Treatment of Acute Wound Infections by Degradable Polymer Nanoparticle with a Synergistic Photothermal and Chemodynamic Strategy

Abstract Mild‐heat photothermal antibacterial therapy avoids heat‐induced damage to normal tissues but causes bacterial tolerance. The use of photothermal therapy in synergy with chemodynamic therapy is expected to address this issue. Herein, two pseudo‐conjugated polymers PM123 with photothermal units and PFc with ferrocene (Fc) units are designed to co‐assemble with DSPE‐mPEG2000 into nanoparticle NPM123/Fc. NPM123/Fc under 1064 nm laser irradiation (NPM123/Fc+NIR‐II) generates mild heat and additionally more toxic ∙OH from endogenous H2O2, displaying a strong synergistic photothermal and chemodynamic effect. NPM123/Fc+NIR‐II gives >90% inhibition rates against MDR ESKAPE pathogens in vitro. Metabolomics analysis unveils that NPM123/Fc+NIR‐II induces bacterial metabolic dysregulation including inhibited nucleic acid synthesis, disordered energy metabolism, enhanced oxidative stress, and elevated DNA damage. Further, NPM123/Fc+NIR‐II possesses >90% bacteriostatic rates at infected wounds in mice, resulting in almost full recovery of infected wounds. Immunodetection and transcriptomics assays disclose that the therapeutic effect is mainly dependent on the inhibition of inflammatory reactions and the promotion of wound healing. What is more, thioketal bonds in NPM123/Fc are susceptible to ROS, making it degradable with highly favorable biosafety in vitro and in vivo. NPM123/Fc+NIR‐II with a unique synergistic antibacterial strategy would be much less prone to select bacterial resistance and represent a promising antibiotics‐alternative anti‐infective measure.


Table of Contents
Experimental Section.Scheme S1.Schematic illustration of preparation of NP M123 and NP Fc .Synthesis of M4.M4 was synthesized as descried previously. [1] was poured into 500 mL water, then extracted with 100 mL ethyl acetate for three times, and separated by column chromatography to obtain a white solid M4 with a yield of 66%.
Characterizations. 5 mg P M123 or P Fc was dissolved in deuterated THF, and 1 H NMR spectra was subsequently measured by a 400 MHz NMR spectrometer (Bruker) at room temperature.20 μg mL -1 NP M123 , NP Fc , or NP M123/Fc aqueous solution was prepared and then TEM and SEM images were observed by Hitachi HT-7700 TEM (Japan) and JEM-ARM200F (Japan) respectively, while STEM and EDXS images were recorded by JEOL JEM-2100F STEM (USA).20 μg mL -1 NP M123/Fc aqueous solution was prepared and then UV-Vis-NIR spectra in wavelength range from f 350 nm to 1250 nm was recorded using a spectrophotometer (TU-

1901).
In Vitro Photothermal Measurement.A series of NP M123 or NP M123/Fc at different concentrations (200, 100, 50, 25, and 0 μg mL -1 , respectively) were prepared and irradiated with 1064 nm laser (1.0 W cm -2 , 5 min), and then the temperature changes were recorded by infrared thermal imaging camera (FLIR A6501, USA).For determination of photothermal conversion efficiency, NP M123 or NP M123/Fc was continuously irradiated by 1064 nm laser (1.0 W cm -2 , 11 min) until the temperatures were increased to a highest steady state, and then the laser was turned off until the temperatures were cooled to a lowest steady state; after that, the aqueous dispersion was cooled at room temperature and the photothermal conversion efficiency (η) of NP M123/Fc was calculated by the following equation: [3] η= Where h was the heat-transfer coefficient, Tmax was the equilibrium temperature, Tsurr was the ambient temperature, I was the density of laser power, Aλ was the absorbance of NP M123/Fc at 1064 nm, and τs was the time constant that was calculated by the fitting curve of time versus −ln obtained from Figure 1e.For photothermal stability measurement, NP M123 or NP M123/Fc (100 μg mL -1 ) was exposed to 1064 nm laser irradiation (1.0 W cm -2 , 5 min) for 4 ON/OFF cycles, and the temperature variation of the solution was recorded by infrared thermal imaging camera (FLIR A6501, USA).
In Vitro Chemodynamic Study.DPBF assay was used to measure the ROS generation efficacy of NP M123 , NP Fc and NP M123/Fc , and all the experiments were carried out at room temperature.100 μg mL -1 NP M123 , NP Fc or NP M123/Fc was mixed with 1 mM H2O2 and 2 mM DPBF, and tested at every half minute for a total duration time of 3.5 min under the NIR-II laser (1064 nm, 1.0 W cm -2 ).UV-vis-NIR spectrophotometer was used to record the degradation curve of DPBF.The absorbance changes of DPBF at 410 nm were used to quantify the decomposition rate.MB assays were used to measure the •OH production efficacy of NP M123 , NP Fc or NP M123/Fc , and all the experiments were carried out at room temperature; 10 µg mL -1 of MB, 1 mM H2O2, and 100 µg mL -1 samples were incubated for different times (0 to 3.5 min, tested every half minute) under the NIR-II laser (1064 nm, 1.0 W cm -2 ), and UV-vis-NIR spectrophotometer was used to record the degradation curve of MB.The absorbance changes of MB at 664 nm were used to quantify the decomposition rate.The Fc release kinetics of NP M123/Fc in vitro was measured by the dialysis method; a sealed dialysis bag (molecular retention of 3500) containing 5 mL of NP M123/Fc (100 μM Fc) was immersed in 200 mL of PBS in the presence or absence of 10 mM H2O2, followed by shaking at 100 rpm at room temperature.At various time points (0, 1, 2, 4, 6, 8, 10, 12, 24, 48 h), 1.5 mL of sample solution was taken from the dialysate and measured by ICP-MS to detect released Fc.XPS examination of P Fc (1 mg mL -1 , 1 mL) was challenged with H2O2 (1 mM, 1 mL) at 37 ℃ for 24 h, and the solution was freeze-dried under reduced pressure and the resulting lyophilized samples were then tested by XPS.
Disintegration of Nanoparticles Triggered by H2O2. 100 μg mL -1 NP M123 , NP Fc or NP M123/Fc was mixed with 1 mM H2O2 at 37 ℃ for 3.5 min.The solution was diluted to 20 μg mL -1 and then TEM and SEM images were recorded.Meanwhile, hydrodynamic diameters and zeta potentials were measured on Malvern Zetasizer Nano ZS90 (Malvern Instruments, UK).
Subsequently, the mixture (30 μL) was inoculated in BHI broth (3 mL) and incubated in a shaker at 37 ℃ for 3 h (200 rpm), which was the second-generation culture at the middle logarithmic phage.Then, this culture was inoculated in BHI broth (3 mL) at a ratio of 1:100 and incubated in a shaker at 37 ℃ for 3 h (200 rpm), which was the third-generation culture at the middle logarithmic phage.Finally, the OD600 value of this culture was adjusted to 0.2 with a concentration of about 10 7 CFU mL -1 for subsequent experiments.For bacterial cultivation using BHI broth (see above) or BHI plate (see below), the following antibiotics were added: methicillin (5 μg mL -1 ) for S. aureus, vancomycin (6 μg mL -1 ) for Ec.faecium, ceftazidime (20 μg mL -1 ) for A. baumannii, and meropenem (4 μg mL -1 ) for P. aeruginosa, K. pneumoniae, and

Eb. hormaechei.
In vitro Antibacterial Assay.The in vitro antibacterial efficacy against ESKAPE bacteria was determined by counting the number of colony-forming units (CFUs).Experiments were divided into 8 treatment groups: Mock (FF1), NIR-II (FF2), NP M123 (FF3), NP M123 +NIR-II (FF4), NP Fc (FF5), NP Fc +NIR-II (FF6), NP M123/Fc (FF7), NP M123/Fc +NIR-II (FF8) as employed as the dispersion solution, and the pure buffer was set as the FF1 treatment group.NIR-II represented 1064 nm laser irradiation (1.0 W cm -2 , 3.5 min).For each treatment group, bacteria (100 µL, 10 7 CFU mL -1 ) were mixed with NP M123 , NP Fc , or NP M123/Fc (100 μg mL -1 ) in 1 mL of phosphate buffer saline (PBS, 0.1 м, pH = 7.4) with the addition of 1 mM H2O2.Subsequently, the mixture was diluted 100 times with PBS (0.1 м, pH = 7.4) and the diluted mixtures (each 100 µL) were plotted on BHI plates and cultured at 37 °C for 18 h to be used for photograph; meanwhile, the mixture was diluted 1000 times by PBS (0.1 м, pH = 7.4), and 10 µL of the diluted mixtures were plotted on BHI plates to be used for CFU counting.Finally, the number of CFUs was counted, followed by the calculation of bacterial viability using the following equation: Where CFU(control) was the CFU value of the FF1 treatment group, and CFU(experimental group) was the CFU value of each of the FF2 to FF8 treatment groups.
Calculation of Q Value: Q value was calculated by the following equation: Where EA and EB were the in vitro antibacterial efficacy mediated by the photothermal effect of NP M123 +NIR-II (FF4) and that attributive to the chemodynamic effect of NP M123/Fc (FF7), respectively, and EA+B was the overall in vitro antibacterial efficacy of NP M123/Fc +NIR-II (FF8) combined with photo-therapy.The Q value reflected the synergistic effect between chemodynamic effect and photothermal effect: Q > 1.15 suggested strong synergism and 0.85 < Q < 1.15 indicated an additive effect, while Q < 0.85 indicated an antagonism effect [8][9][10] .
Live/Dead Fluorescent Staining.The viability of bacteria cells was qualitatively assessed through SYTO-9/PI staining.Bacteria (100 µL, 10 7 CFU mL -1 ) were treated as described in vitro antibacterial assay until incubation in a shaker at 37℃ for 30 min, and then the mixture was incubated with SYTO-9/PI for 20 min in the dark.The stained bacteria were observed with confocal laser scanning microscope (CLSM, ZEISS LSM 880).
Characterization of Bacterial Morphology.Bacteria (100 µL, 10 7 CFU mL -1 ) were treated as described in in vitro antibacterial assay, and then the mixture was centrifuged (6000 rpm) for 5 min, washed twice with PBS (0.1M, pH = 7.4), and fixed by 4% paraformaldehyde for 2 h.Subsequently, the suspension was gradually dehydrated at different concentrations of ethanol (50%, 70%, 90%, and 100%, respectively) for 10 min, respectively, and then SEM or TEM (see above) was used to observe the changes in bacterial morphology and the bacterial adherence to nanoparticles.
Evaluation of DNA, Protein, K + and Na + Leakage.Bacteria (100 µL, 10 7 CFU mL -1 ) were treated as described in in vitro antibacterial assay until incubation in a shaker at 37 °C for 30 min.For the detection of the levels of DNA, protein, K + , and Na + released by bacteria, bacterial supernatant was collected by centrifugation (6000 rpm) for 5 min.OD260nm value determined by Nano Drop UV-Vis spectrophotometer reflected the amount of released DNA.Protein level was determined by using BCA Protein Assay Kit (#23227, Thermo-Fisher, Grand Island, NY).
In addition, K + and Na + levels were measured by ICP-MS.DNA Damage Staining.Bacteria (100 µL, 10 7 CFU mL -1 ) were treated as described in the above in vitro antibacterial assay, and then the mixture was incubated with DAPI for 20 min in the dark, and then stained bacteria were observed with CLSM (see above).
Bacterial Metabolomics Assay.P. aeruginosa (100 µL, 10 8 CFU mL -1 ) was treated in vitro by the NP M123/Fc +NIR-II group (giving a bacterial viability rate of about 30%) or by the Mock group, and then total metabolites were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS).Differentially regulated metabolites (DRMs) were identified as the metabolites with variable importance in the projection (VIP) value > 1, |log2(fold change)| > 1, and student's t-test P-value < 0.05.
In Vivo Antibacterial and Wound Healing Evaluation.BALB/c female mice (6~8 weeks, about 18 g) were purchased from Vital River Laboratories (Beijing, China).A wound approximately 7 mm in diameter was made on the dorsum of each mouse, and bacterial suspension (10 µL, 10 7 CFU mL -1 ) was dropped into each wound.After the wounds were infected for 24 h, the mice were divided randomly into six therapeutic groups: Mock (FF1), NIR-II (FF2), NP M123 (FF3), NP M123 +NIR-II (FF4), NP M123/Fc (FF7), and NP M123/Fc +NIR-II (FF8) as above.NIR represented laser irradiation (1064 nm, 1.0 W cm -2 , 3.5 min), which was used 1.5 cm away from wound so that the light could cover the entire wound.The changes in temperature at infected wounds post therapy were recorded every half minute for a total during time of 3.5 min by an infrared thermal imaging camera (see above).Photographs of wounds were taken on days 0, 2, 4, 6, 8, and 10 post therapy, and the sizes of wounds and the body weights of mice were measured simultaneously.The mice were sacrificed on day 10 post therapy and the primary organs including heart, liver, spleen, lung, kidney, and wound tissues were harvested, fixed in 4% paraformaldehyde overnight, and dehydrated, and then embedded in paraffin for pathological histology, immunohistochemistry, and immunofluorescence staining.
Histology, Immunohistochemistry, Immunofluorescence Staining, and RNA-seq.The organ or wound tissues of infected mice on day 8 post-therapy were sliced.The slices (4 μm in thickness) were gradually dewaxed in xylene for 15 min and washed with 75%, 85% and 95% ethanol, respectively, for 5 min.
For hematoxylin and eosin staining, the dewaxed slices were incubated with hematoxylin for 5 min and washed with water for three times, and gradually dehydrated with 75%, 85% and 95% ethanol, respectively, for 5 min, followed by incubation with eosin for 5 min.Furthermore, the slices were washed with ethanol for 5 min and three times, and then with xylene for 5 min and two times.The results were observed by a light microscope (Nikon Eclipse E100).
The results were observed by microscope (XSP-C204).
For RNA-seq, total RNA samples from P. aeruginosa-infected wound tissues on day 8 posttherapy by the NP M123/Fc +NIR-II group or by the Mock group were extracted, followed by library construction, Illumina sequencing, and data mining, as described previously. [11]A student t-test P-value < 0.05, and a foldchange > 2 were set as the statistical significance.

Figure S3 .
Figure S3.TEM images of NP M123 and NP Fc .

Figure S9 .
Figure S9.Absorption curves of DPBF as a selective trapping agent to detect ROS in the reaction mixture of NP M123 +H2O2, NP Fc +H2O2, or NP M123/Fc +H2O2 after different incubation times.

Figure S10 .
Figure S10.Trends of UV absorption at 410 nm as indicated by the above DPBF-based detection results.

Figure S11 .
Figure S11.a) Absorption curves of MB as a selective trapping agent to detect •OH in the reaction mixture of NP Fc +H2O2 after different incubation times.b) Trends of UV absorption at 410 nm as indicated by the above MB-based detection results.

Figure S12 .
Figure S12.Fc release kinetics of NP M123/Fc in vitro was determined by ICP-MS.

Figure S14 .
Figure S14.TEM images for NP M123 , NP Fc and NP M123/Fc in the presence of H2O2.

Figure S15 .
Figure S15.SEM images for NP M123 , NP Fc and NP M123/Fc in the presence of H2O2.

Figure S16 .
Figure S16.DLS measurement of diameter sizes and PDIs of a) NP M123 and b) NP Fc in the presence or absence of H2O2.c) Average Zeta potentials of NP M123 and NP Fc in the presence or absence of H2O2.

Figure S17 .
Figure S17.TEM images of P. aeruginosa and S. aureus post treatment.

Figure S22 .
Figure S22.Heatmap of selected DRGs involved in proinflammation and wound healing genes.

Figure S24 .
Figure S24.Hemolysis rates of RBCs incubated with different concentrations of NP M123/Fc .

Figure S3 .
Figure S3.TEM images of NP M123 and NP Fc .Scale bar is 100 nm.

Figure S9 .
Figure S9.Absorption curves of DPBF as a selective trapping agent to detect ROS in the reaction mixture of NP M123 +H2O2, NP Fc +H2O2, or NP M123/Fc +H2O2 after different incubation times.

Figure S10 .
Figure S10.Trends of UV absorption at 410 nm as indicated by the above DPBF-based detection results.

Figure S11 .
Figure S11.a) Absorption curves of MB as a selective trapping agent to detect •OH in the reaction mixture of NP Fc +H2O2 after different incubation times.b) Trends of UV absorption at 410 nm as indicated by the above MB-based detection results.

Figure S12 .
Figure S12.Fc release kinetics of NP M123/Fc in vitro was determined by ICP-MS.

Figure S13 .
Figure S13.XPS spectra of P Fc .a) In the absence of H2O2, the peaks at 707 Ev denote ferrous (+2) valence state.b) In the presence H2O2, the peaks at 711 Ev show ferric (+3) valence state.

Figure S14 .
Figure S14.TEM images for NP M123 , NP Fc and NP M123/Fc in the presence of H2O2.Scale bar is 100 nm.

Figure S15 .
Figure S15.SEM images for NP M123 , NP Fc and NP M123/Fc in the presence of H2O2.Scale bar is 100 nm.

Figure S16 .
Figure S16.DLS measurement of diameter sizes and PDIs of a) NP M123 and b) NP Fc in the presence or absence of H2O2.c) Average Zeta potentials of NP M123 and NP Fc in the presence or absence of H2O2.

Figure S23 .
Figure S23.Viability of NIH-3T3 and IOSE-80 cells incubated with NP M123/Fc at different concentrations for 24 h.All data are presented as mean ± SD (n = 6).

Figure S24 .
Figure S24.Hemolysis rates of RBCs incubated with different concentrations of NP M123/Fc .The insert was the optical photograph.PBS and water were used as the negative and positive controls, respectively.All data are presented as mean ± SD (n = 3).UD: undetectable.