabbit ears [190]. Treatment with 1 mL of PRP signif- treatments, the delivery of LLKKK18 in Carbopol hydrogels reduced icantly increased the number of endothelial cells and the number of the wound size faster than the control, especially in the first 6 days. microvessels per field of view upon histological analysis. The expression The level of re-epithelialization was increased in the groups that of VEGF-A also increased significantly in the PRP group in comparison were treated with LLKKK18. Further, LLKKK18 helps to regulate the the control group that did not receive PRP injections. Qui et al. also levels of reactive oxygen species (ROS), which are present in high showed that the controlled release of PRP from a hydrogel composed quantities in the initial stages of healing following burn injury. Finally, of poly(d,l-lactide)-poly(ethylene glycol)-poly(d,l-lactide) (PDLLA- burns treated with LLKKK18 displayed a three-fold increase in the PEG-PDLLA: PLEL) triblock copolymer increased cellular migration and number of new blood vessels at the wound site as determined by α- tube formation significantly over the control [191]. Further, the con- SMA staining. trolled release of 0.8 mL of PRP significantly increased the wound clo- Vasoactive intestinal peptide (VIP) is a 28-amino acid long pep- sure in rabbits over PRP alone and the control. Additionally, PRP tide that has angiogenic and wound healing properties [180,181]. delivered from a PLEL scaffold significantly increased the number of In one study, VIP was loaded into PCL nanospun fibers and deliv- blood vessels in comparison to PRP without the scaffold or the control ered to skin wounds on the backs of mice. The wounds that were groups. Therapy with PRP may also promote angiogenesis by modulat- treated with VIP experienced higher closure percentages than the ing the tissue to secrete inflammatory proteins into the surrounding controls, achieving 96% wound closure after 7 days. As with the de- space [192]. In tendon cells from both healthy and tendons exposed to livery of the AMPs mentioned previously, the wounds experienced IL-1β (tendinopathic conditions), the addition of PRP to the culture re- significant increases in cell proliferation and angiogenesis. The duced the secretion of IL-6, IL-8 and monocyte chemoattractant protein treatment used in this study increased numbers of endothelial 1 (MCP-1). In addition, treatment with PRP increased the production of 104 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125

VEGF-A and hepatocyte growth factor in normal and tendinopathic studies using proteins/peptides other than growth factors for enhancing cells. angiogenesis in wound healing. Portions of PRP called platelet lysates have been shown to promote angiogenesis and help direct cell migration during the wound healing 3. Gene and nucleic acid-based therapies for wound angiogenesis process [193]. A major function of PRP therapy is derived from its effect on mesenchymal stem cell (MSC) homing and migration. Platelet ly- 3.1. MicroRNAs in wound angiogenesis sates have been shown to promote significantly faster adhesion and migration of MSCs in culture over the controls. When exposed to platelet Gene regulation plays a critical role in angiogenesis during wound lysate, MSCs increase their secretion of VEGF-A and PlGF, both impor- healing. MicroRNAs (miRNAs) are roughly 21-25 nucleotide long non- tant chemotactic growth factors for endothelial cells. While platelet ly- coding RNAs that play a powerful role in controlling gene expression sate also promotes endothelial migration, the addition of the secreted by binding to target mRNA and either suppressing mRNA synthesis or proteins from the MSCs in the local environment may even further ac- causing mRNA degradation [196–198]. Following inflammation, gene celerate the endothelial migration. Finally, platelet lysates have been expression is regulated to increase angiogenesis and remodeling of the shown to increase the levels of neovascularization, as shown by the in- woundedtissue[199]. MiRNAs have been shown to regulate many as- creased expression of PECAM-1 in platelet lysate coated scaffolds over pects of wound healing including cell proliferation and migration, colla- the uncoated scaffolds [193]. gen biosynthesis and network maturation, inhibition of Hemoglobin is another protein that researchers have used to im- neovascularization, and improved blood vessel growth [200–206]. prove ischemic wound healing [194,195]. Hemoglobin carries oxygen throughout the body’s circulatory system and oxygen plays a significant role in wound healing, by facilitating aerobic respiration, as well as 3.1.1. MicroRNAs that target angiogenesis in wound healing evolving into ROS. In a rabbit ear ischemic model, the delivery of IKOR With the power to increase or suppress gene expression, miRNAs 2048 (a chemically modified bovine hemoglobin) was shown to en- have become a target of interest in the field of wound healing. A handful hance wound repair and increase cellular survival. The local expression of miRNAs have been shown to specifically regulate angiogenesis within of Ki-67 (a marker of cellular proliferation), PECAM-1, VEGF and colla- wound healing, summarized in Table 3. These miRNAs are involved in gen were also increased upon administration of IKOR 2048 when com- increasing vessel formation and maturity, modulating migration, reduc- pared to the saline control. The delivery of 400 mg/kg of IKOR 2048 ing scar formation, and regulating expression of pro-angiogenic pro- significantly increased the oxygen tension in the tissue over untreated teins VEGF, GATA2, Ang-2 and PDGF-B, as well as the anti-angiogenic ischemic tissue, however, the treated tissue could not return it to nor- protein TSP-1 [200,203,205,207–209]. Overall, it is becoming increas- mal levels. Liposome encapsulated hemoglobin (LEH) delivery to gas- ingly clear that miRNAs play a powerful role in regulating angiogenesis trointestinal wounds in rats yielded an increase in burst pressure two in the context of wound healing. days after delivery, over the control groups, demonstrating increased High expression of various miRNAs has been correlated with de- healing. creased angiogenesis. By targeting these inhibitors of angiogenesis, sev- Blood derived factors present a promising area of research for scien- eral groups have been able increase angiogenesis in wounds. One tists and clinicians. While longer-term studies are needed to assess the method of inhibiting miRNAs is using anti-miRNA or “antagomirs”. safety of these treatments, the autologous nature of the treatments, Antagomirs are inhibitors of miRNAs that function by binding to specific the wide range of therapies available, and the auspicious pre-clinical miRNAs to prevent them from binding to their mRNA targets [210,211]. and clinical results indicate PRP treatments could provide a better alter- Patients with chronic wounds have been found to have a more than native to single growth factor therapies alone. Table 2 summarizes the two-fold greater expression of miR-92a compared to patients with healing skin wounds [212]. Likewise, overexpression of miR-92a has

Table 2 Proteins, peptides, and blood derived factors for enhancing wound healing and angiogenesis.

Treatment Function Exp. model Ref.

Insulin Accelerates wound healing, promotes keratinocyte migration, increases angiogenesis Mice/Rats [136–138] Erythropoietin Delivery increases the rates of neovascularization and increases levels of PECAM-1+ cells at the wound site Mice/Rats [140–143] hGH Increases re-epithelialization, increases angiogenesis, and increases infiltration of T-lymphocytes Diabetic [325,326] Rats Syndecan-4 Enhances the revascularization, wound closure, and angiogenesis in response to treatment with FGF-2 and PDGF-BB ob/ob [159] Mice/Rats Glypican-1 Increases revascularization in hind limb ischemia model and increases FGF-2 activity in endothelial cells ob/ob Mice [48] SDF-1 Promotes recruitment of endothelial progenitor cells and anti-inflammatory monocytes. Helps to accelerate the wound healing Mice [148,153] process and promotes bMSC migration to the wound site. Spidroin Increases proliferation of cells at the wound site to promote wound closure and angiogenesis Mice [156] Tβ4 Promotes faster wound closure rates, greater neovascularization at the wound site, and greater capillary density. Diabetic [157] Rats LL-37 Upregulates VEGF-A production, increases wound closure percentage, increases rates of re-epithelialization Mice [91–92,94] (peptide) CW49 Increases angiogenesis in vivo, HUVEC tube formation in vitro Diabetic [171] (peptide) Mice Tylotoin Accelerated wound closure (comparable to 100 μg/mL EGF dose), induces tube formation in vitro Mice [172] SR-0379 Decreased wound healing time, accelerated wound closure Rats [173] WRL3 Increased angiogenesis in burn model as shown by increase number of blood vessels per field of view Mice [174] Ap-57 Increase wound closure rates and promotes angiogenesis Mice [93,95] (peptide) VIP Increases wound closure percentages, cell proliferation and angiogenesis at the wound site Mice [180,181] PRP Increases the number of PECAM-1+ endothelial cells and the number of microvessels in the wound site. Increases the wound Rabbits [190,191,193] closure rate and MSC migration to the wound site Hemoglobin Increases cell survival, oxygen tension in the tissue, greater macrophage infiltration and tissue granulation. Rats [194,195] A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 105

Table 3 Gene therapies for enhancing wound healing and angiogenesis.

Gene/miRNA Function Exp. model Ref. microRNA

miR-21 Supports wound contraction and collagen network maturation Wound Model in Mice [206] miR-23a Reduction of miR expression, increased MET expression and increased angiogenic potential In vitro [204] miR-26a Inhibiting miR-26a induced angiogenesis, and increased wound closure (by 53%) Wound Model in db/db mice [214] miR-27b Improves bone marrow derived angiogenic cells by suppressing TSP-1. Overexpression reduces Wound Model in db/db mice/Skin burn [200,201] directional migration of mMSCs. model in mice miR-29b Reduces collagen biosynthesis during wound healing via inhibition of HSP47 expression, also Wound and Burn Model in Mice [327] suppressing angiogenesis, leading to reduced scar formation miR-92a Overexpression blocks angiogenesis and antagomirs enhance blood vessel growth. Inhibition improves Hind limb Ischemia Mice and Wound [213,328] wound healing and angiogenesis. Model in db/db Mice miR-184 Reduces neovascularization, reduces FOG2, PDGF-B, and PPAP2B Matrigel plug assay in Mice [203] miR-191 Modulates cell migration and angiogenesis via ZO-1 regulation miR proﬁling in diabetic patients/in vitro [205] tubule formation assay miR-199a-5p Downregulation of miR increases Ets-1- MMP1 pathway expression and angiogenesis Wound model in WT and Ets-1 KO mice [329] miR-200b Inhibition of miR-200b increased GATA2 and VEGFR2 leading to improved wound angiogenesis and Excisional wound model in WT and db/db [208,215] closure mice

been shown to result in decreased angiogenesis, however anti-miR-92a samples from the wound bed, margin, and normal skin were collected antagomir treatment (8 kg/mg injection) enhanced blood vessel growth and total RNA was isolated for miRNA microarray analysis. MiR-31, in an in vivo hind limb ischemia model in mice by over 50% [213]. Inhi- miR-21, miR-203 were upregulated by more than two-fold and miR- bition of miR-92a also improved wound healing and angiogenesis in a 429 was down regulated by more than two-fold, confirmed by qPCR. full thickness excisional wound model, specifically through decreasing MiR-31 has been identified as a regulator of HIF-1α, miR-203 targets activity of integrin subunit α5 (Itga5) and sirtuin-1 (Sirt1) [212,213]. p63 which is a regulates stem cell maintenance. Meanwhile, miR-203 Some antagomir treatments have been modified to be light inducible. and miR-429 regulate genes associated with tumors. Treatment with The addition of photolabile protecting groups prevents binding with 16 μg of miR-21 antagomirs dissolved in 100 μL of PBS injected locally target miRNA until activated by a light source, this light-inducible in the surrounding dermis reduced wound closure and collagen deposi- group has been labeled a “caged” antaogmir [212]. This light inducible tion. In addition, epithelial cell migration into the wound was reduced delivery method enables spatial control of the delivery of the gene ther- with miR-21 targeted antagomirs treatment [206]. In both excisional apy [212]. Treatment of diabetic (db/db) mice with an intradermal in- and burn wound models, expression of miR-29b was decreased by jection of 1 μg of light-inducible anti-miR-92a in 50 μL of PBS resulted over 50% compared to intact skin at day 7 post wounding [202]. How- in double the population of platelet endothelial cell adhesion molecule ever, treatment with oligonucleotide inhibitors of miR-29b increased (PECAM-1) positive cells in the granulation tissue compared to the HSP47 and COL1A1 gene expression in fibroblasts in vitro by more anti-miR-Control at 11 days post injury (Fig. 3A). Wound area closure than two-fold. Furthermore, miR-29b lentivirus treatment to increase for caged antimiR-92a with light was on par with non-caged antimiR- expression of miR-29b (2 x 107 TU/wound) applied either topically in 92a. Meanwhile, caged antimiR-92a was not effective at improving a diluted Matrigel or injected intradermally in an excisional wound wound healing, with closure rates similar to the cage antimiR-control model resulted a decrease in HSP47 and COL1A1 expression by over (Fig. 3B-G). Identifying miRNAs that regulate wound healing enables a 50% compared to the control virus treatment. Treatment with miR- clear path forward with potential antagomir treatment, as seen with 29b also reduced scar tissue formation and increased breaking strength miR92-a. In diabetic mouse wounds, levels of miR-26a have been of newly formed tissue [202]. found to be elevated compared to wild type (3.5-fold increase) [214]. MicroRNA profiling of patients with and without diabetes re- To target miR-26a, Locked Nucleic Acid (LNA) anti-miR-26a inhibitors vealed significantly lower levels of circulating miR-191 and miR- were used. Locked Nucleic Acid (LNA) is a high-affinity RNA analog, 200b in patients with type 2 diabetes. Interestingly, diabetic patients which “locks” to the target RNA. Intra dermal injection of 0.63 mg/kg with chronic wounds and peripheral arterial disease (PAD) had in- of LNA-anti-miR-26a inhibitors induced angiogenesis, accelerated creased levels miR-191 and miR-200b, like that of healthy patients wound closure by 53% and resulted in an approximately 50% increase [205]. Slightly higher concentrations of miR-191 were found at the in endothelial cells (PECAM-1+ cells) in full thickness excisional wound site compared to plasma levels in diabetic patients with wounds. PAD and chronic wounds. To understand the effects of miR-191, tu- It has been reported that temporary downregulation of miR-200b bule formation assays were performed. Tubule formation assays are during wound healing enables angiogenesis [208,215]. MicroR-200b is an in vitro assay to assess angiogenic potential, where more tubule thought to inhibit epithelial to mesenchymal transition and target an- formation is indicative of greater angiogenic potential. Treatment giogenic cytokines and transcription factors. Treatment of full thickness with miR-191 reduced in vitro tubule formation by over 50% mean- excisional wounds in mice with lentiviral particles expressing miR-200b while, treatment with anti-miR-191 increased tubule formation (at a titer of 2 x 107 cfu/mL, injected intradermally 1 mm from the compared to the scramble control. wound) induced slower wound healing and reduced blood flow, com- Another study found significantly reduced levels of miR-27b in pared to the control lentivirus treatment. Additionally, reduced num- mononuclear blood cells in patients with type 2 diabetes compared to bers of PECAM-1+ cells were found in the wound in miR-200b healthy patients [200]. To confirm the role of miR-27b in angiogenesis treatment groups compared to the control [208]. Treatment with anti- during wound healing, bone marrow-derived angiogenic cells (endo- miR-200b resulted in significantly increased microvessel density (mea- thelial progenitor cells selected via LDL uptake and lectin binding via sured via PECAM1+ immunostaining) compared to treatment with the Ulex europeus agglutinin-FITC binding assay) were co-delivered with vehicle control in a diabetic (db/db) mouse full thickness excisional miR-27 mimics (0.25 nmol in a 1% F-127 pluronic gel applied locally wound model [216]. to the surface of the wound). The miR-27 mimic delivery improved MiRNA profiling of wound healing in C57BL mice identified 54 wound perfusion, as measured by laser Doppler, in a full thickness exci- miRNAs that were increased or decreased more than two-fold com- sional wound model in db/db mice. Perfusion was improved by approx- pared to miRNA expression levels in normal skin [206]. Wound tissue imately 60% compared to scramble control treatment, to levels similar 106 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125

Fig. 3. (A) Expression of miR-92a in healthy and diabetic mice, and in acute and chronic human wounds. (B) Percent wound closure over time in full thickness excisional wound model. (C) Photographs of wound area over time for each treatment group. (D) Distance between epithelial tips as a measure of re-epithelialization. (E) Amount of granulated tissue, measured as an area. (F) Distance between the ends of the panniculus carnosus. (G). Representative images of hematoxylin and eosin staining, indicating granulation tissue (gt), epithelium (e), and ends of the panniculus carnosus (arrow heads). Used with permission from [328]. A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 107 to that of heterozygous non-diabetic littermate control mice after 8-10 fibrin scaffold to enable release of eNOS plasmids from the fibrin gel days [200]. followed by a secondary wave of release of Rab18 plasmids from the fi- Many new miRNA therapies are emerging with the potential to im- brin microspheres within the gel. The combination of Rab18-eNOS DNA prove angiogenesis in wounds to enhance healing. These therapies are delivery increased wound closure to approximately 90% after 14 days promising, in large part due to the targetable nature of miRNAs using whereas eNOS delivery alone had only reached approximately 70% antagomir treatments. However, effective delivery and stability in the wound closure [225]. This model is useful in delivering multiple treat- wound environment remain challenges for using miRNA-based thera- ments in sequence to maximize therapeutic effects. peutics in this area. Elastin-like-polypeptide (ELP) systems have also been used for the sequential delivery of genes such as eNOS and IL-10 [226]. IL-10 is an 3.2. Delivery methods anti-inflammatory cytokine that is present in ischemic tissue [227]. Like the fibrin microspheres in a fibrin gel, this system was designed 3.2.1. Free viral vector delivery to deliver IL-10 plasmids quickly followed by delivery of eNOS from Adeno-associated viruses (AAV) are non-enveloped viruses contain- ELP micro-spheres within the delivery platform. Co-delivery of IL-10 ing linear single stranded DNA. These viruses are characterized by low (10 μg) and eNOS (20 μg) and delivery of eNOS alone (20 μg) encoding immunogenicity and the need for a helper virus to replicate [217]. plasmids resulted in increased blood vessel density after two weeks in a AAVs are an attractive method for gene therapy particularly due to subcutaneous implant model compared to IL-10 plasmids. In a hind their safety, as they show little evidence of being pathogenic limb ischemia model in mice, perfusion almost doubled in eNOS and [217,218]. Recombinant adeno-associated viruses (rAAV) encoding IL-10/eNOS treatment groups in comparison to saline controls, ELP scaf- human vascular endothelial growth factor-165 (VEGF165) was deliv- fold alone, or IL-10 treatment [226]. ered in a db/db diabetic mouse wound model in which two full thick- Electrospun poly-L-lactide (PLA) and polycaprolactone (PCL) have ness longitudinal wounds were made on the dorsum of the mice. been used to create scaffolds for gene delivery of keratinocyte growth Recombinant AAV-VEGF165 treatment, delivered by intradermal injec- factor (KGF) [228]. It was found that PLA/PCL composite scaffolds pro- tion of ~1011 particles, resulted in a significant increase in angiogenesis moted the highest levels of cell adhesion to the scaffold, indicating after 7 and 14 days compared to the control delivery of rAAV-LacZ. Re- that the scaffold could support cell growth to enable angiogenesis and combinant AAV-VEGF165 increased angiogenesis, epidermal regenera- wound healing [228]. In a full thickness excisional mouse wound tion, and granulation tissue thickness at the wound site by over two- model, treatment with KGF plasmids (approximately 3.4 μg DNA/mg fold at both 7 and 14-day time points [219]. scaffold) in a PLA/PCL scaffold place on the wound resulted in a two- The angiopoietins are another family of regulators of angiogenesis fold increase in re-epithelization of excisional wounds significantly at and vasculogenesis. Specifically, Ang-1 important for vessel stabiliza- day 5 after wounding compared to untreated wounds. Greater prolifer- tion, maturation, and remodeling [220]. Delivery of rAAV encoding ation, and improved epidermal thickness were seen after 7 days com- Ang-1 more than doubled granulation tissue thickness after both 7 pared to scaffolds with control plasmids [228]. and 14 days in a diabetic mouse incisional wound model, and nearly tri- Bilayer dermal equivalents have also been used for gene delivery in pled capillaries present in the wound samples in rAAV-Ang-1 treated full thickness wounds. Dermal equivalents are useful in providing a mice compared to the delivery of rAVV-LacZ after 14 days [209]. In template for the dermis to use as it heals. Bilayer dermal equivalents these studies, it was demonstrated that VEGFR-2 was more than dou- have been used to deliver VEGF-165 to full thickness burns in a porcine bled as a result of rAAV-Ang-1 treatment compared to rAAV-LacZ treat- wound model [229]. The porous collagen-chitosan/silicone bilayer ment, but VEGF mRNA expression did not differ between treatment membrane dermal equivalents (BDE) loaded with plasmid VEGF-165 groups [209]. Overall, it appears that AAV delivery of VEGF and (50 μg per mg scaffold) demonstrated sustained release for up to 4 angiopoietin are both effective treatments for improving angiogenesis weeks in vitro. In vivo results showed significantly more vessel forma- and wound healing in diabetic mouse models of wound healing. tion in the VEGF-BDE compared to blank BDEs or plasmid DNA alone, with the greatest improvements at 21 days being an 80% increase in ves- 3.2.2. Scaffolds and hydrogels employed in gene delivery sel density compared to the control [230]. As well as significantly more A broad range of scaffold and hydrogel delivery systems have been mature vessels at 7, 14, and 21 days post wounding. This demonstrates employed for delivering gene therapies targeting angiogenesis in the benefits of combinatorial therapies providing both a tissue scaffold wound healing, each delivery system with unique benefits for gene- and gene delivery in the wound. based therapies. The delivery systems include simple fibrin scaffolds, Modification of the HIF1-α gene by truncating the oxygen depen- gel-in-gel systems that enable sequential release, electrospun gels, mul- dent domain from the gene (HIF1-α-ΔODD) has provided a novel tilayer gels, and hydrogels functionalized with peptides to encourage gene to deliver for increasing angiogenesis [231,232]. Under normoxic nuclear translocation. condition, oxygen binds to HIF1-α and eventually results in degradation Nitric oxide (NO) is a regulator of endothelial cell growth, vasomotor of the HIF1-α protein. Removal of the ODD from HIF1-α prevents oxy- tone and angiogenesis [221]. Previously, it has been demonstrated that gen binding and subsequent degradation, thereby stabilizing the pro- there is a decrease in the production of nitric oxide (NO) in wounds of tein [233]. Once stabilized, HIF1-α can induce expression of VEGF patients with diabetes [222]. Endothelial nitric oxide synthase (eNOS) longer, with the goal of increasing angiogenesis. Delivery of HIF1-α can be delivered to increase NO production and enhance wound DNA with a deleted oxygen-sensitive degradation domain (HIF1-α- healing. Delivery of adenoviral eNOS (3 x 107 pfu) in a fibrin scaffold ΔODD) improved vessel maturity as measured by the number of vascu- (60 mg/mL) led to a sustained production of eNOS for two weeks com- lar structures displaying PECAM-1 and smooth muscle actin (SMA) pared to delivery of the free virus in a diabetic rabbit ear excisional [232]. The HIF1-α-ΔODD treatment resulted in the greatest number of wound model [223,224]. Additionally, the fibrin scaffold yielded a re- vessels displaying both PECAM-1 and α-SMA compared to VEGF-A production in the volume of inflammatory cells by approximately half tein treatment or the fibrin matrix alone in an excisional wound mouse from the 7-day time point to the 14-day time point [223]. model at the 7-day time point. In another study, the HIF1-α-ΔODD gene Rab18, a gene involved in trafficking and regulating secretion, had a was delivered with poly (L-lysine)-graft-poly (ethylene glycol) (PLL-g- 40-fold decrease in expression in wounded keratinocytes under high PEG) functionalized with one of two peptides, either a TG-peptide de- glucose conditions [225]. A hyperglycemic rabbit model was used to signed to sustain release of the DNA or a poly-arginine peptide designed test the co-delivery of Rab18 and eNOS plasmid DNA. Fibrin micro- to aid in nuclear translocation [231]. The poly-arginine modified PLL-g- spheres were encapsulated in a fibrin gel (60 mg/to deliver the plasmids PEG delivery of HIF1-α-ΔODD resulted in the greatest increase in VEGF- (10 μg of Rab18 plasmid DNA). This delivery system built on the use of a A, PECAM-1, and smooth muscle alpha-2 actin (ACTA2) in wounds of 108 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 normal mice and diabetic rats seven days after wounding, compared to inflammatory macrophages [252–254]. Macrophages play a significant all other treatment groups. role in the initiation and progression of wound healing, but an uncon- trolled inflammatory response in the long-term can be counterproduc- 3.2.3. Electroporation and sonoporation enhanced delivery tive for tissue repair. Further studies need to be performed to evaluate Electroporation uses electrical pulses to induce temporary pores in long-term dosage response of NO-releasing nanoparticles to determine the cell membrane, allowing the passage of DNA into the cell. long-term therapeutic efficacy. Sonoporation uses ultrasound to create cavitation bubbles, which in turn create pores in the cell membrane [234]. The major benefitofelec- 4.2. Adenosine triphosphate troporation and sonoporation is that they do not require virus particles to deliver DNA to cells [235,236]. Use of electroporation during delivery Most of the energy-requiring processes, including mitotic and bio- of plasmid TGF-β1 significantly increased wound closure three and five synthetic activities of cells during wound healing, either directly or in- days post wounding in a full thickness excisional wound model in dia- directly occur via hydrolysis of adenosine triphosphate (ATP). ATP is betic (db/db) mice [237]. Approximately five-fold more endothelial used throughout the wound healing process during various metabolic cells were observed in the granulation tissue at the center of the processes involving protein and lipid biosynthesis, growth factor pro- wound bed compared to TGF-β1 plasmid treatment without electropo- duction, signal transduction and mitosis. There are changes in enzy- ration, indicative of angiogenesis. matic and metabolic activity throughout the wound healing process. Minicircle DNA is a type of plasmid DNA in which the bacterial back- Reduced blood flow and decreased oxygen supply at wound site bone has been removed to reduce the potential for immunotoxic re- often results in ischemic and potentially hypoxic conditions which sponses [238,239]. Minicircle-VEGF165 DNA (20 μg) delivery via can disrupt the balance of energy production and utilization in cells. sonoporation improved wound healing compared to the null plasmid This imbalance can result in the depletion of high-energy phosphate control diabetic mouse treatment group in a full thickness excisional ATP that in turn can decrease the cellular energy supply, potentially wound model [240]. Wound closure was significantly improved at day hindering the healing process [255]. Although the delivery of ATP is six after wounding, almost to the same level as non-diabetic mice con- a potential strategy to enhance wound healing. Extracellular ATP re- trols. Blood perfusion and endothelial cell staining indicated that leased during cell damage and apoptosis has been shown to activate VEGF165 gene delivery via sonoporation increased capillary density damage-associated molecular patterns (DAMP) signaling eliciting an and blood perfusion in the wound site compared to null plasmid control inflammatory immune response [256]. Therefore it is important to uti- treated diabetic mice. Similar studies came to the same conclusion, ul- lize a vehicle to deliver soluble ATP to mediate an undesired immune trasound delivery of plasmid VEGF165 gene in a diabetic mouse exci- response. sional wound model yielded strong increases in blood perfusion in the Encapsulation of ATP in small unilamellar lipid vesicles (ATP-vesi- wound compared to EGF gene delivery and control diabetic mice (un- cles) accelerated the healing of full-thickness skin wound in nondia- treated) [241]. These studies demonstrate the potential for electropora- betic and diabetic rabbit models [257,258]. Early in vivo studies tion and sonoporation to boost gene delivery once targetable genes evaluating the treatment of full-thickness skin wounds with highly have been identified for angiogenesis in wound healing. fusogenic lipid vesicles containing magnesium–ATP showed that intracellular delivery of ATP significantly enhanced wound healing [257]. 4. Drug and drug-like compounds for enhancing wound Wang et al. created wounds on the ventral side of each ear using a angiogenesis stainless-steel punch. Ischemic ears were generated via artery ligation at the base of ear. Immunostaining for PECAM-1 in the non-ischemic 4.1. Strategies using delivery of small molecules to promote angiogenesis ear wound after six days showed that wounds treated with ATP- and rescue impaired wound healing in animal models vesicles contained significantly higher number of small capillaries compared to saline-treated group 14.3 ± 2.7 capillaries/field versus A dynamic angiogenic response is critical for wound healing. Nu- 5.5 ± 1.2 capillaries/field observed respectively. The delivery of ATP- merous investigators have focused on the development of innovative vesicles (10 mM) to ischemic ear wounds in rabbits initiated early an- approaches for therapeutic angiogenesis to aid in healing of chronic giogenesis in the wound bed to promote faster wound closure and wounds [242–245]. Recent advances in our understanding of chronic generation of better developed granular tissue and re- wound biology have led to the development of small-molecular thera- epithelialization compared to nontreated rabbits [258]. In addition to pies that promote angiogenic activity and can modulate the repair pro- the presence of capillary loops in the deeper layers of the wound cess with exciting potential for clinical application. Previously, small bed in the ATP-vesicles–treated group histological analysis confirmed molecules have been used to remedy diabetic impaired wound healing an increased presence of neutrophils, macrophages and lymphocytes in animal models [246–249]. Nitric oxide (NO) has been identified as in the ATP-vesicle treated group in comparison to saline-treated one molecule that plays a vital role in wound healing. NO levels increase group six days after injury. significantly following skin injury and then gradually decrease as A follow up study by Wang J. et al. performed in diabetic rabbits wound healing progresses [250,251]. Han et al. synthesized nitric observed similar results to the study published a year earlier [258]. oxide releasing nanoparticles (NO-np) by encapsulating in a hydrogel- Full thickness and ischemic ear wounds were created in alloxan- glass composite a mixture of tetramethyl orthosilicate, polyethylene induced diabetic mice. There was a significant difference in the glycol, chitosan, glucose and sodium nitrite in sodium phosphate buffer wound closure times between ATP-vesicle treated groups in both is- (0.5 mol/L) [250]. The nitrite within the matrix reduced to NO to allow chemic and non-ischemic ears. Like their first paper, treatment with for stable release of NO from nanoparticles. Han et al. found that topical ATP-vesicles increased closure rate of wound significantly exhibiting application of NO-releasing nanoparticles increased angiogenesis in wound closure as early as day 9 for ATP-vesicle treated mice com- mice excisional skin wound model utilizing implanted silicone ring. pared to day 12 for mice treated with saline only. When ischemic Generation of dense vascularization of wounded area was observed, wounds of diabetic mice were treated with ATP-vesicles, the average while non-treated wounds experienced minimal new blood vessel for- closure time was 15.3 days in comparison to 19.3 days for control mation by measuring expression of CD34. NO-releasing nanoparticles wounds [259]. Histological analysis after two days showed early an- also increased levels of TGF-β in wounds known to play an important giogenic activity the presence of a few small capillaries and numerous role in promoting angiogenesis during wound repair. In addition to an- inflammatory cells including neutrophils, lymphocytes, and macro- giogenesis, NO also mediates inflammatory response in wound healing phages were present in granulation tissue of ATP-vesicle treated regulating recruitment and infiltration of inflammatory cells like wounds. Unfortunately, Wang et al. did not measure the formation A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 109 of mature vascular networks. After 17 days, saline-treated wounds studies suggest that statins angiogenic activity is mediated via the Akt/ showed incomplete re-epithelialization and less granulation tissue for- PI3K pathway to regulate proliferation of endothelial cells and capillary mation, while the ATP-vesicles treated group showed full granular tis- morphogenesis, and to modulate secretion of critical factors such as sue coverage and complete re-epithelialization of the wound VEGF-A in multiple cell types [261]. In an early study evaluating the indicating ATP-vesicles have the potential to initiate formation of a therapeutic potential of simvastatin to enhance wound healing, Bitto new vasculature network in injured tissue to restore blood circulation et al. used simvastatin to treat full-thickness wounds in female db/db in wound area critical to facilitate ECM production and epithelization, mice. Intraperitoneal injection of diabetic mice with simvastatin but this was not confirmed. [259]. These findings indicate that the in- (5 mg/kg) increased VEGF-A protein and gene expression in diabetic tracellular delivery of ATP enhanced wound healing in normal and di- mice with excisional wounds, following 3 and 6 days of treatment abetic wounds and offers a potential option for treating chronic non- [262]. Improved wound healing and increased angiogenesis were ob- healing wounds. These studies evaluated the angiogenic potential of served in simvastatin treated diabetic wounds by day 12. Immunostain- ATP-vesicles several days post injury. Future studies that investigate ing used to detect PECAM-1 in injured tissue sections revealed an the therapeutic potential of ATP-vesicles in wound healing should fur- increase in PECAM-1 positive staining and nearly double the microvas- ther evaluate the long-term effect of ATP-vesicle treatment on angio- culature density in simvastatin-treated group compared to the un- genesis in wound healing, and how presence or lack of a stable treated control group [262]. Additionally, daily treatment with vasculature alters progress of wound healing. Although the mecha- simvastatin also improved the tensile strength of damaged tissue and nisms by which intracellular ATP delivery enhances wound healing enhanced production of nitric oxide in wound, nitric oxide as previously are not fully understood, it is apparent that the availability of ATP is discussed is a molecule shown to play a significant role in wound an important aspect of the healing process. healing process [250,251]. A similar study conducted a few years later by Asai et al. observed 4.3. Statins that the wounds of db/db mice treated topically with simvastatin (50 μg/wound) exhibited more than 90% re-epithelization of injured tissue Statins are 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) and accelerated wound closure with significantly smaller wound areas reductase inhibitors primarily used to treat hypercholesterolemia and observed by day 7 compared to control. Immunostaining for PECAM-1 atherosclerosis. Over the past decade, researchers have shown that, in showed a marked increase in neovascularization in treated wounds on addition to their lipid-lowering effect, statins possess pro-angiogenic day 14 (Fig. 4A, B). In addition to enhanced wound vascularity, immu- properties that can potentially protect against ischemic injury and stim- nostaining for lymphatic endothelial cell specific marker lymphatic en- ulate angiogenesis [260]. Although still under investigation, early dothelial hyaluronan (HA) receptor-1 (LYVE-1) displayed the presence

Fig. 4. Effects of simvastatin on vascularity and lymphangiogenesis in full-thickness excision skin wound of db/db mice. (A) Neovascularization 14 days post-wounding at the wound margin of simvastatin-treated or vehicle-treated db/db mice. (B) Quantification of percent of vascularity. (C) Lymphangiogenesis 14 days post-wounding at the wound margin of simvastatin-treated or vehicle-treated db/db mice. (D) Quantification of percent of lymphatic vascularity. Green corresponds to LYVE-1-positive, newly formed lymphatic vessels. Blue fluorescence indicates DAPI-labeled nuclei. Scale bar = 100 μm. Used with permission from [246]. 110 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 of new lymphatic vessel at the margins of the wound (Fig. 4C, D) [246]. controlling hypoxia-mediated events in wound healing [247,269]. Dur- Presence of new lymphatic vessels suggests recovery of the ing wound repair, the active subunit HIF-1α regulates various processes lymphangiogenic function, which is important for maintaining tissue including cell metabolism, proliferation, survival, and angiogenesis homeostasis and macrophage infiltration during an immune response targeting important pro-angiogenic genes such as VEGF-A and stromal [263]. Treatment with simvastatin resulted in an increased population cell-derived factor 1-α (SDF-1α)[247]. SDF-1α also known as C-X-C of macrophages M2 (alternatively activate) macrophages expressing motif chemokine 12 (CXCL12), has been shown to be beneficial in pro- IL-13 with a significantly smaller population of TNF-α expressing M1 moting wound healing in in vivo models.[155] The chemokine CXL12 macrophages indicating that simivastatin treatment did not elicit an un- binds receptors on surface of immune cells to regulate inflammatory re- desired inflammatory response that could potentially hinder wound sponse during healing process. Lactobacillus reuteri bacteria (L. reuteri) healing. Simvastatin treated group also exhibited increased protein ex- transformed with a plasmid encoding for the chemokine CXCL12 to pro- pression of VEGF-C in simvastatin treated wounds. Gene expression for duce prolong overexpression of CXCL12 by sustaining a pH regulated PDGF-B, eNOS and FGF-2 were also significantly upregulated by simva- hypoxic wound microenvironment were shown to be beneficial to statin treatment [246]. wound healing when topically administered to full-thickness wounds More recently, improved drug delivery systems have been utilized to in mice with peripheral ischemic following hind-limb ischemia proce- enhance the angiogenic and wound healing potential of statins for dure and hyperglycemia.[155] Improvements in wound healing and treating chronic wounds. Lyophilized wafers composed of sodium blood perfusion were observed in both hyperglycemic and ischemic carboxymethyl cellulose and methyl cellulose have been developed as mice. Therefore, downstream effectors of HIF-1 particularly CXCL12 or potential wound dressings for administering simvastatin and other SDF-1α are promising targets for wound healing therapeutics. small molecules to chronic wound sites [263]. Moisture retentive dress- Deferoxamine (DFO) is an iron chelator, which has been used to ings are desired for treating chronic wounds to absorb wound exudate mimic oxygen deprivation in cells and induce the accumulation of and manage the moisture balance [264]. Lyophilized wafers are a viable HIF-1α. Hou et al. explored the effect of DFO treatment on neovascular- candidate for wound dressings that require a moist environment such ization in diabetic wounds, and the expression of HIF-1α and down- as mucosal wounds and venous ulcers [265]. When applied to wound stream genes. Full-thickness excision cutaneous wounds were sites, they absorb wound exudate and form a gel to maintain a moist en- generated on dorsal skin of rats with streptozotocin-induced diabetes vironment [263,265]. Hydrogels have also been developed that possess and treated with the vehicle control group (PBS), DFO (10 mg/mL) or desired mechanical properties that maintain optimum moisture in the VEGF-A (0.5 nM) [247]. The DFO-treated group exhibited accelerated wound area and aid in wound healing process. An ideal wound dressing wound closure at 7, 10 and 14 days compared to the vehicle and would exhibit slower drug release from the medicated dressing to help VEGF-A treated groups [247]. Histological analysis showed that DFO prolong the action and release of the active drug at wound site. A recent treatment stimulated formation of microvessels around the newly study by Yasasvini et al. evaluated the in vivo wound healing efficacy of formed granulation tissue seven days after wounding. Vessel density polyvinyl alcohol (PVA) hydrogels loaded with simvastatin encapsu- was assessed via vWF immunostaining at 7 and 14 days. The number lated chitosan microparticles. Simvastatin loaded chitosan microparti- of vessels per field was approximately four times greater in DFO treated cles were generated via modified ionic gelation method and loaded wounds compared to the vehicle only group, and slightly higher than hydrogels were prepared by chemical cross linking method.[266]Cuta- the VEGF-A treated groups with 20 and 15 vessels/field respectively. neous wounds were created in rats followed by treatment with varying Western blot analysis of tissue lysates showed that HIF-1α was associ- concentration of simvastatin-chitosan microparticle loaded PVA ated with DFO treatment enhancing neovascularization. Protein expres- hydrogels (2.5, 5 and 10 mg). Treatment with low dose simvastatin sion of HIF-α was significantly elevated in DFO treated wounds and hydrogels (2.5 mg/patch) every 7 days for 21 days displayed increased VEFG protein expression was also found to be higher in the DFO- rate of wound closure compared to control non-treated wounds and treated group compared to vehicle control group. Protein expression medium and high dose treated wounds (5 mg and 10 mg respectively) levels of SDF-1α were elevated at day 7 compared to both VEGF-A and [266]. In rat wounds treated with low dose hydrogels, histological anal- vehicle treated groups. These results suggest the potential of DFO to en- ysis revealed granulation tissue with complete epithelialization 21 days hance neovascularization in wound healing through increasing HIF-1α after wounding. Recent studies have shown that utilizing drug delivery activity. systems to administer statins have increased drug efficacy as a thera- A later study by Ram et al. found that treating wounds of diabetic in- peutic in wound healing. Unfortunately, there is still very little research duced rats with DFO ointment (0.1%) also enhanced wound healing and that explores how these systems enhance statins angiogenic potential angiogenesis. Wounds treated with DFO experienced greater wound in vivo. Previous studies exploring the angiogenic potential of free closure from day 7 to 19 compared to control group. Real time PCR statins have shown that topical administration of statins promotes vas- and western blot analysis showed that gene and protein expression of cular formation in wound injury. The field would further benefitfrom HIF-1α, VEGF-A, SDF-1α,TGF-β1 and IL-10 were significantly upregu- exploring how target drug delivery systems enhance this effect. Addi- lated in wounds treated with DFO from days 3 to 14 [270]. In contrast, tionally, although novel drug delivery systems offer a strategy for sus- the relative mRNA expressions of pro-inflammatory cytokines TNF-α, tainable controlled drug release to facilitate a prolonged therapeutic MMP-9 and IL-1β were significantly downregulated at days 7 and 14 effect of drugs and aid in long-term wound recovery, further character- post-wounding. Additionally, DFO treatment was observed to decrease ization of the in vitro and in vivo toxicity and efficacy of simvastatin- protein levels of tumor necrosis factor alpha (TNF-α) post-wounding loaded delivery systems is needed. from day 3 onward and increased protein levels of IL-10 compared to controls at day 3 and onward. Indicating that treatment with DFO 4.4. Deferoxamine does not induce an early inflammatory response in vivo. DFO also induced angiogenesis in newly formed granulation tissue [270]. Although In chronic wounds the healing process is often impaired and many density of microvessels was not quantified, an overall larger number of growth factors like VEGF, which are essential to angiogenesis and the small blood vessels were observed in regenerated tissue of DFO-treated wound repair process are downregulated [267,268]. As part of the group throughout the 19-day study compared to control group. wound repair mechanism, the wound microenvironment can become Researchers have tried to enhance the therapeutic potential of DFO hypoxic due to injury of the microvasculature. This hypoxia can induce using transdermal delivery systems that can obtain deeper penetration cytokine production and cell recruitment to accelerate wound closure into the wound bed. Duscher et al. developed a transdermal drug deliv- [247,269]. Hypoxia-inducible factor-1 (HIF-1) is involved in regulating ery system that used reverse micelle encapsulation of DFO by nonionic the response of cells to altered oxygen levels and plays a key role in surfactants (polysorbate 80 and sorbitan monolaurete) within a A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 111

Fig. 5. Deferoxamine-treated diabetic ulcers exhibit increased neovascularization and improved dermal thickness. (A) Immunohistochemical staining for newly formed capillaries via endothelial cell marker PECAM-1 (red). Scale bar = 10 μm. (B) Quantification of PECAM-1+ pixels per high-power field (HPF). (C) Picrosirius red staining used to assess dermal thickness of completely healed diabetic mice ulcers. Scale bar = 10 μm. (D) Quantification of picrosirius red-positive pixels per HPF. Used with permission [271]. polyvinyprrolidone (PVP) biodegradable matrix and disperse in ethyl (Fig. 6C). Consistent with the reduction in scarring, AS-IV treatment cellulose for slow release [271]. Pressure ulcers were induced in db/db stimulated angiogenesis at wound site. A few blood vessels could be ob- mice by placing two ceramic magnets for three separate ischemia/reserved in healed tissue of AS-IV treated group 30 days after wounding perfusion cycles. Each cycle consisted of placement of magnets for 3 or (Fig. 6A-B) [272]. 6 hours followed by release or reperfusion for 3 or 6 hours. Treatment To reduce the dosing frequency and enhance therapeutic efficacy, of pressure ulcers with transdermal DFO (1%) improved healing and pharmaceutical carriers for the topical application of AS-IV have been stimulated complete wound closure by day 27 compared to day 39 for investigated. One study examined the use of solid lipid nanoparticle- control group. Histological analysis showed that localized treatment enriched hydrogel (SLN-gel) prepared by a solvent evaporation method with DFO transdermal delivery improved the dermal thickness of to encapsulate AS-IV (0.5% AS-IV/gel) and locally delivered it to full- healed diabetic ulcer and promoted neovascularization within the thickness skin excision wounds in rats [273]. They found that treating wound bed (Fig. 5A-D). Immunostaining for PECAM-1 showed that wounds with solid lipid nanoparticle-enriched hydrogel loaded with treatment with transdermal DFO had more than twice the number of AS-IV (AS-based SLN-gel) dramatically enhanced wound healing capa- PECAM-1 positive capillaries. Sustained local delivery of DFO treatment bilities compared to a blank SLN-gel, and free AS-IV solution treated was also found to increase early VEGF protein expression in diabetic ul- groups at days 4 and 12 after wounding. AS-IV solution and AS-based cers at 24 and 48 hours. Pretreatment for 48 h with transdermal deliv- SLN-gel both stimulated angiogenesis at the wound site. Histological ered DFO prior to ulcer induction visibly reduced tissue necrosis and analysis of the wound beds showed a higher density of newly formed prevented ulcer formation later. Dysregulation of the HIF-1α/VEGF sig- blood vessels in both AS-IV solution and AS-based SLN-gel, while no naling can be caused by excessive ROS production a central problem in blood vessels were observed in control and only a few microvessels in diabetic wound healing Levels of ROS were negatively affected by the SLN-gel carrier only treated group [273]. pretreating with DFO. Dihydroethidium (DHE) is a superoxide indicator In addition, AS-IV has been used to functionalize wound dressings to used to measure in situ generation of ROS. DHE immunofluorescent promote healing and prevent scar formation in burn wounds. Shan et al. staining showed that ROS levels decreased dramatically in DFO developed silk fibroin/gelatin electrospun nanofibrous dressings loaded pretreated db/db mice. Thus, DFO is a promising therapy for rescuing with AS-IV (0.5 mg). A homogeneous solution of a suitable blending impaired wound healing in diabetic patients and novel delivery systems ratio of SF/gelatin and AS-IV was flowed through a needle at a fixed offer a potential strategy for improving treatment efficacy. rate of 0.01 mL/min. under a high applied voltage, 15kV, and electrospun to produce nanofibrous dressing. Treatment with AS-IV 4.5. Natural compounds loaded dressing significantly accelerated wound healing compared to blank control (normal saline) groups at 7 and 15 days post-wounding In addition to tissue healing and regeneration, another goal of in a partial-thickness burn wound model in rats [274]. Minimal scarring wound therapy is to promote adequate healing while minimizing scar was observed in repaired tissue of both AS-IV loaded nanofibrous dress- complications such as imbalanced collagen synthesis [272]. ing and AS-IV solution treated wounds. In addition, immunostaining for Astragaloside IV (AS-IV) one of the main active ingredients in Astragali PECAM-1 showed increased blood vessels in both treatment groups. Radix derived from the root of Astragalus membranaceus has long been Newly formed vessels were observed in the newly formed granulation exploited in Traditional Chinese Medicine for its healing and anti- tissue 31 days post-wounding in both AS-IV treatment groups. Treat- scarring effects [272,273]. Chen et al. observed that AS-IV (0.5%) locally ment with AS-IV enhanced the number and dimensions of blood vessels delivered at the wound site of full-thickness skin excision wounds in compared to no vasculature formed in blank control and very few micro rats stimulated quicker recovery of wounds with minimal scarring at vessels in blank nanofibrous dressing [274]. AS-IV is a promising candi- day 30 compared to blank control wounds. A lower ratio of type I/III date for treating impaired wound healing. Previous studies have shown was observed in the AS-IV group compared to that of the blank control its ability to promote wound re-epithelization, angiogenesis and regu- group demonstrating treatment was able to regulate collagen synthesis late extracellular matrix remodeling in chronic and burn wounds. Al- and reduce scar formation during the wound remodeling process though a poorly water-soluble drug, it has been successfully 112 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125

Fig. 6. Anti-scar effects of astragaloside IV on rat full-thickness skin excision wounds 30 days after wounding. (A) Masson’s trichrome staining of granulation tissue to assess collagen synthesis and blood vessels formation. Collagen and blood vessels identified by red and green arrows, respectively. (B) Effect on angiogenesis assessed by presence of blood vessels in granulation tissue indicated by black arrows. (C) Sirius red staining to evaluate collagen composition in healed tissue. Type I collagen indicated by white arrow, and type III collagen indicated by pink arrows. Blank control refers to the no treatment group. Used with permission from [272]. incorporated into novel biocompatible carriers that allow for sustained macrophages present in wound at 1, 5 and 7 days post treatment com- drug release to induce increased tissue regeneration and inhibit scar for- pared to control. Examination of cytokine levels in the exudates from mation during wound healing. asiaticoside-treated burn wounds showed increased levels of IL-1β at Herbal remedies have been helpful in the treatment of dermatolog- multiple time points compared to control vehicle treated group [278]. ical disorders such as skin lesions, eczema, burns and hypertrophic scars Topical application of asiaticoside also increased level of monocyte [275–277]. One example is the plant, Centella asiatica that contains var- chemoattractant protein-1 (MCP-1) in exudates of burn wound- ious active compounds such as centelloids, asiaticoside, madecasosside, treated mice compared to control group [278]. Histological analysis of asiatic acid and madecassic acid that have been found to be beneficial in regenerating skin of burn wound-treated mice showed increased num- wound healing [275]. Ointments containing varying doses (0.1 to 1%, w/ bers of proliferating cells, as assessed by Ki-67 staining, and increased in w) of these compounds or a combination have previously been shown number of VEGF expressing cells in the healing wound at day 9 [278]. to enhance wound repair [275,278–280]. Asiaticoside derived from Several other studies demonstrated that low doses of asiaticoside stim- Centella asiatica possesses diverse pharmacological effects including an- ulate angiogenesis by enhancing keratinocyte production of MCP-1 that giogenic promoting activity and wound healing enhancing capabilities. in turn increases VEGF-A levels in chronic wounds [278,279]. Kimura et al. observed that topical treatment of burns in mice with The poor solubility of asiaticoside in aqueous medium is a major asiaticoside at lower doses of 10−8 to 10−12 (w/w) enhanced burn limiting factor in the drug’s therapeutic efficacy [279–281]. To im- wound healing. Treating with ointment for 20 days containing various prove the pharmacological effect of free asiaticoside, carriers have doses of asiaticoside significantly reduced burn wound area on days 6 been developed to increase wound healing potential. Phaechamud to 18 compared to vehicle control (petroleum jelly only) with no signif- et al. performed the chick chorioallanotoic membrane assay (CAM) icant difference observed between three concentrations (10−8,10−10 to evaluate the angiogenic effect of chitosan-aluminum monostearate and 10−12 w/w) [278]. Kimura’s group also treated the burn surface composite sponge dressings loaded with varying concentrations of with polyethylene filter pellets containing three different concentra- asiaticoside. Homogenous mixture of chitosan solution and tions of asiaticoside 10 pg, 1 ng, and 100 ng. Asiaticoside (10 pg and 1 asiaticoside was fabricated via lyophilization technique, frozen, freeze ng/filter pellet) decreased polymorphonuclear leukocytes infiltration dried, and treated with dehydrothermal treatment to stabilize struc- at wound site at 1 and 3 days, while treatment with all three doses ture of lyophilized sponge dressing. Phaechamud et al. showed that (10 pg, 1 ng, and 100 ng/filter pellet) increased the number of asiaticoside exhibits dose-dependent angiogenic activity in vitro A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 113

[279]. The average angiogenesis score increased with increasing con- HA-Mediated Motility (RHAMM), hyaluronic acid receptor for endocy- centration of asiaticoside. The average angiogenesis score was calcu- tosis (HARE), lymphatic vessel endothelial hyaluronic acid receptor 1 lated as the ratio of total number of embryos treated to evaluable (LYVE1), layilin, toll-like receptor 2 (TLR2) and toll-like receptor 4 embryos left after a 12-day study. Embryos treated with varying con- (TLR4) to influence receptor activation and their downstream signaling. centration of asiaticoside 40 μg, 80 μg, 160 μg, and 320 μg generated CD44 is a cell-surface receptor for hyaluronan implicated in the regula- the highest overall angiogenesis scores 1.25, 1.25, 1.83, and 2.12 re- tion of endothelial cell functions, and tumor angiogenesis [295,296]. Al- spectively. In comparison to VEGF positive control which at the fol- though the role of CD44 in angiogenesis is still being investigated, CD44 lowing concentrations 100ng, 200ng, and 300ng scored 0.43, 0.17, has been shown to possess anti-angiogenic properties impairing tube and 0.2 respectively. formation in vitro and in vivo [296,297]. CD44 mediates HA-dependent In another study, full-thickness skin wounds in rats were treated cell signaling involved in the regulation of endothelial cell proliferation, with asiaticoside-loaded porous polyvinyl alcohol (PVA) microspheres migration, adhesion, and tube formation required for angiogenesis. The [280]. The asiaticoside-microspheres enhanced wound healing com- interaction between HA and CD44 receptor is, in part, dictated by the pared to untreated wounds or those treated with free asiaticoside solu- size of the HA molecule that can influence CD44 clustering and activation. Asiaticoside microspheres enhanced wound healing accelerating tion. Oligomeric HA can competitively bind to CD44 receptor in place closer rates compared to free drug significantly at day 7 and day 14 of anti-angiogenic high molecular weight HA to act as an antagonist to and minimized scar formation. In the rats treated with asiaticoside mi- CD44, triggering intracellular signaling to promote endothelial cell pro- crospheres, immunohistochemical staining for PECAM-1 showed a liferation, and migration to stimulate neovascularization in vitro higher density of microvessels in comparison to the other treatment [287,291]. groups. Overall, treatment with asiaticoside reduced scar complications, Previous studies have shown that topical delivery of exogenous stimulated re-epithelialization and collagen deposition, and promoted HA oligomers to injured tissue promotes wound repair in vivo wound angiogenesis to improve wound healing. Although free [249,292]. Gao et al. topically treated murine excisional dermal asiaticoside has been previously shown to possess properties that en- wounds with HA oligomers (50 μg/per wound) encapsulated in a hance healing and reduce scar formation, its lack of solubility is a slow-releasing gel of ethylene-vinyl acetate copolymer. Treatment major limitation and new carriers for its delivery would be beneficial with HA oligomers accelerated wound healing, with the treated for improving its bioavailability. wounds exhibiting similar closure rates and recovery as those Materials derived from natural processes have also been shown to treated with VEGF-A (10 μg per wound). The HA oligomer treated have significant pro-angiogenic activity in wound healing. Dextran wounds had increased numbers of blood vessels. Compared to the hydrogel-based dressings were highly proangiogenic in burn injury PBS and HMW-HA treated mice, there was a significantly larger in mice and pigs [282,283]. In comparison to control wounds, there number of blood vessels in the wound bed. The group treated with was increased neutrophil infiltration in wounds treated with dextran HA oligomer encapsulated gel by day 6 had approximately 4 times hydrogels [282]. In addition, there was increased infiltration of endo- the number of blood vessels per field area of those wounds treated thelial cells with treatment with the dextran hydrogel dressings. Bio- with PBS and HMW-HA. By day 8 post wounding HA oligomer gels active glass microfibers doped with cupric oxide also increased contained more PECAM-1 positive blood vessels although this num- angiogenesis and wound healing [284]. These microfibers degrade ber was less significant than at day 6. Additionally, treatment with to hydroxyapatite under physiological conditions in seven days. In HA oligomers induced the formation of new lymphatic endothelium. a rat wound model, these fibers increased wound closure and vessel Immunostaining for LYVE-1, a marker for lymphatic endothelial area during healing. cells, was upregulated and dispersed throughout vessels in the wound bed of mice treated with HA oligomers, VEGF and HMW- 4.6. Hyaluronan oligosaccharides HA [292]. Overall density of lymphatic vessels in HA oligomer- treated mice was significantly greater than PBS-treated mice on Hyaluronic acid (HA), also known as hyaluronan, is a glycosamino- day 4 and 8. Although the lymphatic vessel density was less com- glycan (GAG) that is a long unbranched polymer comprised of repeating pared to high molecular weight HA and VEGF-A, suggesting that D-glucuronic acid and N-acetyl-D-glucosamine disaccharide units, HA oligomers are superior in stimulating angiogenesis over bound through alternating β-1, 4 and β-1, 3 glycosidic bonds. HA is a lymphangiogenesis. Administration of HA oligomers was found to functional component of connective tissue ECM that plays a critical affect the expression of numerous factors involved in wound repair role in cell signaling, tissue development and maintenance of tissue including endothelial cell proliferation, cell migration and collagen health and homeostasis [285,286]. Unlike many other GAGs, HA is not synthesis. Treatment with HA oligomers upregulated gene expres- sulfated or covalently attached to core protein as a proteoglycan [287]. sion of endothelial eNOS, an important atheroprotective molecule, Previous research has implicated hyaluronan as a potential therapeutic and cell adhesion molecules, E-selectin and integrin β3atearly capable of both stimulating and inhibiting angiogenesis depending on time points compared to control [292]. In addition to inducing an- its form and concentration [287,288]. Physiological responses to giogenesis during wound repair of dermal injury, oligomeric HA hyaluronic acid is highly influenced by HA molecular weight, which is treatment effects the mRNA expression of key endothelial mediators classified into low (≤5 kDa), intermediate (60-800 kDa) and high and regulators of collagen deposition. (N800 kDa) molecular weight groups [289]. Physiological concentra- A recent study also supports the use of HA oligomers for treating di- tions of native long-chain, high molecular weight HA have been abetic wounds. Ointment prepared by emulsification containing HA shown to induce anti-inflammatory and anti-angiogenic activity oligomers (0.15%), petroleum jelly (2.4 g), stearic acid (2.4 g), glycerol in vivo and in vitro [249,288,290,291]. In particular, HA oligomers monostearate (1.6 g) and liquid paraffin wax (2.5 g) was topically ad- consisting of 2-10 disaccharides are highly bioactive and capable of ministered to wounds created in rats with streptozotocin-induced dia- stimulating neovascularization in animal wound models [249,292]. Al- betes [249]. Treatment with HA oligomers containing ointment though the size-dependent effects of HA on biological processes are significantly decreased wound size and induced formation of granula- not completely understood, the size of HA can have a considerable influ- tion tissue and re-epithelialization of wound surface by day 14 after ence on receptor activation and its downstream signaling. In an injured wounding [249]. In addition to enhancing wound healing, HA oligomer or disease state, aberrant conditions can cause the degradation of high treatment improved blood flow in diabetic wounds as measured by molecular weight by hyaluronidases and ROS into low molecular weight laser doppler imaging. Histological analysis of wound tissue 14 days HA which further depolymerized to form oligomeric HA [293,294]. post-wounding showed increased numbers of capillaries in the HA olig- Byproducts can then bind several cell surface receptors such as CD44, omer groups in comparison to control wounds [249]. Treatment with 114 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125

Table 4 Small molecules for enhancing wound healing and angiogenesis in mice.

Treatment Results Mechanism of action Exp. model Ref.

Simvastatin ↑ VEGF mRNA Inhibition of HMG-CoA reductase db/db Mice [262] ↑ Endothelial Cells Pleiotropic Effects ↑ NO products ↑ Collagen synthesis Simvastatin ↑ Endothelial Cells Inhibition of HMG-CoA reductase db/db Mice [246] ↑ LYVE-1 Pleiotropic Effects ↑ Macrophage inﬁltration ↑ VEGF-C ↑ PDGF- β, eNOS, FGF-2 mRNA Deferoxamine ↑ VEGF protein Iron chelator db/db Mice [271] ↑ Endothelial Cells Increases HIF-1α transactivation Upregulates angiogenic factors ↓ Skin Necrosis/Apoptosis ↓ ROS Prevented Ulcer Formation Asiaticoside ↑ Macrophage Inﬁltration Inhibition of TGF-β receptors Mice [278] ↑ VEGF, MCP-1, and IL-1β Increased NO production ↑ Cell Proliferation Increased TNF-α production HA oligomers ↑ Endothelial Cells Binds to CD44 receptor promoting pro-mitotic effects on Mice [292] ↑ LYVE-1 endothelial cells and angiogenesis ↑ Fibroblast proliferation ↑ eNOS/procollagen mRNA ↓ MMP-9/MMP-13 mRNA

HA oligomer containing ointment was found to not affect total Src and paracrine effects, immune response modulation, and their ability to dif- ERK1/2 expression in wound tissue, but levels of phospho-Src and ferentiate into vascular lineages. phospho-ERK1/2 increased significantly [249]. Additionally, oligomeric The mode of delivery of cells to the wound can have profound effects HA treatment increased expression of TGF-β1 protein. on the cell state and regenerative activity [298]. Multiple strategies have Overall, hyaluronan has proven to be a versatile bioactive molecule been tested to overcome the general lack of engraftment and cell viabil- playing a significant role in the initiation and progression of biological ity associated with cellular therapeutics [299]. Many researchers have processes and pathological conditions. Shorter HA fragments like oligo- utilized porous scaffolds constructed out of various polymers and mers are crucial mediators of intracellular signaling cascades triggering hydrogels. Primarily, these scaffolds function to improve cellular en- activation of surface receptors such as CD44 to regulate inflammatory graftment so that they can elicit a prolonged effect at the site of injury. and angiogenic responses [287]. Topical administration of oligomeric Ideally, scaffold materials should be non-immunogenic, non-cytotoxic HA is a promising therapeutic strategy to promote wound healing and and facilitate a phenotype in the delivered cells that supports their re- angiogenesis in animal models of chronic and diabetic wounds generative properties. For wound healing applications, resorbable scaf- [249,292]. Evidence suggests treatment with HA oligomers is beneficial folds are often used so that endogenous tissues can grow to replace the in promoting neovascularization in animal wound models, but addi- scaffold area. Furthermore, scaffolds benefit from having a level of po- tional studies are needed to further profile the adverse effects of rosity that can allow for cellular infiltration of their network and migra- prolonged HA oligosaccharide treatment including potential unwanted tion toward the site of injury. To promote this outward migration, cell activation and prolonged inflammation. Tables 4, 5 and 6 summa- integrin-binding/cell adhesion sites can be added to the material sur- rize the results of studies using small molecular therapies to enhance face. While each of these characteristics can help with cellular viability wound healing and angiogenesis. and integration into host tissue, some cellular therapies have found success without the use of a scaffold. The following section summarizes and highlights several differing approaches for promoting angiogenesis and 5. Stem cell based therapies for wound angiogenesis wound healing through therapeutic cellular delivery.

5.1. Challenges and strategies for therapeutic cell delivery 5.2. Adipose derived stem cells

Stem cell therapies are an emerging and promising approach for Adipose-derived stem cells (ASCs) are a promising therapeutic cell treating diseases not well addressed by conventional therapies. Several type for wound healing as they are multipotent and can achieve a vascu- stem cell types have been explored to facilitate angiogenesis and wound lar phenotype, however their success has been hindered due to poor healing. Adult stem cells including adipose-derived stem cells (ASCs), survivability at the wound microenvironment [299]. Numerous studies bone marrow-derived mesenchymal stem cells (MSCs) and induced have been conducted to optimize ASC delivery and achieve a robust pluripotent stem cells (iPSCs) have been explored for their potential therapeutic effect in various wound healing models (Table 7). Zamora

Table 5 Small molecules for enhancing wound healing and angiogenesis in rabbits.

Exp. model Treatment Results Mechanism of action Ref.

Rabbit Mg-ATP delivered in ↑ Endothelial Cells Regulates production of pro- and anti-angiogenic factors that activate [258] unilamellar lipid vesicles ↑ Inﬂammatory Cells various receptors such as A2B receptors ↑ Re-epithelialization Diabetic Rabbit Mg-ATP delivered in ↑ Granulation Tissue Regulates production of pro- and anti-angiogenic factors that activate [259] unilamellar lipid vesicles ↑ Re-epithelialization various receptors such as A2B receptors ↑ Inﬂammatory cells A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 115

Table 6 Small molecules for enhancing wound healing and angiogenesis in rats.

Exp. Treatment Results Mechanism of action Ref model

Rat Simvastatin loaded ↓ Wound size Inhibition of HMG-CoA reductase [266] chitosan microparticles in Complete epithelialization (d21) Pleiotropic Effects PVA hydrogel Rat Astragaloside IV ↓ Scar Formation Alters activity of VEGF receptors regulating pathways [272] Topically applied ↑ Skin Wound Tensile Strength involving PKB/PI3K ↑ Microvessel Density ↓ Ratio of Type I/III collagen Rat Astragaloside IV ↑ Blood Vessels Alters activity of VEGF receptors regulating pathways [273] Solid lipid nanoparticle in hydrogel ↑ type I collagen deposition involving PKB/PI3K Rat Astragaloside IV in ↓ Scar Formation Alters activity of VEGF receptors regulating pathways [274] Silk ﬁbroin/gelatin electrospun nanoﬁbrous dressing ↑ VEGF involving PKB/PI3K ↑ numbers of blood vessels Type I collagen deposition Rat Asiaticoside in ↑ Blood Vessels Inhibition of TGF-β receptors [280] PVA/ethyl cellulose microspheres Minimal Scarring Increases NO and TNF-α Organized Collagen I Fibers Diabetic Rat +/- DFO ↑ Capillary Density Iron chelator [247] +/- VEGF ↑ HIF-1α, SDF-1α and VEGF Increases HIF-1α transactivation Upregulates Intraperitoneal Injection angiogenic factors Diabetic Rat DFO Ointment ↑ HIF-1α, VEGF, SDF-1α, TGF-β1 and IL-10 Iron chelator [270] ↓ TNF-α, MMP-9, and IL-1β Increases HIF-1α transactivation Upregulates angiogenic factors Diabetic Rat HA Oligomers in ↓ Wound Size Binds to CD44 receptor promoting pro-mitotic effects [249] Ointment ↑ Blood Flow on endothelial cells and angiogenesis ↑ Capillary Density ↑ p-Src, p-ERK and TGF-β1

et al aimed to expedite wound healing through the use of human ASCs on the outer layer of medium sized vessels in the wound bed, ostensibly differentiating into a supportive vascular role on PEG-fibrin (FPEG) gels performing a similar role to that of pericytes or vSMCs. In ASCs grown [300]. ASCs were derived from discarded burn skin samples and cul- on FPEG scaffolds in vitro, VEGFA gene expression was found to increase tured on a 3D PEG-fibrin gel. After three days in culture, ASCs formed over the culture period along with VEGF-A protein expression over the tubule-like structures like those created by endothelial cells, however course of 15 days. These results indicate that, under specificconditions, they lacked PECAM-1 and vWF expression. Gene expression of pericyte ASCs can differentiate into cells that both encourage neovascularization markers including for alpha actin-2 (ACTA2), PDGFRB, neural/glial through VEGF secretion and stabilize vessels through pericyte-like antigen-2 (NG2), angiopoietin-1 (ANGPT1), and angiopoietin-2 interactions. (ANGPT2) were detected via RT-PCR. This result was confirmed via im- While particular cellular treatments are meant to facilitate cell sur- munostaining of NG2 and PDGFR-β, suggesting a supportive vascular vival or differentiation so that they might have a direct effect on phenotype. To test the wound healing potential of ASCs, researchers wound healing, others can have an effect via paracrine signaling. In pa- employed a full-thickness excisional wound model in rats. Although pers by Garg et al and Kosaraju et al, pullulan-collagen hydrogels were wounds appeared to close at similar rates, histological analysis revealed created that increased bone marrow-derived mesenchymal progenitor that vascularization of the wound bed occurred earlier and to a higher cell (BM-MPC) recruitment to injury sites [301,302]. Pullulan is a non- degree in the ASC-FPEG treated group. A human specific mitochondrial immunogenic, hemocompatible, material comprised of polysaccha- antigen was used to determine if these differentiated ASCs played a di- rides. To test progenitor cell recruitment, Kosaraju et al used a parabio- rect role in wound healing and vascularization. Human cells were found sis model in which the vasculature of a GFP-expressing reporter mouse

Table 7 Adipose-derived stem cell therapies for enhancing wound angiogenesis.

Cell type Scaffold Treatment Result Exp. model Ref.

mASCs Pullulan-collagen hydrogel None ↑Recruitment of BM-MPCs Mice [302] ↑Provasculogenic gene expression hASCs Pullulan-collagen hydrogel Capillary Seeding ↑Wound healing speed Mice [301] ↑Vascularity hASCs PEG-fibrin gel None ↑Remodeling speed Rats [300] ↑Vessel formation pASCs Integra collagen scaffold None ↑Vascular density Pigs [330] ↑Number of mature vessels rASCs Cell sheet Ascorbic acid ↑Wound closure speed Rats [331] ↑Vessel density hASCs None Low-Level Light Therapy ↑VEGF and HIF-1α secretion, Mice [332] ↑Cell survival ↑Perfusion recovery rASCs ncl-PADM None ↑Elastic modulus of graft Rats [333] ↑Cellular and vascular infiltration 116 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 and a wild-type mouse were connected. This model allows for the trans- as opposed to fibrosis [303–306]. In one study, researchers used a full- fer of cells between mice to be observed. Two dorsal excisional wounds thickness cutaneous wound model in rats to study the wound healing were created on the backs of the wild-type mice. These wounds were potential of rat MSCs [307]. The cells were treated with dexamethasone treated with ASC-seeded hydrogels, an injection of ASCs in PBS, or a and ascorbic acid (vitamin C) and cultured to create a tightly packed, PBS control injection. Cells from the unwounded, GFP expressing, mice confluent, sheet of cells. After 12 days, the cell-aggregate layer began then migrated to the injury sites on the WT mice. BM-MPCs comprised to detach from the culture surface and was used in place of a scaffold of 23%, 8.4%, and 2.1% of the total cells recruited to the injury site in ASC for therapeutic delivery. Evaluation of gene expression showed that hydrogel implant, ASC injection, and PBS control treatments respec- pro-wound healing markers collagen 1 (COL1) and transforming tively. Furthermore, a subpopulation of these BM-MPCs was further growth factor beta (TGF-β)weresignificantly elevated in cells that identified using single cell gene expression profile analysis. This popula- grew in the aggregate form, suggesting superior wound healing poten- tion included the expression of known provasculogenic and remodeling tial. A full thickness cutaneous wound model was performed in rats to genes such as angiogenin (ANG), endosialin (CD248), NANOG, NFKB1, test wound healing. The cell aggregate was placed on the wound bed stem cell antigen-1 (SCA1), and extracellular superoxide dismutase with three layers of sterilized porcine small intestine submucosa to pro- (SOD3). This enhanced wound healing gene profile was expressed by tect the cells from the wound dressing. Wound size after four weeks was a greater amount of BM-MPCs for the ASC hydrogel condition compared significantly smaller (~50% smaller) for the aggregate group compared to ASC injection and PBS control. to the topically delivered cell suspension and intravenously delivered Conditioned media was collected from ASCs cultured on hydrogels cells. Furthermore, capillary density was found to be greatest in the ag- or ASCs cultured using glass or plastic cell culture surfaces[302]. Condi- gregate group. tioned media harvested from hydrogel-cultured ASCs significantly in- Inflammation was assessed for each group through RT-PCR and im- creased migration and proliferation in isolated BM-MPCs. Expression munofluorescence staining. After week two, gene expression showed of genes relating to wound healing, cellular recruitment, and neo- significantly lower TNF-α and IL-1β levels compared to controls. Addi- vascular genes including chemokine ligand 2 (CCL2), matrix tionally, cell aggregate groups showed lower inflammatory marker metalloproteinase-3 (MMP3), hepatocyte growth factor (HGF), hypoxia levels compared to the other two treatment groups. Gene expression inducible factor 1α (HIF1α), and endoglin (ENG) was significantly ele- for inducible nitric oxide synthase (iNOS) was found to be higher for vated in BM-MPC cultures with hydrogel cultured conditioned media the aggregate group compared to both the control and other treatment compared to control media. In addition, they observed increased pro- groups. Taken together, these results indicate a lower level of inflamma- tein levels of HGF and MMP3. Conditioned media from ASCs grown in tion and a pro-healing wound environment. Furthermore, the presence hydrogels increased the tube formation by BM-MPCs in comparison to of CD45+ lymphocytes was reduced and BM-MSC engraftment was im- control groups. Overall, this study showed that ASCs grown on this proved at the wound bed after two weeks. This study did not show that pullulan-collagen scaffold enhanced angiogenesis through paracrine MSCs were directly involved in the formation of new vasculature, in- mechanisms and increased recruitment of circulating BM-MPCs to the stead the authors attribute the improved wound healing to inflamma- wound area. tory regulation. Furthermore, this work represents a departure from a substantial portion of tissue engineering research which utilizes an in- dependent scaffold for cell survival and engraftment by achieving a pos- 5.3. Bone marrow derived mesenchymal stem cells itive effect with a relatively simple cell aggregate approach. This method avoids issues that are normally considered with synthetic scaffolds such Bone marrow derived mesenchymal stem cells (MSCs) are also as degradation/absorption, cytotoxicity, and immune response. While promising candidates as a cellular therapy for enhancing wound healing this technique proved effective in dermal wound healing in this work, (Table 8). MSCs have been found to modulate inflammation at wound further investigation has opened the door to other wound healing sites as well as influence surrounding cells to encourage regeneration arenas such as cardiac tissue repair, with areas soon to follow. [308,309]

Table 8 Cell-based therapies for enhancing wound angiogenesis.

Cell Type Scaffold Treatment Result Ref.

rBM-MSCs Cell Aggregate Matrix Dexamethasone/ Ascorbic Acid ↑ Wound closure [307] ↑ Capillary density ↓ Inflammation mBM-MSCs PEG-PU None ↑ Anti-inflammatory cytokines [310] ↑ Wound closure ↓ Oxidative stress ↓ Pro-inflammatory cytokines ↓ ROS at wound site rBM-MSCs Graphene Foam None ↑ Wound closure [334] ↑ Neo-vascularization ↑ VEGF and FGF-2 mRNA ↓ Fibrotic scar formation hiPSCs HA Hydrogel VEGF and ↑ Cellular integration into vasculature [335] SB-431542 hiPSCs None VEGF, BMP-4, and FGF-2 ↑ Tubule formation [313] ↑ Vascular density ↑ Wound closure PMSCs None None ↑ Wound closure ↑ Vascular density ↑ Cellular integration ↑Integrin β1, Integrin β3, VEGF ↓ICAM SKPs Collagen Sponge None ↑ Capillary regeneration [336] Monocytes iMonogrid Gel Monocytes treated toward M2 phenotype ↑ Vascular density [337] A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 117

While MSCs have been shown to act as modulators of immune re- embedded in the scaffold. By day 7, the MSC-scaffold group appeared sponse, they are still susceptible to the potentially cytotoxic environ- to have the greatest level of wound closure, with approximately five ment of the wound bed. Geesala et al sought to compliment the anti- times the number cells at the site of injury than the control. Significant inflammatory, pro-regenerative, function of MSCs by including a scaf- upregulation of pro-inflammatory cytokine gene expression was ob- fold composed of polyethylene glycol and polyurethane (PEG-PU) served in the MSC-only group compared to both the scaffold-only and [310]. Reactive oxygen species can impair stem cell function and reduce MSC-scaffold groups, indicating that the scaffold may reduce inflamma- wound healing [311,312]. This group aimed to modulate ROS levels and tion and could protect transplanted cells an immune response. The increase the efficacy of their cellular treatment through the combined presence of ROS in the tissue was evaluated using oxidation sensitive activity of MSCs and a PEG-PU scaffold. The PEG-PU scaffold was synthe- dihydroethidium dye staining. These results indicated that the sized using castor oil, a substance that contains elevated levels of vita- scaffold-only and MSC-scaffold groups had the least ROS activity while min E. In vehicle control studies, vitamin E has shown to increase the the MSC-only group had the greatest. Consistent with these findings, activity of the antioxidant enzyme, Cu/ZnSOD. Preliminary work was gene expression for the antioxidant enzymes catalase and GPX2, a glu- performed to create the interpenetrating polymer network, test scaffold tathione peroxidase, was significantly elevated for the MSC-scaffold biocompatibility, and to ensure cellular migration into and out of the group compared to free MSCs. scaffold. The protective role of the scaffold was assessed by varying con- To test successful engraftment at the wound site, MSC-indicative centrations of hydrogen peroxide to induce oxidative stress in cells that fluorescent markers including CD133-PE and CD90.2-APC were used were cultured either with or without scaffolds. Each of the cell-only on tissue samples. The MSC-scaffold group showed the largest amount groups exhibited a dose dependent drop in proliferation based on an of fluorescence at days 7 and 10, suggesting better engraftment was MTT assay. All scaffold groups showed increased proliferation compared achieved with the scaffold. The MSC-scaffold group sections contained to the cell-only groups when they were MSCs treated with 1 μMand10 a significantly greater amount of PECAM-1 stained cells compared to

μMH2O2. Hoechst staining showed markedly fewer apoptotic nuclei be- the scaffold and MSC-only groups. In addition, there was increased tween the scaffold and non-scaffold groups. gene expression for angiogenesis related genes including those for The scaffolds were then tested in a full-thickness excisional wound VEGFR2, VEGFR3, and Tie-2 from tissues harvested from the wound model in C57BL/6J mice. The study consisted of four treatment groups site. Overall, the study demonstrates a novel and potentially effective including an untreated control, scaffold alone, free MSCs, and MSCs strategy for combining cellular therapeutics with hydrogel scaffolds to

Fig. 7. The angiogenic potential of hiPSC-derived and hESC-derived ECs (red) and vSMCs (green) to HUVEC/vSMC controls in a co-culture tube formation assay were compared. Equal numbers of cells across each group were cultured on Matrigel and allowed to form tubules overnight. Multiple EC to vSMC ratios were tested across all groups including 100:0, 60:40 and 40:60. (A) Fluorescent microscopy images of the tube formation assay. (B) Quantification of the tube area for the cells in the tube formation assay. *p b 0.05 versus 100:0 HUVEC group. Scale bar = 100 μm. Used with permission [313]. 118 A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 improve the inhospitable area of the wound site to allow for expedited demonstrating expedited closure but also engraftment of PMSCs into healing. the functional tissues at the wound bed. With the relative ease of har- vesting this type of stem cell, PMSCs could seemingly see wider use in 5.4. Induced pluripotent stem cells similar studies and applications in the future. Skin progenitor cells have been explored as a therapy for wound Induced pluripotent stem cells (iPSCs) have recently received signif- healing and angiogenesis [318,319]. Ke et al. sought to examine the ef- icant attention as a cell type for improving angiogenesis and wound fects of skin-derived precursor cells (SKPs) on diabetic wound healing healing. Kim et al differentiated iPSCs into separate populations of endo- in T2D mice. They hypothesized that SKPs could aid in the healing pro- thelial cells and vSMCs [313]. They compared the angiogenic capability cess of chronic wound disease states by improving vascularization in the of these iPSC-derived vascular cells to primary somatic cells of the same area via differentiation and integration of SKPs. A collagen sponge was type. They first performed a co-culture tube formation assay using vas- used as a scaffold to aid the integration of SKPs into the dorsal, full- cular cells differentiated from iPSCs, cells differentiated from ECs, and thickness, skin wound of diabetic mice. Greater wound closure was ob- HUVECs/hvSMCs at varying ratios. Both differentiated cell groups exhib- served for the SKP/collagen as well as the collagen only groups com- ited cooperation between ECs and vSMCs and greater tubule area compared to the saline control at day 7 but slight difference was observed pared to the HUVEC/hvSMC group (Fig. 7A, B). They then injected the between the groups at day 14. Furthermore, greater capillary densities cell groups subcutaneously into athymic nu/nu mice suspended in a were observed in the SKP/collagen group via αSMA and isolectin stain- mixture of Matrigel and collagen I. After two weeks, they found in- ing of histology at the wound bed. The authors also observed potential creased perfused vessel area and vascular density in the groups treated neuronal regeneration alongside vascular formation through the addi- with the iPSC-derived cells in comparison to groups treated with the gel tion of nestin staining. SKPs appear to have a vast potential in the alone or somatic endothelial cell/vSMCs in the gel. Through proteomic wound healing arena as they can differentiate towards three crucial profiling of the secreted factors they found that iPSC-derived vascular would heal cell lineages (vSMCs, ECs, and neurons). Paired with an ef- cell produced more pro-angiogenic soluble factors. They also implanted fective scaffold, difficulties involving engraftment and cell survival can the cells in a dermal wound model in nu/nu mice and found that the be mitigated and the effect of SKPs can be enhanced as seen in the pre- iPSC-derived vascular cells were superior in their ability to induce ciously discussed article. Altogether, these cells present another promis- wound healing and angiogenesis in the wound bed. Lee et al found ing option for an easily sourced adult stem cell therapy for wound that iPSC-derived lymphatic endothelial cells also enhanced wound healing. healing and vascular growth [314]. Using ear and dorsal wound models in nu/nu mice, they observed incorporation of the cells into the lymphatic vessel network and a rise in lymphvasculogenesis and 6. Conclusion lymphangiogenesis. In addition, the iPSC-derived lymphatic endothelial cells induced enhance wound healing in comparison to control (PBS) or The development of effective therapeutics for enhancing wound an- human primary lymphatic endothelial cell-treated wounds. Zhang et al giogenesis, particularly for non-healing or complex wounds, requires also tested the ability of exosomes produced by iPSC-derived MSCs to the ability of the compound to work in the harsh environment of a induced enhanced wound healing and angiogenesis [315]. After poorly healing wound. As discussed in this review, many compounds confirming the activity of the exosomes through proliferation/migration have shown great promise in enhancing wound healing and angiogen- and tube formation assays, exosomes were subcutaneously introduced esis in animal models of wound healing. In the past, many wound in a dorsal cutaneous injury model in rats. Wound closure after two healing treatments that have been successful in preclinical models ulti- weeks was significantly higher for the exosome-treated group com- mately fail to provide benefits to patients when tested in clinical trials. pared to media controls and they observed an increase in collagen and Fundamentally, the field suffers from the lack of a validated preclinical elastin deposition. Furthermore, wound epithelialization and vessel animal model of chronic wound healing that correlates well with density were significantly increased while scar width was significantly human wound healing in the clinic. While many approaches have decreased. Overall, these works support that iPSC-derived cells have po- been used to reduce wound healing in animal models, there is no con- tential for enhancing wound healing and angiogenesis. sensus as to the model with the best correlation to compromised human wound healing [320]. A change in approach and experimental 5.5. Other stem cell populations mindset is needed in how studies are designed for assessing promising wound therapies. In vitro studies should be targeted not solely at A number of other stem cell types have also been investigated in assessing activity in healthy cells but cells either from diseased patients wound healing and angiogenesis. Placenta-derived mesenchymal stem or in combination with treatments that inhibit the angiogenic or wound cells (PMSCs) wound models have been studied in both rats and mice healing response. The baseline assumption should be one of compro- [316,317]. Kong et al. employed a diabetic, Goto-Kakizaki, rat model mised response and therapeutic resistance, rather than healthy healing. with full thickness dorsal skin wounds. Fluorescently labeled PMSCs New treatments should be designed or evaluated in the context of their were injected intradermally at the wound site and monitored until all ability work in compromised wound environments. wounds closed. Compared to non-treated controls, PMSC-treated Ultimately, effective wound healing is a temporal process that can wounds demonstrated expedited wound closure as well as increased potentially be compromised by disease or infection in a number of vascular density as the wound site. Furthermore, PMSCs were con- ways. In diabetic wound healing, studies have identified many firmed to incorporate into the host vascular network visualized through compromising factors for wound healing including the loss of heparan immunofluorescence histology. Similarly, Abd-Allah et al. employed a sulfate proteoglycans [47,48,54], alterations in inflammatory response skin wound model in wild type mice for 7 and 12 days. Like the previous [321], increased proteolysis[322] and alterations in ROS signaling experiment, PMSCs delivered either intraperitoneally or intraregionally [323], to name only a few. It is difficult to imagine therapy with any sin- induced more rapid healing when compared to the untreated group. gle agent to be able to address more than a few of these factors, pointing Additionally, RT-PCR detected higher expression of angiogenic and to the need for combination therapies that address the issues of a pa- wound healing factors including Integrin β1, Integrin β3, and a decrease tient’s particular wound and diagnostic assays to delineate when a par- in ICAM expression. RT-PCR was also used to detect the presence of ticular compound should be used. In this context, the biology and human cells in the form of albumin and GAPDH expression. An ELISA compromised mechanisms of wound healing must be factored into also revealed greater VEGF in the PMSC-treated tissues. Both of these the design and development of treatments and delivery platforms for studies lend merit to PMSCs as wound healing agents by not only addressing chronic wounds. A.P. Veith et al. / Advanced Drug Delivery Reviews 146 (2019) 97–125 119

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