A stretchable wireless wearable bioelectronic system for multiplexed monitoring and combination treatment of infected chronic wounds

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.

Fig. 1.. A wireless stretchable wearable bioelectronic system for multiplexed monitoring and treatment of chronic wounds.

(A) Schematic of a soft wearable patch on an infected chronic nonhealing wound on a diabetic foot. (B) Schematic of layer assembly of the wearable patch, showing the soft and stretchable poly[styrene-b-(ethylene-co-butylene)-b-styrene] (SEBS) substrate, the custom-engineered electrochemical biosensor array, a pair of voltage-modulated electrodes for controlled drug release and electrical stimulation, and an anti-inflammatory and antimicrobial drug-loaded electroactive hydrogel layer. (C) Schematic layout of the smart patch consisting of a temperature (T) sensor, pH, ammonium (NH₄⁺), glucose (Glu), lactate (Lac), and UA sensing electrodes, reference (Ref) and counter electrodes, and a pair of voltage-modulated electrodes for controlled drug release and electrical stimulation. (D and E) Photographs of the fingertip-sized stretchable and flexible wearable patch. Scale bars, 1 cm. (F and G) Schematic diagram (F) and photograph (G) of the fully integrated miniaturized wireless wearable patch. Scale bar, 1 cm. ADC, analog to digital converter; AFE, analog front end; PSoC, programmable system on chip; MUX, multiplexer; BLE, Bluetooth Low Energy. (H) Photograph of a fully integrated wearable patch on a diabetic rat with an open wound. Scale bar, 2 cm.

Fig. 2.. Design and characterization of the sensor array for multiplexed wound analysis.

(A to D) Schematic (A) and chronoamperometric responses of the enzymatic glucose (B), lactate (C), and UA (D) sensors in SWF. Insets in (B) to (D), the calibration plots with a linear fit. PB, Prussian blue; Sub, substrate; Prod, product; CE, counter electrode; WE, working electrode; RE, reference electrode; I, current. (E and F) Schematic (E) and potentiometric response (F) of an NH₄⁺ sensor in SWF. Insets in (F), the calibration plot with a linear fit. ISE, ion-selective electrode; PEDOT, poly(3,4-ethylenedioxythiophene); U, potential. (G) Potentiometric response of a polyaniline-based pH sensor in McIlvaine buffer. Insets, the calibration plot with a linear fit. (H) Resistive response of an Au microwire–based temperature sensor under temperature changes in physiologically relevant range in SWF. Insets, schematic of a temperature sensor and the calibration plot with a linear fit. All error bars in (A) to (H) represent the SD from three sensors. (I) Selectivity study of the multiplexed sensor array in SWF. Ten millimolar glucose, 50 μM UA, 1 mM lactate, and 1 mM NH₄⁺ were added sequentially to the SWF. (J) Responses of the multiplexed sensor array before and during mechanical stretching (15%) in SWF (pH 8) containing 10 mM glucose, 50 μM UA, 1 mM lactate, and 0.25 mM NH₄⁺. (K and L) Representative live (green)/dead (red) images of human dermal fibroblasts (HDFs) (K) and normal human epidermal keratinocytes (NHEKs) (L) cells seeded on the multiplexed sensor array and in PBS (control) after 1-day and 7-day culture. Scale bars, 200 μm. (M and N) Quantitative analysis of cell viability images (M) and cell metabolic activity (N) over a 7-day period after culture. RFUs, relative fluorescence units. Error bars represent the SD (n = 4).

Fig. 3.. Characterization of the therapeutic capabilities of the wearable patch in vitro.

(A to C) Schematic illustration of the therapeutic modules of the wearable patch (A) and the working mechanisms of the controlled drug delivery for antimicrobial treatment (B) and electrical stimulation for tissue regeneration (C). (D) Loading efficiency of dual-functional TCP-25 anti-inflammatory and AMP into CS electroactive hydrogel after 0.5- to 24-hour incubation. (E) Release amount of AMP from the hydrogel under programmed on-off electrical voltage (1 V, 10 min each step). (F) Long-term cumulative release of the AMP under programmed electrical modulation. (G and H) In vitro antimicrobial tests including zone of inhibition (G) and colony forming units (H) assays for electroactive hydrogels with and without TCP-25 AMP against multidrug-resistant Escherichia coli (MDR E. coli), P. aeruginosa, and methicillin-resistant Staphylococcus aureus (MRSA). (I to K) In vitro cytocompatibility assessment of TCP-25–loaded electroactive hydrogels using live/dead staining (I) and quantification of cell viability (J) and metabolic activity (K) for HDF and NHEK cells cultured in the presence of hydrogels. Scale bar, 100 μm. (L and M) Fluorescence images (L) and quantitative wound closure analysis (M) to evaluate the wearable patch’s therapeutic capability via electrical stimulation using an in vitro circular wound healing assay created by HDF cells. ES, electrical stimulation. A pulsed voltage was applied for electrical stimulation (1 V at 50 Hz, 0.01 s voltage on for each cycle). Scale bar, 500 μm. (N) Numerical simulation of the electrical field generated by the custom-designed electrical stimulation electrodes during operation. E, electrical field. Scale bar, 500 μm. Error bars represent the SD (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n ≥ 3). ns, not significant.

Fig. 4.. In vivo evaluation of the wearable patch for multiplexed wound biomarker monitoring in a wound model in diabetic mice.

(A) In vivo multiplexed analysis of the chemical composition of wound fluid using a wearable patch in an infected excisional wound model in a diabetic mouse. Infection and treatment were performed after the sensor recording on days 1 and 4, respectively. (B) In vivo continuous and multiplexed evaluation of wound parameters in a 24-hour fasted mouse before and after glucose administration via tail vein. (C) In vivo assessment of metabolic changes in wound microenvironment in response to fasting and food feeding in a diabetic mouse.

Fig. 5.. Spatial and temporal monitoring of critical-sized full-thickness infected wound defects in diabetic rats using the wearable patch.

(A and B) Schematic (A) and photograph (B) of a soft sensor patch with pH and temperature sensor arrays designed for spatial and temporal monitoring of large and irregular wounds. Scale bar, 1 cm. (C and D) The characterization of pH (C) and temperature (D) sensor arrays on a wearable patch in SWF solutions. (E and F) Dynamic changes in pH (E) and temperature (F) values of each biosensor on a wearable patch for critical-sized noninfected and infected wounds. (G and H) The mapping of daily local pH (G) and temperature (H) sensor readings in the wound area for infected and noninfected wounds on each day over the 7-day study period.

Fig. 6.. In vivo evaluation of wearable patch-facilitated chronic wound healing in full-thickness infected wounds in ZDF diabetic rats.

(A) Schematic of the wearable patch on a diabetic wound and the working diagram of combination therapy. (B and C) Representative images (B) and quantitative analysis of wound closure (C) for the control wound and wounds treated with drug, ES, and combination therapy on days 3 and 14 after application. Scale bar, 500 μm. (D) Representative images of Masson’s trichrome (MTC)–stained sections of the full-thickness skin wounds after 14 days of combination treatment. Scale bars, 500 μm. (E) Representative immunofluorescent stained images for nuclear factor κB (NF-κB) (purple), keratin 14 (Krt14) (green), and phosphatase and tensin homolog (Pten) (red) 14 days after the treatment. Scale bars, 500 μm. (F and G) Quantitative analysis of scar elevation index (SEI) based on MTC images (F) and Krt14 marker based on immunofluorescent images (G). (H) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of a library of wound biomarkers for wound biopsies after 3 and 14 days of treatment. (I to M) Relative expression of Pdgfa (I), Fgf (J), Serpine1 (K), IL-6 (L), and Stat3 (M) genes after 3 and 14 days of treatment. Error bars represent the SD (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; n = 3).