Antimicrobial photodynamic therapy has considerable promise in the fight against bacterial infections. The superior photophysical characteristics of porphyrins have made them effective photosensitizers in the field of phototherapy. Herein, the light-induced antimicrobial effects of three porphyrins with different substituents have been compared. 5,10,15,20-tetrakis(4-hydroxyphenyl) porphyrin (THPP) shows superior photosensitizing activity and antimicrobial ability under irradiation with green light. THPP can also inhibit and destroy mature Staphylococcus aureus biofilms under irradiation. This work provides a reference for the rational design of photosensitizers for application in antimicrobial photodynamic therapy.

Bacterial infections are now the second greatest cause of mortality worldwide,1 placing a huge burden on health systems.2–4 Antibiotics have long been recognized as an effective defensive line against serious infections; however, over-reliance on and abuse of antibiotics have resulted in the evolution of bacterial resistance5,6 and even the emergence of superbugs.7–9 Hence, it is crucial to develop novel antibiotics and explore alternate approaches to combat microbial infections.

In recent years, a multitude of antimicrobial materials have been developed, such as antimicrobial peptides,10–13 phages,14–17 and nano-antibacterial materials.18–21 Antimicrobial photodynamic therapy (APDT) is a novel antimicrobial therapy based on light at specific wavelengths and photosensitizers (PSs),22,23 which produces a photochemical reaction under irradiation, resulting in the generation of reactive oxygen species (ROS).24–26 The generated ROS can oxidatively disrupt the cell walls of bacteria, causing DNA and protein damage.27,28 This mechanism endows APDT with unique advantages over other antimicrobial therapies29–31 and makes it difficult for bacteria to develop resistance.32–34 

Porphyrin, as an endogenous substance with good biosafety and photosensitive activity, has become one of the most frequently used PSs in the biomedical field.35–38 Here, we selected three porphyrin compounds [5,10,15,20-tetrakis(4-hydroxyphenyl) porphyrin (THPP), 5,10,15,20-tetrakis(4-aminophenyl) porphyrin (TAPP), and 5,10,15,20-tetrakis(4-carboxyphenyl) porphyrin (TCPP)] with different substituent groups (–OH, –NH2, and –COOH) and investigated the influence of the molecular structures on their light-induced antimicrobial ability (Fig. 1). The ROS generation capacity, the inhibition effect toward bacterioplankton, and the disruption to biofilms of THPP, TAPP, and TCPP were studied and compared.

FIG. 1.

Schematic diagram of the photodynamic antibacterial performance of the porphyrins toward S. aureus under green light illumination.

FIG. 1.

Schematic diagram of the photodynamic antibacterial performance of the porphyrins toward S. aureus under green light illumination.

Close modal

The bacteria colony was transferred to Luria Broth medium and shaken for 6 h at 37 °C to reach the exponential growth phase. After centrifugation and washing with phosphate buffered saline (PBS), the bacteria were diluted with PBS to a concentration of 109 CFU ml−1 (OD600 of 1.0).

ROS generation in biofilms was investigated using 2,7-dichlorofluorescin diacetate (DCFH-DA) as the indicator. Staphylococcus aureus (S. aureus) (1 ml, 107 CFU ml−1) was cultured in a 24-well plate at 37 °C for 24 h to form biofilms. Subsequently, 5,10,15,20-tetrakis(4-hydroxyphenyl) porphyrin (THPP), 5,10,15,20-tetrakis(4-aminophenyl) porphyrin (TAPP), and 5,10,15,20-tetrakis(4-carboxyphenyl) porphyrin (TCPP) (500 µl, 10 µM) were added, and the biofilms were exposed to green LED light irradiation (18 mW cm−2) for 10 min. Then, they were stained with DCFH-DA for 30 min and washed with PBS. ROS generation in biofilms was visualized using a confocal laser scanning microscope (CLSM).

Various concentrations of THPP, TAPP, and TCPP were added to 5 × 105 CFU bacteria in 96-well plates. Subsequent to irradiation with or without green LED light (18 mW cm−2) for 10 min, the bacterial cultures were incubated at 37 °C for 20 h. Microplate reader measurements determined the OD values.

THPP, TAPP, and TCPP (0.5 µM) were mixed with S. aureus (5 × 105 CFU/ml) for growth profile analysis. The mixture underwent irradiation (18 mW cm−2, 10 min) or not, followed by incubation at 37 °C. During this incubation period, the OD value was measured at specific time points.

S. aureus (107 CFU ml−1) was cultured in 24-well plates at 37 °C for 24 h to form biofilms. Harvested biofilms were treated with THPP, TAPP, and TCPP, followed by 10 min of green LED light irradiation (18 mW cm−2). After 4 h, the bacteria were stained with SYTO 9 (SYTO 9 Green Fluorescent Nucleic Acid Stain) and propidium iodide (PI). Biofilm observations were conducted using CLSM.

THPP and TCPP were synthesized via reported methods.39,40 The structures of THPP and TCPP were validated by 1H nuclear magnetic resonance spectroscopy (Figs. S1 and S2, supplementary material). The absorption spectra of the three porphyrins at the same concentrations are shown in [Fig. 2(a)]. THPP, 5,10,15,20-tetrakis(4-aminophenyl) porphyrin (TAPP), and TCPP have maximum absorption at 425, 439, and 420 nm, respectively, in DMSO [Fig. 2(a)]. The fluorescence spectra of the three porphyrins are also different [Fig. 2(b)].

FIG. 2.

(a) Absorption spectra of THPP, TAPP, and TCPP (2.5 µM) in DMSO. (b) Fluorescence spectra of THPP, TAPP, and TCPP (1 µM) in DMSO. Absorption spectra of (c) THPP + DPBF, (d) TAPP + DPBF, and (e) TCPP + DPBF exposure to green light irradiation (18 mW cm−2) for different times. (f) The absorbance of DPBF at 417 nm varied over time due to ROS generation by the three porphyrins (0.5 µM) under green light irradiation (18 mW cm−2). (g) ROS generation in biofilms incubated with PBS, THPP, TAPP, or TCPP under irradiation (green light, 18 mW cm−2).

FIG. 2.

(a) Absorption spectra of THPP, TAPP, and TCPP (2.5 µM) in DMSO. (b) Fluorescence spectra of THPP, TAPP, and TCPP (1 µM) in DMSO. Absorption spectra of (c) THPP + DPBF, (d) TAPP + DPBF, and (e) TCPP + DPBF exposure to green light irradiation (18 mW cm−2) for different times. (f) The absorbance of DPBF at 417 nm varied over time due to ROS generation by the three porphyrins (0.5 µM) under green light irradiation (18 mW cm−2). (g) ROS generation in biofilms incubated with PBS, THPP, TAPP, or TCPP under irradiation (green light, 18 mW cm−2).

Close modal

The ROS generation ability of porphyrins under green light irradiation was detected by 1,3-diphenylisobenzofuran (DPBF). The absorbance of DPBF at 417 nm exhibited minimal change during green light irradiation (Fig. S3, supplementary material). Due to the generation of ROS by THPP, TAPP, and TCPP during light exposure, the absorbances of DPBF decrease with the increase in illumination time [Figs. 2(c)2(e)]. Under the same conditions, the absorbance of DPBF decreases more significantly after mixing with THPP than that with TAPP or TCPP [Fig. 2(f)], which suggests that THPP results in a higher ROS yield.

Next, we investigated the ROS generation capacities of the three porphyrins in living bacteria. The DCFH-DA probe was employed to detect the generation of ROS in S. aureus biofilms. The CLSM images indicate pronounced green fluorescence signals in the biofilm following treatment with THPP and green light irradiation, and the fluorescence signals are significantly higher than those in other treatment groups [Fig. 2(g)]. Meanwhile, the fluorescence of the biofilms subjected to PBS, TAPP, or TCPP treatment is negligible. These results imply that THPP has the potential to be applied to APDT.

The antimicrobial properties of THPP, TAPP, and TCPP against S. aureus were compared. A broth microdilution susceptibility test was first performed against S. aureus. As shown in Fig. 3(a), THPP significantly inhibits the growth of S. aureus under green light irradiation. At a concentration of 1 µM, the viability rates of TAPP and TCPP treated S. aureus under irradiation are 77.7% and 47%, respectively. However, for the bacteria without light irradiation, the growth of S. aureus was hardly inhibited by the porphyrins [Fig. 3(b)]. Moreover, the above-mentioned results were verified by an agar dilution test. In the dark groups, a substantial quantity of bacterial colonies can be observed. In the light groups, THPP treatment shows superior antimicrobial efficacy with the least number of colonies on agar plates compared to TAPP and TCPP treatments at the same concentration [Fig. 3(c)]. As shown in Fig. 3(c), THPP at a concentration of 1 µM can completely kill S. aureus under light irradiation.

FIG. 3.

The viabilities of S. aureus upon treatment with THPP, TAPP, or TCPP at different concentrations (a) with or (b) without exposure to green light. (c) Pictures of S. aureus colonies on agar plates after treatment with THPP, TAPP, or TCPP (0–1 µM) with/without green light irradiation (abbreviated as L+ and L−). ns: not significant, **p < 0.01, and ****p < 0.0001.

FIG. 3.

The viabilities of S. aureus upon treatment with THPP, TAPP, or TCPP at different concentrations (a) with or (b) without exposure to green light. (c) Pictures of S. aureus colonies on agar plates after treatment with THPP, TAPP, or TCPP (0–1 µM) with/without green light irradiation (abbreviated as L+ and L−). ns: not significant, **p < 0.01, and ****p < 0.0001.

Close modal

Furthermore, we monitored the growth of S. aureus after different treatments for 24 h. Bacteria were treated with THPP, TAPP, TCPP (0.5 µM), or PBS, then exposed to green light irradiation for 10 min. As shown in Fig. 4(a), bacterial proliferation could hardly be observed for the S. aureus treated with THPP and light irradiation, which indicates that the ROS generated by THPP can rapidly inactivate the bacteria and persistently inhibit their growth and reproduction. Meanwhile, in the dark groups, there is no difference in the growth of S. aureus after different treatments [Fig. 4(b)]. The destruction of S. aureus after light treatment was further observed by scanning electron microscopy (SEM). Scanning electron microscopy (SEM) observations further demonstrated the inactivation of S. aureus after light treatment. In Fig. 4(c), the bacteria in the PBS group retain their dense surfaces and intact morphologies. However, the bacteria in the THPP group lose their original morphological structures, and the bacterial membranes are disrupted by leakage of the cytoplasm. Part of the bacterial membranes in the TCPP group show distortion, and the bacteria in the TAPP group are relatively undisturbed. The SEM images are consistent with the results of the antimicrobial tests, which together confirm that the ROS generated by THPP under irradiation disrupts the original membrane structures of the bacteria, thus leading to the inactivation of the bacteria.

FIG. 4.

S. aureus growth curves under THPP, TAPP, or TCPP (0.5 µM) treatment (a) with and (b) without green light irradiation. (c) SEM images of S. aureus under different treatments. ns: not significant, ***p < 0.001, and ****p < 0.0001.

FIG. 4.

S. aureus growth curves under THPP, TAPP, or TCPP (0.5 µM) treatment (a) with and (b) without green light irradiation. (c) SEM images of S. aureus under different treatments. ns: not significant, ***p < 0.001, and ****p < 0.0001.

Close modal

Biofilms are organized bacterial communities consisting of bacterial aggregates and the extracellular matrix they secrete. Bacteria can adhere to biotic and abiotic surfaces to form biofilms under suitable conditions. Bacteria use biofilms for self-defense and further spread, which often leads to enhanced host immunity and antibiotic resistance in organisms.41–43 Considering that S. aureus is widely recognized as a prominent pathogen associated with biofilm infections,42 we further evaluated the ability of the three porphyrins to resist biofilms by crystalline violet staining and live/dead bacteria staining experiments. Biofilm formation of S. aureus in the dark groups was almost unaffected by the treatment of the porphyrins at a concentration of 8 µM. However, 8 µM of THPP could effectively inhibit the formation of S. aureus biofilm when exposed to green light [Fig. 5(a)]. Figure 5(b) is the quantification of crystal violet staining, which shows that the formation of biofilms can also be inhibited by TAPP and TCPP under irradiation to different degrees. We further calculated the inhibition rates of the three porphyrins on S. aureus biofilm formation, and the inhibition rates of THPP, TAPP, and TCPP were 72%, 37%, and 48%, respectively [Fig. 5(c)]. The ability of the porphyrins to eradicate mature biofilms was further analyzed via live/dead bacterial staining assays. In the 3D CLSM images, the biofilm in the THPP group exhibits significant red fluorescence [Fig. 5(d)], indicating the complete destruction of the biofilm after THPP treatment under light irradiation. Red fluorescent signals could also be observed in the biofilm in the TCPP group, whereas TAPP does not seem to be effective in the eradication of S. aureus biofilms. The above-mentioned results indicate that THPP can effectively inhibit the formation of biofilms and destroy mature biofilms after exposure to green light irradiation.

FIG. 5.

(a) Photographs of S. aureus biofilms stained by crystalline violet after treatments with PBS, THPP, TAPP, or TCPP with/without light irradiation. (b) Quantification of crystalline violet staining in (a). (c) Inhibition rate of THPP, TAPP, and TCPP combined with light irradiation toward biofilm formation in a crystalline violet staining assay. The bacteria in the control group were treated with PBS. (d) 3D CLSM images of S. aureus biofilms stained by SYTO and PI after different treatments.

FIG. 5.

(a) Photographs of S. aureus biofilms stained by crystalline violet after treatments with PBS, THPP, TAPP, or TCPP with/without light irradiation. (b) Quantification of crystalline violet staining in (a). (c) Inhibition rate of THPP, TAPP, and TCPP combined with light irradiation toward biofilm formation in a crystalline violet staining assay. The bacteria in the control group were treated with PBS. (d) 3D CLSM images of S. aureus biofilms stained by SYTO and PI after different treatments.

Close modal

Furthermore, we selected NIH 3T3 and L929 cells for (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays to evaluate the cytotoxicity of THPP, TAPP, and TCPP toward normal cells. As shown in Fig. 6, the survival rates of both cell lines exceed 90% when the concentrations of the porphyrins reach 1 µM, which implies that all three porphyrins have good biocompatibility.

FIG. 6.

Cytotoxicity of THPP, TAPP, and TCPP toward (a) L929 and (b) NIH 3T3 cells.

FIG. 6.

Cytotoxicity of THPP, TAPP, and TCPP toward (a) L929 and (b) NIH 3T3 cells.

Close modal

In summary, we evaluated the photodynamic antimicrobial effect of three porphyrin PSs (THPP, TAPP, and TCPP). Under green light irradiation, THPP can effectively eliminate S. aureus and has a smaller inhibitory concentration compared to the other two porphyrins. Furthermore, THPP can inhibit the growth of S. aureus biofilms and destroy the mature biofilms under green light irradiation. The superior photodynamic antimicrobial effect of THPP may stem from its stronger ROS generation efficiency. In view of the unique advantages of APDT, THPP promises to be a candidate PS for combating bacterial infections.

Experimental methods, including methods and instruments, will be described in the supplementary material. In addition, Figs. S1–S3 can be found there.

This work was supported by the Jilin Province Science and Technology Development Plan Project (Grant Nos. YDZJ202201ZYTS523/20230204085YY) and the National Natural Science Foundation of China (Grant No. 32271511).

The authors have no conflicts to disclose.

Shengman Yu: Formal analysis (lead); Investigation (lead); Validation (lead); Writing – original draft (lead). Jingwei Shi: Funding acquisition (lead); Project administration (equal); Supervision (equal). Tingting Sun: Conceptualization (equal); Resources (equal); Supervision (equal); Writing – review & editing (lead). Zhigang Xie: Conceptualization (lead); Resources (lead); Supervision (lead). Liyuan Sun: Funding acquisition (equal); Supervision (equal); Writing – review & editing (equal).

The data that support the findings of this study are available from the corresponding authors upon reasonable request.

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