Supramolecular coordination platinum metallacycle–based multilevel wound dressing for bacteria sensing and wound healing

Significance Non-healing wounds induced by antibiotic resistance have emerged as serious threats to human health. As promising candidates for the treatment of bacterial infections, metal-organic cycles/cages (MOCs) exhibit excellent antibacterial properties. However, the rational application of nanoscale MOCs has been limited due to difficulties in processability and transferability. To address these problems, a centimeter-scale Pt MOC film was constructed via multistage assembly and improved by coating it on N,N′-dimethylated dipyridinium thiazolo[5,4-d]thiazole (MPT)-stained silk fabric for bacterial sensing and wound healing. The as-prepared multilevel wound dressing enabled the monitoring of wound infection in real time and timely treatment with high spatiotemporal precision.

Fabrication of centimeter Pt MOC film 100 μL DCM solution of Pt MOCs (400 μM) was mixed with 100 μL DCM in a vial.Then, 800 μL EA was slowly added to the DCM solution, and the reaction was allowed to proceed for 48 h.For other assemblies, a similar procedure using different amounts of DCM/EA solutions was applied to tune the ratio of DCM/EA to obtain assemblies with controllable size and shape (2).

Preparation of complex I+II
The silk fabric (I) with a diameter of approximately 1 cm was soaked in 20 mL of a 200 μM of MPT (II) solution at room temperature for 5 h.Obvious color change from white to slightly yellow was observed.Thereafter, the complex I+II was obtained and washed 3 times with H2O and dried.

Preparation of complex I+II+III
Complex I+II+III was prepared by directly transferring the centimeter Pt MOC film from DCM/EA solutions to the complex I+II (as a support substrate) with similar diameter, and dried in vacuum.

Bacteria cultivation
Gram-negative E. coli and gram-positive S. aureus were inoculated into nutrient agar by streak plate method.After being cultured in a constant temperature incubator (MJ-150-I) for 15 h, colonies were transferred to 25 mL sterile LB medium at 37 °C and cultured to mid-log phase.After centrifugation and being washed for 3 times, bacterial suspensions with different concentrations in PBS were prepared.

Electron paramagnetic resonance (EPR) measurements
MPT (100 μM) was dissolved in PBS.Then 100 μL of the bacteria suspension (E. coli or S. aureus) was added into 3.5 mL LB medium.The medium was transferred into NMR tube and sealed.The samples were incubated at 37 °C for 12 h and then measured by EPR spectrometer (3).

In vitro photothermal performance
The 660 nm laser was produced by DS660-MFC-2 Laser Light Source, and the photothermal induced temperature change was recorded by an infrared camera.After incubation with bacteria, the solution of MPT showed obvious color change.Then the samples were irradiated by the 660 nm laser (1.0 W/cm 2 ) for 10 min, and obtained the temperature curve.

In vitro photothermal cycles of complex I+II+III
First, 300 μL of S. aureus solution (1.0×10 8 CFU mL -1 ) was taken and added to a 12-well cell culture plate.Subsequently, a circular complex I+II+III with a diameter of 1 cm was immersed in the bacterial solution.After incubation at 37 °C for 3 h, an obvious color change from light yellow to dark purple was observed in complex I+II+III.Then, the color-changed complex I+II+III was gently transferred to a clean white foam board.Under optimal lighting condition (660 nm laser, 0.5 W/cm 2 ), continuous irradiation for 3 min resulted in a rapid increase in the temperature of complex I+II+III with increasing irradiation time.After 3 min, the light source was turned off, and natural cooling occurred leading to a gradual reduction in the temperature of complex I+II+III with increasing cooling time.This heating and cooling process was repeated 5 times, and the temperature changes during heating and cooling were recorded using FLIR A35 FOV 25 (60 Hz) thermal imaging camera to obtain the photothermal cycles curve as shown in Figure 3f.

In vitro antibacterial experiments of complex I+II+III
Firstly, the complex I+II+III and other control groups were transferred on the agarose culture medium plates with S. aureus and E. coli, respectively.After incubation with bacteria, the complex I+II+III showed obvious color change from slightly yellow to deep purple.Then the samples were irradiated by 600 nm light (0.5 W/cm 2 ) for 5 min.After that, the bacteria were further cultured in a constant temperature incubator at 37 °C for 15 h, the color change and zone of inhibition in each group was recorded by camera and analyzed.

Establishment of wound-infection model
3-week-old Female Sprague-Dawley rats were used in the animal experiments.Rat handling and procedures were guided by the Institutional Animal Care and Use Committee.Standard laboratory food and water were provided in the animal facility of Wuhan University of Science and Technology.All animal procedures were approved by the committee of the Laboratory Animal Science Department at Wuhan University of Science and Technology.Rats were anesthetized by inhalation of 1.5% isoflurane and the dorsum was shaved before surgery.One full-thickness round wound (diameter=1.0cm) was created on the dorsum of each rat.Subsequently, 50 μL of S. aureus (1.0×10 8 CFU mL -1 ) was immediately dripped onto the round wounds and evenly smeared over the wound surface.Then the wounds were fixed with elastic medical bandages.The next day, the wound-infection models were established successfully.

In vivo antibacterial effect
The wounds in each rat were treated with different samples: I Dark/Light, I+II Dark/Light, I+III Dark/Light, and I+II+III Dark/Light (0.5 W/cm 2 , 4 min).To quantitatively investigate the antibacterial effect in vivo, the tissue fluid at day 1 was collected and homogenized in normal saline (1.0 mL). 100 μL of the diluted bacterial suspension was uniformly spread onto the fresh LB agar plates.After cultivation overnight, the bacterial colonies on the agar plates were photographed and counted.
The bacteria suspension treated by PBS with or without 660 nm laser irradiation was investigated in the control groups.

Live/Dead staining of bacteria
We studied the antibacterial properties of complex I+II+III by a bacterial live/dead staining assay.Briefly, after different treatments, the bacteria cells were co-stained by PI and FDA for 30 min in the dark, followed by washing thrice with PBS.According to the manufacturer's instructions, all bacteria were labeled by FDA and appeared green fluorescence, while dead bacteria were stained by PI and revealed red fluorescence.Finally, fluorescence images were captured using an inverted laser scanning microscopy (Olympus, FV1000).

Characterization of bacterial morphology
SEM imaging was performed to visualize the bacteria morphological changes after the antibacterial experiments.Specifically, after treatments, all collected bacteria were washed with PBS and then fixed with 2.5% glutaraldehyde solution at 4 °C for 4 h.After fixation, these specimens were serially dehydrated by graded ethanol solutions (10%, 20%, 30%, 50%, 70%, 80%, 90%, 100%).Next, the dried bacteria were sputter-coated with Au to increase conductivity, and their morphologies were observed by SEM.

Photothermal conversion efficiency
The photothermal conversion efficiencies (η) were measured according to a previously described method: (5,6) =[hs(Tmax-Tsurr)-QDis]/I(1-10 -A ) -----Equation (Se1) h is the heat transfer coefficient, s is the surface area of the container, and the value of hs is determined from the equation (Se2).QDis represents heat dissipated from the laser mediated by the solvent and container.We chose 660 nm laser irradiation (Power density: 1.0 W/cm 2 ).For convenience, I is the laser power and A is the absorbance at 660 nm.
hs=mC/τs -----Equation (Se2) m is the mass of the solution containing the photoactive material, C is the specific heat capacity of the solution, and τs is the associated time constant, which can be determined from equation (Se3).

Statistical analysis
All experiments were conducted at least three times unless otherwise noted.The statistical analysis was performed using OriginPro 8.0, followed by a student's t-test and one-way analysis of variance (ANOVA).*P<0.05 was considered statistically significant.**P<0.01 and ***P<0.001were considered highly significant.When adding S. aureus solution (1.0×10 8 CFU mL -1 ), the intensity of absorption peak at 630 nm (OD630) was measured according to a method: At is the absorption peak intensity of PBS or I+II at 630 nm after cocultivation with S. aureus solution (1.0×10 8 CFU mL -1 ), A0 is the absorption peak intensity of the initial S. aureus solution at 630 nm.

Fig. S1 .
Fig. S1.TEM images of (a) elongated nanofibers generated from a nanosphere, (b-c) the fusion among fibers results in the formation of network.

Fig. S3 .
Fig. S3.Bright-field microscopy images of the formation process of complex I+II+III.

Fig. S4 .
Fig. S4.The inverted fluorescent microscopy photographs of the formation process of complex I+II+III under 425 nm light.

Fig. S6 .
Fig. S6.(a, c) UV-Vis spectroscopic analysis of supernatant solution and (b, d) corresponding release relationship of MPT from complex I+II+III with time via different treatments.

Fig. S7 .
Fig. S7.(a) Fluorescence changes of supernatant solution and (b) corresponding release relationship of Pt MOCs from complex I+II+III before heating treatment.

Fig. S10 .
Fig. S10.(a) Color changes of PBS and I+II after repeated addition and removal of the S. aureus solution (1.0×10 8 CFU mL -1 ).(b) UV-Vis spectra of PBS and I+II after cocultivation with S. aureus solution (1.0×10 8 CFU mL -1 ) for 3 h.(c) Corresponding intensity of absorption peak at 630 nm with incubating time.

Fig. S19 .
Fig. S19.Temperature change curves and the calculated time constants of complex I+II+III.

Fig. S20 .
Fig. S20.The inverted fluorescent microscopy photographs of disassembly of Pt MOC film on complex I+II+III after heating treatment at 55 °C under 425 laser irradiation.

Fig. S22 .
Fig. S22.Antibacterial effect of complex I+II+III against E. coli and S. aureus.Disc diffusion assay results with different treatments under dark conditions.

Fig. S24 .
Fig. S24.SEM images of morphological changes of E. coli and S. aureus after different treatments.Scale bar=1 μm.

Fig. S27 .
Fig. S27.Infrared thermal images at the wound sites of rats with different treatments under 660 nm laser irradiation (0.5 W/cm 2 ).

Fig. S28 .
Fig. S28.Photographs of S. aureus-infected wounds treated under dark condition from day 0 to day 12.

Fig. S33 .
Fig. S33.Photographs of bacterial colonies on agar plates from wound sites on the first day with different treatments under dark condition (n=3).

Fig. S34 .
Fig. S34.Corresponding quantitative analysis of bacterial colonies on agar plates from wound sites on the first day after different treatments (n=3, ***P<0.001).

Fig. S37 .
Fig. S37.H&E staining of healed skin tissues after different treatments under dark conditions.