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The ultraviolet spectra of melanin extracted from M. edulis shells were characterized. A maximum absorption peak near 230 nm (Supplementary Figure S1A, available in www.besjournal.com), and the black color of the shell extracts indicated that the extract was melanin[42, 43].
Figure S1. (A) UV absorption spectra of HCl hydrolyzed-melanin and trypsin hydrolyzed-melanin. (B) FTIR spectra of HCl hydrolyzed-melanin and trypsin hydrolyzed-melanin. (C) SEM images of HCl hydrolyzed-melanin and trypsin hydrolyzed-melanin.
We further examined whether the hydrolyzed black extracts from M. edulis shells really were melanin using infrared spectroscopy. The infrared spectra of the hydrolyzed extracts (Supplementary Figure S1B) revealed peaks at 3,300–3,500 cm–1, 1,600–1,670 cm–1 and 1,250–1,465 cm–1, thus confirming that the extracts from the M. edulis shells were indeed melanin. Supplementary Figures S1C and S2 (available in www.besjournal.com) show ultrastructural SEM images and XPS of extracted and T- and H-melanin.
Figure S2. (A) XPS survey spectrum of HCl hydrolyzed-melanin. (B) XPS C1S spectrum of HCl hydrolyzed-melanin. (C) XPS N1s spectrum of HCl hydrolyzed-melanin. (D) XPS O1s spectrum of HCl hydrolyzed-melanin. (E) XPS survey spectrum of trypsin hydrolyzed-melanin. (F) XPS C1S spectrum of trypsin hydrolyzed-melanin. (G) XPS N1s spectrum of trypsin hydrolyzed-melanin. (H) XPS O1s spectrum of trypsin hydrolyzed-melanin.
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We compared the photothermal conversion effects of M. edulis melanin hydrolyzed with concentrated HCl and various proteases (P-melanin). Figure 1A shows the relationship between the temperature of H-melanin and T-melanin, and the length of exposure to NIR irradiation (0.8 W, 808 nm). The temperature of hydrolyzed melanin changed roughly in three stages as follows. During the first 0–1 min, the temperature rapidly increased under near-infrared radiation, and immediately produced a thermal effect. The rate of the temperature increase gradually slowed over the next 1–6 min, then stabilized between 6–7 min. The temperature of T-melanin increased more rapidly, and peaked at 81.4 °C. The H-melanin peaked at 67.4 °C, and the P-melanin reached only 49 °C. These findings indicated that melanin can be photothermally converted by NIR irradiation to promote a local increase in temperature, thus exerting sterilization and inhibition effects, and that T-melanin was the most effective.
Figure 1. Photothermal conversional effects of hydrolyzed melanin. (A) Photothermal conversional effects of various concentrations of hydrolyzed melanin under NIR laser radiation at 0.8 W and 808 nm. Numbers 1 to 7 represent melanin hydrolyzed by pepsase, animal proteinase, neutral protease, alkaline protease, trypsin, compound protease, and HCl, respectively. (B) Increases in temperature of water and of aqueous suspensions of H-melanin according to duration of irradiation. (C) Temperature variations (Δ) in aqueous suspensions of H-melanin over 300 s. (D) Photothermal response of 200 μg/mL of aqueous H-melanin under NIR laser irradiation at 2 W and 808 nm for 5 min and then the laser was shut off. (E) Linear time data vs. −lnθ were acquired based on the cooling period shown in (C).
We assessed temperature changes of 25 to 200 μg/mL of melanin to determine photothermal conversion at various concentrations (Figures 1B and 2A). Since the temperature of melanin suspended in water at 200 μg/mL changed the most, we applied this concentration to calculate photothermal conversion and to correspond with the doses in the antibacterial experiments in vitro and the biocompatibility assays in vivo. After irradiation for 5 min, the temperatures of the H-melanin and T-melanin suspensions and of pure water increased by 20.4 °C (Figure 1C), 23.2 °C (Figure 2B) and 11.2 °C, respectively.
Figure 2. Photothermal responses of T-melanin. (A) Increases in temperature of water and aqueous T-melanin suspensions at various concentrations during irradiation for 5 min. (B) Temperature variations (ΔT) Temperature variations (Δ) in aqueous H-melanin suspension during 300 s. (C) Photothermal response of 200 μg/mL of aqueous T-melanin under NIR laser irradiation at 2 W and 808 nm for 5 min and then later the laser was shut off. (D) Linear time data vs. −lnθ acquired based on cooling period shown in (C).
Next, we measured the photothermal conversion efficiency (η) of H-melanin and T-melanin as described[44]. The η, t, and θ values were calculated as:
$$\eta = \frac{{hA\Delta {T_{\text{max} }} - {Q_s}}}{{I\;(1 - {{10}^{ - {A_\lambda }}})}},$$ (Eq. 1) $$t = - \frac{{\sum\limits_i {{m_i}{C_{p,i}}} }}{{hA}}ln\theta ,$$ (Eq. 2) $$\theta = \frac{{\Delta T}}{{\Delta {T_{\text{max} }}}},$$ (Eq. 3) where h is the heat transfer coefficient, A is the surface area of the container, ΔTmax is the temperature change in the melanin suspension at the maximum steady-state temperature, I is laser power, Aλ is absorbance of the melanin suspension at 808 nm, Qs is the amount of heat associated with the light absorbance of the solvent (pure water; measured independently as 25.2 mW), η is photothermal conversion efficiency, and m and C are the mass and heat capacity of the solvent (water), respectively. According to Equation 1, and Figures 1E and 2D, the η values of H-melanin and T- melanin were 22.89% and 51.35%, respectively.
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We compared the bacteriostatic effects of T- and H-melanin against Gram-negative (E. coli) and positive
(S. aureus) bacteria. Figure 3A shows that the number of bacteria did not significantly change in the control group regardless of NIR irradiation. These findings indicated that NIR irradiation alone does not affect the normal growth of E. coli and S. aureus. After 15 min of incubation without irradiation, the numbers of E. coli and S. aureus did not significantly change in the T- and H-melanin groups compared with the controls, indicating that neither hydrolyzed melanin exerted bactericidal effects without NIR irradiation. In contrast, the numbers of bacteria in the H- and T-melanin groups slightly and significantly decreased respectively, after 15 min of NIR radiation compared with none. These findings indicated H- and T-melanin was weakly, and more strongly bactericidal, respectively, against E. coli and S. aureus.
Figure 3. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) incubated with H-melanin and T-melanin followed by NIR irradiation for 15 min. (A) Photographs show E. coli and S. aureus after incubation with melanin followed by NIR irradiation. (B) Antibacterial ratios against E. coli and S. aureus. Data are presented as means ± SD
(n = 3; **P < 0.01 and ***P < 0.001). (C) TEM images for E. coli and S. aureus incubated T-melanin and H-melanin. NIR-, without, and NIR+, with near infrared radiation. With NIR irradiation, the bacteriostatic rates of H- and T-melanin against E. coli and S. aureus were 35.43% and 29.10% (Figure 3B), and 97.43% and 94.23%, respectively.
Changes in the morphology of individual E. coli and S. aureus cells after different treatments were assessed by TEM. Figure 3C shows that the bacteria in the control group appeared normal, with a complete membrane structure and a normal rod-like or spherical shape. The edges of E. coli and S. aureus cells exposed to NIR irradiation for 15 min and incubated with H-melanin were slightly broken, but the basic shape persisted. In contrast, the cell walls and membranes of these bacteria similarly exposed to NIR irradiation and incubated with T-melanin were completely ruptured, releasing the cytoplasm into the milieu (red arrow). These findings indicated that T-melanin together with NIR irradiation are bacteriostatic and can play a sterilizing role.
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Bacterial viability after incubation with H- and T-melanin and exposure to NIR was determined using LIVE/DEAD BacLight kits (Thermo Fisher Scientific Inc.). Viable and dead bacteria are stained green and red, respectively.
Figure 4 shows that control E. coli and S. aureus incubated without H-melanin stained green with or without NIR irradiation, indicating that NIR alone cannot kill these bacteria. Incubating the bacteria with H-melanin in the absence of NIR irradiation showed that H-melanin was not cytotoxic. Furthermore, NIR irradiation of the H-melanin group killed only a few cells, indicating a slight bactericidal effect against E. coli and S. aureus.
Figure 4. Representative fluorescence images of viability of E. coli and S. aureus cell incubated with T- or H- melanin and with or without NIR irradiation. NIR-, without, and NIR+, with near infrared radiation.
Green cells found after incubating these bacteria with T-melanin but without NIR exposure indicated that these bacteria remained viable. However, most cells exposed to T-melanin together with NIR irradiation were stained red, indicating that T-melanin exerted more powerful bactericidal and sterilization effects than H-melanin.
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Based on the antibacterial findings in vitro, we assessed the wound healing activities of H-melanin and T-melanin in vivo, individually. In brief, a rat model of bacterial infection was constructed by local infection with the resistant S. aureus. Thereafter, the infected rats were randomized as H-melanin, T-melanin, and control groups, separately (Figure 5). On the day 0, the drug-resistant S. aureus infected wounds in the three groups were of equal size. On the day 3, all three groups of wounds were healed to varying degrees. The T-melanin group with NIR+ had the smallest wound area, followed by the H-melanin group, while the control group had the largest wound area. By day 6, the T-melanin group with NIR+ was basically healed, the healing effects were significant compared with H-melanin ad the control group.
Figure 5. Wound tissues and bacterial colonies collected after treatment with melanin and NIR irradiation days 0, 3, and 6.
We quantified the wound healing process by comparing wound sizes on days 3 and 6 with that on day 0 (Figure 6A). The size of wounds significantly differed in the group treated with T-melanin and NIR radiation compared with the control on day 3 (P < 0.01). The wounded area was significantly smaller on day 6 in the group treated with T-melanin and NIR radiation compared with the control (P < 0.05) and the NIR- irradiated H-melanin group (P < 0.001). These findings confirmed that the high temperature of NIR induced the maximal bactericidal effect of T-melanin against these methicillin-resistant bacteria.
Figure 6. Wound sizes, bacterial CFU and thermal images. (A) Wound sizes in rats from different groups after various treatments. (B) Numbers of bacterial CFU in wound tissues on days 0, 3, and 6. Black, red, blue, magenta and green columns in A and B represent control, H-melanin and NIR-, H-melanin and NIR+, H-melanin and NIR-, and H-melanin and NIR+, respectively. (C) Thermal images of rats after various treatments. All data are shown as means ± SD
(n = 3 rats per group. *P < 0.05, **P < 0.01, and ***P < 0.001). Figure 6C shows infrared images of the temperature increase in wounds treated with melanin and NIR irradiation[45]. After 15 min of irradiation at 2 W, local temperatures of the H- and T-melanin groups increased from 36.4 °C to 46.8 °C and 66.3 °C, respectively, indicating better photothermal conversion of T-melanin.
Wound tissues were harvested from each group of rats to further determine the abundance of S. aureus cells on days 0, 3, and 6 by counting CFU (Figure 6B). The number of bacterial colonies was the lowest on days 3 and 6 when treated with T-melanin and NIR irradiation compared with the control (P < 0.05), followed by H-melanin with NIR irradiation. The wound healing capacity on day 6 was better for T- than H-melanin with NIR irradiation.
Wound tissues were stained with H&E on day 6 to evaluate wound healing. Neutrophils stain blue in infected tissues[46]. Figure 7 shows the histological findings of wounds in the control, H-melanin and T-melanin groups without NIR. A blue arrow indicates inflammatory cells, suggesting serious infection of the wound, The skin at the surface of the wound surface is cracked and damaged (black arrow). In contrast, the ratio of inflammatory cells is very small in the wounds treated with T-melanin and NIR irradiation. This combination exerted the optimal healing effect as indicated by a smooth, complete skin surface (yellow arrow) and normal subcutaneous tissue structure (green arrows).
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The above results indicated that T-melanin could offer a tremendous advantage for disinfecting wounds. Therefore, the biosafety of this nanomaterial should be considered.
We evaluated the cytotoxicity of T- and H-melanin in vitro using the MTT method, and tested relative survival by adding different volumes of melanin suspension to L929 cells and HUVEC, respectively. Figure 8A shows that at melanin concentrations of 0–200 μg/mL, the survival rates of these cells remained > 99%. Therefore, we considered that T- and H-melanin are safe and not cytotoxic in vitro.
Figure 8. Cytotoxicity of H-melanin and T-melanin against L929 cells and HUVEC in vitro and in mice in vivo. Values are shown as means and standard deviations of three mice per group. (A) Cytotoxicity of H- and T-melanin against L929 cells and HUVEC in vitro. (B) Sections of heart, liver, spleen, lungs, and kidneys harvested from mice treated with or without (control; PBS) melanin (H&E stain). (C) Blood biochemical findings of mice under treated with (a) PBS, (b) H-melanin, (c) T-melanin. A/G, albumin-to-globulin ratio; ALB, albumin; ALP, alkaline phosphatase; ALT, alanine transaminase; AST, aspartate transaminase; GLOB, globulin; TP, total protein; UREA, urea nitrogen; HUVEC, human umbilical vein endothelial cells
We then assessed the cytotoxicity of H-melanin and T-melanin in mice in vivo. The main organ tissues of mice treated with melanin and stained with H&E showed no obvious organ abnormalities or inflammation, and their morphology and structure were similar to those of normal organs (Figure 8B). We also intravenously injected healthy Sprague-Dawley rats with aqueous H-melanin and T-melanin for 6 d, then assessed hepatorenal functions as blood biochemical values. These indexes did not significantly differ from those in untreated mice (Figure 8C). These results suggest that H-melanin and T-melanin are highly biocompatible, not toxic and do not elicit side effects in vivo and in vitro.
Photothermal Effect-based Cytotoxic Ability of Melanin from Mytilus edulis Shells to Heal Wounds Infected with Drug-resistant Bacteria in vivo
doi: 10.3967/bes2020.052
- Received Date: 2020-03-06
- Accepted Date: 2020-05-21
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Key words:
- Melanin /
- Photothermal conversion /
- Antibacterial /
- Wound healing
Abstract:
Citation: | LIU Ya Mei, MA Wei Shuai, WEI Yu Xi, XU Yuan Hong. Photothermal Effect-based Cytotoxic Ability of Melanin from Mytilus edulis Shells to Heal Wounds Infected with Drug-resistant Bacteria in vivo[J]. Biomedical and Environmental Sciences, 2020, 33(7): 471-483. doi: 10.3967/bes2020.052 |