Abstract
Chronic nonhealing wounds are one of the major and rapidly growing clinical complications all over the world. Current therapies frequently require emergent surgical interventions, while abuse and misapplication of therapeutic drugs often lead to an increased morbidity and mortality rate. Here, we introduce a wearable bioelectronic system that wirelessly and continuously monitors the physiological conditions of the wound bed via a custom-developed multiplexed multimodal electrochemical biosensor array and performs noninvasive combination therapy through controlled anti-inflammatory antimicrobial treatment and electrically stimulated tissue regeneration. The wearable patch is fully biocompatible, mechanically flexible, stretchable, and can conformally adhere to the skin wound throughout the entire healing process. Real-time metabolic and inflammatory monitoring in a series of preclinical in vivo experiments showed high accuracy and electrochemical stability of the wearable patch for multiplexed spatial and temporal wound biomarker analysis. The combination therapy enabled substantially accelerated cutaneous chronic wound healing in a rodent model.
INTRODUCTION
Chronic wounds are characterized by impaired or stagnant healing, prolonged and uncontrolled inflammation, as well as compromised extracellular matrix (ECM) function (1–3). Over 6.7 million people in the United States alone suffer from chronic nonhealing wounds including diabetic ulcers, nonhealing surgical wounds, burns, and venous-related ulcerations (4, 5), causing a staggering financial burden of over $25 billion per year on the health care system (6). Chronic wound healing is a highly complex biological process consisting of four integrated and overlapping phases: hemostasis, inflammation, proliferation, and remodeling (1–3). Current therapies including skin grafts, skin substitutes, negative pressure wound therapy, and others can be beneficial but frequently require procedures or surgical intervention (7). Microbial infection at the wound site can severely prolong the healing process and lead to necrosis, sepsis, and even death (3). Both topical and systemic antibiotics are increasingly prescribed to patients suffering from chronic nonhealing wounds, but the overuse, abuse, and misapplication of antibiotics often lead to an escalating drug resistance in bacteria, causing a drastic increase in morbidity and mortality rates (8). As an alternative therapeutic approach, electrical stimulation has shown to have a substantial effect on the wound healing process, including stimulating fibroblast proliferation and differentiation into myofibroblasts and collagen formation, keratinocyte migration, angiogenesis, and attracting macrophages (9, 10). However, currently reported electrical stimulation devices usually require bulky equipment and wire connections, making them highly challenging for practical clinical use. More effective, fully controllable, and easy-to-implement therapies are critically needed for personalized treatment of chronic wounds with minimal side effects.
At each stage of healing process, the chemical composition of the wound exudate changes substantially, indicating the stage of healing and even the presence of an infection (11–13). For example, increased temperature is associated with bacterial infection, and changes in temperature can provide information on various factors relevant to healing, inflammation, and oxygenation in the wound bed; acidity (pH) indicates a healing state with balanced protease activities and effective ECM remodeling, moreover, elevated pH in wound environment can be a sign of infection; elevated uric acid (UA) indicates wound severity with excessive reactive oxygen species and inflammation and shows immune system responding to inflammatory cytokines (14); lactate and ammonium are crucial markers for soft-tissue infection diagnosis and angiogenesis in diabetic foot ulcers (15); wound exudate glucose has a strong correlation with blood glucose and bacterial activities (16), providing crucial therapeutical guidance for clinical diabetic wound treatment.
Recent advances in digital health and flexible electronics have transformed conventional medicine into remote at-home health care (17–23). Wearable biosensors could allow real-time and continuous monitoring of physical vital signs and physiological biomarkers in various biofluids such as sweat, saliva, and interstitial fluids (18–21, 24–30). In general, an ideal wound dressing should provide a moist wound environment, offer protection from secondary infections, remove wound exudate, and promote tissue regeneration. Despite the promising prospects opened by the wearable technologies (31–37), major challenges exist to realize their full potential toward practical chronic wound management applications: the chronic nonhealing wounds pose high requirement on the flexibility, breathability, and biocompatibility of the wearable devices to protect the wound bed from bacterial infiltrations and infection and modulate wound exudate level; the complex wound exudate matrix could substantially affect the biosensor performance, and thus, there are few reports on prolonged evaluation of biosensors in vivo (13, 31); personalized wound management demands both effective wound therapy and close monitoring of crucial wound healing biomarkers in the wound exudate; the absence of miniaturized user-interactive fully integrated closed-loop wearable systems and the evaluation of such systems in vivo impede their practical use.
To address these challenges, here, we introduce a fully integrated wireless wearable bioelectronic system that effectively monitors the physiological conditions of the wound bed via multiplexed and multimodal wound biomarker analysis and performs combination therapy through electro-responsive controlled drug delivery for anti-inflammatory antimicrobial treatment and exogenous electrical stimulation for tissue regeneration (Fig. 1, A and B). The wearable patch is mechanically flexible, stretchable, and can conformally adhere to the skin wound throughout the entire wound healing process, preventing any undesired discomfort or skin irritation. Because of the wound’s complex pathophysiological environment, compared to previously reported single-analyte sensing, multiplexing analysis of wound exudate biomarkers can provide more comprehensive and personalized information for effective chronic wound management. In this regard, a panel of wound biomarkers including temperature, pH, ammonium, glucose, lactate, and UA were chosen on the basis of their importance in reflecting the infection, metabolic, and inflammatory status of the chronic wounds. Real-time selective monitoring of these biomarkers in complex wound exudate could be realized in situ using custom-engineered electrochemical biosensor arrays (Fig. 1C). The wearable system’s capabilities of multiplexed monitoring, biomarker mapping, and combination therapy were evaluated in vivo over prolonged periods of time in rodent models with infected diabetic wounds. The multiplexed biomarker information collected by the wearable patch revealed both spatial and temporal changes in the microenvironment as well as inflammatory status of the infected wound during different healing stages. In addition, the combination of electrically modulated antibiotic delivery with electrical stimulation on the wearable technology enabled substantially accelerated chronic wound closure.
RESULTS
Design of the fully integrated stretchable wearable bioelectronic system
The disposable wearable patch consists of a multimodal biosensor array for simultaneous and multiplexed electrochemical sensing of wound exudate biomarkers, a stimulus-responsive electroactive hydrogel loaded with a dual-function anti-inflammatory and antimicrobial peptide (AMP), as well as a pair of voltage-modulated electrodes for controlled drug release and electrical stimulation (Fig. 1, B and C). The multiplexed sensor array patch is fabricated via standard microfabrication protocols on a sacrificial layer of copper followed by transfer printing onto a poly[styrene-b-(ethylene-co-butylene)-b-styrene] (SEBS) thermoplastic elastomer substrate (figs. S1 and S2). The serpentine-like design of electronic interconnects, and the highly elastic nature of SEBS enables high stretchability and resilience of the sensor patch against undesirable physical deformations (Fig. 1, D and E). The flexible bandage seamlessly interfaces with a flexible printed circuit board (FPCB) for electrochemical sensor data acquisition, wireless communication, and programmed voltage modulation for controlled drug delivery and electrical stimulation (Fig. 1, F to H, and figs. S3 to S5). The wireless wearable device can be attached to the wound area with firm adhesion, allowing the animals to move freely over a prolonged period (movie S1 and figs. S6 and S7).
Design and characterization of the soft sensor array for multiplexed biomarker analysis
The array of flexible biosensors was custom developed to allow real-time multiplexed monitoring of the biomarkers in complex wound exudate. The continuous and selective measurement of glucose, lactate, and UA is based on amperometric enzymatic electrodes with glucose oxidase, lactate oxidase, and uricase immobilized in a highly permeable, adhesive, and biocompatible chitosan film, respectively (Fig. 2A). Electrodeposited Prussian blue (PB) serves as the electron-transfer redox mediator for the enzymatic reaction, which allows the biosensors to operate at a low potential (~0.0 V) to minimize the interferences of oxygen and other electroactive molecules. Because of the complex and heterogeneous composition of wound fluid (e.g., high protein levels, local and migrated cells, and exogenous factors such as bacteria) (13), previously reported enzymatic sensors suffer from severe matrix effects and fail to accurately measure the target metabolite levels in untreated wound fluid (figs. S8 and S9 and note S1). Moreover, high levels of metabolites in diabetic wound fluid, especially glucose (up to 50 mM), pose another major challenge to obtain linear sensor response in the physiological concentration ranges. To address these issues and achieve accurate wound fluid metabolic monitoring, increase sensor range, and minimize biofouling effects, we explored the use of an outer porous membrane that serves as a diffusion limiting layer to protect the enzyme, tune response, increase operational stability, as well as enhance the linearity and sensitivity magnitude of the sensor. We fabricated our enzymatic glucose oxidase/chitosan/single-walled carbon nanotubes (GOx/CS/MWCNT) glucose sensor with additional porous membrane coatings including CS, poly(ethylene glycol) diglycidyl ether (PEGDGE), Nafion, and polyurethane (PU) (fig. S9). As expected, the addition of diffusion layers indeed improves the sensor’s linear range in simulated wound fluid (SWF). However, CS-, PEGDGE-, and Nafion-coated sensors did not show reliable responses in wound fluid upon the addition of glucose. The PU-based enzymatic sensors showed the highest linearity over the wide physiological concentration range as well as high reproducibility in complex wound fluid matrix (fig. S10). The amperometric current signals generated from the PU-coated enzymatic glucose, lactate, and UA sensors are proportional to the physiologically relevant concentrations of the corresponding metabolites in SWF with sensitivities of 16.34, 41.44, and 189.60 nA mM−1, respectively (Fig. 2, B to D). Continuous monitoring of ammonium is based on a potentiometric ion-selective electrode where the binding of ammonium with its ionophore results in an electrode potential log-linearly corresponding to the target ion concentration with a sensitivity of 59.7 mV decade−1 (Fig. 2, E and F). Similarly, the pH sensor uses an electrodeposited polyaniline film as the pH-sensitive membrane and shows a sensitivity of 59.7 mV per pH (Fig. 2G). For all chemical sensors, a polyvinyl butyral (PVB)–coated Ag/AgCl electrode was used as the reference electrode that provides a stable voltage independent of the variations of wound fluid compositions (24). A gold microwire-based resistive temperature sensor is integrated as part of the sensor array and shows a sensitivity of approximately 0.21% °C−1 in the physiological temperature range of 25° to 45°C (Fig. 2H).
Considering that other electrolytes and metabolites present in wound fluid may negatively affect the sensor outputs, we examined the selectivity of the sensor array consisting of all six sensors. As illustrated in Fig. 2I, the addition of nontarget electrolytes and metabolites did not trigger any substantial interference to the sensor response. Moreover, all biosensors showed high selectivity over nonspecific compounds when evaluated in SWF (fig. S11). It should be noted that while temperature has negligible effects on the potentiometric sensors, it substantiallyinfluences the performance of the enzymatic sensors due to the temperature-dependent enzyme activities (fig. S12). Moreover, our data show that the medium pH could also affect the performance of enzymatic sensors (fig. S13). With pH and temperature sensors integrated into the wearable patch, we are able to perform real-time adjustments and calibration of the enzymatic biosensors based on temperature and pH variations to realize accurate wound metabolite analysis.
Owing to the soft SEBS substrate and the serpentine-like design of electronic interconnects, the wound patch showed excellent mechanical flexibility and stretchability, which are essential to maintaining good contact with the skin in vivo during the chronic wound healing process. Negligible alterations in the sensor responses before and under unidirectional tensile stretching (Fig. 2I) and after repetitive mechanical bending (fig. S14) were observed, indicating highly consistent sensor performance under various physical deformations.
As the sensor patch is designed for long-term in vivo use, its cytocompatibility and biocompatibility are of great importance. Cell viability and metabolic activity of the cells seeded on a multiplexed sensor array were analyzed using a commercial live/dead kit and PrestoBlue assay, respectively (Fig. 2, K to N, and fig. S15). The high cell viabilities shown in the representative live/dead staining images of human dermal fibroblasts (HDFs) and normal human epidermal keratinocytes (NHEK) cells (Fig. 2, K to M, and fig. S15), along with the consistently increased cell metabolic activities (Fig. 2N) over multiday culture periods, indicate the high cytocompatibility of the soft sensor patch.
Characterization of the therapeutic capabilities of the wearable patch in vitro
In addition to the multiplexed and multimodal biosensing, the wearable patch is able to perform combination treatment of chronic wounds through drug release from an electroactive hydrogel layer and electrical stimulation under an exogenic electric field, both controlled by a pair of voltage-modulated electrodes (Fig. 3, A to C). The electroactive hydrogel consists of chondroitin 4-sulfate (CS), a sulfated glycosaminoglycan composed of units of glucosamine, cross-linked with 1,4-butanediol diglycidyl ether (fig. S16). Because of the shear-thinning behavior of the prepolymer solution, the hydrogel can be precisely fabricated via three-dimensional (3D) printing (fig. S17). The negatively charged CS hydrogel is an ideal choice for loading and controlled release of positively charged large biological drug molecules based on an electrically modulated “on/off” drug release mechanism (Fig. 3B). Here, an AMP, thrombin-derived c-terminal peptide-25 (TCP-25) (38), was loaded within the CS hydrogel network through the electrostatic interactions with the polymer backbone, with up to 15% loading efficiency (Fig. 3D). The highly porous hydrogel network under equilibrium swelling could further enhance the drug loading efficiency (fig. S18). Under an applied positive voltage, the electroactive hydrogels will be rapidly protonated, resulting in anisotropic and microscopic contraction followed by syneresis/expelling of water from the gel (39) and consequently allowing a controlled release of the TCP-25 AMP (Fig. 3, E and F, and figs. S19 and S20). In addition, the electrical field will also facilitate the diffusion of positively charged AMP out of the stimuli-sensitive CS hydrogel toward the cathode due to electrophoretic flow (40)….
