A H2S‐Generated Supramolecular Photosensitizer for Enhanced Photodynamic Antibacterial Infection and Relieving Inflammation

Abstract Photodynamic therapy (PDT) is a promising treatment against bacteria‐caused infections. By producing large amounts of reactive oxygen species (ROS), PDT can effectively eliminate pathogenic bacteria, without causing drug resistance. However, excessive ROS may also impose an oxidative stress on surrounding tissues, resulting in local inflammation. To avoid this major drawback and limit pro‐inflammation during PDT, this work prepared a supramolecular photosensitizer (TPP‐CN/CP5) based on host‐guest interactions between a cysteine‐responsive cyano‐tetraphenylporphyrin (TPP‐CN) and a water‐soluble carboxylatopillar[5]arene (CP5). TPP‐CN/CP5 not only possesses excellent photodynamic antibacterial properties, but also shows good anti‐inflammatory and cell protection capabilities. Under 660 nm light irradiation, TPP‐CN/CP5 could rapidly produce abundant ROS for sterilization. After the PDT process, the addition of cysteine (Cys) triggers the release of H2S from TPP‐CN. H2S then stops the induced inflammation by inhibiting the production of related inflammatory factors. Both in vitro and in vivo experiments show the excellent antibacterial effects and anti‐inflammatory abilities of TPP‐CN/CP5. These results will certainly promote the clinical application of PDT in the treatment of bacterial infectious diseases.


Methods
The 1 H nuclear magnetic resonance ( 1 H NMR) spectra were recorded using a BRUKER AV400 Spectrophotometer (400 MHz, 298K) with tetramethylsilane (TMS) as an internal reference.The ultraviolet-visible (UV-vis) absorption spectra were determined on a Thermo Scientific Evolution 220 spectrophotometer, and fluorescence spectra measurements were performed on a Varian's Cary Eclipse fluorescence spectrophotometer.Dynamic light scattering (DLS) and zeta potential measurements were carried out using a Beckman Coulter Delasa Nano C particle analyser and samples were characterized in an aqueous solution.All the measurements were carried out at room temperature.Transmission electron microscopy (TEM) analysis was performed on a JEOL JEM-1400 electron microscope operated at 100 kV.Scanning electron microscopy (SEM) analysis was conducted with Scanning Electron Microscope--Energy Dispersive X-ray Spectroscope (S-3400N).

Synthesis of TPP-CN
Synthesis of TPP-NCS: TPP-NH 2 (150 mg, 0.24 mmol) and DMAP (12.2 mg, 0.1 mmol) were dissolved in dry CH 2 Cl 2 and allowed to stir at room temperature for 20 minutes under a nitrogen atmosphere.Thiophosgene (22 μL, 0.28 mmol) was added to the solution, and the mixture was stirred at room temperature for 1 h.Then the reaction solution was washed with saturated ammonium chloride aqueous solution (for twice) and sodium chloride aqueous solution (for twice).The organic layer concentrated by rotary evaporation and purified by column chromatography to obtain TPP-NCS.Yield: 150 mg, 93.1%.

Synthesis of TPP-CN:
4-Hydroxyphenylacetonitrile (95 mg, 0.71 mmol) was dissolved in 5 mL of dry THF and placed in nitrogen atmosphere, then a certain amount of sodium hydride was added to the solution.After stirring for 30 min, TPP-NCS (95 mg, 0.14 mmol) was dissolved in 5 mL of dry THF and added into the reaction system.The deionized water was slowly added dropwise to quench the reaction after reacting at room temperature for 1 h.Subsequently, the crude product was obtained by extraction with DCM and rotary evaporation, which was further purified by column chromatography (EA: PE = 1:4).Yield: 38 mg, 33.4%.

Figure S7 .
Figure S7.Fluorescence spectra of the TPP-CN and TPP-CN/CP5 assemblies (dissolved in DCM) at room temperature with or without the addition of Cys.

Figure S11 .
Figure S11.UV-vis absorption spectra for H 2 S release detected by the MB method

Figure S14 .
Figure S14.Changes of wound size in different groups of mice with days.

Figure S15 .
Figure S15.The healing rate of wounds in different groups of mice on the 4 th and 7 th day.