miRNA delivery for skin wound healing

Accepted Manuscript miRNA delivery for skin wound healing

Zhao Meng, Dezhong Zhou, Yongsheng Gao, Ming Zeng, Wenxin Wang PII: DOI: Reference:

S0169-409X(17)30314-9 https://doi.org/10.1016/j.addr.2017.12.011 ADR 13229

To appear in:

Advanced Drug Delivery Reviews

Received date: Revised date: Accepted date:

29 September 2017 24 November 2017 16 December 2017

Please cite this article as: Zhao Meng, Dezhong Zhou, Yongsheng Gao, Ming Zeng, Wenxin Wang , miRNA delivery for skin wound healing. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Adr(2017), https://doi.org/10.1016/j.addr.2017.12.011

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ACCEPTED MANUSCRIPT

miRNA delivery for skin wound healing Zhao Meng, Dezhong Zhou*, Yongsheng Gao, Ming Zeng, Wenxin Wang* Charles Institute of Dermatology, School of Medicine, University College Dublin, Dublin

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4, Ireland Corresponding author: Charles Institute of Dermatology, School of Medicine, University

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College Dublin, Dublin 4, Ireland, E-mail address: [email protected] (D. Zhou);

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[email protected] (W. Wang)

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Graphical Abstract:

Abstract: The wound healing has remained a worldwide challenge as one of significant public health problems. Pathological scars and chronic wounds caused by injury, aging or diabetes lead to impaired tissue repair and regeneration. Due to the unique biological wound environment, the wound healing is a highly complicated process, efficient and targeted

ACCEPTED MANUSCRIPT treatments are still lacking. Hence, research-driven to discover more efficient therapeutics is a highly urgent demand. Recently, the research results have revealed that microRNA (miRNA) is a promising tool in therapeutic and diagnostic fields because miRNA is an essential regulator in cellular physiology and pathology. Therefore, new technologies for wound healing based on miRNA have been developed and miRNA delivery has become a significant

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research topic in the field of gene delivery.

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Keywords: cutaneous wound healing, scarring, microRNA, non-viral delivery

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Contents

1. Introduction ........................................................................................................... 1

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2. Wound repair across phylogeny .......................................................................... 2 2.1 The formation of a scar ................................................................................. 2 2.2 Wound repair process .................................................................................... 2

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3. Biogenesis and function of miRNAs .................................................................... 6

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3.1 Biogenesis of miRNA ................................................................................... 6 3.2 Different types of miRNA invovled in wound healing ................................. 8

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4. Development of non-viral miRNA delivery systems ........................................ 11 5. Wound healing based on miRNA delivery ........................................................ 16

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6. Conclusion ........................................................................................................... 21 Acknowledgements ...................................................................................................... 22 References .................................................................................................................... 22

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1.

Introduction

The impaired wound healing, including chronic wound and pathological scars, is a worldwide issue, which causes heavy economic and healthcare burden. Especially with the increasing incidence of diabetes and lengthening of life expectancy [1], chronic wounds which often occur in the elderly people and diabetic patients may cause significant disability even

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increased mortality [2-4]. People with chronic wounds like diabetic foot ulcers, venous ulcers,

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and pressure ulcers are always subject to both physical and mental sufferings. The nature of chronic wounds including pain, disability, and compromised looks also often brings about

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inferiority, depression even social isolation [5]. It is reported that more than 6.5 million

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people in the U.S. are suffering from chronic, non-healing wounds resulting in heavy economic burden, over 25 billion dollars annually [6]. While in Europe, this number could

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take 2-4% of total healthcare expense [7], and it will continuously grow with population aging and increasing prevalence of diabetes and other diseases [6]. The immense economic and social impact of wounds calls for enhancing the understanding of the biological mechanisms

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underlying cutaneous wound complications. Thus, treatment options for chronic wounds or

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scars which are both clinically effective and cost-effective are showing vital to us. However, efficient and targeted treatments for chronic wounds are still lacking, which, in part,

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is due to our limited understanding of the biological mechanisms underlying these diseases [8]. Therefore, to develop effective therapeutics for the treatment of impaired wound healings

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is crucial to understand the possible mechanisms that would promote skin regeneration and function restoration [9]. In addition, the complicated wound environment also increases the difficulty in finding proper therapeutics. For example, free radicals generated by oxidative stress may cause cellular damage even death, therefore, the identification of free radical scavengers (e. g. deferoxamine) would help to develop therapeutics for wound healing. Besides, enzymatic, hypoxia/ischemia, infected and necrotic tissues are all obstacles presented in wound environment that need to overcome [1]. Even if the most basic mechanical movements would lead it difficult to retain the delivered therapeutics locally, let alone the changing of dressings. Therefore, there is an urgent demand to maintain the efficacy 1

ACCEPTED MANUSCRIPT of therapeutics while overcoming the multiple obstacles of wound environment. 2.

Wound repair across phylogeny

2.1 The formation of a scar A wound is defined as all manner of tissue damage resulting in the disorder of the original tissue structure and homeostasis [10]. Skin is the largest organ of human body, accounting for

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approximately 15% of body weight in adults. Because it is directly exposed to air and plays a role in protecting human body, skin is also the most vulnerable and frequent organ to injury

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[11, 12]. After the injury, the human body will active multiple biological pathways that intend

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to restore the tissues and maintain skin integrity. This wound healing process may occur intracellularly, intercellularly and extracellularly simultaneously [10]. Many types of cell,

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including fibroblasts, endothelial cells, keratinocytes and immune cells, undergo marked changes in gene expression and phenotype, leading to cell proliferation, differentiation and

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migration [13]. In this process, a non-functioning mass of fibrotic tissues is produced to protect the wound and known as a scar. The dermal scars often bring inconvenience to normal

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life, such as function loss, movement restriction and disfigurement [14]. Likewise, scar

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formation that occurs during wound repair leads to similar tissue dysfunction wherever it takes place.

2.2 Wound repair process

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For most injuries, the scars usually consist of two parts, a patch of fibroblasts and a

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disorganized extracellular matrix which is mainly collagen [15]. The formation of scars is a complex and tightly regulated process. Studying wound repair in various phyla could improve our understanding of wound repair in humans and might help to identify the pathway that can be targeted to renovation capability of the skin. Generally speaking, it can be divided into three phases: inflammation, proliferation and remodeling [16-18] (Fig. 1). When skin gets injured, inflammatory cells are recruited to sites of the wounded area in response to injury, which is followed by the proliferation of fibroblasts and these cells are responsible for synthesizing various tissue components. The clotting mechanism will be mobilized in the human body first, and clotting factors are immediately 2

ACCEPTED MANUSCRIPT released to activate thrombocytes, which leads to the formation of blood clots with fibrin, vitronectin, fibronectin, and thrombospondin [19, 20]. Large amounts of cytokines and growth factors are also released to promote wound repair [21]. After 3 days, a large number of macrophages with different phenotypes will be gathered at the wound site [22]. During this phase, wound environment may be rather challenging, hypoxia, ischemia and infected or

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necrotic tissue all can affect wound repairing process. Coagulation cascade, inflammatory pathways and immune system are needed to prevent ongoing blood and fluid losses, to

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remove dead and devitalized (dying) tissues and to prevent infection [10]. Haemostasis is

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achieved by platelets and neutrophils are then recruited to the wound in response to injury. After that, during the proliferation phase, the wound matrix formed earlier will be gradually

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replaced by granulation tissue containing fibroblasts capillaries, and collagen bundles which also provides cells with the scaffold to migrate and grow [19]. The proliferation of

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keratinocytes is a vital step in this phase because it favors the re-epithelialization process, which also needs abundant oxygen and nutrient [16]. Keratinocytes migrated to the injuried

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dermis, and then restore the barrier function of the epithelium [10]. Then, rapid cellular

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proliferation occurs and new blood vessels (known as angiogenesis) and epithelium appear. Hereafter, fibroblasts differentiate into myofibroblasts to promote collagen deposition and wound contraction. So, the multiple aggregated cells gather to form a new tissue covering the

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impaired area, which forms a scar. However, the scar remodeling may take a few months. During this stage, all the processes activated after injury wind down. Most of the endothelial

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cells, macrophages and myofibroblasts undergo apoptosis. The wound contains little cells and only collagen and other extracellular-matrix proteins. Remodeling stage may last over 6-12 months, but the complete restoration would never be achieved because scarring is an irreversible process [23]. Usually, a scar can only restore 70 percent of the tensile strength of pre-injury skin, and the function of normal skin will never recover completely [24, 25].

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Fig. 1. | Three stages of wound healing: inflammation (a), proliferation (b), and remodeling

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(c). a), Inflammation. This process may last until 48 hours after injury. One of the characteristics of the wound in this period is that fibrin clots gradually formed in an

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ischaemic/ hypoxic environment. Bacteria, neutrophils, and platelets are abundant in the wound. b), Proliferation. This stage occurs about 2–10 days after injury. During this stage, an

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eschar has formed on the surface of the wound. Most cells have migrated from the wound

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and new blood vessels populate the impaired area. c), Remodeling. This stage may last one year or much longer. Disorganized collagen has been laid down by fibroblasts migrated into the wound. The repaired wound has contracted and is slightly higher than its surrounding

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surface, even the color of regenerated skin is different from the normal skin. Besides, the

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healed region does not contain normal skin appendages (such as hair follicles and sweat duct glands) [10]. Reused with permission, Copyright © 2008, Rights Managed by Nature Publishing Group.

To date, there are no reliable treatments to prevent scarring and millions of patients are suffering from the adverse physiological and psychological effects of scars. Gauze has been the most popular wound dressing for a long time. However, gauze would promote desiccation of the wound and make it susceptible to saturate wound fluid resulting in bacterial invasion [26, 27]. In contrast, wound care requires a warm and moist environment while minimizing 5

ACCEPTED MANUSCRIPT bacterial loading [28]. Furthermore, gauze dressings usually adhere to the wound so that patients always feel pain when removing the dressings. While films do not have such shortcomings, they are thin and semi-permeable, which allows the exchange of oxygen and water vapor. Sometimes antibiotic treatments are applied to wound dressings in order to accelerate wound healing, which may further cause microbial resistance and prolonged

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inflammation [29]. Over the last two decades, a great number of new dressings and drug delivery models have been developed for wound healing. For instance, a new kind of wound

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dressing containing nanovesicles was reported. This system can act as an indicator of

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infection, the existence of pathogenic bacteria would induce a color change and then antimicrobials release would be triggered [30]. Silver-based antimicrobials dressing have also

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been proven effective to facilitate wound decontamination [31].

Extensive studies over past two decades have provided insights into the function of miRNAs

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in a wide range of physiological and pathological conditions. The important roles of miRNAs in skin wound healing have also been revealed recently. Ghatak et al. reported that the

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expression of Dicer which is essiencial role in miRNA biogenesis was increased during the

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wound healing process [32], thus, a reasonable conclusion that miRNA are closely related to wound healing was came up with. Hence, miRNA therapies were introduced into the field of

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wound healing.

Biogenesis and function of miRNAs

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3.1 Biogenesis of miRNA

miRNAs are short endogenous non-coding RNAs with an average length of 22 nucleotides that mediate post-transcriptional regulation of gene expression [33, 34]. The human genome contains more than 500 miRNAs, and each can repress hundreds of genes [35]. miRNAs are involved in nearly all developmental and pathophysiological processes in animals as they target over one-half of protein-coding transcripts [36, 37]. miRNAs are initially transcribed by RNA polymerase II in the nucleus to form primary-miRNA (pri-miRNA) with the stem-loop structure [38-40]. The nuclear ribonuclease (RNase)-III enzymes, Drosha, cleaves pri-miRNA 6

ACCEPTED MANUSCRIPT into precursor-miRNAs (pre-miRNA) [41]. Drosha is a nonspecific RNase that is used to mediate the genesis of miRNAs from the pri-miRNA and DGCR8 is a cofactor to help form protein complex [37]. The resulting pre-miRNAs are then exported to the cytoplasm through the nuclear export factor exportin-5 [40]. Another RNase III enzyme, Dicer, then cleaves the pre-miRNAs into 18 to 24 nt double-stranded RNAs which become miRNA duplex after

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maturation [39]. After cleavage by Dicer, the result 21 to 24 nt miRNA duplex needs to release one of the strands to enter Argonaute for function [42, 43]. Most miRNAs are

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incorporated into the RNA-induced silencing complex (RISC) to form miRNA-containing

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RISC [44]. Usually, one strand, also known as passenger strand disappears, whereas the other strand, termed as guide strand becomes mature miRNA. Passenger strands are subject to rapid

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degradation, however, recent studies have confirmed that these strands could also be loaded on Argonaute and show repression to target mRNAs [43, 45]. They achieve the purpose of

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regulating gene expression by promoting mRNAs degradation and inhibiting mRNAs

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translation [16]. (Fig. 2)

Fig. 2 | The biogenesis of miRNA begins with the transcription of miRNA to pri-miRNA in the nucleus. The pri-miRNA is cleaved by the Drosha/DGCR8 complex to form pre-miRNA, which can then be exported from the nucleus to the cytoplasm by exportin 5 (XPO5). In the 7

ACCEPTED MANUSCRIPT cytoplasm, pre-miRNA is further processed into the mature miRNA duplex by Dicer. One strand (usually the guide strand) of the mature miRNA forms a complex with Dicer and the Argonaute protein, known as the miRNA-containing RISC, where miRNA binds to the 30 UTR of its target mRNA, resulting in the degradation or suppression of the translation of the

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target mRNA [46].

miRNA pairs to the RNA-induced silencing complex and binds to the 3’ untranslated region

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of target mRNA and then repress the translation process and even cause the degradation of

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mRNA [47]. Most protein-coding genes are regulated by miRNAs through this way [48]. Recently, it is reported that miRNAs play key regulatory roles in the diverse biological

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process and are observed dysregulated in many human diseases like cancer, cardiovascular disease and hepatitis C virus (HCV) infection [49]. miRNA-based gene therapy has gradually

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come into view since a single miRNA could regulate multiple targets so that it shows more promising than traditional drug therapy targeting merely single protein.

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3.2 Different types of miRNA invovled in wound healing

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Many reports have revealed the important role that miRNAs play in the wound healing process. Ghatak, S., et al. have demonstrated that in a wound healing model in mice, one of the enzymes involved increased, which indicates a close connection between miRNA and

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wound healing process [32]. A summary of miRNAs and their roles in wound healing is outlined in Table 1. In general, wound healing involves three stages, that is inflammation,

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proliferation, and remodeling. miRNA plays a key role in regulating the polarization of macrophages [50]. Several miRNAs have been identified to be critical at this stage. For example, the expression of miR-146a was observed increase in epidermal keratinocytes simulated by toll-like receptor 2 (TLR2), TLR3, TLR4 and TLR5 [51]. It is found that miR-146a regulated inflammatory response negatively in comparison with intact skin. The downregulation indicates miR-146a may facilitate inflammation resolution[51]. miR-155 is another important one for immune cells. At the inflammation stage, miR-155 is upregulated as verified in a mouse model which showed that treatment with miR-155-specific inhibitors can 8

ACCEPTED MANUSCRIPT effectively reduce the accumulation of the inflammatory cells at the wound site and thus improve the architecture of the regenerated tissues [52-54]. miR-132 plays an anti-inflammatory role in the process, which restricts the overproduction of pro-inflammatory cytokines [55, 56]. Shaked et al. found that inflammation may induce the expression of miR-132 in leukocytes [57] and Li et al. revealed the change of its expression level during the

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transition from inflammation phase to proliferation phase [56]. In addition, miR-21 is also proven to be critical in inflammation resolution [58]. Besides, some miRNAs are reported to

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be related to polarization regulation and inflammatory response such as miR-125b and

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miR-223 [59, 60].

miR-21 has been critical in not only inflammation phase but also proliferation phase. miR-21

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has been proven to promote migration of keratinocytes and fibroblasts [61, 62]. When miR-21 is inhibited, the re-epithelialization process would be delayed, and impaired wound

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contraction would also be suppressed [61]. miR-132 is another crucial one in both inflammation and proliferation. Li et al. found that miR-132 inhibition would lead to severe

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inflammation of wound site, decreased keratinocyte growth and prolong the duration of the

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wound closure [56]. These studies showed that miR-132 is not only an anti-inflammation factor but also a promoter of granulation tissue growth. As for miR-31, it is found to be substantially upregulated in keratinocytes of the impaired area, and studies have also

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suggested that miR-31 makes a great contribution to the migration and proliferation of keratinocytes and the re-epithelialization process [63]. Jin et al. reported that downregulation

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of miR-99 family resulted in promoting the migration and proliferation of keratinocytes, ultimately facilitating wound closure [64]. For hypoxia in ischemic chronic wounds, miR-210 is often found expressed at a high level, because miR-210 was able to repress the mitochondrial metabolism and decrease oxygen cost [65, 66]. Ghatak et al. encapsulated miR-210 inhibitors into lipid nanoparticles and injected them into the murine suffering from an ischemic wound, results showed that accelerated wound healing was achieved, which illustrates the promise of miR-210 in wound healing [67]. During the remodeling phase process, fibroblasts differentiate into myofibroblasts, collagen deposit and wound contract 9

ACCEPTED MANUSCRIPT [68]. It has been verified that miR-29a has a direct influence on the expression of collagen, miR-192, miR-29b, and miR-29c are all highly induced in this process [69-72].

Table 1. miRNAs in wound healing miRNA

Inflammation miR-146a

Function

Reference

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Phase

Suppresses excessive inflammatory response [51]

Regulates development and functions of [52-54]

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miR-155

immune cells

Anti-inflammation and decreases chemokine [55-57]

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miR-132

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in keratinocytes

production by keratinocytes [58]

miR-125b

Polarization regulation

[59]

miR-223

Polarization regulation

[60]

miR-21

Promotes migration of keratinocytes and [61, 62]

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Inhibites inflammatory response

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proliferation

miR-21

fibroblasts;

overexpression

of

miR-21

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inhibits re-epithelialization and granulation

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miR-132

miR-31

tissue formation Promotes

keratinocyte

growth

and [56]

angiogenesis Promotes proliferation and migration of [63] keratinocytes

miR-99

Suppresses

family

proliferation

miR-210

Suppresses

keratinocyte

migration

keratinocyte

and [64]

proliferation, [65-67]

promotes angiogenesis Remodeling

miR-29a

Improves collagen expression 10

[69]

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miR-29b

Improves ECM remodeling

[70]

miR-29c

Improves ECM remodeling

[71]

miR-192

Improves ECM remodeling

[72]

Development of non-viral miRNA delivery systems

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Currently, a variety of gene delivery systems have been developed and viral vectors are the most efficient ones [16, 73-92]. However, viral vectors are usually associated with

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disadvantages, such as mutagenesis, toxicity, and limited capacity for genetic cargo [93], and

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efficient delivery of miRNA for therapeutic purposes still remains challenging. Low cellular uptake of RNA (attributed to its high molecular weight and negative charge), degradation in

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the bloodstream, and rapid renal clearance are the most significant obstacles that hinder the successful delivery of miRNA [94]. Thus, development of safe and efficient miRNA delivery

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systems are of great significance [46]. Alternatively, non-viral delivery systems have gradually become the new focus of research [84, 87, 90, 91, 95]. Different from viral vehicles,

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non-viral miRNA delivery systems are characterized by lower toxicity and immunogenicity,

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increased cellular uptake, water solubility, resistance to endonucleases, and phagocytosis [96, 97]. Diverse cationic polymers, liposomes, and peptides exhibit the capability to package genetic cargos and deliver them into the nucleus of cells [98]. Natural and synthetic lipids,

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polymers such as phosphatidylcholine, PLGA, and chitosan are the most common nanocarriers for miRNA delivery, these vectors can undergo biodegradation into products

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which would be absorbed by the natural biochemical pathways of the body [99]. An ideal miRNA delivery vector should: 1) first be positively charged considering that miRNA molecules are negatively charged [99]; 2) be stable in the circulation, carry the miRNA to arrive at the target site, promote cellular uptake, avoid lysosomal degradation and facilitate endosomal escape [100]; 3) can avoid rapid clearance by the reticuloendothelial system and enhance circulation time when injected into the bloodstream, therefore, the outer surfaces of the carriers are often modified with hydrophilic components such polyethylene glycol [99]. Other considerations that need to be factored in when designing a delivery system include the 11

ACCEPTED MANUSCRIPT knowledge of the tissue architecture, the microenvironment of the wound site, and therapeutically meaningful doses that are required for efficient miRNA inhibition [99]. Generally, non-viral miRNA delivery could be divided into three categories according to the nature of the strategies: complexation, conjugation, and encapsulation [46]. Complexation strategy refers to the process that negatively charged miRNA is condensed by the positively

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charged carriers to form polyplexes via electrostatic interaction. Liposomes which are composed of the bilayer phospholipid membrane are most widely used for miRNA

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complexation. In the conjugation strategy, miRNA binds to vectors covalently by linkers,

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which results in a highly stable delivery system able to provide miRNA with robust protection in the bloodstream circulation. The encapsulation strategy is to put miRNA into biodegradable

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nanoparticles such as poly(lactic-co-glycolic acid) (PLGA) or silica nanoparticles to deliver it to target sites. Compared to the other two methods, this one shows great advantages in terms

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of safety since it does not require use of any potentially toxic cationic materials [46, 99]. In addition, in order to confer nuclease resistance and increase the binding affinity of

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anti-miRNA oligonucleotides to their cognate miRNAs [101], three chemical modification

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methods have also been developed: i) 2’-O-methyl-group-modified oligonucleotides (2’-Ome-RNA); ii) 2’-methoxyethyl-modified oligonucleotides (2’-MOE-RNA); iii) Locked

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nucleic acid (LNA) (Fig. 3) [102].

Fig. 3 | Chemically modified oligonucleotides analogs that have been used in non-viral miRNA delivery system [102]. Reused with permission, Copyright © 2005, Rights Managed 12

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Chemical modification of miRNA molecule can protect naked miRNA from renal clearance and degradation by RNase in circulation. However, some downsides including off-target effects, reduced activity, and toxicity would be triggered [103]. In order to prolong the

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circulation half-life of miRNA, protect it from degradation, and reduce reticule-endothelial system (RES) uptake, water-soluble polymers such as poly(ethylene glycol) (PEG) are usually

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used as shielding agents [104, 105]. In addition, similar to that of DNA, delivery systems that

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are more efficient to release the miRNA mimics or anti-miRNAs from endosomes will further

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improve therapeutic potential.

Fig. 4 | miRNA main delivery systems. Several vehicles from different categories can be used

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for miRNA-based delivery: lipid vesicles (complexation), polymeric nanoparticles (encapsulation),

dendrimers

(complexation),

gold

nanoparticles

(complexation

or

conjugation), and cationic polymeric polyplexes [46]. Reused with permission, Copyright © 2013, Controlled Release Society.

The specific characteristics of miRNA delivery strategies dictate the biocompatibility, targeting capability, specificity, intracellular trafficking, and release or activation mechanism of each system. Various miRNA delivery systems that are currently widely used are illustrated 13

ACCEPTED MANUSCRIPT in Fig. 4 [46], and the specific miRNAs that are delivered are outlined in Table 2. Lipid vesicles are composed of bilayer phospholipid membrane encasing a water compartment can form the most common sort of non-viral RNA vector liposomes. In miRNA delivery, lipid vesicles are usually modified chemically with targeting moieties or coated with PEG, HA to avoid immune system recognition and reticuloendothelial system (RES) uptake

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[46, 106, 107]. Lipid vehicles are good vector candidates for miRNA complexation via the electrostatic interaction between the negatively charged miRNA and the positively charged

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vehicles. miRNA delivery system based on lipid vesicles show advantages in many aspects

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such as easy to the formation and being able to interact with negatively charged biological membranes. However, shortcomings also exist, and the major one is high cytotoxicity. Even

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though, Wu et al. [108, 109] used a cationic lipoplex-based system to deliver miRNA and much higher transfection efficiency was achieved compared to the standard commercial

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miRNA delivery reagents (siPORT NeoFX transfection agent, Ambion) both in vivo and in vitro. Zhang et al. [110] found that the use of lactobionic acid to partially reduce the positive

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surface charge of the liposomes can lower the toxicity of lipid vesicles for miRNA delivery.

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These results indicate that lipid vesicles remain a promising miRNA delivery system as far as the safety issue is solved.

Due to their high flexibility and adaptability, polymers, either natural or synthetic, are

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considered as another type of candidate materials for miRNA delivery. Polymers can be easily tailored in size and charge to maximize the payload potential for nucleic acid [46].

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Traditionally, Polyethylenimine (PEI) is the most frequently used synthetic cationic polymer for nucleic acid delivery. The high charge density of PEI enables formulation of stable complexes with miRNA, the multiple tertiary amines in PEI greatly facilitates miRNA carrying polyplexes escape from endosomes via the proton sponge effect [111]. However, it has also been widely reported that PEI is not an ideal gene delivery vector because of its non-biodegradability and high cytotoxicity [112, 113]. Alternatively, cyclodextrins (CD) has attracted much attentions in gene delivery. As a sort of natural cyclic oligosaccharides, the CD is proven to be nontoxic and nonimmunogenic [114], these characteristics make CD widely 14

ACCEPTED MANUSCRIPT used to reduce cytotoxicity of polymers like PEI in gene delivery via covalent conjugation modification. CD-grafted PEI was synthesized to impart the advantageous properties associated with CD (low toxicity, high water solubility, inclusion complex formation ability) to PEI. After a series of experiments, it is realized that the best CD-bPEI (branched PEI) for plasmid delivery was determined to have 8% PEI amine grafting, while, the best CD-lPEI

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(linear PEI) was synthesized with 12% grafting.When these best CD-PEI polymers were tested for their ability to deliver plasmids to PC3 cells, CD-PEI polymers deliver plasmids

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with higher efficiency than the parent PEI polymers (75% CD-lPEI vs 40% for lPEI and 70%

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for CD-bPEI vs 30% for bPEI). Of significance is the observation that the PEGylated CD-PEI particles can provide good transfection in the absence of chloroquine. After tail vein injection

solutions and better transfection efficiencies.

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in mice, this formulation was confirmed lower toxicity, higher stability in physiologic salt

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Dendrimers are another important potential carriers for miRNA delivery. Dendrimers are synthetic polymers with a unique three-dimensional (3D) structure exhibiting a branched

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spherical morphology with multiple functional end groups at the surface. Dendrimers with

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cationic amine periphery groups are the most often used ones to complex with negatively charged miRNA. Cationic dendrimers usually show high gene transfection efficacy which is attributed to their high buffering capacity facilitating the endosomal escape of polyplexes

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[115]. Ren et al. [116] reported a dendrimer gene vector poly(amidoamine) (PAMAM), which was used to load with antimetabolite 5-fluorouracil (5-FU), a chemotherapeutic drug, and

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anti-miR-21 simultaneously, high gene transfection efficiency was achieved in vitro by this co-delivery system.

Because of the advantageous surface characteristics and relatively low toxicity, gold nanoparticles have received much attention as an emerging miRNA delivery vector [117]. Gold nanoparticles allow for easy functionalization with various chemical and biological molecules, such as thiol and amino groups. Experiments have repeatedly shown that gold nanoparticles are noncytotoxic and nonimmunogenic [118]. Ghosh et al. developed amino-functionalized gold nanoparticles for miRNA delivery. In this system, gold 15

ACCEPTED MANUSCRIPT nanoparticles firstly complexed with miRNA and then PEG was coated. Results demonstrate that this miRNA delivery system was noncytotoxic, able to take up by cells through endocytosis with high efficiency and release the complexed miRNA at the target site in time [119]. Besides, poly(lactic-co-glycolic acid) (PLGA) nanoparticles are also widely used for miRNA delivery. PLGA is a type of biocompatible and biodegradable polymers synthesized

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by copolymerization of lactic acid and glycolic acid [46]. After decorating with cell-penetrating peptide or penetratin, intracellular delivery of miRNA can be further

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enhanced by PLGA nanoparticles [120].

Table 2.

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Various miRNA delivery strategies and vector systems Delivery system

miRNA

Complexation

Cationic lipoplex

miR-29b mimic

Liposomes

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dendrimers

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Lipid nanoparticles

miRNA type Reference

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Category

[108]

miR-132 Anti-miRNA

[121]

miR-155 Anti-miRNA

[122]

miR-21

[116]

Anti-miRNA

miR-210 Anti-miRNA

[67]

Encapsulation PLGA nanoparticle

miR-155 Anti-miRNA

[120, 123]

Silica nanoparticles

miR-34a

mimic

[124]

Gold nanoparticles

miR-29

Anti-miRNA

[125]

Magnetic nanoparticle miR-221 Anti-miRNA

[126]

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Conjugation

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Lipid nanoparticles

5.

Wound healing based on miRNA delivery

Since miRNAs have been identified as promising mediators of wound healing, they are attractive candidates for a broad set of novel therapeutic strategies [96]. The greatest advantage of miRNA therapy is that the miRNA mimics or anti-miRNAs inside the cells could exert their functions even when they are absent from plasma due to the high biological half-lives [127] [128]. 16

ACCEPTED MANUSCRIPT It has been confirmed that many miRNAs expression in the wound healing process is dysregulated, especially in chronic wound healing [40]. In a recent controlled trial in a diabetic rat model, researchers found that among the 83 diabetes-related miRNAs, 18 were upregulated and 63 were downregulated, which further confirms the potential of miRNA for wound healing [129]. Studies have repeatedly pointed out that therapeutics targeting miRNAs

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represent the frontier in the treatment of various diseases that will ultimately lead to chronic wounds such as diabetes and a series of diabetes-associated complications [37]. Chronic

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diabetic wound which results in high mortality and amputation rate has become one of the

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most challenging topics in wound healing [130, 131]. Several RNAs based therapeutics are under development including miRNA mimetics and inhibitors, siRNAs, and antisense

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oligonucleotide [37, 132]. Among these, therapeutics targeting of miRNAs have shown capability to regulate gene networks, therefore, effective delivery of miRNA mimics or

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inhibitors to microvasculature to facilitate cellular signaling of the injured tissues would enhance the wound healing [37]. It is supposed that miRNA therapy could be used for

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diagnosis and prognosis of diabetes-associated complications given the ability of miRNAs

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circulating in exosomes and microvesicles [37]. For example, it was reported after injury, the miR-200b expression in the wound tissue was decreased while angiogenesis was switched on [133]. In diabetic wound of rat models, miR-15b and miR-27b were observed to play critical

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roles in angiogenesis and can accelerate the wound closure significantly [134, 135]. In addition, miRNA therapy has the ability to target multiple genes of a pathway at the same

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time, that plasma levels of an anti-miRNA or miRNA-mimetic can be cleared from plasma within hours by uptake into tissues [127, 136]. Many types of non-viral miRNA delivery systems have been proved efficient in wound healing process. For example, miR-126 is involved in an important stage in wound healing, angiogenesis in vitro. When it is loaded to polyethylene glycol-modified liposomes and is delivered to an ischemic fibrosis model, the angiogenic factor, vascular growth factor (VEGF), is inducted and the blood flow is improved, which helps promote angiogenesis and then wound healing [137]. 17

ACCEPTED MANUSCRIPT However, miRNA therapies still have challenges in effective targeting and delivery. Chemically modified antisense microRNA inhibitors, termed antimiRs, or miRNA mimics can facilitate the modulation of miRNAs, however, naked antimiRs are not stable as predicted, they are easily degraded under endogenous circulation and RNases, so antimiRs protection is necessary in order to enhance target stability, affinity and tissue uptake efficiency [138-140].

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What’s more, one of the major challenges associated with miRNA mimics replacement technology include the ability to target miRNA to a specific cell type and the potential

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requirement of multiple doses to achieve sustained target repression[37].To overcome these

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limitations, Mordorski et al. [141] used lipid nanoparticles as a vector to deliver oligonucleotide antisense inhibitor to inhibit the endogenous messenger RNA (mRNA) in the

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setting of ischemic wounds, which leads to impaired healing. Usually, an ischemic wound will result in an increase of the local level of hypoxia-induced miRNAs (hypoxamiR) which

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refers to miRNA that is sensitive to hypoxia. Since chronic non-healing wounds are usually characterized as hypoxia, hypoxiamiR has been the focus of possible miRNA therapeutic

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target for wound healing and miR-210, miR-21, and miR-203 are the hot candidates [142].

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Ghatak et al. [67] developed antihypoxamiR encapsulated lipid nanoparticles (LNPs) aiming to improve cutaneous wound healing. Soy phosphatidylcholine (SPC) was utilized for the lipid bilayer formation along with cationic lipid with tertiary (DODAP) and quaternary

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(DOTAP) amine headgroups, Gramicidin A was also incorporated to improve endosomal escape and facilitate ion channel formation in the lipid bilayer (Fig. 5) [67, 122]. Injection of

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these nanoparticles intradermally in bipedicle flap wounds created in mice prone to diabetes and atherosclerosis, miR-210 expression in the ischemic wound edge tissue was significantly reduced. Animal studies further indicate that after treatment with AFGLNmiR-210,

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re-epithelialization process was accelerated and accompanied by obvious characteristics of epithelial hyperplasia [67] (Fig. 5).

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Fig. 5 | The composition of antihypoxamiR functionalized lipid nanoparticles (AFGLN) [67],

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and (i) Digital photographs of the murine ischemic wound at days 0, 2, 4 and 6 after delivery

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of empty GLN, AFGLNscramble and AFGLNmiR-210. (ii) H&E images stained sections from ischemic wounds at day 6 post-wounding. (iii) Wound cross-sections were stained with anti-keratin 14 antibody and counterstained with DAPI to show re-epithelialization at day 6 following treatment with GLN, AFGLNscramble and AFGLNmiR-210. (n = 4) [67]. Reused with permission, Copyright © 2016 Elsevier Inc.

Another example of miRNA therapy is diabetic foot wound. Accumulated studies have suggested that some miRNAs may be dysregulated and participate in different pathophysiological phases of diabetic wound healing (Fig.6) [143]. For example, miR-27b 19

ACCEPTED MANUSCRIPT was overexpressed in diabetic condition, which results in increasing of cell proliferation, tube formation, adhesion, and delayed apoptosis. All these regualtions are completed through directly targeting TSP1, TSP2, semaphorin 6A, and p66shc (Src homology 2 domain containing transforming protein 1) in bone marrow–derived angiogenic cells (BMAC). It is observed that after topical transplantation of BMACs in diabetic mice, the overexpression of

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miR-27b led to improved wound healing and wound perfusion. Interestingly, Wang et al. [144] found that direct delivery of miR-27b could only partially improved wound healing, which

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suggested other miRNAs might also be involved in this process. miR-99 family was another

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example. It is found that miR-99 family was reduced in diabetic wounds, signaling path PI3K/Akt was inhibited and keratinocytes migrated and proliferated [64]. Thus, it is

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reasonable to think that miR-99 family played an essential role in re-epithelialization. Furthermore, miR-155, an famous RNA functionally regulating immune response was also

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found to be induced in diabetic mice [53]. miR-155 exerted its function through target genes BCL6, RhoA, SHIP1, and FIZZ1. Deficiency of miR-155 led to a reduced inflammatory

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response and improved wound closure. Moreover, miRNA-26a was also confirmed to be

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induced in diabetic dermal wounds in mice model. When the expression of miR-26a was inhibited, the granulation tissue increased, angiogenesis induced and wound healing promoted via D1/SMAD1 signaling pathway. In addition, local inhibition of miR-26a had no effect on

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leukocyte accumulation and even enhanced the accumulation of myofibroblasts, which are important for wound contraction [145].

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Collective, these studies revealed important effect from the microvasculature of wounds on dermal fibroblast function to diabetic wound healing.

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Fig. 6 | miRNA regulation of diabetic wound healing. In response to diabetic conditions,

6.

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several miRNAs are dysregulated.

Conclusion

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The need for a safe and efficient therapy for wound healing is becoming more and more urgent. Emerging treatment options have embraced the need to address deficits in critical

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signaling pathways, cellular dysfunction, and impaired tissue regeneration associated with

development stage.

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chronic wounds. However, these treatments are largely experimental or at the very early Utilization of miRNAs presents an attractive proposition for the

development of genetic therapeutics for wound healing. miRNA-based therapeutics show

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unique advantages because by modulating a single miRNA, a group of functionally related genes in a pathway can be targeted, which is extremely effective compared to traditional drug

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therapy or DNA therapy. What’s more, miRNAs expression can be efficiently inhibited both in vitro and in vivo. So, the enthusiasm about the therapeutic potential of miRNA in wound healing has been triggered by manipulating miRNAs expression through the delivery of miRNAs inducers or inhibitors [99]. Diverse miRNAs play obviously different roles in the different phases of wound healing process, and accumulated studies have highlighted the essential functions of miRNAs in diverse aspects of chronic wounds associated with diabetes. According to Frost & Sullivan, the U.S. miRNA markets have earned revenues of over $20.3 million in 2008 and $98.6 million in 2015 [99]. All these results indicate that miRNA therapy 21

ACCEPTED MANUSCRIPT has the capability to move from bench to bedside and great potentials in the wound healing field. However, the limitation in the implementation of miRNA therapy is the development of the delivery system. The design of the effective delivery system must overcome multiple extracellular and intercellular barriers to ensure that the therapeutic cargo is successfully delivered to the desired target without causing any immune response. For wound healing, it is

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important to maintain an optimal healing environment. The moist and protected micro-environment in addition to the efficacious delivery of the miRNA are key points of the

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therapeutics. To date, various non-viral delivery systems for miRNA delivery have been

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developed and proven effective, including liposomes, cationic polymers, nanoparticles and even inorganic materials. Therefore, it can be foreseen that miRNA will hold the potential to

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become the new generation of nucleic acid therapeutics for wound healing treatment and

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management along with the successful development of safe and efficient delivery systems.

Acknowledgements

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References

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This work was funded by Science Foundation Ireland (SFI) Principal Investigator Award (13/IA/1962), Investigator Award (12/IP/1688), Health Research Board (HRA-POR-2013-412), National Science Foundation of China (NSFC) (51573129) and Irish Research Council CAROLINE Fellowship (CLNE/2017/358).

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