Hypericin and Pheophorbide a Mediated Photodynamic Therapy Fighting MRSA Wound Infections: A Translational Study from In Vitro to In Vivo

Abstract

High prevalence rates of methicillin-resistant Staphylococcus aureus (MRSA) and lack of effective antibacterial treatments urge discovery of alternative therapeutic modalities. The advent of antibacterial photodynamic therapy (aPDT) is a promising alternative, composing rapid, nonselective cell destruction without generating resistance. We used a panel of clinically relevant MRSA to evaluate hypericin (Hy) and pheophobide a (Pa)-mediated PDT with clinically approved methylene blue (MB). We translated the promising in vitro anti-MRSA activity of selected compounds to a full-thick MRSA wound infection model in mice (in vivo) and the interaction of aPDT innate immune system (cytotoxicity towards neutrophils). Hy-PDT consistently displayed lower minimum bactericidal concentration (MBC) values (0.625-10 µM) against ATCC RN4220/pUL5054 and a whole panel of community-associated (CA)-MRSA compared to Pa or MB. Interestingly, Pa-PDT and Hy-PDT topical application demonstrated encouraging in vivo anti-MRSA activity (>1 log₁₀ CFU reduction). Furthermore, histological analysis showed wound healing via re-epithelization was best in the Hy-PDT group. Importantly, the dark toxicity of Hy was significantly lower (p < 0.05) on neutrophils compared to Pa or MB. Overall, Hy-mediated PDT is a promising alternative to treat MRSA wound infections, and further rigorous mechanistic studies are warranted.

Keywords: hypericin; methicillin-resistant Staphylococcus aureus; photodynamic therapy; wound infection model.

Figure 1

Chemical structures of 3 PSs used in the current investigation.

Figure 2

Timeline for the intravenous treatment.

Figure 3

Timeline for the topical treatment.

Figure 4

Bacterial load of wounds in different groups of mice after intravenous injection treatment with or without PDT. (A) Pa and Hy 2.5 mg/kg, irradiation for 30 s for PDT groups; (B) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (n = 5).

Figure 5

Wound areas in different groups of mice after intravenous injection treatment with or without PDT. (A) Pa and Hy 2.5 mg/kg irradiation for 30 s for PDT groups; (B) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (n = 5).

Figure 6

Body weight of different groups of mice after intravenous injection treatment with or without PDT. (A) Pa and Hy 2.5 mg/kg, irradiation for30 s for PDT groups; (B) Pa and Hy 2.5 mg/kg, irradiation for 10 min for PDT groups. Data are mean ± SEM (n=5).

Figure 7

Bacterial load of wounds in different groups of mice after topical treatment with or without PDT. Data are mean ± SEM (n = 6–10).* p < 0.05 and *** p < 0.001 indicated significant bacterial load difference between No treatment and treatment groups by Student’s t-test.

Figure 8

Wound areas of mice in different groups of mice after topical treatment with or without PDT. Data are mean ± SEM (n = 6–10).

Figure 9

Body weight of different groups of mice before and after treatment with or without PDT. Data are mean ± SEM (n = 6–10).

Figure 10

Representative wound section with haematoxylin and eosin stained and examined at ×100 magnification. Scale bar, 200 μm.

Figure 11

Cytotoxicity of Pa, MB and Hy, without light irradiation on human neutrophils isolated from buffy coat. Data are mean ± SEM (n = 3).

Figure 12

Cytotoxicity of Pa, MB and Hy, with light irradiation, on human neutrophils isolated from buffy coat with. Data are mean ± SEM (n = 3).