Microneedle arrays for the treatment of chronic wounds

Abstract

Introduction:

Chronic wounds are seen frequently in diabetic and bedbound patients. Such skin injuries, which do not heal in a timely fashion, can lead to life-threatening conditions. In an effort to resolve the burdens of chronic wounds, numerous investigations have explored the efficacy of various therapeutics on wound healing. Therapeutics can be topically delivered to cutaneous wounds to reduce the complications associated with systemic drug delivery because the compromised skin barrier is not expected to negatively affect drug distribution. However, researchers have recently demonstrated that the complex environment of chronic wounds could lower the localized availability of the applied therapeutics. Microneedle arrays (MNAs) can be exploited to enhance delivery efficiency and consequently improved healing.

Areas covered:

In this review, we briefly describe the pathophysiology of chronic wounds and current treatment strategies. We further introduce methods and materials commonly used for the fabrication of MNAs. Subsequently, the studies demonstrating the benefits of MNAs in wound care are highlighted.

Expert opinion:

Microneedles have great potential to treat the complicated pathophysiology of chronic wounds. Challenges that will need to be addressed include development of a robust chronic wound model and MNAs that combine complex functionality with simplicity of use.

1. Introduction

Skin injuries usually heal quickly and with minor discomfort due to the excellent regenerative properties of skin[1], though deep or severe wounds have the potential to generate scarring because the connective-tissue contracts aggressively to ensure rapid closure of the injury and prevent infiltration of environmental pathogens[2]. To avoid infection and further injury, various dressings have been developed to cover and protect the wound area and improve the physiological healing process[3,4]. However, some conditions, such as diabetes or the presence of stress on major injuries, can significantly lower the ability of skin to regenerate[5]. In these cases, infections and other complications may occur that further impede the wound healing process. Currently, more than 6 million cases of chronic wounds exist in the United States (US), which imposes a staggering financial burden on patients and the healthcare system[6]. In addition, patients who suffer from these wounds often report severe symptoms. These include physical issues such as constant pain and odor, which can impair sleep and mobility, which in turn can lead to mental health complications like shame, depression, and social isolation[7].

While the essential challenge in acute skin injuries is preventing excessive contraction and scarring, in chronic wounds the major obstacle is promoting wound closure[8]. Complications such as tissue necrosis and infection can place the patient’s life at the risk. In severe cases, highly invasive surgical debridement and even limb amputation may be necessary. It is noteworthy that chronic wounds, specifically diabetic ulcers, are the leading cause of non-traumatic limb amputation in the world[9]. The continuous increase of diabetic and aging populations position chronic wounds as a growing medical challenge[6]. Therefore, numerous research efforts have been dedicated to identifying the causes of chronic wounds as well as therapeutics that can prevent their formation or improve their healing. Researchers have characterized the sequence of events post injury essential for wound healing and the dysfunctional processes leading to formation of chronic wounds[10]. In addition, the therapeutics that can enhance healing and regulate various physiological processes have been identified. These therapeutics can include chemicals such as oxygen, growth factors, small drug molecules, scaffolding materials, and cells. Different therapeutics used in wound care are reviewed in detail elsewhere[8,11].

One important element affecting the efficacy of therapeutics is their point of delivery[12]. Due to complications associated with systemic delivery of therapeutics and the ease-of-access of skin injuries and wounds, the logical strategy has been to apply therapies topically[3]. In cases with significant necrotic tissue, therapies are administered post wound debridement. However, a close look at the wound environment shows that an eschar typically separates healthy cells below from the skin exterior[1]. This means that any therapeutics applied topically must diffuse through the eschar to access healthy cells. In addition, some wounds are continuously exuding, which can wash delivered therapeutics out of the wound bed. As a result, the local bioavailability of delivered drugs is lower than expected when they are applied topically[12]. This fundamental concept has been commonly disregarded in wound care practice. Recently, a few studies have shown the benefit of advanced transdermal and intradermal drug delivery tools for improving wound healing outcomes. Physical systems such as iontophoresis[13], liquid jet injectors[14], and microneedle arrays (MNAs)[15] are easy-to-operate and can overcome some of the challenges listed above to enhance the concentration and bioavailability of therapeutics in the wound bed. Among these, MNAs are one of the most robust platforms available for the delivery of various drugs and cells to the wound bed.

In the following sections, we will briefly discuss the pathophysiology of chronic wounds. Then, various materials and approaches commonly used for fabrication of microneedles will be described. Successful examples of MNA-based wound care systems will be highlighted. Finally, the opportunities and challenges facing the translation of MNA-based wound care products will be discussed.

2. Pathophysiology of chronic wounds

An acute wound heals through a series of generally sequential but overlapping stages[11]: hemostasis, inflammation, proliferation, and remodeling[16,17]. Hemostasis occurs in the immediate aftermath of an injury and involves the production of a clot at the injury site to prevent continued blood loss. Clot formation is precipitated through the secretion of pro-clotting factors from platelets, which are among the first cells to arrive at the area and adhere to collagen exposed in the extracellular matrix (ECM)[17]. In addition to stemming blood flow, the formed fibrin clot serves as a temporary ECM for the inflammatory cells that are moving into the wound at this time[1].

The initial leukocytes release biological factors that increase vasculature permeability and fluid exchange into the wound site, causing the local swelling that is indicative of inflammation and allowing neutrophil infiltration[18]. The key functions of these neutrophils include release of antibacterial proteases, peptides, and reactive oxygen species (ROS)[19,20], as well as phagocytosis of dead cells, bacteria, damaged ECM, etc. to clear the wound bed of detritus that inhibits wound healing[20,21]. As healing progresses, monocytes and lymphocytes are recruited into the wound, where they differentiate into macrophages that continue the neutrophils’ efforts to clear the area[22]. Macrophages also release protease inhibitors to mitigate proteolysis as well as cytokines and growth factors that influence the proliferation of several key cell types and promote creation of permanent ECM[23]. Macrophages in the inflammation stage are of the M1 phenotype, which are pro-inflammatory. During the proliferation stage, an anti-inflammatory M2 phenotype macrophage polarization is observed [24]. At the end of the inflammation stage the level of proinflammatory cytokines is decreased as macrophages phagocytize the remaining neutrophils[1,25,26].

In the proliferation stage, keratinocytes proliferate from the wound edges and cover its surface. Fibroblasts begin creating new, permanent ECM through the secretion of its component proteins such as fibronectin and collagen[27]. To keep up with the high level of cellular function during this phase, several key growth factors promote an increase in angiogenesis to ensure there is sufficient vasculature to supply necessary oxygen and nutrients as well as remove waste[28]. The wound is closed through a combination of re-epithelialization and contraction, executed by myofibroblasts, differentiated from fibroblasts at the edges of the wound[1]. Excessive contraction, which is a reparative process, can result in scarring and low quality wound healing[2].

Remodeling is the final stage of wound healing[29]. During this period, the extra vasculature that populated the wound bed to fuel healing retreats and condenses, leaving behind fewer, more robust and stabilized blood vessels[30–32]. Similarly, the cells that have been functioning to repair the wound and prevent infection become unnecessary and either migrate out of the area or undergo apoptosis[33]. The temporary collagen III ECM that was laid out in the early phases of healing is replaced with the collagen I that forms the permanent matrix[34]. Matrix metalloproteinases (MMPs) direct the alignment of collagen by oxidase, secreted from fibroblasts, in order to strengthen the tissue covering the wounded area[30,31]. The remodeling process continues for months to over a year before wound healing is finally complete[16].

However, skin injuries do not always follow the timely and orchestrated processes described above. In many cases, the healing cascade halts. Wounds which remain open for more than 90 days are described as “chronic”. There are different kinds of chronic wounds, such as diabetic foot ulcers, venous leg ulcers, and pressure ulcers[35], which result from different etiologies; however, they all share some essential features. Chronic wounds fail to move through the outlined progression of healing, becoming trapped in the inflammation stage. In an acute wound, the inflammation period lasts for days to weeks[35,36], while chronic wounds may maintain it for months or years[35,37]. This standstill in healing is perpetuated by a number of factors, including lack of sufficient angiogenesis resulting in excessive hypoxia, ongoing overexpression of pro-inflammatory cytokines and MMPs, and infections. The excess MMPs can deactivate growth factors, break down temporary ECM before it can be infiltrated, and damage cells[38,39]. This necessarily results in the disruption of healing as intracellular signaling is impaired, scaffolding is no longer available to host cells, and damaged immune cells can’t effectively target infection. Poor immune cell function is exacerbated by hypoxia, which is typical in chronic wounds[40]. Hypoxia also contributes to the development of infection by inducing cell death and creating areas of non-viable tissue covering the wound (eschar) that bacteria often eagerly colonize. This generally leads to the development of biofilm that makes it difficult for topically applied drugs to reach the wound bed to help the wound move out of the inflammation cycle it is locked in[8].

3. Wound healing strategies

In order to holistically evaluate and care for chronic wounds, the TIME (Tissue, Infection, Moisture, and Edge of wound) system was described in 2002 to serve as a standard of care[41,42]. The components of this acronym highlight areas of major concern when trying to promote healing: removing necrotic tissue, eliminating bacterial load, keeping the wound moist while wicking excessive exudate, and promoting healthy cellular ingrowth from the wound edge. In addition to meeting these challenges, treatments for chronic wounds should also make an effort to minimize cost and frequency of care for the patient, as well as focus on patient driven outcomes such as pain treatment[6,29,43–45].

A number of techniques have been developed that work to address specific components of chronic wound symptoms. Collectively, these strategies seek to modulate the wound environment through elimination of pathologic conditions and enhancement or regulation of physiologic processes. Removal of wet eschar has been clinically performed to reduce the hospitable environment for pathogens and enhance the accessibility of healthy cells[10,46]. Negative pressure systems and hydrophilic dressings have been widely used for removal of excessive exudates typically rich in proinflammatory cytokines and MMPs[8,47]. It should be noted that moisture balance is important for improving the rate of physiological processes[29,48,49]. The exposure of cutaneous tissue to the environment make them susceptible to pathogen contamination. The presence of eschar and exudate provide any introduced pathogens an ideal environment to grow[10]. The use of antibacterial dressings or prophylactic antibiotics have shown some success in reducing the rate of infection; however, they typically reduce the rate of wound healing and can create resistant pathogens[8]. Therefore, management of bacterial infection has been one of the most challenging tasks in care for patients with chronic wounds. The excessive production of MMPs in chronic wounds disintegrates the temporary ECM essential for the proliferation stage of wound healing[38,39]. In addition, following wound debridement, significant defects are typically generated, which lack any ECM to support cell ingrowth. To overcome this challenge, researchers have developed an array of scaffolds from ECM mimicking materials such as collagen, gelatin, and hyaluronic acid that are biodegradable and support the physiological processes essential for wound healing [50]. Some scaffolds have incorporated biological factors and drugs that further improve the wound environment[20,53–55].

It is now accepted that the localized delivery of exogenous growth factors and molecules can improve the quality of wound healing. This is due to the lack or limited availability of endogenous biological factors resulting from dysfunctional cells[56]. Physiological processes can be modulated by controlling the level of biological factors. For example, angiogenic factors such as vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) can enhance the migration and sprouting of endothelial cells[28]. This is of the utmost importance in diabetic wounds, in which the endothelial cells are dysfunctional[57,58]. Modulation of immune response is also important. Excessive inflammation, especially towards the end of the inflammation stage, can prevent wound healing[59]. Thus, the use of anti-inflammatory drugs and factors has been shown to improve wound healing[60,61]. However, the delivery of a single biological factor or therapeutic has shown only modest improvement due to the multifactorial challenges leading to occurrence of chronic wounds. In addition, these biological factors are needed at specific time points with the correct concentration, as improper use of biological factors can be non-effective or even negatively impact wound healing.

For these reasons, significant attention has been dedicated to identifying and modulating the temporal profile of drug delivery. The research efforts in this area have recently been focused on three major area of interest: 1) active delivery tools, 2) dressings based on smart materials, and 3) dressings with integrated sensors. Active delivery tools ideally enable predictable and programmable release kinetics through the utilization of external stimulation to trigger drug release[9,62]. One notable example was developed by Mostafalu et al.[63] In this study, a fabric dressing was created using flexible heaters as threads, each of which was coated in a layer of hydrogel loaded with thermoresponsive drug carriers. Each thread could be independently loaded with a dose of a specific medication. This platform enabled the active delivery of different drugs with independent release kinetics. The dressing was used for treatment of diabetic wounds in mice and the release of VEGF improved the rate of tissue granulation.

An alternative to active delivery tools is the use of smart biomaterials that can respond to changes in wound conditions. Stimuli-responsive materials can be engineered to trigger drug release in response to change in temperature, pH, oxygen level, and inflammatory enzymes commonly present in the chronic wounds. For example, bacteria-[64] and pH-responsive[60] materials have been implemented to trigger drug release upon infection or chronicity of the wound.

One of the challenges in clinical wound care is the lack of detailed knowledge about the wound environment. To address this issue and assist healthcare providers with treatment decision making, researchers have developed sensors that interface with the wound surface and monitor the environment. pH, temperature, and oxygen have been some of the most popular markers targeted for monitoring, commonly through colorimetric or electrochemical based sensors[9]. In both cases, the stability of the sensors for long term use has been a challenge. In addition, the majority of these sensors are topical or external, which means that they measure the properties of wound exudate rather than the properties of the wound bed itself.

A common problem associated with both drug delivery tools and sensing systems used in wound care is the selection of the point of action. This is more important for chronic wounds because they are often covered with eschar that topically delivered drugs must pass through to reach the healthy cells below while combating the transport of exudate in the reverse direction. Therefore, the actual local bioavailability of drugs near healthy cells can be much lower than the dose delivered. Similarly, the values measured using topical sensors may not precisely reflect the changes in the physiological environment of healthy cells below eschar. There is increasing evidence suggesting the importance of the delivery/sampling point in wound care systems. MNAs have emerged as a minimally invasive tool that reduces the distance between healthy cells and the delivery/sampling point. In the following sections we will discuss strategies used for the fabrication of MNAs and examples showing their successful use in wound care practice.

4. Materials and fabrication technologies for production of MNAs

MNAs have been fabricated from various materials and using an array of different technologies. The utilized materials and fabrication approaches must meet minimum requirements, such as sufficient mechanical properties and geometrical resolution, in order to facilitate minimally invasive penetration into the skin[15,65]. Recent improvements in material science as well as micro- and nano-fabrication strategies offer specific advantages for the development of MNAs in different biological applications. MNAs fabricated with the most widely used materials and approaches are highlighted in Figure 1.

Figure 1.

Common materials and fabrication technologies used for development of MNAs in biomedical applications. (A) Solid[66] and (B) hollow[67] silicon MNAs fabricated using reactive ion etching. (C) Glass microneedles fabricated by micro-pulling fire-polished borosilicate glass pipettes[68]. Inset shows the assembled microneedles in an MNA. (D) Al₂O₃ ceramic MNAs fabricated by micromolding[69]. Inset demonstrates their porous structure. (E) Metal MNAs fabricated by assembly of 31 G stainless steel needless on a polystyrene support backing layer[70]. (F) Methacrylated hyaluronic acid MNAs fabricated by molding precursor followed by UV crosslinking[71]. Inset shows a patch consisting of a 10x10 array of these microneedles. (G) Complex-shaped polymeric MNAs fabricated out of PEGDA using an improved photolithography approach[72]. (H) Long SU-8 MNAs produced by drawing lithography[73]. (I) 3D printed resin-based flexible MNAs using an FDM approach[12]. (J) Polymeric microneedles fabricated using SLA printing (left) followed by inkjet printing of drug loaded polymer coating (right)[74]. (K) PEGDA MNAs with backward-facing barbs fabricated using DLP-based 3D printing for improved adhesion[71]. (L) High resolution MNAs with sharp tips fabricated using TPP[75]. Subfigures were reproduced with permission from National Academy of Sciences[66] (Copyright (2003) National Academy of Sciences, U.S.A.), Elsevier[68,69,74], Public Library of Science[70], Wiley[12,71,73], and Nature[67,72,75] publishing groups.

The selection of an MNA fabrication approach is primarily dependent on the material being used. Silicon was the first material used for fabrication of microneedle structures during the advent of microfabrication technologies in 1990s[76]. Biocompatibility, high mechanical strength, material stability, and well-developed microfabrication strategies make silicon an attractive candidate for MNA production. Solid[77] and hollow[67] silicon MNAs are usually fabricated using various dry and wet etching methods (Figure 1A, B). Although these approaches offer nano-scale resolution, they require clean-room facilities and multiple time-consuming fabrication steps, which renders their fabrication expensive and low throughput. Furthermore, the brittle nature of silicon-based structures makes them susceptible to fracture, leaving residuals in the skin and inducing an immune response. To overcome the limitations of complicated and expensive clean-room microfabrication, glass microneedles were implemented for transdermal drug delivery[68] (Figure 1C). Glass is cost-effective, chemically inert, and has high mechanical strength, though it is brittle. Fabrication of glass microneedles is based on micro-pulling of glass tubes. While this technique does not require the use of expensive tools, it is a low throughput process.

Ceramics are another group of materials used for fabrication of MNAs. Ceramics have been employed not only due to their significant mechanical strength, but also because of their porous structure, which enables therapeutics to be loaded and subsequently delivered upon insertion[69] (Figure 1D). Furthermore, ceramic precursor solutions are easily micromolded, offering a simple and low cost fabrication process[78]. However, tip fracture and the subsequent immune response remain a key challenge in the application of these structures[65]. In contrast to silicon, glass, and ceramics, metals have both significant mechanical strength and ductility. Since metals have been used widely in medical applications, metallic MNAs face a less challenging path toward approval and commercialization[79]. Metallic MNAs have been fabricated mainly by manual assembly of small, commercially available needles on a supporting layer[70] (Figure 1E), laser cutting[80] or etching[81].

Polymeric MNAs have been introduced as a promising alternative to these materials, as they offer various advantages including biodegradability, porous structure, low cost, and compatibility with various microfabrication strategies. Although polymeric MNAs have mainly been fabricated using the simple and low cost micromolding approach[71] (Figure 1F), these structures can be formed in high resolution using photolithography[72] (Figure 1G), with high aspect ratio using drawing lithography[73] (Figure 1H), and with high throughput using hot embossing[82] or microinjection[83]. Recently, 3D printing has been introduced as a powerful strategy for fabrication of polymeric microneedles[84].

3D printing is a versatile approach that utilizes computer aided design (CAD) models for layer by layer fabrication of complex constructs[85]. Various 3D printing approaches including fused deposition molding (FDM)[12], stereolithography (SLA)[74], digital light projecting (DLP)[71], and inkjet 3D printing[74] have been used for fabrication of microneedles (Figure 1I–K). The main challenges in 3D printing MNAs have been the toxicity of utilized resins and the resolution of the printing strategy, but these challenges have been resolved through the development of biocompatible resins and high resolution printing strategies such as two photon polymerization (TPP)[75] (Figure 1L). 3D printing of MNAs allows the realization of structures with complex architectures. For example, the fabrication of arrays with different needle lengths has been reported[86]. In the same study, the fabrication of MNAs with precise control over the distribution of medication has been reported by combining 3D printing of a rigid resin-based exterior and a selectively filled hydrogel interior[86].

Regardless of the fabrication strategy, the structure of the MNAs are designed based on their mechanism of action in either delivery of therapeutics or uptake of biomarkers from interstitial fluid. Different designs include: (i) solid MNAs which pierce the skin and create microchannels to expose lower layers of skin for enhanced delivery of therapeutics[87] or uptake of interstitial fluid[88]; (ii) MNAs coated with an additional layer containing therapeutics to be delivered upon MNA insertion[89] or capture biomolecules for targeted diagnosis[90,91]; (iii) hollow MNAs usually used for the delivery[12] or uptake[92] of large amount of fluids; (iv) dissolving/degrading MNAs which release their encapsulated therapeutics upon dissociation[93]; (v) detachable MNAs for delivery of drugs[94] and cells[95]; and (vi) porous MNAs for release of therapeutics[96] or absorption of interstitial fluid[97] based on diffusion.

5. MNA-based bandages for wound healing

Various MNA designs have been applied for the delivery of different drugs, vaccines, and even cells in medical therapies[100,101]. MNAs enhance the efficiency of therapeutic transportation by bypassing various physical and chemical barriers, delivering treatment to the targeted area with improved spatial distribution[102]. In chronic wounds, the presence of eschar, discharge of exudate, and a harsh chemical microenvironment rich with various enzymes disrupt the delivery of topically administrated therapeutics[8,103]. Therefore, MNA systems have recently been implemented to increase the availability of therapeutics in the wound bed with a controlled spatial distribution by controlling the drug content of individual needles. Similar to other drug delivery tools, the strategies by which MNAs deliver therapeutics to the wound bed can be classified into passive, active, and smart release, based on the level of controllability on the temporal release profile. Using these different strategies, researchers have utilized MNAs to deliver various therapeutics that promote wound healing and resolve the major dysfunctions typically present in chronic wound microenvironments.

5.1. Passive delivery of biological materials

Passive release is the simplest mechanism available to deliver biologics from MNAs. Although release kinetics cannot be altered once a passive release MNA is in place, they can be optimized during development by modifying different components of the system design. This technique has been utilized to address a number of different chronic wound symptoms, including infection and low vascularization. A straightforward approach to creating antibacterial MNAs is the fabrication of MNAs out of an antibacterial material, or encapsulation of an antibiotic agent within the MNA structure. Yi et al. combined both concepts by loading zinc nitrate (Zn²⁺) into chitosan (CS) MNs[104]. The combined antibacterial properties of these compounds outperformed unloaded CS MNs in elimination of S. aureus and E. coli. Importantly, the engineered MNAs were able to kill more bacteria than a topically applied film of the same composition, highlighting the value of piercing the biofilm in eradicating infection.

A technique offering a better control over the drug release from a MNA dressing was developed by Park et al. through the incorporation of green tea extract (GT) as an antibiotic compound into hyaluronic acid (HA)-based microneedles[105]. The degradation rate, and therefore the release kinetics, could be tuned by manipulating the concentration of HA. A 30% HA and 70% GT mixture was found to result in a sustained release of GT for 72 hr, which caused elimination of several gram-positive and -negative bacterial strains while maintaining the viability of CHO-K1, 293T, and C2C12 cell lines. The application of the engineered MNAs in an infected rat wound model further demonstrated a significant decrease in bacterial load, leading to improved wound closure compared to both infected and uninfected control groups.

MNAs can also be used for the delivery of cells to modulate the wound environment. In an important study, dissolvable MNAs were used for the delivery of stem cells, which were intended to regulate the environment through secretion of various growth factors, exosomes, and cytokines for enhanced angiogenesis, recruitment of various cells, immune modulation, and remodeling of ECM[95]. To circumvent the challenges associated with stem cell delivery, while harnessing their potential, Lee et al. created hybrid microneedles consisting of PLGA shells filled with cell-laden GelMA cores on a detachable backing layer[95] (Figure 2). Using this strategy, stem cells were delivered to the wound bed of cutaneous skin injuries on mice in a minimally invasive manner. The stiff PLGA shell facilitated the penetration of microneedles into the mouse skin, while the GelMA core offered a biologically suitable scaffold that supported cellular functionality (Figure 2A). The controlled degradation of the PLGA shell (>2 weeks) further prevented excessive cell migration, ensuring the localized secretion of bioactive factors within the wound bed (Figure 2B). Improved wound healing with enhanced angiogenesis was reported in a mouse skin wound model as a result of the MNA-based stem cell delivery (Figure 2C, D). Although promising, the translation of this strategy could be challenging due to the limited stiffness of the microneedles for insertion into human skin. The study also used a prolonged photocrosslinking duration for enhanced mechanical strength, which could significantly lower the viability of the encapsulated cells.

Figure 2.

Stem-cell laden hydrogel MNAs for enhanced wound healing. (A) The core-shell structure of MNAs offer high mechanical strength for facile insertion as well as cell-favorable scaffolding material for preserving the functionality of stem-cells. (B) The mechanism of MNA-based stem cell delivery action for enhanced regeneration. (C) Improved wound healing characterized by enhanced wound closure and re-epithelialization. (D) Enhanced wound bed angiogenesis as a result of MNA-based stem cell delivery. Reproduced with permission from Wiley[95].

5.2. Actively triggered systems

Although passive MNAs offer simple and easily applicable point of care (POC) systems, they cannot comply with the requirements associated with the dynamic nature of the wound environment throughout the healing process. Therapeutics that can improve healing at one stage of healing may be useless or even harmful during a different stage. A promising alternative could be the application of active MNA systems. An active MNA-based dressing was developed for treatment of infected wounds[106]. In this approach, photosensitising methylene blue was loaded in a dissolving Gantrez® AN-139 MNA to perform photodynamic antimicrobial chemotherapy (PACT). This technique uses light to activate a photosensitizing drug, causing it to release reactive radicals, which are able to break down the targeted bacteria. In this study, a release experiment was conducted to determine the minimum and maximum amounts of methylene blue the MNA could release. Then, cultures of S. aureus, E. coli, and C. albicans were exposed to these concentrations of methylene blue, and it was determined that even the minimum concentration was highly effective at killing all three strains when activated using PACT. Although promising, this study did not examine the direct effect of MNA mediated methylene blue delivery on bacterial growth, instead quantifying release from the MNA and then conducting in vitro studies that utilized direct application of methylene blue on bacteria cultures. Additionally, while the authors state that the photosensitizer is selective to microbial cells, toxicity to human cells was not explored in this study.

In another example of active MNA-based delivery, Zhang et al.[111] developed an MNA system enabling on demand oxygen release for improved wound healing (Figure 3A–E). As mentioned previously, hypoxia is one of the major challenges in chronic wound healing, causing dysregulated cellular behavior and creating a hospitable environment for bacterial infection. To deliver the oxygen required for proper healing to the wound bed, GelMA microneedles were supplemented with black phosphorus quantum dots (BP-QDs) and hemoglobin (Hb), backed with a PVA supporting layer, and inserted into the wounded tissue. Upon insertion, the PVA backing was dissolved, leaving GelMA microneedles inside the wound bed. Near-infrared (NIR) irradiation was then employed to generate heat as a result of the BP-QDs photothermal effect, which consequently decreased the oxygen binding capacity of hemoglobin causing oxygen release into the wound bed (Figure 3A, B). The implemented strategy improved wound healing in diabetic rats, demonstrated by faster wound closure, more organized ECM remodeling, fewer inflammatory signals, and a higher degree of vascularization (Figure 3D, E). However, one limitation of this strategy could be the sensitivity of the release mechanism to body temperature before initiation of the stimuli signal for the active delivery of the therapeutic.

Figure 3.

Active MNA-based drug delivery systems for treatment of diabetic wounds. (A-E) On demand delivery of oxygen into the wound bed using NIR exposure-responsive MNAs[111]. (A) The MNA patch mechanism of action. (B) Molded microneedles (top) and their drug loading capability demonstrated through encapsulation of fluorescent-labeled protein (bottom). The effect of oxygen delivery using the proposed strategy on healing of wounded diabetic rats, indicated by the thickness of regenerated (C) granulation tissue and (D) epithelium, as well as (E) level of angiogenesis. (F-H) Controlled pumping of therapeutics through hollow MNAs into the wound bed using a POC system[12]. (F) A microfluidic flexible patch integrated with MNAs for distribution of various drugs in the wound bed. Inset shows an SEM image of 3D printed hollow microneedles. (G) The pumping system wirelessly controlled by a smartphone to enable on-demand delivery of therapeutics. (H) Enhanced healing of wounds in diabetic mice through MNA-based intradermal delivery of VEGF. Reproduced with permission from American Chemical Society[111] and Wiley[12].

An alternative to stimuli-responsive materials for active drug release is the application of external pumping systems integrated with hollow MNAs. Miniaturized pumps have been integrated into wound dressings to facilitate high-volume, on-demand administration of various therapeutics[9]. To address the fact that multiple therapeutics need to be delivered deep into the wound bed during different stages of chronic wound healing, Derakhshandeh et al.[12] introduced a wirelessly controlled active MNA system (Figure 3F–H). Hollow MNAs were 3D printed from a biocompatible resin and bonded to a flexible microfluidic backing layer (Figure 3F). The flexible microfluidic device was then connected to a wearable control system with integrated miniaturized pumps (Figure 3G). Using this strategy, various therapeutics could be delivered into the wound bed independently. This MNA-based controlled injection could significantly enhance the delivery efficiency compared to topical administration. Given the crucial role of vascularization in physiological wound healing, VEGF was targeted in this study to improve chronic wound treatment. Transdermal VEGF delivery using the proposed strategy demonstrated a faster scratch closure in an in vitro chronic wound model compared to topical VEGF administration. Similarly, VEGF delivery using the developed strategy demonstrated enhanced wound closure rate (Figure 3G), angiogenesis, re-epithelialization and new hair growth in chronic wounds of diabetic mice.

5.3. Stimuli-responsive “smart” material systems

Another strategy in engineering MNA-based systems for enhanced wound healing is the use of smart materials that can respond to changes in the wound environment (Figure 4). Smart systems combine the user simplicity of passive release MNAs with the responsiveness of active systems, able to react dynamically to the fluctuating needs of a chronic wound. An example of this strategy was recently demonstrated in a study from Chi et al., wherein MNAs were fabricated from a combination of antibacterial chitosan and VEGF-loaded NIPAM hydrogel[107]. Since NIPAM is temperature sensitive[112], the increased temperature of an inflamed chronic wound triggers VEGF release from the porous hydrogel network (Figure 4A, B). The combination of chitosan with VEGF loading allows these needles to target both bacterial infection and lack of vascularization in chronic wounds. Antibacterial testing showed the MNA’s ability to kill the majority of bacteria in both S. aureus and E. coli cultures (Figure 4C). When the developed MNA-patch was applied to severely infected wounds in rats, the VEGF loaded MNA group showed the most wound closure and the thickest granulation tissue (Figure 4D) at the end of the study, as well as demonstrating increased angiogenesis and collagen deposition, and downregulated inflammatory response.

Figure 4.

MNA-based smart drug delivery for improved wound healing. (A-D) Antibacterial and thermo-responsive MNAs[107]. (A) The mechanism of VEGF release into the wound bed upon temperature increase during wound inflammation. (B) MNA fabricated by molding chitosan followed by impregnation of VEGF-loaded NIPAM. (C) Antibacterial properties of chitosan. (D) Infected wound healing in rats through the application of the developed smart MNA bandage. (E-G) Bacteria-responsive MNA system for treatment of infected wounds[109]. (E) The MNA patch mechanism of action. (F) Fabricated MNA (top) loaded with fluorescent-labeled drugs (bottom). (G) Application of smart system for elimination of bacterial biofilms. Reproduced with permission from Elsevier[107] and American Chemical Society[109].

Bacteria-responsive smart materials have also been used for the fabrication of MNA-systems for treatment of infected wounds. Mir et al. loaded Carvacro (CAR), an effective antibiotic against resistant bacterial strains, into PCL microparticles, which were then incorporated into PVA/PVP MNAs [108]. The PCL nanoparticles increased antibiotic retention in the wound bed and provided on-demand release as a result of increased degradation in the presence of lipase, which is secreted by bacteria. The integration of drug-loaded PCL nanoparticles into an MNA facilitated their delivery deeper into an ex vivo porcine skin model, allowing the antibiotic direct access to the infection site. Although incorporating CAR into the nanoparticles improved the reduction of bacterial burden, MNAs loaded with CAR directly were still able to kill bacteria. In another study utilizing a MNA containing bacteria-reactive nanoparticles, chloramphenicol (CAM) was loaded into gelatin nanoparticles that would dissociate in response to gelatinase, another enzyme secreted by bacteria[109] (Figure 4E, F). The use of nanoparticles limited off-target toxicity, while the MNA-based delivery strategy significantly increased drug penetration of the biofilm. An in vitro antibacterial test demonstrated that the MNA containing CAM laden nanoparticles performed better than free CAM solution in killing bacteria (Figure 4G). A similar approach was reported by Permana et al. in the development of PLGA/PCL MNAs supplemented with bacteria responsive PCL nanoparticles with a chitosan coating[110]. The PCL nanoparticles were loaded with Doxycycline, an antibacterial drug, which was released when lipase secreted from bacteria increased the degradation of the PCL. An ex vivo pig skin study showed application of the MNA dressing resulted in essentially complete elimination of bacteria from a biofilm. While smart MNAs are exciting for regulating the wound environment, they can carry a limited amount of drug and may be sensitive to premature therapeutic release if they are triggered by some off-target environmental stimuli.

5.4. Mechanically interacting systems

MNAs have also been used to improve wound closure physically by applying mechanical forces. MNAs have been engineered to offer mechanical interlocking upon insertion as a result of swelling[113,114]. Interlocking MNAs can help wound healing by protecting the tissue from mechanical stress and inducing wound closure. To achieve this, MNAs with a hybrid core-shell structure consisting of a swellable hydrogel shell and a non-swellable core were utilized. Upon insertion, the hydrogel absorbs ISF and induces physical entanglement through the swelling of microneedle tips. The MNA patches significantly increased resistance against bacterial infiltration compared to surgical staples[114]. Additionally, insignificant tissue damage and limited scar formation after application of MNAs enhanced the mechanical strength of the healed tissue which consequently decreased the susceptibility of wound reopening[113]. More importantly, application of MNAs enhanced both internal and external wound closure rates compared to suture application in rats. A more organized ECM orientation, fewer inflammatory markers, and increased re-epithelialization were observed as a result of MNA application compared to suturing[113]. In addition to their application as a suture replacement, mechanically self-interlocking needles are appealing for incorporation into chronic wound dressings as they can hold the MNA in place for long term drug delivery. Although MNAs have primarily been considered for delivery of therapeutics with a controlled spatiotemporal profile, there is research demonstrating the benefits of drug-free MNA application on wound healing[98,99]. It has been suggested that “microneedling” can enhance wound debridement and induce mechanical stimulation to promote cell proliferation, which expedites the proliferation stage and promotes remodeling of the ECM, though the exact mechanism and efficacy is still a subject of debate [98,99].

6. Expert opinion on application of MNA-based dressings for chronic wound healing

Treatment of chronic wounds has remained a major healthcare challenge, but the use of MNA-based dressings has drawn noticeable attention as a strategy for addressing it. These systems are robust and can be easily integrated with existing wound dressings and can carry different types of active molecules. The ability of polymers used in the fabrication of some of these MNAs to preserve the activity of drugs over time is an additional advantage of such dressings. There are several studies indicating the benefit of MNA-mediated delivery of therapeutics for the treatment of chronic wounds. However, only a few of them investigated the potential superiority of MNA-based delivery in comparison to the topical delivery of the same therapeutics. In addition, to the best of our knowledge, no one has investigated the effect of MNAs size and density on treatment outcome.

While the use of MNAs for wound treatment shows great promise, the field is very young, with a limited number of studies examining this specific application of the technology. In addition, while many of these studies target treatment of chronic wounds, the chronic wound models used for testing these platforms present limited pathophysiological characteristics, such as infection[107] or diabetic comorbidity[12]. A more holistic and standard model will need to be utilized as the field progresses. MNAs will not be a cure-all for chronic wounds. In some cases, especially those needing significant tissue debridement, MNA dressings alone will not be enough to address all the demands of the wound, which may require treatments such as skin grafts or other tissue engineered constructs that can create a temporary scaffold supporting tissue ingrowth and wound healing. The very micro scale of MNAs that makes them appealing for their painlessness and lack of additional tissue trauma also means that they cannot deliver therapeutics beyond the surface layers of tissue. For this reason, chronic wounds exhibiting deep tissues pathologies, such as infections that have reached bone, cannot be treated with MNAs and likely require more drastic interventions. Another intrinsic limitation of MNAs’ microscale size can be their low total therapeutic loading capacity. This can be overcome in some instances through the use of hollow microneedles and controlled pumping, which provides the dual capability of controlled continuous release and high therapeutic throughput as needed[12]. However, these more complicated systems add complexity and bulk to a technology intended to be compact and easy-to-use, which must be balanced against their improved functionality. As MNA dressings grow more intricate and are able to serve multiple long-term functions in wound healing, the issue of potential immune response will need to be considered. Despite these challenges, MNAs have the potential to overhaul chronic wound treatment, an area of healthcare that has failed to significantly advance for years.

Most of the studies in the field have been focused on the delivery of therapeutics including growth factors, small drug molecules, and stem cells. Stem cells in particular are of interest due to the multifunctionality of their secretome but can carry the risk of immunogenicity and uncontrolled stem cell differentiation. While most of patches have been designed to execute passive, controlled release of compounds, for the treatment of chronic wounds with a dynamic environment, more controllable systems are required. Thus, two strategies have been proposed: smart materials which self-respond to the changes in their environment and active systems that can precisely control the type of drug, time of delivery, and dosage delivered. Smart material-based solutions are exciting to use, but they can carry a limited drug quantity. It is also not clear when the active molecules are consumed within the patch. Active systems are typically more expensive and bulkier than MNAs made of smart materials, but they allow enhanced control over the delivery rate.

In engineering smart materials, it is important to carefully select the environmental signals a MNA will react to. In addition to the temperature and bacteria responsive MNAs discussed above, pH and oxygen sensitive materials have the potential to be incorporated into MNA-based smart dressings for the treatment of chronic wounds. pH is a key indicator of wound progression[115] and as such has been a common target for topical sensors. Skin is normally acidic but after injury damages the skin, bacterial colonization can decrease the acidity of the wound environment, making a high pH a good indicator of infection[116,117]. pH responsive materials have already been utilized in MNAs for drug[118], vaccine[119], and insulin delivery[120] and could be easily translated into use for chronic wound care. Furthermore, the biological relevance of oxygen is well established in numerous cellular functions including collagen production, angiogenesis, fighting infection, and proliferation[121]. MNAs utilizing hypoxia sensitive vesicles for the smart release of insulin have been developed[122]. This technology could also be adapted to create hypoxia responsive MNAs for addressing the impediments to chronic wound healing presented by lack of oxygen.

The effective use of controlled systems requires an understanding of the changes in the wound environment. Thus, the necessity of sensing the wound environment becomes more pronounced. MNA-based sensors have been developed for various applications. Colorimetric MNAs have been used to track cancer antigens[123], glucose[124–126], and estrogen biomarker[91]. Another strategy for MNA based sensing involves sampling ISF and removing it for secondary analysis[88]. This has been applied for measurement of glucose and vancomycin[127], glucose and insulin[71], and RG6[128]. Electrochemical sensors can detect H₂O₂[129], glucose[130–132], temperature and other bio-signals such as ECG[133], and pH[134]. A similar concept has been applied for directly monitoring electrical fields generated when skin is wounded[135]. However, MNA-based sensors have not been used, so far, for wound care applications. This is an area that should see significant progress in the next few years. In addition, the ability to control spatial distribution of different therapeutics within the wound bed to support functions of various cells is important and is expected to be investigated. Perhaps one key challenge in sensor integrated dressings is identification of markers highly specific to chronic wounds. MNA-based wound dressings are easy-to-use and are similar to other FDA-approved MNA-based technologies. The similarity in their fabrication and operation should facilitate their translation of these patches into clinical practice. Despite moderate translational complications, MNAs remain an exciting strategy for the treatment of chronic wound that have the potential to overcome continuing challenges in the field.

Article highlights.

• Chronic wounds are a major healthcare challenges worldwide, imposing devastating human and financial burdens.

• While several therapeutic factors have been introduced for treatment of chronic wounds, their clinical success has been limited due to inefficient delivery to the wound bed.

• MNA-based drug delivery can bypass physiochemical barriers usually present in chronic wound environment, thereby enhancing the local availability of the therapeutics in the wound.

• MNAs have been used for passive delivery of antibacterial agents and even stem cells to improve the quality and speed of wound healing, though the dynamic nature wound environment requires a more flexible delivery system.

• Active and smart drug delivery strategies integrated with MNAs can easily comply with the dynamic pathophysiological conditions of chronic wounds and further enhance wound healing.

• Development of new chronic wound models and multimodal MNAs can provide more opportunities for translation of MNA based drug delivery in clinical treatment of chronic wounds.