Monosubstituted tricationic Zn(II) phthalocyanine enhances antimicrobial photodynamic inactivation (aPDI) of methicillin-resistant Staphylococcus aureus (MRSA) and cytotoxicity evaluation for topical applications: in vitro and in vivo study

Abstract

Antimicrobial photodynamic therapy (aPDT) is an innovative approach to combat multi-drug resistant bacteria. It is known that cationic Zn(II) phthalocyanines (ZnPc) are effective in mediating aPDT against methicillin-resistant Staphylococcus aureus (MRSA). Here we used ZnPc-based photosensitizer named ZnPcE previously reported by our research group to evaluate its aPDT efficacy against broad spectrum of clinically relevant MRSAs. Remarkably, in vitro anti-MRSA activity was achieved using near-infrared (NIR, >610 nm) light with minimal bactericidal concentrations ranging <0.019-0.156 µM against the panel of MRSAs. ZnPcE was not only significantly (p < .05) more potent than methylene blue, which is a clinically approved photosensitizer but also demonstrated low cytotoxicity against human fibroblasts cell line (Hs-27) and human immortalized keratinocytes cell line (HaCaT). The toxicity was further evaluated on human 3-D skin constructs and found ZnPcE did not manifest in vivo skin irritation at ≤7.8 µM concentration. In the murine MRSA wound model, ZnPcE with PDT group demonstrated > 4 log₁₀ CFU reduction and the value is significantly higher (p < .05) than all test groups except positive control. To conclude, results of present study provide a scientific basis for future clinical evaluation of ZnPcE-PDT on MRSA wound infection.

Keywords: Staphylococcus aureus (MRSA); Methicillin-resistant; antimicrobial photodynamic therapy (aPDT); cytotoxicity; murine wound infection model; phthalocyanine.

Figure 1.

Chemical structure of ZnPcE.

Figure 2.

Timeline for in vivo aPDT study. Four treatment cycles were performed at Day 2, 3, 5 and 9. After the 4th treatment, the mice were sacrificed for the CFU count.

Figure 3.

(a) Cytotoxicity of MB and ZnPcE on Hs-27 cells upon PDT (n = 3). (b) Cytotoxicity of MB and ZnPcE on Hs-27 cells under dark (n = 3). (c) Cytotoxicity of MB and ZnPcE on HaCaT cells upon PDT (n = 3). (d) Cytotoxicity of MB and ZnPcE on HaCaT cells under dark (n = 3). The LC₅₀ values of ZnPcE against both Hs-27 and HaCaT is well above 100 µM and the values are significantly higher (p < .05) than MB under dark conditions.

Figure 4.

Cell viability of EPI-200 cells treated with DPBS [Negative control (NC)], 5% SDS [Positive control (PC)], 7.8 µM of ZnPcE [equivalent to 100 × MBC against MRSA RN 4220/pUL5054 (ZnPcE-100)] and 0.78 µM of ZnPcE [equivalent to 10× MBC against MRSA RN 4220/pUL5054 (ZnPcE-10)]. Mean cell viability > 50% for the ZnPcE-100 and ZnPcE-10 implies ZnPcE did not pose any skin irritation for human 3-D skin construct at or below 7.8 µM concentration.

Figure 5.

In vivo aPDT efficiency against MRSA RN4220/pUL5054 infected wound mediated by 7.8 µM concentration of ZnPcE. Experimental data are expressed as mean ± SD (n = 6). Means that do not share a letter are significantly different. ZnPcE + PDT cohort showed significantly lower (p < .05) bacterial load after four treatment cycles, compared to all other treated groups, except positive control (2% Fusidic cream).