miRNA‐encapsulated abiotic materials and biovectors for cutaneous and oral wound healing: Biogenesis, mechanisms, and delivery nanocarriers

Abstract MicroRNAs (miRNAs) as therapeutic agents have attracted increasing interest in the past decade owing to their significant effectiveness in treating a wide array of ailments. These polymerases II‐derived noncoding RNAs act through post‐transcriptional controlling of different proteins and their allied pathways. Like other areas of medicine, researchers have utilized miRNAs for managing acute and chronic wounds. The increase in the number of patients suffering from either under‐healing or over‐healing wound demonstrates the limited efficacy of the current wound healing strategies and dictates the demands for simpler approaches with greater efficacy. Various miRNA can be designed to induce pathway beneficial for wound healing. However, the proper design of miRNA and its delivery system for wound healing applications are still challenging due to their limited stability and intracellular delivery. Therefore, new miRNAs are required to be identified and their delivery strategy needs to be optimized. In this review, we discuss the diverse roles of miRNAs in various stages of wound healing and provide an insight on the most recent findings in the nanotechnology and biomaterials field, which might offer opportunities for the development of new strategies for this chronic condition. We also highlight the advances in biomaterials and delivery systems, emphasizing their challenges and resolutions for miRNA‐based wound healing. We further review various biovectors (e.g., adenovirus and lentivirus) and abiotic materials such as organic and inorganic nanomaterials, along with dendrimers and scaffolds, as the delivery systems for miRNA‐based wound healing. Finally, challenges and opportunities for translation of miRNA‐based strategies into clinical applications are discussed.


| INTRODUCTION
Chronic wounds, particularly venous ulcers, diabetic ulcers, arterial insufficiency ulcers, and pressure ulcers, are arduous and exorbitant to cure. 1 Globally, many people suffer from chronic wounds with a growing prevalence every year. The Considerable but often unrecognized impact on patients and the healthcare system have turned chronic wounds into a silent epidemic. The increase in chronic wounds can be attributed to various factors such as the aging population and the associated rise in comorbidities and lifestyle disorders, including diabetes, obesity, chronic venous hypertension, and peripheral artery diseases. 2,3 Chronic wounds are not only a substantial health issue, but they also have substantial monetary and psychological consequences. According to the presented reports, approximately 6.5 million of the US population are agonized by non-healing, chronic wounds causing hefty monetary distress of 25 billion dollars per annum. 4 The burden of these medical morbidities is vast, thereby driving a substantial interest in novel medicines with better clinical efficacy.
In a normal physiological condition, the cascade of hemostasis, inflammation, proliferation, and remodeling leads to tissue regeneration and wound healing. 5,6 However, if the cascade is interrupted, wound healing will be impaired. If the wound does not heal for more than 90 days, it is referred to as chronic wound. 7 Chronic wounds are typically represented by tissue hypoxia, excessive inflammation, and persistent bacterial infections and biofilms. [8][9][10] To treat chronic wounds, an array of strategies including biological factor and cell therapies and electromechanical stimulation of the injury site have been explored. 11 The harsh environment of chronic wounds has limited the success of exogenous biological factor therapies due to the instability of large proteins and their limited distribution in the healing tissue. 12 miRNA, a small noncoding endogenic RNA, has an average length of 22 nucleotide molecules and regulates the post-transcriptional gene expression. 13 Various miRNAs are being established for tissue regeneration upon injury and promising results are being reported. 14,15 For example, miRNA-155, miRNA-21, miRNA-130a, miRNA-31, miRNA-132, miRNA-378a, and miRNA-198 are the predominant miRNAs demonstrating various roles in the process of wound healing. 16,17 Among several miRNAs known to contribute in tissue regeneration, miRNA-21 is demonstrated to perform two vital roles in the wound healing management, primarily by modulating the inflammatory and by modulating the proliferative phases, the two critical physiological healing stages needed for enhanced healing and reduction of scar formation. 18,19 In rat models with diabetic wounds, miR-27b and miR-15b were witnessed to play a significant part in the process of angiogenesis together with accelerating the closure of wounds.
The promise of miRNAs in healing skin wounds attracted increasing attention toward their expanded applicants for a wide array of modern therapeutic approaches in wound healing. 20 For instance, miRNA-126 is engaged in angiogenesis, one of the most crucial factors in wound healing process. Upon loading the miRNA-126 on liposomes modified using polyethylene glycol and delivering it to ischemic wounds, the angiogenic factor, vascular endothelial growth factor (VEGF), was triggered. The systemic circulation was enhanced, aided to fuel angiogenesis, and ultimately promoted wound healing. 21 However, an efficient delivery is challenging in miRNA therapy due to their short-term stability and limited cellular permeability, which is why various biovectors like adenovirus, lentivirus, and abiotic nanomaterials such as gold and silver nanoparticles (NPs) have been used to surpass the clinical limitations. 22 The current review explores miRNA synthesis, structure, functionality, and its contribution in different stages of wound repair in dermal and oral tissues. The review outlines various mechanisms of miRNA action, its therapeutic capability for oral mucosa, and dermal tissue wound healing. In addition, the delivery platforms including biovectors and nonviral vectors materials (e.g., nanoscale particles, dendrimers, and hydrogels) have been presented. Besides, the current article highlights the benefits of miRNA therapy and thus scrutinizes the success of this versatile therapeutic entity as a developing approach toward chronic wound healing.

| BIOGENY OF miRNA
The genetic makeup of human beings comprises an excess of over 500 miRNAs, and each of them can carry out the suppression of hundreds of genes. 23 miRNAs in animals are strongly associated with almost every pathophysiological process conducive to development as they are proposed to target over 50% of the protein-coding transcript. 24 Their functionalities add to numerous disorders, including malignant growths, cardiovascular diseases, and neurodegenerative disorders, so miRNAs are generally utilized as biomarkers and therapeutics in medicine. [25][26][27] Small RNAs in eukaryotes can suppress genetic material and unwanted cell transcripts. 28,29 Also, small RNAs are usually between 20 and 30 nucleotides in length and are associated with argonaute family proteins. 30 These proteins are divided into miRNA, siRNA, and piwi (an endoribonuclease domain)-interacting RNA. 30 In examining the structure, miRNAs are usually a dominant class of small RNAs consisting of approximately 22 nucleotides and are produced by Dicer, two RNase III proteins, and Drosha. 31 Transcription, processing by Drosha and Dicer, loading onto argonaute family proteins, and turnover are steps during which miRNA is regulated. 32,33 RNA polymerase II/III transcribes miRNA genes, and the primary transcript has a hairpin structure where miRNA sequences are embedded. 34 Succeeding transmission, the miRNA undergoes the initial cleavage step where the miRNA is cleaved into primary-miRNA (a stem of 33-35 bp, a terminal loop and single-stranded RNA segments at both the 5 0 and 3 0 sides) through RNA polymerase II/III. 34 The p53, MYC, zinc finger E-box-binding homeobox 1 and zinc finger E-box-binding homeobox 2, and myoblast determination protein 1 (as transcription factors) positively or negatively regulate miRNA expression. 35 In the next step, Drosha is cropping the stem-loop to release a small hairpin-shaped RNA that is made up of about 65 nucleotides and is called precursor miRNA. 36 Drosha, a nonspecific doublestranded RNA, inserts staggered cuts in every strand of RNA-helix and with DiGeorge syndrome critical region 8 (DGCR8), also known as pasha in drosophila melanogaster and as pash-1 in caenorhabditis elegans forms a complex called microprocessor. 37,38 DGCR8, a vital cofactor for Drosha, comprises two dual-stranded RNA that codes for binding domains and aids in the formation of protein complexes. 39 The interaction of Drosha with DGCR8 takes place in the nucleus. 40 Human recombinant Drosha solely demonstrates nonspecific RNase activity; however, the addition of DGCR8 makes it specific for processing precursor miRNA. 41 Precursor miRNA are preserved in the form of internal loops and bulges that frequently emerge in the definite positions of the miRNA stem. 42 This allows proper enzyme-catalyzed processing, thus resulting in miRNA maturation. 42 The consequential precursor miRNA is migrated to the cytoplasm where maturation can be completed. The protein exportin-5 forms a transport complex with Guanosine triphosphate-binding nuclear protein Ran-GTP and a precursor miRNA. 43,44 The reduction of Guanosine triphosphate impairs the migration of precursor miRNA-bound Ran.
Therefore, it is considered that exportin-5 depends on the nuclear Ran-GTP to perform its functions. 45 Upon cleaving by an RNase III enzyme known as Dicer, precursor miRNA is converted into 18-24 nucleotide double-stranded RNA. 46 After maturation, this cleaves precursor miRNA, thus turning it into a miRNA duplex. 46 The enzyme Dicer works via intramolecular dimerization of its two domains, the double-stranded RNA binding domain and the flanked RNA binding domain, that is, Piwi/Ago/Zwille. 47 After cleavage, the resulting miRNA duplex calls for the release of any one of the strands to receive entry into argonaute for functions. 48 The majority of the miRNAs are encapsulated in the RNA-induced silencing complex to form miRNA-containing RNA-induced silencing complex. 49 RNA-induced silencing complex assembly includes loading RNA duplex and its unwinding steps. 50 In the miRNA duplex, the strand fated to transform into the matured miRNA is referred to as the guide strand, while the remaining second strand of the duplex is termed star or passenger strand. 48 After cleavage, miRNA duplex needs to release the passenger strand to receive entry into argonaute to function. 48 The guide strand of the miRNA duplex is encapsulated and subsequently directs the enzyme to form a miRNA-containing RNA-induced silencing complex. 49 After binding to the mRNA, argonaute recruits the trinucleotide repeat containing six proteins, a scaffold protein tethering effector protein to destabilize and translationally repress target mRNAs by inducing their decapping and deadenylation ( Figure 1).

| ROLE OF miRNA IN WOUND HEALING PHASES AND IMMUNOGENICITY
The wound healing process consists of complex and dynamic physiological pathways that promptly occur to hemotasis and inflammation, proliferation, and regeneration within the wound and tissue. 8,51,52 Various miRNAs have been found to date to undergo either downregulation or upregulation during four phases of wound healing.
Some of the essential miRNAs having a significant involvement in wound repair and immunogenicity pathways are mentioned in Table 1.
DEKA DEY ET AL.

| First phase: miRNA in the inflammatory stage
Inflammation is known as a biological and pathophysiological reaction complex caused by tissue damage or infection. Molecular networks play a role in the functioning of regulatory pathways. In addition to protein regulators, miRNAs appear to be significant regulators of inflammation and modulate the onset and end of inflammation by suppressing or amplifying signaling ( Figure 2). 81 Inflammation is the primary stage of wound healing and commences immediately following the disruption of the stratum corneum and activates the cascade of events associated with the clotting process. 82 Since hemostasis takes place via the development of fibrin clots, immune cells discharge the chemokines and cytokines like platelet-derived growth factor, platelet factor-IV, transforming growth factor-β, and tumor necrosis factor-α (TNF-α) into the wounding area. 83,84 Immune cells like monocytes and neutrophils are then passively released into the wound via the injured blood vessels. 85 The effused monocytes develop into macrophages and perform phagocytic actions. Monocytes might be pro-angiogenic and can be either pro-inflammatory or antiinflammatory. 86 Furthermore, due to infringement of the protective dermal layer, the underlying tissues get exposed to the pathogens, and the chance of infections increases manifold. 87 The released neutrophils then arrive at the wound site via the chemokine signaling and cytokine mentioned above and elicit their role of cleansing and killing the invasive microorganisms. 88 This inflammatory signaling and the expression of receptor genes are several ways in which miRNA regulation of the inflammatory stage is observed. Various miRNA was recognized at this phase to be fundamental. For instance, miRNA-146a negatively regulates the inflammatory stage responses associated with the intact skin. 89 The miRNA-146a expression is enhanced in the epidermal keratinocytes prompted by the toll-like receptors 2, 3, 4, and 5.
Moreover, the negative regulation suggests that miRNA-146a might promote the resolution of inflammation. 90 A significant downregulation in the appearance of miRNA-146a was witnessed in diabetic wounds of mice in contrast to the nondiabetic models. 91 Furthermore, investigations suggested that this miRNA also targets and silences the pro-inflammatory mediators like interleukin-1 receptor-associated kinase one and TNF receptor-associated factor 6. 92 miRNA 155 is another essential miRNA for the immune cell. Both miRNA-155 and miRNA-146a regulate macrophages, which further stimulate the generation of cytokines and the growth factors required for the differentiation of monocytes to macrophages. 93 Moreover, upregulation of miRNA-155 in the inflammatory phase has been F I G U R E 1 Graphic representation of the biogenesis of miRNA. RNA polymerase II/III carries out miRNA transcription from genomic DNA to construct the pri-miRNA in the nucleus. Drosha and its cofactor DGCR8 then cleave the pri-miRNA to form Pre-miRNA. Exportin-5 and Ran-GTP transports the pre-miRNA into the cytoplasm. In the cytoplasm, the Dicer and TRBP recognize pre-miRNA and cut into dsRNA which matures and thus resulting in a miRNA duplex. Matured miRNA duplex is loaded in the RISC to form the mature miRNA molecules. AGO, argonaute; DGCR8, DiGeorge syndrome critical region 8; Pre-miRNA, precursor miRNA; Pri-miRNA, primary miRNA; RISC, RNA-induced silencing complex; TRBP, TAR RNA binding protein audited in mouse models, demonstrating that miRNA-155-specific inhibitor therapy can efficaciously mitigate the inflammatory cellular proliferation at the wound area and, therefore, ameliorate the construction of restored tissues. 51,94,95 miRNA-132 has an antiinflammatory role in this phase and limits the excessive production of pro-inflammatory cytokines. 96,97 Inflammation has stimulated the miRNA-132 expression in the leukocytes 81   The topical administration of miRNA-132 mimics loaded liposomes at the wound area resulted in a rapid recovery of the dermal wound in diabetic mice and re-epithelization of the human ex vivo dermal wounds through the suppression of the pro-inflammatory cytokines in keratinocytes and macrophages. Furthermore, miRNA-132 also improves the changeover from inflammation to the proliferation stage during the healing process via targeting the heparin-binding endothelial growth factor. 99 Additionally, miRNA-21 is also evidenced to be crucial in the resolution of inflammation. 100 Furthermore, miRNAs including miRNA-191, miRNA-200, and several others are intricate in regulating inflammation in wound healing.
Although miRNAs have an essential role in promoting and inducing inflammation, they also downregulate or end the stage. 93 Protraction of this stage leads to damaged tissues and impedes proper wound healing, resulting in chronic wounds. 94  in inflammation. 101 With the termination of the inflammatory stage by the decreased neutrophils count and macrophages, the proliferation phase is initiated.
By increasing miRNA-9 expression in the cerebral cortex, factors associated with the NF-κB signaling pathway such as NF-κB p65, TNF-α, and interleukin (IL)-1β are reduced. Decreased miRNA-9 is associated with increased synthesis of pro-inflammatory mediators such as IL-1β, TNF-α, IL-6, and monocyte chemoattractant protein-1. 102 On the other hand, miRNA-173p and miRNA-31 target E-selectin and intercellular adhesion molecule 1, respectively. miRNA-92a reduces inflammation by targeting mitogen-activated protein kinase 4. miRNA-99b regulates the expression of inflammatory cytokines IL-6, IL-12, and IL-1β. 63 The secretion of IL-6, TNF-α, chemokine monocyte chemo- During the remodeling phase, the blood vessels toward the wound site reduce, and the cells' activities begin to slow down in preparing for termination. In particular, miRNA-29a regulates the fibroblast by monitoring their contractility via targeting tissue inhibitors of metalloproteinase-1. 121 Besides this, miRNA-29a has also been veri- So far, the intricacy of the wound healing process at the genomic extent is distinctly indicated. A comparison between miRNA expression patterns of skin and oral mucosa at normal and wound healing states was conducted. The results displayed the baseline diversity of the site-specific miRNA profile in healthy skin and oral mucosal epithelium. Furthermore, the miRNAs expression pattern was also different with oral mucosa over time, consistent with the variation in wound healing, and implies that the oral mucosa holds an intrinsic and modified genetic reaction that enhances wound healing (Figure 4b,c).

| Nonviral vectored delivery systems
Poor cell membrane permeation (due to higher molecular weight and negative charge), short half-life in blood circulation, and quick removal of the biomolecules from the plasma are the major hurdles that obstruct the fortunate transport of the miRNAs 157 Consequently, developing an effective and secure carrier system for delivering miRNA to the target tissue is of paramount importance. 158 As an alternate, the nonviral delivery vehicles incrementally happened to be the major purpose of the study. 159,160 Unlike viral vectors, nonviral systems are accentuated by low immunogenicity and toxicity profile, high cell uptake, aqueous solubility, and elicit resistivity toward endonucleases and phagocytosis. [161][162][163][164] Gene transfer via a nonviral vectored system is accomplished by establishing the desired plasmid RNA-encoded gene, RNA interference, and miRNA. Different nonviral vectors have been developed so far and some of them include cationic polymers, lipid-based carriers, micro-seeding, naked plasmids, dendrimers, electroporation, and particle bombardment. 165  mediated endocytosis an easier task for uptake of NP together with decreasing the essential dose and the associated adverse effects of the regimen. 186,187 Additionally, the NPs colloidal stability in a complex physiological environment is required for the miRNA delivery toward the target cells. 188 Following administration, NPs must preferably distribute if they arrive at the target site. They must be designed so that they undergo endosomal escape to ensure the suitable miRNA interaction with its target (for instance, by utilizing the Proton Sponge Effect). 189 However, the circulation time is dependent on the interaction of NP with the biological environment, which might further result in a faster clearance.
The biocompatibility and biodegradability of nanomaterials make them an ideal nanocarrier for drug delivery. Consequently, nanomaterials are highly safe in vivo. Their biocompatibility and biodegradability are high since they are the principal components of biological tissues and participate in the body's natural metabolism through dissolution into nontoxic ions. 190 Usually, NPs are expelled out of the body in two main ways that are through the urinary system and through the hepatobiliary system and feces. Heavy metals and particles with a diameter larger than 6 nm can be quickly absorbed by the liver and spleen. Additionally, NPs with sizes less than five nanometers are rapidly metabolized in the urine by the renal system because their filtration sizes are smaller than those required for renal excretion. 191 In recent times, NPs derived from inorganic materials such as gold NPs, graphene oxide, mesoporous silicon, and ferric oxide NPs are Multiple miRNAs and polyethyleneimine complexes loaded in magnetic NPs were examined to allow magnetic targeting of transfected CD105+ human mesenchymal stem cells, gaining higher uptake rates with negligible toxicity. 195 Likewise, a nonviral and nonlipid scaffold approach was presented for the very first time to deliver both mimics as well as anti-miR to the human mesenchymal stem cells. In the experiment, nanohydroxyapatite particles were developed that successfully delivered miRNAs to the human mesenchymal stem cells in both 2D and 3D culture media, thus establishing this strategy as a potential therapeutic choice for a broad spectrum of tissue repairing applications. 196 An experimental study carried out by Krebs research group is of paramount importance in this regard. The Krebs and Liechty laboratories have notably concentrated on the miRNA-146a mimic since it has been observed that miRNA-146a regulates the inflammatory milieu in diabetic wounds. It is believed to work by aiming a target on the key adapter molecules present in the NF-κB inflammatory signal transduction pathway, thereby causing a reduction in the expression of the pro-inflammatory cytokines, including the interleukins. 197,198 Since there is a downregulation of miRNA-146a in diabetic wounds, Liechty's research group injected a combination of the miRNA with cerium oxide NPs as a reactive oxygen species scavenger that may aid in the reduction of total inflammatory condition and thus bring about an improvement in the wound healing process.
During the wounding phase, the mice were provided with a single treatment, and the kinetics of release was not noted in the study. A considerable reduction in the wounded area was witnessed at the interval of 7 days of miRNA-cerium oxide NPs administration. This outcome highlighted the significance of an appropriate carrier system for miRNA delivery (Figure 7). 198,199 Furthermore, the miRNA-146a-cerium oxide NPs were laden in a zwitterionic hydrogel system to present a persistent release for almost a month, with approximately 50% of the NPs released in the first couple of days. In in vivo studies, the release of miRNA-146a-cerium oxide NPs from the hydrogel elicited a complete wound closure by 2 weeks in contrast to around 20 days in the case of mice solely treated with hydrogel. 200 Apart from these inorganic material-based NPs, poly(lactic-coglycolic acid) NPs are also being exploited as miRNA delivery vehicles on a larger basis. Poly(lactic-co-glycolic acid) is a polymer synthesized by copolymerization of glycolic acid and lactic acid and owns biodegradability besides biocompatibility. 158 The intracellular miRNA delivery can further be improved using poly(lactic-co-glycolic acid) NPs after ornamenting them with peptides with cell-penetrating properties.
Based on those mentioned earlier and several other experimental outcomes, it can be proposed that compared to free molecules, biomolecules delivered using NPs demonstrate better effects in chronic wound healing. [201][202][203] There are, however, some limitations to NP-

| Lipid-based NPs
Lipoidal vesicles, which are pockets consisting of phospholipid bilayer membrane enclosing an aqueous chamber, 204 are the finest candidate vehicles for the complexation of miRNA. In the delivery of miRNA, lipid vesicles either undergo chemical modification with target-specific groups or are PEGylated to prevent recognition by the immune system and reticuloendothelial system uptake. 158,205 Cationic lipids, which are amphiphiles consisting of a hydrophilic head and lipophilic tail, 206

| Organic-based nanomaterials
Natural and synthetic polymers are perceived as another potential material for miRNA delivery on account of greater adaptability and flexibility. Polymer's facile size and charge adjustability are authorized to optimize the loading potential for nucleic acids. 158 Conventionally, Polyethylenimine is the most popular synthetic cationic polymer for the delivery of nucleic acids. They possess abundant amine moieties and carry a positive charge. Therefore, they can bind to the smaller RNAs to create nanoscale complexes that obstruct the degradation of RNA and thus enhance the uptake by the cells and promote its release intracellularly. 207 Additionally, the myriad of tertiary amines in polyethylenimine greatly expedites miRNA-loaded polyplexes' endosomal escape through the proton sponge effect. 208  Chitosan is another linear biopolymer that has been frequently utilized for gene transport. It comprises an amino moiety and two hydroxyl groups within the glucoside residue. Furthermore, the preparation method can affect its structure. [211][212][213] Chitosan, as a cationic polymer plays an important role in providing some fascinating characteristics regarding oppositely charged units, particularly, nucleic acids and surfaces of mucosa (through sugar moieties like sialic acid). 214,215 Together with lower immunogenicity, biodegradability, and biocompatibility, these exceptional characters present chitosan as a better agent for gene delivery in bedside use. 13,216 Several derivatives of chitosan can be employed for the targeted biomolecular delivery.
Chitosan with cationic polyelectrolyte character offers a stronger electrostatic interaction with the negatively charged nucleic acids and thus safeguards it from degradation by nuclease. 217,218 Nevertheless, due to its poor transfection efficacy, the in vivo applications of this polymer are restricted. As a result, scientists used different strategies to enhance the biomolecular transfection efficiency of chitosan.

| Dendrimers
As an alternative to the previously known polymers, cyclodextrin has grabbed much of the attention in the delivery of biomolecules. 219 As a form of naturally found cyclic oligosaccharide, cyclodextrin is expressed as nonimmunogenic and nontoxic to cells. 220

| 3D bioengineered scaffolds and hydrogels
Over the past few years, several advancements have been achieved in scaffolds and hydrogel-based dressings to tackle the issues related to chronic wounds. For proper cell growth, proliferation, and differentiation at the wounded site, these novel 3D scaffolds and hydrogelbased dressing provide a beneficial micro-requisite environment around the wounded site. Hydrogels have been universally used in tissue engineering for the local release of growth factors, 221 drug delivery applications, 222 and as a 3D recyclable material for controlled cell growth and soft tissue regeneration. 223 Recently, hydrogels have also found application in miRNA delivery for chronic wound management. 224 In this work, a new hyaluronic acid and oxidized hydroxymethyl propyl cellulose loaded with siRNA-29a hydrogel has been designed and integrated with oridonin micro/nanostructures, crosslinked via Schiff base bonds (Figure 9). 225 The synthesized nanointegrated hydrogels showed reasonable mechanical properties, excellent swelling, remarkable biodegradation, enhanced stability, and controlled release of oridonin and siRNA-29a,

| CHALLENGES IN TRANSLATIONAL MEDICINE
The naked miRNAs are susceptible to degradation by endonucleases, 228 have poor cellular uptake due to surface negative charge, 229 face entrapment issues in endosomes, 230 have low binding efficacy, 231 and sometimes undergo off-target delivery. Inadequate stability and consistency of the released miRNA molecule after local application is observed in blood circulation. However, degradation and endosomal escape are the most challenging task and hence several strategies have been developed such as using pH-sensitive liposomes, 230 cationic liposomes/NPs, 228 light-sensitive molecules, 232 and bioengineered polymeric scaffolds and hydrogels. 233 For proper cell proliferation, and cell differentiation at the wound site, the polymeric hydrogels and scaffolds provide an appropriate 3D micro-requisite environment for local tissue repair and regeneration for controlled release of miRNA, thereby producing a suitable set of stimuli. 234 Atelocollagen ® is a type of collagen gel employed to treat skin disorders and other cosmetic surgeries. 233 Atelocollagen ® itself has been proposed for the controlled delivery of siRNA as well as miRNA. Moreover, Atelocollagen ® is more effective for in vivo gene delivery. When it is confounded along with siRNA or miRNA, it is impervious to serum RNase nucleases and can be further proficiently transduced into cells. 235 However, such hurdles are being presented in numerous ways to deliver potentially therapeutic miRNA modality using diverse carriers. 236  The key stimulating constraint in miRNA delivery using nanotechnology is its low entrapment efficiency. 240 Due to high-water affinity, they undergo rapid diffusion into the aqueous phase when nanoprecipitation or emulsion-based approach is being used, thus leading to decreased entrapment efficiency. 240 Interestingly, NPs enhance the tissue and site-specific distribution of miRNA, but the extent of enhancement is usually not sufficient. To date, numerous studies have been directed to fabricate proper surface functionalization of the nanomaterials with a specific ligand to realize receptor-mediated endocytosis, thus dropping the necessary dose and adverse effects associated with the use of delivery systems. 241 Apart from this, nanomaterial's colloidal stability in the biological milieu is useful for targeting miRNA delivery to a particular cell or tissue. 188 Ideally, after administration, NPs should circulate in the blood flow until they get delivered to the targeted site. In addition, they should be premeditated in such a way that they undergo endosomal escape for appropriate interaction between the miRNA and its targeted cell/tissues. 242 Numerous studies reveal that highly charged particles are predisposed to opsonization, especially positively charged, compared with neutral particles. 243  Another major challenge in the clinical translation of nanotechnology-based vehicles for miRNA delivery involves their associated toxicity development due to the accumulated nanomaterial in the nonspecific sites. When the nanocompounds enter the body, they penetrate through the cell boundaries and damages mitochondria, causing a mutation in DNA and ultimately resulting in cell death. 247 Moreover, the underlying mechanism for nanomedicine toxicity also involves the release of reactive oxygen species which might lead to oxidative stress and consecutive inflammatory processes thus affecting protein synthesis, cell membrane, and DNA. 248 For example, toxicity due to gold NPs may be attributed to their physicochemical characteristics leading to the production of reactive oxygen species and oxidative stress. Their shape is considered a factor behind the toxic effect that is gold nanostars are highly toxic compared to gold nanospheres. 249 Silver NPs also show toxicity in tissues at high doses due to the nonspecific release of silver ions in the healthy tissues. 250 Regulatory issues are yet another concern regarding the gap between laboratory research and clinical use. Inorganic nanomaterials are frequently denied regulatory approval despite positive clinical results owing to concerns from the government, including inappropriate design of endpoints, inadequate justification on clinical comparator selection, and insufficient analysis methodology for clinical data. Thus, pharmaceutical companies must solicit feedback from regulatory agencies during the whole nanomaterial development process so as to prevent delay or denial of regulatory approval. 245

| CONCLUSION AND FUTURE PERSPECTIVES
During the past decades, a tremendous amount of research and clinical trials have been performed on the identification and development of biological factors that modulate the physiological processes throughout wound healing to enhance the healing rate and quality.
However, the growing population of patients suffering from chronic wounds or scarring demonstrates the need for more efficient strategies in wound healing. miRNAs present attractive therapeutic candidates to develop safe, simple, and effective strategies, capable of modulating various biological mechanisms for improved wound healing. However, due to their limited stability and membrane permeability, the main limitation of miRNA-based therapy is their inefficient delivery to the target site. Therefore, future investigations need to develop novel delivery systems for the efficient delivery of miRNAs without an adverse immune response.
To date, multiple drug carriers have been identified and developed for the successful delivery of miRNAs such as liposomes, cationic polymeric NPs, scaffolds, hydrogels, and even inorganic materials. However, in addition to the drug carrier, the delivery strategy that controls the spatiotemporal release profile is a very important factor. However, less attention has been paid to the proper delivery tool. An optimal drug delivery system should release the specific therapeutic agents corresponding to the spatiotemporal requirements throughout wound healing stages. 251 Various drug delivery systems, which can control the temporal release profile, including passive, active, and smart systems can be used for miRNA-based wound therapy. 11 Smart systems can either incorporate smart materials reacting to wound biomarkers, or integrated sensing/delivery systems.
Furthermore, to control spatial distribution of the miRNA in the wound bed, two main approaches including topical and intradermal/ transdermal delivery can be implemented. 7  Writingreview and editing (supporting). Sumit Jamwal: Writingoriginal draft (equal). Aziz Maleki: writingreview and editing (supporting).