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Introduction

The overuse of antibiotics has resulted in the development of antibiotic resistance in pathogenic bacteria, making the treatment of bacterial infectious diseases more challenging.[1] In recent years, photodynamic antimicrobial therapy has garnered extensive attention for its non-invasiveness, light-controllable features, low systemic toxicity, and minimal drug resistance.[2] Photosensitizers, which play a key role in photodynamic therapy (PDT),[3] can transfer energy to surrounding oxygen molecules under the excitation of light at a specific wavelength, producing highly cytotoxic reactive oxygen species (ROS) that destroy biological molecules such as proteins and nucleic acids.[4] The instantaneous production of large amounts of ROS achieves a potent antibacterial effect, reducing the risk of producing drug-resistant bacteria.[5] However, the excessive amount ROS acts as an exogenous stimulus, inducing oxidative stress in the surrounding tissues. This, in turn, triggers the release of a series of cytokines, including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), which causes local tissue inflammation, damaging proteins, nucleic acids, and lipids, and eventually resulting in cell death and tissue damage.[6] Although the pro-inflammatory side effects stemming from elevated ROS levels during PDT can seriously impede the development of clinical applications, they are often overlooked.[7]

Hydrogen sulfide (H2S) serves as a crucial endogenous gas transmitter, regulating physiological and pathological processes across the gastrointestinal, cardiovascular, nervous, and endocrine systems.[8] Being an abundant physiological gaseous medium, H2S can effortlessly penetrate cell membranes without the need for specific carriers.[9] Its roles and mechanism in cardiovascular protection, immune response, anti-inflammatory, and anticancer activities have been closely investigated.[10] Various platforms and advanced materials utilizing H2S donors have been designed for therapeutic and clinical purposes.[8a,11] For example, Wan et al. introduced a zwitterion-based H2S nanomotor designed to induce multiple acidosis in tumor cells, effectively inhibiting tumor growth.[12] Lin and colleagues used dendritic mesoporous organosilica as a nanocarrier and a new kind of H2S gas generator, that they loaded with chloroperoxidase and sodium-hyaluronate-modified calcium peroxide nanoparticles to construct a tumor-microenvironment-responsive nanocomposite for H2S gas and trimodal-enhanced enzyme dynamic therapy.[13] In physiological environment, H2S suppresses the expression of inflammatory cytokines, including TNF-α and IL-6, by inhibiting crucial pro-inflammatory transcription factors like phosphodiesterase and nuclear factor B (NF-κB).[14] Consequently, H2S can trigger endogenous antioxidant stress defense mechanisms, safeguarding cells against infection-related oxidative stress.[14a] Research and development of H2S-based therapy is, thus, of great significance for treating inflammation-related diseases.

In this study, we have developed a novel supramolecular porphyrin photosensitizer specifically triggered by cysteine (Cys) to release H2S, achieving the synergistic effect of photodynamic antibacterial and H2S anti-inflammatory therapies. As illustrated in Scheme 1, the photosensitizer consists of a cyano-tetraphenyl porphyrin with a Cys-responsive group (TPP-CN) and the water-soluble carboxylatopillar[5]arene (CP5). Functioning as a near-infrared photosensitizer, TPP-CN exhibits excellent ROS generation capability under 660 nm light excitation. Furthermore, the stability and biocompatibility of TPP-CN could significantly be improved by forming the supramolecular porphyrin photosensitizer (TPP-CN/CP5) through the host-guest complex interaction between the cyano group and the water-soluble macrocyclic host CP5. At the site of bacterial infection, the supramolecular photosensitizer TPP-CN/CP5 produces a substantial amount of ROS under light radiation, achieving the effect of photodynamic sterilization. Subsequently, in the presence of Cys, TPP-CN releases H2S, which plays a role in resisting oxidative stress and relieving inflammation. Thus, TPP-CN/CP5 can simultaneously achieve bactericidal and anti-inflammatory responses, facilitating the rapid recovery of the infected site. By intelligently combining antibacterial and anti-inflammatory capacities, this strategy overcomes the pro-inflammatory side effects of PDT, offering a novel prospect for PDT in the clinical treatment of infectious diseases.

Scheme 1. Schematic illustration of supramolecular porphyrin photosensitizer TPP-CN/CP5 and its antibacterial and anti-inflammatory activity.
Results and Discussion Preparation and Characterization of Supramolecular Photosensitizer

The detailed synthetic procedures for the synthesis of cyano-tetraphenyl porphyrin with a cysteine (Cys)-responsive group (TPP-CN) and carboxylatopillar[5]arene (CP5) are provided in Supporting Information (Scheme S1, Supporting Information). The host-guest complexation of CP5 and TPP-CN was investigated through 1H NMR titration experiments. Owing to the poor solubility of TPP-CN in D2O, the model guest GM (4-hydroxyphenyl acetonitrile) was employed to form a complex with host CP5, as shown in Figure 1a. Because of the shielding effect of the electron-rich cavity of CP5, the signals related to the protons on GM (H1, H2, and H3) shifted upfield. Simultaneously, the phenyl protons (H4) and methylene protons (H5 and H6) on CP5 slightly shifted upfield. These results confirmed that the host-guest interaction between CP5 and GM was robust enough to facilitate the versatile construction of supramolecular complexes.[15]

Figure 1. a) 1H NMR spectra of solutions of GM at a fixed concentration of 3.0 mm mixed with different concentrations of CP5: A) 0 mm, B) 1.0 mm, C) 2.0 mm, D) 3.0 mm, E) 5.0 mm, and F) 1H NMR spectrum of CP5. A schematic illustration of CP5 (host) complexed with cyano group (guest) is shown on the right side of the 1H NMR spectra. b) UV–vis absorption spectra of TPP-CN (1 × 10−2 mm) in aqueous solution in presence of different equivalents of CP5. c) Fluorescence spectra of TPP-CN (1 × 10−2 mm) mixed with different equivalent of CP5 (0-3 equiv) in aqueous solution at room temperature. d) Job's plot for CP5 and TPP-CN in aqueous solution showed a 1:1 stoichiometry (according to fluorescence titration). e) Plot of fluorescence (FL) intensity as a function of concentration of CP5, where the red solid line was obtained from the non-linear curve-fitting.

The formation of host-guest complexation was further confirmed by UV–vis and fluorescence titration experiments. As the concentration of CP5 increased, the absorption and fluorescence intensity of porphyrins gradually rose (Figure 1b,c), indicating that host-guest complexation improved the dispersibility of TPP-CN in aqueous solution. Simultaneously, the stoichiometry of complexation between CP5 and TPP-CN in aqueous solution was investigated using Job's plot method, which revealed a 1:1 stoichiometry between CP5 and TPP-CN (Figure 1d and Figure S6, Supporting Information). According to the non-linear curve-fitting, the association constant (Ka) of CP5 and TPP-CN was calculated to be (7.43 ± 1.75) × 104 m−1 (Figure 1e), highlighting a strong binding affinity between CP5 and cyano units. The excellent complexation of this host-guest system was primarily attributed to hydrophilic-hydrophobic interactions.

Subsequently, supramolecular assemblies (TPP-CN/CP5) were prepared by combining TPP-CN and CP5 in aqueous solution via a precipitation strategy. The hydrodynamic size and morphology of TPP-CN/CP5 assemblies were characterized by dynamic laser scattering (DLS) and transmission electron microscopy (TEM). As shown in Figure 2a,b, TPP-CN/CP5 assemblies exhibited a uniform spherical morphology with a hydrodynamic diameter of ≈140 nm. Upon incubation with Cys, both the size and polydispersity index of the assemblies slightly increased. Besides, the fluorescence spectra in Figure 2c and Figure S7, Supporting Information, revealed that the fluorescence intensity of TPP-CN/CP5 assemblies near 660 nm was stronger than that of TPP-CN, which indicated that host CP5 hindered the aggregation of porphyrins to some extent and enhanced the fluorescence of porphyrin molecules. The addition of Cys had minimal effect on the fluorescence properties of TPP-CN/CP5. The UV–vis absorption spectra (Figure 2d) displayed the characteristic absorption of porphyrin, featuring a strong absorption peak near 425 nm (Soret band) and four weak absorption peaks between 500 and 670 nm (Q band).

Figure 2. a) DLS results of TPP-CN/CP5 assemblies in aqueous solution with or without Cys. b) TEM image of TPP-CN/CP5 supramolecular nanoparticles. c) Fluorescence spectra of the TPP-CN, TPP-CN/CP5 assemblies in aqueous solution at room temperature with or without Cys. d) UV–vis absorption spectra of TPP-CN and TPP-CN/CP5 in aqueous solution with or without Cys. e) UV–vis spectra of 1,3-diphenylisobenzofuran (DPBF) (detection of singlet oxygen production) in a TPP-CN/CP5 aqueous solution under 660 nm light irradiation. f) Percentage of degradation of DPBF in different solutions upon light irradiation.

Furthermore, the singlet oxygen (1O2) generation property of TPP-CN/CP5 under light irradiation was measured by using 1,3-diphenylisobenzofuran (DPBF), a singlet oxygen scavenger. The absorbance of DPBF at 425 nm in a TPP-CN/CP5 solution gradually decreased with increasing irradiation time (Figure 2e). To evaluate the yield of 1O2 formation more quantitatively, we plotted the change in DPBF absorbance against irradiation time (Figure 2f). As shown in this plot, TPP-CN/CP5 possessed an outstanding 1O2 generation capacity, with a percentage of DPBF degradation reaching 48% within 30 s. It is also worth noting that the addition of Cys in a TPP-CN/CP5 solution significantly inhibited DPBF degradation under light irradiation. Interestingly, a similar observation could be made when we evaluated the change in DPBF absorbance of a TPP-CN/CP5 solution in presence of H2S (Figure S8, Supporting Information) for different irradiation time. We attributed this inhibition to the generation of H2S from the reaction between TPP-CN/CP5 and Cys, thereby quenching most of the ROS. To further confirm that this phenomenon was due to the antioxidant capacity of H2S rather than Cys, sodium tetrakis-(4-carboxyphenyl)-porphyrin (TCPP(Na4)) was employed as a model photosensitizer to detect the degradation of DPBF with or without Cys (Figure S9, Supporting Information). The results showed that TCPP(Na4) demonstrated the same degradation ability for DPBF under light radiation regardless of the presence of Cys (Figure S10, Supporting Information). This indicated that the amount of ROS captured by DPBF was not affected by Cys, further supporting the conclusion that the low DPBF degradation capacity of TPP-CN/CP5 in the presence of Cys could be attributed to the release of H2S in response to Cys.

Cys-Responsiveness and H2S Release from TPP-CN/CP5 Assemblies

Because H2S is the third endogenous gas transmitter besides carbon monoxide and nitric oxide, its controlled release has attracted increasing attention.[8a] The release of H2S can be triggered by specific stimuli such as thiols, light, enzymes, and ROS.[16] In the present study, TPP-CN reacted with Cys to release H2S, as depicted in Figure 3a.

Figure 3. Evaluation of responsive release capacity of H2S. a) Proposed mechanism of H2S release from TPP-CN triggered by Cys. b) UV–vis absorption spectra for detecting H2S release (MB method) at different incubation times. The concentration of TPP-CN was 10 µg mL−1 and that of Cys was 100 µm. c) Plot of UV–vis absorption at 665 nm as a function of incubation time of TPP-CN with Cys. d) Changes in UV–vis absorbance (at 665 nm) of H2S detection solutions (containing TPP-CN) in the absence of Cys at different pH values at room temperature. e) Bacterial intracellular H2S detection using WSP-1 as a probe after different treatments. Scale bar is 20 µm. f) Mean fluorescence intensities (MFI) of H2S probe fluorescence images. Results are expressed as mean ± SD (n = 3). One-way ANOVA with a post-hoc bonferroni's test, ***p ≤ 0.001; ****p ≤ 0.0001.

The colorimetric methylene blue (MB) assay was utilized to determine the Cys-triggered H2S release of TPP-CN and TPP-CN/CP5. Figure 3b shows an obvious increase in UV–vis absorption as a function of incubation time of TPP-CN with Cys, indicating an enhanced H2S release. The slow-release process of H2S can further be observed in Figure 3c which shows the time-dependent H2S release curve performed within 3 h. As mentioned in a previous study, the ideal H2S release process should be slow, continuous and controllable without disturbing the internal environment and homeostasis, or producing undesirable bioactive byproducts.[17] TPP-CN/CP5 supramolecular assemblies presented a similar H2S release behavior triggered by Cys (Figure S11, Supporting Information). However, in the absence of Cys, no H2S was generated from TPP-CN assemblies under neither acidic nor alkaline environments (Figure 3d and Figure S12, Supporting Information), indicating the good stability of TPP-CN assemblies in different pH conditions. Similar behavior as a function of pH could also be observed for the supramolecular TPP-CN/CP5 assemblies in absence of Cys (Figure S13, Supporting Information). These results demonstrate that Cys plays an essential role in H2S production by TPP-CN and TPP-CN/CP5 assemblies. For further investigation, we detected the responsive release of H2S from TPP-CN/CP5 in live bacterial cells using the H2S fluorescent probe Washington State Probe-1 (WSP-1).[18] After pretreating bacteria with different solutions and WSP-1, the channel images of the H2S probe were monitored by fluorescence microscopy (Figure 3e). As expected, almost no fluorescence was observed in the control group, and the bacteria exhibited weak fluorescence in the TPP-CN/CP5 groups due to the release of a small amount of H2S triggered by endogenous Cys. However, the green fluorescence became brighter, and the mean fluorescence intensity (MFI) strongly increased with addition of Cys (100 µm) (Figure 3e,f). These results validate that TPP-CN/CP5 can react with Cys for efficient H2S release.

In Vitro Antibacterial Activity and Biofilm Dissipation

Inspired by the high 1O2 generation efficiency of TPP-CN/CP5, the minimum inhibitory concentration (MIC) assay and the spread plate method were employed to evaluate the in vitro antibacterial activity against Gram-positive bacteria (S. aureus) and Gram-negative bacteria (E. coli). As depicted in Figure 4a,b, TPP-CN/CP5 displayed a dose-dependent antibacterial efficiency toward both S. aureus and E. coli under 660 nm light irradiation (600 mW cm−2, 15 min). Over 90% of S. aureus and 80% of E. coli were eliminated at a concentration of 60 µg mL−1 under light irradiation, while TPP-CN/CP5 displayed weak bacterial cytotoxicity in the absence of irradiation. Moreover, the addition of Cys after light irradiation had almost no effect on the photodynamic antibacterial performance of TPP-CN/CP5. The disparity in survival rates between S. aureus and E. coli is mainly due to E. coli being Gram-negative bacteria with an external cellular membrane linked by lipopolysaccharides, which reduces its sensitivity to reactive oxygen species.[1c] Additionally, results from the spread plate method (Figure 4c) showed that the number of bacterial colonies was significantly low on the agar under 660 nm irradiation for both S. aureus and E. coli, showcasing the excellent PDT antibacterial ability of TPP-CN/CP5.

Figure 4. In vitro studies of antibacterial activity of TPP-CN/CP5. a) In vitro toxicity of S. aureus (S) and E. coli (E) treated with TPP-CN/CP5 in the presence of Cys (100 µm). b) Bacterial viability of S. aureus (S) and E. coli (E) treated with TPP-CN/CP5. L is short for light irradiation, representing that the sample was irradiated under light conditions. Values are mean ± standard deviation (SD) (n = 3). Student's t-test was used for statistically significant analysis, **p ≤ 0.01; ***p ≤ 0.001. c) Plates images of S. aureus and E. coli colonies with different treatments. d) SEM images of S. aureus and E. coli with different treatments. Yellow arrows point to the obvious breakages of S. aureus and E. coli. Scale bar: 50 nm.

To further visualize the destructive effect of PDT on bacteria, scanning electron microscopy (SEM) analysis was employed to observe the morphological changes of S. aureus and E. coli (Figure 4d). In both the control and non-irradiated groups, the bacteria had regular body shapes with smooth surfaces and intact cell walls. In contrast, the cell membranes of bacteria treated with TPP-CN/CP5 and light were significantly wrinkled and collapsed, indicating the impaired or destroyed bacterial cellular integrity. Furthermore, the results of live-dead staining analysis by confocal laser scanning microscope (CLSM) (Figure 5a) confirmed that there was significantly more red fluorescence (staining dead bacterial cells) in light-irradiated groups compared to the control and non-irradiated groups, which primarily exhibited green fluorescence (staining live bacterial cells). Quantitative analysis of red-green fluorescence intensity (Figure 5b) also showed that the number of bacterial deaths in the light-irradiated groups were much larger than in the non-irradiated groups. These findings were consistent with the above antibacterial results, emphasizing the remarkable PDT antibacterial efficiency of the TPP-CN/CP5 nanoparticles.

Figure 5. a) Live-dead images of S. aureus and E. coli treated with PBS (control), TPP-CN/CP5 and TPP-CN/CP5+L with or without Cys (100 µm). All bacterial cells were indicated by green fluorescence (SYTO9 channel), and the disruption of cell membranes was indicated by red fluorescence (PI channel). Scale bar: 10 µm. b) Quantitative analysis of red/green fluorescence signals, showing the dead/alive ratio of bacteria. Results are expressed as mean ± SD (n = 3), **p ≤ 0.01; ***p ≤ 0.001. c) The photographs of crystal violet (CV) stained MRSA biofilm in plates. d) OD590 nm value of the remaining biofilm biomass after different treatment. e) Dispersion rate of biofilms with different treatments. Results are expressed as mean ± SD (n = 3), One-way ANOVA with a post-hoc bonferroni's test, **p ≤ 0.01; ***p ≤ 0.001. f) 3D CLSM images of biofilm treated with PBS (the control), TPP-CN/CP5 and TPP-CN/CP5+L with or without Cys (100 µm). The polysaccharide on the biofilm and dead bacteria were stained with ConA-FITC (green fluorescence) and PI (red fluorescence), respectively. Light irradiation: 660 nm laser light, 600 mW cm−2, 10 min. Scale bar: 20 µm.

The formation of bacterial biofilm is a critical factor contributing to the development of bacterial resistance, which significantly hinders the recovery of infected site.[19] Therefore, we further investigated the photodynamic biofilm dissipation capacity of TPP-CN/CP5. The crystal violet (CV) staining method was employed to directly observe the effect of the supramolecular photosensitizers on biofilm dissipation (Figure 5c) and quantify the biofilm mass (Figure 5d). The stained biofilm plates and the residual biofilm biomass revealed that there was only a small amount of residual biofilm in the light groups (TPP-CN/CP5+L and TPP-CN/CP5+L+Cys), while the biofilm was relatively intact, with a certain thickness in all the non-irradiated groups. Particularly, the dissipation efficiency of the biofilm treated by TPP-CN/CP5+L and TPP-CN/CP5+L+Cys both reached 84% (Figure 5e), demonstrating that ROS generated by TPP-CN/CP5 under light irradiation could effectively destroy the biofilm. The thickness and breakage of biofilms in different groups were observed by 3D images obtained by confocal microscopy (Figure 5f). Consistently, the biofilms treated with TPP-CN/CP5+L and TPP-CN/CP5+L+Cys exhibited fragmentation, and the bacterial density within the biofilm decreased dramatically. In contrast, the biofilms in other groups remained relatively dense and thick. The outstanding photodynamic biofilm dissipation capacity of TPP-CN/CP5 significantly contributed to its photodynamic antibacterial capability.

Anti-inflammatory Activity of TPP-CN/CP5

Previous studies have reported that H2S can inhibit the production of inflammatory cytokines, achieving an anti-inflammatory effect in the cell microenvironment.[14a] We, thus, decided to take advantage of the excellent ability of TPP-CN/CP5 to release H2S in presence of Cys to alleviate inflammation induced by pro-inflammatory cytokines and ROS. As a proof-of-concept, the anti-inflammatory effect of TPP-CN/CP5 triggered by Cys in RAW264.7 cells was investigated. Lipopolysaccharide (LPS) was selected as the stimulus to establish the cellular inflammation model.[6] The inflammatory response of RAW264.7 was evaluated through the expression levels of tumor necrosis factor-α (TNF-α) and chemokine interleukin-6 (IL-6) monitored by ELISA Kit. In comparison to the control group, the expression levels of TNF-α and IL-6 in RAW264.7 cells pretreated with LPS significantly increased. After incubating the pretreated cells with TPP-CN/CP5 and Cys, a dose-dependent inhibition of TNF-α and IL-6 was observed. Moreover, the anti-inflammatory effects of TPP-CN/CP5 without Cys and that of Cys alone were also evaluated; none of them attenuated the generation of TNF-α and IL-6 (Figure 6a,b). These results indicate that TPP-CN/CP5 exhibited good anti-inflammatory activity after being triggered by Cys, most likely due to the release of H2S.

Figure 6. Expression of inflammatory cytokines a) TNF-α and b) IL-6 in LPS-pretreated macrophage RAW264.7 cells. Results are expressed as mean ± SD (n = 3), ###p ≤ 0.001 versus the control group; ***p ≤ 0.001 and ****p ≤ 0.0001 versus the 0 µg mL−1 of TPP-CN/CP5 group. The levels of c) TNF-α and d) IL-6 in light-irradiated RAW264.7 cells (n = 3, mean ± SD). One-way ANOVA with a post-hoc Bonferroni's test, #p ≤ 0.05, ##p ≤ 0.01, and ###p ≤ 0.001 versus the control group; ***p ≤ 0.001 versus the TPP-CN/CP5 group. e) Cell viability of L929 treated with TPP-CN/CP5 and TPP-CN/CP5+Cys (n = 3, mean ± SD). f) Cell viability of L929 treated with different concentrations of Cys (n = 3, mean ± SD).

Furthermore, solutions of TPP-CN/CP5, Cys, and TPP-CN/CP5+Cys with different concentrations of TPP-CN/CP5 upon 660 nm light irradiation were used to comparatively investigate the anti-inflammatory protection capabilities against PDT-aggravated ROS-induced inflammation (Figure 6c,d). TNF-α and IL-6 were overexpressed after light irradiation in RAW264.7 cells incubated with TPP-CN/CP5 alone. In the absence of photosensitizers (the Cys group and the group of Cys 0 µg/mL + TPP-CN/CP5 0 µm), the production of inflammatory factors induced by light irradiation was very limited. After the administration of TPP-CN/CP5+Cys, the levels of TNF-α and IL-6 gradually decreased to reach those of the control groups, indicating an excellent anti-inflammatory protection ability.

In Vivo MRSA-Infected Therapy

Considering the desirable antibacterial properties and anti-inflammatory effects mentioned earlier, the efficacy of the antibacterial and anti-inflammatory performance of TPP-CN/CP5 was further evaluated in an MRSA-infected mouse wound model. Before conducting in vivo antibacterial experiments, we first tested the biocompatibility of TPP-CN/CP5 and Cys by MTT cell assay. As shown in Figure 6e, the TPP-CN/CP5 assembly exhibited a certain level of toxicity to L929 cells as the concentration increased. However, the cell survival rate was significantly improved after the addition of Cys, possibly because the generation of H2S could promote anti-apoptotic effects to protect the cells.[17] The results in Figure 6e,f confirmed that the treatment of TPP-CN/CP5+Cys was biocompatible and could be applied to subsequent in vivo studies.

The animal experiments were performed according to the protocol schematized in Figure 7a. After wound modeling and bacterial infection, mice were randomly divided into five groups, which were given different treatments: phosphate buffered saline (PBS) (control group), TPP-CN/CP5 (dark group), TPP-CN/CP5+L (light), TPP-CN/CP5+Cys (dark group), and TPP-CN/CP5+L+Cys (light). The evolution of the MRSA infection sites of mice in each group was monitored over a period of 7 days, and photographs of the wound sites are shown in Figure 7b. To better illustrate the variation in the wound healing effects of the different treatments, the corresponding wound sizes were measured. The wound healing trend could be observed through the change in wound size (Figure 7c and Figure S14, Supporting Information). In the group treated with TPP-CN/CP5+L+Cys, there was no obvious suppuration in the wound, which gradually healed from day 0 to day 7, leaving a negligible small scar on the skin. However, the wounds of mice in other groups showed a certain degree of suppuration on the second day after MRSA infection. Additionally, suppuration in MRSA-infected wounds for the control or dark groups became severe on day 4, and large wounds were still visible by day 7. Figure 7d illustrates that on the seventh day the infected wound healed by more than 90% after treatment with TPP-CN/CP5 under laser irradiation, especially in the TPP-CN/CP5+L+Cys group, where the wound healing rate reached 98%, while the wound healing rate of other groups was relatively low. Wound healing rates of mice in each group on day 4 and day 7 were recorded (Figure S15, Supporting Information). Although TPP-CN/CP5+L could also facilitate the wound healing process, the degree of wound recovery and relative wound size in mice showed that the bacterial inhibitory effect was enhanced when combined with Cys. This improvement was due to the release of hydrogen sulfide triggered by the addition of Cys to TPP-CN/CP5, effectively inhibiting the expression of inflammatory cytokines in wound tissues after antibacterial PDT by TPP-CN/CP5. Thus, the dual antibacterial and anti-inflammatory effects jointly promoted wound healing.

Figure 7. In vivo antibacterial efficacy study using mice wound model of MRSA infection. a) Schematic diagram of the in vivo antibacterial experiment. b) Photographs of MRSA infected wound sites on mice taken over the 7-day post-treatment experiments, for different treatments; Note: the scale bar for wound size is given at the bottom. c) Changes of infected wound sizes in mice of the different groups. d) Wound healing rate of mice in different groups. Values are mean ± standard deviation (SD) (n = 3), One-way ANOVA with a post-hoc Bonferroni's test,**p ≤ 0.01; ***p ≤ 0.001. (600 mW cm−2, 15 min).

Histological analyses using hematoxylin and eosin (H&E) staining were performed to evaluate the wound-healing efficacy. As shown in Figure 8a, the skin tissues of the control group and dark groups exhibited acute and extensive inflammatory states with a significant infiltration of inflammatory cells. Conversely, in the TPP-CN/CP5+L group, there was a reduction in inflammatory cells, and the inflammatory foci became localized, which indicates that the inflammation was alleviated due to the elimination of pathogenic bacteria by PDT. The TPP-CN/CP5+L+Cys group displayed fewer inflammatory cells and better-formed fibers were observed, exhibiting healthy morphological features and suggesting excellent antibacterial and wound healing ability.

Figure 8. Histological and immunohistochemical evaluation of skin tissues after 7-day treatments. a) H&E staining images of the infected skins harvested from different mice on seventh day post-treatment. Immunohistochemical staining for b) TNF-α and c) IL-6 in the skin tissues of the infected areas. d) Immunostaining images of CD68+ expressed in the phagocytic macrophages. Scale bar: 50 µm. The corresponding quantification of positive cells with e) TNF-α, f) IL-6, g) CD68+. n = 3, One-way ANOVA with a post-hoc Bonferroni's test, **p [less than] 0.01, ***p [less than] 0.001.

Subsequently, we further analyzed the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), crucial inflammatory markers, in the wound tissues by immunohistochemical (IHC) evaluation. A trend similar to that observed with H&E staining can be seen in Figure 8b,c, where the relevant inflammatory factors were stained brown. The control group, dark groups, as well and TPP-CN/CP5+L group, exhibited a wide range of TNF-α and IL-6 positive expression cells. In contrast, the expression of these pro-inflammatory cytokines was significantly restrained in the TPP-CN/CP5+L+Cys group. Similarly, consistent results were observed in skin slice images for each group by using CD68 as a reliable marker of macrophages with pro-inflammatory activity (Figure 8d). Compared with the other four groups, the expression level of CD68+ in the TPP-CN/CP5+L+Cys group was extremely limited, indicating that the mice from this group basically recovered to a healthy state without any obvious inflammatory reaction in the wound sites.

From the above experimental results, we can conclude that TPP-CN/CP5+L could effectively eliminate local pathogens through PDT. However, the photodynamic antibacterial process can exacerbate inflammation, limiting the application of PDT in infectious diseases. PDT performed with TPP-CN/CP5+L+Cys, on the other hand, decreases effectively the external stimuli of bacteria. Simultaneously, TPP-CN/CP5+L+Cys alleviates the side effects of PDT by releasing H2S to inhibit the expression of pro-inflammatory factors and regulate local inflammation.

Conclusion

In summary, a supramolecular porphyrin photosensitizer TPP-CN/CP5 with antibacterial and anti-inflammatory functions was successfully designed and constructed, which solved the contradiction between ROS sterilization and pro-inflammatory effect in PDT applications. CP5 endowed the supramolecular TPP-CN/CP5 assemblies with good water solubility and biocompatibility, improving its ROS generation capability. In addition, TPP-CN could be triggered by Cys to release H2S, which has excellent anti-inflammatory ability. Therefore, TPP-CN/CP5+Cys nanoplatform achieved the perfect combination of PDT sterilization and H2S anti-inflammatory effects. The PDT antibacterial activity of TPP-CN/CP5 was confirmed by in vitro antibacterial experiments and MRSA-infected wound experiments. Moreover, the results of the macrophage inflammation model and immunohistochemical evaluation showed that TPP-CN/CP5 significantly inhibited the expression of related inflammatory factors (TNF-α and IL-6) in the presence of Cys. The integration of PDT antibacterial and H2S anti-inflammatory properties was effective, with excellent antibacterial activity, protecting normal cells from oxidative stress and inflammatory reactions. Thus, this antibacterial system sheds light on the future design and development of photodynamic antibacterial materials and provides a broad prospect for its future clinical application.

Experimental Section Self-Assembly of TPP-CN/CP5 in Aqueous Solution

TPP-CN (1.0 mg, 0.001 mmol) was dissolved in 0.2 mL of DMF and then injected dropwise into a CP5 aqueous solution (5 mL, 0.15 mm) under stirring. After stirring for 4 h, DMF was removed by dialysis (MWCO = 12 kDa) against deionized water (renewed fresh water four times). Finally, the concentration of the assembly was 0.36 mg mL−1.

In Vitro Antibacterial Experiments

A bacteria suspension was diluted to 106 CFU mL−1 with a sterile medium. Then a volume of 150 µL of the diluted bacterial solution was added into 96-well plates and mixed with 50 µL of sample solutions with varying concentrations (dissolved in pure liquid medium). The irradiation duration and intensity were set as 660 nm laser light on the power of 600 mW cm−2 for 15 min (shortened as “+L” in group labeling). The 96-well plates were incubated at 37 °C for 24 h after different treatments. A control group with only bacterial solution and medium was carried out. The group TPP-CN/CP5 +Cys referred to the bacteria treated by both TPP-CN/CP5 and Cys, but no light irradiation. After light irradiation, ʟ-cysteine (100 µm) was immediately added to the TPP-CN/CP5+L+Cys group. Subsequently, the bacteria suspension was diluted with PBS 1000 times and spread on the solid LB agar plate, followed by culturing at 37 °C for 12 h before colony forming units (CFU) counting and taking photos.

Triplicate analyses of each sample were performed and each experiment was carried out in duplicate.

SEM Analysis

The bacterial suspension (109 CFU mL−1) was washed with PBS, centrifuged (6500 rpm for 4 min), and dispersed in 1 mL of PBS. Next, the bacterial suspension alone or treated with TPP-CN/CP5 assembly (80 µg mL−1) was incubated in a shaking incubator (37 °C, 180 rpm). After 30 min, the latter was irradiated by 660 nm light for 15 min or not. After further incubation with Cys or not for 1 h, the bacteria were centrifuged to remove PBS and then fixed with 2.5% glutaraldehyde overnight. The glutaraldehyde was removed by centrifugation and the bacteria were washed with PBS three times. Subsequently, the obtained samples were dehydrated with ethanol solution in a graded series (30%, 50%, 70%, 80%, 90%, and 100%) for 15 min, respectively, and followed by being dispersed in ethanol. Ultimately, the specimens were dropped onto clean silicon slices and dried in the air before being observed by SEM.

Live/Dead Bacterial Staining Assay

Bacteria were incubated in an LB medium overnight with shaking. Then 0.5 mL bacteria suspension was centrifuged to remove the medium and then dispersed in PBS containing TPP-CN/CP5 nanoparticles of 80 µg mL−1. The suspensions were irradiated by 660 nm light for 15 min or not after incubation for 1 h, and further treated with or without Cys (100 µm). The live/dead dye SYTO9 and PI were added in the dark and kept for 20 min. Finally, CLSM was used to observe the viability of bacteria.

Statistical Analysis

One-way ANOVA with a post-hoc bonferroni's test was used for statistical analysis. The statistically significant difference between different groups was marked as ∗p, ∗∗p, and ∗∗∗p for p < 0.01, 0.05, and 0.001, respectively.

Acknowledgements

J.T. and B.H. contributed equally to this work. The authors acknowledge the financial support from the National Natural Science Foundation of China (52333014, 22075079, 52203009), and the Science and Technology Commission of Shanghai Municipality (21ZR1417800, 21520713400 and 22142201100). The authors acknowledge the help of Prof. Gerald Guerin (East China University of Science and Technology, China) on the language polish and scientific discussion. All animal experiments in this study were performed in accordance with institutional guidelines and approved by the Laboratory Animal Centre of East China University of Science and Technology (ECUST-2022-010).

Conflict of Interest

The authors declare no conflict of interest.

Data Availability Statement

The data that support the findings of this study are available in the supplementary material of this article.

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© 2024. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

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.

Details

Title
A H2S-Generated Supramolecular Photosensitizer for Enhanced Photodynamic Antibacterial Infection and Relieving Inflammation
Author
Tian, Jia 1   VIAFID ORCID Logo  ; Huang, Baoxuan 1 ; Xia, Lei 1 ; Zhu, Yucheng 1 ; Zhang, Weian 1   VIAFID ORCID Logo 

1  Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai, China 
Section
Research Articles
Publication year
2024
Publication date
Mar 2024
Publisher
John Wiley & Sons, Inc.
e-ISSN
21983844
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.1002/advs.202305183
ProQuest document ID
2937665849
Copyright
© 2024. This work is published under http://creativecommons.org/licenses/by/4.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.