Detection of Redox State Evolution during Wound Healing Process

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Detection of redox state evolution during wound healing process based on a redox-sensitive wound dressing Jie Sun, Shuyan Han, Ying Wang, Guanyu Zhao, Weiping Qian, and Jian Dong Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00471 • Publication Date (Web): 14 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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Analytical Chemistry

Detection of redox state evolution during wound healing process based on a redox-sensitive wound dressing Jie Sun,a Shuyan Han,a Ying Wang,a Guanyu Zhao,a Weiping Qian,a,* and Jian Donga,b,* a

State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing, China. b Laboratory of Environment and Biosafety, Research Institute of Southeast University in Suzhou, Suzhou, China. ABSTRACT: To detect the redox state evolution during wound healing process, a redox-sensitive surface-enhanced Raman scattering (SERS) probe was constructed by attaching anthraquinone as a redox-sensitive molecule onto gold nanoshells, and the redoxsensitive SERS probes were loaded on one surface of a chitosan membrane as a redox-sensitive wound dressing. The redoxsensitive wound dressing covered an acute wound as both a wound dressing and a redox state sensor. The spatiotemporal evolution of the redox states of the healing wound was obtained by collecting the SERS spectra of the SERS probes in situ and noninvasively. The domains with the lowest redox potential moved from the edge to the center of a wound during normal wound healing process, and high concentration of glucose blocked the movement of the domains and the healing process. The redox-sensitive wound dressing and the method of detecting redox states of the wound provide a new path for the detection in vivo, which would benefit the understanding and therapy of wound healing and other pathophysiological processes.

Introduction The change in redox state is related to many physiological and pathological processes (such as the behaviors of cells, inflammation, and several severe diseases) and the favorite redox states are varied for different physiological and pathological processes.1-4 It is mainly dependent on the reaction of reactive oxygen species (ROS), reactive nitrogen species (RNS), antioxidant molecules, and corresponding enzymes. Wound healing is a complicated and ordered biological process involving vasoconstriction and coagulation, inflammatory reactions, cellular proliferation, wound remodeling, and the overlapping of these pathophysiological processes.5-7 In previous reports, the outbreak of ROS (exceeding 300 µM of H2O2) during early wound healing process changed the redox states of the wound,10 and the amounts of ROS varied at different pathophysiological stages and triggered related cellular signaling pathways, autonomous defense, and antibacterial radicals for wound healing.8 However, the evolution of redox states during wound healing process is unclear because no given method can meet the requirement. For invasive detection, the sampling of wound fluids would destroy the healing wound and make a new wound on the old wound, changing the redox state of the healing wound. For noninvasive detection, such as that the electrochemical methods only provided the redox states of one dot of the wound and electron paramagnetic resonance spectroscopy only provided the redox states of the whole wound.9-10 Therefore, it is necessary to develop alternative methods to detect the redox states spatiotemporally without destroying the healing wound, which would be contributed to the understanding of mechanism of wound healing.

short time consuming, high sensitivity and so on.11-12 Using the infrared excitation light transmitting through the skin, the signal of SERS probes in subcutaneous tissues can be collected with noninvasion.13-14 Thus, the SERS techniques would become potential tools in biomedical fields for groundbreaking work. Here, a SERS method was developed to detect the evolution of redox states during early wound healing process. First, anthraquinone derivatives, as reversible sensitive molecules for redox states, were functionalized on gold nanoshells (GNSs) to construct redox-sensitive SERS probes.15 Second, the SERS probes were loaded on one surface of a chitosan membrane (a common wound dressing) as a redox-sensitive wound dressing.16 Finally, the redox-sensitive wound dressing covered an acute wound with the surface loaded the SERS probes toward the wound, and SERS spectra obtained in vivo were used to indicate the redox state of the wound.

Materials and Methods Materials and Chemicals The silica colloidal particles of ~110 nm in diameter were obtained from Nissan Chemical Corporation (Japan). Anthraquinone-2-carboxylic acid, cystamine dihydrochloride, dicyclohexylcarbodiimide, N-Hydroxysuccinimide, and chitosan were obtained from Sinopharm Chemical Reagent Co., Ltd. All of the other chemicals were of analytical grade and used without further purification. Pure water used in the experiments was prepared by Milli-Q water from Milli-Q system (resistivity > 18 MΩ·cm).

The development of a novel detection method would carry out a novel research that cannot be carried out previously, and in vivo and noninvasive detected methods are desirable for biological and medical research. Surface-enhanced Raman scattering (SERS) techniques have gotten much attention due to their noninvasion,

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collected to assess the influence of the matrix of chitosan membranes on the detected SERS signal, with the loaded surface forward and backward the objective lens, respectively. SERS spectra of 100 random points of a redox-sensitive wound dressing and 100 random points of five wound dressings were collected to assess SERS signal uniformity of the redox-sensitive wound dressing. SERS spectra of a redoxsensitive wound dressing immersed in PBS (0.2 M, pH 7.4) were collected to assess the stability of the redox-sensitive wound dressing. Relative standard deviation (RSD) was calculated using Microsoft EXCEL.

Fig.1. An illustration of detecting the evolution of the redox state of a wound based on a redox-sensitive wound dressing.

Animal samples 30 ICR mice (male, 4 to 5 weeks old, 26-32 g) used were of the specific pathogen free (SPF) class, obtained from Nanjing animal breeding grounds (China). Animal experiments were performed with the permission of the Science and Technology Department of Jiangsu Province (SYXK 2016-0014). Animal breeding, care and all experiments were carried out in adherence to the Jiangsu animal experiment center guidelines. Fabrication of redox-sensitive wound dressing Fabrication of redox-sensitive SERS probes. To structure SERS probes, as shown in Fig. 1, the gold nanoshells (GNSs, Au@SiO2) with a diameter of 170±5 nm were fabricated as previous report.17 First, 100 mg of anthraquinone-2-carboxylic acid, 80 mg of dicyclohexylcarbodiimide, and 115 mg of NHydroxysuccinimide were dissolved into 50 mL of dimethyl sulfoxide, stirring for 3 hours at room temperature; second, after 22.5 mg of cystamine dihydrochloride was dissolved into the solution above, the mixed solution was kept at 4 °C for 10 h; third, the supernatant was collected and diluted 100 times in ethanol, and 1 mL of the GNSs suspension (OD700 nm = 2.0) was added into 10 mL of supernatant dilution, mixed by ultrasonic for 5 min, and kept at 4 °C for 6 h; finally, the mixture was centrifuged for 5 min at 4500 rpm and the precipitation was resuspended in water (OD700 nm = 2.0) for use. Fabrication of chitosan membrane. First, 200 mg chitosan powder was dissolved in 10 ml of 1 % (v/v) acetic aqueous solution; second, 2 ml of chitosan solution was added into a Petri dish of 8 cm in diameter to form a dried chitosan membrane at room temperature; third, 20 ml of 2 % (w/v) sodium hydroxide solution was added to the Petri dish to soak overnight; finally, the chitosan membranes were remove from the Petri dish and washed three times with water and ethanol, respectively. Fabrication of redox-sensitive wound dressing. Excessive redox-sensitive SERS probes were added on one surface of a chitosan membrane, and dried at room temperature; then, the chitosan membrane was washed with water thoroughly to remove unabsorbed SERS probes. Before used, the redoxsensitive wound dressing was cut to round shape. In vitro evaluation of redox-sensitive wound dressings SERS spectra of the redox-sensitive wound dressing were

In vivo detection of redox state evolution during wound healing process of mice Redox-sensitive SERS probes were injected subcutaneously of mice to assess its feasibility of detecting the redox states of mice and physiological redox state of mice. The skin of the back of mice was cut to form an acute wound with a diameter of about 6 mm, and the same sized round-shaped redoxsensitive wound dressing covered the acute wound immediately. The SERS spectra from the edge to the center of the redoxsensitive wound dressing were collected (each point is 200 µm apart) at 15:00 per day. To investigate the influence of glucose on the evolution of the redox state of wound healing process, 0.45 ml of 1.5 % or 15 % glucose aqueous solution were injected intraperitoneally at 8:00 and 20:00 per day. Characterizations and Measurements Scanning Electron Microscopy (SEM) images of morphology of GNSs, chitosan membrane, and GNSs loaded on membranes were carried out on a Zeiss ULTRA-plus scanning electron microscope. Transmission electron microscopy (TEM) images of GNSs were carried out on a HITACHI H8100 electron microscope (Hitachi, Tokyo, Japan) with an accelerating applied potential of 200 kV. SERS spectra were obtained at room temperature with a Renishaw Invia microRaman spectroscopy using a 785 nm excitation laser, under in focus mode. The laser was focused onto the sample surface by using a 50× long working distance objective. The extinction power was 2.4 µW for wounds and 240 µW for skin, and the acquisition time was 10 s. As previous reports,15 the intensity ratios at 1606 cm-1 and 1666 cm-1 (I1606 cm-1/I1666 cm1 ) of the SERS spectra were used as indicators of redox, which were calculated by the ratio of the intensity values at 1606 cm1 and 1666 cm-1 minus the intensity values at 1700 cm-1 (as background value), respectively.

Results and discussion Fabrication and assessment of the redox-sensitive wound dressing Chitosan is a common wound dressing due to its good biological consistent, absorbent, ventilated and antibacterial properties. The chitosan membrane loaded with SERS probes covered wound as a wound dressing for wound healing and as a redox sensor for detection the redox states of the healing wound, and the SERS spectra can be collected without invasion during the wound healing process. For constructing SERS probes, the GNSs were fabricated as SERS substrates. SEM and TEM images of GNSs are shown in Fig. 2A and 2B, and the diameter is about 170 ± 5 nm. Fig. 2C is a SEM image of


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Analytical Chemistry a chitosan membrane with porous structures. Fig. 2D shows SERS probes closely packed and absorbed on a chitosan membrane after washed thoroughly.

Fig.2. (A) a SEM image of GNSs; (B) a TEM image of GNSs; (C) a SEM image of chitosan membrane; (D) a SEM image of SERS probes loaded on a chitosan membrane.

From the Raman spectra of the chitosan membrane and the chitosan membrane loaded with GNSs shown in Fig. 3A, with 2.4 µW of extinction power no normal Raman line appeared and only two weak SERS line appeared, suggesting that the chitosan is an ideal carrier of SERS probes. Fig. 3B shows SERS spectra of a redox-sensitive wound dressing with the loaded surface forward and backward the objective lens, respectively. Due to the existence of the chitosan membrane, the SERS intensity of the backward spectrum decreased to about 14 % of that of the forward spectrum. But there is no difference between signals of the two spectra except their strength, indicating that the chitosan has no influence on the SERS spectra of the probes except the intensity. To assess the homogeneity of the redox-sensitive wound dressing, backward SERS spectra of random 100 points of a redox-sensitive wound dressing and random 100 points of five redox-sensitive wound dressings were collected, and the intensities ratios at 1606 cm-1 and 1666 cm-1 (I1606 cm-1/I1666 cm-1) of the SERS spectra, used to indicate the redox state, were shown in Fig. 3C. This result shows that the redox-sensitive wound dressing has good homogeneity (0.1562±0.0118 and 0.1571±0.0143, respectively). To assess its stability, the redox-sensitive wound dressing was immersed in PBS buffer (0.2 M, pH 7.4) for 8 days and backward Raman spectra were collected. As shown in Fig. 3D, the SERS intensities do not decrease in 8 days, and the values of I1606 cm-1/I1666 cm-1 of the SERS spectra are between 0.1589 ± 0.0070, indicating that the redox-sensitive wound dressing has good stability in PBS buffer in 8 days.

Fig.3. (A ) the Raman spectra of the chitosan membrane and the chitosan membrane loaded with GNSs; (B) forward and backward SERS spectra of a redox-sensitive wound dressing (the position of lines represent 1606 cm-1 and 1666 cm-1, respectively); (C) the values of I1606 cm-1/I1666 cm-1 of backward SERS spectra of a redoxsensitive wound dressing and different wound dressings; (D) the values of I1606 cm-1/I1666 cm-1 of SERS spectra of a redox-sensitive wound dressing in PBS (0.2 M, pH 7.4).

The redox potential of anthraquinone is also dependent on pH. The pH in wounds may fluctuate during the wound healing process. That is to say, the change of I1606 cm-1/I1666 cm-1 of SERS spectra may be from the change in pH and redox state of their environment. On the one hand, it is difficult to SERS detect pH and redox state on a wound dressing simultaneously, and on the other hand, when the value of pH changed from 7.0 to 5.0, the changed amount of value of I1606 cm-1/I1666 cm-1 is about 0.05. Thus, in the following experiments the influence of pH on the I1606 cm-1/I1666 cm-1 of SERS spectra did not consider. Before detecting the evolution of redox states of wound healing process, the SERS probes were injected subcutaneously to detect the physiological redox states of mice and the influence of the extra injected glucose on the physiological redox states of the mice. As shown in Fig. 3C, the values of I1606 cm-1/I1666 cm1 are about 0.1562 ± 0.0018 when the redox-sensitive wound dressing were exposed to air. However, as the black line shown in Fig. 4A, when the SERS probes were exposed to the body environment, the values of I1606 cm-1/I1666 cm-1 increase to 0.8274 ± 0.0103 (calculated from the corresponding SERS spectra) after one day, and decrease to 0.5200 ± 0.0070 (124.38 ±1.2711 mV and -56.662 ± 2.9787 mV, calculated according to scatter plot and linear fitting result of Fig. S2, respectively) at the fourth day and remain in next several days. The results suggest that the potential of the SERS probes change with their environment, that at the physiological redox potential of the mice should be -124.38 ± 1.2711 mV, and that the redox potential in the early 3 days was lower than the physiological redox potential. The potential change in 24 h after injection of SERS probes are shown in Fig. 4B. At 3 h after injection the detected redox state was up to the peak of oxidized state due to the outbreak of ROS and interestingly, in next time the detected redox state began to turn to reduced state and was up to the peak of reduced state at about 13 h after injection. And at fourth day, the detected redox state recovered to the physiological ones.



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tial at the center of the wound is highest (highly oxidized state) and higher than the physiological redox potential. In next days, the redox potential at the edge increased continuously, and up to higher than the physiological redox potential and meanwhile, the domains with the lowest redox potential moved continuously from the edge to the center (as the green domains in Fig. 5A). In the early four days, the redox potentials at the center were higher than the physiological redox potential. At last, the wound healed completely after the redox-sensitive wound dressing was torn down.

Fig.4. (A) the redox state evolution after subcutaneous injection of SERS probes with and without the injection of glucose in 14 days (the right indicates the relationship between the SERS intensity ratio and the redox state, the grey line represents the physiological redox state); (B) the redox state evolution after subcutaneous injection of SERS probes in 24 h. The error bars are from three spectra of the same injected dot of a mouse (The potentials were calculated according to the linear fitting result of Fig. S2).

After the potential the SERS probes was stable, 0.45 mL of 15 % (w/v) of glucose solution was intraperitoneally injected at 8:00 and 20:00 per day, and the SERS spectra were collected at 15:00 per day. As the red line shown in Fig. 4A, after a mouse was injected extra glucose, the potential of the injection dot increased compared to physiological redox potential, indicating that the redox potential increased after the injection of extra glucose. This suggests that excessive glucose may disturb the redox homeostasis of the mice. Detection of redox state evolution during wound healing process of mice In order to detect the evolution of the redox states of healing wound, a round-shaped redox-sensitive wound dressing covered an acute wound, and the influence of wound dressing with or without SERS probes on the wound healing was assessed first. After the wound dressings with or without SERS probes were torn down at 8th day, the wound of all mice healed completely, while after the scabs of natural healed wound were torn down at 8th day, the wounds of two mice healed completely and the wounds of two mice were bleeding, which suggested that the loaded SERS probes did not decrease the property of chitosan wound dressing improving the wound healing. The SERS spectra from the edge to the center of the redox-sensitive wound dressing were collected. The heatmap of redox potential in 8 days are shown in Fig. 5A. At the first day after covered, the detected redox potential at the edge of the wound is lowest (highly reduced state) and lower than the physiological redox potential and meanwhile, the redox poten-

Fig. 5. (A) the heatmap of the redox state evolution during wound healing process; (B) the redox state evolution of a redox-sensitive wound dressing after torn down and exposed to air. The error bars are from nine spectra of three mice (three spectra from different dots with the same distance to the center of the same wound per mouse and the potentials were calculated according to the linear fitting result of Fig. S2).

In previous reports, the outbreak of ROS usually sustains one to two days.18-19 As shown in Fig. 4B, due to the outbreak of ROS the redox potential increased and became higher than physiological redox potential for 4 h after injection of SERS probes. But as shown in Fig. 5A, the farther away from the edge, the longer the sustained time of the higher redox potentials than the physiological redox potential is, even up to four days at the center of a wound. Based on the knowledge that the increase of redox potential of a wound results from the outbreak of ROS and its decrease results from the expression of antioxidative enzymes and the production of antioxidant molecules, we speculated that the cells produced ROS are recruited from the injured sites, and that the cells produced antioxidative enzymes or antioxidant molecules are recruited from the healthy tissues of the edge of the wound or differentiated from the recruited cells. The speculation can also explain the phenomenon that the movement of the domains with the highly reduced state is from the edge to the center of the wound (as shown in Fig. 5A), instead of from the center to the edge of the wound. From 5th to 8th day, the detected redox potential at the edge of the redox-sensitive wound dressing in-


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Analytical Chemistry creased and became higher than the physiological redox potential, and the reason is that its edge peeled away from the healed edge of the wound and exposed to air completely. To verify that the decrease of redox potential (highly reduced state) result from the evolution of the wound other than the exposure to air, a redox-sensitive wound dressing was torn down from the wound at the third day after it covered a wound. As shown in Fig. 5B, after torn down and exposed to air completely, the detected redox potentials at the whole redoxsensitive wound dressing increased rapidly, suggesting that the detected redox potentials lower than physiological redox potential result from the decrease of redox potential of the wound during wound healing process. Glucose at low concentration is good for wound healing while that at high concentration would suppress wound healing. To assess the feasibility of the redox-sensitive wound dressing further, after redox-sensitive wound dressing covered an acute wound, the glucose solution was injected intraperitoneally at 8:00 and 20:00 per day, and the SERS spectra were collected at 15:00 per day. As shown in Fig. 6A, when 0.45 mL of 1.5 % of glucose was injected, the evolution of the redox potential during wound healing process is similar to that shown in Fig. 5A, and at eighth day when the redox-sensitive wound dressing was torn down, the wound healed and did not breed (shown in Fig. 6a), indicating that low concentration of glucose has little influence on wound healing. As shown in Fig. 6B, when 0.45 mL of 15 % of glucose was injected, besides that of the edge of the wound, the redox potentials of the acute wound almost keep under the physiological redox potential in 8 days, and at the eighth day when the redox-sensitive wound dressing was torn down, the wound did not heal and also did not breed (shown in Fig. 6b). We speculated that high concentration of glucose disturbed the normal wound healing process and blocked the conversion from oxidized state to reduced ones. Additionally, biological and physiological researches suggested that excessive glucose not only participates in the process of mitochondrial respiration to produce excessive ROSs disturbing the hemostasis, but also inhibits signaling pathway to attenuate wound healing, such as the focal adhesion kinase-mediated wound healing and epidermal growth factor receptor/phosphatidylinositol 3-Kinase/Akt-mediated wound healing.20,21 To verify the influence of the high concentration of glucose on the wound healing further, 0.45 mL of 15 % of glucose was injected intraperitoneally at the fifth day after a redoxsensitive wound dressing covered an acute wound. As shown in Fig. 6C, before injection of glucose, the evolution of the redox potentials of the wound is similar to that shown in Fig. 5A, but after injection the domains with the lowest redox potential almost did not move to the center of the wound, and the detected redox potentials increased. At the eighth day, when the redox-sensitive wound dressing was torn down, the wound did not heal completely and was breeding (shown in Fig. 6c). Combined Fig. 6A, 6B, and 6C, we conclude that it is necessary for wound healing to undergo the stages of outbreak of ROS and the lowest redox potential.

Fig.6. (A) The heatmap of the redox state evolution during wound healing process after injection of 0.45 mL of 1.5% glucose; (B) the heatmap of the redox state evolution during wound healing process after injection of 0.45 mL of 15% glucose; (C) the heatmap of the redox state evolution during wound healing process after injection of 0.45 mL of 15% glucose from fifth day; (a, b, c) The pictures of the wound recovery results corresponding to the above conditions, respectively (The bars indicate 6 mm). The results are from nine spectra of three mice (three spectra from different dots with the same distance to the center of the same wound per mouse) and the potentials were calculated according to the linear fitting result of Fig. S2.

Conclusion A redox-sensitive wound dressing has been fabricated by loading redox-sensitive SERS probes on one surface of a chitosan membrane. An acute wound was covered with the redoxsensitive wound dressing for wound healing with its surface loaded with SERS probes forward the wound, and its redox states during the wound healing process were reported by the SERS probes. The SERS spectra of the SERS probes have been collected in situ and noninvasively and the spatiotemporal evolution of the redox states of the healing wound has been obtained. The influence of glucose on the healing of an acute wound has been investigated, and the stage of the lowest redox potential may be a necessary stage for the wound healing. The fabricated redox-sensitive wound dressing would benefit the understanding of the mechanism of the wound healing and the therapy of the complicated wound of advanced age, diabetes, or immunosuppression.

AUTHOR INFORMATION Corresponding Author [email protected], [email protected].



Present Addresses State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, Southeast University, Nanjing, China.

ACKNOWLEDGMENT We gratefully acknowledge supports from the National key research and development program of China (2017YFA0205303, 2017YFE0100200), the National Natural Science Foundation of Jiangsu Province (EB2017771), the Science-technology Foundation of Suzhou (grants SYN201722 and SNG201603).

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Analytical Chemistry

for TOC only

To detect the redox state evolution during wound healing process, the redox-sensitive wound dressing covered an acute wound as both a wound dressing and a redox state sensor. The spatiotemporal evolution of the redox states of the healing wound was obtained by collecting the SERS spectra of the SERS probes in situ and noninvasively. The redox-sensitive wound dressing and the method of detecting redox states of the wound provide a new path for the detection in vivo, which would benefit the understanding and therapy of wound healing and other pathophysiological processes.


Detection of Redox State Evolution during Wound Healing Process

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